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United States Patent Application 20180169193
Kind Code A1
KHALILI; Kamel ;   et al. June 21, 2018

METHODS AND COMPOSITIONS FOR RNA-GUIDED TREATMENT OF HIV INFECTION

Abstract

Methods of inactivating a proviral DNA genome or a DNA genome integrated into the genome of a host cell latently infected with a retrovirus, by treating the host cell with a composition comprising a Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)-associated endonuclease, and two or more different multiplex guide RNAs (gRNAs), wherein each of the at least two gRNAs is complementary to a different target nucleic acid sequence in a long terminal repeat (LTR) of the proviral DNA that is unique from the genome of the host cell, cleaving a double strand of the proviral DNA at a first target protospacer sequence with the CRISPR-associated endonuclease, cleaving a double strand of the proviral DNA at a second target protospacer sequence with the CRISPR-associated endonuclease, excising an entire proviral genome of the proviral DNA, and eradicating the proviral DNA from the host cell.


Inventors: KHALILI; Kamel; (BalaCynwyd, PA) ; HU; Wenhui; (Cherry Hill, NJ)
Applicant:
Name City State Country Type

Temple University of the Commonwealth System of Higher Education

Philadelphia

PA

US
Assignee: Temple University of the Commonwealth System of Higher Education
Philadelphia
PA

Family ID: 1000003176618
Appl. No.: 15/879877
Filed: January 25, 2018


Related U.S. Patent Documents

Application NumberFiling DatePatent Number
14838057Dec 11, 20159925248
15879877
PCT/US14/53441Aug 29, 2014
14838057
61871626Aug 29, 2013
62018441Jun 27, 2014
62026103Jul 18, 2014

Current U.S. Class: 1/1
Current CPC Class: C12N 15/111 20130101; C12N 2320/30 20130101; C12N 2310/20 20170501; A61K 38/465 20130101; C12Y 301/21 20130101; A61K 35/12 20130101; A61K 48/00 20130101; C12N 7/00 20130101; C12N 9/22 20130101; A61K 9/0034 20130101; A61K 48/005 20130101; C12N 2740/16063 20130101; A61K 45/06 20130101
International Class: A61K 38/46 20060101 A61K038/46; C12N 15/11 20060101 C12N015/11; A61K 35/12 20060101 A61K035/12; A61K 48/00 20060101 A61K048/00; C12N 7/00 20060101 C12N007/00; C12N 9/22 20060101 C12N009/22; A61K 9/00 20060101 A61K009/00; A61K 45/06 20060101 A61K045/06

Goverment Interests



STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

[0001] This invention was made with U.S. government support under grant numbers R01MH093271, R01NS087971, and P30MH092177 awarded by the National Institutes of Health. The U.S. government may have certain rights in the invention.
Claims



1. A method of inactivating a proviral DNA genome integrated into the genome of a host cell latently infected with a retrovirus, including the steps of: treating the host cell with a composition comprising a Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)-associated endonuclease, and two or more different multiplex guide RNAs (gRNAs), wherein each of the at least two gRNAs is complementary to a different target nucleic acid sequence in a long terminal repeat (LTR) of the proviral DNA that is unique from the genome of the host cell; cleaving a double strand of the proviral DNA at a first target protospacer sequence with the CRISPR-associated endonuclease; cleaving a double strand of the proviral DNA at a second target protospacer sequence with the CRISPR-associated endonuclease; excising an entire proviral genome of the proviral DNA; and eradicating the proviral DNA from the host cell.

2. The method of claim 1, wherein said step of treating the host cell includes the steps of: exposing the host cell to a composition including an isolated nucleic acid encoding the CRISPR-associated endonuclease; an isolated nucleic acid sequence encoding a first gRNA having a first spacer sequence that is complementary to a first target protospacer sequence in a proviral DNA; and an isolated nucleic acid encoding a second gRNA having a second spacer sequence that is complementary to a second target protospacer sequence in the proviral DNA; expressing in the host cell the CRISPR-associated endonuclease, the first gRNA, and the second gRNA; assembling, in the host cell, a first gene editing complex including the CRISPR-associated endonuclease and the first gRNA; and a second gene editing complex including the CRISPR-associated endonuclease and the second gRNA; directing the first gene editing complex to the first target protospacer sequence by complementary base pairing between the first spacer sequence and the first target protospacer sequence; and directing the second gene editing complex to the second target protospacer sequence by complementary base pairing between the second spacer sequence and the second target protospacer sequence.

3. The method of claim 2, wherein at least one of the first target protospacer sequence and the second target protospacer sequence is situated within the U3 region of the LTR.

4. The method of claim 3, wherein the retrovirus is selected from the group consisting of human immunodeficiency virus-1 (HIV-1), HIV-2, simian immunodeficiency virus (SIV), feline immunodeficiency virus (FIV), bovine immunodeficiency virus (BIV), equine infectious anemia virus (EIAV), and caprine arthritis/encephalitis virus (CAEV).

5. The method of claim 4, wherein the retrovirus is HIV-1, and the first spacer sequence and the second spacer sequence each include a sequence complementary to a target protospacer sequence selected from the group consisting of SEQ ID NO: 96, SEQ ID NO: 121, SEQ ID NO: 87, and SEQ ID NO: 110.

6. The method of claim 4, wherein the retrovirus is HIV-1, and the first spacer sequence and the second spacer sequence include, respectively, a sequence complementary to the target protospacer sequences SEQ ID NO: 96 and SEQ ID NO: 121.

7. The method of claim 4, wherein the retrovirus is HIV-1, and the first spacer sequence and the second spacer sequence each include, respectively, a sequence complementary to the target protospacer sequences SEQ ID NO: 87 and SEQ ID NO: 110.

8. The method of claim 1, wherein the CRISPR-associated endonuclease is Cas9 or a human-optimized Cas9.

9. The method of claim 2, wherein the isolated nucleic acids encoding a CRISPR-associated endonuclease, the first gRNA, and the second gRNA, are encoded in at least one expression vector.

10. The method of claim 9, wherein the at least one expression vector is selected from the group consisting of a plasmid vector, a lentiviral vector, an adenoviral vector, and an adeno-associated virus vector.

11. The method of claim 1, wherein at least one of the gRNAs comprises a CRISPR RNA (crRNA) and a trans-activated small RNA (tracrRNA), which are expressed as separate nucleic acids.

12. The method of claim 1, wherein at least one of the gRNAs is engineered as an artificial fusion small guide RNA (sgRNA) comprised of a crRNA and a tracrRNA.

13. The method of claim 1, wherein said step of expressing in the host cell the CRISPR-associated endonuclease, the first gRNA, and the second gRNA, is further defined as stably expressing in the host cell the CRISPR-associated endonuclease, the first gRNA, and the second gRNA, and the method additionally includes the step of immunizing the host cell against new retroviral infection.

14. The method of claim 1, wherein the host cell latently infected with a retrovirus is chosen from the group consisting of a CD4+ T cell, a macrophage, a monocyte, a gut associated lymphoid cell, a microglial cell, and an astrocyte.

15. A method of inactivating a DNA genome integrated into the genome of a host cell, including the steps of: treating the host cell with a composition comprising a Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)-associated endonuclease, and two or more different multiplex guide RNAs (gRNAs), wherein each of the at least two gRNAs is complementary to a different target nucleic acid sequence of the DNA genome that is unique from the genome of the host cell; cleaving a double strand of the proviral DNA at a first target protospacer sequence with the CRISPR-associated endonuclease; cleaving a double strand of the proviral DNA at a second target protospacer sequence with the CRISPR-associated endonuclease; excising an entire DNA genome integrated into the genome of the host cell; and eradicating the DNA genome integrated into the genome of the host cell from the host cell.

16. The method of claim 15, wherein the DNA genome is a retrovirus.

17. The method of claim 15, wherein said step of treating the host cell includes the steps of: exposing the host cell to a composition including an isolated nucleic acid encoding the CRISPR-associated endonuclease; an isolated nucleic acid sequence encoding a first gRNA having a first spacer sequence that is complementary to a first target protospacer sequence in a DNA genome; and an isolated nucleic acid encoding a second gRNA having a second spacer sequence that is complementary to a second target protospacer sequence in the DNA genome; expressing in the host cell the CRISPR-associated endonuclease, the first gRNA, and the second gRNA; assembling, in the host cell, a first gene editing complex including the CRISPR-associated endonuclease and the first gRNA; and a second gene editing complex including the CRISPR-associated endonuclease and the second gRNA; directing the first gene editing complex to the first target protospacer sequence by complementary base pairing between the first spacer sequence and the first target protospacer sequence; and directing the second gene editing complex to the second target protospacer sequence by complementary base pairing between the second spacer sequence and the second target protospacer sequence.

18. The method of claim 17, wherein at least one of the first target protospacer sequence and the second target protospacer sequence is situated within the U3 region of the LTR.

19. The method of claim 16, wherein the retrovirus is selected from the group consisting of human immunodeficiency virus-1 (HIV-1), HIV-2, simian immunodeficiency virus (SIV), feline immunodeficiency virus (FIV), bovine immunodeficiency virus (BIV), equine infectious anemia virus (EIAV), and caprine arthritis/encephalitis virus (CAEV).

20. The method of claim 19, wherein the retrovirus is HIV-1, and the first spacer sequence and the second spacer sequence each include a sequence complementary to a target protospacer sequence selected from the group consisting of SEQ ID NO: 96, SEQ ID NO: 121, SEQ ID NO: 87, and SEQ ID NO: 110.

21. The method of claim 19, wherein the retrovirus is HIV-1, and the first spacer sequence and the second spacer sequence include, respectively, a sequence complementary to the target protospacer sequences SEQ ID NO: 96 and SEQ ID NO: 121.

22. The method of claim 19, wherein the retrovirus is HIV-1, and the first spacer sequence and the second spacer sequence each include, respectively, a sequence complementary to the target protospacer sequences SEQ ID NO: 87 and SEQ ID NO: 110.

23. The method of claim 15, wherein the CRISPR-associated endonuclease is Cas9 or a human-optimized Cas9.

24. The method of claim 17, wherein the isolated nucleic acids encoding a CRISPR-associated endonuclease, the first gRNA, and the second gRNA, are encoded in at least one expression vector.

25. The method of claim 24, wherein the at least one expression vector is selected from the group consisting of a plasmid vector, a lentiviral vector, an adenoviral vector, and an adeno-associated virus vector.

26. The method of claim 15, wherein at least one of the gRNAs comprises a CRISPR RNA (crRNA) and a trans-activated small RNA (tracrRNA), which are expressed as separate nucleic acids.

27. The method of claim 15, wherein at least one of the gRNAs is engineered as an artificial fusion small guide RNA (sgRNA) comprised of a crRNA and a tracrRNA.

28. The method of claim 15, wherein the host cell is chosen from the group consisting of a CD4+ T cell, a macrophage, a monocyte, a gut associated lymphoid cell, a microglial cell, and an astrocyte.

29. A method of inactivating a proviral DNA integrated into the genome of a host cell latently infected with a retrovirus, including the steps of: treating the host cell with a composition comprising a Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)-associated endonuclease, and two or more different guide RNAs (gRNAs), wherein each of the at least two gRNAs is complementary to a different target nucleic acid sequence in a long terminal repeat (LTR) of the proviral DNA; and inactivating the proviral DNA.

30. A method of inactivating a proviral DNA integrated into the genome of a host cell latently infected with a retrovirus, including the steps of: treating the host cell with a composition comprising a Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)-associated endonuclease, and two or more different guide RNAs (gRNAs), wherein each of the at least two gRNAs is complementary to a different target nucleic acid sequence in a long terminal repeat (LTR) of the proviral DNA; and excising the proviral DNA.
Description



BACKGROUND OF THE INVENTION

1. Technical Field

[0002] The present invention relates to compositions that specifically cleave target sequences in retroviruses, for example human immunodeficiency virus (HIV). Such compositions, which can include nucleic acids encoding a Clustered Regularly Interspace Short Palindromic Repeat (CRISPR) associated endonuclease and a guide RNA sequence complementary to a target sequence in a human immunodeficiency virus, can be administered to a subject having or at risk for contracting an HIV infection.

2. Background Art

[0003] For more than three decades since the discovery of HIV-1, AIDS remains a major public health problem affecting greater than 35.3 million people worldwide. AIDS remains incurable due to the permanent integration of HIV-1 into the host genome. Current therapy (highly active antiretroviral therapy or HAART) for controlling HIV-1 infection and impeding AIDS development profoundly reduces viral replication in cells that support HIV-1 infection and reduces plasma viremia to a minimal level. But HAART fails to suppress low level viral genome expression and replication in tissues and fails to target the latently-infected cells, for example, resting memory T cells, brain macrophages, microglia, and astrocytes, gut-associated lymphoid cells, that serve as a reservoir for HIV-1. Persistent HIV-1 infection is also linked to co-morbidities including heart and renal diseases, osteopenia, and neurological disorders. There is a continuing need for curative therapeutic strategies that target persistent viral reservoirs.

SUMMARY OF THE INVENTION

[0004] The present invention provides for a method of inactivating a proviral DNA genome integrated into the genome of a host cell latently infected with a retrovirus, by treating the host cell with a composition comprising a Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)-associated endonuclease, and two or more different multiplex guide RNAs (gRNAs), wherein each of the at least two gRNAs is complementary to a different target nucleic acid sequence in a long terminal repeat (LTR) of the proviral DNA that is unique from the genome of the host cell, cleaving a double strand of the proviral DNA at a first target protospacer sequence with the CRISPR-associated endonuclease, cleaving a double strand of the proviral DNA at a second target protospacer sequence with the CRISPR-associated endonuclease, excising an entire proviral genome of the proviral DNA, and eradicating the proviral DNA from the host cell.

[0005] The present invention also provides for a method of inactivating a DNA genome integrated into the genome of a host cell, by treating the host cell with a composition comprising a Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)-associated endonuclease, and two or more different multiplex guide RNAs (gRNAs), wherein each of the at least two gRNAs is complementary to a different target nucleic acid sequence of the DNA genome that is unique from the genome of the host cell, cleaving a double strand of the proviral DNA at a first target protospacer sequence with the CRISPR-associated endonuclease, cleaving a double strand of the proviral DNA at a second target protospacer sequence with the CRISPR-associated endonuclease, excising an entire DNA genome integrated into the genome of the host cell, and eradicating the DNA genome integrated into the genome of the host cell from the host cell.

[0006] The present invention provides for a method of inactivating a proviral DNA integrated into the genome of a host cell latently infected with a retrovirus, by treating the host cell with a composition comprising a Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)-associated endonuclease, and two or more different guide RNAs (gRNAs), wherein each of the at least two gRNAs is complementary to a different target nucleic acid sequence in a long terminal repeat (LTR) of the proviral DNA, and inactivating the proviral DNA.

[0007] The present invention further provides for a method of inactivating a proviral DNA integrated into the genome of a host cell latently infected with a retrovirus, by treating the host cell with a composition comprising a Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)-associated endonuclease, and two or more different guide RNAs (gRNAs), wherein each of the at least two gRNAs is complementary to a different target nucleic acid sequence in a long terminal repeat (LTR) of the proviral DNA, and excising the proviral DNA.

[0008] The details of one or more embodiments of the invention are set forth in the accompanying drawings and the description below. Other features, objects, and advantages of the invention will be apparent from the description and drawings, and from the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

[0009] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0010] FIG. 1A, FIG. 1B, FIG. 1C, FIG. 1D, FIG. 1E, FIG. 1F, FIG. 1G, and

[0011] FIG. 1H show that Cas9/LTR-gRNA suppresses HIV-1 reporter virus production in CHME5 microglial cells latently infected with HIV-1. FIG. 1A shows a representative gating diagram of EGFP flow cytometry shows a dramatic reduction in TSA-induced reactivation of latent pNL4-3-.DELTA.Gag-d2EGFP reporter virus by stably expressed Cas9 plus LTR-A or -B, vs. empty U6-driven gRNA expression vector (U6-CAG). FIG. 1B shows SURVEYOR Cel-I nuclease assay of PCR product (-453 to +43 within LTR) from selected LTR-A- or -B-expressing stable clones shows dramatic indel mutation patterns (arrows). FIG. 1C shows a PCR fragment analysis of a precise deletion of 190-bp region between LTRs A and B cutting sites (arrowhead and arrow in FIG. 1D), leaving 306-bp fragment (arrow in FIG. 1C) validated by TA-cloning and sequencing results. FIG. 1D discloses SEQ ID NOS 1-3, respectively, in order of appearance. FIG. 1E is a graph showing subcloning of LTR-A/B stable clones reveals complete loss of reporter reactivation determined by EGFP flow cytometry, and FIG. 1F shows elimination of pNL4-3-.DELTA.Gag-d2EGFP proviral genome detected by standard, and FIG. 1G shows real-time PCR amplification of genomic DNA for EGFP and HIV-1 Rev response element (RRE); .beta.-actin is a DNA purification and loading control. FIG. 1H shows PCR genotyping of LTR-A/B subclones (#8, 13) using primers to amplify DNA fragment covering HIV-1 LTR U3/R/U5 regions (-411 to +129) shows indels (a, deletion; c, insertion) and "intact" or combined LTR (b).

[0012] FIG. 2A, FIG. 2B, and FIG. 2C show that Cas9/LTR-gRNA efficiently eradicates latent HIV-1 virus from U1 monocytic cells. FIG. 2A shows a diagram showing excision of HIV-1 entire genome in chromosome Xp11.4. HIV-1 integration sites were identified using a Genome-Walker link PCR kit. Left, analysis of PCR amplicon lengths using a primer pair (P1/P2) targeting chromosome X integration site-flanking sequence reveals elimination of the entire HIV-1 genome (9709-bp), leaving two fragments (833- and 670-bp). FIG. 2B shows TA cloning and sequencing of the LTR fragment (833-bp) showing the host genomic sequence (small letters, 226-bp) and the partial sequences (634-27=607 bp) of 5'-LTR (underlined using dashes) and 3'-LTR (first underlined section) with a 27-bp deletion around the LTR-A targeting site (second underlined section). Bottom, two indel alleles identified from 15 sequenced clonal amplicons. The 670-bp fragment consists of a host sequence (226-bp) and the remaining LTR sequence (634-190=444 bp) after 190-bp excision by simultaneous cutting at LTR-A and B target sites. The underlined and highlighted sequences indicate the gRNA LTR-A target site and PAM.

[0013] FIG. 2B discloses SEQ ID NOS 4-13, respectively, in order of appearance. FIG. 2C shows a functional analysis of LTR-A/B-induced eradication of HIV-1 genome, showing substantial blockade of TSA/PMA reactivation-induced p24 virion release. U1 cells were transfected with pX260-LTRs-A, -B, or -A/B. After 2-week puromycin selection, cells were treated with TSA (250 nM)/PMA for 2 days before p24 Gag ELISA was performed.

[0014] FIG. 3A, FIG. 3B, FIG. 3C, and FIG. 3D show that stable expression of Cas9 plus LTR-A/B vaccinates TZM-bl cells against new HIV-1 virus infection. FIG. 3A shows immunohistochemistry (ICC) and Western blot (WB) analyses with anti-Flag antibody confirm the expression of Flag-Cas9 in TZM-bl stable clones puromycin (2 .mu.g/ml)-selected for 2 weeks. FIG. 3B shows PCR genotyping of Cas9/LTR-A/B stable clones (c1-c7) reveals a close correlation of LTR excision with repression of LTR luciferase reporter activation. Fold changes represent TSA/PMA-induced levels over corresponding non-induction levels. FIG. 3C shows Cas9/LTR-A/B-expressing cells (c4) were infected with pseudotyped-pNL4-3-Nef-EGFP lentivirus at indicated multiplicity of infection (MOI) and infection efficiency measured by EGFP flow cytometry, 2 d post-infection. FIG. 3D shows phase-contrast/fluorescence micrographs show that LTR-A/B stable, but not control (U6-CAG; black) cells, are resistant to new infection (right panel) by pNL4-3-.DELTA.E-EGFP HIV-1 reporter virus (gray).

[0015] FIG. 4A, FIG. 4B, FIG. 4C, and FIG. 4D illustrate the off-target effects of Cas9/LTR-A/B on the human genome. FIG. 4A is a SURVEYOR assay that shows no indel mutations in predicted/potential off-target regions in human TZM-bl and U1 cells. LTR-A on-target region (A) was used as a positive control and empty U6-CAG vector (U6) as a negative control. FIG. 4B shoes whole-genome sequencing of LTR-A/B stable TZM-bl subclone showing the numbers of called indels in the U6-CAG control and LTR-A/B samples, FIG. 4C shows detailed information on 10 called indels near gRNA target sites in both samples, and FIG. 4D shows distribution of off-target called indels. FIG. 4C discloses SEQ ID NOS 14-15, respectively, in order of appearance.

[0016] FIG. 5 shows the LTR U3 sequence of the integrated lentiviral LTR-firefly luciferase reporter identified by TA-cloning and sequencing of PCR product (-411 to -10) from the genomic DNA of human TZM-bl cells. The protospacer and PAM (NGG) sequences of 4 gRNAs (LTR-A to D) and the predicted binding sites of indicated transcription factors are highlighted. The precise cleavage sites are marked with scissors. +1 indicates the transcriptional start site. FIG. 5 discloses SEQ ID NO: 16.

[0017] FIG. 6A, FIG. 6B, and FIG. 6C show that LTR-C and LTR-D remarkably suppress TSA-induced reactivation of latent pNL4-3-.DELTA.Gag-d2EGFP virus in CHME5 microglia cells. FIG. 6A is a diagram schematically showing pNL4-3-.DELTA.Gag-d2EGFP vector containing Tat, Rev, Env, Vpu, and Nef with the reporter gene d2EGFP. FIG. 6B shows a SURVEYOR assay showing indel mutations in the on-target LTR genome of Cas9/LTR-D but not Cas9/LTR-C transfected cells. FIG. 6C shows a representative gating diagram of EGFP flow cytometry showing a dramatic reduction in TSA-induced reactivation of latent pNL4-3-.DELTA.Gag-d2EGFP reporter viruses by stable expression of Cas9/LTR-C or LTR-D as compared with empty U6-driven gRNA expression vector (U6-CAG).

[0018] FIG. 7A, FIG. 7B, FIG. 7C, FIG. 7D, FIG. 7E, and FIG. 7F show that both LTR-C and LTR-D induced indel mutations and significantly decreased constitutive and TSA/PMA-induced luciferase activity in TZM-bl cells stably incorporated with HIV-1 LTR-firefly luciferase reporter gene. FIG. 7A shows a functional luciferase reporter assay revealing a significant reduction of LTR reactivation by LTR-C, LTR-D or both. FIG. 7B shows a SURVEYOR assay showing indel mutation in LTR DNA (-453 to +43) induced by LTR-C and LTR-D (upper arrow). A combination of LTR-C and LTR-D generates a 194 bp fragment (lower arrow) resulting from the deletion of 302 bp region between LTR-C and LTR-D. FIG. 7C and FIG. 7D show Sanger sequencing of 30 clones validating the indel efficiency at 23% for LTR-C and 13% for LTR-D and example chromatograms showing insertion/deletion. FIG. 7C discloses SEQ ID NOS 17-25, respectively, in order of appearance. FIG. 7D discloses SEQ ID NOS 26-30, respectively, in order of appearance. FIG. 7E shows PCR-restriction fragment length polymorphism (RFLP) analysis using BsaJ I to cut 5 sites (96, 102, 372, 386, 482) of the PCR product covering -453 to +43 of LTR showing two major bands (96 bp and 270 bp) in the U6-CAG control sample, but an additional 372 bp band (upper arrow) after LTR-C-induced indel mutation at the 96/102 sites, a 290 bp band (middle arrow) after LTR-D-induced mutations at the 372 site and a 180 bp fragment (lower arrow) after LTR-C/D-induced excision. FIG. 7F shows chromatograms showing the deletion of a 302 bp fragment between LTR-C and LTR-D (top) and an additional 17 bp deletion (bottom). Red arrows indicate the junction sites. *P<0.05 indicates a significant decrease in LTR-C or LTR-D-mediated luciferase activation compared to U6-CAG control. FIG. 7F discloses SEQ ID NOS 31-32, respectively, in order of appearance.

[0019] FIG. 8A, FIG. 8B, and FIG. 8C illustrate the TA cloning and Sanger sequencing of PCR products from CHME5 subclones of LTR-A/B and empty U6-CAG control using primers covering HIV-1 LTR U3/R/U5 regions (-411 to +129). FIG. 8A shows possible combination of LTR-A and LTR-B cuts on both 5'- and 3'-LTRs generating potential fragments a-c as indicated. FIG. 8B shows blasting of fragment a (351 bp) showing 190 bp deletion between LTR-A and LTR-B cut sites. FIG. 8C shows a blast of fragment c (682 bp) showing a 175 bp insertion at the LTR-A cleavage site and a 27 bp deletion at the LTR-B cleavage site. FIG. 8C discloses SEQ ID NOS 33-34, respectively, in order of appearance.

[0020] FIG. 9A, FIG. 9B, FIG. 9C, and FIG. 9D demonstrate that Cas9/LTR-gRNA efficiently eradicates latent HIV-1 virus from U1 monocytic cells. FIG. 9A shows a Sanger sequencing of a 1.1 kb fragment from long-range PCR using a primer pair (T492/T493) targeting a chromosome 2 integration site-flanking sequence (small letters, 467-bp) reveals elimination of the entire HIV-1 genome (9709-bp), leaving combined 5'-LTR (underlined using dashes) and 3'-LTR with a 6-bp insertion (boxed) precisely at the third nucleotide from PAM (TGG) LTR-A targeting site (underlined) and a 4-bp deletion (nnnn). FIG. 9A discloses SEQ ID NO: 35. FIG. 9B is a representative DNA gel picture that shows specific eradication of the HIV-1 genome. NS, non-specific band. FIG. 9C is a graph and FIG. 9D is a graph showing quantitative PCR analysis using the primer pair targeting the Gag gene (T457/T458) shows 85% efficiency of entire HIV-1 genome eradication in Cas9/LTR-A/B-expressing U1 cells. U1 cells were transfected with pX260 empty vector (U6-CAG) or LTRs-A/B-encoding vectors. After 2-week puromycin selection, the cellular genomic DNAs were used for absolute quantitative qPCR analysis using spiked pNL4-3-.DELTA.E-EGFP human genomic DNA as a standard. **P<0.01 indicates a significant decrease compared to the U6-CAG control.

[0021] FIG. 10A, FIG. 10B, and FIG. 10C show that Cas9/LTR gRNAs effectively eradicates HIV-1 provirus in J-Lat latently infected T cells. FIG. 10A shows functional analysis by EGFP flow cytometry reveals approximately 50% reduction of PMA and TNF.alpha.-induced reactivation of EGFP reporter viruses. FIG. 10B is a SURVEYOR assay that shows indel mutations (arrow) in the on-target LTR genome of Cas9/LTR-A/B transfected cells. J-Lat cells were transfected with pX260 empty vector or LTRs-A and -B. After 2-week puromycin selection, cells were treated with PMA or TNF.alpha. for 24 h. The genomic DNAs were subject to PCR using primers covering HIV-1 LTR U3/R/U5 regions (-411 to +129) and the SURVEYOR assay was performed. **P<0.01 indicates a significant decrease compared to the U6-CAG control. FIG. 10C shows a PCR fragment analysis using primers covering HIV-1 LTR (-374 to +43) shows a precise deletion of 190-bp region between LTRs A and B cutting sites, leaving 227-bp fragment (arrow). House-keeping gene .beta.-actin serves as a DNA purification and loading control.

[0022] FIG. 11A, FIG. 11B, FIG. 11C, and FIG. 11D show that genome editing efficiency depends upon the presence of Cas9 and gRNAs. FIG. 11A shows PCR genotyping reveals the absence of a U6-driven LTR-A or LTR-B expression cassette and FIG. 11B shows absence/reduction of CMV-driven Cas9 DNA in puromycin-selected TZM-bl subclones without any indication of genomic editing. Genomic DNAs from indicated subclones were subject to conventional (FIG. 11A) or real-time (FIG. 11B) PCR analyses using a primer pair covering U6 promoter (T351) and LTR-A (T354) or -B (T356), and targeting Cas9 (T477/T491). FIG. 11C and FIG. 11D show Cas9 protein expression is absent in ineffective TZM-bl subclones. FIG. 11C shows that the Flag-tagged Cas9 fusion protein was detected by Western blot (WB) and immunocytochemistry (ICC) with anti-Flag monoclonal antibody. HEK293T cell line stably expressing Flag-Cas9 was used as a positive control for WB. GAPDH serves as a protein loading control. Clone c6 contains Cas9 DNA but no Cas9 protein expression, suggesting a potential mechanism of epigenetic repression after puromycin selection. Clone c5 and c3 may represent a truncated Flag-Cas9 (tCas9). FIG. 11D shows that the nucleus was stained with Hoechst 33258.

[0023] FIG. 12A, FIG. 12B, FIG. 12C, and FIG. 12D demonstrate that stable expression of Cas9/LTR-A/B gRNAs in TZM-bl cells vaccinates against pseudotyped or native HIV-1 viruses. FIG. 12 shows that flow cytometry shows a significant reduction of native pNL4-3-.DELTA.E-EGFP reporter virus infection efficiency in Cas9/LTR-A/B expressing TZM-bl subclones. Real-time PCR analysis reveals suppression or elimination of viral RNA as shown in FIG. 12B and DNA as shown in FIG. 12C by Cas9/LTR-A/B gRNAs. FIG. 12D shows that the firefly-luciferase luminescent assay demonstrates dramatic inhibition of virus infection-stimulated LTR promoter activity by Cas9/LTR-A/B gRNAs. The stable Cas9/LTR-A/B gRNA-expressing TZM-bl cells were infected for 2 hours with indicated native HIV-1 viruses, and washed twice with PBS. At 2 days post-infection, cells were collected, fixed and analyzed by flow cytometry for EGFP expression (in FIG. 12A), or lysed for total RNA extraction and RT-qPCR (in FIG. 12B), genomic DNA purification for qPCR (in FIG. 12C) and luminescence measurement (in FIG. 12D). *P<0.05 and **P<0.01 indicate significant decreases compared to the U6-CAG control.

[0024] FIG. 13 shows the predicted LTR gRNAs and their off-target numbers (100% match). The 5'-LTR sense and antisense sequences (SEQ ID NOS 79-111 and 112-141, respectively) (634 bp) of pHR'-CMV-LacZ lentiviral vector (AF105229) were utilized to search for Cas9/gRNA target sites containing a 20-bp guide sequence (protospacer) plus the protospacer adjacent motif sequence (NGG) using Jack Lin's CRISPR/Cas9 gRNA finder tool (http://spot.colorado.edu/.sup..about.slin/cas9.html). Each gRNA plus NGG (AGG, TGG, GGG, CGG) was blasted against available human genomic and transcript sequences with 1000 aligned sequences being displayed. After pressing Control+F, copy/paste the target sequence (1-23 through 9-23 nucleotides) and find the number of genomic targets with 100% match. The number of off-targets for each searching was divided by 3 because of repeated genome library. The number shown indicates the sum of 4 searches (NGG). The top number (for example, for gRNA sequence (sense): 20, 19, 19, 17, 16, 15, 14, 13, 12) indicates the gRNA target sequences farthest from NGG. The sequence and off-target numbers for the selected LTR-A/B and LTR-C/D are highlighted red and green respectively.

[0025] FIG. 14 depicts the oligonucleotides for gRNA targeting sites and primers (SEQ ID NOS 36-78, respectively, in order of appearance) used for PCR and sequencing.

[0026] FIG. 15 shows the locations of predicted gRNA targeting sites of LTR-A and LTR-B and discloses "query Seq" sequences as SEQ ID NOS 142-252, and "ref Seq" sequences as SEQ ID NOS 253-363, all respectively, in order of appearance.

[0027] FIG. 16A, FIG. 16B, FIG. 16C, FIG. 16D, FIG. 16E, FIG. 16F, FIG. 16G, and FIG. 16H show that both LTR-C and LTR-D decreased constitutive and TSA/PMA-induced luciferase activity in TZMBI cells stably incorporated with HIV-1 LTR firefly luciferase reporter gene and combination induced precise genome excision. FIG. 16A shows that six gRNA targets were designed for the promoter region of HIV-LTR. FIG. 16A discloses SEQ ID NO: 16. TZMBI cells were cotransfected with Cas9-EGFP and chimera gRNA expression cassette (PCR products) by lipofectamine 2000. FIG. 16B is a graph showing that after 3 d, EGFP-positive cells were sorted through FACS and 2000 cells per group were collected for luciferase assay. FIG. 16B discloses SEQ ID: 31. FIG. 16C is a graph showing the population sorted cells were cultured for 2 d and treated with TSA/PMA for 1 d before luciferase assay. The single cells were sorted into 96-well plate and cultured till confluence for luciferase assay in the absence (shown in the graph of FIG. 16D) of TSA/PMA for 1 d or presence (shown in the graph of FIG. 1E) of TSA/PMA for 1 d. FIG. 16F and FIG. 16G show the PCR product from the population sorted cells were analyzed with Surveyor Cel-I nuclease assay and restriction fragment length polymorphism with Bsajl (FIG. 16G) showing mutation (FIG. 16F) or uncut (FIG. 16G) band (red arrow). A 200 bp fragment (FIG. 16F, FIG. 16G, black arrow) resulting from the deletion of 321 bp region between LTR-C and LTR-D as predicted (FIG. 16A, red arrowhead) was validated by TA-cloning and sequencing showing precise genomic excision (FIG. 16H). Sanger sequencing of PCR products from individual LTR-C and -D identified % and % indel mutation efficiency respectively. *p<0.05 indicates statistically significant reduction using a student's t test compared to the corresponding U6-CAG control. Protospace(E), Protospace(C), Protospace(A), Protospace(B), Protospace(D), and Protospace(F) correspond to SEQ ID NOS 365, 367, 369, 371, 373, and 375, respectively, in order of appearance.

[0028] FIG. 17A, FIG. 17B, FIG. 17C, FIG. 17D, FIG. 17E, FIG. 17F, FIG. 17G, and FIG. 17H show that Cas9/LTR-gRNA inhibited constitutive and inducible production of HIV-1 virus measured by EGFP flow cytometry in HIV-1 latently infected CHME5 microglia cell line. The pHR' lentiviral vector containing Tat, Rev, Env, Vpu, and Nef with the reported gene d2EGFP was transduced into human fetal microglia cell line CHME5 and 400 bp deletion in U3 region of 3'-LTR is illustrated (shown in FIG. 17A). FIG. 17B is a graph showing transient transfection of Cas9/gRNA, Human HIV-1 LTR-A, B alone or combination decreased the intensity but not percentage of EGFP due to suppression of LTR promoter activity. FIG. 17C is a graph showing transient transfection of Cas9/gRNA, Human HIV-1 LTR-C, D alone or combination decreased the intensity but not percentage of EGFP due to suppression of LTR promoter activity. FIG. 17D and FIG. 18 are graphs showing that after antibiotic selection for 1-2 weeks, the percentage of EGFP cells was also reduced. FIG. 17F and FIG. 17G show the PCR product from the stable selected clones were analyzed with Surveyor Cel-I nuclease assay showing indel mutation dramatically in LTR-A and LTR-B but weakly in the combination of LTR-A/B (red arrow). A 331 bp fragment (shown in FIG. 17F and FIG. 17G, black arrow) resulting from the deletion of 190 bp region between LTR-A and LTR-B as predicted (FIG. 17H, red arrowhead) was validated by TA-cloning and sequencing showing precise genomic excision (FIG. 17H). FIG. 17H discloses SEQ ID NOS 1-3, respectively, in order of appearance.

[0029] FIG. 18 shows LTR of a representative HIV-1 sequence (SEQ ID NO: 376). The U3 region extends from nucleotide 1 to nucleotide 432 (SEQ ID NO: 377), the R region extends from nucleotide 432 to nucleotide 559 (SEQ ID NO: 378), and the U5 region extends from 560 to nucleotide 634 (SEQ ID NO: 379).

[0030] FIG. 19 shows LTR of a representative SIV sequence (SEQ ID NO: 380). The U3 region extends from nucleotide 1 to nucleotide 517 (SEQ ID NO: 381), the R region extends from nucleotide 518 to nucleotide 693 (SEQ ID NO: 382), and the U5 region extends from 694 to nucleotide 818 (SEQ ID NO: 383).

DETAILED DESCRIPTION OF THE INVENTION

[0031] The present invention is based, in part, on our discovery that we could eliminate the integrated HIV-1 genome from HIV-1 infected cells by using the RNA-guided Clustered Regularly Interspace Short Palindromic Repeat (CRISPR)-Cas 9 nuclease system (Cas9/gRNA) in single and multiplex configurations. We identified highly specific targets within the HIV-1 LTR U3 region that were efficiently edited by Cas9/gRNA, inactivating viral gene expression and replication in latently-infected microglial, promonocytic and T cells. Cas9/gRNAs caused neither genotoxicity nor off-target editing to the host cells, and completely excised a 9709-bp fragment of integrated proviral DNA that spanned from its 5'- to 3'-LTRs. Furthermore, the presence of multiplex gRNAs within Cas9-expressing cells prevented HIV-1 infection. Our results suggest that Cas9/gRNA can be engineered to provide a specific, efficacious prophylactic and therapeutic approach against AIDS.

[0032] Accordingly, the invention features compositions comprising a nucleic acid encoding a CRISPR-associated endonuclease and a guide RNA that is complementary to a target sequence in a retrovirus, e.g., HIV, as well as pharmaceutical formulations comprising a nucleic acid encoding a CRISPR-associated endonuclease and a guide RNA that is complementary to a target sequence in HIV. Also featured are compositions comprising a CRISPR-associated endonuclease polypeptide and a guide RNA that is complementary to a target sequence in HIV, as well as pharmaceutical formulations comprising a CRISPR-associated endonuclease polypeptide and a guide RNA that is complementary to a target sequence in HIV.

[0033] Also featured are methods of administering the compositions to treat a retroviral infection, e.g., HIV infection, methods of eliminating viral replication, and methods of preventing HIV infection. The therapeutic methods described herein can be carried out in connection with other antiretroviral therapies (e.g., HAART).

[0034] The clinical course of HIV infection can vary according to a number of factors, including the subject's genetic background, age, general health, nutrition, treatment received, and the HIV subtype. In general, most individuals develop flu-like symptoms within a few weeks or months of infection. The symptoms can include fever, headache, muscle aches, rash, chills, sore throat, mouth or genital ulcers, swollen lymph glands, joint pain, night sweats, and diarrhea. The intensity of the symptoms can vary from mild to severe depending upon the individual. During the acute phase, the HIV viral particles are attracted to and enter cells expressing the appropriate CD4 receptor molecules. Once the virus has entered the host cell, the HIV encoded reverse transcriptase generates a proviral DNA copy of the HIV RNA and the pro-viral DNA becomes integrated into the host cell genomic DNA. It is this HIV provirus that is replicated by the host cell, resulting in the release of new HIV virions which can then infect other cells. The methods and compositions of the invention are generally and variously useful for excision of integrated HIV proviral DNA, although the invention is not so limited, and the compositions may be administered to a subject at any stage of infection or to an uninfected subject who is at risk for HIV infection.

[0035] The primary HIV infection subsides within a few weeks to a few months, and is typically followed by a long clinical "latent" period which may last for up to 10 years. The latent period is also referred to as asymptomatic HIV infection or chronic HIV infection. The subject's CD4 lymphocyte numbers rebound, but not to pre-infection levels and most subjects undergo seroconversion, that is, they have detectable levels of anti-HIV antibody in their blood, within 2 to 4 weeks of infection. During this latent period, there can be no detectable viral replication in peripheral blood mononuclear cells and little or no culturable virus in peripheral blood. During the latent period, also referred to as the clinical latency stage, people who are infected with HIV may experience no HIV-related symptoms, or only mild ones. But, the HIV virus continues to reproduce at very low levels. In subjects who have treated with anti-retroviral therapies, this latent period may extend for several decades or more. However, subjects at this stage are still able to transmit HIV to others even if they are receiving antiretroviral therapy, although anti-retroviral therapy reduces the risk of transmission. As noted above, anti-retroviral therapy does not suppress low levels of viral genome expression nor does it efficiently target latently infected cells such as resting memory T cells, brain macrophages, microglia, astrocytes and gut associated lymphoid cells.

[0036] Clinical signs and symptoms of AIDS (acquired immunodeficiency syndrome) appear as CD4 lymphocyte numbers decrease, resulting in irreversible damage to the immune system. Many patients also present with AIDS-related complications, including, for example, opportunistic infections such as tuberculosis, salmonellosis, cytomegalovirus, candidiasis, cryptococcal meningitis, toxoplasmosis, and cryptosporidiosis, as well as certain kinds of cancers, including for example, Kaposi's sarcoma, and lymphomas, as well as wasting syndrome, neurological complications, and HIV-associated nephropathy.

[0037] Compositions

[0038] The compositions of the invention include nucleic acids encoding a CRISPR-associated endonuclease, e.g., Cas9, and a guide RNA that is complementary to a target sequence in a retrovirus, e.g., HIV. In bacteria the CRISPR/Cas loci encode RNA-guided adaptive immune systems against mobile genetic elements (viruses, transposable elements and conjugative plasmids). Three types (I-III) of CRISPR systems have been identified. CRISPR clusters contain spacers, the sequences complementary to antecedent mobile elements. CRISPR clusters are transcribed and processed into mature CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) RNA (crRNA). The CRISPR-associated endonuclease, Cas9, belongs to the type II CRISPR/Cas system and has strong endonuclease activity to cut target DNA. Cas9 is guided by a mature crRNA that contains about 20 base pairs (bp) of unique target sequence (called spacer) and a trans-activated small RNA (tracrRNA) that serves as a guide for ribonuclease III-aided processing of pre-crRNA. The crRNA:tracrRNA duplex directs Cas9 to target DNA via complementary base pairing between the spacer on the crRNA and the complementary sequence (called protospacer) on the target DNA. Cas9 recognizes a trinucleotide (NGG) protospacer adjacent motif (PAM) to specify the cut site (the 3rd nucleotide from PAM). The crRNA and tracrRNA can be expressed separately or engineered into an artificial fusion small guide RNA (sgRNA) via a synthetic stem loop (AGAAAU) to mimic the natural crRNA/tracrRNA duplex. Such sgRNA, like shRNA, can be synthesized or in vitro transcribed for direct RNA transfection or expressed from U6 or H1-promoted RNA expression vector, although cleavage efficiencies of the artificial sgRNA are lower than those for systems with the crRNA and tracrRNA expressed separately.

[0039] The compositions of the invention can include a nucleic acid encoding a CRISPR-associated endonuclease. In some embodiments, the CRISPR-associated endonuclease can be a Cas9 nuclease. The Cas9 nuclease can have a nucleotide sequence identical to the wild type Streptococcus pyrogenes sequence. In some embodiments, the CRISPR-associated endonuclease can be a sequence from other species, for example other Streptococcus species, such as thermophilus; Psuedomona aeruginosa, Escherichia coli, or other sequenced bacteria genomes and archaea, or other prokaryotic microorganisms. Alternatively, the wild type Streptococcus pyrogenes Cas9 sequence can be modified. The nucleic acid sequence can be codon optimized for efficient expression in mammalian cells, i.e., "humanized." A humanized Cas9 nuclease sequence can be for example, the Cas9 nuclease sequence encoded by any of the expression vectors listed in Genbank accession numbers KM099231.1 GI:669193757; KM099232.1 GI:669193761; or KM099233.1 GI:669193765. Alternatively, the Cas9 nuclease sequence can be for example, the sequence contained within a commercially available vector such as PX330 or PX260 from Addgene (Cambridge, Mass.). In some embodiments, the Cas9 endonuclease can have an amino acid sequence that is a variant or a fragment of any of the Cas9 endonuclease sequences of Genbank accession numbers KM099231.1 GI:669193757; KM099232.1 GI:669193761; or KM099233.1 GI:669193765 or Cas9 amino acid sequence of PX330 or PX260 (Addgene, Cambridge, Mass.). The Cas9 nucleotide sequence can be modified to encode biologically active variants of Cas9, and these variants can have or can include, for example, an amino acid sequence that differs from a wild type Cas9 by virtue of containing one or more mutations (e.g., an addition, deletion, or substitution mutation or a combination of such mutations). One or more of the substitution mutations can be a substitution (e.g., a conservative amino acid substitution). For example, a biologically active variant of a Cas9 polypeptide can have an amino acid sequence with at least or about 50% sequence identity (e.g., at least or about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity) to a wild type Cas9 polypeptide. Conservative amino acid substitutions typically include substitutions within the following groups: glycine and alanine; valine, isoleucine, and leucine; aspartic acid and glutamic acid; asparagine, glutamine, serine and threonine; lysine, histidine and arginine; and phenylalanine and tyrosine. The amino acid residues in the Cas9 amino acid sequence can be non-naturally occurring amino acid residues. Naturally occurring amino acid residues include those naturally encoded by the genetic code as well as non-standard amino acids (e.g., amino acids having the D-configuration instead of the L-configuration). The present peptides can also include amino acid residues that are modified versions of standard residues (e.g. pyrrolysine can be used in place of lysine and selenocysteine can be used in place of cysteine). Non-naturally occurring amino acid residues are those that have not been found in nature, but that conform to the basic formula of an amino acid and can be incorporated into a peptide. These include D-alloisoleucine(2R,3S)-2-amino-3-methylpentanoic acid and L-cyclopentyl glycine (S)-2-amino-2-cyclopentyl acetic acid. For other examples, one can consult textbooks or the worldwide web (a site is currently maintained by the California Institute of Technology and displays structures of non-natural amino acids that have been successfully incorporated into functional proteins).

[0040] The Cas9 nuclease sequence can be a mutated sequence. For example the Cas9 nuclease can be mutated in the conserved HNH and RuvC domains, which are involved in strand specific cleavage. For example, an aspartate-to-alanine (D10A) mutation in the RuvC catalytic domain allows the Cas9 nickase mutant (Cas9n) to nick rather than cleave DNA to yield single-stranded breaks, and the subsequent preferential repair through HDR can potentially decrease the frequency of unwanted indel mutations from off-target double-stranded breaks.

[0041] In some embodiments, compositions of the invention can include a CRISPR-associated endonuclease polypeptide encoded by any of the nucleic acid sequences described above. The terms "peptide," "polypeptide," and "protein" are used interchangeably herein, although typically they refer to peptide sequences of varying sizes. We may refer to the amino acid-based compositions of the invention as "polypeptides" to convey that they are linear polymers of amino acid residues, and to help distinguish them from full-length proteins. A polypeptide of the invention can "constitute" or "include" a fragment of a CRISPR-associated endonuclease, and the invention encompasses polypeptides that constitute or include biologically active variants of a CRISPR-associated endonuclease. It will be understood that the polypeptides can therefore include only a fragment of a CRISPR-associated endonuclease (or a biologically active variant thereof) but may include additional residues as well. Biologically active variants will retain sufficient activity to cleave target DNA.

[0042] The bonds between the amino acid residues can be conventional peptide bonds or another covalent bond (such as an ester or ether bond), and the polypeptides can be modified by amidation, phosphorylation or glycosylation. A modification can affect the polypeptide backbone and/or one or more side chains. Chemical modifications can be naturally occurring modifications made in vivo following translation of an mRNA encoding the polypeptide (e.g., glycosylation in a bacterial host) or synthetic modifications made in vitro. A biologically active variant of a CRISPR-associated endonuclease can include one or more structural modifications resulting from any combination of naturally occurring (i.e., made naturally in vivo) and synthetic modifications (i.e., naturally occurring or non-naturally occurring modifications made in vitro). Examples of modifications include, but are not limited to, amidation (e.g., replacement of the free carboxyl group at the C-terminus by an amino group); biotinylation (e.g., acylation of lysine or other reactive amino acid residues with a biotin molecule); glycosylation (e.g., addition of a glycosyl group to either asparagines, hydroxylysine, serine or threonine residues to generate a glycoprotein or glycopeptide); acetylation (e.g., the addition of an acetyl group, typically at the N-terminus of a polypeptide); alkylation (e.g., the addition of an alkyl group); isoprenylation (e.g., the addition of an isoprenoid group); lipoylation (e.g. attachment of a lipoate moiety); and phosphorylation (e.g., addition of a phosphate group to serine, tyrosine, threonine or histidine).

[0043] One or more of the amino acid residues in a biologically active variant may be a non-naturally occurring amino acid residue. Naturally occurring amino acid residues include those naturally encoded by the genetic code as well as non-standard amino acids (e.g., amino acids having the D-configuration instead of the L-configuration). The present peptides can also include amino acid residues that are modified versions of standard residues (e.g. pyrrolysine can be used in place of lysine and selenocysteine can be used in place of cysteine). Non-naturally occurring amino acid residues are those that have not been found in nature, but that conform to the basic formula of an amino acid and can be incorporated into a peptide. These include D-alloisoleucine(2R,3S)-2-amino-3-methylpentanoic acid and L-cyclopentyl glycine (S)-2-amino-2-cyclopentyl acetic acid. For other examples, one can consult textbooks or the worldwide web (a site is currently maintained by the California Institute of Technology and displays structures of non-natural amino acids that have been successfully incorporated into functional proteins).

[0044] Alternatively, or in addition, one or more of the amino acid residues in a biologically active variant can be a naturally occurring residue that differs from the naturally occurring residue found in the corresponding position in a wildtype sequence. In other words, biologically active variants can include one or more amino acid substitutions. We may refer to a substitution, addition, or deletion of amino acid residues as a mutation of the wildtype sequence. As noted, the substitution can replace a naturally occurring amino acid residue with a non-naturally occurring residue or just a different naturally occurring residue. Further the substitution can constitute a conservative or non-conservative substitution. Conservative amino acid substitutions typically include substitutions within the following groups: glycine and alanine; valine, isoleucine, and leucine; aspartic acid and glutamic acid; asparagine, glutamine, serine and threonine; lysine, histidine and arginine; and phenylalanine and tyrosine.

[0045] The polypeptides that are biologically active variants of a CRISPR-associated endonuclease can be characterized in terms of the extent to which their sequence is similar to or identical to the corresponding wild-type polypeptide. For example, the sequence of a biologically active variant can be at least or about 80% identical to corresponding residues in the wild-type polypeptide. For example, a biologically active variant of a CRISPR-associated endonuclease can have an amino acid sequence with at least or about 80% sequence identity (e.g., at least or about 85%, 90%, 95%, 97%, 98%, or 99% sequence identity) to a CRISPR-associated endonuclease or to a homolog or ortholog thereof.

[0046] A biologically active variant of a CRISPR-associated endonuclease polypeptide will retain sufficient biological activity to be useful in the present methods. The biologically active variants will retain sufficient activity to function in targeted DNA cleavage. The biological activity can be assessed in ways known to one of ordinary skill in the art and includes, without limitation, in vitro cleavage assays or functional assays.

[0047] Polypeptides can be generated by a variety of methods including, for example, recombinant techniques or chemical synthesis. Once generated, polypeptides can be isolated and purified to any desired extent by means well known in the art. For example, one can use lyophilization following, for example, reversed phase (preferably) or normal phase HPLC, or size exclusion or partition chromatography on polysaccharide gel media such as Sephadex G-25. The composition of the final polypeptide may be confirmed by amino acid analysis after degradation of the peptide by standard means, by amino acid sequencing, or by FAB-MS techniques. Salts, including acid salts, esters, amides, and N-acyl derivatives of an amino group of a polypeptide may be prepared using methods known in the art, and such peptides are useful in the context of the present invention.

[0048] The compositions of the invention include sequence encoding a guide RNA (gRNA) comprising a sequence that is complementary to a target sequence in a retrovirus. The retrovirus can be a lentivirus, for example, a human immunodeficiency virus, a simian immunodeficiency virus, a feline immunodeficiency virus or a bovine immunodeficiency virus. The human immunodeficiency virus can be HIV-1 or HIV-2. The target sequence can include a sequence from any HIV, for example, HIV-1 and HIV-2, and any circulating recombinant form thereof. The genetic variability of HIV is reflected in the multiple groups and subtypes that have been described. A collection of HIV sequences is compiled in the Los Alamos HIV databases and compendiums. The methods and compositions of the invention can be applied to HIV from any of those various groups, subtypes, and circulating recombinant forms. These include for example, the HIV-1 major group (often referred to as Group M) and the minor groups, Groups N, O, and P, as well as but not limited to, any of the following subtypes, A, B, C, D, F, G, H, J and K. or group (for example, but not limited to any of the following Groups, N, O and P) of HIV. The methods and compositions can also be applied to HIV-2 and any of the A, B, C, F or G clades (also referred to as "subtypes" or "groups"), as well as any circulating recombinant form of HIV-2.

[0049] The guide RNA can be a sequence complimentary to a coding or a non-coding sequence. For example, the guide RNA can be an HIV sequence, such as a long terminal repeat (LTR) sequence, a protein coding sequence, or a regulatory sequence. In some embodiments, the guide RNA comprises a sequence that is complementary to an HIV long terminal repeat (LTR) region. The HIV-1 LTR is approximately 640 bp in length. An exemplary HIV-1 LTR is the sequence of SEQ ID NO: 376. An exemplary SIV LTR is the sequence of SEQ ID NO: 380. HIV-1 long terminal repeats (LTRs) are divided into U3, R and U5 regions. Exemplary HIV-1 LTR U3, R and U5 regions are SEQ ID NOs: 377, 378 and 379, respectively. Exemplary SIV LTR U3, R and U5 regions are SEQ ID NOs: 381, 382, and 383, respectively. The configuration of the U1, R, U5 regions for exemplary HIV-1 and SIV sequences are shown in FIGS. 18 and 19, respectively. LTRs contain all of the required signals for gene expression and are involved in the integration of a provirus into the genome of a host cell. For example, the basal or core promoter, a core enhancer and a modulatory region is found within U3 while the transactivation response element is found within R. In HIV-1, the U5 region includes several sub-regions, for example, TAR or trans-acting responsive element, which is involved in transcriptional activation; Poly A, which is involved in dimerization and genome packaging; PBS or primer binding site; Psi or the packaging signal; DIS or dimer initiation site

[0050] Useful guide sequences are complementary to the U3, R, or U5 region of the LTR. Exemplary guide RNA sequences that target the U3 region of HIV-1 are shown in FIG. 13. A guide RNA sequence can comprise, for example, a sequence complementary to the target protospacer sequence of:

TABLE-US-00001 LTR A: (SEQ ID NO: 96) ATCAGATATCCACTGACCTTTGG, LTR B: (SEQ ID NO: 121) CAGCAGTTCTTGAAGTACTCCGG, LTR C: (SEQ ID NO: 87) GATTGGCAGAACTACACACCAGG, or LTR D: (SEQ ID NO: 110) GCGTGGCCTGGGCGGGACTGGGG.

[0051] The locations of LTR A (SEQ ID NO: 96), LTR B (SEQ ID NO: 121), LTR C (SEQ ID NO: 87) and LTR D (SEQ ID NO: 110) within the U3 (SEQ ID NO: 16) region are shown FIG. 5. Additional exemplary guide RNA sequences that target the U3 region are listed in the table shown in FIG. 13 and can have the sequence of any of SEQ ID NOs: 79-111 and SEQ ID NOs: 111-141. In some embodiments, the guide sequence can comprise a sequence having 95% identity to any of SEQ ID NOs: 79-111 and SEQ ID NOs: 111-141. Thus, a guide RNA sequence can comprise, for example, a sequence having 95% identity to a sequence complementary to the target protospacer sequence of:

TABLE-US-00002 LTR A: (SEQ ID NO: 96) ATCAGATATCCACTGACCTTTGG, LTR B: (SEQ ID NO: 121) CAGCAGTTCTTGAAGTACTCCGG, LTR C (SEQ ID NO: 87) GATTGGCAGAACTACACACCAGG, or LTR D: (SEQ ID NO: 110) GCGTGGCCTGGGCGGGACTGGGG.

[0052] We may also be refer to the guide RNA sequence as a spacer, e.g., spacer (A), spacer (B), spacer (C), and spacer (D).

[0053] The guide RNA sequence can be complementary to a sequence found within an HIV-1 U3, R, or U5 region reference sequence or consensus sequence. The invention is not so limiting however, and the guide RNA sequences can be selected to target any variant or mutant HIV sequence. In some embodiments, more than one guide RNA sequence is employed, for example a first guide RNA sequence and a second guide RNA sequence, with the first and second guide RNA sequences being complimentary to target sequences in any of the above mentioned retroviral regions. In some embodiments, the guide RNA can include a variant sequence or quasi-species sequence. In some embodiments, the guide RNA can be a sequence corresponding to a sequence in the genome of the virus harbored by the subject undergoing treatment. Thus for example, the sequence of the particular U3, R, or U5 region in the HIV virus harbored by the subject can be obtained and guide RNAs complementary to the patient's particular sequences can be used.

[0054] In some embodiments, the guide RNA can be a sequence complimentary to a protein coding sequence, for example, a sequence encoding one or more viral structural proteins, (e.g., gag, pol, env and tat). Thus, the sequence can be complementary to sequence within the gag polyprotein, e.g., MA (matrix protein, p17); CA (capsid protein, p24); SP1 (spacer peptide 1, p2); NC (nucleocapsid protein, p7); SP2 (spacer peptide 2, p1) and P6 protein; pol, e.g., reverse transcriptase (RT) and RNase H, integrase (IN), and HIV protease (PR); env, e.g., gp160, or a cleavage product of gp160, e.g., gp120 or SU, and gp41 or TM; or tat, e.g., the 72-amino acid one-exon Tat or the 86-101 amino-acid two-exon Tat. In some embodiments, the guide RNA can be a sequence complementary to a sequence encoding an accessory protein, including for example, vif, nef (negative factor) vpu (Virus protein U) and tev.

[0055] In some embodiments, the sequence can be a sequence complementary to a structural or regulatory element, for example, an LTR, as described above; TAR (Target sequence for viral transactivation), the binding site for Tat protein and for cellular proteins, consists of approximately the first 45 nucleotides of the viral mRNAs in HIV-1 (or the first 100 nucleotides in HIV-2) forms a hairpin stem-loop structure; RRE (Rev responsive element) an RNA element encoded within the env region of HIV-1, consisting of approximately 200 nucleotides (positions 7710 to 8061 from the start of transcription in HIV-1, spanning the border of gp120 and gp41); PE (Psi element), a set of 4 stem-loop structures preceding and overlapping the Gag start codon; SLIP, a TTTTTT "slippery site", followed by a stem-loop structure; CRS (Cis-acting repressive sequences); INS Inhibitory/Instability RNA sequences) found for example, at nucleotides 414 to 631 in the gag region of HIV-1.

[0056] The guide RNA sequence can be a sense or anti-sense sequence. The guide RNA sequence generally includes a proto-spacer adjacent motif (PAM). The sequence of the PAM can vary depending upon the specificity requirements of the CRISPR endonuclease used. In the CRISPR-Cas system derived from S. pyogenes, the target DNA typically immediately precedes a 5'-NGG proto-spacer adjacent motif (PAM). Thus, for the S. pyogenes Cas9, the PAM sequence can be AGG, TGG, CGG or GGG. Other Cas9 orthologs may have different PAM specificities. For example, Cas9 from S. thermophilus requires 5'-NNAGAA for CRISPR 1 and 5'-NGGNG for CRISPR3) and Neiseria menigiditis requires 5'-NNNNGATT). The specific sequence of the guide RNA may vary, but, regardless of the sequence, useful guide RNA sequences will be those that minimize off-target effects while achieving high efficiency and complete ablation of the genomically integrated HIV-1 provirus. The length of the guide RNA sequence can vary from about 20 to about 60 or more nucleotides, for example about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, about 30, about 31, about 32, about 33, about 34, about 35, about 36, about 37, about 38, about 39, about 40, about 45, about 50, about 55, about 60 or more nucleotides. Useful selection methods identify regions having extremely low homology between the foreign viral genome and host cellular genome including endogenous retroviral DNA, include bioinformatic screening using 12-bp+NGG target-selection criteria to exclude off-target human transcriptome or (even rarely) untranslated-genomic sites; avoiding transcription factor binding sites within the HIV-1 LTR promoter (potentially conserved in the host genome); selection of LTR-A- and -B-directed, 30-bp gRNAs and also pre-crRNA system reflecting the original bacterial immune mechanism to enhance specificity/efficiency vs. 20-bp gRNA-, chimeric crRNA-tracRNA-based system and WGS, Sanger sequencing and SURVEYOR assay, to identify and exclude potential off-target effects.

[0057] The guide RNA sequence can be configured as a single sequence or as a combination of one or more different sequences, e.g., a multiplex configuration. Multiplex configurations can include combinations of two, three, four, five, six, seven, eight, nine, ten, or more different guide RNAs, for example any combination of sequences in U3, R, or U5. In some embodiments, combinations of LTR A, LTR B, LTR C and LTR D can be used. In some embodiments, combinations of any of the sequences LTR A (SEQ ID NO: 96), LTR B (SEQ ID NO: 121), LTR C (SEQ ID NO: 87), and LTR D (SEQ ID NO: 110), can be used. In some embodiments, any combinations of the sequences having the sequence of SEQ ID NOs: 79-111 and SEQ ID NOs: 111-141 can be used. When the compositions are administered in an expression vector, the guide RNAs can be encoded by a single vector. Alternatively, multiple vectors can be engineered to each include two or more different guide RNAs. Useful configurations will result in the excision of viral sequences between cleavage sites resulting in the ablation of HIV genome or HIV protein expression. Thus, the use of two or more different guide RNAs promotes excision of the viral sequences between the cleavage sites recognized by the CRISPR endonuclease. The excised region can vary in size from a single nucleotide to several thousand nucleotides. Exemplary excised regions are described in the examples.

[0058] When the compositions are administered as a nucleic acid or are contained within an expression vector, the CRISPR endonuclease can be encoded by the same nucleic acid or vector as the guide RNA sequences. Alternatively or in addition, the CRISPR endonuclease can be encoded in a physically separate nucleic acid from the guide RNA sequences or in a separate vector.

[0059] In some embodiments, the RNA molecules e.g. crRNA, tracrRNA, gRNA are engineered to comprise one or more modified nucleobases. For example, known modifications of RNA molecules can be found, for example, in Genes VI, Chapter 9 ("Interpreting the Genetic Code"), Lewis, ed. (1997, Oxford University Press, New York), and Modification and Editing of RNA, Grosjean and Benne, eds. (1998, ASM Press, Washington D.C.). Modified RNA components include the following: 2'-O-methylcytidine; N.sup.4-methylcytidine; N.sup.4-2'-O-dimethylcytidine; N.sup.4-acetylcytidine; 5-methylcytidine; 5,2'-O-dimethylcytidine; 5-hydroxymethylcytidine; 5-formylcytidine; 2'-O-methyl-5-formaylcytidine; 3-methylcytidine; 2-thiocytidine; lysidine; 2'-O-methyluridine; 2-thiouridine; 2-thio-2'-O-methyluridine; 3,2'-O-dimethyluridine; 3-(3-amino-3-carboxypropyl)uridine; 4-thiouridine; ribosylthymine; 5,2'-O-dimethyluridine; 5-methyl-2-thiouridine; 5-hydroxyuridine; 5-methoxyuridine; uridine 5-oxyacetic acid; uridine 5-oxyacetic acid methyl ester; 5-carboxymethyluridine; 5-methoxycarbonylmethyluridine; 5-methoxycarbonylmethyl-2'-O-methyluridine; 5-methoxycarbonylmethyl-2'-thiouridine; 5-carbamoylmethyluridine; 5-carbamoylmethyl-2'-O-methyluridine; 5-(carboxyhydroxymethyl)uridine; 5-(carboxyhydroxymethyl) uridinemethyl ester; 5-aminomethyl-2-thiouridine; 5-methylaminomethyluridine; 5-methylaminomethyl-2-thiouridine; 5-methylaminomethyl-2-selenouridine; 5-carboxymethylaminomethyluridine; 5-carboxymethylaminomethyl-2'-O-methyl-uridine; 5-ca rboxymethylaminomethyl-2-thiouridine; dihydrouridine; dihydroribosylthymine; 2'-methyladenosine; 2-methyladenosine; N.sup.6N-methyladenosine; N.sup.6, N.sup.6-dimethyladenosine; N.sup.6,2'-O-trimethyladenosine; 2-methylthio-N.sup.6N-isopentenyladenosine; N.sup.6-(cis-hydroxyisopentenyl)-adenosine; 2-methylthio-N.sup.6-(cis-hydroxyisopentenyl)-adenosine; N.sup.6-glycinylcarbamoyl)adenosine; N.sup.6-threonylcarbamoyl adenosine; N.sup.6-methyl-N.sup.6-threonylcarbamoyl adenosine; 2-methylthio-N.sup.6-methyl-N.sup.6-threonylcarbamoyl adenosine; N.sup.6-hydroxynorvalylcarbamoyl adenosine; 2-methylthio-N.sup.6-hydroxnorvalylcarbamoyl adenosine; 2'-O-ribosyladenosine (phosphate); inosine; 2'O-methyl inosine; 1-methyl inosine; 1; 2'-O-dimethyl inosine; 2'-O-methyl guanosine; 1-methyl guanosine; N.sup.2-methyl guanosine; N.sup.2,N.sup.2-dimethyl guanosine; N.sup.2, 2'-O-dimethyl guanosine; N.sup.2, N.sup.2, 2'-O-trimethyl guanosine; 2'-O-ribosyl guanosine (phosphate); 7-methyl guanosine; N.sup.2; 7-dimethyl guanosine; N.sup.2; N.sup.2; 7-trimethyl guanosine; wyosine; methylwyosine; under-modified hydroxywybutosine; wybutosine; hydroxywybutosine; peroxywybutosine; queuosine; epoxyqueuosine; galactosyl-queuosine; mannosyl-queuosine; 7-cyano-7-deazaguanosine; arachaeosine [also called 7-formamido-7-deazaguanosine]; and 7-aminomethyl-7-deazaguanosine.

[0060] We may use the terms "nucleic acid" and "polynucleotide" interchangeably to refer to both RNA and DNA, including cDNA, genomic DNA, synthetic DNA, and DNA (or RNA) containing nucleic acid analogs, any of which may encode a polypeptide of the invention and all of which are encompassed by the invention. Polynucleotides can have essentially any three-dimensional structure. A nucleic acid can be double-stranded or single-stranded (i.e., a sense strand or an antisense strand). Non-limiting examples of polynucleotides include genes, gene fragments, exons, introns, messenger RNA (mRNA) and portions thereof, transfer RNA, ribosomal RNA, siRNA, micro-RNA, ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, and primers, as well as nucleic acid analogs. In the context of the present invention, nucleic acids can encode a fragment of a naturally occurring Cas9 or a biologically active variant thereof and a guide RNA where in the guide RNA is complementary to a sequence in HIV.

[0061] An "isolated" nucleic acid can be, for example, a naturally-occurring DNA molecule or a fragment thereof, provided that at least one of the nucleic acid sequences normally found immediately flanking that DNA molecule in a naturally-occurring genome is removed or absent. Thus, an isolated nucleic acid includes, without limitation, a DNA molecule that exists as a separate molecule, independent of other sequences (e.g., a chemically synthesized nucleic acid, or a cDNA or genomic DNA fragment produced by the polymerase chain reaction (PCR) or restriction endonuclease treatment). An isolated nucleic acid also refers to a DNA molecule that is incorporated into a vector, an autonomously replicating plasmid, a virus, or into the genomic DNA of a prokaryote or eukaryote. In addition, an isolated nucleic acid can include an engineered nucleic acid such as a DNA molecule that is part of a hybrid or fusion nucleic acid. A nucleic acid existing among many (e.g., dozens, or hundreds to millions) of other nucleic acids within, for example, cDNA libraries or genomic libraries, or gel slices containing a genomic DNA restriction digest, is not an isolated nucleic acid.

[0062] Isolated nucleic acid molecules can be produced by standard techniques. For example, polymerase chain reaction (PCR) techniques can be used to obtain an isolated nucleic acid containing a nucleotide sequence described herein, including nucleotide sequences encoding a polypeptide described herein. PCR can be used to amplify specific sequences from DNA as well as RNA, including sequences from total genomic DNA or total cellular RNA. Various PCR methods are described in, for example, PCR Primer: A Laboratory Manual, Dieffenbach and Dveksler, eds., Cold Spring Harbor Laboratory Press, 1995. Generally, sequence information from the ends of the region of interest or beyond is employed to design oligonucleotide primers that are identical or similar in sequence to opposite strands of the template to be amplified. Various PCR strategies also are available by which site-specific nucleotide sequence modifications can be introduced into a template nucleic acid.

[0063] Isolated nucleic acids also can be chemically synthesized, either as a single nucleic acid molecule (e.g., using automated DNA synthesis in the 3' to 5' direction using phosphoramidite technology) or as a series of oligonucleotides. For example, one or more pairs of long oligonucleotides (e.g., >50-100 nucleotides) can be synthesized that contain the desired sequence, with each pair containing a short segment of complementarity (e.g., about 15 nucleotides) such that a duplex is formed when the oligonucleotide pair is annealed. DNA polymerase is used to extend the oligonucleotides, resulting in a single, double-stranded nucleic acid molecule per oligonucleotide pair, which then can be ligated into a vector. Isolated nucleic acids of the invention also can be obtained by mutagenesis of, e.g., a naturally occurring portion of a Cas9-encoding DNA (in accordance with, for example, the formula above).

[0064] Two nucleic acids or the polypeptides they encode may be described as having a certain degree of identity to one another. For example, a Cas9 protein and a biologically active variant thereof may be described as exhibiting a certain degree of identity. Alignments may be assembled by locating short Cas9 sequences in the Protein Information Research (PIR) site, followed by analysis with the "short nearly identical sequences." Basic Local Alignment Search Tool (BLAST) algorithm on the NCBI website.

[0065] As used herein, the term "percent sequence identity" refers to the degree of identity between any given query sequence and a subject sequence. For example, a naturally occurring Cas9 can be the query sequence and a fragment of a Cas9 protein can be the subject sequence. Similarly, a fragment of a Cas9 protein can be the query sequence and a biologically active variant thereof can be the subject sequence.

[0066] To determine sequence identity, a query nucleic acid or amino acid sequence can be aligned to one or more subject nucleic acid or amino acid sequences, respectively, using the computer program ClustalW (version 1.83, default parameters), which allows alignments of nucleic acid or protein sequences to be carried out across their entire length (global alignment). See Chenna et al., Nucleic Acids Res. 31:3497-3500, 2003.

[0067] ClustalW calculates the best match between a query and one or more subject sequences and aligns them so that identities, similarities and differences can be determined. Gaps of one or more residues can be inserted into a query sequence, a subject sequence, or both, to maximize sequence alignments. For fast pair wise alignment of nucleic acid sequences, the following default parameters are used: word size: 2; window size: 4; scoring method: percentage; number of top diagonals: 4; and gap penalty: 5. for multiple alignments of nucleic acid sequences, the following parameters are used: gap opening penalty: 10.0; gap extension penalty: 5.0; and weight transitions: yes. For fast pair wise alignment of protein sequences, the following parameters are used: word size: 1; window size: 5; scoring method: percentage; number of top diagonals: 5; gap penalty: 3. For multiple alignment of protein sequences, the following parameters are used: weight matrix: blosum; gap opening penalty: 10.0; gap extension penalty: 0.05; hydrophilic gaps: on; hydrophilic residues: Gly, Pro, Ser, Asn, Asp, Gln, Glu, Arg, and Lys; residue-specific gap penalties: on. The output is a sequence alignment that reflects the relationship between sequences. ClustalW can be run, for example, at the Baylor College of Medicine Search Launcher site (searchlauncher.bcm.tmc.edu/multi-align/multi-align.html) and at the European Bioinformatics Institute site on the World Wide Web (ebi.ac.uk/clustalw).

[0068] To determine a percent identity between a query sequence and a subject sequence, ClustalW divides the number of identities in the best alignment by the number of residues compared (gap positions are excluded), and multiplies the result by 100. The output is the percent identity of the subject sequence with respect to the query sequence. It is noted that the percent identity value can be rounded to the nearest tenth. For example, 78.11, 78.12, 78.13, and 78.14 are rounded down to 78.1, while 78.15, 78.16, 78.17, 78.18, and 78.19 are rounded up to 78.2.

[0069] The nucleic acids and polypeptides described herein may be referred to as "exogenous". The term "exogenous" indicates that the nucleic acid or polypeptide is part of, or encoded by, a recombinant nucleic acid construct, or is not in its natural environment. For example, an exogenous nucleic acid can be a sequence from one species introduced into another species, i.e., a heterologous nucleic acid. Typically, such an exogenous nucleic acid is introduced into the other species via a recombinant nucleic acid construct. An exogenous nucleic acid can also be a sequence that is native to an organism and that has been reintroduced into cells of that organism. An exogenous nucleic acid that includes a native sequence can often be distinguished from the naturally occurring sequence by the presence of non-natural sequences linked to the exogenous nucleic acid, e.g., non-native regulatory sequences flanking a native sequence in a recombinant nucleic acid construct. In addition, stably transformed exogenous nucleic acids typically are integrated at positions other than the position where the native sequence is found.

[0070] Recombinant constructs are also provided herein and can be used to transform cells in order to express Cas9 and/or a guide RNA complementary to a target sequence in HIV. A recombinant nucleic acid construct comprises a nucleic acid encoding a Cas9 and/or a guide RNA complementary to a target sequence in HIV as described herein, operably linked to a regulatory region suitable for expressing the Cas9 and/or a guide RNA complementary to a target sequence in HIV in the cell. It will be appreciated that a number of nucleic acids can encode a polypeptide having a particular amino acid sequence. The degeneracy of the genetic code is well known in the art. For many amino acids, there is more than one nucleotide triplet that serves as the codon for the amino acid. For example, codons in the coding sequence for Cas9 can be modified such that optimal expression in a particular organism is obtained, using appropriate codon bias tables for that organism.

[0071] Vectors containing nucleic acids such as those described herein also are provided. A "vector" is a replicon, such as a plasmid, phage, or cosmid, into which another DNA segment may be inserted so as to bring about the replication of the inserted segment. Generally, a vector is capable of replication when associated with the proper control elements. Suitable vector backbones include, for example, those routinely used in the art such as plasmids, viruses, artificial chromosomes, BACs, YACs, or PACs. The term "vector" includes cloning and expression vectors, as well as viral vectors and integrating vectors. An "expression vector" is a vector that includes a regulatory region. A wide variety of host/expression vector combinations may be used to express the nucleic acid sequences described herein. Suitable expression vectors include, without limitation, plasmids and viral vectors derived from, for example, bacteriophage, baculoviruses, and retroviruses. Numerous vectors and expression systems are commercially available from such corporations as Novagen (Madison, Wis.), Clontech (Palo Alto, Calif.), Stratagene (La Jolla, Calif.), and Invitrogen/Life Technologies (Carlsbad, Calif.).

[0072] The vectors provided herein also can include, for example, origins of replication, scaffold attachment regions (SARs), and/or markers. A marker gene can confer a selectable phenotype on a host cell. For example, a marker can confer biocide resistance, such as resistance to an antibiotic (e.g., kanamycin, G418, bleomycin, or hygromycin). As noted above, an expression vector can include a tag sequence designed to facilitate manipulation or detection (e.g., purification or localization) of the expressed polypeptide. Tag sequences, such as green fluorescent protein (GFP), glutathione S-transferase (GST), polyhistidine, c-myc, hemagglutinin, or Flag.TM. tag (Kodak, New Haven, Conn.) sequences typically are expressed as a fusion with the encoded polypeptide. Such tags can be inserted anywhere within the polypeptide, including at either the carboxyl or amino terminus.

[0073] Additional expression vectors also can include, for example, segments of chromosomal, non-chromosomal and synthetic DNA sequences. Suitable vectors include derivatives of SV40 and known bacterial plasmids, e.g., E. coli plasmids col E1, pCR1, pBR322, pMal-C2, pET, pGEX, pMB9 and their derivatives, plasmids such as RP4; phage DNAs, e.g., the numerous derivatives of phage 1, e.g., NM989, and other phage DNA, e.g., M13 and filamentous single stranded phage DNA; yeast plasmids such as the 2.mu. plasmid or derivatives thereof, vectors useful in eukaryotic cells, such as vectors useful in insect or mammalian cells; vectors derived from combinations of plasmids and phage DNAs, such as plasmids that have been modified to employ phage DNA or other expression control sequences.

[0074] Yeast expression systems can also be used. For example, the non-fusion pYES2 vector (XbaI, SphI, ShoI, NotI, GstXI, EcoRI, BstXI, BamH1, SacI, KpnI, and HindIII cloning sites; Invitrogen) or the fusion pYESHisA, B, C (XbaI, SphI, ShoI, NotI, BstXI, EcoRI, BamH1, SacI, KpnI, and HindIII cloning sites, N-terminal peptide purified with ProBond resin and cleaved with enterokinase; Invitrogen), to mention just two, can be employed according to the invention. A yeast two-hybrid expression system can also be prepared in accordance with the invention.

[0075] The vector can also include a regulatory region. The term "regulatory region" refers to nucleotide sequences that influence transcription or translation initiation and rate, and stability and/or mobility of a transcription or translation product. Regulatory regions include, without limitation, promoter sequences, enhancer sequences, response elements, protein recognition sites, inducible elements, protein binding sequences, 5' and 3' untranslated regions (UTRs), transcriptional start sites, termination sequences, polyadenylation sequences, nuclear localization signals, and introns.

[0076] As used herein, the term "operably linked" refers to positioning of a regulatory region and a sequence to be transcribed in a nucleic acid so as to influence transcription or translation of such a sequence. For example, to bring a coding sequence under the control of a promoter, the translation initiation site of the translational reading frame of the polypeptide is typically positioned between one and about fifty nucleotides downstream of the promoter. A promoter can, however, be positioned as much as about 5,000 nucleotides upstream of the translation initiation site or about 2,000 nucleotides upstream of the transcription start site. A promoter typically comprises at least a core (basal) promoter. A promoter also may include at least one control element, such as an enhancer sequence, an upstream element or an upstream activation region (UAR). The choice of promoters to be included depends upon several factors, including, but not limited to, efficiency, selectability, inducibility, desired expression level, and cell- or tissue-preferential expression. It is a routine matter for one of skill in the art to modulate the expression of a coding sequence by appropriately selecting and positioning promoters and other regulatory regions relative to the coding sequence.

[0077] Vectors include, for example, viral vectors (such as adenoviruses ("Ad"), adeno-associated viruses (AAV), and vesicular stomatitis virus (VSV) and retroviruses), liposomes and other lipid-containing complexes, and other macromolecular complexes capable of mediating delivery of a polynucleotide to a host cell. Vectors can also comprise other components or functionalities that further modulate gene delivery and/or gene expression, or that otherwise provide beneficial properties to the targeted cells. As described and illustrated in more detail below, such other components include, for example, components that influence binding or targeting to cells (including components that mediate cell-type or tissue-specific binding); components that influence uptake of the vector nucleic acid by the cell; components that influence localization of the polynucleotide within the cell after uptake (such as agents mediating nuclear localization); and components that influence expression of the polynucleotide. Such components also might include markers, such as detectable and/or selectable markers that can be used to detect or select for cells that have taken up and are expressing the nucleic acid delivered by the vector. Such components can be provided as a natural feature of the vector (such as the use of certain viral vectors which have components or functionalities mediating binding and uptake), or vectors can be modified to provide such functionalities. Other vectors include those described by Chen et al; BioTechniques, 34: 167-171 (2003). A large variety of such vectors are known in the art and are generally available.

[0078] A "recombinant viral vector" refers to a viral vector comprising one or more heterologous gene products or sequences. Since many viral vectors exhibit size-constraints associated with packaging, the heterologous gene products or sequences are typically introduced by replacing one or more portions of the viral genome. Such viruses may become replication-defective, requiring the deleted function(s) to be provided in trans during viral replication and encapsidation (by using, e.g., a helper virus or a packaging cell line carrying gene products necessary for replication and/or encapsidation). Modified viral vectors in which a polynucleotide to be delivered is carried on the outside of the viral particle have also been described (see, e.g., Curiel, D T, et al. PNAS 88: 8850-8854, 1991).

[0079] Suitable nucleic acid delivery systems include recombinant viral vector, typically sequence from at least one of an adenovirus, adenovirus-associated virus (AAV), helper-dependent adenovirus, retrovirus, or hemagglutinating virus of Japan-liposome (HVJ) complex. In such cases, the viral vector comprises a strong eukaryotic promoter operably linked to the polynucleotide e.g., a cytomegalovirus (CMV) promoter. The recombinant viral vector can include one or more of the polynucleotides therein, preferably about one polynucleotide. In some embodiments, the viral vector used in the invention methods has a pfu (plague forming units) of from about 10.sup.8 to about 5.times.10.sup.10 pfu. In embodiments in which the polynucleotide is to be administered with a non-viral vector, use of between from about 0.1 nanograms to about 4000 micrograms will often be useful e.g., about 1 nanogram to about 100 micrograms.

[0080] Additional vectors include viral vectors, fusion proteins and chemical conjugates. Retroviral vectors include Moloney murine leukemia viruses and HIV-based viruses. One HIV-based viral vector comprises at least two vectors wherein the gag and pol genes are from an HIV genome and the env gene is from another virus. DNA viral vectors include pox vectors such as orthopox or avipox vectors, herpesvirus vectors such as a herpes simplex I virus (HSV) vector [Geller, A. I. et al., J. Neurochem, 64: 487 (1995); Lim, F., et al., in DNA Cloning: Mammalian Systems, D. Glover, Ed. (Oxford Univ. Press, Oxford England) (1995); Geller, A. I. et al., Proc Natl. Acad. Sci.: U.S.A.:90 7603 (1993); Geller, A. I., et al., Proc Natl. Acad. Sci USA: 87:1149 (1990)], Adenovirus Vectors [LeGal LaSalle et al., Science, 259:988 (1993); Davidson, et al., Nat. Genet. 3: 219 (1993); Yang, et al., J. Virol. 69: 2004 (1995)] and Adeno-associated Virus Vectors [Kaplitt, M. G., et al., Nat. Genet. 8:148 (1994)].

[0081] Pox viral vectors introduce the gene into the cells cytoplasm. Avipox virus vectors result in only a short term expression of the nucleic acid. Adenovirus vectors, adeno-associated virus vectors and herpes simplex virus (HSV) vectors may be an indication for some invention embodiments. The adenovirus vector results in a shorter term expression (e.g., less than about a month) than adeno-associated virus, in some embodiments, may exhibit much longer expression. The particular vector chosen will depend upon the target cell and the condition being treated. The selection of appropriate promoters can readily be accomplished. An example of a suitable promoter is the 763-base-pair cytomegalovirus (CMV) promoter. Other suitable promoters which may be used for gene expression include, but are not limited to, the Rous sarcoma virus (RSV) (Davis, et al., Hum Gene Ther 4:151 (1993)), the SV40 early promoter region, the herpes thymidine kinase promoter, the regulatory sequences of the metallothionein (MMT) gene, prokaryotic expression vectors such as the .beta.-lactamase promoter, the tac promoter, promoter elements from yeast or other fungi such as the Gal 4 promoter, the ADC (alcohol dehydrogenase) promoter, PGK (phosphoglycerol kinase) promoter, alkaline phosphatase promoter; and the animal transcriptional control regions, which exhibit tissue specificity and have been utilized in transgenic animals: elastase I gene control region which is active in pancreatic acinar cells, insulin gene control region which is active in pancreatic beta cells, immunoglobulin gene control region which is active in lymphoid cells, mouse mammary tumor virus control region which is active in testicular, breast, lymphoid and mast cells, albumin gene control region which is active in liver, alpha-fetoprotein gene control region which is active in liver, alpha 1-antitrypsin gene control region which is active in the liver, beta-globin gene control region which is active in myeloid cells, myelin basic protein gene control region which is active in oligodendrocyte cells in the brain, myosin light chain-2 gene control region which is active in skeletal muscle, and gonadotropic releasing hormone gene control region which is active in the hypothalamus. Certain proteins can expressed using their native promoter. Other elements that can enhance expression can also be included such as an enhancer or a system that results in high levels of expression such as a tat gene and tar element. This cassette can then be inserted into a vector, e.g., a plasmid vector such as, pUC19, pUC118, pBR322, or other known plasmid vectors, that includes, for example, an E. coli origin of replication. See, Sambrook, et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory press, (1989). The plasmid vector may also include a selectable marker such as the .beta.-lactamase gene for ampicillin resistance, provided that the marker polypeptide does not adversely affect the metabolism of the organism being treated. The cassette can also be bound to a nucleic acid binding moiety in a synthetic delivery system, such as the system disclosed in WO 95/22618.

[0082] If desired, the polynucleotides of the invention may also be used with a microdelivery vehicle such as cationic liposomes and adenoviral vectors. For a review of the procedures for liposome preparation, targeting and delivery of contents, see Mannino and Gould-Fogerite, BioTechniques, 6:682 (1988). See also, Feigner and Holm, Bethesda Res. Lab. Focus, 11(2):21 (1989) and Maurer, R. A., Bethesda Res. Lab. Focus, 11(2):25 (1989).

[0083] Replication-defective recombinant adenoviral vectors, can be produced in accordance with known techniques. See, Quantin, et al., Proc. Natl. Acad. Sci. USA, 89:2581-2584 (1992); Stratford-Perricadet, et al., J. Clin. Invest., 90:626-630 (1992); and Rosenfeld, et al., Cell, 68:143-155 (1992).

[0084] Another delivery method is to use single stranded DNA producing vectors which can produce the expressed products intracellularly. See for example, Chen et al, BioTechniques, 34: 167-171 (2003), which is incorporated herein, by reference, in its entirety.

[0085] Pharmaceutical Compositions

[0086] As described above, the compositions of the present invention can be prepared in a variety of ways known to one of ordinary skill in the art. Regardless of their original source or the manner in which they are obtained, the compositions of the invention can be formulated in accordance with their use. For example, the nucleic acids and vectors described above can be formulated within compositions for application to cells in tissue culture or for administration to a patient or subject. Any of the pharmaceutical compositions of the invention can be formulated for use in the preparation of a medicament, and particular uses are indicated below in the context of treatment, e.g., the treatment of a subject having an HIV infection or at risk for contracting and HIV infection. When employed as pharmaceuticals, any of the nucleic acids and vectors can be administered in the form of pharmaceutical compositions. These compositions can be prepared in a manner well known in the pharmaceutical art, and can be administered by a variety of routes, depending upon whether local or systemic treatment is desired and upon the area to be treated. Administration may be topical (including ophthalmic and to mucous membranes including intranasal, vaginal and rectal delivery), pulmonary (e.g., by inhalation or insufflation of powders or aerosols, including by nebulizer; intratracheal, intranasal, epidermal and transdermal), ocular, oral or parenteral. Methods for ocular delivery can include topical administration (eye drops), subconjunctival, periocular or intravitreal injection or introduction by balloon catheter or ophthalmic inserts surgically placed in the conjunctival sac. Parenteral administration includes intravenous, intra-arterial, subcutaneous, intraperitoneal or intramuscular injection or infusion; or intracranial, e.g., intrathecal or intraventricular administration. Parenteral administration can be in the form of a single bolus dose, or may be, for example, by a continuous perfusion pump. Pharmaceutical compositions and formulations for topical administration may include transdermal patches, ointments, lotions, creams, gels, drops, suppositories, sprays, liquids, powders, and the like. Conventional pharmaceutical carriers, aqueous, powder or oily bases, thickeners and the like may be necessary or desirable.

[0087] This invention also includes pharmaceutical compositions which contain, as the active ingredient, nucleic acids and vectors described herein in combination with one or more pharmaceutically acceptable carriers. We use the terms "pharmaceutically acceptable" (or "pharmacologically acceptable") to refer to molecular entities and compositions that do not produce an adverse, allergic or other untoward reaction when administered to an animal or a human, as appropriate. The term "pharmaceutically acceptable carrier," as used herein, includes any and all solvents, dispersion media, coatings, antibacterial, isotonic and absorption delaying agents, buffers, excipients, binders, lubricants, gels, surfactants and the like, that may be used as media for a pharmaceutically acceptable substance. In making the compositions of the invention, the active ingredient is typically mixed with an excipient, diluted by an excipient or enclosed within such a carrier in the form of, for example, a capsule, tablet, sachet, paper, or other container. When the excipient serves as a diluent, it can be a solid, semisolid, or liquid material (e.g., normal saline), which acts as a vehicle, carrier or medium for the active ingredient. Thus, the compositions can be in the form of tablets, pills, powders, lozenges, sachets, cachets, elixirs, suspensions, emulsions, solutions, syrups, aerosols (as a solid or in a liquid medium), lotions, creams, ointments, gels, soft and hard gelatin capsules, suppositories, sterile injectable solutions, and sterile packaged powders. As is known in the art, the type of diluent can vary depending upon the intended route of administration. The resulting compositions can include additional agents, such as preservatives. In some embodiments, the carrier can be, or can include, a lipid-based or polymer-based colloid. In some embodiments, the carrier material can be a colloid formulated as a liposome, a hydrogel, a microparticle, a nanoparticle, or a block copolymer micelle. As noted, the carrier material can form a capsule, and that material may be a polymer-based colloid.

[0088] The nucleic acid sequences of the invention can be delivered to an appropriate cell of a subject. This can be achieved by, for example, the use of a polymeric, biodegradable microparticle or microcapsule delivery vehicle, sized to optimize phagocytosis by phagocytic cells such as macrophages. For example, PLGA (poly-lacto-co-glycolide) microparticles approximately 1-10 .mu.m in diameter can be used. The polynucleotide is encapsulated in these microparticles, which are taken up by macrophages and gradually biodegraded within the cell, thereby releasing the polynucleotide. Once released, the DNA is expressed within the cell. A second type of microparticle is intended not to be taken up directly by cells, but rather to serve primarily as a slow-release reservoir of nucleic acid that is taken up by cells only upon release from the micro-particle through biodegradation. These polymeric particles should therefore be large enough to preclude phagocytosis (i.e., larger than 5 .mu.m and preferably larger than 20 .mu.m). Another way to achieve uptake of the nucleic acid is using liposomes, prepared by standard methods. The nucleic acids can be incorporated alone into these delivery vehicles or co-incorporated with tissue-specific antibodies, for example antibodies that target cell types that are commonly latently infected reservoirs of HIV infection, for example, brain macrophages, microglia, astrocytes, and gut-associated lymphoid cells. Alternatively, one can prepare a molecular complex composed of a plasmid or other vector attached to poly-L-lysine by electrostatic or covalent forces. Poly-L-lysine binds to a ligand that can bind to a receptor on target cells. Delivery of "naked DNA" (i.e., without a delivery vehicle) to an intramuscular, intradermal, or subcutaneous site, is another means to achieve in vivo expression. In the relevant polynucleotides (e.g., expression vectors) the nucleic acid sequence encoding the an isolated nucleic acid sequence comprising a sequence encoding a CRISPR-associated endonuclease and a guide RNA is operatively linked to a promoter or enhancer-promoter combination. Promoters and enhancers are described above.

[0089] In some embodiments, the compositions of the invention can be formulated as a nanoparticle, for example, nanoparticles comprised of a core of high molecular weight linear polyethylenimine (LPEI) complexed with DNA and surrounded by a shell of polyethyleneglycol-modified (PEGylated) low molecular weight LPEI.

[0090] The nucleic acids and vectors may also be applied to a surface of a device (e.g., a catheter) or contained within a pump, patch, or other drug delivery device. The nucleic acids and vectors of the invention can be administered alone, or in a mixture, in the presence of a pharmaceutically acceptable excipient or carrier (e.g., physiological saline). The excipient or carrier is selected on the basis of the mode and route of administration. Suitable pharmaceutical carriers, as well as pharmaceutical necessities for use in pharmaceutical formulations, are described in Remington's Pharmaceutical Sciences (E. W. Martin), a well-known reference text in this field, and in the USP/NF (United States Pharmacopeia and the National Formulary).

[0091] In some embodiments, the compositions may be formulated as a topical gel for blocking sexual transmission of HIV. The topical gel can be applied directly to the skin or mucous membranes of the male or female genital region prior to sexual activity. Alternatively or in addition the topical gel can be applied to the surface or contained within a male or female condom or diaphragm.

[0092] In some embodiments, the compositions can be formulated as a nanoparticle encapsulating a nucleic acid encoding Cas9 or a variant Cas9 and a guide RNA sequence complementary to a target HIV or vector comprising a nucleic acid encoding Cas9 and a guide RNA sequence complementary to a target HIV. Alternatively, the compositions can be formulated as a nanoparticle encapsulating a CRISPR-associated endonuclease polypeptide, e.g., Cas9 or a variant Cas9 and a guide RNA sequence complementary to a target.

[0093] The present formulations can encompass a vector encoding Cas9 and a guide RNA sequence complementary to a target HIV. The guide RNA sequence can include a sequence complementary to a single region, e.g. LTR A, B, C, or D or it can include any combination of sequences complementary to LTR A, B, C, and D. Alternatively the sequence encoding Cas9 and the sequence encoding the guide RNA sequence can be on separate vectors.

[0094] Methods of Treatment

[0095] The compositions disclosed herein are generally and variously useful for treatment of a subject having a retroviral infection, e.g., an HIV infection. We may refer to a subject, patient, or individual interchangeably. The methods are useful for targeting any HIV, for example, HIV-1, HIV-2, and any circulating recombinant form thereof. A subject is effectively treated whenever a clinically beneficial result ensues. This may mean, for example, a complete resolution of the symptoms of a disease, a decrease in the severity of the symptoms of the disease, or a slowing of the disease's progression. These methods can further include the steps of a) identifying a subject (e.g., a patient and, more specifically, a human patient) who has an HIV infection; and b) providing to the subject a composition comprising a nucleic acid encoding a CRISPR-associated nuclease, e.g., Cas9, and a guide RNA complementary to an HIV target sequence, e.g. an HIV LTR. A subject can be identified using standard clinical tests, for example, immunoassays to detect the presence of HIV antibodies or the HIV polypeptide p24 in the subject's serum, or through HIV nucleic acid amplification assays. An amount of such a composition provided to the subject that results in a complete resolution of the symptoms of the infection, a decrease in the severity of the symptoms of the infection, or a slowing of the infection's progression is considered a therapeutically effective amount. The present methods may also include a monitoring step to help optimize dosing and scheduling as well as predict outcome. In some methods of the present invention, one can first determine whether a patient has a latent HIV-1 infection, and then make a determination as to whether or not to treat the patient with one or more of the compositions described herein. Monitoring can also be used to detect the onset of drug resistance and to rapidly distinguish responsive patients from nonresponsive patients. In some embodiments, the methods can further include the step of determining the nucleic acid sequence of the particular HIV harbored by the patient and then designing the guide RNA to be complementary to those particular sequences. For example, one can determine the nucleic acid sequence of a subject's LTR U3, R or U5 region and then design one or more guide RNAs to be precisely complementary to the patient's sequences.

[0096] The compositions are also useful for the treatment, for example, as a prophylactic treatment, of a subject at risk for having a retroviral infection, e.g., an HIV infection. These methods can further include the steps of a) identifying a subject at risk for having an HIV infection; b) providing to the subject a composition comprising a nucleic acid encoding a CRISPR-associated nuclease, e.g., Cas9, and a guide RNA complementary to an HIV target sequence, e.g. an HIV LTR. A subject at risk for having an HIV infection can be, for example, any sexually active individual engaging in unprotected sex, i.e., engaging in sexual activity without the use of a condom; a sexually active individual having another sexually transmitted infection; an intravenous drug user; or an uncircumcised man. A subject at risk for having an HIV infection can be, for example, an individual whose occupation may bring him or her into contact with HIV-infected populations, e.g., healthcare workers or first responders. A subject at risk for having an HIV infection can be, for example, an inmate in a correctional setting or a sex worker, that is, an individual who uses sexual activity for income employment or nonmonetary items such as food, drugs, or shelter.

[0097] The compositions can also be administered to a pregnant or lactating woman having an HIV infection in order to reduce the likelihood of transmission of HIV from the mother to her offspring. A pregnant woman infected with HIV can pass the virus to her offspring transplacentally in utero, at the time of delivery through the birth canal or following delivery, through breast milk. The compositions disclosed herein can be administered to the HIV infected mother either prenatally, perinatally or postnatally during the breast-feeding period, or any combination of prenatal, perinatal, and postnatal administration. Compositions can be administered to the mother along with standard antiretroviral therapies as described below. In some embodiments, the compositions of the invention are also administered to the infant immediately following delivery and, in some embodiments, at intervals thereafter. The infant also can receive standard antiretroviral therapy.

[0098] The methods and compositions disclosed herein are useful for the treatment of retroviral infections. Exemplary retroviruses include human immunodeficiency viruses, e.g. HIV-1, HIV-2; simian immunodeficiency virus (SIV); feline immunodeficiency virus (FIV); bovine immunodeficiency virus (BIV); equine infectious anemia virus (EIAV); and caprine arthritis/encephalitis virus (CAEV). The methods disclosed herein can be applied to a wide range of species, e.g., humans, non-human primates (e.g., monkeys), horses or other livestock, dogs, cats, ferrets or other mammals kept as pets, rats, mice, or other laboratory animals.

[0099] The methods of the invention can be expressed in terms of the preparation of a medicament. Accordingly, the invention encompasses the use of the agents and compositions described herein in the preparation of a medicament. The compounds described herein are useful in therapeutic compositions and regimens or for the manufacture of a medicament for use in treatment of diseases or conditions as described herein.

[0100] Any composition described herein can be administered to any part of the host's body for subsequent delivery to a target cell. A composition can be delivered to, without limitation, the brain, the cerebrospinal fluid, joints, nasal mucosa, blood, lungs, intestines, muscle tissues, skin, or the peritoneal cavity of a mammal. In terms of routes of delivery, a composition can be administered by intravenous, intracranial, intraperitoneal, intramuscular, subcutaneous, intramuscular, intrarectal, intravaginal, intrathecal, intratracheal, intradermal, or transdermal injection, by oral or nasal administration, or by gradual perfusion over time. In a further example, an aerosol preparation of a composition can be given to a host by inhalation.

[0101] The dosage required will depend on the route of administration, the nature of the formulation, the nature of the patient's illness, the patient's size, weight, surface area, age, and sex, other drugs being administered, and the judgment of the attending clinicians. Wide variations in the needed dosage are to be expected in view of the variety of cellular targets and the differing efficiencies of various routes of administration. Variations in these dosage levels can be adjusted using standard empirical routines for optimization, as is well understood in the art. Administrations can be single or multiple (e.g., 2- or 3-, 4-, 6-, 8-, 10-, 20-, 50-, 100-, 150-, or more fold). Encapsulation of the compounds in a suitable delivery vehicle (e.g., polymeric microparticles or implantable devices) may increase the efficiency of delivery.

[0102] The duration of treatment with any composition provided herein can be any length of time from as short as one day to as long as the life span of the host (e.g., many years). For example, a compound can be administered once a week (for, for example, 4 weeks to many months or years); once a month (for, for example, three to twelve months or for many years); or once a year for a period of 5 years, ten years, or longer. It is also noted that the frequency of treatment can be variable. For example, the present compounds can be administered once (or twice, three times, etc.) daily, weekly, monthly, or yearly.

[0103] An effective amount of any composition provided herein can be administered to an individual in need of treatment. The term "effective" as used herein refers to any amount that induces a desired response while not inducing significant toxicity in the patient. Such an amount can be determined by assessing a patient's response after administration of a known amount of a particular composition. In addition, the level of toxicity, if any, can be determined by assessing a patient's clinical symptoms before and after administering a known amount of a particular composition. It is noted that the effective amount of a particular composition administered to a patient can be adjusted according to a desired outcome as well as the patient's response and level of toxicity. Significant toxicity can vary for each particular patient and depends on multiple factors including, without limitation, the patient's disease state, age, and tolerance to side effects.

[0104] Any method known to those in the art can be used to determine if a particular response is induced. Clinical methods that can assess the degree of a particular disease state can be used to determine if a response is induced. The particular methods used to evaluate a response will depend upon the nature of the patient's disorder, the patient's age, and sex, other drugs being administered, and the judgment of the attending clinician.

[0105] The compositions may also be administered with another therapeutic agent, for example, an anti-retroviral agent, used in HAART. Exemplary antiretroviral agents include reverse transcriptase inhibitors (e.g., nucleoside/nucleotide reverse transcriptase inhibitors, zidovudine, emtricitibine, lamivudine and tenofivir; and non-nucleoside reverse transcriptase inhibitors such as efavarenz, nevirapine, rilpivirine); protease inhibitors, e.g., tipiravir, darunavir, indinavir; entry inhibitors, e.g., maraviroc; fusion inhibitors, e.g., enfuviritide; or integrase inhibitors e.g., raltegrivir, dolutegravir. Exemplary antiretroviral agents can also include multi-class combination agents for example, combinations of emtricitabine, efavarenz, and tenofivir; combinations of emtricitabine; rilpivirine, and tenofivir; or combinations of elvitegravir, cobicistat, emtricitabine and tenofivir.

[0106] Concurrent administration of two or more therapeutic agents does not require that the agents be administered at the same time or by the same route, as long as there is an overlap in the time period during which the agents are exerting their therapeutic effect. Simultaneous or sequential administration is contemplated, as is administration on different days or weeks. The therapeutic agents may be administered under a metronomic regimen, e.g., continuous low-doses of a therapeutic agent.

[0107] Dosage, toxicity and therapeutic efficacy of such compositions can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD.sub.50 (the dose lethal to 50% of the population) and the ED.sub.50 (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD.sub.50/ED.sub.50.

[0108] The data obtained from the cell culture assays and animal studies can be used in formulating a range of dosage for use in humans. The dosage of such compositions lies preferably within a range of circulating concentrations that include the ED.sub.50 with little or no toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized. For any composition used in the method of the invention, the therapeutically effective dose can be estimated initially from cell culture assays. A dose may be formulated in animal models to achieve a circulating plasma concentration range that includes the IC.sub.50 (i.e., the concentration of the test compound which achieves a half-maximal inhibition of symptoms) as determined in cell culture. Such information can be used to more accurately determine useful doses in humans. Levels in plasma may be measured, for example, by high performance liquid chromatography.

[0109] As described, a therapeutically effective amount of a composition (i.e., an effective dosage) means an amount sufficient to produce a therapeutically (e.g., clinically) desirable result. The compositions can be administered one from one or more times per day to one or more times per week; including once every other day. The skilled artisan will appreciate that certain factors can influence the dosage and timing required to effectively treat a subject, including but not limited to the severity of the disease or disorder, previous treatments, the general health and/or age of the subject, and other diseases present. Moreover, treatment of a subject with a therapeutically effective amount of the compositions of the invention can include a single treatment or a series of treatments.

[0110] The compositions described herein are suitable for use in a variety of drug delivery systems described above. Additionally, in order to enhance the in vivo serum half-life of the administered compound, the compositions may be encapsulated, introduced into the lumen of liposomes, prepared as a colloid, or other conventional techniques may be employed which provide an extended serum half-life of the compositions. A variety of methods are available for preparing liposomes, as described in, e.g., Szoka, et al., U.S. Pat. Nos. 4,235,871, 4,501,728 and 4,837,028 each of which is incorporated herein by reference. Furthermore, one may administer the drug in a targeted drug delivery system, for example, in a liposome coated with a tissue-specific antibody. The liposomes will be targeted to and taken up selectively by the organ.

[0111] Also provided, are methods of inactivating a retrovirus, for example a lentivirus such as a human immunodeficiency virus, a simian immunodeficiency virus, a feline immunodeficiency virus, or a bovine immunodeficiency virus in a mammalian cell. The human immunodeficiency virus can be HIV-1 or HIV-2. The human immunodeficiency virus can be a chromosomally integrated provirus. The mammalian cell can be any cell type infected by HIV, including, but not limited to CD4+ lymphocytes, macrophages, fibroblasts, monocytes, T lymphocytes, B lymphocytes, natural killer cells, dendritic cells such as Langerhans cells and follicular dendritic cells, hematopoietic stem cells, endothelial cells, brain microglial cells, and gastrointestinal epithelial cells. Such cell types include those cell types that are typically infected during a primary infection, for example, a CD4+ lymphocyte, a macrophage, or a Langerhans cell, as well as those cell types that make up latent HIV reservoirs, i.e., a latently infected cell.

[0112] The methods can include exposing the cell to a composition comprising an isolated nucleic acid encoding a gene editing complex comprising a CRISPR-associated endonuclease and one or more guide RNAs wherein the guide RNA is complementary to a target nucleic acid sequence in the retrovirus. In a preferred embodiment, as previously described, the method of inactivating a proviral DNA integrated into the genome of a host cell latently infected with a retrovirus includes the steps of treating the host cell with a composition comprising a CRISPR-associated endonuclease, and two or more different guide RNAs (gRNAs), wherein each of the at least two gRNAs is complementary to a different target nucleic acid sequence in the proviral DNA; and inactivating the proviral DNA. The at least two gRNAs can be configured as a single sequence or as a combination of one or more different sequences, e.g., a multiplex configuration. Multiplex configurations can include combinations of two, three, four, five, six, seven, eight, nine, ten, or more different gRNAs, for example any combination of sequences in U3, R, or U5. In some embodiments, combinations of LTR A, LTR B, LTR C and LTR D can be used. In some embodiments, combinations of any of the sequences LTR A (SEQ ID NO: 96), LTR B (SEQ ID NO: 121), LTR C (SEQ ID NO: 87), and LTR D (SEQ ID NO: 110), can be used. In experiments described in the Examples, the use of two different gRNAs caused the excision of the viral sequences between the cleavage sites recognized by the CRISPR endonuclease. The excised region can include the entire HIV-1 genome. The treating step can take place in vivo, that is, the compositions can be administered directly to a subject having HIV infection. The methods are not so limited however, and the treating step can take place ex vivo. For example, a cell or plurality of cells, or a tissue explant, can be removed from a subject having an HIV infection and placed in culture, and then treated with a composition comprising a CRISPR-associated endonuclease and a guide RNA wherein the guide RNA is complementary to the nucleic acid sequence in the human immunodeficiency virus. As described above, the composition can be a nucleic acid encoding a CRISPR-associated endonuclease and a guide RNA wherein the guide RNA is complementary to the nucleic acid sequence in the human immunodeficiency virus; an expression vector comprising the nucleic acid sequence; or a pharmaceutical composition comprising a nucleic acid encoding a CRISPR-associated endonuclease and a guide RNA wherein the guide RNA is complementary to the nucleic acid sequence in the human immunodeficiency virus; or an expression vector comprising the nucleic acid sequence. In some embodiments, the gene editing complex can comprise a CRISPR-associated endonuclease polypeptide and a guide RNA wherein the guide RNA is complementary to the nucleic acid sequence in the human immunodeficiency virus.

[0113] Regardless of whether compositions are administered as nucleic acids or polypeptides, they are formulated in such a way as to promote uptake by the mammalian cell. Useful vector systems and formulations are described above. In some embodiments the vector can deliver the compositions to a specific cell type. The invention is not so limited however, and other methods of DNA delivery such as chemical transfection, using, for example calcium phosphate, DEAE dextran, liposomes, lipoplexes, surfactants, and perfluoro chemical liquids are also contemplated, as are physical delivery methods, such as electroporation, micro injection, ballistic particles, and "gene gun" systems.

[0114] Standard methods, for example, immunoassays to detect the CRISPR-associated endonuclease, or nucleic acid-based assays such as PCR to detect the gRNA, can be used to confirm that the complex has been taken up and expressed by the cell into which it has been introduced. The engineered cells can then be reintroduced into the subject from whom they were derived as described below.

[0115] The gene editing complex comprises a CRISPR-associated nuclease, e.g., Cas9, and a guide RNA complementary to the retroviral target sequence, for example, an HIV target sequence. The gene editing complex can introduce various mutations into the proviral DNA. The mechanism by which such mutations inactivate the virus can vary, for example the mutation can affect proviral replication, viral gene expression or proviral excision. The mutations may be located in regulatory sequences or structural gene sequences and result in defective production of HIV. The mutation can comprise a deletion. The size of the deletion can vary from a single nucleotide base pair to about 10,000 base pairs. In some embodiments, the deletion can include all or substantially all of the proviral sequence. In some embodiments the deletion can include the entire proviral sequence. The mutation can comprise an insertion; that is the addition of one or more nucleotide base pairs to the pro-viral sequence. The size of the inserted sequence also may vary, for example from about one base pair to about 300 nucleotide base pairs. The mutation can comprise a point mutation, that is, the replacement of a single nucleotide with another nucleotide. Useful point mutations are those that have functional consequences, for example, mutations that result in the conversion of an amino acid codon into a termination codon or that result in the production of a nonfunctional protein.

[0116] In exemplary multiplex methods for inactivating proviral DNA integrated into the genome of a host cell, as demonstrated in Examples 2-5, two different gRNA sequences are deployed, with each gRNA sequence targeting a different site in the proviral DNA. That is, the methods include the steps of exposing the host cell to a composition including an isolated nucleic acid encoding a CRISPR-associated endonuclease; an isolated nucleic acid sequence encoding a first gRNA having a first spacer sequence that is complementary to a first target protospacer sequence in a proviral DNA; and an isolated nucleic acid encoding a second gRNA having a second spacer sequence that is complementary to a second target protospacer sequence in the proviral DNA; expressing in the host cell the CRISPR-associated endonuclease, the first gRNA, and the second gRNA; assembling, in the host cell, a first gene editing complex including the CRISPR-associated endonuclease and the first gRNA; and a second gene editing complex including the CRISPR-associated endonuclease and the second gRNA; directing the first gene editing complex to the first target protospacer sequence by complementary base pairing between the first spacer sequence and the first target protospacer sequence; directing the second gene editing complex to the second target protospacer sequence by complementary base pairing between the second spacer sequence and the second target protospacer sequence; cleaving the proviral DNA at the first target protospacer sequence with the CRISPR-associated endonuclease; cleaving the proviral DNA at the second target protospacer sequence with the CRISPR-associated endonuclease; and inducing at least one mutation in the proviral DNA. The same multiplex method is readily incorporated into methods for treating a subject having a human immunodeficiency virus, and for reducing the risk of a human immunodeficiency virus infection. It will be understood that the term "composition" can include not only a mixture of components, but also separate components that are not necessarily administered simultaneously. As a non-limiting example, a composition according to the present invention can include separate component preparations of nucleic acid sequences encoding a Cas9 nuclease, a first gRNA, and a second gRNA, with each component being administered sequentially in an infusion, during a time frame that results in a host cell being exposed to all three components.

[0117] In other embodiments, the compositions comprise a cell which has been transformed or transfected with one or more Cas/gRNA vectors. In some embodiments, the methods of the invention can be applied ex vivo. That is, a subject's cells can be removed from the body and treated with the compositions in culture to excise HIV sequences and the treated cells returned to the subject's body. The cell can be the subject's cells or they can be haplotype matched or a cell line. The cells can be irradiated to prevent replication. In some embodiments, the cells are human leukocyte antigen (HLA)-matched, autologous, cell lines, or combinations thereof. In other embodiments the cells can be a stem cell. For example, an embryonic stem cell or an artificial pluripotent stem cell (induced pluripotent stem cell (iPS cell)). Embryonic stem cells (ES cells) and artificial pluripotent stem cells (induced pluripotent stem cell, iPS cells) have been established from many animal species, including humans. These types of pluripotent stem cells would be the most useful source of cells for regenerative medicine because these cells are capable of differentiation into almost all of the organs by appropriate induction of their differentiation, with retaining their ability of actively dividing while maintaining their pluripotency. iPS cells, in particular, can be established from self-derived somatic cells, and therefore are not likely to cause ethical and social issues, in comparison with ES cells which are produced by destruction of embryos. Further, iPS cells, which are self-derived cell, make it possible to avoid rejection reactions, which are the biggest obstacle to regenerative medicine or transplantation therapy.

[0118] The gRNA expression cassette can be easily delivered to a subject by methods known in the art, for example, methods which deliver siRNA. In some aspects, the Cas may be a fragment wherein the active domains of the Cas molecule are included, thereby cutting down on the size of the molecule. Thus, the, Cas9/gRNA molecules can be used clinically, similar to the approaches taken by current gene therapy. In particular, a Cas9/multiplex gRNA stable expression stem cell or iPS cells for cell transplantation therapy as well as HIV-1 vaccination will be developed for use in subjects.

[0119] Transduced cells are prepared for reinfusion according to established methods. After a period of about 2-4 weeks in culture, the cells may number between 1.times.10.sup.6 and 1.times.10.sup.10. In this regard, the growth characteristics of cells vary from patient to patient and from cell type to cell type. About 72 hours prior to reinfusion of the transduced cells, an aliquot is taken for analysis of phenotype, and percentage of cells expressing the therapeutic agent. For administration, cells of the present invention can be administered at a rate determined by the LD.sub.50 of the cell type, and the side effects of the cell type at various concentrations, as applied to the mass and overall health of the patient. Administration can be accomplished via single or divided doses. Adult stem cells may also be mobilized using exogenously administered factors that stimulate their production and egress from tissues or spaces that may include, but are not restricted to, bone marrow or adipose tissues.

[0120] Articles of Manufacture

[0121] The compositions described herein can be packaged in suitable containers labeled, for example, for use as a therapy to treat a subject having a retroviral infection, for example, an HIV infection or a subject at for contracting a retroviral infection, for example, an HIV infection. The containers can include a composition comprising a nucleic acid sequence encoding a CRISPR-associated endonuclease, for example, a Cas9 endonuclease, and a guide RNA complementary to a target sequence in a human immunodeficiency virus, or a vector encoding that nucleic acid, and one or more of a suitable stabilizer, carrier molecule, flavoring, and/or the like, as appropriate for the intended use. Accordingly, packaged products (e.g., sterile containers containing one or more of the compositions described herein and packaged for storage, shipment, or sale at concentrated or ready-to-use concentrations) and kits, including at least one composition of the invention, e.g., a nucleic acid sequence encoding a CRISPR-associated endonuclease, for example, a Cas9 endonuclease, and a guide RNA complementary to a target sequence in a human immunodeficiency virus, or a vector encoding that nucleic acid and instructions for use, are also within the scope of the invention. A product can include a container (e.g., a vial, jar, bottle, bag, or the like) containing one or more compositions of the invention. In addition, an article of manufacture further may include, for example, packaging materials, instructions for use, syringes, delivery devices, buffers or other control reagents for treating or monitoring the condition for which prophylaxis or treatment is required.

[0122] In some embodiments, the kits can include one or more additional antiretroviral agents, for example, a reverse transcriptase inhibitor, a protease inhibitor or an entry inhibitor. The additional agents can be packaged together in the same container as a nucleic acid sequence encoding a CRISPR-associated endonuclease, for example, a Cas9 endonuclease, and a guide RNA complementary to a target sequence in a human immunodeficiency virus, or a vector encoding that nucleic acid or they can be packaged separately. The nucleic acid sequence encoding a CRISPR-associated endonuclease, for example, a Cas9 endonuclease, and a guide RNA complementary to a target sequence in a human immunodeficiency virus, or a vector encoding that nucleic acid and the additional agent may be combined just before use or administered separately.

[0123] The product may also include a legend (e.g., a printed label or insert or other medium describing the product's use (e.g., an audio- or videotape)). The legend can be associated with the container (e.g., affixed to the container) and can describe the manner in which the compositions therein should be administered (e.g., the frequency and route of administration), indications therefor, and other uses. The compositions can be ready for administration (e.g., present in dose-appropriate units), and may include one or more additional pharmaceutically acceptable adjuvants, carriers or other diluents and/or an additional therapeutic agent. Alternatively, the compositions can be provided in a concentrated form with a diluent and instructions for dilution.

Example 1: Materials and Methods

[0124] Plasmid Preparation:

[0125] Vectors containing human Cas9 and gRNA expression cassette, pX260, and pX330 (Addgene) were utilized to create various constructs, LTR-A, B, C, and D.

[0126] Cell Culture and Stable Cell Lines:

[0127] TZM-b1 reporter and U1 cell lines were obtained from the NIH AIDS Reagent Program and CHME5 microglial cells are known in the art.

[0128] Immunohistochemistry and Western Blot:

[0129] Standard methods for immunocytochemical observation of the cells and evaluation of protein expression by Western blot were utilized.

[0130] Firefly-Luciferase Assay:

[0131] Cells were lysed 24 h post-treatment using Passive Lysis Buffer (Promega) and assayed with a Luciferase Reporter Gene Assay kit (Promega) according to the manufacturer's protocol. Luciferase activity was normalized to the number of cells determined by a parallel MTT assay (Vybrant, Invitrogen)

[0132] p24 ELISA:

[0133] After infection or reactivation, the levels of HIV-1 viral load in the supernatants were quantified by p24 Gag ELISA (Advanced BioScience Laboratories, Inc) following the manufacturer's protocol. To assess cell viability upon treatments, MTT assay was performed in parallel according to the manufacturer's manual (Vybrant, Invitrogen).

[0134] EGFP Flow Cytometry:

[0135] Cells were trypsinized, washed with PBS and fixed in 2% paraformaldehyde for 10 min at room temperature, then washed twice with PBS and analyzed using a Guava EasyCyte Mini flow cytometer (Guava Technologies).

[0136] HIV-1 Reporter Virus Preparation and Infections:

[0137] HEK293T cells were transfected using Lipofectamine 2000 reagent (Invitrogen) with pNL4-3-.DELTA.E-EGFP (NIH AIDS Research and Reference Reagent Program). After 48 h, the supernatant was collected, 0.45 .mu.m filtered and tittered in HeLa cells using EGFP as an infection marker. For viral infection, stable Cas9/gRNA TZM-bl cells were incubated 2 h with diluted viral stock, and then washed twice with PBS. At 2 and 4 d post-infection, cells were collected, fixed and analyzed by flow cytometry for EGFP expression, or genomic DNA purification was performed for PCR and whole genome sequencing.

[0138] Genomic DNA Amplification, PCR, TA-Cloning, and Sanger Sequencing, GenomeWalker Link PCR:

[0139] Standard methods for DNA manipulation for cloning and sequencing were utilized. For identification of the integration sites of HIV-1, we utilized Lenti-X.TM. integration site analysis kit was used.

[0140] Surveyor Assay:

[0141] The presence of mutations in PCR products was examined using a SURVEYOR Mutation Detection Kit (Transgenomic) according to the protocol from the manufacturer. Briefly heterogeneous PCR product was denatured for 10 min in 95.degree. C. and hybridized by gradual cooling using a thermocycler. Next, 300 ng of hybridized DNA (9 .mu.l) was subjected to digestion with 0.25 .mu.l of SURVEYOR Nuclease in the presence of 0.25 .mu.l SURVEYOR Enhancer S and 15 mM MgCl.sub.2 for 4 h at 42.degree. C. Then Stop Solution was added and samples were resolved in 2% agarose gel together with equal amounts of undigested PCR product controls.

[0142] Some PCR products were used for restriction fragment length polymorphism analysis. Equal amounts of the PCR products were digested with BsaJI. Digested DNA was separated on an ethidium bromide-contained agarose gel (2%). For sequencing, PCR products were cloned using a TA Cloning.RTM. Kit Dual Promoter with pCR.TM.II vector (Invitrogen). The insert was confirmed by digestion with EcoRI and positive clones were sent to Genewiz for Sanger sequencing.

[0143] Selection of LTR Target Sites, Whole Genome Sequencing and Bioinformatics and Statistical Analysis.

[0144] We utilized Jack Lin's CRISPR/Cas9 gRNA finder tool for initial identification of potential target sites within the LTR.

[0145] Plasmid Preparation.

[0146] DNA segment expressing LTR-A or LTR-B for pre-crRNA was cloned into the pX260 vector that contains the puromycin selection gene (Addgene, plasmid #42229). DNA segments expressing LTR-C or LTR-D for the chimeric crRNA-tracrRNA were cloned into the pX330 vector (Addgene, plasmid #42230). Both vectors contain a humanized Cas9 coding sequence driven by a CAG promoter and a gRNA expression cassette driven by a human U6 promoter. The vectors were digested with BbsI and treated with Antarctic Phosphatase, and the linearized vector was purified with a Quick nucleotide removal kit (Qiagen). A pair of oligonucleotides for each targeting site (FIG. 14, AlphaDNA) was annealed, phosphorylated, and ligated to the linearized vector. The gRNA expression cassette was sequenced with U6 sequencing primer (FIG. 14) in GENEWIZ. For pX330 vectors, we designed a pair of universal PCR primers with overhang digestion sites (FIG. 14) that can tease out the gRNA expression cassette (U6-gRNA-crRNA-stem-tracrRNA) for direct transfection or subcloning to other vectors.

[0147] Cell Culture.

[0148] TZM-bl reporter cell line from Dr John C. Kappes, Dr Xiaoyun Wu and Tranzyme Inc, U1/Hiv-1 cell line from Dr. Thomas Folks and J-Lat full length clone from Dr. Eric Verdin were obtained through the NIH AIDS Reagent Program, Division of AIDS, NIAID, NIH. CHME5/HIV fetal microglia cell line were generated as previously described. TZM-bl and CHME5 cells were cultured in Dulbecco's minimal essential medium high glucose supplemented with 10% heat-inactivated fetal bovine serum (FBS) and 1% penicillin/streptomycin. U1 and J-Lat cells were cultured in RPMI 1640 containing 2.0 mM L-glutamine, 10% FBS and 1% penicillin/streptomycin.

[0149] Stable Cell Lines and Subcloning.

[0150] TZM-bl or CHME5/HIV cells were seeded in 6-well plates at 1.5.times.10.sup.5 cells/well and transfected using Lipofectamine 2000 reagent (Invitrogen) with 1 .mu.g of pX260 (for LTR-A and B) or 1 .mu.g/0.1 .mu.g of pX330/pX260 (for LTR-C and D) plasmids. Next day, cells were transferred into 100-mm dishes and incubated with growth medium containing 1 .mu.g/ml of puromycin (Sigma). Two weeks later, surviving cell colonies were isolated using cloning cylinders (Corning). U1 cells (1.5.times.10.sup.5) were electroporated with 1 .mu.g of DNA using 10 .mu.l tip, 3.times.10 ms 1400 V impulses at The Neon.TM. Transfection System (Invitrogen). Cells were selected with 0.5 .mu.g/ml of puromycin for two weeks. The stable clones were subcultured using a limited dilution method in 96-well plates and single cell-derived subclones were maintained for further studies.

[0151] Immunocytochemistry and Western Blot.

[0152] The Cas9/gRNA stable expression TZM-bl cells were cultured in 8-well chamber slides for 2 days and fixed for 10 min in 4% paraformaldehyde/PBS. After three rinses, the cells were treated with 0.5% Triton X-100/PBS for 20 min and blocked in 10% donkey serum for 1 h. Cells were incubated overnight at 4.degree. C. with mouse anti-Flag M2 primary antibody (1:500, Sigma). After rinsing three times, cells were incubated for 1 h with donkey anti-mouse Alexa-Fluor-594 secondary antibodies, and incubated with Hoechst 33258 for 5 min. After three rinses with PBS, the cells were coverslipped with anti-fading aqueous mounting media (Biomeda) and analyzed under a Leica DM16000B fluorescence microscope.

[0153] TZM-bl cells cultured in 6-well plate were solubilized in 200 .mu.l of Triton X-100-based lysis buffer containing 20 mM Tris-HCl (pH 7.4), 1% Triton X-100, 5 mM ethylenediaminetetraacetic acid, 5 mM dithiothreitol, 150 mM NaCl, 1 mM phenylmethylsulfonyl fluoride, 1.times. nuclear extraction proteinase inhibitor cocktail (Cayman Chemical, Ann Arbor, Mich.), 1 mM sodium orthovanadate and 30 mM NaF. Cell lysates were rotated at 4.degree. C. for 30 min. Nuclear and cellular debris was cleared by centrifugation at 20,000 g for 20 min at 4.degree. C. Equal amounts of lysate proteins (20 .mu.g) were denatured by boiling for 5 min in sodium dodecyl sulphate (SDS) sample buffer, fractionated by SDS-polyacrylamide gel electrophoresis in tris-glycine buffer, and transferred to nitrocellulose membrane (BioRad). The SeeBlue prestained standards (Invitrogen) were used as a molecular weight reference. Blots were blocked in 5% BSA/tris-buffered saline (pH 7.6) plus 0.1% Tween-20 (TBS-T) for 1 h and then incubated overnight at 4.degree. C. with mouse anti-Flag M2 monoclonal antibody (1:1000, Sigma) or mouse anti-GAPDH monoclonal antibody (1:3000, Santa Cruz Biotechnology). After washing with TBS-T, the blots were incubated with IRDye 680LT-conjugated anti-mouse antibody for 1 h at room temperature. Membranes were scanned and analyzed using an Odyssey Infrared Imaging System (LI-COR Biosciences).

[0154] Firefly-Luciferase Assay.

[0155] Cells were lysed 24 h post-treatment using Passive Lysis Buffer (Promega) and assayed with a Luciferase Reporter Gene Assay kit (Promega) according to the protocol of the manufacturer. Luciferase activity was normalized to the number of cells determined by parallel MTT assay (Vybrant, Invitrogen).

[0156] p24 ELISA

[0157] After infection or reactivation, the HIV-1 viral load levels in the supernatants were quantified by p24 Gag ELISA (Advanced BioScience Laboratories, Inc) following the manufacturer's protocol. To assess the cell viability upon treatments, MTT assay was performed in parallel according to the manufacturer's protocol (Vybrant, Invitrogen).

[0158] EGFP Flow Cytometry.

[0159] Cells were trypsinized, washed with PBS and fixed in 2% paraformaldehyde for 10 min at room temperature, then washed twice with PBS and analyzed using a Guava EasyCyte Mini flow cytometer (Guava Technologies).

[0160] Hiv-1 Reporter Virus Preparation and Infections.

[0161] HEK293T cells were transfected using Lipofectamine 2000 reagent (Invitrogen) with pNL4-3-.DELTA.E-EGFP, SF162 and JRFL (NIH AIDS Research and Reference Reagent Program). For pseudotyped pNL4-3-.DELTA.E-EGFP, the VSVG vector was cotransfected. After 48 h, the supernatant was collected, 0.45 .mu.m filtered and tittered in HeLa cells using expressed EGFP as an infection marker. For viral infection, stable Cas9/gRNA TZM-bl cells were incubated 2 h with a diluted viral stock, and washed twice with PBS. At 2 and 4 days post-infection, cells were collected, fixed and analyzed by flow cytometry for EGFP expression, or genomic DNA purification was performed for PCR and whole genome sequencing.

[0162] Genomic DNA Purification, PCR, TA-Cloning and Sanger Sequencing.

[0163] Genomic DNA was isolated from cells using an ArchivePure DNA cell/tissue purification kit (SPRIME) according to the protocol recommended by the manufacturer. One hundred ng of extracted DNA were subjected to PCR using a high-fidelity FailSafe PCR kit (Epicentre) using primers listed in FIG. 14. Three steps of standard PCR were carried out for 30 cycles with 55.degree. C. annealing and 72.degree. C. extension. The products were resolved in 2% agarose gel. The bands of interest were gel-purified and cloned into pCRII T-A vector (Invitrogen), and the nucleotide sequence of individual clones was determined by sequencing at Genewiz using universal T7 and/or SP6 primers.

[0164] Conventional and Real-Time Reverse Transcription (RT)-PCR.

[0165] For total RNA extraction, cells were processed with an RNeasy Mini kit (Qiagen) as per manufacturer's instructions. The potentially residual genomic DNA was removed through on-column DNase digestion with an RNase-Free DNase Set (Qiagen). One .mu.g of RNA for each sample was reversely transcribed into cDNAs using random hexanucleotide primers with a High Capacity cDNA Reverse Transcription Kit (Invitrogen, Grand Island, N.Y.). Conventional PCR was performed using a standard protocol. Quantitative PCR (qPCR) analyses were carried out in a LightCycler480 (Roche) using an SYBR.RTM. Green PCR Master Mix Kit (Applied Biosystems). The RT reactions were diluted to 5 ng of total RNA per micro-liter of reactions and 2 .mu.l was used in a 20-.mu.l PCR reaction. For qPCR analysis of HIV-1 proviruses, 50 ng of genomic DNA were used. The primers were synthesized in AlphaDNA and shown in FIG. 14. The primers for human housekeeping genes GAPDH and RPL13A were obtained from RealTimePrimers (Elkins Park, Pa.). Each sample was tested in triplicate. Cycle threshold (Ct) values were obtained graphically for the target genes and house-keeping genes. The difference in Ct values between the housekeeping gene and target gene was represented as .DELTA.Ct values. The .DELTA..DELTA.Ct values were obtained by subtracting the .DELTA.Ct values of control samples from those of experimental samples. Relative fold or percentage change was calculated as 2-.DELTA..DELTA.Ct. In some cases, absolute quantification was performed using the pNL4-3-.DELTA.E-EGFP plasmid spiked in human genomic DNA as a standard. The number of HIV-1 viral copies was calculated based on standard curve after normalization with housekeeping gene.

[0166] GenomeWalker Link PCR and Long-Range PCR.

[0167] The integration sites of HIV-1 in host cells were identified using a Lenti-X.TM. Integration Site Analysis kit (Clontech) following the manufacturer's instruction. Briefly, high quality genomic DNAs were extracted from U1 cells using a NucleoSpin Tissue kit (Clontech). To construct the viral integration libraries, each genomic DNA sample was digested with blunt-end-generating digestion enzymes Dra I, Ssp I or HpaI separately overnight at 37.degree. C. The digestion efficiency was verified by electrophoresis on 0.6% agarose. The digested DNA was purified using a NucleoSpin Gel and PCR Clean-Up kit followed by ligation of the digested genomic DNA fragments to GenomeWalker.TM. Adaptor at 16.degree. C. overnight. The ligation reaction was stopped by incubation at 70.degree. C. for 5 min and diluted 5 times with TE buffer. The primary PCR was performed on the DNA segments with adaptor primer 1 (AP1) and LTR-specific primer 1 (LSP1) using Advantage 2 Polymerase Mix followed by a secondary (nested) PCR using AP2 and LSP2 primers (FIG. 14). The secondary PCR products were separated on 1.5% ethidium bromide-containing agarose gel. The major bands were gel-purified and cloned into pCRII T-A vector (Invitrogen), and the nucleotide sequence of individual clones was determined by sequencing at Genewiz using universal T7 and SP6 primers. The sequence reads were analyzed by NCBI BLAST searching. Two integration sites of HIV-1 in U1 cells were identified in chromosomes X and 2. A pair of primers covering each integration site (FIG. 14) was synthesized in AlphaDNA. Long-range PCR using the U1 genomic DNA was performed with a Phusion High-Fidelity PCR kit (New England Biolabs) following the manufacturer's protocol. The PCR products were visualized on 1% agarose gel and validated by Sanger sequencing.

[0168] Surveyor Assay.

[0169] The presence of mutations in PCR products was tested using a SURVEYOR Mutation Detection Kit (Transgenomic) according to the protocol of the manufacturer. Briefly heterogeneous PCR products were denatured for 10 min in 95.degree. C. and hybridized by gradual cooling using a thermocycler. Next 300 ng of hybridized DNA (9 ul) was subjected to digestion with 0.25 .mu.l of SURVEYOR Nuclease in the presence of 0.25 .mu.l SURVEYOR Enhancer S and 15 mM MgCl.sub.2 for 4 h at 42.degree. C. Then Stop Solution was added and samples were resolved in 2% agarose gel together with equal amounts of undigested PCR products.

[0170] Some PCR products were used for restriction fragment length polymorphism analysis. Equal amount of PCR products were digested with BsaJI. Digested DNA was separated on an ethidium bromide-contained agarose gel (2%). For sequencing, PCR products were cloned using a TA Cloning.RTM. Kit Dual Promoter with pCR.TM.II vector (Invitrogen). The insert was confirmed by digestion with EcoRI and positive clones were sent to Genwiz for Sanger sequencing.

[0171] Selection of LTR Target Sites and Prediction of Potential Off-Target Sites.

[0172] For initial studies, we obtained the LTR promoter sequence (-411 to -10) of the integrated lentiviral LTR-luciferase reporter by TA-cloning sequencing of PCR products from the genome of human TZM-bl cells because of potential mutation of LTR during passaging. This promoter sequence has 100% match to the 5'-LTR of pHR'-CMV-LacZ lentiviral vector (AF105229). Thus, sense and antisense sequences of the full-length pHR' 5'-LTR (634 bp) were utilized to search for Cas9/gRNA target sites containing 20 bp gRNA targeting sequence plus the PAM sequence (NRG) using Jack Lin's CRISPR/Cas9 gRNA finder tool. The number of potential off-targets with exact match was predicted by blasting each gRNA targeting sequence plus NRG (AGG, TGG, GGG and CGG; AAG, TAG, GAG, CAG) against all available human genomic and transcript sequences using the NCBI/blastn suite with E-value cutoff 1,000 and word size 7. After pressing Control+F, copy/paste the target sequence (1-23 through 9-23 nucleotides) and find the number of genomic targets with 100% match to the target sequence. The number of off-targets for each search was divided by 3 because of repeated genome library.

[0173] Whole Genome Sequencing and Bioinformatics Analysis.

[0174] The control subclone C1 and experimental subclone AB7 of TZM-bl cells were validated for target cut efficiency and functional suppression of the LTR-luciferase reporter. The genomic DNA was isolated with NucleoSpin Tissue kit (Clontech). The DNA samples were submitted to the NextGen sequencing facility at Temple University Fox Chase Cancer Center. Duplicated genomic DNA libraries were prepared from each subclone using a NEBNext Ultra DNA Library Prep Kit for Illumina (New England Biolab) following the manufacturer's instruction. All libraries were sequenced with paired-end 141-bp reads in two Illumina Rapid Run flowcells on HiSeq 2500 instrument (Illumina). Demultiplexed read data from the sequenced libraries were sent to AccuraScience, LLC for professional bioinformatics analysis. Briefly, the raw reads were mapped against human genome (hg19) and HIV-1 genome by using Bowtie2. A genomic analysis toolkit (GATK, version 2.8.1) was used for the duplicated read removal, local alignment, base quality recalibration and indel calling. The confidence scores 10 and 30 were the thresholds for low quality (LowQual) and high confidence calling (PASS). The potential off-target sites of LTR-A and LTR-B with various mismatches were predicted by NCBI/blastn suite as described above and by a CRISPR Design Tool. All the potential gRNA target sites (FIG. 15) were used to map the .+-.300 bp regions around each indel identified by GATK. The locations of the overlapped regions in the human genome and HIV-1 genome were compared between the control C1 and experimental AB7.

[0175] Statistical Analysis.

[0176] The quantitative data represented mean.+-.standard deviation from 3-5 independent experiments, and were evaluated by Student's t-test or ANOVA and Newman-Keuls multiple comparison test. A p value that is <0.05 or 0.01 was considered as a statistically significant difference.

Example 2: Cas9/LTR-gRNA Suppresses HIV-1 Reporter Virus Production in CHME5 Microglial Cells Latently Infected with HIV-1

[0177] We assessed the ability of HIV-1-directed guide RNAs (gRNAs) to abrogate LTR transcriptional activity and eradicate proviral DNA from the genomes of latently-infected myeloid cells that serve as HIV-1 reservoirs in the brain, a particularly intractable target population. Our strategy was focused on targeting the HIV-1 LTR promoter U3 region. By bioinformatic screening and efficiency/off-target prediction, we identified four gRNA targets (protospacers; LTRs A-D) that avoid conserved transcription factor binding sites, minimizing the likelihood of altering host gene expression (FIGS. 5 and 13). We inserted DNA fragments complementary to gRNAs A-D into a humanized Cas9 expression vector (A/B in pX260; C/D in pX330) and tested their individual and combined abilities to alter the integrated HIV-1 genome activity. We first utilized the microglial cell line CHME5, which harbors integrated copies of a single round HIV-1 vector that includes the 5' and 3' LTRs, and a gene encoding an enhanced green fluorescent protein (EGFP) reporter replacing Gag (pNL4-3-.DELTA.Gag-d2EGFP). Treating CHME5 cells with trichostatin A (TSA), a histone deacetylase inhibitor, reactivates transcription from the majority of the integrated proviruses and leads to expression of EGFP and the remaining HIV-1 proteome. Expressing of gRNAs plus Cas9 markedly decreased the fraction of TSA-induced EGFP-positive CHME5 cells (FIGS. 1A and 6). We detected insertion/deletion gene mutations (indels) for LTRs A-D (FIGS. 1B and 6B) using a Cel I nuclease-based heteroduplex-specific SURVEYOR assay. Similarly, expressing gRNAs targeting LTRs C and D in HeLa-derived TZM-bl cells, that contain stably incorporated HIV-1 LTR copies driving a firefly-luciferase reporter gene, suppressed viral promoter activity (FIG. 7A), and elicited indels within the LTR U3 region (FIG. 7B-D) demonstrated by SURVEYOR and Sanger sequencing. Moreover, the combined expression of LTR C/D-targeting gRNAs in these cells caused excision of the predicted 302-bp viral DNA sequence, and emergence of the residual 194-bp fragment (FIG. 7E-F).

[0178] Multiplex expression of LTR-A/B gRNAs in mixed clonal CHME5 cells caused deletion of a 190-bp fragment between A and B target sites and led to indels to various extents (FIG. 1C-D). Among >20 puromycin-selected stable subclones, we found cell populations with complete blockade of TSA-induced HIV-1 proviral reactivation determined by flow cytometry for EGFP (FIG. 1E). PCR-based analysis for EGFP and HIV-1 Rev response element (RRE) in the proviral genome validated the eradication of HIV-1 genome (FIG. 1F, G). Furthermore, sequencing of the PCR products revealed the entire 5'-3' LTR-spanning viral genome was deleted, yielding a 351-bp fragment via a 190-bp excision between cleavage sites A and B (FIGS. 1G and 8), and a 682-bp fragment with a 175-bp insertion and a 27-bp deletion at the LTR-A and -B sites respectively (FIG. 8C). The residual HIV-1 genome (FIG. 1F-H) may reflect the presence of trace Cas9/gRNA-negative cells. These results indicate that LTR-targeting Cas9/gRNAs A/B eradicates the HIV-1 genome and blocks its reactivation in latently infected microglial cells.

Example 3: Cas9/LTR-gRNA Efficiently Eradicates Latent HIV-1 Virus from U1 Monocytic Cells

[0179] The promonocytic U-937 cell subclone U1, an HIV-1 latency model for infected perivascular macrophages and monocytes, is chronically HIV-1-infected and exhibits low level constitutive viral gene expression and replication. GenomeWalker mapping detected two integrated proviral DNA copies at chromosomes Xp11-4 (FIG. 2A) and 2p21 (FIG. 9A) in U1 cells. A 9935-bp DNA fragment representing the entire 9709-bp proviral HIV-1 DNA plus a flanking 226-bp X-chromosome-derived sequence (FIG. 2A), and a 10176-bp fragment containing 9709-bp HIV-1 genome plus its flanking 2-chromosome-derived 467-bp (FIG. 9A, B) were identified by the long-range PCR analysis of the parental control or empty-vector (U6-CAG) U1 cells. The 226-bp and 467-bp fragments represent the predicted segment from the other copy of chromosome X and 2 respectively, which lacked the integrated proviral DNA. In U1 cells expressing LTR-A/B gRNAs and Cas9, we found two additional DNA fragments of 833 and 670 bp in chromosome X and one additional 1102-bp fragment in chromosome 2. Thus, gRNAs A/B enabled Cas9 to excise the HIV-1 5'-3' LTR-spanning viral genome segment in both chromosomes. The 833-bp fragment includes the expected 226-bp from the host genome and a 607-bp viral LTR sequence with a 27-bp deletion around the LTR-A site (FIG. 2A-B). The 670-bp fragment encompassed a 226-bp host sequence and residual 444-bp viral LTR sequence after 190-bp fragment excision (FIG. 1D), caused by gRNAs-A/B-guided cleavage at both LTRs (FIG. 2A). The additional fragments did not emerge via circular LTR integration, because it was absent in the parental U1 cells, and such circular LTR viral genome configuration occurs immediately after HIV-1 infection but is short lived and intolerant to repeated passaging. These cells exhibited substantially decreased HIV-1 viral load, shown by the functional p24 ELISA replication assay (FIG. 2C) and real-time PCR analysis (FIG. 9C, D). The detectable but low residual viral load and reactivation may result from cell population heterogeneity and/or incomplete genome editing. We also validated the ablation of HIV-1 genome by Cas9/LTR-A/B gRNAs in latently infected J-Lat T cells harboring integrated HIV-R7/E-/EGFP using flow cytometry analysis, SURVEYOR assay and PCR genotyping (FIG. 10), supporting the results of previous reports on HIV-1 proviral deletion in Jurkat T cells by Cas9/gRNA and ZFN. Taken together, our results suggest that the multiplex LTR-gRNAs/Cas9 system efficiently suppress HIV-1 replication and reactivation in latently HIV-1-infected "reservoir" (microglial, monocytic and T) cells typical of human latent HIV-1 infection, and in TZM-bl cells highly sensitive for detecting HIV-1 transcription and reactivation. Single or multiplex gRNAs targeting 5'- and 3'-LTRs effectively eradicated the entire HIV-1 genome.

Example 4: Stable Expression of Cas9 Plus LTR-A/B Vaccinates TZM-bl Cells Against New HIV-1 Virus Infection

[0180] We next tested whether combined Cas9/LTR gRNAs can immunize cells against HIV-1 infection using stable Cas9/gRNAs-A and -B-expressing TZM-bl-based clones (FIG. 3A). Two of 7 puromycin-selected subclones exhibited efficient excision of the 190-bp LTR-A/B site-spanning DNA fragment (FIG. 3B). However, the remaining 5 subclones exhibited no excision (FIG. 3B) and no indel mutations as verified by Sanger sequencing. PCR genotyping using primers targeting Cas9 and U6-LTR showed that none of these ineffective subclones retained the integrated copies of Cas9/LTR-A/B gRNA expression cassettes. (FIG. 11A, B). As a result, no expression of full-length Cas9 was detected (FIG. 11C, D). The long-term expression of Cas9/LTR-A/B gRNAs did not adversely affect cell growth or viability, suggesting a low occurrence of off-target interference with the host genome or Cas9-induced toxicity in this model. We assessed de novo HIV-1 replication by infecting cells with the VSVG-pseudotyped pNL4-3-.DELTA.E-EGFP reporter virus, with EGFP-positivity by flow cytometry indicating HIV-1 replication. Unlike the control U6-CAG cells, the cells stably expressing Cas9/gRNAs LTRs-A/B failed to support HIV-1 replication at 2 d post infection, indicating that they were immunized effectively against new HIV-1 infection (FIG. 3C-D). A similar immunity against HIV-1 was observed in Cas/LTR-A/B gRNA expressing cells infected with native T-tropic X4 strain pNL4-3-.DELTA.E-EGFP reporter virus (FIG. 12A) or native M-tropic R5 strains such as SF162 and JRFL (FIG. 12B-D).

Example 5: Off-Target Effects of Cas9/LTR-A/B on Human Genome

[0181] The appeal of Cas9/gRNA as an interventional approach rests on its highly specific on-target indel-producing cleavage, but multiplex gRNAs could potentially cause host genome mutagenesis and chromosomal disorders, cytotoxicity, genotoxicity, or oncogenesis. Fairly low viral-human genome homology reduces this risk, but the human genome contains numerous endogenous retroviral genomes that are potentially susceptible to HIV-1-directed gRNAs. Therefore, we assessed off-target effects of selected HIV-1 LTR gRNAs on the human genome. Because the 12-14-bp seed sequence nearest the protospacer-adjacent motif (PAM) region (NGG) is critical for cleavage specificity, we searched >14-bp seed+NGG, and found no off-target candidate sites by LTR gRNAs A-D (FIG. 13). It is not surprising that progressively shorter gRNA segments yielded increasing off-target cleavage sites 100% matched to corresponding on-target sequences (i.e., NGG+13 bp yielded 6, 0, 2 and 9 off-target sites, respectively, whereas NGG+12 bp yielded 16, 5, 16 and 29; FIG. 13). From human genomic DNA we obtained a 500-800-bp sequence covering one of predicted off-target sites using high-fidelity PCR, and analyzed the potential mutations by SURVEYOR and Sanger sequencing. We found no mutations (see representative off-target sites #1, 5 and 6 in TZM-bl and U1 cells; FIG. 4A).

[0182] To assess risk of off-target effects comprehensively, we performed whole genome sequencing (WGS) using the stable Cas9/gRNA A/B-expressing and control U6-CAG TZM-bl cells (FIG. 4B-D). We identified 676,105 indels, using a genome analysis toolkit (GATK, v.2.8.1) with human (hg19) and HIV-1 genomes as reference sequences. Among the indels, 24% occurred in the U6-CAG control, 26% in LTR-A/B subclone, and 50% in both (FIG. 4B). Such substantial inter-sample indel-calling discrepancy suggests the probable off-target effects, but most likely results from its limited confidence, limited WGS coverage (15-30.times.), and cellular heterogeneity. GATK reported only confidently-identified indels: some found in the U6-CAG control but not in the LTR-A/B subclone, and others in the LTR-A/B but not in the U6-CAG. We expected abundant missing indel calls for both samples due to the limited WGS coverage. Such limited indel-calling confidence also implies the possibility of false negatives: missed indels occurring in LTR-A/B but not U6-CAG controls. Cellular heterogeneity may reflect variability of Cas9/gRNA editing efficiency and effects of passaging. Therefore, we tested whether each indel was LTR-A/B gRNA-induced, by analyzing .+-.300 bp flanking each indel against LTRs-A/-B-targeted sites of the HIV-1 genome and predicted/potential gRNA off-target sites of the host genome (FIG. 15). For sequences 100% matched to one containing the seed (12-bp) plus NRG, we identified only 8 overlapped regions of 92 potential off-target sites against 676,105 indels: 6 indels occurring in both samples, and 2 only in the U6-CAG control (FIG. 4C, D). We also identified 2 indels on HIV-1 LTR that occurred only in the LTR-A/B subclone but, as expected, not in the U6-CAG control (FIG. 4C). The results suggest that LTR-A/B gRNAs induce the indicated on-target indels, but no off-target indels, consistent with prior findings using deep sequencing of PCR products covering predicted/potential off-target site.

[0183] Our combined approaches minimized off-target effects while achieving high efficiency and complete ablation of the genomically integrated HIV-1 provirus. In addition to an extremely low homology between the foreign viral genome and host cellular genome including endogenous retroviral DNA, the key design attributes in our study included: bioinformatic screening using the strictest 12-bp+NGG target-selection criteria to exclude off-target human transcriptome or (even rarely) untranslated-genomic sites; avoiding transcription factor binding sites within the HIV-1 LTR promoter (potentially conserved in the host genome); selection of LTR-A- and -B-directed, 30-bp gRNAs and also pre-crRNA system reflecting the original bacterial immune mechanism to enhance specificity/efficiency vs. 20-bp gRNA-, chimeric crRNA-tracRNA-based system; and WGS, Sanger sequencing and SURVEYOR assay, to identify and exclude potential off-target effects. Indeed, the use of newly developed Cas9 double-nicking and RNA-guided FokI nuclease may further assist identification of new targets within the various conserved regions of HIV-1 with reduced off-target effects.

[0184] Our results show that the HIV-1 Cas9/gRNA system has the ability to target more than one copy of the LTR, which are positioned on different chromosomes, suggesting that this genome editing system can alter the DNA sequence of HIV-1 in latently infected patient's cells harboring multiple proviral DNAs. To further ensure high editing efficacy and consistency of our technology, one may consider the most stable region of HIV-1 genome as a target to eradicate HIV-1 in patient samples, which may not harbor only one strain of HIV-1. Alternatively, one may develop personalized treatment modalities based on the data from deep sequencing of the patient-derived viral genome prior to engineering therapeutic Cas9/gRNA molecules.

[0185] Our results also demonstrate that Cas9/gRNA genome editing can be used to immunize cells against HIV-1 infection. The preventative vaccination is independent of HIV-1 strain's diversity because the system targets genomic sequences regardless of how the viruses enter the infected cells. The preexistence of the Cas9/gRNA system in cells led to a rapid elimination of the new HIV-1 before it integrates into the host genome. One may explore various systems for delivery of Cas9/LTR-gRNA for immunizing high-risk subjects, e.g., gene therapies (viral vector and nanoparticle) and transplantation of autologous Cas9/gRNA-modified bone marrow stem/progenitor cells or inducible pluripotent stem cells for eradicating HIV-1 infection.

[0186] Here, we demonstrated the high specificity of Cas9/gRNAs in editing HIV-1 target genome. Results from subclone data revealed the strict dependence of genome editing on the presence of both Cas9 and gRNA. Moreover, only one nucleotide mismatch in the designed gRNA target will disable the editing potency. In addition, all of our 4 designed LTR gRNAs worked well with different cell lines, indicating that the editing is more efficient in the HIV-1 genome than the host cellular genome, wherein not all designed gRNAs are functional, which may be due to different epigenetic regulation, variable genome accessibility, or other reasons. Given the ease and rapidity of Cas9/gRNA development, even if HIV-1 mutations confer resistance to one Cas9/gRNA-based therapy, as described above, HIV-1 variants can be genotyped to enable another personalized therapy for individual patients.

[0187] A number of embodiments of the invention have been described. Nevertheless, it will be understood that various modifications may be made without departing from the spirit and scope of the invention. Accordingly, other embodiments are within the scope of the following claims.

Sequence CWU 1

1

389130DNAHuman immunodeficiency virus 1 1gccagggatc agatatccac tgacctttgg 30234DNAHuman immunodeficiency virus 1 2tccggagtac ttcaagaact gctgacatcg agct 34319DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 3ccactgacta cttcaagaa 194859DNAArtificial SequenceDescription of Artificial Sequence Synthetic polynucleotidemodified_base(289)..(313)a, c, t, g, unknown or othermisc_feature(289)..(313)n is a, c, g, or t 4ctaggtgatt aggatattct acaatccaaa ttcttaccag tttgggatta ttcaaattgg 60gcaccttggc agatatgttt tgaaaactgc taggcaaagc attctggaag aatagacaaa 120gaagtaataa aatataacaa aaagcagtgg aagttacaaa aaaaaatgtt tctcttttgg 180aagggctaat ttggtcccaa agaagacaag atatccttga tctgtggatc taccacacac 240aaggctactt ccctgattgg cagaactaca acaccagggc cagggatcnn nnnnnnnnnn 300nnnnnnnnnn nnnttcaagt tagtaccagt tgagccaggg caggtagaag aggccaatga 360aggagagaac aacaccttgt tacaccctat gagcctgcat gggatggagg acccggaggg 420agaagtatta gtgtggaagt ttgacagcct cctagcattt cgtcacatgg cccgagagct 480gcatccggag tactacaaag actgctgaca tcgagttttc tacaagggac tttccgctgg 540ggactttcca gggaggtgtg gcctgggcgg gactggggag tggcgagccc tcagatgctg 600catataagca gctgcttttt gcctgtactg ggtctctctg gttagaccag atctgagcct 660gggagctctc tggctagcta gggaacccac tgcttaagcc tcaataaagc ttgccttgag 720tgctacaagt agtgtgtgcc cgtctgttgt gtgactctgg taactagaga tccctcagac 780ccttttagtc agtgtggaaa atctctagca tctttaaagt acagaatgcc aaaacaggaa 840ggattgataa gatagtcgt 859510DNAHuman immunodeficiency virus 1 5tcttttggaa 10676DNAHuman immunodeficiency virus 1 6gattggcaga actacacacc agggccaggg atcagatatc cactgacctt tggatggtgc 60ttcaagttag taccag 76710DNAHuman immunodeficiency virus 1 7tctttaaagt 10810DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 8tcttttggaa 10963DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 9gattggcaga actacaacac cagggccagg gatcagatgg atggtgcttc aagttagtac 60cag 631010DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 10tctttaaagt 101110DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 11tcttttggaa 101250DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 12gattggcaga actacaacac cagggccagg gatcttcaag ttagtaccag 501310DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 13tctttaaagt 101424DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 14gagatcctgt ctcaaaaaaa agtt 241517DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 15atctatccat gagggcg 1716402DNAHuman immunodeficiency virus 1 16gatctgtgga tctaccacac acaaggctac ttccctgatt ggcagaacta cacaccaggg 60ccagggatca gatatccact gacctttgga tggtgctaca agctagtacc agttgagcaa 120gagaaggtag aagaagccaa tgaaggagag aacacccgct tgttacaccc tgtgagcctg 180catgggatgg atgacccgga gagagaagta ttagagtgga ggtttgacag ccgcctagca 240tttcatcaca tggcccgaga gctgcatccg gagtacttca agaactgctg acatcgagct 300tgctacaagg gactttccgc tggggacttt ccagggaggc gtggcctggg cgggactggg 360gagtggcgag ccctcagatg ctgcatataa gcagctgctt tt 4021731DNAHuman immunodeficiency virus 1 17ccctgattgg cagaactaca caccagggcc a 311832DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 18ccctgattgg cagaactaca acaccagggc ca 321932DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 19ccctgattgg cagaactaca acaccagggc ca 322032DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 20ccctgattgg cagaactaca acaccagggc ca 322130DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 21ccctgattgg cagaactaca accagggcca 302229DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 22ccctgattgg cagaactaca ccagggcca 292329DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 23ccctgattgg cagaactaca ccagggcca 292426DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 24ccctgattgg cagaactaca gggcca 262529DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 25ccctgattgg cagaactaca gggccaggg 292686DNAHuman immunodeficiency virus 1 26gactttccag ggaggcgtgg cctgggcggg actggggagt ggcgagccct cagatgctgc 60atataagcag cggtgaagcc gaattc 862786DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 27gactttccag ggaggcgtgg cctgggcggg actggggggt ggcgagccct cagatgctgc 60atataagcag cggtgaagcc gaattc 862888DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 28gactttccag ggaggcgtgg cctgggcggg tatctgggga gtggcgagcc ctcagatgct 60gcatataagc agcggtgaag ccgaattc 882985DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 29gactttccag gggggcgtgg cctgggcggg actggggagt ggcgagccct cagatgctgc 60ataaagcagc ggtgaagccg aattc 853023DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 30gactttccag ggaagccgaa ttc 233125DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 31gattggcaga actacactgg ggagt 253226DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 32gattggcaga actacacctc agatgc 263328DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 33catcacatgg cccgctgctg acatcgag 283455DNAHuman immunodeficiency virus 1 34catcacgtgg cccgagagct gcatccggag tacttcaaga actgctgaca tcgag 55351106DNAArtificial SequenceDescription of Artificial Sequence Synthetic polynucleotidemodified_base(152)..(155)a, c, t, g, unknown or othermisc_feature(152)..(155)n is a, c, g, or t 35gctattgtat ctgatcacaa gctgttaaaa gcggtcatgc cacttcttga atgctttgca 60gctggaaggg ctaatttggt cccaaagaag acaagatatc cttgatctgt ggatctacca 120cacacaaggc tacttccctg attggcagaa cnnnncacca gggccaggga tcagatatcc 180actgaccatc cactttggat ggtgcttcaa gttagtacca gttgagccag ggcaggtaga 240agaggccaat gaaggagaga acaacacctt gttacaccct atgagcctgc atgggatgga 300ggacccggag ggagaagtat tagtgtggaa gtttgacagc ctcctagcat ttcgtcacat 360ggcccgagag ctgcatccgg agtactacaa agactgctga catcgagttt tctacaaggg 420actttccgct ggggactttc cagggaggtg tggcctgggc gggactgggg agtggcgagc 480cctcagatgc tgcatataag cagctgcttt ttgcctgtac tgggtctctc tggttagacc 540agatctgagc ctgggagctc tctggctagc tagggaaccc actgcttaag cctcaataaa 600gcttgccttg agtgctacaa gtagtgtgtg cccgtctgtt gtgtgactct ggtaactaga 660gatccctcag acccttttag tcagtgtgga aaatctctag cagcagctta gaaatttttt 720ccaccagagg ccgggcgtgg tggctcacgc ctgtaatccc agcactttgg gaggccgagg 780tgggcggatc acctgaagtc aggagttcga gaccagcctc aacatggaga aaccccatct 840ctactaaaaa tacaaaatta gctgggcgtg gtggtgcatg cctgtaatcc cagctacttg 900ggaggctgag acaggataat tgcttgaacc tggaaggcag aggttgcggt gagccgagat 960tgcgccattg cattccagcc tgggcaacag gagcgaaact tcgtctcaaa aaaaaaaaaa 1020aaagacattt tttccaccag ataccctaga tcatgactgt taagtctggc cttccacgaa 1080gccctaggac ctggacacac aatcaa 11063636DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 36aaacagggcc agggatcaga tatccactga ccttgt 363735DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 37taaacaaggt cagtggatat ctgatccctg gccct 353836DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 38aaacagctcg atgtcagcag ttcttgaagt actcgt 363935DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 39taaacgagta cttcaagaac tgctgacatc gagct 354024DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 40caccgattgg cagaactaca cacc 244124DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 41aaacggtgtg tagttctgcc aatc 244224DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 42caccgcgtgg cctgggcggg actg 244324DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 43aaaccagtcc cgcccaggcc acgc 244424DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 44tggaagggct aattcactcc caac 244524DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 45ccgagagctc ccaggctcag atct 244627DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 46caccgatctg tggatctacc acacaca 274724DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 47aaacgagtca cacaacagac gggc 244837DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 48cgcctcgagg atccgagggc ctatttccca tgattcc 374935DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 49tgtgaattca ggcgggccat ttaccgtaag ttatg 355025DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 50acgactatct tatcaatcct tcctg 255126DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 51ctaggtgatt aggatattct acaatc 265224DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 52gctattgtat ctgatcacaa gctg 245324DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 53ttgattgtgt gtccaggtcc tagg 245423DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 54gcaagggcga ggagctgttc acc 235524DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 55ttgtagttgc cgtcgtcctt gaag 245623DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 56aatggtacat caggccatat cac 235723DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 57cccactgtgt ttagcatggt att 235823DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 58cacagcatca agaagaacct gat 235924DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 59tcttccgtct ggtgtatctt cttc 246028DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 60cgccaagctt gaataggagc tttgttcc 286130DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 61ctaggatcca ggagctgttg atcctttagg 306223DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 62gtggactttg gatggtgaga tag 236323DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 63gcctggcaag agtgaactga gtc 236423DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 64aagataatga gttgtggcag agc 236524DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 65tctacctggt aatccagcat ctgg 246623DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 66ataggaggaa ggcaccaaga ggg 236723DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 67aatgatgctt tggtcctact cct 236824DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 68tgctcttgct actctggcat gtac 246923DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 69aatctacctc tgagagctgc agg 237023DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 70tcagacacag ctgaagcaga ggc 237123DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 71atgccagtgt cagtagatgt cag 237224DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 72tcaagatcag ccagagtgca catg 247323DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 73tgctcttccg agcctctctg gag 237422DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 74atggactatc atatgcttac cg 227528DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 75gcttcagcaa gccgagtcct gcgtcgag 287628DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 76gctcctctgg tttccctttc gctttcaa 287722DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 77gtaatacgac tcactatagg gc 227819DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 78actatagggc acgcgtggt 197923DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 79tcagaccctt ttagtcagtg tgg 238023DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 80ttgcttgtac tgggtctctc tgg 238123DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 81cagctgcttt ttgcttgtac tgg 238223DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 82ctgacatcga gcttgctaca agg 238323DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 83ccgcctagca tttcatcaca tgg 238423DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 84cggagagaga agtattagag tgg 238523DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 85agtaccagtt gagcaagaga agg 238623DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 86gatatccact gacctttgga tgg 238723DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 87gattggcaga actacacacc agg 238823DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 88cacaaggcta cttccctgat tgg 238923DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 89ctgtggatct accacacaca agg 239023DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 90tgggagctct ctggctaact agg 239123DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 91ggttagacca gatctgagcc tgg

239223DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 92tgctacaagg gactttccgc tgg 239323DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 93agagagaagt attagagtgg agg 239423DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 94ttacaccctg tgagcctgca tgg 239523DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 95aaggtagaag aagccaatga agg 239623DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 96atcagatatc cactgacctt tgg 239723DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 97gacaagatat ccttgatctg tgg 239823DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 98gcccgtctgt tgtgtgactc tgg 239923DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 99atctgagcct gggagctctc tgg 2310023DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 100ctttccgctg gggactttcc agg 2310123DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 101cagaactaca caccagggcc agg 2310223DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 102cctgcatggg atggatgacc cgg 2310323DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 103ccctgtgagc ctgcatggga tgg 2310423DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 104ctttccaggg aggcgtggcc tgg 2310523DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 105ggggactttc cagggaggcg tgg 2310623DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 106ccgctgggga ctttccaggg agg 2310723DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 107catggcccga gagctgcatc cgg 2310823DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 108gcctgggcgg gactggggag tgg 2310923DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 109aggcgtggcc tgggcgggac tgg 2311023DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 110gcgtggcctg ggcgggactg ggg 2311123DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 111ccagggaggc gtggcctggg cgg 2311223DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 112tgtggtagat ccacagatca agg 2311323DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 113ggtgtgtagt tctgccaatc agg 2311423DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 114gtcagtggat atctgatccc tgg 2311523DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 115tagcaccatc caaaggtcag tgg 2311623DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 116tagcttgtag caccatccaa agg 2311723DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 117tctaccttct cttgctcaac tgg 2311823DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 118cactctaata cttctctctc cgg 2311923DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 119ccatgtgatg aaatgctagg cgg 2312023DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 120gggccatgtg atgaaatgct agg 2312123DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 121cagcagttct tgaagtactc cgg 2312223DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 122ctgcttatat gcagcatctg agg 2312323DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 123cacactactt gaagcactca agg 2312423DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 124taccagagtc acacaacaga cgg 2312523DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 125acactgacta aaagggtctg agg 2312623DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 126caaggatatc ttgtcttcgt tgg 2312723DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 127cagggaagta gccttgtgtg tgg 2312823DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 128gcgggtgttc tctccttcat tgg 2312923DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 129tagttagcca gagagctccc agg 2313023DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 130ctttattgag gcttaagcag tgg 2313123DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 131actcaaggca agctttattg agg 2313223DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 132ggatatctga tccctggccc tgg 2313323DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 133ggctcacagg gtgtaacaag cgg 2313423DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 134tccatcccat gcaggctcac agg 2313523DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 135agtactccgg atgcagctct cgg 2313623DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 136agagctccca ggctcagatc tgg 2313723DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 137gattttccac actgactaaa agg 2313823DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 138ccgggtcatc catcccatgc agg 2313923DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 139cctccctgga aagtccccag cgg 2314023DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 140gccactcccc agtcccgccc agg 2314123DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 141ccgcccaggc cacgcctccc tgg 2314223DNAHuman immunodeficiency virus 1 142atcagatatc cactgacctt tgg 2314322DNAHuman immunodeficiency virus 1 143tcagatatcc actgaccttt gg 2214422DNAHuman immunodeficiency virus 1 144tcagatatcc actgaccttt gg 2214521DNAHuman immunodeficiency virus 1 145cagatatcca ctgacctttg g 2114621DNAHuman immunodeficiency virus 1 146cagatatcca ctgacctttg g 2114720DNAHuman immunodeficiency virus 1 147agatatccac tgacctttgg 2014820DNAHuman immunodeficiency virus 1 148agatatccac tgacctttgg 2014919DNAHuman immunodeficiency virus 1 149gatatccact gacctttgg 1915019DNAHuman immunodeficiency virus 1 150gatatccact gacctttgg 1915118DNAHuman immunodeficiency virus 1 151atatccactg acctttgg 1815218DNAHuman immunodeficiency virus 1 152atatccactg acctttgg 1815317DNAHuman immunodeficiency virus 1 153tatccactga ccttggg 1715417DNAHuman immunodeficiency virus 1 154tatccactga cctttgg 1715517DNAHuman immunodeficiency virus 1 155tatccactga cctttgg 1715617DNAHuman immunodeficiency virus 1 156tatccactga ccttaag 1715717DNAHuman immunodeficiency virus 1 157tatccactga ccttgag 1715816DNAHuman immunodeficiency virus 1 158atccactgac cttagg 1615916DNAHuman immunodeficiency virus 1 159atccactgac cttagg 1616016DNAHuman immunodeficiency virus 1 160atccactgac cttggg 1616116DNAHuman immunodeficiency virus 1 161atccactgac cttggg 1616216DNAHuman immunodeficiency virus 1 162atccactgac cttggg 1616316DNAHuman immunodeficiency virus 1 163atccactgac cttggg 1616416DNAHuman immunodeficiency virus 1 164atccactgac ctttgg 1616516DNAHuman immunodeficiency virus 1 165atccactgac ctttgg 1616616DNAHuman immunodeficiency virus 1 166atccactgac ctttgg 1616716DNAHuman immunodeficiency virus 1 167atccactgac cttaag 1616816DNAHuman immunodeficiency virus 1 168atccactgac cttaag 1616916DNAHuman immunodeficiency virus 1 169atccactgac cttcag 1617016DNAHuman immunodeficiency virus 1 170atccactgac cttcag 1617116DNAHuman immunodeficiency virus 1 171atccactgac cttgag 1617216DNAHuman immunodeficiency virus 1 172atccactgac cttgag 1617315DNAHuman immunodeficiency virus 1 173tccactgacc ttagg 1517415DNAHuman immunodeficiency virus 1 174tccactgacc ttagg 1517515DNAHuman immunodeficiency virus 1 175tccactgacc ttagg 1517615DNAHuman immunodeficiency virus 1 176tccactgacc ttagg 1517715DNAHuman immunodeficiency virus 1 177tccactgacc ttagg 1517815DNAHuman immunodeficiency virus 1 178tccactgacc ttagg 1517915DNAHuman immunodeficiency virus 1 179tccactgacc ttggg 1518015DNAHuman immunodeficiency virus 1 180tccactgacc ttggg 1518115DNAHuman immunodeficiency virus 1 181tccactgacc ttggg 1518215DNAHuman immunodeficiency virus 1 182tccactgacc ttggg 1518315DNAHuman immunodeficiency virus 1 183tccactgacc ttggg 1518415DNAHuman immunodeficiency virus 1 184tccactgacc ttggg 1518515DNAHuman immunodeficiency virus 1 185tccactgacc ttggg 1518615DNAHuman immunodeficiency virus 1 186tccactgacc ttggg 1518715DNAHuman immunodeficiency virus 1 187tccactgacc tttgg 1518815DNAHuman immunodeficiency virus 1 188tccactgacc tttgg 1518915DNAHuman immunodeficiency virus 1 189tccactgacc tttgg 1519015DNAHuman immunodeficiency virus 1 190tccactgacc tttgg 1519115DNAHuman immunodeficiency virus 1 191tccactgacc tttgg 1519215DNAHuman immunodeficiency virus 1 192tccactgacc tttgg 1519315DNAHuman immunodeficiency virus 1 193tccactgacc tttgg 1519415DNAHuman immunodeficiency virus 1 194tccactgacc tttgg 1519515DNAHuman immunodeficiency virus 1 195tccactgacc tttgg 1519615DNAHuman immunodeficiency virus 1 196tccactgacc ttaag 1519715DNAHuman immunodeficiency virus 1 197tccactgacc ttaag 1519815DNAHuman immunodeficiency virus 1 198tccactgacc ttaag 1519915DNAHuman immunodeficiency virus 1 199tccactgacc ttaag 1520015DNAHuman immunodeficiency virus 1 200tccactgacc ttaag 1520115DNAHuman immunodeficiency virus 1 201tccactgacc ttcag 1520215DNAHuman immunodeficiency virus 1 202tccactgacc ttcag 1520315DNAHuman immunodeficiency virus 1 203tccactgacc ttcag 1520415DNAHuman immunodeficiency virus 1 204tccactgacc ttcag 1520515DNAHuman immunodeficiency virus 1 205tccactgacc ttcag 1520615DNAHuman immunodeficiency virus 1 206tccactgacc ttcag 1520715DNAHuman immunodeficiency virus 1 207tccactgacc ttcag 1520815DNAHuman immunodeficiency virus 1 208tccactgacc ttcag 1520915DNAHuman immunodeficiency virus 1 209tccactgacc ttcag 1521015DNAHuman immunodeficiency virus 1 210tccactgacc ttcag 1521115DNAHuman immunodeficiency virus 1 211tccactgacc ttcag 1521215DNAHuman immunodeficiency virus 1 212tccactgacc ttcag 1521315DNAHuman immunodeficiency virus 1 213tccactgacc ttgag 1521415DNAHuman immunodeficiency virus 1 214tccactgacc ttgag 1521515DNAHuman immunodeficiency virus 1 215tccactgacc ttgag 1521615DNAHuman immunodeficiency virus 1 216tccactgacc ttgag 1521715DNAHuman immunodeficiency virus 1 217tccactgacc ttgag 1521815DNAHuman immunodeficiency virus 1 218tccactgacc ttgag 1521915DNAHuman immunodeficiency virus 1 219tccactgacc ttgag 1522015DNAHuman immunodeficiency virus 1 220tccactgacc ttgag 1522115DNAHuman immunodeficiency virus 1 221tccactgacc ttgag

1522215DNAHuman immunodeficiency virus 1 222tccactgacc tttag 1522315DNAHuman immunodeficiency virus 1 223tccactgacc tttag 1522415DNAHuman immunodeficiency virus 1 224tccactgacc tttag 1522515DNAHuman immunodeficiency virus 1 225tccactgacc tttag 1522615DNAHuman immunodeficiency virus 1 226tccactgacc tttag 1522723DNAHuman immunodeficiency virus 1 227cagcagttct tgaagtactc cgg 2322822DNAHuman immunodeficiency virus 1 228agcagttctt gaagtactcc gg 2222921DNAHuman immunodeficiency virus 1 229gcagttcttg aagtactccg g 2123020DNAHuman immunodeficiency virus 1 230cagttcttga agtactccgg 2023119DNAHuman immunodeficiency virus 1 231agttcttgaa gtactccgg 1923218DNAHuman immunodeficiency virus 1 232gttcttgaag tactccgg 1823317DNAHuman immunodeficiency virus 1 233ttcttgaagt actccgg 1723416DNAHuman immunodeficiency virus 1 234tcttgaagta ctccgg 1623516DNAHuman immunodeficiency virus 1 235tcttgaagta ctctag 1623615DNAHuman immunodeficiency virus 1 236cttgaagtac tcagg 1523715DNAHuman immunodeficiency virus 1 237cttgaagtac tcagg 1523815DNAHuman immunodeficiency virus 1 238cttgaagtac tcagg 1523915DNAHuman immunodeficiency virus 1 239cttgaagtac tcagg 1524015DNAHuman immunodeficiency virus 1 240cttgaagtac tccgg 1524115DNAHuman immunodeficiency virus 1 241cttgaagtac tctgg 1524215DNAHuman immunodeficiency virus 1 242cttgaagtac tcaag 1524315DNAHuman immunodeficiency virus 1 243cttgaagtac tcaag 1524415DNAHuman immunodeficiency virus 1 244cttgaagtac tcaag 1524515DNAHuman immunodeficiency virus 1 245cttgaagtac tcaag 1524615DNAHuman immunodeficiency virus 1 246cttgaagtac tcaag 1524715DNAHuman immunodeficiency virus 1 247cttgaagtac tccag 1524815DNAHuman immunodeficiency virus 1 248cttgaagtac tccag 1524915DNAHuman immunodeficiency virus 1 249cttgaagtac tccag 1525015DNAHuman immunodeficiency virus 1 250cttgaagtac tccag 1525115DNAHuman immunodeficiency virus 1 251cttgaagtac tctag 1525215DNAHuman immunodeficiency virus 1 252cttgaagtac tctag 1525323DNAHuman immunodeficiency virus 1 253atcagatatc cactgacctt tgg 2325422DNAHuman immunodeficiency virus 1 254tcagatatcc actgaccttt gg 2225522DNAHuman immunodeficiency virus 1 255tcagatatcc actgaccttt gg 2225621DNAHuman immunodeficiency virus 1 256cagatatcca ctgacctttg g 2125721DNAHuman immunodeficiency virus 1 257cagatatcca ctgacctttg g 2125820DNAHuman immunodeficiency virus 1 258agatatccac tgacctttgg 2025920DNAHuman immunodeficiency virus 1 259agatatccac tgacctttgg 2026019DNAHuman immunodeficiency virus 1 260gatatccact gacctttgg 1926119DNAHuman immunodeficiency virus 1 261gatatccact gacctttgg 1926218DNAHuman immunodeficiency virus 1 262atatccactg acctttgg 1826318DNAHuman immunodeficiency virus 1 263atatccactg acctttgg 1826417DNAHuman immunodeficiency virus 1 264tatccactga ccttggg 1726517DNAHuman immunodeficiency virus 1 265tatccactga cctttgg 1726617DNAHuman immunodeficiency virus 1 266tatccactga cctttgg 1726717DNAHuman immunodeficiency virus 1 267tatccactga ccttaag 1726817DNAHuman immunodeficiency virus 1 268tatccactga ccttgag 1726916DNAHuman immunodeficiency virus 1 269atccactgac cttagg 1627016DNAHuman immunodeficiency virus 1 270atccactgac cttagg 1627116DNAHuman immunodeficiency virus 1 271atccactgac cttggg 1627216DNAHuman immunodeficiency virus 1 272atccactgac cttggg 1627316DNAHuman immunodeficiency virus 1 273atccactgac cttggg 1627416DNAHuman immunodeficiency virus 1 274atccactgac cttggg 1627516DNAHuman immunodeficiency virus 1 275atccactgac ctttgg 1627616DNAHuman immunodeficiency virus 1 276atccactgac ctttgg 1627716DNAHuman immunodeficiency virus 1 277atccactgac ctttgg 1627816DNAHuman immunodeficiency virus 1 278atccactgac cttaag 1627916DNAHuman immunodeficiency virus 1 279atccactgac cttaag 1628016DNAHuman immunodeficiency virus 1 280atccactgac cttcag 1628116DNAHuman immunodeficiency virus 1 281atccactgac cttcag 1628216DNAHuman immunodeficiency virus 1 282atccactgac cttgag 1628316DNAHuman immunodeficiency virus 1 283atccactgac cttgag 1628415DNAHuman immunodeficiency virus 1 284tccactgacc ttagg 1528515DNAHuman immunodeficiency virus 1 285tccactgacc ttagg 1528615DNAHuman immunodeficiency virus 1 286tccactgacc ttagg 1528715DNAHuman immunodeficiency virus 1 287tccactgacc ttagg 1528815DNAHuman immunodeficiency virus 1 288tccactgacc ttagg 1528915DNAHuman immunodeficiency virus 1 289tccactgacc ttagg 1529015DNAHuman immunodeficiency virus 1 290tccactgacc ttggg 1529115DNAHuman immunodeficiency virus 1 291tccactgacc ttggg 1529215DNAHuman immunodeficiency virus 1 292tccactgacc ttggg 1529315DNAHuman immunodeficiency virus 1 293tccactgacc ttggg 1529415DNAHuman immunodeficiency virus 1 294tccactgacc ttggg 1529515DNAHuman immunodeficiency virus 1 295tccactgacc ttggg 1529615DNAHuman immunodeficiency virus 1 296tccactgacc ttggg 1529715DNAHuman immunodeficiency virus 1 297tccactgacc ttggg 1529815DNAHuman immunodeficiency virus 1 298tccactgacc tttgg 1529915DNAHuman immunodeficiency virus 1 299tccactgacc tttgg 1530015DNAHuman immunodeficiency virus 1 300tccactgacc tttgg 1530115DNAHuman immunodeficiency virus 1 301tccactgacc tttgg 1530215DNAHuman immunodeficiency virus 1 302tccactgacc tttgg 1530315DNAHuman immunodeficiency virus 1 303tccactgacc tttgg 1530415DNAHuman immunodeficiency virus 1 304tccactgacc tttgg 1530515DNAHuman immunodeficiency virus 1 305tccactgacc tttgg 1530615DNAHuman immunodeficiency virus 1 306tccactgacc tttgg 1530715DNAHuman immunodeficiency virus 1 307tccactgacc ttaag 1530815DNAHuman immunodeficiency virus 1 308tccactgacc ttaag 1530915DNAHuman immunodeficiency virus 1 309tccactgacc ttaag 1531015DNAHuman immunodeficiency virus 1 310tccactgacc ttaag 1531115DNAHuman immunodeficiency virus 1 311tccactgacc ttaag 1531215DNAHuman immunodeficiency virus 1 312tccactgacc ttcag 1531315DNAHuman immunodeficiency virus 1 313tccactgacc ttcag 1531415DNAHuman immunodeficiency virus 1 314tccactgacc ttcag 1531515DNAHuman immunodeficiency virus 1 315tccactgacc ttcag 1531615DNAHuman immunodeficiency virus 1 316tccactgacc ttcag 1531715DNAHuman immunodeficiency virus 1 317tccactgacc ttcag 1531815DNAHuman immunodeficiency virus 1 318tccactgacc ttcag 1531915DNAHuman immunodeficiency virus 1 319tccactgacc ttcag 1532015DNAHuman immunodeficiency virus 1 320tccactgacc ttcag 1532115DNAHuman immunodeficiency virus 1 321tccactgacc ttcag 1532215DNAHuman immunodeficiency virus 1 322tccactgacc ttcag 1532315DNAHuman immunodeficiency virus 1 323tccactgacc ttcag 1532415DNAHuman immunodeficiency virus 1 324tccactgacc ttgag 1532515DNAHuman immunodeficiency virus 1 325tccactgacc ttgag 1532615DNAHuman immunodeficiency virus 1 326tccactgacc ttgag 1532715DNAHuman immunodeficiency virus 1 327tccactgacc ttgag 1532815DNAHuman immunodeficiency virus 1 328tccactgacc ttgag 1532915DNAHuman immunodeficiency virus 1 329tccactgacc ttgag 1533015DNAHuman immunodeficiency virus 1 330tccactgacc ttgag 1533115DNAHuman immunodeficiency virus 1 331tccactgacc ttgag 1533215DNAHuman immunodeficiency virus 1 332tccactgacc ttgag 1533315DNAHuman immunodeficiency virus 1 333tccactgacc tttag 1533415DNAHuman immunodeficiency virus 1 334tccactgacc tttag 1533515DNAHuman immunodeficiency virus 1 335tccactgacc tttag 1533615DNAHuman immunodeficiency virus 1 336tccactgacc tttag 1533715DNAHuman immunodeficiency virus 1 337tccactgacc tttag 1533823DNAHuman immunodeficiency virus 1 338cagcagttct tgaagtactc cgg 2333922DNAHuman immunodeficiency virus 1 339agcagttctt gaagtactcc gg 2234021DNAHuman immunodeficiency virus 1 340gcagttcttg aagtactccg g 2134120DNAHuman immunodeficiency virus 1 341cagttcttga agtactccgg 2034219DNAHuman immunodeficiency virus 1 342agttcttgaa gtactccgg 1934318DNAHuman immunodeficiency virus 1 343gttcttgaag tactccgg 1834417DNAHuman immunodeficiency virus 1 344ttcttgaagt actccgg 1734516DNAHuman immunodeficiency virus 1 345tcttgaagta ctccgg 1634616DNAHuman immunodeficiency virus 1 346tcttgaagta ctctag 1634715DNAHuman immunodeficiency virus 1 347cttgaagtac tcagg 1534815DNAHuman immunodeficiency virus 1 348cttgaagtac tcagg 1534915DNAHuman immunodeficiency virus 1 349cttgaagtac tcagg 1535015DNAHuman immunodeficiency virus 1 350cttgaagtac tcagg 1535115DNAHuman immunodeficiency virus 1 351cttgaagtac tccgg 1535215DNAHuman immunodeficiency virus 1 352cttgaagtac tctgg 1535315DNAHuman immunodeficiency virus 1 353cttgaagtac tcaag 1535415DNAHuman immunodeficiency virus 1 354cttgaagtac tcaag 1535515DNAHuman immunodeficiency virus 1 355cttgaagtac tcaag 1535615DNAHuman immunodeficiency virus 1 356cttgaagtac tcaag 1535715DNAHuman immunodeficiency virus 1 357cttgaagtac tcaag 1535815DNAHuman immunodeficiency virus 1 358cttgaagtac tccag 1535915DNAHuman immunodeficiency virus 1 359cttgaagtac tccag 1536015DNAHuman immunodeficiency virus 1 360cttgaagtac tccag 1536115DNAHuman immunodeficiency virus 1 361cttgaagtac tccag 1536215DNAHuman immunodeficiency virus 1 362cttgaagtac tctag 1536315DNAHuman immunodeficiency virus 1 363cttgaagtac tctag 1536423DNAHuman immunodeficiency virus 1 364gatctgtgga tctaccacac aca 2336526DNAHuman immunodeficiency virus 1 365gatctgtgga tctaccacac acaagg 2636620DNAHuman immunodeficiency virus 1 366gattggcaga actacacacc 2036723DNAHuman immunodeficiency virus 1 367gattggcaga actacacacc agg 2336827DNAHuman immunodeficiency virus 1 368gccagggatc agatatccac tgacctt 2736930DNAHuman immunodeficiency virus 1 369gccagggatc agatatccac tgacctttgg 3037030DNAHuman immunodeficiency virus 1 370gagtacttca agaactgctg acatcgagct 3037133DNAHuman immunodeficiency virus 1 371ccggagtact tcaagaactg ctgacatcga gct 3337220DNAHuman immunodeficiency virus 1 372gcgtggcctg ggcgggactg 2037323DNAHuman immunodeficiency virus 1 373gcgtggcctg ggcgggactg ggg 2337422DNAHuman immunodeficiency virus 1 374tcagatgctg catataagca gc 2237525DNAHuman immunodeficiency virus 1 375ccctcagatg ctgcatataa gcagc 25376634DNAArtificial SequenceDescription of Artificial Sequence Synthetic polynucleotide 376tggaagggct aattcactcc caacgaagac aagatatcct tgatctgtgg atctaccaca 60cacaaggcta cttccctgat tggcagaact acacaccagg gccagggatc agatatccac 120tgacctttgg atggtgctac aagctagtac cagttgagca agagaaggta gaagaagcca

180atgaaggaga gaacacccgc ttgttacacc ctgtgagcct gcatgggatg gatgacccgg 240agagagaagt attagagtgg aggtttgaca gccgcctagc atttcatcac atggcccgag 300agctgcatcc ggagtacttc aagaactgct gacatcgagc ttgctacaag ggactttccg 360ctggggactt tccagggagg cgtggcctgg gcgggactgg ggagtggcga gccctcagat 420gctgcatata agcagctgct ttttgcttgt actgggtctc tctggttaga ccagatctga 480gcctgggagc tctctggcta actagggaac ccactgctta agcctcaata aagcttgcct 540tgagtgcttc aagtagtgtg tgcccgtctg ttgtgtgact ctggtaacta gagatccctc 600agaccctttt agtcagtgtg gaaaatctct agca 634377453DNAArtificial SequenceDescription of Artificial Sequence Synthetic polynucleotide 377tggaagggct aattcactcc caacgaagac aagatatcct tgatctgtgg atctaccaca 60cacaaggcta cttccctgat tggcagaact acacaccagg gccagggatc agatatccac 120tgacctttgg atggtgctac aagctagtac cagttgagca agagaaggta gaagaagcca 180atgaaggaga gaacacccgc ttgttacacc ctgtgagcct gcatgggatg gatgacccgg 240agagagaagt attagagtgg aggtttgaca gccgcctagc atttcatcac atggcccgag 300agctgcatcc ggagtacttc aagaactgct gacatcgagc ttgctacaag ggactttccg 360ctggggactt tccagggagg cgtggcctgg gcgggactgg ggagtggcga gccctcagat 420gctgcatata agcagctgct ttttgcttgt act 45337897DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 378gggtctctct ggttagacca gatctgagcc tgggagctct ctggctaact agggaaccca 60ctgcttaagc ctcaataaag cttgccttga gtgcttc 9737984DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 379aagtagtgtg tgcccgtctg ttgtgtgact ctggtaacta gagatccctc agaccctttt 60agtcagtgtg gaaaatctct agca 84380818DNAArtificial SequenceDescription of Artificial Sequence Synthetic polynucleotide 380tggaagggat ttattacagt gcaagaagac atagaatctt agacatatac ttagaaaagg 60aagaaggcat cataccagat tggcaggatt acacctcagg accaggaatt agatacccaa 120agacatttgg ctggctatgg aaattagtcc ctgtaaatgt atcagatgag gcacaggagg 180atgaggagca ttatttaatg catccagctc aaacttccca gtgggatgac ccttggggag 240aggttctagc atggaagttt gatccaactc tggcctacac ttatgaggca tatgttagat 300acccagaaga gtttggaagc aagtcaggcc tgtcagagga agaggttaga agaaggctaa 360ccgcaagagg ccttcttaac atggctgaca agaaggaaac tcgctgaaac agcagggact 420ttccacaagg ggatgttacg gggaggtact ggggaggagc cggtcgggaa cgcccacttt 480cttgatgtat aaatatcact gcatttcgct ctgtattcag tcgctctgcg gagaggctgg 540cagattgagc cctgggaggt tctctccagc actagcaggt agagcctggg tgttccctgc 600tagactctca ccagcacttg gccggtgctg ggcagagtga ctccacgctt gcttgcttaa 660agccctcttc aataaagctg ccattttaga agtaagctag tgtgtgttcc catctctcct 720agccgccgcc tggtcaactc ggtactcaat aataagaaga ccctggtctg ttaggaccct 780ttctgctttg ggaaaccgaa gcaggaaaat ccctagca 818381517DNAArtificial SequenceDescription of Artificial Sequence Synthetic polynucleotide 381tggaagggat ttattacagt gcaagaagac atagaatctt agacatatac ttagaaaagg 60aagaaggcat cataccagat tggcaggatt acacctcagg accaggaatt agatacccaa 120agacatttgg ctggctatgg aaattagtcc ctgtaaatgt atcagatgag gcacaggagg 180atgaggagca ttatttaatg catccagctc aaacttccca gtgggatgac ccttggggag 240aggttctagc atggaagttt gatccaactc tggcctacac ttatgaggca tatgttagat 300acccagaaga gtttggaagc aagtcaggcc tgtcagagga agaggttaga agaaggctaa 360ccgcaagagg ccttcttaac atggctgaca agaaggaaac tcgctgaaac agcagggact 420ttccacaagg ggatgttacg gggaggtact ggggaggagc cggtcgggaa cgcccacttt 480cttgatgtat aaatatcact gcatttcgct ctgtatt 517382176DNAArtificial SequenceDescription of Artificial Sequence Synthetic polynucleotide 382cagtcgctct gcggagaggc tggcagattg agccctggga ggttctctcc agcactagca 60ggtagagcct gggtgttccc tgctagactc tcaccagcac ttggccggtg ctgggcagag 120tgactccacg cttgcttgct taaagccctc ttcaataaag ctgccatttt agaagt 176383125DNAArtificial SequenceDescription of Artificial Sequence Synthetic polynucleotide 383aagctagtgt gtgttcccat ctctcctagc cgccgcctgg tcaactcggt actcaataat 60aagaagaccc tggtctgtta ggaccctttc tgctttggga aaccgaagca ggaaaatccc 120tagca 12538414825DNAHuman immunodeficiency virus 1 384tggaagggct aatttggtcc caaaaaagac aagagatcct tgatctgtgg atctaccaca 60cacaaggcta cttccctgat tggcagaact acacaccagg gccagggatc agatatccac 120tgacctttgg atggtgcttc aagttagtac cagttgaacc agagcaagta gaagaggcca 180atgaaggaga gaacaacagc ttgttacacc ctatgagcca gcatgggatg gaggacccgg 240agggagaagt attagtgtgg aagtttgaca gcctcctagc atttcgtcac atggcccgag 300agctgcatcc ggagtactac aaagactgct gacatcgagc tttctacaag ggactttccg 360ctggggactt tccagggagg tgtggcctgg gcgggactgg ggagtggcga gccctcagat 420gctacatata agcagctgct ttttgcctgt actgggtctc tctggttaga ccagatctga 480gcctgggagc tctctggcta actagggaac ccactgctta agcctcaata aagcttgcct 540tgagtgctca aagtagtgtg tgcccgtctg ttgtgtgact ctggtaacta gagatccctc 600agaccctttt agtcagtgtg gaaaatctct agcagtggcg cccgaacagg gacttgaaag 660cgaaagtaaa gccagaggag atctctcgac gcaggactcg gcttgctgaa gcgcgcacgg 720caagaggcga ggggcggcga ctggtgagta cgccaaaaat tttgactagc ggaggctaga 780aggagagaga tgggtgcgag agcgtcggta ttaagcgggg gagaattaga taaatgggaa 840aaaattcggt taaggccagg gggaaagaaa caatataaac taaaacatat agtatgggca 900agcagggagc tagaacgatt cgcagttaat cctggccttt tagagacatc agaaggctgt 960agacaaatac tgggacagct acaaccatcc cttcagacag gatcagaaga acttagatca 1020ttatataata caatagcagt cctctattgt gtgcatcaaa ggatagatgt aaaagacacc 1080aaggaagcct tagataagat agaggaagag caaaacaaaa gtaagaaaaa ggcacagcaa 1140gcagcagctg acacaggaaa caacagccag gtcagccaaa attaccctat agtgcagaac 1200ctccaggggc aaatggtaca tcaggccata tcacctagaa ctttaaatgc atgggtaaaa 1260gtagtagaag agaaggcttt cagcccagaa gtaataccca tgttttcagc attatcagaa 1320ggagccaccc cacaagattt aaataccatg ctaaacacag tggggggaca tcaagcagcc 1380atgcaaatgt taaaagagac catcaatgag gaagctgcag aatgggatag attgcatcca 1440gtgcatgcag ggcctattgc accaggccag atgagagaac caaggggaag tgacatagca 1500ggaactacta gtacccttca ggaacaaata ggatggatga cacataatcc acctatccca 1560gtaggagaaa tctataaaag atggataatc ctgggattaa ataaaatagt aagaatgtat 1620agccctacca gcattctgga cataagacaa ggaccaaagg aaccctttag agactatgta 1680gaccgattct ataaaactct aagagccgag caagcttcac aagaggtaaa aaattggatg 1740acagaaacct tgttggtcca aaatgcgaac ccagattgta agactatttt aaaagcattg 1800ggaccaggag cgacactaga agaaatgatg acagcatgtc agggagtggg gggacccggc 1860cataaagcaa gagttttggc tgaagcaatg agccaagtaa caaatccagc taccataatg 1920atacagaaag gcaattttag gaaccaaaga aagactgtta agtgtttcaa ttgtggcaaa 1980gaagggcaca tagccaaaaa ttgcagggcc cctaggaaaa agggctgttg gaaatgtgga 2040aaggaaggac accaaatgaa agattgtact gagagacagg ctaatttttt agggaagatc 2100tggccttccc acaagggaag gccagggaat tttcttcaga gcagaccaga gccaacagcc 2160ccaccagaag agagcttcag gtttggggaa gagacaacaa ctccctctca gaagcaggag 2220ccgatagaca aggaactgta tcctttagct tccctcagat cactctttgg cagcgacccc 2280tcgtcacaat aaagataggg gggcaattaa aggaagctct attagataca ggagcagatg 2340atacagtatt agaagaaatg aatttgccag gaagatggaa accaaaaatg atagggggaa 2400ttggaggttt tatcaaagta agacagtatg atcagatact catagaaatc tgcggacata 2460aagctatagg tacagtatta gtaggaccta cacctgtcaa cataattgga agaaatctgt 2520tgactcagat tggctgcact ttaaattttc ccattagtcc tattgagact gtaccagtaa 2580aattaaagcc aggaatggat ggcccaaaag ttaaacaatg gccattgaca gaagaaaaaa 2640taaaagcatt agtagaaatt tgtacagaaa tggaaaagga aggaaaaatt tcaaaaattg 2700ggcctgaaaa tccatacaat actccagtat ttgccataaa gaaaaaagac agtactaaat 2760ggagaaaatt agtagatttc agagaactta ataagagaac tcaagatttc tgggaagttc 2820aattaggaat accacatcct gcagggttaa aacagaaaaa atcagtaaca gtactggatg 2880tgggcgatgc atatttttca gttcccttag ataaagactt caggaagtat actgcattta 2940ccatacctag tataaacaat gagacaccag ggattagata tcagtacaat gtgcttccac 3000agggatggaa aggatcacca gcaatattcc agtgtagcat gacaaaaatc ttagagcctt 3060ttagaaaaca aaatccagac atagtcatct atcaatacat ggatgatttg tatgtaggat 3120ctgacttaga aatagggcag catagaacaa aaatagagga actgagacaa catctgttga 3180ggtggggatt taccacacca gacaaaaaac atcagaaaga acctccattc ctttggatgg 3240gttatgaact ccatcctgat aaatggacag tacagcctat agtgctgcca gaaaaggaca 3300gctggactgt caatgacata cagaaattag tgggaaaatt gaattgggca agtcagattt 3360atgcagggat taaagtaagg caattatgta aacttcttag gggaaccaaa gcactaacag 3420aagtagtacc actaacagaa gaagcagagc tagaactggc agaaaacagg gagattctaa 3480aagaaccggt acatggagtg tattatgacc catcaaaaga cttaatagca gaaatacaga 3540agcaggggca aggccaatgg acatatcaaa tttatcaaga gccatttaaa aatctgaaaa 3600caggaaagta tgcaagaatg aagggtgccc acactaatga tgtgaaacaa ttaacagagg 3660cagtacaaaa aatagccaca gaaagcatag taatatgggg aaagactcct aaatttaaat 3720tacccataca aaaggaaaca tgggaagcat ggtggacaga gtattggcaa gccacctgga 3780ttcctgagtg ggagtttgtc aatacccctc ccttagtgaa gttatggtac cagttagaga 3840aagaacccat aataggagca gaaactttct atgtagatgg ggcagccaat agggaaacta 3900aattaggaaa agcaggatat gtaactgaca gaggaagaca aaaagttgtc cccctaacgg 3960acacaacaaa tcagaagact gagttacaag caattcatct agctttgcag gattcgggat 4020tagaagtaaa catagtgaca gactcacaat atgcattggg aatcattcaa gcacaaccag 4080ataagagtga atcagagtta gtcagtcaaa taatagagca gttaataaaa aaggaaaaag 4140tctacctggc atgggtacca gcacacaaag gaattggagg aaatgaacaa gtagataaat 4200tggtcagtgc tggaatcagg aaagtactat ttttagatgg aatagataag gcccaagaag 4260aacatgagaa atatcacagt aattggagag caatggctag tgattttaac ctaccacctg 4320tagtagcaaa agaaatagta gccagctgtg ataaatgtca gctaaaaggg gaagccatgc 4380atggacaagt agactgtagc ccaggaatat ggcagctaga ttgtacacat ttagaaggaa 4440aagttatctt ggtagcagtt catgtagcca gtggatatat agaagcagaa gtaattccag 4500cagagacagg gcaagaaaca gcatacttcc tcttaaaatt agcaggaaga tggccagtaa 4560aaacagtaca tacagacaat ggcagcaatt tcaccagtac tacagttaag gccgcctgtt 4620ggtgggcggg gatcaagcag gaatttggca ttccctacaa tccccaaagt caaggagtaa 4680tagaatctat gaataaagaa ttaaagaaaa ttataggaca ggtaagagat caggctgaac 4740atcttaagac agcagtacaa atggcagtat tcatccacaa ttttaaaaga aaagggggga 4800ttggggggta cagtgcaggg gaaagaatag tagacataat agcaacagac atacaaacta 4860aagaattaca aaaacaaatt acaaaaattc aaaattttcg ggtttattac agggacagca 4920gagatccagt ttggaaagga ccagcaaagc tcctctggaa aggtgaaggg gcagtagtaa 4980tacaagataa tagtgacata aaagtagtgc caagaagaaa agcaaagatc atcagggatt 5040atggaaaaca gatggcaggt gatgattgtg tggcaagtag acaggatgag gattaacaca 5100tggaaaagat tagtaaaaca ccatatgtat atttcaagga aagctaagga ctggttttat 5160agacatcact atgaaagtac taatccaaaa ataagttcag aagtacacat cccactaggg 5220gatgctaaat tagtaataac aacatattgg ggtctgcata caggagaaag agactggcat 5280ttgggtcagg gagtctccat agaatggagg aaaaagagat atagcacaca agtagaccct 5340gacctagcag accaactaat tcatctgcac tattttgatt gtttttcaga atctgctata 5400agaaatacca tattaggacg tatagttagt cctaggtgtg aatatcaagc aggacataac 5460aaggtaggat ctctacagta cttggcacta gcagcattaa taaaaccaaa acagataaag 5520ccacctttgc ctagtgttag gaaactgaca gaggacagat ggaacaagcc ccagaagacc 5580aagggccaca gagggagcca tacaatgaat ggacactaga gcttttagag gaacttaaga 5640gtgaagctgt tagacatttt cctaggatat ggctccataa cttaggacaa catatctatg 5700aaacttacgg ggatacttgg gcaggagtgg aagccataat aagaattctg caacaactgc 5760tgtttatcca tttcagaatt gggtgtcgac atagcagaat aggcgttact cgacagagga 5820gagcaagaaa tggagccagt agatcctaga ctagagccct ggaagcatcc aggaagtcag 5880cctaaaactg cttgtaccaa ttgctattgt aaaaagtgtt gctttcattg ccaagtttgt 5940ttcatgacaa aagccttagg catctcctat ggcaggaaga agcggagaca gcgacgaaga 6000gctcatcaga acagtcagac tcatcaagct tctctatcaa agcagtaagt agtacatgta 6060atgcaaccta taatagtagc aatagtagca ttagtagtag caataataat agcaatagtt 6120gtgtggtcca tagtaatcat agaatatagg aaaatattaa gacaaagaaa aatagacagg 6180ttaattgata gactaataga aagagcagaa gacagtggca atgagagtga aggagaagta 6240tcagcacttg tggagatggg ggtggaaatg gggcaccatg ctccttggga tattgatgat 6300ctgtagtgct acagaaaaat tgtgggtcac agtctattat ggggtacctg tgtggaagga 6360agcaaccacc actctatttt gtgcatcaga tgctaaagca tatgatacag aggtacataa 6420tgtttgggcc acacatgcct gtgtacccac agaccccaac ccacaagaag tagtattggt 6480aaatgtgaca gaaaatttta acatgtggaa aaatgacatg gtagaacaga tgcatgagga 6540tataatcagt ttatgggatc aaagcctaaa gccatgtgta aaattaaccc cactctgtgt 6600tagtttaaag tgcactgatt tgaagaatga tactaatacc aatagtagta gcgggagaat 6660gataatggag aaaggagaga taaaaaactg ctctttcaat atcagcacaa gcataagaga 6720taaggtgcag aaagaatatg cattctttta taaacttgat atagtaccaa tagataatac 6780cagctatagg ttgataagtt gtaacacctc agtcattaca caggcctgtc caaaggtatc 6840ctttgagcca attcccatac attattgtgc cccggctggt tttgcgattc taaaatgtaa 6900taataagacg ttcaatggaa caggaccatg tacaaatgtc agcacagtac aatgtacaca 6960tggaatcagg ccagtagtat caactcaact gctgttaaat ggcagtctag cagaagaaga 7020tgtagtaatt agatctgcca atttcacaga caatgctaaa accataatag tacagctgaa 7080cacatctgta gaaattaatt gtacaagacc caacaacaat acaagaaaaa gtatccgtat 7140ccagagggga ccagggagag catttgttac aataggaaaa ataggaaata tgagacaagc 7200acattgtaac attagtagag caaaatggaa tgccacttta aaacagatag ctagcaaatt 7260aagagaacaa tttggaaata ataaaacaat aatctttaag caatcctcag gaggggaccc 7320agaaattgta acgcacagtt ttaattgtgg aggggaattt ttctactgta attcaacaca 7380actgtttaat agtacttggt ttaatagtac ttggagtact gaagggtcaa ataacactga 7440aggaagtgac acaatcacac tcccatgcag aataaaacaa tttataaaca tgtggcagga 7500agtaggaaaa gcaatgtatg cccctcccat cagtggacaa attagatgtt catcaaatat 7560tactgggctg ctattaacaa gagatggtgg taataacaac aatgggtccg agatcttcag 7620acctggagga ggcgatatga gggacaattg gagaagtgaa ttatataaat ataaagtagt 7680aaaaattgaa ccattaggag tagcacccac caaggcaaag agaagagtgg tgcagagaga 7740aaaaagagca gtgggaatag gagctttgtt ccttgggttc ttgggagcag caggaagcac 7800tatgggcgca gcgtcaatga cgctgacggt acaggccaga caattattgt ctgatatagt 7860gcagcagcag aacaatttgc tgagggctat tgaggcgcaa cagcatctgt tgcaactcac 7920agtctggggc atcaaacagc tccaggcaag aatcctggct gtggaaagat acctaaagga 7980tcaacagctc ctggggattt ggggttgctc tggaaaactc atttgcacca ctgctgtgcc 8040ttggaatgct agttggagta ataaatctct ggaacagatt tggaataaca tgacctggat 8100ggagtgggac agagaaatta acaattacac aagcttaata cactccttaa ttgaagaatc 8160gcaaaaccag caagaaaaga atgaacaaga attattggaa ttagataaat gggcaagttt 8220gtggaattgg tttaacataa caaattggct gtggtatata aaattattca taatgatagt 8280aggaggcttg gtaggtttaa gaatagtttt tgctgtactt tctatagtga atagagttag 8340gcagggatat tcaccattat cgtttcagac ccacctccca atcccgaggg gacccgacag 8400gcccgaagga atagaagaag aaggtggaga gagagacaga gacagatcca ttcgattagt 8460gaacggatcc ttagcactta tctgggacga tctgcggagc ctgtgcctct tcagctacca 8520ccgcttgaga gacttactct tgattgtaac gaggattgtg gaacttctgg gacgcagggg 8580gtgggaagcc ctcaaatatt ggtggaatct cctacagtat tggagtcagg aactaaagaa 8640tagtgctgtt aacttgctca atgccacagc catagcagta gctgagggga cagatagggt 8700tatagaagta ttacaagcag cttatagagc tattcgccac atacctagaa gaataagaca 8760gggcttggaa aggattttgc tataagatgg gtggcaagtg gtcaaaaagt agtgtgattg 8820gatggcctgc tgtaagggaa agaatgagac gagctgagcc agcagcagat ggggtgggag 8880cagtatctcg agacctagaa aaacatggag caatcacaag tagcaataca gcagctaaca 8940atgctgcttg tgcctggcta gaagcacaag aggaggaaga ggtgggtttt ccagtcacac 9000ctcaggtacc tttaagacca atgacttaca aggcagctgt agatcttagc cactttttaa 9060aagaaaaggg gggactggaa gggctaattc actcccaaag aagacaagat atccttgatc 9120tgtggatcta ccacacacaa ggctacttcc ctgattggca gaactacaca ccagggccag 9180gggtcagata tccactgacc tttggatggt gctacaagct agtaccagtt gagccagata 9240aggtagaaga ggccaataaa ggagagaaca ccagcttgtt acaccctgtg agcctgcatg 9300gaatggatga ccctgagaga gaagtgttag agtggaggtt tgacagccgc ctagcatttc 9360atcacgtggc ccgagagctg catccggagt acttcaagaa ctgctgacat cgagcttgct 9420acaagggact ttccgctggg gactttccag ggaggcgtgg cctgggcggg actggggagt 9480ggcgagccct cagatgctgc atataagcag ctgctttttg cctgtactgg gtctctctgg 9540ttagaccaga tctgagcctg ggagctctct ggctaactag ggaacccact gcttaagcct 9600caataaagct tgccttgagt gcttcaagta gtgtgtgccc gtctgttgtg tgactctggt 9660aactagagat ccctcagacc cttttagtca gtgtggaaaa tctctagcac ccaggaggta 9720gaggttgcag tgagccaaga tcgcgccact gcattccagc ctgggcaaga aaacaagact 9780gtctaaaata ataataataa gttaagggta ttaaatatat ttatacatgg aggtcataaa 9840aatatatata tttgggctgg gcgcagtggc tcacacctgc gcccggccct ttgggaggcc 9900gaggcaggtg gatcacctga gtttgggagt tccagaccag cctgaccaac atggagaaac 9960cccttctctg tgtattttta gtagatttta ttttatgtgt attttattca caggtatttc 10020tggaaaactg aaactgtttt tcctctactc tgataccaca agaatcatca gcacagagga 10080agacttctgt gatcaaatgt ggtgggagag ggaggttttc accagcacat gagcagtcag 10140ttctgccgca gactcggcgg gtgtccttcg gttcagttcc aacaccgcct gcctggagag 10200aggtcagacc acagggtgag ggctcagtcc ccaagacata aacacccaag acataaacac 10260ccaacaggtc caccccgcct gctgcccagg cagagccgat tcaccaagac gggaattagg 10320atagagaaag agtaagtcac acagagccgg ctgtgcggga gaacggagtt ctattatgac 10380tcaaatcagt ctccccaagc attcggggat cagagttttt aaggataact tagtgtgtag 10440ggggccagtg agttggagat gaaagcgtag ggagtcgaag gtgtcctttt gcgccgagtc 10500agttcctggg tgggggccac aagatcggat gagccagttt atcaatccgg gggtgccagc 10560tgatccatgg agtgcagggt ctgcaaaata tctcaagcac tgattgatct taggttttac 10620aatagtgatg ttaccccagg aacaatttgg ggaaggtcag aatcttgtag cctgtagctg 10680catgactcct aaaccataat ttcttttttg tttttttttt tttatttttg agacagggtc 10740tcactctgtc acctaggctg gagtgcagtg gtgcaatcac agctcactgc agcctcaacg 10800tcgtaagctc aagcgatcct cccacctcag cctgcctggt agctgagact acaagcgacg 10860ccccagttaa tttttgtatt tttggtagag gcagcgtttt gccgtgtggc cctggctggt 10920ctcgaactcc tgggctcaag tgatccagcc tcagcctccc aaagtgctgg gacaaccggg 10980gccagtcact gcacctggcc ctaaaccata atttctaatc ttttggctaa tttgttagtc 11040ctacaaaggc agtctagtcc ccaggcaaaa agggggtttg tttcgggaaa gggctgttac 11100tgtctttgtt tcaaactata aactaagttc ctcctaaact tagttcggcc tacacccagg 11160aatgaacaag gagagcttgg aggttagaag cacgatggaa ttggttaggt cagatctctt 11220tcactgtctg agttataatt ttgcaatggt ggttcaaaga ctgcccgctt ctgacaccag 11280tcgctgcatt aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat tgggcgctct 11340tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca 11400gctcactcaa aggcggtaat acggttatcc acagaatcag gggataacgc

aggaaagaac 11460atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt 11520ttccataggc tccgcccccc tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg 11580cgaaacccga caggactata aagataccag gcgtttcccc ctggaagctc cctcgtgcgc 11640tctcctgttc cgaccctgcc gcttaccgga tacctgtccg cctttctccc ttcgggaagc 11700gtggcgcttt ctcatagctc acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc 11760aagctgggct gtgtgcacga accccccgtt cagcccgacc gctgcgcctt atccggtaac 11820tatcgtcttg agtccaaccc ggtaagacac gacttatcgc cactggcagc agccactggt 11880aacaggatta gcagagcgag gtatgtaggc ggtgctacag agttcttgaa gtggtggcct 11940aactacggct acactagaag aacagtattt ggtatctgcg ctctgctgaa gccagttacc 12000ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt 12060ttttttgttt gcaagcagca gattacgcgc agaaaaaaag gatctcaaga agatcctttg 12120atcttttcta cggggtctga cgctcagtgg aacgaaaact cacgttaagg gattttggtc 12180atgagattat caaaaaggat cttcacctag atccttttaa attaaaaatg aagttttaaa 12240tcaatctaaa gtatatatga gtaaacttgg tctgacagtt accaatgctt aatcagtgag 12300gcacctatct cagcgatctg tctatttcgt tcatccatag ttgcctgact ccccgtcgtg 12360tagataacta cgatacggga gggcttacca tctggcccca gtgctgcaat gataccgcga 12420gacccacgct caccggctcc agatttatca gcaataaacc agccagccgg aagggccgag 12480cgcagaagtg gtcctgcaac tttatccgcc tccatccagt ctattaattg ttgccgggaa 12540gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat tgctacaggc 12600atcgtggtgt cacgctcgtc gtttggtatg gcttcattca gctccggttc ccaacgatca 12660aggcgagtta catgatcccc catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg 12720atcgttgtca gaagtaagtt ggccgcagtg ttatcactca tggttatggc agcactgcat 12780aattctctta ctgtcatgcc atccgtaaga tgcttttctg tgactggtga gtactcaacc 12840aagtcattct gagaatagtg tatgcggcga ccgagttgct cttgcccggc gtcaatacgg 12900gataataccg cgccacatag cagaacttta aaagtgctca tcattggaaa acgttcttcg 12960gggcgaaaac tctcaaggat cttaccgctg ttgagatcca gttcgatgta acccactcgt 13020gcacccaact gatcttcagc atcttttact ttcaccagcg tttctgggtg agcaaaaaca 13080ggaaggcaaa atgccgcaaa aaagggaata agggcgacac ggaaatgttg aatactcata 13140ctcttccttt ttcaatatta ttgaagcatt tatcagggtt attgtctcat gagcggatac 13200atatttgaat gtatttagaa aaataaacaa ataggggttc cgcgcacatt tccccgaaaa 13260gtgccacctg acgtctaaga aaccattatt atcatgacat taacctataa aaataggcgt 13320atcacgaggc cctttcgtct cgcgcgtttc ggtgatgacg gtgaaaacct ctgacacatg 13380cagctcccgg agacggtcac agcttgtctg taagcggatg ccgggagcag acaagcccgt 13440cagggcgcgt cagcgggtgt tggcgggtgt cggggctggc ttaactatgc ggcatcagag 13500cagattgtac tgagagtgca ccatatgcgg tgtgaaatac cgcacagatg cgtaaggaga 13560aaataccgca tcaggcgcca ttcgccattc aggctgcgca actgttggga agggcgatcg 13620gtgcgggcct cttcgctatt acgccagggg aggcagagat tgcagtaagc tgagatcgca 13680gcactgcact ccagcctggg cgacagagta agactctgtc tcaaaaataa aataaataaa 13740tcaatcagat attccaatct tttcctttat ttatttattt attttctatt ttggaaacac 13800agtccttcct tattccagaa ttacacatat attctatttt tctttatatg ctccagtttt 13860ttttagacct tcacctgaaa tgtgtgtata caaaatctag gccagtccag cagagcctaa 13920aggtaaaaaa taaaataata aaaaataaat aaaatctagc tcactccttc acatcaaaat 13980ggagatacag ctgttagcat taaataccaa ataacccatc ttgtcctcaa taattttaag 14040cgcctctctc caccacatct aactcctgtc aaaggcatgt gccccttccg ggcgctctgc 14100tgtgctgcca accaactggc atgtggactc tgcagggtcc ctaactgcca agccccacag 14160tgtgccctga ggctgcccct tccttctagc ggctgccccc actcggcttt gctttcccta 14220gtttcagtta cttgcgttca gccaaggtct gaaactaggt gcgcacagag cggtaagact 14280gcgagagaaa gagaccagct ttacaggggg tttatcacag tgcaccctga cagtcgtcag 14340cctcacaggg ggtttatcac attgcaccct gacagtcgtc agcctcacag ggggtttatc 14400acagtgcacc cttacaatca ttccatttga ttcacaattt ttttagtctc tactgtgcct 14460aacttgtaag ttaaatttga tcagaggtgt gttcccagag gggaaaacag tatatacagg 14520gttcagtact atcgcatttc aggcctccac ctgggtcttg gaatgtgtcc cccgaggggt 14580gatgactacc tcagttggat ctccacaggt cacagtgaca caagataacc aagacacctc 14640ccaaggctac cacaatgggc cgccctccac gtgcacatgg ccggaggaac tgccatgtcg 14700gaggtgcaag cacacctgcg catcagagtc cttggtgtgg agggagggac cagcgcagct 14760tccagccatc cacctgatga acagaaccta gggaaagccc cagttctact tacaccagga 14820aaggc 1482538510535DNASimian immunodeficiency virus 385gcatgcacat tttaaaggct tttgctaaat atagccaaaa gtccttctac aaattttcta 60agagttctga ttcaaagcag taacaggcct tgtctcatca tgaactttgg catttcatct 120acagctaagt ttatatcata aatagttctt tacaggcagc accaacttat acccttatag 180catactttac tgtgtgaaaa ttgcatcttt cattaagctt actgtaaatt tactggctgt 240cttccttgca ggtttctgga agggatttat tacagtgcaa gaagacatag aatcttagac 300atatacttag aaaaggaaga aggcatcata ccagattggc aggattacac ctcaggacca 360ggaattagat acccaaagac atttggctgg ctatggaaat tagtccctgt aaatgtatca 420gatgaggcac aggaggatga ggagcattat ttaatgcatc cagctcaaac ttcccagtgg 480gatgaccctt ggggagaggt tctagcatgg aagtttgatc caactctggc ctacacttat 540gaggcatatg ttagataccc agaagagttt ggaagcaagt caggcctgtc agaggaagag 600gttagaagaa ggctaaccgc aagaggcctt cttaacatgg ctgacaagaa ggaaactcgc 660tgaaacagca gggactttcc acaaggggat gttacgggga ggtactgggg aggagccggt 720cgggaacgcc cactttcttg atgtataaat atcactgcat ttcgctctgt attcagtcgc 780tctgcggaga ggctggcaga ttgagccctg ggaggttctc tccagcacta gcaggtagag 840cctgggtgtt ccctgctaga ctctcaccag cacttggccg gtgctgggca gagtgactcc 900acgcttgctt gcttaaagcc ctcttcaata aagctgccat tttagaagta agctagtgtg 960tgttcccatc tctcctagcc gccgcctggt caactcggta ctcaataata agaagaccct 1020ggtctgttag gaccctttct gctttgggaa accgaagcag gaaaatccct agcagattgg 1080cgcctgaaca gggacttgaa ggagagtgag agactcctga gtacggctga gtgaaggcag 1140taagggcggc aggaaccaac cacgacggag tgctcctata aaggcgcggg tcggtaccag 1200acggcgtgag gagcgggaga ggaagaggcc tccggttgca ggtaagtgca acacaaaaaa 1260gaaatagctg tcttttatcc aggaaggggt aataagatag agtgggagat gggcgtgaga 1320aactccgtct tgtcagggaa gaaagcagat gaattagaaa aaattaggct acgacccaac 1380ggaaagaaaa agtacatgtt gaagcatgta gtatgggcag caaatgaatt agatagattt 1440ggattagcag aaagcctgtt ggagaacaaa gaaggatgtc aaaaaatact ttcggtctta 1500gctccattag tgccaacagg ctcagaaaat ttaaaaagcc tttataatac tgtctgcgtc 1560atctggtgca ttcacgcaga agagaaagtg aaacacactg aggaagcaaa acagatagtg 1620cagagacacc tagtggtgga aacaggaaca acagaaacta tgccaaaaac aagtagacca 1680acagcaccat ctagcggcag aggaggaaat tacccagtac aacaaatagg tggtaactat 1740gtccacctgc cattaagccc gagaacatta aatgcctggg taaaattgat agaggaaaag 1800aaatttggag cagaagtagt gccaggattt caggcactgt cagaaggttg caccccctat 1860gacattaatc agatgttaaa ttgtgtggga gaccatcaag cggctatgca gattatcaga 1920gatattataa acgaggaggc tgcagattgg gacttgcagc acccacaacc agctccacaa 1980caaggacaac ttagggagcc gtcaggatca gatattgcag gaacaactag ttcagtagat 2040gaacaaatcc agtggatgta cagacaacag aaccccatac cagtaggcaa catttacagg 2100agatggatcc aactggggtt gcaaaaatgt gtcagaatgt ataacccaac aaacattcta 2160gatgtaaaac aagggccaaa agagccattt cagagctatg tagacaggtt ctacaaaagt 2220ttaagagcag aacagacaga tgcagcagta aagaattgga tgactcaaac actgctgatt 2280caaaatgcta acccagattg caagctagtg ctgaaggggc tgggtgtgaa tcccacccta 2340gaagaaatgc tgacggcttg tcaaggagta ggggggccgg gacagaaggc tagattaatg 2400gcagaagccc tgaaagaggc cctcgcacca gtgccaatcc cttttgcagc agcccaacag 2460aggggaccaa gaaagccaat taagtgttgg aattgtggga aagagggaca ctctgcaagg 2520caatgcagag ccccaagaag acagggatgc tggaaatgtg gaaaaatgga ccatgttatg 2580gccaaatgcc cagacagaca ggcgggtttt ttaggccttg gtccatgggg aaagaagccc 2640cgcaatttcc ccatggctca agtgcatcag gggctgatgc caactgctcc cccagaggac 2700ccagctgtgg atctgctaaa gaactacatg cagttgggca agcagcagag agaaaagcag 2760agagaaagca gagagaagcc ttacaaggag gtgacagagg atttgctgca cctcaattct 2820ctctttggag gagaccagta gtcactgctc atattgaagg acagcctgta gaagtattac 2880tggatacagg ggctgatgat tctattgtaa caggaataga gttaggtcca cattataccc 2940caaaaatagt aggaggaata ggaggtttta ttaatactaa agaatacaaa aatgtagaaa 3000tagaagtttt aggcaaaagg attaaaggga caatcatgac aggggacacc ccgattaaca 3060tttttggtag aaatttgcta acagctctgg ggatgtctct aaattttccc atagctaaag 3120tagagcctgt aaaagtcgcc ttaaagccag gaaaggatgg accaaaattg aagcagtggc 3180cattatcaaa agaaaagata gttgcattaa gagaaatctg tgaaaagatg gaaaaggatg 3240gtcagttgga ggaagctccc ccgaccaatc catacaacac ccccacattt gctataaaga 3300aaaaggataa gaacaaatgg agaatgctga tagattttag ggaactaaat agggtcactc 3360aggactttac ggaagtccaa ttaggaatac cacaccctgc aggactagca aaaaggaaaa 3420gaattacagt actggatata ggtgatgcat atttctccat acctctagat gaagaattta 3480ggcagtacac tgcctttact ttaccatcag taaataatgc agagccagga aaacgataca 3540tttataaggt tctgcctcag ggatggaagg ggtcaccagc catcttccaa tacactatga 3600gacatgtgct agaacccttc aggaaggcaa atccagatgt gaccttagtc cagtatatgg 3660atgacatctt aatagctagt gacaggacag acctggaaca tgacagggta gttttacagt 3720caaaggaact cttgaatagc atagggtttt ctaccccaga agagaaattc caaaaagatc 3780ccccatttca atggatgggg tacgaattgt ggccaacaaa atggaagttg caaaagatag 3840agttgccaca aagagagacc tggacagtga atgatataca gaagttagta ggagtattaa 3900attgggcagc tcaaatttat ccaggtataa aaaccaaaca tctctgtagg ttaattagag 3960gaaaaatgac tctaacagag gaagttcagt ggactgagat ggcagaagca gaatatgagg 4020aaaataaaat aattctcagt caggaacaag aaggatgtta ttaccaagaa ggcaagccat 4080tagaagccac ggtaataaag agtcaggaca atcagtggtc ttataaaatt caccaagaag 4140acaaaatact gaaagtagga aaatttgcaa agataaagaa tacacatacc aatggagtga 4200gactattagc acatgtaata cagaaaatag gaaaggaagc aatagtgatc tggggacagg 4260tcccaaaatt ccacttacca gttgagaagg atgtatggga acagtggtgg acagactatt 4320ggcaggtaac ctggataccg gaatgggatt ttatctcaac accaccgcta gtaagattag 4380tcttcaatct agtgaaggac cctatagagg gagaagaaac ctattataca gatggatcat 4440gtaataaaca gtcaaaagaa gggaaagcag gatatatcac agataggggc aaagacaaag 4500taaaagtgtt agaacagact actaatcaac aagcagaatt ggaagcattt ctcatggcat 4560tgacagactc agggccaaag gcaaatatta tagtagattc acaatatgtt atgggaataa 4620taacaggatg ccctacagaa tcagagagca ggctagttaa tcaaataata gaagaaatga 4680ttaaaaagtc agaaatttat gtagcatggg taccagcaca caaaggtata ggaggaaacc 4740aagaaataga ccacctagtt agtcaaggga ttagacaagt tctcttcttg gaaaagatag 4800agccagcaca agaagaacat gataaatacc atagtaatgt aaaagaattg gtattcaaat 4860ttggattacc cagaatagtg gccagacaga tagtagacac ctgtgataaa tgtcatcaga 4920aaggagaggc tatacatggg caggcaaatt cagatctagg gacttggcaa atggattgta 4980cccatctaga gggaaaaata atcatagttg cagtacatgt agctagtgga ttcatagaag 5040cagaggtaat tccacaagag acaggaagac agacagcact atttctgtta aaattggcag 5100gcagatggcc tattacacat ctacacacag ataatggtgc taactttgct tcgcaagaag 5160taaagatggt tgcatggtgg gcagggatag agcacacctt tggggtacca tacaatccac 5220agagtcaggg agtagtggaa gcaatgaatc accacctgaa aaatcaaata gatagaatca 5280gggaacaagc aaattcagta gaaaccatag tattaatggc agttcattgc atgaatttta 5340aaagaagggg aggaataggg gatatgactc cagcagaaag attaattaac atgatcacta 5400cagaacaaga gatacaattt caacaatcaa aaaactcaaa atttaaaaat tttcgggtct 5460attacagaga aggcagagat caactgtgga agggacccgg tgagctattg tggaaagggg 5520aaggagcagt catcttaaag gtagggacag acattaaggt agtacccaga agaaaggcta 5580aaattatcaa agattatgga ggaggaaaag aggtggatag cagttcccac atggaggata 5640ccggagaggc tagagaggtg gcatagcctc ataaaatatc tgaaatataa aactaaagat 5700ctacaaaagg tttgctatgt gccccatttt aaggtcggat gggcatggtg gacctgcagc 5760agagtaatct tcccactaca ggaaggaagc catttagaag tacaagggta ttggcatttg 5820acaccagaaa aagggtggct cagtacttat gcagtgagga taacctggta ctcaaagaac 5880ttttggacag atgtaacacc aaactatgca gacattttac tgcatagcac ttatttccct 5940tgctttacag cgggagaagt gagaagggcc atcaggggag aacaactgct gtcttgctgc 6000aggttcccga gagctcataa gtaccaggta ccaagcctac agtacttagc actgaaagta 6060gtaagcgatg tcagatccca gggagagaat cccacctgga aacagtggag aagagacaat 6120aggagaggcc ttcgaatggc taaacagaac agtagaggag ataaacagag aggcggtaaa 6180ccacctacca agggagctaa ttttccaggt ttggcaaagg tcttgggaat actggcatga 6240tgaacaaggg atgtcaccaa gctatgtaaa atacagatac ttgtgtttaa tacaaaaggc 6300tttatttatg cattgcaaga aaggctgtag atgtctaggg gaaggacatg gggcaggggg 6360atggagacca ggacctcctc ctcctccccc tccaggacta gcataaatgg aagaaagacc 6420tccagaaaat gaaggaccac aaagggaacc atgggatgaa tgggtagtgg aggttctgga 6480agaactgaaa gaagaagctt taaaacattt tgatcctcgc ttgctaactg cacttggtaa 6540tcatatctat aatagacatg gagacaccct tgagggagca ggagaactca ttagaatcct 6600ccaacgagcg ctcttcatgc atttcagagg cggatgcatc cactccagaa tcggccaacc 6660tgggggagga aatcctctct cagctatacc gccctctaga agcatgctat aacacatgct 6720attgtaaaaa gtgttgctac cattgccagt tttgttttct taaaaaaggc ttggggatat 6780gttatgagca atcacgaaag agaagaagaa ctccgaaaaa ggctaaggct aatacatctt 6840ctgcatcaaa caagtaagta tgggatgtct tgggaatcag ctgcttatcg ccatcttgct 6900tttaagtgtc tatgggatct attgtactct atatgtcaca gtcttttatg gtgtaccagc 6960ttggaggaat gcgacaattc ccctcttttg tgcaaccaag aatagggata cttggggaac 7020aactcagtgc ctaccagata atggtgatta ttcagaagtg gcccttaatg ttacagaaag 7080ctttgatgcc tggaataata cagtcacaga acaggcaata gaggatgtat ggcaactctt 7140tgagacctca ataaagcctt gtgtaaaatt atccccatta tgcattacta tgagatgcaa 7200taaaagtgag acagatagat ggggattgac aaaatcaata acaacaacag catcaacaac 7260atcaacgaca gcatcagcaa aagtagacat ggtcaatgag actagttctt gtatagccca 7320ggataattgc acaggcttgg aacaagagca aatgataagc tgtaaattca acatgacagg 7380gttaaaaaga gacaagaaaa aagagtacaa tgaaacttgg tactctgcag atttggtatg 7440tgaacaaggg aataacactg gtaatgaaag tagatgttac atgaaccact gtaacacttc 7500tgttatccaa gagtcttgtg acaaacatta ttgggatgct attagattta ggtattgtgc 7560acctccaggt tatgctttgc ttagatgtaa tgacacaaat tattcaggct ttatgcctaa 7620atgttctaag gtggtggtct cttcatgcac aaggatgatg gagacacaga cttctacttg 7680gtttggcttt aatggaacta gagcagaaaa tagaacttat atttactggc atggtaggga 7740taataggact ataattagtt taaataagta ttataatcta acaatgaaat gtagaagacc 7800aggaaataag acagttttac cagtcaccat tatgtctgga ttggttttcc actcacaacc 7860aatcaatgat aggccaaagc aggcatggtg ttggtttgga ggaaaatgga aggatgcaat 7920aaaagaggtg aagcagacca ttgtcaaaca tcccaggtat actggaacta acaatactga 7980taaaatcaat ttgacggctc ctggaggagg agatccggaa gttaccttca tgtggacaaa 8040ttgcagagga gagttcctct actgtaaaat gaattggttt ctaaattggg tagaagatag 8100gaatacagct aaccagaagc caaaggaaca gcataaaagg aattacgtgc catgtcatat 8160tagacaaata atcaacactt ggcataaagt aggcaaaaat gtttatttgc ctccaagaga 8220gggagacctc acgtgtaact ccacagtgac cagtctcata gcaaacatag attggattga 8280tggaaaccaa actaatatca ccatgagtgc agaggtggca gaactgtatc gattggaatt 8340gggagattat aaattagtag agatcactcc aattggcttg gcccccacag atgtgaagag 8400gtacactact ggtggcacct caagaaataa aagaggggtc tttgtgctag ggttcttggg 8460ttttctcgca acggcaggtt ctgcaatggg cgcggcgtcg ttgacgctga ccgctcagtc 8520ccgaacttta ttggctggga tagtgcagca acagcaacag ctgttggacg tggtcaagag 8580acaacaagaa ttgttgcgac tgaccgtctg gggaacaaag aacctccaga ctagggtcac 8640tgccatcgag aagtacttaa aggaccaggc gcagctgaat gcttggggat gtgcgtttag 8700acaagtctgc cacactactg taccatggcc aaatgcaagt ctaacaccaa agtggaacaa 8760tgagacttgg caagagtggg agcgaaaggt tgacttcttg gaagaaaata taacagccct 8820cctagaggag gcacaaattc aacaagagaa gaacatgtat gaattacaaa agttgaatag 8880ctgggatgtg tttggcaatt ggtttgacct tgcttcttgg ataaagtata tacaatatgg 8940agtttatata gttgtaggag taatactgtt aagaatagtg atctatatag tacaaatgct 9000agctaagtta aggcaggggt ataggccagt gttctcttcc ccaccctctt atttccagca 9060gacccatatc caacaggacc cggcactgcc aaccagagaa ggcaaagaaa gagacggtgg 9120agaaggcggt ggcaacagct cctggccttg gcagatagaa tatattcatt tcctgatccg 9180ccaactgata cgcctcttga cttggctatt cagcaactgc agaaccttgc tatcgagagt 9240ataccagatc ctccaaccaa tactccagag gctctctgcg accctacaga ggattcgaga 9300agtcctcagg actgaactga cctacctaca atatgggtgg agctatttcc atgaggcggt 9360ccaggccgtc tggagatctg cgacagagac tcttgcgggc gcgtggggag acttatggga 9420gactcttagg agaggtggaa gatggatact cgcaatcccc aggaggatta gacaagggct 9480tgagctcact ctcttgtgag ggacagaaat acaatcaggg acagtatatg aatactccat 9540ggagaaaccc agctgaagag agagaaaaat tagcatacag aaaacaaaat atggatgata 9600tagatgagta agatgatgac ttggtagggg tatcagtgag gccaaaagtt cccctaagaa 9660caatgagtta caaattggca atagacatgt ctcattttat aaaagaaaag gggggactgg 9720aagggattta ttacagtgca agaagacata gaatcttaga catatactta gaaaaggaag 9780aaggcatcat accagattgg caggattaca cctcaggacc aggaattaga tacccaaaga 9840catttggctg gctatggaaa ttagtccctg taaatgtatc agatgaggca caggaggatg 9900aggagcatta tttaatgcat ccagctcaaa cttcccagtg ggatgaccct tggggagagg 9960ttctagcatg gaagtttgat ccaactctgg cctacactta tgaggcatat gttagatacc 10020cagaagagtt tggaagcaag tcaggcctgt cagaggaaga ggttagaaga aggctaaccg 10080caagaggcct tcttaacatg gctgacaaga aggaaactcg ctgaaacagc agggactttc 10140cacaagggga tgttacgggg aggtactggg gaggagccgg tcgggaacgc ccactttctt 10200gatgtataaa tatcactgca tttcgctctg tattcagtcg ctctgcggag aggctggcag 10260attgagccct gggaggttct ctccagcact agcaggtaga gcctgggtgt tccctgctag 10320actctcacca gcacttggcc ggtgctgggc agagtgactc cacgcttgct tgcttaaagc 10380cctcttcaat aaagctgcca ttttagaagt aagctagtgt gtgttcccat ctctcctagc 10440cgccgcctgg tcaactcggt actcaataat aagaagaccc tggtctgtta ggaccctttc 10500tgctttggga aaccgaagca ggaaaatccc tagca 105353869713DNAHuman immunodeficiency virus 2 386agtcgctctg cggagaggct ggcagattga gccctgggag gttctctcca gcactagcag 60gtagagcctg ggtgttccct gctagactct caccggtgct tggccggcac tgggcagacg 120gctccacgct tgcttgctta aaagacctct taataaagct gccagttaga agcaagttaa 180gtgtgtgttc ccatctctcc tagtcgccgc ctggtcattc ggtgttcatc tgaataacaa 240gaccctggtc tgttaggacc ctttctgctt tgggaaacca aagcaggaaa atccctagca 300ggttggcgcc cgaacaggga cttagagaag actgaaaagc cttggaacac ggctgagtga 360aggcagtaag ggcggcagga acaaaccacg acggagtgct cctagaaagg cgcaggccaa 420ggtaccaaag gcggcgtgtg gagcgggagt aaagaggcct ccgggtgaag gtaagtacct 480acaccaaaaa attgtagcca ggaagggctt gttatcctac ctttagacag gtagaagatt 540gtgggagatg ggcgcgagaa actccgtctt gaaagggaaa aaagcagacg aattagaaac 600aattaggtta cggcccggcg gaaagaaaaa atacaggcta aagcatattg tgtgggcagc 660gaatgaattg gacagattcg gattagcaga gagcctgttg gagtcaaaag aaggttgcca 720aagaattctt acagttttag gtccattagt accgacaggt tcagaaaatt taaaaagcct 780ttttaatact gtctgcgtca tttggtgcat acacgcagaa gagaaagtga aagatactga 840aggagcaaaa caaatagtac agagacatct agcggcagaa acaggaactg cagagaaaat 900gccaaataca agtagaccaa cagcaccacc tagcgggaag ggaggaaact tccccgtaca 960acaagtaggc ggcaattata

cccatgtgcc gctgagtcct cgaaccctaa atgcttgggt 1020aaaattagta gaggaaaaga agttcggggc agaggtagtg ccaggatttc aggcactctc 1080agaaggctgc acgccctatg atatcaacca aatgcttaat tgtgtgggcg accatcaagc 1140agctatgcaa ataatcaggg agatcgttaa tgaagaagca gcagattggg atgtgcaaca 1200tccaatacca ggtcccttac cagcggggca gcttagagaa ccaagagggt ctgacatagc 1260agggacaaca agcacagtag atgaacagat ccagtggatg tttaggccac aaaatcccgt 1320accagtggga aacatctata ggagatggat ccagatagga ctgcagaagt gcgtcaggat 1380gtacaacccg accaacatcc tagacataaa acaaggacca aaggaaccat tccaaagtta 1440tgtagataga ttctacaaaa gcttgagggc agaacaaaca gatccagcag tgaagaattg 1500gatgacccag acactactag tacagaatgc caacccagac tgtaaattag tactaaaagg 1560actagggatg aatcctacct tagaagagat gctaaccgcc tgccaagggg taggtgggcc 1620aggccagaaa gctagactaa tggcagaagc cttaaaagag gccttgacac cagcccctat 1680cccatttgca gcagcccagc agaaaaggac aattaaatgc tggaattgtg gaaaggaagg 1740acactcggca agacaatgcc gagcacctag aagacagggc tgctggaagt gtggtaaacc 1800aggacatgtc atagcaaatt gcccagatag acaggtgggt tttttaggga tgggcccccg 1860gggaaagaag ccccgcaact tccccgtggc ccaagtcccg caggggctaa caccaacagc 1920acccccagta gatccagcag tggacctact ggagaattat atgcagcaag gaaaaagaca 1980aagagaacag agagagagac catacaaaga agtgacagag gacttactgc acctcgagca 2040gggagaggca ccatgcagag agacgacaga ggacttgctg cacctcaatt ctctcttttg 2100aaaagaccag tagtcacggc atacgtcgag ggccagccag tagaagttct gctagacacg 2160ggggctgacg actcaatagt agcagggata gagttaggga gcaattatag tccaaagata 2220gtaggaggaa tagggggatt cataaatacc aaggaatata aaaatgtaaa aatagaagtt 2280ttaggtaaaa aggtaagggc caccataatg acaggtgaca ccccaatcaa catttttggc 2340agaaatattc tgacagcctt aggcatgtca ttaaatttac cagtcgccaa aatagaacca 2400ataaaaataa tgttaaagcc aggaaaagat ggaccaaaac tgaggcaatg gcccttaaca 2460aaagaaaaaa tagaggcact aaaagaaatc tgtgaaaaaa tggaaagaga aggccagcta 2520gaggaagcgc ctccaactaa tccttataac acccccacat ttgcaatcaa gaaaaaggac 2580aaaaataaat ggaggatgct aatagatttt agagaactaa acaaggtaac tcaagatttc 2640acagaaattc agttaggaat tccacaccca gcaggattgg ccaagaaaaa aagaattact 2700gtactagata taggggatgc ttacttttcc ataccactac atgaagactt tagacagtat 2760actgcattta ctttaccatc aataaacaat gcagaaccag gaaaaagata tatatataag 2820gtcctgcctc agggatggaa ggggtcacca gcaatttttc aatacacaat gaggcaggtc 2880ttagaaccat tcagaaaagc aaacctagat gtcattatca ttcagtacat ggatgatatc 2940ctaatagcta gtgacaggac agatctagaa catgacaagg tggtcctgca gctaaaggaa 3000cttctaaata acctaggatt ttctacccca gatgagaagt tccaaaagga ccctccatac 3060cactggatgg gctatgaact gtggccaact aagtggaagc tgcagaagat acagttgccc 3120caaaaagatg tatggacagt aaatgacatc caaaagttag tgggtgtctt aaactgggca 3180gcacaaatct acccagggat aaaaaccaga cacttatgta agctaattag aggaaaaatg 3240acactcacag aagaagtaca gtggacagaa ctagcagagg cggagttaga agagaacaag 3300attatcttaa gccaggagca agagggacac tattaccaag aagaaaaaga gttagaagca 3360acagtccaaa aggatcaaga caatcagtgg acatataaag tacaccaggg agagaaaatt 3420ctaaaagtag ggaaatatgc aaagataaaa aatacccata ccaatggggt cagattgtta 3480gcacaagtag ttcaaaagat aggaaaagaa gcactaatca tttggggacg aataccaaaa 3540tttcacctac cagtagaaag agagacatgg gaacagtggt gggatgacta ctggcaggtg 3600acatggatcc ctgactggga cttcgtatct accccgccgc tggtcagact agcatttaac 3660ctggtaaaag atcctatacc aagaacagag actttctaca cagatggatc ctgcaatagg 3720caatcaaagg aaggaaaagc aggatatgta acagatagag ggagagacaa ggtaaggatg 3780ctagaacaaa ctaccaatca gcaagcagaa ttagaagcct ttgcaatggc actaacagac 3840tcaggtccaa aagccaatat tatagtagac tcacagtatg taatggggat agtagcaggc 3900cagccaacag aatcagagag tagaatagta aatcaaatca tagaggagat gataaaaaag 3960gaagcaatct atgttgcatg ggtcccagcc cataaaggca taggagggaa tcaggaggta 4020gatcagttag taagtcaggg catcagacaa gtgttgttcc tggaaaaaat agagcccgct 4080caggaagaac atgagaaata ccatagcaat gtaaaagaac tatcccataa atttggattg 4140cccaaattag tagcaagaca aatagtaaac acatgtgccc aatgtcaaca gaaaggggag 4200gctatacatg ggcaagtaga tgcagaatta ggcacttggc aaatggactg cacacactta 4260gaaggaaaga tcattatagt agcagtacat gttgcaagtg gattcataga agcagaagtc 4320atcccacagg aatcaggaag gcagacagca ctcttcctat taaaactggc cagtaggtgg 4380ccaataacac acttgcacac agataatggt gccaacttca cttcacagga agtaaaaatg 4440gtagcatggt gggtaggtat agaacaatct ttcggagtac cttacaatcc acaaagccaa 4500ggagtagtag aagcaatgaa tcaccaccta aaaaatcaga taagtagaat tagagaacag 4560gcaaatacag tagaaacaat agtactgatg gcaacacact gcatgaattt taaaagaagg 4620ggaggaatag gggatatgac cccagcagaa agactaatca atatgatcac cacagaacaa 4680gaaatacaat tcctccacgc caaaaattca aaattaaaaa attttcgggt ctatttcaga 4740gaaggcagag atcagctgtg gaaaggaccc ggggaactac tgtggaaggg agacggagca 4800gtcatagtca aggtagggac agacataaaa gtagtaccaa ggaggaaagc caagatcatc 4860aaagactatg gaggaaggca agaactggat agtggttccc acttggaggg tgccagggag 4920gatggagaaa tggcatagcc ttgtcaaata tctaaaatac agaacaaaag atctagaaga 4980cgtgtgctat gttccccacc ataaagtagg atgggcatgg tggacttgca gcagggtaat 5040attcccatta aagggaaaca gtcatctaga aatacaggca tattggaacc taacgccaga 5100aaaaggatgg ctctcctctt attcagtaag aatgacttgg tatacggaaa ggttctggac 5160agatgttacc ccagactgtg cagactccct aatacatagc acttatttct cttgctttac 5220agcaggtgaa gtaagaagag ccatcagagg ggaaaagtta ttgtcctgct gcaattatcc 5280ccaagcccat agagcccagg taccgtcact ccaatttttg gccttagtgg tagtgcagca 5340aaatgacaga ccccagagaa acggtacccc caggaaacag tggcgaagag actatcgaag 5400aggccttcaa ttggctagac aggacggtag aagccataaa cagagaggca gtgaatcacc 5460tgccccgaga gcttattttc caggtgtggc agaggtcctg gagatactgg catgatgaac 5520aagggatgtc acaaagttac acaaagtata gatatttgtg cttaatacag aaggctatgt 5580tcacacattg taagagaggg tgcacttgcc tggggggagg acatgggcca ggagggtgga 5640gaccaggacc tccccctcct ccccctccag gtctagtcta atgactgaag caccaacaga 5700gtttcccccg gaggatggga ccccaccgag ggaaccaggg gatgagtgga taatagaaat 5760cctgagaaaa ataaagaaag aagctttaaa gcattttgac cctcgcttgc taactgctct 5820tggcaactat atccatacta gacatggaga cacccttgaa ggcgccagag agctcattaa 5880tgtcctacaa cgagccctct tcatgcactt cagagcggga tgtaggctct caagaattgg 5940ccaaacaggg ggaagaactc ctttcccagc tacatcgacc cctagaacca tgcaataaca 6000aatgctattg taaaggatgc tgcttccact gccagctgtg ttttttaaac aaggggctcg 6060ggatatgtta tgaccggaag ggcagacgaa gaagaactcc gaagaaaact aaggctcatt 6120catcttctgc atcagacaag tgagtatgat gggtggtaga aatcagctgc ttgttgccat 6180tttgctaact agtacttgct tgatatattg caccaattat gtgactgttt tctatggcat 6240acccgcgtgg agaaatgcat ccattcccct cttttgtgca accaagaata gggatacttg 6300gggaaccata cagtgcttgc cagacaatga tgattatcag gagataactt tgaatgtgac 6360agaggctttc gatgcatggg ataatacagt aacagaacaa gcaatagaag atgtctggaa 6420tctatttgag acatcaataa aaccatgtgt caaattaacg cctttatgtg tagcaatgag 6480atgtaacaac acagatgcaa ggaacacaac cacacccaca acagcatccc cgcgtacaat 6540aaaacccgtg acagagataa gtgagaattc ctcatgcata cgcgcaaaca actgctcagg 6600attgggagaa gaagaggtgg tcaattgtca attcaatatg acaggattag agagagataa 6660gaaaaagcaa tatagtgaga catggtactc gaaggatgta gtttgtgaag gaaatggcac 6720cacagataca tgttacatga accattgcaa cacatcggtc atcacagagt catgtgacaa 6780gcactattgg gatgctatga ggtttagata ctgtgcacca ccaggttttg ccctactaag 6840atgcaatgat accaattatt caggctttgc gcccaattgc tctaaggtag tagctgctac 6900atgcaccaga atgatggaaa cgcaaacttc tacatggttt ggctttaatg gcactagagc 6960agaaaataga acatttatct attggcatgg tagggataac agaactatca tcagcttaaa 7020caaatattat aatctcacta tacattgtaa gaggccagga aataagacag tggtaccaat 7080aacacttatg tcagggttaa ggtttcactc ccagccggtc atcaataaaa gacccagaca 7140agcatggtgt tggttcaaag gtgaatggaa gggagccatg caggaggtga aggaaaccct 7200tgcaaaacat cccaggtata aaggaaccaa tgaaacaaag aatattaact ttacagcacc 7260aggaaagggc tcagacccag aggtggcata catgtggact aactgcagag gagaatttct 7320ctactgcaac atgacttggt tcctcaattg gatagaaaat aagacacacc gcaattatgt 7380accgtgccat ataagacaaa taattaacac ctggcataag gtagggaaaa atgtatattt 7440gcctcccagg gaaggggagt tgacctgcaa ctcaacagta actagcataa ttgctaacat 7500tgatgcaaat ggaaataata caaatattac ctttagtgca gaggtggcag aactataccg 7560attagagttg ggagattata aattggtaga aataacacca attggcttcg cacctacagc 7620agaaaaaaga tactcctcta ctccaatgag gaacaagaga ggtgtgttcg tgctagggtt 7680cttgggtttt ctcgcaacag caggctctgc aatgggcgcg gcgtccttaa cgctgtcggc 7740tcagtctcgg actttactgg ccgggatagt gcagcaacag caacagctgt tggacgtggt 7800caagagacaa caggaaatgt tgcgactgac cgtctgggga acaaaaaatc tccaggcaag 7860agtcactgct atcgagaagt acttaaagga ccaggcgcaa ctaaattcat ggggatgtgc 7920atttagacaa gtctgccaca ctactgtacc atgggtaaat gataccttaa cgcctgagtg 7980gaacaatatg acgtggcaag aatgggaagg caaaatccgc gacctggagg caaatatcag 8040tcaacaatta gaacaagcac aaattcagca agagaagaat atgtatgaac tacaaaagtt 8100aaatagctgg gatgtttttg gtaactggtt tgacttaacc tcctggatca agtatattca 8160atatggagtt tatataataa taggaatagt agttcttaga atagtaatat atatagtaca 8220gatgttaagt agacttagaa agggctatag gcctgttttc tcttcccccc ccggttacct 8280ccaacagatc catatccaca aggactggga acagccagcc agagaagaaa cagaagaaga 8340cgttggaaac aacgttggag acagctcgtg gccttggccg ataagatata tacatttcct 8400gatccaccag ctgattcgcc tcttggccgg actatacaac atctgcagga acttactatc 8460caggatctcc ctgaccctcc gaccagtttt ccagagtctt cagagggcac tgacagcaat 8520cagagactgg ctaagaactg acgcagccta cttgcagtat gggtgcgagt ggatccaagg 8580agcgttccag gccttcgcaa gggctacgag agagactctt gcgggcacgt ggagagactt 8640gtggggggca ctgcagcgga tcgggagggg aatacttgca gtcccaagaa gaatcaggca 8700gggagcagag atcgccctcc tatgagggac agcggtatca gcagggagac tttatgaata 8760ccccatggag aaccccagca aaagaagggg agaaagaatt gtacaagcaa caaaatagag 8820atgatgtaga ttcggatgat gatgacctag taggggtctc tgtcacacca agagtaccac 8880taagagaatt gacacataga ttagcaatag atgtgtcaca ttttataaaa gaaaaagggg 8940gactggaagg gatgtattac agtgagagaa gacatagaat cttagacata taccttgaaa 9000aggaagaagg gataattgca gattggcaga actatactca tgggccagga ataagatacc 9060caatgttctt tgggtggcta tggaagctag taccagtaga tgtcacacga caggaggagg 9120acgatgggac tcactgttta ctacacccag cacaaacaag caggtttgat gacccgcatg 9180gggaaacact gatatggaag tttgacccca cgctggctca tgattacaag gcttttatcc 9240tgcacccaga ggaatttggg cataagtcag gcctgccaga agaagactgg aaggcaagac 9300tgaaagcaag agggatacca tttagttaga gacaggaaca gctatatttg gccagggcag 9360gaaataacta ctgaaaacag ctgagactgc agggactttc cgaaggggct gtaaccaggg 9420gagggacatg ggaggagccg gtggggaacg ccctcatact ttctgtataa agatacccgc 9480tgcttgcatt gtacttcagt cgctctgcgg agaggctggc agattgagcc ctgggaggtt 9540ctctccagca ctagcaggta gagcctgggt gttccctgct agactctcac cggtgcttgg 9600ccggcactgg gcagacggct ccacgcttgc ttgcttaaaa gacctcttaa taaagctgcc 9660agttagaagc aagttaagtg tgtgttccca tctctcctag tcgccgcctg gtc 971338711878DNAArtificial SequenceDescription of Artificial Sequence Synthetic polynucleotide 387gcctcactga ttaagcattg gtaactgtca gaccaagttt actcatatat actttagatt 60gatttaaaac ttcattttta atttaaaagg atctaggtga agatcctttt tgataatctc 120atgaccaaaa tcccttaacg tgagttttcg ttccactgag cgtcagaccc cgtagaaaag 180atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa 240aaaccaccgc taccagcggt ggtttgtttg ccggatcaag agctaccaac tctttttccg 300aaggtaactg gcttcagcag agcgcagata ccaaatactg ttcttctagt gtagccgtag 360ttaggccacc acttcaagaa ctctgtagca ccgcctacat acctcgctct gctaatcctg 420ttaccagtgg ctgctgccag tggcgataag tcgtgtctta ccgggttgga ctcaagacga 480tagttaccgg ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac acagcccagc 540ttggagcgaa cgacctacac cgaactgaga tacctacagc gtgagctatg agaaagcgcc 600acgcttcccg aagggagaaa ggcggacagg tatccggtaa gcggcagggt cggaacagga 660gagcgcacga gggagcttcc agggggaaac gcctggtatc tttatagtcc tgtcgggttt 720cgccacctct gacttgagcg tcgatttttg tgatgctcgt caggggggcg gagcctatgg 780aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct tttgctggcc ttttgctcac 840atgttctttc ctgcgttatc ccctgattct gtggataacc gtattaccgc ctttgagtga 900gctgataccg ctcgccgcag ccgaacgacc gagcgcagcg agtcagtgag cgaggaagcg 960gaagagcgcc caatacgcaa accgcctctc cccgcgcgtt ggccgattca ttaatgcagc 1020tggcacgaca ggtttcccga ctggaaagcg ggcagtgagc gcaacgcaat taatgtgagt 1080tagctcactc attaggcacc ccaggcttta cactttatgc ttccggctcg tatgttgtgt 1140ggaattgtga gcggataaca atttcacaca ggaaacagct atgaccatga ttacgccaag 1200ctatttaggt gacactatag aatactcaag cttgggggga tcctctagag tcgacctgca 1260ggcatgctat ttgatgaatt aactacactt aaaataatac aattattatt aaattttttt 1320ttgatttatt tattaatttt taaacttaat catttgtatt tgggaggaat tatatatatc 1380tttataatta ttttattttt ttttattttt ttattttttt attattatta ttttttttta 1440tttttttttt ttactgtatc aaagaaaaac ctttaaaaaa aaaattataa tttccccatc 1500ttactatatt tttaatacat acgttttaag gaattaaatt agacaaaagc tatattatgc 1560tttacatata attagaattt ataaacgttt ggttattaga tatttcatgt ctcagtaaag 1620tctttcaata catatgtaaa aaaatatata tgaatacaca taagttgtta atatatttta 1680tatgcataaa tgtataaata tatatatata tatatatata tgtatgtatg tatatgtgtg 1740tatatgaaat tatttcaatg tttaattttt taaattttaa tttttttttt tttttttttt 1800tttattatgt atattgatct ttattattta aatattactt ttttcgtttt ttcttctttt 1860tattattttt tttttttttt atattttata caaatggtaa ttcaaataaa aggtataaat 1920ttatatttaa ttttctttta tggataaata aaagaaaaat ataaatatat aaaaatataa 1980aaatatatat atgtatattg gggtgatgat aaaatgaaag ataatatata tatatatata 2040tctttatttt tttttttttg tagaccccat tgtgagtaca taaatatatt atataactcg 2100ggagcatcag tcatggaatt cttatttctt tttctttttt gcctggccgg cctttttcgt 2160ggccgccggc cttttgtcgc ctcccagctg agacaggtcg atccgtgtct cgtacaggcc 2220ggtgatgctc tggtggatca gggtggcgtc cagcacctct ttggtgctgg tgtacctctt 2280ccggtcgatg gtggtgtcaa agtacttgaa ggcggcaggg gctcccagat tggtcagggt 2340aaacaggtgg atgatattct cggcctgctc tctgatgggc ttatcccggt gcttgttgta 2400ggcggacagc actttgtcca gattagcgtc ggccaggatc actctcttgg agaactcgct 2460gatctgctcg atgatctcgt ccaggtagtg cttgtgctgt tccacaaaca gctgtttctg 2520ctcattatcc tcgggggagc ccttcagctt ctcatagtgg ctggccaggt acaggaagtt 2580cacatatttg gagggcaggg ccagttcgtt tcccttctgc agttcgccgg cagaggccag 2640cattctcttc cggccgtttt ccagctcgaa cagggagtac ttaggcagct tgatgatcag 2700gtcctttttc acttctttgt agcccttggc ttccagaaag tcgatgggat tcttctcgaa 2760gctgcttctt tccatgatgg tgatccccag cagctctttc acactcttca gtttcttgga 2820cttgcccttt tccactttgg ccaccaccag cacagaatag gccacggtgg ggctgtcgaa 2880gccgccgtac ttcttagggt cccagtcctt ctttctggcg atcagcttat cgctgttcct 2940cttgggcagg atagactctt tgctgaagcc gcctgtctgc acctcggtct ttttcacgat 3000attcacttgg ggcatgctca gcactttccg cacggtggca aaatcccggc ccttatccca 3060cacgatctcc ccggtttcgc cgtttgtctc gatcagaggc cgcttccgga tctcgccgtt 3120ggccagggta atctcggtct tgaaaaagtt catgatgttg ctgtagaaga agtacttggc 3180ggtagccttg ccgatttcct gctcgctctt ggcgatcatc ttccgcacgt cgtacacctt 3240gtagtcgccg tacacgaact cgctttccag cttagggtac tttttgatca gggcggttcc 3300cacgacggcg ttcaggtagg cgtcgtgggc gtggtggtag ttgttgatct cgcgcacttt 3360gtaaaactgg aaatccttcc ggaaatcgga caccagcttg gacttcaggg tgatcacttt 3420cacttcccgg atcagcttgt cattctcgtc gtacttagtg ttcatccggg agtccaggat 3480ctgtgccacg tgctttgtga tctgccgggt ttccaccagc tgtctcttga tgaagccggc 3540cttatccagt tcgctcaggc cgcctctctc ggccttggtc agattgtcga actttctctg 3600ggtaatcagc ttggcgttca gcagctgccg ccagtagttc ttcatcttct tcacgacctc 3660ttcggagggc acgttgtcgc tcttgccccg gttcttgtcg cttctggtca gcaccttgtt 3720gtcgatggag tcgtccttca gaaagctctg aggcacgata tggtccacat cgtagtcgga 3780cagccggttg atgtccagtt cctggtccac gtacatatcc cgcccattct gcaggtagta 3840caggtacagc ttctcgttct gcagctgggt gttttccacg gggtgttctt tcaggatctg 3900gctgcccagc tctttgatgc cctcttcgat ccgcttcatt ctctcgcggc tgttcttctg 3960tcccttctgg gtggtctggt tctctctggc catttcgatc acgatgttct cgggcttgtg 4020ccggcccatc actttcacga gctcgtccac caccttcact gtctgcagga tgcccttctt 4080aatggcgggg ctgccggcca gattggcaat gtgctcgtgc aggctatcgc cctggccgga 4140cacctgggct ttctggatgt cctctttaaa ggtcaggctg tcgtcgtgga tcagctgcat 4200gaagtttctg ttggcgaagc cgtcggactt caggaaatcc aggattgtct tgccggactg 4260cttgtcccgg atgccgttga tcagcttccg gctcagcctg ccccagccgg tgtatctccg 4320ccgcttcagc tgcttcatca ctttgtcgtc gaacaggtgg gcataggttt tcagccgttc 4380ctcgatcatc tctctgtcct caaacagtgt cagggtcagc acgatatctt ccagaatgtc 4440ctcgttttcc tcattgtcca ggaagtcctt gtccttgata attttcagca gatcgtggta 4500tgtgcccagg gaggcgttga accgatcttc cacgccggag atttccacgg agtcgaagca 4560ctcgattttc ttgaagtagt cctctttcag ctgcttcacg gtcactttcc ggttggtctt 4620gaacagcagg tccacgatgg cctttttctg ctcgccgctc aggaaggcgg gctttctcat 4680tccctcggtc acgtatttca ctttggtcag ctcgttatac acggtgaagt actcgtacag 4740caggctgtgc ttgggcagca ccttctcgtt gggcaggttc ttatcgaagt tggtcatccg 4800ctcgatgaag ctctgggcgg aagcgccctt gtccaccact tcctcgaagt tccagggggt 4860gatggtttcc tcgctctttc tggtcatcca ggcgaatctg ctgtttcccc tggccagagg 4920gcccacgtag taggggatgc ggaaggtcag gatcttctcg atcttttccc ggttgtcctt 4980caggaatggg taaaaatctt cctgccgccg cagaatggcg tgcagctctc ccaggtggat 5040ctggtggggg atgctgccgt tgtcgaaggt ccgctgcttc cgcagcaggt cctctctgtt 5100cagcttcacg agcagttcct cggtgccgtc catcttttcc aggatgggct tgatgaactt 5160gtagaactct tcctggctgg ctccgccgtc aatgtagccg gcgtagccgt tcttgctctg 5220gtcgaagaaa atctctttgt acttctcagg cagctgctgc cgcacgagag ctttcagcag 5280ggtcaggtcc tggtggtgct cgtcgtatct cttgatcata gaggcgctca ggggggcctt 5340ggtgatctcg gtgttcactc tcaggatgtc gctcagcagg atggcgtcgg acaggttctt 5400ggcggccaga aacaggtcgg cgtactggtc gccgatctgg gccagcaggt tgtccaggtc 5460gtcgtcgtag gtgtccttgc tcagctgcag tttggcatcc tcggccaggt cgaagttgct 5520cttgaagttg ggggtcaggc ccaggctcag ggcaatcagg tttccgaaca ggccattctt 5580cttctcgccg ggcagctggg cgatcagatt ttccagccgt ctgctcttgc tcagtctggc 5640agacaggatg gccttggcgt ccacgccgct ggcgttgatg gggttttcct cgaacagctg 5700gttgtaggtc tgcaccagct ggatgaacag cttgtccacg tcgctgttgt cggggttcag 5760gtcgccctcg atcaggaagt ggccccggaa cttgatcatg tgggccaggg ccagatagat 5820cagccgcagg tcggccttgt cggtgctgtc caccagtttc tttctcaggt ggtagatggt 5880ggggtacttc tcgtggtagg ccacctcgtc cacgatgttg ccgaagatgg ggtgccgctc 5940gtgcttctta tcctcttcca ccaggaagga ctcttccagt ctgtggaaga agctgtcgtc 6000caccttggcc atctcgttgc tgaagatctc ttgcagatag cagatccggt tcttccgtct 6060ggtgtatctt cttctggcgg ttctcttcag ccgggtggcc tcggctgttt cgccgctgtc 6120gaacagcagg gctccgatca ggttcttctt gatgctgtgc cggtcggtgt tgcccagcac 6180cttgaatttc ttgctgggca ccttgtactc gtcggtgatc acggcccagc ccacagagtt

6240ggtgccgatg tccaggccga tgctgtactt cttgtcggct gctgggactc cgtggatacc 6300gaccttccgc ttcttctttg gggccatctt atcgtcatcg tctttgtaat caatatcatg 6360atccttgtag tctccgtcgt ggtccttata gtccattttt ctcgagggat cctgatatat 6420ttctattagg tatttattat tataaaatat aaatcttgaa tgataataaa taaaatatta 6480gttattcctt ttctagttta aaatatacat attataaata tatatatata tatatatatt 6540tttattgtga caagaatata taattataaa ttatattatt tatttttgta tttttttttt 6600tttttttttt tttttctttt tttgttttat ttttcttttt ttttataaat attatttttt 6660tcttttatca tgcacattgg aataatacat taatatatat atatatatta tattatacat 6720atattgaata atgtttataa aaaatgcata acttatatga atataatttt ttttaaatat 6780gacaaaaaga aaaaaaaaaa aaaccaaaaa aaattaaaat tgaaatgaaa tatataaata 6840tattatttat atatattata cattgtttaa tactactaca tgtatatata tatattatat 6900atatatatat atatcaattt tttcaaaaat aaattaatat aaaaagaggg gaaaaaaaaa 6960aaaaaaaaaa aaaaaagata attaagtaag catttaaaaa tatataaatt gataatatat 7020aaaattaatc acatataaaa gcttataaac actaggttag ctaattcgct tgtaagaggt 7080actctcgttt atgcaaaact atttgatata gcattttaac aagtacacat atatatatgt 7140aatatatata ctatatatat ctattgcatg tgtactaagc atgtgcatgg catccccttt 7200ttctcgtgtt taaaacagtt tgtatgataa aatataaagg atttgaaaaa gagaaaaaaa 7260tatatgatct catcctatat agcgccataa tttttatttg ggttgaataa aattttctac 7320taaatttagg tgtaagtaaa ataatggaat atatataagt acaataaaaa agtgcataaa 7380ttaaaaaatt tttataataa atattttttt taaaaaagtc aataataata ttaaatatat 7440ataacacagg attatatatg ttcactacaa ttttttatat tataatataa attcttttca 7500attttcattt tattttacat acactttcct tttttgtcac tatattttaa tattcacata 7560tttagtttaa atactggcta tttctttcta catttgctag taacaattgt gtagtgctta 7620aatatataca cacacctaaa acttacaaag tatcctagga ccatggccaa gcctttgtct 7680caagaagaat ccaccctcat tgaaagagca acggctacaa tcaacagcat ccccatctct 7740gaagactaca gcgtcgccag cgcagctctc tctagcgacg gccgcatctt cactggtgtc 7800aatgtatatc attttactgg gggaccttgt gcagaactcg tggtgctggg cactgctgct 7860gctgcggcag ctggcaacct gacttgtatc gtcgcgatcg gaaatgagaa caggggcatc 7920ttgagcccct gcggacggtg ccgacaggtg cttctcgatc tgcatcctgg gatcaaagcc 7980atagtgaagg acagtgatgg acagccgacg gcagttggga ttcgtgaatt gctgccctct 8040ggttatgtgt gggagggcta accgcgggta ccccattaaa tttatttaat aatagattaa 8100aaatattata aaaataaaaa cataaacaca gaaattacaa aaaaaataca tatgaatttt 8160ttttttgtaa tcttccttat aaatatagaa taatgaatca tataaaacat atcattattc 8220atttatttac atttaaaatt attgtttcag tatctttaat ttattatgta tatataaaaa 8280taacttacaa ttttattaat aaacaatata tgtttattaa ttcatgtttt gtaatttatg 8340ggatagcgat tttttttact gtctgtattt tcttttttaa ttatgtttta attgtattta 8400ttttattttt attattgttc tttttatagt attattttaa aacaaaatgt attttctaag 8460aacttataat aataataata taaattttaa taaaaattat atttatcttt tacaatatga 8520acataaagta caacattaat atatagcttt taatattttt attcctaatc atgtaaatct 8580taaatttttc tttttaaaca tatgttaaat atttatttct cattatatat aagaacatat 8640ttattacatc tagaggtacc gagctcgttt tcgacactgg atggcggcgt tagtatcgaa 8700tcgacagcag tatagcgacc agcattcaca tacgattgac gcatgatatt actttctgcg 8760cacttaactt cgcatctggg cagatgatgt cgaggcgaaa aaaaatataa atcacgctaa 8820catttgatta aaatagaaca actacaatat aaaaaaacta tacaaatgac aagttcttga 8880aaacaagaat ctttttattg tcagtactga ttagaaaaac tcatcgagca tcaaatgaaa 8940ctgcaattta ttcatatcag gattatcaat accatatttt tgaaaaagcc gtttctgtaa 9000tgaaggagaa aactcaccga ggcagttcca taggatggca agatcctggt atcggtctgc 9060gattccgact cgtccaacat caatacaacc tattaatttc ccctcgtcaa aaataaggtt 9120atcaagtgag aaatcaccat gagtgacgac tgaatccggt gagaatggca aaagcttatg 9180catttctttc cagacttgtt caacaggcca gccattacgc tcgtcatcaa aatcactcgc 9240atcaaccaaa ccgttattca ttcgtgattg cgcctgagcg agacgaaata cgcgatcgct 9300gttaaaagga caattacaaa caggaatcga atgcaaccgg cgcaggaaca ctgccagcgc 9360atcaacaata ttttcacctg aatcaggata ttcttctaat acctggaatg ctgttttgcc 9420ggggatcgca gtggtgagta accatgcatc atcaggagta cggataaaat gcttgatggt 9480cggaagaggc ataaattccg tcagccagtt tagtctgacc atctcatctg taacatcatt 9540ggcaacgcta cctttgccat gtttcagaaa caactctggc gcatcgggct tcccatacaa 9600tcgatagatt gtcgcacctg attgcccgac attatcgcga gcccatttat acccatataa 9660atcagcatcc atgttggaat ttaatcgcgg cctcgaaacg tgagtctttt ccttacccat 9720ggttgtttat gttcggatgt gatgtgagaa ctgtatccta gcaagatttt aaaaggaagt 9780atatgaaaga agaacctcag tggcaaatcc taacctttta tatttctcta caggggcgcg 9840gcgtggggac aattcaacgc gtctgtgagg ggagcgtttc cctgctcgca ggtctgcagc 9900gaggagccgt aatttttgct tcgcgccgtg cggccatcaa aatgtatgga tgcaaatgat 9960tatacatggg gatgtatggg ctaaatgtac gggcgacagt cacatcatgc ccctgagctg 10020cgcacgtcaa gactgtcaag gagggtattc tgggcctcca tgtcgctggc ctaacattag 10080taatgtaggt ctgactttca ctcatataag tcttatggta actaaactaa ggtcttacct 10140ttactgatat atgtcttact ttcactaact taggtattac ttttactaac ttaggtctta 10200aattcagtaa ctaaggtcat acttcgacta actaaggtct tacattcact gatataggtc 10260ttatgattac taacttaggt cctaatttga ctaacataag tcctaacatt agtaatgtag 10320gtcttaactt aactaactta ggtcttacct tcactaatat aggtcttaat attactgact 10380taagtaatta aggtactaac ttaggtcgta aggtaactaa tatataggtc ttaaggtaac 10440taatttaggt cttgacttaa taaatatagg tcctaacata aatagtatag gtcctaatat 10500aagtactata ggccttaact taaccaacat aggtcctaac ataagttata taggtcttaa 10560cgtaactaac ataagtcatt aaggtactaa gtttggtctt aatttaacaa taacatgtcg 10620ctggcctaac attagtaatg taggtctgac tttcactcat ataagtctta tggtaactaa 10680actaaggtct tacctttact gatatatgtc ttactttcac taacttaggt attactttta 10740ctaacttagg tcttaaattc agtaactaag gtcatacttc gactaactaa ggtcttacat 10800tcactgatat aggtcttatg attactaact taggtcctaa tttgactaac ataagtccta 10860acattagtaa tgtaggtctt aacttaacta acttaggtct taccttcact aatataggtc 10920ttaatattac tgacttaagt aattaaggta ctaacttagg tcgtaaggta actaatatat 10980aggtcttaag gtaactaatt taggtcttga cttaataaat ataggtccta acataaatag 11040tataggtcct aatataagta ctataggcct taacttaacc aacataggtc ctaacataag 11100ttatataggt cttaacgtaa ctaacataag tcattaaggt actaagtttg gtcttaattt 11160aacaataacc atgtcgctgg ccgggtggtc ttaatttaac aaatatagac catgtcgctg 11220gccgggtgac ccggcgggga cgaggcaagc taaacagatc ctcgtgatac gcctattttt 11280ataggttaat gtcatgataa taatggtttc ttaggacgga tcgcttgcct gtaacttaca 11340cgcgcctcgt atcttttaat gatggaataa tttgggaatt tactctgtgt ttatttattt 11400ttatgttttg tatttggatt ttagaaagta aataaagaag gtagaagagt tacggaatga 11460agaaaaaaaa ataaacaaag gtttaaaaaa tttcaacaaa aagcgtactt tacatatata 11520tttattagac aagaaaagca gattaaatag atatacattc gattaacgat aagtaaaatg 11580taaaatcaca ggattttcgt gtgtggtctt ctacacagac aagatgaaac aattcggcat 11640taatacctga gagcaggaag agcaagataa aaggtagtat ttgttggcga tccccctaga 11700gtcttttaca tcttcggaaa acaaaaacta ttttttcttt aatttctttt tttactttct 11760atttttaatt tatatattta tattaaaaaa tttaaattat aattattttt atagcacgtg 11820atgaaaagga cccaggtggc acttttcggg gaaatctcga cctgcagcgt acgaagct 1187838812044DNAArtificial SequenceDescription of Artificial Sequence Synthetic polynucleotide 388gcctcactga ttaagcattg gtaactgtca gaccaagttt actcatatat actttagatt 60gatttaaaac ttcattttta atttaaaagg atctaggtga agatcctttt tgataatctc 120atgaccaaaa tcccttaacg tgagttttcg ttccactgag cgtcagaccc cgtagaaaag 180atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa 240aaaccaccgc taccagcggt ggtttgtttg ccggatcaag agctaccaac tctttttccg 300aaggtaactg gcttcagcag agcgcagata ccaaatactg ttcttctagt gtagccgtag 360ttaggccacc acttcaagaa ctctgtagca ccgcctacat acctcgctct gctaatcctg 420ttaccagtgg ctgctgccag tggcgataag tcgtgtctta ccgggttgga ctcaagacga 480tagttaccgg ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac acagcccagc 540ttggagcgaa cgacctacac cgaactgaga tacctacagc gtgagctatg agaaagcgcc 600acgcttcccg aagggagaaa ggcggacagg tatccggtaa gcggcagggt cggaacagga 660gagcgcacga gggagcttcc agggggaaac gcctggtatc tttatagtcc tgtcgggttt 720cgccacctct gacttgagcg tcgatttttg tgatgctcgt caggggggcg gagcctatgg 780aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct tttgctggcc ttttgctcac 840atgttctttc ctgcgttatc ccctgattct gtggataacc gtattaccgc ctttgagtga 900gctgataccg ctcgccgcag ccgaacgacc gagcgcagcg agtcagtgag cgaggaagcg 960gaagagcgcc caatacgcaa accgcctctc cccgcgcgtt ggccgattca ttaatgcagc 1020tggcacgaca ggtttcccga ctggaaagcg ggcagtgagc gcaacgcaat taatgtgagt 1080tagctcactc attaggcacc ccaggcttta cactttatgc ttccggctcg tatgttgtgt 1140ggaattgtga gcggataaca atttcacaca ggaaacagct atgaccatga ttacgccaag 1200ctatttaggt gacactatag aatactcaag cttgggggga tcctctagag tcgactaata 1260cgactcacta taggaacata atctatagcg gcgttttaga gctagaaata gcaagttaaa 1320ataaggctag tccgttatca acttgaaaaa gtggcaccga gtcggtgcta gcataacccc 1380ttggggcctc taaacgggtc ttgaggggtt ttttggtcga cctgcaggca tgctatttga 1440tgaattaact acacttaaaa taatacaatt attattaaat ttttttttga tttatttatt 1500aatttttaaa cttaatcatt tgtatttggg aggaattata tatatcttta taattatttt 1560attttttttt atttttttat ttttttatta ttattatttt tttttatttt ttttttttac 1620tgtatcaaag aaaaaccttt aaaaaaaaaa ttataatttc cccatcttac tatattttta 1680atacatacgt tttaaggaat taaattagac aaaagctata ttatgcttta catataatta 1740gaatttataa acgtttggtt attagatatt tcatgtctca gtaaagtctt tcaatacata 1800tgtaaaaaaa tatatatgaa tacacataag ttgttaatat attttatatg cataaatgta 1860taaatatata tatatatata tatatatgta tgtatgtata tgtgtgtata tgaaattatt 1920tcaatgttta attttttaaa ttttaatttt tttttttttt ttttttttta ttatgtatat 1980tgatctttat tatttaaata ttactttttt cgttttttct tctttttatt attttttttt 2040ttttttatat tttatacaaa tggtaattca aataaaaggt ataaatttat atttaatttt 2100cttttatgga taaataaaag aaaaatataa atatataaaa atataaaaat atatatatgt 2160atattggggt gatgataaaa tgaaagataa tatatatata tatatatctt tatttttttt 2220tttttgtaga ccccattgtg agtacataaa tatattatat aactcgggag catcagtcat 2280ggaattctta tttctttttc ttttttgcct ggccggcctt tttcgtggcc gccggccttt 2340tgtcgcctcc cagctgagac aggtcgatcc gtgtctcgta caggccggtg atgctctggt 2400ggatcagggt ggcgtccagc acctctttgg tgctggtgta cctcttccgg tcgatggtgg 2460tgtcaaagta cttgaaggcg gcaggggctc ccagattggt cagggtaaac aggtggatga 2520tattctcggc ctgctctctg atgggcttat cccggtgctt gttgtaggcg gacagcactt 2580tgtccagatt agcgtcggcc aggatcactc tcttggagaa ctcgctgatc tgctcgatga 2640tctcgtccag gtagtgcttg tgctgttcca caaacagctg tttctgctca ttatcctcgg 2700gggagccctt cagcttctca tagtggctgg ccaggtacag gaagttcaca tatttggagg 2760gcagggccag ttcgtttccc ttctgcagtt cgccggcaga ggccagcatt ctcttccggc 2820cgttttccag ctcgaacagg gagtacttag gcagcttgat gatcaggtcc tttttcactt 2880ctttgtagcc cttggcttcc agaaagtcga tgggattctt ctcgaagctg cttctttcca 2940tgatggtgat ccccagcagc tctttcacac tcttcagttt cttggacttg cccttttcca 3000ctttggccac caccagcaca gaataggcca cggtggggct gtcgaagccg ccgtacttct 3060tagggtccca gtccttcttt ctggcgatca gcttatcgct gttcctcttg ggcaggatag 3120actctttgct gaagccgcct gtctgcacct cggtcttttt cacgatattc acttggggca 3180tgctcagcac tttccgcacg gtggcaaaat cccggccctt atcccacacg atctccccgg 3240tttcgccgtt tgtctcgatc agaggccgct tccggatctc gccgttggcc agggtaatct 3300cggtcttgaa aaagttcatg atgttgctgt agaagaagta cttggcggta gccttgccga 3360tttcctgctc gctcttggcg atcatcttcc gcacgtcgta caccttgtag tcgccgtaca 3420cgaactcgct ttccagctta gggtactttt tgatcagggc ggttcccacg acggcgttca 3480ggtaggcgtc gtgggcgtgg tggtagttgt tgatctcgcg cactttgtaa aactggaaat 3540ccttccggaa atcggacacc agcttggact tcagggtgat cactttcact tcccggatca 3600gcttgtcatt ctcgtcgtac ttagtgttca tccgggagtc caggatctgt gccacgtgct 3660ttgtgatctg ccgggtttcc accagctgtc tcttgatgaa gccggcctta tccagttcgc 3720tcaggccgcc tctctcggcc ttggtcagat tgtcgaactt tctctgggta atcagcttgg 3780cgttcagcag ctgccgccag tagttcttca tcttcttcac gacctcttcg gagggcacgt 3840tgtcgctctt gccccggttc ttgtcgcttc tggtcagcac cttgttgtcg atggagtcgt 3900ccttcagaaa gctctgaggc acgatatggt ccacatcgta gtcggacagc cggttgatgt 3960ccagttcctg gtccacgtac atatcccgcc cattctgcag gtagtacagg tacagcttct 4020cgttctgcag ctgggtgttt tccacggggt gttctttcag gatctggctg cccagctctt 4080tgatgccctc ttcgatccgc ttcattctct cgcggctgtt cttctgtccc ttctgggtgg 4140tctggttctc tctggccatt tcgatcacga tgttctcggg cttgtgccgg cccatcactt 4200tcacgagctc gtccaccacc ttcactgtct gcaggatgcc cttcttaatg gcggggctgc 4260cggccagatt ggcaatgtgc tcgtgcaggc tatcgccctg gccggacacc tgggctttct 4320ggatgtcctc tttaaaggtc aggctgtcgt cgtggatcag ctgcatgaag tttctgttgg 4380cgaagccgtc ggacttcagg aaatccagga ttgtcttgcc ggactgcttg tcccggatgc 4440cgttgatcag cttccggctc agcctgcccc agccggtgta tctccgccgc ttcagctgct 4500tcatcacttt gtcgtcgaac aggtgggcat aggttttcag ccgttcctcg atcatctctc 4560tgtcctcaaa cagtgtcagg gtcagcacga tatcttccag aatgtcctcg ttttcctcat 4620tgtccaggaa gtccttgtcc ttgataattt tcagcagatc gtggtatgtg cccagggagg 4680cgttgaaccg atcttccacg ccggagattt ccacggagtc gaagcactcg attttcttga 4740agtagtcctc tttcagctgc ttcacggtca ctttccggtt ggtcttgaac agcaggtcca 4800cgatggcctt tttctgctcg ccgctcagga aggcgggctt tctcattccc tcggtcacgt 4860atttcacttt ggtcagctcg ttatacacgg tgaagtactc gtacagcagg ctgtgcttgg 4920gcagcacctt ctcgttgggc aggttcttat cgaagttggt catccgctcg atgaagctct 4980gggcggaagc gcccttgtcc accacttcct cgaagttcca gggggtgatg gtttcctcgc 5040tctttctggt catccaggcg aatctgctgt ttcccctggc cagagggccc acgtagtagg 5100ggatgcggaa ggtcaggatc ttctcgatct tttcccggtt gtccttcagg aatgggtaaa 5160aatcttcctg ccgccgcaga atggcgtgca gctctcccag gtggatctgg tgggggatgc 5220tgccgttgtc gaaggtccgc tgcttccgca gcaggtcctc tctgttcagc ttcacgagca 5280gttcctcggt gccgtccatc ttttccagga tgggcttgat gaacttgtag aactcttcct 5340ggctggctcc gccgtcaatg tagccggcgt agccgttctt gctctggtcg aagaaaatct 5400ctttgtactt ctcaggcagc tgctgccgca cgagagcttt cagcagggtc aggtcctggt 5460ggtgctcgtc gtatctcttg atcatagagg cgctcagggg ggccttggtg atctcggtgt 5520tcactctcag gatgtcgctc agcaggatgg cgtcggacag gttcttggcg gccagaaaca 5580ggtcggcgta ctggtcgccg atctgggcca gcaggttgtc caggtcgtcg tcgtaggtgt 5640ccttgctcag ctgcagtttg gcatcctcgg ccaggtcgaa gttgctcttg aagttggggg 5700tcaggcccag gctcagggca atcaggtttc cgaacaggcc attcttcttc tcgccgggca 5760gctgggcgat cagattttcc agccgtctgc tcttgctcag tctggcagac aggatggcct 5820tggcgtccac gccgctggcg ttgatggggt tttcctcgaa cagctggttg taggtctgca 5880ccagctggat gaacagcttg tccacgtcgc tgttgtcggg gttcaggtcg ccctcgatca 5940ggaagtggcc ccggaacttg atcatgtggg ccagggccag atagatcagc cgcaggtcgg 6000ccttgtcggt gctgtccacc agtttctttc tcaggtggta gatggtgggg tacttctcgt 6060ggtaggccac ctcgtccacg atgttgccga agatggggtg ccgctcgtgc ttcttatcct 6120cttccaccag gaaggactct tccagtctgt ggaagaagct gtcgtccacc ttggccatct 6180cgttgctgaa gatctcttgc agatagcaga tccggttctt ccgtctggtg tatcttcttc 6240tggcggttct cttcagccgg gtggcctcgg ctgtttcgcc gctgtcgaac agcagggctc 6300cgatcaggtt cttcttgatg ctgtgccggt cggtgttgcc cagcaccttg aatttcttgc 6360tgggcacctt gtactcgtcg gtgatcacgg cccagcccac agagttggtg ccgatgtcca 6420ggccgatgct gtacttcttg tcggctgctg ggactccgtg gataccgacc ttccgcttct 6480tctttggggc catcttatcg tcatcgtctt tgtaatcaat atcatgatcc ttgtagtctc 6540cgtcgtggtc cttatagtcc atttttctcg agggatcctg atatatttct attaggtatt 6600tattattata aaatataaat cttgaatgat aataaataaa atattagtta ttccttttct 6660agtttaaaat atacatatta taaatatata tatatatata tatattttta ttgtgacaag 6720aatatataat tataaattat attatttatt tttgtatttt tttttttttt tttttttttt 6780tctttttttg ttttattttt cttttttttt ataaatatta tttttttctt ttatcatgca 6840cattggaata atacattaat atatatatat atattatatt atacatatat tgaataatgt 6900ttataaaaaa tgcataactt atatgaatat aatttttttt aaatatgaca aaaagaaaaa 6960aaaaaaaaac caaaaaaaat taaaattgaa atgaaatata taaatatatt atttatatat 7020attatacatt gtttaatact actacatgta tatatatata ttatatatat atatatatat 7080caattttttc aaaaataaat taatataaaa agaggggaaa aaaaaaaaaa aaaaaaaaaa 7140aagataatta agtaagcatt taaaaatata taaattgata atatataaaa ttaatcacat 7200ataaaagctt ataaacacta ggttagctaa ttcgcttgta agaggtactc tcgtttatgc 7260aaaactattt gatatagcat tttaacaagt acacatatat atatgtaata tatatactat 7320atatatctat tgcatgtgta ctaagcatgt gcatggcatc ccctttttct cgtgtttaaa 7380acagtttgta tgataaaata taaaggattt gaaaaagaga aaaaaatata tgatctcatc 7440ctatatagcg ccataatttt tatttgggtt gaataaaatt ttctactaaa tttaggtgta 7500agtaaaataa tggaatatat ataagtacaa taaaaaagtg cataaattaa aaaattttta 7560taataaatat tttttttaaa aaagtcaata ataatattaa atatatataa cacaggatta 7620tatatgttca ctacaatttt ttatattata atataaattc ttttcaattt tcattttatt 7680ttacatacac tttccttttt tgtcactata ttttaatatt cacatattta gtttaaatac 7740tggctatttc tttctacatt tgctagtaac aattgtgtag tgcttaaata tatacacaca 7800cctaaaactt acaaagtatc ctaggaccat ggccaagcct ttgtctcaag aagaatccac 7860cctcattgaa agagcaacgg ctacaatcaa cagcatcccc atctctgaag actacagcgt 7920cgccagcgca gctctctcta gcgacggccg catcttcact ggtgtcaatg tatatcattt 7980tactggggga ccttgtgcag aactcgtggt gctgggcact gctgctgctg cggcagctgg 8040caacctgact tgtatcgtcg cgatcggaaa tgagaacagg ggcatcttga gcccctgcgg 8100acggtgccga caggtgcttc tcgatctgca tcctgggatc aaagccatag tgaaggacag 8160tgatggacag ccgacggcag ttgggattcg tgaattgctg ccctctggtt atgtgtggga 8220gggctaaccg cgggtacccc attaaattta tttaataata gattaaaaat attataaaaa 8280taaaaacata aacacagaaa ttacaaaaaa aatacatatg aatttttttt ttgtaatctt 8340ccttataaat atagaataat gaatcatata aaacatatca ttattcattt atttacattt 8400aaaattattg tttcagtatc tttaatttat tatgtatata taaaaataac ttacaatttt 8460attaataaac aatatatgtt tattaattca tgttttgtaa tttatgggat agcgattttt 8520tttactgtct gtattttctt ttttaattat gttttaattg tatttatttt atttttatta 8580ttgttctttt tatagtatta ttttaaaaca aaatgtattt tctaagaact tataataata 8640ataatataaa ttttaataaa aattatattt atcttttaca atatgaacat aaagtacaac 8700attaatatat agcttttaat atttttattc ctaatcatgt aaatcttaaa tttttctttt 8760taaacatatg ttaaatattt atttctcatt atatataaga acatatttat tacatctaga 8820ggtaccgagc tcgttttcga cactggatgg cggcgttagt atcgaatcga cagcagtata 8880gcgaccagca ttcacatacg attgacgcat gatattactt tctgcgcact taacttcgca 8940tctgggcaga tgatgtcgag gcgaaaaaaa atataaatca cgctaacatt tgattaaaat 9000agaacaacta caatataaaa aaactataca aatgacaagt tcttgaaaac aagaatcttt 9060ttattgtcag tactgattag aaaaactcat cgagcatcaa atgaaactgc aatttattca 9120tatcaggatt atcaatacca tatttttgaa aaagccgttt ctgtaatgaa ggagaaaact 9180caccgaggca gttccatagg atggcaagat cctggtatcg gtctgcgatt ccgactcgtc 9240caacatcaat acaacctatt aatttcccct cgtcaaaaat aaggttatca agtgagaaat 9300caccatgagt gacgactgaa tccggtgaga

atggcaaaag cttatgcatt tctttccaga 9360cttgttcaac aggccagcca ttacgctcgt catcaaaatc actcgcatca accaaaccgt 9420tattcattcg tgattgcgcc tgagcgagac gaaatacgcg atcgctgtta aaaggacaat 9480tacaaacagg aatcgaatgc aaccggcgca ggaacactgc cagcgcatca acaatatttt 9540cacctgaatc aggatattct tctaatacct ggaatgctgt tttgccgggg atcgcagtgg 9600tgagtaacca tgcatcatca ggagtacgga taaaatgctt gatggtcgga agaggcataa 9660attccgtcag ccagtttagt ctgaccatct catctgtaac atcattggca acgctacctt 9720tgccatgttt cagaaacaac tctggcgcat cgggcttccc atacaatcga tagattgtcg 9780cacctgattg cccgacatta tcgcgagccc atttataccc atataaatca gcatccatgt 9840tggaatttaa tcgcggcctc gaaacgtgag tcttttcctt acccatggtt gtttatgttc 9900ggatgtgatg tgagaactgt atcctagcaa gattttaaaa ggaagtatat gaaagaagaa 9960cctcagtggc aaatcctaac cttttatatt tctctacagg ggcgcggcgt ggggacaatt 10020caacgcgtct gtgaggggag cgtttccctg ctcgcaggtc tgcagcgagg agccgtaatt 10080tttgcttcgc gccgtgcggc catcaaaatg tatggatgca aatgattata catggggatg 10140tatgggctaa atgtacgggc gacagtcaca tcatgcccct gagctgcgca cgtcaagact 10200gtcaaggagg gtattctggg cctccatgtc gctggcctaa cattagtaat gtaggtctga 10260ctttcactca tataagtctt atggtaacta aactaaggtc ttacctttac tgatatatgt 10320cttactttca ctaacttagg tattactttt actaacttag gtcttaaatt cagtaactaa 10380ggtcatactt cgactaacta aggtcttaca ttcactgata taggtcttat gattactaac 10440ttaggtccta atttgactaa cataagtcct aacattagta atgtaggtct taacttaact 10500aacttaggtc ttaccttcac taatataggt cttaatatta ctgacttaag taattaaggt 10560actaacttag gtcgtaaggt aactaatata taggtcttaa ggtaactaat ttaggtcttg 10620acttaataaa tataggtcct aacataaata gtataggtcc taatataagt actataggcc 10680ttaacttaac caacataggt cctaacataa gttatatagg tcttaacgta actaacataa 10740gtcattaagg tactaagttt ggtcttaatt taacaataac atgtcgctgg cctaacatta 10800gtaatgtagg tctgactttc actcatataa gtcttatggt aactaaacta aggtcttacc 10860tttactgata tatgtcttac tttcactaac ttaggtatta cttttactaa cttaggtctt 10920aaattcagta actaaggtca tacttcgact aactaaggtc ttacattcac tgatataggt 10980cttatgatta ctaacttagg tcctaatttg actaacataa gtcctaacat tagtaatgta 11040ggtcttaact taactaactt aggtcttacc ttcactaata taggtcttaa tattactgac 11100ttaagtaatt aaggtactaa cttaggtcgt aaggtaacta atatataggt cttaaggtaa 11160ctaatttagg tcttgactta ataaatatag gtcctaacat aaatagtata ggtcctaata 11220taagtactat aggccttaac ttaaccaaca taggtcctaa cataagttat ataggtctta 11280acgtaactaa cataagtcat taaggtacta agtttggtct taatttaaca ataaccatgt 11340cgctggccgg gtggtcttaa tttaacaaat atagaccatg tcgctggccg ggtgacccgg 11400cggggacgag gcaagctaaa cagatcctcg tgatacgcct atttttatag gttaatgtca 11460tgataataat ggtttcttag gacggatcgc ttgcctgtaa cttacacgcg cctcgtatct 11520tttaatgatg gaataatttg ggaatttact ctgtgtttat ttatttttat gttttgtatt 11580tggattttag aaagtaaata aagaaggtag aagagttacg gaatgaagaa aaaaaaataa 11640acaaaggttt aaaaaatttc aacaaaaagc gtactttaca tatatattta ttagacaaga 11700aaagcagatt aaatagatat acattcgatt aacgataagt aaaatgtaaa atcacaggat 11760tttcgtgtgt ggtcttctac acagacaaga tgaaacaatt cggcattaat acctgagagc 11820aggaagagca agataaaagg tagtatttgt tggcgatccc cctagagtct tttacatctt 11880cggaaaacaa aaactatttt ttctttaatt tcttttttta ctttctattt ttaatttata 11940tatttatatt aaaaaattta aattataatt atttttatag cacgtgatga aaaggaccca 12000ggtggcactt ttcggggaaa tctcgacctg cagcgtacga agct 1204438912044DNAArtificial SequenceDescription of Artificial Sequence Synthetic polynucleotide 389gcctcactga ttaagcattg gtaactgtca gaccaagttt actcatatat actttagatt 60gatttaaaac ttcattttta atttaaaagg atctaggtga agatcctttt tgataatctc 120atgaccaaaa tcccttaacg tgagttttcg ttccactgag cgtcagaccc cgtagaaaag 180atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa 240aaaccaccgc taccagcggt ggtttgtttg ccggatcaag agctaccaac tctttttccg 300aaggtaactg gcttcagcag agcgcagata ccaaatactg ttcttctagt gtagccgtag 360ttaggccacc acttcaagaa ctctgtagca ccgcctacat acctcgctct gctaatcctg 420ttaccagtgg ctgctgccag tggcgataag tcgtgtctta ccgggttgga ctcaagacga 480tagttaccgg ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac acagcccagc 540ttggagcgaa cgacctacac cgaactgaga tacctacagc gtgagctatg agaaagcgcc 600acgcttcccg aagggagaaa ggcggacagg tatccggtaa gcggcagggt cggaacagga 660gagcgcacga gggagcttcc agggggaaac gcctggtatc tttatagtcc tgtcgggttt 720cgccacctct gacttgagcg tcgatttttg tgatgctcgt caggggggcg gagcctatgg 780aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct tttgctggcc ttttgctcac 840atgttctttc ctgcgttatc ccctgattct gtggataacc gtattaccgc ctttgagtga 900gctgataccg ctcgccgcag ccgaacgacc gagcgcagcg agtcagtgag cgaggaagcg 960gaagagcgcc caatacgcaa accgcctctc cccgcgcgtt ggccgattca ttaatgcagc 1020tggcacgaca ggtttcccga ctggaaagcg ggcagtgagc gcaacgcaat taatgtgagt 1080tagctcactc attaggcacc ccaggcttta cactttatgc ttccggctcg tatgttgtgt 1140ggaattgtga gcggataaca atttcacaca ggaaacagct atgaccatga ttacgccaag 1200ctatttaggt gacactatag aatactcaag cttgggggga tcctctagag tcgactaata 1260cgactcacta taggaaatga tatggatttt gggttttaga gctagaaata gcaagttaaa 1320ataaggctag tccgttatca acttgaaaaa gtggcaccga gtcggtgcta gcataacccc 1380ttggggcctc taaacgggtc ttgaggggtt ttttggtcga cctgcaggca tgctatttga 1440tgaattaact acacttaaaa taatacaatt attattaaat ttttttttga tttatttatt 1500aatttttaaa cttaatcatt tgtatttggg aggaattata tatatcttta taattatttt 1560attttttttt atttttttat ttttttatta ttattatttt tttttatttt ttttttttac 1620tgtatcaaag aaaaaccttt aaaaaaaaaa ttataatttc cccatcttac tatattttta 1680atacatacgt tttaaggaat taaattagac aaaagctata ttatgcttta catataatta 1740gaatttataa acgtttggtt attagatatt tcatgtctca gtaaagtctt tcaatacata 1800tgtaaaaaaa tatatatgaa tacacataag ttgttaatat attttatatg cataaatgta 1860taaatatata tatatatata tatatatgta tgtatgtata tgtgtgtata tgaaattatt 1920tcaatgttta attttttaaa ttttaatttt tttttttttt ttttttttta ttatgtatat 1980tgatctttat tatttaaata ttactttttt cgttttttct tctttttatt attttttttt 2040ttttttatat tttatacaaa tggtaattca aataaaaggt ataaatttat atttaatttt 2100cttttatgga taaataaaag aaaaatataa atatataaaa atataaaaat atatatatgt 2160atattggggt gatgataaaa tgaaagataa tatatatata tatatatctt tatttttttt 2220tttttgtaga ccccattgtg agtacataaa tatattatat aactcgggag catcagtcat 2280ggaattctta tttctttttc ttttttgcct ggccggcctt tttcgtggcc gccggccttt 2340tgtcgcctcc cagctgagac aggtcgatcc gtgtctcgta caggccggtg atgctctggt 2400ggatcagggt ggcgtccagc acctctttgg tgctggtgta cctcttccgg tcgatggtgg 2460tgtcaaagta cttgaaggcg gcaggggctc ccagattggt cagggtaaac aggtggatga 2520tattctcggc ctgctctctg atgggcttat cccggtgctt gttgtaggcg gacagcactt 2580tgtccagatt agcgtcggcc aggatcactc tcttggagaa ctcgctgatc tgctcgatga 2640tctcgtccag gtagtgcttg tgctgttcca caaacagctg tttctgctca ttatcctcgg 2700gggagccctt cagcttctca tagtggctgg ccaggtacag gaagttcaca tatttggagg 2760gcagggccag ttcgtttccc ttctgcagtt cgccggcaga ggccagcatt ctcttccggc 2820cgttttccag ctcgaacagg gagtacttag gcagcttgat gatcaggtcc tttttcactt 2880ctttgtagcc cttggcttcc agaaagtcga tgggattctt ctcgaagctg cttctttcca 2940tgatggtgat ccccagcagc tctttcacac tcttcagttt cttggacttg cccttttcca 3000ctttggccac caccagcaca gaataggcca cggtggggct gtcgaagccg ccgtacttct 3060tagggtccca gtccttcttt ctggcgatca gcttatcgct gttcctcttg ggcaggatag 3120actctttgct gaagccgcct gtctgcacct cggtcttttt cacgatattc acttggggca 3180tgctcagcac tttccgcacg gtggcaaaat cccggccctt atcccacacg atctccccgg 3240tttcgccgtt tgtctcgatc agaggccgct tccggatctc gccgttggcc agggtaatct 3300cggtcttgaa aaagttcatg atgttgctgt agaagaagta cttggcggta gccttgccga 3360tttcctgctc gctcttggcg atcatcttcc gcacgtcgta caccttgtag tcgccgtaca 3420cgaactcgct ttccagctta gggtactttt tgatcagggc ggttcccacg acggcgttca 3480ggtaggcgtc gtgggcgtgg tggtagttgt tgatctcgcg cactttgtaa aactggaaat 3540ccttccggaa atcggacacc agcttggact tcagggtgat cactttcact tcccggatca 3600gcttgtcatt ctcgtcgtac ttagtgttca tccgggagtc caggatctgt gccacgtgct 3660ttgtgatctg ccgggtttcc accagctgtc tcttgatgaa gccggcctta tccagttcgc 3720tcaggccgcc tctctcggcc ttggtcagat tgtcgaactt tctctgggta atcagcttgg 3780cgttcagcag ctgccgccag tagttcttca tcttcttcac gacctcttcg gagggcacgt 3840tgtcgctctt gccccggttc ttgtcgcttc tggtcagcac cttgttgtcg atggagtcgt 3900ccttcagaaa gctctgaggc acgatatggt ccacatcgta gtcggacagc cggttgatgt 3960ccagttcctg gtccacgtac atatcccgcc cattctgcag gtagtacagg tacagcttct 4020cgttctgcag ctgggtgttt tccacggggt gttctttcag gatctggctg cccagctctt 4080tgatgccctc ttcgatccgc ttcattctct cgcggctgtt cttctgtccc ttctgggtgg 4140tctggttctc tctggccatt tcgatcacga tgttctcggg cttgtgccgg cccatcactt 4200tcacgagctc gtccaccacc ttcactgtct gcaggatgcc cttcttaatg gcggggctgc 4260cggccagatt ggcaatgtgc tcgtgcaggc tatcgccctg gccggacacc tgggctttct 4320ggatgtcctc tttaaaggtc aggctgtcgt cgtggatcag ctgcatgaag tttctgttgg 4380cgaagccgtc ggacttcagg aaatccagga ttgtcttgcc ggactgcttg tcccggatgc 4440cgttgatcag cttccggctc agcctgcccc agccggtgta tctccgccgc ttcagctgct 4500tcatcacttt gtcgtcgaac aggtgggcat aggttttcag ccgttcctcg atcatctctc 4560tgtcctcaaa cagtgtcagg gtcagcacga tatcttccag aatgtcctcg ttttcctcat 4620tgtccaggaa gtccttgtcc ttgataattt tcagcagatc gtggtatgtg cccagggagg 4680cgttgaaccg atcttccacg ccggagattt ccacggagtc gaagcactcg attttcttga 4740agtagtcctc tttcagctgc ttcacggtca ctttccggtt ggtcttgaac agcaggtcca 4800cgatggcctt tttctgctcg ccgctcagga aggcgggctt tctcattccc tcggtcacgt 4860atttcacttt ggtcagctcg ttatacacgg tgaagtactc gtacagcagg ctgtgcttgg 4920gcagcacctt ctcgttgggc aggttcttat cgaagttggt catccgctcg atgaagctct 4980gggcggaagc gcccttgtcc accacttcct cgaagttcca gggggtgatg gtttcctcgc 5040tctttctggt catccaggcg aatctgctgt ttcccctggc cagagggccc acgtagtagg 5100ggatgcggaa ggtcaggatc ttctcgatct tttcccggtt gtccttcagg aatgggtaaa 5160aatcttcctg ccgccgcaga atggcgtgca gctctcccag gtggatctgg tgggggatgc 5220tgccgttgtc gaaggtccgc tgcttccgca gcaggtcctc tctgttcagc ttcacgagca 5280gttcctcggt gccgtccatc ttttccagga tgggcttgat gaacttgtag aactcttcct 5340ggctggctcc gccgtcaatg tagccggcgt agccgttctt gctctggtcg aagaaaatct 5400ctttgtactt ctcaggcagc tgctgccgca cgagagcttt cagcagggtc aggtcctggt 5460ggtgctcgtc gtatctcttg atcatagagg cgctcagggg ggccttggtg atctcggtgt 5520tcactctcag gatgtcgctc agcaggatgg cgtcggacag gttcttggcg gccagaaaca 5580ggtcggcgta ctggtcgccg atctgggcca gcaggttgtc caggtcgtcg tcgtaggtgt 5640ccttgctcag ctgcagtttg gcatcctcgg ccaggtcgaa gttgctcttg aagttggggg 5700tcaggcccag gctcagggca atcaggtttc cgaacaggcc attcttcttc tcgccgggca 5760gctgggcgat cagattttcc agccgtctgc tcttgctcag tctggcagac aggatggcct 5820tggcgtccac gccgctggcg ttgatggggt tttcctcgaa cagctggttg taggtctgca 5880ccagctggat gaacagcttg tccacgtcgc tgttgtcggg gttcaggtcg ccctcgatca 5940ggaagtggcc ccggaacttg atcatgtggg ccagggccag atagatcagc cgcaggtcgg 6000ccttgtcggt gctgtccacc agtttctttc tcaggtggta gatggtgggg tacttctcgt 6060ggtaggccac ctcgtccacg atgttgccga agatggggtg ccgctcgtgc ttcttatcct 6120cttccaccag gaaggactct tccagtctgt ggaagaagct gtcgtccacc ttggccatct 6180cgttgctgaa gatctcttgc agatagcaga tccggttctt ccgtctggtg tatcttcttc 6240tggcggttct cttcagccgg gtggcctcgg ctgtttcgcc gctgtcgaac agcagggctc 6300cgatcaggtt cttcttgatg ctgtgccggt cggtgttgcc cagcaccttg aatttcttgc 6360tgggcacctt gtactcgtcg gtgatcacgg cccagcccac agagttggtg ccgatgtcca 6420ggccgatgct gtacttcttg tcggctgctg ggactccgtg gataccgacc ttccgcttct 6480tctttggggc catcttatcg tcatcgtctt tgtaatcaat atcatgatcc ttgtagtctc 6540cgtcgtggtc cttatagtcc atttttctcg agggatcctg atatatttct attaggtatt 6600tattattata aaatataaat cttgaatgat aataaataaa atattagtta ttccttttct 6660agtttaaaat atacatatta taaatatata tatatatata tatattttta ttgtgacaag 6720aatatataat tataaattat attatttatt tttgtatttt tttttttttt tttttttttt 6780tctttttttg ttttattttt cttttttttt ataaatatta tttttttctt ttatcatgca 6840cattggaata atacattaat atatatatat atattatatt atacatatat tgaataatgt 6900ttataaaaaa tgcataactt atatgaatat aatttttttt aaatatgaca aaaagaaaaa 6960aaaaaaaaac caaaaaaaat taaaattgaa atgaaatata taaatatatt atttatatat 7020attatacatt gtttaatact actacatgta tatatatata ttatatatat atatatatat 7080caattttttc aaaaataaat taatataaaa agaggggaaa aaaaaaaaaa aaaaaaaaaa 7140aagataatta agtaagcatt taaaaatata taaattgata atatataaaa ttaatcacat 7200ataaaagctt ataaacacta ggttagctaa ttcgcttgta agaggtactc tcgtttatgc 7260aaaactattt gatatagcat tttaacaagt acacatatat atatgtaata tatatactat 7320atatatctat tgcatgtgta ctaagcatgt gcatggcatc ccctttttct cgtgtttaaa 7380acagtttgta tgataaaata taaaggattt gaaaaagaga aaaaaatata tgatctcatc 7440ctatatagcg ccataatttt tatttgggtt gaataaaatt ttctactaaa tttaggtgta 7500agtaaaataa tggaatatat ataagtacaa taaaaaagtg cataaattaa aaaattttta 7560taataaatat tttttttaaa aaagtcaata ataatattaa atatatataa cacaggatta 7620tatatgttca ctacaatttt ttatattata atataaattc ttttcaattt tcattttatt 7680ttacatacac tttccttttt tgtcactata ttttaatatt cacatattta gtttaaatac 7740tggctatttc tttctacatt tgctagtaac aattgtgtag tgcttaaata tatacacaca 7800cctaaaactt acaaagtatc ctaggaccat ggccaagcct ttgtctcaag aagaatccac 7860cctcattgaa agagcaacgg ctacaatcaa cagcatcccc atctctgaag actacagcgt 7920cgccagcgca gctctctcta gcgacggccg catcttcact ggtgtcaatg tatatcattt 7980tactggggga ccttgtgcag aactcgtggt gctgggcact gctgctgctg cggcagctgg 8040caacctgact tgtatcgtcg cgatcggaaa tgagaacagg ggcatcttga gcccctgcgg 8100acggtgccga caggtgcttc tcgatctgca tcctgggatc aaagccatag tgaaggacag 8160tgatggacag ccgacggcag ttgggattcg tgaattgctg ccctctggtt atgtgtggga 8220gggctaaccg cgggtacccc attaaattta tttaataata gattaaaaat attataaaaa 8280taaaaacata aacacagaaa ttacaaaaaa aatacatatg aatttttttt ttgtaatctt 8340ccttataaat atagaataat gaatcatata aaacatatca ttattcattt atttacattt 8400aaaattattg tttcagtatc tttaatttat tatgtatata taaaaataac ttacaatttt 8460attaataaac aatatatgtt tattaattca tgttttgtaa tttatgggat agcgattttt 8520tttactgtct gtattttctt ttttaattat gttttaattg tatttatttt atttttatta 8580ttgttctttt tatagtatta ttttaaaaca aaatgtattt tctaagaact tataataata 8640ataatataaa ttttaataaa aattatattt atcttttaca atatgaacat aaagtacaac 8700attaatatat agcttttaat atttttattc ctaatcatgt aaatcttaaa tttttctttt 8760taaacatatg ttaaatattt atttctcatt atatataaga acatatttat tacatctaga 8820ggtaccgagc tcgttttcga cactggatgg cggcgttagt atcgaatcga cagcagtata 8880gcgaccagca ttcacatacg attgacgcat gatattactt tctgcgcact taacttcgca 8940tctgggcaga tgatgtcgag gcgaaaaaaa atataaatca cgctaacatt tgattaaaat 9000agaacaacta caatataaaa aaactataca aatgacaagt tcttgaaaac aagaatcttt 9060ttattgtcag tactgattag aaaaactcat cgagcatcaa atgaaactgc aatttattca 9120tatcaggatt atcaatacca tatttttgaa aaagccgttt ctgtaatgaa ggagaaaact 9180caccgaggca gttccatagg atggcaagat cctggtatcg gtctgcgatt ccgactcgtc 9240caacatcaat acaacctatt aatttcccct cgtcaaaaat aaggttatca agtgagaaat 9300caccatgagt gacgactgaa tccggtgaga atggcaaaag cttatgcatt tctttccaga 9360cttgttcaac aggccagcca ttacgctcgt catcaaaatc actcgcatca accaaaccgt 9420tattcattcg tgattgcgcc tgagcgagac gaaatacgcg atcgctgtta aaaggacaat 9480tacaaacagg aatcgaatgc aaccggcgca ggaacactgc cagcgcatca acaatatttt 9540cacctgaatc aggatattct tctaatacct ggaatgctgt tttgccgggg atcgcagtgg 9600tgagtaacca tgcatcatca ggagtacgga taaaatgctt gatggtcgga agaggcataa 9660attccgtcag ccagtttagt ctgaccatct catctgtaac atcattggca acgctacctt 9720tgccatgttt cagaaacaac tctggcgcat cgggcttccc atacaatcga tagattgtcg 9780cacctgattg cccgacatta tcgcgagccc atttataccc atataaatca gcatccatgt 9840tggaatttaa tcgcggcctc gaaacgtgag tcttttcctt acccatggtt gtttatgttc 9900ggatgtgatg tgagaactgt atcctagcaa gattttaaaa ggaagtatat gaaagaagaa 9960cctcagtggc aaatcctaac cttttatatt tctctacagg ggcgcggcgt ggggacaatt 10020caacgcgtct gtgaggggag cgtttccctg ctcgcaggtc tgcagcgagg agccgtaatt 10080tttgcttcgc gccgtgcggc catcaaaatg tatggatgca aatgattata catggggatg 10140tatgggctaa atgtacgggc gacagtcaca tcatgcccct gagctgcgca cgtcaagact 10200gtcaaggagg gtattctggg cctccatgtc gctggcctaa cattagtaat gtaggtctga 10260ctttcactca tataagtctt atggtaacta aactaaggtc ttacctttac tgatatatgt 10320cttactttca ctaacttagg tattactttt actaacttag gtcttaaatt cagtaactaa 10380ggtcatactt cgactaacta aggtcttaca ttcactgata taggtcttat gattactaac 10440ttaggtccta atttgactaa cataagtcct aacattagta atgtaggtct taacttaact 10500aacttaggtc ttaccttcac taatataggt cttaatatta ctgacttaag taattaaggt 10560actaacttag gtcgtaaggt aactaatata taggtcttaa ggtaactaat ttaggtcttg 10620acttaataaa tataggtcct aacataaata gtataggtcc taatataagt actataggcc 10680ttaacttaac caacataggt cctaacataa gttatatagg tcttaacgta actaacataa 10740gtcattaagg tactaagttt ggtcttaatt taacaataac atgtcgctgg cctaacatta 10800gtaatgtagg tctgactttc actcatataa gtcttatggt aactaaacta aggtcttacc 10860tttactgata tatgtcttac tttcactaac ttaggtatta cttttactaa cttaggtctt 10920aaattcagta actaaggtca tacttcgact aactaaggtc ttacattcac tgatataggt 10980cttatgatta ctaacttagg tcctaatttg actaacataa gtcctaacat tagtaatgta 11040ggtcttaact taactaactt aggtcttacc ttcactaata taggtcttaa tattactgac 11100ttaagtaatt aaggtactaa cttaggtcgt aaggtaacta atatataggt cttaaggtaa 11160ctaatttagg tcttgactta ataaatatag gtcctaacat aaatagtata ggtcctaata 11220taagtactat aggccttaac ttaaccaaca taggtcctaa cataagttat ataggtctta 11280acgtaactaa cataagtcat taaggtacta agtttggtct taatttaaca ataaccatgt 11340cgctggccgg gtggtcttaa tttaacaaat atagaccatg tcgctggccg ggtgacccgg 11400cggggacgag gcaagctaaa cagatcctcg tgatacgcct atttttatag gttaatgtca 11460tgataataat ggtttcttag gacggatcgc ttgcctgtaa cttacacgcg cctcgtatct 11520tttaatgatg gaataatttg ggaatttact ctgtgtttat ttatttttat gttttgtatt 11580tggattttag aaagtaaata aagaaggtag aagagttacg gaatgaagaa aaaaaaataa 11640acaaaggttt aaaaaatttc aacaaaaagc gtactttaca tatatattta ttagacaaga 11700aaagcagatt aaatagatat acattcgatt aacgataagt aaaatgtaaa atcacaggat 11760tttcgtgtgt ggtcttctac acagacaaga tgaaacaatt cggcattaat acctgagagc 11820aggaagagca agataaaagg tagtatttgt tggcgatccc cctagagtct tttacatctt 11880cggaaaacaa aaactatttt ttctttaatt tcttttttta ctttctattt ttaatttata 11940tatttatatt aaaaaattta aattataatt atttttatag cacgtgatga aaaggaccca 12000ggtggcactt ttcggggaaa tctcgacctg cagcgtacga agct 12044

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