U.S. patent application number 15/904730 was filed with the patent office on 2018-08-16 for methods and compositions for rnra-guided treatment of hiv infection.
This patent application is currently assigned to Temple University of the Commonwealth System of Higher Education. The applicant listed for this patent is Temple University of the Commonwealth System of Higher Education. Invention is credited to Wenhui HU, Kamel KHALILI.
Application Number | 20180228874 15/904730 |
Document ID | / |
Family ID | 52587370 |
Filed Date | 2018-08-16 |
United States Patent
Application |
20180228874 |
Kind Code |
A1 |
KHALILI; Kamel ; et
al. |
August 16, 2018 |
METHODS AND COMPOSITIONS FOR RNRA-GUIDED TREATMENT OF HIV
INFECTION
Abstract
A pharmaceutical composition for use in inactivating an HIV-1
proviral DNA integrated into the genome of a host cell latently
infected with a retrovirus including 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 HIV-1 proviral DNA, whereby treating the host cell
with the composition cleaves a double strand of the HIV-1 proviral
DNA at a first target protospacer sequence with the
CRISPR-associated endonuclease and cleaves a double strand of the
HIV-1 proviral DNA at a second target protospacer sequence with the
CRISPR-associated endonuclease and thereby excises an entire HIV-1
proviral genome and eradicates the HIV-1 proviral DNA from the host
cell, and a pharmaceutically acceptable carrier.
Inventors: |
KHALILI; Kamel; (Bala
Cynwyd, 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: |
52587370 |
Appl. No.: |
15/904730 |
Filed: |
February 26, 2018 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15148261 |
May 6, 2016 |
9981020 |
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15904730 |
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14838057 |
Dec 11, 2015 |
9925248 |
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PCT/US14/53441 |
Aug 29, 2014 |
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15148261 |
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61871626 |
Aug 29, 2013 |
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62018441 |
Jun 27, 2014 |
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62026103 |
Jul 18, 2014 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12N 9/22 20130101; C12N
2310/20 20170501; A61P 31/12 20180101; A61K 35/12 20130101; A61K
45/06 20130101; A61K 9/0034 20130101; A61K 38/465 20130101; C12N
7/00 20130101; C12N 15/111 20130101; C12Y 301/21 20130101; A61K
48/00 20130101; C12N 2740/16063 20130101; A61P 31/18 20180101; A61K
48/005 20130101; A61P 31/00 20180101; C12N 2320/30 20130101 |
International
Class: |
A61K 38/46 20060101
A61K038/46; A61K 9/00 20060101 A61K009/00; C12N 9/22 20060101
C12N009/22; C12N 7/00 20060101 C12N007/00; C12N 15/11 20060101
C12N015/11; A61K 45/06 20060101 A61K045/06; A61K 48/00 20060101
A61K048/00; A61K 35/12 20060101 A61K035/12 |
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 pharmaceutical composition for use in inactivating an HIV-1
proviral DNA integrated into the genome of a host cell latently
infected with a retrovirus, 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 HIV-1 proviral DNA, whereby treating the host cell
with the composition cleaves a double strand of the HIV-1 proviral
DNA at a first target protospacer sequence with the
CRISPR-associated endonuclease and cleaves a double strand of the
HIV-1 proviral DNA at a second target protospacer sequence with the
CRISPR-associated endonuclease and thereby excises an entire HIV-1
proviral genome and eradicates the HIV-1 proviral DNA from the host
cell; and a pharmaceutically acceptable carrier.
2. The pharmaceutical composition of claim 1, wherein the
pharmaceutically acceptable carrier comprises a lipid-based or
polymer-based colloid.
3. The pharmaceutical composition of claim 1, wherein said colloid
is chosen from the group consisting of a liposome, a hydrogel, a
microparticle, a nanoparticle, or a block copolymer micelle.
4. The pharmaceutical composition of claim 1, wherein said
composition is formulated for topical application.
5. The pharmaceutical composition of claim 4, wherein said
composition is contained within a condom.
6. The pharmaceutical composition of claim 1, wherein said
CRISPR-associated endonuclease is Cas9.
7. The pharmaceutical composition of claim 1, wherein said
CRISPR-associated endonuclease sequence is optimized for expression
in a human cell.
8. The pharmaceutical composition of claim 1, wherein at least one
of said first target protospacer sequence and said second target
protospacer sequence is situated within the U3 region of said
LTR.
9. The pharmaceutical composition of claim 1, wherein said first
spacer sequence and said 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.
10. The pharmaceutical composition of claim 1, wherein said first
spacer sequence and said second spacer sequence include,
respectively, a sequence complementary to target protospacer
sequences SEQ ID NO: 96 and SEQ ID NO: 121.
11. The pharmaceutical composition of claim 1, wherein said first
spacer sequence and said second spacer sequence each include,
respectively, a sequence complementary to target protospacer
sequences SEQ ID NO: 87 and SEQ ID NO: 110.
12. The pharmaceutical composition of claim 1, wherein said
composition is encoded in at least one expression vector.
13. The pharmaceutical composition of claim 12, wherein said 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.
14. The pharmaceutical composition of claim 1, wherein at least one
of said gRNAs comprises a CRISPR RNA (crRNA) and a trans-activated
small RNA (tracrRNA), which are expressed as separate nucleic
acids.
15. The pharmaceutical composition of claim 1, wherein at least one
of said gRNAs is engineered as an artificial fusion small guide RNA
(sgRNA) comprised of a crRNA and a tracrRNA.
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 pharmaceutical
composition for use in inactivating an HIV-1 proviral DNA
integrated into the genome of a host cell latently infected with a
retrovirus including 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 HIV-1 proviral DNA,
whereby treating the host cell with the composition cleaves a
double strand of the HIV-1 proviral DNA at a first target
protospacer sequence with the CRISPR-associated endonuclease and
cleaves a double strand of the HIV-1 proviral DNA at a second
target protospacer sequence with the CRISPR-associated endonuclease
and thereby excises an entire HIV-1 proviral genome and eradicates
the HIV-1 proviral DNA from the host cell, and a pharmaceutically
acceptable carrier.
[0005] 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
[0006] FIG. 1A, FIG. 1B, FIG. 1C, FIG. 1D, FIG. 1E, FIG. 1F, FIG.
1G, and 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).
[0007] 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. 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.
[0008] 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).
[0009] 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.
[0010] 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.
[0011] 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).
[0012] 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.
[0013] 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.
[0014] 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.
[0015] 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 TNFa-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.
[0016] 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.
[0017] 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.
[0018] 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/.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.
[0019] 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.
[0020] 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.
[0021] 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 TZMBl 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. TZMBl 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.
[0022] 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.
[0023] 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).
[0024] 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
[0025] 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
3T-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.
[0026] 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.
[0027] 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).
[0028] 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.
[0029] 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.
[0030] 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.
[0031] Compositions
[0032] 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.
[0033] 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).
[0034] 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.
[0035] 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.
[0036] 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).
[0037] 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).
[0038] 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.
[0039] 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.
[0040] 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.
[0041] 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.
[0042] 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.
[0043] 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
[0044] 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.
[0045] 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.
[0046] We may also be refer to the guide RNA sequence as a spacer,
e.g., spacer (A), spacer (B), spacer (C), and spacer(D).
[0047] 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.
[0048] 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.
[0049] 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.
[0050] 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.
[0051] 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.
[0052] 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.
[0053] 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 DC). 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-carboxymethylaminomethyl-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.
[0054] 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.
[0055] 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.
[0056] 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.
[0057] 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).
[0058] 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.
[0059] 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.
[0060] 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.
[0061] 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).
[0062] 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.
[0063] 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.
[0064] 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.
[0065] 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.).
[0066] 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.
[0067] 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.
[0068] 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.
[0069] 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.
[0070] 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.
[0071] 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.
[0072] 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).
[0073] 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.
[0074] 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)].
[0075] 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.
[0076] 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).
[0077] 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).
[0078] 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.
[0079] Pharmaceutical Compositions
[0080] 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.
[0081] 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.
[0082] 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.
[0083] 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.
[0084] 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).
[0085] 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.
[0086] 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.
[0087] 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.
[0088] Methods of Treatment
[0089] 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.
[0090] 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.
[0091] 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.
[0092] 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.
[0093] 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.
[0094] 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.
[0095] 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.
[0096] 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.
[0097] 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.
[0098] 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.
[0099] 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.
[0100] 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.
[0101] 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.
[0102] 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.
[0103] 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.
[0104] 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.
[0105] 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.
[0106] 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.
[0107] 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.
[0108] 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.
[0109] 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.
[0110] 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.
[0111] 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.
[0112] 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.
[0113] 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.
[0114] Articles of Manufacture
[0115] 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.
[0116] 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.
[0117] 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
[0118] Plasmid preparation: Vectors containing human Cas9 and gRNA
expression cassette, pX260, and pX330 (Addgene) were utilized to
create various constructs, LTR-A, B, C, and D.
[0119] Cell culture and stable cell lines: TZM-b1 reporter and U1
cell lines were obtained from the NIH AIDS Reagent Program and
CHME5 microglial cells are known in the art.
[0120] Immunohistochemistry and Western Blot: Standard methods for
immunocytochemical observation of the cells and evaluation of
protein expression by Western blot were utilized.
[0121] Firefly-luciferase assay: 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,
lnvitrogen)
[0122] p24 ELISA: 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).
[0123] EGFP Flow cytometry: 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).
[0124] HIV-1 reporter virus preparation and infections: 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.
[0125] Genomic DNA amplification, PCR, TA-cloning, and Sanger
sequencing, GenomeWalker link PCR: 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.
[0126] Surveyor assay: 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.
[0127] 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.TM. 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.
[0128] Selection of LTR target sites, whole genome sequencing and
bioinformatics and statistical analysis. We utilized Jack Lin's
CRISPR/Cas9 gRNA finder tool for initial identification of
potential target sites within the LTR.
[0129] Plasmid preparation. 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.
[0130] Cell culture. 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.
[0131] Stable cell lines and subcloning. 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.
[0132] Immunocytochemistry and western blot. 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 DMI6000B fluorescence
microscope.
[0133] 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).
[0134] Firefly-luciferase assay. 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).
[0135] p24 ELISA 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).
[0136] EGFP Flow cytometry. 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).
[0137] Hiv-1 reporter virus preparation and infections. 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.
[0138] Genomic DNA purification, PCR, TA-cloning and Sanger
sequencing. Genomic DNA was isolated from cells using an
ArchivePure DNA cell/tissue purification kit (5PRIME) 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.
[0139] Conventional and real-time reverse transcription (RT)-PCR.
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.
[0140] GenomeWalker link PCR and long-range PCR. 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.
[0141] Surveyor assay. 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 .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 products.
[0142] 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.
[0143] Selection of LTR target sites and prediction of potential
off-target sites. 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.
[0144] Whole genome sequencing and bioinformatics analysis. 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.
[0145] Statistical analysis. 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
[0146] 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).
[0147] 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
[0148] 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 3T-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
[0149] 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. (FIGS. 11A, B). As a
result, no expression of full-length Cas9 was detected (FIGS. 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-1was 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
[0150] 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).
[0151] To assess risk of off-target effects comprehensively, we
performed whole genome sequencing (WGS) using the stable Cas9/gRNA
AM-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 (FIGS. 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.
[0152] 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.
[0153] 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.
[0154] 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.
[0155] 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.
[0156] 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
* * * * *
References