U.S. patent application number 15/550452 was filed with the patent office on 2018-06-28 for compositions and methods for transient delivery of nucleases.
This patent application is currently assigned to University of Massachusetts. The applicant listed for this patent is University of Massachusetts. Invention is credited to Guangping Gao, Dan Wang, Phillip D. Zamore.
Application Number | 20180179501 15/550452 |
Document ID | / |
Family ID | 56614963 |
Filed Date | 2018-06-28 |
United States Patent
Application |
20180179501 |
Kind Code |
A9 |
Gao; Guangping ; et
al. |
June 28, 2018 |
COMPOSITIONS AND METHODS FOR TRANSIENT DELIVERY OF NUCLEASES
Abstract
The disclosure in some aspects relates to recombinant
adeno-associated viruses having nuclease grafted to one or more
capsid proteins. In some aspects, the disclosure relates to
isolated AAV capsid proteins having terminally grafted nucleases
and isolated nucleic acids encoding the same. Recent approaches to
delivering nucleases to cells for gene editing have focused on
delivering of expression vectors engineered to express the
nucleases in target cells. However, these approaches have proved to
be problematic in many instances due to genotoxicity resulting from
to prolonged expression of gene editing system in vivo. To prevent
such off-target genotoxicity due to prolonged presence of a gene
editing system, several studies explored delivery of mRNA or
protein instead of delivering the gene coding for the nucleases in
cell culture.
Inventors: |
Gao; Guangping;
(Westborough, MA) ; Zamore; Phillip D.;
(Northborough, MA) ; Wang; Dan; (Belchertown,
MA) |
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Applicant: |
Name |
City |
State |
Country |
Type |
University of Massachusetts |
Boston |
MA |
US |
|
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Assignee: |
University of Massachusetts
Boston
MA
|
Prior
Publication: |
|
Document Identifier |
Publication Date |
|
US 20180037877 A1 |
February 8, 2018 |
|
|
Family ID: |
56614963 |
Appl. No.: |
15/550452 |
Filed: |
February 12, 2016 |
PCT Filed: |
February 12, 2016 |
PCT NO: |
PCT/US16/17886 PCKC 00 |
371 Date: |
August 11, 2017 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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62115928 |
Feb 13, 2015 |
|
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C07K 14/005 20130101;
C07K 2319/735 20130101; C12N 15/11 20130101; C12N 2750/14121
20130101; A61K 48/005 20130101; C12N 15/907 20130101; C12N 7/00
20130101; C12N 2750/14122 20130101; C12N 2750/14142 20130101; A61K
38/00 20130101; C12N 2750/14143 20130101; C12N 9/22 20130101; C12N
2310/20 20170501 |
International
Class: |
C12N 9/22 20060101
C12N009/22; C07K 14/005 20060101 C07K014/005; C12N 7/00 20060101
C12N007/00; C12N 15/11 20060101 C12N015/11; C12N 15/90 20060101
C12N015/90; A61K 48/00 20060101 A61K048/00 |
Claims
1. An adeno-associated virus (AAV) capsid protein having a
terminally grafted nuclease.
2. The AAV capsid protein of claim 1, wherein the capsid protein is
a VP2 capsid protein.
3. The AAV capsid protein of claim 2, wherein the terminally
grafted nuclease is grafted to the N-terminus of the VP2 capsid
protein.
4. The AAV capsid protein of claim 2, wherein the terminally
grafted nuclease is grafted to the C-terminus of the VP2 capsid
protein.
5. The AAV capsid protein of claim 1, wherein the nuclease is
selected from: Transcription Activator-like Effector Nucleases
(TALENs), Zinc Finger Nucleases (ZFNs), engineered meganuclease,
re-engineered homing endonucleases and a Cas-family nuclease.
6. The AAV capsid protein of claim 1, wherein the nuclease is a
Cas-family nuclease selected from the group consisting of Cas9 and
Cas7.
10. The AAV capsid protein of claim 1, wherein the nuclease is
represented by SEQ ID NO: 2.
11. The AAV capsid protein of claim 1, wherein the nuclease is a
polypeptide encoded by the nucleic acid sequence represented by SEQ
ID NO: 1.
12. The AAV capsid protein of claim 2, further comprising a linker
conjugated to the C-terminus of the terminally grafted nuclease and
the N-terminus of the VP2 protein.
13. The AAV capsid protein of claim 2, further comprising a linker
conjugated to the N-terminus of the terminally grafted nuclease and
the C-terminus of the VP2 protein.
14. The AAV capsid protein of claim 1, wherein the AAV capsid
protein having an terminally grafted nuclease is of a serotype
derived from a non-human primate.
15. The AAV capsid protein of claim 1, wherein the AAV capsid
protein having an terminally grafted nuclease is selected from:
AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11,
and AAV12.
16. A recombinant adeno-associated virus (rAAV) comprising the
capsid protein of claim 1.
17. The rAAV of claim 16, wherein the rAAV comprises a
transgene.
18. The rAAV of claim 17, wherein the transgene encodes a guide
RNA.
19. The rAAV of claim 18, wherein the guide RNA directs the
nuclease to a cleavage site in a target nucleic acid.
20. The rAAV of claim 16, wherein the AAV is an empty viral
particle with no transgene.
21. A composition comprising the rAAV of any one of claims 16 to
20.
22. The composition of claim 21 further comprising a
pharmaceutically acceptable carrier.
23. A nucleic acid encoding an AAV capsid protein having an
terminally grafted nuclease.
24. A host cell containing the nucleic acid of claim 23.
25. A host cell containing a nucleic acid of claim 23, wherein the
AAV capsid protein having an terminally grafted nuclease is a VP2
capsid protein, and wherein the host cell further comprises one or
more nucleic acids encoding VP1 and VP3 capsid proteins.
26. A composition comprising the host cell of claim 24 and a
sterile cell culture medium.
27. A composition comprising the host cell of claim 25, a sterile
cell culture medium, and at least one recombinant AAV viral
particle comprising the VP2 capsid protein having the terminally
grafted nuclease and the VP1 and VP3 capsid proteins
28. A composition comprising the host cell of claim 24 or 25 and a
cryopreservative.
29. An isolated nucleic acid comprising a sequence represented by
SEQ ID NO: 3.
30. An isolated nucleic acid encoding an AAV capsid protein having
an amino acid sequence selected from the group consisting of: SEQ
ID NOs: 2 and 4.
31. An isolated AAV capsid protein comprising an amino acid
sequence selected from the group consisting of: SEQ ID NOs: 2 and
4.
32. A composition comprising the isolated AAV capsid protein of
claim 31.
33. The composition of claim 32 further comprising a
pharmaceutically acceptable carrier.
34. A kit for producing a rAAV, the kit comprising: a container
housing an isolated nucleic acid having a sequence of SEQ ID NO: 1
or 3.
35. The kit of claim 34 further comprising instructions for
producing the rAAV.
36. The kit of claim 35 further comprising at least one container
housing a recombinant AAV vector, wherein the recombinant AAV
vector comprises a transgene.
37. A kit comprising: a container housing a recombinant AAV having
an isolated AAV capsid protein having an amino acid sequence as set
forth in SEQ ID NO: 2 or 4.
38. A method of targeting genome editing in a cell, the method
comprising: delivering to the cell a first recombinant adeno
associated virus (rAAV) having an terminally-grafted nuclease on at
least one capsid protein, wherein when present in the cell, the
terminally-grafted nuclease is directed to a genomic cleavage site
by a guide RNA.
39. The method of claim 38, wherein the first rAAV comprises a
transgene encoding the guide RNA.
40. The method of claim 39 further comprising administering a
second rAAV having a transgene encoding a guide RNA that directs
the nuclease to a cleavage site in a target nucleic acid.
41. The method of any one of claims 38 to 40, wherein cell is
present in a subject, and the first rAAV or second rAAV is
administered to the subject intravenously, intravascularly,
transdermally, intraocularly, intrathecally, orally,
intramuscularly, subcutaneously, intranasally, or by inhalation,
thereby delivering the first rAAV or second rAAV to the cell.
42. The method of claim 41, wherein the subject is selected from a
mouse, a rat, a rabbit, a dog, a cat, a sheep, a pig, and a
non-human primate.
44. The method of claim 42, wherein the subject is a human.
45. A composition comprising: i.) a first recombinant
adeno-associated virus (rAAV) having an terminally-grafted nuclease
on at least one capsid protein; and ii.) a second rAAV having a
transgene encoding a guide RNA that directs the nuclease to a
cleavage site in a target nucleic acid.
46. The composition of claim 45, wherein the first rAAV is an empty
viral particle.
47. The composition of claim 45, wherein the first rAAV has an
terminally-grafted nuclease that is grafted to the C-terminus of a
VP2 capsid protein of the rAAV.
48. An adeno-associated virus (AAV) capsid protein having a
terminally grafted nuclease or fragment thereof, wherein the
nuclease or fragment thereof comprises a terminally grafted
intein.
49. The AAV capsid protein of claim 48, wherein the capsid protein
is a VP2 capsid protein.
50. The AAV capsid protein of claim 48, wherein the intein is IntN
or IntC.
51. The AAV capsid protein of claim 48, wherein the capsid protein
is represented by any one of SEQ ID NO: 7 to 9.
Description
RELATED APPLICATIONS
[0001] This application is a National Stage Application of
PCT/US2016/017886, filed Feb. 12, 2016, entitled "COMPOSITIONS AND
METHODS FOR TRANSIENT DELIVERY OF NUCLEASES", which claims the
benefit under 35 U.S.C. .sctn.119(e) of U.S. Provisional
Application Ser. No. 62/115,928, entitled "COMPOSITIONS AND METHODS
FOR TRANSIENT DELIVERY OF NUCLEASES" filed on Feb. 13, 2015, the
entire contents of each application which are incorporated herein
by reference.
FIELD OF THE INVENTION
[0002] The disclosure in some aspects relates to isolated nucleic
acids, compositions, and kits useful for protein delivery to
cells.
BACKGROUND
[0003] Recently, gene editing using designer DNA sequence-specific
nucleases emerged as a technology for both basic biomedical
research and therapeutic development. Platforms based on three
distinct types of endonucleases have been developed for gene
editing, namely the zinc finger nuclease (ZFN), the transcription
activator-like effector nuclease (TALEN), and the clustered
regularly interspaced short palindromic repeat (CRISPR) associated
endonuclease 9 (cas9). Each nuclease is capable of inducing a DNA
double-stranded break (DSB) at specific DNA loci, thus triggering
two DNA repair pathways. The non-homologous end joining (NHEJ)
pathway generates random insertion/deletion (indel) mutations at
the DSB, whereas the homology-directed repair (HDR) pathway repairs
the DSB with the genetic information carried on a donor template.
Therefore, these gene editing platforms are capable of manipulating
genes at specific genomic loci in multiple ways, such as disrupting
gene function, repairing a mutant gene to normal, and inserting DNA
material.
[0004] Transforming the gene editing technology into therapeutic
uses encounters several obstacles, including the concern over
safety. Certain gene editing platforms have been shown to induce
off-target DSBs throughout genomes, which is associated with
genotoxicity. Such off-target effects not only stem from the
intrinsic ambiguity of DNA sequence recognition by nucleases, but
also attribute to the prolonged presence of an active gene editing
system in a given cell. As a result, off-target DSBs accumulate
over time, and ultimately lead to genotoxicity.
SUMMARY
[0005] Recent approaches to delivering nucleases to cells for gene
editing have focused on delivering of expression vectors engineered
to express the nucleases in target cells. However, these approaches
have proved to be problematic in many instances due to genotoxicity
resulting from to prolonged expression of gene editing system in
vivo. To prevent such off-target genotoxicity due to prolonged
presence of a gene editing system, several studies explored
delivery of mRNA or protein instead of delivering the gene coding
for the nucleases in cell culture. As a result, the gene editing
system functions only in a short period of time until the nuclease
mRNA or protein is naturally degraded inside cells, which has been
shown to reduce off-target effects. However, delivery of mRNA or
protein in vivo is a significant task, and the delivery efficiency
is very limited with conventional techniques. In contrast, the
present disclosure overcomes such genotoxicity and delivery issues
by using viruses for transiently delivering nucleases to cells
thereby fulfilling the task of inducing permanent gene editing in a
transient manner such that the nucleases will degrade naturally. In
some embodiments, the disclosure relates to the uses of a viral
vector (e.g., an AAV) as a delivery vehicle to carry a nuclease
(e.g., a Cas9 protein or other designer nuclease proteins) to
cells. In some embodiments, to avoid the potential genotoxicity due
to prolonged expression of gene editing system in vivo, methods are
provided herein to transiently deliver an endonuclease protein
using recombinant adeno-associated viruses. In some embodiments,
AAV capsid is used as a delivery vehicle to carry the Cas9 protein
or other designer nuclease proteins.
[0006] In some aspects, the disclosure relates to an
adeno-associated virus (AAV) capsid protein having a terminally
grafted nuclease.
[0007] In some embodiments, the capsid protein is a VP2 capsid
protein. In some embodiments, the terminally grafted nuclease is
grafted to the N-terminus of the VP2 capsid protein. In some
embodiments, the terminally grafted nuclease is grafted to the
C-terminus of the VP2 capsid protein.
[0008] In some embodiments, the nuclease is selected from:
Transcription Activator-like Effector Nucleases (TALENs), Zinc
Finger Nucleases (ZFNs), engineered meganuclease, re-engineered
homing endonucleases and a Cas-family nuclease. In some
embodiments, the nuclease is a Cas-family nuclease selected from
the group consisting of Cas9 and Cas7. In some embodiments, the
nuclease is represented by SEQ ID NO: 2. In some embodiments, the
nuclease is a polypeptide encoded by the nucleic acid sequence
represented by SEQ ID NO: 1.
[0009] In some embodiments, the AAV capsid protein further
comprises a linker conjugated to the C-terminus of the terminally
grafted nuclease and the N-terminus of the VP2 protein. In some
embodiments, the AAV capsid protein further comprises a linker
conjugated to the N-terminus of the terminally grafted nuclease and
the C-terminus of the VP2 protein.
[0010] In some embodiments, the AAV capsid protein hays an
terminally grafted nuclease is of a serotype derived from a
non-human primate. In some embodiments, the AAV capsid protein has
an terminally grafted nuclease is selected from: AAV1, AAV2, AAV3,
AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, and AAV12.
[0011] In some aspects, the disclosure relates to a recombinant
adeno-associated virus (rAAV) comprising an adeno-associated virus
(AAV) capsid protein having a terminally grafted nuclease.
[0012] In some embodiments, the rAAV comprises a transgene. In some
embodiments, the transgene encodes a guide RNA. In some
embodiments, the guide RNA directs the nuclease to a cleavage site
in a target nucleic acid.
[0013] In some embodiments, the AAV is an empty viral particle with
no transgene.
[0014] In some aspects, the disclosure provides a composition
comprising an rAAV as described by this document. In some
embodiments, the composition further comprises a pharmaceutically
acceptable carrier.
[0015] In some aspects, the disclosure relates to a nucleic acid
encoding an AAV capsid protein having an terminally grafted
nuclease. In some embodiments, a host cell contains the nucleic
acid. In some embodiments, the host cell contains a nucleic acid
encodes an AAV VP2 capsid protein having an terminally grafted
nuclease. In some embodiments, the host cell further comprises one
or more nucleic acids encoding VP1 and VP3 capsid proteins.
[0016] In some aspects, the disclosure relates to a composition
comprising a host cell as described by this document and a sterile
cell culture medium. In some aspects, the disclosure relates to a
composition comprising a host cell as described by this document
and a cryopreservative.
[0017] In some aspects, the disclosure relates to an isolated
nucleic acid comprising a sequence represented by SEQ ID NO: 3.
[0018] In some aspects, the disclosure relates to an isolated
nucleic acid encoding an AAV capsid protein having an amino acid
sequence selected from the group consisting of: SEQ ID NOs: 2 and
4.
[0019] In some aspects, the disclosure relates to an isolated AAV
capsid protein comprising an amino acid sequence selected from the
group consisting of: SEQ ID NOs: 2 and 4.
[0020] In some aspects, the disclosure relates to a composition
comprising an isolated AAV capsid protein as described by this
document. In some embodiments, the composition further comprises a
pharmaceutically acceptable carrier.
[0021] In some aspects, the disclosure relates to a kit for
producing a rAAV, the kit comprising: a container housing an
isolated nucleic acid having a sequence of SEQ ID NO: 1 or 3. In
some embodiments, the kit further comprises instructions for
producing the rAAV. In some embodiments, the kit further comprises
at least one container housing a recombinant AAV vector, wherein
the recombinant AAV vector comprises a transgene.
[0022] In some aspects, the disclosure relates to a kit comprising:
a container housing a recombinant AAV having an isolated AAV capsid
protein having an amino acid sequence as set forth in SEQ ID NO: 2
or 4.
[0023] In some aspects, the disclosure relates to a method of
targeting genome editing in a cell, the method comprising:
delivering to the cell a first recombinant adeno associated virus
(rAAV) having an terminally-grafted nuclease on at least one capsid
protein, wherein when present in the cell, the terminally-grafted
nuclease is directed to a genomic cleavage site by a guide RNA.
[0024] In some embodiments of the method, the first rAAV comprises
a transgene encoding the guide RNA.
[0025] In some embodiments, the method further comprises
administering a second rAAV having a transgene encoding a guide RNA
that directs the nuclease to a cleavage site in a target nucleic
acid.
[0026] In some embodiments, the cell is present in a subject, and
the first rAAV or second rAAV is administered to the subject
intravenously, intravascularly, transdermally, intraocularly,
intrathecally, orally, intramuscularly, subcutaneously,
intranasally, or by inhalation, thereby delivering the first rAAV
or second rAAV to the cell. In some embodiments, the subject is
selected from a mouse, a rat, a rabbit, a dog, a cat, a sheep, a
pig, and a non-human primate. In some embodiments, the subject is a
human.
[0027] In some aspects, the disclosure relates to a composition
comprising: i.) a first recombinant adeno-associated virus (rAAV)
having an terminally-grafted nuclease on at least one capsid
protein; and ii.) a second rAAV having a transgene encoding a guide
RNA that directs the nuclease to a cleavage site in a target
nucleic acid.
[0028] In some embodiments, the first rAAV is an empty viral
particle. In some embodiments, the first rAAV has an
terminally-grafted nuclease that is grafted to the C-terminus of a
VP2 capsid protein of the rAAV.
[0029] In some aspects, the disclosure relates to an
adeno-associated virus (AAV) capsid protein having a terminally
grafted nuclease or fragment thereof, wherein the nuclease or
fragment thereof comprises a terminally grafted intein.
[0030] In some embodiments, the capsid protein is a VP2 capsid
protein. In some embodiments, the intein is IntN or IntC. In some
embodiments, the capsid protein is represented by any one of SEQ ID
NO: 7 to 9.
[0031] Each of the limitations of the disclosure can encompass
various embodiments of the disclosure. It is, therefore,
anticipated that each of the limitations of the disclosure
involving any one element or combinations of elements can be
included in each aspect of the disclosure. This disclosure is not
limited in its application to the details of construction and the
arrangement of components set forth in the following description or
illustrated in the drawings. The disclosure is capable of other
embodiments and of being practiced or of being carried out in
various ways.
BRIEF DESCRIPTION OF DRAWINGS
[0032] The accompanying drawings are not intended to be drawn to
scale. In the drawings, each identical or nearly identical
component that is illustrated in various figures is represented by
a like numeral. For purposes of clarity, not every component may be
labeled in every drawing. In the drawings:
[0033] FIGS. 1A-1B shows the SpCas9-VP2 fusion protein is produced
in HEK293 cells.
[0034] FIG. 1A shows the HA tagged SpCas9 is fused to the
N-terminus of VP2. The expression of this fusion protein is driven
by the CMV promoter. BGHpA: bovine growth hormone polyadenylation
signal. FIG. 1B depicts western blotting using anti-HA antibody
showing the HA-tagged fusion protein (.about.230 kD, arrow)
produced from transiently transfected HEK293 cells. HA tagged
SpCas9 (.about.162 kD) is marked by the triangle. Star indicates a
band of unknown origin, a likely degradation product from the
fusion protein.
[0035] FIGS. 2A-2B show the SpCas9-VP2 fusion protein mediates gene
editing in HEK293 cells. FIG. 2A shows the DNA repair reporter
construct. The mutant GFP (GFPmut) carries a disruptive insertion
(Ins), followed by out-of-frame (+3 frame) T2A and mCherry. In the
presence of a functional gene editing system targeting Ins, +1
insertion by NHEJ shifts the T2A and mCherry to in-frame, resulting
mCherry fluorescence. FIG. 2B shows the results of a reporter assay
in HEK293 cells by co-transfection of the reporter construct and
various plasmid as indicated. Both mCherry fluorescence and bright
field images are shown. Scale bar=50 .mu.M.
[0036] FIGS. 3A-3B show alternative strategies utilizing
intein-mediated protein trans-splicing (PTS). FIG. 3A shows the
N-terminus and C-terminus Npu DnaE intein (IntN and IntC,) are
fused with SpCas9 and VP2, respectively. The IntC-VP2 is packaged
into AAV virion. PTS occurs between the SpCas9-IntN fusion protein
and the IntC-AAV chimeric virion to produce the SpCas9-AAV virion.
FIG. 3B shows that in the first AAV vector, the AAV genome encodes
the N-terminal portion of SpCas9 (SpCas9N) fused with IntN. The
second AAV vector carries IntC and the C-terminal portion of SpCas9
fused to VP2. In vivo transduction of the first AAV vector produces
the fusion protein SpCas9N-IntN, which is followed by delivery of
the second vector. PTS occurs to reconstitute the full-length
SpCas9 protein.
[0037] FIG. 4 shows an expression construct comprising a nucleic
acid sequence encoding SpCas9 nuclease N-terminally fused to VP2
capsid protein.
[0038] FIG. 5 shows co-transfection of Split Cas9 parts in HEK293
cells reconstituted SpCas9 and VP2 fusion protein, as measured by
Western blot. Ctrl: pCMV-SpCas9-(EAAAKx3)-VP2; N:
pU1a-Cas9.sub.n-Int.sub.n; C part: Int.sub.cCas9.sub.c-( )-VP2; HA
tag is present in SpCas9 N-terminal. The designation "( )" refers
to a linker sequence (e.g., GS, GGGGSx3, EAAAKx3).
[0039] FIG. 6 shows co-transfection of Split Cas9 parts in HEK293
cells reconstituted gene editing function. Cells were transfected
with EGFP-ON reporter, pU1a-Cas9.sub.n-Int.sub.n, and
Int.sub.c-Cas9.sub.C-( )-VP2. EGFP reports Cas9 cleavage and NHEJ
repair; mCherry is constitutively expressed as control.
[0040] FIG. 7 shows incorporation of Int.sub.C-SpCas9.sub.c
polypeptide onto rAAV2 capsid. Cells were transfected with plasmid
encoding VP1 and VP3 proteins, and a plasmid encoding
Int.sub.c-SpCas9.sub.c-( )-VP2. Purified rAAV particles were
examined by silver staining.
DETAILED DESCRIPTION
[0041] Genome editing is a powerful tool for the interrogation and
manipulation of biological functions within cells. For example,
genome editing allows for the repair of mutant genes to normal
function, disruption of gene function and the insertion of genetic
material (e.g. DNA), all at specific genomic loci. However, several
challenges associated with the delivery and prolonged expression of
nucleases in cells, such as genotoxicity due to off-target cleavage
of DNA, has limited the therapeutic effectiveness of gene editing
platforms. The instant disclosure overcomes current limitations by
providing compositions and methods that improve delivery of genome
editing nucleases. Accordingly, in some aspects, the disclosure
relates to viral proteins comprising a terminally grafted
nucleases.
[0042] As used herein, "genome editing" refers to adding,
disrupting or changing genomic sequences (e.g., a gene sequence).
In some embodiments, genome editing is performed using engineered
proteins and related molecules. In some aspects, genome editing
comprises the use of engineered nucleases to cleave a target
genomic locus. In some embodiments, genome editing further
comprises inserting, deleting, mutating or substituting nucleic
acid residues at a cleaved locus. In some embodiments, inserting,
deleting, mutating or substituting nucleic acid residues at a
cleaved locus is accomplished through endogenous cellular
mechanisms such as homologous recombination (HR) and non-homologous
end joining (NHEJ). Exemplary genome editing technologies include,
but are not limited to Transcription Activator-like Effector
Nucleases (TALENs), Zinc Finger Nucleases (ZFNs), engineered
meganuclease re-engineered homing endonucleases, the CRISPR/Cas
system. In some embodiments, the gene editing technologies are
proteins or molecules related to TALENs, including but not limited
to transcription activator-like effectors (TALEs) and restriction
endonucleases (e.g. FokI). In some embodiments, the gene editing
technologies are proteins or molecules related to ZFNs, including
but not limited to proteins comprising the Cys.sub.2His.sub.2 fold
group (for example Zif268 (EGR1)), and restriction endonucleases
(e.g. FokI). In some embodiments, the gene editing technologies are
proteins or molecules related to the CRISPR/Cas system, including
but not limited to Cas9, Cas6, dCas9, CRISPR RNA (crRNA) and
trans-activating crRNA (tracrRNA).
[0043] As used herein, the terms "endonuclease" and "nuclease"
refer to an enzyme that cleaves a phosphodiester bond or bonds
within a polynucleotide chain. Nucleases may be naturally occurring
or genetically engineered. Genetically engineered nucleases are
particularly useful for genome editing and are generally classified
into four families: zinc finger nucleases (ZFNs), transcription
activator-like effector nucleases (TALENs), engineered
meganucleases and CRISPR-associated proteins (Cas nucleases). In
some embodiments, the nuclease is a ZFN. In some embodiments, the
ZFN comprises a FokI cleavage domain. In some embodiments, the ZFN
comprises Cys.sub.2His.sub.2 fold group. In some embodiments, the
nuclease is a TALEN. In some embodiments, the TALEN comprises a
FokI cleavage domain. In some embodiments, the nuclease is an
engineered meganuclease.
[0044] The term "CRISPR" refers to "clustered regularly interspaced
short palindromic repeats", which are DNA loci containing short
repetitions of base sequences. CRISPR loci form a portion of a
prokaryotic adaptive immune system that confers resistance to
foreign genetic material. Each CRISPR loci is flanked by short
segments of "spacer DNA", which are derived from viral genomic
material. In the Type II CRISPR system, spacer DNA hybridizes to
transactivating RNA (tracrRNA) and is processed into CRISPR-RNA
(crRNA) and subsequently associates with CRISPR-associated
nucleases (Cas nucleases) to form complexes that recognize and
degrade foreign DNA. In certain embodiments, the nuclease is a
CRISPR-associated nuclease (Cas nuclease). Examples of CRISPR
nucleases include, but are not limited to Cas9, Cas6 and dCas9.
dCas9 is an engineered Cas protein that binds to a target locus but
does not cleave said locus. In some embodiments, the nuclease is
Cas9. In some embodiments, the Cas9 is derived from the bacteria S.
pyogenes (SpCas9).
[0045] For the purpose of genome editing, the CRISPR system can be
modified to combine the tracrRNA and crRNA in to a single guide RNA
(sgRNA) or just (gRNA). As used herein, the term "guide RNA" or
"gRNA" refers to a polynucleotide sequence that is complementary to
a target sequence in a cell and associates with a Cas nuclease,
thereby directing the Cas nuclease to the target sequence. In some
embodiments, a gRNA ranges between 1 and 30 nucleotides in length.
In some embodiments, a gRNA ranges between 5 and 25 nucleotides in
length. In some embodiments, a gRNA ranges between 10 and 20
nucleotides in length. In some embodiments, a gRNA ranges between
14 and 18 nucleotides in length.
[0046] Aspects of the disclosure relate to SpCas9 grafted to an
AAV2 capsid protein, VP2. However, in some embodiments, the same
strategy can be applied in other contexts. For example, the SpCas9
can be replaced with any modified SpCas9 such as mutated or
truncated forms, Cas9 proteins from other species and nucleases
used in other gene editing platforms such as ZFNs and TALENs. In
some embodiments, a nuclease terminally grafted to an AAV2 capsid
protein may also be fused to another functional domain, for example
single guide RNA (sgRNA).
[0047] Similarly, the AAV2 capsid protein VP2 may be replaced with
VP2 of other AAV serotypes (e.g., AAV3, AAV3b, AAV4, AAV5, AAV6,
AAV7, AAV8, AAV9, AAVrh8, AAV10, and variants thereof), or a
suitable capsid protein of any viral vector. Thus, in some aspects,
the disclosure relates to the viral delivery of a nuclease.
Examples of viral vectors include retroviral vectors (e.g. Maloney
murine leukemia virus, MML-V), adenoviral vectors (e.g. AD100),
lentiviral vectors (HIV and FIV-based vectors), herpesvirus vectors
(e.g. HSV-2). In some embodiments, the disclosure relates to
adeno-associated viruses (AAVs). In some embodiments, a nuclease is
grafted to or replaces all or a portion of a viral
glycoprotein.
[0048] In some embodiments, SpCas-VP2 is incorporated into AAV2
capsid to form AAV virion. In some embodiments, the start codon of
VP2 is mutated in the cap gene from the trans AAV production
plasmid. In some embodiments, when Cas9 is fused to the N-terminus
of VP2, the resulting Cas9-VP2 fusion protein is functional with
respect to both productive AAV assembly and being an active
component of the CRISPR/Cas9 gene editing system.
[0049] In some embodiments, a catalytically deficient form of the
cas9 protein (dCas9) is fused with a C-terminal peptide domain that
either activates or represses gene expression. In such embodiments,
such a dCas9-effector fusion protein binds DNA in a sgRNA-guided
manner.
[0050] In some aspects, the disclosure relates to the discovery
that inteins can be utilized to rejoin (e.g., reconstitute)
fragments or portions of gene editing proteins to generate a
functional gene editing protein that is grafted onto an AAV capsid
protein. As used herein, "intein" refers to a self-splicing protein
intron (e.g., peptide) that ligates flanking N-terminal and
C-terminal exteins (e.g., fragments to be joined). The use of
certain inteins for joining heterologous protein fragments is
described, for example, in Wood et al., J. Biol. Chem. 289(21);
14512-9 (2014). For example, when fused to separate protein
fragments, the inteins IntN and IntC recognize each other, splice
themselves out and simultaneously ligate the flanking N- and
C-terminal exteins of the protein fragments to which they were
fused, thereby reconstituting a full length protein from the two
protein fragments. Other suitable inteins will be apparent to a
person of skill in the art.
[0051] A nuclease protein fragment (e.g., Cas9 fragment) can vary
in length. In some embodiments, a protein fragment ranges from 2
amino acids to about 1000 amino acids in length. In some
embodiments, a protein fragment ranges from about 5 amino acids to
about 500 amino acids in length. In some embodiments, a protein
fragment ranges from about 20 amino acids to about 200 amino acids
in length. In some embodiments, a protein fragment ranges from
about 10 amino acids to about 100 amino acids in length. Suitable
protein fragments of other lengths will be apparent to a person of
skill in the art.
[0052] In some embodiments, a portion or fragment of a nuclease
(e.g., a fragment of Cas9) is fused to an intein. The nuclease can
be fused to the N-terminus or the C-terminus of the intein. In some
embodiments, a portion or fragment of a nuclease (e.g., a fragment
of Cas9) is fused to an intein and fused to an AAV capsid protein.
The intein, nuclease and capsid protein can be fused together in
any arrangement (e.g., nuclease-intein-capsid,
intein-nuclease-capsid, capsid-intein-nuclease, etc.). In some
embodiments, the N-terminus of an intein is fused to the C-terminus
of a nuclease (e.g., Cas9) and the C-terminus of the intein is
fused to the N-terminus of an AAV capsid protein.
[0053] In some embodiments, the IntN/IntC system is used to join
fragments of a nuclease. In some embodiments, IntC is fused to the
N-terminus of a nuclease (e.g., Cas9) fragment that is grafted to
an AAV capsid protein. In some embodiments, IntN is fused to the
C-terminus of a nuclease (e.g., Cas9) fragment. In some
embodiments, a fragment of a nuclease fused to an intein is
represented by SEQ ID NO: 6. In some embodiments, an AAV capsid
protein comprising an intein fused to a fragment of a nuclease that
has been terminally grafted to the AAV capsid protein is
represented by any one of SEQ ID NO: 7 to 9.
Isolated AAV Capsid Proteins and Nucleic Acids Encoding the
Same
[0054] AAVs disclosed herein are useful for creating vectors that
facilitate delivery of nucleases to cells for human gene editing
applications. Protein and amino acid sequences as well as other
information regarding the AAVs capsid are set forth in the sequence
listing.
[0055] In some embodiments, an AAV capsid having a terminally graft
nuclease is provided that has an amino acid sequence represented by
SEQ ID NO: 4. In some embodiments, an AAV capsid having a
terminally graft nuclease is provided that is encoded by a nucleic
acid sequence represented by SEQ ID NO: 3.
[0056] An example of an isolated nucleic acid that encodes an AAV
capsid protein having a terminally graft nuclease is a nucleic acid
having a sequence of: SEQ ID NO: 3 as well as nucleic acids having
substantial homology thereto. In some embodiments, isolated nucleic
acids that encode AAV capsids are provided that encode the VP2
protein portion of the amino acid sequence represented by SEQ ID
NO: 3.
[0057] In some embodiments, nucleic acids are provided that encode
an AAV capsid having a nuclease grafted within its capsid sequence
(e.g., a AAV9 capsid) and up to 5, up to 10, up to 20, up to 30, up
to 40, up to 50, up to 100 other amino acid alternations.
[0058] In some embodiments, a fragment (portion) of an isolated
nucleic acid encoding a AAV capsid sequence may be useful for
constructing a nucleic acid encoding a desired capsid sequence.
Fragments may be of any appropriate length (e.g., at least 9, at
least 18, at least 36, at least 72, at least 144, at least 288, at
least 576, at least 1152 or more nucleotides in length). For
example, a fragment of nucleic acid sequence encoding a variant
amino acid (compared with a known AAV serotype) may be used to
construct, or may be incorporated within, a nucleic acid sequence
encoding an AAV capsid sequence to alter the properties of the AAV
capsid. For example, a nucleic sequence encoding an AAV variant may
comprise n amino acid variants (e.g., in which n=1, 2, 3, 4, 5, 6,
7, 8, 9, 10 or more) compared with a known AAV serotype (e.g.,
AAV9). A recombinant cap sequence may be constructed having one or
more of the n amino acid variants by incorporating fragments of a
nucleic acid sequence comprising a region encoding a variant amino
acid into the sequence of a nucleic acid encoding the known AAV
serotype. The fragments may be incorporated by any appropriate
method, including using site directed mutagenesis. In some
embodiments, polypeptide fragments that are not normally present in
AAV capsid proteins may be incorporated into a recombinant cap
sequence. In some embodiments, the polypeptide fragment is grafted
onto the recombinant cap sequence. Thus, new AAV variants may be
created having new properties.
[0059] As used herein, "grafting" refers to joining or uniting of
at least two polymeric molecules. In some embodiments, the term
grafting refers joining or uniting of at least two polymeric
molecules such that one of the at least two molecules is inserted
within another of the at least two molecules. In some embodiments,
the term grafting refers to joining or uniting of at least two
polymeric molecules such that one of the at least two molecules is
appended to another of the at least two molecules. In some
embodiments, the term grafting refers joining or uniting of at
least two nucleic acid molecules such that one of the at least two
nucleic acid molecules is inserted within another of the at least
two nucleic acid molecules. In some embodiments, the term grafting
refers to joining or uniting of at least two nucleic acid molecules
such that one of the at least two molecules is appended to another
of the at least two nucleic acid molecules.
[0060] In some embodiments, a grafted nucleic acid molecule encodes
a chimeric protein. In some embodiments, a grafted nucleic acid
molecule encodes a chimeric protein, such that one polypeptide is
effectively inserted into another polypeptide (e.g. not directly
conjugated before the N-terminus or after the C-terminus), thereby
creating a contiguous fusion of two polypeptides. In some
embodiments, a grafted nucleic acid molecule encodes a chimeric
protein, such that one polypeptide is effectively appended to
another polypeptide (e.g. directly conjugated before the N-terminus
or after the C-terminus), thereby creating a contiguous fusion of
two polypeptides. In some embodiments, the term grafting refers to
joining or uniting of at least two polypeptides, or fragments
thereof, such that one of the at least two polypeptides or
fragments thereof is inserted within another of the at least two
polypeptides or fragments thereof. In some embodiments, the term
grafting refers to joining or uniting of at least two polypeptides
or fragments thereof such that one of the at least two polypeptides
or fragments thereof is appended to another of the at least two
polypeptides or fragments thereof.
[0061] In some embodiments, the instant disclosure relates to an
adeno-associated virus (AAV) capsid protein comprising a AAV capsid
protein having an N-terminally grafted nuclease.
[0062] In some embodiments, the AAV capsid protein further
comprises a linker. Non-limiting examples of linkers include
flexible linkers (e.g. glycine-rich linkers), rigid linkers (e.g.
[EAAK].sub.n, where n>2), and cleavable linkers (e.g.
protease-sensitive sequences). Other linkers are disclosed, for
example in Chen et al., Fusion protein linkers: Property, design
and functionality. Advanced drug delivery reviews, 2013. In some
embodiments, the linker is conjugated to the C-terminus of a
terminally grafted nuclease (e.g., an N-terminally grafted
nuclease). In some embodiments, the linker is conjugated to the
N-terminus of the terminally grafted nuclease (e.g., an
N-terminally grafted nuclease). In some embodiments, one linker is
conjugated to the N-terminus of the terminally grafted nuclease and
a second linker is conjugated to the C-terminus of the terminally
grafted nuclease.
[0063] In some embodiments, the linker is a glycine-rich linker. In
some embodiments, the linker comprises at least one polypeptide
repeat, each repeat comprising at least 80% glycine residues. In
some embodiments, the polypeptide repeat comprises GGGS (SEQ ID NO:
5). In some embodiments, the linker comprises a formula selected
from the group consisting of: [G].sub.n, [G].sub.nS, [GS].sub.n,
and [GGSG].sub.n, wherein G is glycine and wherein n is an integer
greater than one (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,
15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or more).
[0064] In some cases, fragments of capsid proteins disclosed herein
are provided. Such fragments may at least 10, at least 20, at least
50, at least 100, at least 200, at least 300, at least 400, at
least 500 or more amino acids in length. In some embodiments,
chimeric capsid proteins are provided that comprise one or more
fragments of one or more capsid proteins disclosed herein.
[0065] "Homology" refers to the percent identity between two
polynucleotides or two polypeptide moieties. The term "substantial
homology", when referring to a nucleic acid, or fragment thereof,
indicates that, when optimally aligned with appropriate nucleotide
insertions or deletions with another nucleic acid (or its
complementary strand), there is nucleotide sequence identity in
about 90 to 100% of the aligned sequences. When referring to a
polypeptide, or fragment thereof, the term "substantial homology"
indicates that, when optimally aligned with appropriate gaps,
insertions or deletions with another polypeptide, there is
nucleotide sequence identity in about 90 to 100% of the aligned
sequences. The term "highly conserved" means at least 80% identity,
preferably at least 90% identity, and more preferably, over 97%
identity. In some cases, highly conserved may refer to 100%
identity. Identity is readily determined by one of skill in the art
by, for example, the use of algorithms and computer programs known
by those of skill in the art.
[0066] As described herein, alignments between sequences of nucleic
acids or polypeptides are performed using any of a variety of
publicly or commercially available Multiple Sequence Alignment
Programs, such as "Clustal W", accessible through Web Servers on
the internet. Alternatively, Vector NTI utilities may also be used.
There are also a number of algorithms known in the art which can be
used to measure nucleotide sequence identity, including those
contained in the programs described above. As another example,
polynucleotide sequences can be compared using BLASTN, which
provides alignments and percent sequence identity of the regions of
the best overlap between the query and search sequences. Similar
programs are available for the comparison of amino acid sequences,
e.g., the "Clustal X" program, BLASTP. Typically, any of these
programs are used at default settings, although one of skill in the
art can alter these settings as needed. Alternatively, one of skill
in the art can utilize another algorithm or computer program which
provides at least the level of identity or alignment as that
provided by the referenced algorithms and programs. Alignments may
be used to identify corresponding amino acids between two proteins
or peptides. A "corresponding amino acid" is an amino acid of a
protein or peptide sequence that has been aligned with an amino
acid of another protein or peptide sequence. Corresponding amino
acids may be identical or non-identical. A corresponding amino acid
that is a non-identical amino acid may be referred to as a variant
amino acid.
[0067] Alternatively for nucleic acids, homology can be determined
by hybridization of polynucleotides under conditions which form
stable duplexes between homologous regions, followed by digestion
with single-stranded-specific nuclease(s), and size determination
of the digested fragments. DNA sequences that are substantially
homologous can be identified in a Southern hybridization experiment
under, for example, stringent conditions, as defined for that
particular system. Defining appropriate hybridization conditions is
within the skill of the art.
[0068] A "nucleic acid" sequence refers to a DNA or RNA sequence.
In some embodiments, proteins and nucleic acids of the disclosure
are isolated. As used herein, the term "isolated" means
artificially produced. As used herein with respect to nucleic
acids, the term "isolated" means: (i) amplified in vitro by, for
example, polymerase chain reaction (PCR); (ii) recombinantly
produced by cloning; (iii) purified, as by cleavage and gel
separation; or (iv) synthesized by, for example, chemical
synthesis. An isolated nucleic acid is one which is readily
manipulable by recombinant DNA techniques well known in the art.
Thus, a nucleotide sequence contained in a vector in which 5' and
3' restriction sites are known or for which polymerase chain
reaction (PCR) primer sequences have been disclosed is considered
isolated but a nucleic acid sequence existing in its native state
in its natural host is not. An isolated nucleic acid may be
substantially purified, but need not be. For example, a nucleic
acid that is isolated within a cloning or expression vector is not
pure in that it may comprise only a tiny percentage of the material
in the cell in which it resides. Such a nucleic acid is isolated,
however, as the term is used herein because it is readily
manipulable by standard techniques known to those of ordinary skill
in the art. As used herein with respect to proteins or peptides,
the term "isolated" refers to a protein or peptide that has been
isolated from its natural environment or artificially produced
(e.g., by chemical synthesis, by recombinant DNA technology,
etc.).
[0069] The skilled artisan will also realize that conservative
amino acid substitutions may be made to provide functionally
equivalent variants, or homologs of the capsid proteins. In some
aspects the disclosure embraces sequence alterations that result in
conservative amino acid substitutions. As used herein, a
conservative amino acid substitution refers to an amino acid
substitution that does not alter the relative charge or size
characteristics of the protein in which the amino acid substitution
is made. Variants can be prepared according to methods for altering
polypeptide sequence known to one of ordinary skill in the art such
as are found in references that compile such methods, e.g.
Molecular Cloning: A Laboratory Manual, J. Sambrook, et al., eds.,
Second Edition, Cold Spring Harbor Laboratory Press, Cold Spring
Harbor, N.Y., 1989, or Current Protocols in Molecular Biology, F.
M. Ausubel, et al., eds., John Wiley & Sons, Inc., New York.
Conservative substitutions of amino acids include substitutions
made among amino acids within the following groups: (a) M, I, L, V;
(b) F, Y, W; (c) K, R, H; (d) A, G; (e) S, T; (f) Q, N; and (g) E,
D. Therefore, one can make conservative amino acid substitutions to
the amino acid sequence of the proteins and polypeptides disclosed
herein.
Recombinant AAVs
[0070] In some aspects, the disclosure provides isolated AAVs. As
used herein with respect to AAVs, the term "isolated" refers to an
AAV that has been artificially produced or obtained. Isolated AAVs
may be produced using recombinant methods. Such AAVs are referred
to herein as "recombinant AAVs". Recombinant AAVs (rAAVs)
preferably have tissue-specific targeting capabilities, such that a
nuclease and/or transgene of the rAAV will be delivered
specifically to one or more predetermined tissue(s). The AAV capsid
is an important element in determining these tissue-specific
targeting capabilities. Thus, an rAAV having a capsid appropriate
for the tissue being targeted can be selected Methods for obtaining
recombinant AAVs having a desired capsid protein are well known in
the art. (See, for example, US 2003/0138772), the contents of which
are incorporated herein by reference in their entirety). Typically
the methods involve culturing a host cell which contains a nucleic
acid sequence encoding an AAV capsid protein; a functional rep
gene; a recombinant AAV vector composed of, AAV inverted terminal
repeats (ITRs) and a transgene; and sufficient helper functions to
permit packaging of the recombinant AAV vector into the AAV capsid
proteins. In some embodiments, capsid proteins are structural
proteins encoded by the cap gene of an AAV. AAVs comprise three
capsid proteins, virion proteins 1 to 3 (named VP1, VP2 and VP3),
all of which are transcribed from a single cap gene via alternative
splicing. In some embodiments, the molecular weights of VP1, VP2
and VP3 are respectively about 87 kDa, about 72 kDa and about 62
kDa. In some embodiments, upon translation, capsid proteins form a
spherical 60-mer protein shell around the viral genome. In some
embodiments, the functions of the capsid proteins are to protect
the viral genome, deliver the genome and interact with the host. In
some aspects, capsid proteins deliver the viral genome to a host in
a tissue specific manner. In some embodiments, the a terminally
grafted nuclease is present on all three capsid proteins (e.g. VP1,
VP2, VP3) of a rAAV. In some embodiments, the terminally grafted
nuclease is present on two of the capsid proteins (e.g. VP2 and
VP3) of a rAAV. In some embodiments, the terminally grafted
nuclease is present on a single capsid protein of a rAAV. In some
embodiments, the terminally grafted nuclease is present on the VP2
capsid protein of the rAAV.
[0071] In some embodiments, the instant disclosure relates to an
adeno-associated virus (AAV) capsid protein comprising: an AAV
capsid protein having an N-terminally grafted nuclease, wherein the
AAV capsid protein is not of an AAV2 serotype. In some embodiments,
the AAV capsid protein is of an AAV serotype selected from the
group consisting of AAV3, AAV4, AAV5, AAV6, AAV8, AAVrh8 AAV9, and
AAV10. In some embodiments, the capsid protein having an
N-terminally grafted nuclease is a viral protein 2 (VP2) capsid
protein. In some embodiments, the AAV capsid protein having a
terminally grafted nuclease is of a serotype derived from a
non-human primate. In some embodiments, the AAV capsid protein
having a terminally grafted nuclease is of a AAVrh8 serotype. In
some embodiments, the AAV capsid protein having an N-terminally
grafted nuclease is of an AAV9, optionally AAV9.47, serotype.
[0072] In some aspects, the instant disclosure relates to the
location within an AAV capsid protein where a nuclease is grafted.
In some embodiments, the nuclease is N-terminally grafted to the
capsid protein. In some embodiments, the nuclease is C-terminally
grafted to a capsid protein. In some embodiments, a nuclease that
is C-terminally grafted to a capsid protein (e.g., VP2) resides
within the viral particle, and the viral particle does not contain
a genome, e.g., a nucleic acid harboring a transgene.
[0073] The components to be cultured in the host cell to package a
rAAV vector in an AAV capsid may be provided to the host cell in
trans. Alternatively, any one or more of the required components
(e.g., recombinant AAV vector, rep sequences, cap sequences, and/or
helper functions) may be provided by a stable host cell which has
been engineered to contain one or more of the required components
using methods known to those of skill in the art. Most suitably,
such a stable host cell will contain the required component(s)
under the control of an inducible promoter. However, the required
component(s) may be under the control of a constitutive promoter.
Examples of suitable inducible and constitutive promoters are
provided herein, in the discussion of regulatory elements suitable
for use with the transgene. In still another alternative, a
selected stable host cell may contain selected component(s) under
the control of a constitutive promoter and other selected
component(s) under the control of one or more inducible promoters.
For example, a stable host cell may be generated which is derived
from 293 cells (which contain E1 helper functions under the control
of a constitutive promoter), but which contain the rep and/or cap
proteins under the control of inducible promoters. Still other
stable host cells may be generated by one of skill in the art.
[0074] In some embodiments, the instant disclosure relates to a
host cell containing a nucleic acid that comprises a coding
sequence encoding a nuclease terminally grafted to a capsid protein
that is operably linked to a promoter. In some embodiments, the
instant disclosure relates to a composition comprising the host
cell described above. In some embodiments, the composition
comprising the host cell above further comprises a
cryopreservative.
[0075] The recombinant AAV vector, rep sequences, cap sequences,
and helper functions required for producing the rAAV of the
disclosure may be delivered to the packaging host cell using any
appropriate genetic element (vector). The selected genetic element
may be delivered by any suitable method, including those described
herein. The methods used to construct any embodiment of this
disclosure are known to those with skill in nucleic acid
manipulation and include genetic engineering, recombinant
engineering, and synthetic techniques. See, e.g., Sambrook et al,
Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press,
Cold Spring Harbor, N.Y. Similarly, methods of generating rAAV
virions are well known and the selection of a suitable method is
not a limitation on the present disclosure. See, e.g., K. Fisher et
al, J. Virol., 70:520-532 (1993) and U.S. Pat. No. 5,478,745.
[0076] In some embodiments, recombinant AAVs may be produced using
the triple transfection method (described in detail in U.S. Pat.
No. 6,001,650). Typically, the recombinant AAVs are produced by
transfecting a host cell with an recombinant AAV vector (comprising
a transgene) to be packaged into AAV particles, an AAV helper
function vector, and an accessory function vector. An AAV helper
function vector encodes the "AAV helper function" sequences (i.e.,
rep and cap), which function in trans for productive AAV
replication and encapsidation. Preferably, the AAV helper function
vector supports efficient AAV vector production without generating
any detectable wild-type AAV virions (i.e., AAV virions containing
functional rep and cap genes). Non-limiting examples of vectors
suitable for use with the present disclosure include pHLP19,
described in U.S. Pat. No. 6,001,650 and pRep6cap6 vector,
described in U.S. Pat. No. 6,156,303, the entirety of both
incorporated by reference herein. The accessory function vector
encodes nucleotide sequences for non-AAV derived viral and/or
cellular functions upon which AAV is dependent for replication
(i.e., "accessory functions"). The accessory functions include
those functions required for AAV replication, including, without
limitation, those moieties involved in activation of AAV gene
transcription, stage specific AAV mRNA splicing, AAV DNA
replication, synthesis of cap expression products, and AAV capsid
assembly. Viral-based accessory functions can be derived from any
of the known helper viruses such as adenovirus, herpesvirus (other
than herpes simplex virus type-1), and vaccinia virus.
[0077] In some aspects, the disclosure provides transfected host
cells. The term "transfection" is used to refer to the uptake of
foreign DNA by a cell, and a cell has been "transfected" when
exogenous DNA has been introduced inside the cell membrane. A
number of transfection techniques are generally known in the art.
See, e.g., Graham et al. (1973) Virology, 52:456, Sambrook et al.
(1989) Molecular Cloning, a laboratory manual, Cold Spring Harbor
Laboratories, New York, Davis et al. (1986) Basic Methods in
Molecular Biology, Elsevier, and Chu et al. (1981) Gene 13:197.
Such techniques can be used to introduce one or more exogenous
nucleic acids, such as a nucleotide integration vector and other
nucleic acid molecules, into suitable host cells.
[0078] A "host cell" refers to any cell that harbors, or is capable
of harboring, a substance of interest. Often a host cell is a
mammalian cell. A host cell may be used as a recipient of an AAV
helper construct, an AAV minigene plasmid, an accessory function
vector, or other transfer DNA associated with the production of
recombinant AAVs. The term includes the progeny of the original
cell which has been transfected. Thus, a "host cell" as used herein
may refer to a cell which has been transfected with an exogenous
DNA sequence. It is understood that the progeny of a single
parental cell may not necessarily be completely identical in
morphology or in genomic or total DNA complement as the original
parent, due to natural, accidental, or deliberate mutation.
[0079] As used herein, the term "cell line" refers to a population
of cells capable of continuous or prolonged growth and division in
vitro. Often, cell lines are clonal populations derived from a
single progenitor cell. It is further known in the art that
spontaneous or induced changes can occur in karyotype during
storage or transfer of such clonal populations. Therefore, cells
derived from the cell line referred to may not be precisely
identical to the ancestral cells or cultures, and the cell line
referred to includes such variants.
[0080] As used herein, the terms "recombinant cell" refers to a
cell into which an exogenous DNA segment, such as DNA segment that
leads to the transcription of a biologically-active polypeptide or
production of a biologically active nucleic acid such as an RNA,
has been introduced.
[0081] As used herein, the term "vector" includes any genetic
element, such as a plasmid, phage, transposon, cosmid, chromosome,
artificial chromosome, virus, virion, etc., which is capable of
replication when associated with the proper control elements and
which can transfer gene sequences between cells. Thus, the term
includes cloning and expression vehicles, as well as viral vectors.
In some embodiments, useful vectors are contemplated to be those
vectors in which the nucleic acid segment to be transcribed is
positioned under the transcriptional control of a promoter. A
"promoter" refers to a DNA sequence recognized by the synthetic
machinery of the cell, or introduced synthetic machinery, required
to initiate the specific transcription of a gene. The phrases
"operatively positioned," "under control" or "under transcriptional
control" means that the promoter is in the correct location and
orientation in relation to the nucleic acid to control RNA
polymerase initiation and expression of the gene. The term
"expression vector or construct" means any type of genetic
construct containing a nucleic acid in which part or all of the
nucleic acid encoding sequence is capable of being transcribed. In
some embodiments, expression includes transcription of the nucleic
acid, for example, to generate a biologically-active polypeptide
product or functional RNA (e.g., guide RNA) from a transcribed
gene.
[0082] The foregoing methods for packaging recombinant vectors in
desired AAV capsids to produce the rAAVs of the disclosure are not
meant to be limiting and other suitable methods will be apparent to
the skilled artisan.
Recombinant AAV Vectors
[0083] "Recombinant AAV (rAAV) vectors" of the disclosure are
typically composed of, at a minimum, a transgene and its regulatory
sequences, and 5' and 3' AAV inverted terminal repeats (ITRs). It
is this recombinant AAV vector which is packaged into a capsid
protein and delivered to a selected target cell. In some
embodiments, the transgene is a nucleic acid sequence, heterologous
to the vector sequences, which encodes a polypeptide, protein,
functional RNA molecule (e.g., gRNA) or other gene product, of
interest. The nucleic acid coding sequence is operatively linked to
regulatory components in a manner which permits transgene
transcription, translation, and/or expression in a cell of a target
tissue.
[0084] In some embodiments, the instant disclosure relates to a
recombinant AAV (rAAV) comprising a capsid protein having an
N-terminally grafted nuclease, wherein the N-terminally grafted
nuclease is present only in the VP2 capsid protein. In some
embodiments, the rAAV comprises a capsid protein having an amino
acid sequence represented by SEQ ID NO: 4.
[0085] The AAV sequences of the vector typically comprise the
cis-acting 5' and 3' inverted terminal repeat sequences (See, e.g.,
B. J. Carter, in "Handbook of Parvoviruses", ed., P. Tijsser, CRC
Press, pp. 155 168 (1990)). The ITR sequences are about 145 bp in
length. Preferably, substantially the entire sequences encoding the
ITRs are used in the molecule, although some degree of minor
modification of these sequences is permissible. The ability to
modify these ITR sequences is within the skill of the art. (See,
e.g., texts such as Sambrook et al, "Molecular Cloning. A
Laboratory Manual", 2d ed., Cold Spring Harbor Laboratory, New York
(1989); and K. Fisher et al., J Virol., 70:520 532 (1996)). An
example of such a molecule employed in the present disclosure is a
"cis-acting" plasmid containing the transgene, in which the
selected transgene sequence and associated regulatory elements are
flanked by the 5' and 3' AAV ITR sequences. The AAV ITR sequences
may be obtained from any known AAV, including presently identified
mammalian AAV types.
[0086] In addition to the major elements identified above for the
recombinant AAV vector, the vector also includes conventional
control elements necessary which are operably linked to the
transgene in a manner which permits its transcription, translation
and/or expression in a cell transfected with the plasmid vector or
infected with the virus produced by the disclosure. As used herein,
"operably linked" sequences include both expression control
sequences that are contiguous with the gene of interest and
expression control sequences that act in trans or at a distance to
control the gene of interest.
[0087] Expression control sequences include appropriate
transcription initiation, termination, promoter and enhancer
sequences; efficient RNA processing signals such as splicing and
polyadenylation (polyA) signals; sequences that stabilize
cytoplasmic mRNA; sequences that enhance translation efficiency
(i.e., Kozak consensus sequence); sequences that enhance protein
stability; and when desired, sequences that enhance secretion of
the encoded product. A great number of expression control
sequences, including promoters which are native, constitutive,
inducible and/or tissue-specific, are known in the art and may be
utilized.
[0088] As used herein, a nucleic acid sequence (e.g., coding
sequence) and regulatory sequences are said to be "operably" linked
when they are covalently linked in such a way as to place the
expression or transcription of the nucleic acid sequence under the
influence or control of the regulatory sequences. If it is desired
that the nucleic acid sequences be translated into a functional
protein, two DNA sequences are said to be operably linked if
induction of a promoter in the 5' regulatory sequences results in
the transcription of the coding sequence and if the nature of the
linkage between the two DNA sequences does not (1) result in the
introduction of a frame-shift mutation, (2) interfere with the
ability of the promoter region to direct the transcription of the
coding sequences, or (3) interfere with the ability of the
corresponding RNA transcript to be translated into a protein. Thus,
a promoter region would be operably linked to a nucleic acid
sequence if the promoter region were capable of effecting
transcription of that DNA sequence such that the resulting
transcript might be translated into the desired protein or
polypeptide. Similarly two or more coding regions are operably
linked when they are linked in such a way that their transcription
from a common promoter results in the expression of two or more
proteins having been translated in frame. In some embodiments,
operably linked coding sequences yield a fusion protein. In some
embodiments, operably linked coding sequences yield a functional
RNA (e.g., gRNA).
[0089] For nucleic acids encoding proteins, a polyadenylation
sequence generally is inserted following the transgene sequences
and before the 3' AAV ITR sequence. A rAAV construct useful in the
present disclosure may also contain an intron, desirably located
between the promoter/enhancer sequence and the transgene. One
possible intron sequence is derived from SV-40, and is referred to
as the SV-40 T intron sequence. Another vector element that may be
used is an internal ribosome entry site (IRES). An IRES sequence is
used to produce more than one polypeptide from a single gene
transcript. An IRES sequence would be used to produce a protein
that contain more than one polypeptide chains. Selection of these
and other common vector elements are conventional and many such
sequences are available [see, e.g., Sambrook et al, and references
cited therein at, for example, pages 3.18 3.26 and 16.17 16.27 and
Ausubel et al., Current Protocols in Molecular Biology, John Wiley
& Sons, New York, 1989]. In some embodiments, a Foot and Mouth
Disease Virus 2A sequence is included in polyprotein; this is a
small peptide (approximately 18 amino acids in length) that has
been shown to mediate the cleavage of polyproteins (Ryan, M D et
al., EMBO, 1994; 4: 928-933; Mattion, N M et al., J Virology,
November 1996; p. 8124-8127; Furler, S et al., Gene Therapy, 2001;
8: 864-873; and Halpin, C et al., The Plant Journal, 1999; 4:
453-459). The cleavage activity of the 2A sequence has previously
been demonstrated in artificial systems including plasmids and gene
therapy vectors (AAV and retroviruses) (Ryan, M D et al., EMBO,
1994; 4: 928-933; Mattion, N M et al., J Virology, November 1996;
p. 8124-8127; Furler, S et al., Gene Therapy, 2001; 8: 864-873; and
Halpin, C et al., The Plant Journal, 1999; 4: 453-459; de Felipe, P
et al., Gene Therapy, 1999; 6: 198-208; de Felipe, P et al., Human
Gene Therapy, 2000; 11: 1921-1931.; and Klump, H et al., Gene
Therapy, 2001; 8: 811-817).
[0090] The precise nature of the regulatory sequences needed for
gene expression in host cells may vary between species, tissues or
cell types, but shall in general include, as necessary, 5'
non-transcribed and 5' non-translated sequences involved with the
initiation of transcription and translation respectively, such as a
TATA box, capping sequence, CAAT sequence, enhancer elements, and
the like. Especially, such 5' non-transcribed regulatory sequences
will include a promoter region that includes a promoter sequence
for transcriptional control of the operably joined gene. Regulatory
sequences may also include enhancer sequences or upstream activator
sequences as desired. The vectors of the disclosure may optionally
include 5' leader or signal sequences. The choice and design of an
appropriate vector is within the ability and discretion of one of
ordinary skill in the art.
[0091] Examples of constitutive promoters include, without
limitation, the retroviral Rous sarcoma virus (RSV) LTR promoter
(optionally with the RSV enhancer), the cytomegalovirus (CMV)
promoter (optionally with the CMV enhancer) [see, e.g., Boshart et
al, Cell, 41:521-530 (1985)], the SV40 promoter, the dihydrofolate
reductase promoter, the .beta.-actin promoter, the phosphoglycerol
kinase (PGK) promoter, and the EF1.alpha. promoter
[Invitrogen].
[0092] Inducible promoters allow regulation of gene expression and
can be regulated by exogenously supplied compounds, environmental
factors such as temperature, or the presence of a specific
physiological state, e.g., acute phase, a particular
differentiation state of the cell, or in replicating cells only.
Inducible promoters and inducible systems are available from a
variety of commercial sources, including, without limitation,
Invitrogen, Clontech and Ariad. Many other systems have been
described and can be readily selected by one of skill in the art.
Examples of inducible promoters regulated by exogenously supplied
promoters include the zinc-inducible sheep metallothionine (MT)
promoter, the dexamethasone (Dex)-inducible mouse mammary tumor
virus (MMTV) promoter, the T7 polymerase promoter system (WO
98/10088); the ecdysone insect promoter (No et al, Proc. Natl.
Acad. Sci. USA, 93:3346-3351 (1996)), the tetracycline-repressible
system (Gossen et al, Proc. Natl. Acad. Sci. USA, 89:5547-5551
(1992)), the tetracycline-inducible system (Gossen et al, Science,
268:1766-1769 (1995), see also Harvey et al, Curr. Opin. Chem.
Biol., 2:512-518 (1998)), the RU486-inducible system (Wang et al,
Nat. Biotech., 15:239-243 (1997) and Wang et al, Gene Ther.,
4:432-441 (1997)) and the rapamycin-inducible system (Magari et al,
J. Clin. Invest., 100:2865-2872 (1997)). Still other types of
inducible promoters which may be useful in this context are those
which are regulated by a specific physiological state, e.g.,
temperature, acute phase, a particular differentiation state of the
cell, or in replicating cells only.
[0093] In another embodiment, the native promoter for the transgene
will be used. The native promoter may be preferred when it is
desired that expression of the transgene should mimic the native
expression. The native promoter may be used when expression of the
transgene must be regulated temporally or developmentally, or in a
tissue-specific manner, or in response to specific transcriptional
stimuli. In a further embodiment, other native expression control
elements, such as enhancer elements, polyadenylation sites or Kozak
consensus sequences may also be used to mimic the native
expression.
[0094] In some embodiments, the regulatory sequences impart
tissue-specific gene expression capabilities. In some cases, the
tissue-specific regulatory sequences bind tissue-specific
transcription factors that induce transcription in a tissue
specific manner. Such tissue-specific regulatory sequences (e.g.,
promoters, enhancers, etc..) are well known in the art. Exemplary
tissue-specific regulatory sequences include, but are not limited
to the following tissue specific promoters: a liver-specific
thyroxin binding globulin (TBG) promoter, an insulin promoter, a
glucagon promoter, a somatostatin promoter, a pancreatic
polypeptide (PPY) promoter, a synapsin-1 (Syn) promoter, a creatine
kinase (MCK) promoter, a mammalian desmin (DES) promoter, a
.alpha.-myosin heavy chain (a-MHC) promoter, or a cardiac Troponin
T (cTnT) promoter. Other exemplary promoters include Beta-actin
promoter, hepatitis B virus core promoter, Sandig et al., Gene
Ther., 3:1002-9 (1996); alpha-fetoprotein (AFP) promoter, Arbuthnot
et al., Hum. Gene Ther., 7:1503-14 (1996)), bone osteocalcin
promoter (Stein et al., Mol. Biol. Rep., 24:185-96 (1997)); bone
sialoprotein promoter (Chen et al., J. Bone Miner. Res., 11:654-64
(1996)), CD2 promoter (Hansal et al., J. Immunol., 161:1063-8
(1998); immunoglobulin heavy chain promoter; T cell receptor
.alpha.-chain promoter, neuronal such as neuron-specific enolase
(NSE) promoter (Andersen et al., Cell. Mol. Neurobiol., 13:503-15
(1993)), neurofilament light-chain gene promoter (Piccioli et al.,
Proc. Natl. Acad. Sci. USA, 88:5611-5 (1991)), and the
neuron-specific vgf gene promoter (Piccioli et al., Neuron,
15:373-84 (1995)), among others which will be apparent to the
skilled artisan.
[0095] In some embodiments, one or more bindings sites for one or
more of miRNAs are incorporated in a transgene of a rAAV vector, to
inhibit the expression of the transgene in one or more tissues of
an subject harboring the transgene. The skilled artisan will
appreciate that binding sites may be selected to control the
expression of a transgene in a tissue specific manner. For example,
binding sites for the liver-specific miR-122 may be incorporated
into a transgene to inhibit expression of that transgene in the
liver. The target sites in the mRNA may be in the 5' UTR, the 3'
UTR or in the coding region. Typically, the target site is in the
3' UTR of the mRNA. Furthermore, the transgene may be designed such
that multiple miRNAs regulate the mRNA by recognizing the same or
multiple sites. The presence of multiple miRNA binding sites may
result in the cooperative action of multiple RISCs and provide
highly efficient inhibition of expression. The target site sequence
may comprise a total of 5-100, 10-60, or more nucleotides. The
target site sequence may comprise at least 5 nucleotides of the
sequence of a target gene binding site.
Recombinant AAV Vector: Transgene Coding Sequences
[0096] The composition of the transgene sequence of the rAAV vector
will depend upon the use to which the resulting vector will be put.
For example, one type of transgene sequence includes a reporter
sequence, which upon expression produces a detectable signal. In
another example, the transgene encodes a therapeutic protein or
therapeutic functional RNA. In another example, the transgene
encodes a protein or functional RNA that is intended to be used for
research purposes, e.g., to create a somatic transgenic animal
model harboring the transgene, e.g., to study the function of the
transgene product. In another example, the transgene encodes a
protein or functional RNA that is intended to be used to create an
animal model of disease. Appropriate transgene coding sequences
will be apparent to the skilled artisan.
[0097] Also contemplated herein are methods of delivering a
transgene to a subject using the rAAVs described herein. In some
embodiments, the instant disclosure relates to a method for
delivering a transgene to a subject comprising administering a rAAV
to a subject, wherein the rAAV comprises: (i) a capsid protein
having a terminally grafted nuclease, e.g., a nuclease having a
sequence set forth as SEQ ID NO: 2, and optionally (ii) at least
one transgene, e.g., a transgene encoding a gRNA, and wherein the
rAAV infects cells of a target tissue of the subject. In some
embodiments of the method, at least one transgene encodes a single
guide RNA, a CRISPR RNA (crRNA), and/or a trans-activating crRNA
(tracrRNA).
[0098] In some embodiments, the rAAV vectors may comprise a
transgene, wherein the transgene is a gRNA. In some embodiments,
the gRNA targets a nucleic acid sequence that causes disease in a
subject. For example, expression of the huntingtin (Htt) gene
causes Huntington's disease. Without wishing to be bound by any
particular theory, a gRNA targeting the Htt gene directs Cas9
cleavage of the gene, thereby preventing its expression. Other
similar genes (disease-associated or otherwise) can be
targeted.
Recombinant AAV Administration Methods
[0099] The rAAVs may be delivered to a subject in compositions
according to any appropriate methods known in the art. The rAAV,
preferably suspended in a physiologically compatible carrier (i.e.,
in a composition), may be administered to a subject, i.e. host
animal, such as a human, mouse, rat, cat, dog, sheep, rabbit,
horse, cow, goat, pig, guinea pig, hamster, chicken, turkey, or a
non-human primate (e.g., Macaque). In some embodiments a host
animal does not include a human.
[0100] Delivery of the rAAVs to a mammalian subject may be by, for
example, intramuscular injection or by administration into the
bloodstream of the mammalian subject. Administration into the
bloodstream may be by injection into a vein, an artery, or any
other vascular conduit. In some embodiments, the rAAVs are
administered into the bloodstream by way of isolated limb
perfusion, a technique well known in the surgical arts, the method
essentially enabling the artisan to isolate a limb from the
systemic circulation prior to administration of the rAAV virions. A
variant of the isolated limb perfusion technique, described in U.S.
Pat. No. 6,177,403, can also be employed by the skilled artisan to
administer the virions into the vasculature of an isolated limb to
potentially enhance transduction into muscle cells or tissue.
Moreover, in certain instances, it may be desirable to deliver the
virions to the CNS of a subject. By "CNS" is meant all cells and
tissue of the brain and spinal cord of a vertebrate. Thus, the term
includes, but is not limited to, neuronal cells, glial cells,
astrocytes, cereobrospinal fluid (CSF), interstitial spaces, bone,
cartilage and the like. Recombinant AAVs may be delivered directly
to the CNS or brain by injection into, e.g., the ventricular
region, as well as to the striatum (e.g., the caudate nucleus or
putamen of the striatum), spinal cord and neuromuscular junction,
or cerebellar lobule, with a needle, catheter or related device,
using neurosurgical techniques known in the art, such as by
stereotactic injection (see, e.g., Stein et al., J Virol
73:3424-3429, 1999; Davidson et al., PNAS 97:3428-3432, 2000;
Davidson et al., Nat. Genet. 3:219-223, 1993; and Alisky and
Davidson, Hum. Gene Ther. 11:2315-2329, 2000).
[0101] Aspects of the instant disclosure relate to compositions
comprising a recombinant AAV comprising a capsid protein having a
terminally grafted (e.g., N-terminally grafted or C-terminally
grafted) nuclease. In some embodiments, the nuclease is terminally
grafted onto a capsid protein. In some embodiments, the a
terminally grafted nuclease is present on all three capsid proteins
(e.g. VP1, VP2, VP3) of the rAAV. In some embodiments, the
terminally grafted nuclease is present on two of the capsid
proteins (e.g. VP2 and VP3) of the rAAV. In some embodiments, the
terminally grafted nuclease is present on a single capsid protein
of the rAAV. In some embodiments, the terminally grafted nuclease
is present on the VP2 capsid protein of the rAAV. In some
embodiments, the composition further comprises a pharmaceutically
acceptable carrier.
[0102] The compositions of the disclosure may comprise an rAAV
alone, or in combination with one or more other viruses (e.g., a
second rAAV encoding having one or more different transgenes). In
some embodiments, a composition comprises 1, 2, 3, 4, 5, 6, 7, 8,
9, 10, or more different rAAVs each having one or more different
transgenes.
[0103] Suitable carriers may be readily selected by one of skill in
the art in view of the indication for which the rAAV is directed.
For example, one suitable carrier includes saline, which may be
formulated with a variety of buffering solutions (e.g., phosphate
buffered saline). Other exemplary carriers include sterile saline,
lactose, sucrose, calcium phosphate, gelatin, dextran, agar,
pectin, peanut oil, sesame oil, and water. The selection of the
carrier is not a limitation of the present disclosure.
[0104] Optionally, the compositions of the disclosure may contain,
in addition to the rAAV and carrier(s), other conventional
pharmaceutical ingredients, such as preservatives, or chemical
stabilizers. Suitable exemplary preservatives include
chlorobutanol, potassium sorbate, sorbic acid, sulfur dioxide,
propyl gallate, the parabens, ethyl vanillin, glycerin, phenol, and
parachlorophenol. Suitable chemical stabilizers include gelatin and
albumin.
[0105] The rAAVS are administered in sufficient amounts to
transfect the cells of a desired tissue and to provide sufficient
levels of gene transfer and expression without undue adverse
effects. Conventional and pharmaceutically acceptable routes of
administration include, but are not limited to, direct delivery to
the selected organ (e.g., intraportal delivery to the liver), oral,
inhalation (including intranasal and intratracheal delivery),
intraocular, intravenous, intramuscular, subcutaneous, intradermal,
intratumoral, and other parental routes of administration. Routes
of administration may be combined, if desired.
[0106] The dose of rAAV virions required to achieve a particular
"therapeutic effect," e.g., the units of dose in genome copies/per
kilogram of body weight (GC/kg), will vary based on several factors
including, but not limited to: the route of rAAV virion
administration, the level of gene or RNA expression required to
achieve a therapeutic effect, the specific disease or disorder
being treated, and the stability of the gene or RNA product. One of
skill in the art can readily determine a rAAV virion dose range to
treat a patient having a particular disease or disorder based on
the aforementioned factors, as well as other factors that are well
known in the art.
[0107] An effective amount of an rAAV is an amount sufficient to
target infect an animal, target a desired tissue. In some
embodiments, an effective amount of an rAAV is an amount sufficient
to produce a stable somatic transgenic animal model. The effective
amount will depend primarily on factors such as the species, age,
weight, health of the subject, and the tissue to be targeted, and
may thus vary among animal and tissue. For example, an effective
amount of the rAAV is generally in the range of from about 1 ml to
about 100 ml of solution containing from about 10.sup.9 to
10.sup.16 genome copies. In some cases, a dosage between about
10.sup.11 to 10.sup.13 rAAV genome copies is appropriate. In
certain embodiments, 10.sup.12 or 10.sup.13 rAAV genome copies is
effective to target heart, liver, and pancreas tissues. In some
cases, stable transgenic animals are produced by multiple doses of
an rAAV.
[0108] In some embodiments, rAAV compositions are formulated to
reduce aggregation of AAV particles in the composition,
particularly where high rAAV concentrations are present (e.g.,
.about.10.sup.13 GC/ml or more). Methods for reducing aggregation
of rAAVs are well known in the art and, include, for example,
addition of surfactants, pH adjustment, salt concentration
adjustment, etc. (See, e.g., Wright F R, et al., Molecular Therapy
(2005) 12, 171-178, the contents of which are incorporated herein
by reference.)
[0109] Formulation of pharmaceutically-acceptable excipients and
carrier solutions is well-known to those of skill in the art, as is
the development of suitable dosing and treatment regimens for using
the particular compositions described herein in a variety of
treatment regimens.
[0110] Typically, these formulations may contain at least about
0.1% of the active compound or more, although the percentage of the
active ingredient(s) may, of course, be varied and may conveniently
be between about 1 or 2% and about 70% or 80% or more of the weight
or volume of the total formulation. Naturally, the amount of active
compound in each therapeutically-useful composition may be prepared
is such a way that a suitable dosage will be obtained in any given
unit dose of the compound. Factors such as solubility,
bioavailability, biological half-life, route of administration,
product shelf life, as well as other pharmacological considerations
will be contemplated by one skilled in the art of preparing such
pharmaceutical formulations, and as such, a variety of dosages and
treatment regimens may be desirable.
[0111] In certain circumstances it will be desirable to deliver the
rAAV-based therapeutic constructs in suitably formulated
pharmaceutical compositions disclosed herein either subcutaneously,
intraopancreatically, intranasally, parenterally, intravenously,
intramuscularly, intrathecally, or orally, intraperitoneally, or by
inhalation. In some embodiments, the administration modalities as
described in U.S. Pat. Nos. 5,543,158; 5,641,515 and 5,399,363
(each specifically incorporated herein by reference in its
entirety) may be used to deliver rAAVs. In some embodiments, a
preferred mode of administration is by portal vein injection.
[0112] The pharmaceutical forms suitable for injectable use include
sterile aqueous solutions or dispersions and sterile powders for
the extemporaneous preparation of sterile injectable solutions or
dispersions. Dispersions may also be prepared in glycerol, liquid
polyethylene glycols, and mixtures thereof and in oils. Under
ordinary conditions of storage and use, these preparations contain
a preservative to prevent the growth of microorganisms. In many
cases the form is sterile and fluid to the extent that easy
syringability exists. It must be stable under the conditions of
manufacture and storage and must be preserved against the
contaminating action of microorganisms, such as bacteria and fungi.
The carrier can be a solvent or dispersion medium containing, for
example, water, ethanol, polyol (e.g., glycerol, propylene glycol,
and liquid polyethylene glycol, and the like), suitable mixtures
thereof, and/or vegetable oils. Proper fluidity may be maintained,
for example, by the use of a coating, such as lecithin, by the
maintenance of the required particle size in the case of dispersion
and by the use of surfactants. The prevention of the action of
microorganisms can be brought about by various antibacterial and
antifungal agents, for example, parabens, chlorobutanol, phenol,
sorbic acid, thimerosal, and the like. In many cases, it will be
preferable to include isotonic agents, for example, sugars or
sodium chloride. Prolonged absorption of the injectable
compositions can be brought about by the use in the compositions of
agents delaying absorption, for example, aluminum monostearate and
gelatin.
[0113] For administration of an injectable aqueous solution, for
example, the solution may be suitably buffered, if necessary, and
the liquid diluent first rendered isotonic with sufficient saline
or glucose. These particular aqueous solutions are especially
suitable for intravenous, intramuscular, subcutaneous and
intraperitoneal administration. In this connection, a sterile
aqueous medium that can be employed will be known to those of skill
in the art. For example, one dosage may be dissolved in 1 ml of
isotonic NaCl solution and either added to 1000 ml of
hypodermoclysis fluid or injected at the proposed site of infusion,
(see for example, "Remington's Pharmaceutical Sciences" 15th
Edition, pages 1035-1038 and 1570-1580). Some variation in dosage
will necessarily occur depending on the condition of the host. The
person responsible for administration will, in any event, determine
the appropriate dose for the individual host.
[0114] Sterile injectable solutions are prepared by incorporating
the active rAAV in the required amount in the appropriate solvent
with various of the other ingredients enumerated herein, as
required, followed by filtered sterilization. Generally,
dispersions are prepared by incorporating the various sterilized
active ingredients into a sterile vehicle which contains the basic
dispersion medium and the required other ingredients from those
enumerated above. In the case of sterile powders for the
preparation of sterile injectable solutions, the preferred methods
of preparation are vacuum-drying and freeze-drying techniques which
yield a powder of the active ingredient plus any additional desired
ingredient from a previously sterile-filtered solution thereof.
[0115] The rAAV compositions disclosed herein may also be
formulated in a neutral or salt form. Pharmaceutically-acceptable
salts, include the acid addition salts (formed with the free amino
groups of the protein) and which are formed with inorganic acids
such as, for example, hydrochloric or phosphoric acids, or such
organic acids as acetic, oxalic, tartaric, mandelic, and the like.
Salts formed with the free carboxyl groups can also be derived from
inorganic bases such as, for example, sodium, potassium, ammonium,
calcium, or ferric hydroxides, and such organic bases as
isopropylamine, trimethylamine, histidine, procaine and the like.
Upon formulation, solutions will be administered in a manner
compatible with the dosage formulation and in such amount as is
therapeutically effective. The formulations are easily administered
in a variety of dosage forms such as injectable solutions,
drug-release capsules, and the like.
[0116] As used herein, "carrier" includes any and all solvents,
dispersion media, vehicles, coatings, diluents, antibacterial and
antifungal agents, isotonic and absorption delaying agents,
buffers, carrier solutions, suspensions, colloids, and the like.
The use of such media and agents for pharmaceutical active
substances is well known in the art. Supplementary active
ingredients can also be incorporated into the compositions. The
phrase "pharmaceutically-acceptable" refers to molecular entities
and compositions that do not produce an allergic or similar
untoward reaction when administered to a host.
[0117] Delivery vehicles such as liposomes, nanocapsules,
microparticles, microspheres, lipid particles, vesicles, and the
like, may be used for the introduction of the compositions of the
present disclosure into suitable host cells. In particular, the
rAAV vector delivered transgenes may be formulated for delivery
either encapsulated in a lipid particle, a liposome, a vesicle, a
nanosphere, or a nanoparticle or the like.
[0118] Such formulations may be preferred for the introduction of
pharmaceutically acceptable formulations of the nucleic acids or
the rAAV constructs disclosed herein. The formation and use of
liposomes is generally known to those of skill in the art.
Recently, liposomes were developed with improved serum stability
and circulation half-times (U.S. Pat. No. 5,741,516). Further,
various methods of liposome and liposome like preparations as
potential drug carriers have been described (U.S. Pat. Nos.
5,567,434; 5,552,157; 5,565,213; 5,738,868 and 5,795,587).
[0119] Liposomes have been used successfully with a number of cell
types that are normally resistant to transfection by other
procedures. In addition, liposomes are free of the DNA length
constraints that are typical of viral-based delivery systems.
Liposomes have been used effectively to introduce genes, drugs,
radiotherapeutic agents, viruses, transcription factors and
allosteric effectors into a variety of cultured cell lines and
animals. In addition, several successful clinical trials examining
the effectiveness of liposome-mediated drug delivery have been
completed.
[0120] Liposomes are formed from phospholipids that are dispersed
in an aqueous medium and spontaneously form multilamellar
concentric bilayer vesicles (also termed multilamellar vesicles
(MLVs). MLVs generally have diameters of from 25 nm to 4 .mu.m.
Sonication of MLVs results in the formation of small unilamellar
vesicles (SUVs) with diameters in the range of 200 to 500 ANG.,
containing an aqueous solution in the core.
[0121] Alternatively, nanocapsule formulations of the rAAV may be
used. Nanocapsules can generally entrap substances in a stable and
reproducible way. To avoid side effects due to intracellular
polymeric overloading, such ultrafine particles (sized around 0.1
.mu.m) should be designed using polymers able to be degraded in
vivo. Biodegradable polyalkyl-cyanoacrylate nanoparticles that meet
these requirements are contemplated for use.
[0122] In addition to the methods of delivery described above, the
following techniques are also contemplated as alternative methods
of delivering the rAAV compositions to a host. Sonophoresis (e.g.,
ultrasound) has been used and described in U.S. Pat. No. 5,656,016
as a device for enhancing the rate and efficacy of drug permeation
into and through the circulatory system. Other drug delivery
alternatives contemplated are intraosseous injection (U.S. Pat. No.
5,779,708), microchip devices (U.S. Pat. No. 5,797,898), ophthalmic
formulations (Bourlais et al., 1998), transdermal matrices (U.S.
Pat. Nos. 5,770,219 and 5,783,208) and feedback-controlled delivery
(U.S. Pat. No. 5,697,899).
Kits and Related Compositions
[0123] The agents described herein may, in some embodiments, be
assembled into pharmaceutical or diagnostic or research kits to
facilitate their use in therapeutic, diagnostic or research
applications. A kit may include one or more containers housing the
components of the disclosure and instructions for use.
Specifically, such kits may include one or more agents described
herein, along with instructions describing the intended application
and the proper use of these agents. In certain embodiments agents
in a kit may be in a pharmaceutical formulation and dosage suitable
for a particular application and for a method of administration of
the agents. Kits for research purposes may contain the components
in appropriate concentrations or quantities for running various
experiments.
[0124] In some embodiments, the instant disclosure relates to a kit
for producing a rAAV, the kit comprising a container housing an
isolated nucleic acid having a sequence of SEQ ID NO: 1 or SEQ ID
NO: 3. In some embodiments, the kit further comprises instructions
for producing the rAAV. In some embodiments, the kit further
comprises at least one container housing a recombinant AAV vector,
wherein the recombinant AAV vector comprises a transgene.
[0125] In some embodiments, the instant disclosure relates to a kit
comprising a container housing a recombinant AAV having an isolated
AAV capsid protein having an amino acid sequence as set forth in
any of SEQ ID NO: 4.
[0126] The kit may be designed to facilitate use of the methods
described herein by researchers and can take many forms. Each of
the compositions of the kit, where applicable, may be provided in
liquid form (e.g., in solution), or in solid form, (e.g., a dry
powder). In certain cases, some of the compositions may be
constitutable or otherwise processable (e.g., to an active form),
for example, by the addition of a suitable solvent or other species
(for example, water or a cell culture medium), which may or may not
be provided with the kit. As used herein, "instructions" can define
a component of instruction and/or promotion, and typically involve
written instructions on or associated with packaging of the
disclosure. Instructions also can include any oral or electronic
instructions provided in any manner such that a user will clearly
recognize that the instructions are to be associated with the kit,
for example, audiovisual (e.g., videotape, DVD, etc.), Internet,
and/or web-based communications, etc. The written instructions may
be in a form prescribed by a governmental agency regulating the
manufacture, use or sale of pharmaceuticals or biological products,
which instructions can also reflects approval by the agency of
manufacture, use or sale for animal administration.
[0127] The kit may contain any one or more of the components
described herein in one or more containers. As an example, in one
embodiment, the kit may include instructions for mixing one or more
components of the kit and/or isolating and mixing a sample and
applying to a subject. The kit may include a container housing
agents described herein. The agents may be in the form of a liquid,
gel or solid (powder). The agents may be prepared sterilely,
packaged in syringe and shipped refrigerated. Alternatively it may
be housed in a vial or other container for storage. A second
container may have other agents prepared sterilely. Alternatively
the kit may include the active agents premixed and shipped in a
syringe, vial, tube, or other container. The kit may have one or
more or all of the components required to administer the agents to
an animal, such as a syringe, topical application devices, or iv
needle tubing and bag, particularly in the case of the kits for
producing specific somatic animal models.
[0128] In some cases, the methods involve transfecting cells with
total cellular DNAs isolated from the tissues that potentially
harbor proviral AAV genomes at very low abundance and supplementing
with helper virus function (e.g., adenovirus) to trigger and/or
boost AAV rep and cap gene transcription in the transfected cell.
In some cases, RNA from the transfected cells provides a template
for RT-PCR amplification of cDNA and the detection of novel AAVs.
In cases where cells are transfected with total cellular DNAs
isolated from the tissues that potentially harbor proviral AAV
genomes, it is often desirable to supplement the cells with factors
that promote AAV gene transcription. For example, the cells may
also be infected with a helper virus, such as an Adenovirus or a
Herpes Virus. In a specific embodiment, the helper functions are
provided by an adenovirus. The adenovirus may be a wild-type
adenovirus, and may be of human or non-human origin, preferably
non-human primate (NHP) origin. Similarly adenoviruses known to
infect non-human animals (e.g., chimpanzees, mouse) may also be
employed in the methods of the disclosure (See, e.g., U.S. Pat. No.
6,083,716). In addition to wild-type adenoviruses, recombinant
viruses or non-viral vectors (e.g., plasmids, episomes, etc.)
carrying the necessary helper functions may be utilized. Such
recombinant viruses are known in the art and may be prepared
according to published techniques. See, e.g., U.S. Pat. No.
5,871,982 and U.S. Pat. No. 6,251,677, which describe a hybrid
Ad/AAV virus. A variety of adenovirus strains are available from
the American Type Culture Collection, Manassas, Va., or available
by request from a variety of commercial and institutional sources.
Further, the sequences of many such strains are available from a
variety of databases including, e.g., PubMed and GenBank.
[0129] Cells may also be transfected with a vector (e.g., helper
vector) which provides helper functions to the AAV. The vector
providing helper functions may provide adenovirus functions,
including, e.g., E1a, E1b, E2a, E4ORF6. The sequences of adenovirus
gene providing these functions may be obtained from any known
adenovirus serotype, such as serotypes 2, 3, 4, 7, 12 and 40, and
further including any of the presently identified human types known
in the art. Thus, in some embodiments, the methods involve
transfecting the cell with a vector expressing one or more genes
necessary for AAV replication, AAV gene transcription, and/or AAV
packaging.
[0130] In some cases, a capsid gene can be used to construct and
package recombinant AAV vectors, using methods well known in the
art, to determine functional characteristics associated with the
novel capsid protein encoded by the gene. For example, novel
isolated capsid genes can be used to construct and package
recombinant AAV (rAAV) vectors comprising a reporter gene (e.g.,
B-Galactosidase, GFP, Luciferase, etc.). The rAAV vector can then
be delivered to an animal (e.g., mouse) and the tissue targeting
properties of the novel isolated capsid gene can be determined by
examining the expression of the reporter gene in various tissues
(e.g., heart, liver, kidneys) of the animal. Other methods for
characterizing the novel isolated capsid genes are disclosed herein
and still others are well known in the art.
[0131] The kit may have a variety of forms, such as a blister
pouch, a shrink wrapped pouch, a vacuum sealable pouch, a sealable
thermoformed tray, or a similar pouch or tray form, with the
accessories loosely packed within the pouch, one or more tubes,
containers, a box or a bag. The kit may be sterilized after the
accessories are added, thereby allowing the individual accessories
in the container to be otherwise unwrapped. The kits can be
sterilized using any appropriate sterilization techniques, such as
radiation sterilization, heat sterilization, or other sterilization
methods known in the art. The kit may also include other
components, depending on the specific application, for example,
containers, cell media, salts, buffers, reagents, syringes,
needles, a fabric, such as gauze, for applying or removing a
disinfecting agent, disposable gloves, a support for the agents
prior to administration etc.
[0132] The instructions included within the kit may involve methods
for detecting a latent AAV in a cell. In addition, kits of the
disclosure may include, instructions, a negative and/or positive
control, containers, diluents and buffers for the sample, sample
preparation tubes and a printed or electronic table of reference
AAV sequence for sequence comparisons.
EXAMPLES
Overview
[0133] To avoid the potential genotoxicity due to prolonged
expression gene editing components, an endonuclease protein is
transiently delivered and degrades naturally in the cell.
Specifically, AAV capsid is used as a delivery vehicle to carry a
Cas9 protein or other designer nuclease protein. AAV capsid
consists of 60 copies of three capsid proteins, VP1, VP2 and VP3,
at a ratio of 1:1:18. Although AAV capsid adopts a tightly packed
structure, it has been shown that the VP2 protein with an
N-terminal fusion protein can be incorporated into AAV capsid, and
such a chimeric AAV is infectious.
Example 1
[0134] Results provided herein indicate that when Cas9 is fused to
the N-terminus of VP2 (FIG. 1A), the resulting Cas9-VP2 fusion
protein is functional.
[0135] A plasmid expressing S. pyogenes Cas9 (SpCas9; SEQ ID NOs: 1
and 2) fused with AAV2 VP2 was constructed (FIG. 1A). The resulting
construct is represented by SEQ ID NO: 3 and the fusion protein is
represented by SEQ ID NO: 4. Transfection of the construct into
HEK293 cells yields a fusion protein product of expected size, as
demonstrated by western blotting (FIG. 1B). A fluorescence reporter
assay as illustrated in FIG. 2A was used to test if SpCas9-VP2 can
function in gene editing. In the reporter construct, the GFP is
disrupted by an insertion. The downstream out-of-frame T2A, when
shifted to in-frame, mediates translation termination and
re-initiation to produce mCherry reporter protein. In the presence
of the sgRNA targeting the insertion in the GFP sequence and a
functional SpCas9, indels by NHEJ shift the downstream T2A and
mCherry to in-frame, thus giving mCherry fluorescence signal. Using
this reporter system, SpCas9-VP2 induction of NHEJ by
co-transfection in HEK293 cells was tested (FIG. 2B). Negative
control cells expressing SpCas9 only or sgRNA only did not induce
mCherry signal. Positive control cells, co-expressing sgRNA and
SpCas9 yielded mCherry signal. When sgRNA and the SpCas9-VP2 fusion
were co-expressed, mCherry fluorescence was also observed,
demonstrating that the SpCas-VP2 fusion protein behaves similarly
as SpCas9 in inducing gene editing and NHEJ (FIG. 2B).
[0136] SpCas-VP2 can be also incorporated into AAV2 capsid to form
AAV virion. The start codon of VP2 is mutated in the cap gene from
the trans AAV production plasmid. The omission of VP2 expression
from this plasmid in HEK293 cells is validated by western blotting
using an antibody targeting a C-terminal epitope shared by VP1, VP2
and VP3. Small-scale AAV production is performed using the VP2
null-trans plasmid and the SpCas9-VP2 in replacement of the
original trans plasmid to examine the presence of Cas9 protein
covalently linked to the outer surface of AAV2 virion. ELISA is
performed using antibodies recognizing a fully assembled AAV2
virion and the HA-tagged SpCas9. Alternatively, immuno electron
microscopy is performed to visualize the presence of HA-tagged
SpCas9 immunoreactivity outside of AAV2 virion. Next, a small-scale
AAV production-infection assay is performed to validate that
SpCas9-AAV delivers the SpCas9 into HEK293 cells and mediates gene
editing. The same reporter system as illustrated in FIG. 2B is used
for this assay.
[0137] Serials of in vivo experiments using SpCas9-AAV2 expressing
EGFP and sgRNA targeting the mouse ROSA26 locus
(SpCas9-AAV2-EGFP-sgROSA26) obtained from large-scale production
are next performed. The tropism of SpCas9-AAV2 is characterized in
mice by systemic delivery. Wild-type C57BL/6J mice are injected
with SpCas9-AAV2-EGFP-sgROSA26 at postnatal day 1 (P1) via facial
vein and at 8 weeks old via tail vein, respectively. The mice are
sacrificed 3 weeks after injection and fixed. Tissues including
liver, heart, skeletal muscle, pancreas, adrenal gland, kidneys,
spleen, brain, and spinal cord are analyzed for EGFP expression by
immunofluorescence staining. The best transduced tissue(s) are
selected to demonstrate SpCas9-AAV mediated gene editing of ROSA26
locus in vivo in another group of mice treated in the same manner,
from which fresh tissues are harvested and genomic DNA extracted.
The gene editing events represented by random indels near the sgRNA
targeting site in the ROSA26 locus are investigated using Surveyor
assay and single DNA molecule sequencing.
[0138] To demonstrate the improved safety profile of the SpCas9
transiently delivered using SpCas9-AAV2 and contrast with prolonged
expression of SpCas9 from a conventional rAAV2 vector, SpCas9-AAV2
are packaged with transgene cassettes expressing sgRNAs with
reported off-target effects in mouse genome, and inject into mice.
The gene editing events at both on- and off-target genomic DNA loci
are analyzed by Surveyor assay and single DNA molecule sequencing.
Transient delivery of SpCas9 significantly reduces the chance of
off-target effect.
Example 2
[0139] Intein-mediated protein trans-splicing (PTS) is used to fuse
SpCas9 protein with VP2 after AAV assembly as illustrated in FIG.
3A. For example, the naturally split intein Npu DnaE, which has the
most robust trans-splicing activity identified so far, is used to
fuse SpCas9 protein with VP2 by PTS. The SpCas9 carries IntN, and
VP2 carries IntC. Since IntC comprises only 36 amino acid residues,
an IntC-VP2 fusion is amenable to AAV assembly. First, IntC-AAV2
virion and SpCas9-IntN protein are produced and purified
separately, and then incubated to allow for PTS to occur in vitro.
Intein-mediated PTS is a spontaneous reaction and does not require
other co-factors. The fast kinetic nature of Npu DnaE split intein
produces SpCas9-AAV2 fusion protein rapidly. The fusion protein is
further purified by dialysis.
[0140] Alternatively, IntC is fused with a truncated C-terminal
portion of SpCas9 onto AAV2 capsid to allow for in vivo
transduction. The rest portion of SpCas9 and IntN are encoded by a
transgene expression cassette as rAAV genome (FIG. 3B). Co-delivery
of the two portions of SpCas9 reconstitutes the full-length,
functional SpCas9. Importantly, as the IntC-SpCas9C protein is
degraded naturally, the long-term expression of SpCas9N-IntN only
is non-functional, thus mitigating off-target effects.
[0141] FIG. 4 shows one example of an expression construct
comprising a nucleic acid sequence encoding SpCas9 nuclease
N-terminally fused to VP2 capsid protein.
Example 3
[0142] Gene editing platforms, such as the Cas9/sgRNA system, have
been shown to induce off-target DNA double-stranded breaks (DSBs)
throughout genomes, which is associated with toxicity. Such
off-target effects not only stem from ambiguity of DNA sequence
recognition by nucleases, but also can be attributed to the
prolonged presence of an active gene editing system in a given
cell. As a result, off-target DSBs accumulate over time, and
ultimately lead to genotoxicity. To mitigate the potential toxicity
due to prolonged expression of gene editing system in vivo,
transient delivery of endonuclease protein, which induces permanent
gene editing followed by natural degradation of the endonuclease,
was examined. Specifically, the VP2 protein of AAV capsid was used
as a protein delivery vehicle to ferry the Cas9 protein in
vivo.
[0143] A sensitive gene editing reporter plasmid was constructed.
Co-transfection of the reporter plasmid and a plasmid expressing
the SpCas9-VP2 fusion protein induced gene editing in HEK293 cells.
An rAAV packaging system was modified to include a plasmid
expressing VP1 and VP3, and another plasmid expressing either the
SpCas9-VP2 fusion protein or the EGFP-VP2 fusion protein. EGFP-AAV2
(EGFP protein grafted on the AAV2 capsid) was successfully
produced. However, rAAV particles carrying SpCas9 protein were not
produced, likely because the large size of SpCas9 protein
interfered with the AAV packaging process.
[0144] The transgene encoding SpCas9 was split into halves to
utilize split intein-mediated protein trans-splicing (PTS) to
transiently reconstitute the full-length SpCas9 (FIG. 3A). When the
two parts of a split intein (termed Int.sub.N and Int.sub.C,
respectively) fuse, the split intein mediates PTS, resulting in the
generation of a fusion protein with the intein being spliced out.
Plasmids expressing the fusion proteins SpCas9.sub.N-Int.sub.N and
Int.sub.C-SpCas9.sub.C-VP2 (pU1a-Cas9.sub.n-Int.sub.n and
Int.sub.cCas9.sub.c-( )-VP2, respectively) were generated. The
designation "( )" in the Int.sub.c-Cas9.sub.c plasmid represents
the presence of a linker sequence (e.g., GS, GGGGSx3 or EAAAKx3).
Results show productive intein-mediated reconstitution of
SpCas9-VP2 protein in HEK293 cells by co-transfection (FIG. 5).
Importantly, co-transfection of plasmids expressing
SpCas9.sub.N-Int.sub.N and Int.sub.C-SpCas9.sub.C-VP2 in HEK293
cells led to gene editing based on the EGFP-ON reporter assay, as
shown in FIG. 6. EGFP fluorescence reports Cas9 cleavage and NHEJ
repair and mCherry is constitutively expressed as a control.
[0145] The Int.sub.C-SpCas9.sub.C protein to be grafted on VP2 is
equal or smaller than EGFP. Since EGFP-AAV2 successfully produced,
rAAV packaging of the Int.sub.C-SpCas9.sub.C-VP2 was investigated.
Guided by structural analysis, SpCas9 split sites close to the
C-terminus of SpCas9 were strategically screened and identified.
Cells were transfected with a plasmid encoding VP1 and VP3
proteins, and a second plasmid encoding Int.sub.c-Cas9.sub.c-(
)-VP2. Results indicate successful incorporation of
Int.sub.c-SpCas9.sub.c-VP2 polypeptide onto rAAV2 capsid (FIG.
7).
TABLE-US-00001 SEQUENCES >SEQ ID NO: 1 SpCas9 nucleic acid
sequence ATGTACCCATACGATGTTCCAGATTACGCTTCGCCGAAGAAAAAGCGCAAGGTC
GAAGCGTCCGACAAGAAGTACAGCATCGGCCTGGACATCGGCACCAACTCTGTG
GGCTGGGCCGTGATCACCGACGAGTACAAGGTGCCCAGCAAGAAATTCAAGGTG
CTGGGCAACACCGACCGGCACAGCATCAAGAAGAACCTGATCGGAGCCCTGCTG
TTCGACAGCGGCGAAACAGCCGAGGCCACCCGGCTGAAGAGAACCGCCAGAAG
AAGATACACCAGACGGAAGAACCGGATCTGCTATCTGCAAGAGATCTTCAGCAA
CGAGATGGCCAAGGTGGACGACAGCTTCTTCCACAGACTGGAAGAGTCCTTCCT
GGTGGAAGAGGATAAGAAGCACGAGCGGCACCCCATCTTCGGCAACATCGTGGA
CGAGGTGGCCTACCACGAGAAGTACCCCACCATCTACCACCTGAGAAAGAAACT
GGTGGACAGCACCGACAAGGCCGACCTGCGGCTGATCTATCTGGCCCTGGCCCA
CATGATCAAGTTCCGGGGCCACTTCCTGATCGAGGGCGACCTGAACCCCGACAA
CAGCGACGTGGACAAGCTGTTCATCCAGCTGGTGCAGACCTACAACCAGCTGTTC
GAGGAAAACCCCATCAACGCCAGCGGCGTGGACGCCAAGGCCATCCTGTCTGCC
AGACTGAGCAAGAGCAGACGGCTGGAAAATCTGATCGCCCAGCTGCCCGGCGAG
AAGAAGAATGGCCTGTTCGGCAACCTGATTGCCCTGAGCCTGGGCCTGACCCCCA
ACTTCAAGAGCAACTTCGACCTGGCCGAGGATGCCAAACTGCAGCTGAGCAAGG
ACACCTACGACGACGACCTGGACAACCTGCTGGCCCAGATCGGCGACCAGTACG
CCGACCTGTTTCTGGCCGCCAAGAACCTGTCCGACGCCATCCTGCTGAGCGACAT
CCTGAGAGTGAACACCGAGATCACCAAGGCCCCCCTGAGCGCCTCTATGATCAA
GAGATACGACGAGCACCACCAGGACCTGACCCTGCTGAAAGCTCTCGTGCGGCA
GCAGCTGCCTGAGAAGTACAAAGAGATTTTCTTCGACCAGAGCAAGAACGGCTA
CGCCGGCTACATTGACGGCGGAGCCAGCCAGGAAGAGTTCTACAAGTTCATCAA
GCCCATCCTGGAAAAGATGGACGGCACCGAGGAACTGCTCGTGAAGCTGAACAG
AGAGGACCTGCTGCGGAAGCAGCGGACCTTCGACAACGGCAGCATCCCCCACCA
GATCCACCTGGGAGAGCTGCACGCCATTCTGCGGCGGCAGGAAGATTTTTACCCA
TTCCTGAAGGACAACCGGGAAAAGATCGAGAAGATCCTGACCTTCCGCATCCCC
TACTACGTGGGCCCTCTGGCCAGGGGAAACAGCAGATTCGCCTGGATGACCAGA
AAGAGCGAGGAAACCATCACCCCCTGGAACTTCGAGGAAGTGGTGGACAAGGGC
GCTTCCGCCCAGAGCTTCATCGAGCGGATGACCAACTTCGATAAGAACCTGCCCA
ACGAGAAGGTGCTGCCCAAGCACAGCCTGCTGTACGAGTACTTCACCGTGTATA
ACGAGCTGACCAAAGTGAAATACGTGACCGAGGGAATGAGAAAGCCCGCCTTCC
TGAGCGGCGAGCAGAAAAAGGCCATCGTGGACCTGCTGTTCAAGACCAACCGGA
AAGTGACCGTGAAGCAGCTGAAAGAGGACTACTTCAAGAAAATCGAGTGCTTCG
ACTCCGTGGAAATCTCCGGCGTGGAAGATCGGTTCAACGCCTCCCTGGGCACATA
CCACGATCTGCTGAAAATTATCAAGGACAAGGACTTCCTGGACAATGAGGAAAA
CGAGGACATTCTGGAAGATATCGTGCTGACCCTGACACTGTTTGAGGACAGAGA
GATGATCGAGGAACGGCTGAAAACCTATGCCCACCTGTTCGACGACAAAGTGAT
GAAGCAGCTGAAGCGGCGGAGATACACCGGCTGGGGCAGGCTGAGCCGGAAGC
TGATCAACGGCATCCGGGACAAGCAGTCCGGCAAGACAATCCTGGATTTCCTGA
AGTCCGACGGCTTCGCCAACAGAAACTTCATGCAGCTGATCCACGACGACAGCC
TGACCTTTAAAGAGGACATCCAGAAAGCCCAGGTGTCCGGCCAGGGCGATAGCC
TGCACGAGCACATTGCCAATCTGGCCGGCAGCCCCGCCATTAAGAAGGGCATCC
TGCAGACAGTGAAGGTGGTGGACGAGCTCGTGAAAGTGATGGGCCGGCACAAGC
CCGAGAACATCGTGATCGAAATGGCCAGAGAGAACCAGACCACCCAGAAGGGA
CAGAAGAACAGCCGCGAGAGAATGAAGCGGATCGAAGAGGGCATCAAAGAGCT
GGGCAGCCAGATCCTGAAAGAACACCCCGTGGAAAACACCCAGCTGCAGAACGA
GAAGCTGTACCTGTACTACCTGCAGAATGGGCGGGATATGTACGTGGACCAGGA
ACTGGACATCAACCGGCTGTCCGACTACGATGTGGACCATATCGTGCCTCAGAGC
TTTCTGAAGGACGACTCCATCGACAACAAGGTGCTGACCAGAAGCGACAAGAAC
CGGGGCAAGAGCGACAACGTGCCCTCCGAAGAGGTCGTGAAGAAGATGAAGAA
CTACTGGCGGCAGCTGCTGAACGCCAAGCTGATTACCCAGAGAAAGTTCGACAA
TCTGACCAAGGCCGAGAGAGGCGGCCTGAGCGAACTGGATAAGGCCGGCTTCAT
CAAGAGACAGCTGGTGGAAACCCGGCAGATCACAAAGCACGTGGCACAGATCCT
GGACTCCCGGATGAACACTAAGTACGACGAGAATGACAAGCTGATCCGGGAAGT
GAAAGTGATCACCCTGAAGTCCAAGCTGGTGTCCGATTTCCGGAAGGATTTCCAG
TTTTACAAAGTGCGCGAGATCAACAACTACCACCACGCCCACGACGCCTACCTGA
ACGCCGTCGTGGGAACCGCCCTGATCAAAAAGTACCCTAAGCTGGAAAGCGAGT
TCGTGTACGGCGACTACAAGGTGTACGACGTGCGGAAGATGATCGCCAAGAGCG
AGCAGGAAATCGGCAAGGCTACCGCCAAGTACTTCTTCTACAGCAACATCATGA
ACTTTTTCAAGACCGAGATTACCCTGGCCAACGGCGAGATCCGGAAGCGGCCTCT
GATCGAGACAAACGGCGAAACCGGGGAGATCGTGTGGGATAAGGGCCGGGATTT
TGCCACCGTGCGGAAAGTGCTGAGCATGCCCCAAGTGAATATCGTGAAAAAGAC
CGAGGTGCAGACAGGCGGCTTCAGCAAAGAGTCTATCCTGCCCAAGAGGAACAG
CGATAAGCTGATCGCCAGAAAGAAGGACTGGGACCCTAAGAAGTACGGCGGCTT
CGACAGCCCCACCGTGGCCTATTCTGTGCTGGTGGTGGCCAAAGTGGAAAAGGG
CAAGTCCAAGAAACTGAAGAGTGTGAAAGAGCTGCTGGGGATCACCATCATGGA
AAGAAGCAGCTTCGAGAAGAATCCCATCGACTTTCTGGAAGCCAAGGGCTACAA
AGAAGTGAAAAAGGACCTGATCATCAAGCTGCCTAAGTACTCCCTGTTCGAGCT
GGAAAACGGCCGGAAGAGAATGCTGGCCTCTGCCGGCGAACTGCAGAAGGGAA
ACGAACTGGCCCTGCCCTCCAAATATGTGAACTTCCTGTACCTGGCCAGCCACTA
TGAGAAGCTGAAGGGCTCCCCCGAGGATAATGAGCAGAAACAGCTGTTTGTGGA
ACAGCACAAGCACTACCTGGACGAGATCATCGAGCAGATCAGCGAGTTCTCCAA
GAGAGTGATCCTGGCCGACGCTAATCTGGACAAAGTGCTGTCCGCCTACAACAA
GCACCGGGATAAGCCCATCAGAGAGCAGGCCGAGAATATCATCCACCTGTTTAC
CCTGACCAATCTGGGAGCCCCTGCCGCCTTCAAGTACTTTGACACCACCATCGAC
CGGAAGAGGTACACCAGCACCAAAGAGGTGCTGGACGCCACCCTGATCCACCAG
AGCATCACCGGCCTGTACGAGACACGGATCGACCTGTCTCAGCTGGGAGGCGAC
AGCCCCAAGAAGAAGAGAAAGGTGGAGGCCAGC >SEQ ID NO: 2 SpCas9 amino
acid sequence
MYPYDVPDYASPKKKRKVEASDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLG
NTDRHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVD
DSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLR
LIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKA
ILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKD
TYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDE
HHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMD
GTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKI
LTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDK
NLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNR
KVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDI
LEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKUNGIRD
KQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAG
SPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEE
GIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVP
QSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDN
LTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVIT
LKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDY
KVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEI
VWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDP
KKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAK
GYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHY
EKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDK
PIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRI
DLSQLGGDSPKKKRKVEAS >SEQ ID NO: 3 SpCas9-VP2 fusion nucleic acid
ATGTACCCATACGATGTTCCAGATTACGCTTCGCCGAAGAAAAAGCGCAAGGTC
GAAGCGTCCGACAAGAAGTACAGCATCGGCCTGGACATCGGCACCAACTCTGTG
GGCTGGGCCGTGATCACCGACGAGTACAAGGTGCCCAGCAAGAAATTCAAGGTG
CTGGGCAACACCGACCGGCACAGCATCAAGAAGAACCTGATCGGAGCCCTGCTG
TTCGACAGCGGCGAAACAGCCGAGGCCACCCGGCTGAAGAGAACCGCCAGAAG
AAGATACACCAGACGGAAGAACCGGATCTGCTATCTGCAAGAGATCTTCAGCAA
CGAGATGGCCAAGGTGGACGACAGCTTCTTCCACAGACTGGAAGAGTCCTTCCT
GGTGGAAGAGGATAAGAAGCACGAGCGGCACCCCATCTTCGGCAACATCGTGGA
CGAGGTGGCCTACCACGAGAAGTACCCCACCATCTACCACCTGAGAAAGAAACT
GGTGGACAGCACCGACAAGGCCGACCTGCGGCTGATCTATCTGGCCCTGGCCCA
CATGATCAAGTTCCGGGGCCACTTCCTGATCGAGGGCGACCTGAACCCCGACAA
CAGCGACGTGGACAAGCTGTTCATCCAGCTGGTGCAGACCTACAACCAGCTGTTC
GAGGAAAACCCCATCAACGCCAGCGGCGTGGACGCCAAGGCCATCCTGTCTGCC
AGACTGAGCAAGAGCAGACGGCTGGAAAATCTGATCGCCCAGCTGCCCGGCGAG
AAGAAGAATGGCCTGTTCGGCAACCTGATTGCCCTGAGCCTGGGCCTGACCCCCA
ACTTCAAGAGCAACTTCGACCTGGCCGAGGATGCCAAACTGCAGCTGAGCAAGG
ACACCTACGACGACGACCTGGACAACCTGCTGGCCCAGATCGGCGACCAGTACG
CCGACCTGTTTCTGGCCGCCAAGAACCTGTCCGACGCCATCCTGCTGAGCGACAT
CCTGAGAGTGAACACCGAGATCACCAAGGCCCCCCTGAGCGCCTCTATGATCAA
GAGATACGACGAGCACCACCAGGACCTGACCCTGCTGAAAGCTCTCGTGCGGCA
GCAGCTGCCTGAGAAGTACAAAGAGATTTTCTTCGACCAGAGCAAGAACGGCTA
CGCCGGCTACATTGACGGCGGAGCCAGCCAGGAAGAGTTCTACAAGTTCATCAA
GCCCATCCTGGAAAAGATGGACGGCACCGAGGAACTGCTCGTGAAGCTGAACAG
AGAGGACCTGCTGCGGAAGCAGCGGACCTTCGACAACGGCAGCATCCCCCACCA
GATCCACCTGGGAGAGCTGCACGCCATTCTGCGGCGGCAGGAAGATTTTTACCCA
TTCCTGAAGGACAACCGGGAAAAGATCGAGAAGATCCTGACCTTCCGCATCCCC
TACTACGTGGGCCCTCTGGCCAGGGGAAACAGCAGATTCGCCTGGATGACCAGA
AAGAGCGAGGAAACCATCACCCCCTGGAACTTCGAGGAAGTGGTGGACAAGGGC
GCTTCCGCCCAGAGCTTCATCGAGCGGATGACCAACTTCGATAAGAACCTGCCCA
ACGAGAAGGTGCTGCCCAAGCACAGCCTGCTGTACGAGTACTTCACCGTGTATA
ACGAGCTGACCAAAGTGAAATACGTGACCGAGGGAATGAGAAAGCCCGCCTTCC
TGAGCGGCGAGCAGAAAAAGGCCATCGTGGACCTGCTGTTCAAGACCAACCGGA
AAGTGACCGTGAAGCAGCTGAAAGAGGACTACTTCAAGAAAATCGAGTGCTTCG
ACTCCGTGGAAATCTCCGGCGTGGAAGATCGGTTCAACGCCTCCCTGGGCACATA
CCACGATCTGCTGAAAATTATCAAGGACAAGGACTTCCTGGACAATGAGGAAAA
CGAGGACATTCTGGAAGATATCGTGCTGACCCTGACACTGTTTGAGGACAGAGA
GATGATCGAGGAACGGCTGAAAACCTATGCCCACCTGTTCGACGACAAAGTGAT
GAAGCAGCTGAAGCGGCGGAGATACACCGGCTGGGGCAGGCTGAGCCGGAAGC
TGATCAACGGCATCCGGGACAAGCAGTCCGGCAAGACAATCCTGGATTTCCTGA
AGTCCGACGGCTTCGCCAACAGAAACTTCATGCAGCTGATCCACGACGACAGCC
TGACCTTTAAAGAGGACATCCAGAAAGCCCAGGTGTCCGGCCAGGGCGATAGCC
TGCACGAGCACATTGCCAATCTGGCCGGCAGCCCCGCCATTAAGAAGGGCATCC
TGCAGACAGTGAAGGTGGTGGACGAGCTCGTGAAAGTGATGGGCCGGCACAAGC
CCGAGAACATCGTGATCGAAATGGCCAGAGAGAACCAGACCACCCAGAAGGGA
CAGAAGAACAGCCGCGAGAGAATGAAGCGGATCGAAGAGGGCATCAAAGAGCT
GGGCAGCCAGATCCTGAAAGAACACCCCGTGGAAAACACCCAGCTGCAGAACGA
GAAGCTGTACCTGTACTACCTGCAGAATGGGCGGGATATGTACGTGGACCAGGA
ACTGGACATCAACCGGCTGTCCGACTACGATGTGGACCATATCGTGCCTCAGAGC
TTTCTGAAGGACGACTCCATCGACAACAAGGTGCTGACCAGAAGCGACAAGAAC
CGGGGCAAGAGCGACAACGTGCCCTCCGAAGAGGTCGTGAAGAAGATGAAGAA
CTACTGGCGGCAGCTGCTGAACGCCAAGCTGATTACCCAGAGAAAGTTCGACAA
TCTGACCAAGGCCGAGAGAGGCGGCCTGAGCGAACTGGATAAGGCCGGCTTCAT
CAAGAGACAGCTGGTGGAAACCCGGCAGATCACAAAGCACGTGGCACAGATCCT
GGACTCCCGGATGAACACTAAGTACGACGAGAATGACAAGCTGATCCGGGAAGT
GAAAGTGATCACCCTGAAGTCCAAGCTGGTGTCCGATTTCCGGAAGGATTTCCAG
TTTTACAAAGTGCGCGAGATCAACAACTACCACCACGCCCACGACGCCTACCTGA
ACGCCGTCGTGGGAACCGCCCTGATCAAAAAGTACCCTAAGCTGGAAAGCGAGT
TCGTGTACGGCGACTACAAGGTGTACGACGTGCGGAAGATGATCGCCAAGAGCG
AGCAGGAAATCGGCAAGGCTACCGCCAAGTACTTCTTCTACAGCAACATCATGA
ACTTTTTCAAGACCGAGATTACCCTGGCCAACGGCGAGATCCGGAAGCGGCCTCT
GATCGAGACAAACGGCGAAACCGGGGAGATCGTGTGGGATAAGGGCCGGGATTT
TGCCACCGTGCGGAAAGTGCTGAGCATGCCCCAAGTGAATATCGTGAAAAAGAC
CGAGGTGCAGACAGGCGGCTTCAGCAAAGAGTCTATCCTGCCCAAGAGGAACAG
CGATAAGCTGATCGCCAGAAAGAAGGACTGGGACCCTAAGAAGTACGGCGGCTT
CGACAGCCCCACCGTGGCCTATTCTGTGCTGGTGGTGGCCAAAGTGGAAAAGGG
CAAGTCCAAGAAACTGAAGAGTGTGAAAGAGCTGCTGGGGATCACCATCATGGA
AAGAAGCAGCTTCGAGAAGAATCCCATCGACTTTCTGGAAGCCAAGGGCTACAA
AGAAGTGAAAAAGGACCTGATCATCAAGCTGCCTAAGTACTCCCTGTTCGAGCT
GGAAAACGGCCGGAAGAGAATGCTGGCCTCTGCCGGCGAACTGCAGAAGGGAA
ACGAACTGGCCCTGCCCTCCAAATATGTGAACTTCCTGTACCTGGCCAGCCACTA
TGAGAAGCTGAAGGGCTCCCCCGAGGATAATGAGCAGAAACAGCTGTTTGTGGA
ACAGCACAAGCACTACCTGGACGAGATCATCGAGCAGATCAGCGAGTTCTCCAA
GAGAGTGATCCTGGCCGACGCTAATCTGGACAAAGTGCTGTCCGCCTACAACAA
GCACCGGGATAAGCCCATCAGAGAGCAGGCCGAGAATATCATCCACCTGTTTAC
CCTGACCAATCTGGGAGCCCCTGCCGCCTTCAAGTACTTTGACACCACCATCGAC
CGGAAGAGGTACACCAGCACCAAAGAGGTGCTGGACGCCACCCTGATCCACCAG
AGCATCACCGGCCTGTACGAGACACGGATCGACCTGTCTCAGCTGGGAGGCGAC
AGCCCCAAGAAGAAGAGAAAGGTGGAGGCCAGCGAATTGGCTCCGGGAAAAAA
GAGGCCGGTAGAGCACTCTCCTGTGGAGCCAGACTCCTCCTCGGGAACCGGAAA
GGCGGGCCAGCAGCCTGCAAGAAAAAGATTGAATTTTGGTCAGACTGGAGACGC
AGACTCAGTACCTGACCCCCAGCCTCTCGGACAGCCACCAGCAGCCCCCTCTGGT
CTGGGAACTAATACGATGGCTACAGGCAGTGGCGCACCAATGGCAGACAATAAC
GAGGGCGCCGACGGAGTGGGTAATTCCTCGGGAAATTGGCATTGCGATTCCACA
TGGATGGGCGACAGAGTCATCACCACCAGCACCCGAACCTGGGCCCTGCCCACC
TACAACAACCACCTCTACAAACAAATTTCCAGCCAATCAGGAGCCTCGAACGAC
AATCACTACTTTGGCTACAGCACCCCTTGGGGGTATTTTGACTTCAACAGATTCC
ACTGCCACTTTTCACCACGTGACTGGCAAAGACTCATCAACAACAACTGGGGATT
CCGACCCAAGAGACTCAACTTCAAGCTCTTTAACATTCAAGTCAAAGAGGTCACG
CAGAATGACGGTACGACGACGATTGCCAATAACCTTACCAGCACGGTTCAGGTG
TTTACTGACTCGGAGTACCAGCTCCCGTACGTCCTCGGCTCGGCGCATCAAGGAT
GCCTCCCGCCGTTCCCAGCAGACGTCTTCATGGTGCCACAGTATGGATACCTCAC
CCTGAACAACGGGAGTCAGGCAGTAGGACGCTCTTCATTTTACTGCCTGGAGTAC
TTTCCTTCTCAGATGCTGCGTACCGGAAACAACTTTACCTTCAGCTACACTTTTGA
GGACGTTCCTTTCCACAGCAGCTACGCTCACAGCCAGAGTCTGGACCGTCTCATG
AATCCTCTCATCGACCAGTACCTGTATTACTTGAGCAGAACAAACACTCCAAGTG
GAACCACCACGCAGTCAAGGCTTCAGTTTTCTCAGGCCGGAGCGAGTGACATTCG
GGACCAGTCTAGGAACTGGCTTCCTGGACCCTGTTACCGCCAGCAGCGAGTATCA
AAGACATCTGCGGATAACAACAACAGTGAATACTCGTGGACTGGAGCTACCAAG
TACCACCTCAATGGCAGAGACTCTCTGGTGAATCCGGGCCCGGCCATGGCAAGC
CACAAGGACGATGAAGAAAAGTTTTTTCCTCAGAGCGGGGTTCTCATCTTTGGGA
AGCAAGGCTCAGAGAAAACAAATGTGGACATTGAAAAGGTCATGATTACAGACG
AAGAGGAAATCAGGACAACCAATCCCGTGGCTACGGAGCAGTATGGTTCTGTAT
CTACCAACCTCCAGAGAGGCAACAGACAAGCAGCTACCGCAGATGTCAACACAC
AAGGCGTTCTTCCAGGCATGGTCTGGCAGGACAGAGATGTGTACCTTCAGGGGC
CCATCTGGGCAAAGATTCCACACACGGACGGACATTTTCACCCCTCTCCCCTCAT
GGGTGGATTCGGACTTAAACACCCTCCTCCACAGATTCTCATCAAGAACACCCCG
GTACCTGCGAATCCTTCGACCACCTTCAGTGCGGCAAAGTTTGCTTCCTTCATCAC
ACAGTACTCCACGGGACAGGTCAGCGTGGAGATCGAGTGGGAGCTGCAGAAGGA
AAACAGCAAACGCTGGAATCCCGAAATTCAGTACACTTCCAACTACAACAAGTC
TGTTAATGTGGACTTTACTGTGGACACTAATGGCGTGTATTCAGAGCCTCGCCCC
ATTGGCACCAGATACCTGACTCGTAATCTGTAA >SEQ ID NO: 4 SpCas9-VP2
fusion protein
MYPYDVPDYASPKKKRKVEASDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLG
NTDRHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVD
DSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLR
LIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINTASGVDAKA
ILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKD
TYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDE
HHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMD
GTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKI
LTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDK
NLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNR
KVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDI
LEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRD
KQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAG
SPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEE
GIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVP
QSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDN
LTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVIT
LKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDY
KVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEI
VWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDP
KKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAK
GYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHY
EKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDK
PIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRI
DLSQLGGDSPKKKRKVEASELAPGKKRPVEHSPVEPDSSSGTGKAGQQPARKRLNFG
QTGDADSVPDPQPLGQPPAAPSGLGTNTMATGSGAPMADNNEGADGVGNSSGNWH
CDSTWMGDRVITTSTRTWALPTYNNHLYKQISSQSGASNDNHYFGYSTPWGYFDFN
RFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTQNDGTTTIANNLTSTVQV
FTDSEYQLPYVLGSAHQGCLPPFPADVFMVPQYGYLTLNNGSQAVGRSSFYCLEYFP
SQMLRTGNNFTFSYTFEDVPFHSSYAHSQSLDRLMNPLIDQYLYYLSRTNTPSGTTTQ
SRLQFSQAGASDIRDQSRNWLPGPCYRQQRVSKTSADNNNSEYSWTGATKYHLNGR
DSLVNPGPAMASHKDDEEKFFPQSGVLIFGKQGSEKTNVDIEKVMITDEEEIRTTNPV
ATEQYGSVSTNLQRGNRQAATADVNTQGVLPGMVWQDRDVYLQGPIWAKIPHTDG
HFHPSPLMGGFGLKHPPPQILIKNTPVPANPSTTFSAAKFASFITQYSTGQVSVEIEWE
LQKENSKRWNPEIQYTSNYNKSVNVDFTVDTNGVYSEPRPIGTRYLTRNL >SEQ ID NO: 5
Linker sequence GGGS >SEQ ID NO: 6 Cas9.sub.n-Int.sub.n
MYPYDVPDYASPKKKRKVEASDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLG
NTDRHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVD
DSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLR
LIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINTASGVDAKA
ILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKD
TYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDE
HHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMD
GTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKI
LTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDK
NLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNR
KVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDI
LEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRD
KQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAG
SPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEE
GIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVP
QSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDN
LTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVIT
LKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDY
KVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEI
VWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDP
KKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAK
GYKEVKKDLIIKLPKYSLFELENGRKCLSYETEILTVEYGLLPIGKIVEKRIECTVYSV
DNNGNIYTQPVAQWHDRGEQEVFEYCLEDGSLIRATKDHKFMTVDGQMLPIDEIFER
ELDLMRVDNLPN >SEQ ID NO: 7 Int.sub.c-Cas9.sub.c-GS-VP2
MIKIATRKYLGKQNVYDIGVERDHNFALKNGFIASNCMLASAGELQKGNELALPSK
YVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDK
VLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLI
HQSITGLYETRIDLSQLGGDSPKKKRKVEASGSAPGKKRPVEHSPVEPDSSSGTGKAG
QQPARKRLNFGQTGDADSVPDPQPLGQPPAAPSGLGTNTMATGSGAPMADNNEGA
DGVGNSSGNWHCDSTWMGDRVITTSTRTWALPTYNNHLYKQISSQSGASNDNHYF
GYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTQNDGT
TTIANNLTSTVQVFTDSEYQLPYVLGSAHQGCLPPFPADVFMVPQYGYLTLNNGSQA
VGRSSFYCLEYFPSQMLRTGNNFTFSYTFEDVPFHSSYAHSQSLDRLMNPLIDQYLYY
LSRTNTPSGTTTQSRLQFSQAGASDIRDQSRNWLPGPCYRQQRVSKTSADNNNSEYS
WTGATKYHLNGRDSLVNPGPAMASHKDDEEKFFPQSGVLIFGKQGSEKTNVDIEKV
MITDEEEIRTTNPVATEQYGSVSTNLQRGNRQAATADVNTQGVLPGMVWQDRDVY
LQGPIWAKIPHTDGHFHPSPLMGGFGLKHPPPQILIKNTPVPANPSTTFSAAKFASFITQ
YSTGQVSVEIEWELQKENSKRWNPEIQYTSNYNKSVNVDFTVDTNGVYSEPRPIGTR YLTRNL
>SEQ ID NO: 8 Int.sub.c-Cas9.sub.c-GGGGSGGGGSGGGGS-VP2
MIKIATRKYLGKQNVYDIGVERDHNFALKNGFIASNCMLASAGELQKGNELALPSK
YVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDK
VLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLI
HQSITGLYETRIDLSQLGGDSPKKKRKVEASGGGGSGGGGSGGGGSAPGKKRPVEHS
PVEPDSSSGTGKAGQQPARKRLNFGQTGDADSVPDPQPLGQPPAAPSGLGTNTMAT
GSGAPMADNNEGADGVGNSSGNWHCDSTWMGDRVITTSTRTWALPTYNNHLYKQI
SSQSGASNDNHYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLNFKLF
NIQVKEVTQNDGTTTIANNLTSTVQVFTDSEYQLPYVLGSAHQGCLPPFPADVFMVP
QYGYLTLNNGSQAVGRSSFYCLEYFPSQMLRTGNNFTFSYTFEDVPFHSSYAHSQSL
DRLMNPLIDQYLYYLSRTNTPSGTTTQSRLQFSQAGASDIRDQSRNWLPGPCYRQQR
VSKTSADNNNSEYSWTGATKYHLNGRDSLVNPGPAMASHKDDEEKFFPQSGVLIFG
KQGSEKTNVDIEKVMITDEEEIRTTNPVATEQYGSVSTNLQRGNRQAATADVNTQG
VLPGMVWQDRDVYLQGPIWAKIPHTDGHFHPSPLMGGFGLKHPPPQILIKNTPVPAN
PSTTFSAAKFASFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYNKSVNVDFTV
DTNGVYSEPRPIGTRYLTRNL >SEQ ID NO: 9
Int.sub.c-Cas9.sub.c-EAAAKEAAAKEAAAK-VP2
MIKIATRKYLGKQNVYDIGVERDHNFALKNGFIASNCMLASAGELQKGNELALPSK
YVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDK
VLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLI
HQSITGLYETRIDLSQLGGDSPKKKRKVEASEAAAKEAAAKEAAAKAPGKKRPVEH
SPVEPDSSSGTGKAGQQPARKRLNFGQTGDADSVPDPQPLGQPPAAPSGLGTNTMAT
GSGAPMADNNEGADGVGNSSGNWHCDSTWMGDRVITTSTRTWALPTYNNHLYKQI
SSQSGASNDNHYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLNFKLF
NIQVKEVTQNDGTTTIANNLTSTVQVFTDSEYQLPYVLGSAHQGCLPPFPADVFMVP
QYGYLTLNNGSQAVGRSSFYCLEYFPSQMLRTGNNFTFSYTFEDVPFHSSYAHSQSL
DRLMNPLIDQYLYYLSRTNTPSGTTTQSRLQFSQAGASDIRDQSRNWLPGPCYRQQR
VSKTSADNNNSEYSWTGATKYHLNGRDSLVNPGPAMASHKDDEEKFFPQSGVLIFG
KQGSEKTNVDIEKVMITDEEEIRTTNPVATEQYGSVSTNLQRGNRQAATADVNTQG
VLPGMVWQDRDVYLQGPIWAKIPHTDGHFHPSPLMGGFGLKHPPPQILIKNTPVPAN
PSTTFSAAKFASFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYNKSVNVDFTV
DTNGVYSEPRPIGTRYLTRNL >SEQ ID NO: 10 pU1a-Cas9.sub.c-Int.sub.c
CCTGCAGGCAGCTGCGCGCTCGCTCGCTCACTGAGGCCGCCCGGGCAAAGCCCG
GGCGTCGGGCGACCTTTGGTCGCCCGGCCTCAGTGAGCGAGCGAGCGCGCAGAG
AGGGAGTGGCCAACTCCATCACTAGGGGTTCCTGCGGCCTCTAGAATGGAGGCG
GTACTATGTAGATGAGAATTCAGGAGCAAACTGGGAAAAGCAACTGCTTCCAAA
TATTTGTGATTTTTACAGTGTAGTTTTGGAAAAACTCTTAGCCTACCAATTCTTCT
AAGTGTTTTAAAATGTGGGAGCCAGTACACATGAAGTTATAGAGTGTTTTAATGA
GGCTTAAATATTTACCGTAACTATGAAATGCTACGCATATCATGCTGTTCAGGCT
CCGTGGCCACGCAACTCATACTACCGGTGCCACCATGTACCCATACGATGTTCCA
GATTACGCTTCGCCGAAGAAAAAGCGCAAGGTCGAAGCGTCCGACAAGAAGTAC
AGCATCGGCCTGGACATCGGCACCAACTCTGTGGGCTGGGCCGTGATCACCGAC
GAGTACAAGGTGCCCAGCAAGAAATTCAAGGTGCTGGGCAACACCGACCGGCAC
AGCATCAAGAAGAACCTGATCGGAGCCCTGCTGTTCGACAGCGGCGAAACAGCC
GAGGCCACCCGGCTGAAGAGAACCGCCAGAAGAAGATACACCAGACGGAAGAA
CCGGATCTGCTATCTGCAAGAGATCTTCAGCAACGAGATGGCCAAGGTGGACGA
CAGCTTCTTCCACAGACTGGAAGAGTCCTTCCTGGTGGAAGAGGATAAGAAGCA
CGAGCGGCACCCCATCTTCGGCAACATCGTGGACGAGGTGGCCTACCACGAGAA
GTACCCCACCATCTACCACCTGAGAAAGAAACTGGTGGACAGCACCGACAAGGC
CGACCTGCGGCTGATCTATCTGGCCCTGGCCCACATGATCAAGTTCCGGGGCCAC
TTCCTGATCGAGGGCGACCTGAACCCCGACAACAGCGACGTGGACAAGCTGTTC
ATCCAGCTGGTGCAGACCTACAACCAGCTGTTCGAGGAAAACCCCATCAACGCC
AGCGGCGTGGACGCCAAGGCCATCCTGTCTGCCAGACTGAGCAAGAGCAGACGG
CTGGAAAATCTGATCGCCCAGCTGCCCGGCGAGAAGAAGAATGGCCTGTTCGGC
AACCTGATTGCCCTGAGCCTGGGCCTGACCCCCAACTTCAAGAGCAACTTCGACC
TGGCCGAGGATGCCAAACTGCAGCTGAGCAAGGACACCTACGACGACGACCTGG
ACAACCTGCTGGCCCAGATCGGCGACCAGTACGCCGACCTGTTTCTGGCCGCCAA
GAACCTGTCCGACGCCATCCTGCTGAGCGACATCCTGAGAGTGAACACCGAGAT
CACCAAGGCCCCCCTGAGCGCCTCTATGATCAAGAGATACGACGAGCACCACCA
GGACCTGACCCTGCTGAAAGCTCTCGTGCGGCAGCAGCTGCCTGAGAAGTACAA
AGAGATTTTCTTCGACCAGAGCAAGAACGGCTACGCCGGCTACATTGACGGCGG
AGCCAGCCAGGAAGAGTTCTACAAGTTCATCAAGCCCATCCTGGAAAAGATGGA
CGGCACCGAGGAACTGCTCGTGAAGCTGAACAGAGAGGACCTGCTGCGGAAGCA
GCGGACCTTCGACAACGGCAGCATCCCCCACCAGATCCACCTGGGAGAGCTGCA
CGCCATTCTGCGGCGGCAGGAAGATTTTTACCCATTCCTGAAGGACAACCGGGA
AAAGATCGAGAAGATCCTGACCTTCCGCATCCCCTACTACGTGGGCCCTCTGGCC
AGGGGAAACAGCAGATTCGCCTGGATGACCAGAAAGAGCGAGGAAACCATCAC
CCCCTGGAACTTCGAGGAAGTGGTGGACAAGGGCGCTTCCGCCCAGAGCTTCAT
CGAGCGGATGACCAACTTCGATAAGAACCTGCCCAACGAGAAGGTGCTGCCCAA
GCACAGCCTGCTGTACGAGTACTTCACCGTGTATAACGAGCTGACCAAAGTGAA
ATACGTGACCGAGGGAATGAGAAAGCCCGCCTTCCTGAGCGGCGAGCAGAAAAA
GGCCATCGTGGACCTGCTGTTCAAGACCAACCGGAAAGTGACCGTGAAGCAGCT
GAAAGAGGACTACTTCAAGAAAATCGAGTGCTTCGACTCCGTGGAAATCTCCGG
CGTGGAAGATCGGTTCAACGCCTCCCTGGGCACATACCACGATCTGCTGAAAATT
ATCAAGGACAAGGACTTCCTGGACAATGAGGAAAACGAGGACATTCTGGAAGAT
ATCGTGCTGACCCTGACACTGTTTGAGGACAGAGAGATGATCGAGGAACGGCTG
AAAACCTATGCCCACCTGTTCGACGACAAAGTGATGAAGCAGCTGAAGCGGCGG
AGATACACCGGCTGGGGCAGGCTGAGCCGGAAGCTGATCAACGGCATCCGGGAC
AAGCAGTCCGGCAAGACAATCCTGGATTTCCTGAAGTCCGACGGCTTCGCCAAC
AGAAACTTCATGCAGCTGATCCACGACGACAGCCTGACCTTTAAAGAGGACATC
CAGAAAGCCCAGGTGTCCGGCCAGGGCGATAGCCTGCACGAGCACATTGCCAAT
CTGGCCGGCAGCCCCGCCATTAAGAAGGGCATCCTGCAGACAGTGAAGGTGGTG
GACGAGCTCGTGAAAGTGATGGGCCGGCACAAGCCCGAGAACATCGTGATCGAA
ATGGCCAGAGAGAACCAGACCACCCAGAAGGGACAGAAGAACAGCCGCGAGAG
AATGAAGCGGATCGAAGAGGGCATCAAAGAGCTGGGCAGCCAGATCCTGAAAG
AACACCCCGTGGAAAACACCCAGCTGCAGAACGAGAAGCTGTACCTGTACTACC
TGCAGAATGGGCGGGATATGTACGTGGACCAGGAACTGGACATCAACCGGCTGT
CCGACTACGATGTGGACCATATCGTGCCTCAGAGCTTTCTGAAGGACGACTCCAT
CGACAACAAGGTGCTGACCAGAAGCGACAAGAACCGGGGCAAGAGCGACAACG
TGCCCTCCGAAGAGGTCGTGAAGAAGATGAAGAACTACTGGCGGCAGCTGCTGA
ACGCCAAGCTGATTACCCAGAGAAAGTTCGACAATCTGACCAAGGCCGAGAGAG
GCGGCCTGAGCGAACTGGATAAGGCCGGCTTCATCAAGAGACAGCTGGTGGAAA
CCCGGCAGATCACAAAGCACGTGGCACAGATCCTGGACTCCCGGATGAACACTA
AGTACGACGAGAATGACAAGCTGATCCGGGAAGTGAAAGTGATCACCCTGAAGT
CCAAGCTGGTGTCCGATTTCCGGAAGGATTTCCAGTTTTACAAAGTGCGCGAGAT
CAACAACTACCACCACGCCCACGACGCCTACCTGAACGCCGTCGTGGGAACCGC
CCTGATCAAAAAGTACCCTAAGCTGGAAAGCGAGTTCGTGTACGGCGACTACAA
GGTGTACGACGTGCGGAAGATGATCGCCAAGAGCGAGCAGGAAATCGGCAAGG
CTACCGCCAAGTACTTCTTCTACAGCAACATCATGAACTTTTTCAAGACCGAGAT
TACCCTGGCCAACGGCGAGATCCGGAAGCGGCCTCTGATCGAGACAAACGGCGA
AACCGGGGAGATCGTGTGGGATAAGGGCCGGGATTTTGCCACCGTGCGGAAAGT
GCTGAGCATGCCCCAAGTGAATATCGTGAAAAAGACCGAGGTGCAGACAGGCGG
CTTCAGCAAAGAGTCTATCCTGCCCAAGAGGAACAGCGATAAGCTGATCGCCAG
AAAGAAGGACTGGGACCCTAAGAAGTACGGCGGCTTCGACAGCCCCACCGTGGC
CTATTCTGTGCTGGTGGTGGCCAAAGTGGAAAAGGGCAAGTCCAAGAAACTGAA
GAGTGTGAAAGAGCTGCTGGGGATCACCATCATGGAAAGAAGCAGCTTCGAGAA
GAATCCCATCGACTTTCTGGAAGCCAAGGGCTACAAAGAAGTGAAAAAGGACCT
GATCATCAAGCTGCCTAAGTACTCCCTGTTCGAGCTGGAAAACGGCCGGAAGTGT
CTGTCGTATGAGACCGAGATCCTGACCGTGGAGTATGGACTGCTGCCGATTGGAA
AGATTGTGGAGAAGCGCATTGAGTGCACCGTGTACAGCGTGGATAACAATGGCA
ACATCTATACACAGCCAGTGGCCCAGTGGCACGACCGCGGAGAGCAGGAGGTCT
TCGAGTACTGCCTGGAGGATGGCAGCCTGATTCGCGCCACCAAGGATCATAAGTT
CATGACGGTGGACGGACAGATGCTGCCCATCGATGAGATTTTTGAGCGCGAGCT
GGATCTGATGCGCGTGGATAACCTGCCGAATTAAGAATTCGATCTTTTTCCCTCT
GCCAAAAATTATGGGGACATCATGAAGCCCCTTGAGCATCTGACTTCTGGCTAAT
AAAGGAAATTTATTTTCATTGCAATAGTGTGTTGGAATTTTTTGTGTCTCTCACTC
GGCGGCCGCAGGAACCCCTAGTGATGGAGTTGGCCACTCCCTCTCTGCGCGCTCG
CTCGCTCACTGAGGCCGGGCGACCAAAGGTCGCCCGACGCCCGGGCTTTGCCCG
GGCGGCCTCAGTGAGCGAGCGAGCGCGCAGCTGCCTGCAGG >SEQ ID NO: 11
Int.sub.c-Cas9.sub.c-GS-VP2
GTTGACATTGATTATTGACTAGTTATTAATAGTAATCAATTACGGGGTCATTAGTT
CATAGCCCATATATGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTG
GCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCAT
AGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGACTATTTACGGTAA
ACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTG
ACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATG
GGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGTGA
TGCGGTTTTGGCAGTACATCAATGGGCGTGGATAGCGGTTTGACTCACGGGGATT
TCCAAGTCTCCACCCCATTGACGTCAATGGGAGTTTGTTTTGGCACCAAAATCAA
CGGGACTTTCCAAAATGTCGTAACAACTCCGCCCCATTGACGCAAATGGGCGGTA
GGCGTGTACGGTGGGAGGTCTATATAAGCAGAGCTCTCTGGCTAACTAGAGAAC
CCACTGCTTACTGGCTTATCGAAATTAATACGACTCACTATAGGGAGACCCAAGC
TGGCTAGCGCCACCATGATCAAGATTGCCACGCGCAAGTACCTGGGCAAGCAGA
ACGTGTACGACATCGGAGTGGAGCGCGATCACAACTTTGCCCTGAAGAATGGCT
TTATTGCCTCGAACTGTATGCTGGCCTCTGCCGGCGAACTGCAGAAGGGAAACGA
ACTGGCCCTGCCCTCCAAATATGTGAACTTCCTGTACCTGGCCAGCCACTATGAG
AAGCTGAAGGGCTCCCCCGAGGATAATGAGCAGAAACAGCTGTTTGTGGAACAG
CACAAGCACTACCTGGACGAGATCATCGAGCAGATCAGCGAGTTCTCCAAGAGA
GTGATCCTGGCCGACGCTAATCTGGACAAAGTGCTGTCCGCCTACAACAAGCACC
GGGATAAGCCCATCAGAGAGCAGGCCGAGAATATCATCCACCTGTTTACCCTGA
CCAATCTGGGAGCCCCTGCCGCCTTCAAGTACTTTGACACCACCATCGACCGGAA
GAGGTACACCAGCACCAAAGAGGTGCTGGACGCCACCCTGATCCACCAGAGCAT
CACCGGCCTGTACGAGACACGGATCGACCTGTCTCAGCTGGGAGGCGACAGCCC
CAAGAAGAAGAGAAAGGTGGAGGCCAGCGGATCCGCTCCGGGAAAAAAGAGGC
CGGTAGAGCACTCTCCTGTGGAGCCAGACTCCTCCTCGGGAACCGGAAAGGCGG
GCCAGCAGCCTGCAAGAAAAAGATTGAATTTTGGTCAGACTGGAGACGCAGACT
CAGTACCTGACCCCCAGCCTCTCGGACAGCCACCAGCAGCCCCCTCTGGTCTGGG
AACTAATACGATGGCTACAGGCAGTGGCGCACCAATGGCAGACAATAACGAGGG
CGCCGACGGAGTGGGTAATTCCTCGGGAAATTGGCATTGCGATTCCACATGGATG
GGCGACAGAGTCATCACCACCAGCACCCGAACCTGGGCCCTGCCCACCTACAAC
AACCACCTCTACAAACAAATTTCCAGCCAATCAGGAGCCTCGAACGACAATCAC
TACTTTGGCTACAGCACCCCTTGGGGGTATTTTGACTTCAACAGATTCCACTGCC
ACTTTTCACCACGTGACTGGCAAAGACTCATCAACAACAACTGGGGATTCCGACC
CAAGAGACTCAACTTCAAGCTCTTTAACATTCAAGTCAAAGAGGTCACGCAGAA
TGACGGTACGACGACGATTGCCAATAACCTTACCAGCACGGTTCAGGTGTTTACT
GACTCGGAGTACCAGCTCCCGTACGTCCTCGGCTCGGCGCATCAAGGATGCCTCC
CGCCGTTCCCAGCAGACGTCTTCATGGTGCCACAGTATGGATACCTCACCCTGAA
CAACGGGAGTCAGGCAGTAGGACGCTCTTCATTTTACTGCCTGGAGTACTTTCCT
TCTCAGATGCTGCGTACCGGAAACAACTTTACCTTCAGCTACACTTTTGAGGACG
TTCCTTTCCACAGCAGCTACGCTCACAGCCAGAGTCTGGACCGTCTCATGAATCC
TCTCATCGACCAGTACCTGTATTACTTGAGCAGAACAAACACTCCAAGTGGAACC
ACCACGCAGTCAAGGCTTCAGTTTTCTCAGGCCGGAGCGAGTGACATTCGGGACC
AGTCTAGGAACTGGCTTCCTGGACCCTGTTACCGCCAGCAGCGAGTATCAAAGAC
ATCTGCGGATAACAACAACAGTGAATACTCGTGGACTGGAGCTACCAAGTACCA
CCTCAATGGCAGAGACTCTCTGGTGAATCCGGGCCCGGCCATGGCAAGCCACAA
GGACGATGAAGAAAAGTTTTTTCCTCAGAGCGGGGTTCTCATCTTTGGGAAGCAA
GGCTCAGAGAAAACAAATGTGGACATTGAAAAGGTCATGATTACAGACGAAGAG
GAAATCAGGACAACCAATCCCGTGGCTACGGAGCAGTATGGTTCTGTATCTACCA
ACCTCCAGAGAGGCAACAGACAAGCAGCTACCGCAGATGTCAACACACAAGGCG
TTCTTCCAGGCATGGTCTGGCAGGACAGAGATGTGTACCTTCAGGGGCCCATCTG
GGCAAAGATTCCACACACGGACGGACATTTTCACCCCTCTCCCCTCATGGGTGGA
TTCGGACTTAAACACCCTCCTCCACAGATTCTCATCAAGAACACCCCGGTACCTG
CGAATCCTTCGACCACCTTCAGTGCGGCAAAGTTTGCTTCCTTCATCACACAGTA
CTCCACGGGACAGGTCAGCGTGGAGATCGAGTGGGAGCTGCAGAAGGAAAACA
GCAAACGCTGGAATCCCGAAATTCAGTACACTTCCAACTACAACAAGTCTGTTAA
TGTGGACTTTACTGTGGACACTAATGGCGTGTATTCAGAGCCTCGCCCCATTGGC
ACCAGATACCTGACTCGTAATCTGTAAGAATTAAACCCGCTGATCAGCCTCGACT
GTGCCTTCTAGTTGCCAGCCATCTGTTGTTTGCCCCTCCCCCGTGCCTTCCTTGAC
CCTGGAAGGTGCCACTCCCACTGTCCTTTCCTAATAAAATGAGGAAATTGCATCG
CATTGTCTGAGTAGGTGTCATTCTATTCTGGGGGGTGGGGTGGGGCAGGACAGCA
AGGGGGAGGATTGGGAAGACAATAGCAGGCATGCTGGGGATGCGGTGGGCTCTA TGG >SEQ
ID NO: 12 Int.sub.c-Cas9.sub.c-GGGGSx3-VP2
GTTGACATTGATTATTGACTAGTTATTAATAGTAATCAATTACGGGGTCATTAGTT
CATAGCCCATATATGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTG
GCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCAT
AGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGACTATTTACGGTAA
ACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTG
ACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATG
GGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGTGA
TGCGGTTTTGGCAGTACATCAATGGGCGTGGATAGCGGTTTGACTCACGGGGATT
TCCAAGTCTCCACCCCATTGACGTCAATGGGAGTTTGTTTTGGCACCAAAATCAA
CGGGACTTTCCAAAATGTCGTAACAACTCCGCCCCATTGACGCAAATGGGCGGTA
GGCGTGTACGGTGGGAGGTCTATATAAGCAGAGCTCTCTGGCTAACTAGAGAAC
CCACTGCTTACTGGCTTATCGAAATTAATACGACTCACTATAGGGAGACCCAAGC
TGGCTAGCGCCACCATGATCAAGATTGCCACGCGCAAGTACCTGGGCAAGCAGA
ACGTGTACGACATCGGAGTGGAGCGCGATCACAACTTTGCCCTGAAGAATGGCT
TTATTGCCTCGAACTGTATGCTGGCCTCTGCCGGCGAACTGCAGAAGGGAAACGA
ACTGGCCCTGCCCTCCAAATATGTGAACTTCCTGTACCTGGCCAGCCACTATGAG
AAGCTGAAGGGCTCCCCCGAGGATAATGAGCAGAAACAGCTGTTTGTGGAACAG
CACAAGCACTACCTGGACGAGATCATCGAGCAGATCAGCGAGTTCTCCAAGAGA
GTGATCCTGGCCGACGCTAATCTGGACAAAGTGCTGTCCGCCTACAACAAGCACC
GGGATAAGCCCATCAGAGAGCAGGCCGAGAATATCATCCACCTGTTTACCCTGA
CCAATCTGGGAGCCCCTGCCGCCTTCAAGTACTTTGACACCACCATCGACCGGAA
GAGGTACACCAGCACCAAAGAGGTGCTGGACGCCACCCTGATCCACCAGAGCAT
CACCGGCCTGTACGAGACACGGATCGACCTGTCTCAGCTGGGAGGCGACAGCCC
CAAGAAGAAGAGAAAGGTGGAGGCCAGCGGTGGCGGCGGTTCAGGCGGAGGTG
GCTCTGGGGGCGGGGGTTCTGCTCCGGGAAAAAAGAGGCCGGTAGAGCACTCTC
CTGTGGAGCCAGACTCCTCCTCGGGAACCGGAAAGGCGGGCCAGCAGCCTGCAA
GAAAAAGATTGAATTTTGGTCAGACTGGAGACGCAGACTCAGTACCTGACCCCC
AGCCTCTCGGACAGCCACCAGCAGCCCCCTCTGGTCTGGGAACTAATACGATGGC
TACAGGCAGTGGCGCACCAATGGCAGACAATAACGAGGGCGCCGACGGAGTGG
GTAATTCCTCGGGAAATTGGCATTGCGATTCCACATGGATGGGCGACAGAGTCAT
CACCACCAGCACCCGAACCTGGGCCCTGCCCACCTACAACAACCACCTCTACAA
ACAAATTTCCAGCCAATCAGGAGCCTCGAACGACAATCACTACTTTGGCTACAGC
ACCCCTTGGGGGTATTTTGACTTCAACAGATTCCACTGCCACTTTTCACCACGTGA
CTGGCAAAGACTCATCAACAACAACTGGGGATTCCGACCCAAGAGACTCAACTT
CAAGCTCTTTAACATTCAAGTCAAAGAGGTCACGCAGAATGACGGTACGACGAC
GATTGCCAATAACCTTACCAGCACGGTTCAGGTGTTTACTGACTCGGAGTACCAG
CTCCCGTACGTCCTCGGCTCGGCGCATCAAGGATGCCTCCCGCCGTTCCCAGCAG
ACGTCTTCATGGTGCCACAGTATGGATACCTCACCCTGAACAACGGGAGTCAGGC
AGTAGGACGCTCTTCATTTTACTGCCTGGAGTACTTTCCTTCTCAGATGCTGCGTA
CCGGAAACAACTTTACCTTCAGCTACACTTTTGAGGACGTTCCTTTCCACAGCAG
CTACGCTCACAGCCAGAGTCTGGACCGTCTCATGAATCCTCTCATCGACCAGTAC
CTGTATTACTTGAGCAGAACAAACACTCCAAGTGGAACCACCACGCAGTCAAGG
CTTCAGTTTTCTCAGGCCGGAGCGAGTGACATTCGGGACCAGTCTAGGAACTGGC
TTCCTGGACCCTGTTACCGCCAGCAGCGAGTATCAAAGACATCTGCGGATAACAA
CAACAGTGAATACTCGTGGACTGGAGCTACCAAGTACCACCTCAATGGCAGAGA
CTCTCTGGTGAATCCGGGCCCGGCCATGGCAAGCCACAAGGACGATGAAGAAAA
GTTTTTTCCTCAGAGCGGGGTTCTCATCTTTGGGAAGCAAGGCTCAGAGAAAACA
AATGTGGACATTGAAAAGGTCATGATTACAGACGAAGAGGAAATCAGGACAACC
AATCCCGTGGCTACGGAGCAGTATGGTTCTGTATCTACCAACCTCCAGAGAGGCA
ACAGACAAGCAGCTACCGCAGATGTCAACACACAAGGCGTTCTTCCAGGCATGG
TCTGGCAGGACAGAGATGTGTACCTTCAGGGGCCCATCTGGGCAAAGATTCCAC
ACACGGACGGACATTTTCACCCCTCTCCCCTCATGGGTGGATTCGGACTTAAACA
CCCTCCTCCACAGATTCTCATCAAGAACACCCCGGTACCTGCGAATCCTTCGACC
ACCTTCAGTGCGGCAAAGTTTGCTTCCTTCATCACACAGTACTCCACGGGACAGG
TCAGCGTGGAGATCGAGTGGGAGCTGCAGAAGGAAAACAGCAAACGCTGGAAT
CCCGAAATTCAGTACACTTCCAACTACAACAAGTCTGTTAATGTGGACTTTACTG
TGGACACTAATGGCGTGTATTCAGAGCCTCGCCCCATTGGCACCAGATACCTGAC
TCGTAATCTGTAAGAATTAAACCCGCTGATCAGCCTCGACTGTGCCTTCTAGTTG
CCAGCCATCTGTTGTTTGCCCCTCCCCCGTGCCTTCCTTGACCCTGGAAGGTGCCA
CTCCCACTGTCCTTTCCTAATAAAATGAGGAAATTGCATCGCATTGTCTGAGTAG
GTGTCATTCTATTCTGGGGGGTGGGGTGGGGCAGGACAGCAAGGGGGAGGATTG
GGAAGACAATAGCAGGCATGCTGGGGATGCGGTGGGCTCTATGG >SEQ ID NO: 13
Int.sub.c-Cas9.sub.c-EAAAKx3-VP2
GTTGACATTGATTATTGACTAGTTATTAATAGTAATCAATTACGGGGTCATTAGTT
CATAGCCCATATATGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTG
GCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCAT
AGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGACTATTTACGGTAA
ACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTG
ACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATG
GGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGTGA
TGCGGTTTTGGCAGTACATCAATGGGCGTGGATAGCGGTTTGACTCACGGGGATT
TCCAAGTCTCCACCCCATTGACGTCAATGGGAGTTTGTTTTGGCACCAAAATCAA
CGGGACTTTCCAAAATGTCGTAACAACTCCGCCCCATTGACGCAAATGGGCGGTA
GGCGTGTACGGTGGGAGGTCTATATAAGCAGAGCTCTCTGGCTAACTAGAGAAC
CCACTGCTTACTGGCTTATCGAAATTAATACGACTCACTATAGGGAGACCCAAGC
TGGCTAGCGCCACCATGATCAAGATTGCCACGCGCAAGTACCTGGGCAAGCAGA
ACGTGTACGACATCGGAGTGGAGCGCGATCACAACTTTGCCCTGAAGAATGGCT
TTATTGCCTCGAACTGTATGCTGGCCTCTGCCGGCGAACTGCAGAAGGGAAACGA
ACTGGCCCTGCCCTCCAAATATGTGAACTTCCTGTACCTGGCCAGCCACTATGAG
AAGCTGAAGGGCTCCCCCGAGGATAATGAGCAGAAACAGCTGTTTGTGGAACAG
CACAAGCACTACCTGGACGAGATCATCGAGCAGATCAGCGAGTTCTCCAAGAGA
GTGATCCTGGCCGACGCTAATCTGGACAAAGTGCTGTCCGCCTACAACAAGCACC
GGGATAAGCCCATCAGAGAGCAGGCCGAGAATATCATCCACCTGTTTACCCTGA
CCAATCTGGGAGCCCCTGCCGCCTTCAAGTACTTTGACACCACCATCGACCGGAA
GAGGTACACCAGCACCAAAGAGGTGCTGGACGCCACCCTGATCCACCAGAGCAT
CACCGGCCTGTACGAGACACGGATCGACCTGTCTCAGCTGGGAGGCGACAGCCC
CAAGAAGAAGAGAAAGGTGGAGGCCAGCGAGGCAGCAGCCAAAGAGGCCGCTG
CCAAGGAGGCAGCGGCTAAAGCTCCGGGAAAAAAGAGGCCGGTAGAGCACTCT
CCTGTGGAGCCAGACTCCTCCTCGGGAACCGGAAAGGCGGGCCAGCAGCCTGCA
AGAAAAAGATTGAATTTTGGTCAGACTGGAGACGCAGACTCAGTACCTGACCCC
CAGCCTCTCGGACAGCCACCAGCAGCCCCCTCTGGTCTGGGAACTAATACGATGG
CTACAGGCAGTGGCGCACCAATGGCAGACAATAACGAGGGCGCCGACGGAGTG
GGTAATTCCTCGGGAAATTGGCATTGCGATTCCACATGGATGGGCGACAGAGTC
ATCACCACCAGCACCCGAACCTGGGCCCTGCCCACCTACAACAACCACCTCTACA
AACAAATTTCCAGCCAATCAGGAGCCTCGAACGACAATCACTACTTTGGCTACAG
CACCCCTTGGGGGTATTTTGACTTCAACAGATTCCACTGCCACTTTTCACCACGTG
ACTGGCAAAGACTCATCAACAACAACTGGGGATTCCGACCCAAGAGACTCAACT
TCAAGCTCTTTAACATTCAAGTCAAAGAGGTCACGCAGAATGACGGTACGACGA
CGATTGCCAATAACCTTACCAGCACGGTTCAGGTGTTTACTGACTCGGAGTACCA
GCTCCCGTACGTCCTCGGCTCGGCGCATCAAGGATGCCTCCCGCCGTTCCCAGCA
GACGTCTTCATGGTGCCACAGTATGGATACCTCACCCTGAACAACGGGAGTCAG
GCAGTAGGACGCTCTTCATTTTACTGCCTGGAGTACTTTCCTTCTCAGATGCTGCG
TACCGGAAACAACTTTACCTTCAGCTACACTTTTGAGGACGTTCCTTTCCACAGC
AGCTACGCTCACAGCCAGAGTCTGGACCGTCTCATGAATCCTCTCATCGACCAGT
ACCTGTATTACTTGAGCAGAACAAACACTCCAAGTGGAACCACCACGCAGTCAA
GGCTTCAGTTTTCTCAGGCCGGAGCGAGTGACATTCGGGACCAGTCTAGGAACTG
GCTTCCTGGACCCTGTTACCGCCAGCAGCGAGTATCAAAGACATCTGCGGATAAC
AACAACAGTGAATACTCGTGGACTGGAGCTACCAAGTACCACCTCAATGGCAGA
GACTCTCTGGTGAATCCGGGCCCGGCCATGGCAAGCCACAAGGACGATGAAGAA
AAGTTTTTTCCTCAGAGCGGGGTTCTCATCTTTGGGAAGCAAGGCTCAGAGAAAA
CAAATGTGGACATTGAAAAGGTCATGATTACAGACGAAGAGGAAATCAGGACAA
CCAATCCCGTGGCTACGGAGCAGTATGGTTCTGTATCTACCAACCTCCAGAGAGG
CAACAGACAAGCAGCTACCGCAGATGTCAACACACAAGGCGTTCTTCCAGGCAT
GGTCTGGCAGGACAGAGATGTGTACCTTCAGGGGCCCATCTGGGCAAAGATTCC
ACACACGGACGGACATTTTCACCCCTCTCCCCTCATGGGTGGATTCGGACTTAAA
CACCCTCCTCCACAGATTCTCATCAAGAACACCCCGGTACCTGCGAATCCTTCGA
CCACCTTCAGTGCGGCAAAGTTTGCTTCCTTCATCACACAGTACTCCACGGGACA
GGTCAGCGTGGAGATCGAGTGGGAGCTGCAGAAGGAAAACAGCAAACGCTGGA
ATCCCGAAATTCAGTACACTTCCAACTACAACAAGTCTGTTAATGTGGACTTTAC
TGTGGACACTAATGGCGTGTATTCAGAGCCTCGCCCCATTGGCACCAGATACCTG
ACTCGTAATCTGTAAGAATTAAACCCGCTGATCAGCCTCGACTGTGCCTTCTAGT
TGCCAGCCATCTGTTGTTTGCCCCTCCCCCGTGCCTTCCTTGACCCTGGAAGGTGC
CACTCCCACTGTCCTTTCCTAATAAAATGAGGAAATTGCATCGCATTGTCTGAGT
AGGTGTCATTCTATTCTGGGGGGTGGGGTGGGGCAGGACAGCAAGGGGGAGGAT
TGGGAAGACAATAGCAGGCATGCTGGGGATGCGGTGGGCTCTATGG
[0146] This disclosure is not limited in its application to the
details of construction and the arrangement of components set forth
in this description or illustrated in the drawings. The disclosure
is capable of other embodiments and of being practiced or of being
carried out in various ways. Also, the phraseology and terminology
used herein is for the purpose of description and should not be
regarded as limiting. The use of "including," "comprising," or
"having," "containing," "involving," and variations thereof herein,
is meant to encompass the items listed thereafter and equivalents
thereof as well as additional items.
[0147] Having thus described several aspects of at least one
embodiment of this disclosure, it is to be appreciated various
alterations, modifications, and improvements will readily occur to
those skilled in the art. Such alterations, modifications, and
improvements are intended to be part of this disclosure, and are
intended to be within the spirit and scope of the disclosure.
Accordingly, the foregoing description and drawings are by way of
example only.
Sequence CWU 1
1
1314197DNAArtificial SequenceSynthetic Polynucleotide 1atgtacccat
acgatgttcc agattacgct tcgccgaaga aaaagcgcaa ggtcgaagcg 60tccgacaaga
agtacagcat cggcctggac atcggcacca actctgtggg ctgggccgtg
120atcaccgacg agtacaaggt gcccagcaag aaattcaagg tgctgggcaa
caccgaccgg 180cacagcatca agaagaacct gatcggagcc ctgctgttcg
acagcggcga aacagccgag 240gccacccggc tgaagagaac cgccagaaga
agatacacca gacggaagaa ccggatctgc 300tatctgcaag agatcttcag
caacgagatg gccaaggtgg acgacagctt cttccacaga 360ctggaagagt
ccttcctggt ggaagaggat aagaagcacg agcggcaccc catcttcggc
420aacatcgtgg acgaggtggc ctaccacgag aagtacccca ccatctacca
cctgagaaag 480aaactggtgg acagcaccga caaggccgac ctgcggctga
tctatctggc cctggcccac 540atgatcaagt tccggggcca cttcctgatc
gagggcgacc tgaaccccga caacagcgac 600gtggacaagc tgttcatcca
gctggtgcag acctacaacc agctgttcga ggaaaacccc 660atcaacgcca
gcggcgtgga cgccaaggcc atcctgtctg ccagactgag caagagcaga
720cggctggaaa atctgatcgc ccagctgccc ggcgagaaga agaatggcct
gttcggcaac 780ctgattgccc tgagcctggg cctgaccccc aacttcaaga
gcaacttcga cctggccgag 840gatgccaaac tgcagctgag caaggacacc
tacgacgacg acctggacaa cctgctggcc 900cagatcggcg accagtacgc
cgacctgttt ctggccgcca agaacctgtc cgacgccatc 960ctgctgagcg
acatcctgag agtgaacacc gagatcacca aggcccccct gagcgcctct
1020atgatcaaga gatacgacga gcaccaccag gacctgaccc tgctgaaagc
tctcgtgcgg 1080cagcagctgc ctgagaagta caaagagatt ttcttcgacc
agagcaagaa cggctacgcc 1140ggctacattg acggcggagc cagccaggaa
gagttctaca agttcatcaa gcccatcctg 1200gaaaagatgg acggcaccga
ggaactgctc gtgaagctga acagagagga cctgctgcgg 1260aagcagcgga
ccttcgacaa cggcagcatc ccccaccaga tccacctggg agagctgcac
1320gccattctgc ggcggcagga agatttttac ccattcctga aggacaaccg
ggaaaagatc 1380gagaagatcc tgaccttccg catcccctac tacgtgggcc
ctctggccag gggaaacagc 1440agattcgcct ggatgaccag aaagagcgag
gaaaccatca ccccctggaa cttcgaggaa 1500gtggtggaca agggcgcttc
cgcccagagc ttcatcgagc ggatgaccaa cttcgataag 1560aacctgccca
acgagaaggt gctgcccaag cacagcctgc tgtacgagta cttcaccgtg
1620tataacgagc tgaccaaagt gaaatacgtg accgagggaa tgagaaagcc
cgccttcctg 1680agcggcgagc agaaaaaggc catcgtggac ctgctgttca
agaccaaccg gaaagtgacc 1740gtgaagcagc tgaaagagga ctacttcaag
aaaatcgagt gcttcgactc cgtggaaatc 1800tccggcgtgg aagatcggtt
caacgcctcc ctgggcacat accacgatct gctgaaaatt 1860atcaaggaca
aggacttcct ggacaatgag gaaaacgagg acattctgga agatatcgtg
1920ctgaccctga cactgtttga ggacagagag atgatcgagg aacggctgaa
aacctatgcc 1980cacctgttcg acgacaaagt gatgaagcag ctgaagcggc
ggagatacac cggctggggc 2040aggctgagcc ggaagctgat caacggcatc
cgggacaagc agtccggcaa gacaatcctg 2100gatttcctga agtccgacgg
cttcgccaac agaaacttca tgcagctgat ccacgacgac 2160agcctgacct
ttaaagagga catccagaaa gcccaggtgt ccggccaggg cgatagcctg
2220cacgagcaca ttgccaatct ggccggcagc cccgccatta agaagggcat
cctgcagaca 2280gtgaaggtgg tggacgagct cgtgaaagtg atgggccggc
acaagcccga gaacatcgtg 2340atcgaaatgg ccagagagaa ccagaccacc
cagaagggac agaagaacag ccgcgagaga 2400atgaagcgga tcgaagaggg
catcaaagag ctgggcagcc agatcctgaa agaacacccc 2460gtggaaaaca
cccagctgca gaacgagaag ctgtacctgt actacctgca gaatgggcgg
2520gatatgtacg tggaccagga actggacatc aaccggctgt ccgactacga
tgtggaccat 2580atcgtgcctc agagctttct gaaggacgac tccatcgaca
acaaggtgct gaccagaagc 2640gacaagaacc ggggcaagag cgacaacgtg
ccctccgaag aggtcgtgaa gaagatgaag 2700aactactggc ggcagctgct
gaacgccaag ctgattaccc agagaaagtt cgacaatctg 2760accaaggccg
agagaggcgg cctgagcgaa ctggataagg ccggcttcat caagagacag
2820ctggtggaaa cccggcagat cacaaagcac gtggcacaga tcctggactc
ccggatgaac 2880actaagtacg acgagaatga caagctgatc cgggaagtga
aagtgatcac cctgaagtcc 2940aagctggtgt ccgatttccg gaaggatttc
cagttttaca aagtgcgcga gatcaacaac 3000taccaccacg cccacgacgc
ctacctgaac gccgtcgtgg gaaccgccct gatcaaaaag 3060taccctaagc
tggaaagcga gttcgtgtac ggcgactaca aggtgtacga cgtgcggaag
3120atgatcgcca agagcgagca ggaaatcggc aaggctaccg ccaagtactt
cttctacagc 3180aacatcatga actttttcaa gaccgagatt accctggcca
acggcgagat ccggaagcgg 3240cctctgatcg agacaaacgg cgaaaccggg
gagatcgtgt gggataaggg ccgggatttt 3300gccaccgtgc ggaaagtgct
gagcatgccc caagtgaata tcgtgaaaaa gaccgaggtg 3360cagacaggcg
gcttcagcaa agagtctatc ctgcccaaga ggaacagcga taagctgatc
3420gccagaaaga aggactggga ccctaagaag tacggcggct tcgacagccc
caccgtggcc 3480tattctgtgc tggtggtggc caaagtggaa aagggcaagt
ccaagaaact gaagagtgtg 3540aaagagctgc tggggatcac catcatggaa
agaagcagct tcgagaagaa tcccatcgac 3600tttctggaag ccaagggcta
caaagaagtg aaaaaggacc tgatcatcaa gctgcctaag 3660tactccctgt
tcgagctgga aaacggccgg aagagaatgc tggcctctgc cggcgaactg
3720cagaagggaa acgaactggc cctgccctcc aaatatgtga acttcctgta
cctggccagc 3780cactatgaga agctgaaggg ctcccccgag gataatgagc
agaaacagct gtttgtggaa 3840cagcacaagc actacctgga cgagatcatc
gagcagatca gcgagttctc caagagagtg 3900atcctggccg acgctaatct
ggacaaagtg ctgtccgcct acaacaagca ccgggataag 3960cccatcagag
agcaggccga gaatatcatc cacctgttta ccctgaccaa tctgggagcc
4020cctgccgcct tcaagtactt tgacaccacc atcgaccgga agaggtacac
cagcaccaaa 4080gaggtgctgg acgccaccct gatccaccag agcatcaccg
gcctgtacga gacacggatc 4140gacctgtctc agctgggagg cgacagcccc
aagaagaaga gaaaggtgga ggccagc 419721399PRTArtificial
SequenceSynthetic Polypeptide 2Met Tyr Pro Tyr Asp Val Pro Asp Tyr
Ala Ser Pro Lys Lys Lys Arg 1 5 10 15 Lys Val Glu Ala Ser Asp Lys
Lys Tyr Ser Ile Gly Leu Asp Ile Gly 20 25 30 Thr Asn Ser Val Gly
Trp Ala Val Ile Thr Asp Glu Tyr Lys Val Pro 35 40 45 Ser Lys Lys
Phe Lys Val Leu Gly Asn Thr Asp Arg His Ser Ile Lys 50 55 60 Lys
Asn Leu Ile Gly Ala Leu Leu Phe Asp Ser Gly Glu Thr Ala Glu 65 70
75 80 Ala Thr Arg Leu Lys Arg Thr Ala Arg Arg Arg Tyr Thr Arg Arg
Lys 85 90 95 Asn Arg Ile Cys Tyr Leu Gln Glu Ile Phe Ser Asn Glu
Met Ala Lys 100 105 110 Val Asp Asp Ser Phe Phe His Arg Leu Glu Glu
Ser Phe Leu Val Glu 115 120 125 Glu Asp Lys Lys His Glu Arg His Pro
Ile Phe Gly Asn Ile Val Asp 130 135 140 Glu Val Ala Tyr His Glu Lys
Tyr Pro Thr Ile Tyr His Leu Arg Lys 145 150 155 160 Lys Leu Val Asp
Ser Thr Asp Lys Ala Asp Leu Arg Leu Ile Tyr Leu 165 170 175 Ala Leu
Ala His Met Ile Lys Phe Arg Gly His Phe Leu Ile Glu Gly 180 185 190
Asp Leu Asn Pro Asp Asn Ser Asp Val Asp Lys Leu Phe Ile Gln Leu 195
200 205 Val Gln Thr Tyr Asn Gln Leu Phe Glu Glu Asn Pro Ile Asn Ala
Ser 210 215 220 Gly Val Asp Ala Lys Ala Ile Leu Ser Ala Arg Leu Ser
Lys Ser Arg 225 230 235 240 Arg Leu Glu Asn Leu Ile Ala Gln Leu Pro
Gly Glu Lys Lys Asn Gly 245 250 255 Leu Phe Gly Asn Leu Ile Ala Leu
Ser Leu Gly Leu Thr Pro Asn Phe 260 265 270 Lys Ser Asn Phe Asp Leu
Ala Glu Asp Ala Lys Leu Gln Leu Ser Lys 275 280 285 Asp Thr Tyr Asp
Asp Asp Leu Asp Asn Leu Leu Ala Gln Ile Gly Asp 290 295 300 Gln Tyr
Ala Asp Leu Phe Leu Ala Ala Lys Asn Leu Ser Asp Ala Ile 305 310 315
320 Leu Leu Ser Asp Ile Leu Arg Val Asn Thr Glu Ile Thr Lys Ala Pro
325 330 335 Leu Ser Ala Ser Met Ile Lys Arg Tyr Asp Glu His His Gln
Asp Leu 340 345 350 Thr Leu Leu Lys Ala Leu Val Arg Gln Gln Leu Pro
Glu Lys Tyr Lys 355 360 365 Glu Ile Phe Phe Asp Gln Ser Lys Asn Gly
Tyr Ala Gly Tyr Ile Asp 370 375 380 Gly Gly Ala Ser Gln Glu Glu Phe
Tyr Lys Phe Ile Lys Pro Ile Leu 385 390 395 400 Glu Lys Met Asp Gly
Thr Glu Glu Leu Leu Val Lys Leu Asn Arg Glu 405 410 415 Asp Leu Leu
Arg Lys Gln Arg Thr Phe Asp Asn Gly Ser Ile Pro His 420 425 430 Gln
Ile His Leu Gly Glu Leu His Ala Ile Leu Arg Arg Gln Glu Asp 435 440
445 Phe Tyr Pro Phe Leu Lys Asp Asn Arg Glu Lys Ile Glu Lys Ile Leu
450 455 460 Thr Phe Arg Ile Pro Tyr Tyr Val Gly Pro Leu Ala Arg Gly
Asn Ser 465 470 475 480 Arg Phe Ala Trp Met Thr Arg Lys Ser Glu Glu
Thr Ile Thr Pro Trp 485 490 495 Asn Phe Glu Glu Val Val Asp Lys Gly
Ala Ser Ala Gln Ser Phe Ile 500 505 510 Glu Arg Met Thr Asn Phe Asp
Lys Asn Leu Pro Asn Glu Lys Val Leu 515 520 525 Pro Lys His Ser Leu
Leu Tyr Glu Tyr Phe Thr Val Tyr Asn Glu Leu 530 535 540 Thr Lys Val
Lys Tyr Val Thr Glu Gly Met Arg Lys Pro Ala Phe Leu 545 550 555 560
Ser Gly Glu Gln Lys Lys Ala Ile Val Asp Leu Leu Phe Lys Thr Asn 565
570 575 Arg Lys Val Thr Val Lys Gln Leu Lys Glu Asp Tyr Phe Lys Lys
Ile 580 585 590 Glu Cys Phe Asp Ser Val Glu Ile Ser Gly Val Glu Asp
Arg Phe Asn 595 600 605 Ala Ser Leu Gly Thr Tyr His Asp Leu Leu Lys
Ile Ile Lys Asp Lys 610 615 620 Asp Phe Leu Asp Asn Glu Glu Asn Glu
Asp Ile Leu Glu Asp Ile Val 625 630 635 640 Leu Thr Leu Thr Leu Phe
Glu Asp Arg Glu Met Ile Glu Glu Arg Leu 645 650 655 Lys Thr Tyr Ala
His Leu Phe Asp Asp Lys Val Met Lys Gln Leu Lys 660 665 670 Arg Arg
Arg Tyr Thr Gly Trp Gly Arg Leu Ser Arg Lys Leu Ile Asn 675 680 685
Gly Ile Arg Asp Lys Gln Ser Gly Lys Thr Ile Leu Asp Phe Leu Lys 690
695 700 Ser Asp Gly Phe Ala Asn Arg Asn Phe Met Gln Leu Ile His Asp
Asp 705 710 715 720 Ser Leu Thr Phe Lys Glu Asp Ile Gln Lys Ala Gln
Val Ser Gly Gln 725 730 735 Gly Asp Ser Leu His Glu His Ile Ala Asn
Leu Ala Gly Ser Pro Ala 740 745 750 Ile Lys Lys Gly Ile Leu Gln Thr
Val Lys Val Val Asp Glu Leu Val 755 760 765 Lys Val Met Gly Arg His
Lys Pro Glu Asn Ile Val Ile Glu Met Ala 770 775 780 Arg Glu Asn Gln
Thr Thr Gln Lys Gly Gln Lys Asn Ser Arg Glu Arg 785 790 795 800 Met
Lys Arg Ile Glu Glu Gly Ile Lys Glu Leu Gly Ser Gln Ile Leu 805 810
815 Lys Glu His Pro Val Glu Asn Thr Gln Leu Gln Asn Glu Lys Leu Tyr
820 825 830 Leu Tyr Tyr Leu Gln Asn Gly Arg Asp Met Tyr Val Asp Gln
Glu Leu 835 840 845 Asp Ile Asn Arg Leu Ser Asp Tyr Asp Val Asp His
Ile Val Pro Gln 850 855 860 Ser Phe Leu Lys Asp Asp Ser Ile Asp Asn
Lys Val Leu Thr Arg Ser 865 870 875 880 Asp Lys Asn Arg Gly Lys Ser
Asp Asn Val Pro Ser Glu Glu Val Val 885 890 895 Lys Lys Met Lys Asn
Tyr Trp Arg Gln Leu Leu Asn Ala Lys Leu Ile 900 905 910 Thr Gln Arg
Lys Phe Asp Asn Leu Thr Lys Ala Glu Arg Gly Gly Leu 915 920 925 Ser
Glu Leu Asp Lys Ala Gly Phe Ile Lys Arg Gln Leu Val Glu Thr 930 935
940 Arg Gln Ile Thr Lys His Val Ala Gln Ile Leu Asp Ser Arg Met Asn
945 950 955 960 Thr Lys Tyr Asp Glu Asn Asp Lys Leu Ile Arg Glu Val
Lys Val Ile 965 970 975 Thr Leu Lys Ser Lys Leu Val Ser Asp Phe Arg
Lys Asp Phe Gln Phe 980 985 990 Tyr Lys Val Arg Glu Ile Asn Asn Tyr
His His Ala His Asp Ala Tyr 995 1000 1005 Leu Asn Ala Val Val Gly
Thr Ala Leu Ile Lys Lys Tyr Pro Lys 1010 1015 1020 Leu Glu Ser Glu
Phe Val Tyr Gly Asp Tyr Lys Val Tyr Asp Val 1025 1030 1035 Arg Lys
Met Ile Ala Lys Ser Glu Gln Glu Ile Gly Lys Ala Thr 1040 1045 1050
Ala Lys Tyr Phe Phe Tyr Ser Asn Ile Met Asn Phe Phe Lys Thr 1055
1060 1065 Glu Ile Thr Leu Ala Asn Gly Glu Ile Arg Lys Arg Pro Leu
Ile 1070 1075 1080 Glu Thr Asn Gly Glu Thr Gly Glu Ile Val Trp Asp
Lys Gly Arg 1085 1090 1095 Asp Phe Ala Thr Val Arg Lys Val Leu Ser
Met Pro Gln Val Asn 1100 1105 1110 Ile Val Lys Lys Thr Glu Val Gln
Thr Gly Gly Phe Ser Lys Glu 1115 1120 1125 Ser Ile Leu Pro Lys Arg
Asn Ser Asp Lys Leu Ile Ala Arg Lys 1130 1135 1140 Lys Asp Trp Asp
Pro Lys Lys Tyr Gly Gly Phe Asp Ser Pro Thr 1145 1150 1155 Val Ala
Tyr Ser Val Leu Val Val Ala Lys Val Glu Lys Gly Lys 1160 1165 1170
Ser Lys Lys Leu Lys Ser Val Lys Glu Leu Leu Gly Ile Thr Ile 1175
1180 1185 Met Glu Arg Ser Ser Phe Glu Lys Asn Pro Ile Asp Phe Leu
Glu 1190 1195 1200 Ala Lys Gly Tyr Lys Glu Val Lys Lys Asp Leu Ile
Ile Lys Leu 1205 1210 1215 Pro Lys Tyr Ser Leu Phe Glu Leu Glu Asn
Gly Arg Lys Arg Met 1220 1225 1230 Leu Ala Ser Ala Gly Glu Leu Gln
Lys Gly Asn Glu Leu Ala Leu 1235 1240 1245 Pro Ser Lys Tyr Val Asn
Phe Leu Tyr Leu Ala Ser His Tyr Glu 1250 1255 1260 Lys Leu Lys Gly
Ser Pro Glu Asp Asn Glu Gln Lys Gln Leu Phe 1265 1270 1275 Val Glu
Gln His Lys His Tyr Leu Asp Glu Ile Ile Glu Gln Ile 1280 1285 1290
Ser Glu Phe Ser Lys Arg Val Ile Leu Ala Asp Ala Asn Leu Asp 1295
1300 1305 Lys Val Leu Ser Ala Tyr Asn Lys His Arg Asp Lys Pro Ile
Arg 1310 1315 1320 Glu Gln Ala Glu Asn Ile Ile His Leu Phe Thr Leu
Thr Asn Leu 1325 1330 1335 Gly Ala Pro Ala Ala Phe Lys Tyr Phe Asp
Thr Thr Ile Asp Arg 1340 1345 1350 Lys Arg Tyr Thr Ser Thr Lys Glu
Val Leu Asp Ala Thr Leu Ile 1355 1360 1365 His Gln Ser Ile Thr Gly
Leu Tyr Glu Thr Arg Ile Asp Leu Ser 1370 1375 1380 Gln Leu Gly Gly
Asp Ser Pro Lys Lys Lys Arg Lys Val Glu Ala 1385 1390 1395 Ser
35997DNAArtificial SequenceSynthetic Polynucleotide 3atgtacccat
acgatgttcc agattacgct tcgccgaaga aaaagcgcaa ggtcgaagcg 60tccgacaaga
agtacagcat cggcctggac atcggcacca actctgtggg ctgggccgtg
120atcaccgacg agtacaaggt gcccagcaag aaattcaagg tgctgggcaa
caccgaccgg 180cacagcatca agaagaacct gatcggagcc ctgctgttcg
acagcggcga aacagccgag 240gccacccggc tgaagagaac cgccagaaga
agatacacca gacggaagaa ccggatctgc 300tatctgcaag agatcttcag
caacgagatg gccaaggtgg acgacagctt cttccacaga 360ctggaagagt
ccttcctggt ggaagaggat aagaagcacg agcggcaccc catcttcggc
420aacatcgtgg acgaggtggc ctaccacgag aagtacccca ccatctacca
cctgagaaag 480aaactggtgg acagcaccga caaggccgac ctgcggctga
tctatctggc cctggcccac 540atgatcaagt tccggggcca cttcctgatc
gagggcgacc tgaaccccga caacagcgac 600gtggacaagc tgttcatcca
gctggtgcag acctacaacc agctgttcga ggaaaacccc 660atcaacgcca
gcggcgtgga cgccaaggcc atcctgtctg ccagactgag caagagcaga
720cggctggaaa atctgatcgc ccagctgccc ggcgagaaga agaatggcct
gttcggcaac 780ctgattgccc tgagcctggg cctgaccccc aacttcaaga
gcaacttcga cctggccgag 840gatgccaaac tgcagctgag caaggacacc
tacgacgacg acctggacaa cctgctggcc 900cagatcggcg accagtacgc
cgacctgttt ctggccgcca agaacctgtc cgacgccatc 960ctgctgagcg
acatcctgag agtgaacacc gagatcacca aggcccccct gagcgcctct
1020atgatcaaga gatacgacga gcaccaccag gacctgaccc tgctgaaagc
tctcgtgcgg 1080cagcagctgc ctgagaagta caaagagatt ttcttcgacc
agagcaagaa cggctacgcc 1140ggctacattg acggcggagc cagccaggaa
gagttctaca agttcatcaa gcccatcctg 1200gaaaagatgg acggcaccga
ggaactgctc gtgaagctga acagagagga cctgctgcgg 1260aagcagcgga
ccttcgacaa cggcagcatc ccccaccaga tccacctggg agagctgcac
1320gccattctgc ggcggcagga agatttttac ccattcctga aggacaaccg
ggaaaagatc 1380gagaagatcc tgaccttccg catcccctac tacgtgggcc
ctctggccag gggaaacagc 1440agattcgcct
ggatgaccag aaagagcgag gaaaccatca ccccctggaa cttcgaggaa
1500gtggtggaca agggcgcttc cgcccagagc ttcatcgagc ggatgaccaa
cttcgataag 1560aacctgccca acgagaaggt gctgcccaag cacagcctgc
tgtacgagta cttcaccgtg 1620tataacgagc tgaccaaagt gaaatacgtg
accgagggaa tgagaaagcc cgccttcctg 1680agcggcgagc agaaaaaggc
catcgtggac ctgctgttca agaccaaccg gaaagtgacc 1740gtgaagcagc
tgaaagagga ctacttcaag aaaatcgagt gcttcgactc cgtggaaatc
1800tccggcgtgg aagatcggtt caacgcctcc ctgggcacat accacgatct
gctgaaaatt 1860atcaaggaca aggacttcct ggacaatgag gaaaacgagg
acattctgga agatatcgtg 1920ctgaccctga cactgtttga ggacagagag
atgatcgagg aacggctgaa aacctatgcc 1980cacctgttcg acgacaaagt
gatgaagcag ctgaagcggc ggagatacac cggctggggc 2040aggctgagcc
ggaagctgat caacggcatc cgggacaagc agtccggcaa gacaatcctg
2100gatttcctga agtccgacgg cttcgccaac agaaacttca tgcagctgat
ccacgacgac 2160agcctgacct ttaaagagga catccagaaa gcccaggtgt
ccggccaggg cgatagcctg 2220cacgagcaca ttgccaatct ggccggcagc
cccgccatta agaagggcat cctgcagaca 2280gtgaaggtgg tggacgagct
cgtgaaagtg atgggccggc acaagcccga gaacatcgtg 2340atcgaaatgg
ccagagagaa ccagaccacc cagaagggac agaagaacag ccgcgagaga
2400atgaagcgga tcgaagaggg catcaaagag ctgggcagcc agatcctgaa
agaacacccc 2460gtggaaaaca cccagctgca gaacgagaag ctgtacctgt
actacctgca gaatgggcgg 2520gatatgtacg tggaccagga actggacatc
aaccggctgt ccgactacga tgtggaccat 2580atcgtgcctc agagctttct
gaaggacgac tccatcgaca acaaggtgct gaccagaagc 2640gacaagaacc
ggggcaagag cgacaacgtg ccctccgaag aggtcgtgaa gaagatgaag
2700aactactggc ggcagctgct gaacgccaag ctgattaccc agagaaagtt
cgacaatctg 2760accaaggccg agagaggcgg cctgagcgaa ctggataagg
ccggcttcat caagagacag 2820ctggtggaaa cccggcagat cacaaagcac
gtggcacaga tcctggactc ccggatgaac 2880actaagtacg acgagaatga
caagctgatc cgggaagtga aagtgatcac cctgaagtcc 2940aagctggtgt
ccgatttccg gaaggatttc cagttttaca aagtgcgcga gatcaacaac
3000taccaccacg cccacgacgc ctacctgaac gccgtcgtgg gaaccgccct
gatcaaaaag 3060taccctaagc tggaaagcga gttcgtgtac ggcgactaca
aggtgtacga cgtgcggaag 3120atgatcgcca agagcgagca ggaaatcggc
aaggctaccg ccaagtactt cttctacagc 3180aacatcatga actttttcaa
gaccgagatt accctggcca acggcgagat ccggaagcgg 3240cctctgatcg
agacaaacgg cgaaaccggg gagatcgtgt gggataaggg ccgggatttt
3300gccaccgtgc ggaaagtgct gagcatgccc caagtgaata tcgtgaaaaa
gaccgaggtg 3360cagacaggcg gcttcagcaa agagtctatc ctgcccaaga
ggaacagcga taagctgatc 3420gccagaaaga aggactggga ccctaagaag
tacggcggct tcgacagccc caccgtggcc 3480tattctgtgc tggtggtggc
caaagtggaa aagggcaagt ccaagaaact gaagagtgtg 3540aaagagctgc
tggggatcac catcatggaa agaagcagct tcgagaagaa tcccatcgac
3600tttctggaag ccaagggcta caaagaagtg aaaaaggacc tgatcatcaa
gctgcctaag 3660tactccctgt tcgagctgga aaacggccgg aagagaatgc
tggcctctgc cggcgaactg 3720cagaagggaa acgaactggc cctgccctcc
aaatatgtga acttcctgta cctggccagc 3780cactatgaga agctgaaggg
ctcccccgag gataatgagc agaaacagct gtttgtggaa 3840cagcacaagc
actacctgga cgagatcatc gagcagatca gcgagttctc caagagagtg
3900atcctggccg acgctaatct ggacaaagtg ctgtccgcct acaacaagca
ccgggataag 3960cccatcagag agcaggccga gaatatcatc cacctgttta
ccctgaccaa tctgggagcc 4020cctgccgcct tcaagtactt tgacaccacc
atcgaccgga agaggtacac cagcaccaaa 4080gaggtgctgg acgccaccct
gatccaccag agcatcaccg gcctgtacga gacacggatc 4140gacctgtctc
agctgggagg cgacagcccc aagaagaaga gaaaggtgga ggccagcgaa
4200ttggctccgg gaaaaaagag gccggtagag cactctcctg tggagccaga
ctcctcctcg 4260ggaaccggaa aggcgggcca gcagcctgca agaaaaagat
tgaattttgg tcagactgga 4320gacgcagact cagtacctga cccccagcct
ctcggacagc caccagcagc cccctctggt 4380ctgggaacta atacgatggc
tacaggcagt ggcgcaccaa tggcagacaa taacgagggc 4440gccgacggag
tgggtaattc ctcgggaaat tggcattgcg attccacatg gatgggcgac
4500agagtcatca ccaccagcac ccgaacctgg gccctgccca cctacaacaa
ccacctctac 4560aaacaaattt ccagccaatc aggagcctcg aacgacaatc
actactttgg ctacagcacc 4620ccttgggggt attttgactt caacagattc
cactgccact tttcaccacg tgactggcaa 4680agactcatca acaacaactg
gggattccga cccaagagac tcaacttcaa gctctttaac 4740attcaagtca
aagaggtcac gcagaatgac ggtacgacga cgattgccaa taaccttacc
4800agcacggttc aggtgtttac tgactcggag taccagctcc cgtacgtcct
cggctcggcg 4860catcaaggat gcctcccgcc gttcccagca gacgtcttca
tggtgccaca gtatggatac 4920ctcaccctga acaacgggag tcaggcagta
ggacgctctt cattttactg cctggagtac 4980tttccttctc agatgctgcg
taccggaaac aactttacct tcagctacac ttttgaggac 5040gttcctttcc
acagcagcta cgctcacagc cagagtctgg accgtctcat gaatcctctc
5100atcgaccagt acctgtatta cttgagcaga acaaacactc caagtggaac
caccacgcag 5160tcaaggcttc agttttctca ggccggagcg agtgacattc
gggaccagtc taggaactgg 5220cttcctggac cctgttaccg ccagcagcga
gtatcaaaga catctgcgga taacaacaac 5280agtgaatact cgtggactgg
agctaccaag taccacctca atggcagaga ctctctggtg 5340aatccgggcc
cggccatggc aagccacaag gacgatgaag aaaagttttt tcctcagagc
5400ggggttctca tctttgggaa gcaaggctca gagaaaacaa atgtggacat
tgaaaaggtc 5460atgattacag acgaagagga aatcaggaca accaatcccg
tggctacgga gcagtatggt 5520tctgtatcta ccaacctcca gagaggcaac
agacaagcag ctaccgcaga tgtcaacaca 5580caaggcgttc ttccaggcat
ggtctggcag gacagagatg tgtaccttca ggggcccatc 5640tgggcaaaga
ttccacacac ggacggacat tttcacccct ctcccctcat gggtggattc
5700ggacttaaac accctcctcc acagattctc atcaagaaca ccccggtacc
tgcgaatcct 5760tcgaccacct tcagtgcggc aaagtttgct tccttcatca
cacagtactc cacgggacag 5820gtcagcgtgg agatcgagtg ggagctgcag
aaggaaaaca gcaaacgctg gaatcccgaa 5880attcagtaca cttccaacta
caacaagtct gttaatgtgg actttactgt ggacactaat 5940ggcgtgtatt
cagagcctcg ccccattggc accagatacc tgactcgtaa tctgtaa
599741998PRTArtificial SequenceSynthetic Polypeptide 4Met Tyr Pro
Tyr Asp Val Pro Asp Tyr Ala Ser Pro Lys Lys Lys Arg 1 5 10 15 Lys
Val Glu Ala Ser Asp Lys Lys Tyr Ser Ile Gly Leu Asp Ile Gly 20 25
30 Thr Asn Ser Val Gly Trp Ala Val Ile Thr Asp Glu Tyr Lys Val Pro
35 40 45 Ser Lys Lys Phe Lys Val Leu Gly Asn Thr Asp Arg His Ser
Ile Lys 50 55 60 Lys Asn Leu Ile Gly Ala Leu Leu Phe Asp Ser Gly
Glu Thr Ala Glu 65 70 75 80 Ala Thr Arg Leu Lys Arg Thr Ala Arg Arg
Arg Tyr Thr Arg Arg Lys 85 90 95 Asn Arg Ile Cys Tyr Leu Gln Glu
Ile Phe Ser Asn Glu Met Ala Lys 100 105 110 Val Asp Asp Ser Phe Phe
His Arg Leu Glu Glu Ser Phe Leu Val Glu 115 120 125 Glu Asp Lys Lys
His Glu Arg His Pro Ile Phe Gly Asn Ile Val Asp 130 135 140 Glu Val
Ala Tyr His Glu Lys Tyr Pro Thr Ile Tyr His Leu Arg Lys 145 150 155
160 Lys Leu Val Asp Ser Thr Asp Lys Ala Asp Leu Arg Leu Ile Tyr Leu
165 170 175 Ala Leu Ala His Met Ile Lys Phe Arg Gly His Phe Leu Ile
Glu Gly 180 185 190 Asp Leu Asn Pro Asp Asn Ser Asp Val Asp Lys Leu
Phe Ile Gln Leu 195 200 205 Val Gln Thr Tyr Asn Gln Leu Phe Glu Glu
Asn Pro Ile Asn Ala Ser 210 215 220 Gly Val Asp Ala Lys Ala Ile Leu
Ser Ala Arg Leu Ser Lys Ser Arg 225 230 235 240 Arg Leu Glu Asn Leu
Ile Ala Gln Leu Pro Gly Glu Lys Lys Asn Gly 245 250 255 Leu Phe Gly
Asn Leu Ile Ala Leu Ser Leu Gly Leu Thr Pro Asn Phe 260 265 270 Lys
Ser Asn Phe Asp Leu Ala Glu Asp Ala Lys Leu Gln Leu Ser Lys 275 280
285 Asp Thr Tyr Asp Asp Asp Leu Asp Asn Leu Leu Ala Gln Ile Gly Asp
290 295 300 Gln Tyr Ala Asp Leu Phe Leu Ala Ala Lys Asn Leu Ser Asp
Ala Ile 305 310 315 320 Leu Leu Ser Asp Ile Leu Arg Val Asn Thr Glu
Ile Thr Lys Ala Pro 325 330 335 Leu Ser Ala Ser Met Ile Lys Arg Tyr
Asp Glu His His Gln Asp Leu 340 345 350 Thr Leu Leu Lys Ala Leu Val
Arg Gln Gln Leu Pro Glu Lys Tyr Lys 355 360 365 Glu Ile Phe Phe Asp
Gln Ser Lys Asn Gly Tyr Ala Gly Tyr Ile Asp 370 375 380 Gly Gly Ala
Ser Gln Glu Glu Phe Tyr Lys Phe Ile Lys Pro Ile Leu 385 390 395 400
Glu Lys Met Asp Gly Thr Glu Glu Leu Leu Val Lys Leu Asn Arg Glu 405
410 415 Asp Leu Leu Arg Lys Gln Arg Thr Phe Asp Asn Gly Ser Ile Pro
His 420 425 430 Gln Ile His Leu Gly Glu Leu His Ala Ile Leu Arg Arg
Gln Glu Asp 435 440 445 Phe Tyr Pro Phe Leu Lys Asp Asn Arg Glu Lys
Ile Glu Lys Ile Leu 450 455 460 Thr Phe Arg Ile Pro Tyr Tyr Val Gly
Pro Leu Ala Arg Gly Asn Ser 465 470 475 480 Arg Phe Ala Trp Met Thr
Arg Lys Ser Glu Glu Thr Ile Thr Pro Trp 485 490 495 Asn Phe Glu Glu
Val Val Asp Lys Gly Ala Ser Ala Gln Ser Phe Ile 500 505 510 Glu Arg
Met Thr Asn Phe Asp Lys Asn Leu Pro Asn Glu Lys Val Leu 515 520 525
Pro Lys His Ser Leu Leu Tyr Glu Tyr Phe Thr Val Tyr Asn Glu Leu 530
535 540 Thr Lys Val Lys Tyr Val Thr Glu Gly Met Arg Lys Pro Ala Phe
Leu 545 550 555 560 Ser Gly Glu Gln Lys Lys Ala Ile Val Asp Leu Leu
Phe Lys Thr Asn 565 570 575 Arg Lys Val Thr Val Lys Gln Leu Lys Glu
Asp Tyr Phe Lys Lys Ile 580 585 590 Glu Cys Phe Asp Ser Val Glu Ile
Ser Gly Val Glu Asp Arg Phe Asn 595 600 605 Ala Ser Leu Gly Thr Tyr
His Asp Leu Leu Lys Ile Ile Lys Asp Lys 610 615 620 Asp Phe Leu Asp
Asn Glu Glu Asn Glu Asp Ile Leu Glu Asp Ile Val 625 630 635 640 Leu
Thr Leu Thr Leu Phe Glu Asp Arg Glu Met Ile Glu Glu Arg Leu 645 650
655 Lys Thr Tyr Ala His Leu Phe Asp Asp Lys Val Met Lys Gln Leu Lys
660 665 670 Arg Arg Arg Tyr Thr Gly Trp Gly Arg Leu Ser Arg Lys Leu
Ile Asn 675 680 685 Gly Ile Arg Asp Lys Gln Ser Gly Lys Thr Ile Leu
Asp Phe Leu Lys 690 695 700 Ser Asp Gly Phe Ala Asn Arg Asn Phe Met
Gln Leu Ile His Asp Asp 705 710 715 720 Ser Leu Thr Phe Lys Glu Asp
Ile Gln Lys Ala Gln Val Ser Gly Gln 725 730 735 Gly Asp Ser Leu His
Glu His Ile Ala Asn Leu Ala Gly Ser Pro Ala 740 745 750 Ile Lys Lys
Gly Ile Leu Gln Thr Val Lys Val Val Asp Glu Leu Val 755 760 765 Lys
Val Met Gly Arg His Lys Pro Glu Asn Ile Val Ile Glu Met Ala 770 775
780 Arg Glu Asn Gln Thr Thr Gln Lys Gly Gln Lys Asn Ser Arg Glu Arg
785 790 795 800 Met Lys Arg Ile Glu Glu Gly Ile Lys Glu Leu Gly Ser
Gln Ile Leu 805 810 815 Lys Glu His Pro Val Glu Asn Thr Gln Leu Gln
Asn Glu Lys Leu Tyr 820 825 830 Leu Tyr Tyr Leu Gln Asn Gly Arg Asp
Met Tyr Val Asp Gln Glu Leu 835 840 845 Asp Ile Asn Arg Leu Ser Asp
Tyr Asp Val Asp His Ile Val Pro Gln 850 855 860 Ser Phe Leu Lys Asp
Asp Ser Ile Asp Asn Lys Val Leu Thr Arg Ser 865 870 875 880 Asp Lys
Asn Arg Gly Lys Ser Asp Asn Val Pro Ser Glu Glu Val Val 885 890 895
Lys Lys Met Lys Asn Tyr Trp Arg Gln Leu Leu Asn Ala Lys Leu Ile 900
905 910 Thr Gln Arg Lys Phe Asp Asn Leu Thr Lys Ala Glu Arg Gly Gly
Leu 915 920 925 Ser Glu Leu Asp Lys Ala Gly Phe Ile Lys Arg Gln Leu
Val Glu Thr 930 935 940 Arg Gln Ile Thr Lys His Val Ala Gln Ile Leu
Asp Ser Arg Met Asn 945 950 955 960 Thr Lys Tyr Asp Glu Asn Asp Lys
Leu Ile Arg Glu Val Lys Val Ile 965 970 975 Thr Leu Lys Ser Lys Leu
Val Ser Asp Phe Arg Lys Asp Phe Gln Phe 980 985 990 Tyr Lys Val Arg
Glu Ile Asn Asn Tyr His His Ala His Asp Ala Tyr 995 1000 1005 Leu
Asn Ala Val Val Gly Thr Ala Leu Ile Lys Lys Tyr Pro Lys 1010 1015
1020 Leu Glu Ser Glu Phe Val Tyr Gly Asp Tyr Lys Val Tyr Asp Val
1025 1030 1035 Arg Lys Met Ile Ala Lys Ser Glu Gln Glu Ile Gly Lys
Ala Thr 1040 1045 1050 Ala Lys Tyr Phe Phe Tyr Ser Asn Ile Met Asn
Phe Phe Lys Thr 1055 1060 1065 Glu Ile Thr Leu Ala Asn Gly Glu Ile
Arg Lys Arg Pro Leu Ile 1070 1075 1080 Glu Thr Asn Gly Glu Thr Gly
Glu Ile Val Trp Asp Lys Gly Arg 1085 1090 1095 Asp Phe Ala Thr Val
Arg Lys Val Leu Ser Met Pro Gln Val Asn 1100 1105 1110 Ile Val Lys
Lys Thr Glu Val Gln Thr Gly Gly Phe Ser Lys Glu 1115 1120 1125 Ser
Ile Leu Pro Lys Arg Asn Ser Asp Lys Leu Ile Ala Arg Lys 1130 1135
1140 Lys Asp Trp Asp Pro Lys Lys Tyr Gly Gly Phe Asp Ser Pro Thr
1145 1150 1155 Val Ala Tyr Ser Val Leu Val Val Ala Lys Val Glu Lys
Gly Lys 1160 1165 1170 Ser Lys Lys Leu Lys Ser Val Lys Glu Leu Leu
Gly Ile Thr Ile 1175 1180 1185 Met Glu Arg Ser Ser Phe Glu Lys Asn
Pro Ile Asp Phe Leu Glu 1190 1195 1200 Ala Lys Gly Tyr Lys Glu Val
Lys Lys Asp Leu Ile Ile Lys Leu 1205 1210 1215 Pro Lys Tyr Ser Leu
Phe Glu Leu Glu Asn Gly Arg Lys Arg Met 1220 1225 1230 Leu Ala Ser
Ala Gly Glu Leu Gln Lys Gly Asn Glu Leu Ala Leu 1235 1240 1245 Pro
Ser Lys Tyr Val Asn Phe Leu Tyr Leu Ala Ser His Tyr Glu 1250 1255
1260 Lys Leu Lys Gly Ser Pro Glu Asp Asn Glu Gln Lys Gln Leu Phe
1265 1270 1275 Val Glu Gln His Lys His Tyr Leu Asp Glu Ile Ile Glu
Gln Ile 1280 1285 1290 Ser Glu Phe Ser Lys Arg Val Ile Leu Ala Asp
Ala Asn Leu Asp 1295 1300 1305 Lys Val Leu Ser Ala Tyr Asn Lys His
Arg Asp Lys Pro Ile Arg 1310 1315 1320 Glu Gln Ala Glu Asn Ile Ile
His Leu Phe Thr Leu Thr Asn Leu 1325 1330 1335 Gly Ala Pro Ala Ala
Phe Lys Tyr Phe Asp Thr Thr Ile Asp Arg 1340 1345 1350 Lys Arg Tyr
Thr Ser Thr Lys Glu Val Leu Asp Ala Thr Leu Ile 1355 1360 1365 His
Gln Ser Ile Thr Gly Leu Tyr Glu Thr Arg Ile Asp Leu Ser 1370 1375
1380 Gln Leu Gly Gly Asp Ser Pro Lys Lys Lys Arg Lys Val Glu Ala
1385 1390 1395 Ser Glu Leu Ala Pro Gly Lys Lys Arg Pro Val Glu His
Ser Pro 1400 1405 1410 Val Glu Pro Asp Ser Ser Ser Gly Thr Gly Lys
Ala Gly Gln Gln 1415 1420 1425 Pro Ala Arg Lys Arg Leu Asn Phe Gly
Gln Thr Gly Asp Ala Asp 1430 1435 1440 Ser Val Pro Asp Pro Gln Pro
Leu Gly Gln Pro Pro Ala Ala Pro 1445 1450 1455 Ser Gly Leu Gly Thr
Asn Thr Met Ala Thr Gly Ser Gly Ala Pro 1460 1465 1470 Met Ala Asp
Asn Asn Glu Gly Ala Asp Gly Val Gly Asn Ser Ser 1475 1480 1485 Gly
Asn Trp His Cys Asp Ser Thr Trp Met Gly Asp Arg Val Ile 1490 1495
1500 Thr Thr Ser Thr Arg Thr Trp Ala Leu Pro Thr Tyr Asn Asn His
1505 1510 1515 Leu Tyr Lys Gln Ile Ser Ser Gln Ser Gly Ala Ser Asn
Asp Asn 1520 1525 1530 His Tyr Phe Gly Tyr Ser Thr Pro Trp Gly Tyr
Phe Asp Phe Asn 1535 1540 1545 Arg Phe His Cys His Phe Ser Pro Arg
Asp Trp Gln Arg Leu Ile 1550 1555 1560 Asn Asn Asn Trp Gly Phe Arg
Pro Lys Arg Leu Asn Phe Lys Leu 1565
1570 1575 Phe Asn Ile Gln Val Lys Glu Val Thr Gln Asn Asp Gly Thr
Thr 1580 1585 1590 Thr Ile Ala Asn Asn Leu Thr Ser Thr Val Gln Val
Phe Thr Asp 1595 1600 1605 Ser Glu Tyr Gln Leu Pro Tyr Val Leu Gly
Ser Ala His Gln Gly 1610 1615 1620 Cys Leu Pro Pro Phe Pro Ala Asp
Val Phe Met Val Pro Gln Tyr 1625 1630 1635 Gly Tyr Leu Thr Leu Asn
Asn Gly Ser Gln Ala Val Gly Arg Ser 1640 1645 1650 Ser Phe Tyr Cys
Leu Glu Tyr Phe Pro Ser Gln Met Leu Arg Thr 1655 1660 1665 Gly Asn
Asn Phe Thr Phe Ser Tyr Thr Phe Glu Asp Val Pro Phe 1670 1675 1680
His Ser Ser Tyr Ala His Ser Gln Ser Leu Asp Arg Leu Met Asn 1685
1690 1695 Pro Leu Ile Asp Gln Tyr Leu Tyr Tyr Leu Ser Arg Thr Asn
Thr 1700 1705 1710 Pro Ser Gly Thr Thr Thr Gln Ser Arg Leu Gln Phe
Ser Gln Ala 1715 1720 1725 Gly Ala Ser Asp Ile Arg Asp Gln Ser Arg
Asn Trp Leu Pro Gly 1730 1735 1740 Pro Cys Tyr Arg Gln Gln Arg Val
Ser Lys Thr Ser Ala Asp Asn 1745 1750 1755 Asn Asn Ser Glu Tyr Ser
Trp Thr Gly Ala Thr Lys Tyr His Leu 1760 1765 1770 Asn Gly Arg Asp
Ser Leu Val Asn Pro Gly Pro Ala Met Ala Ser 1775 1780 1785 His Lys
Asp Asp Glu Glu Lys Phe Phe Pro Gln Ser Gly Val Leu 1790 1795 1800
Ile Phe Gly Lys Gln Gly Ser Glu Lys Thr Asn Val Asp Ile Glu 1805
1810 1815 Lys Val Met Ile Thr Asp Glu Glu Glu Ile Arg Thr Thr Asn
Pro 1820 1825 1830 Val Ala Thr Glu Gln Tyr Gly Ser Val Ser Thr Asn
Leu Gln Arg 1835 1840 1845 Gly Asn Arg Gln Ala Ala Thr Ala Asp Val
Asn Thr Gln Gly Val 1850 1855 1860 Leu Pro Gly Met Val Trp Gln Asp
Arg Asp Val Tyr Leu Gln Gly 1865 1870 1875 Pro Ile Trp Ala Lys Ile
Pro His Thr Asp Gly His Phe His Pro 1880 1885 1890 Ser Pro Leu Met
Gly Gly Phe Gly Leu Lys His Pro Pro Pro Gln 1895 1900 1905 Ile Leu
Ile Lys Asn Thr Pro Val Pro Ala Asn Pro Ser Thr Thr 1910 1915 1920
Phe Ser Ala Ala Lys Phe Ala Ser Phe Ile Thr Gln Tyr Ser Thr 1925
1930 1935 Gly Gln Val Ser Val Glu Ile Glu Trp Glu Leu Gln Lys Glu
Asn 1940 1945 1950 Ser Lys Arg Trp Asn Pro Glu Ile Gln Tyr Thr Ser
Asn Tyr Asn 1955 1960 1965 Lys Ser Val Asn Val Asp Phe Thr Val Asp
Thr Asn Gly Val Tyr 1970 1975 1980 Ser Glu Pro Arg Pro Ile Gly Thr
Arg Tyr Leu Thr Arg Asn Leu 1985 1990 1995 54PRTArtificial
SequenceSynthetic Polypeptide 5Gly Gly Gly Ser 1 61333PRTArtificial
SequenceSynthetic Polypeptide 6Met Tyr Pro Tyr Asp Val Pro Asp Tyr
Ala Ser Pro Lys Lys Lys Arg 1 5 10 15 Lys Val Glu Ala Ser Asp Lys
Lys Tyr Ser Ile Gly Leu Asp Ile Gly 20 25 30 Thr Asn Ser Val Gly
Trp Ala Val Ile Thr Asp Glu Tyr Lys Val Pro 35 40 45 Ser Lys Lys
Phe Lys Val Leu Gly Asn Thr Asp Arg His Ser Ile Lys 50 55 60 Lys
Asn Leu Ile Gly Ala Leu Leu Phe Asp Ser Gly Glu Thr Ala Glu 65 70
75 80 Ala Thr Arg Leu Lys Arg Thr Ala Arg Arg Arg Tyr Thr Arg Arg
Lys 85 90 95 Asn Arg Ile Cys Tyr Leu Gln Glu Ile Phe Ser Asn Glu
Met Ala Lys 100 105 110 Val Asp Asp Ser Phe Phe His Arg Leu Glu Glu
Ser Phe Leu Val Glu 115 120 125 Glu Asp Lys Lys His Glu Arg His Pro
Ile Phe Gly Asn Ile Val Asp 130 135 140 Glu Val Ala Tyr His Glu Lys
Tyr Pro Thr Ile Tyr His Leu Arg Lys 145 150 155 160 Lys Leu Val Asp
Ser Thr Asp Lys Ala Asp Leu Arg Leu Ile Tyr Leu 165 170 175 Ala Leu
Ala His Met Ile Lys Phe Arg Gly His Phe Leu Ile Glu Gly 180 185 190
Asp Leu Asn Pro Asp Asn Ser Asp Val Asp Lys Leu Phe Ile Gln Leu 195
200 205 Val Gln Thr Tyr Asn Gln Leu Phe Glu Glu Asn Pro Ile Asn Ala
Ser 210 215 220 Gly Val Asp Ala Lys Ala Ile Leu Ser Ala Arg Leu Ser
Lys Ser Arg 225 230 235 240 Arg Leu Glu Asn Leu Ile Ala Gln Leu Pro
Gly Glu Lys Lys Asn Gly 245 250 255 Leu Phe Gly Asn Leu Ile Ala Leu
Ser Leu Gly Leu Thr Pro Asn Phe 260 265 270 Lys Ser Asn Phe Asp Leu
Ala Glu Asp Ala Lys Leu Gln Leu Ser Lys 275 280 285 Asp Thr Tyr Asp
Asp Asp Leu Asp Asn Leu Leu Ala Gln Ile Gly Asp 290 295 300 Gln Tyr
Ala Asp Leu Phe Leu Ala Ala Lys Asn Leu Ser Asp Ala Ile 305 310 315
320 Leu Leu Ser Asp Ile Leu Arg Val Asn Thr Glu Ile Thr Lys Ala Pro
325 330 335 Leu Ser Ala Ser Met Ile Lys Arg Tyr Asp Glu His His Gln
Asp Leu 340 345 350 Thr Leu Leu Lys Ala Leu Val Arg Gln Gln Leu Pro
Glu Lys Tyr Lys 355 360 365 Glu Ile Phe Phe Asp Gln Ser Lys Asn Gly
Tyr Ala Gly Tyr Ile Asp 370 375 380 Gly Gly Ala Ser Gln Glu Glu Phe
Tyr Lys Phe Ile Lys Pro Ile Leu 385 390 395 400 Glu Lys Met Asp Gly
Thr Glu Glu Leu Leu Val Lys Leu Asn Arg Glu 405 410 415 Asp Leu Leu
Arg Lys Gln Arg Thr Phe Asp Asn Gly Ser Ile Pro His 420 425 430 Gln
Ile His Leu Gly Glu Leu His Ala Ile Leu Arg Arg Gln Glu Asp 435 440
445 Phe Tyr Pro Phe Leu Lys Asp Asn Arg Glu Lys Ile Glu Lys Ile Leu
450 455 460 Thr Phe Arg Ile Pro Tyr Tyr Val Gly Pro Leu Ala Arg Gly
Asn Ser 465 470 475 480 Arg Phe Ala Trp Met Thr Arg Lys Ser Glu Glu
Thr Ile Thr Pro Trp 485 490 495 Asn Phe Glu Glu Val Val Asp Lys Gly
Ala Ser Ala Gln Ser Phe Ile 500 505 510 Glu Arg Met Thr Asn Phe Asp
Lys Asn Leu Pro Asn Glu Lys Val Leu 515 520 525 Pro Lys His Ser Leu
Leu Tyr Glu Tyr Phe Thr Val Tyr Asn Glu Leu 530 535 540 Thr Lys Val
Lys Tyr Val Thr Glu Gly Met Arg Lys Pro Ala Phe Leu 545 550 555 560
Ser Gly Glu Gln Lys Lys Ala Ile Val Asp Leu Leu Phe Lys Thr Asn 565
570 575 Arg Lys Val Thr Val Lys Gln Leu Lys Glu Asp Tyr Phe Lys Lys
Ile 580 585 590 Glu Cys Phe Asp Ser Val Glu Ile Ser Gly Val Glu Asp
Arg Phe Asn 595 600 605 Ala Ser Leu Gly Thr Tyr His Asp Leu Leu Lys
Ile Ile Lys Asp Lys 610 615 620 Asp Phe Leu Asp Asn Glu Glu Asn Glu
Asp Ile Leu Glu Asp Ile Val 625 630 635 640 Leu Thr Leu Thr Leu Phe
Glu Asp Arg Glu Met Ile Glu Glu Arg Leu 645 650 655 Lys Thr Tyr Ala
His Leu Phe Asp Asp Lys Val Met Lys Gln Leu Lys 660 665 670 Arg Arg
Arg Tyr Thr Gly Trp Gly Arg Leu Ser Arg Lys Leu Ile Asn 675 680 685
Gly Ile Arg Asp Lys Gln Ser Gly Lys Thr Ile Leu Asp Phe Leu Lys 690
695 700 Ser Asp Gly Phe Ala Asn Arg Asn Phe Met Gln Leu Ile His Asp
Asp 705 710 715 720 Ser Leu Thr Phe Lys Glu Asp Ile Gln Lys Ala Gln
Val Ser Gly Gln 725 730 735 Gly Asp Ser Leu His Glu His Ile Ala Asn
Leu Ala Gly Ser Pro Ala 740 745 750 Ile Lys Lys Gly Ile Leu Gln Thr
Val Lys Val Val Asp Glu Leu Val 755 760 765 Lys Val Met Gly Arg His
Lys Pro Glu Asn Ile Val Ile Glu Met Ala 770 775 780 Arg Glu Asn Gln
Thr Thr Gln Lys Gly Gln Lys Asn Ser Arg Glu Arg 785 790 795 800 Met
Lys Arg Ile Glu Glu Gly Ile Lys Glu Leu Gly Ser Gln Ile Leu 805 810
815 Lys Glu His Pro Val Glu Asn Thr Gln Leu Gln Asn Glu Lys Leu Tyr
820 825 830 Leu Tyr Tyr Leu Gln Asn Gly Arg Asp Met Tyr Val Asp Gln
Glu Leu 835 840 845 Asp Ile Asn Arg Leu Ser Asp Tyr Asp Val Asp His
Ile Val Pro Gln 850 855 860 Ser Phe Leu Lys Asp Asp Ser Ile Asp Asn
Lys Val Leu Thr Arg Ser 865 870 875 880 Asp Lys Asn Arg Gly Lys Ser
Asp Asn Val Pro Ser Glu Glu Val Val 885 890 895 Lys Lys Met Lys Asn
Tyr Trp Arg Gln Leu Leu Asn Ala Lys Leu Ile 900 905 910 Thr Gln Arg
Lys Phe Asp Asn Leu Thr Lys Ala Glu Arg Gly Gly Leu 915 920 925 Ser
Glu Leu Asp Lys Ala Gly Phe Ile Lys Arg Gln Leu Val Glu Thr 930 935
940 Arg Gln Ile Thr Lys His Val Ala Gln Ile Leu Asp Ser Arg Met Asn
945 950 955 960 Thr Lys Tyr Asp Glu Asn Asp Lys Leu Ile Arg Glu Val
Lys Val Ile 965 970 975 Thr Leu Lys Ser Lys Leu Val Ser Asp Phe Arg
Lys Asp Phe Gln Phe 980 985 990 Tyr Lys Val Arg Glu Ile Asn Asn Tyr
His His Ala His Asp Ala Tyr 995 1000 1005 Leu Asn Ala Val Val Gly
Thr Ala Leu Ile Lys Lys Tyr Pro Lys 1010 1015 1020 Leu Glu Ser Glu
Phe Val Tyr Gly Asp Tyr Lys Val Tyr Asp Val 1025 1030 1035 Arg Lys
Met Ile Ala Lys Ser Glu Gln Glu Ile Gly Lys Ala Thr 1040 1045 1050
Ala Lys Tyr Phe Phe Tyr Ser Asn Ile Met Asn Phe Phe Lys Thr 1055
1060 1065 Glu Ile Thr Leu Ala Asn Gly Glu Ile Arg Lys Arg Pro Leu
Ile 1070 1075 1080 Glu Thr Asn Gly Glu Thr Gly Glu Ile Val Trp Asp
Lys Gly Arg 1085 1090 1095 Asp Phe Ala Thr Val Arg Lys Val Leu Ser
Met Pro Gln Val Asn 1100 1105 1110 Ile Val Lys Lys Thr Glu Val Gln
Thr Gly Gly Phe Ser Lys Glu 1115 1120 1125 Ser Ile Leu Pro Lys Arg
Asn Ser Asp Lys Leu Ile Ala Arg Lys 1130 1135 1140 Lys Asp Trp Asp
Pro Lys Lys Tyr Gly Gly Phe Asp Ser Pro Thr 1145 1150 1155 Val Ala
Tyr Ser Val Leu Val Val Ala Lys Val Glu Lys Gly Lys 1160 1165 1170
Ser Lys Lys Leu Lys Ser Val Lys Glu Leu Leu Gly Ile Thr Ile 1175
1180 1185 Met Glu Arg Ser Ser Phe Glu Lys Asn Pro Ile Asp Phe Leu
Glu 1190 1195 1200 Ala Lys Gly Tyr Lys Glu Val Lys Lys Asp Leu Ile
Ile Lys Leu 1205 1210 1215 Pro Lys Tyr Ser Leu Phe Glu Leu Glu Asn
Gly Arg Lys Cys Leu 1220 1225 1230 Ser Tyr Glu Thr Glu Ile Leu Thr
Val Glu Tyr Gly Leu Leu Pro 1235 1240 1245 Ile Gly Lys Ile Val Glu
Lys Arg Ile Glu Cys Thr Val Tyr Ser 1250 1255 1260 Val Asp Asn Asn
Gly Asn Ile Tyr Thr Gln Pro Val Ala Gln Trp 1265 1270 1275 His Asp
Arg Gly Glu Gln Glu Val Phe Glu Tyr Cys Leu Glu Asp 1280 1285 1290
Gly Ser Leu Ile Arg Ala Thr Lys Asp His Lys Phe Met Thr Val 1295
1300 1305 Asp Gly Gln Met Leu Pro Ile Asp Glu Ile Phe Glu Arg Glu
Leu 1310 1315 1320 Asp Leu Met Arg Val Asp Asn Leu Pro Asn 1325
1330 7803PRTArtificial SequenceSynthetic Polypeptide 7Met Ile Lys
Ile Ala Thr Arg Lys Tyr Leu Gly Lys Gln Asn Val Tyr 1 5 10 15 Asp
Ile Gly Val Glu Arg Asp His Asn Phe Ala Leu Lys Asn Gly Phe 20 25
30 Ile Ala Ser Asn Cys Met Leu Ala Ser Ala Gly Glu Leu Gln Lys Gly
35 40 45 Asn Glu Leu Ala Leu Pro Ser Lys Tyr Val Asn Phe Leu Tyr
Leu Ala 50 55 60 Ser His Tyr Glu Lys Leu Lys Gly Ser Pro Glu Asp
Asn Glu Gln Lys 65 70 75 80 Gln Leu Phe Val Glu Gln His Lys His Tyr
Leu Asp Glu Ile Ile Glu 85 90 95 Gln Ile Ser Glu Phe Ser Lys Arg
Val Ile Leu Ala Asp Ala Asn Leu 100 105 110 Asp Lys Val Leu Ser Ala
Tyr Asn Lys His Arg Asp Lys Pro Ile Arg 115 120 125 Glu Gln Ala Glu
Asn Ile Ile His Leu Phe Thr Leu Thr Asn Leu Gly 130 135 140 Ala Pro
Ala Ala Phe Lys Tyr Phe Asp Thr Thr Ile Asp Arg Lys Arg 145 150 155
160 Tyr Thr Ser Thr Lys Glu Val Leu Asp Ala Thr Leu Ile His Gln Ser
165 170 175 Ile Thr Gly Leu Tyr Glu Thr Arg Ile Asp Leu Ser Gln Leu
Gly Gly 180 185 190 Asp Ser Pro Lys Lys Lys Arg Lys Val Glu Ala Ser
Gly Ser Ala Pro 195 200 205 Gly Lys Lys Arg Pro Val Glu His Ser Pro
Val Glu Pro Asp Ser Ser 210 215 220 Ser Gly Thr Gly Lys Ala Gly Gln
Gln Pro Ala Arg Lys Arg Leu Asn 225 230 235 240 Phe Gly Gln Thr Gly
Asp Ala Asp Ser Val Pro Asp Pro Gln Pro Leu 245 250 255 Gly Gln Pro
Pro Ala Ala Pro Ser Gly Leu Gly Thr Asn Thr Met Ala 260 265 270 Thr
Gly Ser Gly Ala Pro Met Ala Asp Asn Asn Glu Gly Ala Asp Gly 275 280
285 Val Gly Asn Ser Ser Gly Asn Trp His Cys Asp Ser Thr Trp Met Gly
290 295 300 Asp Arg Val Ile Thr Thr Ser Thr Arg Thr Trp Ala Leu Pro
Thr Tyr 305 310 315 320 Asn Asn His Leu Tyr Lys Gln Ile Ser Ser Gln
Ser Gly Ala Ser Asn 325 330 335 Asp Asn His Tyr Phe Gly Tyr Ser Thr
Pro Trp Gly Tyr Phe Asp Phe 340 345 350 Asn Arg Phe His Cys His Phe
Ser Pro Arg Asp Trp Gln Arg Leu Ile 355 360 365 Asn Asn Asn Trp Gly
Phe Arg Pro Lys Arg Leu Asn Phe Lys Leu Phe 370 375 380 Asn Ile Gln
Val Lys Glu Val Thr Gln Asn Asp Gly Thr Thr Thr Ile 385 390 395 400
Ala Asn Asn Leu Thr Ser Thr Val Gln Val Phe Thr Asp Ser Glu Tyr 405
410 415 Gln Leu Pro Tyr Val Leu Gly Ser Ala His Gln Gly Cys Leu Pro
Pro 420 425 430 Phe Pro Ala Asp Val Phe Met Val Pro Gln Tyr Gly Tyr
Leu Thr Leu 435 440 445 Asn Asn Gly Ser Gln Ala Val Gly Arg Ser Ser
Phe Tyr Cys Leu Glu 450 455 460 Tyr Phe Pro Ser Gln Met Leu Arg Thr
Gly Asn Asn Phe Thr Phe Ser 465 470 475 480 Tyr Thr Phe Glu Asp
Val Pro Phe His Ser Ser Tyr Ala His Ser Gln 485 490 495 Ser Leu Asp
Arg Leu Met Asn Pro Leu Ile Asp Gln Tyr Leu Tyr Tyr 500 505 510 Leu
Ser Arg Thr Asn Thr Pro Ser Gly Thr Thr Thr Gln Ser Arg Leu 515 520
525 Gln Phe Ser Gln Ala Gly Ala Ser Asp Ile Arg Asp Gln Ser Arg Asn
530 535 540 Trp Leu Pro Gly Pro Cys Tyr Arg Gln Gln Arg Val Ser Lys
Thr Ser 545 550 555 560 Ala Asp Asn Asn Asn Ser Glu Tyr Ser Trp Thr
Gly Ala Thr Lys Tyr 565 570 575 His Leu Asn Gly Arg Asp Ser Leu Val
Asn Pro Gly Pro Ala Met Ala 580 585 590 Ser His Lys Asp Asp Glu Glu
Lys Phe Phe Pro Gln Ser Gly Val Leu 595 600 605 Ile Phe Gly Lys Gln
Gly Ser Glu Lys Thr Asn Val Asp Ile Glu Lys 610 615 620 Val Met Ile
Thr Asp Glu Glu Glu Ile Arg Thr Thr Asn Pro Val Ala 625 630 635 640
Thr Glu Gln Tyr Gly Ser Val Ser Thr Asn Leu Gln Arg Gly Asn Arg 645
650 655 Gln Ala Ala Thr Ala Asp Val Asn Thr Gln Gly Val Leu Pro Gly
Met 660 665 670 Val Trp Gln Asp Arg Asp Val Tyr Leu Gln Gly Pro Ile
Trp Ala Lys 675 680 685 Ile Pro His Thr Asp Gly His Phe His Pro Ser
Pro Leu Met Gly Gly 690 695 700 Phe Gly Leu Lys His Pro Pro Pro Gln
Ile Leu Ile Lys Asn Thr Pro 705 710 715 720 Val Pro Ala Asn Pro Ser
Thr Thr Phe Ser Ala Ala Lys Phe Ala Ser 725 730 735 Phe Ile Thr Gln
Tyr Ser Thr Gly Gln Val Ser Val Glu Ile Glu Trp 740 745 750 Glu Leu
Gln Lys Glu Asn Ser Lys Arg Trp Asn Pro Glu Ile Gln Tyr 755 760 765
Thr Ser Asn Tyr Asn Lys Ser Val Asn Val Asp Phe Thr Val Asp Thr 770
775 780 Asn Gly Val Tyr Ser Glu Pro Arg Pro Ile Gly Thr Arg Tyr Leu
Thr 785 790 795 800 Arg Asn Leu 8816PRTArtificial SequenceSynthetic
Polypeptide 8Met Ile Lys Ile Ala Thr Arg Lys Tyr Leu Gly Lys Gln
Asn Val Tyr 1 5 10 15 Asp Ile Gly Val Glu Arg Asp His Asn Phe Ala
Leu Lys Asn Gly Phe 20 25 30 Ile Ala Ser Asn Cys Met Leu Ala Ser
Ala Gly Glu Leu Gln Lys Gly 35 40 45 Asn Glu Leu Ala Leu Pro Ser
Lys Tyr Val Asn Phe Leu Tyr Leu Ala 50 55 60 Ser His Tyr Glu Lys
Leu Lys Gly Ser Pro Glu Asp Asn Glu Gln Lys 65 70 75 80 Gln Leu Phe
Val Glu Gln His Lys His Tyr Leu Asp Glu Ile Ile Glu 85 90 95 Gln
Ile Ser Glu Phe Ser Lys Arg Val Ile Leu Ala Asp Ala Asn Leu 100 105
110 Asp Lys Val Leu Ser Ala Tyr Asn Lys His Arg Asp Lys Pro Ile Arg
115 120 125 Glu Gln Ala Glu Asn Ile Ile His Leu Phe Thr Leu Thr Asn
Leu Gly 130 135 140 Ala Pro Ala Ala Phe Lys Tyr Phe Asp Thr Thr Ile
Asp Arg Lys Arg 145 150 155 160 Tyr Thr Ser Thr Lys Glu Val Leu Asp
Ala Thr Leu Ile His Gln Ser 165 170 175 Ile Thr Gly Leu Tyr Glu Thr
Arg Ile Asp Leu Ser Gln Leu Gly Gly 180 185 190 Asp Ser Pro Lys Lys
Lys Arg Lys Val Glu Ala Ser Gly Gly Gly Gly 195 200 205 Ser Gly Gly
Gly Gly Ser Gly Gly Gly Gly Ser Ala Pro Gly Lys Lys 210 215 220 Arg
Pro Val Glu His Ser Pro Val Glu Pro Asp Ser Ser Ser Gly Thr 225 230
235 240 Gly Lys Ala Gly Gln Gln Pro Ala Arg Lys Arg Leu Asn Phe Gly
Gln 245 250 255 Thr Gly Asp Ala Asp Ser Val Pro Asp Pro Gln Pro Leu
Gly Gln Pro 260 265 270 Pro Ala Ala Pro Ser Gly Leu Gly Thr Asn Thr
Met Ala Thr Gly Ser 275 280 285 Gly Ala Pro Met Ala Asp Asn Asn Glu
Gly Ala Asp Gly Val Gly Asn 290 295 300 Ser Ser Gly Asn Trp His Cys
Asp Ser Thr Trp Met Gly Asp Arg Val 305 310 315 320 Ile Thr Thr Ser
Thr Arg Thr Trp Ala Leu Pro Thr Tyr Asn Asn His 325 330 335 Leu Tyr
Lys Gln Ile Ser Ser Gln Ser Gly Ala Ser Asn Asp Asn His 340 345 350
Tyr Phe Gly Tyr Ser Thr Pro Trp Gly Tyr Phe Asp Phe Asn Arg Phe 355
360 365 His Cys His Phe Ser Pro Arg Asp Trp Gln Arg Leu Ile Asn Asn
Asn 370 375 380 Trp Gly Phe Arg Pro Lys Arg Leu Asn Phe Lys Leu Phe
Asn Ile Gln 385 390 395 400 Val Lys Glu Val Thr Gln Asn Asp Gly Thr
Thr Thr Ile Ala Asn Asn 405 410 415 Leu Thr Ser Thr Val Gln Val Phe
Thr Asp Ser Glu Tyr Gln Leu Pro 420 425 430 Tyr Val Leu Gly Ser Ala
His Gln Gly Cys Leu Pro Pro Phe Pro Ala 435 440 445 Asp Val Phe Met
Val Pro Gln Tyr Gly Tyr Leu Thr Leu Asn Asn Gly 450 455 460 Ser Gln
Ala Val Gly Arg Ser Ser Phe Tyr Cys Leu Glu Tyr Phe Pro 465 470 475
480 Ser Gln Met Leu Arg Thr Gly Asn Asn Phe Thr Phe Ser Tyr Thr Phe
485 490 495 Glu Asp Val Pro Phe His Ser Ser Tyr Ala His Ser Gln Ser
Leu Asp 500 505 510 Arg Leu Met Asn Pro Leu Ile Asp Gln Tyr Leu Tyr
Tyr Leu Ser Arg 515 520 525 Thr Asn Thr Pro Ser Gly Thr Thr Thr Gln
Ser Arg Leu Gln Phe Ser 530 535 540 Gln Ala Gly Ala Ser Asp Ile Arg
Asp Gln Ser Arg Asn Trp Leu Pro 545 550 555 560 Gly Pro Cys Tyr Arg
Gln Gln Arg Val Ser Lys Thr Ser Ala Asp Asn 565 570 575 Asn Asn Ser
Glu Tyr Ser Trp Thr Gly Ala Thr Lys Tyr His Leu Asn 580 585 590 Gly
Arg Asp Ser Leu Val Asn Pro Gly Pro Ala Met Ala Ser His Lys 595 600
605 Asp Asp Glu Glu Lys Phe Phe Pro Gln Ser Gly Val Leu Ile Phe Gly
610 615 620 Lys Gln Gly Ser Glu Lys Thr Asn Val Asp Ile Glu Lys Val
Met Ile 625 630 635 640 Thr Asp Glu Glu Glu Ile Arg Thr Thr Asn Pro
Val Ala Thr Glu Gln 645 650 655 Tyr Gly Ser Val Ser Thr Asn Leu Gln
Arg Gly Asn Arg Gln Ala Ala 660 665 670 Thr Ala Asp Val Asn Thr Gln
Gly Val Leu Pro Gly Met Val Trp Gln 675 680 685 Asp Arg Asp Val Tyr
Leu Gln Gly Pro Ile Trp Ala Lys Ile Pro His 690 695 700 Thr Asp Gly
His Phe His Pro Ser Pro Leu Met Gly Gly Phe Gly Leu 705 710 715 720
Lys His Pro Pro Pro Gln Ile Leu Ile Lys Asn Thr Pro Val Pro Ala 725
730 735 Asn Pro Ser Thr Thr Phe Ser Ala Ala Lys Phe Ala Ser Phe Ile
Thr 740 745 750 Gln Tyr Ser Thr Gly Gln Val Ser Val Glu Ile Glu Trp
Glu Leu Gln 755 760 765 Lys Glu Asn Ser Lys Arg Trp Asn Pro Glu Ile
Gln Tyr Thr Ser Asn 770 775 780 Tyr Asn Lys Ser Val Asn Val Asp Phe
Thr Val Asp Thr Asn Gly Val 785 790 795 800 Tyr Ser Glu Pro Arg Pro
Ile Gly Thr Arg Tyr Leu Thr Arg Asn Leu 805 810 815
9816PRTArtificial SequenceSynthetic Polypeptide 9Met Ile Lys Ile
Ala Thr Arg Lys Tyr Leu Gly Lys Gln Asn Val Tyr 1 5 10 15 Asp Ile
Gly Val Glu Arg Asp His Asn Phe Ala Leu Lys Asn Gly Phe 20 25 30
Ile Ala Ser Asn Cys Met Leu Ala Ser Ala Gly Glu Leu Gln Lys Gly 35
40 45 Asn Glu Leu Ala Leu Pro Ser Lys Tyr Val Asn Phe Leu Tyr Leu
Ala 50 55 60 Ser His Tyr Glu Lys Leu Lys Gly Ser Pro Glu Asp Asn
Glu Gln Lys 65 70 75 80 Gln Leu Phe Val Glu Gln His Lys His Tyr Leu
Asp Glu Ile Ile Glu 85 90 95 Gln Ile Ser Glu Phe Ser Lys Arg Val
Ile Leu Ala Asp Ala Asn Leu 100 105 110 Asp Lys Val Leu Ser Ala Tyr
Asn Lys His Arg Asp Lys Pro Ile Arg 115 120 125 Glu Gln Ala Glu Asn
Ile Ile His Leu Phe Thr Leu Thr Asn Leu Gly 130 135 140 Ala Pro Ala
Ala Phe Lys Tyr Phe Asp Thr Thr Ile Asp Arg Lys Arg 145 150 155 160
Tyr Thr Ser Thr Lys Glu Val Leu Asp Ala Thr Leu Ile His Gln Ser 165
170 175 Ile Thr Gly Leu Tyr Glu Thr Arg Ile Asp Leu Ser Gln Leu Gly
Gly 180 185 190 Asp Ser Pro Lys Lys Lys Arg Lys Val Glu Ala Ser Glu
Ala Ala Ala 195 200 205 Lys Glu Ala Ala Ala Lys Glu Ala Ala Ala Lys
Ala Pro Gly Lys Lys 210 215 220 Arg Pro Val Glu His Ser Pro Val Glu
Pro Asp Ser Ser Ser Gly Thr 225 230 235 240 Gly Lys Ala Gly Gln Gln
Pro Ala Arg Lys Arg Leu Asn Phe Gly Gln 245 250 255 Thr Gly Asp Ala
Asp Ser Val Pro Asp Pro Gln Pro Leu Gly Gln Pro 260 265 270 Pro Ala
Ala Pro Ser Gly Leu Gly Thr Asn Thr Met Ala Thr Gly Ser 275 280 285
Gly Ala Pro Met Ala Asp Asn Asn Glu Gly Ala Asp Gly Val Gly Asn 290
295 300 Ser Ser Gly Asn Trp His Cys Asp Ser Thr Trp Met Gly Asp Arg
Val 305 310 315 320 Ile Thr Thr Ser Thr Arg Thr Trp Ala Leu Pro Thr
Tyr Asn Asn His 325 330 335 Leu Tyr Lys Gln Ile Ser Ser Gln Ser Gly
Ala Ser Asn Asp Asn His 340 345 350 Tyr Phe Gly Tyr Ser Thr Pro Trp
Gly Tyr Phe Asp Phe Asn Arg Phe 355 360 365 His Cys His Phe Ser Pro
Arg Asp Trp Gln Arg Leu Ile Asn Asn Asn 370 375 380 Trp Gly Phe Arg
Pro Lys Arg Leu Asn Phe Lys Leu Phe Asn Ile Gln 385 390 395 400 Val
Lys Glu Val Thr Gln Asn Asp Gly Thr Thr Thr Ile Ala Asn Asn 405 410
415 Leu Thr Ser Thr Val Gln Val Phe Thr Asp Ser Glu Tyr Gln Leu Pro
420 425 430 Tyr Val Leu Gly Ser Ala His Gln Gly Cys Leu Pro Pro Phe
Pro Ala 435 440 445 Asp Val Phe Met Val Pro Gln Tyr Gly Tyr Leu Thr
Leu Asn Asn Gly 450 455 460 Ser Gln Ala Val Gly Arg Ser Ser Phe Tyr
Cys Leu Glu Tyr Phe Pro 465 470 475 480 Ser Gln Met Leu Arg Thr Gly
Asn Asn Phe Thr Phe Ser Tyr Thr Phe 485 490 495 Glu Asp Val Pro Phe
His Ser Ser Tyr Ala His Ser Gln Ser Leu Asp 500 505 510 Arg Leu Met
Asn Pro Leu Ile Asp Gln Tyr Leu Tyr Tyr Leu Ser Arg 515 520 525 Thr
Asn Thr Pro Ser Gly Thr Thr Thr Gln Ser Arg Leu Gln Phe Ser 530 535
540 Gln Ala Gly Ala Ser Asp Ile Arg Asp Gln Ser Arg Asn Trp Leu Pro
545 550 555 560 Gly Pro Cys Tyr Arg Gln Gln Arg Val Ser Lys Thr Ser
Ala Asp Asn 565 570 575 Asn Asn Ser Glu Tyr Ser Trp Thr Gly Ala Thr
Lys Tyr His Leu Asn 580 585 590 Gly Arg Asp Ser Leu Val Asn Pro Gly
Pro Ala Met Ala Ser His Lys 595 600 605 Asp Asp Glu Glu Lys Phe Phe
Pro Gln Ser Gly Val Leu Ile Phe Gly 610 615 620 Lys Gln Gly Ser Glu
Lys Thr Asn Val Asp Ile Glu Lys Val Met Ile 625 630 635 640 Thr Asp
Glu Glu Glu Ile Arg Thr Thr Asn Pro Val Ala Thr Glu Gln 645 650 655
Tyr Gly Ser Val Ser Thr Asn Leu Gln Arg Gly Asn Arg Gln Ala Ala 660
665 670 Thr Ala Asp Val Asn Thr Gln Gly Val Leu Pro Gly Met Val Trp
Gln 675 680 685 Asp Arg Asp Val Tyr Leu Gln Gly Pro Ile Trp Ala Lys
Ile Pro His 690 695 700 Thr Asp Gly His Phe His Pro Ser Pro Leu Met
Gly Gly Phe Gly Leu 705 710 715 720 Lys His Pro Pro Pro Gln Ile Leu
Ile Lys Asn Thr Pro Val Pro Ala 725 730 735 Asn Pro Ser Thr Thr Phe
Ser Ala Ala Lys Phe Ala Ser Phe Ile Thr 740 745 750 Gln Tyr Ser Thr
Gly Gln Val Ser Val Glu Ile Glu Trp Glu Leu Gln 755 760 765 Lys Glu
Asn Ser Lys Arg Trp Asn Pro Glu Ile Gln Tyr Thr Ser Asn 770 775 780
Tyr Asn Lys Ser Val Asn Val Asp Phe Thr Val Asp Thr Asn Gly Val 785
790 795 800 Tyr Ser Glu Pro Arg Pro Ile Gly Thr Arg Tyr Leu Thr Arg
Asn Leu 805 810 815 104700DNAArtificial SequenceSynthetic
Polynucleotide 10cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc
ccgggcaaag cccgggcgtc 60gggcgacctt tggtcgcccg gcctcagtga gcgagcgagc
gcgcagagag ggagtggcca 120actccatcac taggggttcc tgcggcctct
agaatggagg cggtactatg tagatgagaa 180ttcaggagca aactgggaaa
agcaactgct tccaaatatt tgtgattttt acagtgtagt 240tttggaaaaa
ctcttagcct accaattctt ctaagtgttt taaaatgtgg gagccagtac
300acatgaagtt atagagtgtt ttaatgaggc ttaaatattt accgtaacta
tgaaatgcta 360cgcatatcat gctgttcagg ctccgtggcc acgcaactca
tactaccggt gccaccatgt 420acccatacga tgttccagat tacgcttcgc
cgaagaaaaa gcgcaaggtc gaagcgtccg 480acaagaagta cagcatcggc
ctggacatcg gcaccaactc tgtgggctgg gccgtgatca 540ccgacgagta
caaggtgccc agcaagaaat tcaaggtgct gggcaacacc gaccggcaca
600gcatcaagaa gaacctgatc ggagccctgc tgttcgacag cggcgaaaca
gccgaggcca 660cccggctgaa gagaaccgcc agaagaagat acaccagacg
gaagaaccgg atctgctatc 720tgcaagagat cttcagcaac gagatggcca
aggtggacga cagcttcttc cacagactgg 780aagagtcctt cctggtggaa
gaggataaga agcacgagcg gcaccccatc ttcggcaaca 840tcgtggacga
ggtggcctac cacgagaagt accccaccat ctaccacctg agaaagaaac
900tggtggacag caccgacaag gccgacctgc ggctgatcta tctggccctg
gcccacatga 960tcaagttccg gggccacttc ctgatcgagg gcgacctgaa
ccccgacaac agcgacgtgg 1020acaagctgtt catccagctg gtgcagacct
acaaccagct gttcgaggaa aaccccatca 1080acgccagcgg cgtggacgcc
aaggccatcc tgtctgccag actgagcaag agcagacggc 1140tggaaaatct
gatcgcccag ctgcccggcg agaagaagaa tggcctgttc ggcaacctga
1200ttgccctgag cctgggcctg acccccaact tcaagagcaa cttcgacctg
gccgaggatg 1260ccaaactgca gctgagcaag gacacctacg acgacgacct
ggacaacctg ctggcccaga 1320tcggcgacca gtacgccgac ctgtttctgg
ccgccaagaa cctgtccgac gccatcctgc 1380tgagcgacat cctgagagtg
aacaccgaga tcaccaaggc ccccctgagc gcctctatga 1440tcaagagata
cgacgagcac caccaggacc tgaccctgct gaaagctctc gtgcggcagc
1500agctgcctga gaagtacaaa gagattttct tcgaccagag caagaacggc
tacgccggct 1560acattgacgg cggagccagc caggaagagt tctacaagtt
catcaagccc atcctggaaa 1620agatggacgg caccgaggaa ctgctcgtga
agctgaacag agaggacctg ctgcggaagc 1680agcggacctt cgacaacggc
agcatccccc accagatcca cctgggagag ctgcacgcca 1740ttctgcggcg
gcaggaagat ttttacccat tcctgaagga caaccgggaa aagatcgaga
1800agatcctgac cttccgcatc ccctactacg tgggccctct ggccagggga
aacagcagat 1860tcgcctggat gaccagaaag agcgaggaaa ccatcacccc
ctggaacttc gaggaagtgg 1920tggacaaggg cgcttccgcc cagagcttca
tcgagcggat gaccaacttc gataagaacc 1980tgcccaacga gaaggtgctg
cccaagcaca gcctgctgta cgagtacttc accgtgtata 2040acgagctgac
caaagtgaaa tacgtgaccg agggaatgag aaagcccgcc ttcctgagcg
2100gcgagcagaa aaaggccatc gtggacctgc tgttcaagac caaccggaaa
gtgaccgtga 2160agcagctgaa
agaggactac ttcaagaaaa tcgagtgctt cgactccgtg gaaatctccg
2220gcgtggaaga tcggttcaac gcctccctgg gcacatacca cgatctgctg
aaaattatca 2280aggacaagga cttcctggac aatgaggaaa acgaggacat
tctggaagat atcgtgctga 2340ccctgacact gtttgaggac agagagatga
tcgaggaacg gctgaaaacc tatgcccacc 2400tgttcgacga caaagtgatg
aagcagctga agcggcggag atacaccggc tggggcaggc 2460tgagccggaa
gctgatcaac ggcatccggg acaagcagtc cggcaagaca atcctggatt
2520tcctgaagtc cgacggcttc gccaacagaa acttcatgca gctgatccac
gacgacagcc 2580tgacctttaa agaggacatc cagaaagccc aggtgtccgg
ccagggcgat agcctgcacg 2640agcacattgc caatctggcc ggcagccccg
ccattaagaa gggcatcctg cagacagtga 2700aggtggtgga cgagctcgtg
aaagtgatgg gccggcacaa gcccgagaac atcgtgatcg 2760aaatggccag
agagaaccag accacccaga agggacagaa gaacagccgc gagagaatga
2820agcggatcga agagggcatc aaagagctgg gcagccagat cctgaaagaa
caccccgtgg 2880aaaacaccca gctgcagaac gagaagctgt acctgtacta
cctgcagaat gggcgggata 2940tgtacgtgga ccaggaactg gacatcaacc
ggctgtccga ctacgatgtg gaccatatcg 3000tgcctcagag ctttctgaag
gacgactcca tcgacaacaa ggtgctgacc agaagcgaca 3060agaaccgggg
caagagcgac aacgtgccct ccgaagaggt cgtgaagaag atgaagaact
3120actggcggca gctgctgaac gccaagctga ttacccagag aaagttcgac
aatctgacca 3180aggccgagag aggcggcctg agcgaactgg ataaggccgg
cttcatcaag agacagctgg 3240tggaaacccg gcagatcaca aagcacgtgg
cacagatcct ggactcccgg atgaacacta 3300agtacgacga gaatgacaag
ctgatccggg aagtgaaagt gatcaccctg aagtccaagc 3360tggtgtccga
tttccggaag gatttccagt tttacaaagt gcgcgagatc aacaactacc
3420accacgccca cgacgcctac ctgaacgccg tcgtgggaac cgccctgatc
aaaaagtacc 3480ctaagctgga aagcgagttc gtgtacggcg actacaaggt
gtacgacgtg cggaagatga 3540tcgccaagag cgagcaggaa atcggcaagg
ctaccgccaa gtacttcttc tacagcaaca 3600tcatgaactt tttcaagacc
gagattaccc tggccaacgg cgagatccgg aagcggcctc 3660tgatcgagac
aaacggcgaa accggggaga tcgtgtggga taagggccgg gattttgcca
3720ccgtgcggaa agtgctgagc atgccccaag tgaatatcgt gaaaaagacc
gaggtgcaga 3780caggcggctt cagcaaagag tctatcctgc ccaagaggaa
cagcgataag ctgatcgcca 3840gaaagaagga ctgggaccct aagaagtacg
gcggcttcga cagccccacc gtggcctatt 3900ctgtgctggt ggtggccaaa
gtggaaaagg gcaagtccaa gaaactgaag agtgtgaaag 3960agctgctggg
gatcaccatc atggaaagaa gcagcttcga gaagaatccc atcgactttc
4020tggaagccaa gggctacaaa gaagtgaaaa aggacctgat catcaagctg
cctaagtact 4080ccctgttcga gctggaaaac ggccggaagt gtctgtcgta
tgagaccgag atcctgaccg 4140tggagtatgg actgctgccg attggaaaga
ttgtggagaa gcgcattgag tgcaccgtgt 4200acagcgtgga taacaatggc
aacatctata cacagccagt ggcccagtgg cacgaccgcg 4260gagagcagga
ggtcttcgag tactgcctgg aggatggcag cctgattcgc gccaccaagg
4320atcataagtt catgacggtg gacggacaga tgctgcccat cgatgagatt
tttgagcgcg 4380agctggatct gatgcgcgtg gataacctgc cgaattaaga
attcgatctt tttccctctg 4440ccaaaaatta tggggacatc atgaagcccc
ttgagcatct gacttctggc taataaagga 4500aatttatttt cattgcaata
gtgtgttgga attttttgtg tctctcactc ggcggccgca 4560ggaaccccta
gtgatggagt tggccactcc ctctctgcgc gctcgctcgc tcactgaggc
4620cgggcgacca aaggtcgccc gacgcccggg ctttgcccgg gcggcctcag
tgagcgagcg 4680agcgcgcagc tgcctgcagg 4700113338DNAArtificial
SequenceSynthetic Polynucleotide 11gttgacattg attattgact agttattaat
agtaatcaat tacggggtca ttagttcata 60gcccatatat ggagttccgc gttacataac
ttacggtaaa tggcccgcct ggctgaccgc 120ccaacgaccc ccgcccattg
acgtcaataa tgacgtatgt tcccatagta acgccaatag 180ggactttcca
ttgacgtcaa tgggtggact atttacggta aactgcccac ttggcagtac
240atcaagtgta tcatatgcca agtacgcccc ctattgacgt caatgacggt
aaatggcccg 300cctggcatta tgcccagtac atgaccttat gggactttcc
tacttggcag tacatctacg 360tattagtcat cgctattacc atggtgatgc
ggttttggca gtacatcaat gggcgtggat 420agcggtttga ctcacgggga
tttccaagtc tccaccccat tgacgtcaat gggagtttgt 480tttggcacca
aaatcaacgg gactttccaa aatgtcgtaa caactccgcc ccattgacgc
540aaatgggcgg taggcgtgta cggtgggagg tctatataag cagagctctc
tggctaacta 600gagaacccac tgcttactgg cttatcgaaa ttaatacgac
tcactatagg gagacccaag 660ctggctagcg ccaccatgat caagattgcc
acgcgcaagt acctgggcaa gcagaacgtg 720tacgacatcg gagtggagcg
cgatcacaac tttgccctga agaatggctt tattgcctcg 780aactgtatgc
tggcctctgc cggcgaactg cagaagggaa acgaactggc cctgccctcc
840aaatatgtga acttcctgta cctggccagc cactatgaga agctgaaggg
ctcccccgag 900gataatgagc agaaacagct gtttgtggaa cagcacaagc
actacctgga cgagatcatc 960gagcagatca gcgagttctc caagagagtg
atcctggccg acgctaatct ggacaaagtg 1020ctgtccgcct acaacaagca
ccgggataag cccatcagag agcaggccga gaatatcatc 1080cacctgttta
ccctgaccaa tctgggagcc cctgccgcct tcaagtactt tgacaccacc
1140atcgaccgga agaggtacac cagcaccaaa gaggtgctgg acgccaccct
gatccaccag 1200agcatcaccg gcctgtacga gacacggatc gacctgtctc
agctgggagg cgacagcccc 1260aagaagaaga gaaaggtgga ggccagcgga
tccgctccgg gaaaaaagag gccggtagag 1320cactctcctg tggagccaga
ctcctcctcg ggaaccggaa aggcgggcca gcagcctgca 1380agaaaaagat
tgaattttgg tcagactgga gacgcagact cagtacctga cccccagcct
1440ctcggacagc caccagcagc cccctctggt ctgggaacta atacgatggc
tacaggcagt 1500ggcgcaccaa tggcagacaa taacgagggc gccgacggag
tgggtaattc ctcgggaaat 1560tggcattgcg attccacatg gatgggcgac
agagtcatca ccaccagcac ccgaacctgg 1620gccctgccca cctacaacaa
ccacctctac aaacaaattt ccagccaatc aggagcctcg 1680aacgacaatc
actactttgg ctacagcacc ccttgggggt attttgactt caacagattc
1740cactgccact tttcaccacg tgactggcaa agactcatca acaacaactg
gggattccga 1800cccaagagac tcaacttcaa gctctttaac attcaagtca
aagaggtcac gcagaatgac 1860ggtacgacga cgattgccaa taaccttacc
agcacggttc aggtgtttac tgactcggag 1920taccagctcc cgtacgtcct
cggctcggcg catcaaggat gcctcccgcc gttcccagca 1980gacgtcttca
tggtgccaca gtatggatac ctcaccctga acaacgggag tcaggcagta
2040ggacgctctt cattttactg cctggagtac tttccttctc agatgctgcg
taccggaaac 2100aactttacct tcagctacac ttttgaggac gttcctttcc
acagcagcta cgctcacagc 2160cagagtctgg accgtctcat gaatcctctc
atcgaccagt acctgtatta cttgagcaga 2220acaaacactc caagtggaac
caccacgcag tcaaggcttc agttttctca ggccggagcg 2280agtgacattc
gggaccagtc taggaactgg cttcctggac cctgttaccg ccagcagcga
2340gtatcaaaga catctgcgga taacaacaac agtgaatact cgtggactgg
agctaccaag 2400taccacctca atggcagaga ctctctggtg aatccgggcc
cggccatggc aagccacaag 2460gacgatgaag aaaagttttt tcctcagagc
ggggttctca tctttgggaa gcaaggctca 2520gagaaaacaa atgtggacat
tgaaaaggtc atgattacag acgaagagga aatcaggaca 2580accaatcccg
tggctacgga gcagtatggt tctgtatcta ccaacctcca gagaggcaac
2640agacaagcag ctaccgcaga tgtcaacaca caaggcgttc ttccaggcat
ggtctggcag 2700gacagagatg tgtaccttca ggggcccatc tgggcaaaga
ttccacacac ggacggacat 2760tttcacccct ctcccctcat gggtggattc
ggacttaaac accctcctcc acagattctc 2820atcaagaaca ccccggtacc
tgcgaatcct tcgaccacct tcagtgcggc aaagtttgct 2880tccttcatca
cacagtactc cacgggacag gtcagcgtgg agatcgagtg ggagctgcag
2940aaggaaaaca gcaaacgctg gaatcccgaa attcagtaca cttccaacta
caacaagtct 3000gttaatgtgg actttactgt ggacactaat ggcgtgtatt
cagagcctcg ccccattggc 3060accagatacc tgactcgtaa tctgtaagaa
ttaaacccgc tgatcagcct cgactgtgcc 3120ttctagttgc cagccatctg
ttgtttgccc ctcccccgtg ccttccttga ccctggaagg 3180tgccactccc
actgtccttt cctaataaaa tgaggaaatt gcatcgcatt gtctgagtag
3240gtgtcattct attctggggg gtggggtggg gcaggacagc aagggggagg
attgggaaga 3300caatagcagg catgctgggg atgcggtggg ctctatgg
3338123377DNAArtificial SequenceSynthetic Polynucleotide
12gttgacattg attattgact agttattaat agtaatcaat tacggggtca ttagttcata
60gcccatatat ggagttccgc gttacataac ttacggtaaa tggcccgcct ggctgaccgc
120ccaacgaccc ccgcccattg acgtcaataa tgacgtatgt tcccatagta
acgccaatag 180ggactttcca ttgacgtcaa tgggtggact atttacggta
aactgcccac ttggcagtac 240atcaagtgta tcatatgcca agtacgcccc
ctattgacgt caatgacggt aaatggcccg 300cctggcatta tgcccagtac
atgaccttat gggactttcc tacttggcag tacatctacg 360tattagtcat
cgctattacc atggtgatgc ggttttggca gtacatcaat gggcgtggat
420agcggtttga ctcacgggga tttccaagtc tccaccccat tgacgtcaat
gggagtttgt 480tttggcacca aaatcaacgg gactttccaa aatgtcgtaa
caactccgcc ccattgacgc 540aaatgggcgg taggcgtgta cggtgggagg
tctatataag cagagctctc tggctaacta 600gagaacccac tgcttactgg
cttatcgaaa ttaatacgac tcactatagg gagacccaag 660ctggctagcg
ccaccatgat caagattgcc acgcgcaagt acctgggcaa gcagaacgtg
720tacgacatcg gagtggagcg cgatcacaac tttgccctga agaatggctt
tattgcctcg 780aactgtatgc tggcctctgc cggcgaactg cagaagggaa
acgaactggc cctgccctcc 840aaatatgtga acttcctgta cctggccagc
cactatgaga agctgaaggg ctcccccgag 900gataatgagc agaaacagct
gtttgtggaa cagcacaagc actacctgga cgagatcatc 960gagcagatca
gcgagttctc caagagagtg atcctggccg acgctaatct ggacaaagtg
1020ctgtccgcct acaacaagca ccgggataag cccatcagag agcaggccga
gaatatcatc 1080cacctgttta ccctgaccaa tctgggagcc cctgccgcct
tcaagtactt tgacaccacc 1140atcgaccgga agaggtacac cagcaccaaa
gaggtgctgg acgccaccct gatccaccag 1200agcatcaccg gcctgtacga
gacacggatc gacctgtctc agctgggagg cgacagcccc 1260aagaagaaga
gaaaggtgga ggccagcggt ggcggcggtt caggcggagg tggctctggg
1320ggcgggggtt ctgctccggg aaaaaagagg ccggtagagc actctcctgt
ggagccagac 1380tcctcctcgg gaaccggaaa ggcgggccag cagcctgcaa
gaaaaagatt gaattttggt 1440cagactggag acgcagactc agtacctgac
ccccagcctc tcggacagcc accagcagcc 1500ccctctggtc tgggaactaa
tacgatggct acaggcagtg gcgcaccaat ggcagacaat 1560aacgagggcg
ccgacggagt gggtaattcc tcgggaaatt ggcattgcga ttccacatgg
1620atgggcgaca gagtcatcac caccagcacc cgaacctggg ccctgcccac
ctacaacaac 1680cacctctaca aacaaatttc cagccaatca ggagcctcga
acgacaatca ctactttggc 1740tacagcaccc cttgggggta ttttgacttc
aacagattcc actgccactt ttcaccacgt 1800gactggcaaa gactcatcaa
caacaactgg ggattccgac ccaagagact caacttcaag 1860ctctttaaca
ttcaagtcaa agaggtcacg cagaatgacg gtacgacgac gattgccaat
1920aaccttacca gcacggttca ggtgtttact gactcggagt accagctccc
gtacgtcctc 1980ggctcggcgc atcaaggatg cctcccgccg ttcccagcag
acgtcttcat ggtgccacag 2040tatggatacc tcaccctgaa caacgggagt
caggcagtag gacgctcttc attttactgc 2100ctggagtact ttccttctca
gatgctgcgt accggaaaca actttacctt cagctacact 2160tttgaggacg
ttcctttcca cagcagctac gctcacagcc agagtctgga ccgtctcatg
2220aatcctctca tcgaccagta cctgtattac ttgagcagaa caaacactcc
aagtggaacc 2280accacgcagt caaggcttca gttttctcag gccggagcga
gtgacattcg ggaccagtct 2340aggaactggc ttcctggacc ctgttaccgc
cagcagcgag tatcaaagac atctgcggat 2400aacaacaaca gtgaatactc
gtggactgga gctaccaagt accacctcaa tggcagagac 2460tctctggtga
atccgggccc ggccatggca agccacaagg acgatgaaga aaagtttttt
2520cctcagagcg gggttctcat ctttgggaag caaggctcag agaaaacaaa
tgtggacatt 2580gaaaaggtca tgattacaga cgaagaggaa atcaggacaa
ccaatcccgt ggctacggag 2640cagtatggtt ctgtatctac caacctccag
agaggcaaca gacaagcagc taccgcagat 2700gtcaacacac aaggcgttct
tccaggcatg gtctggcagg acagagatgt gtaccttcag 2760gggcccatct
gggcaaagat tccacacacg gacggacatt ttcacccctc tcccctcatg
2820ggtggattcg gacttaaaca ccctcctcca cagattctca tcaagaacac
cccggtacct 2880gcgaatcctt cgaccacctt cagtgcggca aagtttgctt
ccttcatcac acagtactcc 2940acgggacagg tcagcgtgga gatcgagtgg
gagctgcaga aggaaaacag caaacgctgg 3000aatcccgaaa ttcagtacac
ttccaactac aacaagtctg ttaatgtgga ctttactgtg 3060gacactaatg
gcgtgtattc agagcctcgc cccattggca ccagatacct gactcgtaat
3120ctgtaagaat taaacccgct gatcagcctc gactgtgcct tctagttgcc
agccatctgt 3180tgtttgcccc tcccccgtgc cttccttgac cctggaaggt
gccactccca ctgtcctttc 3240ctaataaaat gaggaaattg catcgcattg
tctgagtagg tgtcattcta ttctgggggg 3300tggggtgggg caggacagca
agggggagga ttgggaagac aatagcaggc atgctgggga 3360tgcggtgggc tctatgg
3377133377DNAArtificial SequenceSynthetic Polynucleotide
13gttgacattg attattgact agttattaat agtaatcaat tacggggtca ttagttcata
60gcccatatat ggagttccgc gttacataac ttacggtaaa tggcccgcct ggctgaccgc
120ccaacgaccc ccgcccattg acgtcaataa tgacgtatgt tcccatagta
acgccaatag 180ggactttcca ttgacgtcaa tgggtggact atttacggta
aactgcccac ttggcagtac 240atcaagtgta tcatatgcca agtacgcccc
ctattgacgt caatgacggt aaatggcccg 300cctggcatta tgcccagtac
atgaccttat gggactttcc tacttggcag tacatctacg 360tattagtcat
cgctattacc atggtgatgc ggttttggca gtacatcaat gggcgtggat
420agcggtttga ctcacgggga tttccaagtc tccaccccat tgacgtcaat
gggagtttgt 480tttggcacca aaatcaacgg gactttccaa aatgtcgtaa
caactccgcc ccattgacgc 540aaatgggcgg taggcgtgta cggtgggagg
tctatataag cagagctctc tggctaacta 600gagaacccac tgcttactgg
cttatcgaaa ttaatacgac tcactatagg gagacccaag 660ctggctagcg
ccaccatgat caagattgcc acgcgcaagt acctgggcaa gcagaacgtg
720tacgacatcg gagtggagcg cgatcacaac tttgccctga agaatggctt
tattgcctcg 780aactgtatgc tggcctctgc cggcgaactg cagaagggaa
acgaactggc cctgccctcc 840aaatatgtga acttcctgta cctggccagc
cactatgaga agctgaaggg ctcccccgag 900gataatgagc agaaacagct
gtttgtggaa cagcacaagc actacctgga cgagatcatc 960gagcagatca
gcgagttctc caagagagtg atcctggccg acgctaatct ggacaaagtg
1020ctgtccgcct acaacaagca ccgggataag cccatcagag agcaggccga
gaatatcatc 1080cacctgttta ccctgaccaa tctgggagcc cctgccgcct
tcaagtactt tgacaccacc 1140atcgaccgga agaggtacac cagcaccaaa
gaggtgctgg acgccaccct gatccaccag 1200agcatcaccg gcctgtacga
gacacggatc gacctgtctc agctgggagg cgacagcccc 1260aagaagaaga
gaaaggtgga ggccagcgag gcagcagcca aagaggccgc tgccaaggag
1320gcagcggcta aagctccggg aaaaaagagg ccggtagagc actctcctgt
ggagccagac 1380tcctcctcgg gaaccggaaa ggcgggccag cagcctgcaa
gaaaaagatt gaattttggt 1440cagactggag acgcagactc agtacctgac
ccccagcctc tcggacagcc accagcagcc 1500ccctctggtc tgggaactaa
tacgatggct acaggcagtg gcgcaccaat ggcagacaat 1560aacgagggcg
ccgacggagt gggtaattcc tcgggaaatt ggcattgcga ttccacatgg
1620atgggcgaca gagtcatcac caccagcacc cgaacctggg ccctgcccac
ctacaacaac 1680cacctctaca aacaaatttc cagccaatca ggagcctcga
acgacaatca ctactttggc 1740tacagcaccc cttgggggta ttttgacttc
aacagattcc actgccactt ttcaccacgt 1800gactggcaaa gactcatcaa
caacaactgg ggattccgac ccaagagact caacttcaag 1860ctctttaaca
ttcaagtcaa agaggtcacg cagaatgacg gtacgacgac gattgccaat
1920aaccttacca gcacggttca ggtgtttact gactcggagt accagctccc
gtacgtcctc 1980ggctcggcgc atcaaggatg cctcccgccg ttcccagcag
acgtcttcat ggtgccacag 2040tatggatacc tcaccctgaa caacgggagt
caggcagtag gacgctcttc attttactgc 2100ctggagtact ttccttctca
gatgctgcgt accggaaaca actttacctt cagctacact 2160tttgaggacg
ttcctttcca cagcagctac gctcacagcc agagtctgga ccgtctcatg
2220aatcctctca tcgaccagta cctgtattac ttgagcagaa caaacactcc
aagtggaacc 2280accacgcagt caaggcttca gttttctcag gccggagcga
gtgacattcg ggaccagtct 2340aggaactggc ttcctggacc ctgttaccgc
cagcagcgag tatcaaagac atctgcggat 2400aacaacaaca gtgaatactc
gtggactgga gctaccaagt accacctcaa tggcagagac 2460tctctggtga
atccgggccc ggccatggca agccacaagg acgatgaaga aaagtttttt
2520cctcagagcg gggttctcat ctttgggaag caaggctcag agaaaacaaa
tgtggacatt 2580gaaaaggtca tgattacaga cgaagaggaa atcaggacaa
ccaatcccgt ggctacggag 2640cagtatggtt ctgtatctac caacctccag
agaggcaaca gacaagcagc taccgcagat 2700gtcaacacac aaggcgttct
tccaggcatg gtctggcagg acagagatgt gtaccttcag 2760gggcccatct
gggcaaagat tccacacacg gacggacatt ttcacccctc tcccctcatg
2820ggtggattcg gacttaaaca ccctcctcca cagattctca tcaagaacac
cccggtacct 2880gcgaatcctt cgaccacctt cagtgcggca aagtttgctt
ccttcatcac acagtactcc 2940acgggacagg tcagcgtgga gatcgagtgg
gagctgcaga aggaaaacag caaacgctgg 3000aatcccgaaa ttcagtacac
ttccaactac aacaagtctg ttaatgtgga ctttactgtg 3060gacactaatg
gcgtgtattc agagcctcgc cccattggca ccagatacct gactcgtaat
3120ctgtaagaat taaacccgct gatcagcctc gactgtgcct tctagttgcc
agccatctgt 3180tgtttgcccc tcccccgtgc cttccttgac cctggaaggt
gccactccca ctgtcctttc 3240ctaataaaat gaggaaattg catcgcattg
tctgagtagg tgtcattcta ttctgggggg 3300tggggtgggg caggacagca
agggggagga ttgggaagac aatagcaggc atgctgggga 3360tgcggtgggc tctatgg
3377
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