U.S. patent application number 13/607118 was filed with the patent office on 2014-01-23 for isolation, cloning and characterization of new adeno-associated virus (aav) serotypes.
This patent application is currently assigned to Human Services. The applicant listed for this patent is John A. CHIORINI, Michael SCHMIDT. Invention is credited to John A. CHIORINI, Michael SCHMIDT.
Application Number | 20140024013 13/607118 |
Document ID | / |
Family ID | 37177918 |
Filed Date | 2014-01-23 |
United States Patent
Application |
20140024013 |
Kind Code |
A1 |
SCHMIDT; Michael ; et
al. |
January 23, 2014 |
Isolation, Cloning and Characterization of New Adeno-Associated
Virus (AAV) Serotypes
Abstract
The present invention provides new adeno-associated virus (AAV)
viruses and vectors, and particles derived therefrom. In addition,
the present invention provides methods of delivering a nucleic acid
to a cell using the AAV vectors and particles.
Inventors: |
SCHMIDT; Michael;
(Kensington, MD) ; CHIORINI; John A.; (Dayton,
MD) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
SCHMIDT; Michael
CHIORINI; John A. |
Kensington
Dayton |
MD
MD |
US
US |
|
|
Assignee: |
Human Services
Bethesda
MD
The United States of America, as represented by the Secretary,
Department of Health and
|
Family ID: |
37177918 |
Appl. No.: |
13/607118 |
Filed: |
September 7, 2012 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11912803 |
Jul 8, 2008 |
8283151 |
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PCT/US06/17157 |
May 1, 2006 |
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13607118 |
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60676604 |
Apr 29, 2005 |
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Current U.S.
Class: |
435/5 ;
435/235.1; 435/320.1; 530/350; 530/387.9; 536/23.72 |
Current CPC
Class: |
A61P 31/12 20180101;
C07K 14/005 20130101; C12N 15/86 20130101; G01N 33/56983 20130101;
C12N 2750/14121 20130101; C12N 7/00 20130101; C12N 2750/14122
20130101; C12N 2750/14143 20130101; C07K 16/081 20130101 |
Class at
Publication: |
435/5 ;
536/23.72; 435/320.1; 435/235.1; 530/350; 530/387.9 |
International
Class: |
C07K 14/005 20060101
C07K014/005; G01N 33/569 20060101 G01N033/569; C07K 16/08 20060101
C07K016/08 |
Claims
1-113. (canceled)
114. An isolated nucleic acid molecule comprising a nucleic acid
sequence comprising at least 100 contiguous nucleotides from an
AAV-X1 virus.
115. The isolated nucleic acid molecule of claim 114, wherein the
nucleic acid sequence encodes an immunogenic portion of a protein
selected from the group consisting of an AAV-X1 REP and an AAV-X1
CAP protein.
116. The isolated nucleic acid molecule of claim 115, wherein the
AAV-X1 REP protein is selected from the group consisting of AAV-X1
REP40, AAV-X1 REP52, AAV-X1 REP68 and AAV-X1 REP78.
117. The isolated nucleic acid molecule of claim 115, wherein the
AAV-X1 CAP protein is selected from the group consisting of AAV-X1
VP1, AAV-X1 VP2 and AAV-X1 VP3.
118. The isolated nucleic acid molecule of claim 114, wherein the
nucleic acid sequence encodes a protein comprising at least 50
contiguous amino acids from SEQ ID NO: 21 or SEQ ID NO:49.
119. The isolated nucleic acid molecule of claim 114, wherein the
nucleic acid sequence is selected from the group consisting of: a)
a nucleic acid sequence encoding a polypeptide comprising an amino
acid sequence at least 80% identical to SEQ ID NO:21, wherein the
polypeptide binds an antibody raised against a protein consisting
of SEQ ID NO:21; and, b) a nucleic acid sequence encoding a
polypeptide comprising an amino acid sequence at least 80%
identical to SEQ ID NO:49, wherein the polypeptide binds an
antibody raised against a protein consisting of SEQ ID NO:49.
120. The isolated nucleic acid molecule of claim 114, wherein the
nucleic acid sequence is selected from the group consisting of: a)
a nucleic acid sequence encoding a polypeptide comprising an amino
acid sequence at least 95% identical to SEQ ID NO:21, wherein the
polypeptide binds an antibody raised against a protein consisting
of SEQ ID NO:21; and, b) a nucleic acid sequence encoding a
polypeptide comprising an amino acid sequence at least 95%
identical to SEQ ID NO:49, wherein the polypeptide binds an
antibody raised against a protein consisting of SEQ ID NO:49.
121. The isolated nucleic acid molecule of claim 114, wherein the
nucleic acid encodes a protein comprising SEQ ID NO:21 or SEQ ID
NO:49.
122. The isolated nucleic acid molecule of claim 114, wherein the
molecule is an AAV vector.
123. An isolated AAV particle comprising the nucleic acid molecule
of claim 114.
124. An isolated nucleic acid molecule comprising a nucleic acid
sequence selected from the group consisting of: a) a nucleic acid
sequence at least 95% identical to SEQ ID NO:11; b) a nucleic acid
sequence comprising at least 50 contiguous nucleotides from SEQ ID
NO:11; c) a nucleic acid sequence fully complementary to the
sequence of a) or b), wherein the polypeptide encoded by the
nucleic acid sequence of a) or b) binds an antibody raised against
a protein consisting of SEQ ID NO:11; d) a nucleic acid sequence at
least 95% identical to SEQ ID NO:48; e) a nucleic acid sequence
comprising at least 50 contiguous nucleotides from SEQ ID NO:48;
and, f) a nucleic acid sequence fully complementary to the sequence
of d) or e), wherein the polypeptide encoded by the nucleic acid
sequence of d) or e) binds an antibody raised against a protein
consisting of SEQ ID NO:48.
125. The isolated nucleic acid molecule of claim 124, wherein the
nucleic acid sequence comprises SEQ ID NO: 11 or SEQ ID NO:48.
126. The isolated nucleic acid molecule of claim 124, wherein the
molecule is an AAV vector.
127. An isolated AAV particle comprising the nucleic acid molecule
of claim 124.
128. An isolated protein comprising an amino acid sequence from an
AAV-X1 CAP protein or an AAV-X1 REP protein.
129. The isolated protein of claim 128, wherein the amino acid
sequence is selected from the group consisting of: a) an amino acid
sequence comprising at least 50 contiguous amino acids from SEQ ID
NO:21; b) an amino acid sequence at least 80% identical to SEQ ID
NO:21, wherein the isolated protein binds an antibody raised
against a protein consisting of SEQ ID NO:21; c) an amino acid
sequence comprising at least 50 contiguous amino acids from SEQ ID
NO:49; and, d) an amino acid sequence at least 80% identical to SEQ
ID NO:21, wherein the isolated protein binds an antibody raised
against a protein consisting of SEQ ID NO:49.
130. An isolated antibody that selectively binds the isolated
protein of claim 128.
131. An AAV particle comprising the isolated protein of claim
128.
132. Use of the isolated protein of claim 129 to detect antibodies
to AAV-X1, comprising: a) obtaining an antibody-containing sample;
b) contacting the sample with the isolated protein of claim 128;
and, c) determining if an antibody-protein reaction occurred, the
presence of such a reaction indicating the presence of antibodies
to AAV-X1 virus.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims benefit of U.S. Provisional
Application No. 60/676,604, filed Apr. 29, 2005, which is hereby
incorporated herein by reference in its entirety.
BACKGROUND OF THE INVENTION
[0002] Adeno-associated virus (AAV) is a member of the
Parvoviridae, a virus family characterized by a single stranded
linear DNA genome and a small icosahedral shaped capsid measuring
about 20 nm in diameter. AAV was first described as a contamination
of tissue culture grown simian virus 15, a simian adenovirus and
was found dependent on adenovirus for measurable replication. This
lead to its name, adeno-associated virus, and its classification in
the genus Dependovirus (reviewed in Hoggan et al., 1970). AAV is a
common contaminant of adenovirus samples and has been isolated from
human virus samples (AAV2, AAV3, AAV5), from samples of simian
virus-15 infected cells (AAV1, AAV4) as well as from stocks of
avian (AAAV) (Bossis and Chiorini, 2003), bovine, canine and ovine
adenovirus and laboratory adenovirus type 5 stock (AAV6). DNA
spanning the entire rep-cap ORFs of AAV7 and AAV8 was amplified by
PCR from heart tissue of rhesus monkeys (Gao et al., 2002). With
the exception of AAVs 1 and 6, all cloned AAV isolates appear to be
serologically distinct. Nine isolates have been cloned, and
recombinant viral stocks have been generated from each isolated
virus.
[0003] AAV appears to commonly infect humans. 50%-80% of adults in
North America are seropositive for AAV. A steep rise in antibody
response against AAV 1-3 was observed in the age group between 1-10
years (Blacklow et al., 1968). AAV 2 and 3 were readily isolated
from anal and throat specimens from children (Blacklow et al.,
1967) whereas isolation from adults was not observed. It appears
that AAV spreads primarily in the young population (Hoggan, 1970).
Prevalence of antibodies against AAV was found to be similar in
Europe, Brazil and Japan, which suggests a global spread of AAV
(Erles et al., 1999). Infection with AAV appears to be benign in
man and laboratory animals. Currently, no disease has been
associated with AAV infections.
[0004] AAV2 is the best characterized adeno-associated virus and
will be discussed as an AAV prototype. The AAV2 genome consists of
a linear single stranded DNA of 4,780 nucleotides. Both polarities
of DNA are encapsulated by AAV with equal efficiency. The AAV2
genome contains 2 open reading frames (ORF) named rep and cap. The
rep ORF encodes the non-structural proteins that are essential for
viral DNA replication, packaging and AAV integration. The cap ORF
encodes the capsid proteins. The rep ORF is transcribed from
promoters at map units P5 and P19. The rep transcripts contain an
intron close to the 3' end of the rep ORF and can be alternatively
spliced. The rep ORF is therefore expressed as 4 partially
overlapping proteins, which were termed according to their
molecular weight Rep78, 68, 52 and 40. The cap ORF is expressed
from a single promoter at P40. By alternative splicing and
utilization of an alternative ACG start codon, cap is expressed
into the capsid proteins VP1-3 which range in size from 65-86 kDa.
VP3 is the most abundant capsid protein and constitutes 80% of the
AAV2 capsid. All viral transcripts terminate at a polyA signal at
map unit 96.
[0005] During a productive AAV2 infection, unspliced mRNAs from the
p5 promoter encoding Rep78 are the first detectable viral
transcripts. In the course of infection, expression from P5, P19
and P40 increase to 1:3:18 levels respectively. The levels of
spliced transcripts increased to 50% for P5, P19 products and 90%
of P40 expressed RNA (Mouw and Pintel, 2000).
[0006] The AAV2 genome is terminated on both sides by inverted
terminal repeats (ITRs) of 145 nucleotides (nt). 125 nt of the ITR
constitute a palindrome which contains 2 internal palindromes of 21
nt each. The ITR can fold back on itself to generate a T-shaped
hairpin with only 7 non-paired bases. The stem of the ITR contains
a Rep binding site (RBS) and a sequence that is site and strand
specifically cleaved by Rep--the terminal resolution site (TRS).
The ITR is essential for AAV2 genome replication, integration and
contains the packaging signals.
[0007] The single-stranded AAV2 genome is packaged into a
non-enveloped icosahedral shaped capsid of about 20-25 nm diameter.
The virion consists of 26% DNA and 74% protein and has a density of
1.41 g/cm.sup.3. AAV2 particles are extremely stable and can
withstand heating to 60.degree. C. for 1 hour, extreme ph, and
extraction with organic solvents.
[0008] Rep proteins are involved in almost every step of AAV2
replication including AAV2 genome replication, integration, and
packaging. Rep78 and Rep68 possess ATPase, 3'-5' helicase, ligase
and nicking activities and bind specifically to DNA. Rep52 and
Rep40 appear to be involved in the encapsidation process and encode
ATPase and 3'-5' helicase activities. Mutational analysis suggests
a domain structure for Rep78. The N-terminal 225 aa are involved in
DNA binding, DNA nicking and ligation. Rep78 and Rep68 recognize a
GCTC repeat motif in the ITR as well as in a linear truncated form
of the ITR (Chiorini et al., 1994) with similar efficiencies. Rep78
and Rep68 possess a sequence and strand specific endonuclease
activity, which cleaves the ITR at the terminal resolution site
(TRS). Rep endonuclease activity is dependent on nucleoside
triphosphate hydrolysis and presence of metal cations. Rep 78 and
68 can also bind and cleave single stranded DNA in a NTP
independent matter. In addition, Rep78 catalyzes rejoining of
single stranded DNA substrates originating from the AAV2 origin of
replication--i.e., sequences containing a rep binding and terminal
resolution element.
[0009] The central region of AAV2 Rep78, which represents the
N-terminus of Rep52 and Rep40, contains the ATPase and helicase
activities as well as nuclear localization signals. The helicase
activity unwinds DNA-DNA and DNA-RNA duplexes, but not RNA-RNA. The
ATPase activity is constitutive and independent of a DNA substrate.
The C-terminus of Rep78 contains a potential zinc-finger domain and
can inhibit the cellular serine/threonine kinase activity of PKA as
well as its homolog PRKX by pseudosubstrate inhibition. Rep68 which
is translated from a spliced mRNA that encodes the N-terminal 529
amino acids (aa) of Rep78 fused to 7 aa unique for Rep68, doesn't
inhibit either PKA or PRKX. In addition to these biochemical
activities, Rep can affect intracellular conditions by
protein-protein interactions. Rep78 binds to a variety of cellular
proteins including transcription factors like SP-1,
high-mobility-group non-histone protein 1 (HMG-1) and the
oncosuppressor p53. Overexpression of Rep results in pleiotrophic
effects. Rep78 disrupts cell cycle progression and inhibits
transformation by cellular and viral oncogenes. In susceptible cell
lines, overexpression of Rep resulted in apoptosis and cell death.
Several of Rep78 activities contribute to cytotoxicity, including
its constitutive ATPase activity, interference with cellular gene
expression and protein interactions.
[0010] The first step of an AAV infection is binding to the cell
surface. Receptors and coreceptors for AAV2 include heparan sulfate
proteoglycan, fibroblast growth factor receptor-1, and
.alpha..sub.v.beta..sub.5 integrins whereas N-linked 2,3-linked
sialic acid is required for AAV5 binding and transduction (Walters
et al., 2001). In HeLa cells, fluorescently labeled AAV2 particles
appear to enter the cell via receptor-mediated endocytosis in
clathrin coated pits. More than 60% of bound virus was internalized
within 10 min after infection. Labeled AAV particles are observed
to have escaped from the endosome, been trafficked via the
cytoplasm to the cell nucleus and accumulated perinuclear, before
entering the nucleus, probably via nuclear pore complex (NPC). AAV2
particles have been detected in the nucleus, suggesting that
uncoating takes place in the nucleus (Bartlett et al., 2000;
Sanlioglu et al., 2000). AAV5 is internalized in HeLa cells
predominantly by clathrin coated vesicles, but to a lesser degree
also in noncoated pits. AAV particles can also be trafficked
intercellularly via the Golgi apparatus (Bantel-Schaal et al.,
2002). At least partial uncoating of AAV5 was suggested to take
place before entering the nucleus since intact AAV5 particles could
not be detected in the nucleus (Bantel-Schaal et al., 2002) After
uncoating, the single stranded genome is converted into duplex DNA
either by leading strand synthesis or annealing of input DNA of
opposite polarity. AAV replication takes place within the
nucleus.
[0011] During a co-infection with a helper virus such as
Adenovirus, herpes simplex virus or cytomegalovirus, AAV is capable
of an efficient productive replication. The helper functions
provided by Adenovirus have been studied in great detail. In human
embryonic kidney 293 cells, which constitutively express the
Adenovirus E1A and E1B genes, the early Adenovirus gene products of
E2A, E4 and VA were found sufficient to allow replication of
recombinant AAV. Allen et al. reported that efficient production of
rAAV is possible in 293 cells transfected with only an E4orf6
expression plasmid (Allen et al., 2000). E1A stimulates S phase
entry and induces unscheduled DNA synthesis by inactivating the pRB
checkpoint at the G1/S border by interaction with pRB family
proteins which results in the release of E2F (reviewed in
(Ben-Israel and Kleinberger, 2002). This leads to either induction
or activation of enzymes involved in nucleotide synthesis and DNA
replication. Since unscheduled DNA synthesis is a strong apoptotic
signal, anti-apoptotic functions are required. E1B-19k is a Bcl-2
homolog and E1B-55k is a p53 antagonist. Both proteins have
anti-apoptotic functions. E4orf6 forms a complex with E1B-55k and
results in degradation of p53. It is also reported to cause S-phase
arrest (Ben-Israel and Kleinberger, 2002). E2A encodes a single
strand DNA binding protein, which appears to be non-essential for
DNA replication but effects gene expression (Chang and Shenk, 1990)
(Fields 39, 40). The VA transcription unit affects AAV2 RNA
stability and translation (Janik et al., 1989). E1A has a more
direct effect on AAV2 gene expression. The cellular transcription
factor YY-1 binds and inhibits the viral P5 promoter. E1A relieves
this transcriptional block. None of the late Ad gene products have
been found to be essential for AAV2 replication. The main function
of the helper virus appears to be the generation of a cellular
environment with active DNA replication machinery and blocked
pro-apoptotic functions that allows high-level AAV replication
rather than a direct involvement in AAV replication.
[0012] While AAV is usually dependent on a helper virus for
efficient replication, low level AAV replication was observed under
conditions of genotoxic stress (Yakinoglu et al., 1988; Yakobson et
al., 1989). AAV DNA replication and particle formation was also
observed in differentiating keratinocytes in the absence of helper
virus infection (Meyers et al., 2000). This demonstrates that AAV
is not defective per se but rather depends on the helper virus to
establish the favorable cellular condition and to provide factors
for efficient replication
[0013] The ability of AAV vectors to infect dividing and
non-dividing cells, establish long-term transgene expression, and
the lack of pathogenicity has made them attractive for use in gene
therapy applications. Lack of cross competition in binding
experiments suggests that each AAV serotype may have a distinct
mechanism of cell entry. Comparison of the cap ORFs from different
serotypes has identified blocks of conserved and divergent
sequence, with most of the latter residing on the exterior of the
virion, thus explaining the altered tissue tropism among serotypes
(19-21, 48, 56). Vectors based on new AAV serotypes may have
different host range and different immunological properties, thus
allowing for more efficient transduction in certain cell types. In
addition, characterization of new serotypes will aid in identifying
viral elements required for altered tissue tropism.
BRIEF SUMMARY OF THE INVENTION
[0014] Provided herein are adeno-associated viruses (AAV) and
vectors derived therefrom. Thus, the present invention relates to
AAV vectors for and methods of delivering nucleic acids to cells of
subjects.
[0015] Additional advantages of the disclosed method and
compositions will be set forth in part in the description which
follows, and in part will be understood from the description, or
may be learned by practice of the disclosed method and
compositions. The advantages of the disclosed method and
compositions will be realized and attained by means of the elements
and combinations particularly pointed out in the appended claims.
It is to be understood that both the foregoing general description
and the following detailed description are exemplary and
explanatory only and are not restrictive of the invention as
claimed.
BRIEF DESCRIPTION OF THE DRAWINGS
[0016] The accompanying drawings, which are incorporated in and
constitute a part of this specification, illustrate several
embodiments of the disclosed method and compositions and together
with the description, serve to explain the principles of the
disclosed method and compositions.
[0017] FIG. 1 shows evolutionary relationship among human,
non-human primate AAVs, and AAV contaminants detected in adenovirus
stocks. The phylogenetic tree is based on merged ClustalW
alignments of VP1 sequences. VR numbers are identifiers of the ATCC
virus collection.
[0018] FIG. 2 shows rAAV6, rAAV(VR-195), and rAAV(VR-355) are
neutralized by pooled human IgGs. COS cells were transduced with a
pre-incubation mixture consisting of rAAV-6, rAAV(VR-195), and
rAAV(VR-355) expressing GFP and human IgGs at the indicated
concentrations. Twenty-four hours post-inoculation, transduction
was analyzed by flow cytometry and graphed as percent transduction
of the untreated control. Values are means from three experiments;
error bars represent standard deviations.
[0019] FIG. 3 shows inhibitory effect of heparin on of COS cell
transduction. COS cells were transduced with a pre-incubation
mixture consisting out of rAAV2, rAAV5, rAAV6, rAAV(VR-195), and
rAAV(VR-355) expressing GFP and heparin at the indicated
concentrations. 24 h post-inoculation, transduction was analyzed by
flow cytometry. Values are means from three experiments; error bars
represent standard deviations.
[0020] FIG. 4 shows charge-dependency of AAV6, rAAV(VR-195), and
rAAV(VR-355) transduction of COS cells. COS cells were transduced
with rAAV6, rAAV(VR-195), and rAAV(VR-355) in medium containing the
indicated concentrations of NaCl. Twenty-four hour postinoculation,
transduction was analyzed by flow cytometry. Values are means from
three experiments; error bars represent standard deviations
[0021] FIG. 5 shows neuraminidase treatment blocks rAAV6,
rAAV(VR-195), and rAAV(VR-355) transduction and cell binding. FIG.
5A shows gene transfer mediated by rAAV6, rAAV(VR-195), and
rAAV(VR-355) expressing GFP in COS cells, following neuraminidase
pretreatment. FIG. 5B shows binding. Values are means from three
experiments; error bars represent standard deviations
[0022] FIG. 6 shows effects of lectins on rAAV6, rAAV(VR-195), and
rAAV(VR-355) transduction of COS cells. After pre-incubation with
ECL, LCA, Main or STL, COS cells were transduced with rAAV-6,
rAAV(VR-195), and rAAV(VR-355) expressing GFP in the presence of
the indicated lectin. Twenty-four hour post-inoculation,
transduction was analyzed by flow cytometry. Values are means from
two experiments done in duplicate; error bars represent standard
deviations.
[0023] FIG. 7 shows transduction efficiency in human cancer cell
lines. The indicated cell lines were transduced with
1.times.10.sup.8 particles of rAAV6, rAAV(VR-195), and rAAV(VR-355)
expressing a nuclear localized GFP. Twenty-four hour
post-inoculation, transduction was analyzed by flow cytometry.
Values are means from three experiments; error bars represent
standard deviations. * COS cells transduction is given as
transducing units/2.times.10.sup.7 particles.
[0024] FIG. 8 shows AAV6 competition. COS cells were transduced
with a constant amount of rAAV6, rAAV(VR-195) or rAAV(VR-355)
expressing GFP after 60 min pre-incubation with increasing titers
of rAAV6-lacZ. Forty-eight hour post-inoculation, transduction was
analyzed by flow cytometry. Values are means from three
experiments; error bars represent standard deviations.
[0025] FIG. 9 shows effects of sugars on rAAV mediated
transduction. Cos cells were transduced with GFP encoding rAAVs
that were pre-incubated with various sugar monomers and polymers.
24 h post-inoculation, transduction was analyzed by flow cytometry.
Heparin is the attachment factor for AAV2 that mediates initial
binding of AAV2 to the cell. Extracellular heparin binds to the
virus and blocks the attachment of the virus to the cell. None of
the sugars tested blocked transduction of AAV-X1, AAV-X5, AAV-X25
or AAV-X26.
[0026] FIG. 10 shows effects of lectins on rAAV transduction of COS
cells. After pre-incubation with various lectins, COS cells were
transduced with rAAV-6, AAV-X1 and AAV-X25 expressing GFP in the
presence of the indicated lectin. 24 h post-inoculation,
transduction was analyzed by flow cytometry. All AAVs have a
distinct profile on the lectin panel.
[0027] FIG. 11 shows transduction efficiency of rAAVs in cell
lines. Cells were transduced with various rAAV serotypes encoding
GFP. Two days after inoculation, cells were analyzed for GFP
expression by flow cytometry. The transduction profiles of AAV-X1,
AAV-X5, AAV-X25 and AAV-X26 were different from that of known
AAVs.
[0028] FIG. 12 shows rAAV12 COS cell transduction is not inhibited
by heparin. COS cells were transduced with a pre-incubation mixture
consisting out of rAAV2-GFP, or rAAV12-GFP expressing GFP and
heparin at the indicated concentrations. 24 h post-inoculation,
transduction was analyzed by flow cytometry. Values are means from
three experiments; error bars represent standard deviations.
[0029] FIG. 13 shows rAAV12 transduction is independent of cell
surface sialic acid. COS cells were pretreated with the V. cholera
neuraminidase to remove exposed sialic acids groups before the
cells were transduced with rAAV2, rAAV4, rAAV5 and rAAV12-GFP. Gene
transfer was determined by flow cytometry. Values are means from
three experiments; error bars represent standard deviations.
[0030] FIG. 14 shows rAAV12 transduction is protease sensitive and
does not require glycosphingolipids. COS cells were proteolytically
digested with trypsin (A) or treated with the glycosphingolipids
synthesis inhibitors PPMP (B) prior to transduction with rAAV2-GFP,
rAAV12-GFP and rBAAV-GFP. Gene transfer in these cells was compared
to untreated cultures. Values are means from three experiments;
error bars represent standard deviations.
[0031] FIG. 15 shows extracellular mannosamine inhibits rAAV12
transduction. COS cells were transduced with a pre-incubation
mixture of rAAV2-GFP, or rAAV12-GFP and mannosamine at the
indicated concentrations. 24 h post-inoculation, transduction was
analyzed by flow cytometry. Values are means from three
experiments; error bars represent standard deviations.
[0032] FIG. 16 shows rAAV12 has a broad tropism. Transduction
efficiency of rAAV12 was compared to rAAV4 in 13 human cancer cell
lines. Cells were transduced with particles of either rAAV12-GFP or
rAAV4-GFP. Transduction was analyzed by flow cytometry 28 h after
virus inoculation. Values are means from three experiments; error
bars represent standard deviations.
[0033] FIG. 17 shows rAAV12 is highly resistant to neutralization
by human IgGs. rAAV2-GFP, rAAV6-GFP and rAAV12-GFP were incubated
with pooled human IgGs prior to transduction of COS cells. 24 h
post-inoculation, transduction was analyzed by flow cytometry.
Transduction efficiencies relative to an untreated control were
plotted. Values are means from three experiments; error bars
represent standard deviations.
[0034] FIG. 18 shows rAAV12 transduces salivary glands and skeletal
muscles in vivo. Male Balb/c mice were administered 10.sup.9
particles of either AAV2-hEPO or AAV12-hEPO by retrograde ductal
delivery to both submandibular glands or in both their tibialis
anterior (two injection sites per muscle). Secretion of hEPO in
mouse serum was determined 4 weeks after transduction by an ELISA
test. Error bars represent standard deviations.
DETAILED DESCRIPTION OF THE INVENTION
[0035] The disclosed method and compositions may be understood more
readily by reference to the following detailed description of
particular embodiments and the Example included therein and to the
Figures and their previous and following description.
[0036] It is to be understood that the disclosed methods and
compositions are not limited to specific synthetic methods,
specific analytical techniques, or to particular reagents unless
otherwise specified, and, as such, may vary. It is also to be
understood that the terminology used herein is for the purpose of
describing particular embodiments only and is not intended to be
limiting.
[0037] Disclosed are materials, compositions, and components that
can be used for, can be used in conjunction with, can be used in
preparation for, or are products of the disclosed method and
compositions. These and other materials are disclosed herein, and
it is understood that when combinations, subsets, interactions,
groups, etc. of these materials are disclosed that while specific
reference of each various individual and collective combinations
and permutation of these compounds may not be explicitly disclosed,
each is specifically contemplated and described herein. For
example, if a vector is disclosed and discussed and a number of
modifications that can be made to a number of molecules including
the promoters and ITRs are discussed, then each and every
combination and permutation of the promoters and ITRs, and the
modifications that are possible, are specifically contemplated
unless specifically indicated to the contrary. Thus, if a class of
molecules A, B, and C are disclosed as well as a class of molecules
D, E, and F and an example of a combination molecule, A-D is
disclosed, then even if each is not individually recited, each is
individually and collectively contemplated. Thus, is this example,
each of the combinations A-E, A-F, B-D, B-E, B-F, C-D, C-E, and C-F
are specifically contemplated and should be considered disclosed
from disclosure of A, B, and C; D, E, and F; and the example
combination A-D. Likewise, any subset or combination of these is
also specifically contemplated and disclosed. Thus, for example,
the sub-group of A-E, B-F, and C-E are specifically contemplated
and should be considered disclosed from disclosure of A, B, and C;
D, E, and F; and the example combination A-D. This concept applies
to all aspects of this application including, but not limited to,
steps in methods of making and using the disclosed compositions.
Thus, if there are a variety of additional steps that can be
performed it is understood that each of these additional steps can
be performed with any specific embodiment or combination of
embodiments of the disclosed methods, and that each such
combination is specifically contemplated and should be considered
disclosed.
[0038] It must be noted that as used herein and in the appended
claims, the singular forms "a", "an", and "the" include plural
reference unless the context clearly dictates otherwise. Thus, for
example, reference to "a vector" includes a plurality of such
vectors, reference to "the vector" is a reference to one or more
vectors and equivalents thereof known to those skilled in the art,
and so forth.
[0039] "Optional" or "optionally" means that the subsequently
described event, circumstance, or material may or may not occur or
be present, and that the description includes instances where the
event, circumstance, or material occurs or is present and instances
where it does not occur or is not present.
[0040] Ranges may be expressed herein as from "about" one
particular value, and/or to "about" another particular value. When
such a range is expressed, also specifically contemplated and
considered disclosed is the range from the one particular value
and/or to the other particular value unless the context
specifically indicates otherwise. Similarly, when values are
expressed as approximations, by use of the antecedent "about," it
will be understood that the particular value forms another,
specifically contemplated embodiment that should be considered
disclosed unless the context specifically indicates otherwise. It
will be further understood that the endpoints of each of the ranges
are significant both in relation to the other endpoint, and
independently of the other endpoint unless the context specifically
indicates otherwise. Finally, it should be understood that all of
the individual values and sub-ranges of values contained within an
explicitly disclosed range are also specifically contemplated and
should be considered disclosed unless the context specifically
indicates otherwise. The foregoing applies regardless of whether in
particular cases some or all of these embodiments are explicitly
disclosed.
[0041] Unless defined otherwise, all technical and scientific terms
used herein have the same meanings as commonly understood by one of
skill in the art to which the disclosed method and compositions
belong. Although any methods and materials similar or equivalent to
those described herein can be used in the practice or testing of
the present method and compositions, the particularly useful
methods, devices, and materials are as described. Publications
cited herein and the material for which they are cited are hereby
specifically incorporated by reference. Nothing herein is to be
construed as an admission that the present invention is not
entitled to antedate such disclosure by virtue of prior invention.
No admission is made that any reference constitutes prior art. The
discussion of references states what their authors assert, and
applicants reserve the right to challenge the accuracy and
pertinency of the cited documents. It will be clearly understood
that, although a number of publications are referred to herein,
such reference does not constitute an admission that any of these
documents forms part of the common general knowledge in the
art.
[0042] Throughout the description and claims of this specification,
the word "comprise" and variations of the word, such as
"comprising" and "comprises," means "including but not limited to,"
and is not intended to exclude, for example, other additives,
components, integers or steps.
[0043] Those skilled in the art will recognize, or be able to
ascertain using no more than routine experimentation, many
equivalents to the specific embodiments of the method and
compositions described herein. Such equivalents are intended to be
encompassed by the following claims.
[0044] Provided herein are new recombinant adeno-associated viruses
(AAVs) designated AAV-X1, AAV-X1b, AAV-X5, AAV-X19, AAV-X21,
AAV-X22, AAV-X23, AAV-X24, AAV-X25, and AAV-X26. The term AAVX is
used herein to refer generally to the new AAVs disclosed herein.
Thus, AAVX can refer to one or more or all of AAV-X1, AAV-X1b,
AAV-X5, AAV-X19, AAV-X21, AAV-X22, AAV-X23, AAV-X24, AAV-X25, or
AAV-X26. The application provides the isolation, subcloning, and
sequencing of the disclosed AAVXs. These viruses have one or more
of the characteristics described below. In one embodiment, the
compositions provided herein do not include wild-type AAV. The
methods provided herein can use either wild-type AAV or recombinant
AAV-based delivery. Thus, in one embodiment, the methods provided
herein do not use wild-type AAV.
[0045] Provided herein are recombinant AAV-X1, AAV-X1b, AAV-X5,
AAV-X19, AAV-X21, AAV-X22, AAV-X23, AAV-X24, AAV-X25, and AAV-X26
particles, recombinant AAV-X1, AAV-X1b, AAV-X5, AAV-X19, AAV-X21,
AAV-X22, AAV-X23, AAV-X24, AAV-X25, and AAV-X26 vectors and
recombinant AAV-X1, AAV-X1b, AAV-X5, AAV-X19, AAV-X21, AAV-X22,
AAV-X23, AAV-X24, AAV-X25, and AAV-X26 virions. As used herein,
"recombinant" refers to nucleic acids, vectors, polypeptides, or
proteins that have been generated using DNA recombination (cloning)
methods and are distinguishable from native or wild-type nucleic
acids, vectors, polypeptides, or proteins. An AAVX particle is a
viral particle comprising an AAVX capsid protein. A recombinant
AAVX vector is a nucleic acid construct that comprises at least one
unique, isolated nucleic acid of AAVX. The recombinant AAVX vector
can further comprise at least one non-AAVX nucleic acid. As used
herein, a "virion" refers to an infectious virus particle, and
"infectious" refers to the ability of a virion to deliver genetic
material to a cell. Thus, a recombinant AAVX virion is a particle
containing a recombinant AAVX vector, wherein the particle can be
either an AAVX particle as described herein or a non-AAVX particle.
Alternatively, a recombinant AAVX virion can be an AAVX particle
containing a recombinant vector, wherein the vector can be either
an AAVX vector as described herein or a non-AAVX vector. An AAVX
particle can further be an "empty particle", wherein the particle
does not contain a nucleic acid, vector or plasmid, and is
therefore not infectious. These vectors, particles, virions,
nucleic acids and polypeptides are described below.
[0046] Provided herein are nucleotide sequences of AAVX genomes and
vectors and particles derived therefrom. Specifically provided is
an AAVX nucleic acid vector. Thus, provided is a nucleic acid
vector, comprising an AAVX-specific nucleic acid or a nucleic acid
encoding an AAVX-specific protein. The AAVX-specific nucleic acid
can be a pair of AAVX inverted terminal repeats (ITRs) or an AAVX
promoter. The nucleic acid encoding an AAVX-specific protein can be
an AAVX capsid protein or an AAVX Rep protein. Thus, the provided
AAVX nucleic acid vector need only have an AAVX ITR, an AAVX
promoter, an AAVX Rep or an AAVX capsid to be an AAVX nucleic acid
vector.
[0047] The AAV ITR functions as an origin of replication for
packaging of recombinant AAV particles. The minimum sequence
necessary for this activity is the TRS site where Rep cleaves in
order to replicate the virus. Minor modifications in an ITR are
contemplated and are those that will not interfere with the hairpin
structure formed by the ITR as described herein and known in the
art. Furthermore, to be considered within the term it must retain
the Rep binding site described herein. For example, the D- region
of the AAV2 ITR, a single stranded region of the ITR (the D+ region
is the reverse complement of the D- region), inboard of the TRS
site, has been shown to bind a factor, which, depending on its
phosphorylation state, correlates with the conversion of the AAV2
from a single stranded genome to a transcriptionally active form
that allows for expression of the viral DNA. For example, this
region is conserved between AAV2, AAV3, AAV4, and AAV6 but is
divergent in AAV5 and BAAV. Further, as disclosed herein, the TRS
signal (e.g., aa 176-181 of SEQ ID NO:40) and Rep Binding site
(e.g., aa 195-210 of SEQ ID NO:40) is conserved between AAV2 and
AAV-X26.
[0048] In one aspect, the AAVX nucleic acid vector comprises an
AAVX ITR. Thus, the AAVX nucleic acid vector can comprise an
AAV-X1, AAV-X1b, AAV-X5, AAV-X19, AAV-X21, AAV-X22, AAV-X23,
AAV-X24, AAV-X25, or AAV-X26 ITR. In another aspect, the AAVX
nucleic acid vector comprises an ITR from any AAV serotype. Thus,
the AAVX nucleic acid vector can comprise an AAV1, AAV2, AAV3,
AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAAV, or BAAV
ITR.
[0049] In one aspect, the AAVX nucleic acid vector comprises an
AAVX promoter. Thus, the promoter can be an AAVX p5, p19 or p40
promoter. Thus, the promoter can be an AAV-X1 p5 promoter, an
AAV-X1b p5 promoter, an AAV-X5 p5 promoter, an AAV-X19 p5 promoter,
n AAV-X21 p5 promoter, an AAV-X22 p5 promoter, an AAV-X23 p5
promoter, an AAV-X24 p5 promoter, an AAV-X25 p5 promoter, or an
AAV-X26 p5 promoter.
[0050] In another aspect, the promoter can be a promoter from any
of the AAV serotypes. Thus, the promoter can be an AAV1 p5
promoter, an AAV2 p5 promoter, an AAV3 p5 promoter, an AAV4 p5
promoter, AAV5 p5 promoter, an AAV6 p5 promoter, an AAAV p5
promoter, a BAAV p5 promoter, or an AAVX p5 promoter.
[0051] Furthermore, smaller fragments of an AAV p5 promoter that
retain promoter activity can readily be determined by standard
procedures including, for example, constructing a series of
deletions in the p5 promoter, linking the deletion to a reporter
gene, and determining whether the reporter gene is expressed, i.e.,
transcribed and/or translated.
[0052] In yet another aspect, the promoter of the AAVX nucleic acid
vector can be any desired promoter, selected by known
considerations, such as the level of expression of a nucleic acid
functionally linked to the promoter and the cell type in which the
vector is to be used. That is, the promoter can be
tissue/cell-specific. Promoters can be prokaryotic, eukaryotic,
fungal, nuclear, mitochondrial, viral or plant promoters. Promoters
can be exogenous or endogenous to the cell type being transduced by
the vector. Promoters can include, for example, bacterial
promoters, or known strong promoters such as SV40 or the inducible
metallothionein promoter. Additionally, chimeric regulatory
promoters for targeted gene expression can be utilized. Examples of
these regulatory systems, which are known in the art, include the
tetracycline based regulatory system which utilizes the tet
transactivator protein (tTA), a chimeric protein containing the
VP16 activation domain fused to the tet repressor of Escherichia
coli, the IPTG based regulatory system, the CID based regulatory
system, and the Ecdysone based regulatory system (No, D., et al.,
Proc Natl Acad Sci USA. 93(8):3346-3351 (1996)). Other promoters
include promoters derived from actin genes, immunoglobulin genes,
cytomegalovirus (CMV), adenovirus, bovine papilloma virus,
adenoviral promoters, such as the adenoviral major late promoter,
an inducible heat shock promoter, respiratory syncytial virus, Rous
sarcomas virus (RSV), etc.
[0053] In one aspect, the AAVX nucleic acid vector comprises a
nucleic acid encoding an AAVX Rep protein. Thus, the Rep protein
can be an AAV-X1 Rep protein, an AAV-X1b Rep protein, an AAV-X5 Rep
protein, an AAV-X19 Rep protein, n AAV-X21 Rep protein, an AAV-X22
Rep protein, an AAV-X23 Rep protein, an AAV-X24 Rep protein, an
AAV-X25 Rep protein, or an AAV-X26 Rep protein. In another aspect,
the AAVX nucleic acid vector comprises a nucleic acid encoding a
Rep protein from any AAV serotype. Thus, the AAVX nucleic acid
vector can comprise a nucleic acid encoding an AAV1 Rep protein, an
AAV2 Rep protein, an AAV3 Rep protein, an AAV4 Rep protein, an AAV5
Rep protein, an AAV6 Rep protein, an AAV7 Rep protein, an AAV8 Rep
protein, an AAV9 Rep protein, an AAV 10 Rep protein, an AAV 11 Rep
protein, an AAAV Rep protein, or an BAAV Rep protein. For all AAV
serotypes, the AAV Rep proteins can be selected from a group
consisting of Rep78, Rep68, Rep52 and Rep40.
[0054] The AAV-X1 Rep protein of an AAV-X1 nucleic acid vector can
be encoded by a nucleic acid sequence comprising nucleotides 1-743,
744, 745, 746, 747, 748, 749, 750, 751, 752, 752, 754, 755, 756,
757, 758, 759, 760, 761, 762, or 763 of SEQ ID NO:1. The AAV-X1 Rep
protein can be encoded by a nucleic acid sequence of SEQ ID NO:48.
The AAV-X1 Rep protein can comprise the amino acid sequence of SEQ
ID NO:49. The AAV-X1b Rep protein of an AAV-X1b nucleic acid vector
can be encoded by a nucleic acid sequence comprising nucleotides
1-2016, 2017, 2018, 2019; 2020, 2021, 2022, 23 2024, 2025, 2026,
2027, 2028, 2029, 2030, 2031, 2032, 2033, 2034, 2035, or 2036 of
SEQ ID NO:2. The AAV-X5 Rep protein of an AAV-X5 nucleic acid
vector can be encoded by a nucleic acid sequence comprising
nucleotides 1-1926, 1927, 1928, 1929, 1930, 1931, 1932, 1933, 1934,
1935, 1936, 1937, 1938, 1939, 1940, 1941, 1942, 1943, 1944, 1945,
or 1946 of SEQ ID NO3. The AAV-X19 Rep protein of an AAV-X19
nucleic acid vector can be encoded by a nucleic acid sequence
comprising nucleotides 1-743, 744, 745, 746, 747, 748, 749, 750,
751, 752, 752, 754, 755, 756, 757, 758, 759, 760, 761, 762, or 763
of SEQ ID NO:4. The AAV-X21 Rep protein of an AAV-X21 nucleic acid
vector can be encoded by a nucleic acid sequence comprising
nucleotides 1-752, 753, 754, 755, 756, 757, 758, 759. 760, 761,
762, 763, 764, 765, 766, 767, 768, 769, 770, 771, or 772 of SEQ ID
NO:5. The AAV-X22 Rep protein of an AAV-X22 nucleic acid vector can
be encoded by a nucleic acid sequence comprising nucleotides 1-748,
749, 750, 751, 752, 753, 754, 755, 756, 757, 758, 759. 760, 761,
762, 763, 764, 765, 766, 767, or 768 of SEQ ID NO:6. The AAV-X23
Rep protein of an AAV-X23 nucleic acid vector can be encoded by a
nucleic acid sequence comprising nucleotides 1-746, 747, 748, 749,
750, 751, 752, 753, 754, 755, 756, 757, 758, 759. 760, 761, 762,
763, 764, 765, or 766 of SEQ ID NOS:7. The AAV-X24 Rep protein of
an AAV-X24 nucleic acid vector can be encoded by a nucleic acid
sequence comprising nucleotides 1-752, 753, 754, 755, 756, 757,
758, 759. 760, 761, 762, 763, 764, 765, 766, 767, 768, 769, 770,
771, or 772 of SEQ ID NO:8. The AAV-X25 Rep protein of an AAV-X25
nucleic acid vector can be encoded by the a nucleic acid sequence
comprising nucleotides 1-974, 975, 976, 977, 978, 979, 980, 981,
982, 983, 984, 985, 986, 987, 988, 989, 990, 991, 992, 993, or 994
of SEQ ID NO:9. The AAV-X25 Rep protein can be encoded by a nucleic
acid sequence of SEQ ID NO:50. The AAV-X25 Rep protein can comprise
the amino acid sequence of SEQ ID NO:51. The AAV-X26 Rep protein of
an AAV-X26 nucleic acid vector can be encoded by the a nucleic acid
sequence comprising nucleotides 1-789, 790, 791, 792, 793, 794,
795, 796, 797, 798, 799, 800, 801, 802, 803, 804, 805, 806, 807,
808, or 809 of SEQ ID NO:10. The AAV-X26 Rep protein can be encoded
by a nucleic acid sequence of SEQ ID NO:52. The AAV-X26 Rep protein
can comprise the amino acid sequence of SEQ ID NO:53.
[0055] hi one aspect, the AAVX nucleic acid vector comprises a
nucleic acid encoding an AAVX capsid protein. Thus, the capsid
protein can be an AAV-X1 capsid protein, an AAV-Xlb capsid protein,
an AAV-X5 capsid protein, an AAV-X19 capsid protein, n AAV-X21
capsid protein, an AAV-X22 capsid protein, an AAV-X23 capsid
protein, an AAV-X24 capsid protein, an AAV-X25 capsid protein, or
an AAV-X26 capsid protein. In another aspect, the AAVX nucleic acid
vector comprises a nucleic acid encoding a capsid protein from any
AAV serotype. Thus, the AAVX nucleic acid vector can comprise a
nucleic acid encoding an AAV 1 capsid protein, an AAV2 capsid
protein, an AAV3 capsid protein, an AAV4 capsid protein, an AAV5
capsid protein, an AAV6 capsid protein, an AAV7 capsid protein, an
AAV8 capsid protein, an AAV9 capsid protein, an AAV10 capsid
protein, an AAV 11 capsid protein, an AAAV capsid protein, or an
BAAV capsid protein. For all AAV serotypes, the AAV capsid proteins
can be selected from a group consisting of VP1, VP2 and VP3.
[0056] As an example, the AAV-X1 VP1 capsid protein of an AAV-X1
particle can have the amino acid sequence of SEQ ID NO:21. The
AAV-X1b VP1 capsid protein of an AAV-X1b particle can have the
amino acid sequence of SEQ ID NOS:22. The AAV-X5 VP1 capsid protein
of an AAV-X5 particle can have the amino acid sequence of SEQ ID
NO:23. The AAV-X19 VP1 capsid protein of an AAV-X19 particle can
have the amino acid sequence of SEQ ID NO:24. The AAV-X21 VP1
capsid protein of an AAV-X21 particle can have the amino acid
sequence of SEQ ID NO:25. The AAV-X22 VP1 capsid protein of an
AAV-X22 particle can have the amino acid sequence of SEQ ID NO:26.
The AAV-X23 VP1 capsid protein of an AAV-X23 particle can have the
amino acid sequence of SEQ ID NO:27. The AAV-X24 VP1 capsid protein
of an AAV-X24 particle can have the amino acid sequence of SEQ ID
NO:28. The AAV-X25 capsid protein of an AAV-X25 particle can have
the amino acid sequence of SEQ ID NO:29. The AAV-X26 capsid protein
of an AAV-X26 particle can have the amino acid sequence of SEQ ID
NO:30.
[0057] The AAV-X1 VP1 capsid protein of an AAV-X1 particle can be
encoded by the nucleic acid sequence of SEQ ID NO:11. The AAV-X1b
VP1 capsid protein of an AAV-X1b particle can be encoded by the
nucleic acid sequence of SEQ ID NO:12. The AAV-X5 VP1 capsid
protein of an AAV-X5 particle can be encoded by the nucleic acid
sequence of SEQ ID NO:13. The AAV-X19 VP1 capsid protein of an
AAV-X19 particle can be encoded by the nucleic acid sequence of SEQ
ID NO:14. The AAV-X21 VP1 capsid protein of an AAV-X21 particle can
be encoded by the nucleic acid sequence of SEQ ID NO:15. The
AAV-X22 VP1 capsid protein of an AAV-X22 particle can be encoded by
the nucleic acid sequence of SEQ ID NO:16. The AAV-X23 VP1 capsid
protein of an AAV-X23 particle can be encoded by the nucleic acid
sequence of SEQ ID NOS:17. The AAV-X24 VP1 capsid protein of an
AAV-X24 particle can be encoded by the nucleic acid sequence of SEQ
ID NO:18. The AAV-X25 VP1 capsid protein of an AAV-X25 particle can
be encoded by the nucleic acid sequence of SEQ ID NO:19. The
AAV-X26 VP1 capsid protein of an AAV-X26 particle can be encoded by
the nucleic acid sequence of SEQ ID NO:20.
[0058] It should be recognized that any errors in any of the
nucleotide sequences disclosed herein can be corrected, for
example, by using the hybridization procedure described below with
various probes derived from the described sequences such that the
coding sequence can be re-isolated and re-sequenced. Rapid
screening for point mutations can also be achieved with the use of
polymerase chain reaction-single strand conformation polymorphism
(PCR-SSCP). The corresponding amino acid sequence can then be
corrected accordingly. Also, since the disclosed AAV serotypes
AAV-X1, AAV-X1b, AAV-X5, AAV-X19, AAV-X21, AAV-X22, AAV-X23,
AAV-X24, AAV-X25, and AAV-X26 are disclosed herein to be present in
defined ATCC cultures, the whole virus sequence is provided by
reference to the deposit.
[0059] The AAVX-derived vector provided herein can further comprise
an exogenous nucleic acid functionally linked to the promoter. By
"exogenous" nucleic acid is meant any nucleic acid that is not
normally found in wild-type AAVX that can be inserted into a vector
for transfer into a cell, tissue or organism. The exogenous nucleic
acid can be a nucleic acid not normally found in the target cell,
or it can be an extra copy or copies of a nucleic acid normally
found in the target cell. The terms "exogenous" and "heterologous"
are used herein interchangeably.
[0060] By "functionally linked" is meant that the promoter can
promote expression of the exogenous nucleic acid, as is known in
the art, and can include the appropriate orientation of the
promoter relative to the exogenous nucleic acid. Furthermore, the
exogenous nucleic acid preferably has all appropriate sequences for
expression of the nucleic acid. The nucleic acid can include, for
example, expression control sequences, such as an enhancer, and
necessary information processing sites, such as ribosome binding
sites, RNA splice sites, polyadenylation sites, and transcriptional
terminator sequences.
[0061] The exogenous nucleic acid can encode beneficial proteins or
polypeptides that replace missing or defective proteins required by
the cell or subject into which the vector is transferred or can
encode a cytotoxic polypeptide that can be directed, e.g., to
cancer cells or other cells whose death would be beneficial to the
subject. The exogenous nucleic acid can also encode antisense RNAs
that can bind to, and thereby inactivate, mRNAs made by the subject
that encode harmful proteins. The exogenous nucleic acid can also
encode ribozymes that can effect the sequence-specific inhibition
of gene expression by the cleavage of mRNAs. In one aspect,
antisense polynucleotides can be produced from an exogenous
expression cassette in an AAV5 vector construct where the
expression cassette contains a sequence that promotes cell-type
specific expression (Wirak et al., EMBO 10:289 (1991)). For general
methods relating to antisense polynucleotides, see Antisense RNA
and DNA, D. A. Melton, Ed., Cold Spring Harbor Laboratory, Cold
Spring Harbor, N.Y. (1988).
[0062] Examples of exogenous nucleic acids which can be
administered to a cell or subject as part of the present AAVX
vector can include, but are not limited to the following: nucleic
acids encoding secretory and nonsecretory proteins, nucleic acids
encoding therapeutic agents, such as tumor necrosis factors (TNF),
such as TNF-.alpha.; interferons, such as interferon-.alpha.,
interferon-.beta., and interferon-.gamma., interleukins, such as
IL-1, IL-1.beta., and ILs-2 through -14; GM-CSF; adenosine
deaminase; cellular growth factors, such as lymphokines; soluble
CD4; Factor VIII; Factor IX; T-cell receptors; LDL receptor; ApoE;
ApoC; alpha-1 antitrypsin; ornithine transcarbamylase (OTC); cystic
fibrosis transmembrane receptor (CFTR); insulin; Fc receptors for
antigen binding domains of antibodies, such as immunoglobulins;
anti-HIV decoy tar elements; and antisense sequences which inhibit
viral replication, such as antisense sequences which inhibit
replication of hepatitis B or hepatitis non-A, non-B virus. The
nucleic acid is chosen considering several factors, including the
cell to be transfected. Where the target cell is a blood cell, for
example, particularly useful nucleic acids to use are those which
allow the blood cells to exert a therapeutic effect, such as a gene
encoding a clotting factor for use in treatment of hemophilia.
Another target cell is the lung airway cell, which can be used to
administer nucleic acids, such as those coding for the cystic
fibrosis transmembrane receptor, which could provide a gene
therapeutic treatment for cystic fibrosis. Other target cells
include muscle cells where useful nucleic acids, such as those
encoding cytokines and growth factors, can be transduced and the
protein the nucleic acid encodes can be expressed and secreted to
exert its effects on other cells, tissues and organs, such as the
liver. Furthermore, the nucleic acid can encode more than one gene
product, limited only, if the nucleic acid is to be packaged in a
capsid, by the size of nucleic acid that can be packaged.
[0063] Furthermore, suitable nucleic acids can include those that,
when transferred into a primary cell, such as a blood cell, cause
the transferred cell to target a site in the body where that cell's
presence would be beneficial. For example, blood cells such as TIL
cells can be modified, such as by transfer into the cell of a Fab
portion of a monoclonal antibody, to recognize a selected antigen.
Another example would be to introduce a nucleic acid that would
target a therapeutic blood cell to tumor cells. Nucleic acids
useful in treating cancer cells include those encoding chemotactic
factors which cause an inflammatory response at a specific site,
thereby having a therapeutic effect.
[0064] Cells, particularly blood cells, muscle cells, airway
epithelial cells, brain cells and endothelial cells having such
nucleic acids transferred into them can be useful in a variety of
diseases, syndromes and conditions. For example, suitable nucleic
acids include nucleic acids encoding soluble CD4, used in the
treatment of AIDS and .alpha.-antitrypsin, used in the treatment of
emphysema caused by .alpha.-antitrypsin deficiency. Other diseases,
syndromes and conditions in which such cells can be useful include,
for example, adenosine deaminase deficiency, sickle cell
deficiency, brain disorders such as Alzheimer's disease,
thalassemia, hemophilia, diabetes, phenylketonuria, growth
disorders and heart diseases, such as those caused by alterations
in cholesterol metabolism, and defects of the immune system.
[0065] Other cells in which a gene of interest can be expressed
include, but are not limited to, fibroblasts, neurons, retinal
cells, kidney cells, lung cells, bone marrow stem cells,
hematopoietic stem cells, retinal cells and neurons. The cells in
which the gene of interest can be expressed can be dividing cells
such as MDCK cells, BHI cells, HeLa cells, 3T3 cells, CV1 cells,
COS7 cells, HOS cells and 293 cells. The cells can also be
embryonic stem cells of mouse, rhesus, human, bovine or sheep
origin, as well as stem cells of neural, hematopoietic, muscle,
cardiac, immune or other origin. Non-dividing cells can also be
contacted with a particle provided herein to express a gene of
interest. Such cells include, but are not limited to hematopoietic
stem cells and embryonic stem cells that have been rendered
non-dividing.
[0066] As another example, hepatocytes can be transfected with the
present vectors having useful nucleic acids to treat liver disease.
For example, a nucleic acid encoding OTC can be used to transfect
hepatocytes (ex vivo and returned to the liver or in vivo) to treat
congenital hyperammonemia, caused by an inherited deficiency in
OTC. Another example is to use a nucleic acid encoding LDL to
target hepatocytes ex vivo or in vivo to treat inherited LDL
receptor deficiency. Such transfected hepatocytes can also be used
to treat acquired infectious diseases, such as diseases resulting
from a viral infection. For example, transduced hepatocyte
precursors can be used to treat viral hepatitis, such as hepatitis
B and non-A, non-B hepatitis, for example by transducing the
hepatocyte precursor with a nucleic acid encoding an antisense RNA
that inhibits viral replication. Another example includes
transferring a vector provided herein having a nucleic acid
encoding a protein, such as .gamma.-interferon, which can confer
resistance to the hepatitis virus.
[0067] For a procedure using transfected hepatocytes or hepatocyte
precursors, hepatocyte precursors having a vector provided herein
transferred in can be grown in tissue culture, removed from the
tissue culture vessel, and introduced to the body, such as by a
surgical method. In this example, the tissue would be placed
directly into the liver, or into the body cavity in proximity to
the liver, as in a transplant or graft. Alternatively, the cells
can simply be directly injected into the liver, into the portal
circulatory system, or into the spleen, from which the cells can be
transported to the liver via the circulatory system. Furthermore,
the cells can be attached to a support, such as microcarrier beads,
which can then be introduced, such as by injection, into the
peritoneal cavity. Once the cells are in the liver, by whatever
means, the cells can then express the nucleic acid and/or
differentiate into mature hepatocytes which can express the nucleic
acid.
[0068] The provided viral particles can be administered to cells,
as described herein, with a Multiplicity of Infection (MOI) of 10.
The MOI is the ratio of infectious virus particles to the number of
cells being infected. Thus, an MOI of 0.1 results in the average
inoculation of 1 virus particle for every 10 cells. The general
theory behind MOI is to introduce one infectious virus particle to
every host cell that is present in the culture. However, more than
one virus may infect the same cell which leaves a percentage of
cells uninfected. This occurrence can be reduced by using a higher
MOI to ensure that every cell is infected. The provided viral
particles can therefore be administered to cells, as described
herein, with a MOI of 0.01 to 100, such as for example 1, 2, 3, 4,
5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40,
50, 60, 70, 80, 90, 100.
[0069] The AAVX-derived vector can include any normally occurring
AAVX nucleic acid sequences. The AAVX-derived vector can also
include sequences that are at least 80%, 81%, 82%, 83%, 84%, 85%,
86%, 87%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 98.5%,
99%, 99.5% or 99.9% identical to the AAVX nucleic acids set forth
herein. Examples of vector constructs are provided below.
[0070] The present AAVX vector or AAVX particle or recombinant AAVX
virion can utilize any unique nucleic acid fragment of the AAVX
disclosed herein, including the AAVX nucleic acids set forth in SEQ
ID NOS:1-20. A unique fragment consists of a sequence that is not
present anywhere else on a genome. A fragment is a subpart of the
reference sequence, and thus is identical in sequence to the region
of the parent nucleic acid of which it is a fragment. To be unique,
the fragment must be of sufficient size to distinguish it from
other known sequences, which is most readily determined by
comparing any nucleic acid fragment to the nucleotide sequences of
nucleic acids in computer databases, such as GenBank. Such
comparative searches are standard in the art. Typically, a unique
fragment useful as a primer or probe will be at least about 8 or
10, preferable at least 20 or 25 nucleotides in length, depending
upon the specific nucleotide content of the sequence. Additionally,
fragments can be, for example, at least about 30, 40, 50, 75, 100,
200 or 500 nucleotides in length and can encode polypeptides or be
probes. The nucleic acid can be single or double stranded,
depending upon the purpose for which it is intended. Where desired,
the nucleic acid can be RNA.
[0071] It is understood that as discussed herein the use of the
terms "homology" and "identity" mean the same thing as similarity.
Thus, for example, if the use of the word homology is used to refer
to two non-natural sequences, it is understood that this is not
necessarily indicating an evolutionary relationship between these
two sequences, but rather is looking at the similarity or
relatedness between their nucleic acid sequences. Many of the
methods for determining homology between two evolutionarily related
molecules are routinely applied to any two or more nucleic acids or
proteins for the purpose of measuring sequence similarity
regardless of whether they are evolutionarily related.
[0072] In general, it is understood that one way to define any
known variants and derivatives or those that might arise, of the
disclosed nucleic acids and polypeptides herein, is through
defining the variants and derivatives in terms of homology to
specific known sequences. In general, variants of nucleic acids and
polypeptides herein disclosed typically have at least, about 80%,
81%, 82%, 83%, 84%, 85%, 86%, 87%, 89%, 90%, 91%, 92%, 93%, 94%,
95%, 96%, 97%, 98%, 98.5%, 99%, 99.5% or 99.9% homology to the
stated sequence or the native sequence. Those of skill in the art
readily understand how to determine the homology of two
polypeptides or nucleic acids. For example, the homology can be
calculated after aligning the two sequences so that the homology is
at its highest level.
[0073] Another way of calculating homology can be performed by
published algorithms. Optimal alignment of sequences for comparison
may be conducted by the local homology algorithm of Smith and
Waterman Adv. Appl. Math. 2: 482 (1981), by the homology alignment
algorithm of Needleman and Wunsch, J. Mol. Biol. 48: 443 (1970), by
the search for similarity method of Pearson and Lipman, Proc. Natl.
Acad. Sci. U.S.A. 85: 2444 (1988), by computerized implementations
of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the
Wisconsin Genetics Software Package, Genetics Computer Group, 575
Science Dr., Madison, Wis.; the BLAST algorithm of Tatusova and
Madden FEMS Microbiol. Lett. 174: 247-250 (1999) available from the
National Center for Biotechnology Information
(http://www.ncbi.nlm.nih.gov/blast/b12seq/b12.html), or by
inspection.
[0074] The same types of homology can be obtained for nucleic acids
by for example the algorithms disclosed in Zuker, M. Science
244:48-52, 1989, Jaeger et al. Proc. Natl. Acad. Sci. USA
86:7706-7710, 1989, Jaeger et al. Methods Enzymol. 183:281-306,
1989, which are herein incorporated by reference for at least
material related to nucleic acid alignment. It is understood that
any of the methods typically can be used and that in certain
instances the results of these various methods may differ, but the
skilled artisan understands if identity is found with at least one
of these methods, the sequences would be said to have the stated
identity.
[0075] For example, as used herein, a sequence recited as having a
particular percent homology to another sequence refers to sequences
that have the recited homology as calculated by any one or more of
the calculation methods described above. For example, a first
sequence has 80 percent homology, as defined herein, to a second
sequence if the first sequence is calculated to have 80 percent
homology to the second sequence using the Zuker calculation method
even if the first sequence does not have 80 percent homology to the
second sequence as calculated by any of the other calculation
methods. As another example, a first sequence has 80 percent
homology, as defined herein, to a second sequence if the first
sequence is calculated to have 80 percent homology to the second
sequence using both the Zuker calculation method and the Pearson
and Lipman calculation method even if the first sequence does not
have 80 percent homology to the second sequence as calculated by
the Smith and Waterman calculation method, the Needleman and Wunsch
calculation method, the Jaeger calculation methods, or any of the
other calculation methods. As yet another example, a first sequence
has 80 percent homology, as defined herein, to a second sequence if
the first sequence is calculated to have 80 percent homology to the
second sequence using each of calculation methods (although, in
practice, the different calculation methods will often result in
different calculated homology percentages).
[0076] Further provided herein is an AAVX capsid protein that can
combine with other capsid proteins to form an AAVX particle to
contain the disclosed vectors. Also provided herein is an AAVX
particle, comprising an AAVX capsid protein. The capsid protein can
be selected from a group consisting of VP1, VP2 and VP3.
[0077] The AAV-X1 VP1 capsid protein of an AAV-X1 particle can have
the amino acid sequence of SEQ ID NO:21. The AAV-X1b VP1 capsid
protein of an AAV-X1b particle can have the amino acid sequence of
SEQ ID NO:22. The AAV-X5 VP1 capsid protein of an AAV-X5 particle
can have the amino acid sequence of SEQ ID NO:23. The AAV-X19 VP1
capsid protein of an AAV-X19 particle can have the amino acid
sequence of SEQ ID NO:24. The AAV-X21 VP1 capsid protein of an
AAV-X21 particle can have the amino acid sequence of SEQ ID NO:25.
The AAV-X22 VP1 capsid protein of an AAV-X22 particle can have the
amino acid sequence of SEQ ID NO:26. The AAV-X23 VP1 capsid protein
of an AAV-X23 particle can have the amino acid sequence of SEQ ID
NO:27. The AAV-X24 VP1 capsid protein of an AAV-X24 particle can
have the amino acid sequence of SEQ ID NO:28. The AAV-X25 capsid
protein of an AAV-X25 particle can have the amino acid sequence of
SEQ ID NO:29. The AAV-X26 capsid protein of an AAV-X26 particle can
have the amino acid sequence of SEQ ID NO:30.
[0078] The AAV-X1 VP1 capsid protein of an AAV-X1 particle can be
encoded by the nucleic acid sequence of SEQ ID NO:11. The AAV-X1b
VP1 capsid protein of an AAV-X1b particle can be encoded by the
nucleic acid sequence of SEQ ID NO:12. The AAV-X5 VP1 capsid
protein of an AAV-X5 particle can be encoded by the nucleic acid
sequence of SEQ ID NO:13. The AAV-X19 VP1 capsid protein of an
AAV-X19 particle can be encoded by the nucleic acid sequence of SEQ
ID NO:14. The AAV-X21 VP1 capsid protein of an AAV-X21 particle can
be encoded by the nucleic acid sequence of SEQ ID NO:15. The
AAV-X22 VP1 capsid protein of an AAV-X22 particle can be encoded by
the nucleic acid sequence of SEQ ID NO:16. The AAV-X23 VP1 capsid
protein of an AAV-X23 particle can be encoded by the nucleic acid
sequence of SEQ ID NO:17. The AAV-X24 VP1 capsid protein of an
AAV-X24 particle can be encoded by the nucleic acid sequence of SEQ
ID NO:18. The AAV-X25 VP1 capsid protein of an AAV-X25 particle can
be encoded by the nucleic acid sequence of SEQ ID NO:19. The
AAV-X26 VP1 capsid protein of an AAV-X26 particle can be encoded by
the nucleic acid sequence of SEQ ID NO:20.
[0079] For example, provided is an AAVX particle, comprising all
three AAVX capsid proteins, i.e., VP1, VP2 and VP3. Also provided
is an AAVX particle, comprising each AAVX capsid protein
individually or in combination. Also provided is an AAVX particle
comprising VP1 and VP3 capsid proteins, i.e., lacking any VP2
capsid proteins. Thus, an AAVX particle comprising an AAVX capsid
protein comprises at least one AAVX capsid protein (VP1, VP2 or
VP3) or a functional fragment thereof. One of skill in the art
understands that it is the non-conserved amino acids that are
contributing to the properties of AAVX that make it distinct from
the other serotypes. Provided therefore is a capsid protein
comprising a mutation, deletion or substitution in the conserved
regions, including, for example, a substitution with a homologous
region from another AAV serotype.
[0080] An AAVX particle comprising an AAVX capsid protein can be
utilized to deliver a nucleic acid vector to a cell, tissue or
subject. For example, the herein described AAVX vectors can be
encapsidated in an AAVX capsid-derived particle and utilized in a
gene delivery method. Furthermore, other viral nucleic acids can be
encapsidated in the AAVX particle and utilized in such delivery
methods. For example, an AAV1-11, AAAV, or BAAV vector (e.g.
AAV1-11, AAAV, BAAV or AAVX ITR and nucleic acid of interest) can
be encapsidated in an AAVX particle and administered. Furthermore,
an AAVX chimeric capsid incorporating AAV1-11, AAAV, BAAV or AAVX
capsid sequences and a different AAVX capsid sequences can be
generated, by standard cloning methods, selecting regions from the
known sequences of each protein as desired. For example,
particularly antigenic regions of the AAVX capsid protein can be
replaced with the corresponding region of the AAV2 capsid protein.
In addition to chimeric capsids incorporating AAV2 capsid
sequences, chimeric capsids incorporating AAV1, 3-8, AAAV, BAAV or
AAVX capsid sequences can be generated, by standard cloning
methods, selecting regions from the known sequences of each protein
as desired. Alternatively a chimeric capsid can be made by the
addition of a plasmid that expresses AAV 1-11, AAAV, BAAV or AAVX
capsid proteins at a ratio with the AAVX capsid expression plasmid
that allows only a few capsid proteins to be incorporated into the
AAVX particle. Thus, for example, a chimeric particle may be
constructed that contains 6 AAV2 capsid proteins and 54 AAVX capsid
proteins if the complete capsid contains 60 capsid proteins.
Methods for generating chimeric AAVs are known in the art and can
be found in Rabinowitz J E, et al. J. Virol. 2004 May;
78(9):4421-32, herein incorporated by reference for these methods.
Examples of chimeric capsids would be to combine the VP1, 2, 3
proteins of AAVX and the VP1, 2, 3 proteins of AAV5 such that a new
tropism would arise. The capsids can also be modified to alter
their specific tropism by genetically altering the capsid to encode
a specific ligand to a cell surface receptor.
[0081] Alternatively, the capsid can be chemically modified by
conjugating a ligand to a cell surface receptor. By genetically or
chemically altering the capsids, the tropism can be modified to
direct AAVX to a particular cell or population of cells. The
capsids can also be altered immunologically by conjugating the
capsid to an antibody that recognizes a specific protein on the
target cell or population of cells.
[0082] Provided are three regions in the capsid of AAVX that are on
the virus surface and could tolerate substitution. These three
regions in AAV-X1, AAV-X1b, AAV-X19, AAV-X21, AAV-X22, AAV-X23,
AAV-X24, and AAV-X25 are aa 261-271, aa 450-476, and aa 546-559.
These three regions in AAV-X5 are aa 259-268, aa 448-473, and aa
543-554. These three regions in AAV-X26 are aa 260-274, aa 445-477,
and aa 550-565. Thus, provided is an AAVX VP1 capsid, comprising
amino acid substitutions in aa 261-271, aa 450-476, or aa 546-559
of SEQ ID NOS:21, 22, 24, 25, 26, 27, 28, or 29. Thus, also
provided is an AAVX VP1 capsid, comprising amino acid substitutions
in aa 259-268, aa 448-473, and aa 543-554 of SEQ ID NO:23. Thus,
also provided is an AAVX VP1 capsid, comprising amino acid
substitutions in aa 260-274, aa 445-477, and aa 550-565 of SEQ ID
NO:30.
[0083] Other regions of the AAVX capsid could also accommodate the
substitution of amino acids that would allow for epitope
presentation on the surface of the virus. All of these regions
would have surface exposure and the ability to support a
substitution of sequence to insert the epitope while still allowing
for capsid assembly. The substitutions can include non-AAVX
epitopes and non-AAVX ligands.
[0084] Because of the symmetry of the AAV particles, a substitution
in one subunit of the capsid will appear multiple times on the
capsid surface. For example the capsid is made of approximately 50
VP3 proteins, 5 VP1 and 5 VP2. Therefore an epitope incorporated in
the VP3 protein could be expressed 55 times on the surface of each
particle increasing the likelihood of the epitope forming a stable
interaction with its target. In some cases this may be too high of
a ligand density for functional binding or this high density of
epitope may interfere with capsid formation. The epitope density
could be lowered by introducing another plasmid into the packaging
system for production of recombinant particles and the ratio
between the packaging plasmid with the modified VP3 protein and the
wt VP3 protein altered to balance the epitope density on the virus
surface. Thus, the ratio between the modified VP3 and the wt VP3
can be 0:50 to 50:0, including, for example, 1:49, 2:48, 3:47,
4:46, 5:45, 6:44, 7:43, 8:42, 9:41, 10:40, 11:39, 12:38, 13:37,
14:36, 15:35, 16:34, 17:33, 18:32, 19:31, 20:30, 21:29, 22:28,
23:27, 24:27, 25:25, 26:24, 27:23, 28:22, 29:21, 30:20, 31:19,
32:18, 33:17, 34:16, 35:15, 36:14, 37:13, 38:12, 39:11, 40:10,
41:9, 42:8, 43:7, 44:6, 45:5, 46:4, 47:3, 48:2, or 49:1.
[0085] Epitopes could be incorporated into the virus capsid for the
purpose of 1) altering the tropism of the virus 2) blocking an
immune response directed at the virus 3) developing a host immune
response to the epitope for the purpose of vaccination.
Examples of epitopes that could be added to AAVX capsids include
but are not limited to: LH receptor binding epitope RGD integrin
binding epitope CD13 binding epitope NGRAHA SEQ ID NO:35 The
Retanefpolyprotein vaccine candidate for HIV-1 single chain
antibody fragments directed against tumor cells Endothelial cell
binding epitope SIGYPLP SEQ ID NO:36 serpin receptor ligand,
KFNKPFVFLI SEQ ID NO:37 protective B-cell epitope hemagglutinin
(HA) 91-108 from influenza HA NDV B-cell immunodominant epitope
(IDE) spanning residues 447 to 455 Major immunogenic epitope for
parvovirus B19 (NISLDNPLENPSSLFDLVARIK SEQ ID NO:38) that can
elicit protective antibody titers.
[0086] The capsids can also be assembled into empty particles by
expression in mammalian, bacterial, fungal or insect cells. For
example, AAV2 particles are known to be made from VP3 and VP2
capsid proteins in baculovirus. The same basic protocol can produce
an empty AAVX particle comprising AAVX capsid proteins and also
full particles. The empty AAVX particles can be used to deliver,
for example, antigens, drugs, proteins, or metals to cells or cells
in a subject. Antigens can be directly incorporated into the capsid
of an empty AAVX particle. An antigen can further be coupled via an
antibody-antigen complex to the empty particle. Also disclosed is
the coupling of drugs, proteins, or metals on the inside of the
empty particles.
[0087] The herein described recombinant AAVX nucleic acid derived
vector can be encapsidated in a viral particle. The viral particle
can be a parvovirus particle. The parvovirus particle can be a
dependovirus particle. The viral particle can be an AAV particle.
In particular, the recombinant AAVX nucleic acid derived vector can
be encapsidated in an AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7,
AAV8, AAV9, AAV10, AAV11, AAAV, BAAV, AAV-X1, AAV-X1b, AAV-X5,
AAV-X19, AAV-X21, AAV-X22, AAV-X23, AAV-X24, AAV-X25, or AAV-X26
particle, a particle comprising a portion of any of these capsids,
or a chimeric capsid particle as described above, by standard
methods using the appropriate capsid proteins in the encapsidation
process, as long as the nucleic acid vector fits within the size
limitation of the particle utilized. The encapsidation process
itself is standard in the art. The AAVX replication machinery, i.e.
the rep initiator proteins and other functions required for
replication, can be utilized to produce the AAVX genome that can be
packaged in an AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8,
AAV9, AAV10, AAV11, AAAV, BAAV, AAV-X1, AAV-X1b, AAV-X5, AAV-X19,
AAV-X21, AAV-X22, AAV-X23, AAV-X24, AAV-X25, or AAV-X26 capsid.
[0088] The recombinant AAVX virion containing a vector can also be
produced by recombinant methods utilizing multiple plasmids. In one
example, the AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9,
AAV10, AAV11, AAAV, BAAV, AAV-X1, AAV-X1b, AAV-X5, AAV-X19,
AAV-X21, AAV-X22, AAV-X23, AAV-X24, AAV-X25, or AAV-X26 rep nucleic
acid would be cloned into one plasmid, the AAV1, AAV2, AAV3, AAV4,
AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAAV, BAAV, AAV-X1,
AAV-X1b, AAV-X5, AAV-X19, AAV-X21, AAV-X22, AAV-X23, AAV-X24,
AAV-X25, or AAV-X26 ITR nucleic acid would be cloned into another
plasmid and the AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8,
AAV9, AAV10, AAV11, AAAV, BAAV, AAV-X1, AAV-X1b, AAV-X5, AAV-X19,
AAV-X21, AAV-X22, AAV-X23, AAV-X24, AAV-X25, or AAV-X26 capsid
nucleic acid would be cloned on another plasmid. These plasmids
would then be introduced into cells. The cells that were
efficiently transduced by all three plasmids, would exhibit
specific integration as well as the ability to produce AAVX
recombinant virus. Additionally, two plasmids could be used where
the AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10,
AAV11, AAAV, BAAV, AAV-X1, AAV-X1b, AAV-X5, AAV-X19, AAV-X21,
AAV-X22, AAV-X23, AAV-X24, AAV-X25, or AAV-X26 rep nucleic acid
would be cloned into one plasmid and the AAV1, AAV2, AAV3, AAV4,
AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAAV, BAAV, AAV-X1,
AAV-X1b, AAV-X5, AAV-X19, AAV-X21, AAV-X22, AAV-X23, AAV-X24,
AAV-X25, or AAV-X26 ITR and AAV1, AAV2, AAV3, AAV4, AAV5, AAV6,
AAV7, AAV8, AAV9, AAV10, AAV11, AAAV, BAAV, AAV-X1, AAV-X1b,
AAV-X5, AAV-X19, AAV-X21, AAV-X22, AAV-X23, AAV-X24, AAV-X25, or
AAV-X26 capsid would be cloned into another plasmid. These plasmids
would then be introduced into cells. The cells that were
efficiently transduced by both plasmids, would exhibit specific
integration as well as the ability to produce AAVX recombinant
virus.
[0089] An AAV-X1, AAV-X1b, AAV-X19, AAV-X21, AAV-X22, AAV-X23,
AAV-X24, or AAV-X25 capsid can have about 98%, 98.5%, 99%, 99.1%,
99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% homology
to the polypeptide having the amino acid sequence encoded by
nucleotides in SEQ ID NOS:11, 12, 14, 15, 16, 17, 18, or 19,
respectively. An AAV-X5 capsid can have about 93%, 94%, 95%, 96%,
97%, 98%, 98.5%, or 99% homology to the polypeptide having the
amino acid sequence encoded by nucleotides in SEQ ID NO:13. An
AAV-X26 capsid can have about 83%, 84%, 85%, 86%, 87%, 88%, 89%,
90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 98.5%, or 99% homology
to the polypeptide having the amino acid sequence encoded by
nucleotides in SEQ ID NO:20.
[0090] An AAV-X1, AAV-X1b, AAV-X19, AAV-X21, AAV-X22, AAV-X23,
AAV-X24, or AAV-X25 capsid protein can have about 98%, 98.5%, 99%,
99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9%
homology to the protein having the amino acid sequence encoded by
the nucleotides set forth in SEQ ID NOS:21, 22, 24, 25, 26, 27, 28,
or 29, respectively. An AAV-X5 capsid protein can have about 93%,
94%, 95%, 96%, 97%, 98%, 98.5%, or 99% homology to the protein
having the amino acid sequence encoded by the nucleotides set forth
in SEQ ID NO:23. An AAV-X26 capsid protein can have about 83%, 84%,
85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,
98%, 98.5%, or 99% homology to the protein having the amino acid
sequence encoded by the nucleotides set forth in SEQ ID NO:30.
[0091] The percent homology used to identify proteins herein, can
be based on a nucleotide-by-nucleotide comparison or more
preferable is based on a computerized algorithm as described
herein. Variations in the amino acid sequence of the AAVX capsid
protein are contemplated herein, as long as the resulting particle
comprising an AAVX capsid protein remains antigenically or
immunologically distinct from AAV1-11, AAAV, or BAAV capsid, as can
be routinely determined by standard methods. Specifically, for
example, ELISA and Western blots can be used to determine whether a
viral particle is antigenically or immunologically distinct from
AAV2 or the other serotypes. Furthermore, the AAVX particle
preferably retains tissue tropism distinction from other AAVs, such
as that exemplified in the examples herein. An AAVX chimeric
particle comprising at least one AAVX coat protein may have a
different tissue tropism from that of an AAVX particle consisting
only of AAVX coat proteins, but is still distinct from the tropism
of an AAV2 particle.
[0092] Provided herein is a recombinant AAVX virion, comprising an
AAVX particle containing, i.e., encapsidating, a vector comprising
a pair of AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10,
AAV11, AAAV, BAAV, AAV-X1, AAV-X1b, AAV-X5, AAV-X19, AAV-X21,
AAV-X22, AAV-X23, AAV-X24, AAV-X25, or AAV-X26 inverted terminal
repeats. The recombinant vector can further comprise an AAV1, AAV2,
AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAAV, BAAV,
AAV-X1, AAV-X1b, AAV-X5, AAV-X19, AAV-X21, AAV-X22, AAV-X23,
AAV-X24, AAV-X25, or AAV-X26 Rep-encoding nucleic acid. The vector
encapsidated in the particle can further comprise an exogenous
nucleic acid inserted between the inverted terminal repeats.
[0093] Further contemplated are chimeric recombinant ITRs that
contain a rep binding site and a TRS site recognized by that Rep
protein. By "Rep protein" is meant one or more of the Rep proteins,
Rep 40, Rep 78, Rep 52, Rep 68. Alternatively, "Rep protein" could
be all four of the Rep proteins described herein.
[0094] Examples of the combinations of ITR, Rep protein and Capsids
that will produce recombinant virus include but are not limited to:
[0095] AAVX ITR+AAVX Rep+AAVX Cap=virus [0096] AAV5 ITR+AAVX
Rep+AAVX Cap=virus [0097] AAV5 ITR+AAVX Rep+AAV1 Cap=virus [0098]
AAV5 ITR+AAVX Rep+AAV2 Cap=virus [0099] AAV5 ITR+AAVX Rep+AAV3
Cap=virus [0100] AAV5 ITR+AAVX Rep+AAV4 Cap=virus [0101] AAV5
ITR+AAVX Rep+AAV5 Cap=virus [0102] AAV5 ITR+AAVX Rep+AAV6 Cap=virus
[0103] AAV5 ITR+AAVX Rep+AAV7 Cap=virus [0104] AAV5 ITR+AAVX
Rep+AAV8 Cap=virus [0105] AAV5 ITR+AAVX Rep+AAV9 Cap=virus [0106]
AAV5 ITR+AAVX Rep+AAV10 Cap=virus [0107] AAV5 ITR+AAVX Rep+AAV11
Cap=virus [0108] AAV5 ITR+AAVX Rep+AAAV Cap=virus [0109] AAV5
ITR+AAVX Rep+BAAV Cap=virus [0110] AAVX ITR+AAV5 Rep+AAVX Cap=virus
[0111] AAVX ITR+AAV5 Rep+AAV1 Cap=virus [0112] AAVX ITR+AAV5
Rep+AAV2 Cap=virus [0113] AAVX ITR+AAV5 Rep+AAV3 Cap=virus [0114]
AAVX ITR+AAV5 Rep+AAV4 Cap=virus [0115] AAVX ITR+AAV5 Rep+AAV5
Cap=virus [0116] AAVX ITR+AAV5 Rep+AAV6 Cap=virus [0117] AAVX
ITR+AAV5 Rep+AAV7 Cap=virus [0118] AAVX ITR+AAV5 Rep+AAV8 Cap=virus
[0119] AAVX ITR+AAV5 Rep+AAV9 Cap=virus [0120] AAVX ITR+AAV5
Rep+AAV10 Cap=virus [0121] AAVX ITR+AAV5 Rep+AAV 11 Cap=virus
[0122] AAVX ITR+AAV5 Rep+AAAV Cap=virus [0123] AAVX ITR+AAV5
Rep+BAAV Cap=virus [0124] AAV1 ITR+AAV1 Rep+AAVX Cap=virus [0125]
AAV2 ITR+AAV2 Rep+AAVX Cap=virus [0126] AAV3 ITR+AAV3 Rep+AAVX
Cap=virus [0127] AAV4 ITR+AAV4 Rep+AAVX Cap=virus [0128] AAV5
ITR+AAV5 Rep+AAVX Cap=virus [0129] AAV6 ITR+AAV6 Rep+AAVX Cap=virus
[0130] AAV7 ITR+AAV7 Rep+AAVX Cap=virus [0131] AAV8 ITR+AAV8
Rep+AAVX Cap=virus [0132] AAV9 ITR+AAV9 Rep+AAVX Cap=virus [0133]
AAV10 ITR+AAV10 Rep+AAVX Cap=virus [0134] AAV 11 ITR+AAV 11
Rep+AAVX Cap=virus [0135] AAAV ITR+AAAV Rep+AAVX Cap=virus [0136]
BAAV ITR+BAAV Rep+AAVX Cay-virus [Note that AAVX can be AAV-X1,
AAV-X1b, AAV-X5, AAV-X19, AAV-X21, AAV-X22, AAV-X23, AAV-X24,
AAV-X25, or AAV-X26]
[0137] One of skill in the art would know how to employ standard
techniques to obtain the sequences from any of AAV1-11, AAAV, BAAV
or AAVX in order to combine them with AAVX sequences. Examples of
AAVX sequences that can be utilized in these constructs can be
found herein. Examples of AAV1 sequences that can be utilized in
these constructs can be found in GenBank under Accession No.
AF063497 and these sequences are hereby incorporated in their
entireties by this reference. Examples of AAV2 sequences that can
be utilized in these constructs can be found in GenBank under
Accession No. AF043303 and these sequences are hereby incorporated
in their entireties by this reference. Examples of AAV3 sequences
that can be utilized in these constructs can be found in GenBank
under Accession No. NC 001729 and these sequences are hereby
incorporated in their entireties by this reference. Examples of
AAV4 sequences that can be utilized in these constructs can be
found in GenBank under Accession No. U89790 and these sequences are
hereby incorporated in their entireties by this reference. Examples
of AAV5 sequences that can be utilized in these constructs can be
found in GenBank under Accession No. AF085716 and these sequences
are hereby incorporated in their entireties by this reference.
Examples of AAV6 sequences that can be utilized in these constructs
can be found in GenBank under Accession No. NC 001862 and AF028704
and these sequences are hereby incorporated in their entireties by
this reference. Examples of AAV7 sequences that can be utilized in
these constructs can be found in GenBank under Accession No.
AF513851 and these sequences are hereby incorporated in their
entireties by this reference. Examples of AAV8 sequences that can
be utilized in these constructs can be found in GenBank under
Accession No. AF513852 and these sequences are hereby incorporated
in their entireties by this reference. Examples of AAV9 sequences
that can be utilized in these constructs can be found in GenBank
under Accession No. AY530579 and these sequences are hereby
incorporated in their entireties by this reference. Examples of
AAV10 sequences that can be utilized in these constructs can be
found in GenBank under Accession No. AY631965 and these sequences
are hereby incorporated in their entireties by this reference.
Examples of AAV11 sequences that can be utilized in these
constructs can be found in GenBank under Accession No. AY631966 and
these sequences are hereby incorporated in their entireties by this
reference. Examples of AAAV sequences that can be utilized in these
constructs can be found in GenBank under Accession No. AY186198 and
these sequences are hereby incorporated in their entireties by this
reference. Examples of BAAV sequences that can be utilized in these
constructs can be found in GenBank Accession No. AY388617 and these
sequences are hereby incorporated in their entireties by this
reference.
[0138] In any of the constructs described herein, inclusion of a
promoter is preferred. As used in the constructs herein, unless
otherwise specified, Cap (capsid) refers to any of VP1, VP2, VP3,
combinations thereof, functional fragments of any of VP1, VP2 or
VP3, or chimeric capsids as described herein. The ITRs of the
constructs described herein, can be chimeric recombinant ITRs as
described elsewhere in the application.
[0139] Conjugates of recombinant or wild-type AAVX virions and
nucleic acids or proteins can be used to deliver those molecules to
a cell. For example, the purified AAVX can be used as a vehicle for
delivering DNA bound to the exterior of the virus. Examples of this
are to conjugate the DNA to the virion by a bridge using
poly-L-lysine or other charged molecule. Also contemplated are
virosomes that contain AAVX structural proteins (AAVX capsid
proteins), lipids such as DOTAP, and nucleic acids that are
complexed via charge interaction to introduce DNA into cells.
[0140] Also provided herein are AAVX capsid proteins (e.g. VP1, VP2
or VP3 or combinations thereof), or AAVX particles consisting of
AAVX capsid proteins, wherein the capsid proteins or particles do
not contain AAV nucleic acid, vector or plasmid, and are therefore
not infectious. These capsid proteins and "empty particles" can
comprise other substances such as biologically active molecules
(e.g., small molecules, polypeptides, or non-AAV nucleic acids).
The substances can be conjugated to the capsid proteins or comprise
a fusion protein with an AAVX capsid polypeptide. Alternatively,
the substance can be incorporated within an AAVX empty particle.
AAVX capsid proteins and empty particles can be used to deliver the
substance to a target cell using the targeting ability of the
capsid protein to achieve the desired tissue tropism. In addition,
the empty particles can function to protect the substance from
depredation or immune response.
[0141] Also provided herein are conjugates that utilize the AAVX
capsid or a unique region of the AAVX capsid protein (e.g. VP1, VP2
or VP3 or combinations thereof) to introduce DNA into cells. For
example, the AAVX VP1 protein or fragment thereof, can be
conjugated to a DNA on a plasmid that is conjugated to a lipid.
Cells can be infected using the targeting ability of the VP1 capsid
protein to achieve the desired tissue tropism, specific to the
AAVX. AAVX VP2 and VP3 proteins can also be utilized to introduce
DNA or other molecules into cells. By further incorporating the Rep
protein and the AAV TRS into the DNA-containing conjugate, cells
can be transduced and targeted integration can be achieved. For
example, if AAVX specific targeted integration is desired, a
conjugate composed of the AAVX VP1 capsid, AAVX Rep or a fragment
of AAVX Rep, AAVX TRS, the Rep binding site, the exogenous DNA of
interest, and a lipid, can be utilized to achieve AAVX specific
tropism and AAVX specific targeted integration in the genome.
[0142] Further provided herein are chimeric viruses where AAVX
vectors can be encapsi dated by herpes simplex virus (HSV)
(Heister, T., et al. J. Virol. 2002 July; 76(14):7163-73),
incorporated herein for its teaching of HSV/AAV hybrid vectors),
baculovirus or other viruses to achieve a desired tropism
associated with another virus. For example, the AAVX ITRs could be
encapsidated by HSV and cells could be infected. Post-infection,
the ITRs of AAVX could be acted on by AAVX Rep provided in the
system or in a separate vehicle to rescue AAVX from the genome.
Therefore, the cellular tropism of HSV can be combined with AAVX
Rep mediated targeted integration. Other viruses that could be
utilized to construct chimeric viruses include lentivirus,
retrovirus, pseudotyped retroviral vectors and adenoviral
vectors.
[0143] Provided herein are isolated nucleic acids of AAV-X1,
AAV-X1b, AAV-X5, AAV-X19, AAV-X21, AAV-X22, AAV-X23, AAV-X24,
AAV-X25, and AAV-X26. For example, provided is an isolated nucleic
acid comprising the nucleotide sequence set forth in SEQ ID NO:1
(AAV-X1 partial genome). Also provided is an isolated nucleic acid
comprising the nucleotide sequence set forth in SEQ ID NO:2
(AAV-X1b partial genome). Also provided is an isolated nucleic acid
comprising the nucleotide sequence set forth in SEQ ID NO:3 (AAV-X5
partial genome). Also provided is an isolated nucleic acid
comprising the nucleotide sequence set forth in SEQ ID NO:4
(AAV-X19 partial genome). Also provided is an isolated nucleic acid
comprising the nucleotide sequence set forth in SEQ ID NO:5
(AAV-X21 partial genome). Also provided is an isolated nucleic acid
comprising the nucleotide sequence set forth in SEQ ID NO:6
(AAV-X22 partial genome). Also provided is an isolated nucleic acid
comprising the nucleotide sequence set forth in SEQ ID NO:7
(AAV-X23 partial genome). Also provided is an isolated nucleic acid
comprising the nucleotide sequence set forth in SEQ ID NO:8
(AAV-X24 partial genome). Also provided is an isolated nucleic acid
comprising the nucleotide sequence set forth in SEQ ID NO:9
(AAV-X25 partial genome). Also provided is an isolated nucleic acid
comprising the nucleotide sequence set forth in SEQ ID NO:10
(AAV-X26 partial genome).
[0144] This nucleic acid, or unique portions thereof, can be
inserted into vectors, such as plasmids, yeast artificial
chromosomes, or other viral vector (particle), if desired, by
standard cloning methods. Also provided is an isolated nucleic acid
consisting essentially of the nucleotide sequence set forth in SEQ
ID NOs:1-10.
[0145] The phrase "consisting essentially of" is used herein to
refer to a composition that comprises the essential characteristics
of the identified composition. By "essential" is meant the
characteristics that contribute to the structure or function of the
disclosed molecule. Thus, any substitution, deletion or addition to
the provided composition that does not significantly alter the
defining characteristics of the composition are considered
therein.
[0146] For example, if an amino acid sequence X is disclosed, then
a provided polypeptide consisting essentially of the amino acid
sequence X includes, for example, conservative amino acid
substitutions (as described below) that do not significantly alter
the essential characteristics of the polypeptide, e.g.,
secondary/tertiary structure or function of the protein. The
provided polypeptide can further constitute a fusion protein or
otherwise have additional N-terminal, C-terminal, or intermediate
amino acid sequences, e.g., linkers or tags. "Linker", as used
herein, is an amino acid sequences or insertion that can be used to
connect or separate two distinct polypeptides or polypeptide
fragments, wherein the linker does not otherwise contribute to the
essential function of the composition. A polypeptide provided
herein, can have an amino acid linker comprising, for example, the
amino acids GLS, ALS, or LLA. A "tag", as used herein, refers to a
distinct amino acid sequence that can be used to detect or purify
the provided polypeptide, wherein the tag does not otherwise
contribute to the essential function of the composition. The
provided polypeptide can further have deleted N-terminal,
C-terminal or intermediate amino acids that do not contribute to
the essential activity of the polypeptide.
[0147] As another example, if a nucleic acid X is disclosed, then a
provided nucleic acid consisting essentially of nucleic acid
sequence X, includes, for example, nucleotide substitutions that do
not alter the amino acid sequence of the encoded polypeptide, i.e.,
due to degeneracy. If sequence X comprises introns and exons, then
the provided nucleic acid can further be the cDNA sequence that
lacks the introns but comprises the exons of sequence X. To the
extent that specific genes within a genome are identified herein,
it is further understood that the disclosure of a nucleic acid
consisting essentially of the genome sequence would include
fragments of the genome such as isolated sequences comprising a
gene or genes within the genome.
[0148] Other characteristics of nucleic acid or amino acid
sequences that are not herein considered essential include, for
example, junk DNA between genes or any identifiable sequence unit,
e.g., promoters, enhancers, transmembrane domains, poly-adenylation
sequences, signal sequences, etc., that when substituted or removed
would be presumed by one skilled in the art to not significantly
alter the essential characteristics of the disclosed sequence.
[0149] Thus, the nucleotides of SEQ ID NOS:1-10 can have minor
modifications and still be contemplated herein. For example,
modifications that do not alter the amino acid encoded by any given
codon (such as by modification of the third, "wobble," position in
a codon) can readily be made, and such alterations are known in the
art. Furthermore, modifications that cause a resulting neutral
(conserved) amino acid substitution of a similar amino acid can be
made in a coding region of the genome. Additionally, modifications
as described herein for the AAVX components, such as the ITRs, the
p5 promoter, etc. are contemplated herein. Furthermore,
modifications to regions of SEQ ID NOS:1-10, other than in the ITR,
TRS, Rep binding site and hairpin, are likely to be tolerated
without serious impact on the function of the nucleic acid as a
recombinant vector.
[0150] As used herein, the term "isolated" refers to a nucleic acid
separated or significantly free from at least some of the other
components of the naturally occurring organism, for example, the
cell structural components or viral components commonly found
associated with nucleic acids in the environment of the virus
and/or other nucleic acids. The isolation of the native nucleic
acids can be accomplished, for example, by techniques such as cell
lysis followed by phenol plus chloroform extraction, followed by
ethanol precipitation of the nucleic acids. The nucleic acids
provided herein can be isolated from cells according to any of many
methods well known in the art.
[0151] As used herein, the term "nucleic acid" refers to single- or
multiple-stranded molecules which may be DNA or RNA, or any
combination thereof, including modifications to those nucleic
acids. The nucleic acid may represent a coding strand or its
complement, or any combination thereof. Nucleic acids may be
identical in sequence to the sequences which are naturally
occurring for any of the genes discussed herein or may include
alternative codons which encode the same amino acid as those
provided herein, including that which is found in the naturally
occurring sequence. These nucleic acids can also be modified from
their typical structure. Such modifications include, but are not
limited to, methylated nucleic acids, the substitution of a
non-bridging oxygen on the phosphate residue with either a sulfur
(yielding phosphorothioate deoxynucleotides), selenium (yielding
phosphorselenoate deoxynucleotides), or methyl groups (yielding
methylphosphonate deoxynucleotides).
[0152] Additionally provided is an isolated nucleic acid that
selectively hybridizes with any nucleic acid disclosed herein,
including the AAV-X1, AAV-X1b, AAV-X5, AAV-X19, AAV-X21, AAV-X22,
AAV-X23, AAV-X24, AAV-X25, or AAV-X26 genome (SEQ ID NOS:1-10) and
any unique fragment thereof, including the Rep and capsid encoding
sequences, promoters and ITRs. Specifically, the nucleic acid can
selectively or specifically hybridize to an isolated nucleic acid
consisting of the nucleotide sequence set forth in SEQ ID NO:1, 2,
3, 4, 5, 6, 7, 8, 9, or 10. By "selectively hybridizes" as used
herein is meant a nucleic acid that hybridizes to one of the
disclosed nucleic acids under sufficient stringency conditions
without significant hybridization to a nucleic acid encoding an
unrelated protein, and particularly, without detectably hybridizing
to nucleic acids of other AAVs. Thus, a nucleic acid that
selectively hybridizes with a nucleic acid provided herein will not
selectively hybridize under stringent conditions with a nucleic
acid encoding a different protein or the corresponding protein from
a different serotype of the virus, and vice versa. A "specifically
hybridizing" nucleic acid is one that hybridizes under stringent
conditions to only a nucleic acid found in AAVX. Therefore, nucleic
acids for use, for example, as primers and probes to detect or
amplify the target nucleic acids are contemplated herein. Nucleic
acid fragments that selectively hybridize to any given nucleic acid
can be used, e.g., as primers and or probes for further
hybridization or for amplification methods (e.g., polymerase chain
reaction (PCR), ligase chain reaction (LCR)). Additionally, for
example, a primer or probe can be designed that selectively
hybridizes with both AAVX and a gene of interest carried within the
AAVX vector (i.e., a chimeric nucleic acid).
[0153] Stringency of hybridization is controlled by both
temperature and salt concentration of either or both of the
hybridization and washing steps. Typically, the stringency of
hybridization to achieve selective hybridization involves
hybridization in high ionic strength solution (6.times.SSC or
6.times.SSPE) at a temperature that is about 12-25.degree. C. below
the T.sub.m (the melting temperature at which half of the molecules
dissociate from their hybridization partners) followed by washing
at a combination of temperature and salt concentration chosen so
that the washing temperature is about 5.degree. C. to 20.degree. C.
below the T.sub.m. The temperature and salt conditions are readily
determined empirically in preliminary experiments in which samples
of reference DNA immobilized on filters are hybridized to a labeled
nucleic acid of interest and then washed under conditions of
different stringencies. Hybridization temperatures are typically
higher for DNA-RNA and RNA-RNA hybridizations. The washing
temperatures can be used as described above to achieve selective
stringency, as is known in the art. (Sambrook et al., Molecular
Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor
Laboratory, Cold Spring Harbor, N.Y., 1989; Kunkel et al. Methods
Enzymol. 1987:154:367, 1987). A preferable stringent hybridization
condition for a DNA:DNA hybridization can be at about 68.degree. C.
(in aqueous solution) in 6.times.SSC or 6.times.SSPE followed by
washing at 68.degree. C. Stringency of hybridization and washing,
if desired, can be reduced accordingly as the degree of
complementarity desired is decreased, and further, depending upon
the G-C or A-T richness of any area wherein variability is searched
for. Likewise, stringency of hybridization and washing, if desired,
can be increased accordingly as homology desired is increased, and
further, depending upon the G-C or AT richness of any area wherein
high homology is desired, all as known in the art.
[0154] A nucleic acid that selectively hybridizes to any portion of
the AAVX genome is contemplated herein. Therefore, a nucleic acid
that selectively hybridizes to AAVX can be of longer length than
the AAVX genome, it can be about the same length as the AAVX genome
or it can be shorter than the AAVX genome. The length of the
nucleic acid is limited on the shorter end of the size range only
by its specificity for hybridization to AAVX, i.e., once it is too
short, typically less than about 5 to 7 nucleotides in length, it
will no longer bind specifically to AAVX, but rather will hybridize
to numerous background nucleic acids. Additionally contemplated
herein is a nucleic acid that has a portion that specifically
hybridizes to AAVX and a portion that specifically hybridizes to a
gene of interest inserted within AAVX.
[0155] Provided is an isolated nucleic acid comprising an AAVX p5
promoter. Provided is an isolated nucleic acid comprising an AAVX
p19 promoter. Provided is an isolated nucleic acid comprising an
AAVX p40 promoter. Provided is an isolated nucleic acid comprising
an AAVX ITR. Further provided is an isolated nucleic acid encoding
an AAVX Rep protein. The AAVX Rep proteins are encoded by open
reading frame (ORF) 1 of the AAVX genome. Examples of the AAV Rep
proteins include Rep78, Rep68, Rep52 and Rep40. However, it is
contemplated that the Rep nucleic acid can encode any one, two,
three, or four of the four Rep proteins, in any order.
[0156] Furthermore, minor modifications are contemplated in the
nucleic acid, such as silent mutations in the coding sequences,
mutations that make neutral or conservative changes in the encoded
amino acid sequence, and mutations in regulatory regions that do
not disrupt the expression of the gene. Examples of other minor
modifications are known in the art. Further modifications can be
made in the nucleic acid, such as to disrupt or alter expression of
one or more of the Rep proteins in order to, for example, determine
the effect of such a disruption; such as to mutate one or more of
the Rep proteins to determine the resulting effect, etc.
[0157] Further provided is an isolated nucleic acid encoding an
AAVX Capsid protein. Furthermore, provided is a nucleic acid
encoding each of the three AAVX capsid proteins, VP1, VP2, and VP3.
Thus, provided is an isolated nucleic acid encoding AAVX VP1, a
nucleic acid encoding AAVX VP2, and an isolated nucleic acid
encoding AAVX VP3. Thus, provided is an isolated nucleic acid
encoding the amino acid sequence set forth in SEQ ID NO:21 (AAV-X1
VP1). Thus, provided is an isolated nucleic acid encoding the amino
acid sequence set forth in SEQ ID NO:22 (AAV-X1b VP1). Thus,
provided is an isolated nucleic acid encoding the amino acid
sequence set forth in SEQ ID NO:23 (AAV-X5 VP1). Thus, provided is
an isolated nucleic acid encoding the amino acid sequence set forth
in SEQ ID NO:24 (AAV-X19 VP1). Thus, provided is an isolated
nucleic acid encoding the amino acid sequence set forth in SEQ ID
NO:25 (AAV-X21 VP1). Thus, provided is an isolated nucleic acid
encoding the amino acid sequence set forth in SEQ ID NO:26 (AAV-X22
VP1). Thus, provided is an isolated nucleic acid encoding the amino
acid sequence set forth in SEQ ID NO:27 (AAV-X23 VP1). Thus,
provided is an isolated nucleic acid encoding the amino acid
sequence set forth in SEQ ID NO:28 (AAV-X24 VP1). Thus, provided is
an isolated nucleic acid encoding the amino acid sequence set forth
in SEQ ID NO:29 (AAV-X25 VP1). Thus, provided is an isolated
nucleic acid encoding the amino acid sequence set forth in SEQ ID
NO:30 (AAV-X26 VP1).
[0158] Also specifically provided is an isolated nucleic acid
comprising SEQ ID NO:11 (AAV-X1 VP1). Also specifically provided is
an isolated nucleic acid comprising SEQ ID NO:12 (AAV-X1b VP1).
Also specifically provided is an isolated nucleic acid comprising
SEQ ID NO:13 (AAV-X5 VP1). Also specifically provided is an
isolated nucleic acid comprising SEQ ID NO:14 (AAV-X19 VP1). Also
specifically provided is an isolated nucleic acid comprising SEQ ID
NO:15 (AAV-X21 VP1). Also specifically provided is an isolated
nucleic acid comprising SEQ ID NO:16 (AAV-X22 VP1). Also
specifically provided is an isolated nucleic acid comprising SEQ ID
NO:17 (AAV-X23 VP1). Also specifically provided is an isolated
nucleic acid comprising SEQ ID NO:18 (AAV-X24 VP1). Also
specifically provided is an isolated nucleic acid comprising SEQ ID
NO:19 (AAV-X25 VP1). Also specifically provided is an isolated
nucleic acid comprising SEQ ID NO:20 (AAV-X26 VP1).
[0159] Minor modifications in the nucleotide sequences encoding the
capsid, or coat, proteins are contemplated, as described above for
other AAVX nucleic acids. However, in general, a modified nucleic
acid encoding a capsid protein will have at least about 80%, 81%,
82%, 83%, 84%, 85%, 86%, 87%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,
96%, 97%, 98%, 98.5%, 99%, 99.5% or 99.9% or 100% homology to the
capsid nucleic sequences described herein e.g., SEQ ID NOS:11-20,
and the capsid polypeptide encoded therein will have overall about
80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 89%, 90%, 91%, 92%, 93%,
94%, 95%, 96%, 97%, 98%, 98.5%, 99%, 99.5% or 99.9% or 100%
homology with the amino acid sequence described herein, e.g., SEQ
ID NOS:21-30. Isolated nucleic acids that selectively hybridize
with the nucleic acids of SEQ ID NOS:11-20 under the conditions
described above are also provided.
[0160] Also provided is a cell containing one or more of the herein
described nucleic acids, such as the AAVX genome, AAVX ORF1 and
ORF2, each AAVX Rep protein gene, or each AAVX capsid protein gene.
Such a cell can be any desired cell and can be selected based upon
the use intended. For example, cells can include bacterial cells,
yeast cells, insect cells, human HeLa cells and simian Cos cells as
well as other human and mammalian cells and cell lines. Primary
cultures as well as established cultures and cell lines can be
used. Nucleic acids provided herein can be delivered into cells by
any selected means, in particular depending upon the target cells.
Many delivery means are well-known in the art. For example,
electroporation, calcium phosphate precipitation, microinjection,
cationic or anionic liposomes, and liposomes in combination with a
nuclear localization signal peptide for delivery to the nucleus can
be utilized, as is known in the art. Additionally, if the nucleic
acids are in a viral particle, the cells can simply be transduced
with the virion by standard means known in the art for AAV
transduction. Small amounts of the recombinant AAVX virus can be
made to infect cells and produce more of itself.
[0161] Provided herein are purified AAVX polypeptides. The term
"polypeptide" as used herein refers to a polymer of amino acids and
includes full-length proteins and fragments thereof. Thus,
"protein," polypeptide," and "peptide" are often used
interchangeably herein. Substitutions can be selected by known
parameters to be neutral (see, e.g., Robinson WE Jr, and Mitchell
WM., AIDS 4:S151-S162 (1990)). As will be appreciated by those
skilled in the art, also provided herein are those polypeptides
having slight variations in amino acid sequences or other
properties. Such variations may arise naturally as allelic
variations (e.g., due to genetic polymorphism) or may be produced
by human intervention (e.g., by mutagenesis of cloned DNA
sequences), such as induced point, deletion, insertion and
substitution mutants. Minor changes in amino acid sequence are
generally preferred, such as conservative amino acid replacements,
small internal deletions or insertions, and additions or deletions
at the ends of the molecules. Substitutions may be designed based
on, for example, the model of Dayhoff, et al. (in Atlas of Protein
Sequence and Structure 1978, Nat'l Biomed. Res. Found., Washington,
D.C.). These modifications can result in changes in the amino acid
sequence, provide silent mutations, modify a restriction site, or
provide other specific mutations. The location of any modifications
to the polypeptide will often determine its impact on function.
Particularly, alterations in regions non-essential to protein
function will be tolerated with fewer effects on function.
Elsewhere in the application regions of the AAVX proteins are
described to provide guidance as to where substitutions, additions
or deletions can be made to minimize the likelihood of disturbing
the function of the variant.
[0162] Protein variants and derivatives are well understood to
those of skill in the art and in can involve amino acid sequence
modifications. For example, amino acid sequence modifications
typically fall into one or more of three classes: substitutional,
insertional or deletional variants. Insertions include amino and/or
carboxyl terminal fusions as well as intrasequence insertions of
single or multiple amino acid residues. Insertions ordinarily will
be smaller insertions than those of amino or carboxyl terminal
fusions, for example, on the order of one to four residues.
Deletions are characterized by the removal of one or more amino
acid residues from the protein sequence. Typically, no more than
about from 2 to 6 residues are deleted at any one site within the
protein molecule. These variants ordinarily are prepared by site
specific mutagenesis of nucleotides in the DNA encoding the
protein, thereby producing DNA encoding the variant, and thereafter
expressing the DNA in recombinant cell culture. Techniques for
making substitution mutations at predetermined sites in DNA having
a known sequence are well known, for example M13 primer mutagenesis
and PCR mutagenesis. Amino acid substitutions are typically of
single residues, but can occur at a number of different locations
at once; insertions usually will be on the order of about from 1 to
10 amino acid residues; and deletions will range about from 1 to 30
residues. Deletions or insertions preferably are made in adjacent
pairs, i.e. a deletion of 2 residues or insertion of 2 residues.
Substitutions, deletions, insertions or any combination thereof may
be combined to arrive at a final construct. The mutations must not
place the sequence out of reading frame and preferably will not
create complementary regions that could produce secondary mRNA
structure. Substitutional variants are those in which at least one
residue has been removed and a different residue inserted in its
place. Such substitutions generally are made in accordance with the
following Tables 1 and 2 and are referred to as conservative
substitutions.
TABLE-US-00001 TABLE 1 Amino Acid Abbreviations Amino Acid
Abbreviations alanine Ala A allosoleucine AIle arginine Arg R
asparagine Asn N aspartic acid Asp D cysteine Cys C glutamic acid
Glu E glutamine Gln Q glycine Gly G histidine His H isolelucine Ile
I leucine Leu L lysine Lys K phenylalanine Phe F proline Pro P
pyroglutamic acid pGlu serine Ser S threonine Thr T tyrosine Tyr Y
tryptophan Trp W valine Val V
TABLE-US-00002 TABLE 2 Amino Acid Substitutions Original Residue
Exemplary Conservative Substitutions, others are known in the art.
Ala Ser Arg Lys; Gln Asn Gln; His Asp Glu Cys Ser Gln Asn, Lys Glu
Asp Gly Pro His Asn; Gln Ile Leu; Val Leu Ile; Val Lys Arg; Gln Met
Leu; Ile Phe Met; Leu; Tyr Ser Thr Thr Ser Trp Tyr Tyr Trp; Phe Val
Ile; Leu
[0163] Substantial changes in function or immunological identity
can result from selecting substitutions that are less conservative
than those in Table 2, i.e., selecting residues that differ more
significantly in their effect on maintaining (a) the structure of
the polypeptide backbone in the area of the substitution, for
example as a sheet or helical conformation, (b) the charge or
hydrophobicity of the molecule at the target site or (c) the bulk
of the side chain. The substitutions which in general are expected
to produce the greatest changes in the protein properties will be
those in which (a) a hydrophilic residue, e.g. seryl or threonyl,
is substituted for (or by) a hydrophobic residue, e.g. leucyl,
isoleucyl, phenylalanyl, valyl or alanyl; (b) a cysteine or proline
is substituted for (or by) any other residue; (c) a residue having
an electropositive side chain, e.g., lysyl, arginyl, or histidyl,
is substituted for (or by) an electronegative residue, e.g.,
glutamyl or aspartyl; or (d) a residue having a bulky side chain,
e.g., phenylalanine, is substituted for (or by) one not having a
side chain, e.g., glycine, in this case, (e) by increasing the
number of sites for sulfation and/or glycosylation.
[0164] For example, the replacement of one amino acid residue with
another that is biologically and/or chemically similar is known to
those skilled in the art as a conservative substitution. For
example, a conservative substitution would be replacing one
hydrophobic residue for another, or one polar residue for another.
The substitutions include combinations such as, for example, Gly,
Ala; Val, Ile, Leu; Asp, Glu; Asn, Gin; Ser, Thr; Lys, Arg; and
Phe, Tyr. Such conservatively substituted variations of each
explicitly disclosed sequence are included within the mosaic
polypeptides provided herein.
[0165] Generally, a conservative substitution is a substitution of
an amino acid residue for another amino acid residue having similar
biochemical properties. Typically, conservative substitutions have
little to no impact on the biological activity of a resulting
polypeptide. In a particular example, a conservative substitution
is an amino acid substitution in a peptide that does not
substantially affect the biological function of the peptide. A
peptide can include one or more amino acid substitutions, for
example 2-10 conservative substitutions, 2-5 conservative
substitutions, 4-9 conservative substitutions, such as 2, 5 or 10
conservative substitutions.
[0166] For example, a conservative substitution in an AAVX VP1
peptide (such as a peptides encoded by SEQ ID NOS:21-30) does not
substantially affect the ability of VP1 peptide to confer the
unique tropism of the AAVX particle. A polypeptide can be produced
to contain one or more conservative substitutions by manipulating
the nucleotide sequence that encodes that polypeptide using, for
example, standard procedures such as site-directed mutagenesis or
PCR. Alternatively, a polypeptide can be produced to contain one or
more conservative substitutions by using standard peptide synthesis
methods. An alanine scan can be used to identify which amino acid
residues in a protein can tolerate an amino acid substitution. In
one example, the biological activity of the protein is not
decreased by more than 25%, for example not more than 20%, for
example not more than 10%, when an alanine, or other conservative
amino acid (such as those listed below), is substituted for one or
more native amino acids.
[0167] Examples of amino acids which can be substituted for an
original amino acid in a protein and which are regarded as
conservative substitutions include, but are not limited to: Ser for
Ala; Lys for Arg; Gln or H is for Asn; Glu for Asp; Ser for Cys;
Asn for Gln; Asp for Glu; Pro for Gly; Asn or Gln for H is; Leu or
Val for Ile; Ile or Val for Leu; Arg or Gln for Lys; Leu or Ile for
Met; Met, Leu or Tyr for Phe; Thr for Ser; Ser for Thr; Tyr for
Trp; Trp or Phe for Tyr; and Ile or Leu for Val.
[0168] Further information about conservative substitutions can be
found in, among other locations in, Ben-Bassat et al., (J.
Bacteriol. 169:751-7, 1987), O'Regan et al., (Gene 77:237-51,
1989), Sahin-Toth et al., (Protein Sci. 3:240-7, 1994), Hochuli et
al., (Bio/Technology 6:1321-5, 1988) and in standard textbooks of
genetics and molecular biology.
[0169] Substitutional or deletional mutagenesis can be employed to
insert sites for N-glycosylation (Asn-X-Thr/Ser) or O-glycosylation
(Ser or Thr). Deletions of cysteine or other labile residues also
may be desirable. Deletions or substitutions of potential
proteolysis sites, e.g. Arg, is accomplished for example by
deleting one of the basic residues or substituting one by
glutaminyl or histidyl residues.
[0170] Certain post-translational derivatizations are the result of
the action of recombinant host cells on the expressed polypeptide.
Glutaminyl and asparaginyl residues are frequently
post-translationally deamidated to the corresponding glutamyl and
asparyl residues. Alternatively, these residues are deamidated
under mildly acidic conditions. Other post-translational
modifications include hydroxylation of proline and lysine,
phosphorylation of hydroxyl groups of seryl or threonyl residues,
methylation of the o-amino groups of lysine, arginine, and
histidine side chains (T. E. Creighton, Proteins: Structure and
Molecular Properties, W. H. Freeman & Co., San Francisco pp
79-86 [1983]), acetylation of the N-terminal amine and, in some
instances, amidation of the C-terminal carboxyl.
[0171] It is understood that there are numerous amino acid and
peptide analogs which can be incorporated into the disclosed
compositions. For example, there are numerous D amino acids or
amino acids which have a different functional substituent then the
amino acids shown in Table 1 and Table 2. The opposite stereo
isomers of naturally occurring peptides are disclosed, as well as
the stereo isomers of peptide analogs. These amino acids can
readily be incorporated into polypeptide chains by charging tRNA
molecules with the amino acid of choice and engineering genetic
constructs that utilize, for example, amber codons, to insert the
analog amino acid into a peptide chain in a site specific way
(Thorson et al., Methods in Molec. Biol. 77:43-73 (1991), Zoller,
Current Opinion in Biotechnology, 3:348-354 (1992); Ibba,
Biotechnology & Genetic Enginerring Reviews 13:197-216 (1995),
Cahill et al., TIBS, 14(10):400-403 (1989); Benner, TIB Tech,
12:158-163 (1994); Ibba and Hennecke, Bio/technology, 12:678-682
(1994) all of which are herein incorporated by reference at least
for material related to amino acid analogs).
[0172] Molecules can be produced that resemble peptides, but which
are not connected via a natural peptide linkage. For example,
linkages for amino acids or amino acid analogs can include CH2NH--,
--CH2S--, --CH2-CH2-CH.dbd.CH--(cis and trans), --COCH2-,
--CH(OH)CH2-, and --CHH2SO-- (These and others can be found in
Spatola, A. F. in Chemistry and Biochemistry of Amino Acids,
Peptides, and Proteins, B. Weinstein, eds., Marcel Dekker, New
York, p. 267 (1983); Spatola, A. F., Vega Data (March 1983), Vol.
1, Issue 3, Peptide Backbone Modifications (general review);
Morley, Trends Pharm Sci (1980) pp. 463-468; Hudson, D. et al., Int
J Pept Prot Res 14:177-185 (1979) (--CH2NH--, CH2CH2-); Spatola et
al. Life Sci 38:1243-1249 (1986) (--CHH2-S); Hann J. Chem. Soc
Perkin Trans. I 307-314 (1982) (--CH--CH--, cis and trans);
Almquist et al. J. Med. Chem. 23:1392-1398 (1980) (--COCH2-);
Jennings-White et al. Tetrahedron Lett 23:2533 (1982) (--COCH2-);
Szelke et al. European Appin, EP 45665 CA (1982): 97:39405 (1982)
(--CH(OH)CH2-); Holladay et al. Tetrahedron. Lett 24:4401-4404
(1983) (--C(OH)CH2-); and Hruby Life Sci 31:189-199 (1982)
(--CH2-S--); each of which is incorporated herein by reference. A
particularly preferred non-peptide linkage is --CH2NH--. It is
understood that peptide analogs can have more than one atom between
the bond atoms, such as b-alanine, g-aminobutyric acid, and the
like.
[0173] Amino acid analogs and analogs and peptide analogs often
have enhanced or desirable properties, such as, more economical
production, greater chemical stability, enhanced pharmacological
properties (half-life, absorption, potency, efficacy, etc.),
altered specificity (e.g., a broad-spectrum of biological
activities), reduced antigenicity, and others.
[0174] D-amino acids can be used to generate more stable peptides,
because D amino acids are not recognized by peptidases and such.
Systematic substitution of one or more amino acids of a consensus
sequence with a D-amino acid of the same type (e.g., D-lysine in
place of L-lysine) can be used to generate more stable peptides.
Cysteine residues can be used to cyclize or attach two or more
peptides together. This can be beneficial to constrain peptides
into particular conformations. (Rizo and Gierasch Ann. Rev.
Biochem. 61:387 (1992), incorporated herein by reference).
[0175] A polypeptide provided herein can be readily obtained by any
of several means. For example, the polypeptide of interest can be
synthesized chemically by standard methods. Additionally, the
coding regions of the genes can be recombinantly expressed and the
resulting polypeptide isolated by standard methods. Furthermore, an
antibody specific for the resulting polypeptide can be raised by
standard methods (see, e.g., Harlow and Lane, Antibodies: A
Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring
Harbor, N.Y., 1988), and the protein can be isolated from a cell
expressing the nucleic acid encoding the polypeptide by selective
hybridization with the antibody. This protein can be purified to
the extent desired by standard methods of protein purification
(see, e.g., Sambrook et al., Molecular Cloning: A Laboratory
Manual, 2nd Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor,
N.Y., 1989).
[0176] An antigenic or immunoreactive fragment of the provided
compositions and methods is typically an amino acid sequence of at
least about 5 consecutive amino acids, and it can be derived from
the AAVX polypeptide amino acid sequence. An antigenic AAVX
fragment is any fragment unique to the AAVX protein, as described
herein, against which an AAVX-specific antibody can be raised, by
standard methods. Thus, the resulting antibody-antigen reaction
should be specific for AAVX.
[0177] By "unique fragment thereof" is meant any smaller
polypeptide fragment encoded by an AAVX rep gene that is of
sufficient length to be found only in the Rep polypeptide.
Substitutions and modifications of the amino acid sequence can be
made as described herein and, further, can include protein
processing modifications, such as glycosylation, to the
polypeptide. Typically, to be unique, a polypeptide fragment
provided herein will be at least about 5 amino acids in length;
however, unique fragments can be 6, 7, 8, 9, 10, 20, 30, 40, 50,
60, 70, 80, 90, 100 or more amino acids in length. A unique
polypeptide will typically comprise such a unique fragment;
however, a unique polypeptide can also be determined by its overall
homology. A unique polypeptide can be 6, 7, 8, 9, 10, 20, 30, 40,
50, 60, 70, 80, 90, 100 or more amino acids in length. Uniqueness
of a polypeptide fragment can readily be determined by standard
methods such as searches of computer databases of known peptide or
nucleic acid sequences or by hybridization studies to the nucleic
acid encoding the protein or to the protein itself, as known in the
art. The uniqueness of a polypeptide fragment can also be
determined immunologically as well as functionally. Uniqueness can
be simply determined in an amino acid-by-amino acid comparison of
the polypeptides.
[0178] Provided is an isolated AAVX Rep protein. An AAVX Rep
polypeptide is encoded by ORF1 of AAVX. Also provided is each
individual AAVX Rep protein. Provided is an isolated polypeptide,
comprising AAVX Rep 52, or a unique fragment thereof. Provided is
an isolated polypeptide, comprising AAV Rep 78, or a unique
fragment thereof.
[0179] Further provided is an isolated AAVX Capsid protein or a
unique fragment thereof. AAVX capsid protein is encoded by ORF 2 of
AAVX. Further provided are the individual AAVX capsid proteins,
VP1, VP2 and VP3 or unique fragments thereof. Thus, provided is an
isolated polypeptide having the amino acid sequence set forth in
SEQ ID NO:21 (AAV-X1 VP1). Also provided is an isolated polypeptide
having the amino acid sequence set forth in SEQ ID NO:22 (AAV-X1b
VP1). Also provided is an isolated polypeptide having the amino
acid sequence set forth in SEQ ID NO:23 (AAV-X5 VP1). Also provided
is an isolated polypeptide having the amino acid sequence set forth
in SEQ ID NO:24 (AAV-X19 VP1). Also provided is an isolated
polypeptide having the amino acid sequence set forth in SEQ ID
NO:25 (AAV-X21 VP1). Also provided is an isolated polypeptide
having the amino acid sequence set forth in SEQ ID NO:26 (AAV-X22
VP1). Also provided is an isolated polypeptide having the amino
acid sequence set forth in SEQ ID NO:27 (AAV-X23 VP1). Also
provided is an isolated polypeptide having the amino acid sequence
set forth in SEQ ID NO:28 (AAV-X24 VP1). Also provided is an
isolated polypeptide having the amino acid sequence set forth in
SEQ ID NO:29 (AAV-X25 VP1). Also provided is an isolated
polypeptide having the amino acid sequence set forth in SEQ ID
NO:30 (AAV-X26 VP1). Further provided is an isolated polypeptide
consisting essentially of the amino acid sequence set forth in SEQ
ID NO:21, 22, 23, 24, 25, 26, 27, 28, 29, or 30.
[0180] By "unique fragment thereof" is meant any smaller
polypeptide fragment encoded by any AAVX capsid gene that is of
sufficient length to be found only in the AAVX capsid protein.
Substitutions and modifications of the amino acid sequence can be
made as described above and, further, can include protein
processing modifications, such as glycosylation, to the
polypeptide. However, an AAVX Capsid polypeptide including all
three coat proteins will have greater than about 80%, 81%, 82%,
83%, 84%, 85%, 86%, 87%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,
97%, 98%, 98.5%, 99%, 99.5% or 99.9% overall homology to the
polypeptide encoded by the nucleotides set forth in SEQ ID
NOS:21-30.
[0181] Also provided herein are isolated AAV-X1, AAV-X1b, AAV-X5,
AAV-X19, AAV-X21, AAV-X22, AAV-X23, AAV-X24, AAV-X25, and AAV-X26
viruses. In one aspect, the isolated viruses can be used to produce
antibodies specific for each AAVX. Thus, provided is an isolated
antibody that specifically binds an AAVX-specific protein. The
isolated viruses can be used to detect antibodies specific for each
AAVX.
[0182] Thus, provided is an isolated antibody that specifically
binds an AAVX Rep protein, or a unique epitope thereof. Thus, also
provided is an isolated antibody that specifically bind AAVX Rep 52
or AAVX Rep 78, or a unique fragment thereof. Additionally provided
is an isolated antibody that specifically binds any of the AAVX
capsid proteins (VP1, VP2 or VP3), a unique epitope thereof, or the
polypeptide comprising all three AAVX coat proteins. Also provided
is an isolated antibody that specifically binds the AAVX capsid
protein having the amino acid sequence set forth in SEQ ID NO:21
(AAV-X1 VP1), or that specifically binds a unique fragment thereof.
Also provided is an isolated antibody that specifically binds the
AAVX capsid protein having the amino acid sequence set forth in SEQ
ID NO:22 (AAV-X1b VP1), or that specifically binds a unique
fragment thereof. Also provided is an isolated antibody that
specifically binds the AAVX capsid protein having the amino acid
sequence set forth in SEQ ID NO:23 (AAV-X5 VP1), or that
specifically binds a unique fragment thereof. Also provided is an
isolated antibody that specifically binds the AAVX capsid protein
having the amino acid sequence set forth in SEQ ID NO:24 (AAV-X19
VP1), or that specifically binds a unique fragment thereof. Also
provided is an isolated antibody that specifically binds the AAVX
capsid protein having the amino acid sequence set forth in SEQ ID
NO:25 (AAV-X21 VP1), or that specifically binds a unique fragment
thereof. Also provided is an isolated antibody that specifically
binds the AAVX capsid protein having the amino acid sequence set
forth in SEQ ID NO:26 (AAV-X22 VP1), or that specifically binds a
unique fragment thereof. Also provided is an isolated antibody that
specifically binds the AAVX capsid protein having the amino acid
sequence set forth in SEQ ID NO:27 (AAV-X23 VP1), or that
specifically binds a unique fragment thereof. Also provided is an
isolated antibody that specifically binds the AAVX capsid protein
having the amino acid sequence set forth in SEQ ID NO:28 (AAV-X24
VP1), or that specifically binds a unique fragment thereof. Also
provided is an isolated antibody that specifically binds the AAVX
capsid protein having the amino acid sequence set forth in SEQ ID
NO:29 (AAV-X25 VP1), or that specifically binds a unique fragment
thereof. Also provided is an isolated antibody that specifically
binds the AAVX capsid protein having the amino acid sequence set
forth in SEQ ID NO:30 (AAV-X26 VP1), or that specifically binds a
unique fragment thereof. Again, any given antibody can recognize
and bind one of a number of possible epitopes present in the
polypeptide; thus only a unique portion of a polypeptide (having
the epitope) needs to be present in an assay to determine if the
antibody specifically binds the polypeptide.
[0183] The antibody can be a component of a composition that
comprises an antibody that specifically binds the AAVX protein. The
composition can further comprise, e.g., serum, serum-free medium,
or a pharmaceutically acceptable carrier such as physiological
saline, etc.
[0184] By "an antibody that specifically binds" an AAVX polypeptide
or protein is meant an antibody that selectively binds to an
epitope on any portion of the AAVX peptide such that the antibody
binds specifically to the corresponding AAVX polypeptide without
significant background. Specific binding by an antibody further
means that the antibody can be used to selectively remove the
target polypeptide from a sample comprising the polypeptide or and
can readily be determined by radioimmunoassay (RIA), bioassay, or
enzyme-linked immunosorbant (ELISA) technology. An ELISA method
effective for the detection of the specific antibody-antigen
binding can, for example, be as follows: (1) bind the antibody to a
substrate; (2) contact the bound antibody with a sample containing
the antigen; (3) contact the above with a secondary antibody bound
to a detectable moiety (e.g., horseradish peroxidase enzyme or
alkaline phosphatase enzyme); (4) contact the above with the
substrate for the enzyme; (5) contact the above with a color
reagent; (6) observe the color change.
[0185] An antibody can include antibody fragments such as Fab
fragments which retain the binding activity. Antibodies can be made
as described in, e.g., Harlow and Lane, Antibodies: A Laboratory
Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.
(1988). Briefly, purified antigen can be injected into an animal in
an amount and in intervals sufficient to elicit an immune response.
Antibodies can either be purified directly, or spleen cells can be
obtained from the animal. The cells are then fused with an immortal
cell line and screened for antibody secretion. Individual
hybridomas are then propagated as individual clones serving as a
source for a particular monoclonal antibody.
[0186] Additionally provided is a method of screening a cell for
infectivity by AAVX, comprising contacting the cell with AAVX and
detecting the presence of AAVX in the cells. AAVX particles can be
detected using any standard physical or biochemical methods. For
example, physical methods that can be used for this detection
include DNA based methods such as 1) polymerase chain reaction
(PCR) for viral DNA or RNA or 2) direct hybridization with labeled
probes, and immunological methods such as by 3) antibody directed
against the viral structural or non-structural proteins. Catalytic
methods of viral detection include, but are not limited to,
detection of site and strand specific DNA nicking activity of Rep
proteins or replication of an AAV origin-containing substrate.
Reporter genes can also be utilized to detect cells that transduce
AAVX. For example, .beta.-gal, green fluorescent protein or
luciferase can be inserted into a recombinant AAVX. The cell can
then be contacted with the recombinant AAVX, either in vitro or in
vivo and a colorimetric assay could detect a color change in the
cells that would indicate transduction of AAVX in the cell.
Additional detection methods are outlined in Fields, Virology,
Raven Press, New York, N.Y. 1996.
[0187] Provided is a method of screening a cell for infectivity by
AAVX, wherein the presence of AAVX in the cells is determined by
nucleic acid hybridization methods, a nucleic acid probe for such
detection can comprise, for example, a unique fragment of any of
the AAVX nucleic acids provided herein. The uniqueness of any
nucleic acid probe can readily be determined as described herein.
Additionally, the presence of AAVX in cells can be determined by
fluorescence, antibodies to gene products, focus forming assays,
plaque lifts, Western blots and chromogenic assays. The nucleic
acid can be, for example, the nucleic acid whose nucleotide
sequence is set forth in SEQ ID NOS:1, 2, 3, 4, 5, 6, 7, 8, 9, 10,
11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 or a unique fragment
thereof.
[0188] Provided is a method of determining the suitability of an
AAVX vector for administration to a subject comprising contacting
an antibody-containing sample from the subject with an antigenic
fragment of an isolated AAVX Rep or Capsid protein, and detecting
an antibody-antigen reaction in the sample, the presence of a
neutralizing reaction indicating the AAVX vector to be unsuitable
for use in the subject. Further provided is a method of determining
the presence in a subject of an AAVX-specific antibody comprising
contacting an antibody-containing sample from the subject with an
antigenic fragment of an isolated AAVX Rep or Capsid protein and
detecting an antibody-antigen reaction in the sample, the presence
of a reaction indicating the presence of an AAVX-specific antibody
in the subject. The present methods of determining the suitability
of an AAVX vector for administration to a subject or the presence
of an AAVX-specific antibody in a subject can comprise contacting
an antibody-containing sample from the subject with a unique
antigenic or immunogenic fragment of an AAVX Rep protein (e.g. Rep
52, Rep 78) and detecting an antibody-antigen reaction in the
sample, the presence of a reaction indicating the presence of an
AAVX-specific antibody and therefore the AAVX vector to be
unsuitable for use in the subject. The AAVX Rep proteins are
provided herein, and their antigenic fragments are routinely
determined. The AAVX capsid protein can be used to select an
antigenic or immunogenic fragment, for example from the amino acid
sequence set forth in SEQ ID NO:21 (AAV-X1 VP1), the amino acid
sequence set forth in SEQ ID NO:22 (AAV-X1b VP1), the amino acid
sequence set forth in SEQ ID NO:23 (AAV-X5 VP1), the amino acid
sequence set forth in SEQ ID NO:24 (AAV-X19 VP1), the amino acid
sequence set forth in SEQ ID NO:25 (AAV-X21 VP1), the amino acid
sequence set forth in SEQ ID NO:26 (AAV-X22 VP1), the amino acid
sequence set forth in SEQ ID NO:27 (AAV-X23 VP1, the amino acid
sequence set forth in SEQ ID NO:28 (AAV-X24 VP1), the amino acid
sequence set forth in SEQ ID NO:29 (AAV-X25 VP1), or the amino acid
sequence set forth in SEQ ID NO:30 (AAV-X26 VP1)
[0189] Alternatively, or additionally, an antigenic or immunogenic
fragment of an isolated AAVX Rep protein can be utilized in this
determination method. Any given antibody can recognize and bind one
of a number of possible epitopes present in the polypeptide; thus
only a unique portion of a polypeptide (having the epitope) may
need to be present in an assay to determine if the antibody
specifically binds the polypeptide.
[0190] The AAVX polypeptide fragments can be analyzed to determine
their antigenicity, immunogenicity and/or specificity. Briefly,
various concentrations of a putative immunogenically specific
fragment are prepared and administered to a subject and the
immunological response (e.g., the production of antibodies or cell
mediated immunity) of an animal to each concentration is
determined. The amounts of antigen administered depend on the
subject, e.g. a human, rabbit or a guinea pig, the condition of the
subject, the size of the subject, etc. Thereafter an animal so
inoculated with the antigen can be exposed to the AAVX viral
particle or AAVX protein to test the immunoreactivity or the
antigenicity of the specific immunogenic fragment. The specificity
of a putative antigenic or immunogenic fragment can be ascertained
by testing sera, other fluids or lymphocytes from the inoculated
animal for cross reactivity with other closely related viruses,
such as AAV1-11, AAAV, or BAAV.
[0191] By the "suitability of an AAVX vector for administration to
a subject" is meant a determination of whether the AAVX vector will
elicit a neutralizing immune response upon administration to a
particular subject. A vector that does not elicit a significant
immune response is a potentially suitable vector, whereas a vector
that elicits a significant, neutralizing immune response (e.g. at
least 90%) is thus likely to be unsuitable for use in that subject.
Significance of any detectable immune response is a standard
parameter understood by the skilled artisan in the field. For
example, one can incubate the subject's serum with the virus, then
determine whether that virus retains its ability to transduce cells
in culture. If such virus cannot transduce cells in culture, the
vector likely has elicited a significant immune response.
[0192] Alternatively, or additionally, one skilled in the art could
determine whether or not AAVX administration would be suitable for
a particular cell type of a subject. For example, the artisan could
culture muscle cells in vitro and transduce the cells with AAVX in
the presence or absence of the subject's serum. If there is a
reduction in transduction efficiency, this could indicate the
presence of a neutralizing antibody or other factors that may
inhibit transduction. Normally, greater than 90% inhibition would
have to be observed in order to rule out the use of AAVX as a
vector. However, this limitation could be overcome by treating the
subject with an immunosuppressant that could block the factors
inhibiting transduction.
[0193] As will be recognized by those skilled in the art, numerous
types of immunoassays are available for use in the present methods
to detect binding between an antibody and an AAVX polypeptide as
provided herein. For instance, direct and indirect binding assays,
competitive assays, sandwich assays, and the like, as are generally
described in, e.g., U.S. Pat. Nos. 4,642,285; 4,376,110; 4,016,043;
3,879,262; 3,852,157; 3,850,752; 3,839,153; 3,791,932; and Harlow
and Lane, Antibodies, A Laboratory Manual, Cold Spring Harbor
Publications, N.Y. (1988). For example, enzyme immunoassays such as
immunofluorescence assays (IFA), enzyme linked immunosorbent assays
(ELISA) and immunoblotting can be readily adapted to accomplish the
detection of the antibody. An ELISA method effective for the
detection of the antibody bound to the antigen can, for example, be
as follows: (1) bind the antigen to a substrate; (2) contact the
bound antigen with a fluid or tissue sample containing the
antibody; (3) contact the above with a secondary antibody specific
for the antigen and bound to a detectable moiety (e.g., horseradish
peroxidase enzyme or alkaline phosphatase enzyme); (4) contact the
above with the substrate for the enzyme; (5) contact the above with
a color reagent; (6) observe color change.
[0194] The antibody-containing sample of this method can comprise
any biological sample which would contain the antibody or a cell
containing the antibody, such as blood, plasma, serum, bone marrow,
saliva and urine.
[0195] Also provided is a method of producing the AAVX virus by
transducing a cell with the nucleic acid encoding the virus.
[0196] The present method further provides a method of delivering
an exogenous nucleic acid to a cell comprising administering to the
cell an AAVX particle containing a vector comprising the nucleic
acid inserted between a pair of AAV inverted terminal repeats,
thereby delivering the nucleic acid to the cell.
[0197] The AAV ITRs in the vector for the herein described delivery
methods can be AAVX ITRs. Furthermore, the AAV ITRs in the vector
for the herein described nucleic acid delivery methods can also
comprise AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9,
AAV10, AAV11, AAAV, or BAAV inverted terminal repeats.
[0198] Also provided is a method of delivering an exogenous nucleic
acid to a subject comprising administering to a cell of or from the
subject an AAVX particle containing a vector comprising the nucleic
acid inserted between a pair of AAV inverted terminal repeats, and
returning the cell to the subject, thereby delivering the nucleic
acid to the subject. The AAV ITRs can be any AAV ITRs, including
AAVX ITRs, AAV5 ITRs and AAV2 ITRs. For example, in an ex vivo
administration, cells are isolated from a subject by standard means
according to the cell type and placed in appropriate culture
medium, again according to cell type (see, e.g., ATCC catalog).
Viral particles are then contacted with the cells as described
above, and the virus is allowed to transduce the cells. Cells can
then be transplanted back into the subject's body, again by means
standard for the cell type and tissue (e.g., in general, U.S. Pat.
No. 5,399,346; for neural cells, Dunnett, S. B. and Bjorklund, A.,
eds., Transplantation: Neural Transplantation-A Practical Approach,
Oxford University Press, Oxford (1992)). If desired, prior to
transplantation, the cells can be studied for degree of
transduction by the virus, by known detection means and as
described herein. Cells for ex vivo transduction followed by
transplantation into a subject can be selected from those listed
above, or can be any other selected cell. Preferably, a selected
cell type is examined for its capability to be transfected by AAVX.
Preferably, the selected cell will be a cell readily transduced
with AAVX particles; however, depending upon the application, even
cells with relatively low transduction efficiencies can be useful,
particularly if the cell is from a tissue or organ in which even
production of a small amount of the protein or antisense RNA
encoded by the vector will be beneficial to the subject.
[0199] Further provided is a method of delivering an exogenous
nucleic acid to a cell in a subject comprising administering to the
subject an AAVX particle containing a vector comprising the nucleic
acid inserted between a pair of AAV inverted terminal repeats,
thereby delivering the nucleic acid to a cell in the subject.
Administration can be an ex vivo administration directly to a cell
removed from a subject, such as any of the cells listed above,
followed by replacement of the cell back into the subject, or
administration can be in vivo administration to a cell in the
subject. For ex vivo administration, cells are isolated from a
subject by standard means according to the cell type and placed in
appropriate culture medium, again according to cell type (see,
e.g., ATCC catalog). Viral particles are then contacted with the
cells as described above, and the virus is allowed to transfect the
cells. Cells can then be transplanted back into the subject's body,
again by means standard for the cell type and tissue (e.g., for
neural cells, Dunnett, S. B. and Bjorklund, A., eds.,
Transplantation: Neural Transplantation-A Practical Approach,
Oxford University Press, Oxford (1992)). If desired, prior to
transplantation, the cells can be studied for degree of
transfection by the virus, by known detection means and as
described herein.
[0200] Further provided is a method of delivering a nucleic acid to
a cell in a subject having neutralizing antibodies to AAV1, AAV2,
AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAAV, or
BAAV comprising administering to the subject an AAVX particle
containing a vector comprising the nucleic acid, thereby delivering
the nucleic acid to a cell in the subject. A subject that has
neutralizing antibodies to AAV1, AAV2, AAV3, AAV4, AAV5, AAV6,
AAV7, AAV8, AAV9, AAV10, AAV11, AAAV, or BAAV can readily be
determined by any of several known means, such as contacting AAV 1,
AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAAV,
or BAAV protein(s) with an antibody-containing sample, such as
blood, from a subject and detecting an antigen-antibody reaction in
the sample. Delivery of the AAV1, AAV2, AAV3, AAV4, AAV5, AAV6,
AAV7, AAV8, AAV9, AAV10, AAV11, AAAV, or BAAV particle can be by
either ex vivo or in vivo administration as herein described. Thus,
a subject who might have an adverse immunogenic reaction to a
vector administered in an AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7,
AAV8, AAV9, AAV10, AAV11, AAAV, or BAAV viral particle can have a
desired nucleic acid delivered using an AAVX particle. This
delivery system can be particularly useful for subjects who have
received therapy utilizing AAV1, AAV2, AAV3, AAV4, AAV5, AAV6,
AAV7, AAV8, AAV9, AAV10, AAV11, AAAV, or BAAV particles in the past
and have developed antibodies to AAV1, AAV2, AAV3, AAV4, AAV5,
AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAAV, or BAAV. An AAVX
regimen can now be substituted to deliver the desired nucleic
acid.
[0201] In any of the methods of delivering exogenous nucleic acids
to a cell or subject described herein, the AAVX-conjugated nucleic
acid or AAVX particle-conjugated nucleic acids described herein can
be used.
[0202] In vivo administration to a human subject or an animal model
can be by any of many standard means for administering viruses,
depending upon the target organ, tissue or cell. Virus particles
can be administered orally, parenterally (e.g., intravenously), by
intramuscular injection, intrarectally, by direct tissue or organ
injection, by intraperitoneal injection, topically, transdermally,
via aerosol delivery, via the mucosa or the like. Viral nucleic
acids (non-encapsidated) can also be administered, e.g., as a
complex with cationic liposomes, or encapsulated in anionic
liposomes. The present compositions can include various amounts of
the selected viral particle or non-encapsidated viral nucleic acid
in combination with a pharmaceutically acceptable carrier and, in
addition, if desired, may include other medicinal agents,
pharmaceutical agents, carriers, adjuvants, diluents, etc. Parental
administration, if used, is generally characterized by injection.
Injectables can be prepared in conventional forms, either as liquid
solutions or suspensions, solid forms suitable for solution or
suspension in liquid prior to injection, or as emulsions. Dosages
will depend upon the mode of administration, the disease or
condition to be treated, and the individual subject's condition,
but will be that dosage typical for and used in administration of
other AAV vectors, such as AAV2 vectors. Often a single dose can be
sufficient; however, the dose can be repeated if desirable.
Administration methods for gene delivery to the cochlea are routine
and are described in Jero, J. et al. (Gene Ther. 2001 Mar. 20;
12(5):539-48) and Staecker H, et al. (Acta Otolaryngol. 2001
January; 121(2):157-63), both references herein incorporated by
reference for these methods.
[0203] Administration methods can be used to treat brain disorders
such as Parkinson's disease, Alzheimer's disease, and demyelination
disease. Other diseases that can be treated by these methods
include metabolic disorders such as musculoskeletal diseases,
cardiovascular disease, cancer, and autoimmune disorders.
[0204] Administration of this recombinant AAVX virion to the cell
can be accomplished by any means, including simply contacting the
particle, optionally contained in a desired liquid such as tissue
culture medium, or a buffered saline solution, with the cells. The
virion can be allowed to remain in contact with the cells for any
desired length of time, and typically the virion is administered
and allowed to remain indefinitely. For such in vitro methods, the
virion can be administered to the cell by standard viral
transduction methods, as known in the art and as exemplified
herein. Titers of virus to administer can vary, particularly
depending upon the cell type, but will be typical of that used for
AAV transduction in general which is well known in the art.
Additionally the titers used to transduce the particular cells in
the present examples can be utilized.
[0205] The cells that can be transduced by the present recombinant
AAVX virion can include any desired cell, such as the following
cells and cells derived from the following tissues, human as well
as other mammalian tissues, such as primate, horse, sheep, goat,
pig, dog, rat, and mouse and avian species: Adipocytes, Adenocyte,
Adrenal cortex, Amnion, Aorta, Ascites, Astrocyte, Bladder, Bone,
Bone marrow, Brain, Breast, Bronchus, Cardiac muscle, Cecum,
Cervix, Chorion, Cochlear, Colon, Conjunctiva, Connective tissue,
Cornea, Dermis, Duodenum, Embryonic stem cells, Endometrium,
Endothelium, Endothelial cells, Epithelial tissue, Epithelial
cells, Epidermis, Esophagus, Eye, Fascia, Fibroblasts, Foreskin,
Gastric, Glial cells, Glioblast, Gonad, Hepatic cells, Histocyte,
Hair cells in the inner ear, auditory (organ of Corti) sensory
epithelia, vestibular sensory epithelia, Ileum, Intestine, small
Intestine, Jejunum, Keratinocytes, Kidney, Larynx, Leukocytes,
Lipocyte, Liver, Lung, Lymph node, Lymphoblast, Lymphocytes,
Macrophages, Mammary alveolar nodule, Mammary gland, Mastocyte,
Maxilla, Melanocytes, Mesenchymal, Monocytes, Mouth, Myelin,
Myoblasts Nervous tissue, Neuroblast, Neurons, Neuroglia,
Osteoblasts, Osteogenic cells, Ovary, Palate, Pancreas, Papilloma,
Peritoneum, Pituicytes, Pharynx, Placenta, Plasma cells, Pleura,
Prostate, Rectum, Salivary gland, Skeletal muscle, Skin, Smooth
muscle, Somatic, Spleen, Squamous, Stem cells, Stomach,
Submandibular gland, Submaxillary gland, Synoviocytes, Testis,
Thymus, Thyroid, Trabeculae, Trachea, Turbinate, Umbilical cord,
Ureter, Uterus, and vestibular hair cells.
[0206] For example, provided herein is a method of transducing a
cancer cell (e.g., lung cancer cell, non-small cell lung cancer
cell), colon cell, CNS derived cell, ovarian cell, prostate cell,
breast derived cell, cervical cord cell, kidney cell, salivary
gland cell, or muscle cell using an AAV particle disclosed
herein.
[0207] Also provided herein is a method of delivering a nucleic
acid to a cancer cell (e.g., lung cancer cell, non-small cell lung
cancer cell), colon cell, CNS derived cell, ovarian cell, prostate
cell, breast derived cell, cervical cord cell, kidney cell,
salivary gland cell, or muscle cell comprising administering to the
cell an AAV-X1, AAV-X1b, AAV-X5, AAV-X19, AAV-X21, AAV-X22,
AAV-X23, AAV-X24, AAV-X25, or AAV-X26 particle containing a vector
comprising the nucleic acid inserted between a pair of AAV inverted
terminal repeats, thereby delivering the nucleic acid to the
cell.
[0208] The cell of the provided methods can be an inner ear
epithelial cell. Thus, the cell of the provided method can be an
inner ear hair cell. The cell of the provided methods can be an
inner or outer hair cell of the organ of Corti or a vestibular hair
cell. The cell of the provided methods can be an inner ear
supporting cell such as Hensen's, phalangal, interdental, or
vestibular supporting cells.
[0209] The cell of the provided method can be an airway epithelial
cell. The cell of the provided method can be a columnar, goblet or
basal cell.
[0210] The cell of the provided method can be a cell of the
submandibular gland. The cell of the provided method can be a
ductal or acinar cell.
[0211] Provided are recombinant vectors based on AAVX. Such vectors
may be useful for transducing erythroid progenitor cells or cells
resistant to transduction by other serotypes of AAV. These vectors
may also be useful for transducing cells with a nucleic acid of
interest in order to produce cell lines that could be used to
screen for agents that interact with the gene product of the
nucleic acid of interest. In addition to transduction of other cell
types, transduction of erythroid cells would be useful for the
treatment of cancer and genetic diseases which can be corrected by
bone marrow transplants using matched donors. Some examples of this
type of treatment include, but are not limited to, the introduction
of a therapeutic gene such as genes encoding interferons,
interleukins, tumor necrosis factors, adenosine deaminase, cellular
growth factors such as lymphokines, blood coagulation factors such
as factor VIII and IX, cholesterol metabolism uptake and transport
protein such as EpoE and LDL receptor, and antisense sequences to
inhibit viral replication of, for example, hepatitis or HIV.
[0212] Provided is a vector, comprising the AAVX virus as well as
AAVX viral particles. While AAVX is similar to AAV1-11, the viruses
are found herein to be physically and genetically distinct. These
differences endow AAVX with some unique advantages, which better
suit it as a vector for gene therapy.
[0213] Furthermore, as shown herein, AAVX capsid protein is
distinct from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9,
AAV10, AAV11, AAAV, or BAAV capsid proteins and exhibits different
tissue tropism. AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8,
AAV9, AAV10, AAV11, AAAV, BAAV, and AAVX likely utilize distinct
cellular receptors. AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8,
AAV9, AAV10, AAV11, AAAV, BAAV, and AAVX are serologically distinct
and humans are not reported to have neutralizing antibodies to
AAVX, thus in a gene therapy or gene transfer application, AAVX
would allow for transduction of a patient who already possess
neutralizing antibodies to AAV1, AAV2, AAV3, AAV4, AAV5, AAV6,
AAV7, AAV8, AAV9, AAV10, AAV11, AAAV, or BAAV either as a result of
natural immunological defense or from prior exposure to AAV1, AAV2,
AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAAV, or
BAAV vectors.
[0214] Each of the herein disclosed AAVX vectors, or vectors
comprising AAVX capsid proteins, can have unique or distinct tissue
tropism. As shown in FIG. 11, transduction profiles of AAV-X1,
AAV-X5, AAV-X25 and AAV-X26 were different from that of known AAVs.
AAVX1 and AAVX25 utilize a 2-3 link sialic acid as a cell
attachment factor, whereas AAVX26 does not utilize sialic acid in
the transduction process. Further, AAV12 (AAVX26) can be used to
transduce cells lacking cell surface heparan sulfate or
glycosphingolipids. Instead, mannosamine can be part of the AAV12
receptor or attachment.
[0215] As disclosed herein, cells that can be transduced by AAV12
include, but are not limited to, non-small cell lung cancer cells
(e.g., NSCLC), (e.g., A549, EKVX, NCI-H226), colon cells (e.g.,
HCT-15), CNS derived cells (e.g., SF-268 and SF295), ovarian cells
(e.g., IGROV1), prostate cells (e.g., PC-3), breast derived cells
(e.g., T-47D), kidney cells (e.g., 293T), salivary glands (e.g.,
ductal cells), muscle cells.
[0216] Further, many of the AAV contaminated simian adenoviruses
were originally isolated from pooled primary kidney cell cultures
(Hull et al. 1957, 1956, 1958) originally established as part of a
vaccine development program (VR-195, VR-197, VR-198, VR-200,
VR-202, VR-209 and VR-353), but also from rectal swabs (VR-204),
cervical cord (VR-355) and CNS cultures (VR-207) demonstrating that
the tropism of the AAVs isolated from these adenovirus stocks
include cells of kidney, cervical cord and CNS origin.
[0217] Thus, provided herein is a method of transducing a cancer
cell (e.g., lung cancer cell, non-small cell lung cancer cell),
colon cell, CNS derived cell, ovarian cell, prostate cell, breast
derived cell, cervical cord cell, kidney cell, salivary gland cell,
or muscle cell using an AAV particle disclosed herein.
[0218] Thus, provided herein is a method of delivering a nucleic
acid to a cancer cell (e.g., lung cancer cell, non-small cell lung
cancer cell) comprising administering to the cell an AAV12 particle
containing a vector comprising the nucleic acid inserted between a
pair of AAV inverted terminal repeats, thereby delivering the
nucleic acid to the cell.
[0219] Thus, provided herein is a method of delivering a nucleic
acid to a colon cell comprising administering to the cell an AAV12
particle containing a vector comprising the nucleic acid inserted
between a pair of AAV inverted terminal repeats, thereby delivering
the nucleic acid to the cell.
[0220] Thus, provided herein is a method of delivering a nucleic
acid to a CNS derived cell comprising administering to the cell an
AAV12 particle containing a vector comprising the nucleic acid
inserted between a pair of AAV inverted terminal repeats, thereby
delivering the nucleic acid to the cell.
[0221] Thus, provided herein is a method of delivering a nucleic
acid to an ovarian cell comprising administering to the cell an
AAV12 particle containing a vector comprising the nucleic acid
inserted between a pair of AAV inverted terminal repeats, thereby
delivering the nucleic acid to the cell.
[0222] Thus, provided herein is a method of delivering a nucleic
acid to a prostate cell comprising administering to the cell an
AAV12 particle containing a vector comprising the nucleic acid
inserted between a pair of AAV inverted terminal repeats, thereby
delivering the nucleic acid to the cell.
[0223] Thus, provided herein is a method of delivering a nucleic
acid to a breast derived cell comprising administering to the cell
an AAV12 particle containing a vector comprising the nucleic acid
inserted between a pair of AAV inverted terminal repeats, thereby
delivering the nucleic acid to the cell.
[0224] Thus, provided herein is a method of delivering a nucleic
acid to a cervical cord cell comprising administering to the cell
an AAV12 particle containing a vector comprising the nucleic acid
inserted between a pair of AAV inverted terminal repeats, thereby
delivering the nucleic acid to the cell.
[0225] Thus, provided herein is a method of delivering a nucleic
acid to a kidney cell comprising administering to the cell an
AAV-X1, AAV-X1b, AAV-X5, AAV-X19, AAV-X21, AAV-X22, AAV-X23,
AAV-X24, AAV-X25, or AAV-X26 particle containing a vector
comprising the nucleic acid inserted between a pair of AAV inverted
terminal repeats, thereby delivering the nucleic acid to the
cell.
[0226] Thus, provided herein is a method of delivering a nucleic
acid to a salivary gland cell comprising administering to the cell
an AAV12 particle containing a vector comprising the nucleic acid
inserted between a pair of AAV inverted terminal repeats, thereby
delivering the nucleic acid to the cell.
[0227] Thus, provided herein is a method of delivering a nucleic
acid to a muscle cell comprising administering to the cell an AAV12
particle containing a vector comprising the nucleic acid inserted
between a pair of AAV inverted terminal repeats, thereby delivering
the nucleic acid to the cell.
Vector System
[0228] Provided herein is a vector system for producing infectious
virus particles having a characteristic of AAVX. As used herein, a
"vector system" is a combination of one or more vectors that, when
added to an appropriate cell system, can produce a recombinant AAVX
virion, as provided herein. Thus, the provided vector system
comprises at least one vector comprising a nucleic acid selected
from the group consisting of a pair of AAV ITRs, a nucleic acid
encoding an AAV capsid protein, and a nucleic acid encoding an AAV
Rep protein. In addition, it is understood that an AAV vector
system comprises at least one adenovirus helper plasmid.
[0229] The vector system can comprise one or more unique AAV
vectors. Thus, the vector system can comprise, for example, 1, 2,
3, 4, 5, or 6 unique AAV vectors. In a three-vector system, the
first AAV vector can comprise a nucleic acid encoding an AAV capsid
protein, the second AAV vector can comprise a nucleic acid encoding
an AAV Rep protein, and the third AAV vector can comprise a pair of
AAV ITRs (Table 3). It is understood that Rep and Cap sequences can
be efficiently combined in the same vector. Thus, in an example of
a two-vector vector system, the first AAV vector can comprise a
nucleic acid encoding an AAV capsid protein and a nucleic acid
encoding an AAV Rep protein and the second AAV vector can comprise
a pair of AAV ITRs (Table 3). It is understood that at least one
AAV vector in the provided AAVX vector system comprises an AAVX
capsid, Rep or ITR (Table 3).
[0230] Thus, provided is an AAV vector system, wherein the first
vector can comprise a nucleic acid encoding an AAVX capsid protein
and the second vector can comprise a pair of AAV ITRs. The AAV ITRs
of the second vector can be a pair of AAV1 ITRs. The AAV inverted
terminal repeats can be a pair of AAV2 ITRs. The AAV ITRs can be a
pair of AAV3 ITRs. The AAV ITRs can be a pair of AAV4 ITRs. The AAV
ITRs can be a pair of AAV5 ITRs. The AAV ITRs can be a pair of AAV6
ITRs. The AAV ITRs can be a pair of AAV7 ITRs. The AAV ITRs can be
a pair of AAV8 ITRs. The AAV ITRs can be a pair of AAV9 ITRs. The
AAV ITRs can be a pair of AAV 10 ITRs. The AAV ITRs can be a pair
of AAV 11 ITRs. The AAV ITRs can be a pair of AAAV ITRs. The AAV
ITRs can be a pair of BAAV ITRs. The AAV ITRs can be a pair of AAVX
ITRs.
[0231] Also provided is an AAV vector system, wherein the first
vector can comprise a nucleic acid encoding an AAV capsid protein
and the second vector can comprise a pair of AAVX ITRs. The capsid
protein can be an AAV1 capsid protein. The capsid protein can be an
AAV2 capsid protein. The capsid protein can be an AAV3 capsid
protein. The capsid protein can be an AAV4 capsid protein. The
capsid protein can be an AAV5 capsid protein. The capsid protein
can be an AAV6 capsid protein. The capsid protein can be an AAAV
Rep protein. The capsid protein can be a BAAV Rep protein. The
capsid protein can be an AAVX Rep protein.
[0232] In either of the above vector systems, the first vector or a
third vector can further comprise a nucleic acid encoding an AAV
Rep protein. The AAV Rep protein can be AAV1 Rep protein. The AAV
Rep protein can be AAV2 Rep protein. The AAV Rep protein can be
AAV3 Rep protein. The AAV Rep protein can be AAV4 Rep protein. The
AAV Rep protein can be AAV5 Rep protein. The AAV Rep protein can be
AAV6 Rep protein. The AAV Rep protein can be AAV7 Rep protein. The
AAV Rep protein can be AAV8 Rep protein. The AAV Rep protein can be
AAV9 Rep protein. The AAV Rep protein can be AAV10 Rep protein. The
AAV Rep protein can be AAV11 Rep protein. The AAV Rep protein can
be AAAV Rep protein. The AAV Rep protein can be BAAV Rep protein.
The AAV Rep protein can be AAVX Rep protein.
[0233] Also provided is an AAV vector system, wherein the first
vector can comprise a nucleic acid encoding an AAVX Rep protein and
the second vector can comprise a pair of AAV ITRs. The AAV ITRs of
the second vector can be a pair of AAV1 ITRs. The AAV inverted
terminal repeats can be a pair of AAV2 ITRs. The AAV ITRs can be a
pair of AAV3 ITRs. The AAV ITRs can be a pair of AAV4 ITRs. The AAV
ITRs can be a pair of AAV5 ITRs. The AAV ITRs can be a pair of AAV6
ITRs. The AAV ITRs can be a pair of AAV7 ITRs. The AAV ITRs can be
a pair of AAV8 ITRs. The AAV ITRs can be a pair of AAV9 ITRs. The
AAV ITRs can be a pair of AAV10 ITRs. The AAV ITRs can be a pair of
AAV 11 ITRs. The AAV ITRs can be a pair of AAAV ITRs. The AAV ITRs
can be a pair of BAAV ITRs. The AAV ITRs can be a pair of AAVX
ITRs.
[0234] The first vector or a third vector can further comprise a
nucleic acid encoding an AAV Capsid protein. The capsid protein can
be an AAV1 capsid protein. The capsid protein can be an AAV2 capsid
protein. The capsid protein can be an AAV3 capsid protein. The
capsid protein can be an AAV4 capsid protein. The capsid protein
can be an AAV5 capsid protein. The capsid protein can be an AAV6
capsid protein. The capsid protein can be an AAAV Rep protein. The
capsid protein can be a BAAV Rep protein. The capsid protein can be
an AAVX Rep protein.
TABLE-US-00003 TABLE 3 AAVX Vector Systems First Vector Second
Vector Third Vector AAVX capsid + AAV Rep AAV ITR -- AAV capsid +
AAV Rep AAVX ITR -- AAV capsid + AAVX Rep AAV ITR AAVX capsid AAV
ITR AAV Rep AAV capsid AAVX ITR AAV Rep AAV capsid AAV ITR AAVX Rep
"AAV" includes: AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8,
AAV9, AAV10, AAV11, AAAV, BAAV, AAV-X1, AAV-X1b, AAV-X5, AAV-X19,
AAV-X21, AAV-X22, AAV-X23, AAV-X24, AAV-X25, or AAV-X26. "AAVX"
includes: AAV-X1, AAV-X1b, AAV-X5, AAV-X19, AAV-X21, AAV-X22,
AAV-X23, AAV-X24, AAV-X25, or AAV-X26.
[0235] In either of the above vector systems, the second vector
comprising a pair of AAV ITRs can further comprise a promoter
between the ITRs. The promoter can be AAV2 p5 promoter. The
promoter can be AAV5 p5 promoter. The promoter can be AAVX p5
promoter. Furthermore, smaller fragments of p5 promoter that retain
promoter activity can readily be determined by standard procedures
including, for example, constructing a series of deletions in the
p5 promoter, linking the deletion to a reporter gene, and
determining whether the reporter gene is expressed, i.e.,
transcribed and/or translated. The promoter can be the AAVX p19
promoter. The promoter can be the AAVX p40 promoter. The promoter
can be a promoter of any of the AAV serotypes. The promoter can be
a constitutive promoter. Thus, the promoter can be CMV. The
promoter can be RSV. The promoter can be LTR. The promoter can be
eF1. The promoter can be beta actin promoter. The promoter can be a
tissue specific promoter. The promoter can be an inducible
promoter. The promoter can further be functionally linked to an
exogenous nucleic acid.
[0236] Further provided is any of the disclosed vectors of the
vector systems encapsidated into an AAV particle. The AAV particle
can be an AAV1 virus particle comprising at least one AAV1 capsid
protein. The AAV particle can be an AAV2 virus particle comprising
at least one AAV2 capsid protein. The AAV particle can be an AAV3
virus particle comprising at least one AAV3 capsid protein. The AAV
particle can be an AAV4 virus particle comprising at least one AAV4
capsid protein. The AAV particle can be an AAV5 virus particle
comprising at least one AAV5 capsid protein. The AAV particle can
be an AAV6 virus particle comprising at least one AAV6 capsid
protein. The AAV particle can be an AAV7 virus particle comprising
at least one AAV7 capsid protein. The AAV particle can be an AAV8
virus particle comprising at least one AAV8 capsid protein. The AAV
particle can be an AAV9 virus particle comprising at least one AAV9
capsid protein. The AAV particle can be an AAV10 virus particle
comprising at least one AAV10 capsid protein. The AAV particle can
be an AAV 11 virus particle comprising at least one AAV 11 capsid
protein. The AAV particle can be an AAAV virus particle comprising
at least one AAAV capsid protein. The AAV particle can be a BAAV
virus particle comprising at least one BAAV capsid protein. The AAV
particle can be an AAVX virus particle comprising at least one AAVX
capsid protein. The AAV particle can be a chimeric capsid virus
particle (described above) comprising a capsid protein from more
than one serotype of AAV.
EXAMPLES
[0237] It is understood that the disclosed method and compositions
are not limited to the particular methodology, protocols, and
reagents described as these may vary. It is also to be understood
that the terminology used herein is for the purpose of describing
particular embodiments only, and is not intended to limit the scope
of the present invention which will be limited only by the appended
claims.
Example 1
Identification and Characterization of Novel AAV Isolates in ATCC
Virus Stocks
[0238] Materials and Methods
[0239] Cell Culture and Virus Propagation:
[0240] 293T and COS cells were maintained in DMEM, supplemented
with 10% FBS, 2 mM L-glutamine, 100 U/ml penicillin, and 0.1 mg/ml
streptomycin or RPMI with 5% FBS. SF295, MCF7, EKVX, Igrov1, CAKI,
Ovcar 5 cells were cultured in RPMI supplemented with 5% FBS, 2 mM
L-glutamine, 100 U/ml penicillin, and 0.1 mg/ml streptomycin. Cells
were maintained at 37.degree. C. in a 5% CO.sub.2 humidified
atmosphere.
[0241] Screening for Novel AAVs:
[0242] 109 virus samples, obtained from the ATCC (American Type
Culture Collection) as lysate of infected cells, were analyzed for
the presence of AAV DNA by PCR as described earlier (Katano, H., et
al. 2004. Biotechniques 36:676-80). Briefly, low molecular weight
DNA was purified with High Pure Viral Nucleic Acid Kit (Roche).
These DNA samples were assayed for AAV contamination by PCR using
the GC Rich PCR Kit (Roche). This method detects the presence of
AAV DNA by PCR using degenerative PCR primers, which were shown to
amplify a fragment containing sequences of the rep and vp ORF of
all known AAV serotypes. PCR using DNA isolated from ATCC VR-195,
VR-197, VR-198, VR-200, VR-202, VR-204, VR-207, VR-209, VR-353,
VR-355, VR-942, VR-943 as template resulted in the generation of a
1.4 kb amplification product, which was subsequently cloned using
the TOPO TA Cloning KIT (Invitrogen) and sequenced with an ABI
Prism 3100 Genetic Analyzer (ABI) and FS dye-terminator chemistry
(ABI). The obtained sequences showed homology to AAV6 rep ORF and
cap ORF but were not identical to any known AAV.
[0243] Viral DNA Isolation, Cloning and Sequencing:
[0244] The rep and cap ORF of the new AAVs was PCR amplified and
subcloned. Viral DNA that was isolated from VR-195, VR-197, VR-198,
VR-200, VR-202, VR-204, VR-207, VR-209, VR-353, VR-355, VR-942,
VR-943 was PCR amplified with primers:
AAV1-4 225(+): GCGACAKTTTGCGACACCAYGTGG (SEQ ID NO:31) and
UNI-NC: CCANNNGGAATCGCAATGCCAAT (SEQ ID NO:32), or
UNIC: ATGNTNATNTGGTGGGAGGAGGG (SEQ ID NO:33) and
[0245] AAV1-4 polyA4400(-): CGAATNAAMCGGTTTATTGATTAAC (SEQ ID
NO:34), or
AAV 4500 (+): CAATAAACCGkkTnATTCGTkTCAGT (SEQ ID NO:38) and
AAV 450 (-): ACANNWGAGTCAGAAATKCCNGGCAG (SEQ ID NO:39) (N can be A,
C, G, or T; K can be G or T; Y can be C or T; M can be A or C)
[0246] to amplify the Rep ORF, capsid ORF, and ITR(3'-AAVX
terminus, ITR and 3'-terminus of circular or concatermerized AAVs),
respectively. The PCR products were subcloned using the TOPO TA
Cloning KIT (Invitrogen) and at least three clones of each isolate
were sequenced with an ABI Prism 3100 Genetic Analyzer (ABI) and FS
dye-terminator chemistry (ABI). The obtained sequences showed
homology to AAV1 and AAV6 but were not identical to any known AAV.
As shown in Table 5, there are three naming conventions used
herein. For example, the newly identified AAVs were named according
to the ATCC adenovirus strains from which they were discovered
(e.g., AAV(VR-943)). Also, each unique AAV isolate was assigned a
temporary identifier (e.g., AAV-X26). Finally, . . . (e.g.,
AAV-12).
[0247] Identical AAV sequences were detected in VR-195, VR-197,
VR-198 and VR-202 and named AAV-X1. VR-195 also contained a homolog
of AAV-X1 termed AAV-X1b.
[0248] AAV detected in VR-200 was named AAV-X19. AAV detected in
VR-204 was named AAV-X21. AAV detected in VR-207 was named AAV-X22.
AAV detected in VR-209 was named AAV-X23. AAV detected in VR-353
was named AAV-X24. AAV detected in VR-355 was named AAV-X25. AAV
detected in VR-942 was named AAV-X5. AAV detected in VR-943 was
named AAV-X26. (Table 5).
[0249] Sequence Analysis:
[0250] DNA and protein sequence alignments were performed using the
Clustal W multiple sequence alignment tool of the Biology Workbench
web based software (SDSC), MacVector 7 (Oxford Molecular). The
sequences amplified from ATCC virus stocks were compared to
sequences in GenBank using BLAST (Basic Local Alignment Search Tool
at http://www.ncbi.nlm.nih.gov/BLAST/). DNA alignments were
performed using the ClustalW multiple sequence alignment tool of
the Biology Workbench web based software at http://seqtool.sdsc.edu
(SDSC) and MacVector 7 (Accelrys, Burlington, Mass.). Divergent
amino acids in VP1 were mapped on the capsid by superimposing the
VP1 sequence onto a pseudoatomic structure for AAV-6. The AAV6
crystal structure was predicted by remodeling the AAV2 capsid
structure, obtained from the Virus Particle Explorer, at the
Swiss-Model server with the AAV6 capsid sequence.
[0251] AAV-X17 partial ITR sequence was 98% identical to
corresponding AAV2 ITR sequence. AAV-X22 partial ITR sequence was
99% identical to corresponding AAV2 ITR sequence. AAV-X25 partial
ITR sequence was 96% identical to corresponding AAV1 ITR sequence.
AAV-X26 was only about 80% identical to corresponding AAV2 ITR
sequence. However, the TRS signal (e.g., aa 176-181 of SEQ ID
NO:40) and Rep Binding site (e.g., aa 195-210 of SEQ ID NO:40) were
conserved in AAV-X26 ITR as compared to the AAV2 ITR.
[0252] Generation of Recombinant Virus:
[0253] Recombinant AAV-X1, AAV-X1b, AAV-X19, AAV-X21, AAV-X22,
AAV-X23, AAV-X24, AAV-X25, AAV-X5, and AAV-X26 were generated by
transfecting 293 T cells with AAV2 vector plasmid (AAV2-NLS-GFP)
consisting of an GFP expression cassette flanked by AAV-2 ITRs and
AAV-X1, AAV-X1b, AAV-X19, AAV-X21, AAV-X22, AAV-X23, AAV-X24,
AAV-X25, AAV-X5, or AAV-X26 packaging (subcloned UNI-C/AAV1-4
polyA4400(-) PCR fragment), an AAV-2 like Rep expression plasmid,
AAV-X26_Rep, (subcloned AAV-X26 Rep ORF, AAV1-4 225(+) and UNI-NC
PCR fragment), and Ad helper plasmids, 449B, which provided the
essential adenovirus functions that are required for AAV
replication (Smith, R. H., et al. (2002). Biotechniques 33(1):
204-6, 208, 210-1). Two confluent T175 flasks of 293T cells were
harvested, resuspended in 100 ml DMEM 10% FCS, seeded in five 150
mm plates and incubated at 37.degree. C., 5% CO.sub.2 until cells
are 80% confluent (typically 48h). Cells were transfected with 15
.mu.g pAAV2-NLS-GFP, 10 .mu.g AAV-X1, AAV-X1b, AAV-X19, AAV-X21,
AAV-X22, AAV-X23, AAV-X24, AAV-X25, AAV-X5, or AAV-X26 packaging
plasmid, 5 .mu.g AAV-X26 Rep and 30 .mu.g p449B. 48h after
transfection, cells were harvested, washed with PBS and resuspended
in 11 ml TD buffer (0.14 M NaCl, 5.0 mM KCl, 0.7 mM
K.sub.2HPO.sub.4, 25.0 mM Tris, pH7.4. Cells were lyzed by 3 freeze
thaw cycles and incubated for 30 minutes at 37.degree. C. after
adding benzonase to a final concentration of 20 U/ml sodium
deoxycholate (final concentration of 0.5%). After adding 0.55 g/ml
CsCl, the lysate was fractionated using density gradient
centrifugation in a SW41 rotor for 48 h at 38,000 rpm. The
gradients were harvested in 0.5 ml aliquots. Aliquots were assayed
for infectivity and DNase-resistant genome copy titers of the
vector preparations were determined by quantitative real-time PCR
using the TAQMAN system (Applied Biosystems, Inc. (ABI), Foster
City, Calif.) with probes specific to the CMV promoter.
[0254] Recombinant virus was successfully generated using each of
the AAVX packaging plasmids with AAV2 ITR vector plasmids and
AAV-X26 rep plasmids. Recombinant virus was also successfully
generated using Rep plasmids derived from each of the AAVX viruses
in combination with AAV2 ITR vector plasmids. There is therefore
sufficient homology between AAVX and AAV2 Rep and ITR sequences
that they can be used interchangeably in the herein provided
vectors and vector systems.
[0255] Neutralization Assay:
[0256] Exponentially growing COS cells were plated at a density of
5.times.103/well in a flat-bottom 96-well plate. Twenty-four hours
after seeding, cells were incubated for 60 min with 2.times.106
rAAV particles that were pre-incubated with serial dilutions of
pooled human IgGs (Immune Globuline Intravenous, 10%, Gamunex,
BAYER, Leverkusen, Germany). Twenty-four hours after infection,
cells were analyzed for GFP expression with the Guava PCA-96 (Guava
Technologies, Hayward, Calif.) fluorescent cell counter. GFP
expression was used as a surrogate marker for transduction
efficiency.
[0257] Determination of Tissue Tropism:
[0258] Transduction efficiency of recombinant AAV-X1, AAV-X1b,
AAV-X19, AAV-X21, AAV-X22, AAV-X23, AAV-X24, AAV-X25, AAV-X5, or
AAV-X26 vector containing an expression cassette for GFP was
analyzed in cancer cell lines. Cells were infected with dilutions
ranging from 10.sup.6 to 10.sup.9 particles/well. 24-48h after
infection, cells were analyzed for GFP expression by flow
cytometry.
[0259] Neuraminidase Treatment:
[0260] To analyze if sialic acid is required for transduction of
AAV-X1 and AAV-X25, neuraminidases were used to digest cell surface
sialic acid before infection. Cos cells were seeded at 5,000
cells/well in a 96 well plate. 24h after seeding, cells were
incubated for 60 min with a broad spectrum neuraminidase from
Arthrobacter ureafaciens (0.05-1 U/ml) or a 2-3 linkage specific
neuraminidase from S. pneumoniae (0.04-0.2 U/ml) (Calbiochem, La
Jolla, Calif.). Cells were washed and transduced with a
multiplicity of infection (MOI) of 500 with recombinant AAV6,
AAV-X1 or AAV-X25 particles expressing GFP. Cells were analyzed for
GFP expression by flow cytometry with the Guava PCA-96 (Guava
Technologies) 24 h after transduction.
[0261] Binding Assay:
[0262] COS cells were seeded at 5000 cells/well in a 96-well plate.
Forty-eight hours after seeding, cells were incubated for 60 min at
37.degree. C. with neuraminidases from A. ureafaciens (0.1 U/ml),
S. pneumoniae (0.08 U/ml) (Calbiochem) or mock. Cells were then
chilled for 30 min at 4.degree. C. and then incubated for 60 min at
4.degree. C. with 5.times.10.sup.7 recombinant AAV6, AAV(VR-195),
or AAV(VR-355) particles expressing GFP. Cells were then washed
twice with cold medium and phosphate buffered saline (PBS) and
iysed in 50 .mu.l PCRnGo buffer (Pierce). Copy numbers of
cell-associated vector genomes in the cell lysates were determined
by quantitative real-time PCR using the TAQMAN system (Applied
Biosystems) with probes specific to the CMV promoter.
[0263] Lectin Competition:
[0264] COS cells were seeded at 5,000-10,000 cells/well in a 96
well plate. 16-24h after seeding, cells were precubated for 30 min
at 4.degree. C. with 100 .mu.g/ml of either ConA, MalII, LCA, ECL,
WGA, UEA I, DBA, PNA, SBA, GSL I, PSA, LCA, PHA-E, PHA-L, SJA,
succinylated WGA. Subsequently, cells were washed and transduced
for 60 min at 4.degree. C. with 3000 transducing units or 10.sup.8
recombinant AAV-2, AAV-4, AAV-5, AAV-6, BAAV, AAV-X1, AAV-X5,
AAV-X25 or AAV-X26 particles expressing GFP in 50 .mu.l medium
supplemented with lectins (100 .mu.g/ml). 24h after transduction,
cells were analyzed for GFP expression by flow cytometry.
[0265] Sugar Competition:
[0266] COS cells were seeded at 5,000 cells/well in a 96 well
plate. 24h after seeding, cells were precubated for 30 min at
4.degree. C. before being transduced for 60 min at 4.degree. C.
with 10.sup.8 recombinant AAV-2, AAV-4, AAV-5, AAV-6, BAAV, AAV-X1,
AAV-X5, AAV-X25 or AAV-X26 particles expressing GFP in 50 .mu.l
medium supplemented with various sugars (see FIG. 9). 24h after
transduction, cells were analyzed for GFP expression by flow
cytometry.
[0267] Heparin Competition Assay:
[0268] COS cells were plated at a density of 5.times.10.sup.3/well
in a flat-bottom 96-well plate. After 24 h, 10.sup.7 particles
rAAV6-NLS-GFP, rAAV(VR-195)-NLS-GFP and rAAV(VR-355)-NLSGFP were
preincubated for 1 h at room temperature in medium containing 0 to
2000 .mu.g/ml heparin (Sigma, St. Louis, Mo.). Cells were then
transduced for 1 h at 37.degree. C. with this preincubation
mixture, washed with medium and incubated for 24 h. GFP expression
was detected with a fluorescent cell counter (Guava
Technologies).
[0269] NaCl Competition Assay:
[0270] COS cells were seeded at 5,000 cells/well in a 96 well
plate. 24h after seeding, were transduced for 60 min at 37.degree.
C. with MOI of 500 or 10.sup.8 recombinant AAV-2, AAV-4, AAV-5,
AAV-6, BAAV, AAV-X1, AAV-X5, AAV-X25 or AAV-X26 particles
expressing GFP in 50 .mu.l medium supplemented with NaCl to 150 mM,
300 mM, and 450 mM final NaCl concentration. 24h after
transduction, cells were analyzed for GFP expression by flow
cytometry/fluorescent cell counter (Guava Technologies).
[0271] Viral Cross-Competition:
[0272] COS cells were plated at a density of 5.times.10.sup.3/well
in a flat-bottom 96-well plate. After 24 h, cells were preincubated
for 1 h at 37.degree. C. with increasing titers of rAAV6-lacZ, an
AAV6 derived vector expressing a nuclear localized
.beta.-galactosidase, ranging from 0 to 2.6.times.10.sup.9
particles/well. The cells were then washed with medium and
transduced for 45 min with 2.times.10.sup.6 particles of
rAAV2-NLS-GFP, rAAV6-NLS-GFP, rAAV(VR-195)-NLS-GFP, or
rAAV(VR-355)-NLS-GFP. GFP expression was analyzed 48 h after
transduction by flow cytometry.
[0273] Results
[0274] Identification of AAV Contaminations in ATCC Virus
Isolates:
[0275] Viral stocks, supplied by the ATCC were analyzed for the
presence of AAV DNA by a PCR based assay as described earlier
(Katano, H., et al. 2004). PCR primers in this study were designed
to bind to highly conserved regions in the rep and cap ORFs
resulting in amplification of a 1.5 kb fragment spanning from
nucleotide 1437 to 2904 relative to the AAV-2 genome. This method
is highly sensitive and can detect as few as 10 copies of viral
DNA/.mu.L of sample (Katano, H., et al. 2004). AAV DNA was detected
in 13/137 samples analyzed (Table 4). AAV contaminations were
detected in 26% of adenovirus isolates. Interestingly, no AAV was
detected in herpesvirus, retrovirus, coronavirus, orthomyxovirus,
poxvirus, or reovirus stocks. Many of the AAV contaminated simian
adenoviruses were originally isolated from pooled primary kidney
cell cultures (Hull, R. N., et al. 1957; Hull, R. N., et al. 1958;
Hull, R. N., et al. 1956) originally established as part of a
vaccine development program (VR-195, VR-197, VR-198, VR-200,
VR-202, VR-209, and VR-353), but also from rectal swabs (VR-204),
cervical cord (VR-355), and CNS cultures (VR-207). AAV was also
detected in stocks of human adenovirus type 9 (VR-10) as well as in
bovine adenovirus type 1 (VR-313) and type 2 (VR-314). Ten of
theses isolates have high similarity to AAV1 and AAV6 (>98%),
while AAV-X5 isolated from VR-942 and AAV-X26 isolated from VR-943
showed highest homology in the capsid protein VP-1 to AAV-3B (93%)
and AAV-11 (83%) respectively.
[0276] Construction of Packaging Plasmids and Generation of
Recombinant Virus:
[0277] The entire coding region for Rep and Cap of the AAV
contaminations detected in VR-195 and VR-355 termed AAV(VR-195) and
AAV(VR-355), respectively, as well as the capsid ORF under control
of the viral P40 promoter from the adenovirus isolates VR-10,
VR-195, VR-197, VR-198, VR-200, VR-202, VR-209, VR-353, VR-204,
VR-355, and VR-207 were PCR amplified and subcloned to generate
packaging plasmids.
TABLE-US-00004 TABLE 4 Screening of viral samples for the presence
of AAV Family # tested # AAV positive frequency (%) Adenovirida 53
13 26% primate Adenovirida 16 13 81% Herpesviridae 43 0 0
Coronaviridae 9 0 0 Retrovirida 15 0 0 Reoviridae 4 0 0
Orthomyxovirida 4 0 0
[0278] Recombinant AAV was produced by cotransfecting plasmids
encoding the capsid of the novel AAVs, an AAV type 2 Rep expression
plasmid, an AAV-2 vector plasmid, encoding a nuclear localized GFP
together with an adenovirus helper plasmid. Recombinant viruses
were then assayed for transduction activity. Capsid plasmids
encoding functional VP proteins were sequenced on both strands.
Several clones of each isolate were analyzed. GenBank accession
numbers are as follows: AAV(VR-195): DQ180604(SEQ ID NO:1) and
AAV(VR-355): DQ180605 (SEQ ID NO:9).
[0279] The evolutionary relationship among mammalian AAVs and the
AAV contaminants detected in human and non-human primate adenovirus
stocks was analyzed by ClustalW alignments of VP1 amino acid
sequences and plotted as a rooted phylogenetic tree (FIG. 1). All
AAVs detected in simian adenovirus stocks displayed at least 96%
homology on the DNA level and 98% identity in the capsid amino acid
sequence to either AAV1, AAV6, or to each other. The amino acid
sequence of VP1 encoded by AAV contaminants in VR-197, VR-198, and
VR-202 were identical to each other but distinct from AAV1 and
AAV6. The VP1 sequence of the only positive human adenovirus stock,
VR-10 (adenovirus type 9), was greater than 99.9% identical to
AAV2.
[0280] AAV-X1(97%, 99%), AAV-X1B (96%, 98%), AAV-X19 (97%, 99%),
AAV-X21(97%, 99%), AAV-X22 (97%, 99%), AAV-X23(97%, 98%),
AAV-X24(97%, 97%), and AAV-X25 (97%, 98%) share 96-99% sequence
identity to AAV6 or AAV1 at the DNA level (based on a 3 kb partial
genome fragment) (see FIG. 1). Numbers in brackets indicate
identities to AAV6 and AAV1. The amino acid sequence of AAV-X5
capsid protein shows highest homology to AAV-3B (93%), and AAV-X26
capsid protein shows highest homology to AAV-11 (83%). AAV-X5 and
AAV-X26 DNA sequences show highest homology to AAV-3 (88%) and
AAV-11 (79%) respectively (based on a 3 kb partial genome fragment)
(see FIG. 1).
[0281] Mapping of Sequence Variation:
[0282] AAV contaminations, which were greater than 98% identical to
AAV6 in the amino acid sequence of the capsid protein VP1, were
detected in 10 adenovirus isolates. AAV-X5 isolated from VR-942 and
AAV-X26 isolated from VR-943 showed homology (sequence similarity)
in the capsid protein VP1 to AAV-3B (93%) and AAV-11 (83%),
respectively, and only low homology to AAV6 (87% and 61%,
respectively). AAV-X5 and AAV-X26 are therefore very different from
the other new AAVs disclosed herein. The capsid protein VP1 of
AAV(VR-195) and AAV(VR-355) differs in 7 or 6 amino acids,
respectively, from that of AAV6 (Table 5). To identify the location
of the amino acids that are unique for AAV(VR-195) and AAV(VR-355)
within the capsid, we superimposed the AAV(VR-195) and AAV(VR-355)
VP1 sequence onto a pseudoatomic structure for AAV-6. Divergent
regions in the capsid proteins are located on exposed surface loops
at the threefold axis of symmetry, an area of the capsid that has
been associated with receptor binding (Kern, A., et al. 2003; Opie,
S. R., et al. 2003). AAV(VR-195) specific amino acids of three
different VP3 subunits were clustered in close proximity.
AAV(VR-355) specific changes relative to AAV6 were similarly
organized. Since the amino acid changes among AAV(VR-195),
AAV(VR-355), and AAV6 are surface exposed, we hypothesized they may
effect antigenicity or cell tropism.
TABLE-US-00005 TABLE 5 Novel AAVs were isolated from ATCC
adenovirus samples Name A.K.A. Isolated from Classification Family
AAV-X1, AAV(VR-195) VR-195 Simian virus 1 Adenovirida AAV-X1b
AAV-X1 AAV(VR-197) VR-197 Simian virus 15 Adenovirida AAV-X1
AAV(VR-198) VR-198 Simian virus 17 Adenovirida AAV-X19 AAV(VR-200)
VR-200 Simian virus 23 Adenovirida AAV-X1 AAV(VR-202) VR-202 Simian
virus 27 Adenovirida AAV-X21 AAV(VR-204) VR-204 Simian virus 31
Adenovirida AAV-X22 AAV(VR-207) VR-207 Simian virus 34 Adenovirida
AAV-X23 AAV(VR-209) VR-209 Simian virus 37 Adenovirida AAV-X24
AAV(VR-353) VR-353 Simian virus 39 Adenovirida AAV-X25 AAV(VR-355)
VR-355 Simian virus 38 Adenovirida AAV-X5 AAV(VR-942) VR-942 Simian
Adenovirida adenovirus 17 AAV-X26 AAV(VR-943) VR-943 Simian
Adenovirida AAV-12 adenovirus 18
TABLE-US-00006 TABLE 6 Comparison of the amino acid sequence of VP1
162 198 327 386 418 495 514 531 584 590 AAV6 T V N Q R K L
AAV(VR-195) T V S Q G H F H AAV(VR-355) S L N K R F Numbers on top
indicate the amino acid in AAV6 VP1, where either AAV (VR-195) or
AAV(VR-355) diverges from AAV6. Bold are basic amino acids:
Outlined are acidic; Italic are polar or slightly acidic;
Underlined are aromatic.
[0283] Immunological Characterization:
[0284] To test for a difference in the immunological response to
the isolates, IgGs purified from pooled human serum were assayed to
determine if they contained neutralizing antibodies against the
recombinant AAVs and whether a difference in the neutralization
activity against either AAV6, AAV(VR-195) or AAV(VR-355) existed
(FIG. 2). In this assay, all three viruses displayed similar
sensitivity to neutralization with the purified pooled IgGs. Thus,
the changes in the capsid of AAV(VR-195) or AAV(VR-355) compared to
AAV6 and do not appear to alter their sensitivity to neutralization
by human serum.
[0285] Heparin Competition:
[0286] Heparan sulfate, a ubiquitous cell surface
glycosaminoglycan, is an attachment receptor for AAV2 (Summerford,
C., et al. 1998). AAV2 transduction can be inhibited by heparin, a
heparan sulfate analog. Like AAV2, AAV6 can be purified using a
heparin affinity column; however, transduction is not inhibited by
a low heparin concentration, indicating that heparan sulfate does
not act as an AAV6 receptor (Halbert, C. L., et al. 2001). To
investigate the role of heparan sulfate in AAV(VR-195) or
AAV(VR-355) transduction, competition transduction experiments were
performed with increasing amounts of heparin. At low concentrations
of heparin (25 .mu.g/ml), AAV2 was the only rAAV that was inhibited
(FIG. 3). However, at 2000 .mu.g/ml, approximately 50% inhibition
of AAV6 and AAV(VR-195) was observed. No inhibition was observed
with either AAV(VR-355) or AAV5. Therefore, heparan sulfate does
not appear to be a co-receptor for either AAV(VR-195) or
AAV(VR-355).
[0287] AAV6 and AAV(VR-195) Transduction is Charge Sensitive:
[0288] Heparin is a highly charged molecule. The observed
inhibition of AAV6 and AAV(VR-195) at high concentrations of
heparin could be caused by a charge driven interaction between the
viruses and heparin rather than a specific interaction. Increasing
the ionic strength in the medium during transduction can minimize
charge dependent interactions (Amberg, N., et al. 2002). Salt ions
can bind to charged groups on the cell surface and virus capsid,
thereby reducing these electrostatic interactions. To analyze if
AAV6, AAV(VR-195), and AAV(VR-355) transduction is charge
dependent, the ionic strength was increased during transduction by
adjusting the NaCl concentration in the tissue culture medium from
150 mM up to 450 mM. Transduction of rAAV-6, rAAV(VR-195), and
rAAV(VR-355) was inhibited to different amounts by increasing
concentrations of NaCl (FIG. 4). While 250 mM NaCl was sufficient
to inhibit rAAV6 by 50%, higher NaCl concentrations were required
to inhibit AAV(VR-195) and AAV(VR-355) with IC50 of 350 mM and 450
mM respectively. The relative dependence on charge follows the
inhibition of these viruses by heparin and indicates that the
inhibition of AAV6 and AAV(VR-195) by heparin is charge
dependent.
[0289] Effect of Neuraminidase Treatment on rAAV Transduction and
Virus Binding:
[0290] Dependent parvoviruses are also reported to use sialic acid
as a co-receptor for binding and transduction (Kaludov, N., et al.
2001; Seiler, M. P., et al. 2002; Walters, R. W., et al. 2001). To
analyze if sialic acid is required for transduction with
AAV(VR-195) and AAV(VR-355) vectors, the effect of the removal of
cell surface sialic acids was studied by neuraminidase treatment on
transduction (FIG. 5A) and virus binding (FIG. 5B). Treating COS
cells with a broad-spectrum neuraminidase from A. ureafaciens
inhibited rAAV6, rAAV(VR-195), and rAAV(VR-355) transduction 3-5
fold. Neuraminidase isolated from S. pneumoniae will only remove
.alpha.2-3 linked sialic acid. Treatment of the cells with this
enzyme again inhibited transduction with all three rAAVs. Removal
of cell surface sialic acids with neuraminidases from A.
ureafaciens or S. pneumoniae also resulted in a greater than 90%
reduction in cell binding of rAAV6, rAAV(VR-195) and rAAV(VR-355),
indicating that these viruses utilize a 2-3 link sialic acid as an
cell attachment factor.
[0291] AAV6, AAV(VR-195), and AAV(VR-355) can be Distinguished
Based Upon Lectin Competition:
[0292] Lectins are proteins that recognize and bind
oligosaccharides conjugated to proteins and lipids and can be used
to block virus binding (Summerford, C., et al. 1998). MalII, a
lectin that recognizes .alpha. 2-3 linked sialic acid, inhibited
transduction of rAAV-6, rAAV(VR-195), and rAAV(VR-355) (FIG.
6).
[0293] Many glycoproteins contain a core oligosaccharide structure
which includes .alpha.-linked mannose. To study, if these
"high-mannose" proteins play a role in AAV-X1 or AAV-X25
transduction, these proteins were blocked with ConA, a lectin that
recognizes .alpha.-linked mannose. AAV-6, AAV-X1 or AAV-X25 were
blocked equally by ConA, indicating that proteins with a mannose
core are involved in binding and/or uptake. Lens Culinaris
Agglutinin (LCA) recognizes .alpha.-linked mannose together with
neighboring oligosachharides. It is less broad specific than ConA.
LCA inhibited all recombinant viruses tested and had an
approximately twofold higher inhibitory potential on rAAV6 than on
rAAV(VR-195) or rAAV(VR355). ECL is a lectin specific toward
galactose residues and had the highest binding activity toward
galactosyl (.beta.-1,4) N-acetylglucosamine. ECL blocked AAV-X1
mediated transduction less specific than AAV-6 or AAV-X25. While
ECL, which recognizes .alpha.-mannose in conjugation with
galactosyl (.beta.-1,4) N-acetylglucosamine, did not inhibit
rAAV(VR-195), it reduced rAAV6 and rAAV(VR-355) transduction 7 and
5-fold, respectively. STL, which recognizes Nacetylglucosamine, did
not inhibit rAAV(VR-195), but reduced rAAV6 and rAAV(VR-355)
transduction 30 and 12-fold, respectively. These results indicate
that while all three viruses bind terminal a 2-3 linked sialic
acid, the amino acid changes on the capsid surface affected the
cell binding activity of each isolate.
[0294] These differences observed between AAV-6, AAV-X1 and AAV-X25
in lectin competition experiments and NaCl competition indicates
utilization of different receptors or differences in receptor
interaction that can result in a different cell tropism of vectors
based on these isolates. The transduction efficiency of AAV-6,
AAV-X1 and AAV-X25 based vectors were therefore analyzed in human
cancer cell lines (FIG. 9). Each recombinant virus has a unique
transduction profile indicating that they bind to different
receptors or interact differently with a common receptor.
[0295] AAV-X5 isolated from VR-942 and AAV-X26 isolated from VR-943
showed homology (sequence similarity) in the capsid protein VP1 to
AAV-3B (93%) and AAV-11 (83%), respectively, and only low homology
to AAV6 (87% and 61%, respectively). AAV-X5 and AAV-X26 are
therefore very different from the other new AAVs disclosed herein.
To study the attachment factors that are involved in AAV-X5 and
AAV-X26 mediated transduction, competition with various
carbohydrates was analyzed (FIG. 10). Heparin is a known attachment
factor/receptor for AAV2 and addition of Heparin and homolog sugars
to the medium during transduction resulted in an inhibition of
AAV-2 mediated gene transfer. Heparin had no effect on AAV-X5 and
AAV-X26 mediated gene transfer demonstrating that extracellular
heparin can not block AAV-X5 and AAV-X26 mediated gene transfer.
Heparin and related carbohydrates do not appear to have receptor
function for these viruses.
[0296] AAV-X5 and AAV-X26 therefore utilize unique uptake
pathways/receptors that are different from all other AAVs analyzed
thus far. Transduction efficiencies in four cancer cell lines were
therefore examined (FIG. 11). AAV-X5 and AAV-X26 have unique
transduction properties and tropism. Thus, the application range
and potential use of recombinant vectors based on AAV-X5 and
AAV-X26 are unique and different from other AAVs described thus
far.
[0297] rAAV6, rAAV(VR-195), and MAV(VR-355) Differ in their Cell
Tropism:
[0298] As a result of the few sequence changes on the surface of
the rAAV(VR-195) and rAAV(VR-355) capsid, each virus appears to
exhibit different biological characteristics from each other and
from rAAV6. To analyze if this difference also results in a change
in tropism or transduction activity, six human cancer cell lines
and African green monkey kidney cells, COS, were transduced with
recombinant vectors based on rAAV6, rAAV(VR-195), and rAAV(VR-355)
(FIG. 7). AAV6 and AAV(VR-195) transduced COS cells, the non-small
cell lung cancer cell line EKVX, ovarian IGROV1, and renal CAKI
cells with similar efficiency. However, the transduction rates of
AAV(VR-195) on ovarian Ovcar5 cells and the CNS derived SF295 cell
line were approximately 4 times lower compared to AAV6. AAV(VR-355)
demonstrated efficient gene transfer in COS and EKVX cells, but
transduction of Igrov1, CAKI, Ovcar5 and SF295 was 10 to 17-fold
lower than for AAV6. The different transduction efficiencies of
rAAV6, rAAV(VR-195), and rAAV(VR-355) indicate that each isolate
may have a distinct cell tropism which could be the result of
utilization distinct receptors for attachment or internalization or
have different affinities for a common receptor.
[0299] rAAV6 Competition:
[0300] rAAV6, rAAV(VR-195), and rAAV(VR-355) require 2-3 linked
sialic acid for cell attachment and transduction but differ in
their charge dependency, sensitivity to lectin competition, and
transduction activity on a panel of cells. To investigate if these
viruses use distinct receptors, competition experiments were used
to assay for change in transduction between AAV6 and the other
isolates. COS cells were pre-incubated with increasing doses of
rAAV6-lacZ followed by transduction with identical particle titers
of either rAAV2, rAAV6, rAAV(VR-195), or rAAV(VR-355) expressing
GFP. Changes in GFP expression were detected by flow cytometry
(FIG. 8). rAAV6-LacZ competition had the greatest effect on
rAAV6-NLS-GFP transduction. Fifty percent inhibition of
rAAV6-NLS-GFP transduction was observed at approximately 60-fold
excess of the competitor, whereas a 220-fold excess was required
for the same level of inhibition of rAAV(VR-195) or rAAV(VR-355).
Fifty percent inhibition of rAAV2-NLS-GFP transduction required
greater than 250-fold particle excess. The stronger inhibition of
rAAV6-NLS-GFP by rAAV6-lacZ compared with rAAV(VR-195) or
rAAV(VR-355) suggests, that while rAAV6, rAAV(VR-195) and
rAAV(VR-355) share a common attachment factor and potentially a
common receptors, differences in the attachment factor and receptor
interaction exist.
Example 2
Identification and Characterization of AAV12
[0301] Materials And Methods
[0302] Cells Culture and Virus:
[0303] African green monkey kidney COS cells, obtained from the
American Type Culture Collection (ATCC) (Manassas, Va.), were
cultured in RPMI-1640 medium (Biosource, Camarillo, Calif.),
supplemented with 5% fetal bovine serum (Hyclone, Logan, Utah), 2
mM L-glutamine, 100 U of penicillin/ml, and 0.1 mg of
streptomycin/ml (Invitrogen, Carlsbad, Calif.). Cells were
maintained at 37.degree. C. under a 5% CO.sub.2 humidified
atmosphere. Simian Virus 18 isolate, VR-943, was obtained from the
ATCC as crude lysate of virus-infected cells.
[0304] Subcloning of the AAV12 Rep and Cap Gene:
[0305] The complete coding region of AAV12 rep and cap were PCR
amplified and subcloned. DNA was isolated from lysate of Simian
Virus 18 infected cells with the QIAprep Spin Miniprep kit (Qiagen,
Valencia, Calif.). The rep open reading frame (ORF) was PCR
amplified from this DNA with the primers
AAV225(+):GCGACAKTTTGCGACACCAYGTGG (SEQ ID NO:44) and UNI-NC:
CCANNNGGAATCGCAATGCCAAT (SEQ ID NO:45). AAV12 cap was amplified
with the primers UNI-C: 5'-ATGNTNATNTGGTGGGAGGAGGG-3' (SEQ ID
NO:46) and AAV1-4 polyA4400(-): 5'-CGAATNAAMCGGTTTATTGATTAAC-3'
(SEQ ID NO:47). The PCR fragments were subcloned using the TOPO TA
Cloning KIT (Invitrogen) resulting in the plasmids pAAV12-Rep and
pAAV12-Cap. Three clones, that were capable of generating
recombinant virus, were sequenced with an ABI Prism 3100 Genetic
Analyzer (Applied Biosystems, Foster City, Calif.) and FS
dye-terminator chemistry (Applied Biosystems).
[0306] Sequence Analysis:
[0307] The sequences of AAV12 rep and cap were compared to
sequences in GenBank using BLAST. DNA alignments were performed
using the ClustalW multiple sequence alignment tool of the Biology
Workbench web based software at http://seqtool.sdsc.edu (SDSC) and
MacVector 7 (Accelrys, Burlington, Mass.).
[0308] Generation of Recombinant Virus:
[0309] AAV12 (AAVX26) vectors, expressing a nuclear localized green
fluorescent protein (GFP), rAAV12GFP, were produced as described
above. Briefly, 293T cells were cotransfected with pAAV2-NLS-GFP,
pAAV12Rep and pAAV12Cap and the Ad helper plasmids 449B.
Recombinant particles were purified by CsCl gradient
centrifugation. DNase-resistant genome copy numbers of the vector
stocks were determined by quantitative real-time PCR using the
TAQMAN system (Applied Biosystems) with probes specific to the CMV
promoter. AAV12-Epo expressing human erythropoietin was generated
accordingly by packaging pAAVhEPO with pAAV12Rep and pAAV12Cap.
[0310] Digestion of Cell Surface Sialic Acid:
[0311] Exponentially growing COS cells were plated at a density of
5.times.10.sup.3/well in a flat-bottom 96-well plate. Twenty-four
hours after seeding, cells were incubated for 30 min with 0.1 and 1
mU of the broad-spectrum neuraminidase from Vibrio cholerae
(Calbiochem, La Jolla, Calif.). Cells were then washed with medium
and transduced with 1.times.10.sup.7 particles of rAAV2-GFP,
rAAV4-GFP, rAAV5-GFP or rAAV12-GFP. GFP expression, which serves as
a surrogate marker for transduction, was detected twenty-four hours
later with a fluorescent cell counter (Guava PCA-96, Guava
Technologies, Hayward, Calif.).
[0312] Heparin Competition Assay:
[0313] COS cells were plated at a density of 5.times.10.sup.3/well
in a flat-bottom 96-well plate one day prior to transduction.
2.times.10.sup.7 particles rAAV2-GFP, rAAV12-GFP were preincubated
for 1 h at room temperature in medium supplemented with either
heparin, mannose or mannosamine (Sigma, St. Louis, Mo.). This
pre-incubation mixture was then added and left on the cells for 1 h
at 37.degree. C. Cells were then washed with medium and incubated
for one day before GFP expression was detected with a fluorescent
cell counter (Guava Technologies).
[0314] Protease Treatment:
[0315] COS cells were cultured in a 15-cm diameter culture dish
until cells were 80% confluent. Cells were then washed twice with
PBS, scraped, resuspended in 10 ml PBS and incubated with 0.05%
trypsin (Biosource), or mock (untreated control) for 15 min at
37.degree. C. Cells were then washed twice with medium and seeded
at a density of 10,000 cells/well in a 96-well dish. After 1 h
culture at 37.degree. C., cells were transduced with
2.times.10.sup.7 particles rAAV2-GFP, rAAV12-GFP. Transduction
efficiency was determined 24 h later by GFP expression detection
with a fluorescent cell counter (Guava Technologies).
[0316] Inhibition of Glycolipid Synthesis:
[0317] COS cells were plated at a density of 5.times.10.sup.3/well
in a flat-bottom 96-well plate. Eight hours after seeding, cells
were incubated for 40 h with the glucosylceramide synthase
inhibitors
DL-threo-1-Phenyl-2-palmitoylamino-3-morpholino-1-propanol (PPMP)
(Sigma, St. Louis, Mo.). Cells were then washed with medium and
transduced with 2.times.10.sup.8 particles of rAAV2-GFP, rAAV12-GFP
and rBAAV-GFP for 1 h. GFP expression was analyzed 48 h after
transduction by detection with a fluorescent cell counter (Guava
Technologies).
[0318] Neutralization Assay:
[0319] COS cells were seeded at a density of 5.times.10.sup.3/well
in a 96-well plate 1 day before inoculation. 2.times.10.sup.7
rAAV2-GFP, rAAV6-GFP and rAAV12-GFP particles that were
pre-incubated with serial dilutions of pooled human IgGs (Immune
Globuline Intravenous, 10%, Gamunex, BAYER, Leverkusen, Germany) in
medium for 1 h at room temperature. Cells were then inoculated with
this mixture for 1 h at 37.degree. C. and then washed with medium.
Twenty-four hours after transduction, cells were analyzed for GFP
expression by flow cytometry (Guava Technologies).
[0320] Animal Experiments:
[0321] Male Balb/c mice were obtained from the Division of Cancer
Treatment, NCI, Bethesda, Md. Mice were administered 10.sup.9
particles(suspended in 100 .mu.l of 0.9% NaCl) of either AAV2-hEPO
(n=3) or AAV12-hEPO (n=2) by retrograde ductal delivery to both
submandibular glands (SG). Two additional groups (n=3 and n=2,
respectively) received an equal dose of 10.sup.9
particles(suspended in 100 .mu.l of 0.9% NaCl) in both their
tibialis anterior (two injection sites per muscle). A further group
(n=3) of naive mice (administered with 50 .mu.l of 0.9% NaCl to
each SG) was included. Mild anesthesia was induced to all
participating animals with 1 .mu.l/g body weight of a 60 mg/ml
ketamine (Phoenix Scientific, St. Joseph, Mo.) and 8 mg/ml xylazine
(Phoenix Scientific) solution given intramuscularly. Blood samples
were obtained by orbital bleeding at distinct time points.
Hematocrits (Hcts) were determined using micro-hematocrit capillary
tubes (Fisher Scientific, Pittsburgh, Pa.). Secretion of hEPO in
mouse serum was determined by an ELISA using commercial assay kits
(R&D systems, MN, USA). The lower limit of detection was 0.6
mU/ml. Assays were performed according to the manufacturer's
instructions.
[0322] Results
[0323] Identification of AAV Contaminations in ATCC Virus
Isolates:
[0324] AAV sequences were detected in a Simian Adenovirus 18 strain
C676 stock, VR-943, which was isolated from a Vervet monkey
(Cercopithecus aethiops). The entire rep and cap coding region of
the AAV contamination in VR-943, termed AAV12, has been PCR
amplified and subcloned (FIG. 1). Since the rep and cap encoding
PCR fragments also contain viral promoter elements, these plasmids
could be used as packaging constructs for the generation of
recombinant virus. Vectors based on AAV12 were produced by
cotransfecting an AAV-2 vector plasmid, encoding a nuclear
localized GFP flanked by AAV2 inverted terminal repeats (ITRs), and
plasmids encoding AAV12 rep and cap together with an adenovirus
helper plasmid. Recombinant viruses were then assayed for
transduction activity by inoculating COS cells and assaying for GFP
expression by flow cytometry. Three clones, which were capable of
generating recombinant virus were sequenced.
[0325] Phylogenetic Analysis:
[0326] The evolutionary relationship among mammalian AAVs and AAV12
was analyzed by ClustalW alignments of genomic, Rep78 and VP1
sequences and plotted as a rooted phylogenetic tree (FIG. 1). The
DNA sequence of AAV12 showed highest homology with AAV11 and AAV4,
83% and 81% respectively, whereas lowest similarity was observed
with AAV5 (63%). The Rep78 amino acid sequence of AAV12
demonstrated high homology to AAV13, AAV4, AAV10 and AAV11 with 89%
or 88% identity. For the capsid protein VP1, highest homology was
observed with AAV11 and AAV4, 84% and 78% respectively, whereas
AAV5 VP1 displayed lowest similarity with 53%.
[0327] Heparin Competition:
[0328] Heparan sulfate, a cell surface glycosaminoglycan, which is
expressed by virtually all cells, is an attachment receptor for
AAV2 (Summerford, C., et al. 1998). AAV2 transduction can be
inhibited by heparin, a heparan sulfate analog. Thus, analysis was
conducted to determine whether AAV12 uses heparan sulfate as a
receptor by heparin competition experiments. COS cells were
transduced with rAAV12 in the presence of increasing amounts of
heparin. rAAV2 served as a control. rAAV2 transduction was
inhibited to 75% at a concentration of 12.5 m/ml heparin, whereas
no inhibition of rAAV12 was observed at a 80 hold higher heparin
concentration of 1000 m/ml(FIG. 12). Since heparin has no
inhibitory potential towards AAV12 transduction, heparan sulfate
appears not to be involved in AAV12 transduction.
[0329] Effect of Neuraminidase Treatment on rAAV12
Transduction:
[0330] Sialic acids, a family of monosaccharides based on
N-acetylneuramic acid, are commonly found on the outermost end of
glykans and glycolipids. Sialic acids have been identified to serve
as receptors for several viruses, including influenza virus, AAV4,
AAV5 AAV6 and AAV(VR-355). To analyze, if AAV12 transduction is
sialic acid dependent, the effect of the removal of cell surface
sialic acids was studied by neuraminidase treatment on gene
transfer (FIG. 134). Enzymatic digestion of COS cells with a broad
spectrum neuraminidase from Vibrio cholera inhibited rAAV4 and
rAAV5 transduction dose-dependently and blocked gene transfer up to
99% and 97% respectively. AAV12 transduction was unaffected by the
enzymatic removal of cell surface sialic acids, indicating that
AAV12 does not utilize sialic acid in the transduction process.
[0331] rAAV12 Transduction is Protease Sensitive and does not
Require Glycosphingolipids (GSLs):
[0332] rBAAV cell entry and transduction depend on gangliosides,
glycosphingolipids with sialic acid groups, and are resistant to
protease treatment of the cell. To analyze if the rAAV12 receptor
is a protein and if glycolipids are involved in the transduction
process, the effect of proteolytically digesting cell surface
proteins prior to transduction was studied (FIG. 14A). The
dependency of rAAV12 transduction on GSLs was studied by incubating
cells with
DL-threo-1-Phenyl-2-palmitoylamino-3-morpholino-1-propanol (PPMP),
a glucosylceramide synthase inhibitors, which act to deplete GSLs
from the cell membrane prior to transduction (FIG. 14B). As shown
in FIG. 4, AAV12 transduction is protease sensitive, but AAV12 can
transduce cells lacking glycosphingolipids.
[0333] rAAV12 is Inhibited by Extracellular Mannosamine:
[0334] Since rAAV12 transduction does not depend on either heparin
or sialic acid, a broad panel of carbohydrates was screened for the
ability to interfere with rAAV12 transduction to identify
components that might be involved in the rAAV12 cell interaction.
In this assay, mannosamine was identified as compound with
inhibitory function against rAAV12. Pre-incubation of rAAV12 with
mannosamine prior to transduction resulted in a dose-dependent
inhibition of rAAV12 transduction (FIG. 15), while no specific
inhibition of rAAV2 was observed. This result indicates that
mannosamine could be part of the rAAV12 receptor or attachment
factor.
[0335] rAAV12 has a Broad Tropism in Human Cancer Cell Lines:
[0336] All studied AAVs utilize either sialic acid or heparin as
cellular receptor or attachment factor. In contrast, rAAV12
trasduced cells independently of heparin and sialic acid but was
inhibited by mannosamine, indicating that rAAV12 interacts in a
unique way with the cell. To analyze if unique cell interaction
results in a unique tropism, the transduction efficiency of rAAV12
was studied in 13 human cancer cell lines and compared it to rAAV4,
a virus that shows 78% homology to rAAV12 in the capsid protein VP1
(FIG. 16). rAAV12 and rAAV4 demonstrated both a broad tropism with
overall similar transduction efficiencies, but both viruses had a
unique transduction profile. While only rAAV4 transduced HCT10
cells, rAAV12 gene transfer was specific for A549 and HCT15
cells.
[0337] Immunological Characterization of rAAV12:
[0338] Infections with AAV2 are very common and approximately 80%
of the human population are seropositive. Neutralization of human
AAV serotypes, such as AAV2, AAV3 and AAV5, as well as the simian
AAV6 by human serum have been reported. This pre-existing immunity
against these AAVs might limit their usability as vectors for gene
therapy. To test if rAAV12 is antigentically distinct from rAAV2
and rAAV6, assays were conducted to determine if IgGs, purified
from pooled human serum, contain neutralizing antibodies against
the recombinant AAVs and whether there was a difference in the
neutralization activity against either rAAV2, AAV6, or rAAV12 (FIG.
17). In this assay the human rAAV2 and simian rAAV6 displayed
similar sensitivity to neutralization with the purified pooled IgGs
and transduction was inhibited to 50% at a concentration of 0.01
mg/ml IgGs. In contrast, rAAV12 was highly resistant to
neutralization by pooled human IgGs, and even at the highest
concentration of 0.67 mg/ml, a concentration where 100% inhibition
of rAAV2 and rAAV6 was observed, rAAV12 transduction was only
reduced by 30%.
[0339] rAAV12 Transduces Salivary Glands and Skeletal Muscles In
Vivo:
[0340] rAAV12 transduced salivary glands and skeletal muscles with
similar efficiency as rAAV2 (FIG. 18).
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Sequence CWU 1
1
5414259DNAArtificial SequenceDescription of Artificial Sequence
Note = synthethic construct 1tttgcgacat tttgcgacac cacgtggcca
ttcagggtat atatggccga gtgagcgagc 60aggatcccca ttttgaccgc gaaatttgaa
cgagcagcag ccatgccggg cttctacgag 120atcgtgatca aggtgccgag
cgacctggac gagcacctgc cgggcatttc tgactcgttt 180gtgaactggg
tggccgagaa ggaatgggag ctgcccccgg attctgacat ggatctgaat
240ctgattgagc aggcacccct gaccgtggcc gagaagctgc agcgcgactt
cctggtccaa 300tggcgccgcg tgagtaaggc cccggaggcc ctcttctttg
ttcagttcga gaagggcgag 360tcctacttcc acctccatat tctggtggag
accacggggg tcaaatccat ggtgctgggc 420cgcttcttga gtcagattag
ggacaagctg gtgcagacca tctaccgcgg gatcgagccg 480accctgccca
actggttcgc ggtgaccaag acgcgtaatg gcgccggagg ggggaacaag
540gtggtggacg agtgctacat ccccaactac ctcctgccca agactcagcc
cgagctgcag 600tgggcgtgga ctaacatgga ggagtatata agcgcgtgtt
tgaacctggc cgagcgcaaa 660cggctcgtgg cgcagcacct gacccacgtc
agccagaccc aggagcagaa caaggagaat 720ctgaacccca attctgacgc
gcctgtcatc cggtcaaaaa cctccgcgcg ctacatggag 780ctggtcgggt
ggctggtgga ccggggcatc acctccgaga agcagtggat ccaggaggac
840caggcctcgt acatctcctt caacgccgcc tccaactcgc ggtcccagat
caaggccgct 900ctggacaatg ccggcaagat catggcgctg accaaatccg
cgcccgacta cctggtaggc 960cccgctcctc ccgcggacat taaaaccaac
cgcatctacc gcatcctgga gctgaacggc 1020tacgaccctg cctacgccgg
ctccgtcttt ctcggctggg cccagaaacg gttcgggaag 1080cgcaacacca
tctggctgtt tgggccggcc accacgggca agaccaacat cgcggaagcc
1140atcgcccacg ccgtgccctt ctacggctgc gtcaactgga ccaatgagaa
ctttcccttc 1200aacgattgcg tcgacaagat ggtgatctgg tgggaggagg
gcaagatgac ggccaaggtc 1260gtggagtccg ccaaggccat tctcggcggc
agcaaggtgc gcgtggacca aaagtgcaag 1320tcgtccgccc agatcgatcc
cacccccgtg atcgtcacct ccaacaccaa catgtgcgcc 1380gtgattgacg
ggaacagcac caccttcgag caccagcagc cgttgcagga ccggatgttc
1440aaatttgaac tcacccgccg tctggagcat gactttggca aggtgacaaa
gcaggaagtc 1500aaagagttct tccgctgggc gcaggatcac gtgaccgagg
tggcgcatga gttctacgtc 1560agaaagggtg gagccaacaa aagacccgcc
cccgatgacg cggataaaag cgagcccaag 1620cgggcctgcc cctcagtcgc
ggatccatcg acgtcagacg cggaaggagc tccggtggac 1680tttgccgaca
ggtaccaaaa caaatgttct cgtcacgcgg gcatgcttca gatgctgttt
1740ccctgcaaga catgcgagag aatgaatcag aatttcaaca tttgcttcac
gcacgggacc 1800agagactgtt cagaatgttt ccccggcgtg tcagaatctc
aaccggtcgt cagaaaaagg 1860acgtatcgga aactctgtgc gattcatcat
ctgctggggc gggctcccga gattgcttgc 1920tcggcctgcg atctggtcaa
cgtggacctg gatgactgtg tctctgagca ataaatgact 1980taaaccaggt
atggctgccg atggttatct tccagattgg ctcgaggaca acctctctga
2040gggcattcgc gagtggtggg acttgaaacc tggagccccg aaacccaaag
ccaaccagca 2100aaagcaggac gacggccggg gtctggtgct tcctggctac
aagtacctcg gacccttcaa 2160cggactcgac aagggggagc ccgtcaacgc
ggcggacgca gcggccctcg agcacgacaa 2220ggcctacgac cagcagctca
aagcgggtga caatccgtac ctgcggtata accacgccga 2280cgccgagttt
caggagcgtc tgcaagaaga tacgtctttt gggggcaacc tcgggcgagc
2340agtcttccag gccaagaagc gggttctcga accttttggt ctggttgagg
aaggcgctaa 2400gacggctcct ggaaagaaac gtccggtaga gcagtcgcca
caagagccag actcctcctc 2460gggcatcggc aagacaggcc agcagcccgc
taaaaagaga ctcaattttg gtcagactgg 2520cgactcagag tcagtccccg
acccacaacc tctcggagaa cctccagcaa cccccgctgc 2580tgtgggacct
actacaatgg cttcaggcgg tggcgcacca atggcagaca ataacgaagg
2640cgccgacgga gtgggtaatg cctcaggaaa ttggcattgc gattccacat
ggctgggcga 2700cagagtcatc accaccagca cccgcacctg ggccttgccc
acctacaata accacctcta 2760caagcaaatc tccagtgctt caacgggggc
cagcaacgac aaccactact tcggctacag 2820caccccctgg gggtattttg
atttcaacag attccactgc cacttttcac cgcgtgactg 2880gcagcgactc
atcaacaaca attggggatt ccggcccaag agactcaact tcaagctctt
2940caacatccaa gtcaaggagg tcacgacgag tgatggcgtc acaaccatcg
ctaataacct 3000taccagcacg gttcaagtct tctcggactc ggagtaccag
cttccgtacg tcctcggctc 3060tgcgcaccag ggctgcctcc ctccgttccc
ggcggacgtg ttcatgattc cgcaatacgg 3120ctacctgacg ctcaacaatg
gcagccaagc cgtgggacgt tcatcctttt actgcctgga 3180atatttccct
tctcagatgc tgagaacggg caacaacttt accttcagct acacctttga
3240ggaagtgcct ttccacagca gctacgcgca cagccagagc ctggaccggc
tgatgaatcc 3300tctcatcgac caatacctgt attacctgaa cagaactcaa
aatcagtccg gaagtgccca 3360aaacaaggac ttgctgttta gccgtgggtc
tccagctggc atgtctgttc agcccaaaaa 3420ctggctacct ggaccctgtt
atcggcagca gcgcgtttct aaaacaaaaa caggcaacaa 3480caacagcaat
tttacctgga ctggtgcttc aaaatataac ctcaatgggc atgaatccat
3540catcaaccct ggcactgcta tggcctcaca caaagacgac gaagacaagt
tctttcccat 3600gagcggtgtc atgatttttg gaaaagagag cgccggagct
tcaaacactg cattggacaa 3660tgtcatgatt acagacgaag aggaaattaa
agccactaac cctgtggcca ccgaaagatt 3720tgggaccgtg gcagtcaatt
tccagagcag cagcacacac cctgcgaccg gagatgtgca 3780tgttatggga
gcattacctg gcatggtgtg gcaagataga gacgtgtacc tgcagggtcc
3840catctgggcc aaaattcctc acacagatgg acactttcac ccgtctcctc
ttatgggcgg 3900ctttggactc aagcacccgc ctcctcagat cctcatcaaa
aacacgcctg ttcctgcgaa 3960tcctccggcg gagttttcag ctacaaagtt
tgcttcattc atcacccaat actccacagg 4020acaagtgagt gtggaaattg
aatgggagct gcagaaagaa aacagcaagc gctggaatcc 4080cgaagtgcag
tacacatcca attatgcaaa atctgccaac gttgatttta ccgtggacaa
4140caatggactt tatactgagc ctcgccccat tggcacccgt taccttaccc
gtcccctgta 4200attacgtgtt aatcaataaa ccggttgatt cgtttcagtt
gaactttggt ctcctgtcc 425924305DNAArtificial SequenceDescription of
Artificial Sequence Note = synthethic construct 2accctcacta
aagggactag tcctgcaggt ttaaacgaat tcgccctttg cgacattttg 60cgacaccacg
tggccattca gggtatatat ggccgagtga gcgagcagga tccccatttt
120gaccgcgaaa tttgaacgag cagcagccat gccgggcttc tacgagatcg
tgatcaaggt 180gccgagcgac ctggacgagc acctgccggg catttctgac
tcgtttgtga actgggtggc 240cgagaaggaa tgggagctgc ccccggattc
tgacatggat ctgaatctga ttgagcaggc 300acccctgacc gtggccgaga
agctgcagcg cgacttcctg gtccaatggc gccgcgtgag 360taaggccccg
gaggccctct tctttgttca gttcgagaag ggcgagtcct acttccacct
420ccatattctg gtggagacca cgggggtcaa atccatggtg ctgggccgct
tcttgagtca 480gattagggac aagctggtgc agaccatcta ccgcgggatc
gagccgaccc tgcccaactg 540gttcgcggtg accaagacgc gtaatggcgc
cggagggggg aacaaggtgg tggacgagtg 600ctacatcccc aactacctcc
tgcccaagac tcagcccgag ctgcagtggg cgtggactaa 660catggaggag
tatataagcg cgtgtttgaa cctggccgag cgcaaacggc tcgtggcgca
720gcacctgacc cacgtcagcc agacccagga gcagaacaag gagaatctga
accccaattc 780tgacgcgcct gtcatccggt caaaaacctc cgcgcgctac
atggagctgg tcgggtggct 840ggtggaccgg ggcatcacct ccgagaagca
gtggatccag gaggaccagg cctcgtacat 900ctccttcaac gccgcctcca
actcgcggtc ccagatcaag gccgctctgg acaatgccgg 960caagatcatg
gcgctgacca aatccgcgcc cgactacctg gtaggccccg ctcctcccgc
1020ggacattaaa accaaccgca tctaccgcat cctggagctg aacggctacg
accctgccta 1080cgccggctcc gtctttctcg gctgggccca gaaacggttc
gggaagcgca acaccatctg 1140gctgtttggg ccggccacca cgggcaagac
caacatcgcg gaagccatcg cccacgccgt 1200gcccttctac ggctgcgtca
actggaccaa tgagaacttt cccttcaacg attgcgtcga 1260caagatggtg
atctggtggg aggagggcaa gatgacggcc aaggtcgtgg agtccgccaa
1320ggccattctc ggcggcagca aggtgcgcgt ggaccaaaag tgcaagtcgt
ccgcccagat 1380cgatcccacc cccgtgatcg tcacctccaa caccaacatg
tgcgccgtga ttgacgggaa 1440cagcaccacc ttcgagcacc agcagccgtt
gcaggaccgg atgttcaaat ttgaactcac 1500ccgccgtctg gagcatgact
ttggcaaggt gacaaagcag gaagtcaaag agttcttccg 1560ctgggcgcag
gatcacgtga ccgaggtggc gcatgagttc tacgtcagaa agggtggagc
1620caacaaaaga cccgcccccg atgacgcgga taaaagcgag cccaagcggg
cctgcccctc 1680agtcgcggat ccatcgacgt cagacgcgga aggagctccg
gtggactttg ccgacaggta 1740ccaaaacaaa tgttctcgtc acgcgggcat
gcttcagatg ctgtttccct gcaagacatg 1800cgagagaatg aatcagaatt
tcaacatttg cttcacgcac gggaccagag actgttcaga 1860atgtttcccc
ggcgtgtcag aatctcaacc ggtcgtcaga aaaaggacgt atcggaaact
1920ctgtgcgatt catcatctgc tggggcgggc tcccgagatt gcttgctcgg
cctgcgatct 1980ggtcaacgtg gacctggatg actgtgtctc tgagcaataa
atgacttaaa ccaggtatgg 2040ctgccgatgg ttatcttcca gattggctcg
aggacaacct ctctgagggc attcgcgagt 2100ggtgggactt gaaacctgga
gccccgaaac ccaaagccaa ccagcaaaag caggacgacg 2160gccggggtct
ggtgcttcct ggctacaagt acctcggacc cttcaacgga ctcgacaagg
2220gggagcccgt caacgcggcg gacgcagcgg ccctcgagca cgacaaggcc
tacgaccagc 2280agctcaaagc gggtgacaat ccgtacctgc ggtataacca
cgccgacgcc gagtttcagg 2340agcgtctgca agaagatacg tcttttgggg
gcaacctcgg gcgagcagtc ttccaggcca 2400agaagcgggt tctcgaacct
tttggtctgg ttgaggaagg cgctaagacg gctcctggaa 2460agaaacgtcc
ggtagagcag tcgccacaag agccagactc ctcctcgggc atcggcaaga
2520caggccagca gcccgctaaa aagagactca attttggtca gactggcgac
tcagagtcag 2580tccccgaccc acaacctctc ggagaacctc cagcaacccc
cgctgctgtg ggacctacta 2640caatggcttc aggcggtggc gcaccaatgg
cagacaataa cgaaggcgcc gacggagtgg 2700gtaatgcctc aggaaattgg
cattgcgatt ccacatggct gggcgacaga gtcatcacca 2760ccagcacccg
cacctgggcc ttgcccacct acaataacca cctctacaag caaatctcca
2820gtgcttcaac gggggccagc aacgacaacc actacttcgg ctacagcacc
ccctgggggt 2880attttgattt caacagattc cactgccact tttcaccgcg
tgactggcag cgactcatca 2940acaacaattg gggattccgg cccaagagac
tcaacttcaa gctcttcaac atccaagtca 3000aggaggtcac gacgagtgat
ggcgtcacaa ccatcgctaa taaccttacc agcacggttc 3060aagtcttctc
ggactcggag taccagcttc cgtacgtcct cggctctgcg caccagggct
3120gcctccctcc gttcccggcg gacgtgttca tgattccgca atacggctac
ctgacgctca 3180acaatggcag ccaagccgtg ggacgttcat ccttttactg
cctggaatat ttcccttctc 3240agatgctgag aacgggcaac aactttacct
tcagctacac ctttgaggaa gtgcctttcc 3300acagcagcta cgcgcacagc
cagagcctgg accggctgat gaatcctctc atcgaccaat 3360acctgtatta
cctgaacaga actcaaaatc agtccggaag tgcccaaaac aaggacttgc
3420tgtttagccg tgggtctcca gctggcatgt ctgttcagcc caaaaactgg
ctacctggac 3480cctgttatcg gcagcagcgc gtttctaaaa caaaaacagg
caacaacaac agcaatttta 3540cctggactgg tgcttcaaaa tataacctca
atgggcatga atccatcatc aaccctggca 3600ctgctatggc ctcacacaaa
gacgacgaag acaagttctt tcccatgagc ggtgtcatga 3660tttttggaaa
agagagcgcc ggagcttcaa acactgcatt ggacaatgtc atgattacag
3720acgaagagga aattaaagcc actaaccctg tggccaccga aagatttggg
accgtggcag 3780tcaatttcca gagcagcagc acacaccctg cgaccggaga
tgtgcatgtt atgggagcat 3840tacctggcat ggtgtggcaa gatagagacg
tgtacctgca gggtcccatc tgggccaaaa 3900ttcctcacac agatggacac
tttcacccgt ctcctcttat gggcggcttt ggactcaagc 3960acccgcctcc
tcagatcctc atcaaaaaca cgcctgttcc tgcgaatcct ccggcggagt
4020tttcagctac aaagtttgct tcattcatca cccaatactc cacaggacaa
gtgagtgtgg 4080aaattgaatg ggagctgcag aaagaaaaca gcaagcgctg
gaatcccgaa gtgcagtaca 4140catccaatta tgcaaaatct gccaacgttg
attttaccgt ggacaacaat ggactttata 4200ctgagcctcg ccccattggc
acccgttacc ttacccgtcc cctgtaatta cgtgttaatc 4260aataaaccgg
ttgattcgtt tcagttgaac tttggtctcc tgtcc 430534196DNAArtificial
SequenceDescription of Artificial Sequence Note = synthethic
construct 3ccgcgagtga gcgaaccagg agctccattt tgcccgcgaa ttttgaacga
gcagcagcca 60tgccgggatt ctacgagatt gtcctgaagg tgcccagcga cctggacgag
cacctgcctg 120gcatttctga ctcttttgta aactgggtgg cggagaagga
atgggagctg ccgccggatt 180ctgacatgga tctgaatctg attgagcagg
cacccctaac cgtggccgaa aagctgcaac 240gcgaattcct ggtcgagtgg
cgccgcgtga gtaaggcccc ggaggccctc ttctttgttc 300agttcgagaa
gggggacagc tacttccacc tacacattct ggtggagacc gtgggcgtga
360aatccatggt ggtgggccgc tacgtgagcc agattaaaga gaagctggtg
acccgcatct 420accgcggggt cgagccgcag cttccgaact ggttcgcggt
gaccaagacg cgtaatggcg 480ccggaggcgg gaacaaggtg gtggacgact
gctacatccc caactacctg ctccccaaga 540cccagcccga gctccagtgg
gcgtggacta atatggacca gtatttaagc gcctgtttga 600atctcgcgga
gcgtaaacgg ctggtggcgc agcatctgac gcacgtgtcg cagacgcagg
660agcagaacaa agagaaccag aaccnaattc tgacgcgccg gtgattcgat
caaaacctcc 720gcgaggtaca tggagctggt cgggtggctg gtggacccng
ggatcncgtc agaaaagcaa 780tggantccag gaggaccagg cctcttacat
ctccttcaac gccgcctcca actcgcggtc 840acaaatcaag gccgcactgg
acaatgcctc cnaaattatg agcctgacaa aaacggctcc 900ggactacctg
gtgggaaaca acccgccgga ggacattact canaaccgga tctacaaaat
960cctcgagatg aacgggtacg atccgcagta cgcggcctcc gtcttcctgg
gctgggcgca 1020aaagaagttc gggaagagga acaccatctg gctctttggg
ccggccacga cgggtaaaac 1080caacatcgct gaagctatcg cccacgccgt
gcccttttac ggctgcgtga actggaccaa 1140tgagaacttt ccgttcaacg
attgcgtcga caagatggtg atctggtggg aggagggcaa 1200gatgacggcc
aaggtcgtgg agtccgccaa ggccattctg ggcggaagca aggtgcgcgt
1260ggaccaaaag tgcaagtcat cggcccagat cgacccaact cccgtcatcg
tcacctccaa 1320caccaacatg tgcgcggtca tcgacggaaa ttccaccacc
ttcgagcacc aacaaccact 1380ccaagaccgg atgttcaagt tcgagctcac
caagcgcctg gagcacgact ttggcaaggt 1440caccaagcag gaagtcaagg
actttttccg gtgggcgtca gatcacgtga ctgaggtgtc 1500tcacgagttt
tacgtcagaa agggtggagc tagaaagagg cccgccccca atgacgcaga
1560tataagtgag cccaagcggg cctgtccgtc agttgcgcag ccatcgacgt
cagacgcgga 1620agctccggtg gactacgcgg acaggtacca aaacaaatgt
tctcgtcacg tgggcatgaa 1680tctgatgctt tttccctgcc ggcaatgcga
gagaatgaat cagaatgtgg acatttgctt 1740cacgcacggg gtcatggact
gtgccgagtg cttccccgtg tcagaatctc aacccgtgtc 1800tgtcgtcaga
aagcggacat atcagaaact gtgtccgatt catcacatca tggggagggc
1860gcccgaggtg gcttgttcgg cctgcgatct ggccaatgtg gacttggatg
actgtgacat 1920ggagcaataa atgactcaaa ccagatatga ctgacggtta
ccttccagat tggctagagg 1980acaacctctc tgaaggcgtt cgagagtggt
gggcgctgca acctggagcc cctaaaccca 2040aggcaaatca acaacatcag
gacaacgctc ggggtcttgt gcttccgggt tacaaatacc 2100tcggacccgg
caacggactt gacaaggggg aacccgtcaa cgcagcggac gcggcagccc
2160tcgaacacga caaggcctac gaccagcagc tcaaggccgg tgacaacccc
tacctcaagt 2220acaaccacgc cgacgccgag tttcaggagc gtcttcaaga
agatacgtct tttgggggca 2280acctcggacg agcagtcttc caggccaaaa
agaggatcct tgagcctctg ggtctggttg 2340aggaagcggc taagacggct
cctggaaaaa agagacctgt agagcaatct ccagcagaac 2400cggactcctc
ttcgggcatc ggcaaatcag gccagcagcc cgctagaaaa agactgaatt
2460ttggtcagac tggcgacaca gagtcagtcc cagaccctca accactcgga
caacctcccg 2520cagccccctc tggtgtggga tctactacaa tggcttcagg
cggtggcgca ccaatggcag 2580acaataacga gggtgccgat ggagtgggta
attcctcagg aaattggcat tgcgattccc 2640aatggctggg cgacagagtc
atcaccacca gcacccgcac ctgggccctg cccacctaca 2700acaatcacct
ctacaagcaa atctccagcc aatcaggagc caccaacgac aaccactact
2760ttggctacag caccccctgg gggtattttg acttcaacag attccactgc
cacttttcac 2820cacgtgactg gcaaagactc atcaacaaca actggggatt
ccgacccaag agactcaact 2880tcaagctctt taacattcaa gtcaaagagg
tcacgcagaa tgacggtacg acgacgattg 2940ccaataacct taccagcacg
gttcaggtgt ttactgactc cgagtaccag ctcccgtacg 3000tcctcggctc
ggcgcatcag ggatgcctcc cgccgttccc agcagacgtc ttcatggtcc
3060cacagtatgg atacctcacc ctgaacaacg ggagtcaggc ggtaggacgc
tcttcctttt 3120actgcctgga gtactttcct tctcagatgc tgcgtactgg
aaacaacttt cagtttagct 3180acacttttga agacgtgcct ttccacagca
gctacgctca cagccaaagt ctggaccgtc 3240tcatgaatcc tctgatcgac
cagtacctgt actatctgaa caggacacaa acagccagtg 3300gaactcagca
gtctcggcta ctgtttagcc aagctggacc caccagtatg tctcttcaag
3360ctaaaaactg gctgcctgga ccttgctaca gacagcagcg tctgtcaaag
caggcaaacg 3420acaacaacaa cagcaacttt ccctggactg gtgccaccaa
atatcatctg aatggccggg 3480actcattggt gaacccgggc cctgctatgg
ccagtcacaa ggatgacaaa gaaaagtttt 3540tccccatgca tggaaccctg
atatttggta aagaaggaac aaatgccaac aacgcggatt 3600tggaaaatgt
catgattaca gatgaagaag aaatccgcac caccaatccc gtggctacgg
3660agcagtacgg gactgtgtca aataatttgc aaaactcaaa cgctggtcca
actactggaa 3720ctgtcaatca ccaaggagcg ttacctggta tggtgtggca
ggatcgagac gtgtacctgc 3780agggacccat ttgggccaag attcctcaca
ccgatggaca ctttcatcct tctccactga 3840tgggaggttt tgggctcaaa
cacccgcctc ctcagatcat gatcaaaaac actcccgttc 3900cagccaatcc
tcccacaaac tttagtgcgg caaagtttgc ttccttcatc acacagtact
3960ccacggggca ggtcagcgtg gagatcgagt gggagctgca gaaggagaac
agcaaacgct 4020ggaatcccga aattcagtac acttccaact acaacaaatc
tgttaatgtg gactttactg 4080tggacactaa tggtgtgtat tcagagcctc
gccccattgg caccagatac ctgactcgta 4140atctgtaatt gcttgttaat
caataaaccg gttgattcgt ttcagttgaa ctttgg 419643005DNAArtificial
SequenceDescription of Artificial Sequence Note = synthethic
construct 4tggtgggagg agggcaagat gacggccaag gtcgtggagt ccgccaaggc
cattctcggc 60ggcagcaagg tgcgcgtgga ccaaaagtgc aagtcgtccg cccagatcga
tcccaccccc 120gtgatcgtca cctccaacac caacatgtgc gccgtgattg
acgggaacag caccaccttc 180gagcaccagc agccgttgca ggaccggatg
ttcaaatttg aactcacccg ccgtctggag 240catgactttg gcaaggtgac
aaagcaggaa gtcaaagagt tcttccgctg ggcgcaggat 300cacgtgaccg
aggtggcgca tgagttctac gtcagaaagg gtggagccaa caaaagaccc
360gcccccgatg acgcggataa aagcgagccc aagcgggcct gcccctcagt
cgcggatcca 420tcgacgtcag acgcggaagg agctccggtg gactttgccg
acaggtacca aaacaaatgt 480tctcgtcacg cgggcatgct tcagatgctg
tttccctgca agacatgcga gagaatgaat 540cagaatttca acatttgctt
cacgcacggg accagagact gttcagaatg tttccccggc 600gtgtcagaat
ctcaaccggt cgtcagaaaa aggacgtatc ggaaactctg tgcgattcat
660catctgctgg ggcgggctcc cgagattgct tgctcggcct gcgatctggt
caacgtggac 720ctggatgact gtgtctctga gcaataaatg acttaaacca
ggtatggctg ccgatggtta 780tcttccagat tggctcgagg acaacctctc
tgagggcatt cgcgagtggt gggacttgaa 840acctggagcc ccgaaaccca
aagccaacca gcaaaagcag gacgacggcc ggggtctggt 900gcttcctggc
tacaagtacc tcggaccctt caacggactc gacaaggggg agcccgtcaa
960cgcggcggac gcagcggccc tcgagcacga caaggcctac gaccagcagc
tcaaagcggg 1020tgacaatccg tacctgcggt ataaccacgc cgacgccgag
tttcaggagc gtctgcaaga 1080agatacgtct tttgggggca acctcgggcg
agcagtcttc caggccaaga agcgggttct 1140cgaacctttt ggtctggttg
aggaaggcgc taagacggct cctggaaaga aacgtccggt 1200agagcagtcg
ccacaagagc cagactcctc ctcgggcatc ggcaagacag gccagcagcc
1260cgctaaaaag agactcaatt ttggtcagac tggcgactca gagtcagtcc
ccgacccaca 1320acctctcgga gaacctccag caacccccgc tgctgtggga
cctactacaa tggcttcagg 1380cggtggcgca ccaatggcag acaataacga
aggcgccgac ggagtgggta atgcctcagg 1440aaattggcat tgcgattcca
catggctggg cgacagagtc atcaccacca gcacccgcac 1500ctgggccttg
cccacctaca ataaccacct ctacaagcaa atctccagtg cttcaacggg
1560ggccagcaac gacaaccact acttcggcta cagcaccccc tgggggtatt
ttgatttcaa 1620cagattccac tgccactttt caccacgtga ctggcagcga
ctcatcaaca acaattgggg 1680attccggccc aagagactca acttcaagct
cttcaacatc caagtcaagg aggtcacgac 1740gaatgatggc gtcacaacca
tcgctaataa ccttaccagc acggttcaag tcttctcgga 1800ctcggagtac
cagcttccgt acgtcctcgg ctctgcgcac cagggctgcc tccctccgtt
1860cccggcggac gtgttcatga ttccgcaata cggctacctg acgctcaaca
atggcagcca 1920agccgtggga
cgttcatcct tttactgcct ggaatatttc ccttctcaga tgctgagaac
1980gggcaacaac tttaccttca gctacacctt tgaggaagtg cctttccaca
gcagctacgc 2040gcacagccag agcctggacc ggctgatgaa tcctctcatc
gaccaatacc tgtattacct 2100gaacagaact caaaatcagt ccggaagtgc
ccaaaacaag gacttgctgt ttagccgtgg 2160gtctccagct ggcatgtctg
ttcagcccaa aaactggcta cctggaccct gttatcggca 2220gcagcgcgtt
tctaaaacaa aaacagacaa caacaacagc aattttacct ggactggtgc
2280ttcaaaatat aacctcaatg ggcgtgaatc catcatcaac cctggcactg
ctatggcctc 2340acacaaagac gacgaagaca agttctttcc catgagcggt
gtcatgattt ttggaaaaga 2400gagcgccgga gcttcaaaca ctgcattgga
caatgtcatg attacagacg aagaggaaat 2460taaagccact aaccctgtgg
ccaccgaaag atttgggacc gtggcagtca atttccagag 2520cagcagcaca
gaccctgcga ccggagatgt gcatgttatg ggagcattac ctggcatggt
2580gtggcaagat agagacgtgt acctgcaggg tcccatctgg gccaaaattc
ctcacacaga 2640tggacacttt cacccgtctc ctcttatggg cggctttgga
ctcaagcacc cgcctcctca 2700gatcctcatc aaaaacacgc ctgttcctgc
gaatcctccg gcggagtttt cagctacaaa 2760gtttgcttca ttcatcaccc
aatactccac aggacaagtg agtgtggaaa ttgaatggga 2820gctgcagaaa
gaaaacagca agcgctggaa tcccgaagtg cagtacacat ccaattatgc
2880aaaatctgcc aacgttgatt ttaccgtgga caacaatgga ctttatactg
agcctcgccc 2940cattggcacc cgttacctta cccgtcccct gtaattacgt
gttaatcaat aaaccgtttm 3000attcg 300553014DNAArtificial
SequenceDescription of Artificial Sequence Note = synthethic
construct 5atgctnatgt ggtgggagga gggcaagatg acggccaagg tcgtggagtc
cgccaaggcc 60attctcggcg gcagcaaagt gcgcgtggac caaaagtgca agtcgtccgc
ccagatcgat 120cccacccccg tgatcgtcac ctccaacacc aacatgtgcg
ccgtgattga cgggaacagc 180accaccttcg agcaccagca gccgttgcag
gaccggatgt tcaaatttga actcacccgc 240cgtctggagc atgactttgg
caaggtgaca aaacaggaag tcaaagagtt cttccgctgg 300gcgcaggatc
acgtgaccga ggtggcgcat gagttctacg tcagaaaggg tggagccaac
360aagagacccg cccccgatga cgcggataaa agcgagccca agcgggtctg
cccctcagtc 420gcggatccat cgacgtcaga cgcggaagga gctccggtgg
actttgccga caggtaccaa 480aacaaatgtt ctcgtcacgc gggcatgctt
cagatgctgt ttccctgcaa aacatgcgag 540agaatgaatc agaatttcaa
catttgcttc acgcacggga ccagagactg ttcagaatgt 600ttccccggcg
tgtcagaatc tcaaccggtc gtcagaaaaa ggacgtatcg gaaactctgt
660gccattcatc atctgctggg gcgggctccc gagattgctt gctcggcctg
cgatctggtc 720aacgtggacc tggatgactg tgtttctgag caataaatga
cttaaaccag gtatggctgc 780cgatggttat cttccagatt ggctcgagga
caacctctct gagggcattc gcaagtggtg 840ggacttgaaa cctggagccc
cgaaacccaa agccaaccag caaaagcagg acgacggccg 900gggtctggtg
cttcctggct acaagtacct cggacccttc aacggactcg acaaggggga
960gcccgtcaac gcggcggacg cagcggccct cgagcacgac aaggcctacg
accagcagct 1020caaagcgggt gacaatccgt acctgcggta taaccacgcc
gacgccgagt ttcaggagcg 1080tctgcaagaa gatacgtctt ttgggggcaa
cctcgggaga gcagtcttcc aggccaagaa 1140gcgggttctc gaaccttttg
gtctggttga ggaaggcgct aagacggctc ctggaaagaa 1200acgtccggta
gagcagtcgc cacaagagcc agactcctcc tcgggcatcg gcaagacagg
1260ccagcagccc gctaaaaaga gactcaattt tggtcagact ggcgactcag
agtcagtccc 1320cgacccacaa cctctcggag aacctccagc aacccccgct
gctgtgggac ctactacaat 1380ggcttcaggc ggtggcgcac caatggcaga
caataacgaa ggcgccgacg gagtgggtaa 1440tgcctcagga aattggcatt
gcgattccac atggctgggc gacagagtca tcaccaccag 1500cacccgcacc
tgggccttgc ccacctacaa taaccacctc tacaagcaaa tctccagtgc
1560ttcaacgggg gccagcaacg acaaccacta cttcggctac agcaccccct
gggggtattt 1620tgatttcaac agattccact gccacttttc accacgtgac
tggcagcgac tcatcaacaa 1680caattgggga ttccggccca agagactcaa
cttcaagctc ttcaacatcc aagtcaagga 1740ggtcacgacg aatgatggcg
tcacaaccat cgctaataac cttaccagca cggttcaagt 1800cttctcggac
tcggagtacc agcttccgta cgtcctcggc tctgcgcacc agggctgcct
1860ccctccgttc ccggcggacg tgttcatgat tccgcaatac ggctacctga
cgctcaacaa 1920tggcagccaa gccgtgggac gttcatcctt ttactgcctg
gaatatttcc cttctcagat 1980gctgagaacg ggcaacaact ttaccttcag
ctacaccttt gaggaagtgc ctttccacag 2040cagctacgcg cacagccaga
gcctggaccg gctgatgaat cctctcatcg accaatacct 2100gtattacctg
aacagaactc aaaatcagtc cggaagtgcc caaaacaagg acttgctgtt
2160tagccgtggg tctccagctg gcatgtctgt tcagcccaaa aactggctac
ctggaccctg 2220ttatcggcag cagcgcgttt ctaaaacaaa aacagacaac
aacaacagca attttacctg 2280gactggtgct tcaaaatata acctcaatgg
gcgtgaatcc atcatcaacc ctggcactgc 2340tatggcctca cacaaagacg
acaaagacaa gttctttccc atgagcggtg tcatgatttt 2400tggaaaagag
agcgccggag cttcaaacac tgcattggac aatgtcatga ttacagacga
2460agaggaaatt aaagccacta accctgtggc caccgaaaga tttgggaccg
tggcagtcaa 2520tttccagagc agcagcacag accctgcgac cggagatgtg
catgttatgg gagcattacc 2580tggcatggtg tggcaagata gagacgtgta
cctgcagggt cccatttggg ccaaaattcc 2640tcacacagat ggacactttc
acccgtctcc tcttatgggc ggctttggac tcaagcaccc 2700gcctcctcag
atcctcatca aaaacacacc tgttcctgcg aatcctccgg cggagttttc
2760agctacaaag tttgcttcat tcatcaccca atactccaca ggacaagtga
gtgtggaaat 2820tgaatgggag ctgcagaaag aaaacagcaa gcgctggaat
cccgaagtgc agtacacatc 2880caattatgca aaatctgcca acgttgattt
taccgtggac aacaatggac tttatactga 2940gcctcgcccc attggcaccc
gttaccttac ccgtcccctg taattacgtg ttaatcaata 3000aaccgkttha ttcg
301463010DNAArtificial SequenceDescription of Artificial Sequence
Note = synthethic construct 6tgatttggtg ggaggagggc aagatgacgg
ccaaggtcgt ggagtccgcc aaagccattc 60tcggcggcag caaggtgcgc gtggaccaaa
agtgcaagtc gtccgcccrg atcgatccca 120cccccgtgat cgtcacctcc
aacaccaaca tgtgcgccgt gattgacggg aacagcacca 180ccttcgagca
ccagcagccg ttgcaggacc ggatgttcaa atttgaactc acccgccgtc
240tggagcatga ctttggcaag gtgacaaagc aggaagtcaa agagttcttc
cgctgggcgc 300aggatcacgt gaccgaggtg gcgcatgagt tctacgtcag
aaagggtgga gccaacaaaa 360gacccgcccc cgatgacgcg gataaaagcg
agcccaagcg ggcctgcccc tcagtcgcgg 420atccatcgac gtcagacgcg
gaaggagctc cggtggactt tgccgacagg taccaaaaca 480aatgttctcg
tcacgcgggc atgcttcaga tgctgtttcc ctgcaagaca tgcgagagaa
540tgaatcagaa tttcaacatt tgcttcacgc acgggaccag agactgttca
gaatgtttcc 600ccggcgtgtc agaatctcaa ccggtcgtca gaaaaaggac
gtatcggaaa ctctgtgcga 660ttcatcatct gctggggcgg gctcccgaga
ttgcttgctc ggcctgcgat ctggtcaacg 720tggacctgga tgactgtgtc
tctgagcaat aaatgactta aaccaggtat ggctgccgat 780ggttatcttc
cagattggct cgaggacaac ctctctgagg gcattcgcga gtggtgggac
840ttgaaacctg gagccccgaa acccaaagcc aaccagcaaa agcaggacaa
cggccggggt 900ctggtgcttc ctggctacaa gtacctcgga cccttcaacg
gactcgacaa gggggagccc 960gtcaacgcgg cggacgcagc ggccctcgag
cacgacaagg cctacgacca gcagctcaaa 1020gcgggtgaca atccgtacct
gcggtataac cacgccgacg ccgagtttca ggagcgtctg 1080caagaagata
cgtcttttgg gggcaacctc gggcgagcag tcttccaggc caagaagcgg
1140gttctcgaac cttttggtct ggttgaggaa ggcgctaaga cggctcctgg
aaagaaacgt 1200ccggtagagc agtcgccaca agagccagac tcctcctcgg
gcatcggcaa gacaggccag 1260cagcccgcta aaaagagact caattttggt
cagactggcg actcagagtc agtccccgac 1320ccacaacctc tcggagaacc
tccagcaacc cccgctgctg tgggacctac tacaatggct 1380tcaggcggtg
gcgcaccaat ggcagacaat aacgaaggcg ccgacggagt gggtaatgcc
1440tcaggaaatt ggcattgcga ttccacatgg ctgggcgaca gagtcatcac
caccagcacc 1500cgcacctggg ccttgcccac ctacaataac cacctctaca
agcaaatctc cagtgcttca 1560acgggggcca gcaacgacaa ccactacttc
ggctacagca ccccctgggg gtattttgat 1620ttcaacagat tccactgcca
cttttcacca cgtgactggc agcgactcat caacaacaat 1680tggggattcc
ggcccaagag actcaacttc aagctcttca acatccaagt caaggaggtc
1740acgacgaatg atggcgtcac aaccatcgct aataacctta ccagcacggt
tcaagtcttc 1800tcggactcgg agtaccagct tccgtacgtc ctcggctctg
cgcaccaggg ctgcctccct 1860ccgttcccgg cggacgtgtt catgattccg
caatacggct acctgacgct caacaatggc 1920agccaagccg tgggacgttc
atccttttac tgcctggaat atttcccttc tcagatgctg 1980agaacgggca
acaactttac cttcagctac acctttgagg aagtgccttt ccacagcagc
2040tacgcgcaca gccagagcct ggaccggctg atgaatcctc tcatcgacca
atacctgtat 2100tacctgaaca gaactcaaaa tcagtccgga agtgcccaaa
acaaggactt gctgtttagc 2160cgtgggtctc cagctggcat gtctgttcag
cccaaaaact ggctacctgg accctgttat 2220cggcagcagc gcgtttctaa
aacaaaaaca gacaacaaca acagcaattt tacctggact 2280ggtgcttcaa
aatataacct caatgggcgt gaatccatca tcaaccctgg cactgctatg
2340gcctcacaca aagacgacga agacaagttc tttcccatga gcggtgtcat
gatttttgga 2400aaagagagcg ccggagcttc aaacactgca ttggacaatg
tcatgattac agacgaagag 2460gaaattaaag ccactaaccc tgtggccacc
gaaagatttg ggaccgtggc agtcaatttc 2520cagagcagca gcacacaccc
tgcgaccgga gatgtgcatg ttatgggagc attacctggc 2580atggtgtggc
aagatagaga cgtgtacctg cagggtccca tctgggccaa aattcctcac
2640acagatggac actttcaccc gtctcctctt atgggcggct ttggactcaa
gcacccgcct 2700cctcagatcc tcatcaaaaa cacgcctgtt cctgcgaatc
ctccggcgga gttttcagct 2760acaaagtttg cttcattcat cacccaatac
tccacaggac aagtgagtgt ggaaattgaa 2820tgggagctgc agaaagaaaa
cagcaagcgc tggaatcccg aagtgcagta cacatccaat 2880tatgcaaaat
ctgccaacgt tgattttacc gtggacaaca atggacttta tactgagcct
2940cgccccattg gcacccgtta ccttacccgt cccctgtaat tacgtgttaa
tcaataaacc 3000gtttyattcg 301073008DNAArtificial
SequenceDescription of Artificial Sequence Note = synthethic
construct 7atytggtggg aggagggcaa gatgacggcc aaggtcgtgg agtccgccaa
ggccattctc 60ggcggcagca aggtgcgcgt ggaccaaaag tgcaagtcgt ccgcccagat
cgatcccacc 120cccgtgatcg tcacctccaa caccaacatg tgcgccgtga
ttgacgggaa cagcaccacc 180ttcgagcacc agcagccgtt gcaggaccgg
atgttcaaat ttgaactcac ccgccgtctg 240gagcacgact ttggcaaggt
gacaaagcag gaagtcaaag agttcttccg ctgggcgcag 300gatcacgtga
ccgaggtggc gcatgagttc tacgtcagaa agggtggagc caacaagaga
360cccgcccccg atgacgcgga taaaagcgag cccaagcggg cctgcccctc
agtcgcggat 420ccatcgacgt cagacgcgga aggagctccg gtggactttg
ccgacaggta ccaaaacaaa 480tgttctcgtc acgcgggcat gcttcagatg
ctgtttccct gcaaaacatg cgagagaatg 540aatcagaatt tcaacatttg
cttcacgcac gggaccagag actgttcaga gtgctttccc 600ggcgtgtcag
aatctcaacc ggtcgtcaga aaaaggacgt atcggaaact ctgtgccatt
660catcatctgc tggggcgggc tcccgagatt gcttgctcgg cctgcgatct
ggtcaacgtg 720gacatggatg rctgtgtttc tgagcaataa atgacttaaa
ccaggtatgg ctgccgatgg 780ttatcttcca gattggctcg aggacaacct
ctctgagggc attcgcgagt ggtgggactt 840gaaacctgga gccccgaaac
ccaaagccaa ccagcaaaag caggacgacg gccggggtct 900ggtgcttcct
ggctacaagt acctcggacc cttcaacgga ctcgacaagg gggagcccgt
960caacgcggcg gacgcagcgg ccctcgagca cgacaaggcc tacgaccagc
agctcaaagc 1020gggtgacaat ccgtacctgc ggtataacca cgccgacgcc
gagtttcagg agcgtctgca 1080agaagatacg tcttttgggg gcaacctcgg
gcgagcagtc ttccaggcca aaaagcgggt 1140tctcgaacct tttggtctgg
ttgaggaagg cgctaagacg gctcctggaa agaaacgtcc 1200ggtagagcag
tcgccacaag agccagactc ctcctcgggc atcggcaaga caggccagca
1260gcccgctaaa aagagactca attttggtca gactggcgac tcagagtcag
tccccgaccc 1320acaacctctc ggagaacctc cagcaacccc cgctgctgtg
ggacctacta caatggcttc 1380aggcggtggc gcaccaatgg cagacaataa
cgaaggcgcc gacggagtgg gtaatgcctc 1440aggaaattgg cattgcgatt
ccacatggct gggcgacaga gtcatcacca ccagcacccg 1500cacctgggcc
ttgcccacct acaataacca cctctacaag caaatctcca gtgcttcaac
1560gggggccagc aacgacaacc actacttcgg ctacagcacc ccctgggggt
attttgattt 1620caacagattc cactgccact tttcaccacg tgactggcag
cgactcatca acaacaattg 1680gggattccgg cccaagagac tcaacttcaa
gctcttcaac atccaagtca aggaggtcac 1740gacgaatgat ggcgtcacaa
ccatcgctaa taaccttacc agcacggttc aagtcttctc 1800ggactcggag
taccagcttc cgtacgtcct cggctctgcg caccagggct gcctccctcc
1860gttcccggcg gacgtgttca tgattccgca atacggctac ctgacgctca
acaatggcag 1920ccaagccgtg ggacgttcat ccttttattg cctggaatat
ttcccatcgc agatgctgag 1980aacgggcaat aactttacct tcagctacac
ctttgaggac gtgcctttcc acagcagcta 2040cgcgcacagc cagagcctgg
accggctgat gaatcctctc atcgaccagt acctgtatta 2100cctgaacaga
actcagaacc agtccggaag tgcccaaaac aaggacttgc tgtttagccg
2160ggggtctcca gctggcatgt ctgttcagct caaaaactgg ctacctggac
cctgttatcg 2220gcagcagcgc gtttctaaaa caaaaacaga caacaacaac
agcaatttta cctggactgg 2280tgcttcaaaa tataacctta atgggcgtga
atccatcatc aaccctggca ctgctatggc 2340ctcacacaaa gacgacgaag
acaagttctt tcccatgagc ggtgtcatga tttttggaaa 2400agagagcgcc
ggagcttcaa acactgcatt ggacaatgtc atgattacag acgaagagga
2460aattaaagcc actaaccctg tggccaccga aagatttggg accgtggcag
tcaatttcca 2520gagcagcagc acagaccctg cgaccggaga tgtgcatgtt
atgggagcat tacctggcat 2580ggtgtggcaa gatagagacg tgtacctgca
gggtcccatt tgggccaaaa ttcctcacac 2640agatggacac tttcacccgt
ctcctcttat gggcggcttt ggactcaaac acccgcctcc 2700tcagatcctc
atcaaaaaca cacctgttcc tgcgaatcct ccggcggagt tttcagctac
2760aaagtttgct tcattcatca cccaatactc cacaggacaa gtgagcgtgg
aaattgaatg 2820ggagctgcag aaagaaaaca gcaagcgctg gaatcccgaa
gtgcagtaca catccaatta 2880tgcaaaatct gccaacgttg attttaccgt
ggacaacaat ggactttata ctgagcctcg 2940ccccattggc acccgttacc
ttacccgtcc cctgtaatta cgtgttaatc aataaaccgk 3000ttaattcg
300883021DNAArtificial SequenceDescription of Artificial Sequence
Note = synthethic construct 8atgctdatgt ggtgggagga gggcaagatg
acggccaagg tcgtggagtc cgccaaggcc 60attctcggcg gcagcaaagt gcgcgtggac
caaaagtgca agtcgtccgc ccagatcgac 120cccacccccg tcatcgtcac
ctccaacacc aacatgtgcg ccgtgattga cgggaacagc 180accaccttcg
agcaccagca gccgttgcag gaccggatgt tcaaatttga actcacccgc
240cgtctggagc acgactttgg caaggtgaca aagcaggaag tcaaagagtt
cttccactgg 300gcgcaggatc acgtgaccga ggtggcgcat gagttctacg
tcagaaaggg tggagccaac 360aagagacccg cccccgatga cgcggataaa
agcgagccca agcgggcctg cccctcagtc 420gcggatccat cgacgtcaga
cgcggaagga gctccggtgg actttgccga caggtaccaa 480aacaaatgtt
ctcgtcacgc gggcatgctt cagatgctgt ttccctgcaa aacatgcgag
540agaatgaatc agaatttcaa catttgcttc acgcacggga ccagagactg
ttcagaatgt 600ttccccggcg tgtcagaatc tcaaccggtc gtcagaaaaa
agacgtatcg gaaactctgt 660gccattcatc atctgctggg gcgggctccc
gagattgctt gctcggcctg cgatctggtc 720aatgtggacc tggatgactg
tgtttctgag caataaatga cttaaaccag gtatggctgc 780cgatggttat
cttccagatt ggctcgagga caacctctct gagggcattc gcgagtggtg
840ggacttgaaa cctggagccc cgaaacccaa agccaaccag caaaagcagg
acgacggccg 900gggtctggtg cttcctggct acaagtacct cggacccttc
aacggactcg acaaggggga 960gcccgtcaac gcggcggacg cagcggccct
cgagcacgac aaggcctacg accagcagct 1020caaagcgggt gacaatccgt
acctgcggta taaccacgcc gacgccgagt ttcaggagcg 1080tctgcaagaa
gatacgtctt ttgggggcaa cctcgggcga gcagtcttcc aggccaagaa
1140gcgggttctc gaaccttttg gtctggttga ggaaggtgct aagacggctc
ctggaaagaa 1200gagaccggta gagcagtcgc cccaagaacc agactcctca
tcgggcatcg gcaaatcagg 1260ccagcagccc gctaaaaaga gactcaattt
tggtcagact ggcgactcag agtcagtccc 1320cgacccacaa cctctcggag
aacctccagc aacccccgct gctgtgggac ctactacaat 1380ggcttcaggc
ggtggcgcac caatggcaga caataacgaa ggcgccgacg gagtgggtaa
1440tgcctcagga aattggcatt gcgattccac atggctgggc gacagagtca
ttaccaccag 1500cacccgaacc tgggccctgc ccacctataa caaccacctc
tacaaacaaa tctccagcgc 1560ttcaacgggg gccagcaacg acaaccacta
cttcggctac agcaccccct gggggtattt 1620tgattttaac agattccact
gccacttctc accacgtgac tggcagcgac tcatcaacaa 1680caattgggga
ttccggccca agagactcaa cttcaagctc ttcaacatcc aagtcaagga
1740ggtcacgacg aacgatggcg tcacgaccat cgctaataac cttaccagca
cggttcaagt 1800cttctcggac tcggagtacc agttgccgta cgtcctcggc
tctgcgcacc agggctgcct 1860ccctccgttc ccggcggacg tgttcatgat
tccgcagtac ggctacctaa cactcaacaa 1920tggcagccag gccgtgggac
gttcatcctt ttactgcctg gaatatttcc catcgcagat 1980gctgagaacg
ggcaataact ttaccttcag ctacacattc gaggacgtgc ctttccacag
2040cagctacgcg cacagccaaa gcctggaccg gctgatgaat cctctcatcg
accagtactt 2100gtattaccta aacagaactc aaaatcagtc cggaagtgcc
caaaacaagg acttgctgtt 2160tagccggggg tctccagctg gcatgtctgt
tcagcccaaa aactggctac ctggaccctg 2220ttatcggcag cagcgcgttt
ctaaaacaaa aacagacaac aacaacagca actttacctg 2280gactggtgct
tcaaaatata accttaatgg gcgtgaatct ataatcaacc ctggcactgc
2340tatggcttca cacaaagacg acgaagacaa gttctttcca atgagcggtg
tcatgatttt 2400tggcaaggag agcgccggag cttcaaacac tgcattggac
aatgtcatga ttacagacga 2460agaggaaatt aaagccacta accctgtggc
caccgaaaga tttgggaccg tggcagtcaa 2520tttccagagc agcagcacag
accctgcgac cggagatgtg catgttatgg gagcattacc 2580tggcatggtg
tggcaagata gagacgtgta cctgcagggt ccaatttggg ccaaaattcc
2640tcacacagat ggacactttc acccgtctcc tcttatgggc ggctttggac
ttaagcaccc 2700gcctcctcag atcctcatca aaaacacgcc tgttcctgcg
aatcctccgg cagagttttc 2760ggctacaaag tttgcttcat tcatcaccca
gtattccaca ggacaagtga gtgtggaaat 2820tgaatgggag ttgcagaaag
aaaacagcaa gcgttggaat cccgaagtgc agtacacatc 2880taattatgca
aaatctgcca acgttgattt cactgtggac aacaatggac tttatactga
2940gcctcgcccc attggcaccc gttacctcac ccgtcccctg taattacttg
ttaatcaata 3000aaccgtgtha ttcgtgtcag t 302194222DNAArtificial
SequenceDescription of Artificial Sequence Note = synthethic
construct 9ttgcgacatt ttgcgacacc atgtggccat tcagggtata tatggccgag
tgagcgagca 60ggatctccat tttgaccgcg aaatttgaac gagcagcagc catgccgggc
ttctacgaga 120tcgtgatcaa ggtgccgagc gacctggacg agcacctgcc
gggcatttct gactcgtttg 180tgaactgggt ggccgagaag gaatgggagc
tgcccccgga ttctgacatg gatctgaatc 240tgattgagca ggcacccctg
accgtggccg agaagctgca gcgcgacttc ctggtccaat 300ggcgccgcgt
gagtaaggcc ccggaggccc tcttctttgt tcagttcgag aagggcgagt
360cctacttcca cctccatatt ctggtggaga ccacgggggt caaatccatg
gtgctgggcc 420gcttcctgag tcagattagg gacaagctgg tgcagaccat
ctaccgcggg atcgagccga 480ccctgcccaa ctggttcgcg gtgaccaaga
cgcgtaatgg cgccggaggg gggaacaagg 540tggtggacga gtgctacatc
cccaactacc tcctgcccaa gactcagccc gagctgcagt 600gggcgtggac
taacatggag gagtatataa gcgcgtgttt gaacctggcc gagcgcaaac
660ggctcgtggc gcagcacctg acccacgtca gccagaccca ggagcagaac
aaggagaatc 720tgaacccgaa ttctgacgcg cctgtcatcc ggtcaaaaac
ctccgcgcgc tacatggagc 780tggtcgggtg gctggtggac cggggcatca
cctccgagaa gcagtggatc caggaggacc 840aggcctcgta catctccttc
aacgccgcct ccaactcgcg gtcccagatc aaggccgctc 900tggacaatgc
cggcaagatc atggcgctaa ccaaatccgc gcccgactac ctggtaggcc
960ccgctccgcc cgcggacatt aaaaccaacc gcatttaccg catcctggag
ctgaacggct 1020acgaccctgc ctacgccggc tccgtctttc tcggctgggc
ccagaaaagg ttcgggaagc 1080gcaacaccat ctggctgttt gggccggcca
ccacgggcaa gaccaacatc gcggaagcca 1140tcgcacacgc cgtgcccttc
tacggctgcg tcaactggac caatgaaaac tttcccttca 1200acgactgcgt
cgacaagatg gtgatctggt gggaggaggg
caagatgacg gccaaggtcg 1260tggagtccgc caaggccatt ctcggcggca
gcaaggtgcg cgtggaccaa aagtgcaagt 1320cgtccgccca gatcgatccc
acccccgtga tcgtcacctc caacaccaac atgtgcgccg 1380tgattgacgg
gaacagcacc accttcgagc accagcagcc gttgcaggac cggatgttca
1440aatttgaact cacccgccgt ctggagcacg actttggcaa ggtgacaaag
caggaagtca 1500aagagttctt ccgctgggcg caggatcacg tgaccgaggt
ggcgcatgag ttctacgtca 1560gaaagggtgg agccaacaag agacccgccc
ccgatgacgc ggataaaagc gagcccaagc 1620gggtctgccc ctcagtcgcg
gatccatcga cgtcagacgc ggaaggagct ccggtggact 1680ttgccgacag
gtaccaaaac aaatgttctc gtcacgcggg catgcttcag atgctgtttc
1740cctgcaaaac atgcgagaga atgaatcaga atttcaacat ttgcttcacg
cacgggacca 1800gagactgttc agaatgtttc cccggcgtgt cagaatctca
accggtcgtc agaaaaagga 1860cgtatcggaa actctgtgcc attcatcatc
tgctggggcg ggctcccgag attgcttgct 1920cggcctgcga tctggtcaac
gtggacctgg atgactgtgt ttctgagcaa taaatgactt 1980aaaccaggta
tggctgccga tggttatctt ccagattggc tcgaggacaa cctctctgag
2040ggcattcgcg agtggtggga cttgaaacct ggagccccga aacccaaagc
caaccagcaa 2100aagcaggacg acggccgggg tctggtgctt cctggctaca
agtacctcgg acccttcaac 2160ggactcgaca agggggagcc cgtcaacgcg
gcggacgcag cggccctcga gcacgacaag 2220gcctacgacc agcagctcaa
agcgggtgac aatccgtacc tgcggtataa ccacgccgac 2280gccgagtttc
aggagcgtct gcaagaagat acgtcttttg ggggcaacct cgggcgagca
2340gtcttccagg ccaagaagag ggttctcgaa ccttttggtc tggttgagga
aggtgctaag 2400acggctcctg gaaagaaacg tccggtagag cagtcacccc
aagaaccaga ctcctcctcg 2460ggcattggca aatcaggcca gcagcccgct
aaaaagagac tcaattttgg tcagactggc 2520gactcagagt cagtccccga
cccacaacct ctcggagaac ctccagcaac ccccgctgct 2580ttgggaccta
ctacaatggc ttcaggcggt ggcgcaccaa tggcagacaa taacgaaggc
2640gccgacggag tgggtaatgc ctcaggaaat tggcattgcg attccacatg
gctgggcgac 2700agagtcatca ccaccagcac ccgcacctgg gccttgccca
cctacaataa ccacctctac 2760aagcaaatct ccagtgcttc aacgggggcc
agcaacgaca accactactt cggctacagc 2820accccctggg ggtattttga
tttcaacaga ttccactgcc atttctcacc acgtgactgg 2880cagcgactca
tcaacaacaa ctggggattc cggcccaaga gactcaactt caagctcttc
2940aacatccaag tcaaggaggt cacgacgaac gatggcgtca cgaccatcgc
taataacctt 3000accagcacgg ttcaagtctt ctcggactcg gagtaccagc
ttccgtacgt cctcggctct 3060gcgcaccagg gctgcctccc tccgttcccg
gcggacgtgt tcatgattcc gcagtacggc 3120tacctgacgc tcaacaatgg
cagcaaagcc gtgggacgtt catcctttta ctgcctggaa 3180tatttccctt
ctcagatgct gagaacgggc aacaacttta ccttcagcta cacctttgag
3240gaagtgcctt tccacagcag ctacgcgcac agccagagcc tggaccggct
gatgaatcct 3300ctcatcgacc aatacctgta ttacctgaac agaactcaaa
atcagtccgg aagtgcccaa 3360aacaaggact tgctgtttag ccgtgggtct
ccagctggca tgtctgttca gcccaaaaac 3420tggctacctg gaccctgtta
tcggcagcag cgcgtttcta aaacaaaaac agacaacaac 3480aacagcaatt
ttacctggac tggtgcttca aaatataacc tcaatgggcg tgaatccatc
3540atcaaccctg gcactgctat ggcctcacac aaagacgacg aagacaagtt
ctttcccatg 3600agcggtgtca tgatttttgg aaaagagagc gccggagctt
caaacactgc attggacaat 3660gtcatgatta cagacgaaga ggaaattaaa
gccactaacc ctgtggccac cgaaagattt 3720gggaccgtgg cagtcaattt
ccagagcagc agcacagacc ctgcgaccgg agatgtgcat 3780gttatgggag
cattacctgg catggtgtgg caagatagag acgtgtacct gcagggtccc
3840atttgggcca aaattcctca cacggatgga cactttcacc cgtctcctct
tatgggcggc 3900tttggactca agcacccgcc tcctcagatc ctcatcaaaa
acacacctgt tcctgcgaat 3960cctccggcag agttttcggc tacaaagttt
gcttcattca tcacccagta ctccacagga 4020caagtgagcg tggaaattga
atgggagttg cagaaagaaa acagtaagcg ctggaatccc 4080gaagtgcagt
acacatctaa ttatgcaaaa tctgccaacg ttgacttcac tgtggacaac
4140aatggacttt atactgagcc tcgccccatt ggcacccgtt acctcacccg
tcccctgtaa 4200ttacttgtta atcaataaac cg 4222104213DNAArtificial
SequenceDescription of Artificial Sequence Note = synthethic
construct 10ttgcgacagt ttgcgacacc atgtggtcac aagaggtata taaccgcgag
tgagccagcg 60aggagctcca ttttgcccgc gaagtttgaa cgagcagcag ccatgccggg
gttctacgag 120gtggtgatca aggtgcccag cgacctggac gagcacctgc
ccggcatttc tgactccttt 180gtgaactggg tggccgagaa ggaatgggag
ttgcccccgg attctgacat ggatcagaat 240ctgattgagc aggcacccct
gaccgtggcc gagaagctgc agcgcgagtt cctggtggaa 300tggcgccgag
tgagtaaatt tctggaggcc aagttttttg tgcagtttga aaagggggac
360tcgtactttc atttgcatat tctgattgaa attaccggcg tgaaatccat
ggtggtgggc 420cgctacgtga gtcagattag ggataaactg atccagcgca
tctaccgcgg ggtcgagccc 480cagctgccca actggttcgc ggtcacaaag
acccgaaatg gcgccggagg cgggaacaag 540gtggtggacg agtgctacat
ccccaactac ctgctcccca aggtccagcc cgagcttcag 600tgggcgtgga
ctaacatgga ggagtatata agcgcctgtt tgaacctcgc ggagcgtaaa
660cggctcgtgg cgcagcacct gacgcacgtc tcccagaccc aggagggcga
caaggagaat 720ctgaacccga attctgacgc gccggtgatc cggtcaaaaa
cctccgccag gtacatggag 780ctggtcgggt ggctggtgga caagggcatc
acgtccgaga agcagtggat ccaggaggac 840caggcctcgt acatctcctt
caacgcggcc tccaactccc ggtcgcagat caaggcggcc 900ctggacaatg
cctccaaaat catgagcctc accaaaacgg ctccggacta tctcatcggg
960cagcagcccg tgggggacat taccaccaac cggatctaca aaatcctgga
actgaacggg 1020tacgaccccc agtacgccgc ctccgtcttt ctcggctggg
cccagaaaaa gtttggaaag 1080cgcaacacca tctggctgtt tgggcccgcc
accaccggca agaccaacat cgcggaagcc 1140atcgcccacg cggtcccctt
ctacggctgc gtcaactgga ccaatgagaa ctttcccttc 1200aacgactgcg
tcgacaaaat ggtgatttgg tgggaggagg gcaagatgac cgccaaggtc
1260gtagagtccg ccaaggccat tctgggcggc agcaaggtgc gcgtggacca
aaaatgcaag 1320gcctctgcgc agatcgaccc cacccccgtg atcgtcacct
ccaacaccaa catgtgcgcc 1380gtgattgacg ggaacagcac caccttcgag
caccagcagc ccctgcagga ccggatgttc 1440aagtttgaac tcacccgccg
cctcgaccac gactttggca aggtcaccaa gcaggaagtc 1500aaggactttt
tccggtgggc ggctgatcac gtgactgacg tggctcatga gttttacgtc
1560acaaagggtg gagctaagaa aaggcccgcc ccctctgacg aggatataag
cgagcccaag 1620cggccgcgcg tgtcatttgc gcagccggag acgtcagacg
cggaagctcc cggagacttc 1680gccgacaggt accaaaacaa atgttctcgt
cacgcgggta tgctgcagat gctctttccc 1740tgcaagacgt gcgagagaat
gaatcagaat tccaacgtct gcttcacgca cggtcagaaa 1800gattgcgggg
agtgctttcc cgggtcagaa tctcaaccgg tttctgtcgt cagaaaaacg
1860tatcagaaac tgtgcatcct tcatcagctc cggggggcac ccgagatcgc
ctgctctgct 1920tgcgaccaac tcaaccccga tttggacgat tgccaatttg
agcaataaat gactgaaatc 1980aggtatggct gctgacggtt atcttccaga
ttggctcgag gacaacctct ctgaaggcat 2040tcgcgagtgg tgggcgctga
aacctggagc tccacaaccc aaggccaacc aacagcatca 2100ggacaacggc
aggggtcttg tgcttcctgg gtacaagtac ctcggaccct tcaacggact
2160cgacaaggga gagccggtca acgaggcaga cgccgcggcc ctcgagcacg
acaaggccta 2220cgacaagcag ctcgagcagg gggacaaccc gtatctcaag
tacaaccacg ccgacgccga 2280gttccagcag cgcttggcga ccgacacctc
ttttgggggc aacctcgggc gagcagtctt 2340ccaggccaaa aagaggattc
tcgagcctct gggtctggtt gaagagggcg ttaaaacggc 2400tcctggaaag
aaacgcccat tagaaaagac tccaaatcgg ccgaccaacc cggactctgg
2460gaaggccccg gccaagaaaa agcaaaaaga cggcgaacca gccgactctg
ctagaaggac 2520actcgacttt gaagactctg gagcaggaga cggaccccct
gagggatcat cttccggaga 2580aatgtctcat gatgctgaga tgcgtgcggc
gccaggcgga aatgctgtcg aggcgggaca 2640aggtgccgat ggagtgggta
atgcctccgg tgattggcat tgcgattcca cctggtcaga 2700gggccgagtc
accaccacca gcacccgaac ctgggtccta cccacgtaca acaaccacct
2760gtacctgcga atcggaacaa cggccaacag caacacctac aacggattct
ccaccccctg 2820gggatacttt gactttaacc gcttccactg ccacttttcc
ccacgcgact ggcagcgact 2880catcaacaac aactggggac tcaggccgaa
atcgatgcgt gttaaaatct tcaacataca 2940ggtcaaggag gtcacgacgt
caaacggcga gactacggtc gctaataacc ttaccagcac 3000ggttcagatc
tttgcggatt cgacgtatga actcccatac gtgatggacg ccggtcagga
3060ggggagcttt cctccgtttc ccaacgacgt ctttatggtt ccccaatacg
gatactgcgg 3120agttgtcact ggaaaaaacc agaaccagac agacagaaat
gccttttact gcctggaata 3180ctttccatcc caaatgctaa gaactggcaa
caattttgaa gtcagttacc aatttgaaaa 3240agttcctttc cattcaatgt
acgcgcacag ccagagcctg gacagaatga tgaatccttt 3300actggatcag
tacctgtggc atctgcaatc gaccactacc ggaaattccc ttaatcaagg
3360aacagctacc accacgtacg ggaaaattac cactggagac tttgcctact
acaggaaaaa 3420ctggttgcct ggagcctgca ttaaacaaca aaaattttca
aagaatgcca atcaaaacta 3480caagattccc gccagcgggg gagacgccct
tttaaagtat gacacgcata ccactctaaa 3540tgggcgatgg agtaacatgg
ctcctggacc tccaatggca accgcaggtg ccggggactc 3600ggattttagc
aacagccagc tgatctttgc cggacccaat ccgagcggta acacgaccac
3660atcttcaaac aatttgttgt ttacctcaga agaggagatt gccacaacaa
acccacgaga 3720cacggacatg tttggacaga ttgcagataa taatcaaaat
gccaccaccg cccctcacat 3780cgctaacctg gacgctatgg gaattgttcc
cggaatggtc tggcaaaaca gagacatcta 3840ctaccagggc cctatttggg
ccaaggtccc tcacacggac ggacactttc acccttcgcc 3900gctgatggga
ggatttggac tgaaacaccc gcctccacag attttcatca aaaacacccc
3960cgtacccgcc aatcccaata ctacctttag cgctgcaagg attaattctt
ttctgacgca 4020gtacagcacc ggacaagttg ccgttcagat cgactgggaa
attcagaagg agcattccaa 4080acgctggaat cccgaagttc aatttacttc
aaactacggc actcaaaatt ctatgctgtg 4140ggctcccgac aatgctggca
actaccacga actccgggct attgggtccc gtttcctcac 4200ccaccacttg taa
4213112211DNAArtificial SequenceDescription of Artificial Sequence
Note = synthethic construct 11atggctgccg atggttatct tccagattgg
ctcgaggaca acctctctga gggcattcgc 60gagtggtggg acttgaaacc tggagccccg
aaacccaaag ccaaccagca aaagcaggac 120gacggccggg gtctggtgct
tcctggctac aagtacctcg gacccttcaa cggactcgac 180aagggggagc
ccgtcaacgc ggcggacgca gcggccctcg agcacgacaa ggcctacgac
240cagcagctca aagcgggtga caatccgtac ctgcggtata accacgccga
cgccgagttt 300caggagcgtc tgcaagaaga tacgtctttt gggggcaacc
tcgggcgagc agtcttccag 360gccaagaagc gggttctcga accttttggt
ctggttgagg aaggcgctaa gacggctcct 420ggaaagaaac gtccggtaga
gcagtcgcca caagagccag actcctcctc gggcatcggc 480aagacaggcc
agcagcccgc taaaaagaga ctcaattttg gtcagactgg cgactcagag
540tcagtccccg acccacaacc tctcggagaa cctccagcaa cccccgctgc
tgtgggacct 600actacaatgg cttcaggcgg tggcgcacca atggcagaca
ataacgaagg cgccgacgga 660gtgggtaatg cctcaggaaa ttggcattgc
gattccacat ggctgggcga cagagtcatc 720accaccagca cccgcacctg
ggccttgccc acctacaata accacctcta caagcaaatc 780tccagtgctt
caacgggggc cagcaacgac aaccactact tcggctacag caccccctgg
840gggtattttg atttcaacag attccactgc cacttttcac cgcgtgactg
gcagcgactc 900atcaacaaca attggggatt ccggcccaag agactcaact
tcaagctctt caacatccaa 960gtcaaggagg tcacgacgag tgatggcgtc
acaaccatcg ctaataacct taccagcacg 1020gttcaagtct tctcggactc
ggagtaccag cttccgtacg tcctcggctc tgcgcaccag 1080ggctgcctcc
ctccgttccc ggcggacgtg ttcatgattc cgcaatacgg ctacctgacg
1140ctcaacaatg gcagccaagc cgtgggacgt tcatcctttt actgcctgga
atatttccct 1200tctcagatgc tgagaacggg caacaacttt accttcagct
acacctttga ggaagtgcct 1260ttccacagca gctacgcgca cagccagagc
ctggaccggc tgatgaatcc tctcatcgac 1320caatacctgt attacctgaa
cagaactcaa aatcagtccg gaagtgccca aaacaaggac 1380ttgctgttta
gccgtgggtc tccagctggc atgtctgttc agcccaaaaa ctggctacct
1440ggaccctgtt atcggcagca gcgcgtttct aaaacaaaaa caggcaacaa
caacagcaat 1500tttacctgga ctggtgcttc aaaatataac ctcaatgggc
atgaatccat catcaaccct 1560ggcactgcta tggcctcaca caaagacgac
gaagacaagt tctttcccat gagcggtgtc 1620atgatttttg gaaaagagag
cgccggagct tcaaacactg cattggacaa tgtcatgatt 1680acagacgaag
aggaaattaa agccactaac cctgtggcca ccgaaagatt tgggaccgtg
1740gcagtcaatt tccagagcag cagcacacac cctgcgaccg gagatgtgca
tgttatggga 1800gcattacctg gcatggtgtg gcaagataga gacgtgtacc
tgcagggtcc catctgggcc 1860aaaattcctc acacagatgg acactttcac
ccgtctcctc ttatgggcgg ctttggactc 1920aagcacccgc ctcctcagat
cctcatcaaa aacacgcctg ttcctgcgaa tcctccggcg 1980gagttttcag
ctacaaagtt tgcttcattc atcacccaat actccacagg acaagtgagt
2040gtggaaattg aatgggagct gcagaaagaa aacagcaagc gctggaatcc
cgaagtgcag 2100tacacatcca attatgcaaa atctgccaac gttgatttta
ccgtggacaa caatggactt 2160tatactgagc ctcgccccat tggcacccgt
taccttaccc gtcccctgta a 2211122211DNAArtificial SequenceDescription
of Artificial Sequence Note = synthethic construct 12atggctgccg
atggttatct tccagattgg ctcgaggaca acctctctga gggcattcgc 60gagtggtggg
acttgaaacc tggagccccg aaacccaaag ccaaccagca aaagcaggac
120gacggccggg gtctggtgct tcctggctac aagtacctcg gacccttcaa
cggactcgac 180aagggggagc ccgtcaacgc ggcggacgca gcggccctcg
agcacgacaa ggcctacgac 240cagcagctca aagcgggtga caatccgtac
ctgcggtata accacgccga cgccgagttt 300caggagcgtc tgcaagaaga
tacgtctttt gggggcaacc tcgggcgagc agtcttccag 360gccaagaagc
gggttctcga accttttggt ctggttgagg aaggcgctaa gacggctcct
420ggaaagaaac gtccggtaga gcagtcgcca caagagccag actcctcctc
gggcatcggc 480aagacaggcc agcagcccgc taaaaagaga ctcaattttg
gtcagactgg cgactcagag 540tcagtccccg acccacaacc tctcggagaa
cctccagcaa cccccgctgc tgtgggacct 600actacaatgg cttcaggcgg
tggcgcacca atggcagaca ataacgaagg cgccgacgga 660gtgggtaatg
cctcaggaaa ttggcattgc gattccacat ggctgggcga cagagtcatc
720accaccagca cccgcacctg ggccttgccc acctacaata accacctcta
caagcaaatc 780tccagtgctt caacgggggc cagcaacgac aaccactact
tcggctacag caccccctgg 840gggtattttg atttcaacag attccactgc
cacttttcac cgcgtgactg gcagcgactc 900atcaacaaca attggggatt
ccggcccaag agactcaact tcaagctctt caacatccaa 960gtcaaggagg
tcacgacgag tgatggcgtc acaaccatcg ctaataacct taccagcacg
1020gttcaagtct tctcggactc ggagtaccag cttccgtacg tcctcggctc
tgcgcaccag 1080ggctgcctcc ctccgttccc ggcggacgtg ttcatgattc
cgcaatacgg ctacctgacg 1140ctcaacaatg gcagccaagc cgtgggacgt
tcatcctttt actgcctgga atatttccct 1200tctcagatgc tgagaacggg
caacaacttt accttcagct acacctttga ggaagtgcct 1260ttccacagca
gctacgcgca cagccagagc ctggaccggc tgatgaatcc tctcatcgac
1320caatacctgt attacctgaa cagaactcaa aatcagtccg gaagtgccca
aaacaaggac 1380ttgctgttta gccgtgggtc tccagctggc atgtctgttc
agcccaaaaa ctggctacct 1440ggaccctgtt atcggcagca gcgcgtttct
aaaacaaaaa caggcaacaa caacagcaat 1500tttacctgga ctggtgcttc
aaaatataac ctcaatgggc atgaatccat catcaaccct 1560ggcactgcta
tggcctcaca caaagacgac gaagacaagt tctttcccat gagcggtgtc
1620atgatttttg gaaaagagag cgccggagct tcaaacactg cattggacaa
tgtcatgatt 1680acagacgaag aggaaattaa agccactaac cctgtggcca
ccgaaagatt tgggaccgtg 1740gcagtcaatt tccagagcag cagcacacac
cctgcgaccg gagatgtgca tgttatggga 1800gcattacctg gcatggtgtg
gcaagataga gacgtgtacc tgcagggtcc catctgggcc 1860aaaattcctc
acacagatgg acactttcac ccgtctcctc ttatgggcgg ctttggactc
1920aagcacccgc ctcctcagat cctcatcaaa aacacgcctg ttcctgcgaa
tcctccggcg 1980gagttttcag ctacaaagtt tgcttcattc atcacccaat
actccacagg acaagtgagt 2040gtggaaattg aatgggagct gcagaaagaa
aacagcaagc gctggaatcc cgaagtgcag 2100tacacatcca attatgcaaa
atctgccaac gttgatttta ccgtggacaa caatggactt 2160tatactgagc
ctcgccccat tggcacccgt taccttaccc gtcccctgta a
2211132202DNAArtificial SequenceDescription of Artificial Sequence
Note = synthethic construct 13atgactgacg gttaccttcc agattggcta
gaggacaacc tctctgaagg cgttcgagag 60tggtgggcgc tgcaacctgg agcccctaaa
cccaaggcaa atcaacaaca tcaggacaac 120gctcggggtc ttgtgcttcc
gggttacaaa tacctcggac ccggcaacgg acttgacaag 180ggggaacccg
tcaacgcagc ggacgcggca gccctcgaac acgacaaggc ctacgaccag
240cagctcaagg ccggtgacaa cccctacctc aagtacaacc acgccgacgc
cgagtttcag 300gagcgtcttc aagaagatac gtcttttggg ggcaacctcg
gacgagcagt cttccaggcc 360aaaaagagga tccttgagcc tctgggtctg
gttgaggaag cggctaagac ggctcctgga 420aaaaagagac ctgtagagca
atctccagca gaaccggact cctcttcggg catcggcaaa 480tcaggccagc
agcccgctag aaaaagactg aattttggtc agactggcga cacagagtca
540gtcccagacc ctcaaccact cggacaacct cccgcagccc cctctggtgt
gggatctact 600acaatggctt caggcggtgg cgcaccaatg gcagacaata
acgagggtgc cgatggagtg 660ggtaattcct caggaaattg gcattgcgat
tcccaatggc tgggcgacag agtcatcacc 720accagcaccc gcacctgggc
cctgcccacc tacaacaatc acctctacaa gcaaatctcc 780agccaatcag
gagccaccaa cgacaaccac tactttggct acagcacccc ctgggggtat
840tttgacttca acagattcca ctgccacttt tcaccacgtg actggcaaag
actcatcaac 900aacaactggg gattccgacc caagagactc aacttcaagc
tctttaacat tcaagtcaaa 960gaggtcacgc agaatgacgg tacgacgacg
attgccaata accttaccag cacggttcag 1020gtgtttactg actccgagta
ccagctcccg tacgtcctcg gctcggcgca tcagggatgc 1080ctcccgccgt
tcccagcaga cgtcttcatg gtcccacagt atggatacct caccctgaac
1140aacgggagtc aggcggtagg acgctcttcc ttttactgcc tggagtactt
tccttctcag 1200atgctgcgta ctggaaacaa ctttcagttt agctacactt
ttgaagacgt gcctttccac 1260agcagctacg ctcacagcca aagtctggac
cgtctcatga atcctctgat cgaccagtac 1320ctgtactatc tgaacaggac
acaaacagcc agtggaactc agcagtctcg gctactgttt 1380agccaagctg
gacccaccag tatgtctctt caagctaaaa actggctgcc tggaccttgc
1440tacagacagc agcgtctgtc aaagcaggca aacgacaaca acaacagcaa
ctttccctgg 1500actggtgcca ccaaatatca tctgaatggc cgggactcat
tggtgaaccc gggccctgct 1560atggccagtc acaaggatga caaagaaaag
tttttcccca tgcatggaac cctgatattt 1620ggtaaagaag gaacaaatgc
caacaacgcg gatttggaaa atgtcatgat tacagatgaa 1680gaagaaatcc
gcaccaccaa tcccgtggct acggagcagt acgggactgt gtcaaataat
1740ttgcaaaact caaacgctgg tccaactact ggaactgtca atcaccaagg
agcgttacct 1800ggtatggtgt ggcaggatcg agacgtgtac ctgcagggac
ccatttgggc caagattcct 1860cacaccgatg gacactttca tccttctcca
ctgatgggag gttttgggct caaacacccg 1920cctcctcaga tcatgatcaa
aaacactccc gttccagcca atcctcccac aaactttagt 1980gcggcaaagt
ttgcttcctt catcacacag tactccacgg ggcaggtcag cgtggagatc
2040gagtgggagc tgcagaagga gaacagcaaa cgctggaatc ccgaaattca
gtacacttcc 2100aactacaaca aatctgttaa tgtggacttt actgtggaca
ctaatggtgt gtattcagag 2160cctcgcccca ttggcaccag atacctgact
cgtaatctgt aa 2202142211DNAArtificial SequenceDescription of
Artificial Sequence Note = synthethic construct 14atggctgccg
atggttatct tccagattgg ctcgaggaca acctctctga gggcattcgc 60gagtggtggg
acttgaaacc tggagccccg aaacccaaag ccaaccagca aaagcaggac
120gacggccggg gtctggtgct tcctggctac aagtacctcg gacccttcaa
cggactcgac 180aagggggagc ccgtcaacgc ggcggacgca gcggccctcg
agcacgacaa ggcctacgac 240cagcagctca aagcgggtga caatccgtac
ctgcggtata accacgccga cgccgagttt 300caggagcgtc tgcaagaaga
tacgtctttt gggggcaacc tcgggcgagc agtcttccag 360gccaagaagc
gggttctcga accttttggt ctggttgagg aaggcgctaa gacggctcct
420ggaaagaaac gtccggtaga gcagtcgcca caagagccag actcctcctc
gggcatcggc 480aagacaggcc agcagcccgc taaaaagaga ctcaattttg
gtcagactgg cgactcagag 540tcagtccccg acccacaacc tctcggagaa
cctccagcaa cccccgctgc tgtgggacct 600actacaatgg cttcaggcgg
tggcgcacca atggcagaca ataacgaagg cgccgacgga 660gtgggtaatg
cctcaggaaa ttggcattgc gattccacat ggctgggcga cagagtcatc
720accaccagca cccgcacctg ggccttgccc acctacaata accacctcta
caagcaaatc 780tccagtgctt caacgggggc cagcaacgac aaccactact
tcggctacag caccccctgg 840gggtattttg atttcaacag attccactgc
cacttttcac cacgtgactg gcagcgactc 900atcaacaaca attggggatt
ccggcccaag agactcaact tcaagctctt caacatccaa 960gtcaaggagg
tcacgacgaa tgatggcgtc acaaccatcg ctaataacct taccagcacg
1020gttcaagtct tctcggactc ggagtaccag cttccgtacg tcctcggctc
tgcgcaccag 1080ggctgcctcc ctccgttccc ggcggacgtg ttcatgattc
cgcaatacgg ctacctgacg 1140ctcaacaatg gcagccaagc cgtgggacgt
tcatcctttt actgcctgga atatttccct 1200tctcagatgc tgagaacggg
caacaacttt accttcagct acacctttga ggaagtgcct 1260ttccacagca
gctacgcgca cagccagagc ctggaccggc tgatgaatcc tctcatcgac
1320caatacctgt attacctgaa cagaactcaa aatcagtccg gaagtgccca
aaacaaggac 1380ttgctgttta gccgtgggtc tccagctggc atgtctgttc
agcccaaaaa ctggctacct 1440ggaccctgtt atcggcagca gcgcgtttct
aaaacaaaaa cagacaacaa caacagcaat 1500tttacctgga ctggtgcttc
aaaatataac ctcaatgggc gtgaatccat catcaaccct 1560ggcactgcta
tggcctcaca caaagacgac gaagacaagt tctttcccat gagcggtgtc
1620atgatttttg gaaaagagag cgccggagct tcaaacactg cattggacaa
tgtcatgatt 1680acagacgaag aggaaattaa agccactaac cctgtggcca
ccgaaagatt tgggaccgtg 1740gcagtcaatt tccagagcag cagcacagac
cctgcgaccg gagatgtgca tgttatggga 1800gcattacctg gcatggtgtg
gcaagataga gacgtgtacc tgcagggtcc catctgggcc 1860aaaattcctc
acacagatgg acactttcac ccgtctcctc ttatgggcgg ctttggactc
1920aagcacccgc ctcctcagat cctcatcaaa aacacgcctg ttcctgcgaa
tcctccggcg 1980gagttttcag ctacaaagtt tgcttcattc atcacccaat
actccacagg acaagtgagt 2040gtggaaattg aatgggagct gcagaaagaa
aacagcaagc gctggaatcc cgaagtgcag 2100tacacatcca attatgcaaa
atctgccaac gttgatttta ccgtggacaa caatggactt 2160tatactgagc
ctcgccccat tggcacccgt taccttaccc gtcccctgta a
2211152211DNAArtificial SequenceDescription of Artificial Sequence
Note = synthethic construct 15atggctgccg atggttatct tccagattgg
ctcgaggaca acctctctga gggcattcgc 60aagtggtggg acttgaaacc tggagccccg
aaacccaaag ccaaccagca aaagcaggac 120gacggccggg gtctggtgct
tcctggctac aagtacctcg gacccttcaa cggactcgac 180aagggggagc
ccgtcaacgc ggcggacgca gcggccctcg agcacgacaa ggcctacgac
240cagcagctca aagcgggtga caatccgtac ctgcggtata accacgccga
cgccgagttt 300caggagcgtc tgcaagaaga tacgtctttt gggggcaacc
tcgggagagc agtcttccag 360gccaagaagc gggttctcga accttttggt
ctggttgagg aaggcgctaa gacggctcct 420ggaaagaaac gtccggtaga
gcagtcgcca caagagccag actcctcctc gggcatcggc 480aagacaggcc
agcagcccgc taaaaagaga ctcaattttg gtcagactgg cgactcagag
540tcagtccccg acccacaacc tctcggagaa cctccagcaa cccccgctgc
tgtgggacct 600actacaatgg cttcaggcgg tggcgcacca atggcagaca
ataacgaagg cgccgacgga 660gtgggtaatg cctcaggaaa ttggcattgc
gattccacat ggctgggcga cagagtcatc 720accaccagca cccgcacctg
ggccttgccc acctacaata accacctcta caagcaaatc 780tccagtgctt
caacgggggc cagcaacgac aaccactact tcggctacag caccccctgg
840gggtattttg atttcaacag attccactgc cacttttcac cacgtgactg
gcagcgactc 900atcaacaaca attggggatt ccggcccaag agactcaact
tcaagctctt caacatccaa 960gtcaaggagg tcacgacgaa tgatggcgtc
acaaccatcg ctaataacct taccagcacg 1020gttcaagtct tctcggactc
ggagtaccag cttccgtacg tcctcggctc tgcgcaccag 1080ggctgcctcc
ctccgttccc ggcggacgtg ttcatgattc cgcaatacgg ctacctgacg
1140ctcaacaatg gcagccaagc cgtgggacgt tcatcctttt actgcctgga
atatttccct 1200tctcagatgc tgagaacggg caacaacttt accttcagct
acacctttga ggaagtgcct 1260ttccacagca gctacgcgca cagccagagc
ctggaccggc tgatgaatcc tctcatcgac 1320caatacctgt attacctgaa
cagaactcaa aatcagtccg gaagtgccca aaacaaggac 1380ttgctgttta
gccgtgggtc tccagctggc atgtctgttc agcccaaaaa ctggctacct
1440ggaccctgtt atcggcagca gcgcgtttct aaaacaaaaa cagacaacaa
caacagcaat 1500tttacctgga ctggtgcttc aaaatataac ctcaatgggc
gtgaatccat catcaaccct 1560ggcactgcta tggcctcaca caaagacgac
aaagacaagt tctttcccat gagcggtgtc 1620atgatttttg gaaaagagag
cgccggagct tcaaacactg cattggacaa tgtcatgatt 1680acagacgaag
aggaaattaa agccactaac cctgtggcca ccgaaagatt tgggaccgtg
1740gcagtcaatt tccagagcag cagcacagac cctgcgaccg gagatgtgca
tgttatggga 1800gcattacctg gcatggtgtg gcaagataga gacgtgtacc
tgcagggtcc catttgggcc 1860aaaattcctc acacagatgg acactttcac
ccgtctcctc ttatgggcgg ctttggactc 1920aagcacccgc ctcctcagat
cctcatcaaa aacacacctg ttcctgcgaa tcctccggcg 1980gagttttcag
ctacaaagtt tgcttcattc atcacccaat actccacagg acaagtgagt
2040gtggaaattg aatgggagct gcagaaagaa aacagcaagc gctggaatcc
cgaagtgcag 2100tacacatcca attatgcaaa atctgccaac gttgatttta
ccgtggacaa caatggactt 2160tatactgagc ctcgccccat tggcacccgt
taccttaccc gtcccctgta a 2211162211DNAArtificial SequenceDescription
of Artificial Sequence Note = synthethic construct 16atggctgccg
atggttatct tccagattgg ctcgaggaca acctctctga gggcattcgc 60gagtggtggg
acttgaaacc tggagccccg aaacccaaag ccaaccagca aaagcaggac
120aacggccggg gtctggtgct tcctggctac aagtacctcg gacccttcaa
cggactcgac 180aagggggagc ccgtcaacgc ggcggacgca gcggccctcg
agcacgacaa ggcctacgac 240cagcagctca aagcgggtga caatccgtac
ctgcggtata accacgccga cgccgagttt 300caggagcgtc tgcaagaaga
tacgtctttt gggggcaacc tcgggcgagc agtcttccag 360gccaagaagc
gggttctcga accttttggt ctggttgagg aaggcgctaa gacggctcct
420ggaaagaaac gtccggtaga gcagtcgcca caagagccag actcctcctc
gggcatcggc 480aagacaggcc agcagcccgc taaaaagaga ctcaattttg
gtcagactgg cgactcagag 540tcagtccccg acccacaacc tctcggagaa
cctccagcaa cccccgctgc tgtgggacct 600actacaatgg cttcaggcgg
tggcgcacca atggcagaca ataacgaagg cgccgacgga 660gtgggtaatg
cctcaggaaa ttggcattgc gattccacat ggctgggcga cagagtcatc
720accaccagca cccgcacctg ggccttgccc acctacaata accacctcta
caagcaaatc 780tccagtgctt caacgggggc cagcaacgac aaccactact
tcggctacag caccccctgg 840gggtattttg atttcaacag attccactgc
cacttttcac cacgtgactg gcagcgactc 900atcaacaaca attggggatt
ccggcccaag agactcaact tcaagctctt caacatccaa 960gtcaaggagg
tcacgacgaa tgatggcgtc acaaccatcg ctaataacct taccagcacg
1020gttcaagtct tctcggactc ggagtaccag cttccgtacg tcctcggctc
tgcgcaccag 1080ggctgcctcc ctccgttccc ggcggacgtg ttcatgattc
cgcaatacgg ctacctgacg 1140ctcaacaatg gcagccaagc cgtgggacgt
tcatcctttt actgcctgga atatttccct 1200tctcagatgc tgagaacggg
caacaacttt accttcagct acacctttga ggaagtgcct 1260ttccacagca
gctacgcgca cagccagagc ctggaccggc tgatgaatcc tctcatcgac
1320caatacctgt attacctgaa cagaactcaa aatcagtccg gaagtgccca
aaacaaggac 1380ttgctgttta gccgtgggtc tccagctggc atgtctgttc
agcccaaaaa ctggctacct 1440ggaccctgtt atcggcagca gcgcgtttct
aaaacaaaaa cagacaacaa caacagcaat 1500tttacctgga ctggtgcttc
aaaatataac ctcaatgggc gtgaatccat catcaaccct 1560ggcactgcta
tggcctcaca caaagacgac gaagacaagt tctttcccat gagcggtgtc
1620atgatttttg gaaaagagag cgccggagct tcaaacactg cattggacaa
tgtcatgatt 1680acagacgaag aggaaattaa agccactaac cctgtggcca
ccgaaagatt tgggaccgtg 1740gcagtcaatt tccagagcag cagcacacac
cctgcgaccg gagatgtgca tgttatggga 1800gcattacctg gcatggtgtg
gcaagataga gacgtgtacc tgcagggtcc catctgggcc 1860aaaattcctc
acacagatgg acactttcac ccgtctcctc ttatgggcgg ctttggactc
1920aagcacccgc ctcctcagat cctcatcaaa aacacgcctg ttcctgcgaa
tcctccggcg 1980gagttttcag ctacaaagtt tgcttcattc atcacccaat
actccacagg acaagtgagt 2040gtggaaattg aatgggagct gcagaaagaa
aacagcaagc gctggaatcc cgaagtgcag 2100tacacatcca attatgcaaa
atctgccaac gttgatttta ccgtggacaa caatggactt 2160tatactgagc
ctcgccccat tggcacccgt taccttaccc gtcccctgta a
2211172211DNAArtificial SequenceDescription of Artificial Sequence
Note = synthethic construct 17atggctgccg atggttatct tccagattgg
ctcgaggaca acctctctga gggcattcgc 60gagtggtggg acttgaaacc tggagccccg
aaacccaaag ccaaccagca aaagcaggac 120gacggccggg gtctggtgct
tcctggctac aagtacctcg gacccttcaa cggactcgac 180aagggggagc
ccgtcaacgc ggcggacgca gcggccctcg agcacgacaa ggcctacgac
240cagcagctca aagcgggtga caatccgtac ctgcggtata accacgccga
cgccgagttt 300caggagcgtc tgcaagaaga tacgtctttt gggggcaacc
tcgggcgagc agtcttccag 360gccaaaaagc gggttctcga accttttggt
ctggttgagg aaggcgctaa gacggctcct 420ggaaagaaac gtccggtaga
gcagtcgcca caagagccag actcctcctc gggcatcggc 480aagacaggcc
agcagcccgc taaaaagaga ctcaattttg gtcagactgg cgactcagag
540tcagtccccg acccacaacc tctcggagaa cctccagcaa cccccgctgc
tgtgggacct 600actacaatgg cttcaggcgg tggcgcacca atggcagaca
ataacgaagg cgccgacgga 660gtgggtaatg cctcaggaaa ttggcattgc
gattccacat ggctgggcga cagagtcatc 720accaccagca cccgcacctg
ggccttgccc acctacaata accacctcta caagcaaatc 780tccagtgctt
caacgggggc cagcaacgac aaccactact tcggctacag caccccctgg
840gggtattttg atttcaacag attccactgc cacttttcac cacgtgactg
gcagcgactc 900atcaacaaca attggggatt ccggcccaag agactcaact
tcaagctctt caacatccaa 960gtcaaggagg tcacgacgaa tgatggcgtc
acaaccatcg ctaataacct taccagcacg 1020gttcaagtct tctcggactc
ggagtaccag cttccgtacg tcctcggctc tgcgcaccag 1080ggctgcctcc
ctccgttccc ggcggacgtg ttcatgattc cgcaatacgg ctacctgacg
1140ctcaacaatg gcagccaagc cgtgggacgt tcatcctttt attgcctgga
atatttccca 1200tcgcagatgc tgagaacggg caataacttt accttcagct
acacctttga ggacgtgcct 1260ttccacagca gctacgcgca cagccagagc
ctggaccggc tgatgaatcc tctcatcgac 1320cagtacctgt attacctgaa
cagaactcag aaccagtccg gaagtgccca aaacaaggac 1380ttgctgttta
gccgggggtc tccagctggc atgtctgttc agctcaaaaa ctggctacct
1440ggaccctgtt atcggcagca gcgcgtttct aaaacaaaaa cagacaacaa
caacagcaat 1500tttacctgga ctggtgcttc aaaatataac cttaatgggc
gtgaatccat catcaaccct 1560ggcactgcta tggcctcaca caaagacgac
gaagacaagt tctttcccat gagcggtgtc 1620atgatttttg gaaaagagag
cgccggagct tcaaacactg cattggacaa tgtcatgatt 1680acagacgaag
aggaaattaa agccactaac cctgtggcca ccgaaagatt tgggaccgtg
1740gcagtcaatt tccagagcag cagcacagac cctgcgaccg gagatgtgca
tgttatggga 1800gcattacctg gcatggtgtg gcaagataga gacgtgtacc
tgcagggtcc catttgggcc 1860aaaattcctc acacagatgg acactttcac
ccgtctcctc ttatgggcgg ctttggactc 1920aaacacccgc ctcctcagat
cctcatcaaa aacacacctg ttcctgcgaa tcctccggcg 1980gagttttcag
ctacaaagtt tgcttcattc atcacccaat actccacagg acaagtgagc
2040gtggaaattg aatgggagct gcagaaagaa aacagcaagc gctggaatcc
cgaagtgcag 2100tacacatcca attatgcaaa atctgccaac gttgatttta
ccgtggacaa caatggactt 2160tatactgagc ctcgccccat tggcacccgt
taccttaccc gtcccctgta a 2211182211DNAArtificial SequenceDescription
of Artificial Sequence Note = synthethic construct 18atggctgccg
atggttatct tccagattgg ctcgaggaca acctctctga gggcattcgc 60gagtggtggg
acttgaaacc tggagccccg aaacccaaag ccaaccagca aaagcaggac
120gacggccggg gtctggtgct tcctggctac aagtacctcg gacccttcaa
cggactcgac 180aagggggagc ccgtcaacgc ggcggacgca gcggccctcg
agcacgacaa ggcctacgac 240cagcagctca aagcgggtga caatccgtac
ctgcggtata accacgccga cgccgagttt 300caggagcgtc tgcaagaaga
tacgtctttt gggggcaacc tcgggcgagc agtcttccag 360gccaagaagc
gggttctcga accttttggt ctggttgagg aaggtgctaa gacggctcct
420ggaaagaaga gaccggtaga gcagtcgccc caagaaccag actcctcatc
gggcatcggc 480aaatcaggcc agcagcccgc taaaaagaga ctcaattttg
gtcagactgg cgactcagag 540tcagtccccg acccacaacc tctcggagaa
cctccagcaa cccccgctgc tgtgggacct 600actacaatgg cttcaggcgg
tggcgcacca atggcagaca ataacgaagg cgccgacgga 660gtgggtaatg
cctcaggaaa ttggcattgc gattccacat ggctgggcga cagagtcatt
720accaccagca cccgaacctg ggccctgccc acctataaca accacctcta
caaacaaatc 780tccagcgctt caacgggggc cagcaacgac aaccactact
tcggctacag caccccctgg 840gggtattttg attttaacag attccactgc
cacttctcac cacgtgactg gcagcgactc 900atcaacaaca attggggatt
ccggcccaag agactcaact tcaagctctt caacatccaa 960gtcaaggagg
tcacgacgaa cgatggcgtc acgaccatcg ctaataacct taccagcacg
1020gttcaagtct tctcggactc ggagtaccag ttgccgtacg tcctcggctc
tgcgcaccag 1080ggctgcctcc ctccgttccc ggcggacgtg ttcatgattc
cgcagtacgg ctacctaaca 1140ctcaacaatg gcagccaggc cgtgggacgt
tcatcctttt actgcctgga atatttccca 1200tcgcagatgc tgagaacggg
caataacttt accttcagct acacattcga ggacgtgcct 1260ttccacagca
gctacgcgca cagccaaagc ctggaccggc tgatgaatcc tctcatcgac
1320cagtacttgt attacctaaa cagaactcaa aatcagtccg gaagtgccca
aaacaaggac 1380ttgctgttta gccgggggtc tccagctggc atgtctgttc
agcccaaaaa ctggctacct 1440ggaccctgtt atcggcagca gcgcgtttct
aaaacaaaaa cagacaacaa caacagcaac 1500tttacctgga ctggtgcttc
aaaatataac cttaatgggc gtgaatctat aatcaaccct 1560ggcactgcta
tggcttcaca caaagacgac gaagacaagt tctttccaat gagcggtgtc
1620atgatttttg gcaaggagag cgccggagct tcaaacactg cattggacaa
tgtcatgatt 1680acagacgaag aggaaattaa agccactaac cctgtggcca
ccgaaagatt tgggaccgtg 1740gcagtcaatt tccagagcag cagcacagac
cctgcgaccg gagatgtgca tgttatggga 1800gcattacctg gcatggtgtg
gcaagataga gacgtgtacc tgcagggtcc aatttgggcc 1860aaaattcctc
acacagatgg acactttcac ccgtctcctc ttatgggcgg ctttggactt
1920aagcacccgc ctcctcagat cctcatcaaa aacacgcctg ttcctgcgaa
tcctccggca 1980gagttttcgg ctacaaagtt tgcttcattc atcacccagt
attccacagg acaagtgagt 2040gtggaaattg aatgggagtt gcagaaagaa
aacagcaagc gttggaatcc cgaagtgcag 2100tacacatcta attatgcaaa
atctgccaac gttgatttca ctgtggacaa caatggactt 2160tatactgagc
ctcgccccat tggcacccgt tacctcaccc gtcccctgta a
2211192211DNAArtificial SequenceDescription of Artificial Sequence
Note = synthethic construct 19atggctgccg atggttatct tccagattgg
ctcgaggaca acctctctga gggcattcgc 60gagtggtggg acttgaaacc tggagccccg
aaacccaaag ccaaccagca aaagcaggac 120gacggccggg gtctggtgct
tcctggctac aagtacctcg gacccttcaa cggactcgac 180aagggggagc
ccgtcaacgc ggcggacgca gcggccctcg agcacgacaa ggcctacgac
240cagcagctca aagcgggtga caatccgtac ctgcggtata accacgccga
cgccgagttt 300caggagcgtc tgcaagaaga tacgtctttt gggggcaacc
tcgggcgagc agtcttccag 360gccaagaaga gggttctcga accttttggt
ctggttgagg aaggtgctaa gacggctcct 420ggaaagaaac gtccggtaga
gcagtcaccc caagaaccag actcctcctc gggcattggc 480aaatcaggcc
agcagcccgc taaaaagaga ctcaattttg gtcagactgg cgactcagag
540tcagtccccg acccacaacc tctcggagaa cctccagcaa cccccgctgc
tttgggacct 600actacaatgg cttcaggcgg tggcgcacca atggcagaca
ataacgaagg cgccgacgga 660gtgggtaatg cctcaggaaa ttggcattgc
gattccacat ggctgggcga cagagtcatc 720accaccagca cccgcacctg
ggccttgccc acctacaata accacctcta caagcaaatc 780tccagtgctt
caacgggggc cagcaacgac aaccactact tcggctacag caccccctgg
840gggtattttg atttcaacag attccactgc catttctcac cacgtgactg
gcagcgactc 900atcaacaaca actggggatt ccggcccaag agactcaact
tcaagctctt caacatccaa 960gtcaaggagg tcacgacgaa cgatggcgtc
acgaccatcg ctaataacct taccagcacg 1020gttcaagtct tctcggactc
ggagtaccag cttccgtacg tcctcggctc tgcgcaccag 1080ggctgcctcc
ctccgttccc ggcggacgtg ttcatgattc cgcagtacgg ctacctgacg
1140ctcaacaatg gcagcaaagc cgtgggacgt tcatcctttt actgcctgga
atatttccct 1200tctcagatgc tgagaacggg caacaacttt accttcagct
acacctttga ggaagtgcct 1260ttccacagca gctacgcgca cagccagagc
ctggaccggc tgatgaatcc tctcatcgac 1320caatacctgt attacctgaa
cagaactcaa aatcagtccg gaagtgccca aaacaaggac 1380ttgctgttta
gccgtgggtc tccagctggc atgtctgttc agcccaaaaa ctggctacct
1440ggaccctgtt atcggcagca gcgcgtttct aaaacaaaaa cagacaacaa
caacagcaat 1500tttacctgga ctggtgcttc aaaatataac ctcaatgggc
gtgaatccat catcaaccct 1560ggcactgcta tggcctcaca caaagacgac
gaagacaagt tctttcccat gagcggtgtc 1620atgatttttg gaaaagagag
cgccggagct tcaaacactg cattggacaa tgtcatgatt 1680acagacgaag
aggaaattaa agccactaac cctgtggcca ccgaaagatt tgggaccgtg
1740gcagtcaatt tccagagcag cagcacagac cctgcgaccg gagatgtgca
tgttatggga 1800gcattacctg gcatggtgtg gcaagataga gacgtgtacc
tgcagggtcc catttgggcc 1860aaaattcctc acacggatgg acactttcac
ccgtctcctc ttatgggcgg ctttggactc 1920aagcacccgc ctcctcagat
cctcatcaaa aacacacctg ttcctgcgaa tcctccggca 1980gagttttcgg
ctacaaagtt tgcttcattc atcacccagt actccacagg acaagtgagc
2040gtggaaattg aatgggagtt gcagaaagaa aacagtaagc gctggaatcc
cgaagtgcag 2100tacacatcta attatgcaaa atctgccaac gttgacttca
ctgtggacaa caatggactt 2160tatactgagc ctcgccccat tggcacccgt
tacctcaccc gtcccctgta a 2211202970DNAArtificial SequenceDescription
of Artificial Sequence Note = synthethic construct 20agatgaccgc
caaggtcgta gagtccgcca aggccattct gggcggcagc aaggtgcgcg 60tggaccaaaa
atgcaaggcc tctgcgcaga tcgaccccac ccccgtgatc gtcacctcca
120acaccaacat gtgcgccgtg attgacggga acagcaccac cttcgagcac
cagcagcccc 180tgcaggaccg gatgttcaag tttgaactca cccgccgcct
cgactacgac tttggcaagg 240tcaccaagca ggaagtcaag gactttttcc
ggtgggcggc tgatcacgtg actgacgtgg 300ctcatgagtt ttacgtcaca
aagggtggag ctaagaaaag gcccgccccc tctgacgagg 360atataagcga
gcccaagcgg ccgcgcgtgt catttgcgca gccggagacg tcagacgcgg
420aagctcccgg agacttcgcc gacaggtacc aaaacaaatg ttctcgtcac
gcgggtatgc 480tgcagatgct ctttccctgc aagacgtgcg agagaatgaa
tcagaattcc aacgtctgct 540tcacgcacgg tcagaaagat tgcggggagt
gctttcccgg gtcagaatct caaccggttt 600ctgtcgtcag aaaaacgtat
cagaaactgt gcatccttca tcagctccgg ggggcacccg 660agatcgcctg
ctctgcttgc gaccaactca accccgattt ggacgattgc caatttgagc
720aataaatgac tgaaatcagg tatggctgct gacggttatc ttccagattg
gctcgaggac 780aacctctctg aaggcattcg cgagtggtgg gcgctgaaac
ctggagctcc acaacccaag 840gccaaccaac agcatcagga caacggcagg
ggtcttgtgc ttcctgggta caagtacctc 900ggacccttca acggactcga
caagggagag ccggtcaacg aggcagacgc cgcggccctc 960gagcacgaca
aggcctacga caagcagctc gagcaggggg acaacccgta tctcaagtac
1020aaccacggcg acgccgagtt ccagcagcgc ttggcgaccg acacctcttt
tgggggcaac 1080ctcgggcgag cagtcttcca ggccaaaaag aggattctcg
agcctctggg tctggttgaa 1140gagggcgtta aaacggctcc tggaaagaaa
cgcccattag aaaagactcc aaatcggccg 1200accaacccgg actctgggaa
ggccccggcc aagaaaaagc aaaaagacgg cgaaccagcc 1260gactctgcta
gaaggacact cgactttgaa gactctggag caggagacgg accccctgag
1320ggatcatctt ccggagaaat gtctcatgat gctgagatgc gtgcggcgcc
aggcggaaat 1380gctgtcgagg cgggacaagg tgccgatgga gtgggtaatg
cctccggtga ttggcattgc 1440gattccacct ggtcagaggg ccgagtcacc
accaccagca cccgaacctg ggtcctaccc 1500acgtacaaca accacctgta
cctgcgaatc ggaacaacgg ccaacagcaa cacctacaac 1560ggattctcca
ccccctgggg atactttgac tttaaccgct tccactgcca cttttcccca
1620cgcgactggc agcgactcat caacaacaac tggggactca ggccgaaatc
gatgcgtgtt 1680aaaatcttca acatacaggt caaggaggtc acgacgtcaa
acggcgagac tacggtcgct 1740aataacctta ccagcacggt tcagatcttt
gcggattcga cgtatgaact cccatacgtg 1800atggacgccg gtcaggaggg
gagctttcct ccgtttccca acgacgtctt tatggttccc 1860caatacggat
actgcggagt tgtcactgga aaaaaccaga accagacaga cagaaatgcc
1920ttttactgcc tggaatactt tccatcccaa atgctaagaa ctggcaacaa
ttttgaagtc 1980agttaccaat ttgaaaaagt tcctttccat tcaatgtacg
cgcacagcca gagcctggac 2040agaatgatga atcctttact ggatcagtac
ctgtggcatc tgcaatcgac cactaccgga 2100aattccctta atcaaggaac
agctaccacc acgtacggga aaattaccac tggagacttt 2160gcctactaca
ggaaaaactg gttgcctgga gcctgcatta aacaacaaaa attttcaaag
2220aatgccaatc aaaactacaa gattcccgcc agcgggggag acgccctttt
aaagtatgac 2280acgcatacca ctctaaatgg gcgatggagt aacatggctc
ctggacctcc aatggcaacc 2340gcaggtgccg gggactcgga ttttagcaac
agccagctga tctttgccgg acccaatccg 2400agcggtaaca cgaccacatc
ttcaaacaat ttgttgttta cctcagaaga ggagattgcc 2460acaacaaacc
cacgagacac ggacatgttt ggacagattg cagataataa tcaaaatgcc
2520accaccgccc ctcacatcgc taacctggac gctatgggaa ttgttcccgg
aatggtctgg 2580caaaacagag acatctacta ccagggccct atttgggcca
aggtccctca cacggacgga 2640cactttcacc cttcgccgct gatgggagga
tttggactga aacacccgcc tccacagatt 2700ttcatcaaaa acacccccgt
acccgccaat cccaatacta cctttagcgc tgcaaggatt 2760aattcttttc
tgacgcagta cagcaccgga caagttgccg ttcagatcga ctgggaaatt
2820cagaaggagc attccaaacg ctggaatccc gaagttcaat ttacttcaaa
ctacggcact 2880caaaattcta tgctgtgggc tcccgacaat gctggcaact
accacgaact ccgggctatt 2940gggtcccgtt tcctcaccca ccacttgtaa
297021736PRTArtificial SequenceDescription of Artificial Sequence
Note = synthethic construct 21Met Ala Ala Asp Gly Tyr Leu Pro Asp
Trp Leu Glu Asp Asn Leu Ser1 5 10 15Glu Gly Ile Arg Glu Trp Trp Asp
Leu Lys Pro Gly Ala Pro Lys Pro 20 25 30Lys Ala Asn Gln Gln Lys Gln
Asp Asp Gly Arg Gly Leu Val Leu Pro 35 40 45Gly Tyr Lys Tyr Leu Gly
Pro Phe Asn Gly Leu Asp Lys Gly Glu Pro 50 55 60Val Asn Ala Ala Asp
Ala Ala Ala Leu Glu His Asp Lys Ala Tyr Asp65 70 75 80Gln Gln Leu
Lys Ala Gly Asp Asn Pro Tyr Leu Arg Tyr Asn His Ala 85 90 95Asp Ala
Glu Phe Gln Glu Arg Leu Gln Glu Asp Thr Ser Phe Gly Gly 100 105
110Asn Leu Gly Arg Ala Val Phe Gln Ala Lys Lys Arg Val Leu Glu Pro
115 120 125Phe Gly Leu Val Glu Glu Gly Ala Lys Thr Ala Pro Gly Lys
Lys Arg 130 135 140Pro Val Glu Gln Ser Pro Gln Glu Pro Asp Ser Ser
Ser Gly Ile Gly145 150 155 160Lys Thr Gly Gln Gln Pro Ala Lys Lys
Arg Leu Asn Phe Gly Gln Thr 165 170 175Gly Asp Ser Glu Ser Val Pro
Asp Pro Gln Pro Leu Gly Glu Pro Pro 180 185 190Ala Thr Pro Ala Ala
Val Gly Pro Thr Thr Met Ala Ser Gly Gly Gly 195 200 205Ala Pro Met
Ala Asp Asn Asn Glu Gly Ala Asp Gly Val Gly Asn Ala 210 215 220Ser
Gly Asn Trp His Cys Asp Ser Thr Trp Leu Gly Asp Arg Val Ile225 230
235 240Thr Thr Ser Thr Arg Thr Trp Ala Leu Pro Thr Tyr Asn Asn His
Leu 245 250 255Tyr Lys Gln Ile Ser Ser Ala Ser Thr Gly Ala Ser Asn
Asp Asn His 260 265 270Tyr Phe Gly Tyr Ser Thr Pro Trp Gly Tyr Phe
Asp Phe Asn Arg Phe 275 280 285His Cys His Phe Ser Pro Arg Asp Trp
Gln Arg Leu Ile Asn Asn Asn 290 295 300Trp Gly Phe Arg Pro Lys Arg
Leu Asn Phe Lys Leu Phe Asn Ile Gln305 310 315 320Val Lys Glu Val
Thr Thr Ser Asp Gly Val Thr Thr Ile Ala Asn Asn 325 330 335Leu Thr
Ser Thr Val Gln Val Phe Ser Asp Ser Glu Tyr Gln Leu Pro 340 345
350Tyr Val Leu Gly Ser Ala His Gln Gly Cys Leu Pro Pro Phe Pro Ala
355 360 365Asp Val Phe Met Ile Pro Gln Tyr Gly Tyr Leu Thr Leu Asn
Asn Gly 370 375 380Ser Gln Ala Val Gly Arg Ser Ser Phe Tyr Cys Leu
Glu Tyr Phe Pro385 390 395 400Ser Gln Met Leu Arg Thr Gly Asn Asn
Phe Thr Phe Ser Tyr Thr Phe 405 410 415Glu Glu Val Pro Phe His Ser
Ser Tyr Ala His Ser Gln Ser Leu Asp 420 425 430Arg Leu Met Asn Pro
Leu Ile Asp Gln Tyr Leu Tyr Tyr Leu Asn Arg 435 440 445Thr Gln Asn
Gln Ser Gly Ser Ala Gln Asn Lys Asp Leu Leu Phe Ser 450 455 460Arg
Gly Ser Pro Ala Gly Met Ser Val Gln Pro Lys Asn Trp Leu Pro465 470
475 480Gly Pro Cys Tyr Arg Gln Gln Arg Val Ser Lys Thr Lys Thr Gly
Asn 485 490 495Asn Asn Ser Asn Phe Thr Trp Thr Gly Ala Ser Lys Tyr
Asn Leu Asn 500 505 510Gly His Glu Ser Ile Ile Asn Pro Gly Thr Ala
Met Ala Ser His Lys 515 520 525Asp Asp Glu Asp Lys Phe Phe Pro Met
Ser Gly Val Met Ile Phe Gly 530 535 540Lys Glu Ser Ala Gly Ala Ser
Asn Thr Ala Leu Asp Asn Val Met Ile545 550 555 560Thr Asp Glu Glu
Glu Ile Lys Ala Thr Asn Pro Val Ala Thr Glu Arg 565 570 575Phe Gly
Thr Val Ala Val Asn Phe Gln Ser Ser Ser Thr His Pro Ala 580 585
590Thr Gly Asp Val His Val Met Gly Ala Leu Pro Gly Met Val Trp Gln
595 600 605Asp Arg Asp Val Tyr Leu Gln Gly Pro Ile Trp Ala Lys Ile
Pro His 610 615 620Thr Asp Gly His Phe His Pro Ser Pro Leu Met Gly
Gly Phe Gly Leu625 630 635 640Lys His Pro Pro Pro Gln Ile Leu Ile
Lys Asn Thr Pro Val Pro Ala 645 650 655Asn Pro Pro Ala Glu Phe Ser
Ala Thr Lys Phe Ala Ser Phe Ile Thr 660 665 670Gln Tyr Ser Thr Gly
Gln Val Ser Val Glu Ile Glu Trp Glu Leu Gln 675 680 685Lys Glu Asn
Ser Lys Arg Trp Asn Pro Glu Val Gln Tyr Thr Ser Asn 690 695 700Tyr
Ala Lys Ser Ala Asn Val Asp Phe Thr Val Asp Asn Asn Gly Leu705 710
715 720Tyr Thr Glu Pro Arg Pro Ile Gly Thr Arg Tyr Leu Thr Arg Pro
Leu 725 730 73522736PRTArtificial SequenceDescription of Artificial
Sequence Note = synthethic construct 22Met Ala Ala Asp Gly Tyr Leu
Pro Asp Trp Leu Glu Asp Asn Leu Ser1 5 10 15Glu Gly Ile Arg Glu Trp
Trp Asp Leu Lys Pro Gly Ala Pro Lys Pro 20 25 30Lys Ala Asn Gln Gln
Lys Gln Asp Asp Gly Arg Gly Leu Val Leu Pro 35 40 45Gly Tyr Lys Tyr
Leu Gly Pro Phe Asn Gly Leu Asp Lys Gly Glu Pro 50 55 60Val Asn Ala
Ala Asp Ala Ala Ala Leu Glu His Asp Lys Ala Tyr Asp65 70 75 80Gln
Gln Leu Lys Ala Gly Asp Asn Pro Tyr Leu Arg Tyr Asn His Ala 85 90
95Asp Ala Glu Phe Gln Glu Arg Leu Gln Glu Asp Thr Ser Phe Gly Gly
100 105 110Asn Leu Gly Arg Ala Val Phe Gln Ala Lys Lys Arg Val Leu
Glu Pro 115 120 125Phe Gly Leu Val Glu Glu Gly Ala Lys Thr Ala Pro
Gly Lys Lys Arg 130 135 140Pro Val Glu Gln Ser Pro Gln Glu Pro Asp
Ser Ser Ser Gly Ile Gly145 150 155 160Lys Thr Gly Gln Gln Pro Ala
Lys Lys Arg Leu Asn Phe Gly Gln Thr 165 170 175Gly Asp Ser Glu Ser
Val Pro Asp Pro Gln Pro Leu Gly Glu Pro Pro 180 185 190Ala Thr Pro
Ala Ala Val Gly Pro Thr Thr Met Ala Ser Gly Gly Gly 195 200 205Ala
Pro Met Ala Asp Asn Asn Glu Gly Ala Asp Gly Val Gly Asn Ala 210 215
220Ser Gly Asn Trp His Cys Asp Ser Thr Trp Leu Gly Asp Arg Val
Ile225 230 235 240Thr Thr Ser Thr Arg Thr Trp Ala Leu Pro Thr Tyr
Asn Asn His Leu 245 250 255Tyr Lys Gln Ile Ser Ser Ala Ser Thr Gly
Ala Ser Asn Asp Asn His 260 265 270Tyr Phe Gly Tyr Ser Thr Pro Trp
Gly Tyr Phe Asp Phe Asn Arg Phe 275 280 285His Cys His Phe Ser Pro
Arg Asp Trp Gln Arg Leu Ile Asn Asn Asn 290 295 300Trp Gly Phe Arg
Pro Lys Arg Leu Asn Phe Lys Leu Phe Asn Ile Gln305 310 315 320Val
Lys Glu Val Thr Thr Ser Asp Gly Val Thr Thr Ile Ala Asn Asn 325 330
335Leu Thr Ser Thr Val Gln Val Phe Ser Asp Ser Glu Tyr Gln Leu Pro
340 345 350Tyr Val Leu Gly Ser Ala His Gln Gly Cys Leu Pro Pro Phe
Pro Ala 355 360 365Asp Val Phe Met Ile Pro Gln Tyr Gly Tyr Leu Thr
Leu Asn Asn Gly 370 375 380Ser Gln Ala Val Gly Arg Ser Ser Phe Tyr
Cys Leu Glu Tyr Phe Pro385 390 395 400Ser Gln Met Leu Arg Thr Gly
Asn Asn Phe Thr Phe Ser Tyr Thr Phe 405 410 415Glu Glu Val Pro Phe
His Ser Ser Tyr Ala His Ser Gln Ser Leu Asp 420 425 430Arg Leu Met
Asn Pro Leu Ile Asp Gln Tyr Leu Tyr Tyr Leu Asn Arg 435 440 445Thr
Gln Asn Gln Ser Gly Ser Ala Gln Asn Lys Asp Leu Leu Phe Ser 450 455
460Arg Gly Ser Pro Ala Gly Met Ser Val Gln Pro Lys Asn Trp Leu
Pro465 470 475 480Gly Pro Cys Tyr Arg Gln Gln Arg Val Ser Lys Thr
Lys Thr Gly Asn 485 490 495Asn Asn Ser Asn Phe Thr Trp Thr Gly Ala
Ser Lys Tyr Asn Leu Asn 500 505 510Gly His Glu Ser Ile Ile Asn Pro
Gly Thr Ala Met Ala Ser His Lys 515 520 525Asp Asp Glu Asp Lys Phe
Phe Pro Met Ser Gly Val Met Ile Phe Gly 530 535 540Lys Glu Ser Ala
Gly Ala Ser Asn Thr Ala Leu Asp Asn Val Met Ile545 550 555 560Thr
Asp Glu Glu Glu Ile Lys Ala Thr Asn Pro Val Ala Thr Glu Arg 565 570
575Phe Gly Thr Val Ala Val Asn Phe Gln Ser Ser Ser Thr His Pro Ala
580 585 590Thr Gly Asp Val His Val Met Gly Ala Leu Pro Gly Met Val
Trp Gln 595 600 605Asp Arg Asp Val Tyr Leu Gln Gly Pro Ile Trp Ala
Lys Ile Pro His 610 615 620Thr Asp Gly His Phe His Pro Ser Pro Leu
Met Gly Gly Phe Gly Leu625 630 635 640Lys His Pro Pro Pro Gln Ile
Leu Ile Lys Asn Thr Pro Val Pro Ala 645 650 655Asn Pro Pro Ala Glu
Phe Ser Ala Thr Lys Phe Ala Ser Phe Ile Thr 660 665 670Gln Tyr Ser
Thr Gly Gln Val Ser Val Glu Ile Glu Trp Glu Leu Gln 675 680 685Lys
Glu Asn Ser Lys Arg Trp Asn Pro Glu Val Gln Tyr Thr Ser Asn 690 695
700Tyr Ala Lys Ser Ala Asn Val Asp Phe Thr Val Asp Asn Asn Gly
Leu705 710 715 720Tyr Thr Glu Pro Arg Pro Ile Gly Thr Arg Tyr Leu
Thr Arg Pro Leu 725 730 73523733PRTArtificial SequenceDescription
of Artificial Sequence Note = synthethic construct 23Met Thr Asp
Gly Tyr Leu Pro Asp Trp Leu Glu Asp Asn Leu Ser Glu1 5 10 15Gly Val
Arg Glu Trp Trp Ala Leu Gln Pro Gly Ala Pro Lys Pro Lys 20 25 30Ala
Asn Gln Gln His Gln Asp Asn Ala Arg Gly Leu Val Leu Pro Gly 35 40
45Tyr Lys Tyr Leu Gly Pro Gly Asn Gly Leu Asp Lys Gly Glu Pro Val
50 55 60Asn Ala Ala Asp Ala Ala Ala Leu Glu His Asp Lys Ala Tyr Asp
Gln65 70 75 80Gln Leu Lys Ala Gly Asp Asn Pro Tyr Leu Lys Tyr Asn
His Ala Asp 85 90 95Ala Glu Phe Gln Glu Arg Leu Gln Glu Asp Thr Ser
Phe Gly Gly Asn 100 105 110Leu Gly Arg Ala Val Phe Gln Ala Lys Lys
Arg Ile Leu Glu Pro Leu 115 120 125Gly Leu Val Glu Glu Ala Ala Lys
Thr Ala Pro Gly Lys Lys Arg Pro 130 135 140Val Glu Gln Ser Pro Ala
Glu Pro Asp Ser Ser Ser Gly Ile Gly Lys145 150 155 160Ser Gly Gln
Gln Pro Ala Arg Lys Arg Leu Asn Phe Gly Gln Thr Gly 165 170 175Asp
Thr Glu Ser Val Pro Asp Pro Gln Pro Leu Gly Gln Pro Pro Ala 180 185
190Ala Pro Ser Gly Val Gly Ser Thr Thr Met Ala Ser Gly Gly Gly Ala
195 200 205Pro Met Ala Asp Asn Asn Glu Gly Ala Asp Gly Val Gly Asn
Ser Ser 210 215 220Gly Asn Trp His Cys Asp Ser Gln Trp Leu Gly Asp
Arg Val Ile Thr225 230 235 240Thr Ser Thr Arg Thr Trp Ala Leu Pro
Thr Tyr Asn Asn His Leu Tyr 245 250 255Lys Gln Ile Ser Ser Gln Ser
Gly Ala Thr Asn Asp Asn His Tyr Phe 260 265 270Gly Tyr Ser Thr Pro
Trp Gly Tyr Phe Asp Phe Asn Arg Phe His Cys 275 280 285His Phe Ser
Pro Arg Asp Trp Gln Arg Leu Ile Asn Asn Asn Trp Gly 290 295 300Phe
Arg Pro Lys Arg Leu Asn Phe Lys Leu Phe Asn Ile Gln Val Lys305 310
315 320Glu Val Thr Gln Asn Asp Gly Thr Thr Thr Ile Ala Asn Asn Leu
Thr 325 330 335Ser Thr Val Gln Val Phe Thr Asp Ser Glu Tyr Gln Leu
Pro Tyr Val 340 345 350Leu Gly Ser Ala His Gln Gly Cys Leu Pro Pro
Phe Pro Ala Asp Val 355 360 365Phe Met Val Pro Gln Tyr Gly Tyr Leu
Thr Leu Asn Asn Gly Ser Gln 370 375 380Ala Val Gly Arg Ser Ser Phe
Tyr Cys Leu Glu Tyr Phe Pro Ser Gln385 390 395 400Met Leu Arg Thr
Gly Asn Asn Phe Gln Phe Ser Tyr Thr Phe Glu Asp 405 410 415Val Pro
Phe His Ser Ser Tyr Ala His Ser Gln Ser Leu Asp Arg Leu 420 425
430Met Asn Pro Leu Ile Asp Gln Tyr Leu Tyr Tyr Leu Asn Arg Thr Gln
435 440 445Thr Ala Ser Gly Thr Gln Gln Ser Arg Leu Leu Phe Ser Gln
Ala Gly 450 455 460Pro Thr Ser Met Ser Leu Gln Ala Lys Asn Trp Leu
Pro Gly Pro Cys465 470 475 480Tyr Arg Gln Gln Arg Leu Ser Lys Gln
Ala Asn Asp Asn Asn Asn Ser 485 490 495Asn Phe Pro Trp Thr Gly Ala
Thr Lys Tyr His Leu Asn Gly Arg Asp 500 505 510Ser Leu Val Asn Pro
Gly Pro Ala Met Ala Ser His Lys Asp Asp Lys 515 520 525Glu Lys Phe
Phe Pro Met His Gly Thr Leu Ile Phe Gly Lys Glu Gly 530 535 540Thr
Asn Ala Asn Asn Ala Asp Leu Glu Asn Val Met Ile Thr Asp Glu545 550
555 560Glu Glu Ile Arg Thr Thr Asn Pro Val Ala Thr Glu Gln Tyr Gly
Thr 565 570 575Val Ser Asn Asn Leu Gln Asn Ser Asn Ala Gly Pro Thr
Thr Gly Thr 580 585 590Val Asn His Gln Gly Ala Leu Pro Gly Met Val
Trp Gln Asp Arg Asp 595 600 605Val Tyr Leu Gln Gly Pro Ile Trp Ala
Lys Ile Pro His Thr Asp Gly 610 615 620His Phe His Pro Ser Pro Leu
Met Gly Gly Phe Gly Leu Lys His Pro625 630 635 640Pro Pro Gln Ile
Met Ile Lys Asn Thr Pro Val Pro Ala Asn Pro Pro 645 650 655Thr Asn
Phe Ser Ala Ala Lys Phe Ala Ser Phe Ile Thr Gln Tyr Ser 660 665
670Thr Gly Gln Val Ser Val Glu Ile Glu Trp Glu Leu Gln Lys Glu Asn
675 680 685Ser Lys Arg Trp Asn Pro Glu Ile Gln Tyr Thr Ser Asn Tyr
Asn Lys 690 695 700Ser Val Asn Val Asp Phe Thr Val Asp Thr Asn Gly
Val Tyr Ser Glu705 710 715 720Pro Arg Pro Ile Gly Thr Arg Tyr Leu
Thr Arg Asn Leu 725 73024736PRTArtificial SequenceDescription of
Artificial Sequence Note = synthethic construct 24Met Ala Ala Asp
Gly Tyr Leu Pro Asp Trp Leu Glu Asp Asn Leu Ser1 5 10 15Glu Gly Ile
Arg Glu Trp Trp Asp Leu Lys Pro Gly Ala Pro Lys Pro 20
25 30Lys Ala Asn Gln Gln Lys Gln Asp Asp Gly Arg Gly Leu Val Leu
Pro 35 40 45Gly Tyr Lys Tyr Leu Gly Pro Phe Asn Gly Leu Asp Lys Gly
Glu Pro 50 55 60Val Asn Ala Ala Asp Ala Ala Ala Leu Glu His Asp Lys
Ala Tyr Asp65 70 75 80Gln Gln Leu Lys Ala Gly Asp Asn Pro Tyr Leu
Arg Tyr Asn His Ala 85 90 95Asp Ala Glu Phe Gln Glu Arg Leu Gln Glu
Asp Thr Ser Phe Gly Gly 100 105 110Asn Leu Gly Arg Ala Val Phe Gln
Ala Lys Lys Arg Val Leu Glu Pro 115 120 125Phe Gly Leu Val Glu Glu
Gly Ala Lys Thr Ala Pro Gly Lys Lys Arg 130 135 140Pro Val Glu Gln
Ser Pro Gln Glu Pro Asp Ser Ser Ser Gly Ile Gly145 150 155 160Lys
Thr Gly Gln Gln Pro Ala Lys Lys Arg Leu Asn Phe Gly Gln Thr 165 170
175Gly Asp Ser Glu Ser Val Pro Asp Pro Gln Pro Leu Gly Glu Pro Pro
180 185 190Ala Thr Pro Ala Ala Val Gly Pro Thr Thr Met Ala Ser Gly
Gly Gly 195 200 205Ala Pro Met Ala Asp Asn Asn Glu Gly Ala Asp Gly
Val Gly Asn Ala 210 215 220Ser Gly Asn Trp His Cys Asp Ser Thr Trp
Leu Gly Asp Arg Val Ile225 230 235 240Thr Thr Ser Thr Arg Thr Trp
Ala Leu Pro Thr Tyr Asn Asn His Leu 245 250 255Tyr Lys Gln Ile Ser
Ser Ala Ser Thr Gly Ala Ser Asn Asp Asn His 260 265 270Tyr Phe Gly
Tyr Ser Thr Pro Trp Gly Tyr Phe Asp Phe Asn Arg Phe 275 280 285His
Cys His Phe Ser Pro Arg Asp Trp Gln Arg Leu Ile Asn Asn Asn 290 295
300Trp Gly Phe Arg Pro Lys Arg Leu Asn Phe Lys Leu Phe Asn Ile
Gln305 310 315 320Val Lys Glu Val Thr Thr Asn Asp Gly Val Thr Thr
Ile Ala Asn Asn 325 330 335Leu Thr Ser Thr Val Gln Val Phe Ser Asp
Ser Glu Tyr Gln Leu Pro 340 345 350Tyr Val Leu Gly Ser Ala His Gln
Gly Cys Leu Pro Pro Phe Pro Ala 355 360 365Asp Val Phe Met Ile Pro
Gln Tyr Gly Tyr Leu Thr Leu Asn Asn Gly 370 375 380Ser Gln Ala Val
Gly Arg Ser Ser Phe Tyr Cys Leu Glu Tyr Phe Pro385 390 395 400Ser
Gln Met Leu Arg Thr Gly Asn Asn Phe Thr Phe Ser Tyr Thr Phe 405 410
415Glu Glu Val Pro Phe His Ser Ser Tyr Ala His Ser Gln Ser Leu Asp
420 425 430Arg Leu Met Asn Pro Leu Ile Asp Gln Tyr Leu Tyr Tyr Leu
Asn Arg 435 440 445Thr Gln Asn Gln Ser Gly Ser Ala Gln Asn Lys Asp
Leu Leu Phe Ser 450 455 460Arg Gly Ser Pro Ala Gly Met Ser Val Gln
Pro Lys Asn Trp Leu Pro465 470 475 480Gly Pro Cys Tyr Arg Gln Gln
Arg Val Ser Lys Thr Lys Thr Asp Asn 485 490 495Asn Asn Ser Asn Phe
Thr Trp Thr Gly Ala Ser Lys Tyr Asn Leu Asn 500 505 510Gly Arg Glu
Ser Ile Ile Asn Pro Gly Thr Ala Met Ala Ser His Lys 515 520 525Asp
Asp Glu Asp Lys Phe Phe Pro Met Ser Gly Val Met Ile Phe Gly 530 535
540Lys Glu Ser Ala Gly Ala Ser Asn Thr Ala Leu Asp Asn Val Met
Ile545 550 555 560Thr Asp Glu Glu Glu Ile Lys Ala Thr Asn Pro Val
Ala Thr Glu Arg 565 570 575Phe Gly Thr Val Ala Val Asn Phe Gln Ser
Ser Ser Thr Asp Pro Ala 580 585 590Thr Gly Asp Val His Val Met Gly
Ala Leu Pro Gly Met Val Trp Gln 595 600 605Asp Arg Asp Val Tyr Leu
Gln Gly Pro Ile Trp Ala Lys Ile Pro His 610 615 620Thr Asp Gly His
Phe His Pro Ser Pro Leu Met Gly Gly Phe Gly Leu625 630 635 640Lys
His Pro Pro Pro Gln Ile Leu Ile Lys Asn Thr Pro Val Pro Ala 645 650
655Asn Pro Pro Ala Glu Phe Ser Ala Thr Lys Phe Ala Ser Phe Ile Thr
660 665 670Gln Tyr Ser Thr Gly Gln Val Ser Val Glu Ile Glu Trp Glu
Leu Gln 675 680 685Lys Glu Asn Ser Lys Arg Trp Asn Pro Glu Val Gln
Tyr Thr Ser Asn 690 695 700Tyr Ala Lys Ser Ala Asn Val Asp Phe Thr
Val Asp Asn Asn Gly Leu705 710 715 720Tyr Thr Glu Pro Arg Pro Ile
Gly Thr Arg Tyr Leu Thr Arg Pro Leu 725 730 73525736PRTArtificial
SequenceDescription of Artificial Sequence Note = synthethic
construct 25Met Ala Ala Asp Gly Tyr Leu Pro Asp Trp Leu Glu Asp Asn
Leu Ser1 5 10 15Glu Gly Ile Arg Lys Trp Trp Asp Leu Lys Pro Gly Ala
Pro Lys Pro 20 25 30Lys Ala Asn Gln Gln Lys Gln Asp Asp Gly Arg Gly
Leu Val Leu Pro 35 40 45Gly Tyr Lys Tyr Leu Gly Pro Phe Asn Gly Leu
Asp Lys Gly Glu Pro 50 55 60Val Asn Ala Ala Asp Ala Ala Ala Leu Glu
His Asp Lys Ala Tyr Asp65 70 75 80Gln Gln Leu Lys Ala Gly Asp Asn
Pro Tyr Leu Arg Tyr Asn His Ala 85 90 95Asp Ala Glu Phe Gln Glu Arg
Leu Gln Glu Asp Thr Ser Phe Gly Gly 100 105 110Asn Leu Gly Arg Ala
Val Phe Gln Ala Lys Lys Arg Val Leu Glu Pro 115 120 125Phe Gly Leu
Val Glu Glu Gly Ala Lys Thr Ala Pro Gly Lys Lys Arg 130 135 140Pro
Val Glu Gln Ser Pro Gln Glu Pro Asp Ser Ser Ser Gly Ile Gly145 150
155 160Lys Thr Gly Gln Gln Pro Ala Lys Lys Arg Leu Asn Phe Gly Gln
Thr 165 170 175Gly Asp Ser Glu Ser Val Pro Asp Pro Gln Pro Leu Gly
Glu Pro Pro 180 185 190Ala Thr Pro Ala Ala Val Gly Pro Thr Thr Met
Ala Ser Gly Gly Gly 195 200 205Ala Pro Met Ala Asp Asn Asn Glu Gly
Ala Asp Gly Val Gly Asn Ala 210 215 220Ser Gly Asn Trp His Cys Asp
Ser Thr Trp Leu Gly Asp Arg Val Ile225 230 235 240Thr Thr Ser Thr
Arg Thr Trp Ala Leu Pro Thr Tyr Asn Asn His Leu 245 250 255Tyr Lys
Gln Ile Ser Ser Ala Ser Thr Gly Ala Ser Asn Asp Asn His 260 265
270Tyr Phe Gly Tyr Ser Thr Pro Trp Gly Tyr Phe Asp Phe Asn Arg Phe
275 280 285His Cys His Phe Ser Pro Arg Asp Trp Gln Arg Leu Ile Asn
Asn Asn 290 295 300Trp Gly Phe Arg Pro Lys Arg Leu Asn Phe Lys Leu
Phe Asn Ile Gln305 310 315 320Val Lys Glu Val Thr Thr Asn Asp Gly
Val Thr Thr Ile Ala Asn Asn 325 330 335Leu Thr Ser Thr Val Gln Val
Phe Ser Asp Ser Glu Tyr Gln Leu Pro 340 345 350Tyr Val Leu Gly Ser
Ala His Gln Gly Cys Leu Pro Pro Phe Pro Ala 355 360 365Asp Val Phe
Met Ile Pro Gln Tyr Gly Tyr Leu Thr Leu Asn Asn Gly 370 375 380Ser
Gln Ala Val Gly Arg Ser Ser Phe Tyr Cys Leu Glu Tyr Phe Pro385 390
395 400Ser Gln Met Leu Arg Thr Gly Asn Asn Phe Thr Phe Ser Tyr Thr
Phe 405 410 415Glu Glu Val Pro Phe His Ser Ser Tyr Ala His Ser Gln
Ser Leu Asp 420 425 430Arg Leu Met Asn Pro Leu Ile Asp Gln Tyr Leu
Tyr Tyr Leu Asn Arg 435 440 445Thr Gln Asn Gln Ser Gly Ser Ala Gln
Asn Lys Asp Leu Leu Phe Ser 450 455 460Arg Gly Ser Pro Ala Gly Met
Ser Val Gln Pro Lys Asn Trp Leu Pro465 470 475 480Gly Pro Cys Tyr
Arg Gln Gln Arg Val Ser Lys Thr Lys Thr Asp Asn 485 490 495Asn Asn
Ser Asn Phe Thr Trp Thr Gly Ala Ser Lys Tyr Asn Leu Asn 500 505
510Gly Arg Glu Ser Ile Ile Asn Pro Gly Thr Ala Met Ala Ser His Lys
515 520 525Asp Asp Lys Asp Lys Phe Phe Pro Met Ser Gly Val Met Ile
Phe Gly 530 535 540Lys Glu Ser Ala Gly Ala Ser Asn Thr Ala Leu Asp
Asn Val Met Ile545 550 555 560Thr Asp Glu Glu Glu Ile Lys Ala Thr
Asn Pro Val Ala Thr Glu Arg 565 570 575Phe Gly Thr Val Ala Val Asn
Phe Gln Ser Ser Ser Thr Asp Pro Ala 580 585 590Thr Gly Asp Val His
Val Met Gly Ala Leu Pro Gly Met Val Trp Gln 595 600 605Asp Arg Asp
Val Tyr Leu Gln Gly Pro Ile Trp Ala Lys Ile Pro His 610 615 620Thr
Asp Gly His Phe His Pro Ser Pro Leu Met Gly Gly Phe Gly Leu625 630
635 640Lys His Pro Pro Pro Gln Ile Leu Ile Lys Asn Thr Pro Val Pro
Ala 645 650 655Asn Pro Pro Ala Glu Phe Ser Ala Thr Lys Phe Ala Ser
Phe Ile Thr 660 665 670Gln Tyr Ser Thr Gly Gln Val Ser Val Glu Ile
Glu Trp Glu Leu Gln 675 680 685Lys Glu Asn Ser Lys Arg Trp Asn Pro
Glu Val Gln Tyr Thr Ser Asn 690 695 700Tyr Ala Lys Ser Ala Asn Val
Asp Phe Thr Val Asp Asn Asn Gly Leu705 710 715 720Tyr Thr Glu Pro
Arg Pro Ile Gly Thr Arg Tyr Leu Thr Arg Pro Leu 725 730
73526736PRTArtificial SequenceDescription of Artificial Sequence
Note = synthethic construct 26Met Ala Ala Asp Gly Tyr Leu Pro Asp
Trp Leu Glu Asp Asn Leu Ser1 5 10 15Glu Gly Ile Arg Glu Trp Trp Asp
Leu Lys Pro Gly Ala Pro Lys Pro 20 25 30Lys Ala Asn Gln Gln Lys Gln
Asp Asn Gly Arg Gly Leu Val Leu Pro 35 40 45Gly Tyr Lys Tyr Leu Gly
Pro Phe Asn Gly Leu Asp Lys Gly Glu Pro 50 55 60Val Asn Ala Ala Asp
Ala Ala Ala Leu Glu His Asp Lys Ala Tyr Asp65 70 75 80Gln Gln Leu
Lys Ala Gly Asp Asn Pro Tyr Leu Arg Tyr Asn His Ala 85 90 95Asp Ala
Glu Phe Gln Glu Arg Leu Gln Glu Asp Thr Ser Phe Gly Gly 100 105
110Asn Leu Gly Arg Ala Val Phe Gln Ala Lys Lys Arg Val Leu Glu Pro
115 120 125Phe Gly Leu Val Glu Glu Gly Ala Lys Thr Ala Pro Gly Lys
Lys Arg 130 135 140Pro Val Glu Gln Ser Pro Gln Glu Pro Asp Ser Ser
Ser Gly Ile Gly145 150 155 160Lys Thr Gly Gln Gln Pro Ala Lys Lys
Arg Leu Asn Phe Gly Gln Thr 165 170 175Gly Asp Ser Glu Ser Val Pro
Asp Pro Gln Pro Leu Gly Glu Pro Pro 180 185 190Ala Thr Pro Ala Ala
Val Gly Pro Thr Thr Met Ala Ser Gly Gly Gly 195 200 205Ala Pro Met
Ala Asp Asn Asn Glu Gly Ala Asp Gly Val Gly Asn Ala 210 215 220Ser
Gly Asn Trp His Cys Asp Ser Thr Trp Leu Gly Asp Arg Val Ile225 230
235 240Thr Thr Ser Thr Arg Thr Trp Ala Leu Pro Thr Tyr Asn Asn His
Leu 245 250 255Tyr Lys Gln Ile Ser Ser Ala Ser Thr Gly Ala Ser Asn
Asp Asn His 260 265 270Tyr Phe Gly Tyr Ser Thr Pro Trp Gly Tyr Phe
Asp Phe Asn Arg Phe 275 280 285His Cys His Phe Ser Pro Arg Asp Trp
Gln Arg Leu Ile Asn Asn Asn 290 295 300Trp Gly Phe Arg Pro Lys Arg
Leu Asn Phe Lys Leu Phe Asn Ile Gln305 310 315 320Val Lys Glu Val
Thr Thr Asn Asp Gly Val Thr Thr Ile Ala Asn Asn 325 330 335Leu Thr
Ser Thr Val Gln Val Phe Ser Asp Ser Glu Tyr Gln Leu Pro 340 345
350Tyr Val Leu Gly Ser Ala His Gln Gly Cys Leu Pro Pro Phe Pro Ala
355 360 365Asp Val Phe Met Ile Pro Gln Tyr Gly Tyr Leu Thr Leu Asn
Asn Gly 370 375 380Ser Gln Ala Val Gly Arg Ser Ser Phe Tyr Cys Leu
Glu Tyr Phe Pro385 390 395 400Ser Gln Met Leu Arg Thr Gly Asn Asn
Phe Thr Phe Ser Tyr Thr Phe 405 410 415Glu Glu Val Pro Phe His Ser
Ser Tyr Ala His Ser Gln Ser Leu Asp 420 425 430Arg Leu Met Asn Pro
Leu Ile Asp Gln Tyr Leu Tyr Tyr Leu Asn Arg 435 440 445Thr Gln Asn
Gln Ser Gly Ser Ala Gln Asn Lys Asp Leu Leu Phe Ser 450 455 460Arg
Gly Ser Pro Ala Gly Met Ser Val Gln Pro Lys Asn Trp Leu Pro465 470
475 480Gly Pro Cys Tyr Arg Gln Gln Arg Val Ser Lys Thr Lys Thr Asp
Asn 485 490 495Asn Asn Ser Asn Phe Thr Trp Thr Gly Ala Ser Lys Tyr
Asn Leu Asn 500 505 510Gly Arg Glu Ser Ile Ile Asn Pro Gly Thr Ala
Met Ala Ser His Lys 515 520 525Asp Asp Glu Asp Lys Phe Phe Pro Met
Ser Gly Val Met Ile Phe Gly 530 535 540Lys Glu Ser Ala Gly Ala Ser
Asn Thr Ala Leu Asp Asn Val Met Ile545 550 555 560Thr Asp Glu Glu
Glu Ile Lys Ala Thr Asn Pro Val Ala Thr Glu Arg 565 570 575Phe Gly
Thr Val Ala Val Asn Phe Gln Ser Ser Ser Thr His Pro Ala 580 585
590Thr Gly Asp Val His Val Met Gly Ala Leu Pro Gly Met Val Trp Gln
595 600 605Asp Arg Asp Val Tyr Leu Gln Gly Pro Ile Trp Ala Lys Ile
Pro His 610 615 620Thr Asp Gly His Phe His Pro Ser Pro Leu Met Gly
Gly Phe Gly Leu625 630 635 640Lys His Pro Pro Pro Gln Ile Leu Ile
Lys Asn Thr Pro Val Pro Ala 645 650 655Asn Pro Pro Ala Glu Phe Ser
Ala Thr Lys Phe Ala Ser Phe Ile Thr 660 665 670Gln Tyr Ser Thr Gly
Gln Val Ser Val Glu Ile Glu Trp Glu Leu Gln 675 680 685Lys Glu Asn
Ser Lys Arg Trp Asn Pro Glu Val Gln Tyr Thr Ser Asn 690 695 700Tyr
Ala Lys Ser Ala Asn Val Asp Phe Thr Val Asp Asn Asn Gly Leu705 710
715 720Tyr Thr Glu Pro Arg Pro Ile Gly Thr Arg Tyr Leu Thr Arg Pro
Leu 725 730 73527736PRTArtificial SequenceDescription of Artificial
Sequence Note = synthethic construct 27Met Ala Ala Asp Gly Tyr Leu
Pro Asp Trp Leu Glu Asp Asn Leu Ser1 5 10 15Glu Gly Ile Arg Glu Trp
Trp Asp Leu Lys Pro Gly Ala Pro Lys Pro 20 25 30Lys Ala Asn Gln Gln
Lys Gln Asp Asp Gly Arg Gly Leu Val Leu Pro 35 40 45Gly Tyr Lys Tyr
Leu Gly Pro Phe Asn Gly Leu Asp Lys Gly Glu Pro 50 55 60Val Asn Ala
Ala Asp Ala Ala Ala Leu Glu His Asp Lys Ala Tyr Asp65 70 75 80Gln
Gln Leu Lys Ala Gly Asp Asn Pro Tyr Leu Arg Tyr Asn His Ala 85 90
95Asp Ala Glu Phe Gln Glu Arg Leu Gln Glu Asp Thr Ser Phe Gly Gly
100 105 110Asn Leu Gly Arg Ala Val Phe Gln Ala Lys Lys Arg Val Leu
Glu Pro 115 120 125Phe Gly Leu Val Glu Glu Gly Ala Lys Thr Ala Pro
Gly Lys Lys Arg 130 135 140Pro Val Glu Gln Ser Pro Gln Glu Pro Asp
Ser Ser Ser Gly Ile Gly145 150 155 160Lys Thr Gly Gln Gln Pro Ala
Lys Lys Arg Leu Asn Phe Gly Gln Thr 165 170 175Gly Asp Ser Glu Ser
Val Pro Asp Pro Gln Pro Leu Gly Glu Pro Pro 180 185 190Ala Thr Pro
Ala Ala Val Gly Pro Thr Thr Met Ala Ser Gly Gly Gly 195 200 205Ala
Pro Met Ala Asp Asn Asn Glu Gly Ala Asp Gly Val Gly Asn Ala 210 215
220Ser Gly Asn Trp His Cys Asp Ser Thr Trp Leu Gly Asp Arg Val
Ile225 230 235 240Thr Thr Ser Thr Arg Thr Trp Ala Leu Pro Thr Tyr
Asn Asn His Leu 245
250 255Tyr Lys Gln Ile Ser Ser Ala Ser Thr Gly Ala Ser Asn Asp Asn
His 260 265 270Tyr Phe Gly Tyr Ser Thr Pro Trp Gly Tyr Phe Asp Phe
Asn Arg Phe 275 280 285His Cys His Phe Ser Pro Arg Asp Trp Gln Arg
Leu Ile Asn Asn Asn 290 295 300Trp Gly Phe Arg Pro Lys Arg Leu Asn
Phe Lys Leu Phe Asn Ile Gln305 310 315 320Val Lys Glu Val Thr Thr
Asn Asp Gly Val Thr Thr Ile Ala Asn Asn 325 330 335Leu Thr Ser Thr
Val Gln Val Phe Ser Asp Ser Glu Tyr Gln Leu Pro 340 345 350Tyr Val
Leu Gly Ser Ala His Gln Gly Cys Leu Pro Pro Phe Pro Ala 355 360
365Asp Val Phe Met Ile Pro Gln Tyr Gly Tyr Leu Thr Leu Asn Asn Gly
370 375 380Ser Gln Ala Val Gly Arg Ser Ser Phe Tyr Cys Leu Glu Tyr
Phe Pro385 390 395 400Ser Gln Met Leu Arg Thr Gly Asn Asn Phe Thr
Phe Ser Tyr Thr Phe 405 410 415Glu Asp Val Pro Phe His Ser Ser Tyr
Ala His Ser Gln Ser Leu Asp 420 425 430Arg Leu Met Asn Pro Leu Ile
Asp Gln Tyr Leu Tyr Tyr Leu Asn Arg 435 440 445Thr Gln Asn Gln Ser
Gly Ser Ala Gln Asn Lys Asp Leu Leu Phe Ser 450 455 460Arg Gly Ser
Pro Ala Gly Met Ser Val Gln Leu Lys Asn Trp Leu Pro465 470 475
480Gly Pro Cys Tyr Arg Gln Gln Arg Val Ser Lys Thr Lys Thr Asp Asn
485 490 495Asn Asn Ser Asn Phe Thr Trp Thr Gly Ala Ser Lys Tyr Asn
Leu Asn 500 505 510Gly Arg Glu Ser Ile Ile Asn Pro Gly Thr Ala Met
Ala Ser His Lys 515 520 525Asp Asp Glu Asp Lys Phe Phe Pro Met Ser
Gly Val Met Ile Phe Gly 530 535 540Lys Glu Ser Ala Gly Ala Ser Asn
Thr Ala Leu Asp Asn Val Met Ile545 550 555 560Thr Asp Glu Glu Glu
Ile Lys Ala Thr Asn Pro Val Ala Thr Glu Arg 565 570 575Phe Gly Thr
Val Ala Val Asn Phe Gln Ser Ser Ser Thr Asp Pro Ala 580 585 590Thr
Gly Asp Val His Val Met Gly Ala Leu Pro Gly Met Val Trp Gln 595 600
605Asp Arg Asp Val Tyr Leu Gln Gly Pro Ile Trp Ala Lys Ile Pro His
610 615 620Thr Asp Gly His Phe His Pro Ser Pro Leu Met Gly Gly Phe
Gly Leu625 630 635 640Lys His Pro Pro Pro Gln Ile Leu Ile Lys Asn
Thr Pro Val Pro Ala 645 650 655Asn Pro Pro Ala Glu Phe Ser Ala Thr
Lys Phe Ala Ser Phe Ile Thr 660 665 670Gln Tyr Ser Thr Gly Gln Val
Ser Val Glu Ile Glu Trp Glu Leu Gln 675 680 685Lys Glu Asn Ser Lys
Arg Trp Asn Pro Glu Val Gln Tyr Thr Ser Asn 690 695 700Tyr Ala Lys
Ser Ala Asn Val Asp Phe Thr Val Asp Asn Asn Gly Leu705 710 715
720Tyr Thr Glu Pro Arg Pro Ile Gly Thr Arg Tyr Leu Thr Arg Pro Leu
725 730 73528736PRTArtificial SequenceDescription of Artificial
Sequence Note = synthethic construct 28Met Ala Ala Asp Gly Tyr Leu
Pro Asp Trp Leu Glu Asp Asn Leu Ser1 5 10 15Glu Gly Ile Arg Glu Trp
Trp Asp Leu Lys Pro Gly Ala Pro Lys Pro 20 25 30Lys Ala Asn Gln Gln
Lys Gln Asp Asp Gly Arg Gly Leu Val Leu Pro 35 40 45Gly Tyr Lys Tyr
Leu Gly Pro Phe Asn Gly Leu Asp Lys Gly Glu Pro 50 55 60Val Asn Ala
Ala Asp Ala Ala Ala Leu Glu His Asp Lys Ala Tyr Asp65 70 75 80Gln
Gln Leu Lys Ala Gly Asp Asn Pro Tyr Leu Arg Tyr Asn His Ala 85 90
95Asp Ala Glu Phe Gln Glu Arg Leu Gln Glu Asp Thr Ser Phe Gly Gly
100 105 110Asn Leu Gly Arg Ala Val Phe Gln Ala Lys Lys Arg Val Leu
Glu Pro 115 120 125Phe Gly Leu Val Glu Glu Gly Ala Lys Thr Ala Pro
Gly Lys Lys Arg 130 135 140Pro Val Glu Gln Ser Pro Gln Glu Pro Asp
Ser Ser Ser Gly Ile Gly145 150 155 160Lys Ser Gly Gln Gln Pro Ala
Lys Lys Arg Leu Asn Phe Gly Gln Thr 165 170 175Gly Asp Ser Glu Ser
Val Pro Asp Pro Gln Pro Leu Gly Glu Pro Pro 180 185 190Ala Thr Pro
Ala Ala Val Gly Pro Thr Thr Met Ala Ser Gly Gly Gly 195 200 205Ala
Pro Met Ala Asp Asn Asn Glu Gly Ala Asp Gly Val Gly Asn Ala 210 215
220Ser Gly Asn Trp His Cys Asp Ser Thr Trp Leu Gly Asp Arg Val
Ile225 230 235 240Thr Thr Ser Thr Arg Thr Trp Ala Leu Pro Thr Tyr
Asn Asn His Leu 245 250 255Tyr Lys Gln Ile Ser Ser Ala Ser Thr Gly
Ala Ser Asn Asp Asn His 260 265 270Tyr Phe Gly Tyr Ser Thr Pro Trp
Gly Tyr Phe Asp Phe Asn Arg Phe 275 280 285His Cys His Phe Ser Pro
Arg Asp Trp Gln Arg Leu Ile Asn Asn Asn 290 295 300Trp Gly Phe Arg
Pro Lys Arg Leu Asn Phe Lys Leu Phe Asn Ile Gln305 310 315 320Val
Lys Glu Val Thr Thr Asn Asp Gly Val Thr Thr Ile Ala Asn Asn 325 330
335Leu Thr Ser Thr Val Gln Val Phe Ser Asp Ser Glu Tyr Gln Leu Pro
340 345 350Tyr Val Leu Gly Ser Ala His Gln Gly Cys Leu Pro Pro Phe
Pro Ala 355 360 365Asp Val Phe Met Ile Pro Gln Tyr Gly Tyr Leu Thr
Leu Asn Asn Gly 370 375 380Ser Gln Ala Val Gly Arg Ser Ser Phe Tyr
Cys Leu Glu Tyr Phe Pro385 390 395 400Ser Gln Met Leu Arg Thr Gly
Asn Asn Phe Thr Phe Ser Tyr Thr Phe 405 410 415Glu Asp Val Pro Phe
His Ser Ser Tyr Ala His Ser Gln Ser Leu Asp 420 425 430Arg Leu Met
Asn Pro Leu Ile Asp Gln Tyr Leu Tyr Tyr Leu Asn Arg 435 440 445Thr
Gln Asn Gln Ser Gly Ser Ala Gln Asn Lys Asp Leu Leu Phe Ser 450 455
460Arg Gly Ser Pro Ala Gly Met Ser Val Gln Pro Lys Asn Trp Leu
Pro465 470 475 480Gly Pro Cys Tyr Arg Gln Gln Arg Val Ser Lys Thr
Lys Thr Asp Asn 485 490 495Asn Asn Ser Asn Phe Thr Trp Thr Gly Ala
Ser Lys Tyr Asn Leu Asn 500 505 510Gly Arg Glu Ser Ile Ile Asn Pro
Gly Thr Ala Met Ala Ser His Lys 515 520 525Asp Asp Glu Asp Lys Phe
Phe Pro Met Ser Gly Val Met Ile Phe Gly 530 535 540Lys Gly Ser Ala
Gly Ala Ser Asn Thr Ala Leu Asp Asn Val Met Ile545 550 555 560Thr
Asp Glu Glu Glu Ile Lys Ala Thr Asn Pro Val Ala Thr Glu Arg 565 570
575Phe Gly Thr Val Ala Val Asn Phe Gln Ser Ser Ser Thr Asp Pro Ala
580 585 590Thr Gly Asp Val His Val Met Gly Ala Leu Pro Gly Met Val
Trp Gln 595 600 605Asp Arg Asp Val Tyr Leu Gln Gly Pro Ile Trp Ala
Lys Ile Pro His 610 615 620Thr Asp Gly His Phe His Pro Ser Pro Leu
Met Gly Gly Phe Gly Leu625 630 635 640Lys His Pro Pro Pro Gln Ile
Leu Ile Lys Asn Thr Pro Val Pro Ala 645 650 655Asn Pro Pro Ala Glu
Phe Ser Ala Thr Lys Phe Ala Ser Phe Ile Thr 660 665 670Gln Tyr Ser
Thr Gly Gln Val Ser Val Glu Ile Glu Trp Glu Leu Gln 675 680 685Lys
Glu Asn Ser Lys Arg Trp Asn Pro Glu Val Gln Tyr Thr Ser Asn 690 695
700Tyr Ala Lys Ser Ala Asn Val Asp Phe Thr Val Asp Asn Asn Gly
Leu705 710 715 720Tyr Thr Glu Pro Arg Pro Ile Gly Thr Arg Tyr Leu
Thr Arg Pro Leu 725 730 73529736PRTArtificial SequenceDescription
of Artificial Sequence Note = synthethic construct 29Met Ala Ala
Asp Gly Tyr Leu Pro Asp Trp Leu Glu Asp Asn Leu Ser1 5 10 15Glu Gly
Ile Arg Glu Trp Trp Asp Leu Lys Pro Gly Ala Pro Lys Pro 20 25 30Lys
Ala Asn Gln Gln Lys Gln Asp Asp Gly Arg Gly Leu Val Leu Pro 35 40
45Gly Tyr Lys Tyr Leu Gly Pro Phe Asn Gly Leu Asp Lys Gly Glu Pro
50 55 60Val Asn Ala Ala Asp Ala Ala Ala Leu Glu His Asp Lys Ala Tyr
Asp65 70 75 80Gln Gln Leu Lys Ala Gly Asp Asn Pro Tyr Leu Arg Tyr
Asn His Ala 85 90 95Asp Ala Glu Phe Gln Glu Arg Leu Gln Glu Asp Thr
Ser Phe Gly Gly 100 105 110Asn Leu Gly Arg Ala Val Phe Gln Ala Lys
Lys Arg Val Leu Glu Pro 115 120 125Phe Gly Leu Val Glu Glu Gly Ala
Lys Thr Ala Pro Gly Lys Lys Arg 130 135 140Pro Val Glu Gln Ser Pro
Gln Glu Pro Asp Ser Ser Ser Gly Ile Gly145 150 155 160Lys Ser Gly
Gln Gln Pro Ala Lys Lys Arg Leu Asn Phe Gly Gln Thr 165 170 175Gly
Asp Ser Glu Ser Val Pro Asp Pro Gln Pro Leu Gly Glu Pro Pro 180 185
190Ala Thr Pro Ala Ala Leu Gly Pro Thr Thr Met Ala Ser Gly Gly Gly
195 200 205Ala Pro Met Ala Asp Asn Asn Glu Gly Ala Asp Gly Val Gly
Asn Ala 210 215 220Ser Gly Asn Trp His Cys Asp Ser Thr Trp Leu Gly
Asp Arg Val Ile225 230 235 240Thr Thr Ser Thr Arg Thr Trp Ala Leu
Pro Thr Tyr Asn Asn His Leu 245 250 255Tyr Lys Gln Ile Ser Ser Ala
Ser Thr Gly Ala Ser Asn Asp Asn His 260 265 270Tyr Phe Gly Tyr Ser
Thr Pro Trp Gly Tyr Phe Asp Phe Asn Arg Phe 275 280 285His Cys His
Phe Ser Pro Arg Asp Trp Gln Arg Leu Ile Asn Asn Asn 290 295 300Trp
Gly Phe Arg Pro Lys Arg Leu Asn Phe Lys Leu Phe Asn Ile Gln305 310
315 320Val Lys Glu Val Thr Thr Asn Asp Gly Val Thr Thr Ile Ala Asn
Asn 325 330 335Leu Thr Ser Thr Val Gln Val Phe Ser Asp Ser Glu Tyr
Gln Leu Pro 340 345 350Tyr Val Leu Gly Ser Ala His Gln Gly Cys Leu
Pro Pro Phe Pro Ala 355 360 365Asp Val Phe Met Ile Pro Gln Tyr Gly
Tyr Leu Thr Leu Asn Asn Gly 370 375 380Ser Lys Ala Val Gly Arg Ser
Ser Phe Tyr Cys Leu Glu Tyr Phe Pro385 390 395 400Ser Gln Met Leu
Arg Thr Gly Asn Asn Phe Thr Phe Ser Tyr Thr Phe 405 410 415Glu Glu
Val Pro Phe His Ser Ser Tyr Ala His Ser Gln Ser Leu Asp 420 425
430Arg Leu Met Asn Pro Leu Ile Asp Gln Tyr Leu Tyr Tyr Leu Asn Arg
435 440 445Thr Gln Asn Gln Ser Gly Ser Ala Gln Asn Lys Asp Leu Leu
Phe Ser 450 455 460Arg Gly Ser Pro Ala Gly Met Ser Val Gln Pro Lys
Asn Trp Leu Pro465 470 475 480Gly Pro Cys Tyr Arg Gln Gln Arg Val
Ser Lys Thr Lys Thr Asp Asn 485 490 495Asn Asn Ser Asn Phe Thr Trp
Thr Gly Ala Ser Lys Tyr Asn Leu Asn 500 505 510Gly Arg Glu Ser Ile
Ile Asn Pro Gly Thr Ala Met Ala Ser His Lys 515 520 525Asp Asp Glu
Asp Lys Phe Phe Pro Met Ser Gly Val Met Ile Phe Gly 530 535 540Lys
Glu Ser Ala Gly Ala Ser Asn Thr Ala Leu Asp Asn Val Met Ile545 550
555 560Thr Asp Glu Glu Glu Ile Lys Ala Thr Asn Pro Val Ala Thr Glu
Arg 565 570 575Phe Gly Thr Val Ala Val Asn Phe Gln Ser Ser Ser Thr
Asp Pro Ala 580 585 590Thr Gly Asp Val His Val Met Gly Ala Leu Pro
Gly Met Val Trp Gln 595 600 605Asp Arg Asp Val Tyr Leu Gln Gly Pro
Ile Trp Ala Lys Ile Pro His 610 615 620Thr Asp Gly His Phe His Pro
Ser Pro Leu Met Gly Gly Phe Gly Leu625 630 635 640Lys His Pro Pro
Pro Gln Ile Leu Ile Lys Asn Thr Pro Val Pro Ala 645 650 655Asn Pro
Pro Ala Glu Phe Ser Ala Thr Lys Phe Ala Ser Phe Ile Thr 660 665
670Gln Tyr Ser Thr Gly Gln Val Ser Val Glu Ile Glu Trp Glu Leu Gln
675 680 685Lys Glu Asn Ser Lys Arg Trp Asn Pro Glu Val Gln Tyr Thr
Ser Asn 690 695 700Tyr Ala Lys Ser Ala Asn Val Asp Phe Thr Val Asp
Asn Asn Gly Leu705 710 715 720Tyr Thr Glu Pro Arg Pro Ile Gly Thr
Arg Tyr Leu Thr Arg Pro Leu 725 730 73530742PRTArtificial
SequenceDescription of Artificial Sequence Note = synthethic
construct 30Met Ala Ala Asp Gly Tyr Leu Pro Asp Trp Leu Glu Asp Asn
Leu Ser1 5 10 15Glu Gly Ile Arg Glu Trp Trp Ala Leu Lys Pro Gly Ala
Pro Gln Pro 20 25 30Lys Ala Asn Gln Gln His Gln Asp Asn Gly Arg Gly
Leu Val Leu Pro 35 40 45Gly Tyr Lys Tyr Leu Gly Pro Phe Asn Gly Leu
Asp Lys Gly Glu Pro 50 55 60Val Asn Glu Ala Asp Ala Ala Ala Leu Glu
His Asp Lys Ala Tyr Asp65 70 75 80Lys Gln Leu Glu Gln Gly Asp Asn
Pro Tyr Leu Lys Tyr Asn His Ala 85 90 95Asp Ala Glu Phe Gln Gln Arg
Leu Ala Thr Asp Thr Ser Phe Gly Gly 100 105 110Asn Leu Gly Arg Ala
Val Phe Gln Ala Lys Lys Arg Ile Leu Glu Pro 115 120 125Leu Gly Leu
Val Glu Glu Gly Val Lys Thr Ala Pro Gly Lys Lys Arg 130 135 140Pro
Leu Glu Lys Thr Pro Asn Arg Pro Thr Asn Pro Asp Ser Gly Lys145 150
155 160Ala Pro Ala Lys Lys Lys Gln Lys Asp Gly Glu Pro Ala Asp Ser
Ala 165 170 175Arg Arg Thr Leu Asp Phe Glu Asp Ser Gly Ala Gly Asp
Gly Pro Pro 180 185 190Glu Gly Ser Ser Ser Gly Glu Met Ser His Asp
Ala Glu Met Arg Ala 195 200 205Ala Pro Gly Gly Asn Ala Val Glu Ala
Gly Gln Gly Ala Asp Gly Val 210 215 220Gly Asn Ala Ser Gly Asp Trp
His Cys Asp Ser Thr Trp Ser Glu Gly225 230 235 240Arg Val Thr Thr
Thr Ser Thr Arg Thr Trp Val Leu Pro Thr Tyr Asn 245 250 255Asn His
Leu Tyr Leu Arg Ile Gly Thr Thr Ala Asn Ser Asn Thr Tyr 260 265
270Asn Gly Phe Ser Thr Pro Trp Gly Tyr Phe Asp Phe Asn Arg Phe His
275 280 285Cys His Phe Ser Pro Arg Asp Trp Gln Arg Leu Ile Asn Asn
Asn Trp 290 295 300Gly Leu Arg Pro Lys Ser Met Arg Val Lys Ile Phe
Asn Ile Gln Val305 310 315 320Lys Glu Val Thr Thr Ser Asn Gly Glu
Thr Thr Val Ala Asn Asn Leu 325 330 335Thr Ser Thr Val Gln Ile Phe
Ala Asp Ser Thr Tyr Glu Leu Pro Tyr 340 345 350Val Met Asp Ala Gly
Gln Glu Gly Ser Phe Pro Pro Phe Pro Asn Asp 355 360 365Val Phe Met
Val Pro Gln Tyr Gly Tyr Cys Gly Val Val Thr Gly Lys 370 375 380Asn
Gln Asn Gln Thr Asp Arg Asn Ala Phe Tyr Cys Leu Glu Tyr Phe385 390
395 400Pro Ser Gln Met Leu Arg Thr Gly Asn Asn Phe Glu Val Ser Tyr
Gln 405 410 415Phe Glu Lys Val Pro Phe His Ser Met Tyr Ala His Ser
Gln Ser Leu 420 425 430Asp Arg Met Met Asn Pro Leu Leu Asp Gln Tyr
Leu Trp His Leu Gln 435 440 445Ser Thr Thr Thr Gly Asn Ser Leu Asn
Gln Gly Thr Ala Thr Thr Thr 450 455 460Tyr Gly Lys Ile Thr Thr Gly
Asp Phe Ala Tyr Tyr Arg Lys Asn Trp465 470 475
480Leu Pro Gly Ala Cys Ile Lys Gln Gln Lys Phe Ser Lys Asn Ala Asn
485 490 495Gln Asn Tyr Lys Ile Pro Ala Ser Gly Gly Asp Ala Leu Leu
Lys Tyr 500 505 510Asp Thr His Thr Thr Leu Asn Gly Arg Trp Ser Asn
Met Ala Pro Gly 515 520 525Pro Pro Met Ala Thr Ala Gly Ala Gly Asp
Ser Asp Phe Ser Asn Ser 530 535 540Gln Leu Ile Phe Ala Gly Pro Asn
Pro Ser Gly Asn Thr Thr Thr Ser545 550 555 560Ser Asn Asn Leu Leu
Phe Thr Ser Glu Glu Glu Ile Ala Thr Thr Asn 565 570 575Pro Arg Asp
Thr Asp Met Phe Gly Gln Ile Ala Asp Asn Asn Gln Asn 580 585 590Ala
Thr Thr Ala Pro His Ile Ala Asn Leu Asp Ala Met Gly Ile Val 595 600
605Pro Gly Met Val Trp Gln Asn Arg Asp Ile Tyr Tyr Gln Gly Pro Ile
610 615 620Trp Ala Lys Val Pro His Thr Asp Gly His Phe His Pro Ser
Pro Leu625 630 635 640Met Gly Gly Phe Gly Leu Lys His Pro Pro Pro
Gln Ile Phe Ile Lys 645 650 655Asn Thr Pro Val Pro Ala Asn Pro Asn
Thr Thr Phe Ser Ala Ala Arg 660 665 670Ile Asn Ser Phe Leu Thr Gln
Tyr Ser Thr Gly Gln Val Ala Val Gln 675 680 685Ile Asp Trp Glu Ile
Gln Lys Glu His Ser Lys Arg Trp Asn Pro Glu 690 695 700Val Gln Phe
Thr Ser Asn Tyr Gly Thr Gln Asn Ser Met Leu Trp Ala705 710 715
720Pro Asp Asn Ala Gly Asn Tyr His Glu Leu Arg Ala Ile Gly Ser Arg
725 730 735Phe Leu Thr His His Leu 7403124DNAArtificial
SequenceDescription of Artificial Sequence Note = synthethic
construct 31gcgacakttt gcgacaccay gtgg 243223DNAArtificial
SequenceDescription of Artificial Sequence Note = synthethic
construct 32ccannnggaa tcgcaatgcc aat 233323DNAArtificial
SequenceDescription of Artificial Sequence Note = synthethic
construct 33atgntnatnt ggtgggagga ggg 233425DNAArtificial
SequenceDescription of Artificial Sequence Note = synthethic
construct 34cgaatnaamc ggtttattga ttaac 25356PRTArtificial
SequenceDescription of Artificial Sequence Note = synthethic
construct 35Asn Gly Arg Ala His Ala1 5367PRTArtificial
SequenceDescription of Artificial Sequence Note = synthethic
construct 36Ser Ile Gly Tyr Pro Leu Pro1 53710PRTArtificial
SequenceDescription of Artificial Sequence Note = synthethic
construct 37Lys Phe Asn Lys Pro Phe Val Phe Leu Ile1 5
103822PRTArtificial SequenceDescription of Artificial Sequence Note
= synthethic construct 38Asn Ile Ser Leu Asp Asn Pro Leu Glu Asn
Pro Ser Ser Leu Phe Asp1 5 10 15Leu Val Ala Arg Ile Lys
203926DNAArtificial SequenceDescription of Artificial Sequence Note
= synthethic construct 39caataaaccg kktnattcgt ktcagt
264026DNAArtificial SequenceDescription of Artificial Sequence Note
= synthethic construct 40acannwgagt cagaaatkcc nggcag
2641640DNAArtificial SequenceDescription of Artificial Sequence
Note = synthethic construct 41gcgaattgaa tttagcggcc gcgaattcgc
ccttcaataa accgtttgat tcgtgtcagt 60tgatctttgg cctacttgtc cttcttatct
tatctggtcg ccatggctgc gtagataagc 120agcttggtat gcgcttcgcg
gttaatcatc aactacgcca aaccctagat gatggagttg 180gccactccct
ctatgcgcgc tcgctcgctc ggtggggccg gactgcccgg catttctgac
240tcctttgtga actgggtggc cctccgggct attgggtccc gtttcctcac
ccaccacttg 300taacccttcc tggttaatca atgaaccgtt taattcgttt
cagttgatct ttggcctact 360tgtccttctt atcttatctg gttgccatgg
ctgcgtagat aagcagcttg gtatgcgctt 420cgcggttaat catcaactac
gccaaaccct agatgatgga gttggccact ccctctatgc 480gcgctcgctc
gctcggtggg gccggactgc cgggcatttc tgactcactt gtaagggcga
540aattcgttta aacctgcagg actagtccct ttagtgaggg ttaattctga
gcttggcgta 600atcatggnca tagctgtttc ctgnggaaaa tgttatccgc
64042248DNAArtificial SequenceDescription of Artificial Sequence
Note = synthethic construct 42tggaggggtg gagtcgtgrc gtgaattacg
tcatagggtt agggaggtcc tgtattagag 60gtcacgtgag tgttttgcga cattttgcga
caccatgtgg tcacgctggg tatttaagcc 120cgagtgwgca cgcagggtct
ccattttgaa gcgggaggtt tgaacgcgca gccgccatgc 180cggggtttta
cgagattgtg attaaggtcc ccagcgacct tgacgagcat ctgccaggaa 240tttctgac
24843317DNAArtificial SequenceDescription of Artificial Sequence
Note = synthethic construct 43gcccgggcgg cctcagtgag cgagcgagcg
cgcagagagg gagtggccaa ctccatcact 60aggggttcct ggaggggtgg agtcgtgacg
tgaattacgt catagggtta gggaggtcct 120gtattagagg tcacgtgagt
gttttgcgac attttgcgac accatgtggt cacgctgggt 180atttaagccc
gagtgagcac gcagggtctc cattttgaag cgggaggttt gaacgcgcag
240ccgccatgcc ggggttttac gagattgtga ttaaggtccc cagcgacctt
gacgagcatc 300tgcctggaat ttctgac 31744157DNAArtificial
SequenceDescription of Artificial Sequence Note = synthethic
construct 44gagtgagcga gcagggtctc cattttgacc gcnaaatttg aacgagcagc
atccatgccg 60ggcttctacg agatcgtgat caaggtgccg agcgacctgg acgancacct
gcctggcatt 120tctgactcaa ttgtaagggc naattcgttt aaacctg
1574524DNAArtificial SequenceDescription of Artificial Sequence
Note = synthethic construct 45gcgacakttt gcgacaccay gtgg
244623DNAArtificial SequenceDescription of Artificial Sequence Note
= synthethic construct 46ccannnggaa tcgcaatgcc aat
234723DNAArtificial SequenceDescription of Artificial Sequence Note
= synthethic construct 47atgntnatnt ggtgggagga ggg
234825DNAArtificial SequenceDescription of Artificial Sequence Note
= synthethic construct 48cgaatnaamc ggtttattga ttaac
25491872DNAArtificial SequenceDescription of Artificial Sequence
Note = synthethic construct 49atgccgggct tctacgagat cgtgatcaag
gtgccgagcg acctggacga gcacctgccg 60ggcatttctg actcgtttgt gaactgggtg
gccgagaagg aatgggagct gcccccggat 120tctgacatgg atctgaatct
gattgagcag gcacccctga ccgtggccga gaagctgcag 180cgcgacttcc
tggtccaatg gcgccgcgtg agtaaggccc cggaggccct cttctttgtt
240cagttcgaga agggcgagtc ctacttccac ctccatattc tggtggagac
cacgggggtc 300aaatccatgg tgctgggccg cttcttgagt cagattaggg
acaagctggt gcagaccatc 360taccgcggga tcgagccgac cctgcccaac
tggttcgcgg tgaccaagac gcgtaatggc 420gccggagggg ggaacaaggt
ggtggacgag tgctacatcc ccaactacct cctgcccaag 480actcagcccg
agctgcagtg ggcgtggact aacatggagg agtatataag cgcgtgtttg
540aacctggccg agcgcaaacg gctcgtggcg cagcacctga cccacgtcag
ccagacccag 600gagcagaaca aggagaatct gaaccccaat tctgacgcgc
ctgtcatccg gtcaaaaacc 660tccgcgcgct acatggagct ggtcgggtgg
ctggtggacc ggggcatcac ctccgagaag 720cagtggatcc aggaggacca
ggcctcgtac atctccttca acgccgcctc caactcgcgg 780tcccagatca
aggccgctct ggacaatgcc ggcaagatca tggcgctgac caaatccgcg
840cccgactacc tggtaggccc cgctcctccc gcggacatta aaaccaaccg
catctaccgc 900atcctggagc tgaacggcta cgaccctgcc tacgccggct
ccgtctttct cggctgggcc 960cagaaacggt tcgggaagcg caacaccatc
tggctgtttg ggccggccac cacgggcaag 1020accaacatcg cggaagccat
cgcccacgcc gtgcccttct acggctgcgt caactggacc 1080aatgagaact
ttcccttcaa cgattgcgtc gacaagatgg tgatctggtg ggaggagggc
1140aagatgacgg ccaaggtcgt ggagtccgcc aaggccattc tcggcggcag
caaggtgcgc 1200gtggaccaaa agtgcaagtc gtccgcccag atcgatccca
cccccgtgat cgtcacctcc 1260aacaccaaca tgtgcgccgt gattgacggg
aacagcacca ccttcgagca ccagcagccg 1320ttgcaggacc ggatgttcaa
atttgaactc acccgccgtc tggagcatga ctttggcaag 1380gtgacaaagc
aggaagtcaa agagttcttc cgctgggcgc aggatcacgt gaccgaggtg
1440gcgcatgagt tctacgtcag aaagggtgga gccaacaaaa gacccgcccc
cgatgacgcg 1500gataaaagcg agcccaagcg ggcctgcccc tcagtcgcgg
atccatcgac gtcagacgcg 1560gaaggagctc cggtggactt tgccgacagg
taccaaaaca aatgttctcg tcacgcgggc 1620atgcttcaga tgctgtttcc
ctgcaagaca tgcgagagaa tgaatcagaa tttcaacatt 1680tgcttcacgc
acgggaccag agactgttca gaatgtttcc ccggcgtgtc agaatctcaa
1740ccggtcgtca gaaaaaggac gtatcggaaa ctctgtgcga ttcatcatct
gctggggcgg 1800gctcccgaga ttgcttgctc ggcctgcgat ctggtcaacg
tggacctgga tgactgtgtc 1860tctgagcaat aa 187250623PRTArtificial
SequenceDescription of Artificial Sequence Note = synthethic
construct 50Met Pro Gly Phe Tyr Glu Ile Val Ile Lys Val Pro Ser Asp
Leu Asp1 5 10 15Glu His Leu Pro Gly Ile Ser Asp Ser Phe Val Asn Trp
Val Ala Glu 20 25 30Lys Glu Trp Glu Leu Pro Pro Asp Ser Asp Met Asp
Leu Asn Leu Ile 35 40 45Glu Gln Ala Pro Leu Thr Val Ala Glu Lys Leu
Gln Arg Asp Phe Leu 50 55 60Val Gln Trp Arg Arg Val Ser Lys Ala Pro
Glu Ala Leu Phe Phe Val65 70 75 80Gln Phe Glu Lys Gly Glu Ser Tyr
Phe His Leu His Ile Leu Val Glu 85 90 95Thr Thr Gly Val Lys Ser Met
Val Leu Gly Arg Phe Leu Ser Gln Ile 100 105 110Arg Asp Lys Leu Val
Gln Thr Ile Tyr Arg Gly Ile Glu Pro Thr Leu 115 120 125Pro Asn Trp
Phe Ala Val Thr Lys Thr Arg Asn Gly Ala Gly Gly Gly 130 135 140Asn
Lys Val Val Asp Glu Cys Tyr Ile Pro Asn Tyr Leu Leu Pro Lys145 150
155 160Thr Gln Pro Glu Leu Gln Trp Ala Trp Thr Asn Met Glu Glu Tyr
Ile 165 170 175Ser Ala Cys Leu Asn Leu Ala Glu Arg Lys Arg Leu Val
Ala Gln His 180 185 190Leu Thr His Val Ser Gln Thr Gln Glu Gln Asn
Lys Glu Asn Leu Asn 195 200 205Pro Asn Ser Asp Ala Pro Val Ile Arg
Ser Lys Thr Ser Ala Arg Tyr 210 215 220Met Glu Leu Val Gly Trp Leu
Val Asp Arg Gly Ile Thr Ser Glu Lys225 230 235 240Gln Trp Ile Gln
Glu Asp Gln Ala Ser Tyr Ile Ser Phe Asn Ala Ala 245 250 255Ser Asn
Ser Arg Ser Gln Ile Lys Ala Ala Leu Asp Asn Ala Gly Lys 260 265
270Ile Met Ala Leu Thr Lys Ser Ala Pro Asp Tyr Leu Val Gly Pro Ala
275 280 285Pro Pro Ala Asp Ile Lys Thr Asn Arg Ile Tyr Arg Ile Leu
Glu Leu 290 295 300Asn Gly Tyr Asp Pro Ala Tyr Ala Gly Ser Val Phe
Leu Gly Trp Ala305 310 315 320Gln Lys Arg Phe Gly Lys Arg Asn Thr
Ile Trp Leu Phe Gly Pro Ala 325 330 335Thr Thr Gly Lys Thr Asn Ile
Ala Glu Ala Ile Ala His Ala Val Pro 340 345 350Phe Tyr Gly Cys Val
Asn Trp Thr Asn Glu Asn Phe Pro Phe Asn Asp 355 360 365Cys Val Asp
Lys Met Val Ile Trp Trp Glu Glu Gly Lys Met Thr Ala 370 375 380Lys
Val Val Glu Ser Ala Lys Ala Ile Leu Gly Gly Ser Lys Val Arg385 390
395 400Val Asp Gln Lys Cys Lys Ser Ser Ala Gln Ile Asp Pro Thr Pro
Val 405 410 415Ile Val Thr Ser Asn Thr Asn Met Cys Ala Val Ile Asp
Gly Asn Ser 420 425 430Thr Thr Phe Glu His Gln Gln Pro Leu Gln Asp
Arg Met Phe Lys Phe 435 440 445Glu Leu Thr Arg Arg Leu Glu His Asp
Phe Gly Lys Val Thr Lys Gln 450 455 460Glu Val Lys Glu Phe Phe Arg
Trp Ala Gln Asp His Val Thr Glu Val465 470 475 480Ala His Glu Phe
Tyr Val Arg Lys Gly Gly Ala Asn Lys Arg Pro Ala 485 490 495Pro Asp
Asp Ala Asp Lys Ser Glu Pro Lys Arg Ala Cys Pro Ser Val 500 505
510Ala Asp Pro Ser Thr Ser Asp Ala Glu Gly Ala Pro Val Asp Phe Ala
515 520 525Asp Arg Tyr Gln Asn Lys Cys Ser Arg His Ala Gly Met Leu
Gln Met 530 535 540Leu Phe Pro Cys Lys Thr Cys Glu Arg Met Asn Gln
Asn Phe Asn Ile545 550 555 560Cys Phe Thr His Gly Thr Arg Asp Cys
Ser Glu Cys Phe Pro Gly Val 565 570 575Ser Glu Ser Gln Pro Val Val
Arg Lys Arg Thr Tyr Arg Lys Leu Cys 580 585 590Ala Ile His His Leu
Leu Gly Arg Ala Pro Glu Ile Ala Cys Ser Ala 595 600 605Cys Asp Leu
Val Asn Val Asp Leu Asp Asp Cys Val Ser Glu Gln 610 615
620511872DNAArtificial SequenceDescription of Artificial Sequence
Note = synthethic construct 51atgccgggct tctacgagat cgtgatcaag
gtgccgagcg acctggacga gcacctgccg 60ggcatttctg actcgtttgt gaactgggtg
gccgagaagg aatgggagct gcccccggat 120tctgacatgg atctgaatct
gattgagcag gcacccctga ccgtggccga gaagctgcag 180cgcgacttcc
tggtccaatg gcgccgcgtg agtaaggccc cggaggccct cttctttgtt
240cagttcgaga agggcgagtc ctacttccac ctccatattc tggtggagac
cacgggggtc 300aaatccatgg tgctgggccg cttcctgagt cagattaggg
acaagctggt gcagaccatc 360taccgcggga tcgagccgac cctgcccaac
tggttcgcgg tgaccaagac gcgtaatggc 420gccggagggg ggaacaaggt
ggtggacgag tgctacatcc ccaactacct cctgcccaag 480actcagcccg
agctgcagtg ggcgtggact aacatggagg agtatataag cgcgtgtttg
540aacctggccg agcgcaaacg gctcgtggcg cagcacctga cccacgtcag
ccagacccag 600gagcagaaca aggagaatct gaacccgaat tctgacgcgc
ctgtcatccg gtcaaaaacc 660tccgcgcgct acatggagct ggtcgggtgg
ctggtggacc ggggcatcac ctccgagaag 720cagtggatcc aggaggacca
ggcctcgtac atctccttca acgccgcctc caactcgcgg 780tcccagatca
aggccgctct ggacaatgcc ggcaagatca tggcgctaac caaatccgcg
840cccgactacc tggtaggccc cgctccgccc gcggacatta aaaccaaccg
catttaccgc 900atcctggagc tgaacggcta cgaccctgcc tacgccggct
ccgtctttct cggctgggcc 960cagaaaaggt tcgggaagcg caacaccatc
tggctgtttg ggccggccac cacgggcaag 1020accaacatcg cggaagccat
cgcacacgcc gtgcccttct acggctgcgt caactggacc 1080aatgaaaact
ttcccttcaa cgactgcgtc gacaagatgg tgatctggtg ggaggagggc
1140aagatgacgg ccaaggtcgt ggagtccgcc aaggccattc tcggcggcag
caaggtgcgc 1200gtggaccaaa agtgcaagtc gtccgcccag atcgatccca
cccccgtgat cgtcacctcc 1260aacaccaaca tgtgcgccgt gattgacggg
aacagcacca ccttcgagca ccagcagccg 1320ttgcaggacc ggatgttcaa
atttgaactc acccgccgtc tggagcacga ctttggcaag 1380gtgacaaagc
aggaagtcaa agagttcttc cgctgggcgc aggatcacgt gaccgaggtg
1440gcgcatgagt tctacgtcag aaagggtgga gccaacaaga gacccgcccc
cgatgacgcg 1500gataaaagcg agcccaagcg ggtctgcccc tcagtcgcgg
atccatcgac gtcagacgcg 1560gaaggagctc cggtggactt tgccgacagg
taccaaaaca aatgttctcg tcacgcgggc 1620atgcttcaga tgctgtttcc
ctgcaaaaca tgcgagagaa tgaatcagaa tttcaacatt 1680tgcttcacgc
acgggaccag agactgttca gaatgtttcc ccggcgtgtc agaatctcaa
1740ccggtcgtca gaaaaaggac gtatcggaaa ctctgtgcca ttcatcatct
gctggggcgg 1800gctcccgaga ttgcttgctc ggcctgcgat ctggtcaacg
tggacctgga tgactgtgtt 1860tctgagcaat aa 187252623PRTArtificial
SequenceDescription of Artificial Sequence Note = synthethic
construct 52Met Pro Gly Phe Tyr Glu Ile Val Ile Lys Val Pro Ser Asp
Leu Asp1 5 10 15Glu His Leu Pro Gly Ile Ser Asp Ser Phe Val Asn Trp
Val Ala Glu 20 25 30Lys Glu Trp Glu Leu Pro Pro Asp Ser Asp Met Asp
Leu Asn Leu Ile 35 40 45Glu Gln Ala Pro Leu Thr Val Ala Glu Lys Leu
Gln Arg Asp Phe Leu 50 55 60Val Gln Trp Arg Arg Val Ser Lys Ala Pro
Glu Ala Leu Phe Phe Val65 70 75 80Gln Phe Glu Lys Gly Glu Ser Tyr
Phe His Leu His Ile Leu Val Glu 85 90 95Thr Thr Gly Val Lys Ser Met
Val Leu Gly Arg Phe Leu Ser Gln Ile 100 105 110Arg Asp Lys Leu Val
Gln Thr Ile Tyr Arg Gly Ile Glu Pro Thr Leu 115 120 125Pro Asn Trp
Phe Ala Val Thr Lys Thr Arg Asn Gly Ala Gly Gly Gly 130 135 140Asn
Lys Val Val Asp Glu Cys Tyr Ile Pro Asn Tyr Leu Leu Pro Lys145 150
155 160Thr Gln Pro Glu Leu Gln Trp Ala Trp Thr Asn Met Glu Glu Tyr
Ile 165 170 175Ser Ala Cys Leu Asn Leu Ala Glu Arg Lys Arg Leu Val
Ala Gln His 180 185 190Leu Thr His Val Ser Gln Thr Gln Glu Gln Asn
Lys Glu Asn Leu Asn 195 200 205Pro Asn Ser Asp Ala Pro Val Ile Arg
Ser Lys Thr Ser Ala Arg Tyr 210 215 220Met Glu Leu Val Gly Trp Leu
Val Asp Arg Gly Ile Thr Ser Glu Lys225 230 235 240Gln Trp Ile Gln
Glu Asp Gln Ala Ser Tyr Ile Ser Phe Asn Ala Ala 245 250 255Ser Asn
Ser Arg Ser Gln Ile Lys Ala Ala Leu Asp
Asn Ala Gly Lys 260 265 270Ile Met Ala Leu Thr Lys Ser Ala Pro Asp
Tyr Leu Val Gly Pro Ala 275 280 285Pro Pro Ala Asp Ile Lys Thr Asn
Arg Ile Tyr Arg Ile Leu Glu Leu 290 295 300Asn Gly Tyr Asp Pro Ala
Tyr Ala Gly Ser Val Phe Leu Gly Trp Ala305 310 315 320Gln Lys Arg
Phe Gly Lys Arg Asn Thr Ile Trp Leu Phe Gly Pro Ala 325 330 335Thr
Thr Gly Lys Thr Asn Ile Ala Glu Ala Ile Ala His Ala Val Pro 340 345
350Phe Tyr Gly Cys Val Asn Trp Thr Asn Glu Asn Phe Pro Phe Asn Asp
355 360 365Cys Val Asp Lys Met Val Ile Trp Trp Glu Glu Gly Lys Met
Thr Ala 370 375 380Lys Val Val Glu Ser Ala Lys Ala Ile Leu Gly Gly
Ser Lys Val Arg385 390 395 400Val Asp Gln Lys Cys Lys Ser Ser Ala
Gln Ile Asp Pro Thr Pro Val 405 410 415Ile Val Thr Ser Asn Thr Asn
Met Cys Ala Val Ile Asp Gly Asn Ser 420 425 430Thr Thr Phe Glu His
Gln Gln Pro Leu Gln Asp Arg Met Phe Lys Phe 435 440 445Glu Leu Thr
Arg Arg Leu Glu His Asp Phe Gly Lys Val Thr Lys Gln 450 455 460Glu
Val Lys Glu Phe Phe Arg Trp Ala Gln Asp His Val Thr Glu Val465 470
475 480Ala His Glu Phe Tyr Val Arg Lys Gly Gly Ala Asn Lys Arg Pro
Ala 485 490 495Pro Asp Asp Ala Asp Lys Ser Glu Pro Lys Arg Val Cys
Pro Ser Val 500 505 510Ala Asp Pro Ser Thr Ser Asp Ala Glu Gly Ala
Pro Val Asp Phe Ala 515 520 525Asp Arg Tyr Gln Asn Lys Cys Ser Arg
His Ala Gly Met Leu Gln Met 530 535 540Leu Phe Pro Cys Lys Thr Cys
Glu Arg Met Asn Gln Asn Phe Asn Ile545 550 555 560Cys Phe Thr His
Gly Thr Arg Asp Cys Ser Glu Cys Phe Pro Gly Val 565 570 575Ser Glu
Ser Gln Pro Val Val Arg Lys Arg Thr Tyr Arg Lys Leu Cys 580 585
590Ala Ile His His Leu Leu Gly Arg Ala Pro Glu Ile Ala Cys Ser Ala
595 600 605Cys Asp Leu Val Asn Val Asp Leu Asp Asp Cys Val Ser Glu
Gln 610 615 620532689DNAArtificial SequenceDescription of
Artificial Sequence Note = synthethic construct 53ttgcgacagt
ttgcgacacc atgtggtcac aagaggtata taaccgcgag tgagccagcg 60aggagctcca
ttttgcccgc gaagtttgaa cgagcagcag ccatgccggg gttctacgag
120gtggtgatca aggtgcccag cgacctggac gagcacctgc ccggcatttc
tgactccttt 180gtgaactggg tggccgagaa ggaatgggag ttgcccccgg
attctgacat ggatcagaat 240ctgattgagc aggcacccct gaccgtggcc
gagaagctgc agcgcgagtt cctggtggaa 300tggcgccgag tgagtaaatt
tctggaggcc aagttttttg tgcagtttga aaagggggac 360tcgtactttc
atttgcatat tctgattgaa attaccggcg tgaaatccat ggtggtgggc
420cgctacgtga gtcagattag ggataaactg atccagcgca tctaccgcgg
ggtcgagccc 480cagctgccca actggttcgc ggtcacaaag acccgaaatg
gcgccggagg cgggaacaag 540gtggtggacg agtgctacat ccccaactac
ctgctcccca aggtccagcc cgagcttcag 600tgggcgtgga ctaacatgga
ggagtatata agcgcctgtt tgaacctcgc ggagcgtaaa 660cggctcgtgg
cgcagcacct gacgcacgtc tcccagaccc aggagggcga caaggagaat
720ctgaacccga attctgacgc gccggtgatc cggtcaaaaa cctccgccag
gtacatggag 780ctggtcgggt ggctggtgga caagggcatc acgtccgaga
agcagtggat ccaggaggac 840caggcctcgt acatctcctt caacgcggcc
tccaactccc ggtcgcagat caaggcggcc 900ctggacaatg cctccaaaat
catgagcctc accaaaacgg ctccggacta tctcatcggg 960cagcagcccg
tgggggacat taccaccaac cggatctaca aaatcctgga actgaacggg
1020tacgaccccc agtacgccgc ctccgtcttt ctcggctggg cccagaaaaa
gtttggaaag 1080cgcaacacca tctggctgtt tgggcccgcc accaccggca
agaccaacat cgcggaagcc 1140atcgcccacg cggtcccctt ctacggctgc
gtcaactgga ccaatgagaa ctttcccttc 1200aacgactgcg tcgacaaaat
ggtgatttgg tgggaggagg gcaagatgac cgccaaggtc 1260gtagagtccg
ccaaggccat tctgggcggc agcaaggtgc gcgtggacca aaaatgcaag
1320gcctctgcgc agatcgaccc cacccccgtg atcgtcacct ccaacaccaa
catgtgcgcc 1380gtgattgacg ggaacagcac caccttcgag caccagcagc
ccctgcagga ccggatgttc 1440aagtttgaac tcacccgccg cctcgaccac
gactttggca aggtcaccaa gcaggaagtc 1500aaggactttt tccggtgggc
ggctgatcac gtgactgacg tggctcatga gttttacgtc 1560acaaagggtg
gagctaagaa aaggcccgcc ccctctgacg aggatataag cgagcccaag
1620cggccgcgcg tgtcatttgc gcagccggag acgtcagacg cggaagctcc
cggagacttc 1680gccgacaggt accaaaacaa atgttctcgt cacgcgggta
tgctgcagat gctctttccc 1740tgcaagacgt gcgagagaat gaatcagaat
tccaacgtct gcttcacgca cggtcagaaa 1800gattgcgggg agtgctttcc
cgggtcagaa tctcaaccgg tttctgtcgt cagaaaaacg 1860tatcagaaac
tgtgcatcct tcatcagctc cggggggcac ccgagatcgc ctgctctgct
1920tgcgaccaac tcaaccccga tttggacgat tgccaatttg agcaataaat
gactgaaatc 1980aggtatggct gctgacggtt atcttccaga ttggctcgag
gacaacctct ctgaaggcat 2040tcgcgagtgg tgggcgctga aacctggagc
tccacaaccc aaggccaacc aacagcatca 2100ggacaacggc aggggtcttg
tgcttcctgg gtacaagtac ctcggaccct tcaacggact 2160cgacaaggga
gagccggtca acgaggcaga cgccgcggcc ctcgagcacg acaaggccta
2220cgacaagcag ctcgagcagg gggacaaccc gtatctcaag tacaaccacg
ccgacgccga 2280gttccagcag cgcttggcga ccgacacctc ttttgggggc
aacctcgggc gagcagtctt 2340ccaggccaaa aagaggattc tcgagcctct
gggtctggtt gaagagggcg ttaaaacggc 2400tcctggaaag aaacgcccat
tagaaaagac tccaaatcgg ccgaccaacc cggactctgg 2460gaaggccccg
gccaagaaaa agcaaaaaga cggcgaacca gccgactctg ctagaaggac
2520actcgacttt gaagactctg gagcaggaga cggaccccct gagggatcat
cttccggaga 2580aatgtctcat gatgctgaga tgcgtgcggc gccaggcgga
aatgctgtcg aggcgggaca 2640aggtgccgat ggagtgggta atgcctccgg
tgattggcat tgcgattcc 268954621PRTArtificial SequenceDescription of
Artificial Sequence Note = synthethic construct 54Met Pro Gly Phe
Tyr Glu Val Val Ile Lys Val Pro Ser Asp Leu Asp1 5 10 15Glu His Leu
Pro Gly Ile Ser Asp Ser Phe Val Asn Trp Val Ala Glu 20 25 30Lys Glu
Trp Glu Leu Pro Pro Asp Ser Asp Met Asp Gln Asn Leu Ile 35 40 45Glu
Gln Ala Pro Leu Thr Val Ala Glu Lys Leu Gln Arg Glu Phe Leu 50 55
60Val Glu Trp Arg Arg Val Ser Lys Phe Leu Glu Ala Lys Phe Phe Val65
70 75 80Gln Phe Glu Lys Gly Asp Ser Tyr Phe His Leu His Ile Leu Ile
Glu 85 90 95Ile Thr Gly Val Lys Ser Met Val Val Gly Arg Tyr Val Ser
Gln Ile 100 105 110Arg Asp Lys Leu Ile Gln Arg Ile Tyr Arg Gly Val
Glu Pro Gln Leu 115 120 125Pro Asn Trp Phe Ala Val Thr Lys Thr Arg
Asn Gly Ala Gly Gly Gly 130 135 140Asn Lys Val Val Asp Glu Cys Tyr
Ile Pro Asn Tyr Leu Leu Pro Lys145 150 155 160Val Gln Pro Glu Leu
Gln Trp Ala Trp Thr Asn Met Glu Glu Tyr Ile 165 170 175Ser Ala Cys
Leu Asn Leu Ala Glu Arg Lys Arg Leu Val Ala Gln His 180 185 190Leu
Thr His Val Ser Gln Thr Gln Glu Gly Asp Lys Glu Asn Leu Asn 195 200
205Pro Asn Ser Asp Ala Pro Val Ile Arg Ser Lys Thr Ser Ala Arg Tyr
210 215 220Met Glu Leu Val Gly Trp Leu Val Asp Lys Gly Ile Thr Ser
Glu Lys225 230 235 240Gln Trp Ile Gln Glu Asp Gln Ala Ser Tyr Ile
Ser Phe Asn Ala Ala 245 250 255Ser Asn Ser Arg Ser Gln Ile Lys Ala
Ala Leu Asp Asn Ala Ser Lys 260 265 270Ile Met Ser Leu Thr Lys Thr
Ala Pro Asp Tyr Leu Ile Gly Gln Gln 275 280 285Pro Val Gly Asp Ile
Thr Thr Asn Arg Ile Tyr Lys Ile Leu Glu Leu 290 295 300Asn Gly Tyr
Asp Pro Gln Tyr Ala Ala Ser Val Phe Leu Gly Trp Ala305 310 315
320Gln Lys Lys Phe Gly Lys Arg Asn Thr Ile Trp Leu Phe Gly Pro Ala
325 330 335Thr Thr Gly Lys Thr Asn Ile Ala Glu Ala Ile Ala His Ala
Val Pro 340 345 350Phe Tyr Gly Cys Val Asn Trp Thr Asn Glu Asn Phe
Pro Phe Asn Asp 355 360 365Cys Val Asp Lys Met Val Ile Trp Trp Glu
Glu Gly Lys Met Thr Ala 370 375 380Lys Val Val Glu Ser Ala Lys Ala
Ile Leu Gly Gly Ser Lys Val Arg385 390 395 400Val Asp Gln Lys Cys
Lys Ala Ser Ala Gln Ile Asp Pro Thr Pro Val 405 410 415Ile Val Thr
Ser Asn Thr Asn Met Cys Ala Val Ile Asp Gly Asn Ser 420 425 430Thr
Thr Phe Glu His Gln Gln Pro Leu Gln Asp Arg Met Phe Lys Phe 435 440
445Glu Leu Thr Arg Arg Leu Asp His Asp Phe Gly Lys Val Thr Lys Gln
450 455 460Glu Val Lys Asp Phe Phe Arg Trp Ala Ala Asp His Val Thr
Asp Val465 470 475 480Ala His Glu Phe Tyr Val Thr Lys Gly Gly Ala
Lys Lys Arg Pro Ala 485 490 495Pro Ser Asp Glu Asp Ile Ser Glu Pro
Lys Arg Pro Arg Val Ser Phe 500 505 510Ala Gln Pro Glu Thr Ser Asp
Ala Glu Ala Pro Gly Asp Phe Ala Asp 515 520 525Arg Tyr Gln Asn Lys
Cys Ser Arg His Ala Gly Met Leu Gln Met Leu 530 535 540Phe Pro Cys
Lys Thr Cys Glu Arg Met Asn Gln Asn Ser Asn Val Cys545 550 555
560Phe Thr His Gly Gln Lys Asp Cys Gly Glu Cys Phe Pro Gly Ser Glu
565 570 575Ser Gln Pro Val Ser Val Val Arg Lys Thr Tyr Gln Lys Leu
Cys Ile 580 585 590Leu His Gln Leu Arg Gly Ala Pro Glu Ile Ala Cys
Ser Ala Cys Asp 595 600 605Gln Leu Asn Pro Asp Leu Asp Asp Cys Gln
Phe Glu Gln 610 615 620
* * * * *
References