U.S. patent application number 15/127743 was filed with the patent office on 2017-08-03 for stable gene transfer to proliferating cells.
The applicant listed for this patent is Children's Medical Research Institute, Mount Sinai Hospital, The Sydney Children's Hospitals Network (Randwick and Westmead)(Incorporating the Royal Alexandra. Invention is credited to Ian Alexander, Sharon Cunningham, Andras Nagy.
Application Number | 20170216456 15/127743 |
Document ID | / |
Family ID | 54143555 |
Filed Date | 2017-08-03 |
United States Patent
Application |
20170216456 |
Kind Code |
A1 |
Alexander; Ian ; et
al. |
August 3, 2017 |
Stable Gene Transfer to Proliferating Cells
Abstract
Provided herein are methods for facilitating or inducing stable
transgene integration and expression in a proliferating cell,
comprising administering to the cell (i) a recombinant AAV (rAAV)
vector comprising the transgene flanked by transposon-derived
inverted terminal repeat sequences, which sequences are in turn
flanked by AAV-derived inverted terminal repeat regions, and (ii) a
source of a transposase that recognises said transposon-derived
inverted terminal repeat sequences and directs the genomic
integration of the transgene into the genome of the proliferating
cell. Also provided are methods and transgene delivery systems for
the treatment or prevention of diseases affecting, associated with
or characterised by proliferating cells, including paediatric liver
diseases, bone marrow diseases and cancer.
Inventors: |
Alexander; Ian; (Middle
Dural, AU) ; Cunningham; Sharon; (North Parramatta,
AU) ; Nagy; Andras; (Toronto, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
The Sydney Children's Hospitals Network (Randwick and
Westmead)(Incorporating the Royal Alexandra
Children's Medical Research Institute
Mount Sinai Hospital |
Westmead
Westmead
Toronto |
|
AU
AU
CA |
|
|
Family ID: |
54143555 |
Appl. No.: |
15/127743 |
Filed: |
March 23, 2015 |
PCT Filed: |
March 23, 2015 |
PCT NO: |
PCT/AU2015/050125 |
371 Date: |
September 20, 2016 |
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 48/005 20130101;
C12Y 201/03003 20130101; C12N 15/86 20130101; C12N 2840/007
20130101; C12N 9/14 20130101; C12Y 207/07 20130101; C12N 9/93
20130101; A61K 48/0066 20130101; C12N 2750/14143 20130101; A61K
48/0041 20130101; C12N 9/1241 20130101; C12N 2800/90 20130101; C12N
9/1018 20130101; C12Y 306/03044 20130101; C12Y 603/04005
20130101 |
International
Class: |
A61K 48/00 20060101
A61K048/00; C12N 9/12 20060101 C12N009/12; C12N 9/00 20060101
C12N009/00; C12N 9/14 20060101 C12N009/14; C12N 15/86 20060101
C12N015/86; C12N 9/10 20060101 C12N009/10 |
Foreign Application Data
Date |
Code |
Application Number |
Mar 21, 2014 |
AU |
2014901004 |
Claims
1. A method for stably integrating a transgene into the genome of a
proliferating cell and/or inducing stable transgene expression in a
proliferating cell, the method comprising administering to the
cell: (i) a recombinant AAV (rAAV) vector comprising the transgene
flanked by transposon-derived inverted terminal repeat sequences,
which sequences are in turn flanked by AAV-derived inverted
terminal repeat regions; and (ii) a source of a transposase that
recognises said transposon-derived inverted terminal repeat
sequences and directs the genomic integration of the transgene into
the genome of the proliferating cell.
2. A method according to claim 1, wherein the genomic integration
of the transgene into the genome of the proliferating cell
facilitates or induces the stable transgene expression.
3-4. (canceled)
5. A method according to claim 1, wherein the transgene and
flanking transposon-derived inverted terminal repeat sequences form
a transposon-transgene cassette, optionally comprising one or more
further sequences or genetic elements including, for example, a
promoter, enhancer, post-regulatory element and/or polyadenylation
signal sequence.
6. A method according to claim 1, wherein the transgene is operably
linked to a promoter.
7. A method according to claim 6, wherein the promoter is a
tissue-specific promoter.
8. (canceled)
9. A method according to claim 1, wherein the transposase is
provided to the proliferating cell in a form so as to allow
transient expression of the transposase in the cell.
10. A method according to claim 9, wherein the transposase is
administered to the proliferating cell in the form of mRNA.
11. A method according to claim 1, wherein the transposase is
administered to the proliferating cell via a second rAAV vector
comprising a polynucleotide encoding the transposase, optionally
operably linked to a suitable promoter, and optionally flanked by
AAV-derived inverted terminal repeat regions.
12. A method according to claim 11, wherein the promoter is a
tissue-specific promoter.
13. (canceled)
14. A method according to claim 1, wherein the AAV sequences used
in the rAAV vector(s) are derived from AAV2 or AAV8.
15. (canceled)
16. A method according to claim 1, wherein the transposase is the
piggyBac transposase, and the transposon-derived inverted terminal
repeat sequences are derived from the piggyBac transposon.
17. A method according to claim 1, wherein the proliferating cell
is a rapidly proliferating cell.
18. (canceled)
19. A method according to claim 17, wherein the proliferating cell
is from a neonatal or juvenile liver.
20. A method according to claim 1, wherein the proliferating cell
is a disease cell.
21. (canceled)
22. A method according to claim 1, for the treatment of paediatric
liver diseases.
23. A method for treating a disease of, affecting, or associated
with, a proliferating cell, comprising administering to a subject
in need thereof (i) a recombinant AAV (rAAV) vector comprising a
transgene flanked by transposon-derived inverted terminal repeat
sequences, which sequences are in turn flanked by AAV-derived
inverted terminal repeat regions; and (ii) a source of a
transposase that recognises said transposon-derived inverted
terminal repeat sequences and directs the genomic integration of
the transgene into the genome of the proliferating cell, wherein
said administration results in the stable integration and
expression of the transgene to thereby treat the disease.
24. (canceled)
25. A method according to claim 23, wherein the disease is
associated with the deficiency of one or more gene products in the
proliferating cell, and wherein expression of the transgene
normalises production and activity of the deficient gene
product.
26. A method according to claim 23, wherein the disease is a
paediatric liver disease, a bone marrow disease or a cancer.
27. A method according to claim 26, wherein the paediatric liver
disease is selected from OTC deficiency, ASS deficiency and
progressive familial intrahepatic cholestasis.
28. A method according to claim 27, wherein the progressive
familial intrahepatic cholestasis is progressive familial
intrahepatic cholestasis type 3.
29. A method according to claim 27, wherein: (i) when the disease
is OTC deficiency the transgene comprises a polynucleotide encoding
ornithine transcarbamylase (OTC); (ii) when the disease is ASS
deficiency the transgene comprises a polynucleotide encoding
argininosuccinate synthetase (ASS); or (iii) when the disease is
progressive familial intrahepatic cholestasis the transgene
comprises a polynucleotide encoding ATP-binding cassette subfamily
B member 4 (ABCB4).
30-31. (canceled)
32. A transgene delivery and expression system for inducing stable
transgene expression in a proliferating cell or for treating a
disease of, affecting, or associated with, a proliterating cell,
wherein the system comprises (i) a recombinant AAV (rAAV) vector
comprising the transgene flanked by transposon-derived inverted
terminal repeat sequences, which sequences are in turn flanked by
AAV-derived inverted terminal repeat regions; and (ii) a source of
a transposase that recognises said transposon-derived inverted
terminal repeat sequences and directs the genomic integration of
the transgene into the genome of the proliferating cell.
33-35. (canceled)
Description
TECHNICAL FIELD
[0001] The present invention relates generally to methods for
stably integrating and expressing transgenes in proliferating
cells. The invention also relates to methods and vector systems for
the treatment of genetic diseases associated with, or affecting,
proliferating cells, organs or tissues.
BACKGROUND ART
[0002] Adeno-associated virus (AAV) is a parvovirus having a
single-stranded DNA genome. The AAV genome is relatively simple,
containing two open reading frames (ORFs) flanked by short inverted
terminal repeats (ITRs). The ITRs contain, inter alia, cis-acting
sequences required for virus replication, rescue, packaging and
integration. The integration function of the ITR permits the AAV
genome to integrate into a cellular chromosome after infection.
[0003] Recombinant AAV vectors have been shown to be able to
transduce a wide range of different cell types, such as
hematopoietic cells, epithelial cells and neurons. Interest in AAVs
as vectors for gene therapy results from several advantageous
features of their biology. These include their ability to transduce
non-dividing and dividing cells, their capacity for stable genetic
transformation, and the fact that AAVs do not cause disease (and
low immunogenicity) in humans. The integration of AAV vectors into
the genome of target cells enables long term transgene expression
in transduced cells. At least twelve different AAV serotypes have
been identified and well characterized, including AAV2 and AAV8,
the most widely employed in constructing recombinant AAV vectors
for gene transfer and gene therapy applications.
[0004] Notwithstanding the attractive aspects of AAV-based vectors,
a significant challenge, as yet not overcome, to their widespread
use is maintaining stable levels of therapeutically effective
transgene expression in proliferating cells such as in the juvenile
liver and in bone marrow. For example, despite high efficiency of
transduction of neonatal mouse hepatocytes by recombinant AAV
vectors, episomal vectors are rapidly lost and eliminated within 2
weeks, and stable transgene expression is observed in only a very
small proportion (about 5-10%) of cells (Cunningham et al., 2008,
Molecular Therapy 16:1081-1088). This significantly hampers the
further development of AAV-based gene therapy approaches to the
treatment of, for example, genetic liver diseases, and in
particular paediatric liver diseases.
[0005] There remains a need for vector systems and methods to
increase stable transgene expression in proliferating cells to
therapeutically effective levels so as to enable the development of
gene therapy approaches to treating diseases associated with
cellular proliferation such as cancer, and diseases affecting
proliferating cells, organs and tissues, such as paediatric liver
diseases.
SUMMARY OF THE INVENTION
[0006] According to a first aspect the present invention provides a
method for facilitating or inducing stable transgene expression in
a proliferating cell, the method comprising administering to the
cell: (i) a recombinant AAV (rAAV) vector comprising the transgene
flanked by transposon-derived inverted terminal repeat sequences,
which sequences are in turn flanked by AAV-derived inverted
terminal repeat regions; and (ii) a source of a transposase that
recognises said transposon-derived inverted terminal repeat
sequences and directs the genomic integration of the transgene into
the genome of the proliferating cell.
[0007] Typically the genomic integration of the transgene into the
genome of the proliferating cell in accordance with the present
method facilitates or induces the stable transgene expression.
[0008] A second aspect of the invention provides a method for
stably integrating a transgene into the genome of a proliferating
cell, the method comprising administering to the cell: (i) a
recombinant AAV (rAAV) vector comprising the transgene flanked by
transposon-derived inverted terminal repeat sequences, which
sequences are in turn flanked by AAV-derived inverted terminal
repeat regions; and (ii) a source of a transposase that recognises
said transposon-derived inverted terminal repeat sequences and
directs the genomic integration of the transgene into the genome of
the proliferating cell.
[0009] Typically the stable integration of the transgene into the
genome of the proliferating cell facilitates or induces the stable
expression of the transgene in the cell.
[0010] The method of the first or second aspect may be employed to
treat or prevent a disease in a subject, wherein the stable genomic
integration and expression of the transgene is desired and
beneficial in the treatment or prevention of the disease. Typically
the disease is a disease affecting, or associated with,
proliferating cells.
[0011] Typically the disease is a genetic disease. The disease may
be associated with the deficiency of one or more gene products in
the proliferating cell, typically wherein expression of the
transgene normalises production and activity of the deficient gene
product. In one embodiment the disease may be a paediatric liver
disease. The paediatric liver disease may be selected from OTC
deficiency, ASS deficiency and progressive familial intrahepatic
cholestasis. The progressive intrahepatic cholestasis may be
progressive familial intrahepatic cholestasis type 3. In
alternative embodiments, the disease may be cancer or a bone marrow
disease.
[0012] In an embodiment, wherein the disease is OTC deficiency, the
transgene typically comprises a polynucleotide encoding ornithine
transcarbamylase (OTC). In an embodiment, wherein the disease is
ASS deficiency, the transgene typically comprises a polynucleotide
encoding argininosuccinate synthetase (ASS). In an embodiment,
wherein the disease is progressive familial intrahepatic
cholestasis type 3, the transgene typically comprises a
polynucleotide encoding ATP-binding cassette subfamily B member 4
(ABCB4).
[0013] A third aspect of the invention provides a method for
treating or preventing a disease of, affecting, or associated with,
a proliferating cell, comprising administering to a subject in need
thereof (i) a recombinant AAV (rAAV) vector comprising a transgene
flanked by transposon-derived inverted terminal repeat sequences,
which sequences are in turn flanked by AAV-derived inverted
terminal repeat regions; and (ii) a source of a transposase that
recognises said transposon-derived inverted terminal repeat
sequences and directs the genomic integration of the transgene into
the genome of the proliferating cell, wherein said administration
results in the stable integration and expression of the transgene
to thereby treat the disease.
[0014] A fourth aspect of the invention provides a transgene
delivery and expression system for inducing stable transgene
expression in a proliferating cell, wherein the system comprises
(i) a recombinant AAV (rAAV) vector comprising the transgene
flanked by transposon-derived inverted terminal repeat sequences,
which sequences are in turn flanked by AAV-derived inverted
terminal repeat regions; and (ii) a source of a transposase that
recognises said transposon-derived inverted terminal repeat
sequences and directs the genomic integration of the transgene into
the genome of the proliferating cell.
[0015] A fifth aspect of the invention provides a transgene
delivery and expression system for treating or preventing a disease
of, affecting, or associated with, a proliferating cell, wherein
the system comprises (i) a recombinant AAV (rAAV) vector comprising
the transgene flanked by transposon-derived inverted terminal
repeat sequences, which sequences are in turn flanked by
AAV-derived inverted terminal repeat regions; and (ii) a source of
a transposase that recognises said transposon-derived inverted
terminal repeat sequences and directs the genomic integration of
the transgene into the genome of the proliferating cell.
[0016] A sixth aspect of the invention provides the use of (i) a
recombinant AAV (rAAV) vector comprising a transgene flanked by
transposon-derived inverted terminal repeat sequences, which
sequences are in turn flanked by AAV-derived inverted terminal
repeat regions; and (ii) a source of a transposase that recognises
said transposon-derived inverted terminal repeat sequences and
directs the genomic integration of the transgene into the genome of
a proliferating cell, in the manufacture of a medicament for
inducing stable transgene expression in a proliferating cell.
[0017] A seventh aspect of the invention provides the use of (i) a
recombinant AAV (rAAV) vector comprising a transgene flanked by
transposon-derived inverted terminal repeat sequences, which
sequences are in turn flanked by AAV-derived inverted terminal
repeat regions; and (ii) a source of a transposase that recognises
said transposon-derived inverted terminal repeat sequences and
directs the genomic integration of the transgene into the genome of
a proliferating cell, in the manufacture of a medicament for
treating or preventing a disease of, affecting, or associated with,
a proliferating cell.
[0018] In accordance with the above aspects, typically the disease
is a genetic disease. The disease may be associated with the
deficiency of one or more gene products in the proliferating cell,
typically wherein expression of the transgene normalises production
and activity of the deficient gene product. In one embodiment the
disease may be a paediatric liver disease. The paediatric liver
disease may be selected from OTC deficiency, ASS deficiency and
progressive familial intrahepatic cholestasis. The progressive
intrahepatic cholestasis may be progressive familial intrahepatic
cholestasis type 3. In alternative embodiments, the disease may be
cancer or a bone marrow disease.
[0019] In an embodiment, wherein the disease is OTC deficiency, the
transgene typically comprises a polynucleotide encoding ornithine
transcarbamylase (OTC). In an embodiment, wherein the disease is
ASS deficiency, the transgene typically comprises a polynucleotide
encoding argininosuccinate synthetase (ASS). In an embodiment,
wherein the disease is progressive familial intrahepatic
cholestasis type 3, the transgene typically comprises a
polynucleotide encoding ATP-binding cassette subfamily B member 4
(ABCB4).
[0020] The embodiments and associated disclosure below relate to
each of the aspects described above.
[0021] The transgene may be any gene the expression of which it is
desirable to induce in the proliferating cell. The transgene may be
foreign to the proliferating cell. The transgene may be a gene the
expression of which is absent or reduced in the proliferating cell
in the absence of introduction of the transgene.
[0022] In an embodiment the transgene and flanking
transposon-derived inverted terminal repeat sequences form a
transposon-transgene cassette, optionally comprising one or more
further sequences or genetic elements including, for example, a
promoter, enhancer, post-regulatory element and/or polyadenylation
signal sequence. The cassette may be packaged in a suitable AAV
capsid.
[0023] Typically the transgene is operably linked to a suitable
promoter. The promoter may be a tissue-specific promoter. In an
embodiment the promoter is a liver-specific promoter. In an
exemplary embodiment the liver-specific promoter is the human
alpha-1 antitrypsin promoter.
[0024] The transposase is provided to the proliferating cell in any
form that allows transient expression of the transposase in the
cell. Typically the transposase is administered to the
proliferating cell in the form of a polypeptide, or a
polynucleotide encoding the transposase. The polynucleotide may be
a gene or mRNA. In a particular embodiment, the transposase is
provided to the proliferating cell using a second rAAV vector
comprising a polynucleotide encoding the transposase, optionally
operably linked to a suitable promoter, and optionally flanked by
AAV-derived inverted terminal repeat regions. The promoter may be a
tissue-specific promoter. In an embodiment the promoter is a
liver-specific promoter. In an exemplary embodiment the
liver-specific promoter is the human alpha-1 antitrypsin
promoter.
[0025] In particular embodiments the AAV sequences used in the rAAV
vectors may be derived from AAV2 and/or AAV8. Where the vector
genome is encapsidated, any capsid capable of encapsidating an AAV
genome may be employed.
[0026] In an exemplary embodiment the transposase is the piggyBac
transposase, and the transposon-derived inverted terminal repeat
sequences are derived from the piggyBac transposon.
[0027] The proliferating cell may be a rapidly proliferating cell.
In particular embodiments, the cell is a hepatocyte from a neonatal
or juvenile liver or a bone marrow cell. The proliferating cell may
be a disease cell, such as a cancer cell.
BRIEF DESCRIPTION OF THE DRAWINGS
[0028] Embodiments of the invention are described herein, by way of
example only, with reference to the accompanying drawings.
[0029] FIG. 1. Vector constructs. A. PiggyBac transposase vector.
B. EGFP reporter vector with either the full length (TRSI, TRSII)
or short (I, black box; II, dark gray) piggyBac terminal resolution
sites (TRS). C. Transposon-donor plasmids expressing either murine
OTC, murine ASS or human ABCB4. LSP1, liver specific ApoE/hAAT
enhancer/promoter; LP1*, shortened version of LSP1; ITR, AAV2
inverted terminal repeat regions; PRE, post-transcriptional
regulatory element; pA, polyadenylation signal. Component
nucleotide sequences are defined in Table 1 and sequences are
provided in SEQ ID Nos:1 to 18 of the Sequence Listing. Vector
construct sequences are further described in Example 1 and
sequences provided in SEQ ID Nos:19 to 23 of the Sequence
Listing.
[0030] FIG. 2. Experimental design for testing transposon-donor
transgene vectors encoding EGFP with either full length or short
transposase recognition sites (TRS).
[0031] FIG. 3. A. Representative images of liver sections showing
widespread and numerous EGFP-positive hepatocytes following
concomitant delivery of rAAV-encoded transposase to the newborn
(1-2 days) mouse liver compared with EGFP transposon vector alone.
Scale bar=50 .mu.m. B. Fluorometric analysis of liver lysates
showing higher levels of EGFP expression in 4 week old mice
receiving transposon-encoded EGFP in the presence of piggyBac
transposase (n=4) than transposon alone (n=4). C. Quantitation of
vector genome (vg) copy number per diploid liver cell at 4 weeks of
age with transposon-encoded EGFP (full length TRS vector) in the
presence of piggyBac transposase. D. Relative EGPF mRNA and E.
protein expression per vector genome (vg) copy number (arbitrary
units) at 4 weeks of age with transposon-encoded EGFP (full length
TRS vector) in the presence of piggyBac transposase. F.
Fluorometric quantitation of EGFP in liver lysates with EGFP
transposon rAAV alone (short TIR) (n=3 male, n=3 female) or in
combination with the piggyBac transposase rAAV (n=3 male, n=3
female). G. Quantitation of vector genome (vg) copy number per
diploid liver cell with EGFP transposon rAAV alone (short TIR) or
in combination with the piggyBac transposase rAAV. H. Relative EGFP
protein expression per vector genome copy number (arbitrary units)
with EGFP transposon rAAV alone (short TIR) or in combination with
the piggyBac transposase rAAV. Dot plots in panels C to H show mean
values. EGFP transposon-encoding vector alone and in combination
with the piggyBac transposase-encoding vector are indicated by open
and closed dots, respectively. For statistical comparison of two
experimental groups, the two-tailed Student's unpaired t-test was
used: *P<0.05; **P<0.01; ***P<0.001.
[0032] FIG. 4. Experimental design for testing phenotype correction
following co-delivery of the OTC-encoding transposon-transgene
vector and the piggyBac transposase vector, in the spf.sup.ash
mouse model of OTC deficiency.
[0033] FIG. 5. A. Representative images of liver sections showing
widespread OTC activity (dark brown stain) in mice receiving both
the OTC-encoding transposon-transgene vector and the piggyBac
transposase vector (right panel; 66.+-.5% gene-modified cells),
compared with mice that received OTC-encoding transposon-transgene
vector alone (left panel; 1.+-.0.2% gene-modified cells). B.
Kaplan-Meier survival analysis of mice receiving the OTC-encoding
transposon-transgene vector alone or in combination with the
piggyBac transposase vector, followed by knockdown of residual
endogenous OTC activity at adulthood. C. OTC enzymatic activity in
liver lysates presented as fold-difference over wild-type (wt). D.
Quantitation of vector genome (vg) copy number per diploid liver
cell. E. Relative mOTC mRNA and F. protein expression per vector
genome copy number (arbitrary units). Dot plots in panels C to F
show mean values. OTC transposon-encoding vector alone and in
combination with the piggyBac transposase-encoding vector are
indicated by open and closed dots, respectively. For statistical
comparison of two experimental groups, the two-tailed Student's
unpaired t-test was used: *P<0.05; **P<0.01; ***P<0.001;
****P<0.0001.
[0034] FIG. 6. Experimental design for testing phenotype correction
following co-delivery of the ASS-encoding transposon-transgene
vector and the piggyBac transposase vector, in the citrullinaemic
mouse model of ASS deficiency.
[0035] FIG. 7. A. ASS-deficient pups treated with ASS-encoding
transposon-transgene vector and the piggyBac transposase vector and
wild-type litter mate at 10 days of age (left panel), and treated
ASS-deficient adult (8 week old) mouse (right panel). B.
Microscopic analysis of hair strands from a wild-type (wt) and
treated adult mutant (mut) mouse. Scale bar, 25 .mu.m. C. Plasma
urinary orotic acid in wild-type/heterozygous (open symbols) and
treated mutant adult mice (closed symbols). D. ASS enzyme activity
in liver lysate from wild-type (wt) and heterozygous (het) males
and females and treated mutant (ASS-/-) adult mice. E. Localisation
of ASS protein in liver sections by immunohistochemistry,
co-localised with glutamine synthetase (darkest stained cells) to
identify the hepatic central veins. Representative histological
images from a wild-type control liver (left) and a treated mutant
mouse liver (right). Scale bar, 50 .mu.m. Dot plots in panels C and
D show mean values. For statistical comparison of two experimental
groups, the two-tailed Student's unpaired t-test was used:
**P<0.01; ****P<0.0001.
[0036] FIG. 8. Experimental design for testing phenotype correction
following co-delivery of the hABCB4-encoding transposon-transgene
vector and the piggyBac transposase vector, in mouse model of
progressive familial intrahepatic cholestasis type 3 (PFIC3).
[0037] FIG. 9. A. Homozygous neonates (Abcb4.sup.-/-) that received
a single therapeutic injection of rAAV-piggyBac transposon (with
short TRS) encoding human ABCB4 (rAAV.pB.hABCB4), co-administered
with vector encoding piggyBac tranposase (rAAV.pBase) had stably
increased mean biliary phosphatidylcholine (PC) concentrations at
4, 8, 12 and 16 weeks of age, compared to untreated controls and
those that did not receive concomitant rAAV.pBase. B. Homozygotes
treated at birth had minimal evidence of liver pathology on
representative histology images at early (4 weeks) and late (16
weeks) analysis time-points. Portal tracts (marked with asterisks)
of treated mice are normal, in contrast with those of untreated
controls exhibiting periportal inflammation and features of biliary
obstruction. Infiltration of inflammatory cells also extends
between portal tracts of untreated controls in the haemotoxylin and
eosin (H & E) stained images (arrow) and increased fibrosis
bridges portal tracts in Sirius red stained images, most noticeably
at 16 weeks in the untreated controls (arrowhead). Scale bar=50
.mu.m.
[0038] The present specification contains nucleotide sequence
information prepared using the programme PatentIn Version 3.5,
presented herein in a Sequence Listing.
DETAILED DESCRIPTION OF THE INVENTION
[0039] Throughout this specification and the claims which follow,
unless the context requires otherwise, the word "comprise", and
variations such as "comprises" or "comprising", will be understood
to imply the inclusion of a stated element, integer or step, or
group of elements, integers or steps, but not the exclusion of any
other element, integer or step, or group of elements, integers or
steps.
[0040] The articles "a" and "an" are used herein to refer to one or
to more than one (i.e., to at least one) of the grammatical object
of the article. By way of example, "an element" means one element
or more than one element.
[0041] As used herein the term "derived" in the context of an
AAV-derived or transposon-derived nucleotide sequence means that
the sequence corresponds to, originates from, or otherwise shares
significant sequence homology with a sequence from an AAV or a
transposon. Those skilled in the art will also understand that by
being "derived" from an AAV or transposon, the sequence need not be
physically constructed or generated from a particular AAV or
transposon, but may be chemically synthesised or generated by other
molecular biology techniques known in the art.
[0042] As used herein, the term "transgene" refers to exogenous DNA
or cDNA encoding a gene product. The gene product may be an RNA,
peptide or protein. In addition to the coding region for the gene
product, the transgene may include or be associated with one or
more elements to facilitate or enhance expression, such as a
promoter, enhancer(s), response element(s), repoerter element(s),
insulator element(s), polyadenlyation signal(s) and/or other
functional elements. Embodiments of the invention may utilize any
known suitable promoter, enhancer(s), response element(s),
repoerter element(s), insulator element(s), polyadenlyation
signal(s) and/or other functional elements.
[0043] Suitable elements and sequences will be well known to those
skilled in the art. The transgene integrates into the genome of a
proliferating cell. The transgene may be foreign to the cell or may
represent a gene the expression of which is otherwise absent or
reduced in the proliferating cell in the absence of the
introduction of the transgene.
[0044] It will be understood that as used herein the term
"expression" may refer to expression of a polypeptide or protein,
or to expression of a polynucleotide or gene, depending on the
context. Expression of a polynucleotide may be determined, for
example, by measuring the production of RNA transcript levels using
methods well known to those skilled in the art. Expression of a
protein or polypeptide may be determined, for example, by
immunoassay using an antibody(ies) that bind with the polypeptide
using methods well known to those skilled in the art.
[0045] In the context of this specification, the term "activity" as
it pertains to a protein, polypeptide or polynucleotide means any
cellular function, action, effect or influence exerted by the
protein, polypeptide or polynucleotide, either by a nucleic acid
sequence or fragment thereof, or by the protein or polypeptide
itself or any fragment thereof.
[0046] As used herein the term "effective amount" includes within
its meaning a non-toxic but sufficient amount of an agent or
compound to provide the desired therapeutic effect. The exact
amount required will vary from subject to subject depending on
factors such as the species being treated, the age and general
condition of the subject, the severity of the condition being
treated, the particular agent being administered and the mode of
administration and so forth. Thus, it is not possible to specify an
exact "effective amount". However, for any given case, an
appropriate "effective amount" may be determined by one of ordinary
skill in the art using only routine experimentation.
[0047] As used herein the terms "treating", "treatment",
"preventing" and "prevention" and variations thereof refer to any
and all uses that remedy a disease or one or more symptoms thereof,
prevent the establishment of the disease, or otherwise prevent,
hinder, retard, or reverse the progression of the disease or other
undesirable symptoms in any way whatsoever. Thus the terms
"treating" and "preventing" and the like are to be considered in
their broadest context. For example, treatment does not necessarily
imply that a patient is treated until total recovery. In conditions
which display or a characterized by multiple symptoms, the
treatment or prevention need not necessarily remedy, prevent,
hinder, retard, or reverse all of said symptoms, but may prevent,
hinder, retard, or reverse one or more of said symptoms.
[0048] As used herein the term "associated with" when used in the
context of a disease "associated with" a proliferating cell means
that the disease may result from, result in, be characterised by,
or otherwise associated with cellular proliferation. The
association between the disease and cellular proliferation may be
direct or indirect and may be temporally separated.
[0049] The term "subject" as used herein refers to mammals and
includes humans, primates, livestock animals (eg. sheep, pigs,
cattle, horses, donkeys), laboratory test animals (eg. mice,
rabbits, rats, guinea pigs), companion animals (eg. dogs, cats) and
captive wild animals (eg. foxes, kangaroos, deer). Typically the
mammal is human or a laboratory test animal. Even more typically,
the mammal is a human.
[0050] As described and exemplified herein the inventors have
generated recombinant AAV vectors and vector systems capable of
inducing stable integration and high levels of expression of
transgenes in proliferating cells. The inventors have also
demonstrated the ability of these vectors and vector systems to
correct phenotypes in mouse models of three different diseases of
the paediatric liver.
[0051] Accordingly, provided herein are methods and vector systems
for inducing stable transgene expression in a proliferating cell,
the method comprising administering to the cell: (i) a recombinant
AAV (rAAV) vector comprising the transgene flanked by
transposon-derived inverted terminal repeat sequences, which
sequences are in turn flanked by AAV-derived inverted terminal
repeat regions; and (ii) a source of a transposase that recognises
said transposon-derived inverted terminal repeat sequences and
directs the genomic integration of the transgene into the genome of
the proliferating cell.
[0052] Also provided herein are methods and vector systems for
stably integrating a transgene into the genome of a proliferating
cell, the method comprising administering to the cell: (i) a
recombinant AAV (rAAV) vector comprising the transgene flanked by
transposon-derived inverted terminal repeat sequences, which
sequences are in turn flanked by AAV-derived inverted terminal
repeat regions; and (ii) a source of a transposase that recognises
said transposon-derived inverted terminal repeat sequences and
directs the genomic integration of the transgene into the genome of
the proliferating cell.
[0053] Also provided herein are methods and vector systems for
treating or preventing a disease of, affecting, or associated with,
a proliferating cell, comprising administering to a subject in need
thereof (i) a recombinant AAV (rAAV) vector comprising a transgene
flanked by transposon-derived inverted terminal repeat sequences,
which sequences are in turn flanked by AAV-derived inverted
terminal repeat regions; and (ii) a source of a transposase that
recognises said transposon-derived inverted terminal repeat
sequences and directs the genomic integration of the transgene into
the genome of the proliferating cell, wherein said administration
results in the stable integration and expression of the transgene
to thereby treat the disease.
[0054] In particular embodiments of the present invention, the
proliferating cell may be a liver cell, in particular a cell of a
neonatal or juvenile liver, a bone marrow cell and/or a cancer
cell. However those skilled in the art will appreciate that the
present invention is applicable to any proliferating cell, and thus
to any disease of, affecting, or associated with such proliferating
cells. Non-limiting examples of paediatric liver diseases that may
be treated or prevented in accordance with embodiments of the
invention include OTC deficiency, ASS deficiency, progressive
familial intrahepatic cholestasis, and genetic or metabolic liver
diseases.
[0055] The vector systems of the invention may be used to integrate
and express any transgene in the genome of a proliferating cell,
and the scope of the present disclosure is not to be limited by
reference to any particular transgene exemplified herein. The
transgene may be any gene the expression of which it is desirable
to induce in the proliferating cell. The transgene may be foreign
to the proliferating cell. The transgene may be a gene the
expression of which is absent or reduced in the proliferating cell
in the absence of introduction of the transgene.
[0056] Accordingly, methods and vector systems of the invention
find application in any circumstance or scenario in which it is
desirable to stably integrate and express a transgene in a
proliferating cell. Thus applications of the invention as a
research tool in the investigation of gene expression, the
development of, for example, cell lines and animal models, and
alleviating the need to generate transgenic animals, are
contemplated, in addition to the application of the invention to
the treatment and prevention of diseases of, affecting, or
associated with proliferating cells such as those of the neonatal
or juvenile liver or bone marrow.
[0057] The transposase may be delivered to a proliferating cell in
the form of a polypeptide, or a polynucleotide encoding the
transposase. In a particular embodiment, the transposase is
delivered using a vector comprising a polynucleotide encoding the
transposase, optionally flanked by AAV-derived inverted terminal
repeat regions. However those skilled in the art will recognise
that the transposase may be delivered in any suitable form and by
any suitable means so as to allow for expression of the transposase
in the cell for a sufficient period of time and in sufficient
amount to integrate the transgene or transgene cassette from the
first rAAV vector into the genome of the proliferating cell. It
will therefore be appreciated that transient expression of the
transposase is required to prevent remobilization (excision) of
integrated elements. Accordingly, the transposase may be delivered
to the cell as mRNA encoding the transposase enzyme, or means of
eliminating transposase enzyme from the cell may be employed,
although any means of achieving transient transposase expression is
contemplated.
[0058] Optionally polynucleotides administered to proliferating
cells, encoding the transposase and/or the transgene, may be
operably linked to a promoter. Thus, typically the vectors of the
present invention are expression vectors capable of directing the
transcription of the DNA sequence of the polynucleotide contained
in the vector. The vector may include other expression control and
processing sequences such as, for example, enhancers, internal
ribosome entry sites, 2A elements, translation initiation (e.g.
Kozak) sequences, polyadenylation signals and transcription
termination sequences.
[0059] The promoter operably linked to the transposase and/or the
transgene may be a "ubiquitous" promoter active in essentially all
cells of the host organism (e.g. the beta-actin or cytomegalovirus
promoters) or may be a promoter with expression more or less
specific to the target cells (albumin promoter). Thus, the promoter
may be a tissue-specific promoter that is only (or predominantly)
active in cells of the desired tissue or organ. Thus, in particular
embodiments the promoter may be one that is active primarily in the
hepatic system. The specificity may be absolute or relative.
Similarly, the promoter may be specific for particular cell types,
including for example hepatocytes, Kupffer cells or endothelial
cells. Those of ordinary skill in the art will appreciate that a
tissue-specific promoter for use in an AAV vector in accordance
with the invention may be selected from any of the known
tissue-specific promoters. In an exemplary embodiment a suitable
liver-specific promoter is the human alpha-1 antitrypsin
promoter.
[0060] AAVs of any known serotype may be used in deriving the rAAV
vectors of the invention, such as AAV1, AAV2, AAV3, AAV4, AAV5,
AAV6, AAV7, AAV8, AAV9, AAV10, AAV11 and AAV12, and subtypes
thereof. Exemplary AAVs include AAV2 and AAV8. In exemplary
embodiments described herein the AAV inverted terminal repeat
sequences are derived from AAV2, and comprise the nucleotide
sequences set forth in SEQ ID NO:1 (ITR1) and/or SEQ ID NO:2
(ITR2), or functionally equivalent fragments thereof. The selection
of the most appropriate AAV may be based on various factors
including for example the target cell or cell type, and the
identity or size of the transgene. The skilled addressee can make
the selection without undue experimentation.
[0061] Those skilled in the art will also appreciate that while
exemplified in relation to the piggyBac transposon, the methods and
systems of the present invention may employ sequences derived from
any suitable transposon. By way of example the transposon may be a
member of the piggyBac superfamily or the Tc1/mariner superfamily
(including for example the Sleeping Beauty transposon). The most
appropriate transposon to be employed may be selected depending on
the application of the invention, the target cell or cell type, the
identity of the transgene and the disease to be treated or
prevented. The skilled addressee can make the selection without
undue experimentation. In exemplary embodiments described herein
the transposon-transgene vectors comprise terminal repeat sequences
derived from the piggyBac transposon, which sequences comprise the
nucleotide sequences set forth SEQ ID NO:3, SEQ ID NO:4, SEQ ID
NO:5 and/or SEQ ID NO:6, or functionally equivalent fragments
thereof. Similarly, the transposase may be the piggyBac transposase
encoded by the nucleotide sequence set forth in SEQ ID NO:7, or a
variant or derivative thereof.
[0062] The various polynucleotide and polypeptide sequences used in
the methods and vectors of the invention, including inter alia
AAV-derived inverted terminal repeat regions, transposon-derived
inverted terminal repeats, transposase sequences and transgene
sequences may be natural, recombinant or synthetic and may be
obtained by purification from a suitable source or produced by
standard recombinant DNA techniques such as those well known to
persons skilled in the art, and described in, for example, Sambrook
et al., Molecular Cloning: a Laboratory Manual, Cold Spring Harbor
Laboratory Press (the disclosure of which is incorporated herein by
reference).
[0063] In embodiments in which a first rAAV vector comprising the
transgene and a second vector comprising a polynucleotide encoding
the transposase are co-administered to the proliferating cell, the
ratio of the first vector to the second vector may be between about
50:1 to about 1:50, between about 40:1 to 1:40, between about 30:1
to 1:30, between about 20:1 to 1:20, between about 10:1 to 1:10 or
between about 5:1 to 1:5. In an exemplary embodiment the ratio of
the first vector to the second vector is about 10:1.
[0064] The present invention contemplates the delivery of vectors
and other molecules to proliferating cells by any suitable means.
For administration to subjects requiring treatment, vectors and
other molecules are typically administered in the form of
pharmaceutical compositions, which compositions may comprise one or
more pharmaceutically acceptable carriers, excipients or diluents.
Such compositions may be administered in any convenient or suitable
route such as by parenteral (e.g. subcutaneous, intraarterial,
intravenous, intramuscular), oral (including sublingual), nasal or
topical routes. In circumstances where it is required that
appropriate concentrations of the vectors and molecules are
delivered directly to the site in the body to be treated,
administration may be regional rather than systemic. Regional
administration provides the capability of delivering very high
local concentrations of the vectors and molecules to the required
site and thus is suitable for achieving the desired therapeutic or
preventative effect whilst avoiding exposure of other organs of the
body to the vectors and molecules and thereby potentially reducing
side effects.
[0065] It will be understood that the specific dose level of a
composition of the invention for any particular subject will depend
upon a variety of factors including, for example, the activity of
the specific agents employed, the age, body weight, general health
and diet of the individual to be treated, the time of
administration, rate of excretion, and combination with any other
treatment or therapy. Single or multiple administrations can be
carried out with dose levels and pattern being selected by the
treating physician. A broad range of doses may be applicable.
Considering a patient, for example, from about 0.1 mg to about 1 mg
of agent may be administered per kilogram of body weight per day.
Dosage regimens may be adjusted to provide the optimum therapeutic
response. For example, several divided doses may be administered
daily, weekly, monthly or other suitable time intervals or the dose
may be proportionally reduced as indicated by the exigencies of the
situation.
[0066] Examples of pharmaceutically acceptable carriers or diluents
are demineralised or distilled water; saline solution; vegetable
based oils such as peanut oil, safflower oil, olive oil, cottonseed
oil, maize oil, sesame oil, arachis oil or coconut oil; silicone
oils, including polysiloxanes, such as methyl polysiloxane, phenyl
polysiloxane and methylphenyl polysolpoxane; volatile silicones;
mineral oils such as liquid paraffin, soft paraffin or squalane;
cellulose derivatives such as methyl cellulose, ethyl cellulose,
carboxymethylcellulose, sodium carboxymethylcellulose or
hydroxypropylmethylcellulose; lower alkanols, for example ethanol
or iso-propanol; lower aralkanols; lower polyalkylene glycols or
lower alkylene glycols, for example polyethylene glycol,
polypropylene glycol, ethylene glycol, propylene glycol,
1,3-butylene glycol or glycerin; fatty acid esters such as
isopropyl palmitate, isopropyl myristate or ethyl oleate;
polyvinylpyrridone; agar; carrageenan; gum tragacanth or gum
acacia, and petroleum jelly. Typically, the carrier or carriers
will form from 10% to 99.9% by weight of the compositions.
[0067] The present invention contemplates combination therapies,
wherein vectors and molecules as described herein are
coadministered with other suitable agents that may facilitate the
desired therapeutic or prophylactic outcome. By "coadministered" is
meant simultaneous administration in the same formulation or in two
different formulations via the same or different routes or
sequential administration by the same or different routes. By
"sequential" administration is meant a time difference of from
seconds, minutes, hours or days between the administration of the
agents. Administration may be in any order.
[0068] The reference in this specification to any prior publication
(or information derived from it), or to any matter which is known,
is not, and should not be taken as an acknowledgment or admission
or any form of suggestion that that prior publication (or
information derived from it) or known matter forms part of the
common general knowledge in the field of endeavor to which this
specification relates.
[0069] The present invention will now be described with reference
to the following specific examples, which should not be construed
as in any way limiting the scope of the invention.
Examples
Example 1--AAV/Transposase Vector Constructs
[0070] Transposon-donor vectors and a piggyBac Transposase vector
were constructed using the recombinant adeno-associated viral
vector (rAAV) system. The hybrid AAV/transposase system was
subsequently used (see Examples 2 to 4) to demonstrate phenotype
correction in animal models with genetic metabolic disease
phenotypes. These included the spf.sup.ash mouse model of ornithine
transcarbamylase (OTC) deficiency and the citrullinaemic mouse
model of argininosuccinate synthetase (ASS) deficiency (both urea
cycle disorders), and the PFIC3 mouse model (ABCB4 deficiency) of
progressive familial intrahepatic cholestasis. Each of these
disease phenotypes presents early in life, in neonates or
juveniles.
[0071] The coding sequence of piggyBac transposase was amplified by
PCR from pCAG-PBase. The piggyBac transposase vector was
constructed by inserting the coding region of the piggyBac
transposase into a rAAV2 genome under the transcriptional control
of a liver-specific promoter (pAAV2-LSP1.EGFP), replacing the EGFP
coding sequence to produce pAAV2-LSP1.PBase (FIG. 1A). To construct
the transposon-donor vectors, the 5' (313 bp) and 3' (230 bp)
piggyBac transposon terminal inverted repeats (TIRs) were amplified
by PCR from pPB-CA-GFP (Wolten et al., 2011, Methods Mol. Biol.
767:87-103) and inserted into pAAV2-LSP1.EGFP, immediately internal
to the AAV2 inverted terminal repeats (ITRs) and flanking the
transgene cassette. The resulting vector construct was designated
pAAV2-LSP1.EGFP-TIR (FIG. 1B).
[0072] The piggyBac terminal repeat sequences used were either full
length (TRS I, 313 bp, SEQ ID NO:3; and TRS II, 230 bp, SEQ ID
NO:4) or shortened forms (TRS I, 67 bp, SEQ ID NO:5; and TRS II, 40
bp, SEQ ID NO:6). The genes expressed by the transposon-donor
transgene cassettes included enhanced green fluorescent protein
(EGFP) (FIG. 1B), murine ornithine transcarbamylase (OTC), murine
argininosuccinate synthetase (ASS) and human ATP-binding cassette
subfamily B member 4 (ABCB4) (FIG. 1C). To produce the therapeutic
transposon vector constructs pAAV2-LSP1.mOTC-TIR (Example 3) and
pAAV2-LSP1.mASS-TIR (Example 4), the EGFP cDNA in
pAAV2-LSP1.EGFP-TIR was replaced with the cDNA for either murine
ornithine transcarbamylase (mOTC) (NM 008769.3) or
argininosuccinate synthetase (mASS) (NM 007494).
[0073] The various component sequences of the vectors constructed
are identified below in Table 1 and the nucleotide sequences given
in the Sequence Listing appearing at the end of the
specification.
TABLE-US-00001 TABLE 1 Nucleotide sequences used in vector
construction Name Description SEQ ID NO: AAV2 ITR1 AAV2-derived
inverted terminal repeat 1. 181 bp 1 AAV2 ITR2 AAV2-derived
inverted terminal repeat 2. 181 bp 2 TRS I PiggyBac transposon
terminal repeat sequence I. 3 313 bp TRS II PiggyBac transposon
terminal repeat sequence II. 4 230 bp TRS I (short) PiggyBac
transposon terminal repeat sequence I- 5 shortened (Meir et al.,
2011, BMC Biotechnol 11:28). 67 bp TRS II (short) PiggyBac
transposon terminal repeat sequence II- 6 shortened (Meir et al.,
2011, BMC Biotechnol 11:28). 40 bp PiggyBac PBase Coding region of
PiggyBac transposase. 1785 bp 7 EGFP Coding region of enhanced GFP.
720 bp 8 mOTC Coding region of murine OTC gene. 1066 bp 9 mASS
Coding region of murine ASS gene. 1239 bp 10 hABCB4 var A Coding
region of codon optimised human ABCB4 11 transcript variant A. 3849
bp hApoE enhancer Human apolipoprotein E enhancer. 327 bp 12
hApoE-HCR Human apolipoprotein E hepatic control region 13 enhancer
(derived from apolipoprotein E enhancer above). 192 bp hAAT
promoter Human alpha-1 antitrypsin promoter. 397 bp 14 hAAT*
promoter Truncated human alpha-1 antitrypsin promoter. 254 15 bp
PRE Woodchuck post-regulatory element. 589 bp 16 bGH polyA Bovine
growth hormone polyadenylation signal. 17 276 bp SV40 polyA Simian
virus 40 polyadenylation signal. 134 bp 18 Kozak Kozak sequence. 7
bp (CGCCACC) --
[0074] The vector constructs used in the present study comprised
the above sequences constructed as follows:
[0075] piggyBac Transposase Vector (SEQ ID NO:19)
[0076] AAV2 ITR1
[0077] hApoE enhancer (two copies)
[0078] hAAT promoter
[0079] Kozak
[0080] piggyBac PBase
[0081] PRE
[0082] bGH polyA
[0083] AAV2 ITR2
[0084] EGFP vector (SEQ ID NO:20)
[0085] AAV2 ITR1
[0086] TRS I
[0087] hApoE enhancer (two copies)
[0088] hAAT promoter
[0089] Kozak
[0090] EGFP
[0091] PRE
[0092] bGH polyA
[0093] TRS II
[0094] AAV2 ITR2
[0095] (A smaller version of this vector was also constructed
using: the TRS I (short) and TRS II (short) sequences in place of
TRS I and TRS II; a single copy of hApoE-HCR in place of two copies
of hApoE enhancer; the hAAT* promoter in place of hAAT; and SV40
polyA in place of bGH polyA)
[0096] mOTC Vector (SEQ ID NO:21)
[0097] AAV2 ITR1
[0098] TRS I
[0099] hApoE enhancer (two copies)
[0100] hAAT promoter
[0101] Kozak
[0102] mOTC
[0103] PRE
[0104] bGH polyA
[0105] TRS II
[0106] AAV2 ITR2
[0107] mASS Vector (SEQ ID NO:22)
[0108] AAV2 ITR1
[0109] TRS I
[0110] hApoE enhancer (two copies)
[0111] hAAT promoter
[0112] Kozak
[0113] mASS
[0114] PRE
[0115] bGH polyA
[0116] TRS II
[0117] AAV2 ITR2
[0118] hABCB4 Vector (SEQ ID NO:23)
[0119] AAV2 ITR1
[0120] TRS I (short)
[0121] hApoE-HCR
[0122] hAAT* promoter
[0123] Kozak
[0124] hABCB4 varA (codon optimised)
[0125] PRE
[0126] SV40 polyA
[0127] TRS II (short)
[0128] AAV2 ITR2
[0129] Vector constructs were pseudoserotyped with liver-tropic
vector capsids (AAV8 or AAVrh10), and viral particles were produced
in human embryonic kidney (HEK) 293 cells by standard techniques.
HEK293 cells were cultured in Dulbecco's modified Eagle medium
(Gibco, Invitrogen, Grand Island, N.Y., USA) supplemented with 10%
(v/v) fetal bovine serum (JRH Biosciences, Lenexa, Kans., USA) and
1% (w/v) L-glutamine (Gibco, Invitrogen) and maintained at
37.degree. C. in a humidified 5% CO.sub.2-air atmosphere. Vector
genome titres were assigned by real-time quantitative PCR targeting
the WPRE sequence as previously described (Cunningham et al., 2011,
Mol. Ther. 19:854-859) or the EGFP transgene using the protocol as
described for determining vector copy number (see below).
[0130] DNA was extracted from liver using standard
phenol/chloroform and ethanol precipitation methods (Sambrook et
al., 1989, Molecular cloning: a laboratory manual. Cold Spring
Harbor Laboratory Press) Vector copy number of the transposon rAAVs
was determined using the Quantitect Sybr Green Kit (Qiagen,
Valencia, Calif., USA) and the following oligonucleotide sets
targeting the relevant transgene: EGFP-F/EGFP-R, OTC-F/OTC-R and
ASS-F/ASS-R. Vector copy number of the piggyBac transposase rAAV
was determined using the Takara Sybr Premix Ex Taq Kit (Cat#RR420A)
and oligonucleotides PB-F/PB-R. The PCR reactions were normalized
using the Quantitect Sybr Green Kit (Qiagen, Valencia, Calif., USA)
and oligonucleotide set GAPDH-F/GAPDH-R. Each reaction contained
100-150 ng of genomic DNA. Standards were prepared from linearized
plasmid of the relevant vector, diluted in a background of 100-150
ng human genomic liver DNA per reaction (see Snyder et al., 1996,
in Dracopoli et al. (eds.) Protocols in Human Genetics. John Wiley
& Sons, Inc: Chichester, UK pp 12.0.1-12.1.24).
Example 2--Stable Expression of Hybrid AAV/Transposase Constructs
in Mice
[0131] The ability of the AAV/transposase vector systems described
in Example 1 to stably integrate and express a transgene in a host
genome was determined using transposon-donor vectors encoding
enhanced green fluorescent protein (EGFP) administered to C3H and
FVB.129P2-Abcb4.sup.tm1Bor mice. Animals were housed in a
temperature-controlled environment with 12-hour light/dark cycles
with water and standard rodent chow (18.9% (wt/wt) protein;
Specialty Feeds, Glen Forrest, Australia) supplied ad libitum. All
experimental procedures were evaluated and approved by the
institutional Animal Care and Ethics Committee. The experimental
design is outlined in FIG. 2. Four mice were used for each group.
Constructs were administered by injection via the intraperitoneal
route in 20 .mu.L volumes (diluted in PBS with calcium and
magnesium) in newborn mice, at vector doses of 5.times.10.sup.10
vg/mouse for the transposase vector, and
1.times.10.sup.11-5.times.10.sup.11 vg/mouse for the
transposon-transgene donor vector. Transposon-donor vectors
encoding EGFP with either full length or short transposase
recognition sites were co-administered with vector-encoded piggyBac
transposase at days 0-1. Mice were sacrificed at week 3, 4 or 8,
liver taken and EGFP expression observed via quantitative RT-PCR,
immunofluorescence and fluorometry.
[0132] To measure mRNA expression, total RNA was extracted from
30-50 mg liver tissue using Trizol reagent (Life Technologies)
according to the manufacturer's protocol and treated with DNAse I
(Life Technologies). Reverse transcription using the SuperScript
III First-Strand Synthesis SuperMix (Life Technologies) was used to
generate cDNA. Quantitative RT-PCR was performed using the
Quantitect Sybr Green Kit (Qiagen, Valencia, Calif., USA) and the
relevant oligonucleotide sets.
[0133] For detection of EGFP protein, livers were removed and fixed
in 4% (wt/v) paraformaldehyde (PFA) in phosphate-buffered saline
(PBS), cryoprotected in 10-30% (wt/v) sucrose, and frozen in
Optimum Cutting Temperature (OCT; TissueTek, Sakura Finetek USA,
Torrance, Calif.) in isopentane/liquid Nitrogen for storage at
-80.degree. C. For co-localization of gene-modified cells with
specific regions of liver architecture, the central veins were
identified by immunohistochemical staining of glutamine synthetase.
Frozen sections (5 .mu.m) were permeabilized in methanol at
-20.degree. C. for 10 min, then reacted with a rabbit polyclonal
anti-glutamine synthetase primary antibody (1/150 dilution; Abcam).
Bound primary antibody was detected with an Alexa Fluor 594 donkey
anti-rabbit secondary (1/1,000 dilution; Invitrogen, Carlsbad,
Calif.). Sections were mounted in Immu-Mount (Thermo-Shandon) and
imaged using an Olympus BX51 fluorescent microscope (Olympus,
Centre Vally, Pa.). Direct EGFP fluorescence was detected using
filters D480/30.times. (excitation) and D535/40m (emission), and
Alexa Fluor 594 was detected using filters HQ560/55 (excitation)
and HQ645/75 (emission). The percentage of transduced cells was
determined by counting EGFP-positive cells in three random fields
of view (10.times. magnification).
[0134] FIG. 3A shows widespread and numerous EGFP-positive
hepatocytes following concomitant delivery of rAAV-encoded piggyBac
transposase and transposon-transgene donor vectors compared with
EGFP transposon vector alone. There was a >20-fold increase in
the number of stably gene-modified hepatocytes from 2.4% to 50%,
when the transposase expression vector was co-transfected with the
EGFP transposon-containing vector (FIG. 3A).
[0135] Fluorometric analysis of liver lysates comprised
homogenization of 50 mg liver in 750 uL Lysis Buffer: 0.5% Triton
X-100, 0.01M Hepes, protease inhibitors; incubated 1 hr on ice then
centrifuged at 14000 rpm for 20 min, 4.degree. C. The total protein
content of the lysate was measured (DC Protein assay; Bio-Rad,
Hercules, Calif.) and individual samples adjusted to a final
concentration of 0.5 mg/mL. A standard curve was prepared using
recombinant EGFP protein (BioVision Research Products, Mountain
View, Calif.). Equal volumes of samples and standards were loaded
into black plastic 96-well plates (PerkinElmer, Boston, Mass.) and
EGFP fluorescence quantitated on a VICTOR3 multilabel reader
(PerkinElmer) using an excitation and emission filter set at 485
nm/535 nm. Fluorometry confirmed the observed higher levels of EGFP
expression in EGFP transposon+transposase livers (FIG. 3B).
[0136] Interestingly, the increase in the number of stably
gene-modified hepatocytes correlated with a 43-fold higher level of
EGFP protein expression in liver lysates (FIG. 3B), and a
relatively lower, but nevertheless impressive, 7-fold increase in
vector genome copy number per diploid cell (FIG. 3C). This
indicates significantly higher levels of EGFP mRNA (FIG. 3D) and
protein (FIG. 3E) expression from transposed expression cassettes
than from those stably maintained (presumably through integration)
in the host cell as part of an AAV provirus.
[0137] The system was also tested using minimal piggyBac TIRs to
increase the cargo capacity of the vector-encoded transposable
element (FIG. 1B). Quantitatively and qualitatively equivalent data
were obtained (FIG. 3A bottom panels and FIGS. 3F-H). The hybrid
system performed similarly in male and female mice, delivering an
impressive 70-fold and 103-fold increase in stable EGFP expression
at 3 and 8 weeks of age, respectively, over the transposon vector
delivered alone. The peri-venous and peri-portal zones of the
hepatic lobule, which exhibit metabolic zonation, were targeted
with equivalent efficiency.
Example 3--Gene Therapy in a Mouse Model of OTC Deficiency
[0138] The hybrid AAV/transposase constructs described in Example 1
were used to demonstrate phenotype correction in a mouse model of
OTC deficiency (the spf.sup.ash mouse model). Mice used were strain
B6EiC3Sn a/A-Otc.sup.spf-ash/J (provided by The Jackson
Laboratory). The disease phenotype presents early in life in
neonates or juveniles. As such, vector treatment was delivered to
mice during the neonatal period. Constructs were administered by
injection via the intraperitoneal route in 20 .mu.L volumes
(diluted in PBS with calcium and magnesium) in newborn mice (1-2
days), at vector doses of 5.times.10.sup.10 vg/mouse for the
transposase vector, and 1.times.10.sup.11 vg/mouse for the
transposon-transgene donor vector. The experimental design is
outlined in FIG. 4 with 12 mice receiving OTC-transposon-encoding
AAV2/8 vector alone, and 12 mice receiving OTC transposon-encoding
vector in combination with the piggyBac transposase-encoding AAV2/8
vector.
[0139] Liver sections from mice sacrificed at week 12 were analysed
for OTC activity according to the method described in Ye et al.,
1996, J Biol Chem 271:3639-3646. Liver sections showed widespread
OTC activity in mice that had been administered both the
OTC-encoding transposon-transgene vector and the piggyBac
transposase vector (FIG. 5A, right panel), compared with mice that
received OTC-encoding transposon-transgene vector alone (FIG. 5A,
left panel). Mice treated in the newborn period with the rAAV OTC
transposon alone (n=6 mice) developed severe hyperammonaemia,
necessitating euthanasia, when background endogenous OTC activity
was knocked down in early adulthood, while mice treated with both
the OTC-expressing transposon and piggyBac transposase-encoding
vectors (n=6 mice) survived, as did all cohorts injected with a
rAAV expressing a non-specific (NS) shRNA (data not shown).
Kaplan-Meier survival analysis using Prism (GraphPad software) was
conducted of mice receiving the OTC-encoding transposon-transgene
vector alone or in combination with piggyBac transposase vector,
followed by knockdown of residual endogenous OTC activity at
adulthood (FIG. 5B). Survival curves show, for each plotted time on
the X axis, the portion of all individuals surviving as of that
time. Survival correlated with the approximately 66-fold increase
in the proportion of visibly OTC-positive hepatocytes in liver
sections (FIG. 5A) and a 126-fold increase in OTC activity to
massively supra-physiological levels (FIG. 5C). Consistent with
data generated using the EGFP-encoding constructs, the increase in
stably maintained vector genome copies was less dramatic (FIG. 5D),
again indicating higher levels of transgene expression from
transposed expression cassettes (FIGS. 5E and 5F).
[0140] For ammonia analysis, blood was collected by cardiac
puncture into lithium heparin coated tubes and immediately
centrifuged at 7,500.times.g for 5 minutes at 4.degree. C. The
plasma was frozen in liquid Nitrogen, and stored at -80.degree. C.
Ammonia was measured using the Ammonia Assay Kit (Sigma-Aldrich, St
Louis, Mo.). All mice that received both the OTC-encoding
transposon-transgene vector and the piggyBac transposase vector
survived to the experimental endpoint (28 days), with plasma
ammonia in the normal range (79.7.+-.10.1 .mu.M) while all mice
receiving OTC-encoding transposon-transgene vector alone became
unwell with elevated blood ammonia (954.3.+-.173.3 .mu.M) within 21
days of knockdown.
Example 4--Gene Therapy in Mouse Model of ASS Deficiency
[0141] The hybrid AAV/transposase constructs described in Example 1
were used to demonstrate phenotype correction in a neonatal lethal
knock-out mouse model of ASS deficiency (the citrullinaemic mouse
model). Mice used were strain B6; 12957-Ass1.sup.tm1Bay/J (provided
by The Jackson Laboratory). The disease phenotype presents early in
life in neonates or juveniles. As such, vector treatment was
delivered to mice during the perinatal period. Pregnant females
were anesthetised using isoflurane inhalation anaesthesia.
Buprenorphine (0.01 mg/kg) (Reckitt Benckiser, West Ryde,
Australia) was given by subcutaneous injection as an analgesic. A
laparotomy was performed to expose the uterus and constructs were
administered to each foetus by injection via the intraperitoneal
route in 5 .mu.L volumes (diluted in PBS with calcium and
magnesium) at embryonic day 15, at vector doses of
5.times.10.sup.10 vg/mouse for the transposase vector, and
1.times.10.sup.11 vg/mouse for the transposon-transgene donor
vector. The abdominal incision was then closed with sutures. A
subcutaneous injection of ampicillin was given and mice were
maintained on carprofen (0.14 mL/250 mL) given orally in drinking
water for 7 days. Pups were born around E20, designated Day 0. From
birth, pups were given a daily intra-peritoneal injection of
L-arginine (1 g/kg) until adulthood, after which injections were
reduced to 3 times weekly. Mice were monitored and sacrificed from
3 to 6 months of age. The experimental design is outlined in FIG.
6.
[0142] To determine the ability of the ASS-encoding
transposon-transgene vector to correct the deficient phenotype in
the mice, ammonia and orotic acid levels were analysed. For ammonia
analysis, blood was collected into lithium heparin coated tubes by
cardiac puncture and immediately centrifuged at 7,500.times.g for 5
minutes at 4.degree. C. The plasma was frozen in liquid Nitrogen
and stored at -80.degree. C. Ammonia was measured using the Ammonia
Assay Kit (Sigma-Aldrich, St Louis, Mo.). For orotic acid analysis,
urine was collected over a 24 hour period on Whatman filter paper,
eluted, and analyzed for orotic acid levels using Liquid
Chromatography/Tandem Mass Spectrometry. Results were standardized
against creatinine levels measured by the modified Jaffe
reaction.
[0143] Phenotype correction in the ASS-deficient citrullinaemic
mouse model was successful, with treated mice surviving to
adulthood. Following a single prenatal dose, mutant mice not only
survived the neonatal period, but remained healthy into adulthood,
with one cohort maintained to 6 months of age. Adult treated mutant
males and females were fertile and females carried pups to
full-term. A sparse fur, abnormal skin and hair phenotype was
evident from birth reminiscent of that observed in OTC-deficient
Spf.sup.ash mice, which improved from weaning age (FIGS. 7A and
7B). Orotic acid analysis showed that while normalisation of
urinary orotic acid was variable among treated mice (n=15) (FIG.
7C), treated mice were protected against hyperammonaemia (Table
2).
[0144] ASS activity in the liver was determined using a method
based on the conversion of .sup.14C-aspartate to
.sup.14C-argininosuccinate (Kok et al., 2013, Mol. Ther.
21:1823-1831) All mice exhibited robust control of hyperammonaemia
despite inter-mouse variability in AAS activity (FIG. 7D). Blood
plasma from treated mice was analysed for the amino acids arginine,
citrulline and ornithine. Quantitation plasma was deproteinized by
ultrafiltration before addition of an internal standard, then
analyzed by ion-exchange chromatography with postcolumn ninhydrin
detection on a Biochrom 30 amino acid analyzer (Biochrom,
Cambridge, UK). Table 2 shows that citrulline, arginine and
ornithine were not normalised compared to wildtype controls,
despite ammonia being maintained in the normal range.
TABLE-US-00002 TABLE 2 Biochemical analyses of plasma from treated
ASS-deficient mice. Treated mice WT control mice Citrulline
(.mu.mol/L) 721 .+-. 103 78 .+-. 7.2 Arginine (.mu.mol/L) 88 .+-.
19.6 138 .+-. 17.7 Ammonia (.mu.mol/L) 71 .+-. 11.1 48 .+-. 6.7
Ornithine (.mu.mol/L) 43 .+-. 4.29 66 .+-. 8.1
[0145] ASS protein expression and localization in treated mice was
investigated by immunohistochemistry. Formalin-fixed liver sections
were dewaxed and rehydrated in xylene and an ethanol gradient.
Antigen retrieval was performed using 10 mmol/1 sodium citrate
buffer pH 7.4, followed by blocking with 0.3% (v/v) hydrogen
peroxidize for 30 minutes. Endogenous avidin and biotin were
blocked using the avidin-biotin kit (Vector Laboratories,
Burlingame, Calif.), following the manufacturer's instructions.
Sections were then blocked with 10% (v/v) donkey serum and stained
with a goat antibody against mouse ASS (2.5 .mu.g/ml, ab77590;
Abcam, Cambridge, UK) overnight at 4.degree. C. After washing in
phosphate-buffered saline with 0.05% Tween-20, samples were
incubated with a biotinylated donkey anti-goat secondary antibody
(1.2 .mu.g/ml, 705-065-147; Jackson ImmunoResearch, West Grove,
Pa.) and detected with horseradish peroxidase, using the Vectastain
Elite ABC (PK-7100, Vector Laboratories) and 3,3'-Diaminobenzidine
liquid substrate (D6190, Sigma-Aldrich) as per manufacturer's
instructions. To identify central veins, detection of glutamine
synthetase was carried out using a rabbit polyclonal anti-glutamine
synthetase primary antibody (1/150 dilution, ab16802; Abcam). Bound
primary antibody was detected with a biotinylated donkey
anti-rabbit secondary antibody (1/1000, 711-065-152, Jackson
ImmunoResearch) and the Vectastain Elite ABC system (PK-7100,
Vector Laboratories), followed by application of VIP substrate
(Vector Laboratories) as per the manufacturer's instructions.
Following completion of ASS and glutamine synthetase detection,
sections were counterstained with hematoxylin, and mounted using
Ultramount No. 4 (Fronine) Images were captured using a Zeiss Axio
Imager.A1 microscope and Spot Imaging software.
[0146] As shown in FIG. 7E, ASS protein was localised in the liver
of treated mice, co-localising with glutamine synthetase. The
typical gradient of expression of endogenous ASS activity is shown
in a wildtype untreated mouse (FIG. 7E, left panel), while
widespread high levels of vector-encoded ASS protein can be seen in
a mutant vector-treated mouse (FIG. 7E, right panel).
Example 5--Gene Therapy in Mouse Model of Progressive Intrahepatic
Cholestasis
[0147] Progressive Familial Intrahepatic Cholestasis Type 3 (PFIC3)
is a hereditary chronic liver disease with predominantly childhood
onset. Affected individuals, carrying 2 mutated copies of the ABCB4
gene, have abnormal liver bile production with absent biliary
phosphatidylcholine. Approximately 50% of all patients require
liver transplantation at an average age of 7.5 years. (Jacquemin,
2012, Clinics and Research in Hepatology and Gastroenterology 36
Suppl 1:S26-35). The Abcb4-knockout mouse model shows progressive
liver pathology with early onset, which recapitulates the PFIC3
liver disease phenotype.
[0148] The inventors used this mouse model to demonstrate
correction of the liver disease phenotype using the hybrid
rAAV-piggyBac transposon system (FVB.129P2-Abcb4.sup.tm1Bor/J,
provided by The Jackson Laboratory). The disease phenotype presents
early in life in neonates or juveniles. As such, vector treatment
was delivered to mice during the neonatal period. Constructs were
administered by injection via the intraperitoneal route in 20 uL
volumes (diluted in PBS with calcium and magnesium) in newborn
mice, at vector doses of 5.times.10.sup.10 vg/mouse for the
transposase vector, and 5.times.10.sup.11 vg/mouse for the
transposon-transgene donor vector. The experimental design is
outlined in FIG. 8.
[0149] The piggyBac transposon developed for these sets of
experiments encoded a codon-optimised human ABCB4 sequence (SEQ ID
NO:11) with short transposon recognition sites (SEQ ID NO:5 and SEQ
ID NO:6) flanking the expression cassette, inside the AAV2 inverted
terminal repeat sequences (SEQ ID NO:1 and SEQ ID NO:2).
[0150] A commercial colormetric assay was used to quantify biliary
phosphatidylcholine (EnzyChrom.TM. Phospholipid Assay Kit, BioAssay
Systems). To obtain bile for this analysis, bile was aspirated
directly from the gallbladder in mice that had been fasted for at
least 4 hours, performed as part of the termination procedure.
Histology was performed on 4 .mu.m liver sections that were
formalin-fixed and embedded in paraffin. H+E and Sirius red stains
were performed by the Histology Unit at Westmead Millennium
Institute.
[0151] As shown in FIG. 9, mice that received a single therapeutic
injection of the hABCB4-encoding transposon-transgene donor vector
co-administered with the piggyBac transposase vector, had
significantly raised phosphatidylcholine concentrations in bile and
absence of liver pathology, compared with untreated control
homozygotes.
Sequence CWU 1
1
231181DNAadeno-associated virus 2 1ctgcgcgctc gctcgctcac tgaggccgcc
cgggcaaagc ccgggcgtcg ggcgaccttt 60ggtcgcccgg cctcagtgag cgagcgagcg
cgcagagagg gagtggccaa ctccatcact 120aggggttcct tgtagttaat
gattaacccg ccatgctact tatctacgta gccatgctct 180a
1812181DNAadeno-associated virus 2 2tagagcatgg ctacgtagat
aagtagcatg gcgggttaat cattaactac aaggaacccc 60tagtgatgga gttggccact
ccctctctgc gcgctcgctc gctcactgag gccgggcgac 120caaaggtcgc
ccgacgcccg ggctttgccc gggcggcctc agtgagcgag cgagcgcgca 180g
1813313DNAArtificialpiggyBac transposon 3ttaaccctag aaagatagtc
tgcgtaaaat tgacgcatgc attcttgaaa tattgctctc 60tctttctaaa tagcgcgaat
ccgtcgctgt gcatttagga catctcagtc gccgcttgga 120gctcccgtga
ggcgtgcttg tcaatgcggt aagtgtcact gattttgaac tataacgacc
180gcgtgagtca aaatgacgca tgattatctt ttacgtgact tttaagattt
aactcatacg 240ataattatat tgttatttca tgttctactt acgtgataac
ttattatata tatattttct 300tgttatagat atc
3134230DNAArtificialpiggyBac transposon 4tttgttactt tatagaagaa
attttgagtt tttgtttttt tttaataaat aaataaacat 60aaataaattg tttgttgaat
ttattattag tatgtaagtg taaatataat aaaacttaat 120atctattcaa
attaataaat aaacctcgat atacagaccg ataaaacaca tgcgtcaatt
180ttacgcatga ttatctttaa cgtacgtcac aatatgatta tctttctagg
230567DNAArtificialpiggyBac transposon 5ttaaccctag aaagataatc
atattgtgac gtacgttaaa gataatcatg cgtaaaattg 60acgcatg
67640DNAArtificialpiggyBac transposon 6gcatgcgtca attttacgca
gactatcttt ctagggttaa 4071785DNAArtificialpiggyBac transposon
7atgggtagtt ctttagacga tgagcatatc ctctctgctc ttctgcaaag cgatgacgag
60cttgttggtg aggattctga cagtgaaata tcagatcacg taagtgaaga tgacgtccag
120agcgatacag aagaagcgtt tatagatgag gtacatgaag tgcagccaac
gtcaagcggt 180agtgaaatat tagacgaaca aaatgttatt gaacaaccag
gttcttcatt ggcttctaac 240agaatcttga ccttgccaca gaggactatt
agaggtaaga ataaacattg ttggtcaact 300tcaaagtcca cgaggcgtag
ccgagtctct gcactgaaca ttgtcagatc tcaaagaggt 360ccgacgcgta
tgtgccgcaa tatatatgac ccacttttat gcttcaaact attttttact
420gatgagataa tttcggaaat tgtaaaatgg acaaatgctg agatatcatt
gaaacgtcgg 480gaatctatga caggtgctac atttcgtgac acgaatgaag
atgaaatcta tgctttcttt 540ggtattctgg taatgacagc agtgagaaaa
gataaccaca tgtccacaga tgacctcttt 600gatcgatctt tgtcaatggt
gtacgtctct gtaatgagtc gtgatcgttt tgattttttg 660atacgatgtc
ttagaatgga tgacaaaagt atacggccca cacttcgaga aaacgatgta
720tttactcctg ttagaaaaat atgggatctc tttatccatc agtgcataca
aaattacact 780ccaggggctc atttgaccat agatgaacag ttacttggtt
ttagaggacg gtgtccgttt 840aggatgtata tcccaaacaa gccaagtaag
tatggaataa aaatcctcat gatgtgtgac 900agtggtacga agtatatgat
aaatggaatg ccttatttgg gaagaggaac acagaccaac 960ggagtaccac
tcggtgaata ctacgtgaag gagttatcaa agcctgtgca cggtagttgt
1020cgtaatatta cgtgtgacaa ttggttcacc tcaatccctt tggcaaaaaa
cttactacaa 1080gaaccgtata agttaaccat tgtgggaacc gtgcgatcaa
acaaacgcga gataccggaa 1140gtactgaaaa acagtcgctc caggccagtg
ggaacatcga tgttttgttt tgacggaccc 1200cttactctcg tctcatataa
accgaagcca gctaagatgg tatacttatt atcatcttgt 1260gatgaggatg
cttctatcaa cgaaagtacc ggtaaaccgc aaatggttat gtattataat
1320caaactaaag gcggagtgga cacgctagac caaatgtgtt ctgtgatgac
ctgcagtagg 1380aagacgaata ggtggcctat ggcattattg tacggaatga
taaacattgc ctgcataaat 1440tcttttatta tatacagcca taatgtcagt
agcaagggag aaaaggttca aagtcgcaaa 1500aaatttatga gaaaccttta
catgagcctg acgtcatcgt ttatgcgtaa gcgtttagaa 1560gctcctactt
tgaagagata tttgcgcgat aatatctcta atattttgcc aaatgaagtg
1620cctggtacat cagatgacag tactgaagag ccagtaatga aaaaacgtac
ttactgtact 1680tactgcccct ctaaaataag gcgaaaggca aatgcatcgt
gcaaaaaatg caaaaaagtt 1740atttgtcgag agcataatat tgatatgtgc
caaagttgtt tctga 17858720DNAArtificialSynthetic sequence
8atggtgagca agggcgagga gctgttcacc ggggtggtgc ccatcctggt cgagctggac
60ggcgacgtaa acggccacaa gttcagcgtg tccggcgagg gcgagggcga tgccacctac
120ggcaagctga ccctgaagtt catctgcacc accggcaagc tgcccgtgcc
ctggcccacc 180ctcgtgacca ccctgaccta cggcgtgcag tgcttcagcc
gctaccccga ccacatgaag 240cagcacgact tcttcaagtc cgccatgccc
gaaggctacg tccaggagcg caccatcttc 300ttcaaggacg acggcaacta
caagacccgc gccgaggtga agttcgaggg cgacaccctg 360gtgaaccgca
tcgagctgaa gggcatcgac ttcaaggagg acggcaacat cctggggcac
420aagctggagt acaactacaa cagccacaac gtctatatca tggccgacaa
gcagaagaac 480ggcatcaagg tgaacttcaa gatccgccac aacatcgagg
acggcagcgt gcagctcgcc 540gaccactacc agcagaacac ccccatcggc
gacggccccg tgctgctgcc cgacaaccac 600tacctgagca cccagtccgc
cctgagcaaa gaccccaacg agaagcgcga tcacatggtc 660ctgctggagt
tcgtgaccgc cgccgggatc actctcggca tggacgagct gtacaagtaa
72091066DNAMus musculusmisc_feature(256)..(256)n is a, c, g, or t
9atgctgtcta atttgaggat cctgctcaac aatgcagctc ttagaaaggg tcacacttct
60gtggttcgac atttttggtg tgggaagcca gtccaaagtc aagtacagct gaaaggccgt
120gacctcctca ccttgaagaa cttcacagga gaggagattc agtacatgct
atggctctct 180gcagatctga aattcaggat caagcagaaa ggagaatatt
tacctttatt gcaagggaaa 240tccttaggaa tgattntttg agaaaagaag
tactcgaaca agactgtcca cagaaacagg 300ctttgctctg ctgggaggac
acccttcctt tcttaccaca caagacattc acttgggtgt 360gaatgaaagt
ctcacagaca ccgctcgtgt cttatctagc atgacagatg cagtgttagc
420tcgagtgtat aaacaatcag atctggacac cctggctaaa gaagcatcca
tcccaattgt 480caatggactg tcagacttgt atcatcctat ccagatcctg
gctgattacc ttacactcca 540ggaacactat ggctctctca aaggtcttac
cctcagctgg ataggggatg ggaacaatat 600cttgcactct atcatgatga
gtgctgcaaa attcgggatg caccttcaag cagctactcc 660aaagggttat
gagccagatc ctaatatagt caagctagca gagcagtatg ccaaggagaa
720tggtaccaag ttgtcaatga caaatgatcc actggaagca gcacgtggag
gcaatgtatt 780aattacagat acttggataa gcatgggaca agaggatgag
aagaaaaagc gtcttcaagc 840tttccaaggt taccaggtta cgatgaagac
tgccaaagtg gctgcgtctg actggacatt 900tttacactgt ttgcctagaa
agccagaaga agtggatgat gaagtatttt attctccacg 960gtcattagtg
ttcccagagg cagagaatag aaagtggaca atcatggctg tcatggtatc
1020cctgctgaca gactactcac ctgtgctcca gaagccaaag ttttga
1066101239DNAMus musculus 10atgtccagca agggctctgt ggttctggcc
tacagtggtg gcctggacac ctcctgcatc 60ctcgtgtggc tgaaggaaca aggctatgat
gtcatcgcct acctggccaa cattggccag 120aaggaagact ttgaggaagc
caggaagaag gcgctgaagc ttggggccaa aaaggtgttc 180attgaggatg
tgagcaagga atttgtggaa gagttcatct ggcctgctgt ccagtccagt
240gcactctacg aggaccgcta tctcctgggc acctctctcg ccaggccttg
catagctcgc 300agacaggtgg agattgccca gcgtgaaggg gccaagtatg
tgtctcacgg cgccacggga 360aaggggaatg accaggtccg ctttgagctc
acctgctatt cactggcacc ccagattaag 420gtcatcgctc cctggaggat
gcctgagttt tacaaccggt tcaagggccg aaatgatctg 480atggagtatg
caaagcaaca cggaatcccc atccctgtca cccccaagag cccctggagt
540atggatgaaa acctcatgca catcagctat gaggctggga tcctggaaaa
ccccaagaat 600caagcacctc cgggtctcta cacaaaaact caggaccctg
ccaaagcacc caacagccca 660gatgtccttg agatagaatt caaaaaaggg
gtccctgtga aggtgaccaa catcaaagat 720ggcacaaccc gcaccacatc
cctggaactc ttcatgtacc tgaacgaagt tgcgggcaag 780cacggagtgg
gtcgcattga catcgtggag aaccgcttca ttggaatgaa gtcccgaggt
840atctacgaga ccccagcagg gaccatcctt taccacgctc atttagacat
agaggccttc 900acgatggatc gggaagtacg caaaatcaag cagggcctgg
gcctcaaatt cgcagagctc 960gtatacacag gtttctggca cagccctgaa
tgtgaatttg ttcgccactg tatccagaag 1020tcccaggagc gggtagaagg
gaaggtgcag gtgtctgtct tcaagggcca agtgtacatc 1080ctcggtcggg
agtctccact ttcactctac aatgaagagc tggtgagcat gaacgtgcag
1140ggcgactatg agcccatcga cgccactggc ttcatcaata tcaactcgct
caggctgaag 1200gagtaccatc gccttcagag caaggtcact gccaaatag
1239113849DNAHomo sapiens 11gccgccacca tggacctgga ggcagcaaag
aatggaaccg catggagacc aacatcagca 60gagggggact tcgaactggg tatttcaagc
aagcagaagc gcaagaaaac taagaccgtg 120aaaatgatcg gggtcctcac
cctgttccga tactccgact ggcaggataa gctctttatg 180tctctgggca
caatcatggc cattgctcac gggtctggtc tccctctgat gatgatcgtg
240ttcggggaga tgaccgacaa atttgtcgat acagccggta atttcagctt
tccagtgaac 300ttctctctca gtctgctcaa ccccggcaag atcctggagg
aagagatgac tcgctatgca 360tactattact ctggactggg agctggggtg
ctggtcgcag cttacatcca ggtgagtttc 420tggaccctgg cagctggacg
gcagatccgc aaaattcgac agaagttctt tcatgccatc 480ctgagacagg
agattgggtg gtttgacatc aatgatacca cagaactcaa cacccggctg
540acagacgaca tcagcaaaat ttccgagggt atcggcgata aagtgggaat
gttctttcag 600gcagtcgcca ctttctttgc cggattcatt gtcgggttta
tccggggttg gaagctgacc 660ctggtcatca tggctatttc accaatcctc
gggctgagcg ccgcagtgtg ggcaaagatc 720ctctctgcct tcagtgacaa
agagctggcc gcttatgcta aggcaggagc tgtggctgaa 780gaggcactgg
gagcaattcg aaccgtgatc gcctttggcg gacagaataa ggaactcgag
840aggtaccaga aacacctgga gaacgctaag gaaatcggga ttaagaaagc
tatttccgca 900aacatctcta tgggtattgc tttcctgctc atctatgcat
cttacgcact cgccttttgg 960tatggcagca ccctggtcat cagcaaggag
tacactatcg gaaatgcaat gaccgtcttc 1020ttttctatcc tgattggggc
tttcagtgtg ggtcaggcag ccccctgcat cgacgctttc 1080gcaaatgcac
gcggcgctgc atacgtgatc ttcgacatca ttgataacaa ccctaagatc
1140gactcattca gcgagagggg gcacaaacca gatagcatta agggtaatct
ggaattcaac 1200gacgtgcatt tttcataccc tagcagagcc aatgtcaaga
tcctgaaagg actcaacctg 1260aaagtgcaga gcgggcagac tgtggctctg
gtcggtagct ccggatgcgg gaagtccact 1320accgtgcagc tcattcagcg
gctgtatgac ccagatgagg gcacaatcaa cattgacgga 1380caggacatcc
gcaacttcaa tgtcaactac ctgcgagaga tcattggcgt ggtctcacag
1440gaacccgtgc tgtttagcac aactatcgcc gagaatattt gttatggtag
aggcaacgtg 1500acaatggatg aaattaagaa agctgtcaag gaggctaatg
catacgaatt catcatgaaa 1560ctccctcaga agtttgatac tctggtgggc
gagaggggcg cccagctgag cgggggtcag 1620aaacagcgca tcgccattgc
tcgagcactg gtgaggaacc caaagatcct gctcctggac 1680gaggccacat
ccgctctgga tactgaatct gaggccgaag tgcaggccgc tctggacaag
1740gctagggaag gcagaaccac aatcgtgatt gcccacagac tgagcaccgt
gcggaatgcc 1800gacgtgattg ctggcttcga ggatggagtg atcgtcgaac
agggctccca ttctgagctg 1860atgaagaaag aaggagtgta tttcaagctg
gtcaacatgc agacaagtgg ctcacagatc 1920cagtccgaag agtttgagct
gaatgacgaa aaagcagcca caaggatggc cccaaacgga 1980tggaagagtc
ggctcttccg ccactcaact cagaagaatc tgaaaaacag ccagatgtgc
2040cagaagtccc tcgacgtgga gaccgatggg ctggaagcta atgtgccccc
tgtctccttc 2100ctgaaggtgc tcaaactgaa caagaccgag tggccctact
ttgtggtcgg cacagtctgc 2160gccatcgcta atggcggact gcagcccgcc
ttcagcgtga tcttcagcga aatcattgct 2220atcttcggac ctggggacga
tgcagtgaaa cagcagaagt gtaacatctt tagtctgatt 2280ttcctctttc
tgggcatcat ttcattcttt acattctttc tgcagggatt cacttttgga
2340aaggccgggg agatcctcac caggagactg aggagcatgg cattcaaagc
catgctgaga 2400caggatatgt cctggtttga cgatcataag aattctacag
gcgccctcag tactagactg 2460gctaccgacg ctgcacaggt gcagggtgca
acaggcactc ggctcgctct gatcgcacag 2520aacattgcaa atctcgggac
tggaatcatt atctccttta tctatggttg gcagttaacc 2580ctgctgctgc
tggccgtggt gcccatcatt gccgtgtccg gcatcgtgga aatgaaactg
2640ctggctggaa acgctaagag agataagaaa gaactggagg ctgctggaaa
aatcgctacc 2700gaggctattg agaacattag aaccgtggtc tctctcacac
aggagcggaa gttcgaaagt 2760atgtacgtgg agaaactgta cgggccatat
cgaaacagtg tgcagaaggc ccacatctat 2820ggtattacat tttcaatcag
ccaggccttc atgtacttta gctatgctgg gtgcttccgc 2880tttggtgcat
atctgatcgt gaatggccat atgaggttca gagacgtgat cctcgtcttc
2940agcgccatcg tgttcggagc tgtcgctctg ggacacgcca gctcctttgc
tcccgattac 3000gcaaaggcca aactgtccgc cgctcatctc ttcatgctgt
ttgagagaca gcctctcatc 3060gactcctatt ctgaggaagg cctgaagcca
gataaattcg agggaaacat tacattcaat 3120gaagtggtct ttaactaccc
cactcgggct aatgtgcctg tcctgcaggg actctccctg 3180gaagtgaaga
aagggcagac tctcgccctg gtcggttcta gtgggtgcgg caagtctacc
3240gtggtccagc tgctcgagcg gttttacgac cccctggcag ggactgtgct
gctcgatggt 3300caggaagcta agaaactgaa cgtgcagtgg ctgagagcac
agctgggaat cgtctcacag 3360gagcctattc tgttcgactg tagcatcgca
gaaaacattg cctatggaga caatagtagg 3420gtggtctcac aggatgagat
cgtgtctgca gccaaggctg caaatatcca ccccttcatc 3480gagacactgc
cccataagta cgaaactcgc gtgggcgata aaggaaccca gctgagcggc
3540ggacagaaac agcgaatcgc tattgcacga gccctgatca ggcagcccca
gattctgctc 3600ctggacgagg ctactagcgc actcgatacc gagtccgaaa
aggtggtcca ggaggctctg 3660gacaaagcac gggaaggccg cacctgtatc
gtgattgccc acaggctcag cacaatccag 3720aacgctgatc tgattgtggt
cttccagaat ggcagagtga aggagcacgg aacacatcag 3780cagctcctgg
cacagaaggg aatctatttt tcaatggtct ccgtccaggc aggcactcag
3840aatctctaa 384912327DNAHomo sapiens 12gctcagaggc acacaggagt
ttctgggctc accctgcccc cttccaaccc ctcagttccc 60atcctccagc agctgtttgt
gtgctgcctc tgaagtccac actgaacaaa cttcagccta 120ctcatgtccc
taaaatgggc aaacattgca agcagcaaac agcaaacaca cagccctccc
180tgcctgctga ccttggagct ggggcagagg tcagagacct ctctgggccc
atgccacctc 240caacatccac tcgacccctt ggaatttcgg tggagaggag
cagaggttgt cctggcgtgg 300tttaggtagt gtgagagggt ccggcga
32713192DNAHomo sapiens 13ccctaaaatg ggcaaacatt gcaagcagca
aacagcaaac acacagccct ccctgcctgc 60tgaccttgga gctggggcag aggtcagaga
cctctctggg cccatgccac ctccaacatc 120cactcgaccc cttggaattt
cggtggagag gagcagaggt tgtcctggcg tggtttaggt 180agtgtgagag gg
19214397DNAHomo sapiens 14gatcttgcta ccagtggaac agccactaag
gattctgcag tgagagcaga gggccagcta 60agtggtactc tcccagagac tgtctgactc
acgccacccc ctccaccttg gacacaggac 120gctgtggttt ctgagccagg
tacaatgact cctttcggta agtgcagtgg aagctgtaca 180ctgcccaggc
aaagcgtccg ggcagcgtag gcgggcgact cagatcccag ccagtggact
240tagcccctgt ttgctcctcc gataactggg gtgaccttgg ttaatattca
ccagcagcct 300cccccgttgc ccctctggat ccactgctta aatacggacg
aggacagggc cctgtctcct 360cagcttcagg caccaccact gacctgggac agtgaat
39715254DNAHomo sapiens 15aatgactcct ttcggtaagt gcagtggaag
ctgtacactg cccaggcaaa gcgtccgggc 60agcgtaggcg ggcgactcag atcccagcca
gtggacttag cccctgtttg ctcctccgat 120aactggggtg accttggtta
atattcacca gcagcctccc ccgttgcccc tctggatcca 180ctgcttaaat
acggacgagg acagggccct gtctcctcag cttcaggcac caccactgac
240ctgggacagt gaat 25416589DNAWoodchuck hepatitis virus
16aatcaacctc tggattacaa aatttgtgaa agattgactg gtattcttaa ctatgttgct
60ccttttacgc tatgtggata cgctgcttta atgcctttgt atcatgctat tgcttcccgt
120atggctttca ttttctcctc cttgtataaa tcctggttgc tgtctcttta
tgaggagttg 180tggcccgttg tcaggcaacg tggcgtggtg tgcactgtgt
ttgctgacgc aacccccact 240ggttggggca ttgccaccac ctgtcagctc
ctttccggga ctttcgcttt ccccctccct 300attgccacgg cggaactcat
cgccgcctgc cttgcccgct gctggacagg ggctcggctg 360ttgggcactg
acaattccgt ggtgttgtcg gggaaatcat cgtcctttcc ttggctgctc
420gcctgtgttg ccacctggat tctgcgcggg acgtccttct gctacgtccc
ttcggccctc 480aatccagcgg accttccttc ccgcggcctg ctgccggctc
tgcggcctct tccgcgtctt 540cgccttcgcc ctcagacgag tcggatctcc
ctttgggccg cctccccgc 58917261DNABos taurus 17tcgctgatca gcctcgactg
tgccttctag ttgccagcca tctgttgttt gcccctcccc 60cgtgccttcc ttgaccctgg
aaggtgccac tcccactgtc ctttcctaat aaaatgagga 120aattgcatcg
cattgtctga gtaggtgtca ttctattctg gggggtgggg tggggcagga
180cagcaagggg gaggattggg aagacaatag caggcatgct ggggatgcgg
tgggctctat 240ggcttctgag gcggaaagaa c 26118134DNASimian virus 40
18atgctttatt tgtgaaattt gtgatgctat tgctttattt gtaaccatta taagctgcaa
60taaacaagtt aacaacaaca attgcattca ttttatgttt caggttcagg gggaggtgtg
120ggaggttttt taaa 134197291DNAArtificialSynthetic sequence
19tagctgcgcg ctcgctcgct cactgaggcc gcccgggcaa agcccgggcg tcgggcgacc
60tttggtcgcc cggcctcagt gagcgagcga gcgcgcagag agggagtggc caactccatc
120actaggggtt ccttgtagtt aatgattaac ccgccatgct acttatctac
gtagccatgc 180tctaggtacc gagctcttaa ttaactgcag gctcagaggc
acacaggagt ttctgggctc 240accctgcccc cttccaaccc ctcagttccc
atcctccagc agctgtttgt gtgctgcctc 300tgaagtccac actgaacaaa
cttcagccta ctcatgtccc taaaatgggc aaacattgca 360agcagcaaac
agcaaacaca cagccctccc tgcctgctga ccttggagct ggggcagagg
420tcagagacct ctctgggccc atgccacctc caacatccac tcgacccctt
ggaatttcgg 480tggagaggag cagaggttgt cctggcgtgg tttaggtagt
gtgagagggt ccggcgatta 540actgcaggct cagaggcaca caggagtttc
tgggctcacc ctgccccctt ccaacccctc 600agttcccatc ctccagcagc
tgtttgtgtg ctgcctctga agtccacact gaacaaactt 660cagcctactc
atgtccctaa aatgggcaaa cattgcaagc agcaaacagc aaacacacag
720ccctccctgc ctgctgacct tggagctggg gcagaggtca gagacctctc
tgggcccatg 780ccacctccaa catccactcg accccttgga atttcggtgg
agaggagcag aggttgtcct 840ggcgtggttt aggtagtgtg agagggtccg
gcgaattaag atcttgctac cagtggaaca 900gccactaagg attctgcagt
gagagcagag ggccagctaa gtggtactct cccagagact 960gtctgactca
cgccaccccc tccaccttgg acacaggacg ctgtggtttc tgagccaggt
1020acaatgactc ctttcggtaa gtgcagtgga agctgtacac tgcccaggca
aagcgtccgg 1080gcagcgtagg cgggcgactc agatcccagc cagtggactt
agcccctgtt tgctcctccg 1140ataactgggg tgaccttggt taatattcac
cagcagcctc ccccgttgcc cctctggatc 1200cactgcttaa atacggacga
ggacagggcc ctgtctcctc agcttcaggc accaccactg 1260acctgggaca
gtgaatgcgg ccgctctaga actagtgccg ccaccatggg tagttcttta
1320gacgatgagc atatcctctc tgctcttctg caaagcgatg acgagcttgt
tggtgaggat 1380tctgacagtg aaatatcaga tcacgtaagt gaagatgacg
tccagagcga tacagaagaa 1440gcgtttatag atgaggtaca tgaagtgcag
ccaacgtcaa gcggtagtga aatattagac 1500gaacaaaatg ttattgaaca
accaggttct tcattggctt ctaacagaat cttgaccttg 1560ccacagagga
ctattagagg taagaataaa cattgttggt caacttcaaa gtccacgagg
1620cgtagccgag tctctgcact gaacattgtc agatctcaaa gaggtccgac
gcgtatgtgc 1680cgcaatatat atgacccact tttatgcttc aaactatttt
ttactgatga gataatttcg 1740gaaattgtaa aatggacaaa tgctgagata
tcattgaaac gtcgggaatc tatgacaggt 1800gctacatttc gtgacacgaa
tgaagatgaa atctatgctt tctttggtat tctggtaatg 1860acagcagtga
gaaaagataa ccacatgtcc acagatgacc tctttgatcg atctttgtca
1920atggtgtacg tctctgtaat gagtcgtgat cgttttgatt ttttgatacg
atgtcttaga 1980atggatgaca aaagtatacg gcccacactt cgagaaaacg
atgtatttac tcctgttaga 2040aaaatatggg atctctttat ccatcagtgc
atacaaaatt acactccagg ggctcatttg
2100accatagatg aacagttact tggttttaga ggacggtgtc cgtttaggat
gtatatccca 2160aacaagccaa gtaagtatgg aataaaaatc ctcatgatgt
gtgacagtgg tacgaagtat 2220atgataaatg gaatgcctta tttgggaaga
ggaacacaga ccaacggagt accactcggt 2280gaatactacg tgaaggagtt
atcaaagcct gtgcacggta gttgtcgtaa tattacgtgt 2340gacaattggt
tcacctcaat ccctttggca aaaaacttac tacaagaacc gtataagtta
2400accattgtgg gaaccgtgcg atcaaacaaa cgcgagatac cggaagtact
gaaaaacagt 2460cgctccaggc cagtgggaac atcgatgttt tgttttgacg
gaccccttac tctcgtctca 2520tataaaccga agccagctaa gatggtatac
ttattatcat cttgtgatga ggatgcttct 2580atcaacgaaa gtaccggtaa
accgcaaatg gttatgtatt ataatcaaac taaaggcgga 2640gtggacacgc
tagaccaaat gtgttctgtg atgacctgca gtaggaagac gaataggtgg
2700cctatggcat tattgtacgg aatgataaac attgcctgca taaattcttt
tattatatac 2760agccataatg tcagtagcaa gggagaaaag gttcaaagtc
gcaaaaaatt tatgagaaac 2820ctttacatga gcctgacgtc atcgtttatg
cgtaagcgtt tagaagctcc tactttgaag 2880agatatttgc gcgataatat
ctctaatatt ttgccaaatg aagtgcctgg tacatcagat 2940gacagtactg
aagagccagt aatgaaaaaa cgtacttact gtacttactg cccctctaaa
3000ataaggcgaa aggcaaatgc atcgtgcaaa aaatgcaaaa aagttatttg
tcgagagcat 3060aatattgata tgtgccaaag ttgtttctga aagcttatcg
ataatcaacc tctggattac 3120aaaatttgtg aaagattgac tggtattctt
aactatgttg ctccttttac gctatgtgga 3180tacgctgctt taatgccttt
gtatcatgct attgcttccc gtatggcttt cattttctcc 3240tccttgtata
aatcctggtt gctgtctctt tatgaggagt tgtggcccgt tgtcaggcaa
3300cgtggcgtgg tgtgcactgt gtttgctgac gcaaccccca ctggttgggg
cattgccacc 3360acctgtcagc tcctttccgg gactttcgct ttccccctcc
ctattgccac ggcggaactc 3420atcgccgcct gccttgcccg ctgctggaca
ggggctcggc tgttgggcac tgacaattcc 3480gtggtgttgt cggggaaatc
atcgtccttt ccttggctgc tcgcctgtgt tgccacctgg 3540attctgcgcg
ggacgtcctt ctgctacgtc ccttcggccc tcaatccagc ggaccttcct
3600tcccgcggcc tgctgccggc tctgcggcct cttccgcgtc ttcgccttcg
ccctcagacg 3660agtcggatct ccctttgggc cgcctccccg catcgatacc
gtcgactcgc tgatcagcct 3720cgactgtgcc ttctagttgc cagccatctg
ttgtttgccc ctcccccgtg ccttccttga 3780ccctggaagg tgccactccc
actgtccttt cctaataaaa tgaggaaatt gcatcgcatt 3840gtctgagtag
gtgtcattct attctggggg gtggggtggg gcaggacagc aagggggagg
3900attgggaaga caatagcagg catgctgggg atgcggtggg ctctatggct
tctgaggcgg 3960aaagaaccag ctggggctcg actagagcat ggctacgtag
ataagtagca tggcgggtta 4020atcattaact acaaggaacc cctagtgatg
gagttggcca ctccctctct gcgcgctcgc 4080tcgctcactg aggccgggcg
accaaaggtc gcccgacgcc cgggctttgc ccgggcggcc 4140tcagtgagcg
agcgagcgcg cagagctttt tgcaaaagcc taggcctcca aaaaagcctc
4200ctcactactt ctggaatagc tcagaggccg aggcggcctc ggcctctgca
taaataaaaa 4260aaattagtca gccatggggc ggagaatggg cggaactggg
cggagttagg ggcgggatgg 4320gcggagttag gggcgggact atggttgctg
actaattgag atgcatgctt tgcatacttc 4380tgcctgctgg ggagcctggg
gactttccac acctggttgc tgactaattg agatgcatgc 4440tttgcatact
tctgcctgct ggggagcctg gggactttcc acaccctaac tgacacacat
4500tccacagctg cattaatgaa tcggccaacg cgcggggaga ggcggtttgc
gtattgggcg 4560ctcttccgct tcctcgctca ctgactcgct gcgctcggtc
gttcggctgc ggcgagcggt 4620atcagctcac tcaaaggcgg taatacggtt
atccacagaa tcaggggata acgcaggaaa 4680gaacatgtga gcaaaaggcc
agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc 4740gtttttccat
aggctccgcc cccctgacga gcatcacaaa aatcgacgct caagtcagag
4800gtggcgaaac ccgacaggac tataaagata ccaggcgttt ccccctggaa
gctccctcgt 4860gcgctctcct gttccgaccc tgccgcttac cggatacctg
tccgcctttc tcccttcggg 4920aagcgtggcg ctttctcata gctcacgctg
taggtatctc agttcggtgt aggtcgttcg 4980ctccaagctg ggctgtgtgc
acgaaccccc cgttcagccc gaccgctgcg ccttatccgg 5040taactatcgt
cttgagtcca acccggtaag acacgactta tcgccactgg cagcagccac
5100tggtaacagg attagcagag cgaggtatgt aggcggtgct acagagttct
tgaagtggtg 5160gcctaactac ggctacacta gaagaacagt atttggtatc
tgcgctctgc tgaagccagt 5220taccttcgga aaaagagttg gtagctcttg
atccggcaaa caaaccaccg ctggtagcgg 5280tggttttttt gtttgcaagc
agcagattac gcgcagaaaa aaaggatctc aagaagatcc 5340tttgatcttt
tctacggggt ctgacgctca gtggaacgaa aactcacgtt aagggatttt
5400ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa
aatgaagttt 5460taaatcaatc taaagtatat atgagtaaac ttggtctgac
agttaccaat gcttaatcag 5520tgaggcacct atctcagcga tctgtctatt
tcgttcatcc atagttgcct gactccccgt 5580cgtgtagata actacgatac
gggagggctt accatctggc cccagtgctg caatgatacc 5640gcgagaccca
cgctcaccgg ctccagattt atcagcaata aaccagccag ccggaagggc
5700cgagcgcaga agtggtcctg caactttatc cgcctccatc cagtctatta
attgttgccg 5760ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc
aacgttgttg ccattgctac 5820aggcatcgtg gtgtcacgct cgtcgtttgg
tatggcttca ttcagctccg gttcccaacg 5880atcaaggcga gttacatgat
cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc 5940tccgatcgtt
gtcagaagta agttggccgc agtgttatca ctcatggtta tggcagcact
6000gcataattct cttactgtca tgccatccgt aagatgcttt tctgtgactg
gtgagtactc 6060aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt
tgctcttgcc cggcgtcaat 6120acgggataat accgcgccac atagcagaac
tttaaaagtg ctcatcattg gaaaacgttc 6180ttcggggcga aaactctcaa
ggatcttacc gctgttgaga tccagttcga tgtaacccac 6240tcgtgcaccc
aactgatctt cagcatcttt tactttcacc agcgtttctg ggtgagcaaa
6300aacaggaagg caaaatgccg caaaaaaggg aataagggcg acacggaaat
gttgaatact 6360catactcttc ctttttcaat attattgaag catttatcag
ggttattgtc tcatgagcgg 6420atacatattt gaatgtattt agaaaaataa
acaaataggg gttccgcgca catttccccg 6480aaaagtgcca cctgacgtct
aagaaaccat tattatcatg acattaacct ataaaaatag 6540gcgtatcacg
aggccctttc gtctcgcgcg tttcggtgat gacggtgaaa acctctgaca
6600catgcagctc ccggagacgg tcacagcttg tctgtaagcg gatgccggga
gcagacaagc 6660ccgtcagggc gcgtcagcgg gtgttggcgg gtgtcggggc
tggcttaact atgcggcatc 6720agagcagatt gtactgagag tgcaccattc
gacgctctcc cttatgcgac tcctgcatta 6780ggaagcagcc cagtagtagg
ttgaggccgt tgagcaccgc cgccgcaagg aatggtgcat 6840gcaaggagat
ggcgcccaac agtcccccgg ccacggggcc tgccaccata cccacgccga
6900aacaagcgct catgagcccg aagtggcgag cccgatcttc cccatcggtg
atgtcggcga 6960tataggcgcc agcaaccgca cctgtggcgc cggtgatgcc
ggccacgatg cgtccggcgt 7020agaggatctg gctagcgatg accctgctga
ttggttcgct gaccatttcc gggtgcggga 7080cggcgttacc agaaactcag
aaggttcgtc caaccaaacc gactctgacg gcagtttacg 7140agagagatga
tagggtctgc ttcagtaagc cagatgctac acaattaggc ttgtacatat
7200tgtcgttaga acgcggctac aattaataca taaccttatg tatcatacac
atacgattta 7260ggtgacacta tagaatacac ggaattaatt c
7291206813DNAArtificialSynthetic sequence 20tagctgcgcg ctcgctcgct
cactgaggcc gcccgggcaa agcccgggcg tcgggcgacc 60tttggtcgcc cggcctcagt
gagcgagcga gcgcgcagag agggagtggc caactccatc 120actaggggtt
ccttgtagtt aatgattaac ccgccatgct acttatctac gtagccatgc
180tctagcgatc gcttaaccct agaaagatag tctgcgtaaa attgacgcat
gcattcttga 240aatattgctc tctctttcta aatagcgcga atccgtcgct
gtgcatttag gacatctcag 300tcgccgcttg gagctcccgt gaggcgtgct
tgtcaatgcg gtaagtgtca ctgattttga 360actataacga ccgcgtgagt
caaaatgacg catgattatc ttttacgtga cttttaagat 420ttaactcata
cgataattat attgttattt catgttctac ttacgtgata acttattata
480tatatatttt cttgttatag atatcttaat taactgcagg ctcagaggca
cacaggagtt 540tctgggctca ccctgccccc ttccaacccc tcagttccca
tcctccagca gctgtttgtg 600tgctgcctct gaagtccaca ctgaacaaac
ttcagcctac tcatgtccct aaaatgggca 660aacattgcaa gcagcaaaca
gcaaacacac agccctccct gcctgctgac cttggagctg 720gggcagaggt
cagagacctc tctgggccca tgccacctcc aacatccact cgaccccttg
780gaatttcggt ggagaggagc agaggttgtc ctggcgtggt ttaggtagtg
tgagagggtc 840cggcgattaa ctgcaggctc agaggcacac aggagtttct
gggctcaccc tgcccccttc 900caacccctca gttcccatcc tccagcagct
gtttgtgtgc tgcctctgaa gtccacactg 960aacaaacttc agcctactca
tgtccctaaa atgggcaaac attgcaagca gcaaacagca 1020aacacacagc
cctccctgcc tgctgacctt ggagctgggg cagaggtcag agacctctct
1080gggcccatgc cacctccaac atccactcga ccccttggaa tttcggtgga
gaggagcaga 1140ggttgtcctg gcgtggttta ggtagtgtga gagggtccgg
cgaattaaga tcttgctacc 1200agtggaacag ccactaagga ttctgcagtg
agagcagagg gccagctaag tggtactctc 1260ccagagactg tctgactcac
gccaccccct ccaccttgga cacaggacgc tgtggtttct 1320gagccaggta
caatgactcc tttcggtaag tgcagtggaa gctgtacact gcccaggcaa
1380agcgtccggg cagcgtaggc gggcgactca gatcccagcc agtggactta
gcccctgttt 1440gctcctccga taactggggt gaccttggtt aatattcacc
agcagcctcc cccgttgccc 1500ctctggatcc actgcttaaa tacggacgag
gacagggccc tgtctcctca gcttcaggca 1560ccaccactga cctgggacag
tgaatgcggc cgctctagaa ctagtggatc ccccgggctg 1620caggaattca
ctagtgattt cgccgccacc atggtgagca agggcgagga gctgttcacc
1680ggggtggtgc ccatcctggt cgagctggac ggcgacgtaa acggccacaa
gttcagcgtg 1740tccggcgagg gcgagggcga tgccacctac ggcaagctga
ccctgaagtt catctgcacc 1800accggcaagc tgcccgtgcc ctggcccacc
ctcgtgacca ccctgaccta cggcgtgcag 1860tgcttcagcc gctaccccga
ccacatgaag cagcacgact tcttcaagtc cgccatgccc 1920gaaggctacg
tccaggagcg caccatcttc ttcaaggacg acggcaacta caagacccgc
1980gccgaggtga agttcgaggg cgacaccctg gtgaaccgca tcgagctgaa
gggcatcgac 2040ttcaaggagg acggcaacat cctggggcac aagctggagt
acaactacaa cagccacaac 2100gtctatatca tggccgacaa gcagaagaac
ggcatcaagg tgaacttcaa gatccgccac 2160aacatcgagg acggcagcgt
gcagctcgcc gaccactacc agcagaacac ccccatcggc 2220gacggccccg
tgctgctgcc cgacaaccac tacctgagca cccagtccgc cctgagcaaa
2280gaccccaacg agaagcgcga tcacatggtc ctgctggagt tcgtgaccgc
cgccgggatc 2340actctcggca tggacgagct gtacaagtaa gatatcaagc
ttatcgataa tcaacctctg 2400gattacaaaa tttgtgaaag attgactggt
attcttaact atgttgctcc ttttacgcta 2460tgtggatacg ctgctttaat
gcctttgtat catgctattg cttcccgtat ggctttcatt 2520ttctcctcct
tgtataaatc ctggttgctg tctctttatg aggagttgtg gcccgttgtc
2580aggcaacgtg gcgtggtgtg cactgtgttt gctgacgcaa cccccactgg
ttggggcatt 2640gccaccacct gtcagctcct ttccgggact ttcgctttcc
ccctccctat tgccacggcg 2700gaactcatcg ccgcctgcct tgcccgctgc
tggacagggg ctcggctgtt gggcactgac 2760aattccgtgg tgttgtcggg
gaaatcatcg tcctttcctt ggctgctcgc ctgtgttgcc 2820acctggattc
tgcgcgggac gtccttctgc tacgtccctt cggccctcaa tccagcggac
2880cttccttccc gcggcctgct gccggctctg cggcctcttc cgcgtcttcg
ccttcgccct 2940cagacgagtc ggatctccct ttgggccgcc tccccgcatc
gataccgtcg actcgctgat 3000cagcctcgac tgtgccttct agttgccagc
catctgttgt ttgcccctcc cccgtgcctt 3060ccttgaccct ggaaggtgcc
actcccactg tcctttccta ataaaatgag gaaattgcat 3120cgcattgtct
gagtaggtgt cattctattc tggggggtgg ggtggggcag gacagcaagg
3180gggaggattg ggaagacaat agcaggcatg ctggggatgc ggtgggctct
atggcttctg 3240aggcggaaag aacggccggc ctttgttact ttatagaaga
aattttgagt ttttgttttt 3300ttttaataaa taaataaaca taaataaatt
gtttgttgaa tttattatta gtatgtaagt 3360gtaaatataa taaaacttaa
tatctattca aattaataaa taaacctcga tatacagacc 3420gataaaacac
atgcgtcaat tttacgcatg attatcttta acgtacgtca caatatgatt
3480atctttctag ggttaacctg caggtagagc atggctacgt agataagtag
catggcgggt 3540taatcattaa ctacaaggaa cccctagtga tggagttggc
cactccctct ctgcgcgctc 3600gctcgctcac tgaggccggg cgaccaaagg
tcgcccgacg cccgggcttt gcccgggcgg 3660cctcagtgag cgagcgagcg
cgcagagctt tttgcaaaag cctaggcctc caaaaaagcc 3720tcctcactac
ttctggaata gctcagaggc cgaggcggcc tcggcctctg cataaataaa
3780aaaaattagt cagccatggg gcggagaatg ggcggaactg ggcggagtta
ggggcgggat 3840gggcggagtt aggggcggga ctatggttgc tgactaattg
agatgcatgc tttgcatact 3900tctgcctgct ggggagcctg gggactttcc
acacctggtt gctgactaat tgagatgcat 3960gctttgcata cttctgcctg
ctggggagcc tggggacttt ccacacccta actgacacac 4020attccacagc
tgcattaatg aatcggccaa cgcgcgggga gaggcggttt gcgtattggg
4080cgctcttccg cttcctcgct cactgactcg ctgcgctcgg tcgttcggct
gcggcgagcg 4140gtatcagctc actcaaaggc ggtaatacgg ttatccacag
aatcagggga taacgcagga 4200aagaacatgt gagcaaaagg ccagcaaaag
gccaggaacc gtaaaaaggc cgcgttgctg 4260gcgtttttcc ataggctccg
cccccctgac gagcatcaca aaaatcgacg ctcaagtcag 4320aggtggcgaa
acccgacagg actataaaga taccaggcgt ttccccctgg aagctccctc
4380gtgcgctctc ctgttccgac cctgccgctt accggatacc tgtccgcctt
tctcccttcg 4440ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc
tcagttcggt gtaggtcgtt 4500cgctccaagc tgggctgtgt gcacgaaccc
cccgttcagc ccgaccgctg cgccttatcc 4560ggtaactatc gtcttgagtc
caacccggta agacacgact tatcgccact ggcagcagcc 4620actggtaaca
ggattagcag agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg
4680tggcctaact acggctacac tagaagaaca gtatttggta tctgcgctct
gctgaagcca 4740gttaccttcg gaaaaagagt tggtagctct tgatccggca
aacaaaccac cgctggtagc 4800ggtggttttt ttgtttgcaa gcagcagatt
acgcgcagaa aaaaaggatc tcaagaagat 4860cctttgatct tttctacggg
gtctgacgct cagtggaacg aaaactcacg ttaagggatt 4920ttggtcatga
gattatcaaa aaggatcttc acctagatcc ttttaaatta aaaatgaagt
4980tttaaatcaa tctaaagtat atatgagtaa acttggtctg acagttacca
atgcttaatc 5040agtgaggcac ctatctcagc gatctgtcta tttcgttcat
ccatagttgc ctgactcccc 5100gtcgtgtaga taactacgat acgggagggc
ttaccatctg gccccagtgc tgcaatgata 5160ccgcgagacc cacgctcacc
ggctccagat ttatcagcaa taaaccagcc agccggaagg 5220gccgagcgca
gaagtggtcc tgcaacttta tccgcctcca tccagtctat taattgttgc
5280cgggaagcta gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt
tgccattgct 5340acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt
cattcagctc cggttcccaa 5400cgatcaaggc gagttacatg atcccccatg
ttgtgcaaaa aagcggttag ctccttcggt 5460cctccgatcg ttgtcagaag
taagttggcc gcagtgttat cactcatggt tatggcagca 5520ctgcataatt
ctcttactgt catgccatcc gtaagatgct tttctgtgac tggtgagtac
5580tcaaccaagt cattctgaga atagtgtatg cggcgaccga gttgctcttg
cccggcgtca 5640atacgggata ataccgcgcc acatagcaga actttaaaag
tgctcatcat tggaaaacgt 5700tcttcggggc gaaaactctc aaggatctta
ccgctgttga gatccagttc gatgtaaccc 5760actcgtgcac ccaactgatc
ttcagcatct tttactttca ccagcgtttc tgggtgagca 5820aaaacaggaa
ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa atgttgaata
5880ctcatactct tcctttttca atattattga agcatttatc agggttattg
tctcatgagc 5940ggatacatat ttgaatgtat ttagaaaaat aaacaaatag
gggttccgcg cacatttccc 6000cgaaaagtgc cacctgacgt ctaagaaacc
attattatca tgacattaac ctataaaaat 6060aggcgtatca cgaggccctt
tcgtctcgcg cgtttcggtg atgacggtga aaacctctga 6120cacatgcagc
tcccggagac ggtcacagct tgtctgtaag cggatgccgg gagcagacaa
6180gcccgtcagg gcgcgtcagc gggtgttggc gggtgtcggg gctggcttaa
ctatgcggca 6240tcagagcaga ttgtactgag agtgcaccat tcgacgctct
cccttatgcg actcctgcat 6300taggaagcag cccagtagta ggttgaggcc
gttgagcacc gccgccgcaa ggaatggtgc 6360atgcaaggag atggcgccca
acagtccccc ggccacgggg cctgccacca tacccacgcc 6420gaaacaagcg
ctcatgagcc cgaagtggcg agcccgatct tccccatcgg tgatgtcggc
6480gatataggcg ccagcaaccg cacctgtggc gccggtgatg ccggccacga
tgcgtccggc 6540gtagaggatc tggctagcga tgaccctgct gattggttcg
ctgaccattt ccgggtgcgg 6600gacggcgtta ccagaaactc agaaggttcg
tccaaccaaa ccgactctga cggcagttta 6660cgagagagat gatagggtct
gcttcagtaa gccagatgct acacaattag gcttgtacat 6720attgtcgtta
gaacgcggct acaattaata cataacctta tgtatcatac acatacgatt
6780taggtgacac tatagaatac acggaattaa ttc
6813217147DNAArtificialSynthetic sequence 21tagctgcgcg ctcgctcgct
cactgaggcc gcccgggcaa agcccgggcg tcgggcgacc 60tttggtcgcc cggcctcagt
gagcgagcga gcgcgcagag agggagtggc caactccatc 120actaggggtt
ccttgtagtt aatgattaac ccgccatgct acttatctac gtagccatgc
180tctagcgatc gcttaaccct agaaagatag tctgcgtaaa attgacgcat
gcattcttga 240aatattgctc tctctttcta aatagcgcga atccgtcgct
gtgcatttag gacatctcag 300tcgccgcttg gagctcccgt gaggcgtgct
tgtcaatgcg gtaagtgtca ctgattttga 360actataacga ccgcgtgagt
caaaatgacg catgattatc ttttacgtga cttttaagat 420ttaactcata
cgataattat attgttattt catgttctac ttacgtgata acttattata
480tatatatttt cttgttatag atatcttaat taactgcagg ctcagaggca
cacaggagtt 540tctgggctca ccctgccccc ttccaacccc tcagttccca
tcctccagca gctgtttgtg 600tgctgcctct gaagtccaca ctgaacaaac
ttcagcctac tcatgtccct aaaatgggca 660aacattgcaa gcagcaaaca
gcaaacacac agccctccct gcctgctgac cttggagctg 720gggcagaggt
cagagacctc tctgggccca tgccacctcc aacatccact cgaccccttg
780gaatttcggt ggagaggagc agaggttgtc ctggcgtggt ttaggtagtg
tgagagggtc 840cggcgattaa ctgcaggctc agaggcacac aggagtttct
gggctcaccc tgcccccttc 900caacccctca gttcccatcc tccagcagct
gtttgtgtgc tgcctctgaa gtccacactg 960aacaaacttc agcctactca
tgtccctaaa atgggcaaac attgcaagca gcaaacagca 1020aacacacagc
cctccctgcc tgctgacctt ggagctgggg cagaggtcag agacctctct
1080gggcccatgc cacctccaac atccactcga ccccttggaa tttcggtgga
gaggagcaga 1140ggttgtcctg gcgtggttta ggtagtgtga gagggtccgg
cgaattaaga tcttgctacc 1200agtggaacag ccactaagga ttctgcagtg
agagcagagg gccagctaag tggtactctc 1260ccagagactg tctgactcac
gccaccccct ccaccttgga cacaggacgc tgtggtttct 1320gagccaggta
caatgactcc tttcggtaag tgcagtggaa gctgtacact gcccaggcaa
1380agcgtccggg cagcgtaggc gggcgactca gatcccagcc agtggactta
gcccctgttt 1440gctcctccga taactggggt gaccttggtt aatattcacc
agcagcctcc cccgttgccc 1500ctctggatcc actgcttaaa tacggacgag
gacagggccc tgtctcctca gcttcaggca 1560ccaccactga cctgggacag
tgaatgcggc cgctctagaa ctagtggatc ccccgggctg 1620caggaattcg
ccgccaccat gctgtctaat ttgaggatcc tgctcaacaa tgcagctctt
1680agaaagggtc acacttctgt ggttcgacat ttttggtgtg ggaagccagt
ccaaagtcaa 1740gtacagctga aaggccgtga cctcctcacc ttgaagaact
tcacaggaga ggagattcag 1800tacatgctat ggctctctgc agatctgaaa
ttcaggatca agcagaaagg agaatattta 1860cctttattgc aagggaaatc
cttaggaatg attntttgag aaaagaagta ctcgaacaag 1920actgtccaca
gaaacaggct ttgctctgct gggaggacac ccttcctttc ttaccacaca
1980agacattcac ttgggtgtga atgaaagtct cacagacacc gctcgtgtct
tatctagcat 2040gacagatgca gtgttagctc gagtgtataa acaatcagat
ctggacaccc tggctaaaga 2100agcatccatc ccaattgtca atggactgtc
agacttgtat catcctatcc agatcctggc 2160tgattacctt acactccagg
aacactatgg ctctctcaaa ggtcttaccc tcagctggat 2220aggggatggg
aacaatatct tgcactctat catgatgagt gctgcaaaat tcgggatgca
2280ccttcaagca gctactccaa agggttatga gccagatcct aatatagtca
agctagcaga 2340gcagtatgcc aaggagaatg gtaccaagtt gtcaatgaca
aatgatccac tggaagcagc 2400acgtggaggc aatgtattaa ttacagatac
ttggataagc atgggacaag aggatgagaa 2460gaaaaagcgt cttcaagctt
tccaaggtta ccaggttacg atgaagactg ccaaagtggc 2520tgcgtctgac
tggacatttt tacactgttt gcctagaaag ccagaagaag tggatgatga
2580agtattttat tctccacggt cattagtgtt cccagaggca gagaatagaa
agtggacaat 2640catggctgtc atggtatccc tgctgacaga ctactcacct
gtgctccaga agccaaagtt 2700ttgagatatc aagcttatcg ataatcaacc
tctggattac aaaatttgtg aaagattgac 2760tggtattctt aactatgttg
ctccttttac gctatgtgga tacgctgctt taatgccttt 2820gtatcatgct
attgcttccc gtatggcttt cattttctcc tccttgtata aatcctggtt
2880gctgtctctt tatgaggagt
tgtggcccgt tgtcaggcaa cgtggcgtgg tgtgcactgt 2940gtttgctgac
gcaaccccca ctggttgggg cattgccacc acctgtcagc tcctttccgg
3000gactttcgct ttccccctcc ctattgccac ggcggaactc atcgccgcct
gccttgcccg 3060ctgctggaca ggggctcggc tgttgggcac tgacaattcc
gtggtgttgt cggggaaatc 3120atcgtccttt ccttggctgc tcgcctgtgt
tgccacctgg attctgcgcg ggacgtcctt 3180ctgctacgtc ccttcggccc
tcaatccagc ggaccttcct tcccgcggcc tgctgccggc 3240tctgcggcct
cttccgcgtc ttcgccttcg ccctcagacg agtcggatct ccctttgggc
3300cgcctccccg catcgatacc gtcgactcgc tgatcagcct cgactgtgcc
ttctagttgc 3360cagccatctg ttgtttgccc ctcccccgtg ccttccttga
ccctggaagg tgccactccc 3420actgtccttt cctaataaaa tgaggaaatt
gcatcgcatt gtctgagtag gtgtcattct 3480attctggggg gtggggtggg
gcaggacagc aagggggagg attgggaaga caatagcagg 3540catgctgggg
atgcggtggg ctctatggct tctgaggcgg aaagaacggc cggcctttgt
3600tactttatag aagaaatttt gagtttttgt ttttttttaa taaataaata
aacataaata 3660aattgtttgt tgaatttatt attagtatgt aagtgtaaat
ataataaaac ttaatatcta 3720ttcaaattaa taaataaacc tcgatataca
gaccgataaa acacatgcgt caattttacg 3780catgattatc tttaacgtac
gtcacaatat gattatcttt ctagggttaa cctgcaggta 3840gagcatggct
acgtagataa gtagcatggc gggttaatca ttaactacaa ggaaccccta
3900gtgatggagt tggccactcc ctctctgcgc gctcgctcgc tcactgaggc
cgggcgacca 3960aaggtcgccc gacgcccggg ctttgcccgg gcggcctcag
tgagcgagcg agcgcgcaga 4020gctttttgca aaagcctagg cctccaaaaa
agcctcctca ctacttctgg aatagctcag 4080aggccgaggc ggcctcggcc
tctgcataaa taaaaaaaat tagtcagcca tggggcggag 4140aatgggcgga
actgggcgga gttaggggcg ggatgggcgg agttaggggc gggactatgg
4200ttgctgacta attgagatgc atgctttgca tacttctgcc tgctggggag
cctggggact 4260ttccacacct ggttgctgac taattgagat gcatgctttg
catacttctg cctgctgggg 4320agcctgggga ctttccacac cctaactgac
acacattcca cagctgcatt aatgaatcgg 4380ccaacgcgcg gggagaggcg
gtttgcgtat tgggcgctct tccgcttcct cgctcactga 4440ctcgctgcgc
tcggtcgttc ggctgcggcg agcggtatca gctcactcaa aggcggtaat
4500acggttatcc acagaatcag gggataacgc aggaaagaac atgtgagcaa
aaggccagca 4560aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt
ttccataggc tccgcccccc 4620tgacgagcat cacaaaaatc gacgctcaag
tcagaggtgg cgaaacccga caggactata 4680aagataccag gcgtttcccc
ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc 4740gcttaccgga
tacctgtccg cctttctccc ttcgggaagc gtggcgcttt ctcatagctc
4800acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc aagctgggct
gtgtgcacga 4860accccccgtt cagcccgacc gctgcgcctt atccggtaac
tatcgtcttg agtccaaccc 4920ggtaagacac gacttatcgc cactggcagc
agccactggt aacaggatta gcagagcgag 4980gtatgtaggc ggtgctacag
agttcttgaa gtggtggcct aactacggct acactagaag 5040aacagtattt
ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa gagttggtag
5100ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt
gcaagcagca 5160gattacgcgc agaaaaaaag gatctcaaga agatcctttg
atcttttcta cggggtctga 5220cgctcagtgg aacgaaaact cacgttaagg
gattttggtc atgagattat caaaaaggat 5280cttcacctag atccttttaa
attaaaaatg aagttttaaa tcaatctaaa gtatatatga 5340gtaaacttgg
tctgacagtt accaatgctt aatcagtgag gcacctatct cagcgatctg
5400tctatttcgt tcatccatag ttgcctgact ccccgtcgtg tagataacta
cgatacggga 5460gggcttacca tctggcccca gtgctgcaat gataccgcga
gacccacgct caccggctcc 5520agatttatca gcaataaacc agccagccgg
aagggccgag cgcagaagtg gtcctgcaac 5580tttatccgcc tccatccagt
ctattaattg ttgccgggaa gctagagtaa gtagttcgcc 5640agttaatagt
ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc
5700gtttggtatg gcttcattca gctccggttc ccaacgatca aggcgagtta
catgatcccc 5760catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg
atcgttgtca gaagtaagtt 5820ggccgcagtg ttatcactca tggttatggc
agcactgcat aattctctta ctgtcatgcc 5880atccgtaaga tgcttttctg
tgactggtga gtactcaacc aagtcattct gagaatagtg 5940tatgcggcga
ccgagttgct cttgcccggc gtcaatacgg gataataccg cgccacatag
6000cagaacttta aaagtgctca tcattggaaa acgttcttcg gggcgaaaac
tctcaaggat 6060cttaccgctg ttgagatcca gttcgatgta acccactcgt
gcacccaact gatcttcagc 6120atcttttact ttcaccagcg tttctgggtg
agcaaaaaca ggaaggcaaa atgccgcaaa 6180aaagggaata agggcgacac
ggaaatgttg aatactcata ctcttccttt ttcaatatta 6240ttgaagcatt
tatcagggtt attgtctcat gagcggatac atatttgaat gtatttagaa
6300aaataaacaa ataggggttc cgcgcacatt tccccgaaaa gtgccacctg
acgtctaaga 6360aaccattatt atcatgacat taacctataa aaataggcgt
atcacgaggc cctttcgtct 6420cgcgcgtttc ggtgatgacg gtgaaaacct
ctgacacatg cagctcccgg agacggtcac 6480agcttgtctg taagcggatg
ccgggagcag acaagcccgt cagggcgcgt cagcgggtgt 6540tggcgggtgt
cggggctggc ttaactatgc ggcatcagag cagattgtac tgagagtgca
6600ccattcgacg ctctccctta tgcgactcct gcattaggaa gcagcccagt
agtaggttga 6660ggccgttgag caccgccgcc gcaaggaatg gtgcatgcaa
ggagatggcg cccaacagtc 6720ccccggccac ggggcctgcc accataccca
cgccgaaaca agcgctcatg agcccgaagt 6780ggcgagcccg atcttcccca
tcggtgatgt cggcgatata ggcgccagca accgcacctg 6840tggcgccggt
gatgccggcc acgatgcgtc cggcgtagag gatctggcta gcgatgaccc
6900tgctgattgg ttcgctgacc atttccgggt gcgggacggc gttaccagaa
actcagaagg 6960ttcgtccaac caaaccgact ctgacggcag tttacgagag
agatgatagg gtctgcttca 7020gtaagccaga tgctacacaa ttaggcttgt
acatattgtc gttagaacgc ggctacaatt 7080aatacataac cttatgtatc
atacacatac gatttaggtg acactataga atacacggaa 7140ttaattc
7147227284DNAArtificialSynthetic sequence 22tagctgcgcg ctcgctcgct
cactgaggcc gcccgggcaa agcccgggcg tcgggcgacc 60tttggtcgcc cggcctcagt
gagcgagcga gcgcgcagag agggagtggc caactccatc 120actaggggtt
ccttgtagtt aatgattaac ccgccatgct acttatctac gtagccatgc
180tctagcgatc gcttaaccct agaaagatag tctgcgtaaa attgacgcat
gcattcttga 240aatattgctc tctctttcta aatagcgcga atccgtcgct
gtgcatttag gacatctcag 300tcgccgcttg gagctcccgt gaggcgtgct
tgtcaatgcg gtaagtgtca ctgattttga 360actataacga ccgcgtgagt
caaaatgacg catgattatc ttttacgtga cttttaagat 420ttaactcata
cgataattat attgttattt catgttctac ttacgtgata acttattata
480tatatatttt cttgttatag atatcttaat taactgcagg ctcagaggca
cacaggagtt 540tctgggctca ccctgccccc ttccaacccc tcagttccca
tcctccagca gctgtttgtg 600tgctgcctct gaagtccaca ctgaacaaac
ttcagcctac tcatgtccct aaaatgggca 660aacattgcaa gcagcaaaca
gcaaacacac agccctccct gcctgctgac cttggagctg 720gggcagaggt
cagagacctc tctgggccca tgccacctcc aacatccact cgaccccttg
780gaatttcggt ggagaggagc agaggttgtc ctggcgtggt ttaggtagtg
tgagagggtc 840cggcgattaa ctgcaggctc agaggcacac aggagtttct
gggctcaccc tgcccccttc 900caacccctca gttcccatcc tccagcagct
gtttgtgtgc tgcctctgaa gtccacactg 960aacaaacttc agcctactca
tgtccctaaa atgggcaaac attgcaagca gcaaacagca 1020aacacacagc
cctccctgcc tgctgacctt ggagctgggg cagaggtcag agacctctct
1080gggcccatgc cacctccaac atccactcga ccccttggaa tttcggtgga
gaggagcaga 1140ggttgtcctg gcgtggttta ggtagtgtga gagggtccgg
cgaattaaga tcttgctacc 1200agtggaacag ccactaagga ttctgcagtg
agagcagagg gccagctaag tggtactctc 1260ccagagactg tctgactcac
gccaccccct ccaccttgga cacaggacgc tgtggtttct 1320gagccaggta
caatgactcc tttcggtaag tgcagtggaa gctgtacact gcccaggcaa
1380agcgtccggg cagcgtaggc gggcgactca gatcccagcc agtggactta
gcccctgttt 1440gctcctccga taactggggt gaccttggtt aatattcacc
agcagcctcc cccgttgccc 1500ctctggatcc actgcttaaa tacggacgag
gacagggccc tgtctcctca gcttcaggca 1560ccaccactga cctgggacag
tgaatgcggc cgcgccgcca ccatgtccag caagggctct 1620gtggttctgg
cctacagtgg tggcctggac acctcctgca tcctcgtgtg gctgaaggaa
1680caaggctatg atgtcatcgc ctacctggcc aacattggcc agaaggaaga
ctttgaggaa 1740gccaggaaga aggcgctgaa gcttggggcc aaaaaggtgt
tcattgagga tgtgagcaag 1800gaatttgtgg aagagttcat ctggcctgct
gtccagtcca gtgcactcta cgaggaccgc 1860tatctcctgg gcacctctct
cgccaggcct tgcatagctc gcagacaggt ggagattgcc 1920cagcgtgaag
gggccaagta tgtgtctcac ggcgccacgg gaaaggggaa tgaccaggtc
1980cgctttgagc tcacctgcta ttcactggca ccccagatta aggtcatcgc
tccctggagg 2040atgcctgagt tttacaaccg gttcaagggc cgaaatgatc
tgatggagta tgcaaagcaa 2100cacggaatcc ccatccctgt cacccccaag
agcccctgga gtatggatga aaacctcatg 2160cacatcagct atgaggctgg
gatcctggaa aaccccaaga atcaagcacc tccgggtctc 2220tacacaaaaa
ctcaggaccc tgccaaagca cccaacagcc cagatgtcct tgagatagaa
2280ttcaaaaaag gggtccctgt gaaggtgacc aacatcaaag atggcacaac
ccgcaccaca 2340tccctggaac tcttcatgta cctgaacgaa gttgcgggca
agcacggagt gggtcgcatt 2400gacatcgtgg agaaccgctt cattggaatg
aagtcccgag gtatctacga gaccccagca 2460gggaccatcc tttaccacgc
tcatttagac atagaggcct tcacgatgga tcgggaagta 2520cgcaaaatca
agcagggcct gggcctcaaa ttcgcagagc tcgtatacac aggtttctgg
2580cacagccctg aatgtgaatt tgttcgccac tgtatccaga agtcccagga
gcgggtagaa 2640gggaaggtgc aggtgtctgt cttcaagggc caagtgtaca
tcctcggtcg ggagtctcca 2700ctttcactct acaatgaaga gctggtgagc
atgaacgtgc agggcgacta tgagcccatc 2760gacgccactg gcttcatcaa
tatcaactcg ctcaggctga aggagtacca tcgccttcag 2820agcaaggtca
ctgccaaata ggatatcaag cttatcgata atcaacctct ggattacaaa
2880atttgtgaaa gattgactgg tattcttaac tatgttgctc cttttacgct
atgtggatac 2940gctgctttaa tgcctttgta tcatgctatt gcttcccgta
tggctttcat tttctcctcc 3000ttgtataaat cctggttgct gtctctttat
gaggagttgt ggcccgttgt caggcaacgt 3060ggcgtggtgt gcactgtgtt
tgctgacgca acccccactg gttggggcat tgccaccacc 3120tgtcagctcc
tttccgggac tttcgctttc cccctcccta ttgccacggc ggaactcatc
3180gccgcctgcc ttgcccgctg ctggacaggg gctcggctgt tgggcactga
caattccgtg 3240gtgttgtcgg ggaaatcatc gtcctttcct tggctgctcg
cctgtgttgc cacctggatt 3300ctgcgcggga cgtccttctg ctacgtccct
tcggccctca atccagcgga ccttccttcc 3360cgcggcctgc tgccggctct
gcggcctctt ccgcgtcttc gccttcgccc tcagacgagt 3420cggatctccc
tttgggccgc ctccccgcat cgataccgtc gactcgctga tcagcctcga
3480ctgtgccttc tagttgccag ccatctgttg tttgcccctc ccccgtgcct
tccttgaccc 3540tggaaggtgc cactcccact gtcctttcct aataaaatga
ggaaattgca tcgcattgtc 3600tgagtaggtg tcattctatt ctggggggtg
gggtggggca ggacagcaag ggggaggatt 3660gggaagacaa tagcaggcat
gctggggatg cggtgggctc tatggcttct gaggcggaaa 3720gaacggccgg
cctttgttac tttatagaag aaattttgag tttttgtttt tttttaataa
3780ataaataaac ataaataaat tgtttgttga atttattatt agtatgtaag
tgtaaatata 3840ataaaactta atatctattc aaattaataa ataaacctcg
atatacagac cgataaaaca 3900catgcgtcaa ttttacgcat gattatcttt
aacgtacgtc acaatatgat tatctttcta 3960gggttaacct gcaggtagag
catggctacg tagataagta gcatggcggg ttaatcatta 4020actacaagga
acccctagtg atggagttgg ccactccctc tctgcgcgct cgctcgctca
4080ctgaggccgg gcgaccaaag gtcgcccgac gcccgggctt tgcccgggcg
gcctcagtga 4140gcgagcgagc gcgcagagct ttttgcaaaa gcctaggcct
ccaaaaaagc ctcctcacta 4200cttctggaat agctcagagg ccgaggcggc
ctcggcctct gcataaataa aaaaaattag 4260tcagccatgg ggcggagaat
gggcggaact gggcggagtt aggggcggga tgggcggagt 4320taggggcggg
actatggttg ctgactaatt gagatgcatg ctttgcatac ttctgcctgc
4380tggggagcct ggggactttc cacacctggt tgctgactaa ttgagatgca
tgctttgcat 4440acttctgcct gctggggagc ctggggactt tccacaccct
aactgacaca cattccacag 4500ctgcattaat gaatcggcca acgcgcgggg
agaggcggtt tgcgtattgg gcgctcttcc 4560gcttcctcgc tcactgactc
gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct 4620cactcaaagg
cggtaatacg gttatccaca gaatcagggg ataacgcagg aaagaacatg
4680tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct
ggcgtttttc 4740cataggctcc gcccccctga cgagcatcac aaaaatcgac
gctcaagtca gaggtggcga 4800aacccgacag gactataaag ataccaggcg
tttccccctg gaagctccct cgtgcgctct 4860cctgttccga ccctgccgct
taccggatac ctgtccgcct ttctcccttc gggaagcgtg 4920gcgctttctc
atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag
4980ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct gcgccttatc
cggtaactat 5040cgtcttgagt ccaacccggt aagacacgac ttatcgccac
tggcagcagc cactggtaac 5100aggattagca gagcgaggta tgtaggcggt
gctacagagt tcttgaagtg gtggcctaac 5160tacggctaca ctagaagaac
agtatttggt atctgcgctc tgctgaagcc agttaccttc 5220ggaaaaagag
ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggtggtttt
5280tttgtttgca agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga
tcctttgatc 5340ttttctacgg ggtctgacgc tcagtggaac gaaaactcac
gttaagggat tttggtcatg 5400agattatcaa aaaggatctt cacctagatc
cttttaaatt aaaaatgaag ttttaaatca 5460atctaaagta tatatgagta
aacttggtct gacagttacc aatgcttaat cagtgaggca 5520cctatctcag
cgatctgtct atttcgttca tccatagttg cctgactccc cgtcgtgtag
5580ataactacga tacgggaggg cttaccatct ggccccagtg ctgcaatgat
accgcgagac 5640ccacgctcac cggctccaga tttatcagca ataaaccagc
cagccggaag ggccgagcgc 5700agaagtggtc ctgcaacttt atccgcctcc
atccagtcta ttaattgttg ccgggaagct 5760agagtaagta gttcgccagt
taatagtttg cgcaacgttg ttgccattgc tacaggcatc 5820gtggtgtcac
gctcgtcgtt tggtatggct tcattcagct ccggttccca acgatcaagg
5880cgagttacat gatcccccat gttgtgcaaa aaagcggtta gctccttcgg
tcctccgatc 5940gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg
ttatggcagc actgcataat 6000tctcttactg tcatgccatc cgtaagatgc
ttttctgtga ctggtgagta ctcaaccaag 6060tcattctgag aatagtgtat
gcggcgaccg agttgctctt gcccggcgtc aatacgggat 6120aataccgcgc
cacatagcag aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg
6180cgaaaactct caaggatctt accgctgttg agatccagtt cgatgtaacc
cactcgtgca 6240cccaactgat cttcagcatc ttttactttc accagcgttt
ctgggtgagc aaaaacagga 6300aggcaaaatg ccgcaaaaaa gggaataagg
gcgacacgga aatgttgaat actcatactc 6360ttcctttttc aatattattg
aagcatttat cagggttatt gtctcatgag cggatacata 6420tttgaatgta
tttagaaaaa taaacaaata ggggttccgc gcacatttcc ccgaaaagtg
6480ccacctgacg tctaagaaac cattattatc atgacattaa cctataaaaa
taggcgtatc 6540acgaggccct ttcgtctcgc gcgtttcggt gatgacggtg
aaaacctctg acacatgcag 6600ctcccggaga cggtcacagc ttgtctgtaa
gcggatgccg ggagcagaca agcccgtcag 6660ggcgcgtcag cgggtgttgg
cgggtgtcgg ggctggctta actatgcggc atcagagcag 6720attgtactga
gagtgcacca ttcgacgctc tcccttatgc gactcctgca ttaggaagca
6780gcccagtagt aggttgaggc cgttgagcac cgccgccgca aggaatggtg
catgcaagga 6840gatggcgccc aacagtcccc cggccacggg gcctgccacc
atacccacgc cgaaacaagc 6900gctcatgagc ccgaagtggc gagcccgatc
ttccccatcg gtgatgtcgg cgatataggc 6960gccagcaacc gcacctgtgg
cgccggtgat gccggccacg atgcgtccgg cgtagaggat 7020ctggctagcg
atgaccctgc tgattggttc gctgaccatt tccgggtgcg ggacggcgtt
7080accagaaact cagaaggttc gtccaaccaa accgactctg acggcagttt
acgagagaga 7140tgatagggtc tgcttcagta agccagatgc tacacaatta
ggcttgtaca tattgtcgtt 7200agaacgcggc tacaattaat acataacctt
atgtatcata cacatacgat ttaggtgaca 7260ctatagaata cacggaatta attc
7284238086DNAArtificialSynthetic sequence 23tagctgcgcg ctcgctcgct
cactgaggcc gcccgggcaa agcccgggcg tcgggcgacc 60tttggtcgcc cggcctcagt
gagcgagcga gcgcgcagag agggagtggc caactccatc 120actaggggtt
ccttgtagtt aatgattaac ccgccatgct acttatctac gtagccatgc
180tctaggatat cggttaaccc tagaaagata atcatattgt gacgtacgtt
aaagataatc 240atgcgtaaaa ttgacgcatg aatcactagt ccctaaaatg
ggcaaacatt gcaagcagca 300aacagcaaac acacagccct ccctgcctgc
tgaccttgga gctggggcag aggtcagaga 360cctctctggg cccatgccac
ctccaacatc cactcgaccc cttggaattt cggtggagag 420gagcagaggt
tgtcctggcg tggtttaggt agtgtgagag gggaatgact cctttcggta
480agtgcagtgg aagctgtaca ctgcccaggc aaagcgtccg ggcagcgtag
gcgggcgact 540cagatcccag ccagtggact tagcccctgt ttgctcctcc
gataactggg gtgaccttgg 600ttaatattca ccagcagcct cccccgttgc
ccctctggat ccactgctta aatacggacg 660aggacagggc cctgtctcct
cagcttcagg caccaccact gacctgggac agtgaattgc 720tctagagccg
ccaccatgga cctggaggca gcaaagaatg gaaccgcatg gagaccaaca
780tcagcagagg gggacttcga actgggtatt tcaagcaagc agaagcgcaa
gaaaactaag 840accgtgaaaa tgatcggggt cctcaccctg ttccgatact
ccgactggca ggataagctc 900tttatgtctc tgggcacaat catggccatt
gctcacgggt ctggtctccc tctgatgatg 960atcgtgttcg gggagatgac
cgacaaattt gtcgatacag ccggtaattt cagctttcca 1020gtgaacttct
ctctcagtct gctcaacccc ggcaagatcc tggaggaaga gatgactcgc
1080tatgcatact attactctgg actgggagct ggggtgctgg tcgcagctta
catccaggtg 1140agtttctgga ccctggcagc tggacggcag atccgcaaaa
ttcgacagaa gttctttcat 1200gccatcctga gacaggagat tgggtggttt
gacatcaatg ataccacaga actcaacacc 1260cggctgacag acgacatcag
caaaatttcc gagggtatcg gcgataaagt gggaatgttc 1320tttcaggcag
tcgccacttt ctttgccgga ttcattgtcg ggtttatccg gggttggaag
1380ctgaccctgg tcatcatggc tatttcacca atcctcgggc tgagcgccgc
agtgtgggca 1440aagatcctct ctgccttcag tgacaaagag ctggccgctt
atgctaaggc aggagctgtg 1500gctgaagagg cactgggagc aattcgaacc
gtgatcgcct ttggcggaca gaataaggaa 1560ctcgagaggt accagaaaca
cctggagaac gctaaggaaa tcgggattaa gaaagctatt 1620tccgcaaaca
tctctatggg tattgctttc ctgctcatct atgcatctta cgcactcgcc
1680ttttggtatg gcagcaccct ggtcatcagc aaggagtaca ctatcggaaa
tgcaatgacc 1740gtcttctttt ctatcctgat tggggctttc agtgtgggtc
aggcagcccc ctgcatcgac 1800gctttcgcaa atgcacgcgg cgctgcatac
gtgatcttcg acatcattga taacaaccct 1860aagatcgact cattcagcga
gagggggcac aaaccagata gcattaaggg taatctggaa 1920ttcaacgacg
tgcatttttc ataccctagc agagccaatg tcaagatcct gaaaggactc
1980aacctgaaag tgcagagcgg gcagactgtg gctctggtcg gtagctccgg
atgcgggaag 2040tccactaccg tgcagctcat tcagcggctg tatgacccag
atgagggcac aatcaacatt 2100gacggacagg acatccgcaa cttcaatgtc
aactacctgc gagagatcat tggcgtggtc 2160tcacaggaac ccgtgctgtt
tagcacaact atcgccgaga atatttgtta tggtagaggc 2220aacgtgacaa
tggatgaaat taagaaagct gtcaaggagg ctaatgcata cgaattcatc
2280atgaaactcc ctcagaagtt tgatactctg gtgggcgaga ggggcgccca
gctgagcggg 2340ggtcagaaac agcgcatcgc cattgctcga gcactggtga
ggaacccaaa gatcctgctc 2400ctggacgagg ccacatccgc tctggatact
gaatctgagg ccgaagtgca ggccgctctg 2460gacaaggcta gggaaggcag
aaccacaatc gtgattgccc acagactgag caccgtgcgg 2520aatgccgacg
tgattgctgg cttcgaggat ggagtgatcg tcgaacaggg ctcccattct
2580gagctgatga agaaagaagg agtgtatttc aagctggtca acatgcagac
aagtggctca 2640cagatccagt ccgaagagtt tgagctgaat gacgaaaaag
cagccacaag gatggcccca 2700aacggatgga agagtcggct cttccgccac
tcaactcaga agaatctgaa aaacagccag 2760atgtgccaga agtccctcga
cgtggagacc gatgggctgg aagctaatgt gccccctgtc 2820tccttcctga
aggtgctcaa actgaacaag accgagtggc cctactttgt ggtcggcaca
2880gtctgcgcca tcgctaatgg cggactgcag cccgccttca gcgtgatctt
cagcgaaatc 2940attgctatct tcggacctgg ggacgatgca gtgaaacagc
agaagtgtaa catctttagt 3000ctgattttcc tctttctggg catcatttca
ttctttacat tctttctgca gggattcact 3060tttggaaagg ccggggagat
cctcaccagg agactgagga gcatggcatt caaagccatg 3120ctgagacagg
atatgtcctg gtttgacgat cataagaatt ctacaggcgc cctcagtact
3180agactggcta ccgacgctgc acaggtgcag ggtgcaacag gcactcggct
cgctctgatc 3240gcacagaaca ttgcaaatct cgggactgga atcattatct
cctttatcta tggttggcag 3300ttaaccctgc tgctgctggc
cgtggtgccc atcattgccg tgtccggcat cgtggaaatg 3360aaactgctgg
ctggaaacgc taagagagat aagaaagaac tggaggctgc tggaaaaatc
3420gctaccgagg ctattgagaa cattagaacc gtggtctctc tcacacagga
gcggaagttc 3480gaaagtatgt acgtggagaa actgtacggg ccatatcgaa
acagtgtgca gaaggcccac 3540atctatggta ttacattttc aatcagccag
gccttcatgt actttagcta tgctgggtgc 3600ttccgctttg gtgcatatct
gatcgtgaat ggccatatga ggttcagaga cgtgatcctc 3660gtcttcagcg
ccatcgtgtt cggagctgtc gctctgggac acgccagctc ctttgctccc
3720gattacgcaa aggccaaact gtccgccgct catctcttca tgctgtttga
gagacagcct 3780ctcatcgact cctattctga ggaaggcctg aagccagata
aattcgaggg aaacattaca 3840ttcaatgaag tggtctttaa ctaccccact
cgggctaatg tgcctgtcct gcagggactc 3900tccctggaag tgaagaaagg
gcagactctc gccctggtcg gttctagtgg gtgcggcaag 3960tctaccgtgg
tccagctgct cgagcggttt tacgaccccc tggcagggac tgtgctgctc
4020gatggtcagg aagctaagaa actgaacgtg cagtggctga gagcacagct
gggaatcgtc 4080tcacaggagc ctattctgtt cgactgtagc atcgcagaaa
acattgccta tggagacaat 4140agtagggtgg tctcacagga tgagatcgtg
tctgcagcca aggctgcaaa tatccacccc 4200ttcatcgaga cactgcccca
taagtacgaa actcgcgtgg gcgataaagg aacccagctg 4260agcggcggac
agaaacagcg aatcgctatt gcacgagccc tgatcaggca gccccagatt
4320ctgctcctgg acgaggctac tagcgcactc gataccgagt ccgaaaaggt
ggtccaggag 4380gctctggaca aagcacggga aggccgcacc tgtatcgtga
ttgcccacag gctcagcaca 4440atccagaacg ctgatctgat tgtggtcttc
cagaatggca gagtgaagga gcacggaaca 4500catcagcagc tcctggcaca
gaagggaatc tatttttcaa tggtctccgt ccaggcaggc 4560actcagaatc
tctaaaagct tgcagttatc gatatgcttt atttgtgaaa tttgtgatgc
4620tattgcttta tttgtaacca ttataagctg caataaacaa gttaacaaca
acaattgcat 4680tcattttatg tttcaggttc agggggaggt gtgggaggtt
ttttaaacgc gtgcatgcgt 4740caattttacg cagactatct ttctagggtt
aagcaccggt agcatggcta cgtagataag 4800tagcatggcg ggttaatcat
taactacaag gaacccctag tgatggagtt ggccactccc 4860tctctgcgcg
ctcgctcgct cactgaggcc gggcgaccaa aggtcgcccg acgcccgggc
4920tttgcccggg cggcctcagt gagcgagcga gcgcgcagag ctttttgcaa
aagcctaggc 4980ctccaaaaaa gcctcctcac tacttctgga atagctcaga
ggccgaggcg gcctcggcct 5040ctgcataaat aaaaaaaatt agtcagccat
ggggcggaga atgggcggaa ctgggcggag 5100ttaggggcgg gatgggcgga
gttaggggcg ggactatggt tgctgactaa ttgagatgca 5160tgctttgcat
acttctgcct gctggggagc ctggggactt tccacacctg gttgctgact
5220aattgagatg catgctttgc atacttctgc ctgctgggga gcctggggac
tttccacacc 5280ctaactgaca cacattccac agctgcatta atgaatcggc
caacgcgcgg ggagaggcgg 5340tttgcgtatt gggcgctctt ccgcttcctc
gctcactgac tcgctgcgct cggtcgttcg 5400gctgcggcga gcggtatcag
ctcactcaaa ggcggtaata cggttatcca cagaatcagg 5460ggataacgca
ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa
5520ggccgcgttg ctggcgtttt tccataggct ccgcccccct gacgagcatc
acaaaaatcg 5580acgctcaagt cagaggtggc gaaacccgac aggactataa
agataccagg cgtttccccc 5640tggaagctcc ctcgtgcgct ctcctgttcc
gaccctgccg cttaccggat acctgtccgc 5700ctttctccct tcgggaagcg
tggcgctttc tcatagctca cgctgtaggt atctcagttc 5760ggtgtaggtc
gttcgctcca agctgggctg tgtgcacgaa ccccccgttc agcccgaccg
5820ctgcgcctta tccggtaact atcgtcttga gtccaacccg gtaagacacg
acttatcgcc 5880actggcagca gccactggta acaggattag cagagcgagg
tatgtaggcg gtgctacaga 5940gttcttgaag tggtggccta actacggcta
cactagaaga acagtatttg gtatctgcgc 6000tctgctgaag ccagttacct
tcggaaaaag agttggtagc tcttgatccg gcaaacaaac 6060caccgctggt
agcggtggtt tttttgtttg caagcagcag attacgcgca gaaaaaaagg
6120atctcaagaa gatcctttga tcttttctac ggggtctgac gctcagtgga
acgaaaactc 6180acgttaaggg attttggtca tgagattatc aaaaaggatc
ttcacctaga tccttttaaa 6240ttaaaaatga agttttaaat caatctaaag
tatatatgag taaacttggt ctgacagtta 6300ccaatgctta atcagtgagg
cacctatctc agcgatctgt ctatttcgtt catccatagt 6360tgcctgactc
cccgtcgtgt agataactac gatacgggag ggcttaccat ctggccccag
6420tgctgcaatg ataccgcgag acccacgctc accggctcca gatttatcag
caataaacca 6480gccagccgga agggccgagc gcagaagtgg tcctgcaact
ttatccgcct ccatccagtc 6540tattaattgt tgccgggaag ctagagtaag
tagttcgcca gttaatagtt tgcgcaacgt 6600tgttgccatt gctacaggca
tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag 6660ctccggttcc
caacgatcaa ggcgagttac atgatccccc atgttgtgca aaaaagcggt
6720tagctccttc ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt
tatcactcat 6780ggttatggca gcactgcata attctcttac tgtcatgcca
tccgtaagat gcttttctgt 6840gactggtgag tactcaacca agtcattctg
agaatagtgt atgcggcgac cgagttgctc 6900ttgcccggcg tcaatacggg
ataataccgc gccacatagc agaactttaa aagtgctcat 6960cattggaaaa
cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag
7020ttcgatgtaa cccactcgtg cacccaactg atcttcagca tcttttactt
tcaccagcgt 7080ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa
aagggaataa gggcgacacg 7140gaaatgttga atactcatac tcttcctttt
tcaatattat tgaagcattt atcagggtta 7200ttgtctcatg agcggataca
tatttgaatg tatttagaaa aataaacaaa taggggttcc 7260gcgcacattt
ccccgaaaag tgccacctga cgtctaagaa accattatta tcatgacatt
7320aacctataaa aataggcgta tcacgaggcc ctttcgtctc gcgcgtttcg
gtgatgacgg 7380tgaaaacctc tgacacatgc agctcccgga gacggtcaca
gcttgtctgt aagcggatgc 7440cgggagcaga caagcccgtc agggcgcgtc
agcgggtgtt ggcgggtgtc ggggctggct 7500taactatgcg gcatcagagc
agattgtact gagagtgcac cattcgacgc tctcccttat 7560gcgactcctg
cattaggaag cagcccagta gtaggttgag gccgttgagc accgccgccg
7620caaggaatgg tgcatgcaag gagatggcgc ccaacagtcc cccggccacg
gggcctgcca 7680ccatacccac gccgaaacaa gcgctcatga gcccgaagtg
gcgagcccga tcttccccat 7740cggtgatgtc ggcgatatag gcgccagcaa
ccgcacctgt ggcgccggtg atgccggcca 7800cgatgcgtcc ggcgtagagg
atctggctag cgatgaccct gctgattggt tcgctgacca 7860tttccgggtg
cgggacggcg ttaccagaaa ctcagaaggt tcgtccaacc aaaccgactc
7920tgacggcagt ttacgagaga gatgataggg tctgcttcag taagccagat
gctacacaat 7980taggcttgta catattgtcg ttagaacgcg gctacaatta
atacataacc ttatgtatca 8040tacacatacg atttaggtga cactatagaa
tacacggaat taattc 8086
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