U.S. patent application number 16/136926 was filed with the patent office on 2019-01-10 for aadc polynucleotides for the treatment of parkinson's disease.
The applicant listed for this patent is VOYAGER THERAPEUTICS, INC.. Invention is credited to Adrian Philip Kells, Robert Kotin, Bernard Ravina.
Application Number | 20190008931 16/136926 |
Document ID | / |
Family ID | 55910029 |
Filed Date | 2019-01-10 |
United States Patent
Application |
20190008931 |
Kind Code |
A1 |
Kotin; Robert ; et
al. |
January 10, 2019 |
AADC POLYNUCLEOTIDES FOR THE TREATMENT OF PARKINSON'S DISEASE
Abstract
The disclosure relates to compositions and methods for the
preparation, manufacture and therapeutic use of polynucleotides
encoding AADC for the treatment of Parkinson's Disease.
Inventors: |
Kotin; Robert; (Bethesda,
MD) ; Kells; Adrian Philip; (Cambridge, MA) ;
Ravina; Bernard; (Newton, MA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
VOYAGER THERAPEUTICS, INC. |
CAMBRIDGE |
MA |
US |
|
|
Family ID: |
55910029 |
Appl. No.: |
16/136926 |
Filed: |
September 20, 2018 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15524986 |
May 5, 2017 |
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PCT/US2015/059201 |
Nov 5, 2015 |
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16136926 |
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62075298 |
Nov 5, 2014 |
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62155692 |
May 1, 2015 |
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62199578 |
Jul 31, 2015 |
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62243537 |
Oct 19, 2015 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12N 2830/42 20130101;
C12N 7/00 20130101; C12N 9/88 20130101; A61K 38/51 20130101; C12N
2830/50 20130101; C12N 15/86 20130101; C12Y 401/01028 20130101;
A61K 35/76 20130101; A61P 25/14 20180101; C12N 2750/14143 20130101;
C12N 2750/14121 20130101; C12N 2800/22 20130101; A61K 48/005
20130101 |
International
Class: |
A61K 38/51 20060101
A61K038/51; C12N 9/88 20060101 C12N009/88; A61P 25/14 20060101
A61P025/14; A61K 35/76 20060101 A61K035/76; C12N 7/00 20060101
C12N007/00; A61K 48/00 20060101 A61K048/00; C12N 15/86 20060101
C12N015/86 |
Claims
1. An AAV vector genome comprising, in order: a) a 5' inverted
terminal repeat (ITR), wherein said 5' ITR is 141 nucleotides in
length; b) an AADC sequence region, said AADC sequence region
comprising a nucleotide sequence encoding SEQ ID NO: 1, c) a 3'
ITR, wherein said 3' ITR is 141 nucleotides in length.
2. The AAV vector genome of claim 1, wherein the 5' ITR and the 3'
ITR are derived from AAV2.
3. The AAV vector genome of claim 2, wherein the AAV vector genome
comprises a cytomegalovirus (CMV) sequence region derived from a
CMV gene; wherein the CMV sequence region comprises an enhancer
region and promoter region.
4. The AAV vector genome of claim 3, wherein the CMV sequence
region is 507 nucleotides in length.
5. The AAV vector genome of claim 2, wherein the AAV vector genome
comprises an immediate early 1 (IE1) sequence region derived from
an IE1 gene.
6. The AAV vector genome of claim 5, wherein the IE1 sequence
region comprises a nucleotide sequence from IE1 exon1.
7. The AAV vector genome of claim 6, wherein the IE1 sequence
region comprises a nucleotide sequence from IE1 intron 1 or a
fragment thereof.
8. The AAV vector genome of claim 7, wherein the IE1 sequence
region is 166 nucleotides in length.
9. The AAV vector genome of claim 2, wherein the AAV vector genome
comprises a human beta globin (BB) sequence region derived from a
HB gene.
10. The AAV vector genome of claim 9, wherein the HB sequence
region comprises a nucleotide sequence from HB intron 2.
11. The AAV vector genome of claim 10, wherein the HB sequence
region comprises a nucleotide sequence from HB exon 3.
12. The AAV vector genome of claim 11, wherein the HB sequence
region is 400 nucleotides in length.
13. The AAV vector genome of claim 2, wherein the AAV vector genome
comprises a poly(A) signal sequence region derived from the human
growth hormone (hGH) gene.
14. The AAV vector genome of claim 13, wherein the poly(A) signal
sequence region is 477 nucleotides in length.
15. The AAV vector genome of claim 1, wherein the AAV vector genome
comprises a sequence selected from the group consisting of SEQ ID
NO: 10-13, 15, 17-20 and 22 and variants having at least 95%
identity thereto.
16. The AAV vector genome of claim 2, wherein, the AADC region
comprises a codon optimized nucleotide sequence encoding SEQ ID NO:
1.
17. The AAV vector genome of claim 16, wherein the AAV vector
genome comprises a sequence selected from the group consisting of
SEQ ID NO: 7 and 12 or variants having at least 95% identity
thereto.
18. A recombinant AAV virus comprising the AAV vector genome of
claim 1.
19. The recombinant AAV virus of claim 18, comprising a capsid
serotype selected from the group consisting of AAV2, AAV1, AAV3,
AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV9.47, AAV9(hu14), AAV10,
AAV11, AAV12, AAVrh8, AAVrh10, AAV-DJ, and AAV-DJ8.
20. The recombinant AAV virus of claim 19, wherein the capsid
serotype is AAV2.
21. A pharmaceutical composition comprising the recombinant AAV
virus of claim 20.
22. The pharmaceutical composition of claim 21, wherein at least
70% of the recombinant AAV vectors contain an AAV vector genome.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation application which claims
the benefit of U.S. patent application Ser. No. 15/524,986 filed
May 5, 2017, and entitled AADC Polynucleotides for the Treatment of
Parkinson's Disease; which is a national stage filing under 35
U.S.C. .sctn. 371 of International Application No.
PCT/US2015/059201 filed Nov. 5, 2015, and entitled AADC
Polynucleotides for the Treatment of Parkinson's Disease; which
claims priority to U.S. Provisional Patent Application No.
62/075,298 filed Nov. 5, 2014, entitled AADC Polynucleotides for
the Treatment of Parkinson's Disease, and U.S. Provisional Patent
Application No. 62/155,692 filed May 1, 2015, entitled AADC
Polynucleotides for the Treatment of Parkinson's Disease, and U.S.
Provisional Patent Application No. 62/199,578 filed Jul. 31, 2015,
entitled AADC Polynucleotides for the Treatment of Parkinson's
Disease, and U.S. Provisional Patent Application No. 62/243,537
filed Oct. 19, 2015, entitled AADC Polynucleotides for the
Treatment of Parkinson's Disease; the contents of each of which is
herein incorporated by reference in its entirety.
REFERENCE TO SEQUENCE LISTING
[0002] The present application includes a Sequence Listing which is
filed in electronic format. The Sequence Listing file, entitled
2057-1010USCON_SEQLST.txt, was created on Sep. 20, 2018 and is
178,260 bytes in size. The information in electronic format of the
Sequence Listing is incorporated herein by reference in its
entirety.
FIELD OF THE DISCLOSURE
[0003] The invention relates to compositions, particularly nucleic
acid molecules, e.g., polynucleotides encoding AADC, for use in the
treatment of Parkinson's disease. In some embodiments such AADC
polynucleotides may be encoded by or within recombinant
adeno-associated viruses (AAVs).
BACKGROUND
[0004] Aromatic L-amino acid decarboxylase (AADC) is a homodimeric
pyridoxal phosphate-dependent enzyme responsible for the synthesis
of dopamine and serotonin. The encoded protein catalyzes the
decarboxylation of L-3,4-dihydroxyphenylalanine (L-DOPA or
levodopa) to dopamine; L-5-hydroxytryptophan to serotonin; and
L-tryptophan to tryptamine. Defects in this gene are the cause of
aromatic L-amino-acid decarboxylase deficiency (AADCD), which is an
inborn error in neurotransmitter metabolism leading to combined
serotonin and catecholamine deficiency that results in severe motor
and autonomic dysfunctions.
[0005] Parkinson's Disease (PD) is a progressive neurodegenerative
disease of the central nervous system (CNS) producing sensory and
motor symptoms. Dopamine replacement (i.e., levodopa) has been the
standard pharmacotherapy for motor impairment in PD. However, the
benefit of dopamine therapy becomes less marked over time, due, in
part, to the progressive death of dopamine-generating cells and
corresponding loss of AADC activity. Furthermore, systemic
administration of high-dose dopamine is complicated by side
effects, such as fluctuations in motor performance, dyskinesias,
and hallucinations, resulting from dopaminergic stimulation of the
mesolimbic system. One strategy to restore dopaminergic function
and minimize side effects is the use of gene therapy to deliver
AADC directly to a targeted region of the CNS.
[0006] The adeno-associated virus (AAV) has emerged as an
attractive vector for gene therapy due to its long-term gene
expression, the inability to autonomously replicate without a
helper virus, the ability to transduce dividing and non-diving
cells, and the lack of pathogenicity from wild-type infections (See
e.g., Hadaczek et al. Mol. Ther. 18(8), 1458-1461, August 2010).
AAV is a helper-dependent DNA parvovirus which belongs to the genus
Dependovirus.
[0007] The present invention provides such improved nucleic acid
constructs, e.g., polynucleotides, for use with AAV-derived vectors
comprising dopa carboxylase ("DDC") gene sequence which encodes a
full-length AADC protein for the purpose of gene therapy in the
treatment of Parkinson's Disease.
[0008] The nucleic acid constructs described herein comprise at
least a 5'-ITR and a 3'-ITR, each or both of which may be derived
from an AAV, positioned about a DDC gene sequence, as well as
additional components required for gene expression and clone
selection.
SUMMARY
[0009] Described herein are compositions, methods, processes, kits
and devices for the design, preparation, manufacture and/or
formulation of AADC polynucleotides.
[0010] In some embodiments such AADC polynucleotides may be encoded
by or contained within plasmids or vectors or recombinant
adeno-associated viruses (AAV).
[0011] The details of various embodiments of the invention are set
forth in the description below. Other features, objects, and
advantages of the invention will be apparent from the description
and from the claims.
DETAILED DESCRIPTION
Compositions
[0012] According to the present invention, AADC polynucleotides are
provided which function alone or in combination with additional
nucleic acid sequence(s) to encode the AADC protein. As used herein
an "AADC polynucleotide" is any nucleic acid polymer which encodes
an AADC protein and when present in a vector, plasmid or
translatable construct, expresses such AADC protein in a cell,
tissue, organ or organism.
[0013] AADC polynucleotides include precursor molecules which are
processed inside the cell. AADC polynucleotides or the processed
forms thereof may be encoded in a plasmid, vector, genome or other
nucleic acid expression vector for delivery to a cell.
[0014] In some embodiments AADC polynucleotides are designed as
components of AAV viral genomes and packaged in AAV viral particles
which are processed within the cell to produce the wild type AADC
protein.
[0015] As used herein, the wild type AADC protein may be any of the
naturally occurring isoforms or variants from the DDC gene.
Multiple alternatively spliced transcript variants encoding
different isoforms of AADC have been identified. Specifically, the
DDC gene produces seven transcript variants that encode six
distinct isoforms. DDC transcript variants 1 and 2 both encode AADC
isoform 1. In some embodiments, the AADC polynucleotides encode DDC
transcript variant 2, thereby encoding a native AADC isoform 1
(NCBI Reference Sequence: NP_000781.1). This sequence is given
here:
TABLE-US-00001 (SEQ ID NO: 1)
MNASEFRRRGKEMVDYVANYMEGIEGRQVYPDVEPGYLRPLIPAAAPQEP
DTFEDIINDVEKIIMPGVTHWHSPYFFAYFPTASSYPAMLADMLCGAIGC
IGFSWAASPACTELETVMMDWLGKMLELPKAFLNEKAGEGGGVIQGSASE
ATLVALLAARTKVIHRLQAASPELTQAAIMEKLVAYSSDQAHSSVERAGL
IGGVKLKAIPSDGNFAMRASALQEALERDKAAGLIPFFMVATLGTTTCCS
FDNLLEVGPICNKEDIWLHVDAAYAGSAFICPEFRHLLNGVEFADSFNFN
PHKWLLVNFDCSAMWVKKRTDLTGAFRLDPTYLKHSHQDSGLITDYRHWQ
IPLGRRFRSLKMWFVFRMYGVKGLQAYIRKHVQLSHEFESLVRQDPRFEI
CVEVILGLVCFRLKGSNKVNEALLQRINSAKKIHLVPCHLRDKFVLRFAI
CSRTVESAHVQRAWEHIKELAADVLRAERE
[0016] The AADC polynucleotides of the invention, may be engineered
to contain modular elements and/or sequence motifs assembled to
create AADC polynucleotide constructs.
AADC Polynucleotide Constructs
[0017] According to the present invention, AADC polynucleotides are
provided. Such polynucleotides comprise nucleic acid polymers which
comprise a region of linked nucleosides encoding one or more
isoforms or variants of the AADC protein.
[0018] In some embodiments, the AADC polynucleotide comprises a
codon optimized transcript encoding an AADC protein.
[0019] In some embodiments, the AADC polynucleotide comprises a
sequence region encoding one or more wild type isoforms or variants
of an AADC protein. Such polynucleotides may also comprise a
sequence region encoding any one or more of the following: a 5'
ITR, a cytomegalovirus (CMV) Enhancer, a CMV Promoter, an ie1 exon
1, an ie1 intron1, an hbBglobin intron2, an hBglobin exon 3, a 5'
UTR, a 3' UTR, an hGH poly(A) signal, and/or a 3' ITR. Such
sequence regions are taught herein or may be any of those known in
the art.
[0020] In some embodiments, the AADC polynucleotide comprises a
sequence of any of SEQ ID NOs. 2-23.
[0021] In one embodiment, the AADC polynucleotide comprises a
sequence which has a percent identity to any of SEQ ID NOs: 2-23.
The AADC polynucleotide may have 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%,
9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%,
70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% identity to any of SEQ ID
NOs: 2-23. The AADC polynucleotide may have 1-10%, 10-20%, 30-40%,
50-60%, 50-70%, 50-80%, 50-90%, 50-99%, 50-100%, 60-70%, 60-80%,
60-90%, 60-99%, 60-100%, 70-80%, 70-90%, 70-99%, 70-100%, 80-85%,
80-90%, 80-95%, 80-99%, 80-100%, 90-95%, 90-99%, or 90-100% to any
of SEQ ID NOs: 2-23. As a non-limiting example, the AADC
polynucleotide comprises a sequence which as 80% identity to any of
SEQ ID NOs 6, 7, 8, 9, 17, 18, 19, 20, 21, 22, and 23. As another
non-limiting example, the AADC polynucleotide comprises a sequence
which as 85% identity to any of SEQ ID NOs 6, 7, 8, 9, 17, 18, 19,
20, 21, 22, and 23. As another non-limiting example, the AADC
polynucleotide comprises a sequence which as 90% identity to any of
SEQ ID NOs 6, 7, 8, 9, 17, 18, 19, 20, 21, 22, and 23. As another
non-limiting example, the AADC polynucleotide comprises a sequence
which as 95% identity to any of SEQ ID NOs 6, 7, 8, 9, 17, 18, 19,
20, 21, 22, and 23. As another non-limiting example, the AADC
polynucleotide comprises a sequence which as 99% identity to any of
SEQ ID NOs 6, 7, 8, 9, 17, 18, 19, 20, 21, 22, and 23.
[0022] In some embodiments the AADC coding region is 1440
nucleotides in length. Such an AADC polynucleotide may be codon
optimized over all or a portion of the polynucleotide.
[0023] In some embodiments the AADC coding region is 1443
nucleotides in length. In such case, an additional codon may be
present at the 3' end of the polynucleotide.
[0024] In some embodiments the AADC coding region is 1449
nucleotides in length. In such case, additional codons may be
present at the 3' end of the polynucleotide.
[0025] In some embodiments, the AADC polynucleotide comprises any
of SEQ ID NOs 6-9, 17-23 but lacking the 5' and/or 3' ITRs. Such a
polynucleotide may be incorporated into a plasmid or vector and
utilized to express the encoded AADC protein.
[0026] In one embodiment, the AADC polynucleotides may be produced
in insect cells (e.g., Sf9 cells).
[0027] In one embodiment, the AADC polynucleotides may be produced
using triple transfection.
[0028] In one embodiment, the AADC polynucleotide may comprise a
codon optimized open reading frame of an AADC mRNA, at least one
5'ITR and at least one 3'UTR where the one or more of the 5'ITRs
may be located at the 5' end of the promoter region and one or more
3' ITRs may be located at the 3' end of the poly(A) signal. The
AADC mRNA may comprise a promoter region, a 5'untranslated region
(UTR), a 3'UTR and a poly(A) signal. The promoter region may
inclue, but is not limited to, enhancer element, a promoter
element, a first exon region, a first intron region, a second
intron region and a second exon region. As a non-limiting example,
the enhancer element and the promoter element are derived from CMV.
As another non-limiting example, the first exon region is ie1 exon
1 or fragments thereof, the first intron region is ie1 intron 1 or
fragments thereof, the second intron region is hbBglobin intron 2
or fragments thereof and the second exon region is hbBglobin exon 3
or fragments thereof. As yet another non-limiting example, the
poly(A) signal is derived from human growth hormone.
[0029] In one embodiment, the AADC polynucleotide is encoded in a
plasmid or vector, which may be derived from an adeno-associated
virus (AAV). The AAV may be a recombinant AAV virus and may
comprise a capsid serotype such as, but not limited to, of AAV1,
AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV9.47,
AAV9(hu14), AAV10, AAV11, AAV12, AAVrh8, AAVrh10, AAV-DJ, and
AAV-DJ8. As a non-limiting example, the capsid of the recombinant
AAV virus is AAV2. As a non-limiting example, the capsid of the
recombinant AAV virus is AAVrh10. As a non-limiting example, the
capsid of the recombinant AAV virus is AAV9(hu14). As a
non-limiting example, the capsid of the recombinant AAV virus is
AAV-DJ. As a non-limiting example, the capsid of the recombinant
AAV virus is AAV9.47. As a non-limiting example, the capsid of the
recombinant AAV virus is AAV-DJ8.
Promoters
[0030] A person skilled in the art may recognize that a target cell
may require a specific promoter including but not limited to a
promoter that is species specific, inducible, tissue-specific, or
cell cycle-specific Parr et al., Nat. Med. 3:1145-9 (1997); the
contents of which are herein incorporated by reference in its
entirety).
[0031] In one embodiment, the promoter is a promoter deemed to be
efficient for the AADC polynucleotide.
[0032] In one embodiment, the promoter is a promoter deemed to be
efficient for the cell being targeted.
[0033] In one embodiment, the promoter is a weak promoter which
provides expression of a payload for a period of time in targeted
tissues such as, but not limited to, nervous system tissues.
Expression may be for a period of 1 hour, 2, hours, 3 hours, 4
hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11
hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours,
18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, 1 day,
2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 8 days, 9 days, 10
days, 11 days, 12 days, 13 days, 2 weeks, 15 days, 16 days, 17
days, 18 days, 19 days, 20 days, 3 weeks, 22 days, 23 days, 24
days, 25 days, 26 days, 27 days, 28 days, 29 days, 30 days, 31
days, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7
months, 8 months, 9 months, 10 months, 11 months, 1 year, 13
months, 14 months, 15 months, 16 months, 17 months, 18 months, 19
months, 20 months, 21 months, 22 months, 23 months, 2 years, 3
years, 4 years, 5 years, 6 years, 7 years, 8 years, 9 years, 10
years or more than 10 years. Expression may be for 1-5 hours, 1-12
hours, 1-2 days, 1-5 days, 1-2 weeks, 1-3 weeks, 1-4 weeks, 1-2
months, 1-4 months, 1-6 months, 2-6 months, 3-6 months, 3-9 months,
4-8 months, 6-12 months, 1-2 years, 1-5 years, 2-5 years, 3-6
years, 3-8 years, 4-8 years or 5-10 years. As a non-limiting
example, the promoter is a weak promoter for sustained expression
of a payload in nervous tissues. As another non-limiting example,
the promoter is a weak promoter for sustained frataxin expression
in nervous system tissue such as, but not limited to, neuronal
tissue and glial tissue.
[0034] In one embodiment, the FRDA promoter is used with the AADC
polynucleotides described herein.
[0035] In one embodiment, there is a region located approximately
.about.5 kb upstream of the first exon of the payload. As a
non-limiting example, there is a 17 bp region located approximately
4.9 kb upstream of the first exon of the frataxin gene in order to
allow for expression with the FRDA promoter (See e.g., Puspasari et
al. Long Range Regulation of Human FXN Gene Expression, PLOS ONE,
2011; the contents of which is herein incorporated by reference in
its entirety).
[0036] In one embodiment, the promoter may be a promoter which is
less than 1 kb. The promoter may have a length of 200, 210, 220,
230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350,
360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480,
490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610,
620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740,
750, 760, 770, 780, 790, 800 or more than 800. The promoter may
have a length between 200-300, 200-400, 200-500, 200-600, 200-700,
200-800, 300-400, 300-500, 300-600, 300-700, 300-800, 400-500,
400-600, 400-700, 400-800, 500-600, 500-700, 500-800, 600-700,
600-800 or 700-800.
[0037] In one embodiment, the promoter may be a combination of two
or more components such as, but not limited to, CMV and CBA. Each
component may have a length of 200, 210, 220, 230, 240, 250, 260,
270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 381,
382, 383, 384, 385, 386, 387, 388, 389, 390, 400, 410, 420, 430,
440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560,
570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690,
700, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800 or more than
800. Each component may have a length between 200-300, 200-400,
200-500, 200-600, 200-700, 200-800, 300-400, 300-500, 300-600,
300-700, 300-800, 400-500, 400-600, 400-700, 400-800, 500-600,
500-700, 500-800, 600-700, 600-800 or 700-800. As a non-limiting
example, the promoter is a combination of a 382 nucleotide
CMV-enhancer sequence and a 260 nucleotide CBA-promoter
sequence.
[0038] In one embodiment, at least one element may be used with the
AADC polynucleotides described herein to enhance the transgene
target specificity and expression (See e.g., Powell et al. Viral
Expression Cassette Elements to Enhance Transgene Target
Specificity and Expression in Gene Therapy, 2015; the contents of
which are herein incorporated by reference in its entirety).
Non-limiting examples of elements to enhance the transgene target
specificity and expression include promoters, endogenous miRNAs,
post-transcriptional regulatory elements (PREs), polyadenylation
(PolyA) signal sequences and upstream enhancers (USEs), CMV
enhancers and introns.
[0039] In one embodiment, at least one element may be used with the
AADC polynucleotides described herein to enhance the transgene
target specificity and expression (See e.g., Powell et al. Viral
Expression Cassette Elements to Enhance Transgene Target
Specificity and Expression in Gene Therapy, 2015; the contents of
which are herein incorporated by reference in its entirety) such as
promoters. Promoters for which promote expression in most tissues
include, but are not limited to, human elongation factor
1.alpha.-subunit (EF1.alpha.), immediate-early cytomegalovirus
(CMV), chicken .beta.-actin (CBA) and its derivative CAG, the
.beta. glucuronidase (GUSB), or ubiquitin C (UBC). Tissue-specific
expression elements can be used to restrict expression to certain
cell types such as, but not limited to, nervous system promoters
which can be used to restrict expression to neurons, astrocytes, or
oligodendrocytes. Non-limiting example of tissue-specific
expression elements for neurons include neuron-specific enolase
(NSE), platelet-derived growth factor (PDGF), platelet-derived
growth factor B-chain (PDGF-.beta.), the synapsin (Syn), the
methyl-CpG binding protein 2 (MeCP2), CaMKII, mGluR2, NFL, NFH,
n.beta.2, PPE, Enk and EAAT2 promoters. A non-limiting example of a
tissue-specific expression elements for astrocytes include the
glial fibrillary acidic protein (GFAP) and EAAT2 promoters. A
non-limiting example of a tissue-specific expression element for
oligodendrocytes include the myelin basic protein (MBP)
promoter.
[0040] In one embodiment, a ubiquitous promoter may be used with
the AADC polynucleotides described herein. Non-limiting examples of
ubiquitous promoters include CMV, CBA (including derivatives CAG,
CBh, etc.), EF-1.alpha., PGK, UBC, GUSB (hGBp), and UCOE (promoter
of HNRPA2B1-CBX3). Yu et al. (Molecular Pain 2011, 7:63; the
contents of which are herein incorporated by reference in its
entirety) evaluated the expression of eGFP under the CAG,
EFI.alpha., PGK and UBC promoters in rat DRG cells and primary DRG
cells using lentiviral vectors and found that UBC showed weaker
expression than the other 3 promoters and there was only 10-12%
glia expression seen for all promoters. Soderblom et al. (E. Neuro
2015; the contents of which are herein incorporated by reference in
its entirety) the expression of eGFP in AAV8 with CMV and UBC
promoters and AAV2 with the CMV promoter after injection in the
motor cortex. Intranasal administration of a plasmid containing a
UBC or EFI.alpha. promoter showed a sustained airway expression
greater than the expression with the CMV promoter (See e.g., Gill
et al., Gene Therapy 2001, Vol. 8, 1539-1546; the contents of which
are herein incorporated by reference in its entirety). Husain et
al. (Gene Therapy 2009; the contents of which are herein
incorporated by reference in its entirety) evaluated a H.beta.H
construct with a hGUSB promoter, a HSV-1LAT promoter and a NSE
promoter and found that the H.beta.H construct showed weaker
expression than NSE in mice brain. Passini and Wolfe (J. Virol.
2001, 12382-12392, the contents of which are herein incorporated by
reference in its entirety) evaluated the long term effects of the
H.beta.H vector following an intraventricular injection in neonatal
mice and found that there was sustained expression for at least 1
year. Low expression in all brain regions was found by Xu et al.
(Gene Therapy 2001, 8, 1323-1332; the contents of which are herein
incorporated by reference in its entirety) when NF-L and NF-H
promoters were used as compared to the CMV-lacZ, CMV-luc, EF, GFAP,
hENK, nAChR, PPE, PPE+wpre, NSE (0.3 kb), NSE (1.8 kb) and NSE (1.8
kb+wpre). Xu et al. found that the promoter activity in descending
order was NSE (1.8 kb), EF, NSE (0.3 kb), GFAP, CMV, hENK, PPE, NFL
and NFH. NFL is a 650 nucleotide promoter and NFH is a 920
nucleotide promoter which are both absent in the liver but NFH is
abundant in the sensory proprioceptive neurons, brain and spinal
cord and NFH is present in the heart. Scn8a is a 470 nucleotide
promoter which expresses throughout the DRG, spinal cord and brain
with particularly high expression seen in the hippocampal neurons
and cerebellar Purkinje cells, cortex, thalamus and hypothalamus
(See e.g., Drews et al. 2007 and Raymond et al. 2004; the contents
of each of which are herein incorporated by reference in their
entireties).
[0041] In one embodiment, an UBC promoter may be used with the AADC
polynucleotides described herein. The UBC promoter may have a size
of 300-350 nucleotides. As a non-limiting example, the UBC promoter
is 332 nucleotides.
[0042] In one embodiment, a GUSB promoter may be used with the AADC
polynucleotides described herein. The GUSB promoter may have a size
of 350-400 nucleotides. As a non-limiting example, the GUSB
promoter is 378 nucleotides. As a non-limiting example, the
construct may be AAV-promoter-CMV/globin intron-hFXN-RBG, where the
AAV may be self-complementary and the AAV may be the DJ
serotype.
[0043] In one embodiment, a NFL promoter may be used with the AADC
polynucleotides described herein. The NFL promoter may have a size
of 600-700 nucleotides. As a non-limiting example, the NFL promoter
is 650 nucleotides. As a non-limiting example, the construct may be
AAV-promoter-CMV/globin intron-hFXN-RBG, where the AAV may be
self-complementary and the AAV may be the DJ serotype.
[0044] In one embodiment, a NFH promoter may be used with the AADC
polynucleotides described herein. The NFH promoter may have a size
of 900-950 nucleotides. As a non-limiting example, the NFH promoter
is 920 nucleotides. As a non-limiting example, the construct may be
AAV-promoter-CMV/globin intron-hFXN-RBG, where the AAV may be
self-complementary and the AAV may be the DJ serotype.
[0045] In one embodiment, a scn8a promoter may be used with the
AADC polynucleotides described herein. The scn8a promoter may have
a size of 450-500 nucleotides. As a non-limiting example, the scn8a
promoter is 470 nucleotides. As a non-limiting example, the
construct may be AAV-promoter-CMV/globin intron-hFXN-RBG, where the
AAV may be self-complementary and the AAV may be the DJ
serotype.
[0046] In one embodiment, a FXN promoter may be used with the AADC
polynucleotides described herein.
[0047] In one embodiment, a PGK promoter may be used with the AADC
polynucleotides described herein.
[0048] In one embodiment, a CBA promote may be used with the AADC
polynucleotides described herein.
[0049] In one embodiment, a CMV promoter may be used with the AADC
polynucleotides described herein.
[0050] In one embodiment, a liver or a skeletal muscle promoter may
be used with the AADC polynucleotides described herein.
Non-limiting examples of liver promoters include hAAT and TBG.
Non-limiting examples of skeletal muscle promoters include Desmin,
MCK and C5-12.
[0051] In one embodiment, an enhancer element, a promoter and/or a
5'UTR intron may be used with the AADC polynucleotides described
herein. The enhancer may be, but is not limited to, a CMV enhancer,
the promoter may be, but is not limited to, a CMV, CBA, UBC, GUSB,
NSE, Sunapsin, MeCP2, and GFAP promoter and the 5'UTR/intron may
be, but is not limited to, SV40, and CBA-MVM. As a non-limiting
example, the enhancer, promoter and/or intron used in combination
may be: (1) CMV enhancer, CMV promoter, SV40 5'UTR intron; (2) CMV
enhancer, CBA promoter, SV 40 5'UTR intron; (3) CMV enhancer, CBA
promoter, CBA-MVM 5'UTR intron; (4) UBC promoter; (5) GUSB
promoter; (6) NSE promoter; (7) Synapsin promoter; (8) MeCP2
promoter and (9) GFAP promoter.
[0052] In one embodiment, an engineered promoter may be used with
the AADC polynucleotides described herein.
Introns
[0053] In one embodiment, at least one element may be used with the
AADC polynucleotides described herein to enhance the transgene
target specificity and expression (See e.g., Powell et al. Viral
Expression Cassette Elements to Enhance Transgene Target
Specificity and Expression in Gene Therapy, 2015; the contents of
which are herein incorporated by reference in its entirety) such as
an intron. Non-limiting examples of introns include, MVM (67-97
bps), F.IX truncated intron 1 (300 bps), .beta.-globin
SD/immunoglobulin heavy chain splice acceptor (250 bps), adenovirus
splice donor/immunoglobin splice acceptor (500 bps), SV40 late
splice donor/splice acceptor (19S/16S) (180 bps) and hybrid
adenovirus splice donor/IgG splice acceptor (230 bps).
[0054] In one embodiment, the intron may be 100-500 nucleotides in
length. The intron may have a length of 80, 90, 100, 110, 120, 130,
140, 150, 160, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179,
180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300,
310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430,
440, 450, 460, 470, 480, 490 or 500. The intron may have a length
between 80-100, 80-120, 80-140, 80-160, 80-180, 80-200, 80-250,
80-300, 80-350, 80-400, 80-450, 80-500, 200-300, 200-400, 200-500,
300-400, 300-500, or 400-500.
Introduction into Cells
[0055] The AADC polynucleotides of the invention can be introduced
into host cells using any of a variety of approaches. Infection
with a viral vector comprising the AADC polynucleotide can be
effected. Examples of suitable viral vectors include replication
defective retroviral vectors, adenoviral vectors, adeno-associated
vectors and lentiviral vectors.
[0056] According to the present invention, viral vectors for use in
therapeutics and/or diagnostics comprise a virus that has been
distilled or reduced to the minimum components necessary for
transduction of a nucleic acid payload or cargo of interest.
[0057] In this manner, viral vectors are engineered as vehicles for
specific delivery while lacking the deleterious replication and/or
integration features found in wild-type virus.
[0058] As used herein, a "vector" is any molecule or moiety which
transports, transduces or otherwise acts as a carrier of a
heterologous molecule such as the polynucleotides of the invention.
A "viral vector" is a vector which comprises one or more
polynucleotide regions encoding or comprising payload molecule of
interest, e.g., a transgene, a polynucleotide encoding a
polypeptide or multi-polypeptide. Viral vectors of the present
invention may be produced recombinantly and may be based on
adeno-associated virus (AAV) parent or reference sequence.
Serotypes which may be useful in the present invention include any
of those arising from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7,
AAV8, AAV9, AAV9.47, AAV9(hu14), AAV10, AAV11, AAV12, AAVrh8,
AAVrh10, AAV-DJ, and AAV-DJ8.
[0059] In one embodiment, the serotype which may be useful in the
present invention may be AAV-DJ8. The amino acid sequence of
AAV-DJ8 may comprise two or more mutations in order to remove the
heparin binding domain (HBD). As a non-limiting example, the AAV-DJ
sequence described as SEQ ID NO: 1 in U.S. Pat. No. 7,588,772, the
contents of which are herein incorporated by reference in its
entirety, may comprise two mutations: (1) R587Q where arginine (R;
arg) at amino acid 587 is changed to glutamine (Q; gln) and (2)
R590T where arginine (R; arg) at amino acid 590 is changed to
threonine (T; thr). As another non-limiting example, may comprise
three mutations: (1) K406R where lysine (K; lys) at amino acid 406
is changed to arginine (R; arg), (2) R587Q where arginine (R; arg)
at amino acid 587 is changed to glutamine (Q; gln) and (3) R590T
where arginine (R; arg) at amino acid 590 is changed to threonine
(T; thr).
[0060] AAV vectors may also comprise self-complementary AAV vectors
(scAAVs). scAAV vectors contain both DNA strands which anneal
together to form double stranded DNA. By skipping second strand
synthesis, scAAVs allow for rapid expression in the cell.
Pharmaceutical Compositions
[0061] Although the descriptions of pharmaceutical compositions,
e.g., those polynucleotides (including the encoding plasmids or
expression vectors, such as viruses, e.g., AAV) comprising a
payload, e.g., AADC encoding sequences, to be delivered, provided
herein are principally directed to pharmaceutical compositions
which are suitable for administration to humans, it will be
understood by the skilled artisan that such compositions are
generally suitable for administration to any other animal, e.g., to
non-human animals, e.g. non-human mammals. Modification of
pharmaceutical compositions suitable for administration to humans
in order to render the compositions suitable for administration to
various animals is well understood, and the ordinarily skilled
veterinary pharmacologist can design and/or perform such
modification with merely ordinary, if any, experimentation.
Subjects to which administration of the pharmaceutical compositions
is contemplated include, but are not limited to, humans and/or
other primates; mammals, including commercially relevant mammals
such as cattle, pigs, horses, sheep, cats, dogs, mice, and/or rats;
and/or birds, including commercially relevant birds such as
poultry, chickens, ducks, geese, and/or turkeys.
[0062] In some embodiments, compositions are administered to
humans, human patients or subjects. For the purposes of the present
disclosure, the phrase "active ingredient" generally refers either
to the viral vector carrying the payload or to the polynucleotide
payload molecule delivered by a viral vector as described
herein.
[0063] Formulations of the pharmaceutical compositions described
herein may be prepared by any method known or hereafter developed
in the art of pharmacology. In general, such preparatory methods
include the step of bringing the active ingredient into association
with an excipient and/or one or more other accessory ingredients,
and then, if necessary and/or desirable, dividing, shaping and/or
packaging the product into a desired single- or multi-dose
unit.
[0064] Relative amounts of the active ingredient, the
pharmaceutically acceptable excipient, and/or any additional
ingredients in a pharmaceutical composition in accordance with the
invention will vary, depending upon the identity, size, and/or
condition of the subject treated and further depending upon the
route by which the composition is to be administered.
[0065] In one embodiment, the pharmaceutical composition comprises
a recombinant adeno-associated virus (AAV) vector comprising an AAV
capsid and an AAV vector genome. The AAV vector genome may comprise
at least one AADC polynucleotide described herein, such as, but not
limited to, SEQ ID NOs 6, 7, 8, 9, 17, 18, 19, 20, 21, 22, and 23
or variants having at least 95% identity thereto. The recombinant
AAV vectors in the pharmaceutical composition may have at least 70%
which contain an AAV vector genome.
Formulation
[0066] The AADC polynucleotides or viral vectors encoding them can
be formulated using one or more excipients to: (1) increase
stability; (2) increase cell transfection or transduction; (3)
permit the sustained or delayed release; or (4) alter the
biodistribution (e.g., target the viral vector to specific tissues
or cell types).
[0067] Formulations of the present invention can include, without
limitation, saline, lipidoids, liposomes, lipid nanoparticles,
polymers, lipoplexes, core-shell nanoparticles, peptides, proteins,
cells transfected with viral vectors (e.g., for transplantation
into a subject), nanoparticle mimics and combinations thereof.
Further, the viral vectors of the present invention may be
formulated using self-assembled nucleic acid nanoparticles.
[0068] Formulations of the pharmaceutical compositions described
herein may be prepared by any method known or hereafter developed
in the art of pharmacology. In general, such preparatory methods
include the step of associating the active ingredient with an
excipient and/or one or more other accessory ingredients.
[0069] A pharmaceutical composition in accordance with the present
disclosure may be prepared, packaged, and/or sold in bulk, as a
single unit dose, and/or as a plurality of single unit doses. As
used herein, a "unit dose" refers to a discrete amount of the
pharmaceutical composition comprising a predetermined amount of the
active ingredient. The amount of the active ingredient is generally
equal to the dosage of the active ingredient which would be
administered to a subject and/or a convenient fraction of such a
dosage such as, for example, one-half or one-third of such a
dosage.
[0070] Relative amounts of the active ingredient, the
pharmaceutically acceptable excipient, and/or any additional
ingredients in a pharmaceutical composition in accordance with the
present disclosure may vary, depending upon the identity, size,
and/or condition of the subject being treated and further depending
upon the route by which the composition is to be administered. For
example, the composition may comprise between 0.1% and 99% (w/w) of
the active ingredient. By way of example, the composition may
comprise between 0.1% and 100%, e.g., between 0.5 and 50%, between
1-30%, between 5-80%, at least 80% (w/w) active ingredient.
[0071] In some embodiments, the formulations described herein may
contain at least one payload molecule, e.g., an AADC
polynucleotide. As a non-limiting example, the formulations may
contain 1, 2, 3, 4 or 5 AADC polynucleotide payload molecules.
[0072] In some embodiments, a pharmaceutically acceptable excipient
may be at least 95%, at least 96%, at least 97%, at least 98%, at
least 99%, or 100% pure. In some embodiments, an excipient is
approved for use for humans and for veterinary use. In some
embodiments, an excipient may be approved by United States Food and
Drug Administration. In some embodiments, an excipient may be of
pharmaceutical grade. In some embodiments, an excipient may meet
the standards of the United States Pharmacopoeia (USP), the
European Pharmacopoeia (EP), the British Pharmacopoeia, and/or the
International Pharmacopoeia.
[0073] Excipients, which, as used herein, includes, but is not
limited to, any and all solvents, dispersion media, diluents, or
other liquid vehicles, dispersion or suspension aids, surface
active agents, isotonic agents, thickening or emulsifying agents,
preservatives, and the like, as suited to the particular dosage
form desired. Various excipients for formulating pharmaceutical
compositions and techniques for preparing the composition are known
in the art (see Remington: The Science and Practice of Pharmacy,
21.sup.st Edition, A. R. Gennaro, Lippincott, Williams &
Wilkins, Baltimore, Md., 2006; incorporated herein by reference in
its entirety). The use of a conventional excipient medium may be
contemplated within the scope of the present disclosure, except
insofar as any conventional excipient medium may be incompatible
with a substance or its derivatives, such as by producing any
undesirable biological effect or otherwise interacting in a
deleterious manner with any other component(s) of the
pharmaceutical composition.
[0074] Exemplary diluents include, but are not limited to, calcium
carbonate, sodium carbonate, calcium phosphate, dicalcium
phosphate, calcium sulfate, calcium hydrogen phosphate, sodium
phosphate lactose, sucrose, cellulose, microcrystalline cellulose,
kaolin, mannitol, sorbitol, inositol, sodium chloride, dry starch,
cornstarch, powdered sugar, etc., and/or combinations thereof.
[0075] In one embodiment, the AADC polynucleotides may be
formulated in a hydrogel prior to administration. Hydrogels have a
degree of flexibility which is similar to natural tissue as a
result of their significant water content.
[0076] In another embodiment, a hydrogel may be administered to a
subject prior to the administration of an AADC polynucleotide
formulation. As a non-limiting example, the site of administration
of the hydrogel may be within 3 inches (e.g., within 2.9, 2.8, 2.7,
2.6, 2.5, 2.4, 2.3, 2.2, 2.1, 2.0, 1.9, 1.8, 1.7, 1.6, 1.5, 1.4,
1.3, 1.2, 1.1, 1.0, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, 0.1 or
less than 0.1 inches) of the site of administration of the AADC
polynucleotide formulation.
[0077] In one embodiment, the AADC polynucleotides may be
administered in saline. As a non-limiting example, the formulation
may be phosphate buffered saline (PBS) with 0.001% Pluronic acid
(F-68). Additionally the formulation may be sterilized.
Inactive Ingredients
[0078] In some embodiments, AADC polynucleotide formulations may
comprise at least one excipient which is an inactive ingredient. As
used herein, the term "inactive ingredient" refers to one or more
inactive agents included in formulations. In some embodiments, all,
none or some of the inactive ingredients which may be used in the
formulations of the present invention may be approved by the US
Food and Drug Administration (FDA).
[0079] Formulations of viral vectors carrying AADC polynucleotides
disclosed herein may include cations or anions. In one embodiment,
the formulations include metal cations such as, but not limited to,
Zn2+, Ca2+, Cu2+, Mg+ and combinations thereof. As a non-limiting
example, formulations may include polymers and AADC polynucleotides
complexed with a metal cation (See e.g., U.S. Pat. Nos. 6,265,389
and 6,555,525, each of which is herein incorporated by reference in
its entirety).
Administration
[0080] The viral vectors comprising AADC polynucleotides of the
present invention may be administered by any route which results in
a therapeutically effective outcome. These include, but are not
limited to epidural (into the dura matter), oral (by way of the
mouth), transdermal, peridural, intracerebral (into the cerebrum),
intracerebroventricular (into the cerebral ventricles),
subcutaneous (under the skin), nasal administration (through the
nose), intravenous (into a vein), intravenous bolus, intravenous
drip, intraarterial (into an artery), intrathecal (into the spinal
canal), endocervical, intracaudal (within the cauda equine),
intracisternal (within the cisterna magna cerebellomedularis),
intradiscal (within a disc), intradural (within or beneath the
dura), intrameningeal (within the meninges), intrapleural (within
the pleura), intraspinal (within the vertebral column), intrathecal
(within the cerebrospinal fluid at any level of the cerebrospinal
axis), intrathoracic (within the thorax), intrastriatal (within the
striatum, caudate nucleus and/or putamen), caudal block, nerve
block, or spinal. In specific embodiments, compositions may be
administered in a way which allows them cross the blood-brain
barrier, vascular barrier, or other epithelial barrier. In one
embodiment, a formulation for a route of administration may include
at least one inactive ingredient.
[0081] In one embodiment, the viral vectors comprising AADC
polynucleotides of the present invention may be administered to the
right putamen and/or the left putamen. The administration may be at
one or more sites in the putamen such as, but not limited to, 2
sites, 3 sites, 4 sites or more than 4 sites. As a non-limiting
example, the viral vectors comprising AADC polynucleotides of the
present invention are delivered to 2 sites in the left putamen and
2 sites in the right putamen.
[0082] In one embodiment, the administration of the formulation of
the viral vectors comprising the AADC polynucleotides of the
present invention to a subject provides coverage of the putamen of
a subject (e.g., the left and/or right putamen). In one aspect, the
administration of the viral vectors comprising the AADC
polynucleotides may provide at least 8%, 9%, 10%, 13%, 14%, 15%,
19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%,
32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%,
45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 60%, 65%,
70%, 75%, 80%, 85%, 90%, 95% or more than 95% to the left and/or
right putamen of a subject. As a non-limiting example, the coverage
is at least 20%. As a non-limiting example, the coverage is at
least 40%. In another aspect, the administration of the viral
vectors comprising the AADC polynucleotides may provide at least
8%, 9%, 10%, 13%, 14%, 15%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%,
27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%,
40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%,
53%, 54%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more than
95% coverage of the surface area of the left and/or right putamen
of a subject. As a non-limiting example, the total coverage is at
least 20%. As a non-limiting example, the total coverage is at
least 40%. In yet another aspect, the administration of the viral
vectors comprising the AADC polynucleotides may provide 10-40%,
20-40%, 20-30%, 20-35%, 20-50%, 30-40%, 35-40%, 30-60%, 40-70%,
50-80% or 60-90% coverage to the left and/or right putamen of a
subject or to the total surface area of the left and/or right
putamen of a subject.
[0083] In one embodiment, the administration of the formulation of
the viral vectors comprising the AADC polynucleotides of the
present invention to a subject provides coverage of the posterior
putamen of a subject (e.g., the left and/or right posterior
putamen). In one aspect, the administration of the viral vectors
comprising the AADC polynucleotides may provide at least 10%, 15%,
20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%,
33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%,
46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 60%, 65%, 70%,
75%, 80%, 85%, 90%, 95% or more than 95% to the left and/or right
posterior putamen of a subject. As a non-limiting example, the
coverage is at least 20%. As a non-limiting example, the coverage
is at least 40%. In another aspect, the administration of the viral
vectors comprising the AADC polynucleotides may provide at least
10%, 15%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%,
31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%,
44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 60%,
65%, 70%, 75%, 80%, 85%, 90%, 95% or more than 95% coverage of the
surface area of the left and/or right posterior putamen of a
subject. As a non-limiting example, the total coverage is at least
20%. As a non-limiting example, the total coverage is at least 40%.
In yet another aspect, the administration of the viral vectors
comprising the AADC polynucleotides may provide 10-40%, 20-50%,
30-60%, 40-70%, 50-80% or 60-90% coverage to the left and/or right
posterior putamen of a subject or to the total surface area of the
left and/or right putamen of a subject.
[0084] In one embodiment, a subject may be administered the
viral-vectors comprising AADC polynucleotides of the present
invention safely delivered to substantia nigra pars compacta (SNpc)
and ventral tegmental area (VTA) via bilateral infusions, or
alternatively, intrastriatally (into the caudate nucleus and
putamen), or into the subthalamic nucleus (STN).
[0085] In one embodiment, the AADC polynucleotides described herein
may be administered using acute bilateral placement of catheters
into each putamen. The placement may use magnetic resonance image
(MRI)-guided stereotactic neurosurgical techniques known in the art
or described herein. Additionally, a contrast agent such as, but
not limited to a gadolinium based contrast agent (e.g.,
PROHANCE.RTM.) may be used in the formulation to monitor and
confirm the distribution of the formulation.
[0086] In one embodiment, a subject may be administered the viral
vectors comprising AADC polynucleotides of the present invention in
a bilateral stereotactic CED-assisted step infusion into the
putamen (e.g., the post commissural putamen).
[0087] In one embodiment, delivery of viral vector pharmaceutical
compositions in accordance with the present invention to cells of
the central nervous system (e.g., parenchyma) comprises a rate of
delivery defined by [VG/hour=mL/hour*VG/mL] wherein VG is viral
genomes, VG/mL is composition concentration, and mL/hour is rate of
prolonged infusion.
[0088] In one embodiment, delivery of viral vector pharmaceutical
compositions in accordance with the present invention to cells of
the central nervous system (e.g., parenchyma) comprises infusion of
up to 1 mL. In one embodiment, delivery of viral vector
pharmaceutical compositions in accordance with the present
invention to cells of the central nervous system (e.g., parenchyma)
may comprise infusion of 0.001, 0.002, 0.003, 0.004, 0.005, 0.010,
0.015, 0.020, 0.025, 0.030, 0.040, 0.050, 0.060, 0.070, 0.080,
0.090, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, or 0.9 mL.
[0089] In one embodiment, delivery of viral vector pharmaceutical
compositions in accordance with the present invention to cells of
the central nervous system (e.g., parenchyma) comprises infusion of
between about 1 mL to about 120 mL. In one embodiment, delivery of
viral vector pharmaceutical compositions in accordance with the
present invention to cells of the central nervous system (e.g.,
parenchyma) may comprise infusion of 0.1, 1, 1.1, 1.2, 1.3, 1.4,
1.5, 1.6, 1.7, 1.8, 1.9, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,
14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30,
31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47,
48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64,
65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81,
82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98,
99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111,
112, 113, 114, 115, 116, 117, 118, 119, or 120 mL. In one
embodiment, delivery of AAV particles to cells of the central
nervous system (e.g., parenchyma) comprises infusion of at least 3
mL. In one embodiment, delivery of AAV particles to cells of the
central nervous system (e.g., parenchyma) consists of infusion of 3
mL. In one embodiment, delivery of AAV particles to cells of the
central nervous system (e.g., parenchyma) comprises infusion of at
least 10 mL. In one embodiment, delivery of AAV particles to cells
of the central nervous system (e.g., parenchyma) consists of
infusion of 10 mL.
[0090] In one embodiment, the volume of the viral vector
pharmaceutical composition delivered to the cells of the central
nervous system (e.g., parenchyma) of a subject is 50 ul, 100 ul,
200 ul, 300 ul, 400 ul, 500 ul, 600 ul, 700 ul, 800 ul, 900 ul,
1000 ul, 1100 ul, 1200 ul, 1300 ul, 1400 ul, 1500 ul, 1600 ul, 1700
ul, 1800 ul, 1900 ul, 2000 ul or more than 2000 ul.
[0091] In one embodiment, the volume of the viral vector
pharmaceutical composition delivered to a region in both
hemispheres of a subject brain is 50 ul, 100 ul, 200 ul, 300 ul,
400 ul, 500 ul, 600 ul, 700 ul, 800 ul, 900 ul, 1000 ul, 1100 ul,
1200 ul, 1300 ul, 1400 ul, 1500 ul, 1600 ul, 1700 ul, 1800 ul, 1900
ul, 2000 ul or more than 2000 ul. As a non-limiting example, the
volume delivered to a region in both hemispheres is 200 ul. As
another non-limiting example, the volume delivered to a region in
both hemispheres is 900 ul. As yet another non-limiting example,
the volume delivered to a region in both hemispheres is 1800
ul.
[0092] In one embodiment, the volume of the viral vector
pharmaceutical composition delivered to the putamen in both
hemispheres of a subject brain is 50 ul, 100 ul, 200 ul, 300 ul,
400 ul, 450 ul, 500 ul, 600 ul, 700 ul, 800 ul, 900 ul, 1000 ul,
1100 ul, 1200 ul, 1300 ul, 1400 ul, 1500 ul, 1600 ul, 1700 ul, 1800
ul, 1900 ul, 2000 ul or more than 2000 ul. As a non-limiting
example, the volume delivered to the putamen in both hemispheres is
100 ul. As another non-limiting example, the volume delivered to
the putamen in both hemispheres is 200 ul. As a non-limiting
example, the volume delivered to the putamen in both hemispheres is
300 ul. As another non-limiting example, the volume delivered to
the putamen in both hemispheres is 450 ul. As another non-limiting
example, the volume delivered to the putamen in both hemispheres is
900 ul. As yet another non-limiting example, the volume delivered
to the putamen both hemispheres is 1800 ul.
[0093] In one embodiment, the total volume delivered to a subject
may be split between one or more administration sites e.g., 1, 2,
3, 4, 5 or more than 5 sites. As a non-limiting example, the total
volume is split between administration to the left and right
putamen. As another non-limiting example, the total volume is split
between two sites of administration to each of the left and right
putamen.
[0094] In one embodiment, the viral vector pharmaceutical
composition is administered using a fenestrated needle.
Non-limiting examples of fenestrated needles are described in U.S.
Pat. Nos. 8,333,734, 7,135,010, 7,575,572, 7,699,852, 4,411,657,
6,890,319, 6,613,026, 6,726,659, 6,565,572, 6,520,949, 6,382,212,
5,848,996, 5,759,179, 5,674,267, 5,588,960, 5,484,401, 5,199,441,
5,012,818, 4,474,569, 3,766,907, 3,552,394, the contents of each of
which are herein incorporated by reference in its entirety.
[0095] In one embodiment, a composition comprises AADC
polynucleotides described herein and the AADC polynucleotides are
components of an AAV viral genome packaged in an AAV viral
particle. The percent (%) ratio of AAV viral particles comprising
the AADC polynucleotide (also referred to herein and AADC
particles) to the AAV viral particles without the AADC
polynucleotide (also referred to herein as empty capsids) in the
composition may be 0:100, 1:99, 0:90, 15:85, 25:75, 30:70, 50:50,
70:30, 85:15, 90:10, 99:1 or 100:0. As a non-limiting example, the
percent ratio of AADC particles to empty capsids is 50:50. As
another non-limiting example, the percent ratio of AADC particles
to empty capsids is 70:30. As another non-limiting example, the
percent ratio of AADC particles to empty capsids is 85:15. As
another non-limiting example, the percent ratio of AADC particles
to empty capsids is 100:0.
[0096] In one embodiment, the composition described herein
comprises at least 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 51,
52, 53, 54, 55, 60, 65, 70, 75, 80, 81, 82, 83, 84, 85, 86, 87, 88,
89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or greater than 99% AADC
particles. As a non-limiting example, the composition comprises at
least 50% AADC particles. As another non-limiting example, the
composition comprises at least 52% AADC particles. As another
non-limiting example, the composition comprises at least 58% AADC
particles. As another non-limiting example, the composition
comprises at least 70% AADC particles. As another non-limiting
example, the composition comprises at least 83% AADC particles. As
another non-limiting example, the composition comprises at least
85% AADC particles. As another non-limiting example, the
composition comprises at least 99% AADC particles. As another
non-limiting example, the composition comprises 100% AADC
particles.
[0097] In one embodiment, the composition described herein
comprises 1-10%, 10-20%, 30-40%, 50-60%, 50-70%, 50-80%, 50-90%,
50-99%, 50-100%, 60-70%, 60-80%, 60-90%, 60-99%, 60-100%, 70-80%,
70-90%, 70-99%, 70-100%, 80-85%, 80-90%, 80-95%, 80-99%, 80-100%,
90-95%, 90-99%, or 90-100% AADC particles. As a non-limiting
example, the composition described herein comprises 50-100% AADC
particles. As another non-limiting example, the composition
described herein comprises 50-60% AADC particles. As another
non-limiting example, the composition described herein comprises
80-99% AADC particles. As another non-limiting example, the
composition described herein comprises 80-90% AADC particles. As a
non-limiting example, the composition described herein comprises
80-95% AADC particles. As a non-limiting example, the composition
described herein comprises 80-85% AADC particles.
[0098] In one embodiment, the composition described herein
comprises less than 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 51,
52, 53, 54, 55, 60, 65, 70, 75, 80, 81, 82, 83, 84, 85, 86, 87, 88,
89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% empty particles.
As a non-limiting example, the composition comprises less than 50%
empty particles. As a non-limiting example, the composition
comprises less than 45% empty particles. As a non-limiting example,
the composition comprises less than 40% empty particles. As a
non-limiting example, the composition comprises less than 35% empty
particles. As a non-limiting example, the composition comprises
less than 30% empty particles. As a non-limiting example, the
composition comprises less than 25% empty particles. As a
non-limiting example, the composition comprises less than 20% empty
particles. As a non-limiting example, the composition comprises
less than 15% empty particles. As a non-limiting example, the
composition comprises less than 10% empty particles. As a
non-limiting example, the composition comprises less than 5% empty
particles. As a non-limiting example, the composition comprises
less than 1% empty particles.
[0099] In the composition described herein comprises 1-10%, 10-20%,
30-40%, 50-60%, 50-70%, 50-80%, 50-90%, 50-99%, 50-100%, 60-70%,
60-80%, 60-90%, 60-99%, 60-100%, 70-80%, 70-90%, 70-99%, 70-100%,
80-85%, 80-90%, 80-95%, 80-99%, 80-100%, 90-95%, 90-99%, or 90-100%
empty particles. As a non-limiting example, the composition
described herein comprises 30-40% empty particles. As another
non-limiting example, the composition described herein comprises
30-50% empty particles. As another non-limiting example, the
composition described herein comprises 30-60% empty particles. As
another non-limiting example, the composition described herein
comprises 30-70% empty particles. As a non-limiting example, the
composition described herein comprises 30-80% empty particles. As a
non-limiting example, the composition described herein comprises
30-90% empty particles.
[0100] In one embodiment, the AADC polynucleotides described herein
may be administered to a subject who is also undergoing levodopa
therapy. As a non-limiting example, the subject may have a positive
response to levodopa therapy and at least one symptom of PD is
reduced. As another non-limiting example, the subject may have a
response to levodopa therapy where the symptoms of PD experienced
by the subject are stable. As yet another non-limiting example, the
subject may have a negative response to levodopa therapy where the
symptoms of PD experienced by the subject are increasing.
[0101] In one embodiment, the dose of levodopa administered to the
subject prior to the AADC polynucleotides is 1, 2, 3, 4, 5, 6, 7,
8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24,
25 or more than 25 mg/kg. As a non-limiting example, the dose is 3
mg/kg. As another non-limiting example, the dose is 10 mg/kg. As
yet another non-limiting example, the dose is 20 mg/kg. The
subject's response (e.g., behavioral response) to levodopa may be
assessed prior to administration of the AADC polynucleotides.
Additionally, the subject may be administered levodopa again after
the administration of the AADC polynucleotides (e.g., 1 week, 2,
weeks, 3 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6
months, 7 months, 8 months, 9 months, 10 months, 11 months, 1 year
or more than 1 year after the administration of AADC
polynucleotides). The behavioral response can be re-assessed and
compared to the initial response to determine the effects of the
AADC polynucleotides. The subject may have 1%, 2%, 3%, 4%, 5%, 6%,
7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%,
65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% behavioral
improvement.
[0102] In one embodiment, Levodopa may be administered multiple
times after the administration of the AADC polynucleotides.
Levodopa may be administered on a repeating schedule (e.g., every 5
days, weekly, every 10 days, every 15 days, every 30 days, monthly,
bimonthly, every 3 months, every 4 months, every 5 months, every 6
months, every 7 months, every 8 months, every 9 months, every 10
months, every 11 months or yearly) or as symptoms arise. As a
non-limiting example, 3 years post administration of AADC
polynucleotides a subject may have 1-10%, 5-15%, 10-20%, 15-30%,
20-40%, 25-50%, 30-50%, 40-50%, 40-60%, 50-70%, 50- 80%, 60-70%,
60-75%, 60-80%, 60-90%, 70-80%, 70-90%, 75-90%, 80-90%, 90-100% of
the striatal neurons within the infused region of the putamen to be
AADC-immunoreactive. As a non-limiting example, 6 years post
administration of AADC polynucleotides a subject may have 1-10%,
5-15%, 10-20%, 15-30%, 20-40%, 25-50%, 30-50%, 40-50%, 40-60%,
50-70%, 50-80%, 60-70%, 60-75%, 60-80%, 60-90%, 70-80%, 70-90%,
75-90%, 80-90%, 90-100% of the striatal neurons within the infused
region of the putamen to be AADC-immunoreactive. As a non-limiting
example, 9 years post administration of AADC polynucleotides a
subject may have 1-10%, 5-15%, 10-20%, 15-30%, 20-40%, 25-50%,
30-50%, 40-50%, 40-60%, 50-70%, 50-80%, 60-70%, 60-75%, 60-80%,
60-90%, 70-80%, 70-90%, 75-90%, 80-90%, 90-100% of the striatal
neurons within the infused region of the putamen to be
AADC-immunoreactive.
[0103] In one embodiment, a subject who may be administered the
AADC polynucleotides described herein have a documented response to
levodopa therapy but have medically refractory fluctuations and are
considered good surgical candidates. The determination if a subject
is a good surgical candidate may be made by the physician treating
the subject for PD or the physician administering the AADC
polynucleotides who takes into consideration the overall risk to
benefit ratio for the patient for the surgical intervention
required for delivery of the AADC polynucleotides.
[0104] In one embodiment, the ratio of distribution volume in the
parenchyma of an area of a subject to the infusion volume of an
area of a subject may be 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8,
0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1,
2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4,
3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7,
4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0 or
more than 6.0. As a non-limiting example, the ratio of distribution
volume in the parenchyma to infusion volume was 1.6 in the caudate
nucleus. As a non-limiting example, the ratio of distribution
volume in the parenchyma to infusion volume was 3.1 in the putamen.
As a non-limiting example, the distribution of the AADC
polynucleotides in the putamen may be 2-3 times the volume
infused.
Dosing
[0105] The present invention provides methods comprising
administering viral vectors and their AADC polynucleotide payload
or complexes in accordance with the invention to a subject in need
thereof. Viral vector pharmaceutical, imaging, diagnostic, or
prophylactic compositions thereof, may be administered to a subject
using any amount and any route of administration effective for
preventing, treating, diagnosing, or imaging a disease, disorder,
and/or condition (e.g., a disease, disorder, and/or condition
relating to working memory deficits). The exact amount required
will vary from subject to subject, depending on the species, age,
and general condition of the subject, the severity of the disease,
the particular composition, its mode of administration, its mode of
activity, and the like. Compositions in accordance with the
invention are typically formulated in unit dosage form for ease of
administration and uniformity of dosage. It will be understood,
however, that the total daily usage of the compositions of the
present invention may be decided by the attending physician within
the scope of sound medical judgment. The specific therapeutically
effective, prophylactically effective, or appropriate imaging dose
level for any particular patient will depend upon a variety of
factors including the disorder being treated and the severity of
the disorder; the activity of the specific compound employed; the
specific composition employed; the age, body weight, general
health, sex and diet of the patient; the time of administration,
route of administration, and rate of excretion of the specific
polynucleotide payload employed; the duration of the treatment;
drugs used in combination or coincidental with the specific
compound employed; and like factors well known in the medical
arts.
[0106] In certain embodiments, viral vector pharmaceutical
compositions in accordance with the present invention may be
administered at AADC polynucleotide dosage levels sufficient to
deliver from about 0.0001 mg/kg to about 100 mg/kg, from about
0.001 mg/kg to about 0.05 mg/kg, from about 0.005 mg/kg to about
0.05 mg/kg, from about 0.001 mg/kg to about 0.005 mg/kg, from about
0.05 mg/kg to about 0.5 mg/kg, from about 0.01 mg/kg to about 50
mg/kg, from about 0.1 mg/kg to about 40 mg/kg, from about 0.5 mg/kg
to about 30 mg/kg, from about 0.01 mg/kg to about 10 mg/kg, from
about 0.1 mg/kg to about 10 mg/kg, or from about 1 mg/kg to about
25 mg/kg, of subject body weight per day, one or more times a day,
to obtain the desired therapeutic, diagnostic, prophylactic, or
imaging effect. The desired AADC polynucleotide dosage may be
delivered three times in a single day, two times in a single day,
once in a single day or in a period of 24 hours the dosage may be
delivered once, twice, three times or more than three times. In
certain embodiments, the desired AADC polynucleotide dosage may be
delivered using multiple administrations (e.g., two, three, four,
five, six, seven, eight, nine, ten, eleven, twelve, thirteen,
fourteen, or more administrations). When multiple administrations
are employed, split dosing regimens such as those described herein
may be used. As used herein, a "split dose" is the division of
single unit dose or total dose into two or more doses, e.g., two or
more administrations of the single unit dose. As used herein, a
"single unit dose" is a dose of any polynucleotide therapeutic
administered in one dose/at one time/single route/single point of
contact, i.e., single administration event. As used herein, a
"total daily dose" is an amount given or prescribed in 24 hr
period. It may be administered as a single unit dose. In one
embodiment, the viral vectors comprising the AADC polynucleotides
of the present invention are administered to a subject in split
doses. They may be formulated in buffer only or in a formulation
described herein.
[0107] In one embodiment, delivery of viral vector pharmaceutical
compositions in accordance with the present invention to cells of
the central nervous system (e.g., parenchyma) may comprise a total
concentration between about 1.times.10.sup.6 VG/mL and about
1.times.10.sup.16 VG/mL. In some embodiments, delivery may comprise
a composition concentration of about 1.times.10.sup.6,
2.times.10.sup.6, 3.times.10.sup.6, 4.times.10.sup.6,
5.times.10.sup.6, 6.times.10.sup.6, 7.times.10.sup.6,
8.times.10.sup.6, 9.times.10.sup.6, 1.times.10.sup.7,
2.times.10.sup.7, 3.times.10.sup.7, 4.times.10.sup.7,
5.times.10.sup.7, 6.times.10.sup.7, 7.times.10.sup.7,
8.times.10.sup.7, 9.times.10.sup.7, 1.times.10.sup.8,
2.times.10.sup.8, 3.times.10.sup.8, 4.times.10.sup.8,
5.times.10.sup.8, 6.times.10.sup.8, 7.times.10.sup.8,
8.times.10.sup.8, 9.times.10.sup.8, 1.times.10.sup.9,
2.times.10.sup.9, 3.times.10.sup.9, 4.times.10.sup.9,
5.times.10.sup.9, 6.times.10.sup.9, 7.times.10.sup.9,
8.times.10.sup.9, 9.times.10.sup.9, 1.times.10.sup.10,
2.times.10.sup.10, 3.times.10.sup.10, 4.times.10.sup.10,
5.times.10.sup.10, 6.times.10.sup.10, 7.times.10.sup.10,
8.times.10.sup.10, 9.times.10.sup.10, 1.times.10.sup.11,
1.8.times.10.sup.11, 2.times.10.sup.11, 3.times.10.sup.11,
4.times.10.sup.11, 5.times.10.sup.11, 5.5.times.10.sup.11,
6.times.10.sup.11, 7.times.10.sup.11, 8.times.10.sup.11,
9.times.10.sup.11, 0.8.times.10.sup.12, 0.83.times.10.sup.12,
1.times.10.sup.12, 1.1.times.10.sup.12, 1.2.times.10.sup.12,
1.3.times.10.sup.12, 1.4.times.10.sup.12, 1.5.times.10.sup.12,
1.6.times.10.sup.12, 1.7.times.10.sup.12, 1.8.times.10.sup.12,
1.9.times.10.sup.12, 2.times.10.sup.12, 2.1.times.10.sup.12,
2.2.times.10.sup.12, 2.3.times.10.sup.12, 2.4.times.10.sup.12,
2.5.times.10.sup.12, 2.6.times.10.sup.12, 2.7.times.10.sup.12,
2.8.times.10.sup.12, 2.9.times.10.sup.12, 3.times.10.sup.12,
3.1.times.10.sup.12, 3.2.times.10.sup.12, 3.3.times.10.sup.12,
3.4.times.10.sup.12, 3.5.times.10.sup.12, 3.6.times.10.sup.12,
3.7.times.10.sup.12, 3.8.times.10.sup.12, 3.9.times.10.sup.12,
4.times.10.sup.12, 4.1.times.10.sup.12, 4.2.times.10.sup.12,
4.3.times.10.sup.12, 4.4.times.10.sup.12, 4.5.times.10.sup.12,
4.6.times.10.sup.12, 4.7.times.10.sup.12, 4.8.times.10.sup.12,
4.9.times.10.sup.12, 5.times.10.sup.12, 6.times.10.sup.12,
7.times.10.sup.12, 8.times.10.sup.12, 9.times.10.sup.12,
1.times.10.sup.13, 2.times.10.sup.13, 3.times.10.sup.13,
4.times.10.sup.13, 5.times.10.sup.13, 6.times.10.sup.13,
7.times.10.sup.13, 8.times.10.sup.13, 9.times.10.sup.13,
1.times.10.sup.14, 2.times.10.sup.14, 3.times.10.sup.14,
4.times.10.sup.14, 5.times.10.sup.14, 6.times.10.sup.14,
7.times.10.sup.14, 8.times.10.sup.14, 9.times.10.sup.14,
1.times.10.sup.15, 2.times.10.sup.15, 3.times.10.sup.15,
4.times.10.sup.15, 5.times.10.sup.15, 6.times.10.sup.15,
7.times.10.sup.15, 8.times.10.sup.15, 9.times.10.sup.15, or
1.times.10.sup.16 VG/mL. In one embodiment, the concentration of
the viral vector in the composition is 1.times.10.sup.13 VG/mL. In
one embodiment, the concentration of the viral vector in the
composition is 3.times.10.sup.12 VG/mL. In one embodiment, the
concentration of the viral vector in the composition is
1.1.times.10.sup.12 VG/mL. In one embodiment, the concentration of
the viral vector in the composition is 3.7.times.10.sup.12 VG/mL.
In one embodiment, the concentration of the viral vector in the
composition is 8.times.10.sup.11 VG/mL. In one embodiment, the
concentration of the viral vector in the composition is
2.6.times.10.sup.12 VG/mL. In one embodiment, the concentration of
the viral vector in the composition is 4.9.times.10.sup.12 VG/mL.
In one embodiment, the concentration of the viral vector in the
composition is 0.8.times.10.sup.12 VG/mL. In one embodiment, the
concentration of the viral vector in the composition is
0.83.times.10.sup.12 VG/mL. In one embodiment, the concentration of
the viral vector in the composition is the maximum final dose which
can be contained in a vial.
[0108] In one embodiment, delivery of viral vector pharmaceutical
compositions in accordance with the present invention to cells of
the central nervous system (e.g., parenchyma) may comprise a total
concentration per subject between about 1.times.10.sup.6 VG and
about 1.times.10.sup.16 VG. In some embodiments, delivery may
comprise a composition concentration of about 1.times.10.sup.6,
2.times.10.sup.6, 3.times.10.sup.6, 4.times.10.sup.6,
5.times.10.sup.6, 6.times.10.sup.6, 7.times.10.sup.6,
8.times.10.sup.6, 9.times.10.sup.6, 1.times.10.sup.7,
2.times.10.sup.7, 3.times.10.sup.7, 4.times.10.sup.7,
5.times.10.sup.7, 6.times.10.sup.7, 7.times.10.sup.7,
8.times.10.sup.7, 9.times.10.sup.7, 1.times.10.sup.8,
2.times.10.sup.8, 3.times.10.sup.8, 4.times.10.sup.8,
5.times.10.sup.8, 6.times.10.sup.8, 7.times.10.sup.8,
8.times.10.sup.8, 9.times.10.sup.8, 1.times.10.sup.9,
2.times.10.sup.9, 3.times.10.sup.9, 4.times.10.sup.9,
5.times.10.sup.9, 6.times.10.sup.9, 7.times.10.sup.9,
8.times.10.sup.9, 9.times.10.sup.9, 1.times.10.sup.10,
1.5.times.10.sup.10, 2.times.10.sup.10, 3.times.10.sup.10,
4.times.10.sup.10, 5.times.10.sup.10, 6.times.10.sup.10,
7.times.10.sup.10, 8.times.10.sup.1.degree., 9.times.10.sup.10,
1.times.10.sup.11, 1.3.times.10.sup.11, 2.times.10.sup.11,
2.1.times.10.sup.11, 2.2.times.10.sup.11, 2.3.times.10.sup.11,
2.4.times.10.sup.11, 2.5.times.10.sup.11, 2.6.times.10.sup.11,
2.7.times.10.sup.11, 2.8.times.10.sup.11, 2.9.times.10.sup.11,
3.times.10.sup.11, 4.times.10.sup.11, 5.times.10.sup.11,
5.4.times.10.sup.11, 6.times.10.sup.11, 7.times.10.sup.11,
7.1.times.10.sup.11, 7.2.times.10.sup.11, 7.3.times.10.sup.11,
7.4.times.10.sup.11, 7.5.times.10.sup.11, 7.6.times.10.sup.11,
7.7.times.10.sup.11, 7.8.times.10.sup.11, 7.9.times.10.sup.11,
8.times.10.sup.11, 9.times.10.sup.11, 9.4.times.10.sup.11,
1.times.10.sup.12, 1.1.times.10.sup.12, 1.2.times.10.sup.12,
1.3.times.10.sup.12, 1.4.times.10.sup.12, 1.5.times.10.sup.12,
1.6.times.10.sup.12, 1.7.times.10.sup.12, 1.8.times.10.sup.12,
1.9.times.10.sup.12, 2.times.10.sup.12, 2.3.times.10.sup.12,
2.4.times.10.sup.12, 3.times.10.sup.12, 4.times.10.sup.12,
4.1.times.10.sup.12, 4.2.times.10.sup.12, 4.3.times.10.sup.12,
4.4.times.10.sup.12, 4.5.times.10.sup.12, 4.6.times.10.sup.12,
4.7.times.10.sup.12, 4.8.times.10.sup.12, 4.9.times.10.sup.12,
5.times.10.sup.12, 6.times.10.sup.12, 7.times.10.sup.12,
8.times.10.sup.12, 8.1.times.10.sup.12, 8.2.times.10.sup.12,
8.3.times.10.sup.12, 8.4.times.10.sup.12, 8.5.times.10.sup.12,
8.6.times.10.sup.12, 8.7.times.10.sup.12, 8.8.times.10.sup.12,
8.9.times.10.sup.12, 9.times.10.sup.12, 1.times.10.sup.13,
2.times.10.sup.13, 3.times.10.sup.13, 4.times.10.sup.13,
5.times.10.sup.13, 6.times.10.sup.13, 7.times.10.sup.13,
8.times.10.sup.13, 9.times.10.sup.13, 1.times.10.sup.14,
2.times.10.sup.14, 3.times.10.sup.14, 4.times.10.sup.14,
5.times.10.sup.14, 6.times.10.sup.14, 7.times.10.sup.14,
8.times.10.sup.14, 9.times.10.sup.14, 1.times.10.sup.15,
2.times.10.sup.15, 3.times.10.sup.15, 4.times.10.sup.15,
5.times.10.sup.15, 6.times.10.sup.15, 7.times.10.sup.15,
8.times.10.sup.15, 9.times.10.sup.15, or 1.times.10.sup.16
VG/subject. In one embodiment, the concentration of the viral
vector in the composition is 1.times.10.sup.13 VG/subject. In one
embodiment, the concentration of the viral vector in the
composition is 3.times.10.sup.12 VG/subject. As a non-limiting
example, the composition administered to the subject has a
concentration of about 3.times.10.sup.11 VG/subject. As a
non-limiting example, the composition administered to the subject
has a concentration of about 9.times.10.sup.11 VG/subject. In one
embodiment, the concentration of the viral vector in the
composition is 2.3.times.10.sup.11 VG/subject. In one embodiment,
the concentration of the viral vector in the composition is
7.2.times.10.sup.11 VG/subject. In one embodiment, the
concentration of the viral vector in the composition is
7.5.times.10.sup.11 VG/subject. In one embodiment, the
concentration of the viral vector in the composition is
1.4.times.10.sup.12 VG/subject. In one embodiment, the
concentration of the viral vector in the composition is
4.8.times.10.sup.12 VG/subject. In one embodiment, the
concentration of the viral vector in the composition is
8.8.times.10.sup.12 VG/subject. In one embodiment, the
concentration of the viral vector in the composition is
2.3.times.10.sup.12 VG/subject.
[0109] In one embodiment, the effectiveness of the dose, route of
administration and/or volume of administration may be evaluated
using various methods described herein such as, but not limited to,
PET imaging, L-DOPA challenge test (e.g., see Forsayeth et al.
2006, Mol. Ther. 14(4): 571-577), UPDRS scores and patient diaries.
As a non-limiting example, a subject may have decreased dyskinesia
or periods of decreased dyskinesia after administration of the AADC
polynucleotide composition. As another non-limiting example, a
subject may have a decrease in Parkinson's Disease related symptoms
including limited mobility and dyskinesia. As yet another
non-limiting example, a subject may show improvement in off time
and motor fluctuations. The improvement may be at least 10%, at
least 20%, at least 30%, at least 40%, at least 50%, at least 60%,
at least 70%, at least 80%, at least 90% or greater than 90%. The
improvement may last for minutes (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9,
10, 15, 20, 25, 30, 35, 40, 45, 50, 55 or more than 55), hours
(e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,
18, 19, 20, 21, 22, 23 or more than 24), days (e.g., 1, 2, 3, 4, 5,
6 or more than 7), weeks (1, 2, 3, 4, 5, 6, 7 or more than 7),
months (1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or more than 11) or years
(e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or more than 9).
[0110] In one embodiment, the selection of subjects for
administration of the viral vectors described herein and/or the
effectiveness of the dose, route of administration and/or volume of
administration may be evaluated using imaging of the perivascular
spaces (PVS) which are also known as Virchow-Robin spaces. PVS
surround the arterioles and venules as they perforate brain
parenchyma and are filled with cerebrospinal fluid
(CSF)/interstitial fluid. PVS are common in the midbrain, BG, and
centrum semiovale. While not wishing to be bound by theory, PVS may
play a role in the normal clearance of metabolites and have been
associated with worse cognition and several disease states
including Parkinson's disease. PVS are usually normal in size but
they can increase in size in a number of disease states. Potter et
al. (Cerebrovasc Dis. 2015 January; 39(4): 224-231; the contents of
which are herein incorporated by reference in its entirety)
developed a grading method where they studied a full range of PVS
and rated basal ganglia, centrum semiovale and midbrain PVS. They
used the frequency and range of PVS used by Mac and Lullich et al.
(J Neurol Neurosurg Psychiatry. 2004 November; 75(11):1519-23; the
contents of which are herein incorporated by reference in its
entirety) and Potter et al. gave 5 ratings to basal ganglia and
centrum semiovale PVS: 0 (none), 1 (1-10), 2 (11-20), 3 (21-40) and
4 (>40) and 2 ratings to midbrain PVS: 0 (non visible) or 1
(visible). The user guide for the rating system by Potter et al.
can be found at:
www.sbirc.ed.ac.uk/documents/epvs-rating-scale-user-guide.pdf.
[0111] In one embodiment, the selection of subjects for
administration of the viral vectors described herein and/or the
effectiveness of the dose, route of administration and/or volume of
administration may be evaluated using positron emission tomography
(PET) measurements of neuroimaging biomarkers such as, but not
limited to [.sup.18F]FDOPA. Neuroimaging biomarkers such as
[.sup.18F]FDOPA may be used to identify affected individuals and/or
may be used to detect a nigrostriatal defect prior to the onset of
clinical manifestations. Further, PET-based criteria may be used to
categorize subjects based on their nigrostriatal neuronal integrity
(e.g., abnormal, normal or uncertain nigrostriatal neuronal
integrity) (Rachette et al. Am J Med Genet B Neuropsychiatr Genet.
2006 Apr. 5; 141B(3): 245-249; the contents of which are herein
incorporated by reference in its entirety).
[0112] In one embodiment, a subject who may be administered a dose
of the AADC polynucleotides described herein may have advanced PD
and still respond to levodopa therapy but the subject also
experiences medically refractory motor complications (e.g., sever
motor fluctuations and/or dyskinesias that occur during levodopa
and other dopaminergic therapies despite adjustments in and
optimization of medication). The subject may be healthy enough to
undergo a neurosurgical procedure which may be determined by
methods known in the art. As a non-limiting example, the subject
may meet the selection criteria for deep brain stimulation (DBS).
The subject may have idiopathic PD, younger than 69 years of age,
have pronounced responses to levodopa, have medication-refractory
symptoms (e.g., motor fluctuation and/or dyskinesia) and/or have
little or no cognitive dysfunction.
[0113] In one embodiment, a subject who may be administered a dose
of the AADC polynucleotides described herein may also suffer from
dementia or cognitive impairment.
[0114] In one embodiment, a subject who may be administered a dose
of the AADC polynucleotides described herein may have been
previously treated with the same or similar therapeutic. In another
embodiment, a subject may have been treated with a therapeutic
which has been shown to reduce the symptoms of Parkinson's
Disease.
[0115] In one embodiment, a subject who may be administered a dose
of the AADC polynucleotides described herein may have failed to
derive adequate benefit from standard medical therapy. As a
non-limiting example, the subject may not have responded to
treatment. As another non-limiting example, a subject may have
residual disability despite treatment.
[0116] In one embodiment, a subject who may be administered a dose
of the AADC polynucleotides described herein may undergo testing to
evaluate the levels of neurotransmitter analytes to determine the
effectiveness of the dose. As a non-limiting example, CSF
neurotransmitters, plasma AADC activity and/or urine VLA may be
analyzed.
[0117] In one embodiment, a subject who may be administered a dose
of the AADC polynucleotide described herein may be videotaped or
recorded in order to monitor the progress of the subject during the
course of treatment.
Combinations
[0118] The viral vectors comprising the AADC polynucleotide may be
used in combination with one or more other therapeutic,
prophylactic, diagnostic, or imaging agents. By "in combination
with," it is not intended to imply that the agents must be
administered at the same time and/or formulated for delivery
together, although these methods of delivery are within the scope
of the present disclosure. Compositions can be administered
concurrently with, prior to, or subsequent to, one or more other
desired therapeutics or medical procedures. In general, each agent
will be administered at a dose and/or on a time schedule determined
for that agent. In some embodiments, the present disclosure
encompasses the delivery of pharmaceutical, prophylactic,
diagnostic, or imaging compositions in combination with agents that
may improve their bioavailability, reduce and/or modify their
metabolism, inhibit their excretion, and/or modify their
distribution within the body.
Delivery
[0119] In one embodiment, the viral vector comprising an AADC
polynucleotide may be administered or delivered using the methods
for the delivery of AAV virions described in European Patent
Application No. EP1857552, the contents of which are herein
incorporated by reference in its entirety.
[0120] In one embodiment, the viral vector comprising an AADC
polynucleotide may be administered or delivered using the methods
for delivering proteins using AAV vectors described in European
Patent Application No. EP2678433, the contents of which are herein
incorporated by reference in its entirety.
[0121] In one embodiment, the viral vector comprising an AADC
polynucleotide may be administered or delivered using the methods
for delivering DNA molecules using AAV vectors described in U.S.
Pat. No. 5,858,351, the contents of which are herein incorporated
by reference in its entirety.
[0122] In one embodiment, the viral vector comprising an AADC
polynucleotide may be administered or delivered using the methods
for delivering DNA to the bloodstream described in U.S. Pat. No.
6,211,163, the contents of which are herein incorporated by
reference in its entirety.
[0123] In one embodiment, the viral vector comprising an AADC
polynucleotide may be administered or delivered using the methods
for delivering AAV virions described in U.S. Pat. No. 6,325,998,
the contents of which are herein incorporated by reference in its
entirety.
[0124] In one embodiment, the viral vector comprising an AADC
polynucleotide may be administered or delivered using the methods
for delivering a payload to the central nervous system described in
U.S. Pat. No. 7,588,757, the contents of which are herein
incorporated by reference in its entirety.
[0125] In one embodiment, the viral vector comprising an AADC
polynucleotide may be administered or delivered using the methods
for delivering a payload described in U.S. Pat. No. 8,283,151, the
contents of which are herein incorporated by reference in its
entirety.
[0126] In one embodiment, the viral vector comprising an AADC
polynucleotide may be administered or delivered using the methods
for delivering a payload using a glutamic acid decarboxylase (GAD)
delivery vector described in International Patent Publication No.
WO2001089583, the contents of which are herein incorporated by
reference in its entirety.
[0127] In one embodiment, the viral vector comprising an AADC
polynucleotide may be administered or delivered using the methods
for delivering a payload to neural cells described in International
Patent Publication No. WO2012057363, the contents of which are
herein incorporated by reference in its entirety.
[0128] The pharmaceutical compositions of viral vectors described
herein may be characterized by one or more of bioavailability,
therapeutic window and/or volume of distribution.
Bioavailability
[0129] Viral vectors comprising an AADC polynucleotide of the
present invention, when formulated into compositions with
delivery/formulation agents or vehicles as described herein, may
exhibit increased bioavailability as compared to compositions
lacking delivery agents as described herein. As used herein, the
term "bioavailability" refers to the systemic availability of a
given amount of a particular agent administered to a subject.
Bioavailability may be assessed by measuring the area under the
curve (AUC) or the maximum serum or plasma concentration
(C.sub.max) of the unchanged form of a compound following
administration of the compound to a mammal. AUC is a determination
of the area under the curve plotting the serum or plasma
concentration of a compound along the ordinate (Y-axis) against
time along the abscissa (X-axis). Generally, the AUC for a
particular compound may be calculated using methods known to those
of ordinary skill in the art and as described in G. S. Banker,
Modern Pharmaceutics, Drugs and the Pharmaceutical Sciences, v. 72,
Marcel Dekker, New York, Inc., 1996, the contents of which are
herein incorporated by reference in their entirety.
[0130] C.sub.max values are maximum concentrations of compounds
achieved in serum or plasma of a subject following administration
of compounds to the subject. C.sub.max values of particular
compounds may be measured using methods known to those of ordinary
skill in the art. As used herein, the phrases "increasing
bioavailability" or "improving the pharmacokinetics," refer to
actions that may increase the systemic availability of a viral
vector of the present invention (as measured by AUC, C.sub.max, or
C.sub.min) in a subject. In some embodiments, such actions may
comprise co-administration with one or more delivery agents as
described herein. In some embodiments, the bioavailability of viral
vectors may increase by at least about 2%, at least about 5%, at
least about 10%, at least about 15%, at least about 20%, at least
about 25%, at least about 30%, at least about 35%, at least about
40%, at least about 45%, at least about 50%, at least about 55%, at
least about 60%, at least about 65%, at least about 70%, at least
about 75%, at least about 80%, at least about 85%, at least about
90%, at least about 95% or about 100%.
Therapeutic Window
[0131] Viral vectors comprising an AADC polynucleotide of the
present invention, when formulated with one or more delivery agents
as described herein, may exhibit increases in the therapeutic
window of compound and/or composition administration as compared to
the therapeutic window of viral vectors administered without one or
more delivery agents as described herein. As used herein, the term
"therapeutic window" refers to the range of plasma concentrations,
or the range of levels of therapeutically active substance at the
site of action, with a high probability of eliciting a therapeutic
effect. In some embodiments, therapeutic windows of viral vectors
when administered in a formulation may increase by at least about
2%, at least about 5%, at least about 10%, at least about 15%, at
least about 20%, at least about 25%, at least about 30%, at least
about 35%, at least about 40%, at least about 45%, at least about
50%, at least about 55%, at least about 60%, at least about 65%, at
least about 70%, at least about 75%, at least about 80%, at least
about 85%, at least about 90%, at least about 95% or about
100%.
Volume of Distribution
[0132] Viral vectors comprising an AADC polynucleotide of the
present invention, when formulated with one or more delivery agents
as described herein, may exhibit an improved volume of distribution
(V.sub.dist), e.g., reduced or targeted, relative to formulations
lacking one or more delivery agents as described herein. V.sub.dist
relates the amount of an agent in the body to the concentration of
the same agent in the blood or plasma. As used herein, the term
"volume of distribution" refers to the fluid volume that would be
required to contain the total amount of an agent in the body at the
same concentration as in the blood or plasma: V.sub.dist equals the
amount of an agent in the body/concentration of the agent in blood
or plasma. For example, for a 10 mg dose of a given agent and a
plasma concentration of 10 mg/L, the volume of distribution would
be 1 liter. The volume of distribution reflects the extent to which
an agent is present in the extravascular tissue. Large volumes of
distribution reflect the tendency of agents to bind to the tissue
components as compared with plasma proteins. In clinical settings,
V.sub.dist may be used to determine loading doses to achieve steady
state concentrations. In some embodiments, volumes of distribution
of viral vector compositions of the present invention when
co-administered with one or more delivery agents as described
herein may decrease at least about 2%, at least about 5%, at least
about 10%, at least about 15%, at least about 20%, at least about
25%, at least about 30%, at least about 35%, at least about 40%, at
least about 45%, at least about 50%, at least about 55%, at least
about 60%, at least about 65%, at least about 70%.
Kits and Devices
[0133] The invention provides a variety of kits for conveniently
and/or effectively carrying out methods of the present invention.
Typically kits will comprise sufficient amounts and/or numbers of
components to allow a user to perform multiple treatments of a
subject(s) and/or to perform multiple experiments.
[0134] Any of the AADC vectors, AADC constructs, AADC
polynucleotides, or AADC polypeptides of the present invention may
be comprised in a kit. In some embodiments, kits may further
include reagents and/or instructions for creating and/or
synthesizing compounds and/or compositions of the present
invention. In some embodiments, kits may also include one or more
buffers. In some embodiments, kits of the invention may include
components for making protein or nucleic acid arrays or libraries
and thus, may include, for example, solid supports.
[0135] In some embodiments, kit components may be packaged either
in aqueous media or in lyophilized form. The container means of the
kits will generally include at least one vial, test tube, flask,
bottle, syringe or other container means, into which a component
may be placed, and preferably, suitably aliquoted. Where there are
more than one kit component, (labeling reagent and label may be
packaged together), kits may also generally contain second, third
or other additional containers into which additional components may
be separately placed. In some embodiments, kits may also comprise
second container means for containing sterile, pharmaceutically
acceptable buffers and/or other diluents. In some embodiments,
various combinations of components may be comprised in one or more
vial. Kits of the present invention may also typically include
means for containing compounds and/or compositions of the present
invention, e.g., proteins, nucleic acids, and any other reagent
containers in close confinement for commercial sale. Such
containers may include injection or blow-molded plastic containers
into which desired vials are retained.
[0136] In some embodiments, kit components are provided in one
and/or more liquid solutions. In some embodiments, liquid solutions
are aqueous solutions, with sterile aqueous solutions being
particularly preferred. In some embodiments, kit components may be
provided as dried powder(s). When reagents and/or components are
provided as dry powders, such powders may be reconstituted by the
addition of suitable volumes of solvent. In some embodiments, it is
envisioned that solvents may also be provided in another container
means. In some embodiments, labeling dyes are provided as dried
powders. In some embodiments, it is contemplated that 10, 20, 30,
40, 50, 60, 70, 80, 90, 100, 120, 120, 130, 140, 150, 160, 170,
180, 190, 200, 300, 400, 500, 600, 700, 800, 900, 1000 micrograms
or at least or at most those amounts of dried dye are provided in
kits of the invention. In such embodiments, dye may then be
resuspended in any suitable solvent, such as DMSO.
[0137] In some embodiments, kits may include instructions for
employing kit components as well the use of any other reagent not
included in the kit. Instructions may include variations that may
be implemented.
Devices
[0138] In some embodiments, AADC compounds and/or AADC compositions
of the present invention may be combined with, coated onto or
embedded in a device. Devices may include, but are not limited to
stents, pumps, and/or other implantable therapeutic device.
Additionally AADC compounds and/or AADC compositions may be
delivered to a subject while the subject is using a compression
device such as, but not limited to, a compression device to reduce
the chances of deep vein thrombosis (DVT) in a subject.
[0139] The present invention provides for devices which may
incorporate viral vectors that encode one or more AADC
polynucleotide payload molecules. These devices contain in a stable
formulation the viral vectors which may be immediately delivered to
a subject in need thereof, such as a human patient.
[0140] Devices for administration may be employed to deliver the
viral vectors comprising an AADC polynucleotide of the present
invention according to single, multi- or split-dosing regimens
taught herein.
[0141] Method and devices known in the art for multi-administration
to cells, organs and tissues are contemplated for use in
conjunction with the methods and compositions disclosed herein as
embodiments of the present invention. These include, for example,
those methods and devices having multiple needles, hybrid devices
employing for example lumens or catheters as well as devices
utilizing heat, electric current or radiation driven
mechanisms.
[0142] In some embodiments, AADC compounds and/or AADC compositions
of the present invention may be delivered using a device such as,
but not limited to, a stent, a tube, a catheter, a pipe, a straw,
needle and/or a duct. Methods of using these devices are described
herein and are known in the art.
[0143] In one embodiment, the AADC polynucleotides of the present
invention may be administered to a subject using delivery systems
which integrate image guided therapy and integrate imaging such as,
but not limited to, laser, MRgFUS, endoscopic and robotic surgery
devices.
[0144] In one embodiment, the AADC polynucleotides of the present
invention may be administered to a subject using the
CLEARPOINT.RTM. neuro intervention system by MRI Interventions,
Inc. The CLEARPOINT.RTM. neuro intervention system may be used
alone or in combination with any of the other administration
methods and devices described herein. The CLEARPOINT.RTM. neuro
intervention system helps to provide stereotactic guidance in the
placement and operation of instruments or devices during the
planning and operation of neurological procedures.
[0145] In one embodiment, the AADC polynucleotides of the present
invention may be administered to a subject using the NEUROMATE.RTM.
stereotactic robot system by Renishaw PLC. The NEUROMATE.RTM.
system may be used alone or in combination with any of the other
administration methods and devices described herein. As a
non-limiting example, the NEUROMATE.RTM. system may be used with
head holders, CT image localizers, frame attachments, remote
controls and software.
[0146] In one embodiment, the AADC polynucleotides of the present
invention may be administered to a subject using the Elekta
MICRODRIVE.TM. device by Elekta AB. The MICRODRIVE.TM. device may
be used alone or in combination with any of the other
administration methods and devices described herein. As a
non-limiting example, the MICRODRIVE.TM. device may be used to
position electrodes (e.g., for micro electrode recording (MER),
macro stimulation and deep brain stimulation (DBS) electrode
implantation), implantation of catheters, tubes or DBS electrodes
using cross-hair and A-P holders to verify position, biopsies,
injections and aspirations, brain lesioning, endoscope guidance and
GAMMA KNIFE.RTM. radiosurgery.
[0147] In one embodiment, the AADC polynucleotides of the present
invention may be administered to a subject using the AXIIIS.RTM.
stereotactic miniframe by MONTERIS.RTM. Medical, Inc. The
AXIIIS.RTM. stereotactic miniframe may be used alone or in
combination with any of the other administration methods and
devices described herein. The AXIIIS.RTM. stereotactic miniframe is
a trajectory alignment device which may be used for laser
coagulation, biopsies, catheter placement, electrode implant,
endoscopy, and clot evacuation. The miniframe allows for 360 degree
interface and provides access to multiple intracranial targets with
a simple adjustment. Further, the miniframe is compatible with
MM.
[0148] In one embodiment, the AADC polynucleotides of the present
invention may be administered to a subject using the INTEGRA.TM.
CRW.RTM. system by Integra LifeSciences Corporation. The
INTEGRA.TM. CRW.RTM. system may be used alone or in combination
with any of the other administration methods and devices described
herein. The CRW.RTM. system may be used for various applications
such as, but not limited to, stereotactic surgery, microsurgery,
catheterization and biopsy. The CRW.RTM. system is designed to
provide accuracy to those who use the system (e.g., thumb lock
screws, Vernier scaling, double bolt fixation, and a solid
frame).
[0149] In one embodiment, the AADC polynucleotides of the present
invention may be administered to a subject using the EPOCH.RTM.
solution system by Stereotaxis, Inc. which may include the
NIOBE.RTM. ES magnetic navigation system, the VDRIVE.RTM. robotic
navigation system and/or the ODYSSEY.RTM. information solution (all
by Stereotaxis, Inc.). The EPOCH.RTM. solution system may be used
alone or in combination with any of the other administration
methods and devices described herein. As a non-limiting example,
the NIOBE.RTM. ES magnetic navigation system may be used to
accurately contact a subject. As another non-limiting example the
NIOBE.RTM. ES magnetic system may be used with the VDRIVE.RTM.
robotic navigation system to provide precise movement and
stability.
[0150] In one embodiment, the AADC polynucleotides of the present
invention may be administered to a subject using a NeuroStation
workstation which uses frameless stereotactic methods to provide
image-guidance for applications such as, but not limited to,
surgical planning, biopsies, craniotomies, endoscopy,
intra-operative ultrasound and radiation therapy.
[0151] In one embodiment, the AADC polynucleotides of the present
invention may be administered to a subject using a robotic
stereotaxis system such as, but not limited to the device described
in U.S. Pat. No. 5,078,140, the contents of which are herein
incorporated by reference in its entirety. The robotic arm of the
device may be used to precisely orient the surgical tools or other
implements used to conduct a procedure.
[0152] In one embodiment, the AADC polynucleotides of the present
invention may be administered to a subject using an automatic
delivery system such as, but not limited to the device described in
U.S. Pat. No. 5,865,744, the contents of which are herein
incorporated by reference in its entirety. Based on the images
gathered by the delivery system, the computer adjusts the
administration of the needle to be the appropriate depth for the
particular subject.
[0153] In one embodiment, the AADC polynucleotides of the present
invention may be administered to a subject who is simultaneously
using during administration, and/or uses for a period of time
before and/or after administration a compression device such as,
but not limited to, a compression device which reduces the chances
of deep vein thrombosis (DVT) in a subject. The compression device
may be used for at least 5 minutes, 15 minutes, 30 minutes, 45
minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7
hours, 8 hours, or more than 8 hours before a subject is
administered the AADC polynucleotides. The compression device may
be used for at least 5 minutes, 15 minutes, 30 minutes, 45 minutes,
1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8
hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours,
15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21
hours, 22 hours, 23 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6
days, 1 week, 2 weeks, 3 weeks or a month after the AADC
polynucleotides are administered. As a non-limiting example, the
compression device is used simultaneously during the procedure of
the delivery of the AADC polynucleotides. As another non-limiting
example, the compression device is used before the administration
of the AADC polynucleotides. As another non-limiting example, the
compression device is used after administration of the AADC
polynucleotides. As another non-limiting example, the compression
device is used before, during and after administration of the AADC
polynucleotides.
[0154] Non-limiting examples, of compression devices include
ActiveCare+S.F.T. intermittent compression device, ActiveCare+S.F.T
pneumatic compression device, DVTlite's Venowave, KCl system
compression pump, Aircast VenaFlow system, SCD Express Compression
System or Bio Compression Systems, Inc. pneumatic compression
therapy equipment (e.g., the pump may be selected from Model
SC-2004, Model SC-2004-FC, Model SC-3004, Model SC-3004-FC, Model
SC-2008, Model SC-2008-DL, Model SC-3008-T, the BioCryo system,
Model IC-BAP-DL or multi-flo DVT combo IC 1545-DL and the garment
used with the pump may be a 4 chamber, 8 chamber, BioCryo,
Multi-Flo or BioArterial garment).
CNS Diseases
[0155] The polynucleotides of the present invention may be used in
the treatment, prophylaxis or amelioration of any disease or
disorder characterized by aberrant or undesired target expression.
In one embodiment, the invention relates to compositions,
particularly nucleic acid molecules, e.g., polynucleotides encoding
AADC, for use in the treatment of Parkinson's disease.
[0156] In some embodiments, the polynucleotides of the invention
may be used in the treatment, prophylaxis or amelioration of any
disease or disorder characterized by aberrant or undesired target
expression wherein the payload, i.e. AADC, is swapped for an
alternate payload.
[0157] The present disclosure provides a method for treating a
disease, disorder and/or condition in a mammalian subject,
including a human subject, comprising administering to the subject
any of the viral particles e.g., AAV, AAV polynucleotides or AAV
genomes described herein (i.e., viral genomes or "VG") or
administering to the subject a particle comprising said AAV
polynucleotide or AAV genome, or administering to the subject any
of the described compositions, including pharmaceutical
compositions.
[0158] In one embodiment, the disease, disorder and/or condition is
a neurological disease, disorder and/or condition. The CNS diseases
may be diseases that affect any component of the brain (including
the cerebral hemispheres, diencephalon, brain stem, and cerebellum)
or the spinal cord.
[0159] In some embodiments, viral particles of the present
invention, through delivery of a functional payload that is a
therapeutic product that can modulate the level or function of a
gene product in the CNS, may be used to treat a neurodegenerative
diseases and/or diseases or disorders that are characteristic with
neurodegeneration, neuromuscular diseases, lysosomal diseases,
trauma, bone marrow injuries, pain (including neuropathic pain),
cancers of the nervous system, demyelinating diseases, autoimmune
diseases of the nervous system, neurotoxic syndromes, sleeping
disorders, genetic brain disorders and developmental CNS disorders.
A functional payload may alleviate or reduce symptoms that result
from abnormal level and/or function of a gene product (e.g., an
absence or defect in a protein) in a subject in need thereof or
that otherwise confers a benefit to a CNS disorder in a subject in
need thereof.
[0160] As non-limiting examples, therapeutic products delivered by
viral particles of the present invention may include, but are not
limited to, growth and trophic factors, cytokines, hormones,
neurotransmitters, enzymes, anti-apoptotic factors, angiogenic
factors, and any protein known to be mutated in pathological
disorders such as the "survival of motor neuron" protein (SMN);
antisense RNA or RNAi targeting messenger RNAs coding for proteins
having a therapeutic interest in any of CNS diseases discussed
herein; or microRNAs that function in gene silencing and
post-transcriptionally regulation of gene expression in the CNS
(e.g., brain specific Mir-128.alpha., See Adlakha and Saini,
Molecular cancer, 2014, 13:33). For example, an RNAi targeting the
superoxide dismutase enzyme may be packaged by viral particles of
the present invention, for the treatment of ALS.
[0161] The growth and trophic factors may include, but are not
limited to brain-derived growth factor (BDNF), epidermal growth
factor (EGF), basic Fibroblast growth factor (bFGF), Ciliary
neurotrophic factor (CNTF), corticotropin-releasing factor (CRF),
Glial cell line derived growth factor (GDNF), Insulin-like growth
factor-1 (IGF-1), nerve growth factor (NGF), neurotrophin-3 (NT-3),
neurotrophin-4 (NT-4), and vascular endothelial growth factor
(VEGF). Cytokines may include interleukin-10 (IL-10),
interleukin-6, Interleukin-8, chemokine CXCL12 (SDF-1), TGF-beta,
and Growth and differentiation factor (GDF-1/10).
[0162] In some embodiments, the neurological disorders may be
neurodegenerative disorders including, but not limited to,
Alzheimer's Diseases (AD); Amyotrophic lateral sclerosis (ALS);
Creutzfeldt-Jakob Disease (CJD); Huntingtin's disease (HD);
Friedreich's ataxia (FA); Parkinson Disease (PD); Multiple System
Atrophy (MSA); Spinal Muscular Atrophy (SMA), Multiple Sclerosis
(MS); Primary progressive aphasia; Progressive supranuclear palsy
(PSP); Dementia; Brain Cancer, Degenerative Nerve Diseases,
Encephalitis, Epilepsy, Genetic Brain Disorders that cause
neurodegeneration, Retinitis pigmentosa (RP), Head and Brain
Malformations, Hydrocephalus, Stroke, Prion disease, Infantile
neuronal ceroid lipofuscinosis (INCL) (a neurodegenerative disease
of children caused by a deficiency in the lysosomal enzyme
palmitoyl protein thioesterase-1 (PPT1)), and others.
[0163] In some embodiments, viral particles of the present
invention may be used to treat diseases that are associated with
impairments of the growth and development of the CNS, i.e.,
neurodevelopmental disorders. In some aspects, such
neurodevelopmental disorders may be caused by genetic mutations,
including but not limited to, Fragile X syndrome (caused by
mutations in FMR1 gene), Down syndrome (caused by trisomy of
chromosome 21), Rett syndrome, Williams syndrome, Angelman
syndrome, Smith-Magenis syndrome, ATR-X syndrome, Barth syndrome,
Immune dysfunction and/or infectious diseases during infancy such
as Sydenham's chorea, Schizophrenia Congenital toxoplasmosis,
Congenital rubella syndrome, Metabolic disorders such as diabetes
mellitus and phenylketonuria; nutritional defects and/or brain
trauma, Autism and autism spectrum.
[0164] In some embodiments, viral particles of the present
invention, may be used to treat a tumor in the CNS, including but
not limited to, acoustic neuroma, Astrocytoma (Grades I, II, III
and IV), Chordoma, CNS Lymphoma, Craniopharyngioma, Gliomas (e.g.,
brain stem glioma, ependymoma, optical nerve glioma,
subependymoma), Medulloblastoma, Meningioma, Metastatic brain
tumors, Oligodendroglioma, Pituitary Tumors, Primitive
neuroectodermal (PNET), and Schwannoma.
[0165] In some embodiments, the neurological disorders may be
functional neurological disorders with motor and/or sensory
symptoms which have neurological origin in the CNS. As non-limiting
examples, functional neurological disorders may be chronic pain,
seizures, speech problems, involuntary movements, and sleep
disturbances.
[0166] In some embodiments, the neurological disorders may be white
matter disorders (a group of diseases that affects nerve fibers in
the CNS) including but not limited to, Pelizaeus-Merzbacher
disease, Hypomyelination with atrophy of basal ganglia and
cerebellum, Aicardi-Goutieres syndrome, Megalencephalic
leukoencephalopathy with subcortical cysts, Congenital muscular
dystrophies, Myotonic dystrophy, Wilson disease, Lowe syndrome,
Sjogren-Larsson syndrome, PIBD or Tay syndrome, Cockayne's disease,
erebrotendinous xanthomatosis, Zellweger syndrome, Neonatal
adrenoleukodystrophy, Infantile Refsum disease, Zellweger-like
syndrome, Pseudo-Zellweger syndrome, Pseudo-neonatal
adrenoleukodystrophy, Bifunctional protein deficiency, X-linked
adrenoleukodystrophy and adrenomyeloneuropathy and Refsum
disease.
[0167] In some embodiments, the neurological disorders may be
lysosomal storage disorders (LSDs) caused by the inability of cells
in the CNS to break down metabolic end products, including but not
limited to Niemann-Pick disease (a LSD resulting from inherited
deficiency in acid sphingomyelinase (ASM); Metachromatic
leukodystrophy (MLD) (a LSD characterized by accumulation of
sulfatides in glial cells and neurons, the result of an inherited
deficiency of arylsulfatase A (ARSA)); Globoid-cell leukodystrophy
(GLD) (a LSD caused by mutations in galactosylceramidase); Fabry
disease (a LSD caused by mutations in the alpha-galactosidase A
(GLA) gene); Gaucher disease (caused by mutations in the
beta-glucocerebrosidase (GBA) gene); GM1/GM2 gangliosidosis;
Mucopolysaccharidoses disorder; Pompe disease; and Neuronal ceroid
lipofuscinosis.
[0168] In one embodiment, the neurological disease, disorder and/or
condition is Parkinson's disease. In one embodiment the
polynucleotide used to treat Parkinson's disease comprises any one
of SEQ ID NOs 2-23, such as, but not limited to SEQ ID NOs: 6-9 and
17-23, wherein the payload is replaced by AADC or any other payload
known in the art for treating Parkinson's disease. As a
non-limiting example, the condition is early stage Parkinson's
disease. As another non-limiting example, the condition is late
stage Parkinson's disease.
[0169] In one embodiment, the subject is a human patient who has a
minimum motor score of about 30 to a maximum score of about 100,
about 10 to a maximum score of about 100, about 20 to a maximum
score of about 100 in the Unified Parkinson's Disease Rating
Scale.
[0170] In one embodiment, the subject has been diagnosed with
Parkinson's disease within the past 5 years prior to treatment with
the compositions described herein. As a non-limiting example, the
subject may have been diagnosed with Parkinson's disease within a
week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8
weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7
months, 8 months, 9 months, 10 months, 11 months, 12 months, 13
months, 14 months, 15 months, 16 months, 17 months, 18 months, 1
year, 2 years, 3 years, 4 years or less than 5 years prior to
treatment with the compositions described herein.
[0171] In one embodiment, the subject has been diagnosed with
Parkinson's disease between 5 and 10 years prior to treatment with
the compositions described herein. As a non-limiting example, the
subject may have been diagnosed with Parkinson's disease 5, 5.5, 6,
6.5, 7, 7.5, 8, 8.5, 9, 9.5 or years prior to treatment with the
compositions described herein.
[0172] In one embodiment, the subject has been diagnosed with
Parkinson's disease more than 10 years prior to treatment with the
compositions described herein. As a non-limiting example, the
subject may have been diagnosed with Parkinson's disease 10.5, 11,
11.5, 12, 12.5, 13, 13.5, 14, 14.5, 15, 15.5, 16, 16.5, 17, 17.5,
18, 18.5, 19, 19.5, 20, 20.5, 21, 21.5, 22, 22.5, 23, 23.5, 24 or
more than 24 years prior to treatment with the compositions
described herein.
[0173] In one embodiment, a subject has seen a change in motor
symptoms such as tremors and movements prior to administration of
the composition described herein. Non-limiting examples of tremors
include, unilateral or bilateral mild tremors, bilateral or midline
moderate tremors or intractable tremors. Non-limiting examples of
movements include mild bradykinesia, moderate bradykinesia, severe
bradykinesia and morning akinesia.
[0174] In one embodiment, a subject may have changes in balance
such as, but not limited to, impaired balance, impaired righting
reflexes, significant balance disorder or falling.
[0175] In one embodiment, a subject may have a reduced quality of
life. As a non-limiting example, the subject may have a moderate
impact on their quality of life such as experiencing some
limitations to activities of daily living. As another non-limiting
example, the subject may have a quality of life which has been
diminished by illness.
[0176] In one embodiment, a subject has seen a change in non-motor
symptoms prior to administration of the composition described
herein. As a non-limiting example, the subject may have mild to
moderate cognitive impairment prior to administration to the
composition described herein. As another non-limiting example, the
subject may have significant cognitive impairment such as dementia
which may also include behavioral disturbances such as
hallucinations.
[0177] In one embodiment, a subject may have a satisfactory
response with limited fluctuations on one or more dopaminergic
medications prior to administration of the compositions described
herein.
[0178] In one embodiment, a subject may have motor fluctuations
causing mild to moderate disability on one or more dopaminergic
medications prior to administration of the compositions described
herein.
[0179] In one embodiment, a subject may have medically refractory
motor fluctuations consisting of "wearing off" and/or
levodopa-induced dyskinesias causing significant disability prior
to administration of the compositions described herein.
[0180] In one embodiment, a subject may have mild symptoms
associated with Parkinson's disease such as, but not limited to, no
cognitive impairment, diagnosed within the past 5 years,
satisfactory response with limited fluctuations on one or more
dopaminergic medications, unilateral or bilateral mild tremors,
little to no impact on the quality of life, and/or no balance
impairment.
[0181] In one embodiment, a subject may have moderate symptoms
associated with Parkinson's disease such as, but not limited to,
mild to moderate cognitive impairment, first signs of impaired
balance and righting reflexes, motor fluctuations causing
mild-moderate disability on one or more dopaminergic medications,
diagnosed within the past 5 to 10 years, bilateral or midline
moderate tremors, moderate bradykinesia and/or subject experiencing
some limitations to activities of daily living.
[0182] In one embodiment, a subject may have advanced symptoms
associated with Parkinson's disease such as, but not limited to,
being diagnosed with Parkinson's more than 10 years, medium
refractory motor fluctuations wearing off and/or levodopa-induced
dyskinesia causing significant disability, intractable tremors,
significant balance disorder and/or falling, significant cognitive
impairment (such as dementia with or without behavioral
disturbances), sever bradykinesia, quality of life markedly
diminished by illness and/or morning akinesia.
[0183] In one embodiment, a subject has been referred to a movement
disorder specialist (MDS) but has not undergone deep brain
stimulation.
[0184] In one embodiment, a subject is using DUOPA.TM. in
combination with the compositions described herein. As a
non-limiting example, the subject may have success with using
DUOPA.TM. alone. As a non-limiting example, the subject may not
have any success or limited success using DUOPA.TM. alone.
[0185] In another embodiment, the neurological disease, disorder
and/or condition is Friedreich's Ataxia. In one embodiment the
polynucleotide used to treat Friedreich's Ataxia comprises any one
of SEQ ID NOs 2-23, such as, but not limited to SEQ ID NOs: 6-9 and
17-23, wherein the payload is replaced by Frataxin or any other
payload known in the art for treating Friedreich's Ataxia.
[0186] In another embodiment, the neurological disease, disorder
and/or condition is Amyotrophic lateral sclerosis (ALS). In one
embodiment the polynucleotide used to treat ALS comprises any one
of SEQ ID NOs 2-23, such as, but not limited to SEQ ID NOs: 6-9 and
17-23, wherein the payload is replaced by an shRNA, miRNA, siRNA,
RNAi for SOD1 or any other payload known in the art for treating
ALS.
[0187] In another embodiment, the neurological disease, disorder
and/or condition is Huntington's disease. In one embodiment the
polynucleotide used to treat Huntington's disease comprises any one
of SEQ ID NOs 2-23, such as, but not limited to SEQ ID NOs: 6-9 and
17-23, wherein the payload is replaced by an shRNA, miRNA, siRNA,
RNAi for Htt or any other payload known in the art for treating
Huntington's disease.
[0188] In another embodiment, the neurological disease, disorder or
condition is spinal muscular atrophy (SMA). In one embodiment the
polynucleotide used to treat SMA comprises any one of SEQ ID NOs
2-23, such as, but not limited to SEQ ID NOs: 6-9 and 17-23,
wherein the payload is replaced by SMN or any other payload known
in the art for treating SMA.
Circadian Rhythm and Sleep-Wake Cycles
[0189] Circadian rhythms are physical, mental and behavioral
changes that tend to follow a 24 hour cycle. Circadian rhythms can
influence sleep-wake cycles, hormone release, body temperature and
other bodily functions. Changes in the circadian rhythm can cause
conditions and/or disorder such as, but not limited to sleep
disorders (e.g., insomnia), depression, bipolar disorder, seasonal
affective disorder, obesity and diabetes.
[0190] In one embodiment, the AADC polynucleotides described herein
may be used to treat insomnia.
[0191] The sleep-wake cycle comprises periods of sleep and periods
of wake. Generally, in a 24 hour period the total hours of sleep
are less than the total hours of wakefulness. As a non-limiting
example, the sleep-wake cycle comprises 7-9 hours of sleep and
15-17 hours of wakefulness. As a non-limiting example, the
sleep-wake cycle comprises 8 hours of sleep and 16 hours of
wakefulness. As a non-limiting example, the sleep-wake cycle
comprises 8-10 hours of sleep and 14-16 hours of wakefulness.
[0192] In one embodiment, the sleep-wake cycle of a subject is
improved by administered to the subject the AADC polynucleotides
described herein.
[0193] In one embodiment, the sleep-wake cycle of a subject is
regulated by administering to the subject the AADC polynucleotides
described herein. As a non-limiting example, the regulation may be
the correction of more periods of sleep occurring at night and less
periods of sleep occurring
[0194] In one embodiment, the sleep-wake cycle of a subject
administered the AADC polynucleotides described herein improves as
compared to the sleep-wake cycle of the subject prior to
administration of the AADC polynucleotides. As a non-limiting
example, the subject has an increased period of sleep and a
decreased period of wakefulness. As another non-limiting example,
the subject has a decreased period of sleep and an increased period
of wakefulness.
[0195] In one embodiment, the sleep-wake cycle of a subject
administered the AADC polynucleotides described herein is regulated
as compared to the sleep-wake cycle of the subject prior to
administration of the AADC polynucleotides. As a non-limiting
example, the length of the periods of sleep and the periods of
wakefulness may be about the same (e.g., +/-1 hour) for at least 2
days. As another non-limiting example, the length of the periods of
sleep and the periods of wakefulness if a 24 hours period may be
within 10 minutes, 15 minutes, 20 minutes, 25 minutes, 30 minutes,
35 minutes, 40 minutes, 45 minutes, 50 minutes, 55 minutes, 1 hour,
1.5 hours, or 2 hours of the previous 24 hour period.
[0196] In one embodiment, the amount of rapid eye movement (REM)
sleep a subject experiences in a 24 hour period is altered after
the subject is administered the AADC polynucleotides described
herein. REM sleep is generally considered an active period of sleep
marked by intense brain activity where brain waves are fast and
desynchronized. An adult, on average, spends about 20-25% of their
total daily sleep period in REM sleep. As a non-limiting example,
the amount of REM sleep is decreased by 1%, 2%, 3%, 4%, 5%, 6%, 7%,
8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 20%, 25%, 30%, 35%, 40%, 45%,
50%, 55%, 60%, 65% or more than 65%. As a non-limiting example, the
amount of REM sleep is decreased by 1-10%, 5-10%, 5-15%, 10-15%,
15-20%, 15-25%, 20-25%, 20-30%, 25-30%, 25-35%, 30-35%, 30-40%,
35-40%, 40-50% or 40-60%. As a non-limiting example, the amount of
REM sleep is increased by 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%,
11%, 12%, 13%, 14%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%,
60%, 65% or more than 65%. As a non-limiting example, the amount of
REM sleep is increased by 1-5%, 1-10%, 5-10%, 5-15%, 10-15%,
15-20%, 15-25%, 20-25%, 20-30%, 25-30%, 25-35%, 30-35%, 30-40%,
35-40%, 40-50% or 40-60%.
[0197] In one embodiment, the amount of non-REM (NREM) sleep a
subject experiences in a 24 hour period is altered after the
subject is administered the AADC polynucleotides described herein.
NREM sleep is generally characterized by a reduction in
physiological activity since as the brain waves, measured by EEG,
get slower and have greater amplitude. NREM has four stages: Stage
1 is the time of drowsiness or transition from being awake to
falling asleep where the brain waves and muscle activity begin to
slow; Stage 2 is a period of light sleep during which eye movements
stop and brain waves become slower with occasional bursts of rapid
waves (sometimes called sleep spindles); Stage 3 and Stage 4
(collectively referred to as slow wave sleep) are characterized by
the presence of slow brain waves (delta waves) interspersed with
smaller faster waves where there are no eye movements. An adult, on
average, spends about 75-80% of their total daily sleep period in
NREM sleep with about half of their total daily sleep time in NREM
stage 2 sleep.
[0198] In one embodiment, the amount of NREM sleep a subject
experiences is altered after the subject is administered the AADC
polynucleotides described herein. As a non-limiting example, the
amount of NREM sleep is decreased by 1%, 2%, 3%, 4%, 5%, 6%, 7%,
8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 20%, 25%, 30%, 35%, 40%, 45%,
50%, 55%, 60%, 65% or more than 65%. As a non-limiting example, the
amount of NREM sleep is decreased by 1-10%, 5-10%, 5-15%, 10-15%,
15-20%, 15-25%, 20-25%, 20-30%, 25-30%, 25-35%, 30-35%, 30-40%,
35-40%, 40-50% or 40-60%. As a non-limiting example, the amount of
NREM sleep is increased by 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%,
11%, 12%, 13%, 14%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%,
60%, 65% or more than 65%. As a non-limiting example, the amount of
NREM sleep is increased by 1-5%, 1-10%, 5-10%, 5-15%, 10-15%,
15-20%, 15-25%, 20-25%, 20-30%, 25-30%, 25-35%, 30-35%, 30-40%,
35-40%, 40-50% or 40-60%.
[0199] In one embodiment, the amount of NREM Stage 1 sleep a
subject experiences is altered after the subject is administered
the AADC polynucleotides described herein. As a non-limiting
example, the amount of NREM Stage 1 sleep is decreased by 1%, 2%,
3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 20%, 25%,
30%, 35%, 40%, 45%, 50%, 55%, 60%, 65% or more than 65%. As a
non-limiting example, the amount of NREM Stage 1 sleep is decreased
by 1-10%, 5-10%, 5-15%, 10-15%, 15-20%, 15-25%, 20-25%, 20-30%,
25-30%, 25-35%, 30- 35%, 30-40%, 35-40%, 40-50% or 40-60%. As a
non-limiting example, the amount of NREM Stage 1 sleep is increased
by 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%,
15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65% or more than
65%. As a non-limiting example, the amount of NREM Stage 1 sleep is
increased by 1-5%, 1-10%, 5-10%, 5-15%, 10-15%, 15-20%, 15-25%,
20-25%, 20-30%, 25-30%, 25-35%, 30-35%, 30-40%, 35-40%, 40-50% or
40-60%.
[0200] In one embodiment, the amount of NREM Stage 2 sleep a
subject experiences is altered after the subject is administered
the AADC polynucleotides described herein. As a non-limiting
example, the amount of NREM Stage 2 sleep is decreased by 1%, 2%,
3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 20%, 25%,
30%, 35%, 40%, 45%, 50%, 55%, 60%, 65% or more than 65%. As a
non-limiting example, the amount of NREM Stage 2 sleep is decreased
by 1-10%, 5-10%, 5-15%, 10-15%, 15-20%, 15-25%, 20-25%, 20-30%,
25-30%, 25-35%, 30- 35%, 30-40%, 35-40%, 40-50% or 40-60%. As a
non-limiting example, the amount of NREM Stage 2 sleep is increased
by 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%,
15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65% or more than
65%. As a non-limiting example, the amount of NREM Stage 2 sleep is
increased by 1-5%, 1-10%, 5-10%, 5-15%, 10-15%, 15-20%, 15-25%,
20-25%, 20-30%, 25-30%, 25-35%, 30-35%, 30-40%, 35-40%, 40-50% or
40-60%.
[0201] In one embodiment, the amount of NREM Stage 3 and 4 sleep a
subject experiences is altered after the subject is administered
the AADC polynucleotides described herein. As a non-limiting
example, the amount of NREM Stage 3 and 4 sleep is decreased by 1%,
2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 20%,
25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65% or more than 65%. As a
non-limiting example, the amount of NREM Stage 3 and 4 sleep is
decreased by 1-10%, 5-10%, 5-15%, 10-15%, 15-20%, 15-25%, 20-25%,
20-30%, 25-30%, 25-35%, 30-35%, 30-40%, 35-40%, 40-50% or 40-60%.
As a non-limiting example, the amount of NREM Stage 3 and 4 sleep
is increased by 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%,
13%, 14%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65% or
more than 65%. As a non-limiting example, the amount of NREM Stage
3 and 4 sleep is increased by 1-5%, 1-10%, 5-10%, 5-15%, 10-15%,
15-20%, 15-25%, 20-25%, 20-30%, 25-30%, 25-35%, 30-35%, 30-40%,
35-40%, 40-50% or 40-60%.
[0202] In one embodiment, periods of NREM and REM cycles are more
consistent in a subject after the subject is administered the AADC
polynucleotides described herein. Generally NREM and REM cycles
alternate every 90 to 110 minutes four to six times per night.
Definitions
[0203] At various places in the present specification, substituents
of compounds of the present disclosure are disclosed in groups or
in ranges. It is specifically intended that the present disclosure
include each and every individual subcombination of the members of
such groups and ranges.
[0204] About: As used herein, the term "about" means+/-10% of the
recited value.
[0205] Activity: As used herein, the term "activity" refers to the
condition in which things are happening or being done. Compositions
described herein may have activity and this activity may involve
one or more biological events.
[0206] Adeno-associated virus: The term "adeno-associated virus" or
"AAV" as used herein refers to members of the dependovirus genus
comprising any particle, sequence, gene, protein, or component
derived therefrom. The term "AAV particle" as used herein comprises
a capsid and a polynucleotide. The AAV particle may be derived from
any serotype, described herein or known in the art, including
combinations of serotypes (i.e., "pseudotyped" AAV) or from various
genomes (e.g., single stranded or self-complementary). In addition,
the AAV particle may be replication defective and/or targeted.
[0207] Administered in combination: As used herein, the term
"administered in combination" or "combined administration" means
that two or more agents (e.g., AAV) are administered to a subject
at the same time or within an interval such that there may be an
overlap of an effect of each agent on the patient and/or the
subject is at some point in time simultaneously exposed to both. In
some embodiments, they are administered within about 60, 30, 15,
10, 5, or 1 minutes of one another or within about 24 hours, 12
hours, 6 hours, 3 hours of at least one dose of one or more other
agents. In some embodiments, administration occurs in overlapping
dosage regimens. As used herein, the term "dosage regimen" refers
to a plurality of doses spaced apart in time. Such doses may occur
at regular intervals or may include one or more hiatus in
administration. In some embodiments, the administrations of the
agents are spaced sufficiently closely together such that a
combinatorial (e.g., a synergistic) effect is achieved.
[0208] Amelioration: As used herein, the term "amelioration" or
"ameliorating" refers to a lessening of severity of at least one
indicator of a condition or disease. For example, in the context of
neurodegeneration disorder, amelioration includes the reduction of
neuron loss.
[0209] Animal: As used herein, the term "animal" refers to any
member of the animal kingdom. In some embodiments, "animal" refers
to humans at any stage of development. In some embodiments,
"animal" refers to non-human animals at any stage of development.
In certain embodiments, the non-human animal is a mammal (e.g., a
rodent, a mouse, a rat, a rabbit, a monkey, a dog, a cat, a sheep,
cattle, a primate, or a pig). In some embodiments, animals include,
but are not limited to, mammals, birds, reptiles, amphibians, fish,
and worms. In some embodiments, the animal is a transgenic animal,
genetically-engineered animal, or a clone.
[0210] Antisense strand: As used herein, the term "the antisense
strand" or "the first strand" or "the guide strand" of a siRNA
molecule refers to a strand that is substantially complementary to
a section of about 10-50 nucleotides, e.g., about 15-30, 16-25,
18-23 or 19-22 nucleotides of the mRNA of the gene targeted for
silencing. The antisense strand or first strand has sequence
sufficiently complementary to the desired target mRNA sequence to
direct target-specific silencing, e.g., complementarity sufficient
to trigger the destruction of the desired target mRNA by the RNAi
machinery or process.
[0211] Approximately: As used herein, the term "approximately" or
"about," as applied to one or more values of interest, refers to a
value that is similar to a stated reference value. In certain
embodiments, the term "approximately" or "about" refers to a range
of values that fall within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%,
13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less in
either direction (greater than or less than) of the stated
reference value unless otherwise stated or otherwise evident from
the context (except where such number would exceed 100% of a
possible value).
[0212] Associated with: As used herein, the terms "associated
with," "conjugated," "linked," "attached," and "tethered," when
used with respect to two or more moieties, means that the moieties
are physically associated or connected with one another, either
directly or via one or more additional moieties that serves as a
linking agent, to form a structure that is sufficiently stable so
that the moieties remain physically associated under the conditions
in which the structure is used, e.g., physiological conditions. An
"association" need not be strictly through direct covalent chemical
bonding. It may also suggest ionic or hydrogen bonding or a
hybridization based connectivity sufficiently stable such that the
"associated" entities remain physically associated.
[0213] Bifunctional: As used herein, the term "bifunctional" refers
to any substance, molecule or moiety which is capable of or
maintains at least two functions. The functions may affect the same
outcome or a different outcome. The structure that produces the
function may be the same or different.
[0214] Biologically active: As used herein, the phrase
"biologically active" refers to a characteristic of any substance
(e.g., AAV) that has activity in a biological system and/or
organism. For instance, a substance that, when administered to an
organism, has a biological effect on that organism, is considered
to be biologically active. In particular embodiments, a
polynucleotide of the present invention may be considered
biologically active if even a portion of the polynucleotides is
biologically active or mimics an activity considered biologically
relevant.
[0215] Biological system: As used herein, the term "biological
system" refers to a group of organs, tissues, cells, intracellular
components, proteins, nucleic acids, molecules (including, but not
limited to biomolecules) that function together to perform a
certain biological task within cellular membranes, cellular
compartments, cells, tissues, organs, organ systems, multicellular
organisms, or any biological entity. In some embodiments,
biological systems are cell signaling pathways comprising
intracellular and/or extracellular cell signaling biomolecules. In
some embodiments, biological systems comprise growth factor
signaling events within the extracellular/cellular matrix and/or
cellular niches.
[0216] Biomolecule: As used herein, the term "biomolecule" is any
natural molecule which is amino acid-based, nucleic acid-based,
carbohydrate-based or lipid-based, and the like.
[0217] Complementary and substantially complementary: As used
herein, the term "complementary" refers to the ability of
polynucleotides to form base pairs with one another. Base pairs are
typically formed by hydrogen bonds between nucleotide units in
antiparallel polynucleotide strands. Complementary polynucleotide
strands can form base pairs in the Watson-Crick manner (e.g., A to
T, A to U, C to G), or in any other manner that allows for the
formation of duplexes. As persons skilled in the art are aware,
when using RNA as opposed to DNA, uracil rather than thymine is the
base that is considered to be complementary to adenosine. However,
when a U is denoted in the context of the present invention, the
ability to substitute a T is implied, unless otherwise stated.
Perfect complementarity or 100% complementarity refers to the
situation in which each nucleotide unit of one polynucleotide
strand can form a hydrogen bond with a nucleotide unit of a second
polynucleotide strand. Less than perfect complementarity refers to
the situation in which some, but not all, nucleotide units of two
strands can form hydrogen bonds with each other. For example, for
two 20-mers, if only two base pairs on each strand can form
hydrogen bonds with each other, the polynucleotide strands exhibit
10% complementarity. In the same example, if 18 base pairs on each
strand can form hydrogen bonds with each other, the polynucleotide
strands exhibit 90% complementarity. As used herein, the term
"substantially complementary" means that the siRNA has a sequence
(e.g., in the antisense strand) which is sufficient to bind the
desired target mRNA, and to trigger the RNA silencing of the target
mRNA.
[0218] Compound: As used herein, the term "compound," refers to a
distinct chemical entity. In some embodiments, a particular
compound may exist in one or more isomeric or isotopic forms
(including, but not limited to stereoisomers, geometric isomers and
isotopes). In some embodiments, a compound is provided or utilized
in only a single such form. In some embodiments, a compound is
provided or utilized as a mixture of two or more such forms
(including, but not limited to a racemic mixture of stereoisomers).
Those of skill in the art appreciate that some compounds exist in
different such forms, show different properties and/or activities
(including, but not limited to biological activities). In such
cases it is within the ordinary skill of those in the art to select
or avoid particular forms of the compound for use in accordance
with the present invention. For example, compounds that contain
asymmetrically substituted carbon atoms can be isolated in
optically active or racemic forms. Methods on how to prepare
optically active forms from optically active starting materials are
known in the art, such as by resolution of racemic mixtures or by
stereoselective synthesis. Many geometric isomers of olefins,
C.dbd.N double bonds, and the like can also be present in the
compounds described herein, and all such stable isomers are
contemplated in the present disclosure. Cis and trans geometric
isomers of the compounds of the present disclosure are described
and may be isolated as a mixture of isomers or as separated
isomeric forms.
[0219] Compounds of the present disclosure also include tautomeric
forms. Tautomeric forms result from the swapping of a single bond
with an adjacent double bond and the concomitant migration of a
proton. Tautomeric forms include prototropic tautomers which are
isomeric protonation states having the same empirical formula and
total charge.
[0220] Compounds of the present disclosure also include all of the
isotopes of the atoms occurring in the intermediate or final
compounds. "Isotopes" refers to atoms having the same atomic number
but different mass numbers resulting from a different number of
neutrons in the nuclei. For example, isotopes of hydrogen include
tritium and deuterium.
[0221] The compounds and salts of the present disclosure can be
prepared in combination with solvent or water molecules to form
solvates and hydrates by routine methods.
[0222] Conserved: As used herein, the term "conserved" refers to
nucleotides or amino acid residues of a polynucleotide sequence or
polypeptide sequence, respectively, that are those that occur
unaltered in the same position of two or more sequences being
compared. Nucleotides or amino acids that are relatively conserved
are those that are conserved amongst more related sequences than
nucleotides or amino acids appearing elsewhere in the
sequences.
[0223] In some embodiments, two or more sequences are said to be
"completely conserved" if they are 100% identical to one another.
In some embodiments, two or more sequences are said to be "highly
conserved" if they are at least 70% identical, at least 80%
identical, at least 90% identical, or at least 95% identical to one
another. In some embodiments, two or more sequences are said to be
"highly conserved" if they are about 70% identical, about 80%
identical, about 90% identical, about 95%, about 98%, or about 99%
identical to one another. In some embodiments, two or more
sequences are said to be "conserved" if they are at least 30%
identical, at least 40% identical, at least 50% identical, at least
60% identical, at least 70% identical, at least 80% identical, at
least 90% identical, or at least 95% identical to one another. In
some embodiments, two or more sequences are said to be "conserved"
if they are about 30% identical, about 40% identical, about 50%
identical, about 60% identical, about 70% identical, about 80%
identical, about 90% identical, about 95% identical, about 98%
identical, or about 99% identical to one another. Conservation of
sequence may apply to the entire length of an oligonucleotide, a
polynucleotide or polypeptide or may apply to a portion, region or
feature thereof.
[0224] In one embodiment, conserved sequences are not contiguous.
Those skilled in the art are able to appreciate how to achieve
alignment when gaps in contiguous alignment are present between
sequences, and to align corresponding residues not withstanding
insertions or deletions present.
[0225] In one embodiment, conserved sequences are not contiguous.
Those skilled in the art are able to appreciate how to achieve
alignment when gaps in contiguous alignment are present between
sequences, and to align corresponding residues not withstanding
insertions or deletions present.
[0226] Delivery: As used herein, "delivery" refers to the act or
manner of delivering a compound such as a parvovirus, e.g. an AAV
and/or AAV compound, substance, entity, moiety, cargo or payload to
a target. Such target may be a cell, tissue, organ, organism, or
system (whether biological or production).
[0227] Delivery Agent: As used herein, "delivery agent" refers to
any agent or substance which facilitates, at least in part, the in
vivo and/or in vitro delivery of a polynucleotide and/or one or
more substances (including, but not limited to a compounds and/or
compositions of the present invention, e.g., viral particles or
expression vectors) to targeted cells.
[0228] Destabilized: As used herein, the term "destable,"
"destabilize," or "destabilizing region" means a region or molecule
that is less stable than a starting, reference, wild-type or native
form of the same region or molecule.
[0229] Detectable label: As used herein, "detectable label" refers
to one or more markers, signals, or moieties which are attached,
incorporated or associated with another entity that is readily
detected by methods known in the art including radiography,
fluorescence, chemiluminescence, enzymatic activity, absorbance
immunological detection, and the like. Detectable labels may
include radioisotopes, fluorophores, chromophores, enzymes, dyes,
metal ions, ligands such as biotin, avidin, streptavidin and
haptens, quantum dots, and the like. Detectable labels may be
located at any position in the entity with which they are attached,
incorporated or associated. For example, when attached,
incorporated in or associated with a peptide or protein, they may
be within the amino acids, the peptides, or proteins, or located at
the N- or C-termini.
[0230] Dosing regimen: As used herein, a "dosing regimen" is a
schedule of administration or physician determined regimen of
treatment, prophylaxis, or palliative care.
[0231] Effective Amount: As used herein, the term "effective
amount" of an agent is that amount sufficient to effect beneficial
or desired results, for example, upon single or multiple dose
administration to a subject cell, in curing, alleviating, relieving
or improving one or more symptoms of a disorder, clinical results,
and, as such, an "effective amount" depends upon the context in
which it is being applied. For example, in the context of
administering an agent that treats Parkinson's Disease, an
effective amount of an agent is, for example, an amount sufficient
to achieve treatment, as defined herein, of Parkinson's Disease, as
compared to the response obtained without administration of the
agent.
[0232] Encapsulate: As used herein, the term "encapsulate" means to
enclose, surround or encase.
[0233] Engineered: As used herein, embodiments are "engineered"
when they are designed to have a feature or property, whether
structural or chemical, that varies from a starting point,
wild-type or native molecule. Thus, engineered agents or entities
are those whose design and/or production include an act of the hand
of man.
[0234] Epitope: As used herein, an "epitope" refers to a surface or
region on a molecule that is capable of interacting with a
biomolecule. For example a protein may contain one or more amino
acids, e.g., an epitope, which interacts with an antibody, e.g., a
biomolecule. In some embodiments, when referring to a protein or
protein module, an epitope may comprise a linear stretch of amino
acids or a three dimensional structure formed by folded amino acid
chains.
[0235] Expression: As used herein, "expression" of a nucleic acid
sequence refers to one or more of the following events: (1)
production of an RNA template from a DNA sequence (e.g., by
transcription); (2) processing of an RNA transcript (e.g., by
splicing, editing, 5' cap formation, and/or 3' end processing); (3)
translation of an RNA into a polypeptide or protein; (4) folding of
a polypeptide or protein; and (5) post-translational modification
of a polypeptide or protein.
[0236] Feature: As used herein, a "feature" refers to a
characteristic, a property, or a distinctive element.
[0237] Formulation: As used herein, a "formulation" includes at
least one polynucleotide and/or compound and/or composition of the
present disclosure (e.g., a vector, AAV particle, etc.) and a
delivery agent.
[0238] Fragment: A "fragment," as used herein, refers to a
contiguous portion of a whole. For example, fragments of proteins
may comprise polypeptides obtained by digesting full-length protein
isolated from cultured cells. In some embodiments, a fragment of a
protein includes at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,
15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75,
80, 85, 90, 95, 100, 150, 200, 250 or more amino acids. In some
embodiments, fragments of an antibody include portions of an
antibody subjected to enzymatic digestion or synthesized as
such.
[0239] Functional: As used herein, a "functional" biological
molecule is a biological molecule and/or entity with a structure
and in a form in which it exhibits a property and/or activity by
which it is characterized.
[0240] Gene expression: The term "gene expression" refers to the
process by which a nucleic acid sequence undergoes successful
transcription and in most instances translation to produce a
protein or peptide. For clarity, when reference is made to
measurement of "gene expression", this should be understood to mean
that measurements may be of the nucleic acid product of
transcription, e.g., RNA or mRNA or of the amino acid product of
translation, e.g., polypeptides or peptides. Methods of measuring
the amount or levels of RNA, mRNA, polypeptides and peptides are
well known in the art.
[0241] Homology: As used herein, the term "homology" refers to the
overall relatedness between polymeric molecules, e.g. between
nucleic acid molecules (e.g. DNA molecules and/or RNA molecules)
and/or between polypeptide molecules. In some embodiments,
polymeric molecules are considered to be "homologous" to one
another if their sequences are at least 25%, 30%, 35%, 40%, 45%,
50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% identical
or similar. The term "homologous" necessarily refers to a
comparison between at least two sequences (polynucleotide or
polypeptide sequences). In accordance with the invention, two
polynucleotide sequences are considered to be homologous if the
polypeptides they encode are at least about 50%, 60%, 70%, 80%,
90%, 95%, or even 99% for at least one stretch of at least about 20
amino acids. In some embodiments, homologous polynucleotide
sequences are characterized by the ability to encode a stretch of
at least 4-5 uniquely specified amino acids. For polynucleotide
sequences less than 60 nucleotides in length, homology is typically
determined by the ability to encode a stretch of at least 4-5
uniquely specified amino acids. In accordance with the invention,
two protein sequences are considered to be homologous if the
proteins are at least about 50%, 60%, 70%, 80%, or 90% identical
for at least one stretch of at least about 20 amino acids. In many
embodiments, homologous protein may show a large overall degree of
homology and a high degree of homology over at least one short
stretch of at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,
16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50 or more amino acids. In
many embodiments, homologous proteins share one or more
characteristic sequence elements. As used herein, the term
"characteristic sequence element" refers to a motif present in
related proteins. In some embodiments, the presence of such motifs
correlates with a particular activity (such as biological
activity).
[0242] Identity: As used herein, the term "identity" refers to the
overall relatedness between polymeric molecules, e.g., between
oligonucleotide and/or polynucleotide molecules (e.g. DNA molecules
and/or RNA molecules) and/or between polypeptide molecules.
Calculation of the percent identity of two polynucleotide
sequences, for example, may be performed by aligning the two
sequences for optimal comparison purposes (e.g., gaps can be
introduced in one or both of a first and a second nucleic acid
sequences for optimal alignment and non-identical sequences can be
disregarded for comparison purposes). In certain embodiments, the
length of a sequence aligned for comparison purposes is at least
30%, at least 40%, at least 50%, at least 60%, at least 70%, at
least 80%, at least 90%, at least 95%, or 100% of the length of the
reference sequence. The nucleotides at corresponding nucleotide
positions are then compared. When a position in the first sequence
is occupied by the same nucleotide as the corresponding position in
the second sequence, then the molecules are identical at that
position. The percent identity between the two sequences is a
function of the number of identical positions shared by the
sequences, taking into account the number of gaps, and the length
of each gap, which needs to be introduced for optimal alignment of
the two sequences. The comparison of sequences and determination of
percent identity between two sequences can be accomplished using a
mathematical algorithm. For example, the percent identity between
two nucleotide sequences can be determined using methods such as
those described in Computational Molecular Biology, Lesk, A. M.,
ed., Oxford University Press, New York, 1988; Biocomputing:
Informatics and Genome Projects, Smith, D. W., ed., Academic Press,
New York, 1993; Sequence Analysis in Molecular Biology, von Heinje,
G., Academic Press, 1987; Computer Analysis of Sequence Data, Part
I, Griffin, A. M., and Griffin, H. G., eds., Humana Press, New
Jersey, 1994; and Sequence Analysis Primer, Gribskov, M. and
Devereux, J., eds., M Stockton Press, New York, 1991; each of which
is incorporated herein by reference in its entirety. For example,
the percent identity between two nucleotide sequences can be
determined, for example using the algorithm of Meyers and Miller
(CABIOS, 1989, 4:11-17), which has been incorporated into the ALIGN
program (version 2.0) using a PAM120 weight residue table, a gap
length penalty of 12 and a gap penalty of 4. The percent identity
between two nucleotide sequences can, alternatively, be determined
using the GAP program in the GCG software package using an
NWSgapdna.CMP matrix. Methods commonly employed to determine
percent identity between sequences include, but are not limited to
those disclosed in Carillo, H., and Lipman, D., SIAM J Applied
Math., 48:1073 (1988); incorporated herein by reference in its
entirety. Techniques for determining identity are codified in
publicly available computer programs. Exemplary computer software
to determine homology between two sequences include, but are not
limited to, GCG program package, Devereux, J., et al., Nucleic
Acids Research, 12(1), 387 (1984)), BLASTP, BLASTN, and FASTA
Altschul, S. F. et al., J. Molec. Biol., 215, 403 (1990)).
[0243] Inhibit expression of a gene: As used herein, the phrase
"inhibit expression of a gene" means to cause a reduction in the
amount of an expression product of the gene. The expression product
may be RNA transcribed from the gene (e.g. mRNA) or a polypeptide
translated from mRNA transcribed from the gene. Typically a
reduction in the level of mRNA results in a reduction in the level
of a polypeptide translated therefrom. The level of expression may
be determined using standard techniques for measuring mRNA or
protein.
[0244] In vitro: As used herein, the term "in vitro" refers to
events that occur in an artificial environment, e.g., in a test
tube or reaction vessel, in cell culture, in a Petri dish, etc.,
rather than within an organism (e.g., animal, plant, or
microbe).
[0245] In vivo: As used herein, the term "in vivo" refers to events
that occur within an organism (e.g., animal, plant, or microbe or
cell or tissue thereof).
[0246] Isolated: As used herein, the term "isolated" is synonymous
with "separated", but carries with it the inference separation was
carried out by the hand of man. In one embodiment, an isolated
substance or entity is one that has been separated from at least
some of the components with which it was previously associated
(whether in nature or in an experimental setting). Isolated
substances may have varying levels of purity in reference to the
substances from which they have been associated. Isolated
substances and/or entities may be separated from at least about
10%, about 20%, about 30%, about 40%, about 50%, about 60%, about
70%, about 80%, about 90%, or more of the other components with
which they were initially associated. In some embodiments, isolated
agents are more than about 80%, about 85%, about 90%, about 91%,
about 92%, about 93%, about 94%, about 95%, about 96%, about 97%,
about 98%, about 99%, or more than about 99% pure. As used herein,
a substance is "pure" if it is substantially free of other
components.
[0247] Substantially isolated: By "substantially isolated" is meant
that the compound is substantially separated from the environment
in which it was formed or detected. Partial separation can include,
for example, a composition enriched in the compound of the present
disclosure. Substantial separation can include compositions
containing at least about 50%, at least about 60%, at least about
70%, at least about 80%, at least about 90%, at least about 95%, at
least about 97%, or at least about 99% by weight of the compound of
the present disclosure, or salt thereof. Methods for isolating
compounds and their salts are routine in the art. In some
embodiments, isolation of a substance or entity includes disruption
of chemical associations and/or bonds. In some embodiments,
isolation includes only the separation from components with which
the isolated substance or entity was previously combined and does
not include such disruption.
[0248] Modified: As used herein, the term "modified" refers to a
changed state or structure of a molecule or entity of the invention
as compared with a parent or reference molecule or entity.
Molecules may be modified in many ways including chemically,
structurally, and functionally. In some embodiments, compounds
and/or compositions of the present disclosure are modified by the
introduction of non-natural amino acids, or non-natural
nucleotides.
[0249] Mutation: As used herein, the term "mutation" refers to a
change and/or alteration. In some embodiments, mutations may be
changes and/or alterations to proteins (including peptides and
polypeptides) and/or nucleic acids (including polynucleic acids).
In some embodiments, mutations comprise changes and/or alterations
to a protein and/or nucleic acid sequence. Such changes and/or
alterations may comprise the addition, substitution and or deletion
of one or more amino acids (in the case of proteins and/or
peptides) and/or nucleotides (in the case of nucleic acids and or
polynucleic acids). In embodiments wherein mutations comprise the
addition and/or substitution of amino acids and/or nucleotides,
such additions and/or substitutions may comprise 1 or more amino
acid and/or nucleotide residues and may include modified amino
acids and/or nucleotides.
[0250] Naturally occurring: As used herein, "naturally occurring"
means existing in nature without artificial aid or involvement of
the hand of man
[0251] Non-human vertebrate: As used herein, a "non-human
vertebrate" includes all vertebrates except Homo sapiens, including
wild and domesticated species. Examples of non-human vertebrates
include, but are not limited to, mammals, such as alpaca, banteng,
bison, camel, cat, cattle, deer, dog, donkey, gayal, goat, guinea
pig, horse, llama, mule, pig, rabbit, reindeer, sheep water
buffalo, and yak.
[0252] Nucleic acid: As used herein, the term "nucleic acid",
"polynucleotide" and `oligonucleotide" refer to any nucleic acid
polymers composed of either polydeoxyribonucleotides (containing
2-deoxy-D-ribose), or polyribonucleotides (containing D-ribose), or
any other type of polynucleotide which is an N glycoside of a
purine or pyrimidine base, or modified purine or pyrimidine bases.
There is no intended distinction in length between the term
"nucleic acid", "polynucleotide" and "oligonucleotide", and these
terms will be used interchangeably. These terms refer only to the
primary structure of the molecule. Thus, these terms include
double- and single-stranded DNA, as well as double- and single
stranded RNA.
[0253] Off-target: As used herein, "off target" refers to any
unintended effect on any one or more target, gene and/or cellular
transcript.
[0254] Open reading frame: As used herein, "open reading frame" or
"ORF" refers to a sequence which does not contain a stop codon in a
given reading frame.
[0255] Operably linked: As used herein, the phrase "operably
linked" refers to a functional connection between two or more
molecules, constructs, transcripts, entities, moieties or the
like.
[0256] Particle: As used herein, a "particle" is a virus comprised
of at least two components, a protein capsid and a polynucleotide
sequence enclosed within the capsid.
[0257] Patient: As used herein, "patient" refers to a subject who
may seek or be in need of treatment, requires treatment, is
receiving treatment, will receive treatment, or a subject who is
under care by a trained professional for a particular disease or
condition, such as for example Parkinson's Disease.
[0258] Payload: As used herein, "payload" refers to one or more
polynucleotides or polynucleotide regions encoded by or within a
viral genome or an expression product of such polynucleotide or
polynucleotide region, e.g., a transgene, a polynucleotide encoding
a polypeptide or multi-polypeptide or a modulatory nucleic acid or
regulatory nucleic acid.
[0259] Payload construct: As used herein, "payload construct" is
one or more polynucleotide regions encoding or comprising a payload
that is flanked on one or both sides by an inverted terminal repeat
(ITR) sequence. The payload construct is a template that is
replicated in a viral production cell to produce a viral
genome.
[0260] Payload construct vector: As used herein, "payload construct
vector" is a vector encoding or comprising a payload construct, and
regulatory regions for replication and expression in bacterial
cells.
[0261] Payload construct expression vector: As used herein, a
"payload construct expression vector" is a vector encoding or
comprising a payload construct and which further comprises one or
more polynucleotide regions encoding or comprising components for
viral expression in a viral replication cell.
[0262] Peptide: As used herein, "peptide" is less than or equal to
50 amino acids long, e.g., about 5, 10, 15, 20, 25, 30, 35, 40, 45,
or 50 amino acids long.
[0263] Pharmaceutically acceptable: The phrase "pharmaceutically
acceptable" is employed herein to refer to those compounds,
materials, compositions, and/or dosage forms which are, within the
scope of sound medical judgment, suitable for use in contact with
the tissues of human beings and animals without excessive toxicity,
irritation, allergic response, or other problem or complication,
commensurate with a reasonable benefit/risk ratio.
[0264] Pharmaceutically acceptable excipients: The phrase
"pharmaceutically acceptable excipient," as used herein, refers to
any ingredient other than the compounds and/or active agents (e.g.
as described herein) present in pharmaceutical compositions and
having the properties of being substantially nontoxic and
non-inflammatory in a subject such as a patient. In some
embodiments, pharmaceutically acceptable excipients are vehicles
capable of suspending and/or dissolving active agents. Excipients
may include, for example: antiadherents, antioxidants, binders,
coatings, compression aids, disintegrants, dyes (colors),
emollients, emulsifiers, fillers (diluents), film formers or
coatings, flavors, fragrances, glidants (flow enhancers),
lubricants, preservatives, printing inks, sorbents, suspension or
dispersing agents, sweeteners, and waters of hydration. Exemplary
excipients include, but are not limited to: butylated
hydroxytoluene (BHT), calcium carbonate, calcium phosphate
(dibasic), calcium stearate, croscarmellose, crosslinked polyvinyl
pyrrolidone, citric acid, crospovidone, cysteine, ethylcellulose,
gelatin, hydroxypropyl cellulose, hydroxypropyl methylcellulose,
lactose, magnesium stearate, maltitol, mannitol, methionine,
methylcellulose, methyl paraben, microcrystalline cellulose,
polyethylene glycol, polyvinyl pyrrolidone, povidone,
pregelatinized starch, propyl paraben, retinyl palmitate, shellac,
silicon dioxide, sodium carboxymethyl cellulose, sodium citrate,
sodium starch glycolate, sorbitol, starch (corn), stearic acid,
sucrose, talc, titanium dioxide, vitamin A, vitamin E, vitamin C,
and xylitol.
[0265] Pharmaceutically acceptable salts: Pharmaceutically
acceptable salts of the compounds described herein. As used herein,
"pharmaceutically acceptable salts" refers to derivatives or forms
of the disclosed compounds wherein the parent compound is modified
by converting an existing acid or base moiety to its salt form
(e.g., as generated by reacting the free base group with a suitable
organic acid). Examples of pharmaceutically acceptable salts
include, but are not limited to, mineral or organic acid salts of
basic residues such as amines; alkali or organic salts of acidic
residues such as carboxylic acids; and the like. Representative
acid addition salts include acetate, acetic acid, adipate,
alginate, ascorbate, aspartate, benzenesulfonate, benzene sulfonic
acid, benzoate, bisulfate, borate, butyrate, camphorate,
camphorsulfonate, citrate, cyclopentanepropionate, digluconate,
dodecylsulfate, ethanesulfonate, fumarate, glucoheptonate,
glycerophosphate, hemisulfate, heptonate, hexanoate, hydrobromide,
hydrochloride, hydroiodide, 2-hydroxy-ethanesulfonate,
lactobionate, lactate, laurate, lauryl sulfate, malate, maleate,
malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate,
nitrate, oleate, oxalate, palmitate, pamoate, pectinate,
persulfate, 3-phenylpropionate, phosphate, picrate, pivalate,
propionate, stearate, succinate, sulfate, tartrate, thiocyanate,
toluenesulfonate, undecanoate, valerate salts, and the like.
Representative alkali or alkaline earth metal salts include sodium,
lithium, potassium, calcium, magnesium, and the like, as well as
nontoxic ammonium, quaternary ammonium, and amine cations,
including, but not limited to ammonium, tetramethylammonium,
tetraethylammonium, methylamine, dimethylamine, trimethylamine,
triethylamine, ethylamine, and the like. The pharmaceutically
acceptable salts of the present disclosure include the conventional
non-toxic salts of the parent compound formed, for example, from
non-toxic inorganic or organic acids. In some embodiments a
pharmaceutically acceptable salt of the present disclosure can be
synthesized salt prepared from the parent compound which contains a
basic or acidic moiety by conventional chemical methods. Generally,
such salts can be prepared by reacting the free acid or base forms
of these compounds with a stoichiometric amount of the appropriate
base or acid in water or in an organic solvent, or in a mixture of
the two; generally, nonaqueous media like ether, ethyl acetate,
ethanol, isopropanol, or acetonitrile are preferred. Lists of
suitable salts are found in Remington's Pharmaceutical Sciences,
17th ed., Mack Publishing Company, Easton, Pa., 1985, p. 1418,
Pharmaceutical Salts: Properties, Selection, and Use, P. H. Stahl
and C. G. Wermuth (eds.), Wiley-VCH, 2008, and Berge et al.,
Journal of Pharmaceutical Science, 66, 1-19 (1977), each of which
is incorporated herein by reference in its entirety.
[0266] Pharmaceutically acceptable solvate: The term
"pharmaceutically acceptable solvate," as used herein, refers to a
crystalline form of a compound of the invention wherein molecules
of a suitable solvent are incorporated in the crystal lattice. A
suitable solvent is physiologically tolerable at the dosage
administered. For example, solvates may be prepared by
crystallization, recrystallization, or precipitation from a
solution that includes organic solvents, water, or a mixture
thereof. Examples of suitable solvents are ethanol, water (for
example, mono-, di-, and tri-hydrates), N-methylpyrrolidinone
(NMP), dimethyl sulfoxide (DMSO), N,N'-dimethylformamide (DMF),
N,N'-dimethylacetamide (DMAC), 1,3-dimethyl-2-imidazolidinone
(DMEU), 1,3-dimethyl-3,4,5,6-tetrahydro-2-(1H)-pyrimidinone (DMPU),
acetonitrile (ACN), propylene glycol, ethyl acetate, benzyl
alcohol, 2-pyrrolidone, benzyl benzoate, and the like. When water
is the solvent, the solvate is referred to as a "hydrate." In some
embodiments, the solvent incorporated into a solvate is of a type
or at a level that is physiologically tolerable to an organism to
which the solvate is administered (e.g., in a unit dosage form of a
pharmaceutical composition).
[0267] Pharmacokinetic: As used herein, "pharmacokinetic" refers to
any one or more properties of a molecule or compound as it relates
to the determination of the fate of substances administered to a
living organism. Pharmacokinetics is divided into several areas
including the extent and rate of absorption, distribution,
metabolism and excretion. This is commonly referred to as ADME
where: (A) Absorption is the process of a substance entering the
blood circulation; (D) Distribution is the dispersion or
dissemination of substances throughout the fluids and tissues of
the body; (M) Metabolism (or Biotransformation) is the irreversible
transformation of parent compounds into daughter metabolites; and
(E) Excretion (or Elimination) refers to the elimination of the
substances from the body. In rare cases, some drugs irreversibly
accumulate in body tissue.
[0268] Physicochemical: As used herein, "physicochemical" means of
or relating to a physical and/or chemical property.
[0269] Preventing: As used herein, the term "preventing" refers to
partially or completely delaying onset of an infection, disease,
disorder and/or condition; partially or completely delaying onset
of one or more symptoms, features, or clinical manifestations of a
particular infection, disease, disorder, and/or condition;
partially or completely delaying onset of one or more symptoms,
features, or manifestations of a particular infection, disease,
disorder, and/or condition; partially or completely delaying
progression from an infection, a particular disease, disorder
and/or condition; and/or decreasing the risk of developing
pathology associated with the infection, the disease, disorder,
and/or condition, such as for example Parkinson's Disease.
[0270] Prodrug: The present disclosure also includes prodrugs of
the compounds described herein. As used herein, "prodrugs" refer to
any substance, molecule or entity which is in a form predicate for
that substance, molecule or entity to act as a therapeutic upon
chemical or physical alteration. Prodrugs may by covalently bonded
or sequestered in some way and which release or are converted into
the active drug moiety prior to, upon or after administered to a
mammalian subject. Preparation and use of prodrugs is discussed in
T. Higuchi and V. Stella, "Pro-drugs as Novel Delivery Systems,"
Vol. 14 of the A.C.S. Symposium Series, and in Bioreversible
Carriers in Drug Design, ed. Edward B. Roche, American
Pharmaceutical Association and Pergamon Press, 1987, both of which
are hereby incorporated by reference in their entirety.
[0271] Proliferate: As used herein, the term "proliferate" means to
grow, expand, replicate or increase or cause to grow, expand,
replicate or increase. "Proliferative" means having the ability to
proliferate. "Anti-proliferative" means having properties counter
to or in opposition to proliferative properties.
[0272] Prophylactic: As used herein, "prophylactic" refers to a
therapeutic or course of action used to prevent the spread of
disease.
[0273] Prophylaxis: As used herein, a "prophylaxis" refers to a
measure taken to maintain health and prevent the spread of
disease.
[0274] Protein of interest: As used herein, the terms "proteins of
interest" or "desired proteins" include those provided herein and
fragments, mutants, variants, and alterations thereof.
[0275] Purified: As used herein, "purify," "purified,"
"purification" means to make substantially pure or clear from
unwanted components, material defilement, admixture or
imperfection. "Purified" refers to the state of being pure.
"Purification" refers to the process of making pure.
[0276] Region: As used herein, the term "region" refers to a zone
or general area. In some embodiments, when referring to a protein
or protein module, a region may comprise a linear sequence of amino
acids along the protein or protein module or may comprise a three
dimensional area, an epitope and/or a cluster of epitopes. In some
embodiments, regions comprise terminal regions. As used herein, the
term "terminal region" refers to regions located at the ends or
termini of a given agent. When referring to proteins, terminal
regions may comprise N- and/or C-termini. N-termini refer to the
end of a protein comprising an amino acid with a free amino group.
C-termini refer to the end of a protein comprising an amino acid
with a free carboxyl group. N- and/or C-terminal regions may
therefore comprise the N- and/or C-termini as well as surrounding
amino acids. In some embodiments, N- and/or C-terminal regions
comprise from about 3 amino acid to about 30 amino acids, from
about 5 amino acids to about 40 amino acids, from about 10 amino
acids to about 50 amino acids, from about 20 amino acids to about
100 amino acids and/or at least 100 amino acids. In some
embodiments, N-terminal regions may comprise any length of amino
acids that includes the N-terminus, but does not include the
C-terminus. In some embodiments, C-terminal regions may comprise
any length of amino acids, which include the C-terminus, but do not
comprise the N-terminus.
[0277] In some embodiments, when referring to a polynucleotide, a
region may comprise a linear sequence of nucleic acids along the
polynucleotide or may comprise a three dimensional area, secondary
structure, or tertiary structure. In some embodiments, regions
comprise terminal regions. As used herein, the term "terminal
region" refers to regions located at the ends or termini of a given
agent. When referring to polynucleotides, terminal regions may
comprise 5' and 3' termini. 5' termini refer to the end of a
polynucleotide comprising a nucleic acid with a free phosphate
group. 3' termini refer to the end of a polynucleotide comprising a
nucleic acid with a free hydroxyl group. 5' and 3' regions may
therefore comprise the 5' and 3' termini as well as surrounding
nucleic acids. In some embodiments, 5' and 3' terminal regions
comprise from about 9 nucleic acids to about 90 nucleic acids, from
about 15 nucleic acids to about 120 nucleic acids, from about 30
nucleic acids to about 150 nucleic acids, from about 60 nucleic
acids to about 300 nucleic acids and/or at least 300 nucleic acids.
In some embodiments, 5' regions may comprise any length of nucleic
acids that includes the 5' terminus, but does not include the 3'
terminus. In some embodiments, 3' regions may comprise any length
of nucleic acids, which include the 3' terminus, but does not
comprise the 5' terminus.
[0278] RNA or RNA molecule: As used herein, the term "RNA" or "RNA
molecule" or "ribonucleic acid molecule" refers to a polymer of
ribonucleotides; the term "DNA" or "DNA molecule" or
"deoxyribonucleic acid molecule" refers to a polymer of
deoxyribonucleotides. DNA and RNA can be synthesized naturally,
e.g., by DNA replication and transcription of DNA, respectively; or
be chemically synthesized. DNA and RNA can be single-stranded
(i.e., ssRNA or ssDNA, respectively) or multi-stranded (e.g.,
double stranded, i.e., dsRNA and dsDNA, respectively). The term
"mRNA" or "messenger RNA", as used herein, refers to a single
stranded RNA that encodes the amino acid sequence of one or more
polypeptide chains.
[0279] RNA interference: As used herein, the term "RNA
interference" or "RNAi" refers to a sequence specific regulatory
mechanism mediated by RNA molecules which results in the inhibition
or interference or "silencing" of the expression of a corresponding
protein-coding gene.
[0280] Sample: As used herein, the term "sample" refers to an
aliquot, subset or portion taken from a source and/or provided for
analysis or processing. In some embodiments, a sample is from a
biological source such as a tissue, cell or component part (e.g. a
body fluid, including but not limited to blood, mucus, lymphatic
fluid, synovial fluid, cerebrospinal fluid, saliva, amniotic fluid,
amniotic cord blood, urine, vaginal fluid and semen). In some
embodiments, a sample may be or comprise a homogenate, lysate or
extract prepared from a whole organism or a subset of its tissues,
cells or component parts, or a fraction or portion thereof,
including but not limited to, for example, plasma, serum, spinal
fluid, lymph fluid, the external sections of the skin, respiratory,
intestinal, and genitourinary tracts, tears, saliva, milk, blood
cells, tumors, or organs. In some embodiments, a sample is or
comprises a medium, such as a nutrient broth or gel, which may
contain cellular components, such as proteins or nucleic acid
molecule. In some embodiments, a "primary" sample is an aliquot of
the source. In some embodiments, a primary sample is subjected to
one or more processing (e.g., separation, purification, etc.) steps
to prepare a sample for analysis or other use.
[0281] Self-complementary viral particle: As used herein, a
"self-complementary viral particle" is a particle comprised of at
least two components, a protein capsid and a polynucleotide
sequence encoding a self-complementary genome enclosed within the
capsid.
[0282] Sense strand: As used herein, the term "the sense strand" or
"the second strand" or "the passenger strand" of a siRNA molecule
refers to a strand that is complementary to the antisense strand or
first strand. The antisense and sense strands of a siRNA molecule
are hybridized to form a duplex structure. As used herein, a "siRNA
duplex" includes a siRNA strand having sufficient complementarity
to a section of about 10-50 nucleotides of the mRNA of the gene
targeted for silencing and a siRNA strand having sufficient
complementarity to form a duplex with the siRNA strand.
[0283] Signal Sequences: As used herein, the phrase "signal
sequences" refers to a sequence which can direct the transport or
localization.
[0284] Single unit dose: As used herein, a "single unit dose" is a
dose of any therapeutic administered in one dose/at one time/single
route/single point of contact, i.e., single administration event.
In some embodiments, a single unit dose is provided as a discrete
dosage form (e.g., a tablet, capsule, patch, loaded syringe, vial,
etc.).
[0285] Similarity: As used herein, the term "similarity" refers to
the overall relatedness between polymeric molecules, e.g. between
polynucleotide molecules (e.g. DNA molecules and/or RNA molecules)
and/or between polypeptide molecules. Calculation of percent
similarity of polymeric molecules to one another can be performed
in the same manner as a calculation of percent identity, except
that calculation of percent similarity takes into account
conservative substitutions as is understood in the art.
[0286] Small/short interfering RNA: As used herein, the term
"small/short interfering RNA" or "siRNA" refers to an RNA molecule
(or RNA analog) comprising between about 5-60 nucleotides (or
nucleotide analogs) which is capable of directing or mediating
RNAi. Preferably, a siRNA molecule comprises between about 15-30
nucleotides or nucleotide analogs, more preferably between about
16-25 nucleotides (or nucleotide analogs), even more preferably
between about 18-23 nucleotides (or nucleotide analogs), and even
more preferably between about 19-22 nucleotides (or nucleotide
analogs) (e.g., 19, 20, 21 or 22 nucleotides or nucleotide
analogs). The term "short" siRNA refers to a siRNA comprising 5-23
nucleotides, preferably 21 nucleotides (or nucleotide analogs), for
example, 19, 20, 21 or 22 nucleotides. The term "long" siRNA refers
to a siRNA comprising 24-60 nucleotides, preferably about 24-25
nucleotides, for example, 23, 24, 25 or 26 nucleotides. Short
siRNAs may, in some instances, include fewer than 19 nucleotides,
e.g., 16, 17 or 18 nucleotides, or as few as 5 nucleotides,
provided that the shorter siRNA retains the ability to mediate
RNAi. Likewise, long siRNAs may, in some instances, include more
than 26 nucleotides, e.g., 27, 28, 29, 30, 35, 40, 45, 50, 55, or
even 60 nucleotides, provided that the longer siRNA retains the
ability to mediate RNAi or translational repression absent further
processing, e.g., enzymatic processing, to a short siRNA. siRNAs
can be single stranded RNA molecules (ss-siRNAs) or double stranded
RNA molecules (ds-siRNAs) comprising a sense strand and an
antisense strand which hybridized to form a duplex structure called
siRNA duplex.
[0287] Split dose: As used herein, a "split dose" is the division
of single unit dose or total daily dose into two or more doses.
[0288] Stable: As used herein "stable" refers to a compound or
entity that is sufficiently robust to survive isolation to a useful
degree of purity from a reaction mixture, and preferably capable of
formulation into an efficacious therapeutic agent.
[0289] Stabilized: As used herein, the term "stabilize",
"stabilized," "stabilized region" means to make or become stable.
In some embodiments, stability is measured relative to an absolute
value. In some embodiments, stability is measured relative to a
reference compound or entity.
[0290] Subject: As used herein, the term "subject" or "patient"
refers to any organism to which a composition in accordance with
the invention may be administered, e.g., for experimental,
diagnostic, prophylactic, and/or therapeutic purposes. Typical
subjects include animals (e.g., mammals such as mice, rats,
rabbits, non-human primates, and humans) and/or plants. In some
embodiments, the subject may be an infant, neonate, or a child
under the age of 12 years old. In some embodiments, the subject may
be in utero.
[0291] Substantially: As used herein, the term "substantially"
refers to the qualitative condition of exhibiting total or
near-total extent or degree of a characteristic or property of
interest. One of ordinary skill in the biological arts will
understand that biological and chemical phenomena rarely, if ever,
go to completion and/or proceed to completeness or achieve or avoid
an absolute result. The term "substantially" is therefore used
herein to capture the potential lack of completeness inherent in
many biological and chemical phenomena.
[0292] Substantially equal: As used herein as it relates to time
differences between doses, the term means plus/minus 2%.
[0293] Substantially simultaneously: As used herein and as it
relates to plurality of doses, the term typically means within
about 2 seconds.
[0294] Suffering from: An individual who is "suffering from" a
disease, disorder, and/or condition has been diagnosed with or
displays one or more symptoms of a disease, disorder, and/or
condition such as for example Parkinson's Disease.
[0295] Susceptible to: An individual who is "susceptible to" a
disease, disorder, and/or condition has not been diagnosed with
and/or may not exhibit symptoms of the disease, disorder, and/or
condition but harbors a propensity to develop a disease or its
symptoms. In some embodiments, an individual who is susceptible to
a disease, disorder, and/or condition (for example, cancer) may be
characterized by one or more of the following: (1) a genetic
mutation associated with development of the disease, disorder,
and/or condition; (2) a genetic polymorphism associated with
development of the disease, disorder, and/or condition; (3)
increased and/or decreased expression and/or activity of a protein
and/or nucleic acid associated with the disease, disorder, and/or
condition; (4) habits and/or lifestyles associated with development
of the disease, disorder, and/or condition; (5) a family history of
the disease, disorder, and/or condition; and (6) exposure to and/or
infection with a microbe associated with development of the
disease, disorder, and/or condition. In some embodiments, an
individual who is susceptible to a disease, disorder, and/or
condition will develop the disease, disorder, and/or condition. In
some embodiments, an individual who is susceptible to a disease,
disorder, and/or condition will not develop the disease, disorder,
and/or condition.
[0296] Sustained release: As used herein, the term "sustained
release" refers to a pharmaceutical composition or compound release
profile that conforms to a release rate over a specific period of
time.
[0297] Synthetic: The term "synthetic" means produced, prepared,
and/or manufactured by the hand of man. Synthesis of
polynucleotides or polypeptides or other molecules of the present
invention may be chemical or enzymatic.
[0298] Targeting: As used herein, "targeting" means the process of
design and selection of nucleic acid sequence that will hybridize
to a target nucleic acid and induce a desired effect.
[0299] Targeted Cells: As used herein, "targeted cells" refers to
any one or more cells of interest. The cells may be found in vitro,
in vivo, in situ or in the tissue or organ of an organism. The
organism may be an animal, preferably a mammal, more preferably a
human and most preferably a patient.
[0300] Therapeutic Agent: The term "therapeutic agent" refers to
any agent that, when administered to a subject, has a therapeutic,
diagnostic, and/or prophylactic effect and/or elicits a desired
biological and/or pharmacological effect.
[0301] Therapeutically effective amount: As used herein, the term
"therapeutically effective amount" means an amount of an agent to
be delivered (e.g., nucleic acid, drug, therapeutic agent,
diagnostic agent, prophylactic agent, etc.) that is sufficient,
when administered to a subject suffering from or susceptible to an
infection, disease, disorder, and/or condition, to treat, improve
symptoms of, diagnose, prevent, and/or delay the onset of the
infection, disease, disorder, and/or condition such as for example
Parkinson's Disease. In some embodiments, a therapeutically
effective amount is provided in a single dose. In some embodiments,
a therapeutically effective amount is administered in a dosage
regimen comprising a plurality of doses. Those skilled in the art
will appreciate that in some embodiments, a unit dosage form may be
considered to comprise a therapeutically effective amount of a
particular agent or entity if it comprises an amount that is
effective when administered as part of such a dosage regimen.
[0302] Therapeutically effective outcome: As used herein, the term
"therapeutically effective outcome" means an outcome that is
sufficient in a subject suffering from or susceptible to an
infection, disease, disorder, and/or condition, to treat, improve
symptoms of, diagnose, prevent, and/or delay the onset of the
infection, disease, disorder, and/or condition.
[0303] Total daily dose: As used herein, a "total daily dose" is an
amount given or prescribed in 24 hr period. It may be administered
as a single unit dose.
[0304] Transfection: As used herein, the term "transfection" refers
to methods to introduce exogenous nucleic acids into a cell.
Methods of transfection include, but are not limited to, chemical
methods, physical treatments and cationic lipids or mixtures.
[0305] Treating: As used herein, the term "treating" refers to
partially or completely alleviating, ameliorating, improving,
relieving, delaying onset of, inhibiting progression of, reducing
severity of, and/or reducing incidence of one or more symptoms or
features of a particular infection, disease, disorder, and/or
condition. For example, "treating" cancer may refer to inhibiting
survival, growth, and/or spread of a tumor. Treatment may be
administered to a subject who does not exhibit signs of a disease,
disorder, and/or condition and/or to a subject who exhibits only
early signs of a disease, disorder, and/or condition for the
purpose of decreasing the risk of developing pathology associated
with the disease, disorder, and/or condition such as for example
Parkinson's Disease.
[0306] Unmodified: As used herein, "unmodified" refers to any
substance, compound or molecule prior to being changed in any way.
Unmodified may, but does not always, refer to the wild type or
native form of a biomolecule or entity. Molecules or entities may
undergo a series of modifications whereby each modified substance,
compound, molecule or entity may serve as the "unmodified" starting
molecule for a subsequent modification.
[0307] Vector: As used herein, a "vector" is any molecule or moiety
which transports, transduces or otherwise acts as a carrier of a
heterologous molecule. Vectors of the present disclosure may be
produced recombinantly and may be based on and/or may comprise
adeno-associated virus (AAV) parent or reference sequence. Such
parent or reference AAV sequences may serve as an original, second,
third or subsequent sequence for engineering vectors. In
non-limiting examples, such parent or reference AAV sequences may
comprise any one or more of the following sequences: a
polynucleotide sequence encoding a polypeptide or
multi-polypeptide, which sequence may be wild-type or modified from
wild-type and which sequence may encode full-length or partial
sequence of a protein, protein domain, or one or more subunits of a
protein; a polynucleotide comprising a modulatory or regulatory
nucleic acid which sequence may be wild-type or modified from
wild-type; and a transgene that may or may not be modified from
wild-type sequence. These AAV sequences may serve as either the
"donor" sequence of one or more codons (at the nucleic acid level)
or amino acids (at the polypeptide level) or "acceptor" sequences
of one or more codons (at the nucleic acid level) or amino acids
(at the polypeptide level).
[0308] Viral construct vector: As used herein, a "viral construct
vector" is a vector which comprises one or more polynucleotide
regions encoding or comprising Rep and or Cap protein.
[0309] Viral construct expression vector: As used herein, a "viral
construct expression vector" is a vector which comprises one or
more polynucleotide regions encoding or comprising Rep and or Cap
that further comprises one or more polynucleotide regions encoding
or comprising components for viral expression in a viral
replication cell.
[0310] Viral genome: As used herein, a "viral genome" is a
polynucleotide encoding at least one inverted terminal repeat
(ITR), at least one regulatory sequence, and at least one payload.
The viral genome is derived by replication of a payload construct
from the payload construct expression vector. A viral genome
encodes at least one copy of the payload construct.
EQUIVALENTS AND SCOPE
[0311] Those skilled in the art will recognize, or be able to
ascertain using no more than routine experimentation, many
equivalents to the specific embodiments in accordance with the
invention described herein. The scope of the present invention is
not intended to be limited to the above Description, but rather is
as set forth in the appended claims.
[0312] In the claims, articles such as "a," "an," and "the" may
mean one or more than one unless indicated to the contrary or
otherwise evident from the context. Claims or descriptions that
include "or" between one or more members of a group are considered
satisfied if one, more than one, or all of the group members are
present in, employed in, or otherwise relevant to a given product
or process unless indicated to the contrary or otherwise evident
from the context. The invention includes embodiments in which
exactly one member of the group is present in, employed in, or
otherwise relevant to a given product or process. The invention
includes embodiments in which more than one, or all of the group
members are present in, employed in, or otherwise relevant to a
given product or process.
[0313] It is also noted that the term "comprising" is intended to
be open and permits but does not require the inclusion of
additional elements or steps. When the term "comprising" is used
herein, the term "consisting of" is thus also encompassed and
disclosed.
[0314] Unless otherwise defined, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this invention belongs. Methods
and materials are described herein for use in the present
disclosure; other, suitable methods and materials known in the art
can also be used.
[0315] Where ranges are given, endpoints are included. Furthermore,
it is to be understood that unless otherwise indicated or otherwise
evident from the context and understanding of one of ordinary skill
in the art, values that are expressed as ranges can assume any
specific value or subrange within the stated ranges in different
embodiments of the invention, to the tenth of the unit of the lower
limit of the range, unless the context clearly dictates
otherwise.
[0316] In addition, it is to be understood that any particular
embodiment of the present invention that falls within the prior art
may be explicitly excluded from any one or more of the claims.
Since such embodiments are deemed to be known to one of ordinary
skill in the art, they may be excluded even if the exclusion is not
set forth explicitly herein. Any particular embodiment of the
compositions of the invention (e.g., any nucleic acid or protein
encoded thereby; any method of production; any method of use; etc.)
can be excluded from any one or more claims, for any reason,
whether or not related to the existence of prior art.
[0317] All cited sources, for example, references, publications,
databases, database entries, and art cited herein, are incorporated
into this application by reference, even if not expressly stated in
the citation. In case of conflicting statements of a cited source
and the instant application, the statement in the instant
application shall control.
[0318] Section and table headings are not intended to be
limiting.
EXAMPLES
Example 1. Design of AADC Polynucleotides
[0319] AADC polynucleotides are designed to comprise at a minimum a
nucleic acid sequence encoding an AADC protein.
[0320] Once designed, the sequence is engineered or synthesized or
inserted in a plasmid or vector and administered to a cell or
organism. Suitable plasmids or vectors are any which transduce or
transfect the target cell.
[0321] Adeno-associated viral vectors (AAV), viral particles or
entire viruses may be used.
[0322] Administration results in the processing of the AADC
polynucleotide to generate the AADC protein which alters the
etiology of the disease, in this case Parkinson's Disease.
[0323] In one non-limiting example, plasmids containing an AADC
polynucleotide of the invention are given in Table 1. These AADC
polynucleotides in the table are contained in a Fastback plasmid
and have a CMV promoter and encode AADC. In some embodiments the
open reading frame of the AADC protein mRNA is codon optimized
(e.g., codop).
TABLE-US-00002 TABLE 1 AADC polynucleotide-containing
plasmids/vectors. SEQ ID Construct NO pFB CMV hAADC-1 2 pFB CMV
hAADC-2 3 pFB CMV hAADC-3 4 pFB CMV hAADC-4 5
Example 2. AADC Polynucleotides: ITR to ITR
[0324] AADC polynucleotides suitable for use in an AAV viral vector
include those in Table 2.
[0325] Given in Table 2 are the ITR to ITR sequences from Table
1.
TABLE-US-00003 TABLE 2 ITR to ITR AADC polynucleotides SEQ ID
Construct NO pFB CMV hAADC-1 (ITR to ITR) 6 pFB CMV hAADC-2 (ITR to
ITR) 7 pFB CMV hAADC-3 (ITR to ITR) 8 pFB CMV hAADC-4 (ITR to ITR)
9
Example 3. Relative to the ITR to ITR Parent Sequence
[0326] AADC polynucleotides are designed according to Table 3 and
Table 4. The start and stop positions given are relative to the ITR
to ITR AADC polynucleotides described in Table 2.
TABLE-US-00004 TABLE 3 Component modules or sequence regions of
AADC polynucleotides pFB CMV pFB CMV pFB CMV pFB CMV hAADC-1
hAADC-2 hAADC-3 hAADC-4 (ITR to ITR) (ITR to ITR) (ITR to ITR) (ITR
to ITR) Start Stop Start Stop Start Stop Start Stop 5' ITR 1 130 1
130 1 130 1 130 CMV Enhancer 263 566 263 566 263 566 296 599 CMV
Promoter 567 769 567 769 567 769 600 802 ie1 exon 1 784 917 784 917
784 917 817 950 ie1 intron1 918 949 918 949 918 949 951 982
hbBglobin 950 1296 950 1296 950 1296 983 1329 intron2 hBglobin 1297
1349 1297 1349 1297 1349 1330 1382 exon 3 5' UTR -- -- -- -- -- --
1398 1468 hAADC 1374 2822 -- -- 1374 2822 1473 2921 hAADC codop --
-- 1374 2816 -- -- -- -- 3' UTR -- -- -- -- 2823 3221 2922 3361 hGH
poly(A) 2841 3317 2835 3311 3240 3716 3380 3856 signal 3' ITR 3417
3535 3416 3534 3822 3940 3961 4079
TABLE-US-00005 TABLE 4 Component modules or sequence regions of
AADC polynucleotides hAADC_1k hAADC_2k hAADC_3k hAADC_4 Start Stop
Start Stop Start Stop Start Stop 5' ITR 1 141 1 141 1 141 1 141 CMV
Enhancer 245 548 245 548 245 548 245 548 CMV Promoter 549 751 549
751 549 751 549 751 ie1 exon 1 766 899 766 899 766 899 766 899 ie1
intron1 900 931 900 931 900 931 900 931 hbBglobin 932 1278 932 1278
932 1278 932 1278 intron2 hBglobin 1279 1331 1279 1331 1279 1331
1279 1331 exon 3 5' UTR -- -- -- -- -- -- 1347 3310 hAADC 1356 2804
1356 2804 -- -- 1422 2864 hAADC codop -- -- -- -- 1356 2798 -- --
3' UTR -- -- -- -- -- -- 2865 3310 hGH poly(A) 2823 3299 2823 3299
2817 3293 3329 3805 signal 3' ITR 3357 3497 3357 3497 3351 3491
3863 4003
TABLE-US-00006 TABLE 5 Component modules or sequence regions of
AADC polynucleotides hAADC_5k hAADC_6k hAADC_9k Start Stop Start
Stop Start Stop 5' ITR 1 145 1 141 1 130 CMV Enhancer 249 552 245
548 234 537 CMV Promoter 553 755 549 751 538 740 ie1 exon 1 770 903
766 899 755 888 ie1 intron1 904 935 900 931 889 920 hbBglobin
intron2 936 1282 932 1278 921 1267 hBglobin exon 3 1283 1335 1279
1331 1268 1320 5' UTR -- -- -- -- -- -- hAADC 1356 2798 1345 2793
hAADC codop 1360 2802 -- -- -- -- 3' UTR -- -- 2799 3203 -- -- hGH
poly(A) signal 2821 3297 3222 3698 2812 3288 3' ITR 3355 3499 3756
3896 3346 3475
Example 4. Design of AADC Polynucleotides
[0327] AADC polynucleotides are designed to comprise at a minimum a
nucleic acid sequence encoding an AADC protein.
[0328] Once designed, the sequence is engineered or synthesized or
inserted in a plasmid or vector and administered to a cell or
organism. Suitable plasmids or vectors are any which transduce or
transfect the target cell.
[0329] Adeno-associated viral vectors (AAV), viral particles or
entire viruses may be used.
[0330] Administration results in the processing of the AADC
polynucleotide to generate the AADC protein which alters the
etiology of the disease, in this case Parkinson's Disease.
[0331] In one non-limiting example, plasmids containing an AADC
polynucleotide of the invention are given in Table 6. These AADC
polynucleotides in the table are contained in a Fastback plasmid
and have a CMV promoter and encode AADC. In some embodiments the
open reading frame of the AADC protein mRNA is codon optimized
(e.g., codop).
TABLE-US-00007 TABLE 6 AADC polynucleotide-containing
plasmids/vectors. SEQ ID Construct NO phAADC_1k 10 phAADC_2k 11
phAADC_3k 12 phAADC_4 13 phAADC_5k 14 phAADC_6k 15 phAADC_9k 16
[0332] Given in Table 7 are the ITR to ITR sequences from Table
6.
TABLE-US-00008 TABLE 7 ITR to ITR AADC polynucleotides SEQ ID
Construct NO phAADC_1k (ITR to ITR) 17 phAADC_2k (ITR to ITR) 18
phAADC_3k (ITR to ITR) 19 phAADC_4 (ITR to ITR) 20 phAADC_5k (ITR
to ITR) 21 phAADC_6k (ITR to ITR) 22 phAADC_9k (ITR to ITR) 23
Example 5. Administration of AADC Polynucleotide Compositions to
Patients for Gene Therapy
[0333] AADC polynucleotide-containing recombinant AAV vector
compositions are infused into the substantia nigra, and in
particular, the substantia nigra pars compacta (SNpc) and ventral
tegmental area (VTA) of patients having Parkinson's Disease and
identified as qualified for treatment according to methods known in
the art.
[0334] One method of administration contemplated for use in the
methods described herein is real-time convection-enhanced delivery
(RCD) of AADC polynucleotide-containing AAV vector compositions by
co-infusion of gadoteridol (a magnetic resonance (MR) contrast
agent) and T1 or T2 magnetic resonance imaging (MRI), which can
predict areas of subsequent AADC gene expression. As described in
Richardson, et al., 2011, the accuracy of cannula placement and
initial infusate distribution may be safely determined by saline
infusion without significantly altering the subsequent distribution
of the tracer agent (Richardson, et al., 2011, Neurosurgery,
69(1):154-163). T2 RCD provides detection of intraparenchymal
convection-enhanced delivery in the uninjured brain and may predict
subsequent distribution of a transgene after viral vector infusion.
Subjects undergo saline infusion/T2 acquisition, immediately
followed by gadoteridol infusion/T1 acquisition in the putamen and
brainstem. Distribution volumes and spatial patterns are analyzed.
Gadoteridol and AAV-encoded AADC are co-infused under alternating
T2/T1 acquisition in the thalamus, and hyperintense areas are
compared with areas of subsequent transgene expression. Ratios of
distribution volume to infusion volume are expected to be similar
between saline and gadoteridol RCD. Spatial overlap should
correlate well between T2 and T1 images. The second infusate will
follow a spatiotemporal pattern similar to that of the first,
filling the target area before developing extra-target
distribution. Areas of AADC expression should correlate well with
areas of both T1 and T2 hyperintensity observed during RCD
(Richardson, et al., 2011, Neurosurgery, 69(1):154-163).
[0335] Convection-enhanced delivery (CED) of macromolecules
directly into the brain parenchyma has been known for over two
decades. CED is a term that denotes the use of a pressure gradient
to generate bulk flow within the brain parenchyma, i.e. convection
of macromolecules within the interstitial fluid driven by infusing
a solution through a cannula placed directly in the targeted
structure. This method allows therapeutic agents to be homogenously
distributed through large volumes of brain tissue by bypassing the
blood brain barrier and surpassing simple diffusion (Richardson, et
al., 2011, Stereotact. Funct. Neurosurg. 89:141-151).
[0336] Salegio, et al. recently demonstrated the distribution of
nanoparticles of different sizes, including micelles (.about.15 nm
in size), AAV (.about.20-25 nm) and liposomes (.about.65 nm),
within the CNS of rodents and NHPs (Salegio et al., 2014, Frontiers
in Neuroanatomy, vol. 8, article 9: pp. 1-8). Simple injections
cannot engage the perivascular system, and specialized infusion
cannulae are required, enabling constant pressures to be exerted at
the tip of the cannula such that the interstitial hydrostatic
pressure is exceeded and infusate can flow out into the tissue.
Simple needles generate significant reflux; thus, reflux-resistant
cannulas have been developed to counter this tendency. The advent
of platforms for MM-guided convection-enhanced infusions further
refined understanding of the mechanics of perivascular flow, and it
was demonstrated that perivascular distribution of liposomes was
linear with respect to time, the slope of the curve was increased
in myelinated regions, and cessation of infusion prevented further
expansion in the volume of distribution. (Richardson, et al., 2011,
Stereotact. Funct. Neurosurg. 89:141-151; Salegio et al., 2014,
Frontiers in Neuroanatomy, vol. 8, article 9: pp. 1-8).
[0337] Intraparenchymal rAAV injections are known to result in
robust but relatively local transduction. Such local delivery
methods are advantageous when attempting gene therapy for
neurological disorders that result from neuropathology that is
localized to a specific anatomical region or anatomical circuitry
such as in the case of Parkinson's disease. However, in treatments
requiring more widespread CNS transduction, intraparenchymal
injections are impractical. Treatment of neurological disorders
attributable to inborn errors of metabolism and/or single-gene
defects, or those that affect motor neurons of the spinal cord can
require transduction of large proportions of the brain or spinal
cord, respectively. Development of less invasive trans-BBB delivery
methods for vectors is an extremely important endeavor. Numerous
attempts to use molecules that are known to interact with various
active transport mechanisms (probably receptor-mediated) to convey
proteins across the BBB have been reported with varying results.
Given the large number of AAV serotypes available, one or more
serotypes may bind a cell-entry receptor capable of transporting
the AAV capsid across the BBB (Manfredsson, et al., 2009, "AAV9: a
potential blood-brain barrier buster." Molecular Therapy
17(3):403-405).
Vector and Stereotaxic Infusion
[0338] A stereotactic approach may be used to surgically deliver
the AADC polynucleotides. Although individuals with AADC deficiency
lack epinephrine and norepinephrine, these patients should maintain
stable blood pressure and heart rates during the surgery. There
should be no notable intracerebral hemorrhages in the postoperative
computed tomography (CT) or MRI scans. The needle tracts, as shown
on the MM scans, should show accurate injection into the substantia
nigra pars compacta (SNpc) and ventral tegmental area (VTA). The
patients will be discharged from the hospital about one week after
the surgery (Hwu, W. L., et al., 2012. Gene therapy for aromatic
L-amino acid decarboxylase deficiency. Sci. Transl. Med. Vol. 4,
134ra61).
[0339] Subjects of treatment receive the AAV-vector composition
vector, safely delivered to substantia nigra pars compacta (SNpc)
and ventral tegmental area (VTA) via bilateral infusions, or
alternatively, intrastriatally (into the caudate nucleus and
putamen), or into the subthalamic nucleus (STN), for example
optionally using the FDA-approved SMARTFLOW.RTM. neuroventricular
cannula (SurgiVision, Inc.) specifically designed for clinical
application, with or without the aid of the CLEARPOINT.RTM. system
to help the treating neurosurgeon(s) target and observe the
delivery of the therapeutic agent in the brain (See, for example,
San Sebastian, et al., 2014, Mol. Ther. Methods Clin. Dev. 3:
14049; See, for example, Feng and Maguire-Zeiss, 2010, CNS Drugs
24(3):177-192).
[0340] For example, during the surgery, two target points are
determined in the substantia nigra pars compacta (SNpc) and ventral
tegmental area (VTA) that are sufficiently separated from each
other in dorsolateral directions and identified on a magnetic
resonance image. One burr hole is trepanned in each side of the
cranial bone, through which the vector is injected into the two
target points via the two-track insertion route. The
AAV-vector-containing solution is prepared to a concentration of
1.5.times.10.sup.12 vector genome/ml, and 50 .mu.l per point of the
solution is injected at 1 .mu.l/min; each patient receives
3.times.10.sup.11 vector genome of the AAV-vector construct.
[0341] Neutralizing antibody titers against AAV2 are determined by
measuring .beta.-galactosidase activities in HEK293 cells
transduced with 5.times.10.sup.3 vector genome/cell of AAV2 vectors
expressing .beta.-galactosidase in various dilutions of sera.
PET
[0342] The AADC expression level in the substantia nigra are
assessed on PET imaging with FMT six days before surgery and at
one- and six-months after gene transfer. All patients cease taking
dopaminergic medications 18 hours before PET and take 2.5 mg/kg of
carbidopa orally one hour before FMT injection. Subsequently, 0.12
mCi/kg of FMT in saline is infused into an antecubital vein, and a
90-minute dynamic acquisition sequence is obtained. The PET and
magnetic resonance imaging data are co-registered with a fusion
processing program (Syntegra; Philips, Amsterdam, The Netherlands)
to produce the fusion images. Radioactivities within volumes of
interest drawn in the nigrostriatal pathway are calculated between
80 and 90 minutes after tracer injection. A change in nigrostriatal
pathway FMT uptake from baseline to 24 weeks is assessed using the
sub stantia nigra to striatal ratio of radioactivities.
Statistical Analysis
[0343] Values at baseline and 6 months after gene transfer are
compared using Student's t-test (paired analyses). A two-sided P
value <0.05 is taken to indicate significant differences.
Two-way analysis of variance with Bonferroni correction of P values
is used for the short-duration response to levodopa. (See, for
example, Muramatsu, et al., 2010, "A phase I study of aromatic
L-amino acid decarboxylase gene therapy for Parkinson's disease."
Mol. Ther. 18:1731-1735).
[0344] Safety and tolerability of bilateral administration of
AAV-vector compositions using real-time image-guided infusion into
the brains of Parkinson's Disease subjects may be monitored for up
to or after 9 months post-surgery. Broad coverage of targeted areas
(substantia nigra pars compacta (SNpc) and ventral tegmental area
(VTA)) and widespread AADC protein distribution in the striatum
should be achieved without inducing any adverse effects.
[0345] Changes in Growth and Motor Skills:
[0346] The patients should gain weight and exhibit improvement in
their motor scores after gene transfer, within a year,
post-treatment. Weight will be measured at 3 to 6 months after gene
transfer. All patients initially should have raw scores of zero on
the Alberta Infant Motor Scale (AIMS) and very low raw scores for
the Peabody Developmental Motor Scale, Second Edition (PDMS-II).
After the gene transfer, all of the patients should show continuous
increases in their raw scores on these two scales, which indicates
that their motor functions have improved. The Comprehensive
Developmental Inventory for Infants and Toddlers (CDIIT) covers
both cognition and motor development. All of the patients should
show low raw CDIIT scores before gene transfer, and the subsequent
increase in scores demonstrate improvement in both motor and
cognitive functions.
Subjective Improvements after Gene Transfer
[0347] To document the symptoms that are more difficult to
quantify, spouses, guardians or caretakers of the patients are
asked to fill out a questionnaire at the end of the study. The
symptoms of the oculogyric crises should lessen, and eye deviations
and sleep disruptions, for example, are some mild symptoms of the
oculogyric crises that may remain after gene therapy. Subjects may
experience increased emotional stability, and/or some improvements
in sweating and hyperthermia (a common manifestation of body
temperature instability in hot weather). There should be no
detectable abnormality in heart rate variability as assessed by
24-hour Holter monitoring either before or after gene transfer.
Before gene therapy, patients that were bedridden and showed little
spontaneous movement may exhibit less severe ptosis (drooping of
the upper eyelid) one to two weeks after the gene transfer.
According to previous studies, dyskinesia may occur one month after
gene transfer, but upon observation of a decrease in dyskinesia,
motor development should start (Hwu, W. L., et al., 2012. Gene
therapy for aromatic L-amino acid decarboxylase deficiency. Sci.
Transl. Med. Vol. 4, 134ra61). Subjects may exhibit increased head
control after three months, sitting with support after six to nine
months, sitting up from the prone position after thirteen months,
and holding toys and standing with support sixteen months after the
gene transfer, for example. Anti-AAV2 antibodies should be negative
in the patients before gene therapy, and the titers may increase
slightly after gene transfer.
PET Scans and CSF Analyses
[0348] PET scans and CSF analyses are completed for the treated
patients. Six months after gene transfer, PET scans should reveal
that uptake of 6-[18F] fluorodopa (FDOPA) increase from baseline in
the combined (right and left) treatment sites. The CSF analysis
should reveal increases in the levels of homovanillic acid (HVA, a
metabolite of dopamine) and 5-hydroxyindoleacetic acid (HIAA, a
metabolite of serotonin). However, the levels of L-DOPA and
3-O-methyldopa may remain elevated (Hwu, W. L., et al., 2012. Gene
therapy for aromatic L-amino acid decarboxylase deficiency. Sci.
Transl. Med. Vol. 4, 134ra61).
Example 6. Administration of AADC Polynucleotides
[0349] AADC polynucleotide-containing recombinant AAV vector
compositions are infused into the putamen of patients having
Parkinson's Disease using the administration methods described in
Example 5. The dose, number of patients and volume are outlined in
Table 8.
TABLE-US-00009 TABLE 8 Study Design Number of Study No. Patients
Dose Volume 1 6 3 .times. 10.sup.11 vg 100 ul per putamen 2 6 9
.times. 10.sup.11 vg 300 ul per putamen 3 10 2.3 .times. 10.sup.11
vg 100 ul per putamen 4 10 7.5 .times. 10.sup.11 vg 100 ul per
putamen 5 5 7.5 .times. 10.sup.11 vg 450 ul per putamen 6 Up to 20
1.4 .times. 10.sup.12 vg Up to 900 ul per putamen 7 Up to 20 4.8
.times. 10.sup.12 vg Up to 900 ul per putamen 8 Up to 20 8.8
.times. 10.sup.12 vg Up to 900 ul per putamen
[0350] During the course of the study the safety and tolerability
of the infusion of the AADC polynucleotide-containing recombinant
adeno-associated virus (AAV) vector compositions in human patients
diagnosed with Parkinson's Disease is evaluated. Patients are
evaluated preoperatively and monthly postoperatively for six
months, using multiple measures, including the Global Systonia
Scale (GDS) (see Comella, et al., 2003, Movement Disorders,
18(3):303-312), L-DOPA challenge test, UPDRS scores, motor state
diaries, and laboratory tests. Using diaries that separate the day
into half-hour segments, the caregivers of the patients will record
their mobility during the four days before admission and for
another four days at six months after admission to the study site.
The patient caregivers are trained to rate subject's condition as
sleeping, immobile, mobile without troublesome dyskinesias, or
mobile with troublesome dyskinesias. The total number of hours
spent in each of these categories is calculated, and the
differences between the baseline and the six-month scores are
compared between the groups. The short-duration response to
levodopa is evaluated at baseline and 6 months after gene transfer;
subjects take 100 mg of levodopa orally with 25 mg benserazide
after 20 hours without dopaminergic medication. Motor symptoms
based on GDS and plasma levodopa concentrations are assessed at
baseline and 30 minutes, 1, 2, 3, and 4 hours after levodopa intake
(See, for example, Muramatsu, et al., 2010, "A phase I study of
aromatic L-amino acid decarboxylase gene therapy for Parkinson's
disease." Mol. Ther. 18:1731-1735).
Example 7. In Vivo Administration of AADC Polynucleotides
[0351] Two AADC polynucleotide-containing recombinant AAV vector
compositions (plasmid SEQ ID NO: 10 and 12; ITR to ITR SEQ ID NO:
17 and 19), a control and a standard were administered to rats
(n=5) by bilateral intrastriation (10 ul/side) at a dose level of
2.times.10.sup.12 vg/ml. The expression of AADC in rat striatum was
determined by ELISA after 4 and 8 weeks. Variation was seen between
the animals and the hemispheres due to variable delivery between
the infusion sites. Both constructs expressed AADC, but the
phAADC_3k construct (plasmid SEQ ID NO: 12; ITR to ITR SEQ ID NO:
19) showed up to 200% increase of expression as compared to the
standard construct.
Example 8. Dose Response Study of AADC Polynucleotides
[0352] Compositions of AADC polynucleotide-containing recombinant
AAV vectors (plasmid SEQ ID NO: 10; ITR to ITR SEQ ID NO: 17) at
five different dose levels ranging from 1.times.10.sup.11 vg/ml to
1.times.10.sup.13 vg/ml and a control are administered to 6-OHDA
lesioned rats. The behavioral response to low-dose levodopa
administration is quantified before and after (week 3 and 4)
delivery of the composition. 5 weeks after dosing, necropsy is
conducted and the AADC enzymatic activity is measured in ex vivo
striatal tissue assay and the distribution of AADC in the brain is
determined by immunohistochemical (IHC) staining.
Example 9. Effect of Empty Particles on Intrastriatal
Transduction
[0353] Adult rats (n=6) were administered varying ratios of
full:empty vector particles at: 0% full, 50% full, 85% full or 99%
full. AADC polynucleotide-containing recombinant AAV vector
(plasmid SEQ ID NO: 10; ITR to ITR SEQ ID NO: 17) at a constant
dose and volume (5 ul and 1.times.10 vg) was administered
intrastriatally. The rats were evaluated 4 weeks after
administration. The low vector dose resulted in limited AADC vector
expression. The volume of distribution for the particles is shown
in Table 9 (ELISA) and the striatal levels of AADC expression is
shown in Table 10 (Histology).
TABLE-US-00010 TABLE 9 Volume of Distribution % Ratio of full
AAV2-AADC Approximate Volume of particle to empty capsids
Distribution (mm.sup.3) 50:50 2 70:30 2.4 85:15 2.5 100:0 3
TABLE-US-00011 TABLE 10 Striatal Levels % full of AAV2-AADC
particles AADC pg/ug protein 0 0.4 52 1.1 58 1.7 83 2 >99
2.1
[0354] Distribution was comparable for all groups. Relatively low
vector dose resulted in limited AADC expression. There was also a
trend to lower AADC expression levels with >30% empty
particles.
[0355] While the present invention has been described at some
length and with some particularity with respect to the several
described embodiments, it is not intended that it should be limited
to any such particulars or embodiments or any particular
embodiment, but it is to be construed with references to the
appended claims so as to provide the broadest possible
interpretation of such claims in view of the prior art and,
therefore, to effectively encompass the intended scope of the
invention.
[0356] All publications, patent applications, patents, and other
references mentioned herein are incorporated by reference in their
entirety. In case of conflict, the present specification, including
definitions, will control. In addition, section headings, the
materials, methods, and examples are illustrative only and not
intended to be limiting.
Sequence CWU 1
1
231480PRTHomo sapiens 1Met Asn Ala Ser Glu Phe Arg Arg Arg Gly Lys
Glu Met Val Asp Tyr 1 5 10 15 Val Ala Asn Tyr Met Glu Gly Ile Glu
Gly Arg Gln Val Tyr Pro Asp 20 25 30 Val Glu Pro Gly Tyr Leu Arg
Pro Leu Ile Pro Ala Ala Ala Pro Gln 35 40 45 Glu Pro Asp Thr Phe
Glu Asp Ile Ile Asn Asp Val Glu Lys Ile Ile 50 55 60 Met Pro Gly
Val Thr His Trp His Ser Pro Tyr Phe Phe Ala Tyr Phe 65 70 75 80 Pro
Thr Ala Ser Ser Tyr Pro Ala Met Leu Ala Asp Met Leu Cys Gly 85 90
95 Ala Ile Gly Cys Ile Gly Phe Ser Trp Ala Ala Ser Pro Ala Cys Thr
100 105 110 Glu Leu Glu Thr Val Met Met Asp Trp Leu Gly Lys Met Leu
Glu Leu 115 120 125 Pro Lys Ala Phe Leu Asn Glu Lys Ala Gly Glu Gly
Gly Gly Val Ile 130 135 140 Gln Gly Ser Ala Ser Glu Ala Thr Leu Val
Ala Leu Leu Ala Ala Arg 145 150 155 160 Thr Lys Val Ile His Arg Leu
Gln Ala Ala Ser Pro Glu Leu Thr Gln 165 170 175 Ala Ala Ile Met Glu
Lys Leu Val Ala Tyr Ser Ser Asp Gln Ala His 180 185 190 Ser Ser Val
Glu Arg Ala Gly Leu Ile Gly Gly Val Lys Leu Lys Ala 195 200 205 Ile
Pro Ser Asp Gly Asn Phe Ala Met Arg Ala Ser Ala Leu Gln Glu 210 215
220 Ala Leu Glu Arg Asp Lys Ala Ala Gly Leu Ile Pro Phe Phe Met Val
225 230 235 240 Ala Thr Leu Gly Thr Thr Thr Cys Cys Ser Phe Asp Asn
Leu Leu Glu 245 250 255 Val Gly Pro Ile Cys Asn Lys Glu Asp Ile Trp
Leu His Val Asp Ala 260 265 270 Ala Tyr Ala Gly Ser Ala Phe Ile Cys
Pro Glu Phe Arg His Leu Leu 275 280 285 Asn Gly Val Glu Phe Ala Asp
Ser Phe Asn Phe Asn Pro His Lys Trp 290 295 300 Leu Leu Val Asn Phe
Asp Cys Ser Ala Met Trp Val Lys Lys Arg Thr 305 310 315 320 Asp Leu
Thr Gly Ala Phe Arg Leu Asp Pro Thr Tyr Leu Lys His Ser 325 330 335
His Gln Asp Ser Gly Leu Ile Thr Asp Tyr Arg His Trp Gln Ile Pro 340
345 350 Leu Gly Arg Arg Phe Arg Ser Leu Lys Met Trp Phe Val Phe Arg
Met 355 360 365 Tyr Gly Val Lys Gly Leu Gln Ala Tyr Ile Arg Lys His
Val Gln Leu 370 375 380 Ser His Glu Phe Glu Ser Leu Val Arg Gln Asp
Pro Arg Phe Glu Ile 385 390 395 400 Cys Val Glu Val Ile Leu Gly Leu
Val Cys Phe Arg Leu Lys Gly Ser 405 410 415 Asn Lys Val Asn Glu Ala
Leu Leu Gln Arg Ile Asn Ser Ala Lys Lys 420 425 430 Ile His Leu Val
Pro Cys His Leu Arg Asp Lys Phe Val Leu Arg Phe 435 440 445 Ala Ile
Cys Ser Arg Thr Val Glu Ser Ala His Val Gln Arg Ala Trp 450 455 460
Glu His Ile Lys Glu Leu Ala Ala Asp Val Leu Arg Ala Glu Arg Glu 465
470 475 480 28150DNAArtificial SequenceDescription of Artificial
Sequence Synthetic polynucleotide 2cattcgccat tcaggctgca aataagcgtt
gatattcagt caattacaaa cattaataac 60gaagagatga cagaaaaatt ttcattctgt
gacagagaaa aagtagccga agatgacggt 120ttgtcacatg gagttggcag
gatgtttgat taaaaacata acaggaagaa aaatgccccg 180ctgtgggcgg
acaaaatagt tgggaactgg gaggggtgga aatggagttt ttaaggatta
240tttagggaag agtgacaaaa tagatgggaa ctgggtgtag cgtcgtaagc
taatacgaaa 300attaaaaatg acaaaatagt ttggaactag atttcactta
tctggttcgg atctcctagg 360cgatatcagt gatcagatcc agacatgata
agatacattg atgagtttgg acaaaccaca 420actagaatgc agtgaaaaaa
atgctttatt tgtgaaattt gtgatgctat tgctttattt 480gtaaccatta
taagctgcaa taaacaagtt aacaacaaca attgcattca ttttatgttt
540caggttcagg gggaggtgtg ggaggttttt taaagcaagt aaaacctcta
caaatgtggt 600atggctgatt atgatcctct agtacttctc gacaagctta
cattattgaa gcatttatca 660gggttattgt ctcagacctg caggcagctg
cgcgctcgct cgctcactga ggccgcccgg 720gcaaagcccg ggcgtcgggc
gacctttggt cgcccggcct cagtgagcga gcgagcgcgc 780agagagggag
tggccaactc catcactagg ggttccttgt agttaatgat taacccgcca
840tgctacttat ctacgtagcc atgctctaga gcggccgcac gcgtggagct
agttattaat 900agtaatcaat tacggggtca ttagttcata gcccatatat
ggagttccgc gttacataac 960ttacggtaaa tggcccgcct ggctgaccgc
ccaacgaccc ccgcccattg acgtcaataa 1020tgacgtatgt tcccatagta
acgtcaatag ggactttcca ttgacgtcaa tgggtggagt 1080atttacggta
aactgcccac ttggcagtac atcaagtgta tcatatgcca agtacgcccc
1140ctattgacgt caatgacggt aaatggcccg cctggcatta tgcccagtac
atgaccttat 1200gggactttcc tacttggcag tacatctacg tattagtcat
cgctattacc atggtgatgc 1260ggttttggca gtacatcaat gggcgtggat
agcggtttga ctcacgggga tttccaagtc 1320tccaccccat tgacgtcaat
gggagtttgt tttgcaccaa aatcaacggg actttccaaa 1380atgtcgtaac
aactccgccc cattgacgca aatgggcggt aggcgtgtac ggtgggaggt
1440ctatataagc agagctcgtt tagtgaaccg tcagatcgcc tggagacgcc
atccacgctg 1500ttttgacctc catagaagac accgggaccg atccagcctc
cgcggattcg aatcccggcc 1560gggaacggtg cattggaacg cggattcccc
gtgccaagag tgacgtaagt accgcctata 1620gagtctatag gcccacaaaa
aatgctttct tcttttaata tacttttttg tttatcttat 1680ttctaatact
ttccctaatc tctttctttc agggcaataa tgatacaatg tatcatgcct
1740ctttgcacca ttctaaagaa taacagtgat aatttctggg ttaaggcaat
agcaatattt 1800ctgcatataa atatttctgc atataaattg taactgatgt
aagaggtttc atattgctaa 1860tagcagctac aatccagcta ccattctgct
tttattttat ggttgggata aggctggatt 1920attctgagtc caagctaggc
ccttttgcta atcatgttca tacctcttat cttcctccca 1980cagctcctgg
gcaacgtgct ggtctgtgtg ctggcccatc actttggcaa agaattggga
2040ttcgaacatc ggccgccacc atgaacgcaa gtgaattccg aaggagaggg
aaggagatgg 2100tggattacgt ggccaactac atggaaggca ttgagggacg
ccaggtctac cctgacgtgg 2160agcccgggta cctgcggccg ctgatccctg
ccgctgcccc tcaggagcca gacacgtttg 2220aggacatcat caacgacgtt
gagaagataa tcatgcctgg ggtgacgcac tggcacagcc 2280cctacttctt
cgcctacttc cccactgcca gctcgtaccc ggccatgctt gcggacatgc
2340tgtgcggggc cattggctgc atcggcttct cctgggcggc aagcccagca
tgcacagagc 2400tggagactgt gatgatggac tggctcggga agatgctgga
actaccaaag gcatttttga 2460atgagaaagc tggagaaggg ggaggagtga
tccagggaag tgccagtgaa gccaccctgg 2520tggccctgct ggccgctcgg
accaaagtga tccatcggct gcaggcagcg tccccagagc 2580tcacacaggc
cgctatcatg gagaagctgg tggcttactc atccgatcag gcacactcct
2640cagtggaaag agctgggtta attggtggag tgaaattaaa agccatcccc
tcagatggca 2700acttcgccat gcgtgcgtct gccctgcagg aagccctgga
gagagacaaa gcggctggcc 2760tgattccttt ctttatggtt gccaccctgg
ggaccacaac atgctgctcc tttgacaatc 2820tcttagaagt cggtcctatc
tgcaacaagg aagacatatg gctgcacgtt gatgcagcct 2880acgcaggcag
tgcattcatc tgccctgagt tccggcacct tctgaatgga gtggagtttg
2940cagattcatt caactttaat ccccacaaat ggctattggt gaattttgac
tgttctgcca 3000tgtgggtgaa aaagagaaca gacttaacgg gagcctttag
actggacccc acttacctga 3060agcacagcca tcaggattca gggcttatca
ctgactaccg gcattggcag ataccactgg 3120gcagaagatt tcgctctttg
aaaatgtggt ttgtatttag gatgtatgga gtcaaaggac 3180tgcaggctta
tatccgcaag catgtccagc tgtcccatga gtttgagtca ctggtgcgcc
3240aggatccccg ctttgaaatc tgtgtggaag tcattctggg gcttgtctgc
tttcggctaa 3300agggttccaa caaagtgaat gaagctcttc tgcaaagaat
aaacagtgcc aaaaaaatcc 3360acttggttcc atgtcacctc agggacaagt
ttgtcctgcg ctttgccatc tgttctcgca 3420cggtggaatc tgcccatgtg
cagcgggcct gggaacacat caaagagctg gcggccgacg 3480tgctgcgagc
agagagggag taggagtgag ctgctcgaga gatctacggg tggcatccct
3540gtgacccctc cccagtgcct ctcctggccc tggaagttgc cactccagtg
cccaccagcc 3600ttgtcctaat aaaattaagt tgcatcattt tgtctgacta
ggtgtccttc tataatatta 3660tggggtggag gggggtggta tggagcaagg
ggcaagttgg gaagacaacc tgtagggcct 3720gcggggtcta ttgggaacca
agctggagtg cagtggcaca atcttggctc actgcaatct 3780ccgcctcctg
ggttcaagcg attctcctgc ctcagcctcc cgagttgttg ggattccagg
3840catgcatgac caggctcagc taatttttgt ttttttggta gagacggggt
ttcaccatat 3900tggccaggct ggtctccaac tcctaatctc aggtgatcta
cccaccttgg cctcccaaat 3960tgctgggatt acaggcgtga accactgctc
ccttccctgt ccttctgatt ttgtaggtaa 4020ccccggacca cgtgcggacc
gagcggccgc tctagagcat ggctacgtag ataagtagca 4080tggcgggtta
atcattaact acaaggaacc cctagtgatg gagttggcca ctccctctct
4140gcgcgctcgc tcgctcactg aggccgggcg accaaaggtc gcccgacgcc
cgggcggcct 4200cagtgagcga gcgagcgcgc agctgcctgc aggtctgaga
caataaccct gataaatgct 4260tcaataatgt agccaaccac tagaactata
gctagagtcc tgggcgaaca aacgatgctc 4320gccttccaga aaaccgagga
tgcgaaccac ttcatccggg gtcagcacca ccggcaagcg 4380ccgcgacggc
cgaggtcttc cgatctcctg aagccagggc agatccgtgc acagcacctt
4440gccgtagaag aacagcaagg ccgccaatgc ctgacgatgc gtggagaccg
aaaccttgcg 4500ctcgttcgcc agccaggaca gaaatgcctc gacttcgctg
ctgcccaagg ttgccgggtg 4560acgcacaccg tggaaacgga tgaaggcacg
aacccagttg acataagcct gttcggttcg 4620taaactgtaa tgcaagtagc
gtatgcgctc acgcaactgg tccagaacct tgaccgaacg 4680cagcggtggt
aacggcgcag tggcggtttt catggcttgt tatgactgtt tttttgtaca
4740gtctatgcct cgggcatcca agcagcaagc gcgttacgcc gtgggtcgat
gtttgatgtt 4800atggagcagc aacgatgtta cgcagcagca acgatgttac
gcagcagggc agtcgcccta 4860aaacaaagtt aggtggctca agtatgggca
tcattcgcac atgtaggctc ggccctgacc 4920aagtcaaatc catgcgggct
gctcttgatc ttttcggtcg tgagttcgga gacgtagcca 4980cctactccca
acatcagccg gactccgatt acctcgggaa cttgctccgt agtaagacat
5040tcatcgcgct tgctgccttc gaccaagaag cggttgttgg cgctctcgcg
gcttacgttc 5100tgcccaagtt tgagcagccg cgtagtgaga tctatatcta
tgatctcgca gtctccggcg 5160agcaccggag gcagggcatt gccaccgcgc
tcatcaatct cctcaagcat gaggccaacg 5220cgcttggtgc ttatgtgatc
tacgtgcaag cagattacgg tgacgatccc gcagtggctc 5280tctatacaaa
gttgggcata cgggaagaag tgatgcactt tgatatcgac ccaagtaccg
5340ccacctaaca attcgttcaa gccgagatcg gcttcccggc cgcggagttg
ttcggtaaat 5400tgtcacaacg ccgcgaatat agtctttacc atgcccttgg
ccacgcccct ctttaatacg 5460acgggcaatt tgcacttcag aaaatgaaga
gtttgcttta gccataacaa aagtccagta 5520tgctttttca cagcataact
ggactgattt cagtttacaa ctattctgtc tagtttaaga 5580ctttattgtc
atagtttaga tctattttgt tcagtttaag actttattgt ccgcccacac
5640ccgcttacgc agggcatcca tttattactc aaccgtaacc gattttgcca
ggttacgcgg 5700ctggtctatg cggtgtgaaa taccgcacag atgcgtaagg
agaaaatacc gcatcaggcg 5760ctcttccgct tcctcgctca ctgactcgct
gcgctcggtc gttcggctgc ggcgagcggt 5820atcagctcac tcaaaggcgg
taatacggtt atccacagaa tcaggggata acgcaggaaa 5880gaacatgtga
gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc
5940gtttttccat aggctccgcc cccctgacga gcatcacaaa aatcgacgct
caagtcagag 6000gtggcgaaac ccgacaggac tataaagata ccaggcgttt
ccccctggaa gctccctcgt 6060gcgctctcct gttccgaccc tgccgcttac
cggatacctg tccgcctttc tcccttcggg 6120aagcgtggcg ctttctcata
gctcacgctg taggtatctc agttcggtgt aggtcgttcg 6180ctccaagctg
ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg ccttatccgg
6240taactatcgt cttgagtcca acccggtaag acacgactta tcgccactgg
cagcagccac 6300tggtaacagg attagcagag cgaggtatgt aggcggtgct
acagagttct tgaagtggtg 6360gcctaactac ggctacacta gaagaacagt
atttggtatc tgcgctctgc tgaagccagt 6420taccttcgga aaaagagttg
gtagctcttg atccggcaaa caaaccaccg ctggtagcgg 6480tggttttttt
gtttgcaagc agcagattac gcgcagaaaa aaaggatctc aagaagatcc
6540tttgatcttt tctacggggt ctgacgctca gtggaacgaa aactcacgtt
aagggatttt 6600ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt
ttaaattaaa aatgaagttt 6660taaatcaatc taaagtatat atgagtaaac
ttggtctgac agttaccaat gcttaatcag 6720tgaggcacct atctcagcga
tctgtctatt tcgttcatcc atagttgcct gactccccgt 6780cgtgtagata
actacgatac gggagggctt accatctggc cccagtgctg caatgatacc
6840gcgagaccca cgctcaccgg ctccagattt atcagcaata aaccagccag
ccggaagggc 6900cgagcgcaga agtggtcctg caactttatc cgcctccatc
cagtctatta attgttgccg 6960ggaagctaga gtaagtagtt cgccagttaa
tagtttgcgc aacgttgttg ccattgctac 7020aggcatcgtg gtgtcacgct
cgtcgtttgg tatggcttca ttcagctccg gttcccaacg 7080atcaaggcga
gttacatgat cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc
7140tccgatcgtt gtcagaagta agttggccgc agtgttatca ctcatggtta
tggcagcact 7200gcataattct cttactgtca tgccatccgt aagatgcttt
tctgtgactg gtgagtactc 7260aaccaagtca ttctgagaat agtgtatgcg
gcgaccgagt tgctcttgcc cggcgtcaat 7320acgggataat accgcgccac
atagcagaac tttaaaagtg ctcatcattg gaaaacgttc 7380ttcggggcga
aaactctcaa ggatcttacc gctgttgaga tccagttcga tgtaacccac
7440tcgtgcaccc aactgatctt cagcatcttt tactttcacc agcgtttctg
ggtgagcaaa 7500aacaggaagg caaaatgccg caaaaaaggg aataagggcg
acacggaaat gttgaatact 7560catactcttc ctttttcaat attattgaag
catttatcag ggttattgtc tcatgagcgg 7620atacatattt gaatgtattt
agaaaaataa acaaataggg gttccgcgca catttccccg 7680aaaagtgcca
cctaaattgt aagcgttaat attttgttaa aattcgcgtt aaatttttgt
7740taaatcagct cattttttaa ccaataggcc gaaatcggca aaatccctta
taaatcaaaa 7800gaatagaccg agatagggtt gagtgttgtt ccagtttgga
acaagagtcc actattaaag 7860aacgtggact ccaacgtcaa agggcgaaaa
accgtctatc agggcgatgg cccactacgt 7920gaaccatcac cctaatcaag
ttttttgggg tcgaggtgcc gtaaagcact aaatcggaac 7980cctaaaggga
gcccccgatt tagagcttga cggggaaagc cggcgaacgt ggcgagaaag
8040gaagggaaga aagcgaaagg agcgggcgct agggcgctgg caagtgtagc
ggtcacgctg 8100cgcgtaacca ccacacccgc cgcgcttaat gcgccgctac
agggcgcgtc 815038149DNAArtificial SequenceDescription of Artificial
Sequence Synthetic polynucleotide 3cattcgccat tcaggctgca aataagcgtt
gatattcagt caattacaaa cattaataac 60gaagagatga cagaaaaatt ttcattctgt
gacagagaaa aagtagccga agatgacggt 120ttgtcacatg gagttggcag
gatgtttgat taaaaacata acaggaagaa aaatgccccg 180ctgtgggcgg
acaaaatagt tgggaactgg gaggggtgga aatggagttt ttaaggatta
240tttagggaag agtgacaaaa tagatgggaa ctgggtgtag cgtcgtaagc
taatacgaaa 300attaaaaatg acaaaatagt ttggaactag atttcactta
tctggttcgg atctcctagg 360cgatatcagt gatcagatcc agacatgata
agatacattg atgagtttgg acaaaccaca 420actagaatgc agtgaaaaaa
atgctttatt tgtgaaattt gtgatgctat tgctttattt 480gtaaccatta
taagctgcaa taaacaagtt aacaacaaca attgcattca ttttatgttt
540caggttcagg gggaggtgtg ggaggttttt taaagcaagt aaaacctcta
caaatgtggt 600atggctgatt atgatcctct agtacttctc gacaagctta
cattattgaa gcatttatca 660gggttattgt ctcagacctg caggcagctg
cgcgctcgct cgctcactga ggccgcccgg 720gcaaagcccg ggcgtcgggc
gacctttggt cgcccggcct cagtgagcga gcgagcgcgc 780agagagggag
tggccaactc catcactagg ggttccttgt agttaatgat taacccgcca
840tgctacttat ctacgtagcc atgctctaga gcggccgcac gcgtggagct
agttattaat 900agtaatcaat tacggggtca ttagttcata gcccatatat
ggagttccgc gttacataac 960ttacggtaaa tggcccgcct ggctgaccgc
ccaacgaccc ccgcccattg acgtcaataa 1020tgacgtatgt tcccatagta
acgtcaatag ggactttcca ttgacgtcaa tgggtggagt 1080atttacggta
aactgcccac ttggcagtac atcaagtgta tcatatgcca agtacgcccc
1140ctattgacgt caatgacggt aaatggcccg cctggcatta tgcccagtac
atgaccttat 1200gggactttcc tacttggcag tacatctacg tattagtcat
cgctattacc atggtgatgc 1260ggttttggca gtacatcaat gggcgtggat
agcggtttga ctcacgggga tttccaagtc 1320tccaccccat tgacgtcaat
gggagtttgt tttgcaccaa aatcaacggg actttccaaa 1380atgtcgtaac
aactccgccc cattgacgca aatgggcggt aggcgtgtac ggtgggaggt
1440ctatataagc agagctcgtt tagtgaaccg tcagatcgcc tggagacgcc
atccacgctg 1500ttttgacctc catagaagac accgggaccg atccagcctc
cgcggattcg aatcccggcc 1560gggaacggtg cattggaacg cggattcccc
gtgccaagag tgacgtaagt accgcctata 1620gagtctatag gcccacaaaa
aatgctttct tcttttaata tacttttttg tttatcttat 1680ttctaatact
ttccctaatc tctttctttc agggcaataa tgatacaatg tatcatgcct
1740ctttgcacca ttctaaagaa taacagtgat aatttctggg ttaaggcaat
agcaatattt 1800ctgcatataa atatttctgc atataaattg taactgatgt
aagaggtttc atattgctaa 1860tagcagctac aatccagcta ccattctgct
tttattttat ggttgggata aggctggatt 1920attctgagtc caagctaggc
ccttttgcta atcatgttca tacctcttat cttcctccca 1980cagctcctgg
gcaacgtgct ggtctgtgtg ctggcccatc actttggcaa agaattggga
2040ttcgaacatc ggccgccacc atgaacgcca gcgagttcag gaggaggggc
aaggagatgg 2100tggactacgt ggccaactac atggagggca tcgagggcag
gcaggtgtac cccgacgtgg 2160agcccggcta cctgaggccc ctgatccccg
ccgccgcccc ccaggagccc gacaccttcg 2220aggacatcat caacgacgtg
gagaagatca tcatgcccgg cgtgacccac tggcacagcc 2280cctacttctt
cgcctacttc cccaccgcca gcagctaccc cgccatgctg gccgacatgc
2340tgtgcggcgc catcggctgc atcggcttca gctgggccgc cagccccgcc
tgcaccgagc 2400tggagaccgt gatgatggac tggctgggca agatgctgga
gctgcccaag gccttcctga 2460acgagaaggc cggcgagggc ggcggcgtga
tccagggcag cgccagcgag gccaccctgg 2520tggccctgct ggccgccagg
accaaggtga tccacaggct gcaggccgcc agccccgagc 2580tgacccaggc
cgccatcatg gagaagctgg tggcctacag cagcgaccag gcccacagca
2640gcgtggagag ggccggcctg atcggcggcg tgaagctgaa ggccatcccc
agcgacggca 2700acttcgccat gagggccagc gccctgcagg aggccctgga
gagggacaag gccgccggcc 2760tgatcccctt cttcatggtg gccaccctgg
gcaccaccac ctgctgcagc ttcgacaacc 2820tgctggaggt gggccccatc
tgcaacaagg aggacatctg gctgcacgtg gacgccgcct 2880acgccggcag
cgccttcatc tgccccgagt tcaggcacct gctgaacggc gtggagttcg
2940ccgacagctt caacttcaac ccccacaagt ggctgctggt gaacttcgac
tgcagcgcca 3000tgtgggtgaa gaagaggacc gacctgaccg gcgccttcag
gctggacccc acctacctga 3060agcacagcca ccaggacagc ggcctgatca
ccgactacag gcactggcag atccccctgg 3120gcaggaggtt caggagcctg
aagatgtggt tcgtgttcag gatgtacggc gtgaagggcc 3180tgcaggccta
catcaggaag cacgtgcagc tgagccacga gttcgagagc ctggtgaggc
3240aggaccccag gttcgagatc tgcgtggagg tgatcctggg cctggtgtgc
ttcaggctga 3300agggcagcaa caaggtgaac gaggccctgc tgcagaggat
caacagcgcc aagaagatcc 3360acctggtgcc ctgccacctg agggacaagt
tcgtgctgag gttcgccatc tgcagcagga 3420ccgtggagag cgcccacgtg
cagagggcct gggagcacat caaggagctg gccgccgacg 3480tgctgagggc
cgagagggag tgagctgctc gagagatcta cgggtggcat ccctgtgacc
3540cctccccagt gcctctcctg gccctggaag
ttgccactcc agtgcccacc agccttgtcc 3600taataaaatt aagttgcatc
attttgtctg actaggtgtc cttctataat attatggggt 3660ggaggggggt
ggtatggagc aaggggcaag ttgggaagac aacctgtagg gcctgcgggg
3720tctattggga accaagctgg agtgcagtgg cacaatcttg gctcactgca
atctccgcct 3780cctgggttca agcgattctc ctgcctcagc ctcccgagtt
gttgggattc caggcatgca 3840tgaccaggct cagctaattt ttgttttttt
ggtagagacg gggtttcacc atattggcca 3900ggctggtctc caactcctaa
tctcaggtga tctacccacc ttggcctccc aaattgctgg 3960gattacaggc
gtgaaccact gctcccttcc ctgtccttct gattttgtag gtaacgtaac
4020cccggaccac gtgcggaccg agcggccgct ctagagcatg gctacgtaga
taagtagcat 4080ggcgggttaa tcattaacta caaggaaccc ctagtgatgg
agttggccac tccctctctg 4140cgcgctcgct cgctcactga ggccgggcga
ccaaaggtcg cccgacgccc gggcggcctc 4200agtgagcgag cgagcgcgca
gctgcctgca ggtctgagac aataaccctg ataaatgctt 4260caataatgta
gccaaccact agaactatag ctagagtcct gggcgaacaa acgatgctcg
4320ccttccagaa aaccgaggat gcgaaccact tcatccgggg tcagcaccac
cggcaagcgc 4380cgcgacggcc gaggtcttcc gatctcctga agccagggca
gatccgtgca cagcaccttg 4440ccgtagaaga acagcaaggc cgccaatgcc
tgacgatgcg tggagaccga aaccttgcgc 4500tcgttcgcca gccaggacag
aaatgcctcg acttcgctgc tgcccaaggt tgccgggtga 4560cgcacaccgt
ggaaacggat gaaggcacga acccagttga cataagcctg ttcggttcgt
4620aaactgtaat gcaagtagcg tatgcgctca cgcaactggt ccagaacctt
gaccgaacgc 4680agcggtggta acggcgcagt ggcggttttc atggcttgtt
atgactgttt ttttgtacag 4740tctatgcctc gggcatccaa gcagcaagcg
cgttacgccg tgggtcgatg tttgatgtta 4800tggagcagca acgatgttac
gcagcagcaa cgatgttacg cagcagggca gtcgccctaa 4860aacaaagtta
ggtggctcaa gtatgggcat cattcgcaca tgtaggctcg gccctgacca
4920agtcaaatcc atgcgggctg ctcttgatct tttcggtcgt gagttcggag
acgtagccac 4980ctactcccaa catcagccgg actccgatta cctcgggaac
ttgctccgta gtaagacatt 5040catcgcgctt gctgccttcg accaagaagc
ggttgttggc gctctcgcgg cttacgttct 5100gcccaagttt gagcagccgc
gtagtgagat ctatatctat gatctcgcag tctccggcga 5160gcaccggagg
cagggcattg ccaccgcgct catcaatctc ctcaagcatg aggccaacgc
5220gcttggtgct tatgtgatct acgtgcaagc agattacggt gacgatcccg
cagtggctct 5280ctatacaaag ttgggcatac gggaagaagt gatgcacttt
gatatcgacc caagtaccgc 5340cacctaacaa ttcgttcaag ccgagatcgg
cttcccggcc gcggagttgt tcggtaaatt 5400gtcacaacgc cgcgaatata
gtctttacca tgcccttggc cacgcccctc tttaatacga 5460cgggcaattt
gcacttcaga aaatgaagag tttgctttag ccataacaaa agtccagtat
5520gctttttcac agcataactg gactgatttc agtttacaac tattctgtct
agtttaagac 5580tttattgtca tagtttagat ctattttgtt cagtttaaga
ctttattgtc cgcccacacc 5640cgcttacgca gggcatccat ttattactca
accgtaaccg attttgccag gttacgcggc 5700tggtctatgc ggtgtgaaat
accgcacaga tgcgtaagga gaaaataccg catcaggcgc 5760tcttccgctt
cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta
5820tcagctcact caaaggcggt aatacggtta tccacagaat caggggataa
cgcaggaaag 5880aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta
aaaaggccgc gttgctggcg 5940tttttccata ggctccgccc ccctgacgag
catcacaaaa atcgacgctc aagtcagagg 6000tggcgaaacc cgacaggact
ataaagatac caggcgtttc cccctggaag ctccctcgtg 6060cgctctcctg
ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga
6120agcgtggcgc tttctcatag ctcacgctgt aggtatctca gttcggtgta
ggtcgttcgc 6180tccaagctgg gctgtgtgca cgaacccccc gttcagcccg
accgctgcgc cttatccggt 6240aactatcgtc ttgagtccaa cccggtaaga
cacgacttat cgccactggc agcagccact 6300ggtaacagga ttagcagagc
gaggtatgta ggcggtgcta cagagttctt gaagtggtgg 6360cctaactacg
gctacactag aagaacagta tttggtatct gcgctctgct gaagccagtt
6420accttcggaa aaagagttgg tagctcttga tccggcaaac aaaccaccgc
tggtagcggt 6480ggtttttttg tttgcaagca gcagattacg cgcagaaaaa
aaggatctca agaagatcct 6540ttgatctttt ctacggggtc tgacgctcag
tggaacgaaa actcacgtta agggattttg 6600gtcatgagat tatcaaaaag
gatcttcacc tagatccttt taaattaaaa atgaagtttt 6660aaatcaatct
aaagtatata tgagtaaact tggtctgaca gttaccaatg cttaatcagt
6720gaggcaccta tctcagcgat ctgtctattt cgttcatcca tagttgcctg
actccccgtc 6780gtgtagataa ctacgatacg ggagggctta ccatctggcc
ccagtgctgc aatgataccg 6840cgagacccac gctcaccggc tccagattta
tcagcaataa accagccagc cggaagggcc 6900gagcgcagaa gtggtcctgc
aactttatcc gcctccatcc agtctattaa ttgttgccgg 6960gaagctagag
taagtagttc gccagttaat agtttgcgca acgttgttgc cattgctaca
7020ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat tcagctccgg
ttcccaacga 7080tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag
cggttagctc cttcggtcct 7140ccgatcgttg tcagaagtaa gttggccgca
gtgttatcac tcatggttat ggcagcactg 7200cataattctc ttactgtcat
gccatccgta agatgctttt ctgtgactgg tgagtactca 7260accaagtcat
tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata
7320cgggataata ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg
aaaacgttct 7380tcggggcgaa aactctcaag gatcttaccg ctgttgagat
ccagttcgat gtaacccact 7440cgtgcaccca actgatcttc agcatctttt
actttcacca gcgtttctgg gtgagcaaaa 7500acaggaaggc aaaatgccgc
aaaaaaggga ataagggcga cacggaaatg ttgaatactc 7560atactcttcc
tttttcaata ttattgaagc atttatcagg gttattgtct catgagcgga
7620tacatatttg aatgtattta gaaaaataaa caaatagggg ttccgcgcac
atttccccga 7680aaagtgccac ctaaattgta agcgttaata ttttgttaaa
attcgcgtta aatttttgtt 7740aaatcagctc attttttaac caataggccg
aaatcggcaa aatcccttat aaatcaaaag 7800aatagaccga gatagggttg
agtgttgttc cagtttggaa caagagtcca ctattaaaga 7860acgtggactc
caacgtcaaa gggcgaaaaa ccgtctatca gggcgatggc ccactacgtg
7920aaccatcacc ctaatcaagt tttttggggt cgaggtgccg taaagcacta
aatcggaacc 7980ctaaagggag cccccgattt agagcttgac ggggaaagcc
ggcgaacgtg gcgagaaagg 8040aagggaagaa agcgaaagga gcgggcgcta
gggcgctggc aagtgtagcg gtcacgctgc 8100gcgtaaccac cacacccgcc
gcgcttaatg cgccgctaca gggcgcgtc 814948555DNAArtificial
SequenceDescription of Artificial Sequence Synthetic polynucleotide
4cattcgccat tcaggctgca aataagcgtt gatattcagt caattacaaa cattaataac
60gaagagatga cagaaaaatt ttcattctgt gacagagaaa aagtagccga agatgacggt
120ttgtcacatg gagttggcag gatgtttgat taaaaacata acaggaagaa
aaatgccccg 180ctgtgggcgg acaaaatagt tgggaactgg gaggggtgga
aatggagttt ttaaggatta 240tttagggaag agtgacaaaa tagatgggaa
ctgggtgtag cgtcgtaagc taatacgaaa 300attaaaaatg acaaaatagt
ttggaactag atttcactta tctggttcgg atctcctagg 360cgatatcagt
gatcagatcc agacatgata agatacattg atgagtttgg acaaaccaca
420actagaatgc agtgaaaaaa atgctttatt tgtgaaattt gtgatgctat
tgctttattt 480gtaaccatta taagctgcaa taaacaagtt aacaacaaca
attgcattca ttttatgttt 540caggttcagg gggaggtgtg ggaggttttt
taaagcaagt aaaacctcta caaatgtggt 600atggctgatt atgatcctct
agtacttctc gacaagctta cattattgaa gcatttatca 660gggttattgt
ctcagacctg caggcagctg cgcgctcgct cgctcactga ggccgcccgg
720gcaaagcccg ggcgtcgggc gacctttggt cgcccggcct cagtgagcga
gcgagcgcgc 780agagagggag tggccaactc catcactagg ggttccttgt
agttaatgat taacccgcca 840tgctacttat ctacgtagcc atgctctaga
gcggccgcac gcgtggagct agttattaat 900agtaatcaat tacggggtca
ttagttcata gcccatatat ggagttccgc gttacataac 960ttacggtaaa
tggcccgcct ggctgaccgc ccaacgaccc ccgcccattg acgtcaataa
1020tgacgtatgt tcccatagta acgtcaatag ggactttcca ttgacgtcaa
tgggtggagt 1080atttacggta aactgcccac ttggcagtac atcaagtgta
tcatatgcca agtacgcccc 1140ctattgacgt caatgacggt aaatggcccg
cctggcatta tgcccagtac atgaccttat 1200gggactttcc tacttggcag
tacatctacg tattagtcat cgctattacc atggtgatgc 1260ggttttggca
gtacatcaat gggcgtggat agcggtttga ctcacgggga tttccaagtc
1320tccaccccat tgacgtcaat gggagtttgt tttgcaccaa aatcaacggg
actttccaaa 1380atgtcgtaac aactccgccc cattgacgca aatgggcggt
aggcgtgtac ggtgggaggt 1440ctatataagc agagctcgtt tagtgaaccg
tcagatcgcc tggagacgcc atccacgctg 1500ttttgacctc catagaagac
accgggaccg atccagcctc cgcggattcg aatcccggcc 1560gggaacggtg
cattggaacg cggattcccc gtgccaagag tgacgtaagt accgcctata
1620gagtctatag gcccacaaaa aatgctttct tcttttaata tacttttttg
tttatcttat 1680ttctaatact ttccctaatc tctttctttc agggcaataa
tgatacaatg tatcatgcct 1740ctttgcacca ttctaaagaa taacagtgat
aatttctggg ttaaggcaat agcaatattt 1800ctgcatataa atatttctgc
atataaattg taactgatgt aagaggtttc atattgctaa 1860tagcagctac
aatccagcta ccattctgct tttattttat ggttgggata aggctggatt
1920attctgagtc caagctaggc ccttttgcta atcatgttca tacctcttat
cttcctccca 1980cagctcctgg gcaacgtgct ggtctgtgtg ctggcccatc
actttggcaa agaattggga 2040ttcgaacatc ggccgccacc atgaacgcaa
gtgaattccg aaggagaggg aaggagatgg 2100tggattacgt ggccaactac
atggaaggca ttgagggacg ccaggtctac cctgacgtgg 2160agcccgggta
cctgcggccg ctgatccctg ccgctgcccc tcaggagcca gacacgtttg
2220aggacatcat caacgacgtt gagaagataa tcatgcctgg ggtgacgcac
tggcacagcc 2280cctacttctt cgcctacttc cccactgcca gctcgtaccc
ggccatgctt gcggacatgc 2340tgtgcggggc cattggctgc atcggcttct
cctgggcggc aagcccagca tgcacagagc 2400tggagactgt gatgatggac
tggctcggga agatgctgga actaccaaag gcatttttga 2460atgagaaagc
tggagaaggg ggaggagtga tccagggaag tgccagtgaa gccaccctgg
2520tggccctgct ggccgctcgg accaaagtga tccatcggct gcaggcagcg
tccccagagc 2580tcacacaggc cgctatcatg gagaagctgg tggcttactc
atccgatcag gcacactcct 2640cagtggaaag agctgggtta attggtggag
tgaaattaaa agccatcccc tcagatggca 2700acttcgccat gcgtgcgtct
gccctgcagg aagccctgga gagagacaaa gcggctggcc 2760tgattccttt
ctttatggtt gccaccctgg ggaccacaac atgctgctcc tttgacaatc
2820tcttagaagt cggtcctatc tgcaacaagg aagacatatg gctgcacgtt
gatgcagcct 2880acgcaggcag tgcattcatc tgccctgagt tccggcacct
tctgaatgga gtggagtttg 2940cagattcatt caactttaat ccccacaaat
ggctattggt gaattttgac tgttctgcca 3000tgtgggtgaa aaagagaaca
gacttaacgg gagcctttag actggacccc acttacctga 3060agcacagcca
tcaggattca gggcttatca ctgactaccg gcattggcag ataccactgg
3120gcagaagatt tcgctctttg aaaatgtggt ttgtatttag gatgtatgga
gtcaaaggac 3180tgcaggctta tatccgcaag catgtccagc tgtcccatga
gtttgagtca ctggtgcgcc 3240aggatccccg ctttgaaatc tgtgtggaag
tcattctggg gcttgtctgc tttcggctaa 3300agggttccaa caaagtgaat
gaagctcttc tgcaaagaat aaacagtgcc aaaaaaatcc 3360acttggttcc
atgtcacctc agggacaagt ttgtcctgcg ctttgccatc tgttctcgca
3420cggtggaatc tgcccatgtg cagcgggcct gggaacacat caaagagctg
gcggccgacg 3480tgctgcgagc agagagggag taggagtgaa gccagctgca
ggaatcaaaa attgaagaga 3540gatatatctg aaaactggaa taagaagcaa
ataaatatca tcctgccttc atggaactca 3600gctgtctgtg gcttcccatg
tctttctcca aagttatcca gagggttgtg attttgtctg 3660cttagtatct
catcaacaaa gaaatattat ttgctaatta aaaagttaat cttcatggcc
3720atagctttta ttcattagct gtgatttttg ttgattaaaa cattatagat
tttcatgttc 3780ttgcagtcat cagaagtggt aggaaagcct cactgatata
ttttccaggg caatcaatgt 3840tcacgcaact tgaaattata tctgtggtct
tcaaattgtc ttttgtcatg tggctaaatg 3900cctaataagc tgctcgagag
atctacgggt ggcatccctg tgacccctcc ccagtgcctc 3960tcctggccct
ggaagttgcc actccagtgc ccaccagcct tgtcctaata aaattaagtt
4020gcatcatttt gtctgactag gtgtccttct ataatattat ggggtggagg
ggggtggtat 4080ggagcaaggg gcaagttggg aagacaacct gtagggcctg
cggggtctat tgggaaccaa 4140gctggagtgc agtggcacaa tcttggctca
ctgcaatctc cgcctcctgg gttcaagcga 4200ttctcctgcc tcagcctccc
gagttgttgg gattccaggc atgcatgacc aggctcagct 4260aatttttgtt
tttttggtag agacggggtt tcaccatatt ggccaggctg gtctccaact
4320cctaatctca ggtgatctac ccaccttggc ctcccaaatt gctgggatta
caggcgtgaa 4380ccactgctcc cttccctgtc cttctgattt tgtaggtaac
cgtaaccccg gaccacgtgc 4440ggaccgagcg gccgctctag agcatggcta
cgtagataag tagcatggcg ggttaatcat 4500taactacaag gaacccctag
tgatggagtt ggccactccc tctctgcgcg ctcgctcgct 4560cactgaggcc
gggcgaccaa aggtcgcccg acgcccgggc ggcctcagtg agcgagcgag
4620cgcgcagctg cctgcaggtc tgagacaata accctgataa atgcttcaat
aatgtagcca 4680accactagaa ctatagctag agtcctgggc gaacaaacga
tgctcgcctt ccagaaaacc 4740gaggatgcga accacttcat ccggggtcag
caccaccggc aagcgccgcg acggccgagg 4800tcttccgatc tcctgaagcc
agggcagatc cgtgcacagc accttgccgt agaagaacag 4860caaggccgcc
aatgcctgac gatgcgtgga gaccgaaacc ttgcgctcgt tcgccagcca
4920ggacagaaat gcctcgactt cgctgctgcc caaggttgcc gggtgacgca
caccgtggaa 4980acggatgaag gcacgaaccc agttgacata agcctgttcg
gttcgtaaac tgtaatgcaa 5040gtagcgtatg cgctcacgca actggtccag
aaccttgacc gaacgcagcg gtggtaacgg 5100cgcagtggcg gttttcatgg
cttgttatga ctgttttttt gtacagtcta tgcctcgggc 5160atccaagcag
caagcgcgtt acgccgtggg tcgatgtttg atgttatgga gcagcaacga
5220tgttacgcag cagcaacgat gttacgcagc agggcagtcg ccctaaaaca
aagttaggtg 5280gctcaagtat gggcatcatt cgcacatgta ggctcggccc
tgaccaagtc aaatccatgc 5340gggctgctct tgatcttttc ggtcgtgagt
tcggagacgt agccacctac tcccaacatc 5400agccggactc cgattacctc
gggaacttgc tccgtagtaa gacattcatc gcgcttgctg 5460ccttcgacca
agaagcggtt gttggcgctc tcgcggctta cgttctgccc aagtttgagc
5520agccgcgtag tgagatctat atctatgatc tcgcagtctc cggcgagcac
cggaggcagg 5580gcattgccac cgcgctcatc aatctcctca agcatgaggc
caacgcgctt ggtgcttatg 5640tgatctacgt gcaagcagat tacggtgacg
atcccgcagt ggctctctat acaaagttgg 5700gcatacggga agaagtgatg
cactttgata tcgacccaag taccgccacc taacaattcg 5760ttcaagccga
gatcggcttc ccggccgcgg agttgttcgg taaattgtca caacgccgcg
5820aatatagtct ttaccatgcc cttggccacg cccctcttta atacgacggg
caatttgcac 5880ttcagaaaat gaagagtttg ctttagccat aacaaaagtc
cagtatgctt tttcacagca 5940taactggact gatttcagtt tacaactatt
ctgtctagtt taagacttta ttgtcatagt 6000ttagatctat tttgttcagt
ttaagacttt attgtccgcc cacacccgct tacgcagggc 6060atccatttat
tactcaaccg taaccgattt tgccaggtta cgcggctggt ctatgcggtg
6120tgaaataccg cacagatgcg taaggagaaa ataccgcatc aggcgctctt
ccgcttcctc 6180gctcactgac tcgctgcgct cggtcgttcg gctgcggcga
gcggtatcag ctcactcaaa 6240ggcggtaata cggttatcca cagaatcagg
ggataacgca ggaaagaaca tgtgagcaaa 6300aggccagcaa aaggccagga
accgtaaaaa ggccgcgttg ctggcgtttt tccataggct 6360ccgcccccct
gacgagcatc acaaaaatcg acgctcaagt cagaggtggc gaaacccgac
6420aggactataa agataccagg cgtttccccc tggaagctcc ctcgtgcgct
ctcctgttcc 6480gaccctgccg cttaccggat acctgtccgc ctttctccct
tcgggaagcg tggcgctttc 6540tcatagctca cgctgtaggt atctcagttc
ggtgtaggtc gttcgctcca agctgggctg 6600tgtgcacgaa ccccccgttc
agcccgaccg ctgcgcctta tccggtaact atcgtcttga 6660gtccaacccg
gtaagacacg acttatcgcc actggcagca gccactggta acaggattag
6720cagagcgagg tatgtaggcg gtgctacaga gttcttgaag tggtggccta
actacggcta 6780cactagaaga acagtatttg gtatctgcgc tctgctgaag
ccagttacct tcggaaaaag 6840agttggtagc tcttgatccg gcaaacaaac
caccgctggt agcggtggtt tttttgtttg 6900caagcagcag attacgcgca
gaaaaaaagg atctcaagaa gatcctttga tcttttctac 6960ggggtctgac
gctcagtgga acgaaaactc acgttaaggg attttggtca tgagattatc
7020aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga agttttaaat
caatctaaag 7080tatatatgag taaacttggt ctgacagtta ccaatgctta
atcagtgagg cacctatctc 7140agcgatctgt ctatttcgtt catccatagt
tgcctgactc cccgtcgtgt agataactac 7200gatacgggag ggcttaccat
ctggccccag tgctgcaatg ataccgcgag acccacgctc 7260accggctcca
gatttatcag caataaacca gccagccgga agggccgagc gcagaagtgg
7320tcctgcaact ttatccgcct ccatccagtc tattaattgt tgccgggaag
ctagagtaag 7380tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt
gctacaggca tcgtggtgtc 7440acgctcgtcg tttggtatgg cttcattcag
ctccggttcc caacgatcaa ggcgagttac 7500atgatccccc atgttgtgca
aaaaagcggt tagctccttc ggtcctccga tcgttgtcag 7560aagtaagttg
gccgcagtgt tatcactcat ggttatggca gcactgcata attctcttac
7620tgtcatgcca tccgtaagat gcttttctgt gactggtgag tactcaacca
agtcattctg 7680agaatagtgt atgcggcgac cgagttgctc ttgcccggcg
tcaatacggg ataataccgc 7740gccacatagc agaactttaa aagtgctcat
cattggaaaa cgttcttcgg ggcgaaaact 7800ctcaaggatc ttaccgctgt
tgagatccag ttcgatgtaa cccactcgtg cacccaactg 7860atcttcagca
tcttttactt tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa
7920tgccgcaaaa aagggaataa gggcgacacg gaaatgttga atactcatac
tcttcctttt 7980tcaatattat tgaagcattt atcagggtta ttgtctcatg
agcggataca tatttgaatg 8040tatttagaaa aataaacaaa taggggttcc
gcgcacattt ccccgaaaag tgccacctaa 8100attgtaagcg ttaatatttt
gttaaaattc gcgttaaatt tttgttaaat cagctcattt 8160tttaaccaat
aggccgaaat cggcaaaatc ccttataaat caaaagaata gaccgagata
8220gggttgagtg ttgttccagt ttggaacaag agtccactat taaagaacgt
ggactccaac 8280gtcaaagggc gaaaaaccgt ctatcagggc gatggcccac
tacgtgaacc atcaccctaa 8340tcaagttttt tggggtcgag gtgccgtaaa
gcactaaatc ggaaccctaa agggagcccc 8400cgatttagag cttgacgggg
aaagccggcg aacgtggcga gaaaggaagg gaagaaagcg 8460aaaggagcgg
gcgctagggc gctggcaagt gtagcggtca cgctgcgcgt aaccaccaca
8520cccgccgcgc ttaatgcgcc gctacagggc gcgtc 855558694DNAArtificial
SequenceDescription of Artificial Sequence Synthetic polynucleotide
5cattcgccat tcaggctgca aataagcgtt gatattcagt caattacaaa cattaataac
60gaagagatga cagaaaaatt ttcattctgt gacagagaaa aagtagccga agatgacggt
120ttgtcacatg gagttggcag gatgtttgat taaaaacata acaggaagaa
aaatgccccg 180ctgtgggcgg acaaaatagt tgggaactgg gaggggtgga
aatggagttt ttaaggatta 240tttagggaag agtgacaaaa tagatgggaa
ctgggtgtag cgtcgtaagc taatacgaaa 300attaaaaatg acaaaatagt
ttggaactag atttcactta tctggttcgg atctcctagg 360cgatatcagt
gatcagatcc agacatgata agatacattg atgagtttgg acaaaccaca
420actagaatgc agtgaaaaaa atgctttatt tgtgaaattt gtgatgctat
tgctttattt 480gtaaccatta taagctgcaa taaacaagtt aacaacaaca
attgcattca ttttatgttt 540caggttcagg gggaggtgtg ggaggttttt
taaagcaagt aaaacctcta caaatgtggt 600atggctgatt atgatcctct
agtacttctc gacaagctta cattattgaa gcatttatca 660gggttattgt
ctcagacctg caggcagctg cgcgctcgct cgctcactga ggccgcccgg
720gcaaagcccg ggcgtcgggc gacctttggt cgcccggcct cagtgagcga
gcgagcgcgc 780agagagggag tggccaactc catcactagg ggttccttgt
agttaatgat taacccgcca 840tgctacttat ctacgtagcc atgctctaga
gcggccgcaa cgcgtaaact tcgtcgacga 900tctgcggccg cacgcgtgga
gctagttatt aatagtaatc aattacgggg tcattagttc 960atagcccata
tatggagttc cgcgttacat aacttacggt aaatggcccg cctggctgac
1020cgcccaacga cccccgccca ttgacgtcaa taatgacgta tgttcccata
gtaacgtcaa 1080tagggacttt ccattgacgt caatgggtgg agtatttacg
gtaaactgcc cacttggcag 1140tacatcaagt gtatcatatg ccaagtacgc
cccctattga cgtcaatgac ggtaaatggc 1200ccgcctggca ttatgcccag
tacatgacct tatgggactt tcctacttgg cagtacatct 1260acgtattagt
catcgctatt accatggtga tgcggttttg gcagtacatc aatgggcgtg
1320gatagcggtt tgactcacgg ggatttccaa gtctccaccc cattgacgtc
aatgggagtt 1380tgttttgcac caaaatcaac gggactttcc aaaatgtcgt
aacaactccg ccccattgac 1440gcaaatgggc ggtaggcgtg tacggtggga
ggtctatata agcagagctc gtttagtgaa 1500ccgtcagatc gcctggagac
gccatccacg ctgttttgac ctccatagaa gacaccggga 1560ccgatccagc
ctccgcggat tcgaatcccg gccgggaacg gtgcattgga acgcggattc
1620cccgtgccaa gagtgacgta agtaccgcct atagagtcta taggcccaca
aaaaatgctt 1680tcttctttta atatactttt ttgtttatct
tatttctaat actttcccta atctctttct 1740ttcagggcaa taatgataca
atgtatcatg cctctttgca ccattctaaa gaataacagt 1800gataatttct
gggttaaggc aatagcaata tttctgcata taaatatttc tgcatataaa
1860ttgtaactga tgtaagaggt ttcatattgc taatagcagc tacaatccag
ctaccattct 1920gcttttattt tatggttggg ataaggctgg attattctga
gtccaagcta ggcccttttg 1980ctaatcatgt tcatacctct tatcttcctc
ccacagctcc tgggcaacgt gctggtctgt 2040gtgctggccc atcactttgg
caaagaattg ggattcgaac atcgaattcg ggcacgaggg 2100aggacagaga
gcaagtcact cccggctgcc tttttcacct ctgacagagc ccagacacca
2160tgaacgcaag tgaattccga aggagaggga aggagatggt ggattacgtg
gccaactaca 2220tggaaggcat tgagggacgc caggtctacc ctgacgtgga
gcccgggtac ctgcggccgc 2280tgatccctgc cgctgcccct caggagccag
acacgtttga ggacatcatc aacgacgttg 2340agaagataat catgcctggg
gtgacgcact ggcacagccc ctacttcttc gcctacttcc 2400ccactgccag
ctcgtacccg gccatgcttg cggacatgct gtgcggggcc attggctgca
2460tcggcttctc ctgggcggca agcccagcat gcacagagct ggagactgtg
atgatggact 2520ggctcgggaa gatgctggaa ctaccaaagg catttttgaa
tgagaaagct ggagaagggg 2580gaggagtgat ccagggaagt gccagtgaag
ccaccctggt ggccctgctg gccgctcgga 2640ccaaagtgat ccatcggctg
caggcagcgt ccccagagct cacacaggcc gctatcatgg 2700agaagctggt
ggcttactca tccgatcagg cacactcctc agtggaaaga gctgggttaa
2760ttggtggagt gaaattaaaa gccatcccct cagatggcaa cttcgccatg
cgtgcgtctg 2820ccctgcagga agccctggag agagacaaag cggctggcct
gattcctttc tttatggttg 2880ccaccctggg gaccacaaca tgctgctcct
ttgacaatct cttagaagtc ggtcctatct 2940gcaacaagga agacatatgg
ctgcacgttg atgcagccta cgcaggcagt gcattcatct 3000gccctgagtt
ccggcacctt ctgaatggag tggagtttgc agattcattc aactttaatc
3060cccacaaatg gctattggtg aattttgact gttctgccat gtgggtgaaa
aagagaacag 3120acttaacggg agcctttaga ctggacccca cttacctgaa
gcacagccat caggattcag 3180ggcttatcac tgactaccgg cattggcaga
taccactggg cagaagattt cgctctttga 3240aaatgtggtt tgtatttagg
atgtatggag tcaaaggact gcaggcttat atccgcaagc 3300atgtccagct
gtcccatgag tttgagtcac tggtgcgcca ggatccccgc tttgaaatct
3360gtgtggaagt cattctgggg cttgtctgct ttcggctaaa gggttccaac
aaagtgaatg 3420aagctcttct gcaaagaata aacagtgcca aaaaaatcca
cttggttcca tgtcacctca 3480gggacaagtt tgtcctgcgc tttgccatct
gttctcgcac ggtggaatct gcccatgtgc 3540agcgggcctg ggaacacatc
aaagagctgg cggccgacgt gctgcgagca gagagggagt 3600aggagtgaag
ccagctgcag gaatcaaaaa ttgaagagag atatatctga aaactggaat
3660aagaagcaaa taaatatcat cctgccttca tggaactcag ctgtctgtgg
cttcccatgt 3720ctttctccaa agttatccag agggttgtga ttttgtctgc
ttagtatctc atcaacaaag 3780aaatattatt tgctaattaa aaagttaatc
ttcatggcca tagcttttat tcattagctg 3840tgatttttgt tgattaaaac
attatagatt ttcatgttct tgcagtcatc agaagtggta 3900ggaaagcctc
actgatatat tttccagggc aatcaatgtt cacgcaactt gaaattatat
3960ctgtggtctt caaattgtct tttgtcatgt ggctaaatgc ctaataaaca
attcaagtga 4020aatactaaaa aaaaaaaaaa aaaaaaaagc tgctcgagag
atctacgggt ggcatccctg 4080tgacccctcc ccagtgcctc tcctggccct
ggaagttgcc actccagtgc ccaccagcct 4140tgtcctaata aaattaagtt
gcatcatttt gtctgactag gtgtccttct ataatattat 4200ggggtggagg
ggggtggtat ggagcaaggg gcaagttggg aagacaacct gtagggcctg
4260cggggtctat tgggaaccaa gctggagtgc agtggcacaa tcttggctca
ctgcaatctc 4320cgcctcctgg gttcaagcga ttctcctgcc tcagcctccc
gagttgttgg gattccaggc 4380atgcatgacc aggctcagct aatttttgtt
tttttggtag agacggggtt tcaccatatt 4440ggccaggctg gtctccaact
cctaatctca ggtgatctac ccaccttggc ctcccaaatt 4500gctgggatta
caggcgtgaa ccactgctcc cttccctgtc cttctgattt tgtaggtaac
4560gtaaccccgg accacgtgcg gaccgagcgg ccgctctaga gcatggctac
gtagataagt 4620agcatggcgg gttaatcatt aactacaagg aacccctagt
gatggagttg gccactccct 4680ctctgcgcgc tcgctcgctc actgaggccg
ggcgaccaaa ggtcgcccga cgcccgggcg 4740gcctcagtga gcgagcgagc
gcgcagctgc ctgcaggtct gagacaataa ccctgataaa 4800tgcttcaata
atgtagccaa ccactagaac tatagctaga gtcctgggcg aacaaacgat
4860gctcgccttc cagaaaaccg aggatgcgaa ccacttcatc cggggtcagc
accaccggca 4920agcgccgcga cggccgaggt cttccgatct cctgaagcca
gggcagatcc gtgcacagca 4980ccttgccgta gaagaacagc aaggccgcca
atgcctgacg atgcgtggag accgaaacct 5040tgcgctcgtt cgccagccag
gacagaaatg cctcgacttc gctgctgccc aaggttgccg 5100ggtgacgcac
accgtggaaa cggatgaagg cacgaaccca gttgacataa gcctgttcgg
5160ttcgtaaact gtaatgcaag tagcgtatgc gctcacgcaa ctggtccaga
accttgaccg 5220aacgcagcgg tggtaacggc gcagtggcgg ttttcatggc
ttgttatgac tgtttttttg 5280tacagtctat gcctcgggca tccaagcagc
aagcgcgtta cgccgtgggt cgatgtttga 5340tgttatggag cagcaacgat
gttacgcagc agcaacgatg ttacgcagca gggcagtcgc 5400cctaaaacaa
agttaggtgg ctcaagtatg ggcatcattc gcacatgtag gctcggccct
5460gaccaagtca aatccatgcg ggctgctctt gatcttttcg gtcgtgagtt
cggagacgta 5520gccacctact cccaacatca gccggactcc gattacctcg
ggaacttgct ccgtagtaag 5580acattcatcg cgcttgctgc cttcgaccaa
gaagcggttg ttggcgctct cgcggcttac 5640gttctgccca agtttgagca
gccgcgtagt gagatctata tctatgatct cgcagtctcc 5700ggcgagcacc
ggaggcaggg cattgccacc gcgctcatca atctcctcaa gcatgaggcc
5760aacgcgcttg gtgcttatgt gatctacgtg caagcagatt acggtgacga
tcccgcagtg 5820gctctctata caaagttggg catacgggaa gaagtgatgc
actttgatat cgacccaagt 5880accgccacct aacaattcgt tcaagccgag
atcggcttcc cggccgcgga gttgttcggt 5940aaattgtcac aacgccgcga
atatagtctt taccatgccc ttggccacgc ccctctttaa 6000tacgacgggc
aatttgcact tcagaaaatg aagagtttgc tttagccata acaaaagtcc
6060agtatgcttt ttcacagcat aactggactg atttcagttt acaactattc
tgtctagttt 6120aagactttat tgtcatagtt tagatctatt ttgttcagtt
taagacttta ttgtccgccc 6180acacccgctt acgcagggca tccatttatt
actcaaccgt aaccgatttt gccaggttac 6240gcggctggtc tatgcggtgt
gaaataccgc acagatgcgt aaggagaaaa taccgcatca 6300ggcgctcttc
cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag
6360cggtatcagc tcactcaaag gcggtaatac ggttatccac agaatcaggg
gataacgcag 6420gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa
ccgtaaaaag gccgcgttgc 6480tggcgttttt ccataggctc cgcccccctg
acgagcatca caaaaatcga cgctcaagtc 6540agaggtggcg aaacccgaca
ggactataaa gataccaggc gtttccccct ggaagctccc 6600tcgtgcgctc
tcctgttccg accctgccgc ttaccggata cctgtccgcc tttctccctt
6660cgggaagcgt ggcgctttct catagctcac gctgtaggta tctcagttcg
gtgtaggtcg 6720ttcgctccaa gctgggctgt gtgcacgaac cccccgttca
gcccgaccgc tgcgccttat 6780ccggtaacta tcgtcttgag tccaacccgg
taagacacga cttatcgcca ctggcagcag 6840ccactggtaa caggattagc
agagcgaggt atgtaggcgg tgctacagag ttcttgaagt 6900ggtggcctaa
ctacggctac actagaagaa cagtatttgg tatctgcgct ctgctgaagc
6960cagttacctt cggaaaaaga gttggtagct cttgatccgg caaacaaacc
accgctggta 7020gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag
aaaaaaagga tctcaagaag 7080atcctttgat cttttctacg gggtctgacg
ctcagtggaa cgaaaactca cgttaaggga 7140ttttggtcat gagattatca
aaaaggatct tcacctagat ccttttaaat taaaaatgaa 7200gttttaaatc
aatctaaagt atatatgagt aaacttggtc tgacagttac caatgcttaa
7260tcagtgaggc acctatctca gcgatctgtc tatttcgttc atccatagtt
gcctgactcc 7320ccgtcgtgta gataactacg atacgggagg gcttaccatc
tggccccagt gctgcaatga 7380taccgcgaga cccacgctca ccggctccag
atttatcagc aataaaccag ccagccggaa 7440gggccgagcg cagaagtggt
cctgcaactt tatccgcctc catccagtct attaattgtt 7500gccgggaagc
tagagtaagt agttcgccag ttaatagttt gcgcaacgtt gttgccattg
7560ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc
tccggttccc 7620aacgatcaag gcgagttaca tgatccccca tgttgtgcaa
aaaagcggtt agctccttcg 7680gtcctccgat cgttgtcaga agtaagttgg
ccgcagtgtt atcactcatg gttatggcag 7740cactgcataa ttctcttact
gtcatgccat ccgtaagatg cttttctgtg actggtgagt 7800actcaaccaa
gtcattctga gaatagtgta tgcggcgacc gagttgctct tgcccggcgt
7860caatacggga taataccgcg ccacatagca gaactttaaa agtgctcatc
attggaaaac 7920gttcttcggg gcgaaaactc tcaaggatct taccgctgtt
gagatccagt tcgatgtaac 7980ccactcgtgc acccaactga tcttcagcat
cttttacttt caccagcgtt tctgggtgag 8040caaaaacagg aaggcaaaat
gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa 8100tactcatact
cttccttttt caatattatt gaagcattta tcagggttat tgtctcatga
8160gcggatacat atttgaatgt atttagaaaa ataaacaaat aggggttccg
cgcacatttc 8220cccgaaaagt gccacctaaa ttgtaagcgt taatattttg
ttaaaattcg cgttaaattt 8280ttgttaaatc agctcatttt ttaaccaata
ggccgaaatc ggcaaaatcc cttataaatc 8340aaaagaatag accgagatag
ggttgagtgt tgttccagtt tggaacaaga gtccactatt 8400aaagaacgtg
gactccaacg tcaaagggcg aaaaaccgtc tatcagggcg atggcccact
8460acgtgaacca tcaccctaat caagtttttt ggggtcgagg tgccgtaaag
cactaaatcg 8520gaaccctaaa gggagccccc gatttagagc ttgacgggga
aagccggcga acgtggcgag 8580aaaggaaggg aagaaagcga aaggagcggg
cgctagggcg ctggcaagtg tagcggtcac 8640gctgcgcgta accaccacac
ccgccgcgct taatgcgccg ctacagggcg cgtc 869463535DNAArtificial
SequenceDescription of Artificial Sequence Synthetic polynucleotide
6ctgcgcgctc gctcgctcac tgaggccgcc cgggcaaagc ccgggcgtcg ggcgaccttt
60ggtcgcccgg cctcagtgag cgagcgagcg cgcagagagg gagtggccaa ctccatcact
120aggggttcct tgtagttaat gattaacccg ccatgctact tatctacgta
gccatgctct 180agagcggccg cacgcgtgga gctagttatt aatagtaatc
aattacgggg tcattagttc 240atagcccata tatggagttc cgcgttacat
aacttacggt aaatggcccg cctggctgac 300cgcccaacga cccccgccca
ttgacgtcaa taatgacgta tgttcccata gtaacgtcaa 360tagggacttt
ccattgacgt caatgggtgg agtatttacg gtaaactgcc cacttggcag
420tacatcaagt gtatcatatg ccaagtacgc cccctattga cgtcaatgac
ggtaaatggc 480ccgcctggca ttatgcccag tacatgacct tatgggactt
tcctacttgg cagtacatct 540acgtattagt catcgctatt accatggtga
tgcggttttg gcagtacatc aatgggcgtg 600gatagcggtt tgactcacgg
ggatttccaa gtctccaccc cattgacgtc aatgggagtt 660tgttttgcac
caaaatcaac gggactttcc aaaatgtcgt aacaactccg ccccattgac
720gcaaatgggc ggtaggcgtg tacggtggga ggtctatata agcagagctc
gtttagtgaa 780ccgtcagatc gcctggagac gccatccacg ctgttttgac
ctccatagaa gacaccggga 840ccgatccagc ctccgcggat tcgaatcccg
gccgggaacg gtgcattgga acgcggattc 900cccgtgccaa gagtgacgta
agtaccgcct atagagtcta taggcccaca aaaaatgctt 960tcttctttta
atatactttt ttgtttatct tatttctaat actttcccta atctctttct
1020ttcagggcaa taatgataca atgtatcatg cctctttgca ccattctaaa
gaataacagt 1080gataatttct gggttaaggc aatagcaata tttctgcata
taaatatttc tgcatataaa 1140ttgtaactga tgtaagaggt ttcatattgc
taatagcagc tacaatccag ctaccattct 1200gcttttattt tatggttggg
ataaggctgg attattctga gtccaagcta ggcccttttg 1260ctaatcatgt
tcatacctct tatcttcctc ccacagctcc tgggcaacgt gctggtctgt
1320gtgctggccc atcactttgg caaagaattg ggattcgaac atcggccgcc
accatgaacg 1380caagtgaatt ccgaaggaga gggaaggaga tggtggatta
cgtggccaac tacatggaag 1440gcattgaggg acgccaggtc taccctgacg
tggagcccgg gtacctgcgg ccgctgatcc 1500ctgccgctgc ccctcaggag
ccagacacgt ttgaggacat catcaacgac gttgagaaga 1560taatcatgcc
tggggtgacg cactggcaca gcccctactt cttcgcctac ttccccactg
1620ccagctcgta cccggccatg cttgcggaca tgctgtgcgg ggccattggc
tgcatcggct 1680tctcctgggc ggcaagccca gcatgcacag agctggagac
tgtgatgatg gactggctcg 1740ggaagatgct ggaactacca aaggcatttt
tgaatgagaa agctggagaa gggggaggag 1800tgatccaggg aagtgccagt
gaagccaccc tggtggccct gctggccgct cggaccaaag 1860tgatccatcg
gctgcaggca gcgtccccag agctcacaca ggccgctatc atggagaagc
1920tggtggctta ctcatccgat caggcacact cctcagtgga aagagctggg
ttaattggtg 1980gagtgaaatt aaaagccatc ccctcagatg gcaacttcgc
catgcgtgcg tctgccctgc 2040aggaagccct ggagagagac aaagcggctg
gcctgattcc tttctttatg gttgccaccc 2100tggggaccac aacatgctgc
tcctttgaca atctcttaga agtcggtcct atctgcaaca 2160aggaagacat
atggctgcac gttgatgcag cctacgcagg cagtgcattc atctgccctg
2220agttccggca ccttctgaat ggagtggagt ttgcagattc attcaacttt
aatccccaca 2280aatggctatt ggtgaatttt gactgttctg ccatgtgggt
gaaaaagaga acagacttaa 2340cgggagcctt tagactggac cccacttacc
tgaagcacag ccatcaggat tcagggctta 2400tcactgacta ccggcattgg
cagataccac tgggcagaag atttcgctct ttgaaaatgt 2460ggtttgtatt
taggatgtat ggagtcaaag gactgcaggc ttatatccgc aagcatgtcc
2520agctgtccca tgagtttgag tcactggtgc gccaggatcc ccgctttgaa
atctgtgtgg 2580aagtcattct ggggcttgtc tgctttcggc taaagggttc
caacaaagtg aatgaagctc 2640ttctgcaaag aataaacagt gccaaaaaaa
tccacttggt tccatgtcac ctcagggaca 2700agtttgtcct gcgctttgcc
atctgttctc gcacggtgga atctgcccat gtgcagcggg 2760cctgggaaca
catcaaagag ctggcggccg acgtgctgcg agcagagagg gagtaggagt
2820gagctgctcg agagatctac gggtggcatc cctgtgaccc ctccccagtg
cctctcctgg 2880ccctggaagt tgccactcca gtgcccacca gccttgtcct
aataaaatta agttgcatca 2940ttttgtctga ctaggtgtcc ttctataata
ttatggggtg gaggggggtg gtatggagca 3000aggggcaagt tgggaagaca
acctgtaggg cctgcggggt ctattgggaa ccaagctgga 3060gtgcagtggc
acaatcttgg ctcactgcaa tctccgcctc ctgggttcaa gcgattctcc
3120tgcctcagcc tcccgagttg ttgggattcc aggcatgcat gaccaggctc
agctaatttt 3180tgtttttttg gtagagacgg ggtttcacca tattggccag
gctggtctcc aactcctaat 3240ctcaggtgat ctacccacct tggcctccca
aattgctggg attacaggcg tgaaccactg 3300ctcccttccc tgtccttctg
attttgtagg taaccccgga ccacgtgcgg accgagcggc 3360cgctctagag
catggctacg tagataagta gcatggcggg ttaatcatta actacaagga
3420acccctagtg atggagttgg ccactccctc tctgcgcgct cgctcgctca
ctgaggccgg 3480gcgaccaaag gtcgcccgac gcccgggcgg cctcagtgag
cgagcgagcg cgcag 353573534DNAArtificial SequenceDescription of
Artificial Sequence Synthetic polynucleotide 7ctgcgcgctc gctcgctcac
tgaggccgcc cgggcaaagc ccgggcgtcg ggcgaccttt 60ggtcgcccgg cctcagtgag
cgagcgagcg cgcagagagg gagtggccaa ctccatcact 120aggggttcct
tgtagttaat gattaacccg ccatgctact tatctacgta gccatgctct
180agagcggccg cacgcgtgga gctagttatt aatagtaatc aattacgggg
tcattagttc 240atagcccata tatggagttc cgcgttacat aacttacggt
aaatggcccg cctggctgac 300cgcccaacga cccccgccca ttgacgtcaa
taatgacgta tgttcccata gtaacgtcaa 360tagggacttt ccattgacgt
caatgggtgg agtatttacg gtaaactgcc cacttggcag 420tacatcaagt
gtatcatatg ccaagtacgc cccctattga cgtcaatgac ggtaaatggc
480ccgcctggca ttatgcccag tacatgacct tatgggactt tcctacttgg
cagtacatct 540acgtattagt catcgctatt accatggtga tgcggttttg
gcagtacatc aatgggcgtg 600gatagcggtt tgactcacgg ggatttccaa
gtctccaccc cattgacgtc aatgggagtt 660tgttttgcac caaaatcaac
gggactttcc aaaatgtcgt aacaactccg ccccattgac 720gcaaatgggc
ggtaggcgtg tacggtggga ggtctatata agcagagctc gtttagtgaa
780ccgtcagatc gcctggagac gccatccacg ctgttttgac ctccatagaa
gacaccggga 840ccgatccagc ctccgcggat tcgaatcccg gccgggaacg
gtgcattgga acgcggattc 900cccgtgccaa gagtgacgta agtaccgcct
atagagtcta taggcccaca aaaaatgctt 960tcttctttta atatactttt
ttgtttatct tatttctaat actttcccta atctctttct 1020ttcagggcaa
taatgataca atgtatcatg cctctttgca ccattctaaa gaataacagt
1080gataatttct gggttaaggc aatagcaata tttctgcata taaatatttc
tgcatataaa 1140ttgtaactga tgtaagaggt ttcatattgc taatagcagc
tacaatccag ctaccattct 1200gcttttattt tatggttggg ataaggctgg
attattctga gtccaagcta ggcccttttg 1260ctaatcatgt tcatacctct
tatcttcctc ccacagctcc tgggcaacgt gctggtctgt 1320gtgctggccc
atcactttgg caaagaattg ggattcgaac atcggccgcc accatgaacg
1380ccagcgagtt caggaggagg ggcaaggaga tggtggacta cgtggccaac
tacatggagg 1440gcatcgaggg caggcaggtg taccccgacg tggagcccgg
ctacctgagg cccctgatcc 1500ccgccgccgc cccccaggag cccgacacct
tcgaggacat catcaacgac gtggagaaga 1560tcatcatgcc cggcgtgacc
cactggcaca gcccctactt cttcgcctac ttccccaccg 1620ccagcagcta
ccccgccatg ctggccgaca tgctgtgcgg cgccatcggc tgcatcggct
1680tcagctgggc cgccagcccc gcctgcaccg agctggagac cgtgatgatg
gactggctgg 1740gcaagatgct ggagctgccc aaggccttcc tgaacgagaa
ggccggcgag ggcggcggcg 1800tgatccaggg cagcgccagc gaggccaccc
tggtggccct gctggccgcc aggaccaagg 1860tgatccacag gctgcaggcc
gccagccccg agctgaccca ggccgccatc atggagaagc 1920tggtggccta
cagcagcgac caggcccaca gcagcgtgga gagggccggc ctgatcggcg
1980gcgtgaagct gaaggccatc cccagcgacg gcaacttcgc catgagggcc
agcgccctgc 2040aggaggccct ggagagggac aaggccgccg gcctgatccc
cttcttcatg gtggccaccc 2100tgggcaccac cacctgctgc agcttcgaca
acctgctgga ggtgggcccc atctgcaaca 2160aggaggacat ctggctgcac
gtggacgccg cctacgccgg cagcgccttc atctgccccg 2220agttcaggca
cctgctgaac ggcgtggagt tcgccgacag cttcaacttc aacccccaca
2280agtggctgct ggtgaacttc gactgcagcg ccatgtgggt gaagaagagg
accgacctga 2340ccggcgcctt caggctggac cccacctacc tgaagcacag
ccaccaggac agcggcctga 2400tcaccgacta caggcactgg cagatccccc
tgggcaggag gttcaggagc ctgaagatgt 2460ggttcgtgtt caggatgtac
ggcgtgaagg gcctgcaggc ctacatcagg aagcacgtgc 2520agctgagcca
cgagttcgag agcctggtga ggcaggaccc caggttcgag atctgcgtgg
2580aggtgatcct gggcctggtg tgcttcaggc tgaagggcag caacaaggtg
aacgaggccc 2640tgctgcagag gatcaacagc gccaagaaga tccacctggt
gccctgccac ctgagggaca 2700agttcgtgct gaggttcgcc atctgcagca
ggaccgtgga gagcgcccac gtgcagaggg 2760cctgggagca catcaaggag
ctggccgccg acgtgctgag ggccgagagg gagtgagctg 2820ctcgagagat
ctacgggtgg catccctgtg acccctcccc agtgcctctc ctggccctgg
2880aagttgccac tccagtgccc accagccttg tcctaataaa attaagttgc
atcattttgt 2940ctgactaggt gtccttctat aatattatgg ggtggagggg
ggtggtatgg agcaaggggc 3000aagttgggaa gacaacctgt agggcctgcg
gggtctattg ggaaccaagc tggagtgcag 3060tggcacaatc ttggctcact
gcaatctccg cctcctgggt tcaagcgatt ctcctgcctc 3120agcctcccga
gttgttggga ttccaggcat gcatgaccag gctcagctaa tttttgtttt
3180tttggtagag acggggtttc accatattgg ccaggctggt ctccaactcc
taatctcagg 3240tgatctaccc accttggcct cccaaattgc tgggattaca
ggcgtgaacc actgctccct 3300tccctgtcct tctgattttg taggtaacgt
aaccccggac cacgtgcgga ccgagcggcc 3360gctctagagc atggctacgt
agataagtag catggcgggt taatcattaa ctacaaggaa 3420cccctagtga
tggagttggc cactccctct ctgcgcgctc gctcgctcac tgaggccggg
3480cgaccaaagg tcgcccgacg cccgggcggc ctcagtgagc gagcgagcgc gcag
353483940DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 8ctgcgcgctc gctcgctcac tgaggccgcc
cgggcaaagc ccgggcgtcg ggcgaccttt 60ggtcgcccgg cctcagtgag cgagcgagcg
cgcagagagg gagtggccaa ctccatcact 120aggggttcct tgtagttaat
gattaacccg ccatgctact tatctacgta gccatgctct 180agagcggccg
cacgcgtgga gctagttatt aatagtaatc aattacgggg tcattagttc
240atagcccata tatggagttc cgcgttacat aacttacggt aaatggcccg
cctggctgac 300cgcccaacga cccccgccca ttgacgtcaa taatgacgta
tgttcccata gtaacgtcaa 360tagggacttt ccattgacgt caatgggtgg
agtatttacg gtaaactgcc cacttggcag 420tacatcaagt gtatcatatg
ccaagtacgc cccctattga cgtcaatgac ggtaaatggc 480ccgcctggca
ttatgcccag tacatgacct tatgggactt tcctacttgg cagtacatct
540acgtattagt catcgctatt accatggtga tgcggttttg gcagtacatc
aatgggcgtg 600gatagcggtt tgactcacgg ggatttccaa gtctccaccc
cattgacgtc aatgggagtt 660tgttttgcac caaaatcaac gggactttcc
aaaatgtcgt aacaactccg ccccattgac
720gcaaatgggc ggtaggcgtg tacggtggga ggtctatata agcagagctc
gtttagtgaa 780ccgtcagatc gcctggagac gccatccacg ctgttttgac
ctccatagaa gacaccggga 840ccgatccagc ctccgcggat tcgaatcccg
gccgggaacg gtgcattgga acgcggattc 900cccgtgccaa gagtgacgta
agtaccgcct atagagtcta taggcccaca aaaaatgctt 960tcttctttta
atatactttt ttgtttatct tatttctaat actttcccta atctctttct
1020ttcagggcaa taatgataca atgtatcatg cctctttgca ccattctaaa
gaataacagt 1080gataatttct gggttaaggc aatagcaata tttctgcata
taaatatttc tgcatataaa 1140ttgtaactga tgtaagaggt ttcatattgc
taatagcagc tacaatccag ctaccattct 1200gcttttattt tatggttggg
ataaggctgg attattctga gtccaagcta ggcccttttg 1260ctaatcatgt
tcatacctct tatcttcctc ccacagctcc tgggcaacgt gctggtctgt
1320gtgctggccc atcactttgg caaagaattg ggattcgaac atcggccgcc
accatgaacg 1380caagtgaatt ccgaaggaga gggaaggaga tggtggatta
cgtggccaac tacatggaag 1440gcattgaggg acgccaggtc taccctgacg
tggagcccgg gtacctgcgg ccgctgatcc 1500ctgccgctgc ccctcaggag
ccagacacgt ttgaggacat catcaacgac gttgagaaga 1560taatcatgcc
tggggtgacg cactggcaca gcccctactt cttcgcctac ttccccactg
1620ccagctcgta cccggccatg cttgcggaca tgctgtgcgg ggccattggc
tgcatcggct 1680tctcctgggc ggcaagccca gcatgcacag agctggagac
tgtgatgatg gactggctcg 1740ggaagatgct ggaactacca aaggcatttt
tgaatgagaa agctggagaa gggggaggag 1800tgatccaggg aagtgccagt
gaagccaccc tggtggccct gctggccgct cggaccaaag 1860tgatccatcg
gctgcaggca gcgtccccag agctcacaca ggccgctatc atggagaagc
1920tggtggctta ctcatccgat caggcacact cctcagtgga aagagctggg
ttaattggtg 1980gagtgaaatt aaaagccatc ccctcagatg gcaacttcgc
catgcgtgcg tctgccctgc 2040aggaagccct ggagagagac aaagcggctg
gcctgattcc tttctttatg gttgccaccc 2100tggggaccac aacatgctgc
tcctttgaca atctcttaga agtcggtcct atctgcaaca 2160aggaagacat
atggctgcac gttgatgcag cctacgcagg cagtgcattc atctgccctg
2220agttccggca ccttctgaat ggagtggagt ttgcagattc attcaacttt
aatccccaca 2280aatggctatt ggtgaatttt gactgttctg ccatgtgggt
gaaaaagaga acagacttaa 2340cgggagcctt tagactggac cccacttacc
tgaagcacag ccatcaggat tcagggctta 2400tcactgacta ccggcattgg
cagataccac tgggcagaag atttcgctct ttgaaaatgt 2460ggtttgtatt
taggatgtat ggagtcaaag gactgcaggc ttatatccgc aagcatgtcc
2520agctgtccca tgagtttgag tcactggtgc gccaggatcc ccgctttgaa
atctgtgtgg 2580aagtcattct ggggcttgtc tgctttcggc taaagggttc
caacaaagtg aatgaagctc 2640ttctgcaaag aataaacagt gccaaaaaaa
tccacttggt tccatgtcac ctcagggaca 2700agtttgtcct gcgctttgcc
atctgttctc gcacggtgga atctgcccat gtgcagcggg 2760cctgggaaca
catcaaagag ctggcggccg acgtgctgcg agcagagagg gagtaggagt
2820gaagccagct gcaggaatca aaaattgaag agagatatat ctgaaaactg
gaataagaag 2880caaataaata tcatcctgcc ttcatggaac tcagctgtct
gtggcttccc atgtctttct 2940ccaaagttat ccagagggtt gtgattttgt
ctgcttagta tctcatcaac aaagaaatat 3000tatttgctaa ttaaaaagtt
aatcttcatg gccatagctt ttattcatta gctgtgattt 3060ttgttgatta
aaacattata gattttcatg ttcttgcagt catcagaagt ggtaggaaag
3120cctcactgat atattttcca gggcaatcaa tgttcacgca acttgaaatt
atatctgtgg 3180tcttcaaatt gtcttttgtc atgtggctaa atgcctaata
agctgctcga gagatctacg 3240ggtggcatcc ctgtgacccc tccccagtgc
ctctcctggc cctggaagtt gccactccag 3300tgcccaccag ccttgtccta
ataaaattaa gttgcatcat tttgtctgac taggtgtcct 3360tctataatat
tatggggtgg aggggggtgg tatggagcaa ggggcaagtt gggaagacaa
3420cctgtagggc ctgcggggtc tattgggaac caagctggag tgcagtggca
caatcttggc 3480tcactgcaat ctccgcctcc tgggttcaag cgattctcct
gcctcagcct cccgagttgt 3540tgggattcca ggcatgcatg accaggctca
gctaattttt gtttttttgg tagagacggg 3600gtttcaccat attggccagg
ctggtctcca actcctaatc tcaggtgatc tacccacctt 3660ggcctcccaa
attgctggga ttacaggcgt gaaccactgc tcccttccct gtccttctga
3720ttttgtaggt aaccgtaacc ccggaccacg tgcggaccga gcggccgctc
tagagcatgg 3780ctacgtagat aagtagcatg gcgggttaat cattaactac
aaggaacccc tagtgatgga 3840gttggccact ccctctctgc gcgctcgctc
gctcactgag gccgggcgac caaaggtcgc 3900ccgacgcccg ggcggcctca
gtgagcgagc gagcgcgcag 394094079DNAArtificial SequenceDescription of
Artificial Sequence Synthetic polynucleotide 9ctgcgcgctc gctcgctcac
tgaggccgcc cgggcaaagc ccgggcgtcg ggcgaccttt 60ggtcgcccgg cctcagtgag
cgagcgagcg cgcagagagg gagtggccaa ctccatcact 120aggggttcct
tgtagttaat gattaacccg ccatgctact tatctacgta gccatgctct
180agagcggccg caacgcgtaa acttcgtcga cgatctgcgg ccgcacgcgt
ggagctagtt 240attaatagta atcaattacg gggtcattag ttcatagccc
atatatggag ttccgcgtta 300cataacttac ggtaaatggc ccgcctggct
gaccgcccaa cgacccccgc ccattgacgt 360caataatgac gtatgttccc
atagtaacgt caatagggac tttccattga cgtcaatggg 420tggagtattt
acggtaaact gcccacttgg cagtacatca agtgtatcat atgccaagta
480cgccccctat tgacgtcaat gacggtaaat ggcccgcctg gcattatgcc
cagtacatga 540ccttatggga ctttcctact tggcagtaca tctacgtatt
agtcatcgct attaccatgg 600tgatgcggtt ttggcagtac atcaatgggc
gtggatagcg gtttgactca cggggatttc 660caagtctcca ccccattgac
gtcaatggga gtttgttttg caccaaaatc aacgggactt 720tccaaaatgt
cgtaacaact ccgccccatt gacgcaaatg ggcggtaggc gtgtacggtg
780ggaggtctat ataagcagag ctcgtttagt gaaccgtcag atcgcctgga
gacgccatcc 840acgctgtttt gacctccata gaagacaccg ggaccgatcc
agcctccgcg gattcgaatc 900ccggccggga acggtgcatt ggaacgcgga
ttccccgtgc caagagtgac gtaagtaccg 960cctatagagt ctataggccc
acaaaaaatg ctttcttctt ttaatatact tttttgttta 1020tcttatttct
aatactttcc ctaatctctt tctttcaggg caataatgat acaatgtatc
1080atgcctcttt gcaccattct aaagaataac agtgataatt tctgggttaa
ggcaatagca 1140atatttctgc atataaatat ttctgcatat aaattgtaac
tgatgtaaga ggtttcatat 1200tgctaatagc agctacaatc cagctaccat
tctgctttta ttttatggtt gggataaggc 1260tggattattc tgagtccaag
ctaggccctt ttgctaatca tgttcatacc tcttatcttc 1320ctcccacagc
tcctgggcaa cgtgctggtc tgtgtgctgg cccatcactt tggcaaagaa
1380ttgggattcg aacatcgaat tcgggcacga gggaggacag agagcaagtc
actcccggct 1440gcctttttca cctctgacag agcccagaca ccatgaacgc
aagtgaattc cgaaggagag 1500ggaaggagat ggtggattac gtggccaact
acatggaagg cattgaggga cgccaggtct 1560accctgacgt ggagcccggg
tacctgcggc cgctgatccc tgccgctgcc cctcaggagc 1620cagacacgtt
tgaggacatc atcaacgacg ttgagaagat aatcatgcct ggggtgacgc
1680actggcacag cccctacttc ttcgcctact tccccactgc cagctcgtac
ccggccatgc 1740ttgcggacat gctgtgcggg gccattggct gcatcggctt
ctcctgggcg gcaagcccag 1800catgcacaga gctggagact gtgatgatgg
actggctcgg gaagatgctg gaactaccaa 1860aggcattttt gaatgagaaa
gctggagaag ggggaggagt gatccaggga agtgccagtg 1920aagccaccct
ggtggccctg ctggccgctc ggaccaaagt gatccatcgg ctgcaggcag
1980cgtccccaga gctcacacag gccgctatca tggagaagct ggtggcttac
tcatccgatc 2040aggcacactc ctcagtggaa agagctgggt taattggtgg
agtgaaatta aaagccatcc 2100cctcagatgg caacttcgcc atgcgtgcgt
ctgccctgca ggaagccctg gagagagaca 2160aagcggctgg cctgattcct
ttctttatgg ttgccaccct ggggaccaca acatgctgct 2220cctttgacaa
tctcttagaa gtcggtccta tctgcaacaa ggaagacata tggctgcacg
2280ttgatgcagc ctacgcaggc agtgcattca tctgccctga gttccggcac
cttctgaatg 2340gagtggagtt tgcagattca ttcaacttta atccccacaa
atggctattg gtgaattttg 2400actgttctgc catgtgggtg aaaaagagaa
cagacttaac gggagccttt agactggacc 2460ccacttacct gaagcacagc
catcaggatt cagggcttat cactgactac cggcattggc 2520agataccact
gggcagaaga tttcgctctt tgaaaatgtg gtttgtattt aggatgtatg
2580gagtcaaagg actgcaggct tatatccgca agcatgtcca gctgtcccat
gagtttgagt 2640cactggtgcg ccaggatccc cgctttgaaa tctgtgtgga
agtcattctg gggcttgtct 2700gctttcggct aaagggttcc aacaaagtga
atgaagctct tctgcaaaga ataaacagtg 2760ccaaaaaaat ccacttggtt
ccatgtcacc tcagggacaa gtttgtcctg cgctttgcca 2820tctgttctcg
cacggtggaa tctgcccatg tgcagcgggc ctgggaacac atcaaagagc
2880tggcggccga cgtgctgcga gcagagaggg agtaggagtg aagccagctg
caggaatcaa 2940aaattgaaga gagatatatc tgaaaactgg aataagaagc
aaataaatat catcctgcct 3000tcatggaact cagctgtctg tggcttccca
tgtctttctc caaagttatc cagagggttg 3060tgattttgtc tgcttagtat
ctcatcaaca aagaaatatt atttgctaat taaaaagtta 3120atcttcatgg
ccatagcttt tattcattag ctgtgatttt tgttgattaa aacattatag
3180attttcatgt tcttgcagtc atcagaagtg gtaggaaagc ctcactgata
tattttccag 3240ggcaatcaat gttcacgcaa cttgaaatta tatctgtggt
cttcaaattg tcttttgtca 3300tgtggctaaa tgcctaataa acaattcaag
tgaaatacta aaaaaaaaaa aaaaaaaaaa 3360agctgctcga gagatctacg
ggtggcatcc ctgtgacccc tccccagtgc ctctcctggc 3420cctggaagtt
gccactccag tgcccaccag ccttgtccta ataaaattaa gttgcatcat
3480tttgtctgac taggtgtcct tctataatat tatggggtgg aggggggtgg
tatggagcaa 3540ggggcaagtt gggaagacaa cctgtagggc ctgcggggtc
tattgggaac caagctggag 3600tgcagtggca caatcttggc tcactgcaat
ctccgcctcc tgggttcaag cgattctcct 3660gcctcagcct cccgagttgt
tgggattcca ggcatgcatg accaggctca gctaattttt 3720gtttttttgg
tagagacggg gtttcaccat attggccagg ctggtctcca actcctaatc
3780tcaggtgatc tacccacctt ggcctcccaa attgctggga ttacaggcgt
gaaccactgc 3840tcccttccct gtccttctga ttttgtaggt aacgtaaccc
cggaccacgt gcggaccgag 3900cggccgctct agagcatggc tacgtagata
agtagcatgg cgggttaatc attaactaca 3960aggaacccct agtgatggag
ttggccactc cctctctgcg cgctcgctcg ctcactgagg 4020ccgggcgacc
aaaggtcgcc cgacgcccgg gcggcctcag tgagcgagcg agcgcgcag
4079109266DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 10gagaggcggt ttgcgtattg ggcgctcttc
cgcttcctcg ctcactgact cgctgcgctc 60ggtcgttcgg ctgcggcgag cggtatcagc
tcactcaaag gcggtaatac ggttatccac 120agaatcaggg gataacgcag
gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa 180ccgtaaaaag
gccgcgttgc tggcgttttg agatcctttt tttctgcgcg taatctgctg
240cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc
aagagctacc 300aactcttttt ccgaaggtaa ctggcttcag cagagcgcag
ataccaaata ctgttcttct 360agtgtagccg tagttaggcc accacttcaa
gaactctgta gcaccgccta catacctcgc 420tctgctaatc ctgttaccag
tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt 480ggactcaaga
cgatagttac cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg
540cacacagccc agcttggagc gaacgaccta caccgaactg agatacctac
agcgtgagct 600atgagaaagc gccacgcttc ccgaagggag aaaggcggac
aggtatccgg taagcggcag 660ggtcggaaca ggagagcgca cgagggagct
tccaggggga aacgcctggt atctttatag 720tcctgtcggg tttcgccacc
tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg 780gcggagccta
tggaaagaag atcctttgat cttttctacg gggtctgacg ctcagtggaa
840cgaaaactca cgttaaggga ttttggtcat gagattatca aaaaggatct
tcacctagat 900ccttttaaat taaaaatgaa gttttaaatc aagcccaatc
tgaataatgt tacaaccaat 960taaccaattc tgaaaacgcg cgatgcagct
ctggcccgtg tctcaaaatc tctgatgtta 1020cattgcacaa gataaaaata
tatcatcatg aacaataaaa ctgtctgctt acataaacag 1080taatacaagg
ggtgttatga gccatattca acgggaaacg tcgaggccgc gattaaattc
1140caacatggat gctgatttat atgggtataa atgggctcgc gataatgtcg
ggcaatcagg 1200tgcgacaatc tatcgcttgt atgggaagcc cgatgcgcca
gagttgtttc tgaaacatgg 1260caaaggtagc gttgccaatg atgttacaga
tgagatggtc agactaaact ggctgacgga 1320atttatgcct cttccgacca
tcaagcattt tatccgtact cctgatgatg catggttact 1380caccactgcg
atccccggaa aaacagcatt ccaggtatta gaagaatatc ctgattcagg
1440tgaaaatatt gttgatgcgc tggcagtgtt cctgcgccgg ttgcattcga
ttcctgtttg 1500taattgtcct tttaacagcg atcgcgtatt tcgtctcgct
caggcgcaat cacgaatgaa 1560taacggtttg gttgatgcga gtgattttga
tgacgagcgt aatggctggc ctgttgaaca 1620agtctggaaa gaaatgcata
aacttttgcc attctcaccg gattcagtcg tcactcatgg 1680tgatttctca
cttgataacc ttatttttga cgaggggaaa ttaataggtt gtattgatgt
1740tggacgagtc ggaatcgcag accgatacca ggatcttgcc atcctatgga
actgcctcgg 1800tgagttttct ccttcattac agaaacggct ttttcaaaaa
tatggtattg ataatcctga 1860tatgaataaa ttgcagtttc atttgatgct
cgatgagttt ttctaatcag aattggttaa 1920ttggttgtaa cattattcag
attgggcttg atttaaaact tcatttttaa tttaaaagga 1980tctaggtgaa
gatccttttt gataatctca tgaccaaaat cccttaacgt gagttttcgt
2040tccactgagc gtcagacccc gtagaaaaga tcaaaggatc ttcaacaccc
cttgtattac 2100tgtttatgta agcagacagt tttattgttc atgatgatat
atttttatct tgtgcaatgt 2160aacatcagag attttgagac acgggccaga
gctgcatcgc gcgtttcggt gatgacggtg 2220aaaacctctg acacatgcag
ctcccgtatc tggaggctat aaatgattac gatacttcga 2280aaacatcctt
atcctggaaa actccccgtt catttcccac tctcctgcca ttcgacactc
2340gtgcgtccat tcaagtctgt gctaatttct acgcatttcc ctctgccatt
gactttgctt 2400gcgtgggata ataactatta agccaacttg attcaactca
ggcattcaaa aggcattttg 2460attagtcgaa aagtggcacg gctgacgagt
ggagctgcca tttcacgttt atttcgaact 2520tttgattaca tggtatattt
agtaatacac atatttttta gccagcaggt taatacttca 2580aagttgtttc
gattttcaac gaataaaagg aatttggcat ggtgtttttt ctggtctttg
2640gtattgacaa atcagggata tacaatatgt attaacaaaa ttggattgct
acatatattt 2700aatgattttc attattagaa tgggtggaat aaagttttaa
attacattat taatttcatt 2760agggttggct gattgattac tatttcctca
ttgaattgca ttcgatatta gcagtattta 2820tctggcccat tttggaagtt
acttaaattt cgaaccttaa gtcattatta gagctaatat 2880acaacagagt
tgtcaacttg gtttgatcag cattgaaatt aaactgattt cttgttcgtt
2940cttatttcgt ttggttttgt atctcgtatt ttatatgcag gagggcatat
attggcacaa 3000gacaaataat cacagcaccc attctcaact ttgtcctgtt
tctcagttta gtgtaaatag 3060aatgttgcaa aaaattgaat ataagcatta
tcatagtgtc aaattcgaga ggatgagttt 3120gctgtgggtc ttcgaggttc
tttccctgtg tttgccacgg ttcgttgcac gttgccactg 3180attggacaca
tgtccgcagt gaaaaatcta cggtcaggta ccacccaact aaaccggaag
3240gcggcacgtg tgtgggcgtt gagcggtgtg ggcgtggcag ccatataatg
agacatttat 3300ttaaaaagaa cattcgaaca tttttgatgg gccatatctt
ggcttgtcaa gctccagggc 3360agacattttc ctcgactcca ccgccttttt
catttgaatg gaggaggtgt agggcacaaa 3420aaacgtttcc tgccttttta
agcagcacaa aggatatgag ccattaaatt agttaatttc 3480gagtgcattt
cattttaaat aagtgttgga aagggaataa aaaagcgact gatagaaaag
3540ttgcagacac caagagaatg ttaaatattt taataatagc tacatttttc
tatctctgtg 3600acaaggcctg tgtactttaa tgttatatta cttctttgtg
tttgatttct gttaaaagtt 3660tatgattttg gcatgtacaa atcttacaac
ccttttttgt catcatgaca aaatcaaaat 3720cgtgcaatat ttccattctc
atcttttagc cgattcataa cattcgtaga aattaatatg 3780aaatcaatca
tgtataattt tcatgcacgt tatgccgatt tggtatcgtc gatttgccgg
3840ctcatcgtcg ccattgtgtt aatggaaaag ttatgctttt cgcaggaaaa
aagagttgaa 3900aattccaatt tttgtcatgc tcatcaaaat gttatacatt
cggtaatttg agctggccaa 3960acgttaaccc cttatcgccc cgcgcaagca
ttcattaact gcccccagag cgtaattaat 4020ctctttctcg cagaatttcc
ttcacagcca cgtcttgggt aaaagtcacc cctcaaagtg 4080gcagttgcat
gttgtaaatt agattttcgt ccgtcatgac aaccctcgtg cgagggttgc
4140tgcagcattt gaatgcaaaa ctgcttttgg tcattaacct gtctgaggca
ctttgctgca 4200aaacttctat tagaattcgg tttggactcg aattcggatt
tcttccccca aaatgcgatg 4260gctacagcaa aatgctttca gcttccgaag
ttttgcaacc ccaaagtcca gagtttccac 4320gctgtcacga ttccaaagcg
gcgccatttt tggctcacgc aaaacttatt aatgaacttt 4380aagcagacta
cgaggaaagg gctataatgg ccacacaaaa gggttaagca agtgttaagg
4440gctggtggga gggaaaaatc atcaatcata ttttctgcat agctgaatca
tttgcgccga 4500tcagttgggg ttacttaatg ggtttatttc aagaagaaaa
ctgtgggaaa ctaactgact 4560ctaaacttaa tcagattaaa tgtaacctgg
agatgatttt caaaatatta aaagagtttt 4620atggaggtaa ttttaaagaa
aactgaaaaa ctttgaagaa gtctataata gatccagtct 4680taatcgtagc
actgattatt tgtttttaaa ttggaaaaaa ttaaatgata catcatgctt
4740aaatgcgatg tccaacaaat cccaagacgg taatttccgt gcgttccaaa
ctcatctgaa 4800gaatcttaca aatacatttc gtgtatcccc aggctcagcg
tgcaaataaa tcttttggat 4860ctttgcaaat gcgaaatgct tatttttatg
cgttttcaat gccaattcga tggcaaaacc 4920aacaaaatca catggaaata
atggaaaagc aatttccatt tctacaaggg aggcagtgag 4980acacaagagc
agcgtataca attccccagc agcgatttga ccattaaaat tatacccaca
5040ggacgagcag gcataaaaag cagaggatgc gaggtgttca ggggactgtc
tatatctcca 5100acttgaatat atgaaatcgt ttggaaatag ccaagcgacc
aaattggcat ttggaaaatt 5160gcacaacgac agaagaagat gtttgaaaag
agaaattgaa ataataaacc aagatatatg 5220taaacggttg gtcacatttg
ggttctgaga cggtcacagc ttgtctgtaa gcggatgccg 5280ggagcagaca
agcccgtcag ggcgcgtcag cgggtgttgg cgggtgtcgg ggctggctta
5340actatgcggc atcagagcag attgtactga gagtgcacca tatgcggtgt
gaaataccgc 5400acagatgcgt aaggagaaaa taccgcatca ggcgccattc
gccattcagg ctgcgcaact 5460gttgggaagg gcgatcggtg cgggcctctt
cgctattacg ccagctggcg aaagggggat 5520gtgctgcaag gcgattaagt
tgggtaacgc cagggttttc ccagtcacga cgttgtaaaa 5580cgacggccag
tgaattcgag ctcggtacca tttaaatcgt cttggccact ccctctctgc
5640gcgctcgctc gctcactgag gccgcccggg caaagcccgg gcgtcgggcg
acctttggtc 5700gcccggcctc agtgagcgag cgagcgcgca gagagggagt
ggccaactcc atcactaggg 5760gttccttcgc gagcttaatt aactgcggcc
gctcggtccg cacgtggtta cctacaaaat 5820cagaaggaca gggaagggag
cagtggttca cgcctgtaat cccagcaatt tgggaggcca 5880aggtgggtag
atcacctgag attaggagtt ggagaccagc ctggccaata tggtgaaacc
5940ccgtctctac caaaaaaaca aaaattagct gagcctggtc atgcatgcct
ggaatcccaa 6000caactcggga ggctgaggca ggagaatcgc ttgaacccag
gaggcggaga ttgcagtgag 6060ccaagattgt gccactgcac tccagcttgg
ttcccaatag accccgcagg ccctacaggt 6120tgtcttccca acttgcccct
tgctccatac cacccccctc caccccataa tattatagaa 6180ggacacctag
tcagacaaaa tgatgcaact taattttatt aggacaaggc tggtgggcac
6240tggagtggca acttccaggg ccaggagagg cactggggag gggtcacagg
gatgccaccc 6300gtagatctct cgagcagctc actcctactc cctctctgct
cgcagcacgt cggccgccag 6360ctctttgatg tgttcccagg cccgctgcac
atgggcagat tccaccgtgc gagaacagat 6420ggcaaagcgc aggacaaact
tgtccctgag gtgacatgga accaagtgga tttttttggc 6480actgtttatt
ctttgcagaa gagcttcatt cactttgttg gaacccttta gccgaaagca
6540gacaagcccc agaatgactt ccacacagat ttcaaagcgg ggatcctggc
gcaccagtga 6600ctcaaactca tgggacagct ggacatgctt gcggatataa
gcctgcagtc ctttgactcc 6660atacatccta aatacaaacc acattttcaa
agagcgaaat cttctgccca gtggtatctg 6720ccaatgccgg tagtcagtga
taagccctga atcctgatgg ctgtgcttca ggtaagtggg 6780gtccagtcta
aaggctcccg ttaagtctgt tctctttttc acccacatgg cagaacagtc
6840aaaattcacc aatagccatt tgtggggatt aaagttgaat gaatctgcaa
actccactcc 6900attcagaagg tgccggaact cagggcagat gaatgcactg
cctgcgtagg ctgcatcaac 6960gtgcagccat atgtcttcct tgttgcagat
aggaccgact tctaagagat tgtcaaagga 7020gcagcatgtt gtggtcccca
gggtggcaac cataaagaaa ggaatcaggc cagccgcttt 7080gtctctctcc
agggcttcct gcagggcaga cgcacgcatg gcgaagttgc catctgaggg
7140gatggctttt aatttcactc caccaattaa cccagctctt tccactgagg
agtgtgcctg 7200atcggatgag taagccacca gcttctccat gatagcggcc
tgtgtgagct ctggggacgc 7260tgcctgcagc cgatggatca ctttggtccg
agcggccagc agggccacca gggtggcttc 7320actggcactt ccctggatca
ctcctccccc ttctccagct ttctcattca aaaatgcctt 7380tggtagttcc
agcatcttcc cgagccagtc catcatcaca gtctccagct ctgtgcatgc
7440tgggcttgcc gcccaggaga agccgatgca gccaatggcc ccgcacagca
tgtccgcaag 7500catggccggg tacgagctgg cagtggggaa gtaggcgaag
aagtaggggc tgtgccagtg
7560cgtcacccca ggcatgatta tcttctcaac gtcgttgatg atgtcctcaa
acgtgtctgg 7620ctcctgaggg gcagcggcag ggatcagcgg ccgcaggtac
ccgggctcca cgtcagggta 7680gacctggcgt ccctcaatgc cttccatgta
gttggccacg taatccacca tctccttccc 7740tctccttcgg aattcacttg
cgttcatggt ggcggccgat gttcgaatcc caattctttg 7800ccaaagtgat
gggccagcac acagaccagc acgttgccca ggagctgtgg gaggaagata
7860agaggtatga acatgattag caaaagggcc tagcttggac tcagaataat
ccagccttat 7920cccaaccata aaataaaagc agaatggtag ctggattgta
gctgctatta gcaatatgaa 7980acctcttaca tcagttacaa tttatatgca
gaaatattta tatgcagaaa tattgctatt 8040gccttaaccc agaaattatc
actgttattc tttagaatgg tgcaaagagg catgatacat 8100tgtatcatta
ttgccctgaa agaaagagat tagggaaagt attagaaata agataaacaa
8160aaaagtatat taaaagaaga aagcattttt tgtgggccta tagactctat
aggcggtact 8220tacgtcactc ttggcacggg gaatccgcgt tccaatgcac
cgttcccggc cgggattcga 8280atccgcggag gctggatcgg tcccggtgtc
ttctatggag gtcaaaacag cgtggatggc 8340gtctccaggc gatctgacgg
ttcactaaac gagctctgct tatatagacc tcccaccgta 8400cacgcctacc
gcccatttgc gtcaatgggg cggagttgtt acgacatttt ggaaagtccc
8460gttgattttg gtgcaaaaca aactcccatt gacgtcaatg gggtggagac
ttggaaatcc 8520ccgtgagtca aaccgctatc cacgcccatt gatgtactgc
caaaaccgca tcaccatggt 8580aatagcgatg actaatacgt agatgtactg
ccaagtagga aagtcccata aggtcatgta 8640ctgggcataa tgccaggcgg
gccatttacc gtcattgacg tcaatagggg gcgtacttgg 8700catatgatac
acttgatgta ctgccaagtg ggcagtttac cgtaaatact ccacccattg
8760acgtcaatgg aaagtcccta ttgacgttac tatgggaaca tacgtcatta
ttgacgtcaa 8820tgggcggggg tcgttgggcg gtcagccagg cgggccattt
accgtaagtt atgtaacgcg 8880gaactccata tatgggctat gaactaatga
ccccgtaatt gattactatt aataactagc 8940tccacgcgtg cggccgcaga
tcgtcgacga agtttaaact caggaacccc tagtgatgga 9000gttggccact
ccctctctgc gcgctcgctc gctcactgag gccgcccggg caaagcccgg
9060gcgtcgggcg acctttggtc gcccggcctc agtgagcgag cgagcgcgca
gagagggagt 9120ggccaacgtc atttaaatgc atgcaagctt ggcgtaatca
tggtcatagc tgtttcctgt 9180gtgaaaattg cgttgcgctc actgcccgct
ttccagtcgg gaaacctgtc gtgccagctg 9240cattaatgaa tcggccaacg cgcggg
9266119236DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 11cccgcgcgtt ggccgattca ttaatgcagc
tggcacgaca ggtttcccga ctggaaagcg 60ggcagtgagc gcaacgcaat tttcacacag
gaaacagcta tgaccatgat tacgccaagc 120ttgcatgcat ttaaatgacg
cagctgcgcg ctcgctcgct cactgaggcc gcccgggcaa 180agcccgggcg
tcgggcgacc tttggtcgcc cggcctcagt gagcgagcga gcgcgcagag
240agggagtggc caactccatc actaggggtt gagtttaaac ttcgtcgacg
atctgcggcc 300gcacgcgtgg agctagttat taatagtaat caattacggg
gtcattagtt catagcccat 360atatggagtt ccgcgttaca taacttacgg
taaatggccc gcctggctga ccgcccaacg 420acccccgccc attgacgtca
ataatgacgt atgttcccat agtaacgtca atagggactt 480tccattgacg
tcaatgggtg gagtatttac ggtaaactgc ccacttggca gtacatcaag
540tgtatcatat gccaagtacg ccccctattg acgtcaatga cggtaaatgg
cccgcctggc 600attatgccca gtacatgacc ttatgggact ttcctacttg
gcagtacatc tacgtattag 660tcatcgctat taccatggtg atgcggtttt
ggcagtacat caatgggcgt ggatagcggt 720ttgactcacg gggatttcca
agtctccacc ccattgacgt caatgggagt ttgttttgca 780ccaaaatcaa
cgggactttc caaaatgtcg taacaactcc gccccattga cgcaaatggg
840cggtaggcgt gtacggtggg aggtctatat aagcagagct cgtttagtga
accgtcagat 900cgcctggaga cgccatccac gctgttttga cctccataga
agacaccggg accgatccag 960cctccgcgga ttcgaatccc ggccgggaac
ggtgcattgg aacgcggatt ccccgtgcca 1020agagtgacgt aagtaccgcc
tatagagtct ataggcccac aaaaaatgct ttcttctttt 1080aatatacttt
tttgtttatc ttatttctaa tactttccct aatctctttc tttcagggca
1140ataatgatac aatgtatcat gcctctttgc accattctaa agaataacag
tgataatttc 1200tgggttaagg caatagcaat atttctgcat ataaatattt
ctgcatataa attgtaactg 1260atgtaagagg tttcatattg ctaatagcag
ctacaatcca gctaccattc tgcttttatt 1320ttatggttgg gataaggctg
gattattctg agtccaagct aggccctttt gctaatcatg 1380ttcatacctc
ttatcttcct cccacagctc ctgggcaacg tgctggtctg tgtgctggcc
1440catcactttg gcaaagaatt gggattcgaa catcggccgc caccatgaac
gcaagtgaat 1500tccgaaggag agggaaggag atggtggatt acgtggccaa
ctacatggaa ggcattgagg 1560gacgccaggt ctaccctgac gtggagcccg
ggtacctgcg gccgctgatc cctgccgctg 1620cccctcagga gccagacacg
tttgaggaca tcatcaacga cgttgagaag ataatcatgc 1680ctggggtgac
gcactggcac agcccctact tcttcgccta cttccccact gccagctcgt
1740acccggccat gcttgcggac atgctgtgcg gggccattgg ctgcatcggc
ttctcctggg 1800cggcaagccc agcatgcaca gagctggaga ctgtgatgat
ggactggctc gggaagatgc 1860tggaactacc aaaggcattt ttgaatgaga
aagctggaga agggggagga gtgatccagg 1920gaagtgccag tgaagccacc
ctggtggccc tgctggccgc tcggaccaaa gtgatccatc 1980ggctgcaggc
agcgtcccca gagctcacac aggccgctat catggagaag ctggtggctt
2040actcatccga tcaggcacac tcctcagtgg aaagagctgg gttaattggt
ggagtgaaat 2100taaaagccat cccctcagat ggcaacttcg ccatgcgtgc
gtctgccctg caggaagccc 2160tggagagaga caaagcggct ggcctgattc
ctttctttat ggttgccacc ctggggacca 2220caacatgctg ctcctttgac
aatctcttag aagtcggtcc tatctgcaac aaggaagaca 2280tatggctgca
cgttgatgca gcctacgcag gcagtgcatt catctgccct gagttccggc
2340accttctgaa tggagtggag tttgcagatt cattcaactt taatccccac
aaatggctat 2400tggtgaattt tgactgttct gccatgtggg tgaaaaagag
aacagactta acgggagcct 2460ttagactgga ccccacttac ctgaagcaca
gccatcagga ttcagggctt atcactgact 2520accggcattg gcagatacca
ctgggcagaa gatttcgctc tttgaaaatg tggtttgtat 2580ttaggatgta
tggagtcaaa ggactgcagg cttatatccg caagcatgtc cagctgtccc
2640atgagtttga gtcactggtg cgccaggatc cccgctttga aatctgtgtg
gaagtcattc 2700tggggcttgt ctgctttcgg ctaaagggtt ccaacaaagt
gaatgaagct cttctgcaaa 2760gaataaacag tgccaaaaaa atccacttgg
ttccatgtca cctcagggac aagtttgtcc 2820tgcgctttgc catctgttct
cgcacggtgg aatctgccca tgtgcagcgg gcctgggaac 2880acatcaaaga
gctggcggcc gacgtgctgc gagcagagag ggagtaggag tgagctgctc
2940gagagatcta cgggtggcat ccctgtgacc cctccccagt gcctctcctg
gccctggaag 3000ttgccactcc agtgcccacc agccttgtcc taataaaatt
aagttgcatc attttgtctg 3060actaggtgtc cttctataat attatggggt
ggaggggggt ggtatggagc aaggggcaag 3120ttgggaagac aacctgtagg
gcctgcgggg tctattggga accaagctgg agtgcagtgg 3180cacaatcttg
gctcactgca atctccgcct cctgggttca agcgattctc ctgcctcagc
3240ctcccgagtt gttgggattc caggcatgca tgaccaggct cagctaattt
ttgttttttt 3300ggtagagacg gggtttcacc atattggcca ggctggtctc
caactcctaa tctcaggtga 3360tctacccacc ttggcctccc aaattgctgg
gattacaggc gtgaaccact gctcccttcc 3420ctgtccttct gattttgtag
gtaaccacgt gcggaccgag cggccgcagt taattaagct 3480cgcgaaaccc
ctagtgatgg agttggccac tccctctctg cgcgctcgct cgctcactga
3540ggccgggcga ccaaaggtcg cccgacgccc gggctttgcc cgggcggcct
cagtgagcga 3600gcgagcgcgc agctggacga tttaaatggt accgagctcg
aattcactgg ccgtcgtttt 3660acaacgtcgt gactgggaaa accctggcgt
tacccaactt aatcgccttg cagcacatcc 3720ccctttcgcc agctggcgta
atagcgaaga ggcccgcacc gatcgccctt cccaacagtt 3780gcgcagcctg
aatggcgaat ggcgcctgat gcggtatttt ctccttacgc atctgtgcgg
3840tatttcacac cgcatatggt gcactctcag tacaatctgc tctgatgccg
catagttaag 3900ccagccccga cacccgccaa cacccgctga cgcgccctga
cgggcttgtc tgctcccggc 3960atccgcttac agacaagctg tgaccgtctc
agaacccaaa tgtgaccaac cgtttacata 4020tatcttggtt tattatttca
atttctcttt tcaaacatct tcttctgtcg ttgtgcaatt 4080ttccaaatgc
caatttggtc gcttggctat ttccaaacga tttcatatat tcaagttgga
4140gatatagaca gtcccctgaa cacctcgcat cctctgcttt ttatgcctgc
tcgtcctgtg 4200ggtataattt taatggtcaa atcgctgctg gggaattgta
tacgctgctc ttgtgtctca 4260ctgcctccct tgtagaaatg gaaattgctt
ttccattatt tccatgtgat tttgttggtt 4320ttgccatcga attggcattg
aaaacgcata aaaataagca tttcgcattt gcaaagatcc 4380aaaagattta
tttgcacgct gagcctgggg atacacgaaa tgtatttgta agattcttca
4440gatgagtttg gaacgcacgg aaattaccgt cttgggattt gttggacatc
gcatttaagc 4500atgatgtatc atttaatttt ttccaattta aaaacaaata
atcagtgcta cgattaagac 4560tggatctatt atagacttct tcaaagtttt
tcagttttct ttaaaattac ctccataaaa 4620ctcttttaat attttgaaaa
tcatctccag gttacattta atctgattaa gtttagagtc 4680agttagtttc
ccacagtttt cttcttgaaa taaacccatt aagtaacccc aactgatcgg
4740cgcaaatgat tcagctatgc agaaaatatg attgatgatt tttccctccc
accagccctt 4800aacacttgct taaccctttt gtgtggccat tatagccctt
tcctcgtagt ctgcttaaag 4860ttcattaata agttttgcgt gagccaaaaa
tggcgccgct ttggaatcgt gacagcgtgg 4920aaactctgga ctttggggtt
gcaaaacttc ggaagctgaa agcattttgc tgtagccatc 4980gcattttggg
ggaagaaatc cgaattcgag tccaaaccga attctaatag aagttttgca
5040gcaaagtgcc tcagacaggt taatgaccaa aagcagtttt gcattcaaat
gctgcagcaa 5100ccctcgcacg agggttgtca tgacggacga aaatctaatt
tacaacatgc aactgccact 5160ttgaggggtg acttttaccc aagacgtggc
tgtgaaggaa attctgcgag aaagagatta 5220attacgctct gggggcagtt
aatgaatgct tgcgcggggc gataaggggt taacgtttgg 5280ccagctcaaa
ttaccgaatg tataacattt tgatgagcat gacaaaaatt ggaattttca
5340actctttttt cctgcgaaaa gcataacttt tccattaaca caatggcgac
gatgagccgg 5400caaatcgacg ataccaaatc ggcataacgt gcatgaaaat
tatacatgat tgatttcata 5460ttaatttcta cgaatgttat gaatcggcta
aaagatgaga atggaaatat tgcacgattt 5520tgattttgtc atgatgacaa
aaaagggttg taagatttgt acatgccaaa atcataaact 5580tttaacagaa
atcaaacaca aagaagtaat ataacattaa agtacacagg ccttgtcaca
5640gagatagaaa aatgtagcta ttattaaaat atttaacatt ctcttggtgt
ctgcaacttt 5700tctatcagtc gcttttttat tccctttcca acacttattt
aaaatgaaat gcactcgaaa 5760ttaactaatt taatggctca tatcctttgt
gctgcttaaa aaggcaggaa acgttttttg 5820tgccctacac ctcctccatt
caaatgaaaa aggcggtgga gtcgaggaaa atgtctgccc 5880tggagcttga
caagccaaga tatggcccat caaaaatgtt cgaatgttct ttttaaataa
5940atgtctcatt atatggctgc cacgcccaca ccgctcaacg cccacacacg
tgccgccttc 6000cggtttagtt gggtggtacc tgaccgtaga tttttcactg
cggacatgtg tccaatcagt 6060ggcaacgtgc aacgaaccgt ggcaaacaca
gggaaagaac ctcgaagacc cacagcaaac 6120tcatcctctc gaatttgaca
ctatgataat gcttatattc aattttttgc aacattctat 6180ttacactaaa
ctgagaaaca ggacaaagtt gagaatgggt gctgtgatta tttgtcttgt
6240gccaatatat gccctcctgc atataaaata cgagatacaa aaccaaacga
aataagaacg 6300aacaagaaat cagtttaatt tcaatgctga tcaaaccaag
ttgacaactc tgttgtatat 6360tagctctaat aatgacttaa ggttcgaaat
ttaagtaact tccaaaatgg gccagataaa 6420tactgctaat atcgaatgca
attcaatgag gaaatagtaa tcaatcagcc aaccctaatg 6480aaattaataa
tgtaatttaa aactttattc cacccattct aataatgaaa atcattaaat
6540atatgtagca atccaatttt gttaatacat attgtatatc cctgatttgt
caataccaaa 6600gaccagaaaa aacaccatgc caaattcctt ttattcgttg
aaaatcgaaa caactttgaa 6660gtattaacct gctggctaaa aaatatgtgt
attactaaat ataccatgta atcaaaagtt 6720cgaaataaac gtgaaatggc
agctccactc gtcagccgtg ccacttttcg actaatcaaa 6780atgccttttg
aatgcctgag ttgaatcaag ttggcttaat agttattatc ccacgcaagc
6840aaagtcaatg gcagagggaa atgcgtagaa attagcacag acttgaatgg
acgcacgagt 6900gtcgaatggc aggagagtgg gaaatgaacg gggagttttc
caggataagg atgttttcga 6960agtatcgtaa tcatttatag cctccagata
cgggagctgc atgtgtcaga ggttttcacc 7020gtcatcaccg aaacgcgcga
tgcagctctg gcccgtgtct caaaatctct gatgttacat 7080tgcacaagat
aaaaatatat catcatgaac aataaaactg tctgcttaca taaacagtaa
7140tacaaggggt gttgaagatc ctttgatctt ttctacgggg tctgacgctc
agtggaacga 7200aaactcacgt taagggattt tggtcatgag attatcaaaa
aggatcttca cctagatcct 7260tttaaattaa aaatgaagtt ttaaatcaag
cccaatctga ataatgttac aaccaattaa 7320ccaattctga ttagaaaaac
tcatcgagca tcaaatgaaa ctgcaattta ttcatatcag 7380gattatcaat
accatatttt tgaaaaagcc gtttctgtaa tgaaggagaa aactcaccga
7440ggcagttcca taggatggca agatcctggt atcggtctgc gattccgact
cgtccaacat 7500caatacaacc tattaatttc ccctcgtcaa aaataaggtt
atcaagtgag aaatcaccat 7560gagtgacgac tgaatccggt gagaatggca
aaagtttatg catttctttc cagacttgtt 7620caacaggcca gccattacgc
tcgtcatcaa aatcactcgc atcaaccaaa ccgttattca 7680ttcgtgattg
cgcctgagcg agacgaaata cgcgatcgct gttaaaagga caattacaaa
7740caggaatcga atgcaaccgg cgcaggaaca ctgccagcgc atcaacaata
ttttcacctg 7800aatcaggata ttcttctaat acctggaatg ctgtttttcc
ggggatcgca gtggtgagta 7860accatgcatc atcaggagta cggataaaat
gcttgatggt cggaagaggc ataaattccg 7920tcagccagtt tagtctgacc
atctcatctg taacatcatt ggcaacgcta cctttgccat 7980gtttcagaaa
caactctggc gcatcgggct tcccatacaa gcgatagatt gtcgcacctg
8040attgcccgac attatcgcga gcccatttat acccatataa atcagcatcc
atgttggaat 8100ttaatcgcgg cctcgacgtt tcccgttgaa tatggctcat
aacacccctt gtattactgt 8160ttatgtaagc agacagtttt attgttcatg
atgatatatt tttatcttgt gcaatgtaac 8220atcagagatt ttgagacacg
ggccagagct gcatcgcgcg ttttcagaat tggttaattg 8280gttgtaacat
tattcagatt gggcttgatt taaaacttca tttttaattt aaaaggatct
8340aggtgaagat cctttttgat aatctcatga ccaaaatccc ttaacgtgag
ttttcgttcc 8400actgagcgtc agaccccgta gaaaagatca aaggatcttc
tttccatagg ctccgccccc 8460ctgacgagca tcacaaaaat cgacgctcaa
gtcagaggtg gcgaaacccg acaggactat 8520aaagatacca ggcgtttccc
cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc 8580cgcttaccgg
atacctgtcc gcctttctcc cttcgggaag cgtggcgctt tctcatagct
8640cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc caagctgggc
tgtgtgcacg 8700aaccccccgt tcagcccgac cgctgcgcct tatccggtaa
ctatcgtctt gagtccaacc 8760cggtaagaca cgacttatcg ccactggcag
cagccactgg taacaggatt agcagagcga 8820ggtatgtagg cggtgctaca
gagttcttga agtggtggcc taactacggc tacactagaa 8880gaacagtatt
tggtatctgc gctctgctga agccagttac cttcggaaaa agagttggta
8940gctcttgatc cggcaaacaa accaccgctg gtagcggtgg tttttttgtt
tgcaagcagc 9000agattacgcg cagaaaaaaa ggatctcaaa acgccagcaa
cgcggccttt ttacggttcc 9060tggccttttg ctggcctttt gctcacatgt
tctttcctgc gttatcccct gattctgtgg 9120ataaccgtat taccgccttt
gagtgagctg ataccgctcg ccgcagccga acgaccgagc 9180gcagcgagtc
agtgagcgag gaagcggaag agcgcccaat acgcaaaccg cctctc
9236126260DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 12cccgcgcgtt ggccgattca ttaatgcagc
tggcacgaca ggtttcccga ctggaaagcg 60ggcagtgagc gcaacgcaat tttcacacag
gaaacagcta tgaccatgat tacgccaagc 120ttgcatgcat ttaaatgacg
ttggccactc cctctctgcg cgctcgctcg ctcactgagg 180ccgggcgacc
aaaggtcgcc cgacgcccgg gctttgcccg ggcggcctca gtgagcgagc
240gagcgcgcag agagggagtg gccaactcca tcactagggg ttcctgagtt
taaacttcgt 300cgacgatctg cggccgcacg cgtggagcta gttattaata
gtaatcaatt acggggtcat 360tagttcatag cccatatatg gagttccgcg
ttacataact tacggtaaat ggcccgcctg 420gctgaccgcc caacgacccc
cgcccattga cgtcaataat gacgtatgtt cccatagtaa 480cgtcaatagg
gactttccat tgacgtcaat gggtggagta tttacggtaa actgcccact
540tggcagtaca tcaagtgtat catatgccaa gtacgccccc tattgacgtc
aatgacggta 600aatggcccgc ctggcattat gcccagtaca tgaccttatg
ggactttcct acttggcagt 660acatctacgt attagtcatc gctattacca
tggtgatgcg gttttggcag tacatcaatg 720ggcgtggata gcggtttgac
tcacggggat ttccaagtct ccaccccatt gacgtcaatg 780ggagtttgtt
ttgcaccaaa atcaacggga ctttccaaaa tgtcgtaaca actccgcccc
840attgacgcaa atgggcggta ggcgtgtacg gtgggaggtc tatataagca
gagctcgttt 900agtgaaccgt cagatcgcct ggagacgcca tccacgctgt
tttgacctcc atagaagaca 960ccgggaccga tccagcctcc gcggattcga
atcccggccg ggaacggtgc attggaacgc 1020ggattccccg tgccaagagt
gacgtaagta ccgcctatag agtctatagg cccacaaaaa 1080atgctttctt
cttttaatat acttttttgt ttatcttatt tctaatactt tccctaatct
1140ctttctttca gggcaataat gatacaatgt atcatgcctc tttgcaccat
tctaaagaat 1200aacagtgata atttctgggt taaggcaata gcaatatttc
tgcatataaa tatttctgca 1260tataaattgt aactgatgta agaggtttca
tattgctaat agcagctaca atccagctac 1320cattctgctt ttattttatg
gttgggataa ggctggatta ttctgagtcc aagctaggcc 1380cttttgctaa
tcatgttcat acctcttatc ttcctcccac agctcctggg caacgtgctg
1440gtctgtgtgc tggcccatca ctttggcaaa gaattgggat tcgaacatcg
gccgccacca 1500tgaacgccag cgagttcagg aggaggggca aggagatggt
ggactacgtg gccaactaca 1560tggagggcat cgagggcagg caggtgtacc
ccgacgtgga gcccggctac ctgaggcccc 1620tgatccccgc cgccgccccc
caggagcccg acaccttcga ggacatcatc aacgacgtgg 1680agaagatcat
catgcccggc gtgacccact ggcacagccc ctacttcttc gcctacttcc
1740ccaccgccag cagctacccc gccatgctgg ccgacatgct gtgcggcgcc
atcggctgca 1800tcggcttcag ctgggccgcc agccccgcct gcaccgagct
ggagaccgtg atgatggact 1860ggctgggcaa gatgctggag ctgcccaagg
ccttcctgaa cgagaaggcc ggcgagggcg 1920gcggcgtgat ccagggcagc
gccagcgagg ccaccctggt ggccctgctg gccgccagga 1980ccaaggtgat
ccacaggctg caggccgcca gccccgagct gacccaggcc gccatcatgg
2040agaagctggt ggcctacagc agcgaccagg cccacagcag cgtggagagg
gccggcctga 2100tcggcggcgt gaagctgaag gccatcccca gcgacggcaa
cttcgccatg agggccagcg 2160ccctgcagga ggccctggag agggacaagg
ccgccggcct gatccccttc ttcatggtgg 2220ccaccctggg caccaccacc
tgctgcagct tcgacaacct gctggaggtg ggccccatct 2280gcaacaagga
ggacatctgg ctgcacgtgg acgccgccta cgccggcagc gccttcatct
2340gccccgagtt caggcacctg ctgaacggcg tggagttcgc cgacagcttc
aacttcaacc 2400cccacaagtg gctgctggtg aacttcgact gcagcgccat
gtgggtgaag aagaggaccg 2460acctgaccgg cgccttcagg ctggacccca
cctacctgaa gcacagccac caggacagcg 2520gcctgatcac cgactacagg
cactggcaga tccccctggg caggaggttc aggagcctga 2580agatgtggtt
cgtgttcagg atgtacggcg tgaagggcct gcaggcctac atcaggaagc
2640acgtgcagct gagccacgag ttcgagagcc tggtgaggca ggaccccagg
ttcgagatct 2700gcgtggaggt gatcctgggc ctggtgtgct tcaggctgaa
gggcagcaac aaggtgaacg 2760aggccctgct gcagaggatc aacagcgcca
agaagatcca cctggtgccc tgccacctga 2820gggacaagtt cgtgctgagg
ttcgccatct gcagcaggac cgtggagagc gcccacgtgc 2880agagggcctg
ggagcacatc aaggagctgg ccgccgacgt gctgagggcc gagagggagt
2940gagctgctcg agagatctac gggtggcatc cctgtgaccc ctccccagtg
cctctcctgg 3000ccctggaagt tgccactcca gtgcccacca gccttgtcct
aataaaatta agttgcatca 3060ttttgtctga ctaggtgtcc ttctataata
ttatggggtg gaggggggtg gtatggagca 3120aggggcaagt tgggaagaca
acctgtaggg cctgcggggt ctattgggaa ccaagctgga 3180gtgcagtggc
acaatcttgg ctcactgcaa tctccgcctc ctgggttcaa gcgattctcc
3240tgcctcagcc tcccgagttg ttgggattcc aggcatgcat gaccaggctc
agctaatttt 3300tgtttttttg gtagagacgg ggtttcacca tattggccag
gctggtctcc aactcctaat 3360ctcaggtgat ctacccacct tggcctccca
aattgctggg attacaggcg tgaaccactg 3420ctcccttccc tgtccttctg
attttgtagg taaccacgtg cggaccgagc ggccgcagtt 3480aattaagctc
gcgaaggaac ccctagtgat ggagttggcc actccctctc tgcgcgctcg
3540ctcgctcact gaggccgggc gaccaaaggt cgcccgacgc ccgggctttg
cccgggcggc 3600ctcagtgagc gagcgagcgc gcagagaggg agtggccaag
acgatttaaa tggtaccgag 3660ctcgaattca ctggccgtcg ttttacaacg
tcgtgactgg gaaaaccctg gcgttaccca 3720acttaatcgc cttgcagcac
atcccccttt cgccagctgg cgtaatagcg aagaggcccg 3780caccgatcgc
ccttcccaac agttgcgcag cctgaatggc gaatggcgcc tgatgcggta
3840ttttctcctt acgcatctgt gcggtatttc acaccgcata tggtgcactc
tcagtacaat
3900ctgctctgat gccgcatagt taagccagcc ccgacacccg ccaacacccg
ctgacgcgcc 3960ctgacgggct tgtctgctcc cggcatccgc ttacagacaa
gctgtgaccg tctccgggag 4020ctgcatgtgt cagaggtttt caccgtcatc
accgaaacgc gcgatgcagc tctggcccgt 4080gtctcaaaat ctctgatgtt
acattgcaca agataaaaat atatcatcat gaacaataaa 4140actgtctgct
tacataaaca gtaatacaag gggtgttgaa gatcctttga tcttttctac
4200ggggtctgac gctcagtgga acgaaaactc acgttaaggg attttggtca
tgagattatc 4260aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga
agttttaaat caagcccaat 4320ctgaataatg ttacaaccaa ttaaccaatt
ctgattagaa aaactcatcg agcatcaaat 4380gaaactgcaa tttattcata
tcaggattat caataccata tttttgaaaa agccgtttct 4440gtaatgaagg
agaaaactca ccgaggcagt tccataggat ggcaagatcc tggtatcggt
4500ctgcgattcc gactcgtcca acatcaatac aacctattaa tttcccctcg
tcaaaaataa 4560ggttatcaag tgagaaatca ccatgagtga cgactgaatc
cggtgagaat ggcaaaagtt 4620tatgcatttc tttccagact tgttcaacag
gccagccatt acgctcgtca tcaaaatcac 4680tcgcatcaac caaaccgtta
ttcattcgtg attgcgcctg agcgagacga aatacgcgat 4740cgctgttaaa
aggacaatta caaacaggaa tcgaatgcaa ccggcgcagg aacactgcca
4800gcgcatcaac aatattttca cctgaatcag gatattcttc taatacctgg
aatgctgttt 4860ttccggggat cgcagtggtg agtaaccatg catcatcagg
agtacggata aaatgcttga 4920tggtcggaag aggcataaat tccgtcagcc
agtttagtct gaccatctca tctgtaacat 4980cattggcaac gctacctttg
ccatgtttca gaaacaactc tggcgcatcg ggcttcccat 5040acaagcgata
gattgtcgca cctgattgcc cgacattatc gcgagcccat ttatacccat
5100ataaatcagc atccatgttg gaatttaatc gcggcctcga cgtttcccgt
tgaatatggc 5160tcataacacc ccttgtatta ctgtttatgt aagcagacag
ttttattgtt catgatgata 5220tatttttatc ttgtgcaatg taacatcaga
gattttgaga cacgggccag agctgcatcg 5280cgcgttttca gaattggtta
attggttgta acattattca gattgggctt gatttaaaac 5340ttcattttta
atttaaaagg atctaggtga agatcctttt tgataatctc atgaccaaaa
5400tcccttaacg tgagttttcg ttccactgag cgtcagaccc cgtagaaaag
atcaaaggat 5460cttctttcca taggctccgc ccccctgacg agcatcacaa
aaatcgacgc tcaagtcaga 5520ggtggcgaaa cccgacagga ctataaagat
accaggcgtt tccccctgga agctccctcg 5580tgcgctctcc tgttccgacc
ctgccgctta ccggatacct gtccgccttt ctcccttcgg 5640gaagcgtggc
gctttctcat agctcacgct gtaggtatct cagttcggtg taggtcgttc
5700gctccaagct gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc
gccttatccg 5760gtaactatcg tcttgagtcc aacccggtaa gacacgactt
atcgccactg gcagcagcca 5820ctggtaacag gattagcaga gcgaggtatg
taggcggtgc tacagagttc ttgaagtggt 5880ggcctaacta cggctacact
agaagaacag tatttggtat ctgcgctctg ctgaagccag 5940ttaccttcgg
aaaaagagtt ggtagctctt gatccggcaa acaaaccacc gctggtagcg
6000gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct
caaaacgcca 6060gcaacgcggc ctttttacgg ttcctggcct tttgctggcc
ttttgctcac atgttctttc 6120ctgcgttatc ccctgattct gtggataacc
gtattaccgc ctttgagtga gctgataccg 6180ctcgccgcag ccgaacgacc
gagcgcagcg agtcagtgag cgaggaagcg gaagagcgcc 6240caatacgcaa
accgcctctc 6260136442DNAArtificial SequenceDescription of
Artificial Sequence Synthetic polynucleotide 13cccgcgcgtt
ggccgattca ttaatgcagc tggcacgaca ggtttcccga ctggaaagcg 60ggcagtgagc
gcaacgcaat tttcacacag gaaacagcta tgaccatgat tacgccaagc
120ttgcatgcat ttaaatgacg cagctgcgcg ctcgctcgct cactgaggcc
gcccgggcaa 180agcccgggcg tcgggcgacc tttggtcgcc cggcctcagt
gagcgagcga gcgcgcagag 240agggagtggc caactccatc actaggggtt
gagtttaaac ttcgtcgacg atctgcggcc 300gcacgcgtgg agctagttat
taatagtaat caattacggg gtcattagtt catagcccat 360atatggagtt
ccgcgttaca taacttacgg taaatggccc gcctggctga ccgcccaacg
420acccccgccc attgacgtca ataatgacgt atgttcccat agtaacgtca
atagggactt 480tccattgacg tcaatgggtg gagtatttac ggtaaactgc
ccacttggca gtacatcaag 540tgtatcatat gccaagtacg ccccctattg
acgtcaatga cggtaaatgg cccgcctggc 600attatgccca gtacatgacc
ttatgggact ttcctacttg gcagtacatc tacgtattag 660tcatcgctat
taccatggtg atgcggtttt ggcagtacat caatgggcgt ggatagcggt
720ttgactcacg gggatttcca agtctccacc ccattgacgt caatgggagt
ttgttttgca 780ccaaaatcaa cgggactttc caaaatgtcg taacaactcc
gccccattga cgcaaatggg 840cggtaggcgt gtacggtggg aggtctatat
aagcagagct cgtttagtga accgtcagat 900cgcctggaga cgccatccac
gctgttttga cctccataga agacaccggg accgatccag 960cctccgcgga
ttcgaatccc ggccgggaac ggtgcattgg aacgcggatt ccccgtgcca
1020agagtgacgt aagtaccgcc tatagagtct ataggcccac aaaaaatgct
ttcttctttt 1080aatatacttt tttgtttatc ttatttctaa tactttccct
aatctctttc tttcagggca 1140ataatgatac aatgtatcat gcctctttgc
accattctaa agaataacag tgataatttc 1200tgggttaagg caatagcaat
atttctgcat ataaatattt ctgcatataa attgtaactg 1260atgtaagagg
tttcatattg ctaatagcag ctacaatcca gctaccattc tgcttttatt
1320ttatggttgg gataaggctg gattattctg agtccaagct aggccctttt
gctaatcatg 1380ttcatacctc ttatcttcct cccacagctc ctgggcaacg
tgctggtctg tgtgctggcc 1440catcactttg gcaaagaatt gggattcgaa
catcgaattc gggcacgagg gaggacagag 1500agcaagtcac tcccggctgc
ctttttcacc tctgacagag cccagacacc atgaacgcaa 1560gtgaattccg
aaggagaggg aaggagatgg tggattacgt ggccaactac atggaaggca
1620ttgagggacg ccaggtctac cctgacgtgg agcccgggta cctgcggccg
ctgatccctg 1680ccgctgcccc tcaggagcca gacacgtttg aggacatcat
caacgacgtt gagaagataa 1740tcatgcctgg ggtgacgcac tggcacagcc
cctacttctt cgcctacttc cccactgcca 1800gctcgtaccc ggccatgctt
gcggacatgc tgtgcggggc cattggctgc atcggcttct 1860cctgggcggc
aagcccagca tgcacagagc tggagactgt gatgatggac tggctcggga
1920agatgctgga actaccaaag gcatttttga atgagaaagc tggagaaggg
ggaggagtga 1980tccagggaag tgccagtgaa gccaccctgg tggccctgct
ggccgctcgg accaaagtga 2040tccatcggct gcaggcagcg tccccagagc
tcacacaggc cgctatcatg gagaagctgg 2100tggcttactc atccgatcag
gcacactcct cagtggaaag agctgggtta attggtggag 2160tgaaattaaa
agccatcccc tcagatggca acttcgccat gcgtgcgtct gccctgcagg
2220aagccctgga gagagacaaa gcggctggcc tgattccttt ctttatggtt
gccaccctgg 2280ggaccacaac atgctgctcc tttgacaatc tcttagaagt
cggtcctatc tgcaacaagg 2340aagacatatg gctgcacgtt gatgcagcct
acgcaggcag tgcattcatc tgccctgagt 2400tccggcacct tctgaatgga
gtggagtttg cagattcatt caactttaat ccccacaaat 2460ggctattggt
gaattttgac tgttctgcca tgtgggtgaa aaagagaaca gacttaacgg
2520gagcctttag actggacccc acttacctga agcacagcca tcaggattca
gggcttatca 2580ctgactaccg gcattggcag ataccactgg gcagaagatt
tcgctctttg aaaatgtggt 2640ttgtatttag gatgtatgga gtcaaaggac
tgcaggctta tatccgcaag catgtccagc 2700tgtcccatga gtttgagtca
ctggtgcgcc aggatccccg ctttgaaatc tgtgtggaag 2760tcattctggg
gcttgtctgc tttcggctaa agggttccaa caaagtgaat gaagctcttc
2820tgcaaagaat aaacagtgcc aaaaaaatcc acttggttcc atgtcacctc
agggacaagt 2880ttgtcctgcg ctttgccatc tgttctcgca cggtggaatc
tgcccatgtg cagcgggcct 2940gggaacacat caaagagctg gcggccgacg
tgctgcgagc agagagggag taggagtgaa 3000gccagctgca ggaatcaaaa
attgaagaga gatatatctg aaaactggaa taagaagcaa 3060ataaatatca
tcctgccttc atggaactca gctgtctgtg gcttcccatg tctttctcca
3120aagttatcca gagggttgtg attttgtctg cttagtatct catcaacaaa
gaaatattat 3180ttgctaatta aaaagttaat cttcatggcc atagctttta
ttcattagct gtgatttttg 3240ttgattaaaa cattatagat tttcatgttc
ttgcagtcat cagaagtggt aggaaagcct 3300cactgatata ttttccaggg
caatcaatgt tcacgcaact tgaaattata tctgtggtct 3360tcaaattgtc
ttttgtcatg tggctaaatg cctaataaac aattcaagtg aaatactaaa
3420aaaaaaaaaa aaaaaaaaag ctgctcgaga gatctacggg tggcatccct
gtgacccctc 3480cccagtgcct ctcctggccc tggaagttgc cactccagtg
cccaccagcc ttgtcctaat 3540aaaattaagt tgcatcattt tgtctgacta
ggtgtccttc tataatatta tggggtggag 3600gggggtggta tggagcaagg
ggcaagttgg gaagacaacc tgtagggcct gcggggtcta 3660ttgggaacca
agctggagtg cagtggcaca atcttggctc actgcaatct ccgcctcctg
3720ggttcaagcg attctcctgc ctcagcctcc cgagttgttg ggattccagg
catgcatgac 3780caggctcagc taatttttgt ttttttggta gagacggggt
ttcaccatat tggccaggct 3840ggtctccaac tcctaatctc aggtgatcta
cccaccttgg cctcccaaat tgctgggatt 3900acaggcgtga accactgctc
ccttccctgt ccttctgatt ttgtaggtaa ccacgtgcgg 3960accgagcggc
cgcagttaat taagctcgcg aaacccctag tgatggagtt ggccactccc
4020tctctgcgcg ctcgctcgct cactgaggcc gggcgaccaa aggtcgcccg
acgcccgggc 4080tttgcccggg cggcctcagt gagcgagcga gcgcgcagct
ggacgattta aatggtaccg 4140agctcgaatt cactggccgt cgttttacaa
cgtcgtgact gggaaaaccc tggcgttacc 4200caacttaatc gccttgcagc
acatccccct ttcgccagct ggcgtaatag cgaagaggcc 4260cgcaccgatc
gcccttccca acagttgcgc agcctgaatg gcgaatggcg cctgatgcgg
4320tattttctcc ttacgcatct gtgcggtatt tcacaccgca tatggtgcac
tctcagtaca 4380atctgctctg atgccgcata gttaagccag ccccgacacc
cgccaacacc cgctgacgcg 4440ccctgacggg cttgtctgct cccggcatcc
gcttacagac aagctgtgac cgtctccggg 4500agctgcatgt gtcagaggtt
ttcaccgtca tcaccgaaac gcgcgatgca gctctggccc 4560gtgtctcaaa
atctctgatg ttacattgca caagataaaa atatatcatc atgaacaata
4620aaactgtctg cttacataaa cagtaataca aggggtgtta tgagccatat
tcaacgggaa 4680acgtcgaggc cgcgattaaa ttccaacatg gatgctgatt
tatatgggta taaatgggct 4740cgcgataatg tcgggcaatc aggtgcgaca
atctatcgct tgtatgggaa gcccgatgcg 4800ccagagttgt ttctgaaaca
tggcaaaggt agcgttgcca atgatgttac agatgagatg 4860gtcagactaa
actggctgac ggaatttatg cctcttccga ccatcaagca ttttatccgt
4920actcctgatg atgcatggtt actcaccact gcgatccccg gaaaaacagc
attccaggta 4980ttagaagaat atcctgattc aggtgaaaat attgttgatg
cgctggcagt gttcctgcgc 5040cggttgcatt cgattcctgt ttgtaattgt
ccttttaaca gcgatcgcgt atttcgtctc 5100gctcaggcgc aatcacgaat
gaataacggt ttggttgatg cgagtgattt tgatgacgag 5160cgtaatggct
ggcctgttga acaagtctgg aaagaaatgc ataaactttt gccattctca
5220ccggattcag tcgtcactca tggtgatttc tcacttgata accttatttt
tgacgagggg 5280aaattaatag gttgtattga tgttggacga gtcggaatcg
cagaccgata ccaggatctt 5340gccatcctat ggaactgcct cggtgagttt
tctccttcat tacagaaacg gctttttcaa 5400aaatatggta ttgataatcc
tgatatgaat aaattgcagt ttcatttgat gctcgatgag 5460tttttctaat
cagaattggt taattggttg taacattatt cagattgggc ttgatttaaa
5520acttcatttt taatttaaaa ggatctaggt gaagatcctt tttgataatc
tcatgaccaa 5580aatcccttaa cgtgagtttt cgttccactg agcgtcagac
cccgtagaaa agatcaaagg 5640atcttcttga gatccttttt ttctgcgcgt
aatctgctgc ttgcaaacaa aaaaaccacc 5700gctaccagcg gtggtttgtt
tgccggatca agagctacca actctttttc cgaaggtaac 5760tggcttcagc
agagcgcaga taccaaatac tgttcttcta gtgtagccgt agttaggcca
5820ccacttcaag aactctgtag caccgcctac atacctcgct ctgctaatcc
tgttaccagt 5880ggctgctgcc agtggcgata agtcgtgtct taccgggttg
gactcaagac gatagttacc 5940ggataaggcg cagcggtcgg gctgaacggg
gggttcgtgc acacagccca gcttggagcg 6000aacgacctac accgaactga
gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc 6060cgaagggaga
aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcac
6120gagggagctt ccagggggaa acgcctggta tctttatagt cctgtcgggt
ttcgccacct 6180ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg
cggagcctat ggaaaaacgc 6240cagcaacgcg gcctttttac ggttcctggc
cttttgctgg ccttttgctc acatgttctt 6300tcctgcgtta tcccctgatt
ctgtggataa ccgtattacc gcctttgagt gagctgatac 6360cgctcgccgc
agccgaacga ccgagcgcag cgagtcagtg agcgaggaag cggaagagcg
6420cccaatacgc aaaccgcctc tc 6442149260DNAArtificial
SequenceDescription of Artificial Sequence Synthetic polynucleotide
14cccgcgcgtt ggccgattca ttaatgcagc tggcacgaca ggtttcccga ctggaaagcg
60ggcagtgagc gcaacgcaat tttcacacag gaaacagcta tgaccatgat tacgccaagc
120ttgcatgcat ttaaatgacg ttggccactc cctctctgcg cgctcgctcg
ctcactgagg 180ccgggcgacc aaaggtcgcc cgacgcccgg gctttgcccg
ggcggcctca gtgagcgagc 240gagcgcgcag agagggagtg gccaactcca
tcactagggg ttcctgagtt taaacttcgt 300cgacgatctg cggccgcacg
cgtggagcta gttattaata gtaatcaatt acggggtcat 360tagttcatag
cccatatatg gagttccgcg ttacataact tacggtaaat ggcccgcctg
420gctgaccgcc caacgacccc cgcccattga cgtcaataat gacgtatgtt
cccatagtaa 480cgtcaatagg gactttccat tgacgtcaat gggtggagta
tttacggtaa actgcccact 540tggcagtaca tcaagtgtat catatgccaa
gtacgccccc tattgacgtc aatgacggta 600aatggcccgc ctggcattat
gcccagtaca tgaccttatg ggactttcct acttggcagt 660acatctacgt
attagtcatc gctattacca tggtgatgcg gttttggcag tacatcaatg
720ggcgtggata gcggtttgac tcacggggat ttccaagtct ccaccccatt
gacgtcaatg 780ggagtttgtt ttgcaccaaa atcaacggga ctttccaaaa
tgtcgtaaca actccgcccc 840attgacgcaa atgggcggta ggcgtgtacg
gtgggaggtc tatataagca gagctcgttt 900agtgaaccgt cagatcgcct
ggagacgcca tccacgctgt tttgacctcc atagaagaca 960ccgggaccga
tccagcctcc gcggattcga atcccggccg ggaacggtgc attggaacgc
1020ggattccccg tgccaagagt gacgtaagta ccgcctatag agtctatagg
cccacaaaaa 1080atgctttctt cttttaatat acttttttgt ttatcttatt
tctaatactt tccctaatct 1140ctttctttca gggcaataat gatacaatgt
atcatgcctc tttgcaccat tctaaagaat 1200aacagtgata atttctgggt
taaggcaata gcaatatttc tgcatataaa tatttctgca 1260tataaattgt
aactgatgta agaggtttca tattgctaat agcagctaca atccagctac
1320cattctgctt ttattttatg gttgggataa ggctggatta ttctgagtcc
aagctaggcc 1380cttttgctaa tcatgttcat acctcttatc ttcctcccac
agctcctggg caacgtgctg 1440gtctgtgtgc tggcccatca ctttggcaaa
gaattgggat tcgaacatcg gccgccacca 1500tgaacgccag cgagttcagg
aggaggggca aggagatggt ggactacgtg gccaactaca 1560tggagggcat
cgagggcagg caggtgtacc ccgacgtgga gcccggctac ctgaggcccc
1620tgatccccgc cgccgccccc caggagcccg acaccttcga ggacatcatc
aacgacgtgg 1680agaagatcat catgcccggc gtgacccact ggcacagccc
ctacttcttc gcctacttcc 1740ccaccgccag cagctacccc gccatgctgg
ccgacatgct gtgcggcgcc atcggctgca 1800tcggcttcag ctgggccgcc
agccccgcct gcaccgagct ggagaccgtg atgatggact 1860ggctgggcaa
gatgctggag ctgcccaagg ccttcctgaa cgagaaggcc ggcgagggcg
1920gcggcgtgat ccagggcagc gccagcgagg ccaccctggt ggccctgctg
gccgccagga 1980ccaaggtgat ccacaggctg caggccgcca gccccgagct
gacccaggcc gccatcatgg 2040agaagctggt ggcctacagc agcgaccagg
cccacagcag cgtggagagg gccggcctga 2100tcggcggcgt gaagctgaag
gccatcccca gcgacggcaa cttcgccatg agggccagcg 2160ccctgcagga
ggccctggag agggacaagg ccgccggcct gatccccttc ttcatggtgg
2220ccaccctggg caccaccacc tgctgcagct tcgacaacct gctggaggtg
ggccccatct 2280gcaacaagga ggacatctgg ctgcacgtgg acgccgccta
cgccggcagc gccttcatct 2340gccccgagtt caggcacctg ctgaacggcg
tggagttcgc cgacagcttc aacttcaacc 2400cccacaagtg gctgctggtg
aacttcgact gcagcgccat gtgggtgaag aagaggaccg 2460acctgaccgg
cgccttcagg ctggacccca cctacctgaa gcacagccac caggacagcg
2520gcctgatcac cgactacagg cactggcaga tccccctggg caggaggttc
aggagcctga 2580agatgtggtt cgtgttcagg atgtacggcg tgaagggcct
gcaggcctac atcaggaagc 2640acgtgcagct gagccacgag ttcgagagcc
tggtgaggca ggaccccagg ttcgagatct 2700gcgtggaggt gatcctgggc
ctggtgtgct tcaggctgaa gggcagcaac aaggtgaacg 2760aggccctgct
gcagaggatc aacagcgcca agaagatcca cctggtgccc tgccacctga
2820gggacaagtt cgtgctgagg ttcgccatct gcagcaggac cgtggagagc
gcccacgtgc 2880agagggcctg ggagcacatc aaggagctgg ccgccgacgt
gctgagggcc gagagggagt 2940gagctgctcg agagatctac gggtggcatc
cctgtgaccc ctccccagtg cctctcctgg 3000ccctggaagt tgccactcca
gtgcccacca gccttgtcct aataaaatta agttgcatca 3060ttttgtctga
ctaggtgtcc ttctataata ttatggggtg gaggggggtg gtatggagca
3120aggggcaagt tgggaagaca acctgtaggg cctgcggggt ctattgggaa
ccaagctgga 3180gtgcagtggc acaatcttgg ctcactgcaa tctccgcctc
ctgggttcaa gcgattctcc 3240tgcctcagcc tcccgagttg ttgggattcc
aggcatgcat gaccaggctc agctaatttt 3300tgtttttttg gtagagacgg
ggtttcacca tattggccag gctggtctcc aactcctaat 3360ctcaggtgat
ctacccacct tggcctccca aattgctggg attacaggcg tgaaccactg
3420ctcccttccc tgtccttctg attttgtagg taaccacgtg cggaccgagc
ggccgcagtt 3480aattaagctc gcgaaggaac ccctagtgat ggagttggcc
actccctctc tgcgcgctcg 3540ctcgctcact gaggccgggc gaccaaaggt
cgcccgacgc ccgggctttg cccgggcggc 3600ctcagtgagc gagcgagcgc
gcagagaggg agtggccaag acgatttaaa tggtaccgag 3660ctcgaattca
ctggccgtcg ttttacaacg tcgtgactgg gaaaaccctg gcgttaccca
3720acttaatcgc cttgcagcac atcccccttt cgccagctgg cgtaatagcg
aagaggcccg 3780caccgatcgc ccttcccaac agttgcgcag cctgaatggc
gaatggcgcc tgatgcggta 3840ttttctcctt acgcatctgt gcggtatttc
acaccgcata tggtgcactc tcagtacaat 3900ctgctctgat gccgcatagt
taagccagcc ccgacacccg ccaacacccg ctgacgcgcc 3960ctgacgggct
tgtctgctcc cggcatccgc ttacagacaa gctgtgaccg tctcagaacc
4020caaatgtgac caaccgttta catatatctt ggtttattat ttcaatttct
cttttcaaac 4080atcttcttct gtcgttgtgc aattttccaa atgccaattt
ggtcgcttgg ctatttccaa 4140acgatttcat atattcaagt tggagatata
gacagtcccc tgaacacctc gcatcctctg 4200ctttttatgc ctgctcgtcc
tgtgggtata attttaatgg tcaaatcgct gctggggaat 4260tgtatacgct
gctcttgtgt ctcactgcct cccttgtaga aatggaaatt gcttttccat
4320tatttccatg tgattttgtt ggttttgcca tcgaattggc attgaaaacg
cataaaaata 4380agcatttcgc atttgcaaag atccaaaaga tttatttgca
cgctgagcct ggggatacac 4440gaaatgtatt tgtaagattc ttcagatgag
tttggaacgc acggaaatta ccgtcttggg 4500atttgttgga catcgcattt
aagcatgatg tatcatttaa ttttttccaa tttaaaaaca 4560aataatcagt
gctacgatta agactggatc tattatagac ttcttcaaag tttttcagtt
4620ttctttaaaa ttacctccat aaaactcttt taatattttg aaaatcatct
ccaggttaca 4680tttaatctga ttaagtttag agtcagttag tttcccacag
ttttcttctt gaaataaacc 4740cattaagtaa ccccaactga tcggcgcaaa
tgattcagct atgcagaaaa tatgattgat 4800gatttttccc tcccaccagc
ccttaacact tgcttaaccc ttttgtgtgg ccattatagc 4860cctttcctcg
tagtctgctt aaagttcatt aataagtttt gcgtgagcca aaaatggcgc
4920cgctttggaa tcgtgacagc gtggaaactc tggactttgg ggttgcaaaa
cttcggaagc 4980tgaaagcatt ttgctgtagc catcgcattt tgggggaaga
aatccgaatt cgagtccaaa 5040ccgaattcta atagaagttt tgcagcaaag
tgcctcagac aggttaatga ccaaaagcag 5100ttttgcattc aaatgctgca
gcaaccctcg cacgagggtt gtcatgacgg acgaaaatct 5160aatttacaac
atgcaactgc cactttgagg ggtgactttt acccaagacg tggctgtgaa
5220ggaaattctg cgagaaagag attaattacg ctctgggggc agttaatgaa
tgcttgcgcg 5280gggcgataag gggttaacgt ttggccagct caaattaccg
aatgtataac attttgatga 5340gcatgacaaa aattggaatt ttcaactctt
ttttcctgcg aaaagcataa cttttccatt 5400aacacaatgg cgacgatgag
ccggcaaatc gacgatacca aatcggcata acgtgcatga 5460aaattataca
tgattgattt catattaatt tctacgaatg ttatgaatcg gctaaaagat
5520gagaatggaa atattgcacg attttgattt tgtcatgatg acaaaaaagg
gttgtaagat 5580ttgtacatgc caaaatcata aacttttaac agaaatcaaa
cacaaagaag taatataaca 5640ttaaagtaca caggccttgt cacagagata
gaaaaatgta gctattatta aaatatttaa 5700cattctcttg gtgtctgcaa
cttttctatc agtcgctttt ttattccctt tccaacactt 5760atttaaaatg
aaatgcactc gaaattaact aatttaatgg ctcatatcct ttgtgctgct
5820taaaaaggca ggaaacgttt tttgtgccct acacctcctc cattcaaatg
aaaaaggcgg 5880tggagtcgag gaaaatgtct gccctggagc ttgacaagcc
aagatatggc ccatcaaaaa 5940tgttcgaatg ttctttttaa ataaatgtct
cattatatgg ctgccacgcc cacaccgctc
6000aacgcccaca cacgtgccgc cttccggttt agttgggtgg tacctgaccg
tagatttttc 6060actgcggaca tgtgtccaat cagtggcaac gtgcaacgaa
ccgtggcaaa cacagggaaa 6120gaacctcgaa gacccacagc aaactcatcc
tctcgaattt gacactatga taatgcttat 6180attcaatttt ttgcaacatt
ctatttacac taaactgaga aacaggacaa agttgagaat 6240gggtgctgtg
attatttgtc ttgtgccaat atatgccctc ctgcatataa aatacgagat
6300acaaaaccaa acgaaataag aacgaacaag aaatcagttt aatttcaatg
ctgatcaaac 6360caagttgaca actctgttgt atattagctc taataatgac
ttaaggttcg aaatttaagt 6420aacttccaaa atgggccaga taaatactgc
taatatcgaa tgcaattcaa tgaggaaata 6480gtaatcaatc agccaaccct
aatgaaatta ataatgtaat ttaaaacttt attccaccca 6540ttctaataat
gaaaatcatt aaatatatgt agcaatccaa ttttgttaat acatattgta
6600tatccctgat ttgtcaatac caaagaccag aaaaaacacc atgccaaatt
ccttttattc 6660gttgaaaatc gaaacaactt tgaagtatta acctgctggc
taaaaaatat gtgtattact 6720aaatatacca tgtaatcaaa agttcgaaat
aaacgtgaaa tggcagctcc actcgtcagc 6780cgtgccactt ttcgactaat
caaaatgcct tttgaatgcc tgagttgaat caagttggct 6840taatagttat
tatcccacgc aagcaaagtc aatggcagag ggaaatgcgt agaaattagc
6900acagacttga atggacgcac gagtgtcgaa tggcaggaga gtgggaaatg
aacggggagt 6960tttccaggat aaggatgttt tcgaagtatc gtaatcattt
atagcctcca gatacgggag 7020ctgcatgtgt cagaggtttt caccgtcatc
accgaaacgc gcgatgcagc tctggcccgt 7080gtctcaaaat ctctgatgtt
acattgcaca agataaaaat atatcatcat gaacaataaa 7140actgtctgct
tacataaaca gtaatacaag gggtgttgaa gatcctttga tcttttctac
7200ggggtctgac gctcagtgga acgaaaactc acgttaaggg attttggtca
tgagattatc 7260aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga
agttttaaat caagcccaat 7320ctgaataatg ttacaaccaa ttaaccaatt
ctgattagaa aaactcatcg agcatcaaat 7380gaaactgcaa tttattcata
tcaggattat caataccata tttttgaaaa agccgtttct 7440gtaatgaagg
agaaaactca ccgaggcagt tccataggat ggcaagatcc tggtatcggt
7500ctgcgattcc gactcgtcca acatcaatac aacctattaa tttcccctcg
tcaaaaataa 7560ggttatcaag tgagaaatca ccatgagtga cgactgaatc
cggtgagaat ggcaaaagtt 7620tatgcatttc tttccagact tgttcaacag
gccagccatt acgctcgtca tcaaaatcac 7680tcgcatcaac caaaccgtta
ttcattcgtg attgcgcctg agcgagacga aatacgcgat 7740cgctgttaaa
aggacaatta caaacaggaa tcgaatgcaa ccggcgcagg aacactgcca
7800gcgcatcaac aatattttca cctgaatcag gatattcttc taatacctgg
aatgctgttt 7860ttccggggat cgcagtggtg agtaaccatg catcatcagg
agtacggata aaatgcttga 7920tggtcggaag aggcataaat tccgtcagcc
agtttagtct gaccatctca tctgtaacat 7980cattggcaac gctacctttg
ccatgtttca gaaacaactc tggcgcatcg ggcttcccat 8040acaagcgata
gattgtcgca cctgattgcc cgacattatc gcgagcccat ttatacccat
8100ataaatcagc atccatgttg gaatttaatc gcggcctcga cgtttcccgt
tgaatatggc 8160tcataacacc ccttgtatta ctgtttatgt aagcagacag
ttttattgtt catgatgata 8220tatttttatc ttgtgcaatg taacatcaga
gattttgaga cacgggccag agctgcatcg 8280cgcgttttca gaattggtta
attggttgta acattattca gattgggctt gatttaaaac 8340ttcattttta
atttaaaagg atctaggtga agatcctttt tgataatctc atgaccaaaa
8400tcccttaacg tgagttttcg ttccactgag cgtcagaccc cgtagaaaag
atcaaaggat 8460cttctttcca taggctccgc ccccctgacg agcatcacaa
aaatcgacgc tcaagtcaga 8520ggtggcgaaa cccgacagga ctataaagat
accaggcgtt tccccctgga agctccctcg 8580tgcgctctcc tgttccgacc
ctgccgctta ccggatacct gtccgccttt ctcccttcgg 8640gaagcgtggc
gctttctcat agctcacgct gtaggtatct cagttcggtg taggtcgttc
8700gctccaagct gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc
gccttatccg 8760gtaactatcg tcttgagtcc aacccggtaa gacacgactt
atcgccactg gcagcagcca 8820ctggtaacag gattagcaga gcgaggtatg
taggcggtgc tacagagttc ttgaagtggt 8880ggcctaacta cggctacact
agaagaacag tatttggtat ctgcgctctg ctgaagccag 8940ttaccttcgg
aaaaagagtt ggtagctctt gatccggcaa acaaaccacc gctggtagcg
9000gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct
caaaacgcca 9060gcaacgcggc ctttttacgg ttcctggcct tttgctggcc
ttttgctcac atgttctttc 9120ctgcgttatc ccctgattct gtggataacc
gtattaccgc ctttgagtga gctgataccg 9180ctcgccgcag ccgaacgacc
gagcgcagcg agtcagtgag cgaggaagcg gaagagcgcc 9240caatacgcaa
accgcctctc 9260159665DNAArtificial SequenceDescription of
Artificial Sequence Synthetic polynucleotide 15cccgcgcgtt
ggccgattca ttaatgcagc tggcacgaca ggtttcccga ctggaaagcg 60ggcagtgagc
gcaacgcaat tttcacacag gaaacagcta tgaccatgat tacgccaagc
120ttgcatgcat ttaaatgacg ttggccactc cctctctgcg cgctcgctcg
ctcactgagg 180ccgggcgacc aaaggtcgcc cgacgcccgg gctttgcccg
ggcggcctca gtgagcgagc 240gagcgcgcag agagggagtg gccaactcca
tcactagggg ttcctgagtt taaacttcgt 300cgacgatctg cggccgcacg
cgtggagcta gttattaata gtaatcaatt acggggtcat 360tagttcatag
cccatatatg gagttccgcg ttacataact tacggtaaat ggcccgcctg
420gctgaccgcc caacgacccc cgcccattga cgtcaataat gacgtatgtt
cccatagtaa 480cgtcaatagg gactttccat tgacgtcaat gggtggagta
tttacggtaa actgcccact 540tggcagtaca tcaagtgtat catatgccaa
gtacgccccc tattgacgtc aatgacggta 600aatggcccgc ctggcattat
gcccagtaca tgaccttatg ggactttcct acttggcagt 660acatctacgt
attagtcatc gctattacca tggtgatgcg gttttggcag tacatcaatg
720ggcgtggata gcggtttgac tcacggggat ttccaagtct ccaccccatt
gacgtcaatg 780ggagtttgtt ttgcaccaaa atcaacggga ctttccaaaa
tgtcgtaaca actccgcccc 840attgacgcaa atgggcggta ggcgtgtacg
gtgggaggtc tatataagca gagctcgttt 900agtgaaccgt cagatcgcct
ggagacgcca tccacgctgt tttgacctcc atagaagaca 960ccgggaccga
tccagcctcc gcggattcga atcccggccg ggaacggtgc attggaacgc
1020ggattccccg tgccaagagt gacgtaagta ccgcctatag agtctatagg
cccacaaaaa 1080atgctttctt cttttaatat acttttttgt ttatcttatt
tctaatactt tccctaatct 1140ctttctttca gggcaataat gatacaatgt
atcatgcctc tttgcaccat tctaaagaat 1200aacagtgata atttctgggt
taaggcaata gcaatatttc tgcatataaa tatttctgca 1260tataaattgt
aactgatgta agaggtttca tattgctaat agcagctaca atccagctac
1320cattctgctt ttattttatg gttgggataa ggctggatta ttctgagtcc
aagctaggcc 1380cttttgctaa tcatgttcat acctcttatc ttcctcccac
agctcctggg caacgtgctg 1440gtctgtgtgc tggcccatca ctttggcaaa
gaattgggat tcgaacatcg gccgccacca 1500tgaacgcaag tgaattccga
aggagaggga aggagatggt ggattacgtg gccaactaca 1560tggaaggcat
tgagggacgc caggtctacc ctgacgtgga gcccgggtac ctgcggccgc
1620tgatccctgc cgctgcccct caggagccag acacgtttga ggacatcatc
aacgacgttg 1680agaagataat catgcctggg gtgacgcact ggcacagccc
ctacttcttc gcctacttcc 1740ccactgccag ctcgtacccg gccatgcttg
cggacatgct gtgcggggcc attggctgca 1800tcggcttctc ctgggcggca
agcccagcat gcacagagct ggagactgtg atgatggact 1860ggctcgggaa
gatgctggaa ctaccaaagg catttttgaa tgagaaagct ggagaagggg
1920gaggagtgat ccagggaagt gccagtgaag ccaccctggt ggccctgctg
gccgctcgga 1980ccaaagtgat ccatcggctg caggcagcgt ccccagagct
cacacaggcc gctatcatgg 2040agaagctggt ggcttactca tccgatcagg
cacactcctc agtggaaaga gctgggttaa 2100ttggtggagt gaaattaaaa
gccatcccct cagatggcaa cttcgccatg cgtgcgtctg 2160ccctgcagga
agccctggag agagacaaag cggctggcct gattcctttc tttatggttg
2220ccaccctggg gaccacaaca tgctgctcct ttgacaatct cttagaagtc
ggtcctatct 2280gcaacaagga agacatatgg ctgcacgttg atgcagccta
cgcaggcagt gcattcatct 2340gccctgagtt ccggcacctt ctgaatggag
tggagtttgc agattcattc aactttaatc 2400cccacaaatg gctattggtg
aattttgact gttctgccat gtgggtgaaa aagagaacag 2460acttaacggg
agcctttaga ctggacccca cttacctgaa gcacagccat caggattcag
2520ggcttatcac tgactaccgg cattggcaga taccactggg cagaagattt
cgctctttga 2580aaatgtggtt tgtatttagg atgtatggag tcaaaggact
gcaggcttat atccgcaagc 2640atgtccagct gtcccatgag tttgagtcac
tggtgcgcca ggatccccgc tttgaaatct 2700gtgtggaagt cattctgggg
cttgtctgct ttcggctaaa gggttccaac aaagtgaatg 2760aagctcttct
gcaaagaata aacagtgcca aaaaaatcca cttggttcca tgtcacctca
2820gggacaagtt tgtcctgcgc tttgccatct gttctcgcac ggtggaatct
gcccatgtgc 2880agcgggcctg ggaacacatc aaagagctgg cggccgacgt
gctgcgagca gagagggagt 2940aggagtgaag ccagctgcag gaatcaaaaa
ttgaagagag atatatctga aaactggaat 3000aagaagcaaa taaatatcat
cctgccttca tggaactcag ctgtctgtgg cttcccatgt 3060ctttctccaa
agttatccag agggttgtga ttttgtctgc ttagtatctc atcaacaaag
3120aaatattatt tgctaattaa aaagttaatc ttcatggcca tagcttttat
tcattagctg 3180tgatttttgt tgattaaaac attatagatt ttcatgttct
tgcagtcatc agaagtggta 3240ggaaagcctc actgatatat tttccagggc
aatcaatgtt cacgcaactt gaaattatat 3300ctgtggtctt caaattgtct
tttgtcatgt ggctaaatgc ctaataagct gctcgagaga 3360tctacgggtg
gcatccctgt gacccctccc cagtgcctct cctggccctg gaagttgcca
3420ctccagtgcc caccagcctt gtcctaataa aattaagttg catcattttg
tctgactagg 3480tgtccttcta taatattatg gggtggaggg gggtggtatg
gagcaagggg caagttggga 3540agacaacctg tagggcctgc ggggtctatt
gggaaccaag ctggagtgca gtggcacaat 3600cttggctcac tgcaatctcc
gcctcctggg ttcaagcgat tctcctgcct cagcctcccg 3660agttgttggg
attccaggca tgcatgacca ggctcagcta atttttgttt ttttggtaga
3720gacggggttt caccatattg gccaggctgg tctccaactc ctaatctcag
gtgatctacc 3780caccttggcc tcccaaattg ctgggattac aggcgtgaac
cactgctccc ttccctgtcc 3840ttctgatttt gtaggtaacc acgtgcggac
cgagcggccg cagttaatta agctcgcgaa 3900ggaaccccta gtgatggagt
tggccactcc ctctctgcgc gctcgctcgc tcactgaggc 3960cgggcgacca
aaggtcgccc gacgcccggg ctttgcccgg gcggcctcag tgagcgagcg
4020agcgcgcaga gagggagtgg ccaagacgat ttaaatggta ccgagctcga
attcactggc 4080cgtcgtttta caacgtcgtg actgggaaaa ccctggcgtt
acccaactta atcgccttgc 4140agcacatccc cctttcgcca gctggcgtaa
tagcgaagag gcccgcaccg atcgcccttc 4200ccaacagttg cgcagcctga
atggcgaatg gcgcctgatg cggtattttc tccttacgca 4260tctgtgcggt
atttcacacc gcatatggtg cactctcagt acaatctgct ctgatgccgc
4320atagttaagc cagccccgac acccgccaac acccgctgac gcgccctgac
gggcttgtct 4380gctcccggca tccgcttaca gacaagctgt gaccgtctca
gaacccaaat gtgaccaacc 4440gtttacatat atcttggttt attatttcaa
tttctctttt caaacatctt cttctgtcgt 4500tgtgcaattt tccaaatgcc
aatttggtcg cttggctatt tccaaacgat ttcatatatt 4560caagttggag
atatagacag tcccctgaac acctcgcatc ctctgctttt tatgcctgct
4620cgtcctgtgg gtataatttt aatggtcaaa tcgctgctgg ggaattgtat
acgctgctct 4680tgtgtctcac tgcctccctt gtagaaatgg aaattgcttt
tccattattt ccatgtgatt 4740ttgttggttt tgccatcgaa ttggcattga
aaacgcataa aaataagcat ttcgcatttg 4800caaagatcca aaagatttat
ttgcacgctg agcctgggga tacacgaaat gtatttgtaa 4860gattcttcag
atgagtttgg aacgcacgga aattaccgtc ttgggatttg ttggacatcg
4920catttaagca tgatgtatca tttaattttt tccaatttaa aaacaaataa
tcagtgctac 4980gattaagact ggatctatta tagacttctt caaagttttt
cagttttctt taaaattacc 5040tccataaaac tcttttaata ttttgaaaat
catctccagg ttacatttaa tctgattaag 5100tttagagtca gttagtttcc
cacagttttc ttcttgaaat aaacccatta agtaacccca 5160actgatcggc
gcaaatgatt cagctatgca gaaaatatga ttgatgattt ttccctccca
5220ccagccctta acacttgctt aacccttttg tgtggccatt atagcccttt
cctcgtagtc 5280tgcttaaagt tcattaataa gttttgcgtg agccaaaaat
ggcgccgctt tggaatcgtg 5340acagcgtgga aactctggac tttggggttg
caaaacttcg gaagctgaaa gcattttgct 5400gtagccatcg cattttgggg
gaagaaatcc gaattcgagt ccaaaccgaa ttctaataga 5460agttttgcag
caaagtgcct cagacaggtt aatgaccaaa agcagttttg cattcaaatg
5520ctgcagcaac cctcgcacga gggttgtcat gacggacgaa aatctaattt
acaacatgca 5580actgccactt tgaggggtga cttttaccca agacgtggct
gtgaaggaaa ttctgcgaga 5640aagagattaa ttacgctctg ggggcagtta
atgaatgctt gcgcggggcg ataaggggtt 5700aacgtttggc cagctcaaat
taccgaatgt ataacatttt gatgagcatg acaaaaattg 5760gaattttcaa
ctcttttttc ctgcgaaaag cataactttt ccattaacac aatggcgacg
5820atgagccggc aaatcgacga taccaaatcg gcataacgtg catgaaaatt
atacatgatt 5880gatttcatat taatttctac gaatgttatg aatcggctaa
aagatgagaa tggaaatatt 5940gcacgatttt gattttgtca tgatgacaaa
aaagggttgt aagatttgta catgccaaaa 6000tcataaactt ttaacagaaa
tcaaacacaa agaagtaata taacattaaa gtacacaggc 6060cttgtcacag
agatagaaaa atgtagctat tattaaaata tttaacattc tcttggtgtc
6120tgcaactttt ctatcagtcg cttttttatt ccctttccaa cacttattta
aaatgaaatg 6180cactcgaaat taactaattt aatggctcat atcctttgtg
ctgcttaaaa aggcaggaaa 6240cgttttttgt gccctacacc tcctccattc
aaatgaaaaa ggcggtggag tcgaggaaaa 6300tgtctgccct ggagcttgac
aagccaagat atggcccatc aaaaatgttc gaatgttctt 6360tttaaataaa
tgtctcatta tatggctgcc acgcccacac cgctcaacgc ccacacacgt
6420gccgccttcc ggtttagttg ggtggtacct gaccgtagat ttttcactgc
ggacatgtgt 6480ccaatcagtg gcaacgtgca acgaaccgtg gcaaacacag
ggaaagaacc tcgaagaccc 6540acagcaaact catcctctcg aatttgacac
tatgataatg cttatattca attttttgca 6600acattctatt tacactaaac
tgagaaacag gacaaagttg agaatgggtg ctgtgattat 6660ttgtcttgtg
ccaatatatg ccctcctgca tataaaatac gagatacaaa accaaacgaa
6720ataagaacga acaagaaatc agtttaattt caatgctgat caaaccaagt
tgacaactct 6780gttgtatatt agctctaata atgacttaag gttcgaaatt
taagtaactt ccaaaatggg 6840ccagataaat actgctaata tcgaatgcaa
ttcaatgagg aaatagtaat caatcagcca 6900accctaatga aattaataat
gtaatttaaa actttattcc acccattcta ataatgaaaa 6960tcattaaata
tatgtagcaa tccaattttg ttaatacata ttgtatatcc ctgatttgtc
7020aataccaaag accagaaaaa acaccatgcc aaattccttt tattcgttga
aaatcgaaac 7080aactttgaag tattaacctg ctggctaaaa aatatgtgta
ttactaaata taccatgtaa 7140tcaaaagttc gaaataaacg tgaaatggca
gctccactcg tcagccgtgc cacttttcga 7200ctaatcaaaa tgccttttga
atgcctgagt tgaatcaagt tggcttaata gttattatcc 7260cacgcaagca
aagtcaatgg cagagggaaa tgcgtagaaa ttagcacaga cttgaatgga
7320cgcacgagtg tcgaatggca ggagagtggg aaatgaacgg ggagttttcc
aggataagga 7380tgttttcgaa gtatcgtaat catttatagc ctccagatac
gggagctgca tgtgtcagag 7440gttttcaccg tcatcaccga aacgcgcgat
gcagctctgg cccgtgtctc aaaatctctg 7500atgttacatt gcacaagata
aaaatatatc atcatgaaca ataaaactgt ctgcttacat 7560aaacagtaat
acaaggggtg ttgaagatcc tttgatcttt tctacggggt ctgacgctca
7620gtggaacgaa aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa
ggatcttcac 7680ctagatcctt ttaaattaaa aatgaagttt taaatcaagc
ccaatctgaa taatgttaca 7740accaattaac caattctgat tagaaaaact
catcgagcat caaatgaaac tgcaatttat 7800tcatatcagg attatcaata
ccatattttt gaaaaagccg tttctgtaat gaaggagaaa 7860actcaccgag
gcagttccat aggatggcaa gatcctggta tcggtctgcg attccgactc
7920gtccaacatc aatacaacct attaatttcc cctcgtcaaa aataaggtta
tcaagtgaga 7980aatcaccatg agtgacgact gaatccggtg agaatggcaa
aagtttatgc atttctttcc 8040agacttgttc aacaggccag ccattacgct
cgtcatcaaa atcactcgca tcaaccaaac 8100cgttattcat tcgtgattgc
gcctgagcga gacgaaatac gcgatcgctg ttaaaaggac 8160aattacaaac
aggaatcgaa tgcaaccggc gcaggaacac tgccagcgca tcaacaatat
8220tttcacctga atcaggatat tcttctaata cctggaatgc tgtttttccg
gggatcgcag 8280tggtgagtaa ccatgcatca tcaggagtac ggataaaatg
cttgatggtc ggaagaggca 8340taaattccgt cagccagttt agtctgacca
tctcatctgt aacatcattg gcaacgctac 8400ctttgccatg tttcagaaac
aactctggcg catcgggctt cccatacaag cgatagattg 8460tcgcacctga
ttgcccgaca ttatcgcgag cccatttata cccatataaa tcagcatcca
8520tgttggaatt taatcgcggc ctcgacgttt cccgttgaat atggctcata
acaccccttg 8580tattactgtt tatgtaagca gacagtttta ttgttcatga
tgatatattt ttatcttgtg 8640caatgtaaca tcagagattt tgagacacgg
gccagagctg catcgcgcgt tttcagaatt 8700ggttaattgg ttgtaacatt
attcagattg ggcttgattt aaaacttcat ttttaattta 8760aaaggatcta
ggtgaagatc ctttttgata atctcatgac caaaatccct taacgtgagt
8820tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa aggatcttct
ttccataggc 8880tccgcccccc tgacgagcat cacaaaaatc gacgctcaag
tcagaggtgg cgaaacccga 8940caggactata aagataccag gcgtttcccc
ctggaagctc cctcgtgcgc tctcctgttc 9000cgaccctgcc gcttaccgga
tacctgtccg cctttctccc ttcgggaagc gtggcgcttt 9060ctcatagctc
acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc aagctgggct
9120gtgtgcacga accccccgtt cagcccgacc gctgcgcctt atccggtaac
tatcgtcttg 9180agtccaaccc ggtaagacac gacttatcgc cactggcagc
agccactggt aacaggatta 9240gcagagcgag gtatgtaggc ggtgctacag
agttcttgaa gtggtggcct aactacggct 9300acactagaag aacagtattt
ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa 9360gagttggtag
ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt
9420gcaagcagca gattacgcgc agaaaaaaag gatctcaaaa cgccagcaac
gcggcctttt 9480tacggttcct ggccttttgc tggccttttg ctcacatgtt
ctttcctgcg ttatcccctg 9540attctgtgga taaccgtatt accgcctttg
agtgagctga taccgctcgc cgcagccgaa 9600cgaccgagcg cagcgagtca
gtgagcgagg aagcggaaga gcgcccaata cgcaaaccgc 9660ctctc
9665166236DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 16cccgcgcgtt ggccgattca ttaatgcagc
tggcacgaca ggtttcccga ctggaaagcg 60ggcagtgagc gcaacgcaat tttcacacag
gaaacagcta tgaccatgat tacgccaagc 120ttgcatgcat ttaaatgacg
cagctgcgcg ctcgctcgct cactgaggcc gcccgggcaa 180agcccgggcg
tcgggcgacc tttggtcgcc cggcctcagt gagcgagcga gcgcgcagag
240agggagtggc caactccatc actaggggtt gagtttaaac ttcgtcgacg
atctgcggcc 300gcacgcgtgg agctagttat taatagtaat caattacggg
gtcattagtt catagcccat 360atatggagtt ccgcgttaca taacttacgg
taaatggccc gcctggctga ccgcccaacg 420acccccgccc attgacgtca
ataatgacgt atgttcccat agtaacgtca atagggactt 480tccattgacg
tcaatgggtg gagtatttac ggtaaactgc ccacttggca gtacatcaag
540tgtatcatat gccaagtacg ccccctattg acgtcaatga cggtaaatgg
cccgcctggc 600attatgccca gtacatgacc ttatgggact ttcctacttg
gcagtacatc tacgtattag 660tcatcgctat taccatggtg atgcggtttt
ggcagtacat caatgggcgt ggatagcggt 720ttgactcacg gggatttcca
agtctccacc ccattgacgt caatgggagt ttgttttgca 780ccaaaatcaa
cgggactttc caaaatgtcg taacaactcc gccccattga cgcaaatggg
840cggtaggcgt gtacggtggg aggtctatat aagcagagct cgtttagtga
accgtcagat 900cgcctggaga cgccatccac gctgttttga cctccataga
agacaccggg accgatccag 960cctccgcgga ttcgaatccc ggccgggaac
ggtgcattgg aacgcggatt ccccgtgcca 1020agagtgacgt aagtaccgcc
tatagagtct ataggcccac aaaaaatgct ttcttctttt 1080aatatacttt
tttgtttatc ttatttctaa tactttccct aatctctttc tttcagggca
1140ataatgatac aatgtatcat gcctctttgc accattctaa agaataacag
tgataatttc 1200tgggttaagg caatagcaat atttctgcat ataaatattt
ctgcatataa attgtaactg 1260atgtaagagg tttcatattg ctaatagcag
ctacaatcca gctaccattc tgcttttatt 1320ttatggttgg gataaggctg
gattattctg agtccaagct aggccctttt gctaatcatg 1380ttcatacctc
ttatcttcct cccacagctc ctgggcaacg tgctggtctg tgtgctggcc
1440catcactttg gcaaagaatt gggattcgaa catcggccgc caccatgaac
gcaagtgaat 1500tccgaaggag agggaaggag atggtggatt acgtggccaa
ctacatggaa ggcattgagg 1560gacgccaggt ctaccctgac gtggagcccg
ggtacctgcg gccgctgatc cctgccgctg 1620cccctcagga gccagacacg
tttgaggaca tcatcaacga cgttgagaag ataatcatgc 1680ctggggtgac
gcactggcac agcccctact tcttcgccta cttccccact gccagctcgt
1740acccggccat gcttgcggac atgctgtgcg gggccattgg ctgcatcggc
ttctcctggg 1800cggcaagccc agcatgcaca gagctggaga ctgtgatgat
ggactggctc gggaagatgc
1860tggaactacc aaaggcattt ttgaatgaga aagctggaga agggggagga
gtgatccagg 1920gaagtgccag tgaagccacc ctggtggccc tgctggccgc
tcggaccaaa gtgatccatc 1980ggctgcaggc agcgtcccca gagctcacac
aggccgctat catggagaag ctggtggctt 2040actcatccga tcaggcacac
tcctcagtgg aaagagctgg gttaattggt ggagtgaaat 2100taaaagccat
cccctcagat ggcaacttcg ccatgcgtgc gtctgccctg caggaagccc
2160tggagagaga caaagcggct ggcctgattc ctttctttat ggttgccacc
ctggggacca 2220caacatgctg ctcctttgac aatctcttag aagtcggtcc
tatctgcaac aaggaagaca 2280tatggctgca cgttgatgca gcctacgcag
gcagtgcatt catctgccct gagttccggc 2340accttctgaa tggagtggag
tttgcagatt cattcaactt taatccccac aaatggctat 2400tggtgaattt
tgactgttct gccatgtggg tgaaaaagag aacagactta acgggagcct
2460ttagactgga ccccacttac ctgaagcaca gccatcagga ttcagggctt
atcactgact 2520accggcattg gcagatacca ctgggcagaa gatttcgctc
tttgaaaatg tggtttgtat 2580ttaggatgta tggagtcaaa ggactgcagg
cttatatccg caagcatgtc cagctgtccc 2640atgagtttga gtcactggtg
cgccaggatc cccgctttga aatctgtgtg gaagtcattc 2700tggggcttgt
ctgctttcgg ctaaagggtt ccaacaaagt gaatgaagct cttctgcaaa
2760gaataaacag tgccaaaaaa atccacttgg ttccatgtca cctcagggac
aagtttgtcc 2820tgcgctttgc catctgttct cgcacggtgg aatctgccca
tgtgcagcgg gcctgggaac 2880acatcaaaga gctggcggcc gacgtgctgc
gagcagagag ggagtaggag tgagctgctc 2940gagagatcta cgggtggcat
ccctgtgacc cctccccagt gcctctcctg gccctggaag 3000ttgccactcc
agtgcccacc agccttgtcc taataaaatt aagttgcatc attttgtctg
3060actaggtgtc cttctataat attatggggt ggaggggggt ggtatggagc
aaggggcaag 3120ttgggaagac aacctgtagg gcctgcgggg tctattggga
accaagctgg agtgcagtgg 3180cacaatcttg gctcactgca atctccgcct
cctgggttca agcgattctc ctgcctcagc 3240ctcccgagtt gttgggattc
caggcatgca tgaccaggct cagctaattt ttgttttttt 3300ggtagagacg
gggtttcacc atattggcca ggctggtctc caactcctaa tctcaggtga
3360tctacccacc ttggcctccc aaattgctgg gattacaggc gtgaaccact
gctcccttcc 3420ctgtccttct gattttgtag gtaaccacgt gcggaccgag
cggccgcagt taattaagct 3480cgcgaaaccc ctagtgatgg agttggccac
tccctctctg cgcgctcgct cgctcactga 3540ggccgggcga ccaaaggtcg
cccgacgccc gggctttgcc cgggcggcct cagtgagcga 3600gcgagcgcgc
agctggacga tttaaatggt accgagctcg aattcactgg ccgtcgtttt
3660acaacgtcgt gactgggaaa accctggcgt tacccaactt aatcgccttg
cagcacatcc 3720ccctttcgcc agctggcgta atagcgaaga ggcccgcacc
gatcgccctt cccaacagtt 3780gcgcagcctg aatggcgaat ggcgcctgat
gcggtatttt ctccttacgc atctgtgcgg 3840tatttcacac cgcatatggt
gcactctcag tacaatctgc tctgatgccg catagttaag 3900ccagccccga
cacccgccaa cacccgctga cgcgccctga cgggcttgtc tgctcccggc
3960atccgcttac agacaagctg tgaccgtctc cgggagctgc atgtgtcaga
ggttttcacc 4020gtcatcaccg aaacgcgcga tgcagctctg gcccgtgtct
caaaatctct gatgttacat 4080tgcacaagat aaaaatatat catcatgaac
aataaaactg tctgcttaca taaacagtaa 4140tacaaggggt gttgaagatc
ctttgatctt ttctacgggg tctgacgctc agtggaacga 4200aaactcacgt
taagggattt tggtcatgag attatcaaaa aggatcttca cctagatcct
4260tttaaattaa aaatgaagtt ttaaatcaag cccaatctga ataatgttac
aaccaattaa 4320ccaattctga ttagaaaaac tcatcgagca tcaaatgaaa
ctgcaattta ttcatatcag 4380gattatcaat accatatttt tgaaaaagcc
gtttctgtaa tgaaggagaa aactcaccga 4440ggcagttcca taggatggca
agatcctggt atcggtctgc gattccgact cgtccaacat 4500caatacaacc
tattaatttc ccctcgtcaa aaataaggtt atcaagtgag aaatcaccat
4560gagtgacgac tgaatccggt gagaatggca aaagtttatg catttctttc
cagacttgtt 4620caacaggcca gccattacgc tcgtcatcaa aatcactcgc
atcaaccaaa ccgttattca 4680ttcgtgattg cgcctgagcg agacgaaata
cgcgatcgct gttaaaagga caattacaaa 4740caggaatcga atgcaaccgg
cgcaggaaca ctgccagcgc atcaacaata ttttcacctg 4800aatcaggata
ttcttctaat acctggaatg ctgtttttcc ggggatcgca gtggtgagta
4860accatgcatc atcaggagta cggataaaat gcttgatggt cggaagaggc
ataaattccg 4920tcagccagtt tagtctgacc atctcatctg taacatcatt
ggcaacgcta cctttgccat 4980gtttcagaaa caactctggc gcatcgggct
tcccatacaa gcgatagatt gtcgcacctg 5040attgcccgac attatcgcga
gcccatttat acccatataa atcagcatcc atgttggaat 5100ttaatcgcgg
cctcgacgtt tcccgttgaa tatggctcat aacacccctt gtattactgt
5160ttatgtaagc agacagtttt attgttcatg atgatatatt tttatcttgt
gcaatgtaac 5220atcagagatt ttgagacacg ggccagagct gcatcgcgcg
ttttcagaat tggttaattg 5280gttgtaacat tattcagatt gggcttgatt
taaaacttca tttttaattt aaaaggatct 5340aggtgaagat cctttttgat
aatctcatga ccaaaatccc ttaacgtgag ttttcgttcc 5400actgagcgtc
agaccccgta gaaaagatca aaggatcttc tttccatagg ctccgccccc
5460ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg
acaggactat 5520aaagatacca ggcgtttccc cctggaagct ccctcgtgcg
ctctcctgtt ccgaccctgc 5580cgcttaccgg atacctgtcc gcctttctcc
cttcgggaag cgtggcgctt tctcatagct 5640cacgctgtag gtatctcagt
tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg 5700aaccccccgt
tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc
5760cggtaagaca cgacttatcg ccactggcag cagccactgg taacaggatt
agcagagcga 5820ggtatgtagg cggtgctaca gagttcttga agtggtggcc
taactacggc tacactagaa 5880gaacagtatt tggtatctgc gctctgctga
agccagttac cttcggaaaa agagttggta 5940gctcttgatc cggcaaacaa
accaccgctg gtagcggtgg tttttttgtt tgcaagcagc 6000agattacgcg
cagaaaaaaa ggatctcaaa acgccagcaa cgcggccttt ttacggttcc
6060tggccttttg ctggcctttt gctcacatgt tctttcctgc gttatcccct
gattctgtgg 6120ataaccgtat taccgccttt gagtgagctg ataccgctcg
ccgcagccga acgaccgagc 6180gcagcgagtc agtgagcgag gaagcggaag
agcgcccaat acgcaaaccg cctctc 6236173497DNAArtificial
SequenceDescription of Artificial Sequence Synthetic polynucleotide
17cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc ccgggcaaag cccgggcgtc
60gggcgacctt tggtcgcccg gcctcagtga gcgagcgagc gcgcagagag ggagtggcca
120actccatcac taggggttcc tgagtttaaa cttcgtcgac gatctgcggc
cgcacgcgtg 180gagctagtta ttaatagtaa tcaattacgg ggtcattagt
tcatagccca tatatggagt 240tccgcgttac ataacttacg gtaaatggcc
cgcctggctg accgcccaac gacccccgcc 300cattgacgtc aataatgacg
tatgttccca tagtaacgtc aatagggact ttccattgac 360gtcaatgggt
ggagtattta cggtaaactg cccacttggc agtacatcaa gtgtatcata
420tgccaagtac gccccctatt gacgtcaatg acggtaaatg gcccgcctgg
cattatgccc 480agtacatgac cttatgggac tttcctactt ggcagtacat
ctacgtatta gtcatcgcta 540ttaccatggt gatgcggttt tggcagtaca
tcaatgggcg tggatagcgg tttgactcac 600ggggatttcc aagtctccac
cccattgacg tcaatgggag tttgttttgc accaaaatca 660acgggacttt
ccaaaatgtc gtaacaactc cgccccattg acgcaaatgg gcggtaggcg
720tgtacggtgg gaggtctata taagcagagc tcgtttagtg aaccgtcaga
tcgcctggag 780acgccatcca cgctgttttg acctccatag aagacaccgg
gaccgatcca gcctccgcgg 840attcgaatcc cggccgggaa cggtgcattg
gaacgcggat tccccgtgcc aagagtgacg 900taagtaccgc ctatagagtc
tataggccca caaaaaatgc tttcttcttt taatatactt 960ttttgtttat
cttatttcta atactttccc taatctcttt ctttcagggc aataatgata
1020caatgtatca tgcctctttg caccattcta aagaataaca gtgataattt
ctgggttaag 1080gcaatagcaa tatttctgca tataaatatt tctgcatata
aattgtaact gatgtaagag 1140gtttcatatt gctaatagca gctacaatcc
agctaccatt ctgcttttat tttatggttg 1200ggataaggct ggattattct
gagtccaagc taggcccttt tgctaatcat gttcatacct 1260cttatcttcc
tcccacagct cctgggcaac gtgctggtct gtgtgctggc ccatcacttt
1320ggcaaagaat tgggattcga acatcggccg ccaccatgaa cgcaagtgaa
ttccgaagga 1380gagggaagga gatggtggat tacgtggcca actacatgga
aggcattgag ggacgccagg 1440tctaccctga cgtggagccc gggtacctgc
ggccgctgat ccctgccgct gcccctcagg 1500agccagacac gtttgaggac
atcatcaacg acgttgagaa gataatcatg cctggggtga 1560cgcactggca
cagcccctac ttcttcgcct acttccccac tgccagctcg tacccggcca
1620tgcttgcgga catgctgtgc ggggccattg gctgcatcgg cttctcctgg
gcggcaagcc 1680cagcatgcac agagctggag actgtgatga tggactggct
cgggaagatg ctggaactac 1740caaaggcatt tttgaatgag aaagctggag
aagggggagg agtgatccag ggaagtgcca 1800gtgaagccac cctggtggcc
ctgctggccg ctcggaccaa agtgatccat cggctgcagg 1860cagcgtcccc
agagctcaca caggccgcta tcatggagaa gctggtggct tactcatccg
1920atcaggcaca ctcctcagtg gaaagagctg ggttaattgg tggagtgaaa
ttaaaagcca 1980tcccctcaga tggcaacttc gccatgcgtg cgtctgccct
gcaggaagcc ctggagagag 2040acaaagcggc tggcctgatt cctttcttta
tggttgccac cctggggacc acaacatgct 2100gctcctttga caatctctta
gaagtcggtc ctatctgcaa caaggaagac atatggctgc 2160acgttgatgc
agcctacgca ggcagtgcat tcatctgccc tgagttccgg caccttctga
2220atggagtgga gtttgcagat tcattcaact ttaatcccca caaatggcta
ttggtgaatt 2280ttgactgttc tgccatgtgg gtgaaaaaga gaacagactt
aacgggagcc tttagactgg 2340accccactta cctgaagcac agccatcagg
attcagggct tatcactgac taccggcatt 2400ggcagatacc actgggcaga
agatttcgct ctttgaaaat gtggtttgta tttaggatgt 2460atggagtcaa
aggactgcag gcttatatcc gcaagcatgt ccagctgtcc catgagtttg
2520agtcactggt gcgccaggat ccccgctttg aaatctgtgt ggaagtcatt
ctggggcttg 2580tctgctttcg gctaaagggt tccaacaaag tgaatgaagc
tcttctgcaa agaataaaca 2640gtgccaaaaa aatccacttg gttccatgtc
acctcaggga caagtttgtc ctgcgctttg 2700ccatctgttc tcgcacggtg
gaatctgccc atgtgcagcg ggcctgggaa cacatcaaag 2760agctggcggc
cgacgtgctg cgagcagaga gggagtagga gtgagctgct cgagagatct
2820acgggtggca tccctgtgac ccctccccag tgcctctcct ggccctggaa
gttgccactc 2880cagtgcccac cagccttgtc ctaataaaat taagttgcat
cattttgtct gactaggtgt 2940ccttctataa tattatgggg tggagggggg
tggtatggag caaggggcaa gttgggaaga 3000caacctgtag ggcctgcggg
gtctattggg aaccaagctg gagtgcagtg gcacaatctt 3060ggctcactgc
aatctccgcc tcctgggttc aagcgattct cctgcctcag cctcccgagt
3120tgttgggatt ccaggcatgc atgaccaggc tcagctaatt tttgtttttt
tggtagagac 3180ggggtttcac catattggcc aggctggtct ccaactccta
atctcaggtg atctacccac 3240cttggcctcc caaattgctg ggattacagg
cgtgaaccac tgctcccttc cctgtccttc 3300tgattttgta ggtaaccacg
tgcggaccga gcggccgcag ttaattaagc tcgcgaagga 3360acccctagtg
atggagttgg ccactccctc tctgcgcgct cgctcgctca ctgaggccgg
3420gcgaccaaag gtcgcccgac gcccgggctt tgcccgggcg gcctcagtga
gcgagcgagc 3480gcgcagctgc ctgcagg 3497183497DNAArtificial
SequenceDescription of Artificial Sequence Synthetic polynucleotide
18cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc ccgggcaaag cccgggcgtc
60gggcgacctt tggtcgcccg gcctcagtga gcgagcgagc gcgcagagag ggagtggcca
120actccatcac taggggttcc tgagtttaaa cttcgtcgac gatctgcggc
cgcacgcgtg 180gagctagtta ttaatagtaa tcaattacgg ggtcattagt
tcatagccca tatatggagt 240tccgcgttac ataacttacg gtaaatggcc
cgcctggctg accgcccaac gacccccgcc 300cattgacgtc aataatgacg
tatgttccca tagtaacgtc aatagggact ttccattgac 360gtcaatgggt
ggagtattta cggtaaactg cccacttggc agtacatcaa gtgtatcata
420tgccaagtac gccccctatt gacgtcaatg acggtaaatg gcccgcctgg
cattatgccc 480agtacatgac cttatgggac tttcctactt ggcagtacat
ctacgtatta gtcatcgcta 540ttaccatggt gatgcggttt tggcagtaca
tcaatgggcg tggatagcgg tttgactcac 600ggggatttcc aagtctccac
cccattgacg tcaatgggag tttgttttgc accaaaatca 660acgggacttt
ccaaaatgtc gtaacaactc cgccccattg acgcaaatgg gcggtaggcg
720tgtacggtgg gaggtctata taagcagagc tcgtttagtg aaccgtcaga
tcgcctggag 780acgccatcca cgctgttttg acctccatag aagacaccgg
gaccgatcca gcctccgcgg 840attcgaatcc cggccgggaa cggtgcattg
gaacgcggat tccccgtgcc aagagtgacg 900taagtaccgc ctatagagtc
tataggccca caaaaaatgc tttcttcttt taatatactt 960ttttgtttat
cttatttcta atactttccc taatctcttt ctttcagggc aataatgata
1020caatgtatca tgcctctttg caccattcta aagaataaca gtgataattt
ctgggttaag 1080gcaatagcaa tatttctgca tataaatatt tctgcatata
aattgtaact gatgtaagag 1140gtttcatatt gctaatagca gctacaatcc
agctaccatt ctgcttttat tttatggttg 1200ggataaggct ggattattct
gagtccaagc taggcccttt tgctaatcat gttcatacct 1260cttatcttcc
tcccacagct cctgggcaac gtgctggtct gtgtgctggc ccatcacttt
1320ggcaaagaat tgggattcga acatcggccg ccaccatgaa cgcaagtgaa
ttccgaagga 1380gagggaagga gatggtggat tacgtggcca actacatgga
aggcattgag ggacgccagg 1440tctaccctga cgtggagccc gggtacctgc
ggccgctgat ccctgccgct gcccctcagg 1500agccagacac gtttgaggac
atcatcaacg acgttgagaa gataatcatg cctggggtga 1560cgcactggca
cagcccctac ttcttcgcct acttccccac tgccagctcg tacccggcca
1620tgcttgcgga catgctgtgc ggggccattg gctgcatcgg cttctcctgg
gcggcaagcc 1680cagcatgcac agagctggag actgtgatga tggactggct
cgggaagatg ctggaactac 1740caaaggcatt tttgaatgag aaagctggag
aagggggagg agtgatccag ggaagtgcca 1800gtgaagccac cctggtggcc
ctgctggccg ctcggaccaa agtgatccat cggctgcagg 1860cagcgtcccc
agagctcaca caggccgcta tcatggagaa gctggtggct tactcatccg
1920atcaggcaca ctcctcagtg gaaagagctg ggttaattgg tggagtgaaa
ttaaaagcca 1980tcccctcaga tggcaacttc gccatgcgtg cgtctgccct
gcaggaagcc ctggagagag 2040acaaagcggc tggcctgatt cctttcttta
tggttgccac cctggggacc acaacatgct 2100gctcctttga caatctctta
gaagtcggtc ctatctgcaa caaggaagac atatggctgc 2160acgttgatgc
agcctacgca ggcagtgcat tcatctgccc tgagttccgg caccttctga
2220atggagtgga gtttgcagat tcattcaact ttaatcccca caaatggcta
ttggtgaatt 2280ttgactgttc tgccatgtgg gtgaaaaaga gaacagactt
aacgggagcc tttagactgg 2340accccactta cctgaagcac agccatcagg
attcagggct tatcactgac taccggcatt 2400ggcagatacc actgggcaga
agatttcgct ctttgaaaat gtggtttgta tttaggatgt 2460atggagtcaa
aggactgcag gcttatatcc gcaagcatgt ccagctgtcc catgagtttg
2520agtcactggt gcgccaggat ccccgctttg aaatctgtgt ggaagtcatt
ctggggcttg 2580tctgctttcg gctaaagggt tccaacaaag tgaatgaagc
tcttctgcaa agaataaaca 2640gtgccaaaaa aatccacttg gttccatgtc
acctcaggga caagtttgtc ctgcgctttg 2700ccatctgttc tcgcacggtg
gaatctgccc atgtgcagcg ggcctgggaa cacatcaaag 2760agctggcggc
cgacgtgctg cgagcagaga gggagtagga gtgagctgct cgagagatct
2820acgggtggca tccctgtgac ccctccccag tgcctctcct ggccctggaa
gttgccactc 2880cagtgcccac cagccttgtc ctaataaaat taagttgcat
cattttgtct gactaggtgt 2940ccttctataa tattatgggg tggagggggg
tggtatggag caaggggcaa gttgggaaga 3000caacctgtag ggcctgcggg
gtctattggg aaccaagctg gagtgcagtg gcacaatctt 3060ggctcactgc
aatctccgcc tcctgggttc aagcgattct cctgcctcag cctcccgagt
3120tgttgggatt ccaggcatgc atgaccaggc tcagctaatt tttgtttttt
tggtagagac 3180ggggtttcac catattggcc aggctggtct ccaactccta
atctcaggtg atctacccac 3240cttggcctcc caaattgctg ggattacagg
cgtgaaccac tgctcccttc cctgtccttc 3300tgattttgta ggtaaccacg
tgcggaccga gcggccgcag ttaattaagc tcgcgaagga 3360acccctagtg
atggagttgg ccactccctc tctgcgcgct cgctcgctca ctgaggccgg
3420gcgaccaaag gtcgcccgac gcccgggctt tgcccgggcg gcctcagtga
gcgagcgagc 3480gcgcagctgc ctgcagg 3497193491DNAArtificial
SequenceDescription of Artificial Sequence Synthetic polynucleotide
19cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc ccgggcaaag cccgggcgtc
60gggcgacctt tggtcgcccg gcctcagtga gcgagcgagc gcgcagagag ggagtggcca
120actccatcac taggggttcc tgagtttaaa cttcgtcgac gatctgcggc
cgcacgcgtg 180gagctagtta ttaatagtaa tcaattacgg ggtcattagt
tcatagccca tatatggagt 240tccgcgttac ataacttacg gtaaatggcc
cgcctggctg accgcccaac gacccccgcc 300cattgacgtc aataatgacg
tatgttccca tagtaacgtc aatagggact ttccattgac 360gtcaatgggt
ggagtattta cggtaaactg cccacttggc agtacatcaa gtgtatcata
420tgccaagtac gccccctatt gacgtcaatg acggtaaatg gcccgcctgg
cattatgccc 480agtacatgac cttatgggac tttcctactt ggcagtacat
ctacgtatta gtcatcgcta 540ttaccatggt gatgcggttt tggcagtaca
tcaatgggcg tggatagcgg tttgactcac 600ggggatttcc aagtctccac
cccattgacg tcaatgggag tttgttttgc accaaaatca 660acgggacttt
ccaaaatgtc gtaacaactc cgccccattg acgcaaatgg gcggtaggcg
720tgtacggtgg gaggtctata taagcagagc tcgtttagtg aaccgtcaga
tcgcctggag 780acgccatcca cgctgttttg acctccatag aagacaccgg
gaccgatcca gcctccgcgg 840attcgaatcc cggccgggaa cggtgcattg
gaacgcggat tccccgtgcc aagagtgacg 900taagtaccgc ctatagagtc
tataggccca caaaaaatgc tttcttcttt taatatactt 960ttttgtttat
cttatttcta atactttccc taatctcttt ctttcagggc aataatgata
1020caatgtatca tgcctctttg caccattcta aagaataaca gtgataattt
ctgggttaag 1080gcaatagcaa tatttctgca tataaatatt tctgcatata
aattgtaact gatgtaagag 1140gtttcatatt gctaatagca gctacaatcc
agctaccatt ctgcttttat tttatggttg 1200ggataaggct ggattattct
gagtccaagc taggcccttt tgctaatcat gttcatacct 1260cttatcttcc
tcccacagct cctgggcaac gtgctggtct gtgtgctggc ccatcacttt
1320ggcaaagaat tgggattcga acatcggccg ccaccatgaa cgccagcgag
ttcaggagga 1380ggggcaagga gatggtggac tacgtggcca actacatgga
gggcatcgag ggcaggcagg 1440tgtaccccga cgtggagccc ggctacctga
ggcccctgat ccccgccgcc gccccccagg 1500agcccgacac cttcgaggac
atcatcaacg acgtggagaa gatcatcatg cccggcgtga 1560cccactggca
cagcccctac ttcttcgcct acttccccac cgccagcagc taccccgcca
1620tgctggccga catgctgtgc ggcgccatcg gctgcatcgg cttcagctgg
gccgccagcc 1680ccgcctgcac cgagctggag accgtgatga tggactggct
gggcaagatg ctggagctgc 1740ccaaggcctt cctgaacgag aaggccggcg
agggcggcgg cgtgatccag ggcagcgcca 1800gcgaggccac cctggtggcc
ctgctggccg ccaggaccaa ggtgatccac aggctgcagg 1860ccgccagccc
cgagctgacc caggccgcca tcatggagaa gctggtggcc tacagcagcg
1920accaggccca cagcagcgtg gagagggccg gcctgatcgg cggcgtgaag
ctgaaggcca 1980tccccagcga cggcaacttc gccatgaggg ccagcgccct
gcaggaggcc ctggagaggg 2040acaaggccgc cggcctgatc cccttcttca
tggtggccac cctgggcacc accacctgct 2100gcagcttcga caacctgctg
gaggtgggcc ccatctgcaa caaggaggac atctggctgc 2160acgtggacgc
cgcctacgcc ggcagcgcct tcatctgccc cgagttcagg cacctgctga
2220acggcgtgga gttcgccgac agcttcaact tcaaccccca caagtggctg
ctggtgaact 2280tcgactgcag cgccatgtgg gtgaagaaga ggaccgacct
gaccggcgcc ttcaggctgg 2340accccaccta cctgaagcac agccaccagg
acagcggcct gatcaccgac tacaggcact 2400ggcagatccc cctgggcagg
aggttcagga gcctgaagat gtggttcgtg ttcaggatgt 2460acggcgtgaa
gggcctgcag gcctacatca ggaagcacgt gcagctgagc cacgagttcg
2520agagcctggt gaggcaggac cccaggttcg agatctgcgt ggaggtgatc
ctgggcctgg 2580tgtgcttcag gctgaagggc agcaacaagg tgaacgaggc
cctgctgcag aggatcaaca 2640gcgccaagaa gatccacctg gtgccctgcc
acctgaggga caagttcgtg ctgaggttcg 2700ccatctgcag caggaccgtg
gagagcgccc acgtgcagag ggcctgggag cacatcaagg 2760agctggccgc
cgacgtgctg agggccgaga gggagtgagc tgctcgagag atctacgggt
2820ggcatccctg tgacccctcc ccagtgcctc tcctggccct ggaagttgcc
actccagtgc 2880ccaccagcct tgtcctaata aaattaagtt gcatcatttt
gtctgactag gtgtccttct 2940ataatattat ggggtggagg ggggtggtat
ggagcaaggg gcaagttggg aagacaacct 3000gtagggcctg cggggtctat
tgggaaccaa gctggagtgc agtggcacaa tcttggctca 3060ctgcaatctc
cgcctcctgg gttcaagcga ttctcctgcc tcagcctccc gagttgttgg
3120gattccaggc atgcatgacc aggctcagct aatttttgtt tttttggtag
agacggggtt 3180tcaccatatt ggccaggctg gtctccaact cctaatctca
ggtgatctac ccaccttggc 3240ctcccaaatt gctgggatta caggcgtgaa
ccactgctcc cttccctgtc cttctgattt 3300tgtaggtaac cacgtgcgga
ccgagcggcc
gcagttaatt aagctcgcga aggaacccct 3360agtgatggag ttggccactc
cctctctgcg cgctcgctcg ctcactgagg ccgggcgacc 3420aaaggtcgcc
cgacgcccgg gctttgcccg ggcggcctca gtgagcgagc gagcgcgcag
3480ctgcctgcag g 3491204003DNAArtificial SequenceDescription of
Artificial Sequence Synthetic polynucleotide 20cctgcaggca
gctgcgcgct cgctcgctca ctgaggccgc ccgggcaaag cccgggcgtc 60gggcgacctt
tggtcgcccg gcctcagtga gcgagcgagc gcgcagagag ggagtggcca
120actccatcac taggggttcc tgagtttaaa cttcgtcgac gatctgcggc
cgcacgcgtg 180gagctagtta ttaatagtaa tcaattacgg ggtcattagt
tcatagccca tatatggagt 240tccgcgttac ataacttacg gtaaatggcc
cgcctggctg accgcccaac gacccccgcc 300cattgacgtc aataatgacg
tatgttccca tagtaacgtc aatagggact ttccattgac 360gtcaatgggt
ggagtattta cggtaaactg cccacttggc agtacatcaa gtgtatcata
420tgccaagtac gccccctatt gacgtcaatg acggtaaatg gcccgcctgg
cattatgccc 480agtacatgac cttatgggac tttcctactt ggcagtacat
ctacgtatta gtcatcgcta 540ttaccatggt gatgcggttt tggcagtaca
tcaatgggcg tggatagcgg tttgactcac 600ggggatttcc aagtctccac
cccattgacg tcaatgggag tttgttttgc accaaaatca 660acgggacttt
ccaaaatgtc gtaacaactc cgccccattg acgcaaatgg gcggtaggcg
720tgtacggtgg gaggtctata taagcagagc tcgtttagtg aaccgtcaga
tcgcctggag 780acgccatcca cgctgttttg acctccatag aagacaccgg
gaccgatcca gcctccgcgg 840attcgaatcc cggccgggaa cggtgcattg
gaacgcggat tccccgtgcc aagagtgacg 900taagtaccgc ctatagagtc
tataggccca caaaaaatgc tttcttcttt taatatactt 960ttttgtttat
cttatttcta atactttccc taatctcttt ctttcagggc aataatgata
1020caatgtatca tgcctctttg caccattcta aagaataaca gtgataattt
ctgggttaag 1080gcaatagcaa tatttctgca tataaatatt tctgcatata
aattgtaact gatgtaagag 1140gtttcatatt gctaatagca gctacaatcc
agctaccatt ctgcttttat tttatggttg 1200ggataaggct ggattattct
gagtccaagc taggcccttt tgctaatcat gttcatacct 1260cttatcttcc
tcccacagct cctgggcaac gtgctggtct gtgtgctggc ccatcacttt
1320ggcaaagaat tgggattcga acatcgaatt cgggcacgag ggaggacaga
gagcaagtca 1380ctcccggctg cctttttcac ctctgacaga gcccagacac
catgaacgca agtgaattcc 1440gaaggagagg gaaggagatg gtggattacg
tggccaacta catggaaggc attgagggac 1500gccaggtcta ccctgacgtg
gagcccgggt acctgcggcc gctgatccct gccgctgccc 1560ctcaggagcc
agacacgttt gaggacatca tcaacgacgt tgagaagata atcatgcctg
1620gggtgacgca ctggcacagc ccctacttct tcgcctactt ccccactgcc
agctcgtacc 1680cggccatgct tgcggacatg ctgtgcgggg ccattggctg
catcggcttc tcctgggcgg 1740caagcccagc atgcacagag ctggagactg
tgatgatgga ctggctcggg aagatgctgg 1800aactaccaaa ggcatttttg
aatgagaaag ctggagaagg gggaggagtg atccagggaa 1860gtgccagtga
agccaccctg gtggccctgc tggccgctcg gaccaaagtg atccatcggc
1920tgcaggcagc gtccccagag ctcacacagg ccgctatcat ggagaagctg
gtggcttact 1980catccgatca ggcacactcc tcagtggaaa gagctgggtt
aattggtgga gtgaaattaa 2040aagccatccc ctcagatggc aacttcgcca
tgcgtgcgtc tgccctgcag gaagccctgg 2100agagagacaa agcggctggc
ctgattcctt tctttatggt tgccaccctg gggaccacaa 2160catgctgctc
ctttgacaat ctcttagaag tcggtcctat ctgcaacaag gaagacatat
2220ggctgcacgt tgatgcagcc tacgcaggca gtgcattcat ctgccctgag
ttccggcacc 2280ttctgaatgg agtggagttt gcagattcat tcaactttaa
tccccacaaa tggctattgg 2340tgaattttga ctgttctgcc atgtgggtga
aaaagagaac agacttaacg ggagccttta 2400gactggaccc cacttacctg
aagcacagcc atcaggattc agggcttatc actgactacc 2460ggcattggca
gataccactg ggcagaagat ttcgctcttt gaaaatgtgg tttgtattta
2520ggatgtatgg agtcaaagga ctgcaggctt atatccgcaa gcatgtccag
ctgtcccatg 2580agtttgagtc actggtgcgc caggatcccc gctttgaaat
ctgtgtggaa gtcattctgg 2640ggcttgtctg ctttcggcta aagggttcca
acaaagtgaa tgaagctctt ctgcaaagaa 2700taaacagtgc caaaaaaatc
cacttggttc catgtcacct cagggacaag tttgtcctgc 2760gctttgccat
ctgttctcgc acggtggaat ctgcccatgt gcagcgggcc tgggaacaca
2820tcaaagagct ggcggccgac gtgctgcgag cagagaggga gtaggagtga
agccagctgc 2880aggaatcaaa aattgaagag agatatatct gaaaactgga
ataagaagca aataaatatc 2940atcctgcctt catggaactc agctgtctgt
ggcttcccat gtctttctcc aaagttatcc 3000agagggttgt gattttgtct
gcttagtatc tcatcaacaa agaaatatta tttgctaatt 3060aaaaagttaa
tcttcatggc catagctttt attcattagc tgtgattttt gttgattaaa
3120acattataga ttttcatgtt cttgcagtca tcagaagtgg taggaaagcc
tcactgatat 3180attttccagg gcaatcaatg ttcacgcaac ttgaaattat
atctgtggtc ttcaaattgt 3240cttttgtcat gtggctaaat gcctaataaa
caattcaagt gaaatactaa aaaaaaaaaa 3300aaaaaaaaaa gctgctcgag
agatctacgg gtggcatccc tgtgacccct ccccagtgcc 3360tctcctggcc
ctggaagttg ccactccagt gcccaccagc cttgtcctaa taaaattaag
3420ttgcatcatt ttgtctgact aggtgtcctt ctataatatt atggggtgga
ggggggtggt 3480atggagcaag gggcaagttg ggaagacaac ctgtagggcc
tgcggggtct attgggaacc 3540aagctggagt gcagtggcac aatcttggct
cactgcaatc tccgcctcct gggttcaagc 3600gattctcctg cctcagcctc
ccgagttgtt gggattccag gcatgcatga ccaggctcag 3660ctaatttttg
tttttttggt agagacgggg tttcaccata ttggccaggc tggtctccaa
3720ctcctaatct caggtgatct acccaccttg gcctcccaaa ttgctgggat
tacaggcgtg 3780aaccactgct cccttccctg tccttctgat tttgtaggta
accacgtgcg gaccgagcgg 3840ccgcagttaa ttaagctcgc gaaggaaccc
ctagtgatgg agttggccac tccctctctg 3900cgcgctcgct cgctcactga
ggccgggcga ccaaaggtcg cccgacgccc gggctttgcc 3960cgggcggcct
cagtgagcga gcgagcgcgc agctgcctgc agg 4003213499DNAArtificial
SequenceDescription of Artificial Sequence Synthetic polynucleotide
21ttggccactc cctctctgcg cgctcgctcg ctcactgagg ccgggcgacc aaaggtcgcc
60cgacgcccgg gctttgcccg ggcggcctca gtgagcgagc gagcgcgcag agagggagtg
120gccaactcca tcactagggg ttcctgagtt taaacttcgt cgacgatctg
cggccgcacg 180cgtggagcta gttattaata gtaatcaatt acggggtcat
tagttcatag cccatatatg 240gagttccgcg ttacataact tacggtaaat
ggcccgcctg gctgaccgcc caacgacccc 300cgcccattga cgtcaataat
gacgtatgtt cccatagtaa cgtcaatagg gactttccat 360tgacgtcaat
gggtggagta tttacggtaa actgcccact tggcagtaca tcaagtgtat
420catatgccaa gtacgccccc tattgacgtc aatgacggta aatggcccgc
ctggcattat 480gcccagtaca tgaccttatg ggactttcct acttggcagt
acatctacgt attagtcatc 540gctattacca tggtgatgcg gttttggcag
tacatcaatg ggcgtggata gcggtttgac 600tcacggggat ttccaagtct
ccaccccatt gacgtcaatg ggagtttgtt ttgcaccaaa 660atcaacggga
ctttccaaaa tgtcgtaaca actccgcccc attgacgcaa atgggcggta
720ggcgtgtacg gtgggaggtc tatataagca gagctcgttt agtgaaccgt
cagatcgcct 780ggagacgcca tccacgctgt tttgacctcc atagaagaca
ccgggaccga tccagcctcc 840gcggattcga atcccggccg ggaacggtgc
attggaacgc ggattccccg tgccaagagt 900gacgtaagta ccgcctatag
agtctatagg cccacaaaaa atgctttctt cttttaatat 960acttttttgt
ttatcttatt tctaatactt tccctaatct ctttctttca gggcaataat
1020gatacaatgt atcatgcctc tttgcaccat tctaaagaat aacagtgata
atttctgggt 1080taaggcaata gcaatatttc tgcatataaa tatttctgca
tataaattgt aactgatgta 1140agaggtttca tattgctaat agcagctaca
atccagctac cattctgctt ttattttatg 1200gttgggataa ggctggatta
ttctgagtcc aagctaggcc cttttgctaa tcatgttcat 1260acctcttatc
ttcctcccac agctcctggg caacgtgctg gtctgtgtgc tggcccatca
1320ctttggcaaa gaattgggat tcgaacatcg gccgccacca tgaacgccag
cgagttcagg 1380aggaggggca aggagatggt ggactacgtg gccaactaca
tggagggcat cgagggcagg 1440caggtgtacc ccgacgtgga gcccggctac
ctgaggcccc tgatccccgc cgccgccccc 1500caggagcccg acaccttcga
ggacatcatc aacgacgtgg agaagatcat catgcccggc 1560gtgacccact
ggcacagccc ctacttcttc gcctacttcc ccaccgccag cagctacccc
1620gccatgctgg ccgacatgct gtgcggcgcc atcggctgca tcggcttcag
ctgggccgcc 1680agccccgcct gcaccgagct ggagaccgtg atgatggact
ggctgggcaa gatgctggag 1740ctgcccaagg ccttcctgaa cgagaaggcc
ggcgagggcg gcggcgtgat ccagggcagc 1800gccagcgagg ccaccctggt
ggccctgctg gccgccagga ccaaggtgat ccacaggctg 1860caggccgcca
gccccgagct gacccaggcc gccatcatgg agaagctggt ggcctacagc
1920agcgaccagg cccacagcag cgtggagagg gccggcctga tcggcggcgt
gaagctgaag 1980gccatcccca gcgacggcaa cttcgccatg agggccagcg
ccctgcagga ggccctggag 2040agggacaagg ccgccggcct gatccccttc
ttcatggtgg ccaccctggg caccaccacc 2100tgctgcagct tcgacaacct
gctggaggtg ggccccatct gcaacaagga ggacatctgg 2160ctgcacgtgg
acgccgccta cgccggcagc gccttcatct gccccgagtt caggcacctg
2220ctgaacggcg tggagttcgc cgacagcttc aacttcaacc cccacaagtg
gctgctggtg 2280aacttcgact gcagcgccat gtgggtgaag aagaggaccg
acctgaccgg cgccttcagg 2340ctggacccca cctacctgaa gcacagccac
caggacagcg gcctgatcac cgactacagg 2400cactggcaga tccccctggg
caggaggttc aggagcctga agatgtggtt cgtgttcagg 2460atgtacggcg
tgaagggcct gcaggcctac atcaggaagc acgtgcagct gagccacgag
2520ttcgagagcc tggtgaggca ggaccccagg ttcgagatct gcgtggaggt
gatcctgggc 2580ctggtgtgct tcaggctgaa gggcagcaac aaggtgaacg
aggccctgct gcagaggatc 2640aacagcgcca agaagatcca cctggtgccc
tgccacctga gggacaagtt cgtgctgagg 2700ttcgccatct gcagcaggac
cgtggagagc gcccacgtgc agagggcctg ggagcacatc 2760aaggagctgg
ccgccgacgt gctgagggcc gagagggagt gagctgctcg agagatctac
2820gggtggcatc cctgtgaccc ctccccagtg cctctcctgg ccctggaagt
tgccactcca 2880gtgcccacca gccttgtcct aataaaatta agttgcatca
ttttgtctga ctaggtgtcc 2940ttctataata ttatggggtg gaggggggtg
gtatggagca aggggcaagt tgggaagaca 3000acctgtaggg cctgcggggt
ctattgggaa ccaagctgga gtgcagtggc acaatcttgg 3060ctcactgcaa
tctccgcctc ctgggttcaa gcgattctcc tgcctcagcc tcccgagttg
3120ttgggattcc aggcatgcat gaccaggctc agctaatttt tgtttttttg
gtagagacgg 3180ggtttcacca tattggccag gctggtctcc aactcctaat
ctcaggtgat ctacccacct 3240tggcctccca aattgctggg attacaggcg
tgaaccactg ctcccttccc tgtccttctg 3300attttgtagg taaccacgtg
cggaccgagc ggccgcagtt aattaagctc gcgaaggaac 3360ccctagtgat
ggagttggcc actccctctc tgcgcgctcg ctcgctcact gaggccgggc
3420gaccaaaggt cgcccgacgc ccgggctttg cccgggcggc ctcagtgagc
gagcgagcgc 3480gcagagaggg agtggccaa 3499223896DNAArtificial
SequenceDescription of Artificial Sequence Synthetic polynucleotide
22cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc ccgggcaaag cccgggcgtc
60gggcgacctt tggtcgcccg gcctcagtga gcgagcgagc gcgcagagag ggagtggcca
120actccatcac taggggttcc tgagtttaaa cttcgtcgac gatctgcggc
cgcacgcgtg 180gagctagtta ttaatagtaa tcaattacgg ggtcattagt
tcatagccca tatatggagt 240tccgcgttac ataacttacg gtaaatggcc
cgcctggctg accgcccaac gacccccgcc 300cattgacgtc aataatgacg
tatgttccca tagtaacgtc aatagggact ttccattgac 360gtcaatgggt
ggagtattta cggtaaactg cccacttggc agtacatcaa gtgtatcata
420tgccaagtac gccccctatt gacgtcaatg acggtaaatg gcccgcctgg
cattatgccc 480agtacatgac cttatgggac tttcctactt ggcagtacat
ctacgtatta gtcatcgcta 540ttaccatggt gatgcggttt tggcagtaca
tcaatgggcg tggatagcgg tttgactcac 600ggggatttcc aagtctccac
cccattgacg tcaatgggag tttgttttgc accaaaatca 660acgggacttt
ccaaaatgtc gtaacaactc cgccccattg acgcaaatgg gcggtaggcg
720tgtacggtgg gaggtctata taagcagagc tcgtttagtg aaccgtcaga
tcgcctggag 780acgccatcca cgctgttttg acctccatag aagacaccgg
gaccgatcca gcctccgcgg 840attcgaatcc cggccgggaa cggtgcattg
gaacgcggat tccccgtgcc aagagtgacg 900taagtaccgc ctatagagtc
tataggccca caaaaaatgc tttcttcttt taatatactt 960ttttgtttat
cttatttcta atactttccc taatctcttt ctttcagggc aataatgata
1020caatgtatca tgcctctttg caccattcta aagaataaca gtgataattt
ctgggttaag 1080gcaatagcaa tatttctgca tataaatatt tctgcatata
aattgtaact gatgtaagag 1140gtttcatatt gctaatagca gctacaatcc
agctaccatt ctgcttttat tttatggttg 1200ggataaggct ggattattct
gagtccaagc taggcccttt tgctaatcat gttcatacct 1260cttatcttcc
tcccacagct cctgggcaac gtgctggtct gtgtgctggc ccatcacttt
1320ggcaaagaat tgggattcga acatcggccg ccaccatgaa cgcaagtgaa
ttccgaagga 1380gagggaagga gatggtggat tacgtggcca actacatgga
aggcattgag ggacgccagg 1440tctaccctga cgtggagccc gggtacctgc
ggccgctgat ccctgccgct gcccctcagg 1500agccagacac gtttgaggac
atcatcaacg acgttgagaa gataatcatg cctggggtga 1560cgcactggca
cagcccctac ttcttcgcct acttccccac tgccagctcg tacccggcca
1620tgcttgcgga catgctgtgc ggggccattg gctgcatcgg cttctcctgg
gcggcaagcc 1680cagcatgcac agagctggag actgtgatga tggactggct
cgggaagatg ctggaactac 1740caaaggcatt tttgaatgag aaagctggag
aagggggagg agtgatccag ggaagtgcca 1800gtgaagccac cctggtggcc
ctgctggccg ctcggaccaa agtgatccat cggctgcagg 1860cagcgtcccc
agagctcaca caggccgcta tcatggagaa gctggtggct tactcatccg
1920atcaggcaca ctcctcagtg gaaagagctg ggttaattgg tggagtgaaa
ttaaaagcca 1980tcccctcaga tggcaacttc gccatgcgtg cgtctgccct
gcaggaagcc ctggagagag 2040acaaagcggc tggcctgatt cctttcttta
tggttgccac cctggggacc acaacatgct 2100gctcctttga caatctctta
gaagtcggtc ctatctgcaa caaggaagac atatggctgc 2160acgttgatgc
agcctacgca ggcagtgcat tcatctgccc tgagttccgg caccttctga
2220atggagtgga gtttgcagat tcattcaact ttaatcccca caaatggcta
ttggtgaatt 2280ttgactgttc tgccatgtgg gtgaaaaaga gaacagactt
aacgggagcc tttagactgg 2340accccactta cctgaagcac agccatcagg
attcagggct tatcactgac taccggcatt 2400ggcagatacc actgggcaga
agatttcgct ctttgaaaat gtggtttgta tttaggatgt 2460atggagtcaa
aggactgcag gcttatatcc gcaagcatgt ccagctgtcc catgagtttg
2520agtcactggt gcgccaggat ccccgctttg aaatctgtgt ggaagtcatt
ctggggcttg 2580tctgctttcg gctaaagggt tccaacaaag tgaatgaagc
tcttctgcaa agaataaaca 2640gtgccaaaaa aatccacttg gttccatgtc
acctcaggga caagtttgtc ctgcgctttg 2700ccatctgttc tcgcacggtg
gaatctgccc atgtgcagcg ggcctgggaa cacatcaaag 2760agctggcggc
cgacgtgctg cgagcagaga gggagtagga gtgaagccag ctgcaggaat
2820caaaaattga agagagatat atctgaaaac tggaataaga agcaaataaa
tatcatcctg 2880ccttcatgga actcagctgt ctgtggcttc ccatgtcttt
ctccaaagtt atccagaggg 2940ttgtgatttt gtctgcttag tatctcatca
acaaagaaat attatttgct aattaaaaag 3000ttaatcttca tggccatagc
ttttattcat tagctgtgat ttttgttgat taaaacatta 3060tagattttca
tgttcttgca gtcatcagaa gtggtaggaa agcctcactg atatattttc
3120cagggcaatc aatgttcacg caacttgaaa ttatatctgt ggtcttcaaa
ttgtcttttg 3180tcatgtggct aaatgcctaa taagctgctc gagagatcta
cgggtggcat ccctgtgacc 3240cctccccagt gcctctcctg gccctggaag
ttgccactcc agtgcccacc agccttgtcc 3300taataaaatt aagttgcatc
attttgtctg actaggtgtc cttctataat attatggggt 3360ggaggggggt
ggtatggagc aaggggcaag ttgggaagac aacctgtagg gcctgcgggg
3420tctattggga accaagctgg agtgcagtgg cacaatcttg gctcactgca
atctccgcct 3480cctgggttca agcgattctc ctgcctcagc ctcccgagtt
gttgggattc caggcatgca 3540tgaccaggct cagctaattt ttgttttttt
ggtagagacg gggtttcacc atattggcca 3600ggctggtctc caactcctaa
tctcaggtga tctacccacc ttggcctccc aaattgctgg 3660gattacaggc
gtgaaccact gctcccttcc ctgtccttct gattttgtag gtaaccacgt
3720gcggaccgag cggccgcagt taattaagct cgcgaaggaa cccctagtga
tggagttggc 3780cactccctct ctgcgcgctc gctcgctcac tgaggccggg
cgaccaaagg tcgcccgacg 3840cccgggcttt gcccgggcgg cctcagtgag
cgagcgagcg cgcagctgcc tgcagg 3896233475DNAArtificial
SequenceDescription of Artificial Sequence Synthetic polynucleotide
23cagctgcgcg ctcgctcgct cactgaggcc gcccgggcaa agcccgggcg tcgggcgacc
60tttggtcgcc cggcctcagt gagcgagcga gcgcgcagag agggagtggc caactccatc
120actaggggtt gagtttaaac ttcgtcgacg atctgcggcc gcacgcgtgg
agctagttat 180taatagtaat caattacggg gtcattagtt catagcccat
atatggagtt ccgcgttaca 240taacttacgg taaatggccc gcctggctga
ccgcccaacg acccccgccc attgacgtca 300ataatgacgt atgttcccat
agtaacgtca atagggactt tccattgacg tcaatgggtg 360gagtatttac
ggtaaactgc ccacttggca gtacatcaag tgtatcatat gccaagtacg
420ccccctattg acgtcaatga cggtaaatgg cccgcctggc attatgccca
gtacatgacc 480ttatgggact ttcctacttg gcagtacatc tacgtattag
tcatcgctat taccatggtg 540atgcggtttt ggcagtacat caatgggcgt
ggatagcggt ttgactcacg gggatttcca 600agtctccacc ccattgacgt
caatgggagt ttgttttgca ccaaaatcaa cgggactttc 660caaaatgtcg
taacaactcc gccccattga cgcaaatggg cggtaggcgt gtacggtggg
720aggtctatat aagcagagct cgtttagtga accgtcagat cgcctggaga
cgccatccac 780gctgttttga cctccataga agacaccggg accgatccag
cctccgcgga ttcgaatccc 840ggccgggaac ggtgcattgg aacgcggatt
ccccgtgcca agagtgacgt aagtaccgcc 900tatagagtct ataggcccac
aaaaaatgct ttcttctttt aatatacttt tttgtttatc 960ttatttctaa
tactttccct aatctctttc tttcagggca ataatgatac aatgtatcat
1020gcctctttgc accattctaa agaataacag tgataatttc tgggttaagg
caatagcaat 1080atttctgcat ataaatattt ctgcatataa attgtaactg
atgtaagagg tttcatattg 1140ctaatagcag ctacaatcca gctaccattc
tgcttttatt ttatggttgg gataaggctg 1200gattattctg agtccaagct
aggccctttt gctaatcatg ttcatacctc ttatcttcct 1260cccacagctc
ctgggcaacg tgctggtctg tgtgctggcc catcactttg gcaaagaatt
1320gggattcgaa catcggccgc caccatgaac gcaagtgaat tccgaaggag
agggaaggag 1380atggtggatt acgtggccaa ctacatggaa ggcattgagg
gacgccaggt ctaccctgac 1440gtggagcccg ggtacctgcg gccgctgatc
cctgccgctg cccctcagga gccagacacg 1500tttgaggaca tcatcaacga
cgttgagaag ataatcatgc ctggggtgac gcactggcac 1560agcccctact
tcttcgccta cttccccact gccagctcgt acccggccat gcttgcggac
1620atgctgtgcg gggccattgg ctgcatcggc ttctcctggg cggcaagccc
agcatgcaca 1680gagctggaga ctgtgatgat ggactggctc gggaagatgc
tggaactacc aaaggcattt 1740ttgaatgaga aagctggaga agggggagga
gtgatccagg gaagtgccag tgaagccacc 1800ctggtggccc tgctggccgc
tcggaccaaa gtgatccatc ggctgcaggc agcgtcccca 1860gagctcacac
aggccgctat catggagaag ctggtggctt actcatccga tcaggcacac
1920tcctcagtgg aaagagctgg gttaattggt ggagtgaaat taaaagccat
cccctcagat 1980ggcaacttcg ccatgcgtgc gtctgccctg caggaagccc
tggagagaga caaagcggct 2040ggcctgattc ctttctttat ggttgccacc
ctggggacca caacatgctg ctcctttgac 2100aatctcttag aagtcggtcc
tatctgcaac aaggaagaca tatggctgca cgttgatgca 2160gcctacgcag
gcagtgcatt catctgccct gagttccggc accttctgaa tggagtggag
2220tttgcagatt cattcaactt taatccccac aaatggctat tggtgaattt
tgactgttct 2280gccatgtggg tgaaaaagag aacagactta acgggagcct
ttagactgga ccccacttac 2340ctgaagcaca gccatcagga ttcagggctt
atcactgact accggcattg gcagatacca 2400ctgggcagaa gatttcgctc
tttgaaaatg tggtttgtat ttaggatgta tggagtcaaa 2460ggactgcagg
cttatatccg caagcatgtc cagctgtccc atgagtttga gtcactggtg
2520cgccaggatc cccgctttga aatctgtgtg gaagtcattc tggggcttgt
ctgctttcgg 2580ctaaagggtt ccaacaaagt gaatgaagct cttctgcaaa
gaataaacag tgccaaaaaa 2640atccacttgg ttccatgtca cctcagggac
aagtttgtcc tgcgctttgc catctgttct 2700cgcacggtgg aatctgccca
tgtgcagcgg gcctgggaac acatcaaaga gctggcggcc 2760gacgtgctgc
gagcagagag ggagtaggag tgagctgctc gagagatcta cgggtggcat
2820ccctgtgacc cctccccagt gcctctcctg gccctggaag ttgccactcc
agtgcccacc 2880agccttgtcc taataaaatt aagttgcatc attttgtctg
actaggtgtc cttctataat 2940attatggggt ggaggggggt ggtatggagc
aaggggcaag ttgggaagac aacctgtagg 3000gcctgcgggg tctattggga
accaagctgg
agtgcagtgg cacaatcttg gctcactgca 3060atctccgcct cctgggttca
agcgattctc ctgcctcagc ctcccgagtt gttgggattc 3120caggcatgca
tgaccaggct cagctaattt ttgttttttt ggtagagacg gggtttcacc
3180atattggcca ggctggtctc caactcctaa tctcaggtga tctacccacc
ttggcctccc 3240aaattgctgg gattacaggc gtgaaccact gctcccttcc
ctgtccttct gattttgtag 3300gtaaccacgt gcggaccgag cggccgcagt
taattaagct cgcgaaaccc ctagtgatgg 3360agttggccac tccctctctg
cgcgctcgct cgctcactga ggccgggcga ccaaaggtcg 3420cccgacgccc
gggctttgcc cgggcggcct cagtgagcga gcgagcgcgc agctg 3475
* * * * *
References