U.S. patent application number 15/407037 was filed with the patent office on 2017-05-11 for linkage modified gapped oligomeric compounds and uses thereof.
This patent application is currently assigned to Ionis Pharmaceuticals, Inc.. The applicant listed for this patent is Ionis Pharmaceuticals, Inc.. Invention is credited to Michael Oestergaard, Thazha P. Prakash, Punit P. Seth, Eric E. Swayze.
Application Number | 20170130224 15/407037 |
Document ID | / |
Family ID | 46717938 |
Filed Date | 2017-05-11 |
United States Patent
Application |
20170130224 |
Kind Code |
A1 |
Oestergaard; Michael ; et
al. |
May 11, 2017 |
LINKAGE MODIFIED GAPPED OLIGOMERIC COMPOUNDS AND USES THEREOF
Abstract
The present invention provides gapped oligomeric compounds. More
particularly the gapped oligomeric compounds provided herein
comprise at least one modified internucleoside linkage in the gap
region. Such gapped oligomeric compounds have one or more improved
properties such as selectivity, potency, improved toxicity profile
and or improved proinflammatory profile. Certain such oligomeric
compounds are useful for hybridizing to a complementary nucleic
acid, including but not limited, to nucleic acids in a cell. In
certain embodiments, hybridization results in modulation of the
amount activity or expression of the target nucleic acid in a
cell.
Inventors: |
Oestergaard; Michael;
(Calrsbad, CA) ; Prakash; Thazha P.; (Carlsbad,
CA) ; Seth; Punit P.; (Carlsbad, CA) ; Swayze;
Eric E.; (Encinitas, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Ionis Pharmaceuticals, Inc. |
Carlsbad |
CA |
US |
|
|
Assignee: |
Ionis Pharmaceuticals, Inc.
Carlsbad
CA
|
Family ID: |
46717938 |
Appl. No.: |
15/407037 |
Filed: |
January 16, 2017 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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14237967 |
May 7, 2014 |
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PCT/US2012/049987 |
Aug 8, 2012 |
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15407037 |
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61522659 |
Aug 11, 2011 |
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61596723 |
Feb 8, 2012 |
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61603196 |
Feb 24, 2012 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12N 2310/11 20130101;
C12N 15/111 20130101; C12N 2310/3341 20130101; C12N 2310/3231
20130101; C12N 2310/316 20130101; C12N 15/113 20130101; C12N
2310/321 20130101; C12N 2310/315 20130101; C12N 2320/34 20130101;
C12N 2310/341 20130101; C12N 2310/351 20130101; C12N 2310/312
20130101; C12N 2310/3125 20130101; C12N 2310/321 20130101; C12N
2310/3525 20130101 |
International
Class: |
C12N 15/113 20060101
C12N015/113 |
Claims
1. A gapped oligomeric compound comprising a contiguous sequence of
linked monomer subunits having a gap region located between a
5'-region and a 3'-region wherein the 5' and 3'-regions each,
independently, have from 2 to 8 contiguous RNA-like modified
nucleosides, each independently selected from a bicyclic nucleoside
comprising a bicyclic furanosyl sugar moiety and a modified
nucleoside comprising a furanosyl sugar moiety having at least one
substituent group that each adopt a 3'-endo conformational geometry
when put into an oligomeric compound and wherein the gap region has
from 8 to 10 contiguous .beta.-D-2'-deoxyribonucleosides and
wherein from one to three of the internucleoside linking groups
located between .beta.-D-2'-deoxyribonucleosides in the gap region
have Formula I: ##STR00023## wherein: each X is O or S; each Q is,
independently, selected from C.sub.1-C.sub.6 alkyl, substituted
C.sub.1-C.sub.6 alkyl, C.sub.2-C.sub.6 alkenyl, C(.dbd.O)OH and
CH.sub.2C(.dbd.O)OH; and each substituted group comprises one or
more optionally protected substituent groups independently selected
from halogen, OJ.sub.1, SJ.sub.1 and OC(.dbd.O)J.sub.1; each
J.sub.1 is, independently, H or C.sub.1-C.sub.6 alkyl; and wherein
each internucleoside linking group other than internucleoside
linking groups having Formula I is, independently, a phosphodiester
or a phosphorothioate internucleoside linking group.
2. The gapped oligomeric compound of claim 1 having only one
internucleoside linking group of Formula I.
3. The gapped oligomeric compound of claim 2 wherein the
internucleoside linking group of Formula I is located between
nucleosides 1 and 2, 2 and 3, or 3 and 4, counting from the 5' gap
junction.
4. The gapped oligomeric compound of claim 1 having only two
internucleoside linking groups of Formula I.
5. The gapped oligomeric compound of claim 4 wherein the
internucleoside linking groups of Formula I are located between
nucleosides 1 and 3, or 2 and 4, counting from the 5' gap
junction.
6. The gapped oligomeric compound of claim 1 wherein each
internucleoside linking group other than internucleoside linking
groups having Formula I is a phosphorothioate internucleoside
linking group.
7. The gapped oligomeric compound of claim 1 wherein each monomer
subunit comprises a heterocyclic base independently selected from
uracil, thymine, cytosine, 4-N-benzoylcytosine, 5-methylcytosine,
4-N-benzoyl-5-methylcytosine, adenine, 6-N-benzoyladenine, guanine
and 2-N-isobutyrylguanine.
8. The gapped oligomeric compound of claim 1 wherein each Q is,
independently, selected from CH.sub.3, C(.dbd.O)OH,
CH.sub.2C(.dbd.O)OH, (CH.sub.2).sub.2OCH.sub.3, CH.dbd.CH.sub.2,
CH.sub.2CH.dbd.CH.sub.2 and C.ident.CH.
9. The gapped oligomeric compound of claim 1 wherein each Q is,
independently, selected from C.sub.1-C.sub.6 alkyl and substituted
C.sub.1-C.sub.6 alkyl.
10. The gapped oligomeric compound of claim 9 wherein each Q is,
independently, substituted C.sub.1-C.sub.6 alkyl wherein the
substituent group is OJ.sub.1 and J.sub.1 is C.sub.1-C.sub.6
alkyl.
11. The gapped oligomeric compound of claim 9 wherein each Q is
CH.sub.3.
12. The gapped oligomeric compound of claim 1 wherein each X is
S.
13. The gapped oligomeric compound of claim 1 wherein one or more
of the RNA-like modified nucleosides each comprising a
2'-substituted furanosyl sugar moiety wherein each 2'-substituent
group is, independently, selected from halogen, OCH.sub.3,
OCH.sub.2F, OCHF.sub.2, OCF.sub.3, OCH.sub.2CH.sub.3,
O(CH.sub.2).sub.2F, OCH.sub.2CHF.sub.2, OCH.sub.2CF.sub.3,
OCH.sub.2--CH.dbd.CH.sub.2, O(CH.sub.2).sub.2--OCH.sub.3,
O(CH.sub.2).sub.2--SCH.sub.3, O(CH.sub.2).sub.2--OCF.sub.3,
O(CH.sub.2).sub.3--N(R.sub.3)(R.sub.4),
O(CH.sub.2).sub.2--ON(R.sub.3)(R.sub.4),
O(CH.sub.2).sub.2--O(CH.sub.2).sub.2--N(R.sub.3)(R.sub.4),
OCH.sub.2C(.dbd.O)--N(R.sub.4)(R.sub.4),
OCH.sub.2C(.dbd.O)--N(R.sub.5)--(CH.sub.2).sub.2--N(R.sub.3)(R.sub.4)
and
O(CH.sub.2).sub.2--N(R.sub.5)--C(.dbd.NR.sub.6)[N(R.sub.3)(R.sub.4)]
wherein R.sub.3, R.sub.4, R.sub.5 and R.sub.6 are each,
independently, H or C.sub.1-C.sub.6 alkyl.
14. The gapped oligomeric compound of claim 13 wherein each
2'-substituent group is independently selected from F, OCH.sub.3,
O(CH.sub.2).sub.2--OCH.sub.3 and
OCH.sub.2C(.dbd.O)--N(H)CH.sub.3.
15. The gapped oligomeric compound of claim 14 wherein each
2'-substituent group is O(CH.sub.2).sub.2--OCH.sub.3.
16. The gapped oligomeric compound of claim 1 wherein one or more
of the RNA-like modified nucleosides comprises a bicyclic furanosyl
sugar moiety each having a bridging group independently selected
from 4'-(CH.sub.2)--O-2', 4'-(CH.sub.2)--S-2',
4'-(CH.sub.2).sub.2--O-2', 4'-CH(CH.sub.3)--O-2',
4'-C--H(CH.sub.2OCH.sub.3)--O-2', 4'-C(CH.sub.3).sub.2--O-2',
4'-CH.sub.2--N(OCH.sub.3)-2', 4'-CH.sub.2--O--N(CH.sub.3)-2',
4'-CH.sub.2--C(H)(CH.sub.3)-2' and
4'-CH.sub.2--C(.dbd.CH.sub.2)-2'.
17. The gapped oligomeric compound of claim 18 wherein each
bridging group is 4'-CH[(S)--(CH.sub.3)]--O-2' or
4'-(CH.sub.2)--O-2'.
18. The gapped oligomeric compound of claim 1 wherein the sugar
moiety of each RNA-like modified nucleoside is the same.
19. The gapped oligomeric compound of claim 1 comprising at least
two different types of RNA-like modified nucleosides wherein the
different types of modified nucleosides have at least different
modified sugar moieties.
20. The gapped oligomeric compound of claim 19 wherein the
different types of RNA-like modified nucleosides include
4'-CH[(S)--(CH.sub.3)]--O-2' bicyclic nucleosides and
2'-O(CH.sub.2).sub.2--OCH.sub.3 substituted nucleosides.
21. The gapped oligomeric compound of claim 1 wherein the 5' and
3'-regions each, independently, have from 3 to 6 RNA-like modified
nucleosides.
21. The gapped oligomeric compound of claim 1 further comprising at
least one 5' or 3'-conjugate group.
23. A method of inhibiting gene expression comprising contacting
one or more cells, a tissue or an animal with an oligomeric
compound of claim 1 wherein said oligomeric compound is
complementary to a target RNA.
Description
FIELD OF THE INVENTION
[0001] The present invention pertains generally to
chemically-modified oligonucleotides for use in research,
diagnostics, and/or therapeutics.
SEQUENCE LISTING
[0002] The present application is being filed along with a Sequence
Listing in electronic format. The Sequence Listing is provided as a
file entitled CHEM0084USC1SEQ_ST25.txt, created Jan. 16, 2016,
which is 284 Kb in size. The information in the electronic format
of the sequence listing is incorporated herein by reference in its
entirety.
BACKGROUND OF THE INVENTION
[0003] Antisense compounds have been used to modulate target
nucleic acids. Antisense compounds comprising a variety of chemical
modifications and motifs have been reported. In certain instances,
such compounds are useful as research tools, diagnostic reagents,
and as therapeutic agents. In certain instances antisense compounds
have been shown to modulate protein expression by binding to a
target messenger RNA (mRNA) encoding the protein. In certain
instances, such binding of an antisense compound to its target mRNA
results in cleavage of the mRNA. Antisense compounds that modulate
processing of a pre-mRNA have also been reported. Such antisense
compounds alter splicing, interfere with polyadenlyation or prevent
formation of the 5'-cap of a pre-mRNA.
[0004] The synthesis and biochemical properties of oligonucleotides
containing phosphorus-modified phosphonoacetate and
thio-phosphonoacetate deoxyribonucleotides have been described in
scientific journals and patent literature (see Dellinger et al., J.
Am. Chem. Soc., 2003, 125, 940-950; Nucleic Acids Research, 2003,
31(14), 4109-4118); also see published US patent applications (US
2004/0116687 and US 2002/0058802) and U.S. Pat. No. 6,693,187.
[0005] DNA or RNA containing oligonucleotides comprising
alkylphosphonate internucleoside linkage backbone have been
disclosed (see U.S. Pat. Nos. 5,264,423 and 5,286,717).
[0006] The synthesis of oligodeoxyribonucleotides containing a
methyl phosphonate locked nucleic acid (LNA) thymine monomer has
been described. The Tm values of the duplexes with their DNA or RNA
complements have also been reported (see Lauritsen et al., Bioorg.
Med. Chem. Lett., 2003, 253-256).
[0007] Various dephosphono linkages (linkages without the
phosphorus atom) modifications have been synthesized and studied
for their antisense properties. Nonionic, achiral amide linkages
(De Mesmaeker et al., Chem. Int. Ed. Engl., 1994, 33, 226-229; Just
et al., Synlett, 1994, 137-139) were disclosed. A full account of
the synthesis and properties of the five isomeric amide
modifications was described (De Mesmaeker et al., 1994, Novel
Backbone Replacements for Oligonucleotides, In Carbohydrate
Modifications in Antisense Research; Y. S. Sanghvi and P. D. Cook
Eds. ACS Symposium Series 580:24-39). Gogoi et al. presented the
synthesis of thioacetamido nucleic acids (TANA) backbone and
thermal stability studies with complementary DNA and RNA sequences
(Gogoi et al., Chem. Commun., 2006, 2373-2375).
[0008] Several nitrogen containing backbone modifications similar
to the amides were evaluated as dimeric nucleosides (Sanghvi et
al., Nucleosides Nucleotides, 1997, 16, pp. 907-916). Peoc'h
reported the synthesis of four methylene(methylimino) (MMI) linked
oligodeoxyribonucleotide dimers modified at the 2'-position with
fluoro and/or methoxy groups and their incorporation into different
sequences (Peoc'h et al., Nucleosides, Nucleotides & Nucleic
Acids, 23, 411-438, 2004). Amino linkages have been synthesized and
studied for enhanced cellular absorption (Saha et. al., Tet. Lett.,
1993, 34, 6017-6020; De Mesmaeker et al., J. Bioorg. Med. Chem.
Lett. 1994, 4, 395-398; Caulfield et al, Bioorg. Med. Chem. Lett.
1993, 3, 2771-2776). Other nitrogen containing backbones include
oxime (Sanghvi et al., In Nucleosides and Nucleotides as Antitumor
and Antiviral Agents; C. K. Chu and D. C. Baker Eds.: Plenum Press:
New York, 1993, 311-324), methyleneimino (ibid), methyleneoxy
(methylimino) (MOMI) (ibid), methylene(dimethylhydrazo) (MDH)
(Sanghvi et al., Collect. Czech. Chem. Commun., 1993, 58, pp.
158-162), hydroxyl (methyliminomethylene) (HMIM) (Sanghvi et al.,
11.sup.th IRT Nucleosides & Nucleotides, Leuven, Belgium, Sep.
7-11, 1994 (poster presentation)), carbamate (Dabkowski et al., J
Chem. Soc. Perkin Trans. 1, 1994, 817-829), oxyamide linkage
(Burgess et al., J. Chem. Soc. Chem. Commun., 1994, 915-916),
N-substituted guanidine (Vandendrissche et al., J. Chem. Soc.,
1993, 1567-1575; Pannecouque et al., Tetrahedron, 1994, 50,
7231-7246), urea (Kutterer et al., Bioorg. Med. Chem. Lett., 1994,
3, 435-438) and thiourea linkages (Vandendrissche et al., J. Chem.
Soc. 1993, 1567-1575).
[0009] Synthesis of sulfur-containing backbone modifications, such
as sulfonamide (McElroy et al., Bioorg. Med. Chem. Lett., 1994, 4,
1071-1076), sulfamoyl (Dewynter et al., Acad. Sci., 1992, 315,
1675-1682), sulfonate (Huang et al., Synlett, 1993, 83-84), sulfide
(Wang et al., Chin. Chem. Lett., 1993, 4, 101-104; Huang et al.,
Synlett, 1993, 83-84; Kawai et al., Nucleic Acids Res., 1993, 21,
1473-1479; Meng et al., Angew. Chem. Int. Ed. Engl., 1993, 32,
729-731; Just et al. (1994), Synthesis and Hybridization Properties
of DNA Oligomers Containing Sulfide-Linked Dinucleosides. In
Carbohydrate Modifications in Antisense Research; Y. S. Sanghvi and
P. D. Cook Eds. ACS Symposium Series 580; (pp. 52-65)), and sulfone
linkages (Just et al. (1994), Synthesis and Hybridization
Properties of DNA Oligomers Containing Sulfide-Linked
Dinucleosides. In Carbohydrate Modifications in Antisense Research;
Y. S. Sanghvi and P. D. Cook Eds. ACS Symposium Series 580; (pp.
62-65)) have been accomplished by several research groups.
[0010] Another backbone substitution is the formacetal and the
related thioformacetal (Jones et al., J. Org. Chem., 1993, 58,
2983-2991). Matteucci reported the synthesis of oligonucleotide
analogs with one or more phosphodiester linkages that are replaced
by a formacetal/ketal type linkage (see U.S. Pat. No.
5,264,562).
[0011] Oligomeric compounds have been prepared using Click
chemistry wherein alkynyl phosphonate internucleoside linkages on
an oligomeric compound attached to a solid support are converted
into the 1,2,3-triazolylphosphonate internucleoside linkages and
then cleaved from the solid support (Krishna et al., J. Am. Chem.
Soc., DOI: 101021/ja3026714, published online May 21, 2012).
SUMMARY OF THE INVENTION
[0012] Provided herein are gapped oligomeric compounds comprising
at least one modified internucleoside linkage having Formula I in
the gap region. In certain embodiments, the oligomeric compounds
provided herein hybridize to a portion of a target RNA resulting in
loss of normal function of the target RNA. In certain embodiments,
the oligomeric compounds disclosed herein provide improved
selectivity for a target RNA. In certain embodiments, the
oligomeric compounds provide improved potency for a target RNA. In
certain embodiments, the oligomeric compounds provided herein
provide an improvement in the toxicity profile. In certain
embodiments, the oligomeric compounds provided herein provide an
improvement in the proinflammatory profile. In certain embodiments,
the oligomeric compounds provide improved potency and selectivity
for a target RNA. In certain embodiments, the oligomeric compounds
provide improved potency, selectivity and an improvement in the
proinflammatory profile.
[0013] The variables are defined individually in further detail
herein. It is to be understood that the oligomeric compounds
provided herein include all combinations of the embodiments
disclosed and variables defined herein.
[0014] In certain embodiments, gapped oligomeric compounds are
provided comprising a contiguous sequence of linked monomer
subunits having a gap region located between a 5'-region and a
3'-region wherein the 5' and 3'-regions each, independently, have
from 2 to 8 contiguous modified nucleosides wherein essentially
each modified nucleoside in the 5' and 3'-regions are RNA-like and
the gap region has from 6 to 14 contiguous monomer subunits
selected from .beta.-D-2'-deoxyribonucleosides and modified
nucleosides that are DNA-like and wherein at least one of the
internucleoside linking groups in the gap region or linking the gap
region and the 5'-region or the 3'-region has Formula I:
##STR00001##
wherein independently for each internucleoside linking group having
Formula I:
[0015] X is O or S;
[0016] Q is C.sub.1-C.sub.6 alkyl, substituted C.sub.1-C.sub.6
alkyl, C.sub.2-C.sub.6 alkenyl, substituted C.sub.2-C.sub.6
alkenyl, C.sub.2-C.sub.6 alkynyl, substituted C.sub.2-C.sub.6
alkynyl, C(.dbd.O)OH or CH.sub.2C(.dbd.O)OH;
[0017] each substituted group comprises one or more optionally
protected substituent groups independently selected from halogen,
OJ.sub.1, SJ.sub.1 and OC(.dbd.O)J.sub.1; and
[0018] each J.sub.1 is, independently, H or C.sub.1-C.sub.6
alkyl.
[0019] In certain embodiments, gapped oligomeric compounds are
provided consisting of a contiguous sequence of linked monomer
subunits having a gap region located between a 5'-region and a
3'-region wherein the 5' and 3'-regions each, independently, have
from 2 to 8 contiguous modified nucleosides wherein essentially
each modified nucleoside in the 5' and 3'-regions 5' and 3'-regions
are RNA-like and the gap region has from 6 to 14 contiguous monomer
subunits selected from .beta.-D-2'-deoxyribonucleosides and
modified nucleosides that are DNA-like and wherein at least one of
the internucleoside linking groups in the gap region or linking the
gap region and the 5'-region or the 3'-region has Formula I:
##STR00002##
wherein independently for each internucleoside linking group having
Formula I:
[0020] X is O or S;
[0021] Q is C.sub.1-C.sub.6 alkyl, substituted C.sub.1-C.sub.6
alkyl, C.sub.2-C.sub.6 alkenyl, substituted C.sub.2-C.sub.6
alkenyl, C.sub.2-C.sub.6 alkynyl, substituted C.sub.2-C.sub.6
alkynyl, C(.dbd.O)OH or CH.sub.2C(.dbd.O)OH;
[0022] each substituted group comprises one or more optionally
protected substituent groups independently selected from halogen,
OJ.sub.1, SJ.sub.1 and OC(.dbd.O)J.sub.1; and
[0023] each J.sub.1 is, independently, H or C.sub.1-C.sub.6
alkyl.
[0024] In certain embodiments, gapped oligomeric compounds are
provided consisting of a contiguous sequence of linked monomer
subunits having a gap region located between a 5'-region and a
3'-region wherein the 5' and 3'-regions each, independently, have
from 2 to 8 contiguous modified nucleosides wherein each modified
nucleoside in the 5' and 3'-regions are RNA-like and the gap region
has from 6 to 14 contiguous monomer subunits selected from
.beta.-D-2'-deoxyribonucleosides and modified nucleosides that are
DNA-like and wherein at least one of the internucleoside linking
groups in the gap region or linking the gap region and the
5'-region or the 3'-region has Formula I:
##STR00003##
wherein independently for each internucleoside linking group having
Formula I:
[0025] X is O or S;
[0026] Q is C.sub.1-C.sub.6 alkyl, substituted C.sub.1-C.sub.6
alkyl, C.sub.2-C.sub.6 alkenyl, substituted C.sub.2-C.sub.6
alkenyl, C.sub.2-C.sub.6 alkynyl, substituted C.sub.2-C.sub.6
alkynyl, C(.dbd.O)OH or CH.sub.2C(.dbd.O)OH;
[0027] each substituted group comprises one or more optionally
protected substituent groups independently selected from halogen,
OJ.sub.1, SJ.sub.1 and OC(.dbd.O)J.sub.1; and
[0028] each J.sub.1 is, independently, H or C.sub.1-C.sub.6
alkyl.
[0029] In certain embodiments, gapped oligomeric compounds are
provided consisting of a contiguous sequence of linked monomer
subunits having a gap region located between a 5'-region and a
3'-region wherein the 5' and 3'-regions each, independently, have
from 2 to 8 contiguous modified nucleosides wherein each modified
nucleoside in the 5' and 3'-regions are RNA-like and the gap region
has from 6 to 14 contiguous monomer subunits selected from
.beta.-D-2'-deoxyribonucleosides and wherein at least one of the
internucleoside linking groups in the gap region or linking the gap
region and the 5'-region or the 3'-region has Formula I:
##STR00004##
wherein independently for each internucleoside linking group having
Formula I:
[0030] X is O or S;
[0031] Q is C.sub.1-C.sub.6 alkyl, substituted C.sub.1-C.sub.6
alkyl, C.sub.2-C.sub.6 alkenyl, substituted C.sub.2-C.sub.6
alkenyl, C.sub.2-C.sub.6 alkynyl, substituted C.sub.2-C.sub.6
alkynyl, C(.dbd.O)OH or CH.sub.2C(.dbd.O)OH;
[0032] each substituted group comprises one or more optionally
protected substituent groups independently selected from halogen,
OJ.sub.1, SJ.sub.1 and OC(.dbd.O)J.sub.1; and
[0033] each J.sub.1 is, independently, H or C.sub.1-C.sub.6
alkyl.
[0034] In certain embodiments, gapped oligomeric compounds are
provided having only one internucleoside linking group of Formula I
in the gap region. In certain embodiments, gapped oligomeric
compounds are provided having only two internucleoside linking
groups of Formula I in the gap region. In certain embodiments,
gapped oligomeric compounds are provided having only three
internucleoside linking groups of Formula I in the gap region. In
certain embodiments, the internucleoside linking groups having
Formula I are contiguous. In certain embodiments, gapped oligomeric
compounds are provide having at least two internucleoside linking
groups of Formula I in the gap region that are separated by at
least one phosphorothioate or phosphodiester internucleoside
linking group.
[0035] In certain embodiments, gapped oligomeric compounds are
provided wherein the internucleoside linking group linking the
5'-region and the gap region has Formula I. In certain embodiments,
gapped oligomeric compounds are provided wherein the
internucleoside linking group linking the 5'-region and the gap
region and the adjacent internucleoside linkage in the gap region
has Formula I. In certain embodiments, the internucleoside linking
group linking the 3'-region and the gap region has Formula I. In
certain embodiments, gapped oligomeric compounds are provided
wherein the internucleoside linking group linking the 3'-region and
the gap region and the adjacent internucleoside linkage in the gap
region has Formula I. In certain embodiments, the internucleoside
linking group linking the 5'-region and the gap region has Formula
I and the internucleoside linking group linking the 3'-region and
the gap region has Formula I. In certain embodiments, only one
internucleoside linking group of Formula I is located in the gap
region.
[0036] In certain embodiments, each internucleoside linking group
in the 5' and 3'-regions and each internucleoside linking group in
the gap region other than internucleoside linking groups having
Formula I is a phosphodiester or a phosphorothioate internucleoside
linking group. In certain embodiments, each internucleoside linking
group in the 5' and 3'-regions and each internucleoside linking
group in the gap region other than internucleoside linking groups
having Formula I is a phosphorothioate internucleoside linking
group. In certain embodiments, each internucleoside linking group
in the 5' and 3'-regions and each internucleoside linking group in
the gap region other than internucleoside linking groups having
Formula I is a phosphodiester internucleoside linking group.
[0037] In certain embodiments, gapped oligomeric compounds are
provided wherein each monomer subunit comprises a heterocyclic base
independently selected from an optionally protected purine,
substituted purine, pyrimidine or substituted pyrimidine. In
certain embodiments, each monomer subunit comprises a heterocyclic
base independently selected from uracil, thymine, cytosine,
4-N-benzoylcytosine, 5-methylcytosine,
4-N-benzoyl-5-methylcytosine, adenine, 6-N-benzoyladenine, guanine
or 2-N-isobutyrylguanine.
[0038] In certain embodiments, gapped oligomeric compounds are
provided wherein each Q is, independently, selected from
C.sub.1-C.sub.6 alkyl, substituted C.sub.1-C.sub.6 alkyl,
C.sub.2-C.sub.6 alkenyl, C(.dbd.O)OH and CH.sub.2C(.dbd.O)OH. In
certain embodiments, each Q is, independently, selected from
CH.sub.3, C(.dbd.O)OH, CH.sub.2C(.dbd.O)OH,
(CH.sub.2).sub.2OCH.sub.3, CH.dbd.CH.sub.2, CH.sub.2CH.dbd.CH.sub.2
and C.ident.CH. In certain embodiments, each Q is
CH.sub.2C(.dbd.O)OH. In certain embodiments, each Q is CH.sub.3. In
certain embodiments, each Q is C.ident.CH.
[0039] In certain embodiments, gapped oligomeric compounds are
provided wherein each X is O. In certain embodiments, each X is
S.
[0040] In certain embodiments, gapped oligomeric compounds are
provided wherein the chirality of each internucleoside linking
group having Formula I is R.sub.P. In certain embodiments, the
chirality of each internucleoside linking group having Formula I is
S.sub.P.
[0041] In certain embodiments, each modified nucleoside in the 5'
and 3'-regions provides enhanced hybridization affinity for an RNA
target as compared to an unmodified nucleoside. In certain
embodiments, each modified nucleoside in the 5' and 3'-regions
comprises a modified sugar moiety. In certain embodiments, each
modified nucleoside in the 5' and 3'-regions is, independently, a
bicyclic nucleoside comprising a bicyclic furanosyl sugar moiety or
a modified nucleoside comprising a furanosyl sugar moiety having at
least one substituent group.
[0042] In certain embodiments, gapped oligomeric compounds are
provided wherein the 5' and 3'-regions comprise one or more
2'-modified nucleosides that each have a 2'-substituent group
independently selected from halogen, OCH.sub.3, OCH.sub.2F,
OCHF.sub.2, OCF.sub.3, OCH.sub.2CH.sub.3, O(CH.sub.2).sub.2F,
OCH.sub.2CHF.sub.2, OCH.sub.2CF.sub.3, OCH.sub.2--CH.dbd.CH.sub.2,
O(CH.sub.2).sub.2--OCH.sub.3, O(CH.sub.2).sub.2--SCH.sub.3,
O(CH.sub.2).sub.2--OCF.sub.3,
O(CH.sub.2).sub.3--N(R.sub.3)(R.sub.4),
O(CH.sub.2).sub.2--ON(R.sub.3)(R.sub.4),
O(CH.sub.2).sub.2--O(CH.sub.2).sub.2--N(R.sub.3)(R.sub.4),
OCH.sub.2C(.dbd.O)--N(R.sub.4)(R.sub.4),
OCH.sub.2C(.dbd.O)--N(R.sub.5)--(CH.sub.2).sub.2--N(R.sub.3)(R.sub.4)
and
O(CH.sub.2).sub.2--N(R.sub.5)--C(.dbd.NR.sub.6)[N(R.sub.3)(R.sub.4)]
wherein R.sub.3, R.sub.4, R.sub.5 and R.sub.6 are each,
independently, H and C.sub.1-C.sub.6 alkyl. In certain embodiments,
each 2'-substituent group is independently selected from F,
OCH.sub.3, OCF.sub.3, OCH.sub.2CH.sub.3, OCH.sub.2CF.sub.3,
OCH.sub.2--CH.dbd.CH.sub.2, O(CH.sub.2).sub.2--OCH.sub.3,
O(CH.sub.2).sub.2--O(CH.sub.2).sub.2--N(CH.sub.3).sub.2,
OCH.sub.2C(.dbd.O)--N(H)CH.sub.3,
OCH.sub.2C(.dbd.O)--N(H)--(CH.sub.2).sub.2--N(CH.sub.3).sub.2 and
OCH.sub.2--N(H)--C(.dbd.NH)NH.sub.2. In certain embodiments, each
2'-substituent group is independently selected from F, OCH.sub.3,
O(CH.sub.2).sub.2--OCH.sub.3 and OCH.sub.2C(.dbd.O)--N(H)CH.sub.3.
In certain embodiments, each 2'-substituent group is
O(CH.sub.2).sub.2--OCH.sub.3.
[0043] In certain embodiments, gapped oligomeric compounds are
provided wherein the 5' and 3'-regions comprise one or more
bicyclic nucleosides that each have a bridging group between the 4'
and 2' carbon atoms of the furanosyl ring independently selected
from 4'-(CH.sub.2)--O-2', 4'-(CH.sub.2)--S-2',
4'-(CH.sub.2).sub.2--O-2', 4'-CH(CH.sub.3)--O-2',
4'-CH(CH.sub.2OCH.sub.3)--O-2', 4'-C(CH.sub.3).sub.2--O-2',
4'-CH.sub.2--N(OCH.sub.3)-2', 4'-CH.sub.2--O--N(CH.sub.3)-2',
4'-CH.sub.2--C(H)(CH.sub.3)-2' and
4'-CH.sub.2--C(.dbd.CH.sub.2)-2'. In certain embodiments, each of
the bridging groups is independently selected from
4'-(CH.sub.2)--O-2', 4'-(CH.sub.2).sub.2--O-2',
4'-CH(CH.sub.3)--O-2', 4'-CH.sub.2--C(H)(CH.sub.3)-2' and
4'-CH.sub.2--C(.dbd.CH.sub.2)-2'. In certain embodiments, each
bridging group is 4'-CH[(S)--(CH.sub.3)]--O-2'.
[0044] In certain embodiments, gapped oligomeric compounds are
provided wherein the sugar moieties of each modified nucleoside in
the 5' and 3'-regions are the same. In certain embodiments, gapped
oligomeric compounds are provided comprising at least two different
types of modified nucleosides in the 5' and 3'-regions wherein the
different types of modified nucleosides have at least different
modified sugar moieties. In certain embodiments, the different
types of modified nucleosides include bicyclic nucleosides
comprising bicyclic furanosyl sugar moieties and modified
nucleosides comprising furanosyl sugar moieties having at least one
substituent group. In certain embodiments, the different types of
modified nucleosides include 4'-CH[(S)--(CH.sub.3)]--O-2' bicyclic
nucleosides and 2'-O(CH.sub.2).sub.2--OCH.sub.3 substituted
nucleosides. In certain embodiments, the 5' and 3'-regions include
only 4'-CH[(S)--(CH.sub.3)]--O-2' bicyclic nucleosides and
2'-O(CH.sub.2).sub.2--OCH.sub.3 substituted nucleosides.
[0045] In certain embodiments, gapped oligomeric compounds are
provided wherein one or more modified nucleosides in the 5' and
3'-regions comprise a sugar surrogate.
[0046] In certain embodiments, gapped oligomeric compounds are
provided wherein each monomer subunit in the gap region is a
.beta.-D-2'-deoxyribonucleoside. In certain embodiments, at least
one monomer subunit in the gap region is a modified nucleoside that
is DNA-like. In certain embodiments, each modified nucleoside that
is DNA-like is a 2'-(ara)-F modified nucleoside.
[0047] In certain embodiments, gapped oligomeric compounds are
provided wherein the 5' and 3'-regions each, independently, have
from 2 to 8 monomer subunits. In certain embodiments, the 5' and
3'-regions each, independently, have from 3 to 6 monomer subunits.
In certain embodiments, the gap region has from 6 to 14 monomer
subunits. In certain embodiments, the gap region has from 8 to 10
monomer subunits. In certain embodiments, the 5' and 3'-regions
each, independently, have from 3 to 6 monomer subunits and the gap
region has from 8 to 14 monomer subunits. In certain embodiments,
the 5' and 3'-regions each, independently, have from 3 to 6 monomer
subunits and the gap region has from 6 to 10 monomer subunits. In
certain embodiments, the 5' and 3'-regions each, independently,
have from 3 to 6 monomer subunits and the gap region has from 6 to
8 monomer subunits. In certain embodiments, the 5' and 3'-regions
each, independently, have from 4 to 5 monomer subunits and the gap
region has from 7 to 8 monomer subunits.
[0048] In certain embodiments, gapped oligomeric compounds are
provided wherein at least one modified nucleoside in the 5' and
3'-regions is other than a 2'-OCH.sub.3 substituted nucleoside or a
2'-O--CH.sub.2-4' bridged bicyclic nucleoside.
[0049] In certain embodiments, gapped oligomeric compounds are
provided that include one 5'-terminal group. In certain
embodiments, gapped oligomeric compounds are provided that include
one 3'-terminal group. In certain embodiments, gapped oligomeric
compounds are provided that include at least one 5' or 3'-terminal
group.
[0050] In certain embodiments, the gapped oligomeric compounds
provided herein are other than the gapped oligomeric compounds
listed below:
TABLE-US-00001 SEQ ID NO. Gap wings ISIS # Sequence Chemistry 5'/3'
05/558257 T.sub.eA.sub.kA.sub.kATTpGTCATCA.sub.kC.sub.kC.sub.e
-(P(=O)(CH.sub.3))- ekk kke 05/558256
T.sub.eA.sub.kA.sub.kAT.sub.pTGTCATCA.sub.kC.sub.kC.sub.e
-(P(=O)(CH.sub.3))- ekk kke 05/558255
T.sub.eA.sub.kA.sub.kA.sub.pTTGTCATCA.sub.kC.sub.kC.sub.e
-(P(=O)(CH.sub.3))- ekk kke 05/571123
T.sub.eA.sub.eA.sub.eA.sub.kT.sub.kT.sub.pGT.sup.mCAT.sup.mCA.su-
b.k.sup.mC.sub.k.sup.mC.sub.e -(P(=O)(CH.sub.3))- eeekk kke
05/571124
T.sub.eA.sub.eA.sub.eA.sub.k.sup.xTT.sub.pGT.sup.mCAT.sup.mC.sub-
.eA.sub.k.sup.mC.sub.k.sup.mC -(P(=O)(CH.sub.3))- eeek kke
05/579854
T.sub.eA.sub.eA.sub.eA.sub.kTT.sub.pGT.sup.mCAT.sup.mCA.sub.k.su-
p.mC.sub.k.sup.mC.sub.e -(P(=O)(CH.sub.3))- eeek kke 09/571171
A.sub.eT.sub.kA.sub.eA.sub.kATT.sub.pGT.sup.mCAT.sup.mCA.sub.k.s-
up.mC.sub.e.sup.mC.sub.kA.sub.e -(P(=O)(CH.sub.3))- ekek keke
09/571041
A.sub.eT.sub.kA.sub.eA.sub.kA.sup.xTT.sub.pGT.sup.mCAT.sup.mCA.s-
ub.k.sup.mC.sub.e.sup.mC.sub.kA.sub.e -(P(=O)(CH.sub.3))- ekek
keke
[0051] Wherein unless indicated otherwise each internucleoside
linkage is a phosphorothioate. Each ".sup.xT" is a 2-thio-thymidine
modified nucleoside. A subscript "p" indicates a methyl phosphonate
internucleoside linkage (--(P(.dbd.O)(CH.sub.3))--). Nucleosides
not followed by a subscript are .beta.-D-2'-deoxyribonucleosides.
Nucleosides followed by a subscript "e" are 2'-O-methoxyethyl (MOE)
modified nucleosides. Nucleosides followed by a subscript "k" are
6'-(S)--CH.sub.3 (cEt) bicyclic modified nucleosides. Each
".sup.mC" is a 5-methyl cytosine modified nucleoside.
[0052] In certain embodiments, the gapped oligomeric compounds
provided herein are other than gapped oligomeric compounds
complementary to at least a region of a nucleic acid that is a
Huntingtin gene transcript. In certain embodiments, the gapped
oligomeric compounds provided herein are other than gapped
oligomeric compounds complementary to at least a region of a
nucleic acid comprising a single-nucleotide polymorphism. In
certain embodiments, the gapped oligomeric compounds provided
herein are other than gapped oligomeric compounds complementary to
at least a region of a nucleic acid comprising a single-nucleotide
polymorphism-containing-target nucleic acid of a Huntingtin gene
transcript. In certain embodiments, the gapped oligomeric compounds
provided herein are other than gapped oligomeric compounds
complementary to at least a region of a nucleic acid comprising a
single-nucleotide polymorphism-containing-target nucleic acid of a
gene transcript other than Huntingtin.
[0053] In certain embodiments, methods of inhibiting gene
expression are provided comprising contacting one or more cells, a
tissue or an animal with an oligomeric compound as provided herein
wherein said oligomeric compound is complementary to a target RNA.
In certain embodiments, the cells are in a human. In certain
embodiments, the target RNA is human mRNA. In certain embodiments,
the target RNA is cleaved thereby inhibiting its function.
[0054] In certain embodiments, in vitro methods of inhibiting gene
expression are provided comprising contacting one or more cells or
a tissue with an oligomeric compound as provided herein.
[0055] In certain embodiments, oligomeric compound as provided
herein are used in an in vivo method of inhibiting gene expression
said method comprising contacting one or more cells, a tissue or an
animal with an oligomeric compound as provided herein.
[0056] In certain embodiments, oligomeric compounds as provided
herein are used in medical therapy.
DETAILED DESCRIPTION OF THE INVENTION
[0057] Provided herein are linkage modified gapped oligomeric
compounds. Such gapped oligomeric compounds comprise a contiguous
sequence of linked monomer subunits having a gap region located
between a 5'-region and a 3'-region wherein the 5' and 3'-regions
each, independently, have from 2 to 8 contiguous modified
nucleosides wherein essentially each modified nucleoside in the 5'
and 3'-regions are RNA-like and the gap region has from 6 to 14
contiguous monomer subunits selected from
.beta.-D-2'-deoxyribonucleosides and modified nucleosides that are
DNA-like and wherein at least one of the internucleoside linking
groups in the gap region or between the gap region and the
5'-region or the 3'-region has Formula I:
##STR00005##
wherein independently for each internucleoside linking group having
Formula I:
[0058] X is O or S;
[0059] Q is C.sub.1-C.sub.6 alkyl, substituted C.sub.1-C.sub.6
alkyl, C.sub.2-C.sub.6 alkenyl, substituted C.sub.2-C.sub.6
alkenyl, C.sub.2-C.sub.6 alkynyl, substituted C.sub.2-C.sub.6
alkynyl, C(.dbd.O)OH or CH.sub.2C(.dbd.O)OH;
[0060] each substituted group comprises one or more optionally
protected substituent groups independently selected from halogen,
OJ.sub.1, SJ.sub.1 and OC(.dbd.O)J.sub.1; and
[0061] each J.sub.1 is, independently, H or C.sub.1-C.sub.6
alkyl.
[0062] The gapped oligomeric compounds provided herein have been
shown to have improved properties. In certain embodiments, the
activity of an otherwise unmodified gapped oligomeric compound
against a target nucleic acid is enhanced by incorporation of at
least one internucleoside linking group having Formula I in the gap
region. In certain embodiments, at least one internucleoside
linking group having Formula I is located at the gap junction on
the 5' side wherein the internucleoside linkage separates the gap
region from the wing region. In certain embodiments, at least one
internucleoside linking group having Formula I is located at the
gap junction on the 3' side. As indicated in the in vitro and in
vivo data provided in the example section herein, such properties
include selectivity, potency, improved toxicity profile and or an
improved proinflammatory profile.
[0063] In certain embodiments, a gapped oligomeric compound of
interest is identified and then a series of identical oligomeric
compounds are prepared with a single internucleoside linking group
having Formula I walked across the gap region. If there are 8
monomer subunits in the gap then there will be 8 oligomeric
compounds prepared having the internucleoside linking group having
Formula I located at a different position in each of the oligomeric
compounds which are subsequently assayed in one or more assays as
illustrated herein to determine the lead from the series.
[0064] In certain embodiments, additional internucleoside linking
groups having Formula I are incorporated into the gap region of the
lead oligomeric compound and assayed in one or more assays as
illustrated herein. In certain embodiments, the lead compound is
further functionalized with one or more terminal groups such as for
example a conjugate group. In certain embodiments, a gapped
oligomeric compound of interest is identified and then a series of
identical oligomeric compounds are prepared with blocks of at least
two internucleoside linking group having Formula I walked across
the gap region.
[0065] In certain embodiments, gapped oligomeric compounds are
provided having a reduced proinflammatory response when compared to
unmodified gapped oligomeric compounds. As shown using an in vitro
hPBMC assay (Example 21), a gapped oligomeric compound having
alternating methyl thiophosphonate (--P(CH.sub.3)(.dbd.S)--)
internucleoside linkages in the gap reduced the proinflammatory
response compared to the an identical oligomeric compound without
the modified internucleoside linkages.
[0066] In certain embodiments, gapped oligomeric compounds are
provided having enhanced potency (IC.sub.50) and selectivity when
compared to unmodified gapped oligomeric compounds. As shown using
an in vitro HTT SNP assay (Example 23), a gapped oligomeric
compound having a single methyl phosphonate
(--P(CH.sub.3)(.dbd.O)--) internucleoside linkage in the gap, at
either position 4 or 5 from the 5'-end, showed lower IC.sub.50 and
increased selectivity when compared to the an identical oligomeric
compound without the modified internucleoside linkages.
[0067] In certain embodiments, gapped oligomeric compounds are
provided having enhanced selectivity with comparable potency
(IC.sub.50) when compared to unmodified gapped oligomeric
compounds. As shown using an in vitro HTT SNP assay (Example 24), a
gapped oligomeric compound having one or two methyl phosphonate
(--P(CH.sub.3)(.dbd.O)--) or one phosphonoacetate
(--P(CH.sub.2CO.sub.2.sup.-)(.dbd.O)--) internucleoside linkages in
the gap typically showed enhanced selectivity with comparable or
slightly lower potency.
[0068] In certain embodiments, gapped oligomeric compounds are
provided having enhanced selectivity with comparable potency
(IC.sub.50) when compared to an unmodified gapped oligomeric
compound. As shown using an in vitro HTT SNP assay (Example 25), a
gapped oligomeric compound having one or two methyl phosphonate
(--P(CH.sub.3)(.dbd.O)--) internucleoside linkages in the gap
typically showed enhanced selectivity with comparable or slightly
lower potency.
[0069] In certain embodiments, gapped oligomeric compounds are
provided having improved hepatotoxicity profiles when compared to
unmodified gapped oligomeric compounds. As shown in in vivo PTEN
and SRB-1 assays (Example 26), a gapped oligomeric compound having
two methyl phosphonate (--P(CH.sub.3)(.dbd.O)--) internucleoside
linkages at the 5'-end of the gap and linking the gap to the
5'-wing lowered the ALT as compared to the unmodified oligomeric
compounds. The activity and organ weights are similar for the SRB-1
assay. The activity is reduced for the modified oligomeric compound
in the PTEN assay. Also for the PTEN assay the liver is slightly
elevated for the modified gapped oligomeric compound whereas for
the unmodified oligomeric compound the liver and the spleen weights
are both elevated.
[0070] It is to be understood that both the foregoing general
description and the following detailed description are exemplary
and explanatory only and are not restrictive of the invention, as
claimed. Herein, the use of the singular includes the plural unless
specifically stated otherwise. As used herein, the use of "or"
means "and/or" unless stated otherwise. Furthermore, the use of the
term "including" as well as other forms, such as "includes" and
"included", is not limiting. Also, terms such as "element" or
"component" encompass both elements and components comprising one
unit and elements and components that comprise more than one
subunit, unless specifically stated otherwise.
[0071] The section headings used herein are for organizational
purposes only and are not to be construed as limiting the subject
matter described. All documents, or portions of documents, cited in
this application, including, but not limited to, patents, patent
applications, articles, books, and treatises, are hereby expressly
incorporated by reference in their entirety for any purpose.
A. Definitions
[0072] Unless specific definitions are provided, the nomenclature
used in connection with, and the procedures and techniques of,
analytical chemistry, synthetic organic chemistry, and medicinal
and pharmaceutical chemistry described herein are those well known
and commonly used in the art. Standard techniques may be used for
chemical synthesis, and chemical analysis. Certain such techniques
and procedures may be found for example in "Carbohydrate
Modifications in Antisense Research" Edited by Sangvi and Cook,
American Chemical Society, Washington D.C., 1994; "Remington's
Pharmaceutical Sciences," Mack Publishing Co., Easton, Pa.,
21.sup.st edition, 2005; and "Antisense Drug Technology,
Principles, Strategies, and Applications" Edited by Stanley T.
Crooke, CRC Press, Boca Raton, Fla.; and Sambrook et al.,
"Molecular Cloning, A laboratory Manual," 2' Edition, Cold Spring
Harbor Laboratory Press, 1989, which are hereby incorporated by
reference for any purpose. Where permitted, all patents,
applications, published applications and other publications and
other data referred to throughout in the disclosure are
incorporated by reference herein in their entirety.
[0073] Unless otherwise indicated, the following terms have the
following meanings:
[0074] As used herein, "chemical modification" means a chemical
difference in a compound when compared to a naturally occurring
counterpart. Chemical modifications of oligonucleotides include
nucleoside modifications (including sugar moiety modifications and
nucleobase modifications) and internucleoside linkage
modifications. In reference to an oligonucleotide, chemical
modification does not include differences only in nucleobase
sequence.
[0075] As used herein, "furanosyl" means a structure comprising a
5-membered ring comprising four carbon atoms and one oxygen
atom.
[0076] As used herein, "naturally occurring sugar moiety" means a
ribofuranosyl as found in naturally occurring RNA or a
2'-deoxyribofuranosyl as found in naturally occurring DNA.
[0077] As used herein, "sugar moiety" means a naturally occurring
sugar moiety or a modified sugar moiety of a nucleoside.
[0078] As used herein, "modified sugar moiety" means a substituted
sugar moiety or a sugar surrogate.
[0079] As used herein, "substituted sugar moiety" means a furanosyl
that is not a naturally occurring sugar moiety. Substituted sugar
moieties include, but are not limited to furanosyls comprising
substituents at the 2'-position, the 3'-position, the 5'-position
and/or the 4'-position. Certain substituted sugar moieties are
bicyclic sugar moieties.
[0080] As used herein, "2'-substituted sugar moiety" means a
furanosyl comprising a substituent at the 2'-position other than H
or OH. Unless otherwise indicated, a 2'-substituted sugar moiety is
not a bicyclic sugar moiety (i.e., the 2'-substituent of a
2'-substituted sugar moiety does not form a bridge to another atom
of the furanosyl ring.
[0081] As used herein, "MOE" means
--OCH.sub.2CH.sub.2OCH.sub.3.
[0082] As used herein, "2'-F nucleoside" refers to a nucleoside
comprising a sugar comprising fluorine at the 2' position. Unless
otherwise indicated, the fluorine in a 2'-F nucleoside is in the
ribo position (replacing the OH of a natural ribose).
[0083] As used herein, "2'-(ara)-F" refers to a 2'-F substituted
nucleoside, wherein the fluoro group is in the arabino
position.
[0084] As used herein the term "sugar surrogate" means a structure
that does not comprise a furanosyl ring and that is capable of
replacing the naturally occurring sugar moiety of a nucleoside,
such that the resulting nucleoside sub-units or monomer subunits
are capable of linking together and/or linking to other nucleosides
or other monomer subunits to form an oligomeric compound which is
capable of hybridizing to a complementary oligomeric compound such
as a nucleic acid target. Such structures include rings comprising
a different number of atoms than furanosyl (e.g., 4, 6, or
7-membered rings); replacement of the oxygen atom of a furanosyl
with a non-oxygen atom (e.g., carbon, sulfur, or nitrogen, wherein
replacement of the oxygen atom with sulfur in furanose is generally
considered a modified nucleoside as opposed to a sugar surrogate
but can be considered both); or both a change in the number of
atoms and a replacement of the oxygen. Such structures may also
comprise substitutions corresponding to those described for
substituted sugar moieties (e.g., 6-membered carbocyclic bicyclic
sugar surrogates optionally comprising additional substituents).
Sugar surrogates also include more complex sugar replacements
(e.g., the non-ring systems of peptide nucleic acid). Sugar
surrogates include without limitation morpholinos, cyclohexenyls
and cyclohexitols.
[0085] As used herein, "bicyclic sugar moiety" means a modified
sugar moiety comprising a 4 to 7 membered ring (including but not
limited to a furanosyl) comprising a bridge connecting two atoms of
the 4 to 7 membered ring to form a second ring, resulting in a
bicyclic structure. In certain embodiments, the 4 to 7 membered
ring is a sugar ring. In certain embodiments the 4 to 7 membered
ring is a furanosyl. In certain such embodiments, the bridge
connects the 2'-carbon and the 4'-carbon of the furanosyl.
[0086] As used herein, "nucleoside" means a compound comprising a
nucleobase moiety and a sugar moiety. Nucleosides include, but are
not limited to, naturally occurring nucleosides (as found in DNA
and RNA) and modified nucleosides. Nucleosides may be linked to a
phosphate moiety.
[0087] As used herein, "nucleotide" means a nucleoside further
comprising a phosphate linking group. As used herein, "linked
nucleosides" may or may not be linked by phosphate linkages and
thus includes, but is not limited to "linked nucleotides." As used
herein, "linked nucleosides" are nucleosides that are connected in
a continuous sequence (i.e. no additional nucleosides are present
between those that are linked).
[0088] As used herein the term "nucleobase" generally refers to the
nucleobase of a nucleoside or modified nucleoside. The term
"heterocyclic base moiety" is broader than the term nucleobase in
that it includes any heterocyclic base that can be attached to a
sugar to prepare a nucleoside or modified nucleoside. Such
heterocyclic base moieties include but are not limited to naturally
occurring nucleobases (adenine, guanine, thymine, cytosine and
uracil) and protected forms of unmodified nucleobases (4-N-b
enzoylcytosine, 6-N-benzoyladenine and 2-N-isobutyrylguanine) as
well as modified (5-methyl cytosine) or non-naturally occurring
heterocyclic base moieties and synthetic mimetics thereof (such as
for example phenoxazines).
[0089] As used herein the term "modified nucleoside" refers to a
nucleoside comprising a modified heterocyclic base and or a sugar
moiety other than ribose and 2'-deoxyribose. In certain
embodiments, a modified nucleoside comprises a modified
heterocyclic base moiety. In certain embodiments, a modified
nucleoside comprises a sugar moiety other than ribose and
2'-deoxyribose. In certain embodiments, a modified nucleoside
comprises a modified heterocyclic base moiety and a sugar moiety
other than ribose and 2'-deoxyribose. The term "modified
nucleoside" is intended to include all manner of modified
nucleosides that can be incorporated into an oligomeric compound
using standard oligomer synthesis protocols. Modified nucleosides
include abasic nucleosides but in general a heterocyclic base
moiety is included for hybridization to a complementary nucleic
acid target.
[0090] In certain embodiments, modified nucleosides include a
furanose or modified furanose sugar group such as a 4'-S analog
(4'-S-modified nucleoside and 4'-S-ribonucleoside refer to
replacement of the furanose oxygen atom with S). Such modified
nucleosides include without limitation, substituted nucleosides
(such as 2', 5', and/or 4' substituted nucleosides) 4'-S-modified
nucleosides, (such as 4'-5-ribonucleosides,
4'-S-2'-deoxyribonucleosides and 4'-5-2'-substituted
ribonucleosides), bicyclic modified nucleosides (such as
2'-O--CH(CH.sub.3)-4', 2'-O--CH.sub.2-4' or
2'-O--(CH.sub.2).sub.2-4' bridged furanose analogs) and base
modified nucleosides. The sugar can be modified with more than one
of these modifications listed such as for example a bicyclic
modified nucleoside further including a 5'-substitution or a 5' or
4' substituted nucleoside further including a 2' substituent. The
term modified nucleoside also includes combinations of these
modifications such as base and sugar modified nucleosides. These
modifications are meant to be illustrative and not exhaustive as
other modifications are known in the art and are also envisioned as
possible modifications for the modified nucleosides described
herein.
[0091] In certain embodiments, modified nucleosides comprise a
sugar surrogate wherein the furanose ring has been replaced with a
mono or polycyclic ring system or a non-cyclic sugar surrogate such
as that used in peptide nucleic acids. Illustrative examples of
sugar moieties for such modified nucleosides includes without
limitation morpholino, hexitol, cyclohexenyl, 2.2.2 and 3.2.1
cyclohexose and open non-cyclic groups.
[0092] In certain embodiments, modified nucleosides comprise a
non-naturally occurring sugar moiety and a modified heterocyclic
base moiety. Such modified nucleosides include without limitation
modified nucleosides wherein the heterocyclic base moiety is
replaced with a phenoxazine moiety (for example the
9-(2-aminoethoxy)-1,3-diazaphenoxazine-2-one group, also referred
to as a G-clamp which forms four hydrogen bonds when hybridized
with a guanosine base) and further replacement of the sugar moiety
with a sugar surrogate group such as for example a morpholino, a
cyclohexenyl or a bicyclo[3.1.0]hexyl.
[0093] As used herein, "bicyclic nucleoside" or "BNA" means a
nucleoside comprising a bicyclic sugar moiety.
[0094] As used herein, "constrained ethyl nucleoside" or "cEt"
means a nucleoside comprising a bicyclic sugar moiety comprising a
4'-CH(CH.sub.3)--O-2' bridge. In certain embodiments, the cEt
comprises a comprising a 4'-CH((S)--CH.sub.3)--O-2' bridge. In
certain embodiments, the cEt comprises a comprising a
4'-CH((R)--CH.sub.3)--O-2' bridge.
[0095] As used herein, "locked nucleic acid nucleoside" or "LNA"
means a nucleoside comprising a bicyclic sugar moiety comprising a
4'-CH.sub.2--O-2' bridge.
[0096] As used herein, "2'-substituted nucleoside" means a
ribofuranosyl nucleoside comprising a substituent at the
2'-position other than H or OH. Unless otherwise indicated, a
2'-substituted nucleoside is not a bicyclic nucleoside.
[0097] As used herein, "2'-deoxynucleoside" means a nucleoside
comprising 2'-H(H) furanosyl sugar moiety, as found in naturally
occurring deoxyribonucleosides (DNA). In certain embodiments, a
2'-deoxynucleoside may comprise a modified nucleobase or may
comprise an RNA nucleobase (e.g., uracil).
[0098] As used herein, "RNA-like nucleoside" means a modified
nucleoside other than a .beta.-D-ribose nucleoside that provides an
A-form (northern) duplex when incorporated into an oligomeric
compound and duplexed with a complementary RNA. RNA-like
nucleosides are used as replacements for RNA nucleosides in
oligomeric compounds to enhance one or more properties such as, for
example, nuclease resistance and or hybridization affinity.
RNA-like nucleosides include, but are not limited to modified
furanosyl nucleosides that adopt a 3'-endo conformational geometry
when put into an oligomeric compound. RNA-like nucleosides also
include RNA surrogates such as F-HNA. RNA-like nucleosides include
but are not limited to modified nucleosides comprising a
2'-substituent group selected from F, O(CH.sub.2).sub.2OCH.sub.3
(MOE) and OCH.sub.3. RNA-like nucleosides also include but are not
limited to modified nucleosides comprising bicyclic furanosyl sugar
moiety comprising a 4'-CH.sub.2--O-2', 4'-(CH.sub.2).sub.2--O-2',
4'-C(H)[(R)--CH.sub.3]--O-2' or 4'-C(H)[(S)--CH.sub.3]--O-2'
bridging group.
[0099] As used herein, "DNA-like nucleoside" means a modified
nucleoside other than a .beta.-D-2'-doxyribose nucleoside that
provides a B-form (southern) duplex when incorporated into an
oligomeric compound and duplexed with a complementary DNA. DNA-like
nucleosides provide an intermediate duplex when incorporated into
an oligomeric compound and duplexed with a complementary RNA that
is between A-form and B-form. DNA-like nucleosides are used as
replacements for DNA nucleosides in oligomeric compounds to enhance
one or more properties. DNA-like nucleosides include, but are not
limited to modified nucleosides that adopt a 2'-endo conformational
geometry when put into an oligomeric compound.
[0100] As used herein, "oligonucleotide" means a compound
comprising a plurality of linked nucleosides. In certain
embodiments, an oligonucleotide comprises one or more unmodified
ribonucleosides (RNA) and/or unmodified deoxyribonucleosides (DNA)
and/or one or more modified nucleosides.
[0101] As used herein "oligonucleoside" means an oligonucleotide in
which none of the internucleoside linkages contains a phosphorus
atom. As used herein, oligonucleotides include
oligonucleosides.
[0102] As used herein, "modified oligonucleotide" means an
oligonucleotide comprising at least one modified nucleoside and/or
at least one modified internucleoside linkage.
[0103] As used herein "internucleoside linkage" means a covalent
linkage between adjacent nucleosides in an oligonucleotide.
[0104] As used herein "naturally occurring internucleoside linkage"
means a 3' to 5' phosphodiester linkage.
[0105] As used herein, "modified internucleoside linkage" means any
internucleoside linkage other than a naturally occurring
internucleoside linkage.
[0106] As used herein the term "monomer subunit" is meant to
include all manner of monomers that are amenable to oligomer
synthesis. In general a monomer subunit includes at least a sugar
moiety having at least two reactive sites that can form linkages to
further monomer subunits. Essentially all monomer subunits include
a heterocyclic base moiety that is hybridizable to a complementary
site on a nucleic acid target. Reactive sites on monomer subunits
located on the termini of an oligomeric compound can be protected
or unprotected (generally OH) or can form an attachment to a
terminal group (conjugate or other group). Monomer subunits
include, without limitation, nucleosides and modified nucleosides.
In certain embodiments, monomer subunits include nucleosides such
as .beta.-D-ribonucleosides and .beta.-D-2'-deoxyribnucleosides and
modified nucleosides including but not limited to substituted
nucleosides (such as 2', 5' and bis substituted nucleosides),
4'-S-modified nucleosides (such as 4'-S-ribonucleosides,
4'-S-2'-deoxyribonucleosides and 4'-S-2'-substituted
ribonucleosides), bicyclic modified nucleosides (such as bicyclic
nucleosides wherein the sugar moiety has a 2'-O--CHR.sub.a-4'
bridging group, wherein R.sub.a is H, alkyl or substituted alkyl),
other modified nucleosides and nucleosides having sugar
surrogates.
[0107] As used herein, "conjugate" means an atom or group of atoms
bound to an oligonucleotide or oligomeric compound. In general,
conjugate groups modify one or more properties of the compound to
which they are attached, including, but not limited to
pharmacodynamic, pharmacokinetic, binding, absorption, cellular
distribution, cellular uptake, charge and/or clearance
properties.
[0108] As used herein, "conjugate linking group" means any atom or
group of atoms used to attach a conjugate to an oligonucleotide or
oligomeric compound.
[0109] As used herein, "antisense compound" means a compound
comprising or consisting of an oligonucleotide or oligomeric
compound wherein at least a portion of which is complementary to a
target nucleic acid to which it is capable of hybridizing,
resulting in at least one antisense activity.
[0110] As used herein, "antisense activity" means any detectable
and/or measurable change attributable to the hybridization of an
antisense compound to its target nucleic acid.
[0111] As used herein, "detecting" or "measuring" means that a test
or assay for detecting or measuring is performed. Such detection
and/or measuring may result in a value of zero. Thus, if a test for
detection or measuring results in a finding of no activity
(activity of zero), the step of detecting or measuring the activity
has nevertheless been performed.
[0112] As used herein, "detectable and/or measureable activity"
means a statistically significant activity that is not zero.
[0113] As used herein, "essentially unchanged" means little or no
change in a particular parameter, particularly relative to another
parameter which changes much more. In certain embodiments, a
parameter is essentially unchanged when it changes less than 5%. In
certain embodiments, a parameter is essentially unchanged if it
changes less than two-fold while another parameter changes at least
ten-fold. For example, in certain embodiments, an antisense
activity is a change in the amount of a target nucleic acid. In
certain such embodiments, the amount of a non-target nucleic acid
is essentially unchanged if it changes much less than the target
nucleic acid does, but the change need not be zero.
[0114] As used herein, "expression" means the process by which a
gene ultimately results in a protein. Expression includes, but is
not limited to, transcription, post-transcriptional modification
(e.g., splicing, polyadenlyation, addition of 5'-cap), and
translation.
[0115] As used herein, "target nucleic acid" means a nucleic acid
molecule to which an antisense compound hybridizes.
[0116] As used herein, "mRNA" means an RNA molecule that encodes a
protein.
[0117] As used herein, "pre-mRNA" means an RNA transcript that has
not been fully processed into mRNA. Pre-RNA includes one or more
intron.
[0118] As used herein, "object RNA" means an RNA molecule other
than a target RNA, the amount, activity, splicing, and/or function
of which is modulated, either directly or indirectly, by a target
nucleic acid. In certain embodiments, a target nucleic acid
modulates splicing of an object RNA. In certain such embodiments,
an antisense compound modulates the amount or activity of the
target nucleic acid, resulting in a change in the splicing of an
object RNA and ultimately resulting in a change in the activity or
function of the object RNA.
[0119] As used herein, "microRNA" means a naturally occurring,
small, non-coding RNA that represses gene expression of at least
one mRNA. In certain embodiments, a microRNA represses gene
expression by binding to a target site within a 3' untranslated
region of an mRNA. In certain embodiments, a microRNA has a
nucleobase sequence as set forth in miRBase, a database of
published microRNA sequences found at
http://microrna.sanger.ac.uk/sequences/. In certain embodiments, a
microRNA has a nucleobase sequence as set forth in miRBase version
12.0 released September 2008, which is herein incorporated by
reference in its entirety.
[0120] As used herein, "microRNA mimic" means an oligomeric
compound having a sequence that is at least partially identical to
that of a microRNA. In certain embodiments, a microRNA mimic
comprises the microRNA seed region of a microRNA. In certain
embodiments, a microRNA mimic modulates translation of more than
one target nucleic acids. In certain embodiments, a microRNA mimic
is double-stranded.
[0121] As used herein, "differentiating nucleobase" means a
nucleobase that differs between two nucleic acids. In certain
instances, a target region of a target nucleic acid differs by 1-4
nucleobases from a non-target nucleic acid. Each of those
differences is referred to as a differentiating nucleobase. In
certain instances, a differentiating nucleobase is a
single-nucleotide polymorphism.
[0122] As used herein, "target-selective nucleoside" means a
nucleoside of an anti sense compound that corresponds to a
differentiating nucleobase of a target nucleic acid.
[0123] As used herein, "allele" means one of a pair of copies of a
gene existing at a particular locus or marker on a specific
chromosome, or one member of a pair of nucleobases existing at a
particular locus or marker on a specific chromosome, or one member
of a pair of nucleobase sequences existing at a particular locus or
marker on a specific chromosome. For a diploid organism or cell or
for autosomal chromosomes, each allelic pair will normally occupy
corresponding positions (loci) on a pair of homologous chromosomes,
one inherited from the mother and one inherited from the father. If
these alleles are identical, the organism or cell is said to be
"homozygous" for that allele; if they differ, the organism or cell
is said to be "heterozygous" for that allele. "Wild-type allele"
refers to the genotype typically not associated with disease or
dysfunction of the gene product. "Mutant allele" refers to the
genotype associated with disease or dysfunction of the gene
product.
[0124] As used herein, "allelic variant" means a particular
identity of an allele, where more than one identity occurs. For
example, an allelic variant may refer to either the mutant allele
or the wild-type allele.
[0125] As used herein, "single nucleotide polymorphism" or "SNP"
means a single nucleotide variation between the genomes of
individuals of the same species. In some cases, a SNP may be a
single nucleotide deletion or insertion. In general, SNPs occur
relatively frequently in genomes and thus contribute to genetic
diversity. The location of a SNP is generally flanked by highly
conserved sequences. An individual may be homozygous or
heterozygous for an allele at each SNP site.
[0126] As used herein, "single nucleotide polymorphism site" or
"SNP site" refers to the nucleotides surrounding a SNP contained in
a target nucleic acid to which an antisense compound is
targeted.
[0127] As used herein, "targeting" or "targeted to" means the
association of an antisense compound to a particular target nucleic
acid molecule or a particular region of a target nucleic acid
molecule. An antisense compound targets a target nucleic acid if it
is sufficiently complementary to the target nucleic acid to allow
hybridization under physiological conditions.
[0128] As used herein, "nucleobase complementarity" or
"complementarity" when in reference to nucleobases means a
nucleobase that is capable of base pairing with another nucleobase.
For example, in DNA, adenine (A) is complementary to thymine (T).
For example, in RNA, adenine (A) is complementary to uracil (U). In
certain embodiments, complementary nucleobase means a nucleobase of
an antisense compound that is capable of base pairing with a
nucleobase of its target nucleic acid. For example, if a nucleobase
at a certain position of an antisense compound is capable of
hydrogen bonding with a nucleobase at a certain position of a
target nucleic acid, then the position of hydrogen bonding between
the oligonucleotide and the target nucleic acid is considered to be
complementary at that nucleobase pair. Nucleobases comprising
certain modifications may maintain the ability to pair with a
counterpart nucleobase and thus, are still capable of nucleobase
complementarity.
[0129] As used herein, "non-complementary" in reference to
nucleobases means a pair of nucleobases that do not form hydrogen
bonds with one another.
[0130] As used herein, "complementary" in reference to oligomeric
compounds (e.g., linked nucleosides, oligonucleotides, or nucleic
acids) means the capacity of such oligomeric compounds or regions
thereof to hybridize to another oligomeric compound or region
thereof through nucleobase complementarity under stringent
conditions. Complementary oligomeric compounds need not have
nucleobase complementarity at each nucleoside. Rather, some
mismatches are tolerated. In certain embodiments, complementary
oligomeric compounds or regions are complementary at 70% of the
nucleobases (70% complementary). In certain embodiments,
complementary oligomeric compounds or regions are 80%
complementary. In certain embodiments, complementary oligomeric
compounds or regions are 90% complementary. In certain embodiments,
complementary oligomeric compounds or regions are 95%
complementary. In certain embodiments, complementary oligomeric
compounds or regions are 100% complementary.
[0131] As used herein, "mismatch" means a nucleobase of a first
oligomeric compound that is not capable of pairing with a
nucleobase at a corresponding position of a second oligomeric
compound, when the first and second oligomeric compound are
aligned. Either or both of the first and second oligomeric
compounds may be oligonucleotides.
[0132] As used herein, "hybridization" means the pairing of
complementary oligomeric compounds (e.g., an antisense compound and
its target nucleic acid). While not limited to a particular
mechanism, the most common mechanism of pairing involves hydrogen
bonding, which may be Watson-Crick, Hoogsteen or reversed Hoogsteen
hydrogen bonding, between complementary nucleobases.
[0133] As used herein, "specifically hybridizes" means the ability
of an oligomeric compound to hybridize to one nucleic acid site
with greater affinity than it hybridizes to another nucleic acid
site. In certain embodiments, an antisense oligonucleotide
specifically hybridizes to more than one target site.
[0134] As used herein, "fully complementary" in reference to an
oligonucleotide or portion thereof means that each nucleobase of
the oligonucleotide or portion thereof is capable of pairing with a
nucleobase of a complementary nucleic acid or contiguous portion
thereof. Thus, a fully complementary region comprises no mismatches
or unhybridized nucleobases in either strand.
[0135] As used herein, "percent complementarity" means the
percentage of nucleobases of an oligomeric compound that are
complementary to an equal-length portion of a target nucleic acid.
Percent complementarity is calculated by dividing the number of
nucleobases of the oligomeric compound that are complementary to
nucleobases at corresponding positions in the target nucleic acid
by the total length of the oligomeric compound.
[0136] As used herein, "percent identity" means the number of
nucleobases in a first nucleic acid that are the same type
(independent of chemical modification) as nucleobases at
corresponding positions in a second nucleic acid, divided by the
total number of nucleobases in the first nucleic acid.
[0137] As used herein, "modulation" means a change of amount or
quality of a molecule, function, or activity when compared to the
amount or quality of a molecule, function, or activity prior to
modulation. For example, modulation includes the change, either an
increase (stimulation or induction) or a decrease (inhibition or
reduction) in gene expression. As a further example, modulation of
expression can include a change in splice site selection of
pre-mRNA processing, resulting in a change in the absolute or
relative amount of a particular splice-variant compared to the
amount in the absence of modulation.
[0138] As used herein, "modification motif" means a pattern of
chemical modifications in an oligomeric compound or a region
thereof. Motifs may be defined by modifications at certain
nucleosides and/or at certain linking groups of an oligomeric
compound.
[0139] As used herein, "nucleoside motif" means a pattern of
nucleoside modifications in an oligomeric compound or a region
thereof. The linkages of such an oligomeric compound may be
modified or unmodified. Unless otherwise indicated, motifs herein
describing only nucleosides are intended to be nucleoside motifs.
Thus, in such instances, the linkages are not limited.
[0140] As used herein, "sugar motif" means a pattern of sugar
modifications in an oligomeric compound or a region thereof.
[0141] As used herein, "linkage motif" means a pattern of linkage
modifications in an oligomeric compound or region thereof. The
nucleosides of such an oligomeric compound may be modified or
unmodified. Unless otherwise indicated, motifs herein describing
only linkages are intended to be linkage motifs. Thus, in such
instances, the nucleosides are not limited.
[0142] As used herein, "nucleobase modification motif" means a
pattern of modifications to nucleobases along an oligonucleotide.
Unless otherwise indicated, a nucleobase modification motif is
independent of the nucleobase sequence.
[0143] As used herein, "sequence motif" means a pattern of
nucleobases arranged along an oligonucleotide or portion thereof.
Unless otherwise indicated, a sequence motif is independent of
chemical modifications and thus may have any combination of
chemical modifications, including no chemical modifications.
[0144] As used herein, "type of modification" in reference to a
nucleoside or a nucleoside of a "type" means the chemical
modification of a nucleoside and includes modified and unmodified
nucleosides. Accordingly, unless otherwise indicated, a "nucleoside
having a modification of a first type" may be an unmodified
nucleoside.
[0145] As used herein, "differently modified" mean chemical
modifications or chemical substituents that are different from one
another, including absence of modifications. Thus, for example, a
MOE nucleoside and an unmodified DNA nucleoside are "differently
modified," even though the DNA nucleoside is unmodified. Likewise,
DNA and RNA are "differently modified," even though both are
naturally-occurring unmodified nucleosides. Nucleosides that are
the same but for comprising different nucleobases are not
differently modified. For example, a nucleoside comprising a 2'-OMe
modified sugar and an unmodified adenine nucleobase and a
nucleoside comprising a 2'-OMe modified sugar and an unmodified
thymine nucleobase are not differently modified.
[0146] As used herein, "the same type of modifications" refers to
modifications that are the same as one another, including absence
of modifications. Thus, for example, two unmodified DNA nucleoside
have "the same type of modification," even though the DNA
nucleoside is unmodified. Such nucleosides having the same type
modification may comprise different nucleobases.
[0147] As used herein, "pharmaceutically acceptable carrier or
diluent" means any substance suitable for use in administering to
an animal. In certain embodiments, a pharmaceutically acceptable
carrier or diluent is sterile saline. In certain embodiments, such
sterile saline is pharmaceutical grade saline.
[0148] As used herein, "substituent" and "substituent group," means
an atom or group that replaces the atom or group of a named parent
compound. For example a substituent of a modified nucleoside is any
atom or group that differs from the atom or group found in a
naturally occurring nucleoside (e.g., a modified 2'-substituent is
any atom or group at the 2'-position of a nucleoside other than H
or OH). Substituent groups can be protected or unprotected. In
certain embodiments, compounds of the present invention have
substituents at one or at more than one position of the parent
compound. Substituents may also be further substituted with other
substituent groups and may be attached directly or via a linking
group such as an alkyl or hydrocarbyl group to a parent
compound.
[0149] Likewise, as used herein, "substituent" in reference to a
chemical functional group means an atom or group of atoms differs
from the atom or a group of atoms normally present in the named
functional group. In certain embodiments, a substituent replaces a
hydrogen atom of the functional group (e.g., in certain
embodiments, the substituent of a substituted methyl group is an
atom or group other than hydrogen which replaces one of the
hydrogen atoms of an unsubstituted methyl group). Unless otherwise
indicated, groups amenable for use as substituents include without
limitation, halogen, hydroxyl, alkyl, alkenyl, alkynyl, acyl
(--C(O)R.sub.aa), carboxyl (--C(O)O--R.sub.aa), aliphatic groups,
alicyclic groups, alkoxy, substituted oxy (--O--R.sub.aa), aryl,
aralkyl, heterocyclic radical, heteroaryl, heteroarylalkyl, amino
(--N(R.sub.bb)(R.sub.cc)), imino (.dbd.NR.sub.bb), amido
(--C(O)N(R.sub.bb)(R.sub.cc) or --N(R.sub.bb)C(O)R.sub.aa), azido
(--N.sub.3), nitro (--NO.sub.2), cyano (--CN), carbamido
(--OC(O)N(R.sub.bb)(R.sub.cc) or --N(R.sub.bb)C(O)OR.sub.aa),
ureido (--N(R.sub.bb)C(O)N(R.sub.bb)(R.sub.cc)), thioureido
(--N(R.sub.bb)C(S)N(R.sub.bb)(R.sub.cc)), guanidinyl
(--N(R.sub.bb)C(.dbd.NR.sub.bb)N(R.sub.bb)(R.sub.cc)), amidinyl
(--C(.dbd.NR.sub.bb)N(R.sub.bb)(R.sub.cc) or
--N(R.sub.bb)C(.dbd.NR.sub.bb)(R.sub.aa)), thiol (--SR.sub.bb),
sulfinyl (--S(O)R.sub.bb), sulfonyl (--S(O).sub.2R.sub.bb) and
sulfonamidyl (--S(O).sub.2N(R.sub.bb)(R.sub.cc) or
--N(R.sub.bb)S(O).sub.2R.sub.bb). Wherein each R.sub.aa, R.sub.bb
and R.sub.cc is, independently, H, an optionally linked chemical
functional group or a further substituent group with a preferred
list including without limitation, alkyl, alkenyl, alkynyl,
aliphatic, alkoxy, acyl, aryl, aralkyl, heteroaryl, alicyclic,
heterocyclic and heteroarylalkyl. Selected substituents within the
compounds described herein are present to a recursive degree.
[0150] As used herein, "alkyl," as used herein, means a saturated
straight or branched hydrocarbon radical containing up to twenty
four carbon atoms. Examples of alkyl groups include without
limitation, methyl, ethyl, propyl, butyl, isopropyl, n-hexyl,
octyl, decyl, dodecyl and the like. Alkyl groups typically include
from 1 to about 24 carbon atoms, more typically from 1 to about 12
carbon atoms (C.sub.1-C.sub.12 alkyl) with from 1 to about 6 carbon
atoms being more preferred.
[0151] As used herein, "alkenyl," means a straight or branched
hydrocarbon chain radical containing up to twenty four carbon atoms
and having at least one carbon-carbon double bond. Examples of
alkenyl groups include without limitation, ethenyl, propenyl,
butenyl, 1-methyl-2-buten-1-yl, dienes such as 1,3-butadiene and
the like. Alkenyl groups typically include from 2 to about 24
carbon atoms, more typically from 2 to about 12 carbon atoms with
from 2 to about 6 carbon atoms being more preferred. Alkenyl groups
as used herein may optionally include one or more further
substituent groups.
[0152] As used herein, "alkynyl," means a straight or branched
hydrocarbon radical containing up to twenty four carbon atoms and
having at least one carbon-carbon triple bond. Examples of alkynyl
groups include, without limitation, ethynyl, 1-propynyl, 1-butynyl,
and the like. Alkynyl groups typically include from 2 to about 24
carbon atoms, more typically from 2 to about 12 carbon atoms with
from 2 to about 6 carbon atoms being more preferred. Alkynyl groups
as used herein may optionally include one or more further
substituent groups.
[0153] As used herein, "acyl," means a radical formed by removal of
a hydroxyl group from an organic acid and has the general Formula
--C(O)--X where X is typically aliphatic, alicyclic or aromatic.
Examples include aliphatic carbonyls, aromatic carbonyls, aliphatic
sulfonyls, aromatic sulfinyls, aliphatic sulfinyls, aromatic
phosphates, aliphatic phosphates and the like. Acyl groups as used
herein may optionally include further substituent groups.
[0154] As used herein, "alicyclic" means a cyclic ring system
wherein the ring is aliphatic. The ring system can comprise one or
more rings wherein at least one ring is aliphatic. Preferred
alicyclics include rings having from about 5 to about 9 carbon
atoms in the ring. Alicyclic as used herein may optionally include
further substituent groups.
[0155] As used herein, "aliphatic" means a straight or branched
hydrocarbon radical containing up to twenty four carbon atoms
wherein the saturation between any two carbon atoms is a single,
double or triple bond. An aliphatic group preferably contains from
1 to about 24 carbon atoms, more typically from 1 to about 12
carbon atoms with from 1 to about 6 carbon atoms being more
preferred. The straight or branched chain of an aliphatic group may
be interrupted with one or more heteroatoms that include nitrogen,
oxygen, sulfur and phosphorus. Such aliphatic groups interrupted by
heteroatoms include without limitation, polyalkoxys, such as
polyalkylene glycols, polyamines, and polyimines. Aliphatic groups
as used herein may optionally include further substituent
groups.
[0156] As used herein, "alkoxy" means a radical formed between an
alkyl group and an oxygen atom wherein the oxygen atom is used to
attach the alkoxy group to a parent molecule. Examples of alkoxy
groups include without limitation, methoxy, ethoxy, propoxy,
isopropoxy, n-butoxy, sec-butoxy, tert-butoxy, n-pentoxy,
neopentoxy, n-hexoxy and the like. Alkoxy groups as used herein may
optionally include further substituent groups.
[0157] As used herein, "aminoalkyl" means an amino substituted
C.sub.1-C.sub.12 alkyl radical. The alkyl portion of the radical
forms a covalent bond with a parent molecule. The amino group can
be located at any position and the aminoalkyl group can be
substituted with a further substituent group at the alkyl and/or
amino portions.
[0158] As used herein, "aralkyl" and "arylalkyl" mean an aromatic
group that is covalently linked to a C.sub.1-C.sub.12 alkyl
radical. The alkyl radical portion of the resulting aralkyl (or
arylalkyl) group forms a covalent bond with a parent molecule.
Examples include without limitation, benzyl, phenethyl and the
like. Aralkyl groups as used herein may optionally include further
substituent groups attached to the alkyl, the aryl or both groups
that form the radical group.
[0159] As used herein, "aryl" and "aromatic" mean a mono- or
polycyclic carbocyclic ring system radicals having one or more
aromatic rings. Examples of aryl groups include without limitation,
phenyl, naphthyl, tetrahydronaphthyl, indanyl, idenyl and the like.
Preferred aryl ring systems have from about 5 to about 20 carbon
atoms in one or more rings. Aryl groups as used herein may
optionally include further substituent groups.
[0160] As used herein, "halo" and "halogen," mean an atom selected
from fluorine, chlorine, bromine and iodine.
[0161] As used herein, "heteroaryl," and "heteroaromatic," mean a
radical comprising a mono- or poly-cyclic aromatic ring, ring
system or fused ring system wherein at least one of the rings is
aromatic and includes one or more heteroatoms. Heteroaryl is also
meant to include fused ring systems including systems where one or
more of the fused rings contain no heteroatoms. Heteroaryl groups
typically include one ring atom selected from sulfur, nitrogen or
oxygen. Examples of heteroaryl groups include without limitation,
pyridinyl, pyrazinyl, pyrimidinyl, pyrrolyl, pyrazolyl, imidazolyl,
thiazolyl, oxazolyl, isooxazolyl, thiadiazolyl, oxadiazolyl,
thiophenyl, furanyl, quinolinyl, isoquinolinyl, benzimidazolyl,
benzooxazolyl, quinoxalinyl and the like. Heteroaryl radicals can
be attached to a parent molecule directly or through a linking
moiety such as an aliphatic group or hetero atom. Heteroaryl groups
as used herein may optionally include further substituent
groups.
B. Oligomeric Compounds
[0162] As used herein, the term "oligomeric compound" refers to a
contiguous sequence of linked monomer subunits. Each linked monomer
subunit normally includes a heterocyclic base moiety but monomer
subunits also includes those without a heterocyclic base moiety
such as abasic monomer subunits. At least some and generally most
if not essentially all of the heterocyclic bases in an oligomeric
compound are capable of hybridizing to a nucleic acid molecule,
normally a preselected RNA target. The term "oligomeric compound"
therefore includes oligonucleotides, oligonucleotide analogs and
oligonucleosides. It also includes polymers having one or a
plurality of nucleosides having sugar surrogate groups.
[0163] In certain embodiments, oligomeric compounds comprise a
plurality of monomer subunits independently selected from naturally
occurring nucleosides, non-naturally occurring nucleosides,
modified nucleosides and nucleosides having sugar surrogate groups.
In certain embodiments, oligomeric compounds are single stranded.
In certain embodiments, oligomeric compounds are double stranded
comprising a double-stranded duplex. In certain embodiments,
oligomeric compounds comprise one or more conjugate groups and/or
terminal groups.
[0164] In certain embodiments, the oligomeric compounds as provided
herein can be modified by covalent attachment of one or more
terminal groups to the 5' or 3'-terminal groups. A terminal group
can also be attached at any other position at one of the terminal
ends of the oligomeric compound. As used herein the terms
"5'-terminal group", "3'-terminal group", "terminal group" and
combinations thereof are meant to include useful groups known to
the art skilled that can be placed on one or both of the terminal
ends, including but not limited to the 5' and 3'-ends of an
oligomeric compound respectively, for various purposes such as
enabling the tracking of the oligomeric compound (a fluorescent
label or other reporter group), improving the pharmacokinetics or
pharmacodynamics of the oligomeric compound (such as for example:
uptake and/or delivery) or enhancing one or more other desirable
properties of the oligomeric compound (a group for improving
nuclease stability or binding affinity). In certain embodiments, 5'
and 3'-terminal groups include without limitation, modified or
unmodified nucleosides; two or more linked nucleosides that are
independently, modified or unmodified; conjugate groups; capping
groups; phosphate moieties; and protecting groups.
[0165] In certain embodiments, the present invention provides
oligomeric compounds. In certain embodiments, such oligomeric
compounds comprise oligonucleotides optionally comprising one or
more conjugate and/or terminal groups. In certain embodiments, an
oligomeric compound consists of an oligonucleotide. In certain
embodiments, oligonucleotides comprise one or more chemical
modifications. Such chemical modifications include modifications of
one or more nucleoside (including modifications to the sugar moiety
and/or the nucleobase) and/or modifications to one or more
internucleoside linkage.
[0166] a. Certain Modified Nucleosides
[0167] Provided herein are oligomeric compounds comprising modified
nucleosides. Such modified nucleosides comprise a modified sugar
moeity, a modified nucleobase, or both a modified sugar moiety and
a modified nucleobase.
[0168] i. Certain Modified Sugar Moieties
[0169] In certain embodiments, compounds of the invention comprise
one or more modified nucleosides comprising a modified sugar
moiety. Such compounds comprising one or more sugar-modified
nucleosides may have desirable properties, such as enhanced
nuclease stability or increased binding affinity with a target
nucleic acid relative to an oligonucleotide comprising only
nucleosides comprising naturally occurring sugar moieties. In
certain embodiments, modified sugar moieties are substituted sugar
moieties. In certain embodiments, modified sugar moieties are sugar
surrogates. Such sugar surrogates may comprise one or more
substitutions corresponding to those of substituted sugar
moieties.
[0170] In certain embodiments, modified sugar moieties are
substituted sugar moieties comprising one or more non-bridging
sugar substituents, including but not limited to substituents at
the 2' and/or 5' positions. Examples of sugar substituents suitable
for the 2'-position, include, but are not limited to: 2'-F,
2'-OCH.sub.3 ("OMe" or "O-methyl"), and
2'-O(CH.sub.2).sub.2OCH.sub.3 ("MOE"). In certain embodiments,
sugar substituents at the 2' position are selected from allyl,
amino, azido, thio, O-allyl, O--C.sub.1-C.sub.10 alkyl,
O--C.sub.1-C.sub.10 substituted alkyl; OCF.sub.3,
O(CH.sub.2).sub.2SCH.sub.3, O(CH.sub.2).sub.2--O--N(Rm)(Rn), and
O--CH.sub.2--C(.dbd.O)--N(Rm)(Rn), where each Rm and Rn is,
independently, H or substituted or unsubstituted C.sub.1-C.sub.10
alkyl. Examples of sugar substituents at the 5'-position, include,
but are not limited to: 5'-methyl (R or S); 5'-vinyl, and
5'-methoxy. In certain embodiments, substituted sugars comprise
more than one non-bridging sugar substituent, for example,
2'-F-5'-methyl sugar moieties (see, e.g., PCT International
Application WO 2008/101157, for additional 5', 2'-bis substituted
sugar moieties and nucleosides).
[0171] Nucleosides comprising 2'-substituted sugar moieties are
referred to as 2'-substituted nucleosides. In certain embodiments,
a 2'-substituted nucleoside comprises a 2'-substituent group
selected from halo, allyl, amino, azido, SH, CN, OCN, CF.sub.3,
OCF.sub.3, O, S, or N(R.sub.m)-alkyl; O, S, or N(R.sub.m)-alkenyl;
O, S or N(R.sub.m)-alkynyl; O-alkylenyl-O-alkyl, alkynyl, alkaryl,
aralkyl, O-alkaryl, O-aralkyl, O(CH.sub.2).sub.2SCH.sub.3,
O--(CH.sub.2).sub.2--O--N(R.sub.m)(R.sub.n) or
O--CH.sub.2--C(.dbd.O)--N(R.sub.m)(R.sub.n), where each R.sub.m and
R.sub.n is, independently, H, an amino protecting group or
substituted or unsubstituted C.sub.1-C.sub.10 alkyl. These
2'-substituent groups can be further substituted with one or more
substituent groups independently selected from hydroxyl, amino,
alkoxy, carboxy, benzyl, phenyl, nitro (NO.sub.2), thiol,
thioalkoxy (S-alkyl), halogen, alkyl, aryl, alkenyl and
alkynyl.
[0172] In certain embodiments, a 2'-substituted nucleoside
comprises a 2'-substituent group selected from F, NH.sub.2,
N.sub.3, OCF.sub.3, O--CH.sub.3, O(CH.sub.2).sub.3NH.sub.2,
CH.sub.2--CH.dbd.CH.sub.2, O--CH.sub.2--CH.dbd.CH.sub.2,
OCH.sub.2CH.sub.2OCH.sub.3, O(CH.sub.2).sub.2SCH.sub.3,
O--(CH.sub.2).sub.2--O--N(R.sub.m)(R.sub.n),
O(CH.sub.2).sub.2O(CH.sub.2).sub.2N(CH.sub.3).sub.2, and
N-substituted acetamide
(O--CH.sub.2--C(.dbd.O)--N(R.sub.m)(R.sub.n) where each R.sub.m and
R.sub.n is, independently, H, an amino protecting group or
substituted or unsubstituted C.sub.1-C.sub.10 alkyl.
[0173] In certain embodiments, a 2'-substituted nucleoside
comprises a sugar moiety comprising a 2'-substituent group selected
from F, OCF.sub.3, O--CH.sub.3, OCH.sub.2CH.sub.2OCH.sub.3,
O(CH.sub.2).sub.2SCH.sub.3,
O--(CH.sub.2).sub.2--O--N(CH.sub.3).sub.2,
--O(CH.sub.2).sub.2O(CH.sub.2).sub.2N(CH.sub.3).sub.2, and
O--CH.sub.2--C(.dbd.O)--N(H)CH.sub.3.
[0174] In certain embodiments, a 2'-substituted nucleoside
comprises a sugar moiety comprising a 2'-substituent group selected
from F, O--CH.sub.3, and OCH.sub.2CH.sub.2OCH.sub.3.
[0175] Certain modifed sugar moieties comprise a bridging sugar
substituent that forms a second ring resulting in a bicyclic sugar
moiety. In certain such embodiments, the bicyclic sugar moiety
comprises a bridge between the 4' and the 2' furanose ring atoms.
Examples of such 4' to 2' sugar substituents, include, but are not
limited to: --[C(R.sub.a)(R.sub.b)].sub.n--,
--[C(R.sub.a)(R.sub.b)].sub.n--O--, --C(R.sub.aR.sub.b)--N(R)--O--
or, --C(R.sub.aR.sub.b)--O--N(R)--; 4'-(CH.sub.2).sub.2-2',
4'-(CH.sub.2)--O-2' (LNA); 4'-(CH.sub.2)--S-2;
4'-(CH.sub.2).sub.2--O-2' (ENA); 4'-CH(CH.sub.3)--O-2' (cEt) and
4'-CH(CH.sub.2OCH.sub.3)--O-2', and analogs thereof (see, e.g.,
U.S. Pat. No. 7,399,845, issued on Jul. 15, 2008);
4'-C(CH.sub.3)(CH.sub.3)--O-2' and analogs thereof, (see, e.g.,
WO2009/006478, published Jan. 8, 2009);
4'-CH.sub.2--N(OCH.sub.3)-2' and analogs thereof (see, e.g.,
WO2008/150729, published Dec. 11, 2008);
4'-CH.sub.2--O--N(CH.sub.3)-2' (see, e.g., US2004/0171570,
published Sep. 2, 2004); 4'-CH.sub.2--O--N(R)-2', and
4'-CH.sub.2--N(R)--O-2'-, wherein each R is, independently, H, a
protecting group, or C.sub.1-C.sub.12 alkyl;
4'-CH.sub.2--N(R)--O-2', wherein R is H, C.sub.1-C.sub.12 alkyl, or
a protecting group (see, U.S. Pat. No. 7,427,672, issued on Sep.
23, 2008); 4'-CH.sub.2--C(H)(CH.sub.3)-2' (see, e.g.,
Chattopadhyaya, et al., J. Org. Chem., 2009, 74, 118-134); and
4'-CH.sub.2--C(.dbd.CH.sub.2)-2' and analogs thereof (see,
published PCT International Application WO 2008/154401, published
on Dec. 8, 2008).
[0176] In certain embodiments, such 4' to 2' bridges independently
comprise from 1 to 4 linked groups (generally forming a 4 to 6
membered ring with the parent sugar moiety) independently selected
from --[C(R.sub.a)(R.sub.b)].sub.n--,
--C(R.sub.a).dbd.C(R.sub.b)--, --C(R.sub.a).dbd.N--,
--C(.dbd.NR.sub.a)--, --C(.dbd.O)--, --C(.dbd.S)--, --O--,
--Si(R.sub.a).sub.2--, --S(.dbd.O).sub.x--, and --N(R.sub.a)--;
[0177] wherein:
[0178] x is 0, 1, or 2;
[0179] n is 1, 2, 3, or 4;
[0180] each R.sub.a and R.sub.b is, independently, H, a protecting
group, hydroxyl, C.sub.1-C.sub.12 alkyl, substituted
C.sub.1-C.sub.12 alkyl, C.sub.2-C.sub.12 alkenyl, substituted
C.sub.2-C.sub.12 alkenyl, C.sub.2-C.sub.12 alkynyl, substituted
C.sub.2-C.sub.12 alkynyl, C.sub.5-C.sub.20 aryl, substituted
C.sub.5-C.sub.20 aryl, heterocycle radical, substituted heterocycle
radical, heteroaryl, substituted heteroaryl, C.sub.5-C.sub.7
alicyclic radical, substituted C.sub.5-C.sub.7 alicyclic radical,
halogen, OJ.sub.1, NJ.sub.1J.sub.2, SJ.sub.1, N.sub.3, COOJ.sub.1,
acyl (C(.dbd.O)--H), substituted acyl, CN, sulfonyl
(S(.dbd.O).sub.2-J.sub.1), or sulfoxyl (S(.dbd.O)-J.sub.1); and
[0181] each J.sub.1 and J.sub.2 is, independently, H,
C.sub.1-C.sub.12 alkyl, substituted C.sub.1-C.sub.12 alkyl,
C.sub.2-C.sub.12 alkenyl, substituted C.sub.2-C.sub.12 alkenyl,
C.sub.2-C.sub.12 alkynyl, substituted C.sub.2-C.sub.12 alkynyl,
C.sub.5-C.sub.20 aryl, substituted C.sub.5-C.sub.20 aryl, acyl
(C(.dbd.O)--H), substituted acyl, a heterocycle radical, a
substituted heterocycle radical, C.sub.1-C.sub.12 aminoalkyl,
substituted C.sub.1-C.sub.12 aminoalkyl, or a protecting group.
[0182] Nucleosides comprising bicyclic sugar moieties are referred
to as bicyclic nucleosides or BNAs. Bicyclic nucleosides include,
but are not limited to, (A) .alpha.-L-Methyleneoxy
(4'-CH.sub.2--O-2') BNA, (B) .beta.-D-Methyleneoxy
(4'-CH.sub.2--O-2') BNA (also referred to as locked nucleic acid or
LNA), (C) Ethyleneoxy (4'-(CH.sub.2).sub.2--O-2') BNA, (D) Aminooxy
(4'-CH.sub.2--O--N(R)-2') BNA, (E) Oxyamino
(4'-CH.sub.2--N(R)--O-2') BNA, (F) Methyl(methyleneoxy)
(4'-CH(CH.sub.3)--O-2') BNA (also referred to as constrained ethyl
or cEt), (G) methylene-thio (4'-CH.sub.2--S-2') BNA, (H)
methylene-amino (4'-CH.sub.2--N(R)-2') BNA, (I) methyl carbocyclic
(4'-CH.sub.2--CH(CH.sub.3)-2') BNA, (J) propylene carbocyclic
(4'-(CH.sub.2).sub.3-2') BNA, and (K) Ethylene(methoxy)
(4'-(CH(CH.sub.2OMe)-O-2') BNA (also referred to as constrained MOE
or cMOE) as depicted below.
##STR00006## ##STR00007##
wherein Bx is a nucleobase moiety and R is, independently, H, a
protecting group, or C.sub.1-C.sub.12 alkyl.
[0183] Additional bicyclic sugar moieties are known in the art, for
example: Singh et al., Chem. Commun., 1998, 4, 455-456; Koshkin et
al., Tetrahedron, 1998, 54, 3607-3630; Wahlestedt et al., Proc.
Natl. Acad. Sci. U.S.A, 2000, 97, 5633-5638; Kumar et al., Bioorg.
Med. Chem. Lett., 1998, 8, 2219-2222; Singh et al., J. Org. Chem.,
1998, 63, 10035-10039; Srivastava et al., J. Am. Chem. Soc.,
129(26) 8362-8379 (Jul. 4, 2007); Elayadi et al., Curr. Opinion
Invens. Drugs, 2001, 2, 558-561; Braasch et al., Chem. Biol., 2001,
8, 1-7; Orum et al., Curr. Opinion Mol. Ther., 2001, 3, 239-243;
U.S. Pat. Nos. 7,053,207, 6,268,490, 6,770,748, 6,794,499,
7,034,133, 6,525,191, 6,670,461, and 7,399,845; WO 2004/106356, WO
1994/14226, WO 2005/021570, and WO 2007/134181; U.S. Patent
Publication Nos. US2004/0171570, US2007/0287831, and
US2008/0039618; U.S. patent Ser. Nos. 12/129,154, 60/989,574,
61/026,995, 61/026,998, 61/056,564, 61/086,231, 61/097,787, and
61/099,844; and PCT International Applications Nos.
PCT/US2008/064591, PCT/US2008/066154, and PCT/US2008/068922.
[0184] In certain embodiments, bicyclic sugar moieties and
nucleosides incorporating such bicyclic sugar moieties are further
defined by isomeric configuration. For example, a nucleoside
comprising a 4'-2' methylene-oxy bridge, may be in the .alpha.-L
configuration or in the .beta.-D configuration. Previously,
.alpha.-L-methyleneoxy (4'-CH.sub.2--O-2') bicyclic nucleosides
have been incorporated into antisense oligonucleotides that showed
antisense activity (Frieden et al., Nucleic Acids Research, 2003,
21, 6365-6372).
[0185] In certain embodiments, substituted sugar moieties comprise
one or more non-bridging sugar substituent and one or more bridging
sugar substituent (e.g., 5'-substituted and 4'-2' bridged sugars).
(see, PCT International Application WO 2007/134181, published on
Nov. 22, 2007, wherein LNA is substituted with, for example, a
5'-methyl or a 5'-vinyl group).
[0186] In certain embodiments, modified sugar moieties are sugar
surrogates. In certain such embodiments, the oxygen atom of the
naturally occurring sugar is substituted, e.g., with a sulfer,
carbon or nitrogen atom. In certain such embodiments, such modified
sugar moiety also comprises bridging and/or non-bridging
substituents as described above. For example, certain sugar
surrogates comprise a 4'-sulfer atom and a substitution at the
2'-position (see, e.g., published U.S. Patent Application
US2005/0130923, published on Jun. 16, 2005) and/or the 5' position.
By way of additional example, carbocyclic bicyclic nucleosides
having a 4'-2' bridge have been described (see, e.g., Freier et
al., Nucleic Acids Research, 1997, 25(22), 4429-4443 and Albaek et
al., J. Org. Chem., 2006, 71, 7731-7740).
[0187] In certain embodiments, sugar surrogates comprise rings
having other than 5-atoms. For example, in certain embodiments, a
sugar surrogate comprises a six-membered tetrahydropyran. Such
tetrahydropyrans may be further modified or substituted.
Nucleosides comprising such modified tetrahydropyrans include, but
are not limited to, hexitol nucleic acid (HNA), anitol nucleic acid
(ANA), manitol nucleic acid (MNA) (see Leumann, CJ. Bioorg. &
Med. Chem. (2002) 10:841-854), fluoro HNA (F-HNA), and those
compounds having Formula VII:
##STR00008##
wherein independently for each of said at least one tetrahydropyran
nucleoside analog of Formula VII:
[0188] Bx is a nucleobase moiety;
[0189] T.sub.3 and T.sub.4 are each, independently, an
internucleoside linking group linking the tetrahydropyran
nucleoside analog to the antisense compound or one of T.sub.3 and
T.sub.4 is an internucleoside linking group linking the
tetrahydropyran nucleoside analog to the antisense compound and the
other of T.sub.3 and T.sub.4 is H, a hydroxyl protecting group, a
linked conjugate group, or a 5' or 3'-terminal group;
q.sub.1, q.sub.2, q.sub.3, q.sub.4, q.sub.5, q.sub.6 and q.sub.7
are each, independently, H, C.sub.1-C.sub.6 alkyl, substituted
C.sub.1-C.sub.6 alkyl, C.sub.2-C.sub.6 alkenyl, substituted
C.sub.2-C.sub.6 alkenyl, C.sub.2-C.sub.6 alkynyl, or substituted
C.sub.2-C.sub.6 alkynyl; and
[0190] each of R.sub.1 and R.sub.2 is independently selected from
among: hydrogen, halogen, substituted or unsubstituted alkoxy,
NJ.sub.1J.sub.2, SJ.sub.1, N.sub.3, OC(.dbd.X)J.sub.1,
OC(.dbd.X)NJ.sub.1J.sub.2, NJ.sub.3C(.dbd.X)NJ.sub.1J.sub.2, and
CN, wherein X is O, S or NJ.sub.1, and each J.sub.1, J.sub.2, and
J.sub.3 is, independently, H or C.sub.1-C.sub.6 alkyl.
[0191] In certain embodiments, the modified THP nucleosides of
Formula VII are provided wherein q.sub.1, q.sub.2, q.sub.3,
q.sub.4, q.sub.5, q.sub.6 and q.sub.7 are each H. In certain
embodiments, at least one of q.sub.1, q.sub.2, q.sub.3, q.sub.4,
q.sub.5, q.sub.6 and q.sub.7 is other than H. In certain
embodiments, at least one of q.sub.1, q.sub.2, q.sub.3, q.sub.4,
q.sub.5, q.sub.6 and q.sub.7 is methyl. In certain embodiments, THP
nucleosides of Formula VII are provided wherein one of R.sub.1 and
R.sub.2 is F. In certain embodiments, R.sub.1 is fluoro and R.sub.2
is H, R.sub.1 is methoxy and R.sub.2 is H, and R.sub.1 is
methoxyethoxy and R.sub.2 is H.
[0192] Many other bicyclo and tricyclo sugar surrogate ring systems
are also known in the art that can be used to modify nucleosides
for incorporation into antisense compounds (see, e.g., review
article: Leumann, J. C, Bioorganic & Medicinal Chemistry, 2002,
10, 841-854).
[0193] Combinations of modifications are also provided without
limitation, such as 2'-F-5'-methyl substituted nucleosides (see PCT
International Application WO 2008/101157 Published on Aug. 21, 2008
for other disclosed 5', 2'-bis substituted nucleosides) and
replacement of the ribosyl ring oxygen atom with S and further
substitution at the 2'-position (see published U.S. Patent
Application US2005-0130923, published on Jun. 16, 2005) or
alternatively 5'-substitution of a bicyclic nucleic acid (see PCT
International Application WO 2007/134181, published on Nov. 22,
2007 wherein a 4'-CH.sub.2--O-2' bicyclic nucleoside is further
substituted at the 5' position with a 5'-methyl or a 5'-vinyl
group). The synthesis and preparation of carbocyclic bicyclic
nucleosides along with their oligomerization and biochemical
studies have also been described (see, e.g., Srivastava et al., J.
Am. Chem. Soc. 2007, 129(26), 8362-8379).
[0194] In certain embodiments, the present invention provides
oligonucleotides comprising modified nucleosides. Those modified
nucleotides may include modified sugars, modified nucleobases,
and/or modified linkages. The specific modifications are selected
such that the resulting oligonucleotides possess desirable
characteristics. In certain embodiments, oligonucleotides comprise
one or more RNA-like nucleosides. In certain embodiments,
oligonucleotides comprise one or more DNA-like nucleosides.
[0195] In certain embodiments, the oligomeric compounds provided
herein include RNA-like nucleosides that have been modified to
influence the sugar conformation to have predominantly 3'-endo
conformational geometry. In certain embodiments, such modified
nucleosides include synthetic modifications of the heterocyclic
base moiety, the sugar moiety or both to induce a 3'-endo sugar
conformation. In certain embodiments, RNA-like nucleosides are
selected from RNA surrogates such as including, but not limited to,
F-HNA or cyclohexenyl nucleic acid. RNA-like nucleosides are used
to replace and mimic RNA nucleosides in an oligomeric compound so
that particular properties of the oligomeric compound can be
enhanced. Typically RNA-like nucleosides are used in the 5' and
3'-regions (wings) of gapped oligomeric compounds to improve
stability in the presence of nucleases and also to increase the
affinity for nucleic a nucleic acid target. Other properties that
can also be enhanced by using RNA-like nucleosides include but
aren't limited to modulation of pharmacokinetic properties through
modification of protein binding, protein off-rate, absorption and
clearance as well as chemical stability and specificity of the
oligomeric compound (affinity and specificity for enzymes as well
as for complementary sequences); and increasing efficacy of RNA
cleavage.
[0196] In certain embodiments, RNA-like nucleosides include
modified nucleosides comprising one or more 2', 3', 4' and 5'
substituent groups, bicyclic nucleosides and RNA-surrogates. In
certain embodiments, RNA-like nucleosides include, but are not
limited to modified nucleosides comprising 2'-ribo-substituent
groups selected from: F, OCH.sub.3, O--C.sub.2-C.sub.4 alkyl,
O--CH.sub.2CH.dbd.CH.sub.2, O--(CH.sub.2).sub.2--O--CH.sub.3 (MOE),
O--(CH.sub.2).sub.3--NH.sub.2,
O--(CH.sub.2).sub.2--O--N(R.sub.1).sub.2,
O--CH.sub.2C(O)--N(R.sub.1).sub.2,
O--(CH.sub.2).sub.2--O--(CH.sub.2).sub.2--N(R.sub.1).sub.2,
O--(CH.sub.2).sub.3--NHR.sub.1 and
O--CH.sub.2--N(H)--C(.dbd.NR.sub.1)[N(R.sub.1).sub.2] wherein each
R.sub.1 is, typically H, C.sub.1-C.sub.12 alkyl or a protecting
group. RNA-like nucleosides also include but are not limited to
modified nucleosides having a bicyclic furanosyl sugar moiety
(bicyclic nucleosides) comprising a bridging group between the 4'
and 2'-carbon atoms. Such bicyclic nucleosides include, but are not
limited to bridging groups consisting of from 1 to 3 linked
biradical groups selected from O, S, NR.sub.a, C(R.sub.b)(R.sub.c),
C.dbd.O, C(R.sub.b).dbd.C(R.sub.c) and C[.dbd.C(R.sub.b)(R.sub.c)]
wherein C(R.sub.b).dbd.C(R.sub.c) counts as 2 of said biradical
groups wherein each R.sub.a, R.sub.b and R.sub.c is, independently,
H, C.sub.1-C.sub.6 alkyl, C.sub.1-C.sub.6 alkoxy, C.sub.2-C.sub.6
alkenyl or C.sub.2-C.sub.6 alkynyl. In certain embodiments, the
bridging groups include, but are not limited to
4'-(CH.sub.2)--O-2', 4'-(CH.sub.2)--S-2',
4'-(CH.sub.2).sub.2--O-2', 4'-CH(CH.sub.3)--O-2',
4'-CH(CH.sub.2OCH.sub.3)--O-2', 4'-C(CH.sub.3).sub.2--O-2',
4'-CH.sub.2--N(OCH.sub.3)-2', 4'-CH.sub.2--O--N(CH.sub.3)-2',
4'-CH.sub.2--NCH.sub.3--O-2', 4'-CH.sub.2--C(H)(CH.sub.3)-2' and
4'-CH.sub.2--C(.dbd.CH.sub.2)-2'. In certain embodiments, the
bridging groups include, but are not limited to 4'-CH.sub.2--O-2',
4'-(CH.sub.2).sub.2--O-2', 4'-C(H)[(R)--CH.sub.3]--O-2' and
4'-C(H)[(S)--CH.sub.3]--O-2'.
[0197] In certain embodiments, the oligomeric compounds provided
herein include DNA-like nucleosides that have been modified to
influence the sugar conformation to have predominantly 2'-endo
conformational geometry. Such modified nucleosides can include
synthetic modifications of the heterocyclic base moiety, the sugar
moiety or both to induce the desired 2'-endo sugar conformation.
These modified nucleosides are used to mimic RNA nucleosides so
that particular properties of an oligomeric compound can be
enhanced while maintaining the desirable 2'-endo conformational
geometry.
[0198] In certain embodiments, DNA-like nucleosides include, but
are not limited to 2'-substituted furanosyl nucleosides comprising:
2'=CH.sub.2, 2'-ara-CN, 2'-ara-F, 2'-ara-Br or 2'-ara-Cl,
2'-ara-N.sub.3, 2'-ara-OH, 2'-ara-O--CH.sub.3 or
2'-dehydro-2'-ara-CH.sub.3.
[0199] The C3'-endo and C2'-endo conformational geometries are
shown below:
##STR00009##
[0200] ii Certain Modified Nucleobases
[0201] In certain embodiments, nucleosides of the present invention
comprise one or more unmodified nucleobases. In certain
embodiments, nucleosides of the present invention comprise one or
more modifed nucleobases (heterocyclic base moieties).
[0202] In one embodiment, a heterocyclic base moiety is any
heterocyclic system that contains one or more atoms or groups of
atoms capable of hydrogen bonding to a heterocyclic base of a
nucleic acid. In certain embodiments, nucleobase refers to purines,
modified purines, pyrimidines and modified pyrimidines. In certain
embodiments, nucleobase refers to unmodified or naturally occurring
nucleobases which include, but are not limited to, the purine bases
adenine (A) and guanine (G), and the pyrimidine bases thymine (T),
cytosine (C) and uracil (U) and analogs thereof such as 5-methyl
cytosine. The terms nucleobase and heterocyclic base moiety also
include optional protection for any reactive functional groups such
as 4-N-benzoylcytosine, 4-N-benzoyl-5-methyl-cytosine,
6-N-benzoyladenine or 2-N-isobutyrylguanine.
[0203] In certain embodiments, heterocyclic base moieties include
without limitation modified nucleobases such as 5-methylcytosine
(5-me-C), 5-hydroxymethyl cytosine, xanthine, hypoxanthine,
2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and
guanine, 2-propyl and other alkyl derivatives of adenine and
guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine,
5-halouracil and cytosine, 5-propynyl (--C.ident.C--CH.sub.3)
uracil and cytosine and other alkynyl derivatives of pyrimidine
bases, 6-azo uracil, cytosine and thymine, 5-uracil (pseudouracil),
4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and
other 8-substituted adenines and guanines, 5-halo particularly
5-bromo, 5-trifluoromethyl and other 5-substituted uracils and
cytosines, 7-methylguanine and 7-methyladenine, 2-F-adenine,
2-amino-adenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and
7-deazaadenine, 3-deazaguanine and 3-deazaadenine, universal bases,
hydrophobic bases, promiscuous bases, size-expanded bases, and
fluorinated bases as defined herein.
[0204] In certain embodiments, heterocyclic base moieties include
without limitation tricyclic pyrimidines such as
1,3-diazaphenoxazine-2-one, 1,3-diazaphenothiazine-2-one and
9-(2-aminoethoxy)-1,3-diazaphenoxazine-2-one (G-clamp).
Heterocyclic base moieties also include those in which the purine
or pyrimidine base is replaced with other heterocycles, for example
7-deaza-adenine, 7-deazaguanosine, 2-aminopyridine and 2-pyridone.
Further heterocyclic base moieties include without limitation those
known to the art skilled (see for example: U.S. Pat. No. 3,687,808;
Swayze et al., The Medicinal Chemistry of Oligonucleotides in
Antisense a Drug Technology, Chapter 6, pages 143-182, Crooke, S.
T., ed., 2008); The Concise Encyclopedia Of Polymer Science And
Engineering, Kroschwitz, J. I., Ed., John Wiley & Sons, 1990,
858-859; Englisch et al., Angewandte Chemie, International Edition,
1991, 30, 613; Sanghvi, Y. S., Chapter 15, Antisense Research and
Applications, Crooke, S. T. and Lebleu, B., Eds., CRC Press, 1993,
273-302).
[0205] Modified polycyclic heterocyclic compounds useful as
heterocyclic base moieties are disclosed in the above noted U.S.
Pat. No. 3,687,808, as well as U.S. Pat. Nos. 4,845,205; 5,130,302;
5,134,066; 5,175,273; 5,367,066; 5,432,272; 5,434,257; 5,457,187;
5,459,255; 5,484,908; 5,502,177; 5,525,711; 5,552,540; 5,587,469;
5,594,121, 5,596,091; 5,614,617; 5,645,985; 5,646,269; 5,681,941;
5,750,692; 5,763,588; 5,830,653; 6,005,096; and U.S. Patent
Application Publication 20030158403, each of which is incorporated
herein by reference in its entirety.
[0206] b. Certain Internucleoside Linkages
[0207] In certain embodiments, nucleosides may be linked together
using any internucleoside linkage to form oligonucleotides. The two
main classes of internucleoside linking groups are defined by the
presence or absence of a phosphorus atom. Representative phosphorus
containing internucleoside linkages include, but are not limited
to, phosphodiesters (P.dbd.O), phosphotriesters,
methylphosphonates, phosphoramidate, and phosphorothioates
(P.dbd.S). Representative non-phosphorus containing internucleoside
linking groups include, but are not limited to,
methylenemethylimino (--CH.sub.2--N(CH.sub.3)--O--CH.sub.2--),
thiodiester (--O--C(O)--S--), thionocarbamate (--O--C(O)(NH)--S--);
siloxane (--O--Si(H).sub.2--O--); and N,N'-dimethylhydrazine
(--CH.sub.2--N(CH.sub.3)--N(CH.sub.3)--). Modified linkages,
compared to natural phosphodiester linkages, can be used to alter,
typically increase, nuclease resistance of the oligonucleotide. In
certain embodiments, internucleoside linkages having a chiral atom
can be prepared as a racemic mixture, or as separate enantiomers.
Representative chiral linkages include, but are not limited to,
alkylphosphonates and phosphorothioates. Methods of preparation of
phosphorous-containing and non-phosphorous-containing
internucleoside linkages are well known to those skilled in the
art.
[0208] The oligonucleotides described herein contain one or more
asymmetric centers and thus give rise to enantiomers,
diastereomers, and other stereoisomeric configurations that may be
defined, in terms of absolute stereochemistry, as (R) or (S),
.alpha. or .beta. such as for sugar anomers, or as (D) or (L) such
as for amino acids etc. Included in the antisense compounds
provided herein are all such possible isomers, as well as their
racemic and optically pure forms.
[0209] Neutral internucleoside linkages include without limitation,
phosphotriesters, methylphosphonates, MMI
(3'-CH.sub.2--N(CH.sub.3)--O-5'), amide-3
(3'-CH.sub.2--C(.dbd.O)--N(H)-5'), amide-4
(3'-CH.sub.2--N(H)--C(.dbd.O)-5'), formacetal
(3'-O--CH.sub.2--O-5'), and thioformacetal (3'-S--CH.sub.2--O-5').
Further neutral internucleoside linkages include nonionic linkages
comprising siloxane (dialkylsiloxane), carboxylate ester,
carboxamide, sulfide, sulfonate ester and amides (See for example:
Carbohydrate Modifications in Antisense Research; Y. S. Sanghvi and
P. D. Cook, Eds., ACS Symposium Series 580; Chapters 3 and 4,
40-65). Further neutral internucleoside linkages include nonionic
linkages comprising mixed N, O, S and CH.sub.2 component parts.
[0210] c. Certain Motifs
[0211] In certain embodiments, oligomeric compounds comprise or
consist of oligonucleotides. In certain embodiments, such
oligonucleotides comprise one or more chemical modifications. In
certain embodiments, chemically modified oligonucleotides comprise
one or more modified sugars. In certain embodiments, chemically
modified oligonucleotides comprise one or more modified
nucleobases. In certain embodiments, chemically modified
oligonucleotides comprise one or more modified internucleoside
linkages. In certain embodiments, the chemical modifications (sugar
modifications, nucleobase modifications, and/or linkage
modifications) define a pattern or motif. In certain embodiments,
the patterns of chemical modifications of sugar moieties,
internucleoside linkages, and nucleobases are each independent of
one another. Thus, an oligonucleotide may be described by its sugar
modification motif, internucleoside linkage motif and/or nucleobase
modification motif (as used herein, nucleobase modification motif
describes the chemical modifications to the nucleobases independent
of the sequence of nucleobases).
[0212] i. Certain Sugar Motifs
[0213] In certain embodiments, oligonucleotides comprise one or
more type of modified sugar moieties and/or naturally occurring
sugar moieties arranged along an oligonucleotide or region thereof
in a defined pattern or sugar motif. Such sugar motifs include but
are not limited to any of the sugar modifications discussed
herein.
[0214] In certain embodiments, the oligomeric compounds provided
herein comprise a gapmer sugar motif, which comprises two external
regions or "wings" and a central or internal region or "gap" (also
referred to as 5'-region and 3'-region). The three regions of a
gapmer sugar motif (the 5'-wing, the gap, and the 3'-wing) form a
contiguous sequence of nucleosides wherein at least some of the
sugar moieties of the nucleosides of each of the wings differ from
at least some of the sugar moieties of the nucleosides of the gap.
Specifically, at least the sugar moieties of the nucleosides of
each wing that are closest to the gap (the 3'-most nucleoside of
the 5'-wing and the 5'-most nucleoside of the 3'-wing) differ from
the sugar moiety of the neighboring gap nucleosides, thus defining
the boundary between the wings and the gap. In certain embodiments,
the sugar moieties within the gap are the same as one another. In
certain embodiments, the gap includes one or more nucleoside having
a sugar moiety that differs from the sugar moiety of one or more
other nucleosides of the gap. In certain embodiments, the sugar
moieties of the two wings are the same as one another (symmetric
sugar gapmer). In certain embodiments, the sugar moieties of the
5'-wing differs from the sugar moieties of the 3'-wing (asymmetric
sugar gapmer). In certain embodiments, the sugar moieties in the
two wings are selected from at least two different types that are
different from the sugar moieties in the gap and at least one of
each are in each wing.
[0215] In certain embodiments, the term "gapped oligomeric
compound" refers to an oligomeric compound having two external
regions or wings and an internal region or gap (also referred to as
5'-region and 3'-region). The three regions form a contiguous
sequence of monomer subunits with the sugar moieties of the
external regions (wings) being different than the sugar moieties of
the internal region (gap). In certain embodiments, the sugar
moieties of each monomer subunit within a particular region is
essentially the same. In certain embodiments, the sugar moieties of
each monomer subunit within each wing region is selected
independently from 2 different types of modified nucleosides. In
certain embodiments, the sugar moieties of each monomer subunit
within each wing region is selected independently from 3 different
types of modified nucleosides. In certain embodiments, the sugar
moieties of each monomer subunit within each wing region is
selected independently from 4 different types of modified
nucleosides. In certain embodiments, the sugar moiety of
essentially each monomer subunit within the internal region is
essentially the same. In certain embodiments, the sugar moiety of
each monomer subunit within the internal region is a
.beta.-D-2'-deoxyribonucleoside, a nucleoside that is DNA-like
and/or a nucleoside that supports RNaseH when in the gap
region.
[0216] In certain embodiments, each monomer subunit within a
particular region has the same sugar moiety. When the sugar
moieties of the external regions are the same the gapmer is a
symmetric gapmer and when the sugar moiety used in the 5'-external
region is different from the sugar moiety used in the 3'-external
region, the gapmer is an asymmetric gapmer. In certain embodiments,
the external regions are small (each independently 2, 3, 4, 5 or
about 6 monomer subunits) and the monomer subunits comprise
non-naturally occurring sugar moieties with the internal region
comprising .beta.-D-2'-deoxyribonucleosides. In certain
embodiments, the external regions each, independently, comprise
from 2 to about 8 monomer subunits having non-naturally occurring
sugar moieties and the internal region comprises from 6 to 14
unmodified nucleosides. The internal region or the gap generally
comprises .beta.-D-2'-deoxyribonucleosides but can comprise
non-naturally occurring sugar moieties. The heterocyclic base and
internucleoside linkage is independently variable at each position
of a gapped oligomeric compound. A gapped oligomeric compound can
further include one or more additional groups including but not
limited to capping groups, conjugate groups and other 5' or
3'-terminal groups.
[0217] In certain embodiments, gapped oligomeric compounds comprise
an internal region of .beta.-D-2'-deoxyribonucleosides with a
single internucleoside linkage having Formula I. In certain
embodiments, gapped oligomeric compounds comprise an internal
region of .beta.-D-2'-deoxyribonucleosides having two
internucleoside linkages having Formula I. In certain embodiments,
gapped oligomeric compounds comprise an internal region of
.beta.-D-2'-deoxyribonucleosides having three internucleoside
linkages having Formula I.
[0218] In certain embodiments, the 5' and 3'-wing regions of gapped
oligomeric compounds comprise modified nucleosides wherein all the
sugar moieties have the same type of modification such as cEt or
MOE. In certain embodiments, the 5' and 3'-wing regions of gapped
oligomeric compounds comprise two types of modified nucleosides
having sugar moieties independently selected from 2'-substituted
sugar moieties and furanosyl bicyclic sugar moieties. In certain
embodiments, the 5' and 3'-wing regions of gapped oligomeric
compounds comprise two types of modified nucleosides having sugar
moieties independently selected from 2'-MOE substituted sugar
moieties and furanosyl bicyclic sugar moieties each having a
4'-CH((S)--CH.sub.3)--O-2' bridge.
[0219] In certain embodiments, gapped oligomeric compounds are
provided that are from about 10 to about 30 monomer subunits in
length. In certain embodiments, gapped oligomeric compounds are
provided that are from about 12 to about 20 monomer subunits in
length. In certain embodiments, gapped oligomeric compounds are
provided that are from about 14 to about 20 monomer subunits in
length. In certain embodiments, gapped oligomeric compounds are
provided that are from about 14 to about 18 monomer subunits in
length.
[0220] ii. Certain Nucleobase Modification Motifs
[0221] In certain embodiments, oligonucleotides comprise chemical
modifications to nucleobases arranged along the oligonucleotide or
region thereof in a defined pattern or nucleobases modification
motif. In certain embodiments, each nucleobase is modified. In
certain embodiments, none of the nucleobases is chemically
modified.
[0222] In certain embodiments, oligonucleotides comprise a block of
modified nucleobases. In certain such embodiments, the block is at
the 3'-end of the oligonucleotide. In certain embodiments the block
is within 3 nucleotides of the 3'-end of the oligonucleotide. In
certain such embodiments, the block is at the 5'-end of the
oligonucleotide. In certain embodiments the block is within 3
nucleotides of the 5'-end of the oligonucleotide.
[0223] In certain embodiments, nucleobase modifications are a
function of the natural base at a particular position of an
oligonucleotide. For example, in certain embodiments each purine or
each pyrimidine in an oligonucleotide is modified. In certain
embodiments, each adenine is modified. In certain embodiments, each
guanine is modified. In certain embodiments, each thymine is
modified. In certain embodiments, each cytosine is modified. In
certain embodiments, each uracil is modified.
[0224] In certain embodiments, oligonucleotides comprise one or
more nucleosides comprising a modified nucleobase. In certain
embodiments, oligonucleotides having a gapmer sugar motif comprise
a nucleoside comprising a modified nucleobase. In certain such
embodiments, one nucleoside comprising a modified nucleobases is in
the gap of an oligonucleotide having a gapmer sugar motif. In
certain embodiments, the sugar is an unmodified 2' deoxynucleoside.
In certain embodiments, the modified nucleobase is selected from: a
2-thio pyrimidine and a 5-propyne pyrimidine
[0225] In certain embodiments, some, all, or none of the cytosine
moieties in an oligonucleotide are 5-methyl cytosine moieties.
Herein, 5-methyl cytosine is not a "modified nucleobase."
Accordingly, unless otherwise indicated, unmodified nucleobases
include both cytosine residues having a 5-methyl and those lacking
a 5 methyl. In certain embodiments, the methylation state of all or
some cytosine nucleobases is specified.
[0226] iii. Certain Nucleoside Motifs
[0227] In certain embodiments, oligomeric compounds comprise
nucleosides comprising modified sugar moieties and/or nucleosides
comprising modified nucleobases. Such motifs can be described by
their sugar motif and their nucleobase motif separately or by their
nucleoside motif, which provides positions or patterns of modified
nucleosides (whether modified sugar, nucleobase, or both sugar and
nucleobase) in an oligonucleotide.
[0228] In certain embodiments, the oligomeric compounds comprise or
consist of a region having a gapmer nucleoside motif, which
comprises two external regions or "wings" and a central or internal
region or "gap." The three regions of a gapmer nucleoside motif
(the 5'-wing, the gap, and the 3'-wing) form a contiguous sequence
of nucleosides wherein at least some of the sugar moieties and/or
nucleobases of the nucleosides of each of the wings differ from at
least some of the sugar moieties and/or nucleobase of the
nucleosides of the gap. Specifically, at least the nucleosides of
each wing that are closest to the gap (the 3'-most nucleoside of
the 5'-wing and the 5'-most nucleoside of the 3'-wing) differ from
the neighboring gap nucleosides, thus defining the boundary between
the wings and the gap. In certain embodiments, the nucleosides
within the gap are the same as one another. In certain embodiments,
the gap includes one or more nucleoside that differs from one or
more other nucleosides of the gap. In certain embodiments, the
nucleoside motifs of the two wings are the same as one another
(symmetric gapmer). In certain embodiments, the nucleoside motifs
of the 5'-wing differs from the nucleoside motif of the 3'-wing
(asymmetric gapmer).
[0229] 1. Certain 5'-Wings
[0230] In certain embodiments, the 5'-wing of a gapmer consists of
2 to 8 linked nucleosides. In certain embodiments, the 5'-wing of a
gapmer consists of 2 to 5 linked nucleosides. In certain
embodiments, the 5'-wing of a gapmer consists of 3 to 5 linked
nucleosides. In certain embodiments, the 5'-wing of a gapmer
consists of 4 or 5 linked nucleosides. In certain embodiments, the
5'-wing of a gapmer consists of 1 to 4 linked nucleosides. In
certain embodiments, the 5'-wing of a gapmer consists of 1 to 3
linked nucleosides. In certain embodiments, the 5'-wing of a gapmer
consists of 1 or 2 linked nucleosides. In certain embodiments, the
5'-wing of a gapmer consists of 2 to 4 linked nucleosides. In
certain embodiments, the 5'-wing of a gapmer consists of 2 or 3
linked nucleosides. In certain embodiments, the 5'-wing of a gapmer
consists of 3 or 4 linked nucleosides. In certain embodiments, the
5'-wing of a gapmer consists of 1 nucleoside. In certain
embodiments, the 5'-wing of a gapmer consists of 2 linked
nucleosides. In certain embodiments, the 5'-wing of a gapmer
consists of 3 linked nucleosides. In certain embodiments, the
5'-wing of a gapmer consists of 4 linked nucleosides. In certain
embodiments, the 5'-wing of a gapmer consists of 5 linked
nucleosides.
[0231] In certain embodiments, the 5'-wing of a gapmer consists of
2 to 8 linked nucleosides. In certain embodiments, the 5'-wing of a
gapmer consists of 2 to 5 linked nucleosides. In certain
embodiments, the 5'-wing of a gapmer consists of 2 to 5 linked
nucleosides. In certain embodiments, the 5'-wing of a gapmer
consists of 3 to 5 linked nucleosides. In certain embodiments, the
5'-wing of a gapmer consists of 4 or 5 linked nucleosides. In
certain embodiments, the 5'-wing of a gapmer consists of 2 to 4
linked nucleosides. In certain embodiments, the 5'-wing of a gapmer
consists of 2 to 3 linked nucleosides. In certain embodiments, the
5'-wing of a gapmer consists of 2 linked nucleosides. In certain
embodiments, the 5'-wing of a gapmer consists of 3 or 4 linked
nucleosides. In certain embodiments, the 5'-wing of a gapmer
consists of 2 linked nucleosides. In certain embodiments, the
5'-wing of a gapmer consists of 3 linked nucleosides. In certain
embodiments, the 5'-wing of a gapmer consists of 4 linked
nucleosides. In certain embodiments, the 5'-wing of a gapmer
consists of 5 linked nucleosides. In certain embodiments, the
5'-wing of a gapmer consists of 6 linked nucleosides. In certain
embodiments, the 5'-wing of a gapmer consists of 7 linked
nucleosides. In certain embodiments, the 5'-wing of a gapmer
consists of 8 linked nucleosides.
[0232] In certain embodiments, the 5'-wing of a gapmer comprises at
least one bicyclic nucleoside. In certain embodiments, the 5'-wing
of a gapmer comprises at least two bicyclic nucleosides. In certain
embodiments, the 5'-wing of a gapmer comprises at least three
bicyclic nucleosides. In certain embodiments, the 5'-wing of a
gapmer comprises at least four bicyclic nucleosides. In certain
embodiments, the 5'-wing of a gapmer comprises at least one
constrained ethyl nucleoside. In certain embodiments, the 5'-wing
of a gapmer comprises at least one LNA nucleoside. In certain
embodiments, each nucleoside of the 5'-wing of a gapmer is a
bicyclic nucleoside. In certain embodiments, each nucleoside of the
5'-wing of a gapmer is a constrained ethyl nucleoside. In certain
embodiments, each nucleoside of the 5'-wing of a gapmer is a LNA
nucleoside.
[0233] In certain embodiments, the 5'-wing of a gapmer comprises at
least one non-bicyclic modified nucleoside. In certain embodiments,
the 5'-wing of a gapmer comprises at least one 2'-substituted
nucleoside. In certain embodiments, the 5'-wing of a gapmer
comprises at least one 2'-MOE nucleoside. In certain embodiments,
the 5'-wing of a gapmer comprises at least one 2'-OMe nucleoside.
In certain embodiments, each nucleoside of the 5'-wing of a gapmer
is a non-bicyclic modified nucleoside. In certain embodiments, each
nucleoside of the 5'-wing of a gapmer is a 2'-substituted
nucleoside. In certain embodiments, each nucleoside of the 5'-wing
of a gapmer is a 2'-MOE nucleoside. In certain embodiments, each
nucleoside of the 5'-wing of a gapmer is a 2'-OMe nucleoside.
[0234] In certain embodiments, the 5'-wing of a gapmer comprises at
least one bicyclic nucleoside and at least one non-bicyclic
modified nucleoside. In certain embodiments, the 5'-wing of a
gapmer comprises at least one bicyclic nucleoside and at least one
2'-substituted nucleoside. In certain embodiments, the 5'-wing of a
gapmer comprises at least one bicyclic nucleoside and at least one
2'-MOE nucleoside. In certain embodiments, the 5'-wing of a gapmer
comprises at least one bicyclic nucleoside and at least one 2'-OMe
nucleoside.
[0235] In certain embodiments, the 5'-wing of a gapmer comprises at
least one constrained ethyl nucleoside and at least one
non-bicyclic modified nucleoside. In certain embodiments, the
5'-wing of a gapmer comprises at least one constrained ethyl
nucleoside and at least one 2'-substituted nucleoside. In certain
embodiments, the 5'-wing of a gapmer comprises at least one
constrained ethyl nucleoside and at least one 2'-MOE nucleoside. In
certain embodiments, the 5'-wing of a gapmer comprises at least one
constrained ethyl nucleoside and at least one 2'-OMe
nucleoside.
[0236] In certain embodiments, the 5'-wing of a gapmer has a
nucleoside motif selected from among the following: ABBA; ABB;
ABAA; AABAA; AAABAA; AAAABAA; AAAAABAA; AAABAA; AABAA; ABAB; AAABB;
AAAAA; ABBC; AA; AAA; AAAA; AAAAB; AAAAAAA; AAAAAAAA; ABBB; AB;
ABAB; AAAAB; AABBB; AAAAB; and AABBB, wherein each A is a modified
nucleoside of a first type, each B is a modified nucleoside of a
second type and each C is a modified nucleoside of a third type. In
certain embodiments, such an oligomeric compound is a gapmer. In
certain such embodiments, the 3'-wing of the gapmer may comprise
any nucleoside motif.
[0237] 2. Certain 3'-Wings
[0238] In certain embodiments, the 3'-wing of a gapmer consists of
2 to 8 linked nucleosides. In certain embodiments, the 3'-wing of a
gapmer consists of 2 to 5 linked nucleosides. In certain
embodiments, the 3'-wing of a gapmer consists of 3 to 5 linked
nucleosides. In certain embodiments, the 3'-wing of a gapmer
consists of 4 or 5 linked nucleosides. In certain embodiments, the
3'-wing of a gapmer consists of 1 to 4 linked nucleosides. In
certain embodiments, the 3'-wing of a gapmer consists of 1 to 3
linked nucleosides. In certain embodiments, the 3'-wing of a gapmer
consists of 1 or 2 linked nucleosides. In certain embodiments, the
3'-wing of a gapmer consists of 2 to 4 linked nucleosides. In
certain embodiments, the 3'-wing of a gapmer consists of 2 or 3
linked nucleosides. In certain embodiments, the 3'-wing of a gapmer
consists of 3 or 4 linked nucleosides. In certain embodiments, the
3'-wing of a gapmer consists of 1 nucleoside. In certain
embodiments, the 3'-wing of a gapmer consists of 2 linked
nucleosides. In certain embodiments, the 3'-wing of a gapmer
consists of 3 linked nucleosides. In certain embodiments, the
3'-wing of a gapmer consists of 4 linked nucleosides. In certain
embodiments, the 3'-wing of a gapmer consists of 5 linked
nucleosides.
[0239] In certain embodiments, the 3'-wing of a gapmer consists of
2 to 8 linked nucleosides. In certain embodiments, the 3'-wing of a
gapmer consists of 2 to 5 linked nucleosides. In certain
embodiments, the 3'-wing of a gapmer consists of 2 to 5 linked
nucleosides. In certain embodiments, the 3'-wing of a gapmer
consists of 3 to 5 linked nucleosides. In certain embodiments, the
3'-wing of a gapmer consists of 4 or 5 linked nucleosides. In
certain embodiments, the 3'-wing of a gapmer consists of 2 to 4
linked nucleosides. In certain embodiments, the 3'-wing of a gapmer
consists of 2 to 3 linked nucleosides. In certain embodiments, the
3'-wing of a gapmer consists of 2 linked nucleosides. In certain
embodiments, the 3'-wing of a gapmer consists of 3 or 4 linked
nucleosides. In certain embodiments, the 3'-wing of a gapmer
consists of 2 linked nucleosides. In certain embodiments, the
3'-wing of a gapmer consists of 3 linked nucleosides. In certain
embodiments, the 3'-wing of a gapmer consists of 4 linked
nucleosides. In certain embodiments, the 3'-wing of a gapmer
consists of 5 linked nucleosides. In certain embodiments, the
3'-wing of a gapmer consists of 6 linked nucleosides. In certain
embodiments, the 3'-wing of a gapmer consists of 7 linked
nucleosides. In certain embodiments, the 3'-wing of a gapmer
consists of 8 linked nucleosides.
[0240] In certain embodiments, the 3'-wing of a gapmer comprises at
least one bicyclic nucleoside. In certain embodiments, the 3'-wing
of a gapmer comprises at least one constrained ethyl nucleoside. In
certain embodiments, the 3'-wing of a gapmer comprises at least one
LNA nucleoside. In certain embodiments, each nucleoside of the
3'-wing of a gapmer is a bicyclic nucleoside. In certain
embodiments, each nucleoside of the 3'-wing of a gapmer is a
constrained ethyl nucleoside. In certain embodiments, each
nucleoside of the 3'-wing of a gapmer is a LNA nucleoside.
[0241] In certain embodiments, the 3'-wing of a gapmer comprises at
least one non-bicyclic modified nucleoside. In certain embodiments,
the 3'-wing of a gapmer comprises at least two non-bicyclic
modified nucleosides. In certain embodiments, the 3'-wing of a
gapmer comprises at least three non-bicyclic modified nucleosides.
In certain embodiments, the 3'-wing of a gapmer comprises at least
four non-bicyclic modified nucleosides. In certain embodiments, the
3'-wing of a gapmer comprises at least one 2'-substituted
nucleoside. In certain embodiments, the 3'-wing of a gapmer
comprises at least one 2'-MOE nucleoside. In certain embodiments,
the 3'-wing of a gapmer comprises at least one 2'-OMe nucleoside.
In certain embodiments, each nucleoside of the 3'-wing of a gapmer
is a non-bicyclic modified nucleoside. In certain embodiments, each
nucleoside of the 3'-wing of a gapmer is a 2'-substituted
nucleoside. In certain embodiments, each nucleoside of the 3'-wing
of a gapmer is a 2'-MOE nucleoside. In certain embodiments, each
nucleoside of the 3'-wing of a gapmer is a 2'-OMe nucleoside.
[0242] In certain embodiments, the 3'-wing of a gapmer comprises at
least one bicyclic nucleoside and at least one non-bicyclic
modified nucleoside. In certain embodiments, the 3'-wing of a
gapmer comprises at least one bicyclic nucleoside and at least one
2'-substituted nucleoside. In certain embodiments, the 3'-wing of a
gapmer comprises at least one bicyclic nucleoside and at least one
2'-MOE nucleoside. In certain embodiments, the 3'-wing of a gapmer
comprises at least one bicyclic nucleoside and at least one 2'-OMe
nucleoside.
[0243] In certain embodiments, the 3'-wing of a gapmer comprises at
least one constrained ethyl nucleoside and at least one
non-bicyclic modified nucleoside. In certain embodiments, the
3'-wing of a gapmer comprises at least one constrained ethyl
nucleoside and at least one 2'-substituted nucleoside. In certain
embodiments, the 3'-wing of a gapmer comprises at least one
constrained ethyl nucleoside and at least one 2'-MOE nucleoside. In
certain embodiments, the 3'-wing of a gapmer comprises at least one
constrained ethyl nucleoside and at least one 2'-OMe
nucleoside.
[0244] In certain embodiments, the 3'-wing of a gapmer comprises at
least one LNA nucleoside and at least one non-bicyclic modified
nucleoside. In certain embodiments, the 3'-wing of a gapmer
comprises at least one LNA nucleoside and at least one
2'-substituted nucleoside. In certain embodiments, the 3'-wing of a
gapmer comprises at least one LNA nucleoside and at least one
2'-MOE nucleoside. In certain embodiments, the 3'-wing of a gapmer
comprises at least one LNA nucleoside and at least one 2'-OMe
nucleoside.
[0245] In certain embodiments, the 3'-wing of a gapmer has a
nucleoside motif selected from among the following: ABB; ABAA;
AAABAA, AAAAABAA; AABAA; AAAABAA; AAABAA; ABAB; AAAAA; AAABB;
AAAAAAAA; AAAAAAA; AAAAAA; AAAAB; AAAA; AAA; AA; AB; ABBB; ABAB;
AABBB; wherein each A is a modified nucleoside of a first type,
each B is a modified nucleoside of a second type. In certain
embodiments, an oligonucleotide comprises any 3'-wing motif
provided herein. In certain such embodiments, the 5'-wing of the
gapmer may comprise any nucleoside motif.
[0246] 3. Certain Central Regions (Gap Regions)
[0247] In certain embodiments, the gap of a gapmer consists of 6 to
20 linked nucleosides. In certain embodiments, the gap of a gapmer
consists of 6 to 14 linked nucleosides. In certain embodiments, the
gap of a gapmer consists of 6 to 12 linked nucleosides. In certain
embodiments, the gap of a gapmer consists of 6 to 10 linked
nucleosides. In certain embodiments, the gap of a gapmer consists
of 6 to 9 linked nucleosides. In certain embodiments, the gap of a
gapmer consists of 6 to 8 linked nucleosides. In certain
embodiments, the gap of a gapmer consists of 6 or 7 linked
nucleosides. In certain embodiments, the gap of a gapmer consists
of 7 to 10 linked nucleosides. In certain embodiments, the gap of a
gapmer consists of 7 to 9 linked nucleosides. In certain
embodiments, the gap of a gapmer consists of 7 or 8 linked
nucleosides. In certain embodiments, the gap of a gapmer consists
of 8 to 10 linked nucleosides. In certain embodiments, the gap of a
gapmer consists of 8 or 9 linked nucleosides. In certain
embodiments, the gap of a gapmer consists of 6 linked nucleosides.
In certain embodiments, the gap of a gapmer consists of 7 linked
nucleosides. In certain embodiments, the gap of a gapmer consists
of 8 linked nucleosides. In certain embodiments, the gap of a
gapmer consists of 9 linked nucleosides. In certain embodiments,
the gap of a gapmer consists of 10 linked nucleosides. In certain
embodiments, the gap of a gapmer consists of 11 linked nucleosides.
In certain embodiments, the gap of a gapmer consists of 12 linked
nucleosides. In certain embodiments, the gap of a gapmer consists
of 13 linked nucleosides. In certain embodiments, the gap of a
gapmer consists of 14 linked nucleosides.
[0248] In certain embodiments, each nucleoside of the gap of a
gapmer is a 2'-deoxynucleoside. In certain embodiments, the gap
comprises one or more modified nucleosides. In certain embodiments,
each nucleoside of the gap of a gapmer is a 2'-deoxynucleoside or
is a modified nucleoside that is "DNA-like". In certain
embodiments, "DNA-like" means that the nucleoside has similar
characteristics to DNA, such that a duplex comprising the gapmer
and an RNA molecule is capable of activating RNase H. In certain
embodiments, modified nucleosides that are DNA-like are 2'-endo.
For example, under certain conditions, 2'-(ara)-F have been shown
to support RNase H activation, and thus is DNA-like and further has
2'-endo conformation geometry. In certain embodiments, one or more
nucleosides of the gap of a gapmer is not a 2'-deoxynucleoside and
is not DNA-like. In certain such embodiments, the gapmer
nonetheless supports RNase H activation (e.g., by virtue of the
number or placement of the non-DNA nucleosides).
[0249] In certain embodiments, the gap comprise a stretch of
unmodified 2'-deoxynucleosides interrupted by one or more modified
nucleosides, thus resulting in three sub-regions (two stretches of
one or more 2'-deoxynucleosides and a stretch of one or more
interrupting modified nucleosides). In certain embodiments, no
stretch of unmodified 2'-deoxynucleosides is longer than 5, 6, or 7
nucleosides. In certain embodiments, such short stretches is
achieved by using short gap regions. In certain embodiments, short
stretches are achieved by interrupting a longer gap region.
[0250] 4. Certain Gapmer Motifs
[0251] In certain embodiments, a gapmer comprises a 5'-wing, a gap
comprising at least one internucleoside linkage of Formula I, and a
3' wing, wherein the 5'-wing, gap, and 3' wing are independently
selected from among those discussed above. For example, in certain
embodiments, a gapmer has a 5'-wing, a gap, and a 3'-wing having
features selected from among those listed in the following
non-limiting table:
TABLE-US-00002 TABLE 1 Certain Gapmer Nucleoside Motifs 5'-wing Gap
3'-wing region region region AAAAAAA DDDDDDDDDDD AAA AAAAABB
DDDDDDDD BBAAAAA ABB DDDDDDDDD BBA AABAA DDDDDDDDD AABAA ABB DDDDDD
AABAA AAABAA DDDDDDDDD AAABAA AAABAA DDDDDDDDD AAB ABAB DDDDDDDDD
ABAB AAABB DDDDDDD BBA ABAB DDDDDDDD BBA AA DDDDDDDD BBBBBBBB ABB
DDDDDD ABADB AAAAB DDDDDDD BAAAA ABBB DDDDDDDDD AB AB DDDDDDDDD
BBBA ABBB DDDDDDDDD BBBA AB DDDDDDDD ABA
[0252] wherein each A is a modified nucleoside of a first type,
each B is a modified nucleoside of a second type and each D is a
.beta.-D-2'-deoxyribonucleoside or a nucleoside that is DNA-like.
Each gap region includes at least one internucleoside linkage of
Formula I.
[0253] In certain embodiments, each A comprises a modified sugar
moiety. In certain embodiments, each A comprises a 2'-substituted
sugar moiety. In certain embodiments, each A comprises a
2'-substituted sugar moiety selected from among F, OCH.sub.3,
OCH.sub.2--C(.dbd.O)--N(H)(CH.sub.3) and
O(CH.sub.2).sub.2--OCH.sub.3. In certain embodiments, each A
comprises a bicyclic sugar moiety. In certain embodiments, each A
comprises a bicyclic sugar moiety selected from among cEt, cMOE,
LNA, .alpha.-L-LNA, ENA and 2'-thio LNA. In certain embodiments,
each A comprises a modified nucleobase. In certain embodiments,
each A comprises a modified nucleobase selected from among
2-thio-thymidine nucleoside and 5-propyne uridine nucleoside.
[0254] In certain embodiments, each B comprises a modified sugar
moiety. In certain embodiments, each B comprises a 2'-substituted
sugar moiety. In certain embodiments, each B comprises a
2'-substituted sugar moiety selected from among F, OCH.sub.3,
OCH.sub.2--C(.dbd.O)--N(H)(CH.sub.3) and
O(CH.sub.2).sub.2--OCH.sub.3. In certain embodiments, each B
comprises a bicyclic sugar moiety. In certain embodiments, each B
comprises a bicyclic sugar moiety selected from among cEt, cMOE,
LNA, .alpha.-L-LNA, ENA and 2'-thio LNA. In certain embodiments,
each B comprises a modified nucleobase. In certain embodiments,
each B comprises a modified nucleobase selected from among
2-thio-thymidine nucleoside and 5-propyne uridine nucleoside.
[0255] In certain embodiments, at least one of A or B comprises a
bicyclic sugar moiety, and the other comprises a 2'-substituted
sugar moiety. In certain embodiments, one of A or B is an LNA
nucleoside and the other of A or B comprises a 2'-substituted sugar
moiety. In certain embodiments, one of A or B is a cEt nucleoside
and the other of A or B comprises a 2'-substituted sugar moiety. In
certain embodiments, one of A or B is an .alpha.-L-LNA nucleoside
and the other of A or B comprises a 2'-substituted sugar moiety. In
certain embodiments, one of A or B is an LNA nucleoside and the
other of A or B comprises a 2'-MOE sugar moiety. In certain
embodiments, one of A or B is a cEt nucleoside and the other of A
or B comprises a 2'-MOE sugar moiety. In certain embodiments, one
of A or B is an .alpha.-L-LNA nucleoside and the other of A or B
comprises a 2'-MOE sugar moiety. In certain embodiments, one of A
or B is an LNA nucleoside and the other of A or B comprises a 2'-F
sugar moiety. In certain embodiments, one of A or B is a cEt
nucleoside and the other of A or B comprises a 2'-F sugar moiety.
In certain embodiments, one of A or B is an .alpha.-L-LNA
nucleoside and the other of A or B comprises a 2'-F sugar
moiety.
[0256] In certain embodiments, A comprises a bicyclic sugar moiety,
and B comprises a 2'-substituted sugar moiety. In certain
embodiments, A is an LNA nucleoside and B comprises a
2'-substituted sugar moiety. In certain embodiments, A is a cEt
nucleoside and B comprises a 2'-substituted sugar moiety. In
certain embodiments, A is an .alpha.-L-LNA nucleoside and B
comprises a 2'-substituted sugar moiety.
[0257] In certain embodiments, A comprises a bicyclic sugar moiety,
and B comprises a 2'-MOE sugar moiety. In certain embodiments, A is
an LNA nucleoside and B comprises a 2'-MOE sugar moiety. In certain
embodiments, A is a cEt nucleoside and B comprises a 2'-MOE sugar
moiety. In certain embodiments, A is an .alpha.-L-LNA nucleoside
and B comprises a 2'-MOE sugar moiety.
[0258] In certain embodiments, A comprises a bicyclic sugar moiety,
and B comprises a 2'-F sugar moiety. In certain embodiments, A is
an LNA nucleoside and B comprises a 2'-F sugar moiety. In certain
embodiments, A is a cEt nucleoside and B comprises a 2'-F sugar
moiety. In certain embodiments, A is an .alpha.-L-LNA nucleoside
and B comprises a 2'-F sugar moiety.
[0259] In certain embodiments, B comprises a bicyclic sugar moiety,
and A comprises a 2'-MOE sugar moiety. In certain embodiments, B is
an LNA nucleoside and A comprises a 2'-MOE sugar moiety. In certain
embodiments, B is a cEt nucleoside and A comprises a 2'-MOE sugar
moiety. In certain embodiments, B is an .alpha.-L-LNA nucleoside
and A comprises a 2'-MOE sugar moiety.
[0260] In certain embodiments, B comprises a bicyclic sugar moiety,
and A comprises a 2'-F sugar moiety. In certain embodiments, B is
an LNA nucleoside and A comprises a 2'-F sugar moiety. In certain
embodiments, B is a cEt nucleoside and A comprises a 2'-F sugar
moiety. In certain embodiments, B is an .alpha.-L-LNA nucleoside
and A comprises a 2'-F sugar moiety.
[0261] In certain embodiments, each A and B is, independently, a
modified nucleoside comprising a bicyclic sugar moiety comprising a
4'-CH(CH.sub.3)--O-2' bridge or a modified nucleoside comprising a
2'-OCH.sub.2CH.sub.2OCH.sub.3 (MOE) substituent group. In certain
embodiments, each A and B is, independently, a modified nucleoside
comprising a bicyclic sugar moiety comprising a
4'-CH[(S)--(CH.sub.3)]--O-2' bridge or a modified nucleoside
comprising a 2'-OCH.sub.2CH.sub.2OCH.sub.3 (MOE) substituent group.
In certain embodiments, each A and B is, independently, a modified
nucleoside comprising a bicyclic sugar moiety comprising a
4'-CH[(R)--(CH.sub.3)]--O-2' bridge or a modified nucleoside
comprising a 2'-OCH.sub.2CH.sub.2OCH.sub.3 (MOE) substituent group.
In certain embodiments, at least one modified nucleoside comprising
a 2'-OCH.sub.2CH.sub.2OCH.sub.3 (MOE) substituent group and at
least one modified nucleoside comprising a 4'-CH(CH.sub.3)--O-2'
bridge is located in each of the 3' and 5' wings. In certain
embodiments, at least one modified nucleoside comprising a
2'-OCH.sub.2CH.sub.2OCH.sub.3 (MOE) substituent group and at least
one modified nucleoside comprising a 4'-CH[(S)--(CH.sub.3)]--O-2'
bridge is located in each of the 3' and 5' wings. In certain
embodiments, at least one modified nucleoside comprising a
2'-OCH.sub.2CH.sub.2OCH.sub.3 (MOE) substituent group and at least
one modified nucleoside comprising a 4'-CH[(R)--(CH.sub.3)]--O-2'
bridge is located in each of the 3' and 5' wings.
[0262] iv. Certain Internucleoside Linkage Motifs
[0263] In certain embodiments, oligomeric compounds comprise
modified internucleoside linkages arranged along the oligomeric
compound or region thereof in a defined pattern or modified
internucleoside linkage motif provided that at least one
internucleoside linkage has Formula I. In certain embodiments,
internucleoside linkages are arranged in a gapped motif, as
described above for nucleoside motif. In such embodiments, the
internucleoside linkages in each of two wing regions are different
from the internucleoside linkages in the gap region. In certain
embodiments the internucleoside linkages in the wings are
phosphodiester and the internucleoside linkages in the gap are
phosphorothioate. The nucleoside motif is independently selected,
so such oligomeric compounds having a gapped internucleoside
linkage motif may or may not have a gapped nucleoside motif and if
it does have a gapped nucleoside motif, the wing and gap lengths
may or may not be the same.
[0264] In certain embodiments, oligomeric compounds comprise a
region having an alternating internucleoside linkage motif. In
certain embodiments, oligomeric compounds of the present invention
comprise a region of uniformly modified internucleoside linkages.
In certain such embodiments, the oligomeric compound comprises a
region that is uniformly linked by phosphorothioate internucleoside
linkages. In certain embodiments, the oligonucleotide is uniformly
linked by phosphorothioate. In certain embodiments, each
internucleoside linkage of the oligomeric compound is selected from
phosphodiester and phosphorothioate. In certain embodiments, each
internucleoside linkage of the oligomeric compound is selected from
phosphodiester and phosphorothioate and at least one
internucleoside linkage is phosphorothioate. In certain
embodiments, at least one internucleoside linkage of the oligomeric
compound is selected from other than phosphodiester and
phosphorothioate.
[0265] In certain embodiments, the oligomeric compound comprises at
least 6 phosphorothioate internucleoside linkages. In certain
embodiments, the oligomeric compound comprises at least 8
phosphorothioate internucleoside linkages. In certain embodiments,
the oligomeric compound comprises at least 10 phosphorothioate
internucleoside linkages. In certain embodiments, the oligomeric
compound comprises at least one block of at least 6 consecutive
phosphorothioate internucleoside linkages. In certain embodiments,
the oligomeric compound comprises at least one block of at least 8
consecutive phosphorothioate internucleoside linkages. In certain
embodiments, the oligomeric compound comprises at least one block
of at least 10 consecutive phosphorothioate internucleoside
linkages. In certain embodiments, the oligomeric compound comprises
at least one block of at least one 12 consecutive phosphorothioate
internucleoside linkages. In certain such embodiments, at least one
such block is located at the 3' end of the oligomeric compound. In
certain such embodiments, at least one such block is located within
3 nucleosides of the 3' end of the oligomeric compound. In certain
embodiments, each internucleoside linkage a phosphorothioate
internucleoside linkage.
[0266] v. Certain Modification Motifs
[0267] Modification motifs define oligonucleotides by nucleoside
motif (sugar motif and nucleobase motif) and linkage motif. For
example, certain oligonucleotides have the following modification
motif:
[0268] AAADDDDDDDDDBBB;
[0269] wherein each A is a modified nucleoside comprising a
2'-substituted sugar moiety; each D is a
.beta.-D-2'-deoxyribonucleoside or a modified nucleoside having B
form conformation geometry and each B is a modified nucleoside
comprising a bicyclic sugar moiety wherein at least one
internucleoside linkage had Formula I. The following non-limiting
Table further illustrates certain modification motifs:
TABLE-US-00003 TABLE 2 Certain Modification Motif 5'-wing Gap
3'-wing region region region BB DDDDDDDDD AAAAAAAA ABB DDDDDDDDD
BBA ABB DDDDDDDDD BBA ABBB DDDDDDDDD BBABB ABB DDDDDDDDD BBABB
BBABB DDDDDDDDD BBA ABB DDDDDDDDD BBABBBB AABAA DDDDDDDDD BBA
AAABAA DDDDDDDDD BBA AABAA DDDDDDDDD AABAA AAABAA DDDDDDDDD AABAAA
AAAABAA DDDDDDDDD BBA ABAB DDDDDDDDD BABA ABAB DDDDDDDDD AABAA ABB
DDDDDDDDD BABA BBABBBB DDDDDDDDD BABA AAAAA DDDDDDDDD AAAAA AAAAA
DDDDDDD AAAAA AAAAA DDDDDDDDD BBABBBB AAABB DDDDDDD BBA ABAB
DDDDDDDD BBA ABAB DDDDDDD AAABB AAAAB DDDDDDD BAAAA BB DDDDDDDD AA
AA DDDDDDD AAAAAAAA AAA DDDDDDD AAAAAAA AAA DDDDDDD AAAAAA AB
DDDDDDD BBBA ABBB DDDDDDDDD BA AB DDDDDDDDD BBBA AAABB DDDDDDD
BBAAA AAAAB DDDDDDD BAAAA AABBB DDDDDDD BBBAA AAAAB DDDDDDD AAAAA
AAABB DDDDDDD AAAAA AABBB DDDDDDD AAAAA AAAAA DDDDDDD BAAAA AAAAA
DDDDDDD BBAAA AAAAA DDDDDDD BBBAA
[0270] wherein each A is a modified nucleoside of a first type,
each B is a modified nucleoside of a second type and each D is a
.beta.-D-2'-deoxyribonucleoside or a nucleoside that is DNA-like.
Each gap region includes at least one internucleoside linkage of
Formula I.
[0271] In certain embodiments, each A comprises a modified sugar
moiety. In certain embodiments, each A comprises a 2'-substituted
sugar moiety. In certain embodiments, each A comprises a
2'-substituted sugar moiety selected from among F, OCH.sub.3,
OCH.sub.2--C(.dbd.O)--N(H)(CH.sub.3) and
O(CH.sub.2).sub.2--OCH.sub.3. In certain embodiments, each A
comprises a bicyclic sugar moiety. In certain embodiments, each A
comprises a bicyclic sugar moiety selected from among cEt, cMOE,
LNA, .alpha.-L-LNA, ENA and 2'-thio LNA. In certain embodiments,
each A comprises a modified nucleobase. In certain embodiments,
each A comprises a modified nucleobase selected from among
2-thio-thymidine nucleoside and 5-propyne uridine nucleoside.
[0272] In certain embodiments, each B comprises a modified sugar
moiety. In certain embodiments, each B comprises a 2'-substituted
sugar moiety. In certain embodiments, each B comprises a
2'-substituted sugar moiety selected from among F, OCH.sub.3,
OCH.sub.2--C(.dbd.O)--N(H)(CH.sub.3) and
O(CH.sub.2).sub.2--OCH.sub.3. In certain embodiments, each B
comprises a bicyclic sugar moiety. In certain embodiments, each B
comprises a bicyclic sugar moiety selected from among cEt, cMOE,
LNA, .alpha.-L-LNA, ENA and 2'-thio LNA. In certain embodiments,
each B comprises a modified nucleobase. In certain embodiments,
each B comprises a modified nucleobase selected from among
2-thio-thymidine nucleoside and 5-propyne urindine nucleoside.
[0273] In certain embodiments, at least one of A or B comprises a
bicyclic sugar moiety, and the other comprises a 2'-substituted
sugar moiety. In certain embodiments, one of A or B is an LNA
nucleoside and the other of A or B comprises a 2'-substituted sugar
moiety. In certain embodiments, one of A or B is a cEt nucleoside
and the other of A or B comprises a 2'-substituted sugar moiety. In
certain embodiments, one of A or B is an .alpha.-L-LNA nucleoside
and the other of A or B comprises a 2'-substituted sugar moiety. In
certain embodiments, one of A or B is an LNA nucleoside and the
other of A or B comprises a 2'-MOE sugar moiety. In certain
embodiments, one of A or B is a cEt nucleoside and the other of A
or B comprises a 2'-MOE sugar moiety. In certain embodiments, one
of A or B is an .alpha.-L-LNA nucleoside and the other of A or B
comprises a 2'-MOE sugar moiety. In certain embodiments, one of A
or B is an LNA nucleoside and the other of A or B comprises a 2'-F
sugar moiety. In certain embodiments, one of A or B is a cEt
nucleoside and the other of A or B comprises a 2'-F sugar moiety.
In certain embodiments, one of A or B is an .alpha.-L-LNA
nucleoside and the other of A or B comprises a 2'-F sugar
moiety.
[0274] In certain embodiments, A comprises a bicyclic sugar moiety,
and B comprises a 2'-substituted sugar moiety. In certain
embodiments, A is an LNA nucleoside and B comprises a
2'-substituted sugar moiety. In certain embodiments, A is a cEt
nucleoside and B comprises a 2'-substituted sugar moiety. In
certain embodiments, A is an .alpha.-L-LNA nucleoside and B
comprises a 2'-substituted sugar moiety.
[0275] In certain embodiments, A comprises a bicyclic sugar moiety,
and B comprises a 2'-MOE sugar moiety. In certain embodiments, A is
an LNA nucleoside and B comprises a 2'-MOE sugar moiety. In certain
embodiments, A is a cEt nucleoside and B comprises a 2'-MOE sugar
moiety. In certain embodiments, A is an .alpha.-L-LNA nucleoside
and B comprises a 2'-MOE sugar moiety.
[0276] In certain embodiments, A comprises a bicyclic sugar moiety,
and B comprises a 2'-F sugar moiety. In certain embodiments, A is
an LNA nucleoside and B comprises a 2'-F sugar moiety. In certain
embodiments, A is a cEt nucleoside and B comprises a 2'-F sugar
moiety. In certain embodiments, A is an .alpha.-L-LNA nucleoside
and B comprises a 2'-F sugar moiety.
[0277] In certain embodiments, B comprises a bicyclic sugar moiety,
and A comprises a 2'-MOE sugar moiety. In certain embodiments, B is
an LNA nucleoside and A comprises a 2'-MOE sugar moiety. In certain
embodiments, B is a cEt nucleoside and A comprises a 2'-MOE sugar
moiety. In certain embodiments, B is an .alpha.-L-LNA nucleoside
and A comprises a 2'-MOE sugar moiety.
[0278] In certain embodiments, B comprises a bicyclic sugar moiety,
and A comprises a 2'-F sugar moiety. In certain embodiments, B is
an LNA nucleoside and A comprises a 2'-F sugar moiety. In certain
embodiments, B is a cEt nucleoside and A comprises a 2'-F sugar
moiety. In certain embodiments, B is an .alpha.-L-LNA nucleoside
and A comprises a 2'-F sugar moiety. In certain embodiments, each A
and B is, independently, a modified nucleoside comprising a
bicyclic sugar moiety comprising a 4'-CH(CH.sub.3)--O-2' bridge or
a modified nucleoside comprising a 2'-OCH.sub.2CH.sub.2OCH.sub.3
(MOE) substituent group. In certain embodiments, each A and B is,
independently, a modified nucleoside comprising a bicyclic sugar
moiety comprising a 4'-CH[(S)--(CH.sub.3)]--O-2' bridge or a
modified nucleoside comprising a 2'-OCH.sub.2CH.sub.2OCH.sub.3
(MOE) substituent group. In certain embodiments, each A and B is,
independently, a modified nucleoside comprising a bicyclic sugar
moiety comprising a 4'-CH[(R)--(CH.sub.3)]--O-2' bridge or a
modified nucleoside comprising a 2'-OCH.sub.2CH.sub.2OCH.sub.3
(MOE) substituent group. In certain embodiments, at least one
modified nucleoside comprising a 2'-OCH.sub.2CH.sub.2OCH.sub.3
(MOE) substituent group and at least one modified nucleoside
comprising a 4'-CH(CH.sub.3)--O-2' bridge is located in each of the
3' and 5' wings. In certain embodiments, at least one modified
nucleoside comprising a 2'-OCH.sub.2CH.sub.2OCH.sub.3 (MOE)
substituent group and at least one modified nucleoside comprising a
4'-CH[(S)--(CH.sub.3)]--O-2' bridge is located in each of the 3'
and 5' wings. In certain embodiments, at least one modified
nucleoside comprising a 2'-OCH.sub.2CH.sub.2OCH.sub.3 (MOE)
substituent group and at least one modified nucleoside comprising a
4'-CH[(R)--(CH.sub.3)]--O-2' bridge is located in each of the 3'
and 5' wings.
[0279] d. Certain Overall Lengths
[0280] In certain embodiments, the present invention provides
oligomeric compounds including oligonucleotides of any of a variety
of ranges of lengths. In certain embodiments, the invention
provides oligomeric compounds or oligonucleotides consisting of X
to Y linked nucleosides, where X represents the fewest number of
nucleosides in the range and Y represents the largest number of
nucleosides in the range. In certain such embodiments, X and Y are
each independently selected from 10, 11, 12, 13, 14, 15, 16, 17,
18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 and 30; provided
that X.ltoreq.Y. For example, in certain embodiments, the invention
provides oligomeric compounds which comprise oligonucleotides
consisting of 10 to 11, 10 to 12, 10 to 13, 10 to 14, 10 to 15, 10
to 16, 10 to 17, 10 to 18, 10 to 19, 10 to 20, 10 to 21, 10 to 22,
10 to 23, 10 to 24, 10 to 25, 10 to 26, 10 to 27, 10 to 28, 10 to
29, 10 to 30, 11 to 12, 11 to 13, 11 to 14, 11 to 15, 11 to 16, 11
to 17, 11 to 18, 11 to 19, 11 to 20, 11 to 21, 11 to 22, 11 to 23,
11 to 24, 11 to 25, 11 to 26, 11 to 27, 11 to 28, 11 to 29, 11 to
30, 12 to 13, 12 to 14, 12 to 15, 12 to 16, 12 to 17, 12 to 18, 12
to 19, 12 to 20, 12 to 21, 12 to 22, 12 to 23, 12 to 24, 12 to 25,
12 to 26, 12 to 27, 12 to 28, 12 to 29, 12 to 30, 13 to 14, 13 to
15, 13 to 16, 13 to 17, 13 to 18, 13 to 19, 13 to 20, 13 to 21, 13
to 22, 13 to 23, 13 to 24, 13 to 25, 13 to 26, 13 to 27, 13 to 28,
13 to 29, 13 to 30, 14 to 15, 14 to 16, 14 to 17, 14 to 18, 14 to
19, 14 to 20, 14 to 21, 14 to 22, 14 to 23, 14 to 24, 14 to 25, 14
to 26, 14 to 27, 14 to 28, 14 to 29, 14 to 30, 15 to 16, 15 to 17,
15 to 18, 15 to 19, 15 to 20, 15 to 21, 15 to 22, 15 to 23, 15 to
24, 15 to 25, 15 to 26, 15 to 27, 15 to 28, 15 to 29, 15 to 30, 16
to 17, 16 to 18, 16 to 19, 16 to 20, 16 to 21, 16 to 22, 16 to 23,
16 to 24, 16 to 25, 16 to 26, 16 to 27, 16 to 28, 16 to 29, 16 to
30, 17 to 18, 17 to 19, 17 to 20, 17 to 21, 17 to 22, 17 to 23, 17
to 24, 17 to 25, 17 to 26, 17 to 27, 17 to 28, 17 to 29, 17 to 30,
18 to 19, 18 to 20, 18 to 21, 18 to 22, 18 to 23, 18 to 24, 18 to
25, 18 to 26, 18 to 27, 18 to 28, 18 to 29, 18 to 30, 19 to 20, 19
to 21, 19 to 22, 19 to 23, 19 to 24, 19 to 25, 19 to 26, 19 to 29,
19 to 28, 19 to 29, 19 to 30, 20 to 21, 20 to 22, 20 to 23, 20 to
24, 20 to 25, 20 to 26, 20 to 27, 20 to 28, 20 to 29, 20 to 30, 21
to 22, 21 to 23, 21 to 24, 21 to 25, 21 to 26, 21 to 27, 21 to 28,
21 to 29, 21 to 30, 22 to 23, 22 to 24, 22 to 25, 22 to 26, 22 to
27, 22 to 28, 22 to 29, 22 to 30, 23 to 24, 23 to 25, 23 to 26, 23
to 27, 23 to 28, 23 to 29, 23 to 30, 24 to 25, 24 to 26, 24 to 27,
24 to 28, 24 to 29, 24 to 30, 25 to 26, 25 to 27, 25 to 28, 25 to
29, 25 to 30, 26 to 27, 26 to 28, 26 to 29, 26 to 30, 27 to 28, 27
to 29, 27 to 30, 28 to 29, 28 to 30, or 29 to 30 linked
nucleosides. In embodiments where the number of nucleosides of an
oligomeric compound or oligonucleotide is limited, whether to a
range or to a specific number, the oligomeric compound or
oligonucleotide may, nonetheless further comprise additional other
substituents. For example, an oligonucleotide comprising 8-30
nucleosides excludes oligonucleotides having 31 nucleosides, but,
unless otherwise indicated, such an oligonucleotide may further
comprise, for example one or more conjugates, terminal groups, or
other substituents. In certain embodiments, a gapmer
oligonucleotide has any of the above lengths.
[0281] Further, where an oligonucleotide is described by an overall
length range and by regions having specified lengths, and where the
sum of specified lengths of the regions is less than the upper
limit of the overall length range, the oligonucleotide may have
additional nucleosides, beyond those of the specified regions,
provided that the total number of nucleosides does not exceed the
upper limit of the overall length range.
[0282] e. Certain Oligonucleotides
[0283] In certain embodiments, oligonucleotides of the present
invention are characterized by their modification motif and overall
length. In certain embodiments, such parameters are each
independent of one another. Thus, each internucleoside linkage of
an oligonucleotide having a gapmer sugar motif may be modified or
unmodified and may or may not follow the gapmer modification
pattern of the sugar modifications. For example, the
internucleoside linkages within the wing regions of a sugar-gapmer
may be the same or different from one another and may be the same
or different from the internucleoside linkages of the gap region.
Likewise, such sugar-gapmer oligonucleotides may comprise one or
more modified nucleobase independent of the gapmer pattern of the
sugar modifications. One of skill in the art will appreciate that
such motifs may be combined to create a variety of
oligonucleotides. Herein if a description of an oligonucleotide or
oligomeric compound is silent with respect to one or more
parameter, such parameter is not limited. Thus, an oligomeric
compound described only as having a gapmer sugar motif without
further description may have any length, internucleoside linkage
motif, and nucleobase modification motif. Unless otherwise
indicated, all chemical modifications are independent of nucleobase
sequence.
[0284] f. Certain Conjugate Groups
[0285] In certain embodiments, oligomeric compounds are modified by
attachment of one or more conjugate groups. In general, conjugate
groups modify one or more properties of the attached oligomeric
compound including but not limited to pharmacodynamics,
pharmacokinetics, stability, binding, absorption, cellular
distribution, cellular uptake, charge and clearance. Conjugate
groups are routinely used in the chemical arts and are linked
directly or via an optional conjugate linking moiety or conjugate
linking group to a parent compound such as an oligomeric compound,
such as an oligonucleotide. Conjugate groups includes without
limitation, intercalators, reporter molecules, polyamines,
polyamides, polyethylene glycols, thioethers, polyethers,
cholesterols, thiocholesterols, cholic acid moieties, folate,
lipids, phospholipids, biotin, phenazine, phenanthridine,
anthraquinone, adamantane, acridine, fluoresceins, rhodamines,
coumarins and dyes. Certain conjugate groups have been described
previously, for example: cholesterol moiety (Letsinger et al.,
Proc. Natl. Acad. Sci. USA, 1989, 86, 6553-6556), cholic acid
(Manoharan et al., Bioorg. Med. Chem. Let., 1994, 4, 1053-1060), a
thioether, e.g., hexyl-S-tritylthiol (Manoharan et al., Ann. N.Y.
Acad. Sci., 1992, 660, 306-309; Manoharan et al., Bioorg. Med.
Chem. Let., 1993, 3, 2765-2770), a thiocholesterol (Oberhauser et
al., Nucl. Acids Res., 1992, 20, 533-538), an aliphatic chain,
e.g., do-decan-diol or undecyl residues (Saison-Behmoaras et al.,
EMBO J., 1991, 10, 1111-1118; Kabanov et al., FEBS Lett., 1990,
259, 327-330; Svinarchuk et al., Biochimie, 1993, 75, 49-54), a
phospholipid, e.g., di-hexadecyl-rac-glycerol or triethyl-ammonium
1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate (Manoharan et al.,
Tetrahedron Lett., 1995, 36, 3651-3654; Shea et al., Nucl. Acids
Res., 1990, 18, 3777-3783), a polyamine or a polyethylene glycol
chain (Manoharan et al., Nucleosides & Nucleotides, 1995, 14,
969-973), or adamantane acetic acid (Manoharan et al., Tetrahedron
Lett., 1995, 36, 3651-3654), a palmityl moiety (Mishra et al.,
Biochim. Biophys. Acta, 1995, 1264, 229-237), or an octadecylamine
or hexylamino-carbonyl-oxycholesterol moiety (Crooke et al., J.
Pharmacol. Exp. Ther., 1996, 277, 923-937).
[0286] In certain embodiments, a conjugate group comprises an
active drug substance, for example, aspirin, warfarin,
phenylbutazone, ibuprofen, suprofen, fen-bufen, ketoprofen,
(S)-(+)-pranoprofen, carprofen, dansylsarcosine,
2,3,5-triiodobenzoic acid, flufenamic acid, folinic acid, a
benzothiadiazide, chlorothiazide, a diazepine, indo-methicin, a
barbiturate, a cephalosporin, a sulfa drug, an antidiabetic, an
antibacterial or an antibiotic.
[0287] In certain embodiments, conjugate groups are directly
attached to oligonucleotides in oligomeric compounds. In certain
embodiments, conjugate groups are attached to oligonucleotides by a
conjugate linking group. In certain such embodiments, conjugate
linking groups, including, but not limited to, bifunctional linking
moieties such as those known in the art are amenable to the
compounds provided herein. Conjugate linking groups are useful for
attachment of conjugate groups, such as chemical stabilizing
groups, functional groups, reporter groups and other groups to
selective sites in a parent compound such as for example an
oligomeric compound. In general a bifunctional linking moiety
comprises a hydrocarbyl moiety having two functional groups. One of
the functional groups is selected to bind to a parent molecule or
compound of interest and the other is selected to bind essentially
any selected group such as chemical functional group or a conjugate
group. In some embodiments, the conjugate linker comprises a chain
structure or an oligomer of repeating units such as ethylene glycol
or amino acid units. Examples of functional groups that are
routinely used in a bifunctional linking moiety include, but are
not limited to, electrophiles for reacting with nucleophilic groups
and nucleophiles for reacting with electrophilic groups. In some
embodiments, bifunctional linking moieties include amino, hydroxyl,
carboxylic acid, thiol, unsaturations (e.g., double or triple
bonds), and the like.
[0288] Some nonlimiting examples of conjugate linking moieties
include pyrrolidine, 8-amino-3,6-dioxaoctanoic acid (ADO),
succinimidyl 4-(N-maleimidomethyl) cyclohexane-1-carboxylate (SMCC)
and 6-aminohexanoic acid (AHEX or AHA). Other linking groups
include, but are not limited to, substituted C.sub.1-C.sub.10
alkyl, substituted or unsubstituted C.sub.2-C.sub.10 alkenyl or
substituted or unsubstituted C.sub.2-C.sub.10 alkynyl, wherein a
nonlimiting list of preferred substituent groups includes hydroxyl,
amino, alkoxy, carboxy, benzyl, phenyl, nitro, thiol, thioalkoxy,
halogen, alkyl, aryl, alkenyl and alkynyl.
[0289] Conjugate groups may be attached to either or both ends of
an oligonucleotide (terminal conjugate groups) and/or at any
internal position.
[0290] In certain embodiments, conjugate groups are at the 3'-end
of an oligonucleotide of an oligomeric compound. In certain
embodiments, conjugate groups are near the 3'-end. In certain
embodiments, conjugates are attached at the 3' end of an oligomeric
compound, but before one or more terminal group nucleosides. In
certain embodiments, conjugate groups are placed within a terminal
group.
[0291] In certain embodiments, the present invention provides
oligomeric compounds. In certain embodiments, oligomeric compounds
comprise an oligonucleotide. In certain embodiments, an oligomeric
compound comprises an oligonucleotide and one or more conjugate
and/or terminal groups. Such conjugate and/or terminal groups may
be added to oligonucleotides having any of the motifs discussed
above. Thus, for example, an oligomeric compound comprising an
oligonucleotide having region of alternating nucleosides may
comprise a terminal group.
C. Antisense Compounds
[0292] In certain embodiments, oligomeric compounds provided herein
are antisense compounds. Such antisense compounds are capable of
hybridizing to a target nucleic acid, resulting in at least one
antisense activity. In certain embodiments, antisense compounds
specifically hybridize to one or more target nucleic acid. In
certain embodiments, a specifically hybridizing antisense compound
has a nucleobase sequence comprising a region having sufficient
complementarity to a target nucleic acid to allow hybridization and
result in antisense activity and insufficient complementarity to
any non-target so as to avoid non-specific hybridization to any
non-target nucleic acid sequences under conditions in which
specific hybridization is desired (e.g., under physiological
conditions for in vivo or therapeutic uses, and under conditions in
which assays are performed in the case of in vitro assays).
[0293] In certain embodiments, the present invention provides
antisense compounds comprising oligonucleotides that are fully
complementary to the target nucleic acid over the entire length of
the oligonucleotide. In certain embodiments, oligonucleotides are
99% complementary to the target nucleic acid. In certain
embodiments, oligonucleotides are 95% complementary to the target
nucleic acid. In certain embodiments, such oligonucleotides are 90%
complementary to the target nucleic acid.
[0294] In certain embodiments, such oligonucleotides are 85%
complementary to the target nucleic acid. In certain embodiments,
such oligonucleotides are 80% complementary to the target nucleic
acid. In certain embodiments, an antisense compound comprises a
region that is fully complementary to a target nucleic acid and is
at least 80% complementary to the target nucleic acid over the
entire length of the oligonucleotide. In certain such embodiments,
the region of full complementarity is from 6 to 14 nucleobases in
length.
[0295] a. Certain Antisense Activities and Mechanisms
[0296] In certain antisense activities, hybridization of an
antisense compound results in recruitment of a protein that cleaves
a target nucleic acid. For example, certain antisense compounds
result in RNase H mediated cleavage of target nucleic acid. RNase H
is a cellular endonuclease that cleaves the RNA strand of an
RNA:DNA duplex. The "DNA" in such an RNA:DNA duplex, need not be
unmodified DNA. In certain embodiments, the invention provides
antisense compounds that are sufficiently "DNA-like" to elicit
RNase H activity. Such DNA-like antisense compounds include, but
are not limited to gapmers having unmodified deoxyfuranose sugar
moieties in the nucleosides of the gap and modified sugar moieties
in the nucleosides of the wings.
[0297] Antisense activities may be observed directly or indirectly.
In certain embodiments, observation or detection of an antisense
activity involves observation or detection of a change in an amount
of a target nucleic acid or protein encoded by such target nucleic
acid; a change in the ratio of splice variants of a nucleic acid or
protein; and/or a phenotypic change in a cell or animal.
[0298] In certain embodiments, compounds comprising
oligonucleotides having a gapmer nucleoside motif described herein
have desirable properties compared to non-gapmer oligonucleotides
or to gapmers having other motifs. In certain circumstances, it is
desirable to identify motifs resulting in a favorable combination
of potent antisense activity and relatively low toxicity. In
certain embodiments, compounds of the present invention have a
favorable therapeutic index (measure of potency divided by measure
of toxicity).
[0299] b. Selective Antisense Compounds
[0300] In certain embodiments, antisense compounds provided herein
are selective for a target relative to a non-target nucleic acid.
In certain embodiments, the nucleobase sequences of the target and
non-target nucleic acids differ by no more than 4 differentiating
nucleobases in the targeted region. In certain embodiments, the
nucleobase sequences of the target and non-target nucleic acids
differ by no more than 3 differentiating nucleobases in the
targeted region. In certain embodiments, the nucleobase sequences
of the target and non-target nucleic acids differ by no more than 2
differentiating nucleobases in the targeted region. In certain
embodiments, the nucleobase sequences of the target and non-target
nucleic acids differ by a single differentiating nucleobase in the
targeted region. In certain embodiments, the target and non-target
nucleic acids are transcripts from different genes. In certain
embodiments, the target and non-target nucleic acids are different
alleles for the same gene.
[0301] Selectivity of antisense compounds is achieved, principally,
by nucleobase complementarity. For example, if an antisense
compound has no mismatches for a target nucleic acid and one or
more mismatches for a non-target nucleic acid, some amount of
selectivity for the target nucleic acid will result. In certain
embodiments, provided herein are antisense compounds with enhanced
selectivity (i.e. the ratio of activity for the target to the
activity for non-target is greater). For example, in certain
embodiments, a selective nucleoside comprises a particular feature
or combination of features (e.g., chemical modification, motif,
placement of selective nucleoside, and/or self-complementary
region) that increases selectivity of an antisense compound
compared to an antisense compound not having that feature or
combination of features. In certain embodiments, such feature or
combination of features increases antisense activity for the
target. In certain embodiments, such feature or combination of
features decreases activity for the target, but decreases activity
for the non-target by a greater amount, thus resulting in an
increase in selectivity.
[0302] Without being limited by mechanism, enhanced selectivity may
result from a larger difference in the affinity of an antisense
compound for its target compared to its affinity for the non-target
and/or a larger difference in RNase H activity for the resulting
duplexes. For example, in certain embodiments, a selective
antisense compound comprises a modified nucleoside at that same
position as a differentiating nucleobase (i.e., the selective
nucleoside is modified). That modification may increase the
difference in binding affinity of the antisense compound for the
target relative to the non-target. In addition or in the
alternative, the chemical modification may increase the difference
in RNAse H activity for the duplex formed by the antisense compound
and its target compared to the RNase activity for the duplex formed
by the antisense compound and the non-target. For example, the
modification may exaggerate a structure that is less compatible for
RNase H to bind, cleave and/or release the non-target.
[0303] Antisense compounds having certain specified motifs have
enhanced selectivity, including, but not limited to motifs
described above. In certain embodiments, enhanced selectivity is
achieved by oligonucleotides comprising any one or more of:
[0304] a modification motif comprising a long 5'-wing (longer than
5, 6, or 7 nucleosides);
[0305] a modification motif comprising a long 3'-wing (longer than
5, 6, or 7 nucleosides);
[0306] a modification motif comprising a short gap region (shorter
than 8, 7, or 6 nucleosides); and
[0307] a modification motif comprising an interrupted gap region
(having no uninterrupted stretch of unmodified 2'-deoxynucleosides
longer than 7, 6 or 5).
[0308] i. Certain Selective Nucleobase Sequence Elements
[0309] In certain embodiments, selective antisense compounds
comprise nucleobase sequence elements. Such nucleobase sequence
elements are independent of modification motifs. Accordingly,
oligonucleotides having any of the motifs (modification motifs,
nucleoside motifs, sugar motifs, nucleobase modification motifs,
and/or linkage motifs) may also comprise one or more of the
following nucleobase sequence elements.
[0310] 1. Alignment of Differentiating Nucleobase/Target-Selective
Nucleoside
[0311] In certain embodiments, a target region and a region of a
non-target nucleic acid differ by 1-4 differentiating nucleobase.
In such embodiments, selective antisense compounds have a
nucleobase sequence that aligns with the non-target nucleic acid
with 1-4 mismatches. A nucleoside of the antisense compound that
corresponds to a differentiating nucleobase of the target nucleic
acid is referred to herein as a target-selective nucleoside. In
certain embodiments, selective antisense compounds having a gapmer
motif align with a non-target nucleic acid, such that a
target-selective nucleoside is positioned in the gap. In certain
embodiments, a target-selective nucleoside is the 1.sup.st
nucleoside of the gap from the 5' end. In certain embodiments, a
target-selective nucleoside is the 2.sup.nd nucleoside of the gap
from the 5' end. In certain embodiments, a target-selective
nucleoside is the 3.sup.rd nucleoside of the gap from the 5'-end.
In certain embodiments, a target-selective nucleoside is the
4.sup.th nucleoside of the gap from the 5'-end. In certain
embodiments, a target-selective nucleoside is the 5.sup.th
nucleoside of the gap from the 5'-end. In certain embodiments, a
target-selective nucleoside is the 6.sup.rd nucleoside of the gap
from the 5'-end. In certain embodiments, a target-selective
nucleoside is the 8.sup.th nucleoside of the gap from the 3'-end.
In certain embodiments, a target-selective nucleoside is the
7.sup.th nucleoside of the gap from the 3'-end. In certain
embodiments, a target-selective nucleoside is the 6.sup.th
nucleoside of the gap from the 3'-end. In certain embodiments, a
target-selective nucleoside is the 5.sup.th nucleoside of the gap
from the 3'-end. In certain embodiments, a target-selective
nucleoside is the 4.sup.th nucleoside of the gap from the 3'-end.
In certain embodiments, a target-selective nucleoside is the
3.sup.rd nucleoside of the gap from the 3'-end. In certain
embodiments, a target-selective nucleoside is the 2.sup.nd
nucleoside of the gap from the 3'-end.
[0312] 2. Mismatches to the Target Nucleic Acid
[0313] In certain embodiments, selective antisense compounds
comprise one or more mismatched nucleobases relative to the target
nucleic acid. In certain such embodiments, antisense activity
against the target is reduced by such mismatch, but activity
against the non-target is reduced by a greater amount. Thus, in
certain embodiments selectivity is improved. Any nucleobase other
than the differentiating nucleobase is suitable for a mismatch. In
certain embodiments, however, the mismatch is specifically
positioned within the gap of an oligonucleotide having a gapmer
motif. In certain embodiments, a mismatch relative to the target
nucleic acid is at positions 1, 2, 3, 4, 5, 6, 7, or 8 from the
5'-end of the gap region. In certain embodiments, a mismatch
relative to the target nucleic acid is at positions 9, 8, 7, 6, 5,
4, 3, 2, 1 of the antisense compounds from the 3'-end of the gap
region. In certain embodiments, a mismatch relative to the target
nucleic acid is at positions 1, 2, 3, or 4 of the antisense
compounds from the 5'-end of the wing region. In certain
embodiments, a mismatch relative to the target nucleic acid is at
positions 4, 3, 2, or 1 of the antisense compounds from the 3'-end
of the wing region.
[0314] 3. Self Complementary Regions
[0315] In certain embodiments, selective antisense compounds
comprise a region that is not complementary to the target. In
certain embodiments, such region is complementary to another region
of the antisense compound. Such regions are referred to herein as
self-complementary regions. For example, in certain embodiments, an
antisense compound has a first region at one end that is
complementary to a second region at the other end. In certain
embodiments, one of the first and second regions is complementary
to the target nucleic acid. Unless the target nucleic acid also
includes a self-complementary region, the other of the first and
second region of the antisense compound will not be complementary
to the target nucleic acid. For illustrative purposes, certain
antisense compounds have the following nucleobase motif:
[0316] ABCXXXXXXXXXC'B'A';
[0317] ABCXXXXXXX(X/C')(X/B')(X/A');
[0318] (X/A)(X/B)(X/C)XXXXXXXXXC'B'A'
where each of A, B, and C are any nucleobase; A', B', and C' are
the complementary bases to A, B, and C, respectively; each X is a
nucleobase complementary to the target nucleic acid; and two
letters in parentheses (e.g., (X/C')) indicates that the nucleobase
is complementary to the target nucleic acid and to the designated
nucleoside within the antisense oligonucleotide.
[0319] Without being bound to any mechanism, in certain
embodiments, such antisense compounds are expected to form
self-structure, which is disrupted upon contact with a target
nucleic acid. Contact with a non-target nucleic acid is expected to
disrupt the self-structure to a lesser degree, thus increasing
selectivity compared to the same antisense compound lacking the
self-complementary regions.
[0320] 4. Combinations of Features
[0321] Though it is clear to one of skill in the art, the above
motifs and other elements for increasing selectivity may be used
alone or in combination. For example, a single antisense compound
may include any one, two, three, or more of: self-complementary
regions, a mismatch relative to the target nucleic acid, a short
nucleoside gap, an interrupted gap, and specific placement of the
selective nucleoside.
D. Certain Target Nucleic Acids
[0322] In certain embodiments, antisense compounds comprise or
consist of an oligonucleotide comprising a region that is
complementary to a target nucleic acid. In certain embodiments, the
target nucleic acid is an endogenous RNA molecule. In certain
embodiments, the target nucleic acid is a non-coding RNA. In
certain such embodiments, the target non-coding RNA is selected
from: a long-non-coding RNA, a short non-coding RNA, an intronic
RNA molecule, a snoRNA, a scaRNA, a microRNA (including
pre-microRNA and mature microRNA), a ribosomal RNA, and promoter
directed RNA. In certain embodiments, the target nucleic acid
encodes a protein. In certain such embodiments, the target nucleic
acid is selected from: an mRNA and a pre-mRNA, including intronic,
exonic and untranslated regions. In certain embodiments, oligomeric
compounds are at least partially complementary to more than one
target nucleic acid. For example, antisense compounds of the
present invention may mimic microRNAs, which typically bind to
multiple targets.
[0323] In certain embodiments, the target nucleic acid is a nucleic
acid other than a mature mRNA. In certain embodiments, the target
nucleic acid is a nucleic acid other than a mature mRNA or a
microRNA. In certain embodiments, the target nucleic acid is a
non-coding RNA other than a microRNA. In certain embodiments, the
target nucleic acid is a non-coding RNA other than a microRNA or an
intronic region of a pre-mRNA. In certain embodiments, the target
nucleic acid is a long non-coding RNA. In certain embodiments, the
target RNA is an mRNA. In certain embodiments, the target nucleic
acid is a pre-mRNA. In certain such embodiments, the target region
is entirely within an intron. In certain embodiments, the target
region spans an intron/exon junction. In certain embodiments, the
target region is at least 50% within an intron. In certain
embodiments, the target nucleic acid is selected from among
non-coding RNA, including exonic regions of pre-mRNA. In certain
embodiments, the target nucleic acid is a ribosomal RNA (rRNA). In
certain embodiments, the target nucleic acid is a non-coding RNA
associated with splicing of other pre-mRNAs. In certain
embodiments, the target nucleic acid is a nuclear-retained
non-coding RNA.
[0324] In certain embodiments, antisense compounds described herein
are complementary to a target nucleic acid comprising a
single-nucleotide polymorphism. In certain such embodiments, the
antisense compound is capable of modulating expression of one
allele of the single-nucleotide polymorphism-containing-target
nucleic acid to a greater or lesser extent than it modulates
another allele. In certain embodiments an antisense compound
hybridizes to a single-nucleotide polymorphism-containing-target
nucleic acid at the single-nucleotide polymorphism site. In certain
embodiments, the target nucleic acid is a Huntingtin gene
transcript. In certain embodiments, the target nucleic acid is a
single-nucleotide polymorphism-containing-target nucleic acid of a
Huntingtin gene transcript. In certain embodiments, the target
nucleic acid is not a Huntingtin gene transcript. In certain
embodiments, the target nucleic acid is a single-nucleotide
polymorphism-containing-target nucleic acid of a gene transcript
other than Huntingtin. In certain embodiments, the target nucleic
acid is any nucleic acid other than a Huntingtin gene
transcript.
[0325] a. Single-Nucleotide Polymorphism
[0326] Embodiments of the present invention provide methods,
compounds, and compositions for selectively inhibiting mRNA and
protein expression of an allelic variant of a particular gene or
DNA sequence. In certain embodiments, the allelic variant contains
a single nucleotide polymorphism (SNP). In certain embodiments, a
SNP is associated with a mutant allele. In certain embodiments, a
mutant SNP is associated with a disease. In certain embodiments a
mutant SNP is associated with a disease, but is not causative of
the disease. In certain embodiments, mRNA and protein expression of
a mutant allele is associated with disease.
[0327] In certain embodiments, the expressed gene product of a
mutant allele results in aggregation of the mutant proteins causing
disease. In certain embodiments, the expressed gene product of a
mutant allele results in gain of function causing disease. In
certain embodiments, genes with an autosomal dominant mutation
resulting in a toxic gain of function of the protein are the APP
gene encoding amyloid precursor protein involved in Alzheimer's
disease (Gene, 371: 68, 2006); the PrP gene encoding prion protein
involved in Creutzfeldt-Jakob disease and in fatal familial
insomnia (Nat. Med. 1997, 3: 1009); GFAP gene encoding glial
fibrillary acidic protein involved in Alexander disease (J.
Neurosci. 2006, 26:111623); alpha-synuclein gene encoding
alpha-synuclein protein involved in Parkinson's disease (J. Clin.
Invest. 2003, 111: 145); SOD-1 gene encoding the SOD-1 protein
involved in amyotrophic lateral sclerosis (Science 1998, 281:
1851); atrophin-1 gene encoding atrophin-1 protein involved in
dentato-rubral and pallido-luysian atrophy (DRPA) (Trends Mol. Med.
2001, 7: 479); SCA1 gene encoding ataxin-1 protein involved in
spino-cerebellar ataxia-1 (SCA1) (Protein Sci. 2003, 12: 953); PLP
gene encoding proteolipid protein involved in Pelizaeus-Merzbacher
disease (Neuro Mol Med. 2007, 4: 73); DYT1 gene encoding torsinA
protein involved in Torsion dystonia (Brain Res. 2000, 877: 379);
and alpha-B crystalline gene encoding alpha-B crystalline protein
involved in protein aggregation diseases, including cardiomyopathy
(Cell 2007, 130: 427); alpha1-antitrypsin gene encoding
alpha1-antitrypsin protein involved in chronic obstructive
pulmonary disease (COPD), liver disease and hepatocellular
carcinoma (New Engl J Med. 2002, 346: 45); Ltk gene encoding
leukocyte tyrosine kinase protein involved in systemic lupus
erythematosus (Hum. Mol. Gen. 2004, 13: 171); PCSK9 gene encoding
PCSK9 protein involved in hypercholesterolemia (Hum Mutat. 2009,
30: 520); prolactin receptor gene encoding prolactin receptor
protein involved in breast tumors (Proc. Natl. Assoc. Sci. 2008,
105: 4533); CCL5 gene encoding the chemokine CCL5 involved in COPD
and asthma (Eur. Respir. J. 2008, 32: 327); PTPN22 gene encoding
PTPN22 protein involved in Type 1 diabetes, Rheumatoid arthritis,
Graves disease, and SLE (Proc. Natl. Assoc. Sci. 2007, 104: 19767);
androgen receptor gene encoding the androgen receptor protein
involved in spinal and bulbar muscular atrophy or Kennedy's disease
(J Steroid Biochem. Mol. Biol. 2008, 108: 245); CHMP4B gene
encoding chromatin modifying protein-4B involved in progressive
childhood posterior subcapsular cataracts (Am. J. Hum. Genet 2007,
81: 596); FXR/NR1H4 gene encoding Farnesoid X receptor protein
involved in cholesterol gallstone disease, arthrosclerosis and
diabetes (Mol. Endocrinol. 2007, 21: 1769); ABCA1 gene encoding
ABCA1 protein involved in cardiovascular disease (Transl. Res.
2007, 149: 205); CaSR gene encoding the calcium sensing receptor
protein involved in primary hypercalciuria (Kidney Int. 2007, 71:
1155); alpha-globin gene encoding alpha-globin protein involved in
alpha-thallasemia (Science 2006, 312: 1215); httlpr gene encoding
HTTLPR protein involved in obsessive compulsive disorder (Am. J.
Hum. Genet. 2006, 78: 815); AVP gene encoding arginine vasopressin
protein in stress-related disorders such as anxiety disorders and
comorbid depression (CNS Neurol. Disord. Drug Targets 2006, 5:
167); GNAS gene encoding G proteins involved in congenital visual
defects, hypertension, metabolic syndrome (Trends Pharmacol. Sci.
2006, 27: 260); APAF1 gene encoding APAF1 protein involved in a
predisposition to major depression (Mol. Psychiatry 2006, 11: 76);
TGF-beta1 gene encoding TGF-beta1 protein involved in breast cancer
and prostate cancer (Cancer Epidemiol. Biomarkers Prev. 2004, 13:
759); AChR gene encoding acetylcholine receptor involved in
congential myasthenic syndrome (Neurology 2004, 62: 1090); P2Y12
gene encoding adenosine diphosphate (ADP) receptor protein involved
in risk of peripheral arterial disease (Circulation 2003, 108:
2971); LQT1 gene encoding LQT1 protein involved in atrial
fibrillation (Cardiology 2003, 100: 109); RET protooncogene
encoding RET protein involved in sporadic pheochromocytoma (J.
Clin. Endocrinol. Metab. 2003, 88: 4911); filamin A gene encoding
filamin A protein involved in various congenital malformations
(Nat. Genet. 2003, 33: 487); TARDBP gene encoding TDP-43 protein
involved in amyotrophic lateral sclerosis (Hum. Mol. Gene. t 2010,
19: 671); SCA3 gene encoding ataxin-3 protein involved in
Machado-Joseph disease (PLoS One 2008, 3: e3341); SCAT gene
encoding ataxin-7 protein involved in spino-cerebellar ataxia-7
(PLoS One 2009, 4: e7232); and HTT gene encoding huntingtin protein
involved in Huntington's disease (Neurobiol Dis. 1996, 3:183); and
the CA4 gene encoding carbonic anhydrase 4 protein, CRX gene
encoding cone-rod homeobox transcription factor protein, FSCN2 gene
encoding retinal fascin homolog 2 protein, IMPDH1 gene encoding
inosine monophosphate dehydrogenase 1 protein, NR2E3 gene encoding
nuclear receptor subfamily 2 group E3 protein, NRL gene encoding
neural retina leucine zipper protein, PRPF3 (RP18) gene encoding
pre-mRNA splicing factor 3 protein, PRPF8 (RP13) gene encoding
pre-mRNA splicing factor 8 protein, PRPF31 (RP11) gene encoding
pre-mRNA splicing factor 31 protein, RDS gene encoding peripherin 2
protein, ROM1 gene encoding rod outer membrane protein 1 protein,
RHO gene encoding rhodopsin protein, RP1 gene encoding RP1 protein,
RPGR gene encoding retinitis pigmentosa GTPase regulator protein,
all of which are involved in Autosomal Dominant Retinitis
Pigmentosa disease (Adv Exp Med Biol. 2008, 613:203)
[0328] In certain embodiments, the mutant allele is associated with
any disease from the group consisting of Alzheimer's disease,
Creutzfeldt-Jakob disease, fatal familial insomnia, Alexander
disease, Parkinson's disease, amyotrophic lateral sclerosis,
dentato-rubral and pallido-luysian atrophy DRPA, spino-cerebellar
ataxia, Torsion dystonia, cardiomyopathy, chronic obstructive
pulmonary disease (COPD), liver disease, hepatocellular carcinoma,
systemic lupus erythematosus, hypercholesterolemia, breast cancer,
asthma, Type 1 diabetes, Rheumatoid arthritis, Graves disease, SLE,
spinal and bulbar muscular atrophy, Kennedy's disease, progressive
childhood posterior subcapsular cataracts, cholesterol gallstone
disease, arthrosclerosis, cardiovascular disease, primary
hypercalciuria, alpha-thallasemia, obsessive compulsive disorder,
Anxiety, comorbid depression, congenital visual defects,
hypertension, metabolic syndrome, prostate cancer, congential
myasthenic syndrome, peripheral arterial disease, atrial
fibrillation, sporadic pheochromocytoma, congenital malformations,
Machado-Joseph disease, Huntington's disease, and Autosomal
Dominant Retinitis Pigmentosa disease.
[0329] i. Certain Huntingtin Targets
[0330] In certain embodiments, an allelic variant of huntingtin is
selectively reduced. Nucleotide sequences that encode huntingtin
include, without limitation, the following: GENBANK Accession No.
NT_006081.18, truncated from nucleotides 1566000 to 1768000
(replaced by GENBANK Accession No. NT_006051), incorporated herein
as SEQ ID NO: 8, and NM_002111.6, incorporated herein as SEQ ID NO:
10.
[0331] Table 3 provides SNPs found in the GM04022, GM04281,
GM02171, and GM02173B cell lines. Also provided are the allelic
variants found at each SNP position, the genotype for each of the
cell lines, and the percentage of HD patients having a particular
allelic variant. For example, the two allelic variants for SNP
rs6446723 are T and C. The GM04022 cell line is heterozygous TC,
the GM02171 cell line is homozygous CC, the GM02173 cell line is
heterozygous TC, and the GM04281 cell line is homozygous TT. Fifty
percent of HD patients have a T at SNP position rs6446723.
TABLE-US-00004 TABLE 3 Allelic Variations for SNPs Associated with
HD SNP Variation GM04022 GM02171 GM02173 GM04281 TargetPOP allele
rs6446723 T/C TC CC TC TT 0.50 T rs3856973 A/G AG AA AG GG 0.50 G
rs2285086 A/G AG GG AG AA 0.50 A rs363092 A/C AC AA AC CC 0.49 C
rs916171 C/G GC GG GC CC 0.49 C rs6844859 T/C TC CC TC TT 0.49 T
rs7691627 A/G AG AA AG GG 0.49 G rs4690073 A/G AG AA AG GG 0.49 G
rs2024115 A/G AG GG AG AA 0.48 A rs11731237 T/C CC CC TC TT 0.43 T
rs362296 A/C CC AC AC AC 0.42 C rs10015979 A/G AA AA AG GG 0.42 G
rs7659144 C/G CG CG CG CC 0.41 C rs363096 T/C CC CC TC TT 0.40 T
rs362273 A/G AA AG AG AA 0.39 A rs16843804 T/C CC TC TC CC 0.38 C
rs362271 A/G GG AG AG GG 0.38 G rs362275 T/C CC TC TC CC 0.38 C
rs3121419 T/C CC TC TC CC 0.38 C rs362272 A/G GG -- AG GG 0.38 G
rs3775061 A/G AA AG AG AA 0.38 A rs34315806 T/C CC TC TC CC 0.38 C
rs363099 T/C CC TC TC CC 0.38 C rs2298967 T/C TT TC TC TT 0.38 T
rs363088 A/T AA TA TA AA 0.38 A rs363064 T/C CC TC TC CC 0.35 C
rs363102 A/G AG AA AA AA 0.23 G rs2798235 A/G AG GG GG GG 0.21 A
rs363080 T/C TC CC CC CC 0.21 T rs363072 A/T TA TA AA AA 0.13 A
rs363125 A/C AC AC CC CC 0.12 C rs362303 T/C TC TC CC CC 0.12 C
rs362310 T/C TC TC CC CC 0.12 C rs10488840 A/G AG AG GG GG 0.12 G
rs362325 T/C TC TC TT TT 0.11 T rs35892913 A/G GG GG GG GG 0.10 A
rs363102 A/G AG AA AA AA 0.09 A rs363096 T/C CC CC TC TT 0.09 C
rs11731237 T/C CC CC TC TT 0.09 C rs10015979 A/G AA AA AG GG 0.08 A
rs363080 T/C TC CC CC CC 0.07 C rs2798235 A/G AG GG GG GG 0.07 G
rs1936032 C/G GC CC CC CC 0.06 C rs2276881 A/G GG GG GG GG 0.06 G
rs363070 A/G AA AA AA AA 0.06 A rs35892913 A/G GG GG GG GG 0.04 G
rs12502045 T/C CC CC CC CC 0.04 C rs6446723 T/C TC CC TC TT 0.04 C
rs7685686 A/G AG GG AG AA 0.04 G rs3733217 T/C CC CC CC CC 0.03 C
rs6844859 T/C TC CC TC TT 0.03 C rs362331 T/C TC CC TC TT 0.03
C
E. Certain Pharmaceutical Compositions
[0332] In certain embodiments, the present invention provides
pharmaceutical compositions comprising one or more antisense
compound. In certain embodiments, such pharmaceutical composition
comprises a suitable pharmaceutically acceptable diluent or
carrier. In certain embodiments, a pharmaceutical composition
comprises a sterile saline solution and one or more antisense
compound. In certain embodiments, such pharmaceutical composition
consists of a sterile saline solution and one or more antisense
compound. In certain embodiments, the sterile saline is
pharmaceutical grade saline. In certain embodiments, a
pharmaceutical composition comprises one or more antisense compound
and sterile water. In certain embodiments, a pharmaceutical
composition consists of one or more antisense compound and sterile
water. In certain embodiments, the sterile saline is pharmaceutical
grade water. In certain embodiments, a pharmaceutical composition
comprises one or more antisense compound and phosphate-buffered
saline (PBS). In certain embodiments, a pharmaceutical composition
consists of one or more antisense compound and sterile
phosphate-buffered saline (PBS). In certain embodiments, the
sterile saline is pharmaceutical grade PBS.
[0333] In certain embodiments, antisense compounds may be admixed
with pharmaceutically acceptable active and/or inert substances for
the preparation of pharmaceutical compositions or formulations.
Compositions and methods for the formulation of pharmaceutical
compositions depend on a number of criteria, including, but not
limited to, route of administration, extent of disease, or dose to
be administered.
[0334] Pharmaceutical compositions comprising antisense compounds
encompass any pharmaceutically acceptable salts, esters, or salts
of such esters. In certain embodiments, pharmaceutical compositions
comprising antisense compounds comprise one or more oligonucleotide
which, upon administration to an animal, including a human, is
capable of providing (directly or indirectly) the biologically
active metabolite or residue thereof. Accordingly, for example, the
disclosure is also drawn to pharmaceutically acceptable salts of
antisense compounds, prodrugs, pharmaceutically acceptable salts of
such prodrugs, and other bioequivalents. Suitable pharmaceutically
acceptable salts include, but are not limited to, sodium and
potassium salts.
[0335] A prodrug can include the incorporation of additional
nucleosides at one or both ends of an oligomeric compound which are
cleaved by endogenous nucleases within the body, to form the active
antisense oligomeric compound.
[0336] Lipid moieties have been used in nucleic acid therapies in a
variety of methods. In certain such methods, the nucleic acid is
introduced into preformed liposomes or lipoplexes made of mixtures
of cationic lipids and neutral lipids. In certain methods, DNA
complexes with mono- or poly-cationic lipids are formed without the
presence of a neutral lipid. In certain embodiments, a lipid moiety
is selected to increase distribution of a pharmaceutical agent to a
particular cell or tissue. In certain embodiments, a lipid moiety
is selected to increase distribution of a pharmaceutical agent to
fat tissue. In certain embodiments, a lipid moiety is selected to
increase distribution of a pharmaceutical agent to muscle
tissue.
[0337] In certain embodiments, pharmaceutical compositions provided
herein comprise one or more modified oligonucleotides and one or
more excipients. In certain such embodiments, excipients are
selected from water, salt solutions, alcohol, polyethylene glycols,
gelatin, lactose, amylase, magnesium stearate, talc, silicic acid,
viscous paraffin, hydroxymethylcellulose and
polyvinylpyrrolidone.
[0338] In certain embodiments, a pharmaceutical composition
provided herein comprises a delivery system. Examples of delivery
systems include, but are not limited to, liposomes and emulsions.
Certain delivery systems are useful for preparing certain
pharmaceutical compositions including those comprising hydrophobic
compounds. In certain embodiments, certain organic solvents such as
dimethylsulfoxide are used.
[0339] In certain embodiments, a pharmaceutical composition
provided herein comprises one or more tissue-specific delivery
molecules designed to deliver the one or more pharmaceutical agents
of the present invention to specific tissues or cell types. For
example, in certain embodiments, pharmaceutical compositions
include liposomes coated with a tissue-specific antibody.
[0340] In certain embodiments, a pharmaceutical composition
provided herein comprises a co-solvent system. Certain of such
co-solvent systems comprise, for example, benzyl alcohol, a
nonpolar surfactant, a water-miscible organic polymer, and an
aqueous phase. In certain embodiments, such co-solvent systems are
used for hydrophobic compounds. A non-limiting example of such a
co-solvent system is the VPD co-solvent system, which is a solution
of absolute ethanol comprising 3% w/v benzyl alcohol, 8% w/v of the
nonpolar surfactant Polysorbate 80.TM. and 65% w/v polyethylene
glycol 300. The proportions of such co-solvent systems may be
varied considerably without significantly altering their solubility
and toxicity characteristics. Furthermore, the identity of
co-solvent components may be varied: for example, other surfactants
may be used instead of Polysorbate 80.TM.; the fraction size of
polyethylene glycol may be varied; other biocompatible polymers may
replace polyethylene glycol, e.g., polyvinyl pyrrolidone; and other
sugars or polysaccharides may substitute for dextrose.
[0341] In certain embodiments, a pharmaceutical composition
provided herein is prepared for oral administration. In certain
embodiments, pharmaceutical compositions are prepared for buccal
administration.
[0342] In certain embodiments, a pharmaceutical composition is
prepared for administration by injection (e.g., intravenous,
subcutaneous, intramuscular, etc.). In certain of such embodiments,
a pharmaceutical composition comprises a carrier and is formulated
in aqueous solution, such as water or physiologically compatible
buffers such as Hanks's solution, Ringer's solution, or
physiological saline buffer. In certain embodiments, other
ingredients are included (e.g., ingredients that aid in solubility
or serve as preservatives). In certain embodiments, injectable
suspensions are prepared using appropriate liquid carriers,
suspending agents and the like. Certain pharmaceutical compositions
for injection are presented in unit dosage form, e.g., in ampoules
or in multi-dose containers. Certain pharmaceutical compositions
for injection are suspensions, solutions or emulsions in oily or
aqueous vehicles, and may contain formulatory agents such as
suspending, stabilizing and/or dispersing agents. Certain solvents
suitable for use in pharmaceutical compositions for injection
include, but are not limited to, lipophilic solvents and fatty
oils, such as sesame oil, synthetic fatty acid esters, such as
ethyl oleate or triglycerides, and liposomes. Aqueous injection
suspensions may contain substances that increase the viscosity of
the suspension, such as sodium carboxymethyl cellulose, sorbitol,
or dextran. Optionally, such suspensions may also contain suitable
stabilizers or agents that increase the solubility of the
pharmaceutical agents to allow for the preparation of highly
concentrated solutions.
[0343] In certain embodiments, a pharmaceutical composition is
prepared for transmucosal administration. In certain of such
embodiments penetrants appropriate to the barrier to be permeated
are used in the formulation. Such penetrants are generally known in
the art.
[0344] In certain embodiments, a pharmaceutical composition
provided herein comprises an oligonucleotide in a therapeutically
effective amount. In certain embodiments, the therapeutically
effective amount is sufficient to prevent, alleviate or ameliorate
symptoms of a disease or to prolong the survival of the subject
being treated. Determination of a therapeutically effective amount
is well within the capability of those skilled in the art.
[0345] In certain embodiments, one or more modified oligonucleotide
provided herein is formulated as a prodrug. In certain embodiments,
upon in vivo administration, a prodrug is chemically converted to
the biologically, pharmaceutically or therapeutically more active
form of an oligonucleotide. In certain embodiments, prodrugs are
useful because they are easier to administer than the corresponding
active form. For example, in certain instances, a prodrug may be
more bioavailable (e.g., through oral administration) than is the
corresponding active form. In certain instances, a prodrug may have
improved solubility compared to the corresponding active form. In
certain embodiments, prodrugs are less water soluble than the
corresponding active form. In certain instances, such prodrugs
possess superior transmittal across cell membranes, where water
solubility is detrimental to mobility. In certain embodiments, a
prodrug is an ester. In certain such embodiments, the ester is
metabolically hydrolyzed to carboxylic acid upon administration. In
certain instances the carboxylic acid containing compound is the
corresponding active form. In certain embodiments, a prodrug
comprises a short peptide (polyaminoacid) bound to an acid group.
In certain of such embodiments, the peptide is cleaved upon
administration to form the corresponding active form.
[0346] In certain embodiments, the present invention provides
compositions and methods for reducing the amount or activity of a
target nucleic acid in a cell. In certain embodiments, the cell is
in an animal. In certain embodiments, the animal is a mammal. In
certain embodiments, the animal is a rodent. In certain
embodiments, the animal is a primate. In certain embodiments, the
animal is a non-human primate. In certain embodiments, the animal
is a human.
[0347] In certain embodiments, the present invention provides
methods of administering a pharmaceutical composition comprising an
oligomeric compound of the present invention to an animal. Suitable
administration routes include, but are not limited to, oral,
rectal, transmucosal, intestinal, enteral, topical, suppository,
through inhalation, intrathecal, intracerebroventricular,
intraperitoneal, intranasal, intraocular, intratumoral, and
parenteral (e.g., intravenous, intramuscular, intramedullary, and
subcutaneous). In certain embodiments, pharmaceutical intrathecals
are administered to achieve local rather than systemic exposures.
For example, pharmaceutical compositions may be injected directly
in the area of desired effect (e.g., into the liver).
Nonlimiting Disclosure and Incorporation by Reference
[0348] While certain compounds, compositions and methods described
herein have been described with specificity in accordance with
certain embodiments, the following examples serve only to
illustrate the compounds described herein and are not intended to
limit the same. Each of the references, GenBank accession numbers,
and the like recited in the present application is incorporated
herein by reference in its entirety.
[0349] Although the sequence listing accompanying this filing
identifies each sequence as either "RNA" or "DNA" as required, in
reality, those sequences may be modified with any combination of
chemical modifications. One of skill in the art will readily
appreciate that such designation as "RNA" or "DNA" to describe
modified oligonucleotides is, in certain instances, arbitrary. For
example, an oligonucleotide comprising a nucleoside comprising a
2'-OH sugar moiety and a thymine base could be described as a DNA
having a modified sugar (2'-OH for the natural 2'-H of DNA) or as
an RNA having a modified base (thymine (methylated uracil) for
natural uracil of RNA).
[0350] Accordingly, nucleic acid sequences provided herein,
including, but not limited to those in the sequence listing, are
intended to encompass nucleic acids containing any combination of
natural or modified RNA and/or DNA, including, but not limited to
such nucleic acids having modified nucleobases. By way of further
example and without limitation, an oligomeric compound having the
nucleobase sequence "ATCGATCG" encompasses any oligomeric compounds
having such nucleobase sequence, whether modified or unmodified,
including, but not limited to, such compounds comprising RNA bases,
such as those having sequence "AUCGAUCG" and those having some DNA
bases and some RNA bases such as "AUCGATCG" and oligomeric
compounds having other modified or naturally occurring bases, such
as "AT.sup.meCGAUCG," wherein .sup.meC indicates a cytosine base
comprising a methyl group at the 5-position.
EXAMPLES
[0351] The following examples illustrate certain embodiments of the
present invention and are not limiting. Moreover, where specific
embodiments are provided, the inventors have contemplated generic
application of those specific embodiments. For example, disclosure
of an oligonucleotide having a particular motif provides reasonable
support for additional oligonucleotides having the same or similar
motif. And, for example, where a particular high-affinity
modification appears at a particular position, other high-affinity
modifications at the same position are considered suitable, unless
otherwise indicated.
Example 1
Synthesis of Nucleoside Phosphoramidites
[0352] The preparation of nucleoside phosphoramidites is performed
following procedures that are illustrated herein and in the art
such as but not limited to U.S. Pat. No. 6,426,220 and published
PCT WO 02/36743.
Example 2
Synthesis of Oligomeric Compounds
[0353] The oligomeric compounds used in accordance with this
invention may be conveniently and routinely made through the
well-known technique of solid phase synthesis. Equipment for such
synthesis is sold by several vendors including, for example,
Applied Biosystems (Foster City, Calif.). Any other means for such
synthesis known in the art may additionally or alternatively be
employed. It is well known to use similar techniques to prepare
oligonucleotides such as alkylated derivatives and those having
phosphorothioate linkages.
[0354] Oligomeric compounds: Unsubstituted and substituted
phosphodiester (P.dbd.O) oligomeric compounds, including without
limitation, oligonucleotides can be synthesized on an automated DNA
synthesizer (Applied Biosystems model 394) using standard
phosphoramidite chemistry with oxidation by iodine.
[0355] In certain embodiments, phosphorothioate internucleoside
linkages (P.dbd.S) are synthesized similar to phosphodiester
internucleoside linkages with the following exceptions: thiation is
effected by utilizing a 10% w/v solution of
3,H-1,2-benzodithiole-3-one 1,1-dioxide in acetonitrile for the
oxidation of the phosphite linkages. The thiation reaction step
time is increased to 180 sec and preceded by the normal capping
step. After cleavage from the CPG column and deblocking in
concentrated ammonium hydroxide at 55.degree. C. (12-16 hr), the
oligomeric compounds are recovered by precipitating with greater
than 3 volumes of ethanol from a 1 M NH.sub.4OAc solution.
Phosphinate internucleoside linkages can be prepared as described
in U.S. Pat. No. 5,508,270.
[0356] Alkyl phosphonate internucleoside linkages can be prepared
as described in U.S. Pat. No. 4,469,863.
[0357] 3'-Deoxy-3'-methylene phosphonate internucleoside linkages
can be prepared as described in U.S. Pat. No. 5,610,289 or U.S.
Pat. No. 5,625,050.
[0358] Phosphoramidite internucleoside linkages can be prepared as
described in U.S. Pat. No. 5,256,775 or U.S. Pat. No.
5,366,878.
[0359] Alkylphosphonothioate internucleoside linkages can be
prepared as described in published PCT applications PCT/US94/00902
and PCT/US93/06976 (published as WO 94/17093 and WO 94/02499,
respectively).
[0360] 3'-Deoxy-3'-amino phosphoramidate internucleoside linkages
can be prepared as described in U.S. Pat. No. 5,476,925.
[0361] Phosphotriester internucleoside linkages can be prepared as
described in U.S. Pat. No. 5,023,243.
[0362] Borano phosphate internucleoside linkages can be prepared as
described in U.S. Pat. Nos. 5,130,302 and 5,177,198.
[0363] Oligomeric compounds having one or more non-phosphorus
containing internucleoside linkages including without limitation
methylenemethylimino linked oligonucleosides, also identified as
MMI linked oligonucleosides, methylenedimethylhydrazo linked
oligonucleosides, also identified as MDH linked oligonucleosides,
methylenecarbonylamino linked oligonucleosides, also identified as
amide-3 linked oligonucleosides, and methyleneaminocarbonyl linked
oligonucleosides, also identified as amide-4 linked
oligonucleosides, as well as mixed backbone oligomeric compounds
having, for instance, alternating MMI and P.dbd.O or P.dbd.S
linkages can be prepared as described in U.S. Pat. Nos. 5,378,825,
5,386,023, 5,489,677, 5,602,240 and 5,610,289.
[0364] Formacetal and thioformacetal internucleoside linkages can
be prepared as described in U.S. Pat. Nos. 5,264,562 and
5,264,564.
[0365] Ethylene oxide internucleoside linkages can be prepared as
described in U.S. Pat. No. 5,223,618.
Example 3
Isolation and Purification of Oligomeric Compounds
[0366] After cleavage from the controlled pore glass solid support
or other support medium and deblocking in concentrated ammonium
hydroxide at 55.degree. C. for 12-16 hours, the oligomeric
compounds, including without limitation oligonucleotides and
oligonucleosides, are recovered by precipitation out of 1 M
NH.sub.4OAc with >3 volumes of ethanol. Synthesized oligomeric
compounds are analyzed by electrospray mass spectroscopy (molecular
weight determination) and by capillary gel electrophoresis. The
relative amounts of phosphorothioate and phosphodiester linkages
obtained in the synthesis is determined by the ratio of correct
molecular weight relative to the -16 amu product (+/-32 +/-48). For
some studies oligomeric compounds are purified by HPLC, as
described by Chiang et al., J. Biol. Chem. 1991, 266, 18162-18171.
Results obtained with HPLC-purified material are generally similar
to those obtained with non-HPLC purified material.
Example 4
Synthesis of Oligomeric Compounds Using the 96 Well Plate
Format
[0367] Oligomeric compounds, including without limitation
oligonucleotides, can be synthesized via solid phase P(III)
phosphoramidite chemistry on an automated synthesizer capable of
assembling 96 sequences simultaneously in a 96-well format.
Phosphodiester internucleoside linkages are afforded by oxidation
with aqueous iodine. Phosphorothioate internucleoside linkages are
generated by sulfurization utilizing 3,H-1,2 benzodithiole-3-one
1,1 dioxide (Beaucage Reagent) in anhydrous acetonitrile. Standard
base-protected beta-cyanoethyl-diiso-propyl phosphoramidites can be
purchased from commercial vendors (e.g. PE-Applied Biosystems,
Foster City, Calif., or Pharmacia, Piscataway, N.J.). Non-standard
nucleosides are synthesized as per standard or patented methods and
can be functionalized as base protected beta-cyanoethyldiisopropyl
phosphoramidites.
[0368] Oligomeric compounds can be cleaved from support and
deprotected with concentrated NH.sub.4OH at elevated temperature
(55-60.degree. C.) for 12-16 hours and the released product then
dried in vacuo. The dried product is then re-suspended in sterile
water to afford a master plate from which all analytical and test
plate samples are then diluted utilizing robotic pipettors.
Example 5
Analysis of Oligomeric Compounds Using the 96-Well Plate Format
[0369] The concentration of oligomeric compounds in each well can
be assessed by dilution of samples and UV absorption spectroscopy.
The full-length integrity of the individual products can be
evaluated by capillary electrophoresis (CE) in either the 96-well
format (Beckman P/ACE.TM. MDQ) or, for individually prepared
samples, on a commercial CE apparatus (e.g., Beckman P/ACE.TM.
5000, ABI 270). Base and backbone composition is confirmed by mass
analysis of the oligomeric compounds utilizing electrospray-mass
spectroscopy. All assay test plates are diluted from the master
plate using single and multi-channel robotic pipettors. Plates are
judged to be acceptable if at least 85% of the oligomeric compounds
on the plate are at least 85% full length.
Example 6
[0370] In Vitro Treatment of Cells with Oligomeric Compounds
[0371] The effect of oligomeric compounds on target nucleic acid
expression is tested in any of a variety of cell types provided
that the target nucleic acid is present at measurable levels. This
can be routinely determined using, for example, PCR or Northern
blot analysis. Cell lines derived from multiple tissues and species
can be obtained from American Type Culture Collection (ATCC,
Manassas, Va.).
[0372] The following cell type is provided for illustrative
purposes, but other cell types can be routinely used, provided that
the target is expressed in the cell type chosen. This can be
readily determined by methods routine in the art, for example
Northern blot analysis, ribonuclease protection assays or
RT-PCR.
[0373] b.END cells: The mouse brain endothelial cell line b.END was
obtained from Dr. Werner Risau at the Max Plank Institute (Bad
Nauheim, Germany). b.END cells are routinely cultured in DMEM, high
glucose (Invitrogen Life Technologies, Carlsbad, Calif.)
supplemented with 10% fetal bovine serum (Invitrogen Life
Technologies, Carlsbad, Calif.). Cells are routinely passaged by
trypsinization and dilution when they reached approximately 90%
confluence. Cells are seeded into 96-well plates (Falcon-Primaria
#353872, BD Biosciences, Bedford, Mass.) at a density of
approximately 3000 cells/well for uses including but not limited to
oligomeric compound transfection experiments.
[0374] Experiments involving treatment of cells with oligomeric
compounds:
[0375] When cells reach appropriate confluency, they are treated
with oligomeric compounds using a transfection method as
described.
[0376] LIPOFECTIN.TM.
[0377] When cells reached 65-75% confluency, they are treated with
one or more oligomeric compounds. The oligomeric compound is mixed
with LIPOFECTIN.TM. Invitrogen Life Technologies, Carlsbad, Calif.)
in Opti-MEM.TM.-1 reduced serum medium (Invitrogen Life
Technologies, Carlsbad, Calif.) to achieve the desired
concentration of the oligomeric compound(s) and a LIPOFECTIN.TM.
concentration of 2.5 or 3 .mu.g/mL per 100 nM oligomeric
compound(s). This transfection mixture is incubated at room
temperature for approximately 0.5 hours. For cells grown in 96-well
plates, wells are washed once with 100 .mu.L OPTI-MEM.TM.-1 and
then treated with 130 .mu.L of the transfection mixture. Cells
grown in 24-well plates or other standard tissue culture plates are
treated similarly, using appropriate volumes of medium and
oligomeric compound(s). Cells are treated and data are obtained in
duplicate or triplicate. After approximately 4-7 hours of treatment
at 37.degree. C., the medium containing the transfection mixture is
replaced with fresh culture medium. Cells are harvested 16-24 hours
after treatment with oligomeric compound(s).
[0378] Other suitable transfection reagents known in the art
include, but are not limited to, CYTOFECTIN.TM., LIPOFECTAMINE.TM.,
OLIGOFECTAMINE.TM., and FUGENE.TM.. Other suitable transfection
methods known in the art include, but are not limited to,
electroporation.
Example 7
[0379] Real-Time Quantitative PCR Analysis of Target mRNA
Levels
[0380] Quantitation of target mRNA levels is accomplished by
real-time quantitative PCR using the ABI PRISM.TM. 7600, 7700, or
7900 Sequence Detection System (PE-Applied Biosystems, Foster City,
Calif.) according to manufacturer's instructions. This is a
closed-tube, non-gel-based, fluorescence detection system which
allows high-throughput quantitation of polymerase chain reaction
(PCR) products in real-time. As opposed to standard PCR in which
amplification products are quantitated after the PCR is completed,
products in real-time quantitative PCR are quantitated as they
accumulate. This is accomplished by including in the PCR reaction
an oligonucleotide probe that anneals specifically between the
forward and reverse PCR primers, and contains two fluorescent dyes.
A reporter dye (e.g., FAM or JOE, obtained from either PE-Applied
Biosystems, Foster City, Calif., Operon Technologies Inc., Alameda,
Calif. or Integrated DNA Technologies Inc., Coralville, Iowa) is
attached to the 5' end of the probe and a quencher dye (e.g.,
TAMRA, obtained from either PE-Applied Biosystems, Foster City,
Calif., Operon Technologies Inc., Alameda, Calif. or Integrated DNA
Technologies Inc., Coralville, Iowa) is attached to the 3' end of
the probe. When the probe and dyes are intact, reporter dye
emission is quenched by the proximity of the 3' quencher dye.
During amplification, annealing of the probe to the target sequence
creates a substrate that can be cleaved by the 5'-exonuclease
activity of Taq polymerase. During the extension phase of the PCR
amplification cycle, cleavage of the probe by Taq polymerase
releases the reporter dye from the remainder of the probe (and
hence from the quencher moiety) and a sequence-specific fluorescent
signal is generated. With each cycle, additional reporter dye
molecules are cleaved from their respective probes, and the
fluorescence intensity is monitored at regular intervals by laser
optics built into the ABI PRISM.TM. Sequence Detection System. In
each assay, a series of parallel reactions containing serial
dilutions of mRNA from untreated control samples generates a
standard curve that is used to quantitate the percent inhibition
after antisense oligonucleotide treatment of test samples.
[0381] Prior to quantitative PCR analysis, primer-probe sets
specific to the target gene being measured are evaluated for their
ability to be "multiplexed" with a GAPDH amplification reaction. In
multiplexing, both the target gene and the internal standard gene
GAPDH are amplified concurrently in a single sample. In this
analysis, mRNA isolated from untreated cells is serially diluted.
Each dilution is amplified in the presence of primer-probe sets
specific for GAPDH only, target gene only ("single-plexing"), or
both (multiplexing). Following PCR amplification, standard curves
of GAPDH and target mRNA signal as a function of dilution are
generated from both the single-plexed and multiplexed samples. If
both the slope and correlation coefficient of the GAPDH and target
signals generated from the multiplexed samples fall within 10% of
their corresponding values generated from the single-plexed
samples, the primer-probe set specific for that target is deemed
multiplexable. Other methods of PCR are also known in the art.
[0382] RT and PCR reagents are obtained from Invitrogen Life
Technologies (Carlsbad, Calif.). RT, real-time PCR is carried out
by adding 20 .mu.L PCR cocktail (2.5.times.PCR buffer minus
MgCl.sub.2, 6.6 mM MgCl.sub.2, 375 .mu.M each of dATP, dCTP, dCTP
and dGTP, 375 nM each of forward primer and reverse primer, 125 nM
of probe, 4 Units RNAse inhibitor, 1.25 Units PLATINUM.RTM. Taq, 5
Units MuLV reverse transcriptase, and 2.5.times.ROX dye) to 96-well
plates containing 30 .mu.L total RNA solution (20-200 ng). The RT
reaction is carried out by incubation for 30 minutes at 48.degree.
C. Following a 10 minute incubation at 95.degree. C. to activate
the PLATINUM.RTM. Taq, 40 cycles of a two-step PCR protocol are
carried out: 95.degree. C. for 15 seconds (denaturation) followed
by 60.degree. C. for 1.5 minutes (annealing/-extension).
[0383] Gene target quantities obtained by RT, real-time PCR are
normalized using either the expression level of GAPDH, a gene whose
expression is constant, or by quantifying total RNA using
RIBOGREEN.TM. (Molecular Probes, Inc. Eugene, Oreg.). GAPDH
expression is quantified by real time RT-PCR, by being run
simultaneously with the target, multiplexing, or separately. Total
RNA is quantified using RiboGreen.TM. RNA quantification reagent
(Molecular Probes, Inc. Eugene, Oreg.). Methods of RNA
quantification by RIBOGREEN.TM. are taught in Jones, L. J., et al,
(Analytical Biochemistry, 1998, 265, 368-374).
[0384] In this assay, 170 .mu.L of RIBOGREEN.TM. working reagent
(RIBOGREEN.TM. reagent diluted 1:350 in 10 mM Tris-HCl, 1 mM EDTA,
pH 7.5) is pipetted into a 96-well plate containing 30 .mu.L
purified, cellular RNA. The plate is read in a CytoFluor 4000 (PE
Applied Biosystems) with excitation at 485 nm and emission at 530
nm.
Example 8
Analysis of Inhibition of Target Expression
[0385] Antisense modulation of a target expression can be assayed
in a variety of ways known in the art. For example, a target mRNA
levels can be quantitated by, e.g., Northern blot analysis,
competitive polymerase chain reaction (PCR), or real-time PCR.
Real-time quantitative PCR is presently desired. RNA analysis can
be performed on total cellular RNA or poly(A)+ mRNA. One method of
RNA analysis of the present disclosure is the use of total cellular
RNA as described in other examples herein. Methods of RNA isolation
are well known in the art. Northern blot analysis is also routine
in the art. Real-time quantitative (PCR) can be conveniently
accomplished using the commercially available ABI PRISM.TM. 7600,
7700, or 7900 Sequence Detection System, available from PE-Applied
Biosystems, Foster City, Calif. and used according to
manufacturer's instructions.
[0386] Protein levels of a target can be quantitated in a variety
of ways well known in the art, such as immunoprecipitation, Western
blot analysis (immunoblotting), enzyme-linked immunosorbent assay
(ELISA) or fluorescence-activated cell sorting (FACS). Antibodies
directed to a target can be identified and obtained from a variety
of sources, such as the MSRS catalog of antibodies (Aerie
Corporation, Birmingham, Mich.), or can be prepared via
conventional monoclonal or polyclonal antibody generation methods
well known in the art. Methods for preparation of polyclonal
antisera are taught in, for example, Ausubel, F. M. et al., Current
Protocols in Molecular Biology, Volume 2, pp. 11.12.1-11.12.9, John
Wiley & Sons, Inc., 1997. Preparation of monoclonal antibodies
is taught in, for example, Ausubel, F. M. et al., Current Protocols
in Molecular Biology, Volume 2, pp. 11.4.1-11.11.5, John Wiley
& Sons, Inc., 1997.
[0387] Immunoprecipitation methods are standard in the art and can
be found at, for example, Ausubel, F. M. et al., Current Protocols
in Molecular Biology, Volume 2, pp. 10.16.1-10.16.11, John Wiley
& Sons, Inc., 1998. Western blot (immunoblot) analysis is
standard in the art and can be found at, for example, Ausubel, F.
M. et al., Current Protocols in Molecular Biology, Volume 2, pp.
10.8.1-10.8.21, John Wiley & Sons, Inc., 1997. Enzyme-linked
immunosorbent assays (ELISA) are standard in the art and can be
found at, for example, Ausubel, F. M. et al., Current Protocols in
Molecular Biology, Volume 2, pp. 11.2.1-11.2.22, John Wiley &
Sons, Inc., 1991.
Example 9
Design of Phenotypic Assays and In Vivo Studies for the Use of
Target Inhibitors
Phenotypic Assays
[0388] Once target inhibitors have been identified by the methods
disclosed herein, the oligomeric compounds are further investigated
in one or more phenotypic assays, each having measurable endpoints
predictive of efficacy in the treatment of a particular disease
state or condition.
[0389] Phenotypic assays, kits and reagents for their use are well
known to those skilled in the art and are herein used to
investigate the role and/or association of a target in health and
disease. Representative phenotypic assays, which can be purchased
from any one of several commercial vendors, include those for
determining cell viability, cytotoxicity, proliferation or cell
survival (Molecular Probes, Eugene, Oreg.; PerkinElmer, Boston,
Mass.), protein-based assays including enzymatic assays (Panvera,
LLC, Madison, Wis.; BD Biosciences, Franklin Lakes, N.J.; Oncogene
Research Products, San Diego, Calif.), cell regulation, signal
transduction, inflammation, oxidative processes and apoptosis
(Assay Designs Inc., Ann Arbor, Mich.), triglyceride accumulation
(Sigma-Aldrich, St. Louis, Mo.), angiogenesis assays, tube
formation assays, cytokine and hormone assays and metabolic assays
(Chemicon International Inc., Temecula, Calif.; Amersham
Biosciences, Piscataway, N.J.).
[0390] In one non-limiting example, cells determined to be
appropriate for a particular phenotypic assay (i.e., MCF-7 cells
selected for breast cancer studies; adipocytes for obesity studies)
are treated with a target inhibitors identified from the in vitro
studies as well as control compounds at optimal concentrations
which are determined by the methods described above. At the end of
the treatment period, treated and untreated cells are analyzed by
one or more methods specific for the assay to determine phenotypic
outcomes and endpoints.
[0391] Phenotypic endpoints include changes in cell morphology over
time or treatment dose as well as changes in levels of cellular
components such as proteins, lipids, nucleic acids, hormones,
saccharides or metals. Measurements of cellular status which
include pH, stage of the cell cycle, intake or excretion of
biological indicators by the cell, are also endpoints of
interest.
[0392] Measurement of the expression of one or more of the genes of
the cell after treatment is also used as an indicator of the
efficacy or potency of the target inhibitors. Hallmark genes, or
those genes suspected to be associated with a specific disease
state, condition, or phenotype, are measured in both treated and
untreated cells.
In Vivo Studies
[0393] The individual subjects of the in vivo studies described
herein are warm-blooded vertebrate animals, which includes
humans.
Example 10
RNA Isolation
[0394] Poly(A)+ mRNA Isolation
[0395] Poly(A)+ mRNA is isolated according to Miura et al., (Clin.
Chem., 1996, 42, 1758-1764). Other methods for poly(A)+ mRNA
isolation are routine in the art. Briefly, for cells grown on
96-well plates, growth medium is removed from the cells and each
well is washed with 200 .mu.L cold PBS. 60 .mu.L lysis buffer (10
mM Tris-HCl, pH 7.6, 1 mM EDTA, 0.5 M NaCl, 0.5% NP-40, 20 mM
vanadyl-ribonucleoside complex) is added to each well, the plate is
gently agitated and then incubated at room temperature for five
minutes. 55 .mu.L of lysate is transferred to Oligo d(T) coated
96-well plates (AGCT Inc., Irvine Calif.). Plates are incubated for
60 minutes at room temperature, washed 3 times with 200 .mu.L of
wash buffer (10 mM Tris-HCl pH 7.6, 1 mM EDTA, 0.3 M NaCl). After
the final wash, the plate is blotted on paper towels to remove
excess wash buffer and then air-dried for 5 minutes. 60 .mu.L of
elution buffer (5 mM Tris-HCl pH 7.6), preheated to 70.degree. C.,
is added to each well, the plate is incubated on a 90.degree. C.
hot plate for 5 minutes, and the eluate is then transferred to a
fresh 96-well plate.
[0396] Cells grown on 100 mm or other standard plates may be
treated similarly, using appropriate volumes of all solutions.
Total RNA Isolation
[0397] Total RNA is isolated using an RNEASY 96.TM. kit and buffers
purchased from Qiagen Inc. (Valencia, Calif.) following the
manufacturer's recommended procedures. Briefly, for cells grown on
96-well plates, growth medium is removed from the cells and each
well is washed with 200 .mu.L cold PBS. 150 .mu.L Buffer RLT is
added to each well and the plate vigorously agitated for 20
seconds. 150 .mu.L of 70% ethanol is then added to each well and
the contents mixed by pipetting three times up and down. The
samples are then transferred to the RNEASY 96.TM. well plate
attached to a QIAVAC.TM. manifold fitted with a waste collection
tray and attached to a vacuum source. Vacuum is applied for 1
minute. 500 .mu.L of Buffer RW1 is added to each well of the RNEASY
96.TM. plate and incubated for 15 minutes and the vacuum is again
applied for 1 minute. An additional 500 .mu.L of Buffer RW1 is
added to each well of the RNEASY 96.TM. plate and the vacuum is
applied for 2 minutes. 1 mL of Buffer RPE is then added to each
well of the RNEASY 96.TM. plate and the vacuum applied for a period
of 90 seconds. The Buffer RPE wash is then repeated and the vacuum
is applied for an additional 3 minutes. The plate is then removed
from the QIAVAC.TM. manifold and blotted dry on paper towels. The
plate is then re-attached to the QIAVAC.TM. manifold fitted with a
collection tube rack containing 1.2 mL collection tubes. RNA is
then eluted by pipetting 140 .mu.L of RNAse free water into each
well, incubating 1 minute, and then applying the vacuum for 3
minutes.
[0398] The repetitive pipetting and elution steps may be automated
using a QIAGEN Bio-Robot 9604 (Qiagen, Inc., Valencia Calif.).
Essentially, after lysing of the cells on the culture plate, the
plate is transferred to the robot deck where the pipetting, DNase
treatment and elution steps are carried out.
Example 11
Target-Specific Primers and Probes
[0399] Probes and primers may be designed to hybridize to a target
sequence, using published sequence information.
[0400] For example, for human PTEN, the following primer-probe set
was designed using published sequence information (GENBANK.TM.
accession number U92436.1, SEQ ID NO: 1).
TABLE-US-00005 Forward primer: (SEQ ID NO: 2)
AATGGCTAAGTGAAGATGACAATCAT Reverse primer: (SEQ ID NO: 3)
TGCACATATCATTACACCAGTTCGT
And the PCR probe:
TABLE-US-00006 (SEQ ID NO: 4)
FAM-TTGCAGCAATTCACTGTAAAGCTGGAAAGG-TAMRA,
where FAM is the fluorescent dye and TAMRA is the quencher dye.
Example 12
Western Blot Analysis of Target Protein Levels
[0401] Western blot analysis (immunoblot analysis) is carried out
using standard methods. Cells are harvested 16-20 h after
oligonucleotide treatment, washed once with PBS, suspended in
Laemmli buffer (100 .mu.l/well), boiled for 5 minutes and loaded on
a 16% SDS-PAGE gel. Gels are run for 1.5 hours at 150 V, and
transferred to membrane for western blotting. Appropriate primary
antibody directed to a target is used, with a radiolabeled or
fluorescently labeled secondary antibody directed against the
primary antibody species. Bands are visualized using a
PHOSPHORIMAGER.TM. (Molecular Dynamics, Sunnyvale Calif.).
Example 13
General Method for the Preparation of Phosphoramidites, Compounds
1-4
##STR00010##
[0403] Bx is a heterocyclic base;
[0404] R.sub.1 and R.sub.2 are each independently H, OH, or a
2'-sugar substituent group
[0405] Compounds 1 and 2 are commercially available from Glen
Research. Compounds 3 and 4 are prepared as per the procedures well
known in the art as described in the specification herein (see Seth
et al., Bioorg. Med. Chem., 2011, 21(4), 1122-1125, J. Org. Chem.,
2010, 75(5), 1569-1581, Nucleic Acids Symposium Series, 2008,
52(1), 553-554); and also see published PCT International
Applications (WO 2011/115818, WO 2010/077578, WO2010/036698,
WO2009/143369, WO 2009/006478, and WO 2007/090071), and U.S. Pat.
No. 7,569,686).
Example 14
Preparation of Compound 10
##STR00011##
[0407] Bis(diisopropylamino) chlorophosphine, Compound 5 and
DMT-protected deoxyribonucleoside, Compound 9 are commercially
available from Sigma-Aldrich, and BOC Biosciences.
[0408] Compounds 7 and 10 are prepared using similar procedures as
published in the literature (Yamada et al., J. Am. Chem. Soc.,
2006, 128(15), 5251-5261).
Example 15
Preparation of Compound 13
##STR00012##
[0410] R.sub.3 is --CH.sub.2CH.sub.3, --CH.dbd.CH.sub.2,
--CH.sub.2CH.dbd.CH.sub.2, --CH.sub.2CH.sub.2OCH.sub.3,
--CH.sub.2CH.sub.2OPG or --CH.sub.2CH.sub.2OCH.sub.2F;
[0411] PG is a protecting group;
[0412] Bx is a heterocyclic base
[0413] Bis(diisopropylamino) chlorophosphine, Compound 5 and
DTM-protected deoxyribonucleoside, Compound 9 are commercially
available from Sigma-Aldrich, and BOC Biosciences. The Grignard
reagent, Compound 11 is commercially available from various sources
or can be prepared using similar procedures as published in the
literature (Oppolzer et al., Tet. Let., 1982, 23(38),
3901-3904).
Example 16
General Method for the Preparation of Compound 16
##STR00013##
[0414] Bx is a heterocyclic base; R.sub.4 is alkyl, substituted
alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl,
acyl or substituted acyl; wherein the substituent groups are
independently selected from halogen, oxo, hydroxyl, amino, thio,
azido, cyano or carboxyl;
[0415] Bis(diisopropylamino) chlorophosphine, Compound 5 and
DTM-protected deoxyribonucleoside, Compound 9 are commercially
available from Sigma-Aldrich, and BOC Biosciences. The Grignard
reagent, Compound 14 is commercially available from various sources
or can be prepared as per the procedures illustrated in Example
15.
[0416] Compound 15 used in the phosphitylation step serves only to
illustrate the compounds described herein and is not intended to be
limiting. Additional phosphitylating reagents known in the art as
described in the specification herein can also be employed to
generate various phosphoramidite analogs of Compound 16 and are
used as building blocks for oligonucleotide synthesis.
Example 17
Preparation of Dimeric Phosphoramidites, Compounds 20 and 21
##STR00014##
[0418] Compounds 1 and 17 are commercially available from Glen
Research and Chemexpress. Compounds 18 and 19 were separated by
column chromatography. Although one of the methylphosphonate
internucleoside linkages in DMT phosphoramidite dimer 20 or 21 is
Rp and the other is Sp, the absolute stereochemistry at the
phosphorus chiral center (P*) of the dimers has not been
determined. The spectral analysis of Compounds 20 and 21 was
consistent with the structures.
Example 18
General Method for the Preparation of Oligomeric Compound 25
##STR00015##
[0420] The Unylinker.TM. 22 is commercially available. Oligomeric
Compound 25 comprising a modified internucleoside linkage (e.g.
methyl phosphonate, phosphonoacetate or phosphonoformate) is
prepared using standard procedures in automated DNA/RNA synthesis
(see Dupouy et al., Angew. Chem. Int. Ed., 2006, 45, 3623-3627).
Phosphoramidite building blocks, Compounds 1, 2, 3, 10 and 13 are
prepared as per the procedures illustrated in Examples 13 to 15.
The synthetic steps illustrated are meant to be representative and
not intended to be limiting as other phosphoramidite building
blocks which are disclosed in Examples 13 to 16a can be used in
place of Compound 1, 2, 3, 10 or 13 to prepare an oligomeric
compound having a predetermined sequence and composition. The order
and quantity of phosphoramidites added to the solid support can be
adjusted to prepare gapped oligomeric compounds as described
herein. Such gapped oligomeric compounds can have predetermined
composition and base sequence as dictated by any given target.
Example 19
General Method for the Preparation of Oligomeric Compounds
Comprising a Modified Phosphorus Containing Internucleoside Linkage
Via Solid Phase Techniques (Preparation of 460209, 558255, 573815,
560221, A01 and A02)
[0421] Unless otherwise stated, all reagents and solutions used for
the synthesis of oligomeric compounds are purchased from commercial
sources. Standard phosphoramidite building blocks and solid support
are used for incorporation nucleoside residues which include for
example T, A, U, G, C and .sup.mC residues. A 0.1 M solution of
phosphoramidite in anhydrous acetonitrile was used for
.beta.-D-2'-deoxyribonucleoside, methyl phosphonate and
phosphonoacetate. For (R)- or (S)-methyl phosphonate dimeric
phosphoramidite, a 0.1 M solution in acetonitrile was used. For
2'-O-MOE phosphoramidite, a 0.2 M solution in acetonitrile was
used. For constrained ethyl (cEt) BNA phosphoramidite, a 0.2 M
solution in a 1:1 (v/v) mixture of acetonitrile and toluene was
used.
[0422] The oligomeric compound was synthesized on VIMAD
UnyLinker.TM. solid support and the appropriate amounts of solid
support were packed in the column for synthesis. Dichloroacetic
acid (3%) in DCM was used as detritylating reagent.
4,5-Dicyanoimidazole in the presence of N-methyl-imidazole or
1H-tetrazole in CH.sub.3CN was used as activator during the
coupling step. The synthesis of oligomeric compounds was performed
on an AB1394 synthesizer (Applied Biosystems) on a 2 .mu.mol scale
using the procedures set forth below.
[0423] A solid support preloaded with the Unylinker.TM. was loaded
into a synthesis column after closing the column bottom outlet and
CH.sub.3CN was added to form a slurry. The swelled support-bound
Unylinker.TM. was treated with a detritylating reagent containing
3% dichloroacetic acid in DCM to provide the free hydroxyl groups.
During the coupling step, four to fourteen equivalents of
phosphoramidite solutions were delivered with coupling for 6
minutes for unmodified deoxyribonucleoside phosphoramidites and 13
minutes for other modifications. All of the other steps followed
standard protocols. Phosphodiester linkages were introduced by
oxidation with 10% t-BuOOH solution in CH.sub.3CN for a contact
time of 10 minutes. Phosphorothioate linkages were introduced by
sulfurization with PADS (0.2 M) in 1:1 pyridine/CH.sub.3CN for a
contact time of 5 minutes.
[0424] After the desired sequence was assembled, the cyanoethyl
phosphate protecting groups were deprotected using a 1:1 (v/v)
mixture of triethylamine and acetonitrile. For phosphonoacetate
containing oligomeric compound, 1.5% DBU solution in CH.sub.3CN was
used. The solid support bound oligomeric compound was washed with
acetonitrile and dried under high vacuum. The solid-support bound
oligomeric compound was then suspended in ammonia (28-30 wt %) at
room temperature for 48 h to remove nucleobase protecting groups
and to cleave from the solid support.
[0425] The unbound oligomeric compound was then filtered and the
support was rinsed and filtered with water:ethanol (1:1) followed
by water. The filtrate was combined and concentrated to dryness.
The residue obtained was purified by cationic ion exchange HPLC
(Source 30Q resin, A--50 mM sodium bicarbonate in
CH.sub.3CN:H.sub.2O 3:7 (v/v), B--50 mM sodium bicarbonate, 1.5 M
sodium bromide in CH.sub.3CN:H.sub.2O 3:7 (v/v), 0-30% in 110 min,
flow 6 mL/min, .lamda.=260 nm). Fractions containing full-length
oligomeric compound were pooled together (assessed by LC/MS
analysis>95%). The residue was desalted by HPLC on a reverse
phase cartridge to yield the desired oligomeric compound.
TABLE-US-00007 SEQ ID NO./ ISIS NO. Composition (5' to 3') Gap
Chemistry (motif) 05/460209
T.sub.eA.sub.kA.sub.kA.sub.dT.sub.dT.sub.dG.sub.dT.sub.d.sup.mC.-
sub.dA.sub.dT.sub.d.sup.mC.sub.dA.sub.k.sup.mC.sub.k.sup.mC.sub.e
Positive control (3/9/3) 05/558255
T.sub.eA.sub.kA.sub.kA.sub.dxT.sub.dT.sub.dG.sub.dT.sub.d.sup.mC-
.sub.dA.sub.dT.sub.d.sup.mC.sub.dA.sub.k.sup.mC.sub.k.sup.mC.sub.e
Single -P(CH.sub.3)(=O)- (3/9/3) 05/573815
T.sub.eA.sub.kA.sub.kA.sub.dT.sub.dyT.sub.dG.sub.dT.sub.d.sup.mC-
.sub.dA.sub.dT.sub.d.sup.mC.sub.dA.sub.k.sup.mC.sub.k.sup.mC.sub.e
Single -P(CH.sub.2CO.sub.2.sup.-)(-O)- (3/9/3) 06/560221
T.sub.e.sup.mC.sub.e.sup.mC.sub.eC.sub.dwA.sub.dT.sub.dwT.sub.dT-
.sub.dw.sup.mC.sub.dA.sub.dwG.sub.dG.sub.dwA.sub.d Alternating
-P(CH.sub.3)(=S)-
G.sub.dwA.sub.dC.sub.dw.sup.mC.sub.dT.sub.eG.sub.eG.sub.e different
motif (3/14/3) 05/A01
T.sub.eA.sub.kA.sub.kA.sub.dT.sub.dzT.sub.dG.sub.dT.sub.d.sup.mC.su-
b.dA.sub.dT.sub.d.sup.mC.sub.dA.sub.k.sup.mC.sub.k.sup.mC.sub.e
Single -P((R)-CH.sub.3)(-O)- (3/9/3) 05/A02
T.sub.eA.sub.kA.sub.kA.sub.dT.sub.dqT.sub.dG.sub.dT.sub.d.sup.mC.su-
b.dA.sub.dT.sub.d.sup.mC.sub.dA.sub.k.sup.mC.sub.k.sup.mC.sub.e
Single -P((S)-CH.sub.3)(-O)- (3/9/3)
[0426] Each internucleoside linkage is a phosphorothioate (P.dbd.S)
except for the internucleoside linkage having a subscript "x", "y",
"w", "z" or "q". Each nucleoside followed by a subscript "x"
indicates a methyl phosphonate internucleoside linkage
(--P(CH.sub.3)(.dbd.O)--). Each nucleoside followed by a subscript
"y" indicates a phosphonoacetate internucleoside linkage
(--P(CH.sub.2CO.sub.2.sup.-)(.dbd.O)--). Each nucleoside followed
by a subscript "w" indicates a methyl thiophosphonate
internucleoside linkage (--P(CH.sub.3)(.dbd.S)--). Each nucleoside
followed by a subscript "z" indicates an (R)-methyl phosphonate
internucleoside linkage (--P--(R)--CH.sub.3)(.dbd.O)--). Each
nucleoside followed by a subscript "q" indicates an (S)-methyl
phosphonate internucleoside linkage
(--P--(S)--CH.sub.3)(.dbd.O)--). Each nucleoside followed by a
subscript "d" is a .beta.-D-2'-deoxyribonucleoside. Each nucleoside
followed by a subscript "e" indicates a 2'-O-methoxyethyl (MOE)
modified nucleoside. Each nucleoside followed by a subscript "k"
indicates a bicyclic nucleoside having a 4'-CH((S)--CH.sub.3)--O-2'
bridge also referred to as a (S)-cEt modified nucleoside. Each
".sup.mC" is a 5-methyl cytosine modified nucleoside. Nucleosides
followed by subscripts "e", or "k" are further illustrated
below.
##STR00016##
Example 20
[0427] Human Peripheral Blood Mononuclear Cells (hPBMC) Assay
Protocol
[0428] The hPBMC assay was performed using BD Vautainer CPT tube
method. A sample of whole blood from volunteered donors with
informed consent at US HealthWorks clinic (Faraday & El Camino
Real, Carlsbad) was obtained and collected in 4-15 BD Vacutainer
CPT 8 ml tubes (VWR Cat.# BD362753). The approximate starting total
whole blood volume in the CPT tubes for each donor was recorded
using the PBMC assay data sheet.
[0429] The blood sample was remixed immediately prior to
centrifugation by gently inverting tubes 8-10 times. CPT tubes were
centrifuged at rt (18-25.degree. C.) in a horizontal (swing-out)
rotor for 30 min. at 1500-1800 RCF with brake off (2700 RPM Beckman
Allegra 6R). The cells were retrieved from the buffy coat interface
(between Ficoll and polymer gel layers); transferred to a sterile
50 ml conical tube and pooled up to 5 CPT tubes/50 ml conical
tube/donor. The cells were then washed twice with PBS (Ca.sup.++,
Mg.sup.++ free; GIBCO). The tubes were topped up to 50 ml and mixed
by inverting several times. The sample was then centrifuged at
330.times.g for 15 minutes at rt (1215 RPM in Beckman Allegra 6R)
and aspirated as much supernatant as possible without disturbing
pellet. The cell pellet was dislodged by gently swirling tube and
resuspended cells in RPMI+10% FBS+pen/strep (.about.1 ml/10 ml
starting whole blood volume). A 60 .mu.l sample was pipette into a
sample vial (Beckman Coulter) with 600 .mu.l VersaLyse reagent
(Beckman Coulter Cat# A09777) and was gently vortexed for 10-15
sec. The sample was allowed to incubate for 10 min. at rt and being
mixed again before counting. The cell suspension was counted on
Vicell XR cell viability analyzer (Beckman Coulter) using PBMC cell
type (dilution factor of 1:11 was stored with other parameters).
The live cell/ml and viability were recorded. The cell suspension
was diluted to 1.times.10.sup.7 live PBMC/ml in RPMI+10%
FBS+pen/strep.
[0430] The cells were plated at 5.times.10.sup.5 in 50 .mu.l/well
of 96-well tissue culture plate (Falcon Microtest). 50 .mu.l/well
of 2.times. concentration oligos/controls diluted in RPMI+10%
FBS+pen/strep. was added according to experiment template (100
.mu.l/well total). Plates were placed on the shaker and allowed to
mix for approx. 1 min. After being incubated for 24 hrs at
37.degree. C.; 5% CO.sub.2, the plates were centrifuged at
400.times.g for 10 minutes before removing the supernatant for MSD
cytokine assay (i.e. human IL-6, IL-10, IL-8 and MCP-1).
Example 21
[0431] Evaluation of the Proinflammatory Effects in hPBMC Assay for
a Modified Oligonucleotide Comprising Methyl Thiophosphonate
Internucleoside Linkages--In Vitro Assay
[0432] A modified oligonucleotide was designed based on the 3/14/3
MOE gapmer, ISIS 353512. This modified oligonucleotide was created
by having alternating methyl thiophosphonate
(--P(CH.sub.3)(.dbd.S)--) internucleoside linkages throughout the
gap region. The proinflammatory effect of the modified
oligonucleotide targeting hCRP was evaluated in hPBMC assay using
the protocol described in Example 20.
[0433] The hPBMCs were isolated from fresh, volunteered donors and
were treated with modified oligonucleotides at 0, 0.0128, 0.064,
0.32, 1.6, 8, 40 and 200 .mu.M concentrations. After a 24 hr
treatment, the cytokine levels were measured.
[0434] The levels of IL-6 were used as the primary readout and
compared to the positive control, oligonucleotide, ISIS 353512 and
negative control, ISIS 104838. The results from two donors denoted
as "Donor 1" and "Donor 2" are presented below.
TABLE-US-00008 SEQ ID NO./ ISIS NO. Composition (5' to 3')
06/353512
T.sub.e.sup.mC.sub.e.sup.mC.sub.e.sup.mC.sub.dA.sub.dT.sub.dT.s-
ub.dT.sub.d.sup.mC.sub.dA.sub.dG.sub.dG.sub.dA.sub.dG.sub.dA.sub.d.sup.m
C.sub.d.sup.mC.sub.dT.sub.eG.sub.eG.sub.e 06/560221
T.sub.e.sup.mC.sub.e.sup.mC.sub.eC.sub.dwA.sub.dT.sub.dwT.sub.d-
T.sub.dw.sup.mC.sub.dA.sub.dwG.sub.dG.sub.dwA.sub.d
G.sub.dwA.sub.dC.sub.dw.sup.mC.sub.dT.sub.eG.sub.eG.sub.e 07/104838
G.sub.e.sup.mC.sub.eT.sub.eG.sub.eA.sub.eT.sub.dT.sub.dA.sub.dG-
.sub.dA.sub.dG.sub.dA.sub.dG.sub.dA.sub.dG.sub.dG.sub.e
T.sub.e.sup.mC.sub.e.sup.mC.sub.e.sup.mC.sub.e
[0435] Each internucleoside linkage is a phosphorothioate (P.dbd.S)
except for nucleosides followed by a subscript "w". Each nucleoside
followed by a subscript "w" indicates a methyl thiophosphonate
internucleoside linkage (--P(CH.sub.3)(.dbd.S)--). Each nucleoside
followed by a subscript "d" is a .beta.-D-2'-deoxyribonucleoside.
Each nucleoside followed by a subscript "e" indicates a
2'-O-methoxyethyl (MOE) modified nucleoside. Each ".sup.mC" is a
5-methyl cytosine modified nucleoside. Nucleosides followed by
subscripts "e" are further illustrated below.
##STR00017##
TABLE-US-00009 SEQ ID NO. IL-6 Chemistry/ Conc. (Donor 1) IL-6
(Donor 2) Gap ISIS NO. (uM) (pg/mL) (pg/mL) (gap motif) 06/353512 0
6.3 7.8 Positive control 0.0128 8.3 10.2 (3/14/3) 0.064 77.2 118.2
0.32 151.9 394.3 1.6 152.4 395.3 8 147.6 337.2 40 122.5 228.4 200
119.7 193.5 06/560221 0 5.6 7.6 Alternating 0.0128 6.4 6.9
P(CH.sub.3)(.dbd.S)-- 0.064 6.7 7.6 (3/14/3) 0.32 7.6 8.9 1.6 9.1
11.8 8 17.5 24.3 40 65.8 50.2 200 60.0 100.4 07/104838 0 5.8 7.3
Negative control 0.0128 7.7 7.9 (5/10/5) 0.064 7.5 11.6 0.32 15.1
22.0 1.6 73.1 112.8 8 29.6 51.5 40 41.6 69.5 200 55.4 4018.
Example 22
Single Nucleotide Polymorphisms (SNPs) in the Huntingtin (HTT) Gene
Sequence
[0436] SNP positions (identified by Hayden et al, WO/2009/135322)
associated with the HTT gene were mapped to the HTT genomic
sequence, designated herein as SEQ ID NO: 08 (NT_006081.18
truncated from nucleotides 1566000 to 1768000). The chart below
provides SNP positions associated with the HTT gene and a reference
SNP ID number from the Entrez SNP database at the National Center
for Biotechnology Information (NCBI,
http://www.ncbi.nlm.nih.gov/sites/-entrez?db=snp), incorporated
herein by reference. The chart below furnishes further details on
each SNP. The `Reference SNP ID number` or `RS number` is the
number designated to each SNP from the Entrez SNP database at NCBI,
incorporated herein by reference. `SNP position` refers to the
nucleotide position of the SNP on SEQ ID NO: 08. `Polymorphism`
indicates the nucleotide variants at that SNP position. `Major
allele` indicates the nucleotide associated with the major allele,
or the nucleotide present in a statistically significant proportion
of individuals in the human population. `Minor allele` indicates
the nucleotide associated with the minor allele, or the nucleotide
present in a relatively small proportion of individuals in the
human population.
TABLE-US-00010 Single Nuclear Polymorphisms (SNPs) and their
positions on SEQ ID NO: 08 SNP Major Minor RS No. position
Polymorphism allele allele rs2857936 1963 C/T C T rs12506200 3707
A/G G A rs762855 14449 A/G G A rs3856973 19826 G/A G A rs2285086
28912 G/A A G rs7659144 37974 C/G C G rs16843804 44043 C/T C T
rs2024115 44221 G/A A G rs10015979 49095 A/G A G rs7691627 51063
A/G G A rs2798235 54485 G/A G A rs4690072 62160 G/T T G rs6446723
66466 C/T T C rs363081 73280 G/A G A rs363080 73564 T/C C T
rs363075 77327 G/A G A rs363064 81063 T/C C T rs3025849 83420 A/G A
G rs6855981 87929 A/G G A rs363102 88669 G/A A G rs11731237 91466
C/T C T rs4690073 99803 A/G G A rs363144 100948 T/G T G rs3025838
101099 C/T C T rs34315806 101687 A/G G A rs363099 101709 T/C C T
rs363096 119674 T/C T C rs2298967 125400 C/T T C rs2298969 125897
A/G G A rs6844859 130139 C/T T C rs363092 135682 C/A C A rs7685686
146795 A/G A G rs363088 149983 A/T A T rs362331 155488 C/T T C
rs916171 156468 G/C C G rs362322 161018 A/G A G rs362275 164255 T/C
C T rs362273 167080 A/G A G rs2276881 171314 G/A G A rs3121419
171910 T/C C T rs362272 174633 G/A G A rs362271 175171 G/A G A
rs3775061 178407 C/T C T rs362310 179429 A/G G A rs362307 181498
T/C C T rs362306 181753 G/A G A rs362303 181960 T/C C T rs362296
186660 C/A C A rs1006798 198026 A/G A G.
Example 23
Modified Oligonucleotides Comprising Methyl Phosphonate
Internucleoside Linkage(s) Targeting HTT SNP--In Vitro Study
[0437] A series of modified oligonucleotides were designed based on
ISIS 460209 wherein the gap region contains nine
.beta.-D-2'-deoxyribonucleosides. The modified oligonucleotides
were designed by introducing a methyl phosphonate internucleoside
linkage within the gap region. The oligonucleotides with modified
phosphorus containing backbone were tested for their ability to
selectively inhibit mutant (mut) HTT mRNA expression levels
targeting rs7685686 while leaving the expression of the wild-type
(wt) intact. The potency and selectivity of the modified
oligonucleotides were evaluated and compared to ISIS 460209. The
position on the oligonucleotides opposite to the SNP position, as
counted from the 5'-terminus is position 8.
[0438] Heterozygous fibroblast GM04022 cell line was used for the
in vitro assay (from Coriell Institute). Cultured GM04022 cells at
a density of 25,000 cells per well were transfected using
electroporation with 0.12, 0.37, 1.1, 3.3 and 10 .mu.M
concentrations of modified oligonucleotides. After a treatment
period of approximately 24 hours, cells were washed with DPBS
buffer and lysed. RNA was extracted using Qiagen RNeasy
purification and mRNA levels were measured by quantitative
real-time PCR using ABI assay C_2229297_10 which measures at dbSNP
rs362303. RT-PCR method in short; A mixture was made using 2020
.mu.L 2.times.PCR buffer, 101 .mu.L primers (300 .mu.M from ABI),
1000 .mu.L water and 40.4 .mu.L RT MIX. To each well was added 15
.mu.L of this mixture and 5 .mu.L of purified RNA. The mutant and
wild-type HTT mRNA levels were measured simultaneously by using two
different fluorophores, FAM for mutant allele and VIC for wild-type
allele. The HTT mRNA levels were adjusted according to total RNA
content, as measured by RIBOGREEN.
Analysis of IC.sub.50's and Selectivity
[0439] The half maximal inhibitory concentration (IC.sub.50) of
each oligonucleotide is presented below and was calculated by
plotting the concentrations of oligonucleotides used versus the
percent inhibition of HTT mRNA expression achieved at each
concentration, and noting the concentration of oligonucleotide at
which 50% inhibition of HTT mRNA expression was achieved compared
to the control. The IC.sub.50 at which each oligonucleotide
inhibits the mutant HTT mRNA expression is denoted as `mut
IC.sub.50`. The IC.sub.50 at which each oligonucleotide inhibits
the wild-type HTT mRNA expression is denoted as `wt IC.sub.50`.
Selectivity as expressed in "fold" was calculated by dividing the
IC.sub.50 for inhibition of the wild-type HTT versus the IC.sub.50
for inhibiting expression of the mutant HTT mRNA and the results
are presented below.
TABLE-US-00011 SEQ ID NO./ ISIS NO. Composition (5'to 3') 05/460209
T.sub.eA.sub.kA.sub.kA.sub.dT.sub.dT.sub.dG.sub.dT.sub.d.sup.mC-
.sub.dA.sub.dT.sub.d.sup.mC.sub.dA.sub.k.sup.mC.sub.k.sup.mC.sub.e
05/558255
T.sub.eA.sub.kA.sub.kA.sub.dxT.sub.dT.sub.dG.sub.dT.sub.d.sup.m-
C.sub.dA.sub.dT.sub.d.sup.mC.sub.dA.sub.k.sup.mC.sub.k.sup.mC.sub.e
05/558256
T.sub.eA.sub.kA.sub.kA.sub.dT.sub.dxT.sub.dG.sub.dT.sub.d.sup.m-
C.sub.dA.sub.dT.sub.d.sup.mC.sub.dA.sub.k.sup.mC.sub.k.sup.mC.sub.e
[0440] Each internucleoside linkage is a phosphorothioate (P.dbd.S)
except for the internucleoside linkage having a subscript "x" which
indicates a methyl phosphonate internucleoside linkage
(--P(CH.sub.3)(.dbd.O)--). Each nucleoside followed by a subscript
"d" is a .beta.-D-2'-deoxyribonucleoside. Each nucleoside followed
by a subscript "e" indicates a 2'-O-methoxyethyl (MOE) modified
nucleoside. Each nucleoside followed by a subscript "k" indicates a
bicyclic nucleoside having a 4'-CH((S)--CH.sub.3)--O-2' bridge also
referred to as a (S)-cEt modified nucleoside. Each ".sup.mC" is a
5-methyl cytosine modified nucleoside. Nucleosides followed by
subscripts "e" or "k" are further illustrated below.
##STR00018##
TABLE-US-00012 SEQ ID NO./ IC.sub.50 Fold Selectivity ISIS NO.
(.mu.M) (mut vs. wt) Gap Chemistry 05/460209 0.30 3.3 Positive
control (3/9/3) 05/558255 0.19 6.8 Single --P(CH.sub.3)(.dbd.O)--
(3/9/3) 05/558256 0.20 6.5 Single --P(CH.sub.3)(.dbd.O)--
(3/9/3)
Example 24
Modified Oligonucleotides Comprising Methyl Phosphonate or
Phosphonoacetate Internucleoside Linkage(s) Targeting HTT SNP--In
Vitro Study
[0441] A series of modified oligonucleotides were designed based on
ISIS 460209 wherein the gap region contains nine
.beta.-D-2'-deoxyribonucleosides. The modified oligonucleotides
were synthesized to include one or more methyl phosphonate or
phosphonoacetate internucleoside linkage modifications within the
gap region. The oligonucleotides with modified phosphorus
containing backbone were tested for their ability to selectively
inhibit mutant (mut) HTT mRNA expression levels targeting rs7685686
while leaving the expression of the wild-type (wt) intact. The
potency and selectivity of the modified oligonucleotides were
evaluated and compared to ISIS 460209.
[0442] The position on the oligonucleotides opposite to the SNP
position, as counted from the 5'-terminus is position 8.
[0443] The modified oligonucleotides were tested in vitro.
Heterozygous fibroblast GM04022 cell line was used (from Coriell
Institute). Cultured GM04022 cells at a density of 25,000 cells per
well were transfected using electroporation with 0.12, 0.37, 1.1,
3.3 and 10 .mu.M concentrations of modified oligonucleotides. After
a treatment period of approximately 24 hours, cells were washed
with DPBS buffer and lysed. RNA was extracted using Qiagen RNeasy
purification and mRNA levels were measured by quantitative
real-time PCR using ABI assay C_2229297_10 which measures at dbSNP
rs362303. RT-PCR method in short; A mixture was made using 2020
.mu.L 2.times.PCR buffer, 101 .mu.L primers (300 .mu.M from ABI),
1000 uL water and 40.4 .mu.L RT MIX. To each well was added 15
.mu.L of this mixture and 5 .mu.L of purified RNA. The mutant and
wild-type HTT mRNA levels were measured simultaneously by using two
different fluorophores, FAM for mutant allele and VIC for wild-type
allele. The HTT mRNA levels were adjusted according to total RNA
content, as measured by RIBOGREEN.
[0444] The IC.sub.50s and selectivities as expressed in "fold" were
measured and calculated using methods described previously in
Example 23. The results are presented below.
TABLE-US-00013 SEQ ID NO./ ISIS NO. Composition (5' to 3')
05/460209
T.sub.eA.sub.kA.sub.kA.sub.dT.sub.dT.sub.dG.sub.dT.sub.d.sup.mC-
.sub.dA.sub.dT.sub.d.sup.mC.sub.dA.sub.k.sup.mC.sub.k.sup.mC.sub.e
05/566276
T.sub.eA.sub.kA.sub.kA.sub.dT.sub.dT.sub.dG.sub.dxT.sub.d.sup.m-
C.sub.dA.sub.dT.sub.d.sup.mC.sub.dA.sub.k.sup.mC.sub.k.sup.mC.sub.e
05/566277
T.sub.eA.sub.kA.sub.kA.sub.dT.sub.dT.sub.dG.sub.dT.sub.dx.sup.m-
C.sub.dA.sub.dT.sub.d.sup.mC.sub.dA.sub.k.sup.mC.sub.k.sup.mC.sub.e
05/566278
T.sub.eA.sub.kA.sub.kA.sub.dT.sub.dT.sub.dG.sub.dT.sub.d.sup.mC-
.sub.dxA.sub.dT.sub.d.sup.mC.sub.dA.sub.k.sup.mC.sub.k.sup.mC.sub.e
05/566279
T.sub.eA.sub.kA.sub.kA.sub.dT.sub.dT.sub.dG.sub.dT.sub.d.sup.mC-
.sub.dA.sub.dxT.sub.d.sup.mC.sub.dA.sub.k.sup.mC.sub.k.sup.mC.sub.e
05/566280
T.sub.eA.sub.kA.sub.kA.sub.dT.sub.dT.sub.dG.sub.dT.sub.d.sup.mC-
.sub.dA.sub.dT.sub.dx.sup.mC.sub.dA.sub.k.sup.mC.sub.k.sup.mC.sub.e
05/566283
T.sub.eA.sub.kA.sub.kA.sub.dT.sub.dxT.sub.dxG.sub.dT.sub.d.sup.-
mC.sub.dA.sub.dT.sub.d.sup.mC.sub.dA.sub.k.sup.mC.sub.k.sup.mC.sub.e
05/573815
T.sub.eA.sub.kA.sub.kA.sub.dT.sub.dyT.sub.dG.sub.dT.sub.d.sup.m-
C.sub.dA.sub.dT.sub.d.sup.mC.sub.dA.sub.k.sup.mC.sub.k.sup.mC.sub.e
05/573816
T.sub.eA.sub.kA.sub.kA.sub.dT.sub.dT.sub.dyG.sub.dT.sub.d.sup.m-
C.sub.dA.sub.dT.sub.d.sup.mC.sub.dA.sub.k.sup.mC.sub.k.sup.mC.sub.e
05/573817
T.sub.eA.sub.kA.sub.kA.sub.dT.sub.dT.sub.dG.sub.dT.sub.dy.sup.m-
C.sub.dA.sub.dT.sub.d.sup.mC.sub.dA.sub.k.sup.mC.sub.k.sup.mC.sub.e
05/573818
T.sub.eA.sub.kA.sub.kA.sub.dT.sub.dT.sub.dG.sub.dT.sub.d.sup.mC-
.sub.dA.sub.dT.sub.dy.sup.mC.sub.dA.sub.k.sup.mC.sub.k.sup.mC.sub.e
[0445] Each internucleoside linkage is a phosphorothioate (P.dbd.S)
except for the internucleoside linkage having a subscript "x" or
"y". Each nucleoside followed by a subscript "x" indicates a methyl
phosphonate internucleoside linkage (--P(CH.sub.3)(.dbd.O)--). Each
nucleoside followed by a subscript "y" indicates a phosphonoacetate
internucleoside linkage (--P(CH.sub.2CO.sub.2.sup.-)(.dbd.O)--).
Each nucleoside followed by a subscript "d" is a
.beta.-D-2'-deoxyribonucleoside. Each nucleoside followed by a
subscript "e" indicates a 2'-O-methoxyethyl (MOE) modified
nucleoside. Each nucleoside followed by a subscript "k" indicates a
bicyclic nucleoside having a 4'-CH((S)--CH.sub.3)--O-2' bridge also
referred to as a (S)-cEt modified nucleoside. Each ".sup.mC" is a
5-methyl cytosine modified nucleoside. Nucleosides followed by
subscripts "e" or "k" are further illustrated below.
##STR00019##
TABLE-US-00014 SEQ Fold ID NO./ IC.sub.50 Selectivity ISIS NO.
(.mu.M) (mut vs. wt) Gap Chemistry 05/460209 0.15 9.4 Positive
control (3/9/3) 05/566276 0.76 12.8 Single --P(CH.sub.3)(.dbd.O)--
(3/9/3) 05/566277 0.20 17 Single --P(CH.sub.3)(.dbd.O)-- (3/9/3)
05/566278 0.25 8.9 Single --P(CH.sub.3)(.dbd.O)-- (3/9/3) 05/566279
0.38 -- Single --P(CH.sub.3)(.dbd.O)-- (3/9/3 05/566280 0.27 47
Single --P(CH.sub.3)(.dbd.O)-- (3/9/3 05/566283 0.8 >100 Two
--P(CH.sub.3)(.dbd.O)-- (3/9/3) 05/573815 0.16 18.8 Single
--P(CH.sub.2CO.sub.2.sup.-)(.dbd.O)-- (3/9/3) 05/573816 0.55 18.1
Single --P(CH.sub.2CO.sub.2.sup.-)(.dbd.O)-- (3/9/3) 05/573817 0.17
22.5 Single --P(CH.sub.2CO.sub.2.sup.-)(.dbd.O)-- (3/9/3) 05/573818
0.24 13.5 Single --P(CH.sub.2CO.sub.2.sup.-)(.dbd.O)-- (3/9/3).
Example 25
Modified Oligonucleotides Comprising Methyl Phosphonate
Internucleoside Linkage(s) Targeting HTT SNP--In Vitro Study
[0446] A series of modified oligonucleotide was designed based on
ISIS 460209 or ISIS 476333, wherein the gap region contains nine
.beta.-D-2'-deoxyribonucleosides. The modified oligonucleotides
were designed by introducing methyl phosphonate internucleoside
linkage within the gap region at a fixed position and using
different wing motifs for 3/9/3 and 4/9/4 gapmer motifs. The
modified oligonucleotides were tested for their ability to
selectively inhibit mutant (mut) HTT mRNA expression levels
targeting rs7685686 while leaving the expression of the wild-type
(wt) intact. The potency and selectivity of the modified
oligonucleotides were evaluated and compared to ISIS 460209 or ISIS
476333.
[0447] The position on the oligonucleotides opposite to the SNP
position, as counted from the 5'-terminus is position 8 for ISIS
460209 or position 9 for ISIS 476333.
[0448] The modified oligonucleotides were tested in vitro.
Heterozygous fibroblast GM04022 cell line was used (from Coriell
Institute). Cultured GM04022 cells at a density of 25,000 cells per
well were transfected using electroporation with 0.1, 0.4, 1.1, 3.3
and 10 .mu.M concentrations of modified oligonucleotides. After a
treatment period of approximately 24 hours, cells were washed with
DPBS buffer and lysed. RNA was extracted using Qiagen RNeasy
purification and mRNA levels were measured by quantitative
real-time PCR using ABI assay C_2229297_10 which measures at dbSNP
rs362303. RT-PCR method in short; A mixture was made using 2020 uL
2.times.PCR buffer, 101 uL primers (300 .mu.M from ABI), 1000 uL
water and 40.4 uL RT MIX. To each well was added 15 uL of this
mixture and 5 uL of purified RNA. The mutant and wild-type HTT mRNA
levels were measured simultaneously by using two different
fluorophores, FAM for mutant allele and VIC for wild-type allele.
The HTT mRNA levels were adjusted according to total RNA content,
as measured by RIBOGREEN.
[0449] The IC.sub.50s and selectivities as expressed in "fold" were
measured and calculated using methods described previously in
Example 23. The results are presented below.
TABLE-US-00015 SEQ ID NO./ ISIS NO. Composition (5' to 3')
05/460209
T.sub.eA.sub.kA.sub.kA.sub.dT.sub.dT.sub.dG.sub.dT.sub.d.sup.mC.-
sub.dA.sub.dT.sub.d.sup.mC.sub.dA.sub.k.sup.mC.sub.k.sup.mC.sub.e
05/558257
T.sub.eA.sub.kA.sub.kA.sub.dT.sub.dT.sub.dxG.sub.dT.sub.d.sup.mC-
.sub.dA.sub.dT.sub.d.sup.mC.sub.dA.sub.k.sup.mC.sub.k.sup.mC.sub.e
05/571123
T.sub.eA.sub.eA.sub.eA.sub.kT.sub.kT.sub.dxG.sub.dT.sub.d.sup.mC-
.sub.dA.sub.dT.sub.d.sup.mC.sub.dA.sub.k.sup.mC.sub.k.sup.mC.sub.e
05/579854
T.sub.eA.sub.eA.sub.eA.sub.kT.sub.dT.sub.dxG.sub.dT.sub.d.sup.mC-
.sub.dA.sub.dT.sub.d.sup.mC.sub.dA.sub.k.sup.mC.sub.k.sup.mC.sub.e
05/566282
T.sub.eA.sub.kA.sub.kA.sub.dxT.sub.dxT.sub.dG.sub.dT.sub.d.sup.m-
C.sub.dA.sub.dT.sub.d.sup.mC.sub.dA.sub.k.sup.mC.sub.k.sup.mC.sub.e
09/476333
A.sub.eT.sub.kA.sub.eA.sub.kA.sub.dT.sub.dT.sub.dG.sub.dT.sub.d.-
sup.mC.sub.dA.sub.dT.sub.d.sup.mC.sub.dA.sub.k.sup.mC.sub.e.sup.mC.sub.kA.-
sub.e 09/571171
A.sub.eT.sub.kA.sub.eA.sub.kA.sub.dT.sub.dT.sub.dxG.sub.dT.sub.d-
.sup.mC.sub.dA.sub.dT.sub.d.sup.mC.sub.dA.sub.k.sup.mC.sub.e.sup.mC.sub.kA-
.sub.e
[0450] Each internucleoside linkage is a phosphorothioate (P.dbd.S)
except for the internucleoside linkage having a subscript "x" which
indicates a methyl phosphonate internucleoside linkage
(--P(CH.sub.3)(.dbd.O)--). Each nucleoside followed by a subscript
"d" is a .beta.-D-2'-deoxyribonucleoside. Each nucleoside followed
by a subscript "e" indicates a 2'-O-methoxyethyl (MOE) modified
nucleoside. Each nucleoside followed by a subscript "k" indicates a
bicyclic nucleoside having a 4'-CH((S)--CH.sub.3)--O-2' bridge also
referred to as a (S)-cEt modified nucleoside. Each ".sup.mC" is a
5-methyl cytosine modified nucleoside. Nucleosides followed by
subscripts "e" or "k" are further illustrated below.
##STR00020##
TABLE-US-00016 SEQ ID NO./ Mut IC.sub.50 Wt IC.sub.50 Fold
Selectivity ISIS NO. (.mu.M) (.mu.M) (mut vs. wt) Gap Chemistry
(motif) 05/460209 0.56 3.8 6.8 Positive control (3/9/3) 05/558257
1.7 >10 >5.9 Single --P(CH.sub.3)(.dbd.O)-- (3/9/3) 05/571123
0.96 >10 >10.4 Single --P(CH.sub.3)(.dbd.O)-- (5/7/3)
05/579854 0.63 >10 >15.9 Single --P(CH.sub.3)(.dbd.O)--
(4/8/3) 05/566282 0.51 6.3 12.4 Two --P(CH.sub.3)(.dbd.O)-- (3/9/3)
09/476333 0.56 3.4 6.1 Positive control (4/9/4) 09/571171 0.54
>10 >18.5 Single --P(CH.sub.3)(.dbd.O)-- (4/9/4).
Example 26
Modified Oligonucleotides Comprising Methyl Phosphonate
Internucleoside Linkages Targeting PTEN and SRB-1--In Vivo
Study
[0451] Additional modified oligonucleotides were designed based on
ISIS 482050 and 449093 wherein the gap region contains ten
.beta.-D-2'-deoxyribonucleosides. The modified oligonucleotides
were designed by introducing two methyl phosphonate internucleoside
linkages at the 5'-end of the gap region with a 3/10/3 motif. The
oligonucleotides were evaluated for reduction in PTEN and SRB-1
mRNA expression levels in vivo. The parent gapmers, ISIS 482050 and
449093 were included in the study for comparison.
Treatment
[0452] Six week old BALB/C mice (purchased from Charles River) were
injected subcutaneously twice a week for three weeks at dosage 10
mg/kg or 20 mg/kg with the modified oligonucleotides shown below or
with saline control. Each treatment group consisted of 3 animals.
The mice were sacrificed 48 hrs following last administration, and
organs and plasma were harvested for further analysis.
mRNA Analysis
[0453] Liver tissues were homogenized and mRNA levels were
quantitated using real-time PCR and normalized to RIBOGREEN as
described herein. The results below are listed as PTEN or SRB-1
mRNA expression for each treatment group relative to saline-treated
control (% UTC). As illustrated, reduction in PTEN or SRB-1 mRNA
expression levels was achieved with the oligonucleotides comprising
two methyl phosphonate internucleoside linkages at the 5'-end of
the gap region, ISIS 582073 and 582074.
Plasma Chemistry Markers
[0454] Plasma chemistry markers such as liver transaminase levels,
alanine aminotranferase (ALT) in serum were measured relative to
saline injected mice and the results are presented below. Treatment
with the oligonucleotides resulted in a reduction in ALT level
compared to treatment with the parent gapmer, ISIS 482050 or
449093. The results suggest that introduction of methyl phosphonate
internucleoside linkage(s) can be useful for reduction of
hepatotoxicity profile of otherwise unmodified parent gapmers.
Body and Organ Weights
[0455] Body weights, as well as liver, kidney and spleen weights
were measured at the end of the study. The results below are
presented as the average percent of body and organ weights for each
treatment group relative to saline-treated control. As illustrated,
treatment with ISIS 582073 resulted in a reduction in liver and
spleen weights compared to treatment with the parent gapmer, ISIS
482050. The remaining oligonucleotide, ISIS 582074 did not cause
any changes in body and organ weights outside the expected range as
compared to ISIS 449093.
TABLE-US-00017 SEQ ID NO./ ISIS NO. Composition (5' to 3')
11/482050
A.sub.kT.sub.k.sup.mC.sub.kA.sub.dT.sub.dG.sub.dG.sub.d.sup.mC.-
sub.dT.sub.dG.sub.d.sup.mC.sub.dA.sub.dG.sub.d.sup.mC.sub.kT.sub.kT.sub.k
11/582073
A.sub.kT.sub.k.sup.mC.sub.kA.sub.dxT.sub.dxG.sub.dG.sub.d.sup.m-
C.sub.dT.sub.dG.sub.d.sup.mC.sub.dA.sub.dG.sub.d.sup.mC.sub.kT.sub.kT.sub.-
k 12/449093
T.sub.kT.sub.k.sup.mC.sub.kA.sub.dG.sub.dT.sub.d.sup.mC.sub.dA.-
sub.dT.sub.dG.sub.dA.sub.d.sup.mC.sub.dT.sub.dT.sub.k.sup.mC.sub.k.sup.mC.-
sub.k 12/582074
T.sub.kT.sub.k.sup.mC.sub.kA.sub.dxG.sub.dxT.sub.d.sup.mC.sub.d-
A.sub.dT.sub.dG.sub.dA.sub.d.sup.mC.sub.dT.sub.dT.sub.k.sup.mC.sub.k.sup.m-
C.sub.k
[0456] Each internucleoside linkage is a phosphorothioate (P.dbd.S)
except for the internucleoside linkage having a subscript "x". Each
nucleoside followed by a subscript "x" indicates a methyl
phosphonate internucleoside linkage (--P(CH.sub.3)(.dbd.O)--). Each
nucleoside followed by a subscript "d" is a
.beta.-D-2'-deoxyribonucleoside. Each nucleoside followed by a
subscript "k" indicates a bicyclic nucleoside having a
4'-CH((S)--CH.sub.3)--O-2' bridge also referred to as a (S)-cEt
modified nucleoside. Each ".sup.mC" is a 5-methyl cytosine modified
nucleoside. Nucleosides followed by subscript "k" are further
illustrated below.
##STR00021##
TABLE-US-00018 SEQ Dosage ID NO./ (mg/ % ALT ISIS NO. Target kg/wk)
UTC (IU/L) Chemistry Saline -- 0 100 30 -- 11/482050 PTEN 10 50 228
Full (P.dbd.S) 11/482050 20 36.1 505 11/582073 10 72.2 47.7 Two
--P(CH.sub.3)(.dbd.O)-- 11/582073 20 57.4 46 12/449093 SRB-1 10 48
543 Full (P.dbd.S) 12/449093 20 18.5 1090 12/582074 10 51.3 58.3
Two --P(CH.sub.3)(.dbd.O)-- 12/582074 20 30.3 126.3
TABLE-US-00019 SEQ ID NO./ Dosage Body wt rel to Liver/Body
Spleen/Body Kidney/Body ISIS NO. Target (mg/kg/wk) predose (%) Wt
(%) Wt (%) Wt (%) Saline -- 0 108.4 100 100 100 11/482050 PTEN 10
107.4 154.9 141.8 115.7 11/482050 20 111.3 176.7 142.3 112.5
11/582073 10 108.9 122.9 111.7 100.0 11/582073 20 107.9 133.8 114.6
102.9 12/449093 SRB-1 10 101.3 105.9 117.9 89.3 12/449093 20 95.3
118.6 129.6 93.0 12/582074 10 107.1 92.2 116.4 89.2 12/582074 20
103.8 95.5 128.8 91.9.
Example 27
Modified Oligonucleotides Comprising Methyl Phosphonate
Internucleoside Linkages Targeting Target-Y--In Vivo Study
[0457] Additional modified oligonucleotides were designed in the
same manner as the antisense oligonucleotides described in Example
25, wherein two methyl phosphonate internucleoside linkages are
introduced at the 5'-end of the gap region. The modified
oligonucleotides were designed based on ISIS 464917, 465178, 465984
and 466456 with a 3/10/3 motif. The oligonucleotides were evaluated
for reduction in Target-Y mRNA expression levels in vivo. The
parent gapmers, ISIS 464917, 465178, 465984 and 466456 were
included in the study for comparison.
Treatment
[0458] Six week old BALB/C mice (purchased from Charles River) were
injected subcutaneously twice a week for three weeks at dosage 10
mg/kg or 20 mg/kg with the modified oligonucleotides shown below or
with saline control. Each treatment group consisted of 3 animals.
The mice were sacrificed 48 hrs following last administration, and
organs and plasma were harvested for further analysis.
mRNA Analysis
[0459] Liver tissues were homogenized and mRNA levels were
quantitated using real-time PCR and normalized to RIBOGREEN as
described herein. The results below are listed as Target-Y mRNA
expression for each treatment group relative to saline-treated
control (% UTC). As illustrated, reduction in Target-Y mRNA
expression levels was achieved with the oligonucleotides comprising
two methyl phosphonate internucleoside linkages at the 5'-end of
the gap region, ISIS 582071, 582072, 582069 and 582070.
Plasma Chemistry Markers
[0460] Plasma chemistry markers such as liver transaminase levels,
alanine aminotranferase (ALT) in serum were measured relative to
saline treated mice and the results are presented below. Treatment
with the oligonucleotides resulted in a reduction in ALT level
compared to treatment with the parent gapmer, ISIS 464917, 465178,
465984 or 466456. The results suggest that introduction of methyl
phosphonate internucleoside linkage(s) can be useful for reducing
the hepatotoxicity profile of otherwise unmodified parent
gapmers.
Body and Organ Weights
[0461] Body weights, as well as liver, kidney and spleen weights
were measured at the end of the study. The results below are
presented as the average percent of body and organ weights for each
treatment group relative to saline-treated control. As illustrated,
treatment with ISIS 582070 resulted in a reduction in liver and
spleen weights compared to treatment with the parent gapmer, ISIS
466456. An increase in body and organ weights was observed for ISIS
582071 as compared to ISIS 464917. The remaining oligonucleotides,
ISIS 582072 and 582069 did not cause any changes in body and organ
weights outside the expected range as compared to ISIS 465178 and
465984.
TABLE-US-00020 SEQ ID NO./ ISIS NO. Composition (5' to 3')
13/464917
N.sub.kN.sub.kN.sub.kN.sub.dN.sub.dN.sub.dN.sub.dN.sub.dN.sub.d-
N.sub.dN.sub.dN.sub.dN.sub.dN.sub.kN.sub.kN.sub.k 13/582071
N.sub.kN.sub.kN.sub.kN.sub.dxN.sub.dxN.sub.dN.sub.dN.sub.dN.sub-
.dN.sub.dN.sub.dN.sub.dN.sub.dNkN.sub.kN.sub.k 14/465178
N.sub.kN.sub.kN.sub.kN.sub.dN.sub.dN.sub.dN.sub.dN.sub.dN.sub.d-
N.sub.dN.sub.dN.sub.dN.sub.dN.sub.kNkN.sub.k 14/582072
N.sub.kN.sub.kN.sub.kN.sub.dxN.sub.dxN.sub.dN.sub.dN.sub.dN.sub-
.dN.sub.dN.sub.dN.sub.dN.sub.dNkN.sub.kN.sub.k 15/465984
N.sub.kN.sub.kN.sub.kN.sub.dN.sub.dN.sub.dN.sub.dN.sub.dN.sub.d-
N.sub.dN.sub.dN.sub.dN.sub.dN.sub.eNeN.sub.e 15/582069
N.sub.kN.sub.kN.sub.kN.sub.dxN.sub.dxN.sub.dN.sub.dN.sub.dN.sub-
.dN.sub.dN.sub.dN.sub.dN.sub.dNkN.sub.kN.sub.k 16/466456
N.sub.kN.sub.dN.sub.kN.sub.dN.sub.kN.sub.dN.sub.dN.sub.dN.sub.d-
N.sub.dN.sub.dN.sub.dN.sub.dN.sub.dN.sub.eN.sub.e 16/582070
N.sub.kN.sub.dN.sub.kN.sub.dxN.sub.dxN.sub.dN.sub.dN.sub.dN.sub-
.dN.sub.dN.sub.dN.sub.dN.sub.dN.sub.dN.sub.eN.sub.e
[0462] Each internucleoside linkage is a phosphorothioate (P.dbd.S)
except for the internucleoside linkage having a subscript "x". Each
nucleoside followed by a subscript "x" indicates a methyl
phosphonate internucleoside linkage (--P(CH.sub.3)(.dbd.O)--). Each
nucleoside followed by a subscript "d" is a
.beta.-D-2'-deoxyribonucleoside. Each nucleoside followed by a
subscript "e" indicates a 2'-O-methoxyethyl (MOE) modified
nucleoside. Each nucleoside followed by a subscript "k" indicates a
bicyclic nucleoside having a 4'-CH((S)--CH.sub.3)--O-2' bridge also
referred to as a (S)-cEt modified nucleoside. Each "N" is a
modified or naturally occurring nucleobases (A, T, C, G, U, or
5-methyl C). Nucleosides followed by subscripts "e" or "k" are
further illustrated below.
##STR00022##
TABLE-US-00021 SEQ ID NO./ Dosage ALT ISIS NO. (mg/kg/wk) % UTC
(IU/L) Chemistry Saline 0 100 30 -- 13/464917 10 29 1244 Full
(P.dbd.S) 13/464917 20 30.1 2335 13/582071 20 10.2 274 Two
--P(CH.sub.3)(.dbd.O)-- 14/465178 10 4.9 1231 Full (P.dbd.S)
14/465178 20 10.6 6731 14/582072 10 36.7 44.7 Two
--P(CH.sub.3)(.dbd.O)-- 14/582072 20 23.6 43.7 15/465984 10 4.7 61
Full (P.dbd.S) 15/465984 20 0.9 57 15/582069 10 11.1 39.7 Two
--P(CH.sub.3)(.dbd.O)-- 15/582069 20 3.3 27.7 16/466456 10 9.5 692
Full (P.dbd.S) 16/466456 20 10.5 2209 16/582070 10 73.9 24 Two
--P(CH.sub.3)(.dbd.O)-- 16/582070 20 51.3 36.7
TABLE-US-00022 SEQ ID NO./ Dosage Body wt rel to Liver/Body
Spleen/Body Kidney/Body ISIS NO. (mg/kg/wk) predose (%) Wt (%) Wt
(%) Wt (%) Saline 0 108 100 100 100 13/464917 10 92.9 125 106.2
102.3 13/464917 20 71.1 110.9 67.2 107.3 13/582071 20 104.6 135.2
142.8 89.8 14/465178 10 94.9 131.3 108.1 85.3 14/465178 20 79.5
147.5 112 95.3 14/582072 10 109.2 117.3 111.7 104.8 14/582072 20
107.1 130.1 107.2 99.8 15/465984 10 111.4 117.6 110.1 98.8
15/465984 20 111.3 122.6 134.5 96.1 15/582069 10 107.8 106.2 97
100.6 15/582069 20 105.4 115.8 106.2 100.4 16/466456 10 109.7 148.6
198.7 105.9 16/466456 20 101.2 182.3 213.7 101.9 16/582070 10 111.2
100.3 116.7 100.8 16/582070 20 111.1 108.9 115.6 95.7.
Sequence CWU 1
1
1613160DNAHomo sapiens 1cctcccctcg cccggcgcgg tcccgtccgc ctctcgctcg
cctcccgcct cccctcggtc 60ttccgaggcg cccgggctcc cggcgcggcg gcggaggggg
cgggcaggcc ggcgggcggt 120gatgtggcag gactctttat gcgctgcggc
aggatacgcg ctcggcgctg ggacgcgact 180gcgctcagtt ctctcctctc
ggaagctgca gccatgatgg aagtttgaga gttgagccgc 240tgtgaggcga
ggccgggctc aggcgaggga gatgagagac ggcggcggcc gcggcccgga
300gcccctctca gcgcctgtga gcagccgcgg gggcagcgcc ctcggggagc
cggccggcct 360gcggcggcgg cagcggcggc gtttctcgcc tcctcttcgt
cttttctaac cgtgcagcct 420cttcctcggc ttctcctgaa agggaaggtg
gaagccgtgg gctcgggcgg gagccggctg 480aggcgcggcg gcggcggcgg
cggcacctcc cgctcctgga gcggggggga gaagcggcgg 540cggcggcggc
cgcggcggct gcagctccag ggagggggtc tgagtcgcct gtcaccattt
600ccagggctgg gaacgccgga gagttggtct ctccccttct actgcctcca
acacggcggc 660ggcggcggcg gcacatccag ggacccgggc cggttttaaa
cctcccgtcc gccgccgccg 720caccccccgt ggcccgggct ccggaggccg
ccggcggagg cagccgttcg gaggattatt 780cgtcttctcc ccattccgct
gccgccgctg ccaggcctct ggctgctgag gagaagcagg 840cccagtcgct
gcaaccatcc agcagccgcc gcagcagcca ttacccggct gcggtccaga
900gccaagcggc ggcagagcga ggggcatcag ctaccgccaa gtccagagcc
atttccatcc 960tgcagaagaa gccccgccac cagcagcttc tgccatctct
ctcctccttt ttcttcagcc 1020acaggctccc agacatgaca gccatcatca
aagagatcgt tagcagaaac aaaaggagat 1080atcaagagga tggattcgac
ttagacttga cctatattta tccaaacatt attgctatgg 1140gatttcctgc
agaaagactt gaaggcgtat acaggaacaa tattgatgat gtagtaaggt
1200ttttggattc aaagcataaa aaccattaca agatatacaa tctttgtgct
gaaagacatt 1260atgacaccgc caaatttaat tgcagagttg cacaatatcc
ttttgaagac cataacccac 1320cacagctaga acttatcaaa cccttttgtg
aagatcttga ccaatggcta agtgaagatg 1380acaatcatgt tgcagcaatt
cactgtaaag ctggaaaggg acgaactggt gtaatgatat 1440gtgcatattt
attacatcgg ggcaaatttt taaaggcaca agaggcccta gatttctatg
1500gggaagtaag gaccagagac aaaaagggag taactattcc cagtcagagg
cgctatgtgt 1560attattatag ctacctgtta aagaatcatc tggattatag
accagtggca ctgttgtttc 1620acaagatgat gtttgaaact attccaatgt
tcagtggcgg aacttgcaat cctcagtttg 1680tggtctgcca gctaaaggtg
aagatatatt cctccaattc aggacccaca cgacgggaag 1740acaagttcat
gtactttgag ttccctcagc cgttacctgt gtgtggtgat atcaaagtag
1800agttcttcca caaacagaac aagatgctaa aaaaggacaa aatgtttcac
ttttgggtaa 1860atacattctt cataccagga ccagaggaaa cctcagaaaa
agtagaaaat ggaagtctat 1920gtgatcaaga aatcgatagc atttgcagta
tagagcgtgc agataatgac aaggaatatc 1980tagtacttac tttaacaaaa
aatgatcttg acaaagcaaa taaagacaaa gccaaccgat 2040acttttctcc
aaattttaag gtgaagctgt acttcacaaa aacagtagag gagccgtcaa
2100atccagaggc tagcagttca acttctgtaa caccagatgt tagtgacaat
gaacctgatc 2160attatagata ttctgacacc actgactctg atccagagaa
tgaacctttt gatgaagatc 2220agcatacaca aattacaaaa gtctgaattt
ttttttatca agagggataa aacaccatga 2280aaataaactt gaataaactg
aaaatggacc tttttttttt taatggcaat aggacattgt 2340gtcagattac
cagttatagg aacaattctc ttttcctgac caatcttgtt ttaccctata
2400catccacagg gttttgacac ttgttgtcca gttgaaaaaa ggttgtgtag
ctgtgtcatg 2460tatatacctt tttgtgtcaa aaggacattt aaaattcaat
taggattaat aaagatggca 2520ctttcccgtt ttattccagt tttataaaaa
gtggagacag actgatgtgt atacgtagga 2580attttttcct tttgtgttct
gtcaccaact gaagtggcta aagagctttg tgatatactg 2640gttcacatcc
tacccctttg cacttgtggc aacagataag tttgcagttg gctaagagag
2700gtttccgaaa ggttttgcta ccattctaat gcatgtattc gggttagggc
aatggagggg 2760aatgctcaga aaggaaataa ttttatgctg gactctggac
catataccat ctccagctat 2820ttacacacac ctttctttag catgctacag
ttattaatct ggacattcga ggaattggcc 2880gctgtcactg cttgttgttt
gcgcattttt ttttaaagca tattggtgct agaaaaggca 2940gctaaaggaa
gtgaatctgt attggggtac aggaatgaac cttctgcaac atcttaagat
3000ccacaaatga agggatataa aaataatgtc ataggtaaga aacacagcaa
caatgactta 3060accatataaa tgtggaggct atcaacaaag aatgggcttg
aaacattata aaaattgaca 3120atgatttatt aaatatgttt tctcaattgt
aaaaaaaaaa 3160226DNAArtificial sequencePrimer 2aatggctaag
tgaagatgac aatcat 26325DNAArtificial sequencePrimer 3tgcacatatc
attacaccag ttcgt 25430DNAArtificial sequenceProbe 4ttgcagcaat
tcactgtaaa gctggaaagg 30515DNAArtificial sequenceSynthetic
oligonucleotide 5taaattgtca tcacc 15620DNAArtificial
sequenceSynthetic oligonucleotide 6tcccatttca ggagacctgg
20720DNAArtificial sequenceSynthetic oligonucleotide 7gctgattaga
gagaggtccc 208202001DNAHomo sapiens 8gcccagcagg tgtcagcctc
attttacccc gcccctattc aagatgaagt tgttctggtt 60ccaacgcctc tgacatatta
gctgcatcat tttacatttc tttttttttt ttccttttaa 120atggggtctt
gctctgtcac ccaggctgga gtgctgtggt atgatctcgg ctcactgcaa
180tctccacctc cgaggttcca gcgattctct tgcctcagcc tcccgagtag
ctgggactac 240aggcacccac catcatactg ggctaatttt tgtgttttta
gtagagatgg ggtttcccca 300tgttgcccag gctgatctca aactcctggg
cttaagcaat acagccgcgt tggcctccca 360aagtgttggg attacaagca
tgagctaccc cacccagctc attttacatt tccacttgtt 420aaactgaaaa
ctggcccgag aaagcttctg tactgccatc cttgcgtcct tgcagatgaa
480tcgtaaccta gcatagtagg taggcagact gaaaacctaa cttagcagta
ggcttctgta 540acaacagctg tgtctcagcc agttcctgca gccagacttc
aaccactcac aggccgcaaa 600ctgttcaaac tgtgttcgga gaaggcgaat
tcatctggct gttaacgtgc ctcacttctg 660ctttctgtgg ccactttccc
ttttctgtcc ataaatttgc tttgaccaca cagcatccct 720agagtctccc
tgaatctgct gtgattctgg gacctgcacc atttgtgaat tgtttttttt
780ttccttgatc agctaaactc tgttcaattc aatttgttgg aagtttttaa
cataccaatg 840gtgcaccaag gttccaattt ctccacttcc tcataaataa
gtcattttaa atggcttttc 900agtattccaa tatttggaag tattaatgtt
tctaccaatt ttctattttt ggacattgag 960gttgtttcat tttttttttc
tttttttgag acagagtctc gctccgtcac ccaggctgga 1020gtgcagtggc
ctgatcccgg cccactgcaa cctccacctc cctcctcagc ctcctgagta
1080gctgggatta caggtgcatg caccaccaca cccagctaat ttttgtattt
ttagtagaga 1140tggggtttca ccatgttggt caggctggtc tcaaactcct
gacctcaggt ggtccacctg 1200ccttggcctc ccaaaatgct gggattacag
gcctgagcca ctgcgcctgg cctcatcttc 1260ttgatattaa tgttgcttta
acatctttgt ccctgtgttt tttgtttttt tttttgagac 1320ggagtctcat
tcattctgtc acccaggctg gagttcagtg gcgtgatctc agctcactgc
1380aacctctgtc tcctgggttc cagtgattct cctgcgtcgg tctcctgagt
agctgtgttc 1440ctgggtcttt cgatggttat ttaatacttc cctacagtaa
tgccctgtgc gtacatgcta 1500agtgtgatga aatggttggc acagttaaat
cttttgaaag acattgccaa gtcactcttc 1560agaaaagtga taggaggtca
tagcaatttt aagaagtcct catttctaca tttccttact 1620aatctcggtt
ggtgtctctt caatctttcc tcacactttt cttgggtttt tcctgaatca
1680tgagtctact acatttacac attttaaagc atctttagaa acaggatctc
attttgttgc 1740ccaggctaga gtttggtggc atgattatag ctcctcatac
tcctgggctc aagtgatcct 1800tccacctctg aaaccccaaa atttgagaaa
ggtctcattt aatttagaaa gtttattttg 1860ccaaggttga gggtgcacac
ctgtgatgat atacgagtta aaaagaaatt atttaggcag 1920atactgaggg
taagaaagtc ctcggtaagg ttttcttttc aatgaaaagc agcccccaag
1980cattttcttt tctaacaaag agcagcctgt aaaatcgagc tgcagacata
cacaagcaag 2040ctggaagctt gcacaggtga atgctggcag ctgtgccaat
aagaaaaggc tacctggggc 2100caggcagatc caacatggcg gctccatctt
ccctttcctt gtcaaccatg tgcacagtaa 2160ggagcaggca acatagtgtc
ccccgagtag agaccaattt gcataataaa aggtgagggt 2220agggtgggca
gcttctttgc atgctatgta aacattatgc ctggtccaac caatctttgg
2280gccctgtgta aattagacac cacctcctca agcctgtcta taaaaccctg
tccattctgc 2340cgcaggctgg aagacccact ggggcacccc tctctctcta
taggagacag ctattcattt 2400ttctctttct ttcacctatt aaagctccac
tcttaacccc actccgtgtg tatctatgtt 2460cttgatttcc ttggcatgag
gcaatgaacc ttgggtatta ccccagaacc ttgggtatta 2520tgccacttca
gtgacacagc ctcaggaaat cctgatgaca tgttcccaag atggtcgggg
2580cacagcttgg ttttatacat tttagggaga catgagacgt caattcatat
atgtaagaag 2640tacattggtt ccgtccagaa aggcggggac aacttgaggc
agggagagag cttctaggtc 2700acaggtagac aaatggttgc attcttttga
atctccgata agcctttcca aaggaggcaa 2760tcagaatatg cgtctattga
ctgggcgcag tggctcatgc ctgtaatgcc agcactttgg 2820gaggcggagg
tgggtggatc acctgaggtc aggagtttga gagcagcccg gccaacatgg
2880tgaaaccctg tctctactaa aaatacaaaa aattagctgg gcgtggtggc
gggcgcctgt 2940aatcccagct actcgggagg ctgaggcagg agaatagctt
gaacccagaa ggaagaggtt 3000gcagtgagct gagatggtgc cattgcactc
cagcctgggc aacaagagtg aaactccatc 3060tcagaaaaaa aaaaaaaagg
cctgggcaaa gtggctcacg cctgtaatcc cagcactttg 3120ggaagccgag
gcgggcaggt cacaaagtca ggagattgag accatcctgg ctaacatgat
3180gaaaccccat ctctactaaa aaatacaaaa aactagctgg gtgtggtggc
gagcacctgt 3240agtcccagct actcggcagg ctgaggcagg agaatggcgt
gaaccgggga ggcggagctt 3300gcagtgagcc gagatcacac cactgcactc
cagcccggac gacagggcaa gactctatct 3360caaattaaaa aaaaaaaaaa
aaaaaaaaaa aaagagagag agaatatgca tctatctcag 3420tgagcagaag
gatgactttg aatggaatgg gagcagttcc tagcttgaac ttccccttta
3480gcttcagtga tttgggggct caaggtatgt tcctttcaca tacctcagcc
tcccaagtag 3540ctgggaccac aagtgcatgc caccacacgt ggctaatgtt
ttattttttt tgtaggaata 3600gggtctcact atgtgtccag gctggtctaa
aacccctgag ctcaaatggt cctcccgcct 3660cagcctcccg aaatgctggg
attacaggca tgagccagca tgcccggcct agtctacatt 3720tttataaatt
gctaattcaa agttccctct ccaaaacctc atggttttcc ctgttctcat
3780cccctgcacc ctcccttccc ctggagtact cacctggcct tggaggtctg
gtgtgagccc 3840ggacttcgat tctaggcaca gcatgtgatg agcgccccca
ggtcaaacac ctcccctctg 3900cggcctgtgc ttcaccgcct tgacagtgag
aaaggtctcc cttcggctca ttctcgaagt 3960ctcaaacttc acttctcctg
tgcgctgatt ctgaattcag cccccgtcca aggtcctggc 4020ccctttctct
tctgcttggc gtgttgttca tcaccactgt gcactgctga gggtaagtgc
4080ggttctctgg acctctgctt tatcattaga acagactctt gcggtttccc
acgacattcc 4140tttcacttct cacttggaag atgagccgtg aggaaatcct
gtgttgtgtg gtatgtgggc 4200tgtgcttctg cttgacttga gggccaagca
gcattgcaag ccatggtttt aaataagaaa 4260gaacatttct aaccttcatc
ttctagtaag gaaacaagtg ggctttagag ttcttgctca 4320ggaaagacct
atgtcccagt ccaaccggac cttttactaa agagatcttc ctgatcctcc
4380tccccaggcc aggggagggg tcctccctgg ggttggagcc tttagtaggg
ggtcggagac 4440acgacgtagc cttcatgaca ttcatagtct agttacacga
tccctgtaag ggtcagttga 4500agtaagtgct acaaaggaag ggaggtgctc
agtggagagg gctctctttt atgtattata 4560tttctttcat ggggagggat
atggatcagg gatcagcaga ggtgtttcag tcccgaggga 4620aagaaagtca
gcgtggcttg ggagttggga gcagcaagac agtggctcaa gatatcttaa
4680gactagtgga gtacaccttg catgttaaaa gccttgctca gggctgcctg
gttcttgtag 4740gacgacagag atggcctagc tctgcatact gcacccccag
gggctcagaa cagtgcaaat 4800gtcagtctat ctgtcagtgg cagagccagc
cttggagcag gggtgcaagg aggtctctgc 4860actggccagg catgcagaac
attctgttca gtagcactgg acagaaggcc ccatctagat 4920gagacagagc
tggtggggca ggacaaagac tcctggcagc tcaaacggcc tggcagatgc
4980ttggagagag ggggcttctt gagacagcac catttctggg aagagagtca
cctgggaggg 5040atgaggccac gctccggctt ggaggtgaag agaggggctg
ctgcaagaaa gaattagaga 5100catgccagcc tttgctgtgt tgcccaggct
ggtcatgaac tcttggcctc aagcaatctt 5160cccacctcag cctccccaag
cgctgggatt atagacatga gcccccatgc tggccaataa 5220aagatgattt
tatggagggg atggtggtga aggttgtggg tggtatgaaa tagtaagaaa
5280tatatattgg tctgcaccca gttcctgcca cagagctcct aaaatcctga
gaacttcctg 5340ggtgagcatc ttttgttcta atgaggtgac tcttggtggc
tcctggatag gagtgaatca 5400ccagaaagat caagccagag ttagaagcag
aaagtgctgg ctataacaca ggaaagctgt 5460aacacaaata ataaagtttt
tttttttttt tttgagatgg agcctcactc tgttgcccag 5520gctggagtgc
aatggtgcaa tctcagctca ctacaagctc tgcctcccag gttcaagtga
5580ttctcctgcc tcagcctcct gagcagttgg gactacaggt gtgtgccacc
acatctggct 5640aatttttgta tttttagcag agacggggtt tcaccatatt
aaccaggctg gcctcaaact 5700ccttaccttg tgatccgcct gcctcagcct
cccaaagtgc tgggattaca ggcatgagcc 5760accgtgcctg gccaaaagac
attgttctta aaagaatcaa ctaactaacc aaataaataa 5820aaatctaacc
taattaagaa actaaaaata cacaaaaatt aatttcaagg ggagaaaaat
5880catgtaaaga gagaaagata atgaatactt tgcagaaatt tatgaacata
aacataaaac 5940ttggatgaaa tgcatttcta ggaaaacata atttatcaaa
actaaccaca agtaaaatag 6000aagcctaaat aggatatttt caagagaaga
agtaaagttg tcaaagtgct acccttcaaa 6060aaaacaccag gctcaaacaa
tctgacatgg gaatgttagc acaccttaga gagcaaataa 6120aactttgaat
gggcttgaaa tattccagac tctagaaaaa caaaacttcc caattctttt
6180tataaagcaa gtataaattg ataccaaaat cttataaaga ccttatacaa
aacttcatac 6240caatctcttt tatgaataca aaacccttaa taaagtatta
ccagacagaa cccaacaata 6300cataaaaatg tcacatcata acatagtggg
gtttatttca ataatgcatg gatggttcaa 6360tacaaggaaa ttcagtaaca
caatataata gatcatgtga atatacccaa agaaaaaata 6420gattattttc
atagatgctg taaaggcatt tgaccaaatt caacacctac tttttaggtg
6480gtcaataaaa taaattagtt actccttctt tagcatgata aaatatattt
atcagcccag 6540aaggcatcat tttacccgat aagggcacac gctggaggga
ataatgttaa aattaggaat 6600aagaggatag ctagtttctt tcttcttttt
tttttttgag acggagtctt gctctgttgc 6660caggctggag tgcagtggtg
caatgttggc tcactgcacg ccccccgcct cccaggttca 6720agcgattctc
ctgcctcagc ctcccgagta gctgggacta caggcgcgca ccaccatgcc
6780cggctaattt ttttttgtat tttagtagag atggggtttc accatgttgg
tcaggctggt 6840cttgaactcc caacctcacg tactgggatt accggtgtga
gccaccacgc cagcccaact 6900actttcaaca ttatccttaa tactgatgct
tattgactta ctatggggtt acctctagat 6960aaatccataa taagttgaaa
atataagtaa aaaatgccct taatacacct aacctaccaa 7020acatcatagc
tgagcccagc ctgccttagc tatgctcaga cactgacgtc agcctacaat
7080tggcaaaatc acacagcagc acagtctact gcagagcatc tgctgtttgc
ccttgtgact 7140gcgtggctgc ctgggagctt cccagcttca caagacagta
ttacgtagca catcactagc 7200ctggggaaag atcaaagttg aaaatttgaa
gtgtggtttc cattgaatgt gtactgcttt 7260tgcaccatca tcaagtcaaa
aaattttagt tgaaccagcc taagtttggg accatcttta 7320ttttcaggag
gaacttccat gtacattgat gacggacgat agaatccgtt tctatcatcc
7380taatgaacat aatgaataaa tccagacaaa cataaacatt aacagagtaa
gcagctttcg 7440gggctggaag ccagaagagg gtgggagcgc agagagagag
gccaaacacc agggctgctt 7500ctgctttgcg ggtatttgct gatctggaca
aggtatctgg aaggctgagc taagcctcct 7560ttttttttga ggtggcgtct
cactctgttg ccaggctgga gtgcaatggt gcgatctcag 7620ctcactgcaa
cctccacctc cctggttcaa gcgattctcc tgcctcagcc tcccgagtag
7680ctgggattac aggctcccgc cactacaccc agctgatttt tgtaatttta
gtagagacgg 7740ggtttcacca tgttggccag gatggtctcg atctcttgac
gtcatgatct gtccacctcg 7800gcctcccaaa gtgctgggat tataggcgtg
acccaccgtg ccccgtctga gctaagcctc 7860ttgagcatag gggactaaaa
atgaaatcta gcgcatgcca agtttagggt cccaggcaat 7920tcctttccac
tttggggtcc actttggggt ccaccccacc caagaagaag gatgacttgg
7980aagtaaacca gctctgaaat atggatggtc ctctgggacc ataccaatcc
cttcatatca 8040accacatcca gttcctcaaa actggaactt ggattaagat
ggcctaggac ttctagtgtc 8100ccaggagcct ggcattgcaa acaaaaatcc
tctccggaag aagataatac cttaagcttc 8160aaatgactct ctaataaatt
tcaaatacaa tgtccagcac acaaacacaa attaccagga 8220acgtgatatg
aggcctgatg gatgggaatt agcagaaact tcaggcatga gaaacatacc
8280ctcagaggcc tagaatctat ctagtgtcta gataatggag atatgaaata
cagacactta 8340aacaactatg tttcccatgt tcaaagagga aatttgcaaa
acttgaaagt gttggcagga 8400aatcagaaac tataaaatgt gacaacagca
tactttagag tcagtataaa ttacggtccc 8460gaaaactgca gaattccaga
acttaatggt aaagcaaggg tttaacagca gaatagaaat 8520agccagagag
aactaggaag taagtcagat gacactaccc agaataaggc actgagaggc
8580caaggaatgg aaaatgcaga agaaaggata tggtgagagg atctaatata
catttatttg 8640gagtaccagg gagagagaga aggagaagaa cagaagccgt
gtttcaagga cggtgactga 8700gaggcttcga aactgatgaa agccatcagt
tcacaaattc aaagcccagt gaattccaag 8760gagaaaaaaa gaaatccata
ctgtgaaagc aagtccagac aatgacaaac accatcaaca 8820atacacagga
caggcataag atgcatttaa tggggacact cagaggcaga gggttatcag
8880aaggaggcac ttctctccca agttctcatc atcccagggc cagggacagc
tggtcacacc 8940ttagggagtt cactaggaga gggatctggc ttcttgtcat
tctgggtatt tgtagggaaa 9000ttggaaggga accgagagca cctagccaat
cgcatagcaa tgggagattt caggctgtgg 9060ggaatgtctt tgctggtgaa
aagaacatcc tgaccttaga aatctttcac cgagggggat 9120ctgcgttcca
gaacttctgg agctggtata ggtaaggctt tgagctttcc tactgagcca
9180gcctgttgct aggttaccaa aggggacctc gagggccatc tggccaacaa
gcagacttgt 9240ctctccttac acccccagac gtatcactgc aaaactacag
aaaaccaaag acagagaaaa 9300tcttaaaagc agccagattt aaaaaatggc
atattagttt caaagcagca gccatgaaat 9360tgacagctga tgtctcaaca
gcaagaatga aaagtggaag acaggccagg tgtggtggct 9420caggcctgta
atcccagcac tttgggaggc cgaggcgggt ggatcacgag gtcaggagac
9480caagaccatc ctggctaaca tggtgaaacc ccgtctctac taaaaataca
aaaaaattag 9540tcgggcatgg tggtgggtgc ctgtagtccc agctactcgg
gaggctgagg caggagaatg 9600gcgtgaaccc gggaggcgga gcttgcagtg
agccgagatt gtgccactgc actccagcct 9660gggtgacaga gcaagactct
gtctcaaaaa aaaaaaaaaa aaaaaaaaaa aaagggtgac 9720gaagcttcaa
tctcctgaaa ggaagcaact gccgcctttg attcgatacc caccaaaatc
9780cgtgaagaag gaaggcaaaa taaaaacact tcctgattga actggaaaga
tttccgcaat 9840agaagaccca ctgtccaagg aattctaaag gatgctttcc
aggcagaaga aaatgacccc 9900agaggaagat cagagattca ggaaagaaat
ggagagtgat aaaaatggaa aattcggggg 9960ccaatttaaa caaaagctga
ctgctctaca actgttgtgt ctctatcttt tgtaacatat 10020atgtgtgtgt
agcttttttt tttttttttg tcaagatgga ttctcactct gtcgcccagg
10080ctacagtgaa atggcacggt ctcggctcac tgcaacctct gccccttggg
ctcaaatgat 10140tctcttgcct cagcctcctg agtagctgag attacaggtg
cctggcacaa tgcctggcta 10200atttttgtat ttttactaga gatgggattt
ctccatgttg gccaggctgg tcttgaacac 10260ctgacctcag gtgatccacc
tgcctgggcc tcccaaagtg ctaggattac aggcgcgagc 10320cactgcatct
ggcctatgtg tgtgtttata tggaattaaa acacatggca ataataccct
10380ccaaattggg agaaaccaaa aatagcattt aaatgttgta agctccctgc
ataatcaaga 10440agagaataga tttacgttag attttgatac ctggaggatg
aatgttgtaa tttctagggt 10500gaccatgaaa agaggagaca acggtgtatg
tttttttttt tttgagatgg agtctcactt 10560tgtcacccag gctggagtgt
tgtggtgtga tcttggctca ctgcaacctc ctcctcttgg 10620gttcaggcca
tcctcccacc taggcctcca gagtaggtgg gatcacaggc acctgccacc
10680acacctggct aatttttttt tttttttaaa tatttagtag agatggggtt
tcaccatgtt 10740ggccaggctg gtcttgaact cctgacctca ggcgatctgc
ctacctctgc ctctcaaagt 10800gctgggatta caggtgtgag ccatcgcgcc
cggccaacag tgatcacttt caaactaaca 10860gaggttcaaa aataaaatca
gacttaacca aaaaccaggt aacagagctg gtaggatata 10920cagaaagact
gacctcacgt atatcaacga ttacagttaa tattaatgaa ggaaatgctc
10980tagtttaaaa acgagggttg tcaaagaccc cacataagaa gctccttacc
agcggtgcac 11040ctagaaccta aggaaacagg acagatgaag gaggacgcgc
ccccgccgct gtcctgcgcc 11100tcagccatcc tatgagacgg gaaaggtttc
tgtctgcagc tgggcccgtg ctctttacca 11160gctcctggct ttcttctctg
gaaggttcct gcctgttttg ccctcacacc tgctcctctc 11220tcagccctct
caggggtggg gctggaggcc
accaaagagc ctcctctgct ctccagttgc 11280tcgactgctc ctcatttccc
cctggggtct gcgtcagggt ttccttcttt tccagcccca 11340ccccgcgtgc
atcccacctg gtctcgggtc ggggctgctc ccgcttactg ccccctgccc
11400aggctggtgt gcaccccctc tggctgcttt caaggcctct tctctcttct
cggcaggaca 11460ggcacaggca ggtggccagg tgtcatgctt agctccccgc
ccagtgagat tctttcattt 11520aacaatcttc ccctgaatag ttcatgttca
ttgctgaaaa tttgaaaaat atggaaaagc 11580acaaagatta agatataaac
cgccctcaat tcccctgccc agagagagtc actgctatga 11640cttggtgact
aggaacctta tttctctctc gctctttttt ttttttttga gacagagtct
11700tgctctgtca cccaggctgg agtgcagtgg ctcgatctca gctcactgca
acctccgcct 11760cctgggttca agcgattctc ctgcctcagc ctcttgagta
gctgggatta caggcacctg 11820ccaccatgcc cggctaattt ttgtattttt
agttgagaga gggtttcatc ttgttggtca 11880ggcggacttg aactcctgac
ctcaggtgat cagcccacct cggcctccca aagtgctggg 11940attacaggtg
tgagccactg cgccttcatc tctcttctgt gtatgtgtac gctgtttttt
12000ctttagaatg ggggacgtta tcaggctcta catggtgtgt agtcggctag
catgttgtaa 12060gcctttccct gtgtcacaag tgctcatctg gaacaggatt
ctaatgactg cctgtggcta 12120tgttgggatt cctttaactc agctccttct
gcccagcatc tatctttttt ccatcttttg 12180tcctaagtgt tgctataata
aatcattgat cacacatgcc tgactgtttg cataggataa 12240attacgggaa
atgtttttgc tgttcaggga ctgtgcccat ttttaggcct cagagacacc
12300atgccagact gcccagtatt gatctttact ctttttagat gatgccaaac
ttttctgtga 12360actttaaaaa cctgtgtctt gacagtccat ttctgtaagt
ctttcacatt agatttcctg 12420tcaggatgat agtcaattct aggcagatga
tgttttctca gccatggctg aagcagttgt 12480gatttgttgt ggccatgtaa
agtcccgatg atccattgcc tccctggatg ggttggaata 12540atttggtttg
ggagcatata acagaatgac ctggagtcac agcagctcag acggaagtgt
12600atttctccct tacagatgaa agaattccag gccaggctgg aatgacaact
gcacacagtc 12660atctgggccc cctccttcca gctcccatca ccccaggatg
tggcttttat gcagatgatc 12720caaaatggct gctcaagtcc cagccaacac
atcccattcc agggagcagg aaaaaggtgt 12780gtctttccct tcattttatg
tgattccttt ctagaagtac tactcattac ttctgcttgc 12840atctccctgg
ctagcactta cttagttata tggccatagc tagctgaagg aaggacaggg
12900actgtcatac actagctaag aggcaaactg cttagataaa aaggtctcta
aagaaggtca 12960gagcggctgc tagggtgcaa ctctattact tattgttatg
ggacgaactg tgtccctcat 13020tcaggttgat gtcctaagcc ccagaacctc
agaatgggat tgtatttgga gacaggttct 13080ttaaggaggt aaggaggcta
aaatgagatc attagggtgg gccataatcc gactgatgtc 13140ttacaagaag
agattaggac acggacatgc tcagagggac ggccacgtga ggacaccaag
13200aaaggcagct gtctgcaagt caaggacagg gctcagggga aaccaacctt
gccaacacct 13260tcatctcgga cttctagcct ctaggaccat gagaagatac
atttctgttg tttaagctgc 13320ccggtctgtg gtactttgtt atggcagccc
aagtaaacaa atacagtcat ctgctgctgg 13380aacaaatcac cccagcactg
tggcttggca gcacacatgt ctagtcatag agttatatgt 13440agttacgtgt
agagccatat gtatcgtcac acgttctgtg ggtcaggaat ttggacccag
13500cttaaccagc tccacttctc gccagggttc agtcaaatac cagctgcctc
ccacctgaga 13560gctcagccgg ggaagggtcc ctttccaatc tcacgtggtg
ttggcaggat ccagttcctc 13620atggcctgct ggactgagaa cctcagttct
cactgcctgt tggccagagg ccgcctttat 13680gtcctcgcca tgtgggcctc
tccaacatgg cagctgactt catcagagca tccatgccaa 13740gaaggcaaca
gagagggcca gggagactga agtcataccc ttttgcgacc tagtcatggg
13800gtgacattcc atcacctttg cccattggtt agaagcaggc caccaggtac
agcccaagct 13860cacggggagg ggtcatacaa gggtgtcaat accaggaggt
gaggggtgct ggggccatct 13920tatgagtctg cccactgagg taactaacaa
ccttgaggcc tgacacagtg gacaaaggcc 13980cttattaaca gcagagaact
gggaacttta tttatttatt tatttttgag acagagtctc 14040actcttgtca
cccaggctgg agtgcaatgg catgatcttg gctcactgca acctccacct
14100cccaggttca agcaattctg cctcagcctc cggaatagct gggactacag
gcatgcacca 14160ctacacccgg ctaatttttg tatttttagt agagacaggg
tttcgccatg ttggccaggc 14220tggtctcgaa ctcctgacct ctggtgatct
gcctgccttg gcctcccaaa gtgctgggat 14280tacaggcgtg agccaccgca
cctcgctgga acttaatttt tttagagaca gtgtcgctct 14340atcacccaag
ctggagtgca gtggtgcaat cctagctcac ttgcagcctc aaattcctgg
14400gttcaggtga tcctcccaca tcagcctccc aagaactggg aactaacagc
tgtttctctg 14460ctgtccttct caagaaaagg gaggctactg ctaccccact
ggggacaatg ctgggtttcc 14520ctttaggaca ggctctgaga caaggcggag
gtgctgtttg tggccacaga gcaggggact 14580ctgggttgca ggtgtggcct
ggctaaagta ggctttactg ggctcctctc tgcctgcatc 14640accccccggc
tgggcggttg tctctgaggc caaccttact ccctgctggg caggctggac
14700agctgccctc tccgtttgcc cctctaccac ccaaaaggca ggaggctctg
gagaccagga 14760ccctgcccgc cacggcctgt gtcccaggcg tgagggggtg
ccccacagac ctctgctgag 14820ctgctgctga atgacgcccc ttgggggtcc
tgccggaagg tcagagcagg ggtgcactcc 14880cataaagaaa cgcccccagg
tcgggactca ttcctgtggg cggcatcttg tggccatagc 14940tgcttctcgc
tgcactaatc acagtgcctc tgtgggcagc aggcgctgac cacccaggcc
15000tgccccagac cctctcctcc cttccggggc gctgcgctgg gaccgatggg
gggcgccagg 15060cctgtggaca ccgccctgca ggggcctctc cagctcactg
ggggtggggt gggggtcaca 15120cttggggtcc tcaggtcgtg ccgaccacgc
gcattctctg cgctctgcgc aggagctcgc 15180ccaccctctc cccgtgcaga
gagccccgca gctggctccc cgcagggctg tccgggtgag 15240tatggctctg
gccacgggcc agtgtggcgg gagggcaaac cccaaggcca cctcggctca
15300gagtccacgg ccggctgtcg ccccgctcca ggcgtcggcg ggggatcctt
tccgcatggg 15360cctgcgcccg cgctcggcgc cccctccacg gccccgcccc
gtccatggcc ccgtccttca 15420tgggcgagcc cctccatggc cctgcccctc
cgcgccccac ccctccctcg ccccacctct 15480caccttcctg ccccgccccc
agcctcccca cccctcaccg gccagtcccc tcccctatcc 15540cgctccgccc
ctcagccgcc ccgcccctca gccggcctgc ctaatgtccc cgtccccagc
15600atcgccccgc cccgcccccg tctcgccccg cccctcaggc ggcctccctg
ctgtgccccg 15660ccccggcctc gccacgcccc tacctcacca cgccccccgc
atcgccacgc cccccgcatc 15720gccacgcctc ccttaccatg cagtcccgcc
ccgtcccttc ctcgtcccgc ctcgccgcga 15780cacttcacac acagcttcgc
ctcaccccat tacagtctca ccacgccccg tcccctctcc 15840gttgagcccc
gcgccttcgc ccgggtgggg cgctgcgctg tcagcggcct tgctgtgtga
15900ggcagaacct gcgggggcag gggcgggctg gttccctggc cagccattgg
cagagtccgc 15960aggctagggc tgtcaatcat gctggccggc gtggccccgc
ctccgccggc gcggccccgc 16020ctccgccggc gcagcgtctg ggacgcaagg
cgccgtgggg gctgccggga cgggtccaag 16080atggacggcc gctcaggttc
tgcttttacc tgcggcccag agccccattc attgccccgg 16140tgctgagcgg
cgccgcgagt cggcccgagg cctccgggga ctgccgtgcc gggcgggaga
16200ccgccatggc gaccctggaa aagctgatga aggccttcga gtccctcaag
tccttccagc 16260agcagcagca gcagcagcag cagcagcagc agcagcagca
gcagcagcag cagcaacagc 16320cgccaccgcc gccgccgccg ccgccgcctc
ctcagcttcc tcagccgccg ccgcaggcac 16380agccgctgct gcctcagccg
cagccgcccc cgccgccgcc cccgccgcca cccggcccgg 16440ctgtggctga
ggagccgctg caccgaccgt gagtttgggc ccgctgcagc tccctgtccc
16500ggcgggtccc aggctacggc ggggatggcg gtaaccctgc agcctgcggg
ccggcgacac 16560gaacccccgg ccccgcagag acagagtgac ccagcaaccc
agagcccatg agggacaccc 16620gccccctcct ggggcgaggc cttcccccac
ttcagccccg ctccctcact tgggtcttcc 16680cttgtcctct cgcgagggga
ggcagagcct tgttggggcc tgtcctgaat tcaccgaggg 16740gagtcacggc
ctcagccctc tcgcccttcg caggatgcga agagttgggg cgagaacttg
16800tttcttttta tttgcgagaa accagggcgg gggttctttt aactgcgttg
tgaagagaac 16860ttggaggagc cgagatttgc tcagtgccac ttccctcttc
tagtctgaga gggaagaggg 16920ctgggggcgc gggacacttc gagaggaggc
ggggtttgga gctggagaga tgtgggggca 16980gtggatgaca taatgctttt
aggacgcctc ggcgggagtg gcggggcagg gggggggcgg 17040ggagtgaggg
cgcgtccaat gggagatttc ttttcctagt ggcacttaaa acagcctgag
17100atttgaggct cttcctacat tgtcaggaca tttcatttag ttcatgatca
cggtggtagt 17160aacacgattt taagcaccac ctaagagatc tgctcatcta
agcctaagtt ggtctgcagg 17220cgtttgaatg agttgtggtt gccaagtaaa
gtggtgaact tacgtggtga ttaatgaaat 17280tatcttaaat attaggaaga
gttgattgaa gttttttgcc tatgtgtgtt gggaataaaa 17340ccaacacgtt
gctgatgggg aggttaattg ccgagggatg aatgaggtgt acattttacc
17400agtattccag tcaggcttgc cagaatacgg ggggtccgca gactccgtgg
gcatctcaga 17460tgtgccagtg aaagggtttc tgtttgcttc attgctgaca
gcttgttact ttttggaagc 17520taggggtttc tgttgcttgt tcttggggag
aatttttgaa acaggaaaag agagaccatt 17580aaaacatcta gcggaacccc
aggactttcc ctggaagtct gtgtgtcgag tgtacagtag 17640gagttaggaa
gtactctggt gcagttcagg cctttctctt acctctcagt attctatttc
17700cgatctggat gtgtcccaga tggcatttgg taagaatatc tctgttaaga
ctgattaatt 17760tttagtaata tttcttgttc tttgtttctg ttatgatcct
tgtctcgtct tcaaagttta 17820attagaaaat gattcggaga gcagtgttag
cttatttgtt ggaataaaat ttaggaataa 17880attattctaa aggatggaaa
aactttttgg atatttggag aaattttaaa acaatttggc 17940ttatctcttc
agtaagtaat ttctcatcca gaaatttact gtagtgcttt tctaggaggt
18000aggtgtcata aaagttcaca cattgcatgt atcttgtgta aacactaaac
agggctcctg 18060atgggaagga agacctttct gctgggctgc ttcagacact
tgatcattct aaaaatatgc 18120cttctctttc ttatgctgat ttgacagaac
ctgcatttgc ttatcttcaa aatatgggta 18180tcaagaaatt tcctttgctg
ccttgacaaa ggagatagat tttgtttcat tactttaagg 18240taatatatga
ttaccttatt taaaaaattt aatcaggact ggcaaggtgg cttacacctt
18300taatccgagc actttgggag gcctaggtgg acgaatcacc tgaggtcagg
agtttgagac 18360cagcctggct aacatggtga aaccctgtct ctactaaaaa
tacaaaaatt agctggtcat 18420ggtggcacgt gcctgtaatc caagctacct
gggaggctga ggcaggaaaa tcgcttgaac 18480ccgggaggca gagtctgcag
tgagttgaga tcacgccact gcactccagc ctgggtgaca 18540gagcgagact
ctatctcaaa aaaaattttt tttaatgtat tatttttgca taagtaatac
18600attgacatga tacaaattct gtaattacaa aagggcaata attaaaatat
cttccttcca 18660cccctttcct ctgagtacct aactttgtcc ccaagaacaa
gcactatttc agttcctcat 18720gtatcctgcc agatataacc tgttcatatt
gtaagataga tttaaaatgc tctaaaaaca 18780aaagtagttt agaataatat
atatctatat attttttgag atgtagtctc acattgtcac 18840ccaggctgga
gtgcagtgat acaatctcgg ctcactgcag tctctgcctc ccaggttcaa
18900atgcttctcc tgcctcagcc ttctgagtag ctgggattac aggcgcccac
caccatgtcc 18960agctaatttt tgtattttta gtagagatgg ggtttcacca
tgttggccag gctggtcttg 19020aactcctgac cttgtgatct gtccacctcg
gcctcccaaa gtgctgggat tacaggtgtg 19080agccaccatg cctggctaga
ataataactt ttaaaggttc ttagcatgct ctgaaatcaa 19140ctgcattagg
tttatttata gttttatagt tattttaaat aaaatgcata tttgtcatat
19200ttctctgtat tttgctgttg agaaaggagg tattcactaa ttttgagtaa
caaacactgc 19260tcacaaagtt tggattttgg cagttctgtt cacgtgcttc
agccaaaaaa tcctcttctc 19320aaagtaagat tgatgaaagc aatttagaaa
gtatctgttc tgtttttatg gctcttgctc 19380tttggtgtgg aactgtggtg
tcacgccatg catgggcctc agtttatgag tgtttgtgct 19440ctgctcagca
tacaggatgc aggagttcct tatggggctg gctgcaggct cagcaaatct
19500agcatgcttg ggagggtcct cacagtaatt aggaggcaat taatacttgc
ttctggcagt 19560ttcttattct ccttcagatt cctatctggt gtttccctga
ctttattcat tcatcagtaa 19620atatttacta aacatgtact atgtgcctgg
cactgttata ggtgcagggc tcagcagtga 19680gcagacaaag ctctgccctc
gtgaagcttt cattctaatg aaggacatag acagtaagca 19740agatagataa
gtaaaatata cagtacgtta atacgtggag gaacttcaaa gcagggaagg
19800ggatagggaa atgtcagggt taatcgagtg ttaacttatt tttattttta
aaaaaattgt 19860taagggcttt ccagcaaaac ccagaaagcc tgctagacaa
attccaaaag agctgtagca 19920ctaagtgttg acatttttat tttattttgt
tttgttttgt tttttttgag acagttcttg 19980ctctatcagc caggctggag
tgcactagtg tgatcttggc tcactgcaac ctctgcctct 20040tgggttcaag
tgattctcat gcctcagcct cctgtttagc tgggattata gacatgcact
20100gccatgcctg ggtaattttt tttttttccc ccgagacgga gtcttgctct
gtcgcccagg 20160ctggagtgca gtggcgcgat ctcagctcac tgcaagctcc
gcttcccgag ttcacgccat 20220tctcctgcct cagtctccca agtagctggg
actacaggcg cctgccacca cgtccagcta 20280atttttttgt atttttaata
gagacggggt ttcaccgtgt tagccaggat gatcttgatc 20340tcctgacctc
gtcatccgcc gaccttgtga tccgcccacc tcggcctccc aaagtgctgg
20400gattacaggc atgagccact gtgcccggcc acgcctgggt aatttttgta
tttttagtag 20460agatggggtt ttgccatgat gagcaggctg gtctcgaact
cccggcctca tgtgatctgc 20520ctgccttggc ctcccaaagt gctaggatta
caggcatgag ccaccatacc tggccagtgt 20580tgatatttta aatacggtgt
tcagggaagg tccactgaga agacagcttt tttttttttt 20640ttttttgggg
ttggggggca aggtcttgct ctttaaccca ggctggaatg cagtatcact
20700atcgtagctc acttcagcct tgaactcctg ggctcaagtg atcctcccac
ctcaacctca 20760caatgtgttg ggactatagg tgtgagccat cacacctggc
cagatgatgg cttttgagta 20820aagacctcaa gcgagttaag agtctagtgt
aagggtgtat gaagtagtgg tattccagat 20880ggggggaaca ggtccaaaat
cttcctgttt caggaatagc aaggatgtca ttttagttgg 20940gtgaattgag
tgagggggac atttgtagta agaagtaagg tccaagaggt caagggagtg
21000ccatatcaga ccaatactac ttgccttgta gatggaataa agatattggc
atttatgtga 21060gtgagatggg atgtcactgg aggattagag cagaggagta
gcatgatctg aatttcaatc 21120ttaagtgaac tctggctgac aacagagtga
aggggaacac cggcaaaagc agaaaccagt 21180taggaagcca ctgcagtgct
cagataagca tggtgggttc tgtcagggta ccggctgtcg 21240gctgtgggca
gtgtgaggaa tgactgactg gattttgaat gcggaaccaa ctgcacttgt
21300tgaactctgc taagtataac aatttagcag tagcttgcgt tatcaggttt
gtattcagct 21360gcaagtaaca gaaaatcctg ctgcaatagc ttaaactggt
aacaagcaag agcttatcag 21420aagacaaaaa taagtctggg gaaattcaac
aataagttaa ggaacccagg ctctttcttt 21480tttttttttt tgaaacggag
tttcgctctt gtcacccggg ctggagtgca atgatgtgat 21540ctcagctcac
taaaacctct acctcctggg ttcaagtgat tcttctgcct cagcctccca
21600agtaactggg attacaggcg tataccacca tgcccagcta atttttgtgt
ttttagtaga 21660gatggggttt caccatgttg gccaggctgg tctcgaactt
ctgacctcag gtgatccact 21720cgcctcagcc tgccaaagtg ctgggattac
aggtttgggc cactgcaccc ggtcagaacc 21780caggctcttt cttatactta
ccttgcaaac ccttgttctc attttttccc tttgtatttt 21840tattgttgaa
ttgtaatagt tctttatata ttctggatac tggattctta tcagatagat
21900gatttgtaaa aactctccct tcctttggat tgtcttttta ctttcttgat
agtgtctttt 21960gaagtgtaaa agtttttaat tttgatgaag tcgagtttat
ctattttgtc tttggttgct 22020gtgcttcaag tgtcatatct aagaaatcat
tgtctaatcc aaagtcaaaa aggtttactc 22080ctatgttttc ttctaagaat
tttagagttt tacatttaag tctgatccat tttgagttaa 22140tttttatata
tggttcaggt agaagtccaa ctttattctt ttccatgtgg ttattcagtt
22200gtcccagcac tgtttgttga agagactatt ctttccccat ggaattatct
tagtaccctt 22260gttgaaaatt aatcgtcctt aattgtataa atttatttct
agactgtcag ttctacctgt 22320tggtctttat gtcgatcctg tgccagtacc
atacagtctt gattactgaa gtttgtgtca 22380cagtttaaat tcatgaaatg
tgagttctcc aactttgttc cttttcaaga ttgatttggc 22440catgctgggt
cccttgcatt tccgtacgaa ttgtaggatc agcttgtcag tttcaacaaa
22500gaagccaagt aggattctga gagggattgt gttgaatctg tagatcaact
tggggagtat 22560tcgcatctta acaatattgt cttccaccta tgaacatggg
caaactttgt gtaaatggtc 22620agattgtaag tatttcgggc tgtgtgggca
cagtgtctct gtcacagcta cgcggctctg 22680ccattgtagc atgaaagtag
ccataagcaa tatgtatgag tgtctgtgtt ccaatagaat 22740tttattaatg
acaaggaagt ttgaatttca tataattttc acctgtcatg agatagtatt
22800tgattatttt ggtcaaccat ttaaaaatgt aaaaacattt cttagcttgt
gaactagcca 22860aaaatatgca ggttatagtt ttcccactcc taggttaaaa
tatgatagga ccacatttgg 22920aaagcatttc tttttttttt tttttttttt
tttttgagac ggagtttcac tcttgttgcc 22980caggctggag tgcagtggcg
cgatctcggc tcactgcaac ctctgcctcc caggttcaag 23040acattctcct
gcacggcctc cctagtagct gggattacag gcatgcgcca ccacacccag
23100ctaattttgt atttttagta gagacggggt ttctccatgt tggtcaggct
ggtcttgaac 23160tcctgacctc aggtgatcca cccgcctcag cctcccaaag
tgctgggatt acagggtgtg 23220agccaccaca ccctgctgga aagcatttct
tttttggctg tttttgtttt ttttttaaac 23280tagttttgaa aattataaaa
gttacacata tacattataa aaatatcttc aagcagcaca 23340gatgaaaaac
aaagcccttc ttgcaagtct gtcatctttg tctaacttcc taagaacaaa
23400agtgtttctt gtgtcttctt cccagatttt aatatgcata tacaagcatt
taaatgtgtc 23460attttttgtt tgcttgactg agatcacatt acatatgtat
ttttttactt aacaatgtgt 23520catagatatt gttccatagc agtacctgta
attcttatta attgctatgt aatattttag 23580aatttctttt taaaagagga
cttttggaga tgtaaaggca aaggtctcac atttttgtgg 23640ctgtagaatg
tgctggtgac atattctctc taccttgaga agtccccatc cccatcacct
23700ccatttcctg taaataagtc aaccacttga taaactacct ttgaatggat
ccacactcaa 23760aacatttagt cttattcaga caacaaggag gaaaaataaa
ataccttata aagcactgtt 23820taatattgta ttaaattgga tcaatttggg
ggctagaatg tatgttagag acatgatatg 23880tccataggtc cttgctatca
cagtgaggtc tcagggacag tcgtttggta tcatttggga 23940tctcataagc
agactctctc tgcttgacct gacaaatcag agtctgtgtt ttaacaggtt
24000cagtgagtga cttacatgca cattggagtt tgggaagctc cactgtaggt
gcttagacct 24060tacctttgtt gttgctaata acaatgcaag catttgggag
gaagacctgt gttgctcata 24120tgtgtccagg tgtagctgag gtggccttgc
ttatctgctg tagggccgtt gagcatttct 24180gtagctgtga tgagtgagct
gaggtgagcc tgcggagagc tcccagccat tggtagtggg 24240actcgcttag
atgaactgga aggacccttt catctgagca gccactatgg agaaaaacaa
24300ccgaatgagg ggagagacaa tgtgcaattt tatttagggc acaaaggaga
gctgtggtta 24360gaaggtgaca tttgagtgga aagggggcaa gccatgtgta
tagcgggaga agagaggtcc 24420aggcagagtt aacagaaggc agaaatgctt
tccatgtttg agaaccagta aggaggccag 24480tggctgaagt aaggtgaagg
gcagaaataa ggatgaggct gcgagagatg agaggttaga 24540gacgagcgtc
ttgtgcacca agataagctt gtgtggtcaa aacaagtagt ttaatttatg
24600tttttaaaag atcattttgg ctgggcacaa tggttcatgc ctgtaatacc
agtagtttga 24660gacggtgtgg tgggaggatt gcctgaggcc agacgaccag
catagccaac atagcagcac 24720ctataaggtc tctacaaaaa actttaaaaa
attagctggg catagtggtg tgtgcctgta 24780gtcccagcta ctcaggaggc
tgaggaggct ggaggattgc ttgagtccag gagtttgagg 24840ctgcagtgag
ctatgattat gccactacac tacaacctgg gcaagagagt gagaccctgt
24900ctctaaatat acacacacac acacacacac acacacacac acacacacac
acacacacac 24960acacacatat atatgtatat atatgcattt agatgaaaag
atcactttga caataccaca 25020tgctggtgag gatttagaaa aactaggtca
cttattgctg gtgggaatat aatatagtac 25080ggccactctg gaaaacagtt
tggcagtttg tcataaaact gaacataccg ttagtataca 25140gcccagcagc
aactacaatc ctgggcatta atcctagaga aatgaaacct taatgttcac
25200ataaaaacct atactcaagt atgcatagca gctttaccca taatatctaa
gaactggaat 25260cagctcagat gtccttcaac aggtgaatgg ttaaactact
cagtaataaa aaggaatgag 25320ctactgatag catgcaacag tttaggtgaa
gttatgctaa tgaaaaaagc caatcccaaa 25380aggttataca tactgtatga
ttctatgttt ttttgcaatg gcacagtttt agggatggag 25440aatagattag
tggttgcctg gggttagaga tggggtagta gagtaggtta gtggtggcag
25500aggagagaaa agagagggag gtgaatgtgg ttataaaagg acaacacagg
ggaatacttg 25560taatggaaat gctttgtctt tttttttttt tttttttttt
tggcgacaga gtcttgctct 25620gttgcccagg ctggagtgca gtggcatgat
cttttctcac tgcaacctct gcctcctggg 25680ttcaagtgat acttgtgtct
cagtctccca tgttcagagt gaaacaaacc agaggtaatg 25740ttcatccaaa
taatccaaca cacatgacat taaaacatca agatcaggtc ggacgtggtg
25800gctcatgcct gtaatcccag cacttttggg aggccaaggt gggcagatca
cttgaggtca 25860ggagttcgag accagccggg ccaacatgat gaaaccccat
cttgactaaa aatacaaaaa 25920ttagccgggc atggtggtgt gcacctgtag
tcccagctac ttgggaggct gaggcaagag 25980aactgcttga acccgagggg
cagaggttgc agtgagctga gagtgcgcca ttgcacttca 26040gcctgtgtga
cagagtaaga ctccatctcc aaaaaaaaaa aaccaagatc aattaaaata
26100cagcattact gggccgggtg tggtggctca cacctgtaat cccagcactt
tgggaggccg 26160agatgggcag atcacgaggt caggagatcc agaccatccc
ggctaacacg gtgaaacccc 26220gtctctacta aaaaatacaa aaaattagcc
gggtatagtg gtgggtgcct gtagtcccag 26280ctacttggga ggctgaagca
ggagaatggt
gtgaacccgg gaggcagagc tggcagtgag 26340ctgagatcgc gccactgcac
tccagcctgg gcgacagagc aagactccgt ctcgggggaa 26400aaaaaaaaat
aaataaatag aatgctgtag tgtccttgag tttacatgcc cctccttacg
26460cttgtgtgcc cgtgcagatt gcttgattac acaattagag gaggctggcg
gaggattgtt 26520ttaatttttt tttttttgag acagtctggc tctgttcccc
aggctagagt gcaatggcgc 26580aatcttggtg cactgcaacc tctgcctcct
gggttcaagc agttcttctg ccgcagcctc 26640ccgagtagct gggattatag
gcgcccgcca ccacgcccaa ctattttttg tatttttagt 26700agagcagcgt
ttcaccatgc tggccaggct ggtctcgaac tcctgacctc agatgatctg
26760ctgccccagc ctcccaaagt gctgggatta caggcgtgag ccacacctgg
ccgtttgttt 26820taattttgaa ggtgaagtga aagtgactac atttaccaaa
agtgattgaa aagccaggac 26880tgttcttacc ctgtttttcc agttcttgct
cagagcaagg tggtttcttt ttcacttaat 26940caccatactt acttttcatg
tagaacaagt cagtttgagt tatcagttca tcatcttaac 27000taaattccat
gggggaagga attagtttta gtttcttaaa cttccaggtt tgcttattgg
27060acaaaatgag atagcaaggc agtgttttta agttagattt tttatttctt
tggtaataca 27120attttctcag aaacttagta gtcttttagt ttagttgttt
ttagttggtc ctatgttttg 27180gatcacccct ctctacttta ttttgatagt
gccaactgtg aagacatctg aagccatagg 27240tttggatggg aaggaggcat
ctttagcctg atcatcttcg ccaggctgtt tatctccttt 27300tgcttggctg
agaagtctta ataggaggct tattcccagc tatttgggga catagaagca
27360gttagccatt gcttatattt tactgaggtc tgtgtggtat gttgattgta
gtcagttaac 27420gattttgaga actgaaggca gcctggtata tatagagtag
gtattagact gtgtttcttc 27480taattgaatt tcccatctct tgtaatctat
gccatcatct tctgtactgc tgagaaagaa 27540agaaagtttc taatcaaact
ataccactgg ttgtaagatg cagtttggct ttagtgatgt 27600taacacatga
ttcaaacgtg aaattgattg agtattggtg aaatacagag gagatttaaa
27660gccagaagac ctgggtttaa atgctggctg tatgacttca tatctgtgtg
atcttgggca 27720tgtcatggtt ggcacttcaa tttcttctct ctataatggg
ggaagtgagg ccagtcatgg 27780tggctcatac ctataatccc agtgctttgg
gaggccaaga tgggaagatc gcttgaggcc 27840aggagtttga gcaattgggc
aacatcgtga ggccccgtct ctacaaaata ttttgaaaaa 27900attagccagg
cccagtggtg cgtgcctgtg gtccgcgcca ctcaggaggc tgagacggga
27960ggatcctttc agcctaggag tttaaggcta aagtgagcca tgattgtgct
atcgtactcc 28020agcctgggca gcagagcaag atcctgactc taaaaaaaag
taaaataaag taaaatgggg 28080gaaatgaact gctttagtaa catcatctgt
tttttctgtg agcagcgtag cttgacagcc 28140attggtgaac tcgtgccctg
tgcttccctg tccagatccc cattctgccc gcaacatgga 28200gtataacggt
ttattcatag tagtcgagaa acactcactg aatgaatgaa tgaggtgtag
28260aactaagtgg agtgggtaat tcaacacata ttaatttcct tctttttttt
atttttagaa 28320agaaagaact ttcagctacc aagaaagacc gtgtgaatca
ttgtctgaca atatgtgaaa 28380acatagtggc acagtctgtc aggtaattgc
actttgaact gtctagagaa aataagaact 28440ttgtatattt tcagtcttaa
tgggctagaa tattctttgt gtcccagcta ttttaaatgg 28500attcagaaat
ccatttaaga tgaagaagga cccttttccc atatttctgg ctatatacaa
28560ggatatccag acactgaaat gaataatgtt ccctttttgt aatcttttat
gcaaaaatta 28620aaaccattat ggtaattgaa caacatgttt atgtttagtt
aacaccctta gcaactatag 28680ttattttaaa accatctatg gtttgatatt
tttgcatttg ttgcaatagt aggaacagca 28740caagacagtt cagtttgtct
ctcttatttg ctttttcttg gcagtttgct gtcctattgt 28800acctctgctc
ctagcagtgg ctggagccca ctcctctgtg cttcgggatt agtggggatc
28860gtggggcatt gactgtaggt cagctttcct tgcttgatct ttctcactgg
gatgaactag 28920cagcaccttc ttttgtagct gctttgcttt tgactatctt
tctgaccgtt gttcctagta 28980gctgtagatg gtaaatatat ttaggcctgt
ttccaatggc tcagtaggag acatattcac 29040ctatgatatc tgaattctgt
tacccacatg ggcatgcgtg aaatagttgc cttgccttac 29100tttcccttgg
aataaataat tcatgttatt ctcctggtag aagctagaaa aagcctttat
29160agtcagtcag aaaaaaattt ttagacaaat aatcttgatt ttagtactga
caaaaacgtg 29220tggtgattct ttttttaatt tttttttgag acggagtttc
actcttgttg cccaggctgg 29280agtgcaatgg cgtgatctcg gctcactgca
acctctgcct cctgggttca agtgattctc 29340ctgcctcagc ctcccaagta
gctggagtta caggcatgtg ctactgtgcc cagctaattt 29400tgtattttta
gtagagatgt tggtcaggct gatctcgaac tcccaacctt aggtgatctg
29460cccgcctcag cctcccaaag tgctgggatt acaggcgtga gccagggcgc
ccggtgattc 29520atttgttttt tcaaaaaatt tcctcttggc cattgctttt
cacttttgtt tttttttttt 29580ttttgagacg gagtcacgat ctgtcaccca
ggctggagtg cagtggcatg atcttggctt 29640actgcaagct ctgcctccca
ggttcacgcc attctcctgc ttcagcctgg cgagtagctg 29700ggactacagg
tgctcgccac cacacccggc taattttttg tatttttagt agagatgggg
29760tttcaccgtg gtcttgatct cctgacctca tgacccgctc aactcagcct
cccaaagtgc 29820tgggattaca ggcgtgagcc accgcgcccg gccctctctt
gtctttttat tgtggtaaaa 29880tgcacataaa attgactgtc ttaaccattt
ttaggggtac agttcagtat atatattcgt 29940aatgttgtac agccatcact
gccatctact tcataagttt ttcttctgtc aaaactgaac 30000atctgtcttc
attaaactcc ctatcatcca ttctttcctg tagtcccttt ctactttctg
30060tctgtatgag tgtaactgct ctggagacct catgtaagtg gattcctaca
ggatttgtgt 30120tttttttttg gtgatctgct tatttttaat gcctctgtgc
atttgtatta tatactttca 30180aagtgatttc acaaaaccgt ttcattttag
gttaactcat ttctgttgtt tgtgaaatac 30240tgtgtatgat tctgttctgt
ttctgtctaa tttgtggaaa tgttgtggga agaaaatgaa 30300ataacaaatg
agcatatgtc ctgaaaataa aaatataaaa attctaagtt agcatgctat
30360tgtagaatac aacgctatga taaaagtagg aaaaaaaaag gtttgaattc
tatctctgct 30420acctgtgtaa gctgggtgac tttagataag ctgtaacgtg
tttgagcctt actggctcat 30480ttttgaaatg taatccctag ttacacagtt
cttgtgggat cagatggtac atgtgaaaca 30540ctgtgaaaaa gcaactgcat
agatatgttc attagccacc tgagcgggaa gcgtatccca 30600ttgcgatgcc
catcatccaa agctatatgt tatctttact tttttttttt tgagacagag
30660tcttgctctg ttgcccaggc tagagtgcag tggtgcaatc tcagctcact
gcaagctcca 30720cctcccgggt tcacgctatt ctcctgcccc agcctcccaa
gtagctggga ctacaggcac 30780ccgccaccat gcctggctaa atttttgtat
ttttagtaga gatggggttt caccgtgtta 30840gccaggatgg tcttgatctc
ctgacctcgt gatccgcccg cctcggcctc ccaaagtgct 30900gggattacag
gcgtgagcca ctgcccctgg ccatctttac tttttttgtg aaatgacttt
30960aaatacttgg caaacatttg gtcattgttc atctgatctc caccatccag
gtctcagaga 31020acataatttc tctctgaaag cttattgacc caggaaataa
gatctctttc aatctgagtg 31080cgtcaggctt tattcttgtc attttgtctt
ttgataattt tcaaatggaa ttcatggaat 31140gttggcttat attcatatat
tagtaaagta tgttgagaca tcttaagatt gatttgtggt 31200tctatatgcc
atattaaatc aaaataatag ctgttaatgg ttttcacatt agtctgtctc
31260ttgtttttat ggagtaatgc tgagagttca ttatgcttgt tctacagaag
agcatgttaa 31320aaggagtttt tggagtcaga gaggttattc ttggtttcat
aggatacact ctatactttt 31380tagggatttc agagtatata gctgaaggtg
atattttatg taaatatgtt ttatggaaac 31440ttattgctca tcgctgtttc
ctgttaactc tcctaaaata taattaaact tttggaactt 31500ttttatagct
tttgtgctag actaattttt gtctctaatg aggttatata aatggcagct
31560tctgacgttt tcaatgtagg aagtcattta aaacttcatg tatattgtga
aaatgtagtc 31620tgctttaagc tctctaaagt ggtctaagtt actggttcct
aagtatggat gagcatcaaa 31680atcatctgga aaatttgtta aaaatacagt
aatgaaggca cctcactgtc ctttttccca 31740aacatacttc tgcattctgt
ttgagtaggt agggactaca catttttcac aagtatcctc 31800ttgggaatac
ccaggaatgc ttacttgagc aacctcttac taatatgtac cttgataagg
31860tggctaggta aacataaata tacaaaaatc catagatctc ccatatatta
gcataaatca 31920gctagaaaat ataacgttta aagatctagt tcacagtagc
accaatatat cgaactctaa 31980ggaatcgata aatatgcaaa aactttataa
aaacttctgt taatgtttct gaaagatata 32040ggtgaccact ttctagatag
gaagatttta tattactaag ttgaattttc tctaaattaa 32100cacagaaatt
taaaataatc ttgatcaaaa ttctagtaga ggtatttttg aacttgttca
32160ctgcaagaat aaatacataa ttgcaaagaa tatctcaaaa tcatcaccag
gcctggtgtg 32220gtggcccatg cctgtaatcc cagcactttg ggaggctgag
gcaggcagat cacctgaggt 32280caagagtttg agaccagctg gaccagtgcg
gtgaaacact gcctctacta aaaatacaaa 32340aattagctgg gtgtggtggt
gcatgcctgt agtcccagct acttgggagg ctgaggcagg 32400agaattgctt
gaacccagga ggtacaggtt gcggtgagcc tagatcgcac cactgcattc
32460cagcctgggc gacaagagca aaattctgtc tcaagaaaaa agagaaaaaa
gaaaaagaaa 32520tcaacactaa tatggtgaga cttaatgtat gtgacattaa
aatagtgatt ggatgttaaa 32580acaggtatag aacagaaaga agagtgtatg
tgtgtatctg tatgaattta tgatgggtgt 32640aacatatatg tattagggaa
atgagggaaa tgatacattt ctctgacttt gggagaacat 32700tatatctcta
cctcatattg caaacaaaca taaagttcag attaattacc taaatgtgaa
32760aaaatgaaat aatttcttta aaaaatgtaa tcttagtttg aggaaggtta
acattataaa 32820ggaaaaaact gttttgagtg gaatatagtt caatatgtca
aaatccacct tcaacaaaat 32880tgaaagtaaa ttgaacttgg ggaaagtatt
gacagcatat agatcaaagg ttactagcct 32940gtgtaaagag cagttataaa
tatcgttaag aaaaacactg tcgacctgtc ggcaccttgt 33000tctccgactc
ccagcctcca gaactgtgac gagtaagtgc ttattgttta aaccacccag
33060tctgtatgtg gtattttgtt atagaaactc aagctgatta ggacactagt
aatcagtaga 33120ctgaaactga aacaaaaata agaacctttt ttacctgtca
aattggcaaa cattaagaat 33180attcagattt ttgtcagagg tgatacaacc
ttctaagaag gcaatttggg aaaatataaa 33240gctttagatt attatatgtc
tgacctagca gttttacctc tagggtgctt acccctagga 33300aagtgtgtaa
tgatattggt gcagtgccct tcatcccatt agaaaattaa aaataacctt
33360aatggcctac cactaaaagg ggattgaaaa tttaagatat atttatttat
gtgtttattg 33420agatggagtc ttgcactgtc cgcctgggcc agagtgcaat
ggtgcgatct cggctcactg 33480caacctctgc ttcccgggtt catgtgattc
tcctgcctca gcctcctgag tagctgggat 33540tacaggctca caccaccgca
cccggctaat tttttgtatt tttagtagag atggggtttc 33600actgtgttgg
ccagactggt ctcgaactcc tgacctcatg atccgcgccc ctcggcctcc
33660cagtgttggg attacaggtg tgagccactg cgcctggcca gatacattta
tacaagagaa 33720tgttagttaa cattcataga tatttatatt ttgtttactt
tttattaaaa aaattttttt 33780tagagacagg atcttactct gtcacccagg
caggatgcag ttgcacaatc atagcccact 33840gcagcctgaa ctcctgggct
taagtgatcc ttctgcctca gccttttgag tacctggggg 33900actttaggca
gtgctactat acctggctaa tttttaaatg ttttatagat gagatcttgc
33960tgtattgccc aggctggtct agaattcctg ggcccaagtg atcctcccac
cttggcctcc 34020caaagcgctg agattacagg catgagccac cacttctgac
caatagatat ttatatttgt 34080gactggaaaa tatattaaca atgtgttaaa
aaattcagtt aaaaaataat gaaagatttt 34140tgcttctggc taagatagaa
taacaaggac agcatttatc ttcttgcctt gaaatagttg 34200aaaacggaag
aaatatatgt aacagtggtt ttcaagttat tgggcatcag gcaaagaaga
34260atagttatcc caggaaaatg aatgtggaga gccctacaat ttccttacat
tactgcctgg 34320tcatggcaag aggaaaaact gagaggagac tgaggctgag
ccagtggttt gctgggttga 34380ggaggcagag ctgggagtgc agagatgcaa
ggtggtgaga gcccatatgg aagaatacca 34440gggaagagag ctgcagaggg
agctccggag acctgcaccc tgccctctca gtaccctgtc 34500atgtgtgtag
ctgagtactg acgagcactt gcttgtgcgg aaatgaccca gggctggagg
34560tagagccacc tgaaaggatt agaaggaaca gttgctgaaa gtcacacagg
gccaggaaga 34620atttctaatc acaccagttg gagtggaaaa cctcagctct
catagagcag gtagggtact 34680cagaagggtt tgcccaccta gccccagact
aagtttcgtt actctgaccc tacctaatat 34740taaaaagaga ttaattaaat
tgttcgcaac aaaaataata tatttcagtg tttgtaacac 34800gtagaagtga
attgtatgac aatagcataa aggctggaag agcagaaatt gacatgtatt
34860tgcgctgggc agaataatgc tcccctcttt ccccaaaaga tatcaagtcc
taatccctgg 34920agcctgtaaa tattacttta tatggaaaat tgttttatga
tgtgattaaa ttcaggatct 34980tgagatgagg gggctatctt ggatgatctg
ggtaggcact aaatgcaatc acatatatat 35040aaaaaggagg cagagggaga
ttttacacac agagagaagg ccctgtgaag atggaacaga 35100aagatttgaa
ggtgctggcc ttgaaaattg gagtgatgaa gctataagcc aaggaatgca
35160gcagccacca aagctggaag aggcacggag cagttctcat ttagagccta
ctccagaggg 35220aatgtggtgc tgccaattcc tttttttttt ttttttttaa
gatatcattt acccctttaa 35280gttggttttt tttttttttt ttttttttta
gtatttattg atcattcttg ggtgtttctt 35340ggagaggggg atttggcagg
gtcataggac aatagtggag ggaaggtcag cagataaaca 35400tgtaaacaaa
ggtctctggt tttcctaggc agagggccct gccacgttct gcagtgtttg
35460tgtccctggg tacttgagat tagggagtgg tgatgactct taacgagtat
gctgccttca 35520agcatctgtt taacaaagca catcttgcac cgcccttaat
ccatttaacc cttagtggac 35580acagcacatg tttcagagag cacggggttg
ggggtaaggt tatagattaa cagcatccca 35640aggcagaaga atttttctta
gtacagaaca aaatggagtg tcctatgtct acttctttct 35700acgcagacac
agtaacaatc tgatctctct ttcttttccc acatttcctc cttttctatt
35760cgacaaaact gccaccgtca tcatggactg ttctcaatga gctattgggt
acacctccca 35820gatggggtgg cggccgggca gaggggctcc tcacttccca
gatggggcgg ccgggcagag 35880gcgcccccca acctcccaga cggggcggcg
gctgggcggg ggctgccccc cacctcccgg 35940acggggcggg tggccgggcg
ggggctgccc accacctccc ggacggggcg gctggccggg 36000cgggggctgc
cccccacctc ccggacgggg cgggtggccg ggcgggggct gccccccacc
36060tcccggacgg ggcggctggc cgggcggggg ctgcccccca cctcccggac
ggagcggctg 36120ccgggcggag gggctcctca cttcccggac ggggcggctg
ctgggcggag gggctcctca 36180cttctcagac ggggcggctg gtcagagacg
ctcctcacct cccagacggg gtggcagtgg 36240ggcagagaca ttcttaagtt
cccagacgga gtcacggccg ggcagaggtg ctcttcacat 36300ctcagacggg
gcggcggggc agaggtgctc cccacttccc agacgatggg cggccgggca
36360gagatgctcc tcacttccta gatgggatga cagccgggaa gaggcgctcc
tcacttccca 36420gactgggcag ccaggcagag gggctcctca catcccagac
gatgggcggc caggcagaaa 36480cgctcctcac ttcctagacg gggtggcggc
tgggcagagg ccgcaatctt ggcactttgg 36540gaggccaagg caggcggctg
ggaggtgaag gttgtagtga cccgagatca cgccactgca 36600ctccagcctg
ggcaacactg agcactgagt gagcgagact ccgtctgcaa tcccggcacc
36660tcgggaggcc gaggctggca gatcacttgc agtcaggagc tggagaccag
cccggccaac 36720acggcgaaac cccgtctcca ccaaaaaaca cgaaaaccag
tcagacatgg cggtgcgtgc 36780ctgcaatccc aggcacttgg caggctgagg
caggagaatc aggtagggag gttgcagtga 36840gtagagatgg tggcagtaca
gtccagcctt ggctcggcat cagagggaga ctgtgcgagg 36900gcgagggcga
gggcgaggga attccttaat ttcagtttag tgatactaat tttggactct
36960ggcctctaaa actgtgaaag aaaaaatttt ttgtttgttt gtttctttta
agccacatag 37020tttgtggtaa tttgttacag cagctgcagg aaactaattt
atgctgcatg tgaaatggtg 37080taataaggta gattgtgatg aagatacata
gtataaacaa ttaagcaaca actaaaagca 37140caacaaggaa ttatagctaa
tgaaccaaaa aaggagatta gaataataaa aatggtgaat 37200cccaaagaag
ccagaaatag gggaagaggc aaataaagga aagaaagagc ttgatggtag
37260atttcaacct aactatgtca aaaaggacat tacatgtaaa aggcagcgat
ttttcagatt 37320gaatggaaaa gtaagactcg gtatatgctg ctgcctgcaa
gaaacacatt ctaaatataa 37380aggcaaaaat aacctacagg taacagaacg
gaaagaagtt cactgtgctt acaagaatta 37440gatgcaagct agactggttc
tgttaatatc agacaaagtg gatttcaaag caaaggctct 37500tgcccaggat
gagatggtca tttcataatg atgaagggga ttcgttcatc agcctggcat
37560agcaagctga aatgtttatg caccggacta cagagctaaa atacatgaag
caaagcctga 37620cagaactaca agtagaaaca gacaaatcca cagtgataga
gatttcagta gccgctctca 37680atgatttgta gaacacgtag ccataatatc
tggatctaga acacttgacc aacactgtcc 37740cctgtgcaac ctcattggca
tttacaggac actccaccca gcaccagcag aagagacact 37800ctctcaagtg
ctcacagaat gtttgccaag atagagcaga tgctgggcca taaaacaagt
37860ctctaaatta aaagcattca aattattcag agtatgtttt ctgacctcag
tatcattaag 37920ttggaatata ttataggaag ataacctgga aaagcctcag
atatgtggaa aaacccattt 37980ccacatggcc catgggtcag aagtgaagtc
aaaagggaaa tttgaaagtc ttttggattg 38040actgatataa aaacaataga
tttctaaact tgtggggtgc tgttacagca tagtaaatgg 38100aaatttctag
cattaaatgc ctgttttagg aaagaaagat ttcaaatcaa tgacctcagc
38160ttctaccttt ggaaacttga aaatgacaag caaatggaat ccagagttac
cagaagggcc 38220aggtacggtg gcttatgcct gcagttctgc cactttggga
ggccgaggca ggtggattgt 38280ttgagactgg cagttgaaga ccagcctggg
cagcctaggg agaccccata tctacaaaaa 38340acaaaaaaat tagccaggtg
tggtggcatg tgcctgtagt cccagctaac caggagtcta 38400aggtgggagg
attgcttgag tctgggaggt tgaggctgca gtgaactgtg attgtgccac
38460tgtgttccat cctgggcaac agaatgagac cctgtctcaa aaacaaaaac
agttactaga 38520agaatggaca tcataaagat aggagcagaa gtcagtaaaa
tagaaaacaa aaatacatag 38580gaaatcaata aaaccaaaag ctggttcatc
aagaacatca ataaattggt aaagctgata 38640ggaaaaacag tgaagtcaca
aattagcaat atcaggaatg agggagatga cagtagtata 38700gattatatag
atattaaaag gactgtatga ggcaggtgtg gtggttcacg cctgtaatcc
38760cagcaccttg ggaggccgag gtggacagat cacctgaggt caggagtttg
ggaccagcct 38820ggccaacatg gtgaaactct gtctctacta aaaatacaaa
aattagttgg tcgtggtgct 38880gtgtgcctgt aatcccagct acttgggagg
ctgaggcagg agaattgctt gaacctggga 38940ggcggaggtt gcagtgagct
gagattgtgc cgttgcactc cagcctgggt gacagagcaa 39000gactccatct
caaaacaaat aaataaataa aaaggactat atggtaatat tatgaacaac
39060tttatgccaa taaatttgac aacttataga tgaaatggat gagttccttg
aaagacacag 39120aaactattaa agctctctca agaagatata gataagctga
ttagccctat atctatttta 39180ttgaatttaa atgtaaaaat caatatttag
ttactggaaa acttttaagt gtggttggaa 39240atggtatacg aactttttca
actgaatttt atgaagtcta atcacaggta aaggttttct 39300gatgaaaatt
tagtgtctga attgagatat actgtaaaaa atgttatata tcttaattat
39360ttcttcacat taattacatg ttgaaataat actttgggtg tattgggtta
aattaaatat 39420tatgaaaatc ttgcctgttt tctttttact tttgatgcgt
cagctaggaa atataaaagt 39480gtagctcaca ttctgtttct gttgacagta
ctgctttgga gcacagtgtt tgaatgatct 39540atcatttcaa agacctttcc
tcagttcgtt attcatggct gtctgtattc cacatagata 39600aggtctgaaa
tactgctaag tggcatgttt tgttttatgc ttttataagt ttgttgatca
39660ttactgatgt ggacttttgg tgcctcttag gctcattgct atcttccaac
cattgtttgc 39720aatttttacc tagagataaa gagaaagaga catttggttt
cagagtagtt agattgggat 39780catgaaagag caacctcatt ttgatgcttc
aaaaatagca catcccccgt attactggga 39840tttgctattc ttgggattac
ttcaagaaca tccttgtgtt actggtttgg atgcttctga 39900atgctgtgaa
gtcagtttca tgtacatggc tcatcagttt agctctctct tggctttgtt
39960tagacagttg gagcatgatg gcctaaacag cttctttcaa ttaaacattt
taaaatagtt 40020tacaaatagt aaacaaactc cagtttttgt gactctttgt
ctcgcacaac aaaaacacaa 40080tctgaccatg atcatctggc atcttagggt
gaaatatggt tatactttgg cccataccga 40140aagcaagatt aaaaaggggc
aggagagata gactgctgaa ctgattttca aggttccaag 40200aatattgtag
gttaagagta aaagtaaact tttggtagaa agcagtgggt tgtctaggat
40260tgaagtatct gaagttttta aacgaaaatt taaaaagaaa aatgagaatt
gccttacaag 40320tacaatctct tcttttttaa aaaataaact ttattttgaa
atagttttag atttatagaa 40380aaaaattaga tagggtagga agttttcata
taccctacat ccagttaccc cagttattat 40440catcctaatt tagtgtgaga
cattttcatg tttaatgaat caatattgat atgctattaa 40500cttaagtcca
gactttattc agattttctt aatttctatg taatgtcctt tttctgttcc
40560agaattccat gcaggacacc ggatacctca ttacatttca ttgtcatgtc
accttaggct 40620cctcttgaca gtttctcttc tttttttgct tagaaattct
ccagaatttc agaaacttct 40680gggcatcgct atggaacttt ttctgctgtg
cagtgatgac gcagagtcag atgtcaggat 40740ggtggctgac gaatgcctca
acaaagttat caaagtaaga accgtgtgga tgatgttctc 40800ctcagagcta
tcattgttgt aggctgagag aagaagcgat cattgagtgt tcttctgttt
40860tgagtccctg aggatgtctg cacttttttc ctttctgatg tatggtttgg
aggtgctctg 40920ttgtatggtt tggaggtgct ctgttgtatg gtttggaggt
gctctattgt atggtttgga 40980ggtgctctgt tgtatggttt ggaggtgctc
ttgtatggtt tggaggtgct cttgtatggt 41040ttggaggtgc tctgttgtat
ggtttggagg tggtcttgta tggtttgcag gtgctctatt 41100gcatggtttg
caggtgctct attgtatggt ttggaagtgc tcttgtatgg tttggaggtg
41160ctcttgtatg gtttggagat gctctattgt atggtttgca ggtgctctat
tgtatggttt 41220ggaagtgctc ttgtatggtt tggaggtgct cttgtatggt
ttggaggtgc tctgttgtat 41280ggtttggagg tgctctgttg tatggtttgg
aggtgctctt gtatggtttg gaggtgctct 41340attgtatggt ttggagatgc
tctggtatct
gcctgcattg cttgccacac ctgcccggtc 41400agaaggcgct atgttgacaa
ttgtgcctgc acggtgccta ggtcaatgaa gggaaccgat 41460ggtagccact
ggatgctcct gggaaaatgt cactacaggc accagagaag ccagagctat
41520gcccaaattt ctatgagtct cagttttctt aaccataaaa tgggatcaat
gtttttgtgg 41580catgtgtatg agtgtgtgtc tgtgtatgtg tgaggattaa
attgtgtatg tgtgaggact 41640aattgccact actggatcct caaagtggta
agaagtgttc ttattaataa tgacatcctt 41700acactcttac ccagcaagat
tgatgggtgt ggcactgctt ctctttttcc atcacatggt 41760ttccatggta
tccttttgcc cagggaatct ttgctttgtg gctagcactt tgttgtttgg
41820ctaatcacgc tttctgtggt caggacgctg gcttctctgg agccatggga
ttctagctcc 41880ctgtcttgtc cctagagtgg tcactgtctt ctctctccgc
ttgcaattcc tgctttgctc 41940gcatctcact tatgcagtga cgtatatcag
tttcaccttg ttctccgtgc ctgctgatca 42000ttggcaccac ttgcatggtg
ccatttaggg cctgcttcca gttaagcttg cttctccaca 42060ggcctaaata
tccttgcttg cttcttttat tctcactggc aggaccaggg cggtctgtct
42120ttgcatgaga cagggtctcg ctcagtcacc caggctggag tgcagtggct
gatcacggct 42180cattgcagcc ttgagctacc gggctcaagc tatcctcctg
gcttggcccc ttgagtagct 42240gggactacag gcgtgcacca ccatgcccag
ctaattttta aaattatttg tagagatggg 42300atctcgccag gttgcccagg
ctggtcttga acgcctgggc tcaagtgatc ctccctcctt 42360ggtttcccaa
agtgctggga tcacaggtgt gagccactgt gcctggccct tgatgtttca
42420gttcttgata tttgatcctc agagtcagaa aatctaaaaa gagggctatc
ccaggttgcc 42480ttggttcatg gcaaatggga cgttaagagg gcagagagaa
tatgaacaga aactgttcta 42540atattggtca tttaatgtgt aagtattgtt
cttttttaaa cctccttcat tttttttcca 42600ggaattgctg gacacagtgg
cttggtgtgt gtctgaggac tgtaggccat ggccctaggt 42660tgtggtttta
ggtctcaggt gctcttcctg gctgtctcct tgcttctttc ccatgtcctc
42720ttctttgttt ccagccattt ctcccttatg cttaagtttg gtgcagcagg
gtttggctgc 42780tctcagattc ctgcttcctc agatgctgta gttgtcaggc
ccagcgggct ggcagcggga 42840tcaggatctg gctaggtttg ctctcactgt
ggcagagtag ggggaggcgt gggagagcac 42900gtgtgacccc aggccagctg
tagggagcat aggcatggtc acgtagcctt caggtcctag 42960actttgtctt
ctcatgagta tggctgtgtg tgtatggtga aaactaggtt ctacttagcc
43020caagaaaatg ggcacatttt gcatgtggtt tctgtagaga aatgcactgg
gtatctgaca 43080tagcctggca gcatgcctcc ctcaggtagg ttagtctcag
gcggtgaagc acgtgtgtcc 43140agcaagaact tcatatgtgg cataaagtct
ccgttctgtg aggtgctggc aaatcaccac 43200caccgtcaag aggctgaagt
gatttttgtc tagggaggca ggaaaggctt cctggagtca 43260gcagccagta
ggtgaaagag tagattggag accttcttaa tcatcaccgc ctcttgtctc
43320aaggggtgcc aggaagctgt ggaggctgaa cccatcttat gctgccagag
agtgggacac 43380catgagggtc aggtcaaggg gttgtacctt gtttggtaga
gaattagggg ctcttgaaga 43440ctttggatgt ggtcagggga gtgtatcatt
taggaagagt gacccggtga ggacgtgggg 43500tagaggagga caggtgggag
ggagtccagg tgggagtgag tagacccagc aggagtgcag 43560ggcctcgagc
caggatggtg gcagggctgt gaggagaggc agccacctgt gtgtctgcgg
43620aagcaggggc aagagggaag aggccagcag cgtgctgcca tcacccagcg
actggcgtag 43680attgtgagag accattccct gctcttagga ggggctgagt
tttagttttc tcttgttata 43740caataagctt ggtatttgtt tacaaaacat
ttgtaaagct aaatcaaggt ttgataaggc 43800ttctagtttt atttaagaag
taatgttgaa ataaatgttt gtccaattcg ctttgctcat 43860ttaaggactt
tcagtacaaa ctgcaacaac aggattagga tttaaacgtt tctgagatgt
43920ttttactcct cagaatttcc cagaatgtga tctggttttg attttcaagc
ttgctgaccc 43980aataggttaa cccacaagtt ttacgaagac catctcagtc
cacttacatc aactgcccat 44040gccacggtta aagagatcat cgactgatgt
ttggcacagc ttcctccctc ttgggtgggc 44100aagcatttgg aagagaaggc
tcctatgggt gagagtgggg caccaaagtc ttccctgtcc 44160catcccctag
cttgagaagc ccttctctaa tgtggacttt gtgccgttag catcgttact
44220agcttgaagt tgaccatctg gacgtacttt ctggtttagc ctcacaagtg
agcaaggagg 44280gttgagagat gtgctgtgag gaatgtgggg ccccagctgg
cagcaggctc tgggtcaggg 44340gggcagggac cacgggcata cctgacagtg
aggaggggcc acacctgcag aaaaggatgc 44400aggactccgc cttgggaagt
gttctaggcc agagcgaggg tctgtggttt ataagtacac 44460ccacagtgct
cgggaccctg cagatgtcca gggtgccgtc tgagcccgta tcatccaaca
44520gaatgttctg ctagtgaaga ttaaagattt actccagggg ctttaggatt
tattatatat 44580atataaatcc tatatatata attttttttt tttttttttt
tgagatggag tttcgctctt 44640gttgcccagg ctggagtgca atggcgtgat
cttggctcac tgcaacctcc gcctcccggg 44700ttcaaactat tctcctgcct
cagcctctcg agtagctggg attacaggcg cccaccacca 44760cacccggcta
atttttgtat tttttagtag agacggagtt tctccatgtt ggtcaggctg
44820gtcttgaact cctgacctca ggtgatctgc ccgccttggc ctcccaaagt
gctgggatta 44880caggcatgag ccaccccacc tggccaggat ttattgtatt
tgaaccatct accattttaa 44940ttttgatgtt atgtagtatt tgatgataat
gaaagttaaa ttgtttttct ttccattttt 45000ctgtttaagt gaatgacctg
tatctagttt attcagtaac ttcctgcata tatttgtttc 45060tttcattctt
aatgaatata ttcttaattt agttgctatt atgttttgct ttgccccaaa
45120attgaaatct tagtttcctt ttagctcgtt ttagaactag tgatgggatg
tgtcttccat 45180aaatctcttg tgatttgttg taggctttga tggattctaa
tcttccaagg ttacagctcg 45240agctctataa ggaaattaaa aaggtgggcc
ttgcttttct tttttaaaaa tgttttaaat 45300tttaaatttt tataggtaca
cgtattttgt aggtacatgt aaatgtatat atttatgggg 45360tacatgagat
attttgatac aggtatacaa tacataataa tcacaccatg gaaagttgga
45420tatccatgcc ctcaagcatt tatcctttgt gttacaaaca atccagttac
atgctttact 45480tattttattt tatttttgag acagagtctt gctttcaccc
atgctagagt acagtggcat 45540gaccttggct cactgcaacc tccgcctccc
gggttcaacc gaactttggg ctggtctcaa 45600actcctgacc tcaggtgatc
cgcccgcctc ggcctcccaa agtgttggga ttacaggcgt 45660gagccactgt
gccgggcctg attgtacatt ttaaaataac taaaacagtc agggcacagt
45720ggctcatgcc tgtaatccca gcattttggg aggctgaggc aggtgatcac
ctgagatcag 45780gagttcgaga ccagcctggc caacatggag aaaccctgtc
tctactaaaa atacaaaaat 45840tagccaagtg tggtggcggg cgcctgtaat
cctggctact cgggaggctg aggtagggga 45900atcgcttgaa cctgggggtg
gaggttgcag tgagccgaga tcacgccact gcattccagc 45960ctgagcgaca
gagtgagact ttgtctcaaa aaataaaaat gaaataaaat tgggccgggt
46020gtggtggctc acaccttagt cccagcactt tgggaacctg aggcaggtgg
atgcttgaga 46080ccaggagttt gagaccagca tgggcaacat ggcaaaacgc
tgtctgtaca gaaattagct 46140gggtgtggtg gtgcacaact atagtctcag
ctacttggga gattgaggtg ggaggattaa 46200ttgagcctgg aaggttgaat
ctataggtag ctgagattgt gccactgccc ttcagcctgg 46260gcgaccaagt
gagaccctgt ctcaaaagaa aaacaaaaaa acaaaaaaca aaccactatt
46320atcgactata tattattgtc tatgatccct ctgctgtgct gtcgaatacc
aggtcttggg 46380cccttatttc catcactgag caaacttcac tctgttaagc
agcaggtgtg ggatttcatc 46440gttattcagt aattcacaat gttagaagga
aatgctgttt ggtagacgat tgctttactt 46500ttcttcaaaa ggttactctt
tattagatga gatgagaatt aaaaatggta acttacttta 46560tatctttata
attgaagccc actagacctt aaagtagtta ccagatgttt tatgcattta
46620aatggccttt tctctaaaat tagaaagtaa caaggaaaga aaatgcttcg
tttctatgca 46680accctcttgg tgactagtat gtgactctta atgcaaccct
cattgcaccc cctcagaatg 46740gtgcccctcg gagtttgcgt gctgccctgt
ggaggtttgc tgagctggct cacctggttc 46800ggcctcagaa atgcaggtaa
gttgtacact ctggatgttg gtttttgtcg ggggccagct 46860gctactgatc
ctttatgtct cagctcagat gtcatttcaa aagtctgctc tgccctctcc
46920aaattgcagt cgaccttgcc ctgtttatgt ttccctcata gcactaatcc
atgtcagaaa 46980ttgtcacgta cagtctatct gtgtgcttgt ttattttcta
tcccaccctt ccgcaagaga 47040cttatgggat gtgtgcccca ggacagcagg
ggtcttactg tcttatgctc tgttgcagcc 47100cagcagcgat aacagtgtct
gcacatagta cttgcttaaa agatacttgc caaattgttg 47160aaggttgagg
taccaatttc attattgctg actataggag ttatagcaaa atatccattt
47220gtctgttaca tgagttaaaa atatggttgt tgcactgtga atagtttggt
ttagtcaaaa 47280cagttgtatc ttaacggatt gagaaacaaa agcaggacca
cttttcatca gctccctcct 47340tctccttaac cagcaataca tgctgatgct
gatatcccat agaccctcag ctccatcctg 47400agtcactggg aatgtggtct
aaaccctcac tattaatatg aactgagttt caataagaat 47460cttatatggg
tcgggcatag tggctcatac ctttgatccc agcacttcag gaggccaagg
47520caggtggatt gcttgaccca gactaggcaa catggtgaaa cgccgcctct
acaaaaaata 47580caaaacttag ccaggcatgg tggtgcgtgc ctgtggtcac
agccactcga gaggctgagg 47640tgggaggatc acttgagcct gggaggtgga
ggtcgtgttg agccaagatc gcaccactgc 47700actccagcct gggcaacaga
gtgagacctg tctcaaaaaa accaaaatcc agaaaagaac 47760ttatatggct
gcagaggtat aatcactaag gaaatttcct tttgtataat cttttttctt
47820ttactatcat ttaaaaaaat gtgttatatt tctgaagcaa cacatccagg
ttctgcacat 47880agcagccaaa gtgaccttaa agaatataac tgggtcttgt
cattccctta tttaaactct 47940tgtacccatt tcccagtgcc gtttagatag
agattccaga ctcgtcaatg gctctgtcac 48000ctcagacacc ctgcattgac
tcattagtct gattagagtc aggtttttct tcctcctgat 48060ggtttttttt
tcccccttag ttctcagcgg aacagtcact tccttaggga ggtttcccca
48120gccaccctct gaggccgtgc ttgttgccag actctgccac tagagggcag
ggctgcacca 48180ctcctggcac ctcgcacccg gcctgccctg tcactctgtg
tgttgggtga attcctgtga 48240tctgtgactc actgctctgt gtcctacaca
ttcggctttt cttctctccc cacaacccca 48300ttttataatt ctcctttttc
aggaaagctt tattcccatt taaaaatttt tgtttttaaa 48360atggtatttt
cttacactta ttttctaatt aaaaatgagt gttttaagaa gtattatgat
48420ttactgcaaa taatttttaa acccagcctt ttagatcctc tgtgatcata
agagaaatga 48480aggatgtctc ccaacacttg agcttcatcc acatttcatc
ctcctgttct ttcagctgag 48540ttttccccat cccattaggg actgttggaa
tataaaactg gcttttccct aacagggaat 48600gaattgcttc tgtttctcct
gaaggagagc tggaagaatg acttgcgttc ttttgcatac 48660acaggcctta
cctggtgaac cttctgccgt gcctgactcg aacaagcaag agacccgaag
48720aatcagtcca ggagaccttg gctgcagctg ttcccaaaat tatggcttct
tttggcaatt 48780ttgcaaatga caatgaaatt aaggtatgat tgttgcctca
ggtcacaaac atgcgagtga 48840tgctgtgagt gagtctgtgg agggtgaggg
cttctgaaca gggagtcctg tgggagtgct 48900tcttggggta tgttgtatgt
cgtaatttag actaccatca tttgtgttat ttttgaggca 48960cctaaggact
tctttccact tctcatttct tactgtgggg tgaagagttg aattgggaga
49020tggtttctag atgcaaattg aaaaggcatt tttccagagc agatttgttt
tcggcgtact 49080agagtgactc tttaacctag ctgcgggaag atgactgtgc
caagactgca ggtaggagaa 49140agctcactga cgaggccttg tgggtctgaa
cgtcctgcag ctatcagagc ctgttggctt 49200cctgttgtgc attccaacaa
atcatcttca aacccacttt agtgttttgt ttataatgtc 49260cagaaatagt
gaccctgtca catgctctac agattacagg attcttagcc tcttcctttt
49320tggtaggtca gtcctgggtt tgagcccaag tgaccctcct gggaggtgat
gatacacact 49380gggtagagtg gaatcagatg gacttggatt agaattctgt
cctctttact agttattttc 49440ctctaggcaa actgcccaac agctctaagc
tatttccttc gtattctgaa aaataagcct 49500taatgggacc catatagggc
aactctgaga gtaaaataaa ggaatatgtg ttagagtgta 49560gcatagtcac
ccacgggaag ggcttagatg ttagctgcta ctgctcttat tagctgaatg
49620atttggaata aactgttagc ctctctcatg ttttttctct tgagcttcga
agttttcttg 49680ttaatactaa ggagatattc aaactagtca tggggttttg
gaatgacgaa gggagatgat 49740gaatctaaag aatttagtgt aatatttctt
catgctcagt aaatggtagt ttctgctgct 49800gttattttta ttaccatctc
tttggaatgg gagtaggtgc tcctttgtgg tcagaggctg 49860tgagagctcc
acagcgccag tttgcccatc tgtacactgg ggtctgttga aggcagtccc
49920ctctgtgata tctctggctg tcagagctca gatgatagat ggtatttttg
tactcttagt 49980tctcatcatt ttcatgattt cgatcaccat ttgagtatga
tgatgctaac actttgttga 50040acgtagaatc cgttaattac ttccttcctg
aacctttggc attaaaaaaa atctattctg 50100ctacctctct gctcatttat
ggttattcaa atttattatc aagagcctgg tacagtggct 50160tgtgcctata
attgtagcta cttgggaggc tgaggtagga ggattgcttg aggccaggag
50220tttgagacca gcctgggcaa gatagtgaga ccctatctct aaaaaaactg
aaaaaaaatt 50280agctggacat gatggcatgt gcctgtggtc ctagctactc
aggaggctga gacaggaggc 50340tcggttgagc ccaggagttg gagttcgagg
ctacactgag ctgtgattgt gccaccacac 50400tccagcatgg gtggtaaaac
aagatgccat ttcttaaaaa aaaaaaatat atatatatat 50460attatcaatg
aaattcagta gtaccaacag gattataaac aaagatagta gttcccttcc
50520tactttttct cttaatcctt gtgtctcaca ggcaaacata actcttagta
tttcttccaa 50580tatttacttt catgtttctt tctttctttc tttttttttc
tttgagatgg agttttgctc 50640ttgttgccaa ggctggagtg caatgacgca
atcttggctc accacaacct ctgtctcccg 50700ggttcaagcg attctcctgc
ctcagcctcc tagtagctgg gattacaggc atgcatcacc 50760acgctcggct
aattttgtac ttttagtaga gatggggttt ctccgggttg gtcaggctgg
50820tctcgaactc ctgacctcag gtgatcctcc cacctcagcc tcccaaagtg
ctgggattac 50880aggcgtgagc cactgcgccc agcaacttcc acatttctaa
ataacatgct tctactgcta 50940tttttttttt caattttaga cattttttta
ctttcactat agttctatca gaattcagtg 51000tgtacgttat tatgcctaag
taaatagtca tggttgctta cgtattatat ttctttgatt 51060gtgtttctta
tttgatgaga aagctgtgtt ttttgctctg ggttgaaact ggagagagga
51120cctggggagg aggaggagga cagatgaagt tggtgactgt accttcatgg
ccatagctgg 51180gttctcagca cccggggatc tgctgatcac ctactcatag
gccaggcccc tatcgaagtt 51240ctaggtgacc cagtgctggg gacggggggg
ccacctgcaa ggtctaatca tggaggtggg 51300ggctacagtg ttggcttgtg
ctggggccag catccttagg aaggcatctt ggaggtggag 51360gagacagccg
cccacttctt gattggggcc ttcagcagca ccagcttctt gggcaggctg
51420gtgctggctt tcatcaccat gtcgtgttca atcttcttcc agatcctgac
ttctaggttc 51480agctttcctc agaccctggt tcctttcaga ggccattgct
gctgccttgc tctttgctgg 51540cttgtgcctt gattatatgt ctttgtacaa
ctttttgttt tcctggagtt aatcttcaca 51600tctgttttct tggagttaat
cgttacctct atatcgcttg cttattattc tttggccttt 51660ttgtcttctc
acaccttcca acttctttgt aatatgtgtt tagtacaatt tttcatgaca
51720ggtagtttac tgaatcagtt tttccccagt gtggtcatcc aacttgagtt
atccagctct 51780ctgccccagt ctgggcaggt tgatcttcag gtctgtagta
cacttgtatc ctaggacttc 51840tctttgccat tagcctggaa tttcctttgc
agttctcccg ttggatgccc agttcctaga 51900tgccatatgt ttttctatcg
tctagtagct tcctgagaga agatgaatgg gagggaaatt 51960gtatgaggtt
ttgcattcat aaaaatgcca ttttttttcc tgtacacttg gctgggtatg
52020gtgttctggg gtagaaatca ttttccctca gaaatgcaaa gtctttgccc
tgttgtctta 52080aaatctccaa cgtgacccga ttccttaacc tatgaatgta
cttttctttg gaagctttcc 52140atttttgggg aggtgaagtg ctaggtactt
agtaggcctt ttaatttgga aacttacatc 52200ccttcagttc tgggaaaatt
ttcttaacat ttctctgaga agttcttgcc ttttattttc 52260tgtgttctct
cctgaaattg gttagttgga tgttggtcct cctagattga ctcacatctt
52320acctttttct tttctttttc tggtactttt tagatatcca tctcaaactc
ttctattcat 52380tgttatgttt ttaacttctt tcttttcttt gtctcttgat
ggggtcttgc cctgttgccc 52440aggttgtggt gcagtggtgc gatcatagct
cactgcagcc tcaaattcct gggctcaagc 52500agctgttctg cctcaccctc
ccaagtagtt gggactacag gtatgcacca ccacgtccag 52560ctattttctt
tacttttttt tttttttttt tgagatggag tcctactctg tcgcccaggc
52620tagagtgcgg tggtgggatt ttggctcact taagcctctg cctcccaggt
tcaagcagtt 52680ctcctgcctc agcctctcaa gtagctggga ttacaggtgt
gcaccaccat gcccggctaa 52740tttttgtatt tttagtagag ccagagtttc
accatgttgg ccaggctggt ctcgaacgcc 52800tgacctcagg tgatccgcct
gccttggcct ccgaaagtgc cgggattaca ggcgtgagcc 52860catcattaga
tctttaaata ccagtatcta taagtctttt cctcttgagt cagctagtat
52920ccctggaagg aaattactca ttttcctgct tggaggctat aagcttggct
atgtttatcc 52980tgcaaccggg gactggaagg gaggggactg acagtgttgc
tggtcagggt gccctcttac 53040tttttgtttt ctgtgtgcat ctcacgtctg
tcctcagcct atgtaaacac ctcttgagat 53100tatccctctc aatctttgcc
ggaggtgggg gaggggctgc ttcctgggct gccttggatt 53160ggagggaaga
cctcaggtga gtgggtggga atttgcccaa ggagccatga gaccagccac
53220tatttcaccc tctccatccc tccactttca gatgtatgtg gcgcctccaa
agcccgagct 53280cttcttggcg tctgtggctt caataagctt gctttttgct
ggtatccctc ctaccctccc 53340ctgtccccag caaagcttgc atttgaactt
cttcctacgg gctaacaaat cagtcagtta 53400tgtagctctt gttacttttt
agcttccgaa gttttgttga cacccgtagt ctgctaatgt 53460ccctgttctg
ttctttctgt tcgtgtaaat atatgcttta tacaacttct ttacatgatt
53520tttgtggggt ttctgggtag cagagcttca caagttcaat ccagcgtgtt
ggattagaaa 53580tctcccaccc tctggtttat tcttattctc aaaattacct
gccaaacact gatactccct 53640tgtttttcct tttcctgaca ggaaatgtac
ataccataca ggacagaaat cattagtgta 53700tcccttggtg aataaccaca
aagtgaactt aacccttgta accgccaccc aggtcaagac 53760agaatattac
caagcactca gaagcctctc ccctattccc ccgtcactgc tcctgccttc
53820ctccccaagg tcatgactgc tggcttctaa ttccagagtc tgtttttaaa
ttctgtgtac 53880atagaccatg gattaagtgt tctttttgtc tggtttattt
tggtcgacat taagttcatg 53940agagtcttct atattatcgt gtgtattagt
attcctgtag ttttaggagc ttcatagcat 54000tccattgtag ggatatacca
cagtttattc attgtattat cactgggttg tttctagttc 54060ttggctattg
cgagcagtgc tactgtgacc actcttaggt gtgtcttttg gagtacatgt
54120gcaggtttcc atcttgcaca gctagaggtg gagttgttgg gtgatagggt
gtgtgcatct 54180cagctgcagt agaaactgcc aaatagcttt ccttgagtgc
ttgtaccagc tcaccctttt 54240gccactgtgt atggggattc caggagctct
ggtcctcgct agcacttgga attgctgatg 54300cttttactct tagccttcct
gatgggtgtt ttctggaatc acattatgat tttaatttcc 54360attccttaaa
gtacccttgg ctctgaagtt taatgattca tgcatctctt cccttttgaa
54420gtactcttac aggtatgttg tgcatgtgtt gaaaagtggc actatctatt
ctaaaataca 54480gtatgcctcc tctgtgtttg aacagttgta gcgtggcctt
ggggcctcct gttagctggc 54540ttggagaagg gattcttggg attgtagaga
ttagacctga ggaggcccct tggagctctc 54600tgactaaatt ttattcttta
ttattccaaa ctatttaagc tcaccgtgtg ctgactcatc 54660ataataatga
gtagctctca ttgtgcttgt ctatttggac tcatacaatg attttttttt
54720tttctttgag acagagtctt gctctgttgc ctaggctgga gtgcagtggc
acaatctcgg 54780ctcactgcag cctccacctc ccaggttcaa gtgattcttg
tgcctcagct tctcaagtag 54840ctgagactgc aggtgcgtac caccatgcct
ggctaatgtt tgtattttta gtagagacgg 54900ggtttcacca tgttggccag
gttggtctca aactcctgac ctcaagtgat ctgccttctt 54960cagcctccca
aagtgctggg attacaggtg tgagccactg agcttggcca aagtagtttt
55020ttaagatgtt agtatctttt cttgcagcta aaaaagtttg tcagagatga
ttctactttg 55080ttctccaggt gttttctcag ggagaaattg gaggcagtaa
gccactgggg gagtcctgtg 55140gctggggggt ggggtagtcc tgtggctcct
tgtcagggag tcctgtggct ggcaaggaga 55200gaagtcctgt ggctgggttg
ggagggagtc ctgtggctgg ggtctcatcc tgtgcctaac 55260agtgtccaga
ggtgccgaga ccagctcagt cggggagacc ctaacccagc agcgctagag
55320gaattaaaga cacacacaca gaaatataga ggtgtgaagt gggaaatcag
gggtctcaca 55380gcctttagag ctgagagccc tgaacagaga tttacccaca
tatttattaa tagcaaacca 55440gtcattagca ttgtttctat agatgttaaa
ttaactaaaa gtatccctta tgggaaacga 55500ggggatgggc cgaattaaaa
gaagaggttg ggctagttaa ccgcagcagg agcatgtcct 55560taaggcacag
atcgctcatg ctattgtttg tggcttaaga atgcctttaa gcggttttcc
55620accctgggtg ggccaggtgt tccttgccct cattcctgtc aacccacaac
cttccagtgt 55680gggcattagg gccattatga acatgttaca gtgcttcaga
gattttgttt atggccagtt 55740ttggggccag tttatggcca gattttgggg
ggcctgctcc caatacagag gtctcgtgta 55800aattccctgg gaggcgataa
gcctctgaga aacagactat gctaaccacg ccatgaaaga 55860gaaacttatt
tataaatcag atgccagtta ctagtttact gcttatttgc ccaggcgtag
55920ctctgacaga gtccccgact catagtgctt gctcagtgca tgctgaacaa
tgattggaat 55980caagtcatgg ctcagagcat agttttgaat aatgggaaat
ggatgttctt aagtaacata 56040gtcaccaaga taatgcgact agctgggtca
ccccttttca attttaggat atttttatca 56100agatttaaat ggccatcatt
agagttatag cactttctcc tttggattgt cctagaggcc 56160catgagaaag
tattccctaa tttcttagga gaacagtttg tgggtagtat gcggtcatgt
56220ccagttaaat tgcagatatt tccgatcgaa gatgttccag tcctgagaac
ttcgtgacat 56280tagcaggact tctacaagcc atctcttagg gtggggcatt
tactgcagtt ggctagtact 56340cttttctcct taactttgtc atttgttgat
ttttttttaa ctgtccccaa atactgtggg 56400cagagtgtat ctagaattga
ggcctccacc
attgcggaga ggacatggat gctgagcagt 56460cccctgagtg aaggttataa
agaagcaaat agactacaca tgtctgtaaa ctgctcttga 56520gtgtcccaaa
tttggggtac ttcagttcag ctgtaggaaa agcctcaaac tgtttatact
56580ttgcaagaat tggaaacttc taattcacgt taagttttat gtaatacatg
ataagcttca 56640taggagcttc atcttttatc tacttggact tttgcttccg
taggttttgt taaaggcctt 56700catagcgaac ctgaagtcaa gctcccccac
cattcggcgg acagcggctg gatcagcagt 56760gagcatctgc cagcactcaa
gaaggacaca atatttctat agttggctac taaatgtgct 56820cttaggtaag
gtggaggcat atgagtggaa gagtctccag catgtactca agatagacct
56880ttgaaataaa taaaaccaga tgatccctca gcttctagac caggctattt
ggcactggtt 56940gattgaatgt gaactgcact ggggctgctg tgagcccgca
tgggtctctg tgaccctgca 57000gatgcagccg tgcccaggga ctgggcagtg
ggtgtgggct ggtgtgagcc ctgtctgcca 57060cccagggcct ggccctctgt
ctgtgtcggc catgactatg gtgagtcttg taggcttgag 57120actgtgcctc
gggttcctgc gggttctctg taggtcagtt gacagtttct cctgttgttt
57180gggtaactgt ggaaacgaac actggcaagt gctgaagcga gcatgtggac
gtgcgatatg 57240aaataacgac ctggctttca aaggcagtga ggctctctgg
aaaggacctt gctgagctag 57300ggatgtgggt gtgtagccat tcccagtggg
cctcatggcg tactcgttca tgatcatgtt 57360tgtgccatct tgatctctca
ggatctcttc ttttttaaca gattaagccg ggaatctcca 57420aacagtgagt
cagatgttaa gatgtcttgc ttccaccccc acaggcttac tcgttcctgt
57480cgaggatgaa cactccactc tgctgattct tggcgtgctg ctcaccctga
ggtatttggt 57540gcccttgctg cagcagcagg tcaaggacac aagcctgaaa
ggcagcttcg gagtgacaag 57600gaaagaaatg gaagtctctc cttctgcaga
gcagcttgtc caggtaggag cacagggttt 57660actctaggcc ctgcatgtga
atgactgaca ttcaaagaac cgattaattt ggaagagaag 57720cggcagaacc
gagagttaga ggtgtggact ctggagctgc gctgctcgtt tccaacccta
57780ggtgctgacc tctagctgtc ttccctctgt atgtccctgt caccgtgagt
caaatgcggg 57840tgatgcctcc tcaggtgccg tgttacctaa gcctctcaga
gaccactgct accctgtttc 57900taaaaccaga ggtcacgata tgtgttcatc
cacccagtaa atactgattg agcacccact 57960gtgtgctagg ctctgggata
ggggctgggt atacaatggt gagtatttca gctgcagctt 58020ctgccccgtg
gaggctgtgg cctagcacac tggtctaggc acggtggtat atgctcactc
58080aaggagatag ggacgtggtc gtttggggtg tcggaacaaa atgtcggaac
ttctctttcc 58140aatgcagaga aaccttgcag taattctaat gtactgtgat
tggcagttga cttcagttct 58200ttgtagcacg cttactcagg ttatttcact
aactatgtaa ccatgcagcc tcattttaag 58260caattggatt ttttgaactt
tacttaaaat gttatgtcag ggtttttatt gtgcttaatg 58320tgtgccattt
agctaagttt tgtaggatac gaaattgtaa gtggcttaaa atgattctta
58380atagaatcat gaattgaaga taatgctaat aatttaagca ctgagttagg
tagtgtttgt 58440aaaatgctta gaatgcttcc tggcacatgt taaggccatg
taagtgctgc gtgttgataa 58500acagctgagc aaaagtggac tcttaagaaa
gtattggggc tgagagttct gttccaacca 58560gctgcccttt ggttattttt
cagaataaaa gcagagtctc atgggatatg acatttatat 58620ttccttcaca
aaaaacactg ctgagtgttt tgttgagtaa aaagggtgta gccatggtaa
58680taatacattt aaaatatagt ttatttcatc tttaccttgc cttgtttttt
ttttaagcta 58740gctttttatt gagaattcca cacatacaaa agtatcaact
catgaccagt tatatttcat 58800ttataatcct acttctccct ttttttatta
tttgaaagca aaccccaatt atcctcttat 58860ttcatctata agtatttcag
tatctctata gatgaggact cttctttatt tttaaaactt 58920tatttttaaa
atgatggtca gatgcagtgt tcatgcctgt aatcccagaa ctttgggagg
58980ccaagctggg cggatcactt gaacctggga gtttgagacc agcccgggaa
acatggcgaa 59040accccatgtc ttaaagaaaa aaatcagcca agtgtggtga
tgcatgcctg tagtcccagc 59100tacttgggag gctgagatgg gagggtcaca
tgagcctgga agatcaaggc tgcagtgatc 59160catgattgta ccactgcact
ccatcctggg tgatggagca agattctgtc tcaaaaaaac 59220aaaactgcaa
aacaacgtca caaaacagtg ccattgttag acctgaaaat attaaacatt
59280tcctacatca aatacccacc aactcattat caatttttct ctctactctt
ttggaatcag 59340catctaaata aaattggtcg ataaggattg taaatctctt
tgatgaactg gttcccctcc 59400atcccagttt ttttccctta gagttcattt
attgagaaac cagattgttt gtcttctaag 59460ttttcctgtg gtctgatata
ctgcttccat ctccactgtg taaattaaca cctttttctc 59520ttctctgtat
ttcctgtaaa tcaataattg gaggaaaagc cttgtcagat ttagtgtata
59580ttttatatct gagtccagta tttcttatat aatattttaa gataagtgta
ctcttttaaa 59640aagtattgaa actatatgct caattttttt taactgatgc
ttttaagaag gctgcttgat 59700cataaaagtt tagagatcat tggtctgatg
ggaaaagcaa ataattacta aaccgtttag 59760caaggttgag gtgcacatgg
tggggcctgg agaagttcag tcatgagccg tcacttatgg 59820gcacgtggaa
tctgacccgg cacagagttg ggagaagaca ggagctttat agacagaaaa
59880tgtggtcttt gctaagtccc aggagtgaaa gggtgagaca gtgctcacag
cacacgagtg 59940tgggtgcgta gacagagcaa gggtgggtcc tgaaaaggcc
tgcaggcttt ctcatagatt 60000agcaagagtg ctggttacgg aggtttctaa
catttgtgaa cagatcgaaa ctgtgttaaa 60060ttgggattgc agtaatcctg
gaaggacagg gatagagggt gaaggggaaa aaagggtatg 60120gatgtgagac
ttaattgctg attttcttaa gacctttctc caaagtaaat aaatgatgtg
60180gcacattttt gaactggcaa attctaaact ctagatatga ttatctctat
aacatatctt 60240actccatctt cttttgacta aaaactgttc ttaattaaat
taccatgaga cgttcaattc 60300agcaaatgta gtttggctaa ccatatttaa
ttagaattta atataatcct aggcctggcc 60360aaactattaa gcaagtgtgg
gcaaaatatt gataatttta gatatgcagg aacttagttt 60420gctttccatg
tgtgcttttc gaaaaaggaa taaattgaaa aatagaggaa gccctgaaat
60480ccaagaagca aactctctca cctaggcatg cagtaaaagc aattctagga
tgattgctgt 60540ttggcgcgta gttcgtatta gaaaccattc ttcttgaata
aatagtatgt ttaagaagct 60600gggcagaggg aaggcatatg catatattat
caacaaggag ggagaaaaag gcaattagta 60660accatccata ggagggtcag
caagatttat aaaggaaatt tgtgatccaa gtatgaagca 60720aaataaggtg
cagaataaat tttaagcaag taatagatta gagtaagaga acccatttga
60780ccattaacct tgggacattc tctttcaaat gacatggagt agtactgaaa
tctttctttc 60840tttctgagtc taggttattg tgactggact cagaaagaaa
tatttcatta ttgcagtgaa 60900taacatttgt gaacattatt gttcataaat
tatgcagtga ataacattta tgaacacgtg 60960atgtgtaaga tacatactgt
ttatttttag ttaagttttt tggctcaact tctaggcaga 61020gaacattaaa
tgtaaatagt gttacctagg agcatgtaaa tggaaatctc catagtatga
61080aagcagtgct gttgctaaca gaatttagga gggggcagat gaggtgaagg
aaatgtgggt 61140gctgatttcc ttattacatt gagaggagcc aggagattct
ttgttcaaaa tggatggctt 61200aagaagtcaa agtataagct gattacgtag
agcaggtacc caaaaatgtt ttgtgtaagg 61260ggccagatag taaatatttt
cagtcttgca ggccatccca agtctgtggc agctactcaa 61320cactaccttt
gtagcatgaa agcagccaca ggcagcccat aaatgtggct ctgttccggt
61380gaaactttag gtacaaaagc aggtgcaggc cagacctgac ctgtgcactg
tggtttgctg 61440acctgggatt caggggtata gaagttacca tcagaagagc
taaaagtgag actttttact 61500ttatactctt ctacactgtc tgattttgaa
aaaaagaaac atgtatttta taatattaaa 61560gatagggttg gcaaatagca
aataaaaata cagaatacca gtgaaatttg aacttcagat 61620acattatgag
taattttatg gtgtaagtat attccaaatc atgtgggaca tacttacact
61680acaaaattat ttgttgtttg tttacagttt aaatttgagt gccttgtatt
ttatctggca 61740actgtaatta aagggaaaaa gaataaattc attatgttca
tataatgtga tatagcaggg 61800gtccccaacc cccaggctgc agagtggtac
tggtccatgg gtccccaacc cccaggctgc 61860agagcggtat tggtccatgg
cctgttagga accaggctgc ccagcaggaa gtgagcagca 61920ggtgagctgg
cattcccacc tgagcaccgc ctcctgtcag atcagtggca gcattagatt
61980cccataggag tgcaaaccct attgtgaact gcacatgtga ggggtctagg
ttgtgcgctc 62040cttatgagaa tctaatgcct gatgatctga ggtggaacag
tctcgtcttg aaaccatccc 62100ctggccctgt ggaaaaattg tctcccatga
aaccagtctc tggtgccaga aaggttgggt 62160agcactgtga tatagtatta
aaagtgctaa taaatatggc atactgcctt taaaatgtct 62220ggtagctctt
tctcagtggc actcataata gtgttttttg atttttaaat gtgtgtcaag
62280ctgactctcc cctccgtgta tgctgggctt tattttccct ttcctagtca
ccagttttgg 62340gaaatagaga tcttcattct catgctgctc ctctagtgca
agtgctccat ttatttttaa 62400ggaattaata taacaaaaaa tcatgggaat
ttagaaaaca acatggaagc taatgatcac 62460attggtggaa gtgataggga
aatatttagg gggagaagtt aaggtataaa ctttgtcaat 62520gaagtcctat
taaaaacaac aaaaaagtga agcttaggat gcattttata aactctgacc
62580agaacacctg tgtttctctg tttctaggtt tatgaactga cgttacatca
tacacagcac 62640caagaccaca atgttgtgac cggagccctg gagctgttgc
agcagctctt cagaacgcct 62700ccacccgagc ttctgcaaac cctgaccgca
gtcgggggca ttgggcagct caccgctgct 62760aaggaggagt ctggtggccg
aagccgtagt gggagtattg tggaacttat aggcaagtta 62820ttagcaaggt
ctactcttac aattaacttt gcagtaatac tagttacact ctattgatta
62880tgggcctgcc ctgtgctaag cagtctgcat tccatcttcc ttgccaaaac
ttataataca 62940aatttcatct ttattttata aataggggag ttgggctggg
tgtggtggct cacgcctgta 63000atttcagcac tttggaagga tcgcttcagc
ccaggagttt gagacaacct ggccaagtga 63060gaccctgtct ctacaaaaaa
aaaaaaaaaa aaaaaattag ctgggcatgg tggcacatgc 63120ctgtagtccc
agctgctttg gaggctgagg tggtaggatt gcttaagccc aagaggttga
63180ggctgcagtg aatcttgatg gcagctgcac tgagcctggt gacagagcaa
gatgctgtct 63240caaaataaat ttaaaaataa aataagagaa ttaaagttta
gcaggttggg tggcaaaatg 63300aggccacaca tttaaagccc ctcctcctga
ttcttttctc tgccttggct gcctcctgtg 63360gcattttagg tgctgagaaa
tgaaaacagt agggaaaata gttccaggat cctcatgtta 63420atttgccaga
aatggcatct tcaagtcgtc agagggatct gagagttcct tcctggcctg
63480acttgagaaa atccgtctgt ccccagctct gcgtctgcct ccactgccca
gtcacctcct 63540ctccatgctc ttggggctgg gccctacccc accatgcagt
gctgccctgg agcagtgagc 63600ttggtgggtc ctgtctggca tgagagctgc
ctttgggagc tggatcccag cctctaccac 63660tgggtctggt gcctagcagg
ctatggataa acttctgctg actccggcct ctcctaagcc 63720actgcaacgt
ggtcggtgta gtgcacagtg tgtgtgcagc gtggccttac tcacagcctc
63780cacattagag agaatctgac tgaagtctta ctgctgcctc gtgtgaacat
aaatgtttgc 63840cagaaccatg agcaggaaat gttaatctgc cttgtttcct
gtcctttaca cggaagaatt 63900tttttctgta tggaatgcgt gccttacaaa
taatgagtgg aaatacccat cgctaatgaa 63960aagttatact tgactgttag
tcagctaaat aatctgagat ttctaatact tttaatttgg 64020cttttacaat
gcaatttatc ttagcttttt tgatttctta ggtcatatct ttagaactat
64080atatttgaat gttaatgtaa ttttcatatt gaaattaaaa tgttgaactg
cgatgttaag 64140tgtttcctgt ggaaaaacgt tcacattttc tctagtttta
aagttgaatc aagctgtttg 64200aagattttca catttcttct agattttatc
agcttgttac tttatctgtc actttctgtg 64260atttgcagct ggagggggtt
cctcatgcag ccctgtcctt tcaagaaaac aaaaaggtga 64320ttatttcaga
aatcagagtc ttgtgttgaa tcttactgat tttcttgtat ttctgtaatg
64380taatgtatct tgtatttctt gtaatactgt attggactct gtgtatatct
cttctcagat 64440gagtgattat atgtgtgaat gttgctggaa tctgataacc
aggcctgaat agttttgtag 64500ggtggctttt aaaaattact ttcatatcag
aattgctttg tcataaattt tgaacgcatc 64560ataaatttct aatgttcggg
gtcagcagac tttttttgta aagggacaga gtgtaaacat 64620cttagcttta
tgggccatat ggtctctttt gcaacattca gctctgccct gtgacaggaa
64680tgcagttgta aagacatgag ctactggcca gctatgttcc agtagaactt
tacttacaga 64740aacagacagg ctgtagtttg ccaatacctg ccttagggaa
tgtgttgtta tattttgtga 64800gttaccttct cagtaaattt tatttagtat
tagtcaggaa tattattaag tagcttcttt 64860tccagcctgg tcaacatagt
gagacccggt ctctaccaaa acaaaacaaa acaaaaaaac 64920agccacgcat
gtggcatgtg cctgtagcct cagctgctgc tcagggggct gaggcaagag
64980gattgtttga gcccaggagt ttgaggtcac agtgagctgt agtcatgcca
ctgcactcca 65040gcctaggcaa cagaatgaga ccttgtgtct taaaaaaaaa
aagtttcctt tgttgggtta 65100ttttaatttg gacctggtta tcatttttca
gccatattta actttgtaca tatcagaatg 65160ttctgataaa acttaacttt
tattaaagtg tttgtgatat aatctgctag ttttggtaca 65220cattatcttt
tgcaatgcca gttattttct tttccagtgt gggtttgcat aggaaaagaa
65280ttgctgtcac tttctatttt gaaatcttaa aagactgatc cttttttgtg
tcatgatttg 65340agtatttaat tgagagccta atgcctaata ttatttgcag
tattaaatgg gatcttaaca 65400ggaatagcat tctagccttc attgaattaa
gtaaacattt cttaagagaa cttggaatct 65460ataatatttg cgtcatcata
gtatgagata cttaatcaag tttgagattt tagtgaaaca 65520ttgtttagaa
gccaaaagga ttctaggaaa aattaatgtc tatattcttg aattaggaga
65580gattttggga cgtgtgacta agttacgctg acacttgttt gtttcttagt
cgctttttcc 65640agtggcggtg agaacgaaga tgactgattc acattgctca
gatgagttta tcctcttctg 65700gctgggacat gggatatatc ctgtctcttt
taagcctttt tggtattttt cccccattga 65760gagctgtgtc ttcaaactct
tctgttatag ctggaaaatc ctttttaagt gaaatctgcc 65820caaattataa
gacagatgaa ggtagagttg tgttggatat aggattaggg tgaaagtagt
65880gggggtgtcc tggagcctct cttctggtgg cagcctagct cttgtgcctt
tgaggaaatt 65940accctgggga cggctctgtg gaacatattt gcaaaccact
gatttggaag atagagatgg 66000cttttgttaa gatctgaatt cacctttttg
gcattttatt tgatttctca aggtaaagaa 66060cttattttgt aataaagttt
cctattattt agtagatagg ccaagttgct gtgttaattc 66120catgtagatt
ttgggtttcc tttgctcatt ttttcactct taatctcaca tcattgtaag
66180tttatggaag ttatcatact tctgactttt tctttgaaga gcagaaatta
gaaattccca 66240ataattattt tgatagtgtc atttaatgac actcacatgt
gatgtagcca caaagattta 66300atgagttcag ttttaaatca tattaagact
gttggtttca tttgttctca ttaatgtaat 66360tctgaagatg aacaataaaa
tgtattttta gaactttcaa atgaaatatt atttcatcct 66420tccagatcat
ataatgctta agttctgatt gttaatcata aagtctagaa aattaaaaga
66480taataaaatg aaagtgactt ttaggtatta gagttttatt ataaattctg
gtgtgtcatt 66540ggagctatga catgaatatt tcaaaggcca atagcattgg
atctttacag ttataactta 66600ccatttttaa gtttaagtag taatatagat
tatttaataa tcaaaatcaa taaatattaa 66660ttattaaaat gttttgtggt
atagtttgag aatcattgct tttaactttt tccatatagg 66720tttattgact
ttaatagcat tctaaacata acatctctac attctttgtg tttaatactg
66780tggaggtata aaaatactta tatatgatga taaactatat tagagtaaat
taaatattct 66840tatgagtttc attttagagt gcatttactt aattttgaag
tccttatttt tagcaaacta 66900aaaggaatgt tggtacatta tttactaggc
aaagtgctct taggagaaga agaagccttg 66960gaggatgact ctgaatcgag
atcggatgtc agcagctctg ccttaacagg tagttctcac 67020tagttagccg
ctggtgtgga ccttcactgt ctgccttcca ccccttgccc ttcctgctcg
67080tccccctgca cctggtggac agcacgactg ggggcagcag tggagccagg
ttgcttaaat 67140ggggcatatt cgggcttctt ttataatact tactctgaag
cttgtgtgtc tgtggtgttt 67200gcatcatata tttgttgttt tccatggttt
aggctgtttt aaaattaggt ttatggcttg 67260agcatagggc tttgtgagta
ggggatggca ggtcgaaaca tctcatgagt tggatgggtt 67320atgctggggg
ttgggaaatg ggatgaaaaa ttatgggatg aaaaattgcc tatggatagt
67380ttaacttgaa agaatctgcc tttgtttaca gatagttatc ttttttcttt
tttgagatag 67440agtctcacac tgtcacccag tgcagatacc cagtgtcact
ggagtgcagt ggtgtgctct 67500tggtgcactg cagcctccgc cttctgggtt
ccagcgattc tcctgcctca gcctcccaag 67560tagctgggac tacaggtgcc
cgccaccacg cttggctaat ttttgtattt ttttgtggag 67620acgggttttt
gccatgttgg tcaggctggt cttgaactcc tgacctcaag tgatctgcct
67680gcctcagcct cccacagtgc cgggattaca ggagtgagcc actgtgcccg
gccagttaca 67740gatacttatc taatgaaatt ctctgtgtac tttataaaag
atgaggatta actgaaggta 67800ctaataactg gattatatga gggtggtttt
ggttgtataa tcctatctaa aagaatattt 67860tagctataac tgaaagtaag
acttaaatat ttagagagga aaatctgaat aattctagta 67920gtaattattt
atttacaaaa taaaaataga tttttttttg attacacaaa ttaaacaaca
67980ataaaacatc acagcaatcc ggatactata aagctcacat gcttaccgac
ccaactgccc 68040caggagtgac cactgccaac agcttcatgt cgaccttttt
gccataattt ttatatagcc 68100ttttttgttt ttaaatggta atttagaaag
tcaactagga aaatgtgtta caggtttatc 68160ttccaggaga ataggactgg
agtcgagatc ttgaatgtgg cttggaagaa ggcaagccca 68220ccccagagag
atgagttgac agttgtttct gaccactgct tgcttagagg gcctgcgtgt
68280ctgtgaccgc ctagctttgc gcccctgact aggctgcccc ttaattacaa
atgtctttat 68340atattgctcc agctaaggct tggagtagtc ggttaagaac
ttgaacttcg gtttttgcag 68400tgaaacagca tttgagaata tcaccttctg
ataagcctta ttttataagg tgggtactgt 68460agtgggaggc agtgtgagag
atgcttgaag gatgcactgc tgtcctgcat ttcagcatct 68520tcaggatgct
gtgcagctga aacatttgat aacggtggaa ctgttcgtta ttttgcaagc
68580ctgtgattcc ctattgaatg ttttctctcg ccatttgaca aatgagtgtt
tctctgtctt 68640cagcctcagt gaaggatgag atcagtggag agctggctgc
ttcttcaggg gtttccactc 68700cagggtcagc aggtcatgac atcatcacag
aacagccacg gtcacagcac acactgcagg 68760cggactcagt ggatctggcc
agctgtgact tgacaagctc tgccactgat ggggatgagg 68820aggatatctt
gagccacagc tccagccagg tcagcgccgt cccatctgac cctgccatgg
68880acctgaatga tgggacccag gcctcgtcgc ccatcagcga cagctcccag
accaccaccg 68940aagggcctga ttcagctgtt accccttcag acagttctga
aattgtaagt gggcagaggg 69000gcctgacatc ttttttttta ttttttattt
gagacagagt ctcactccat agtgcagtgg 69060aggccgggca caggggctca
tgcctgtaat cccagcactt tgggagactg aggcaggcgg 69120atcacttgag
gtcaggagtt cgagaccagc ctggccaaca tggtgaaacc ctgtctctac
69180taaaaataca aaaattagtt gggcgtggtg gcacatgtct gtagtcccag
ctgttaggga 69240ggctgaggca ggagaattgc ttgagcctgg gaggcagagg
ttgcaatgag ccgagatcgt 69300gacactgcac tccagcccgg gcaacagagc
aagactccat ttcaaaaaaa ataaaaaaat 69360aaagtgcagt ggctcgttct
cagcccactg caacttctgc ctcccaggct cgagcgattc 69420tcccgcctca
gcctcctgag taggtgggat tacaggtggg caccaccaca ctcagctaat
69480gtttgtattt tcagtagaga cagggtttca ccatgttggc caggctggtc
tcaaactcct 69540gaccttagat gatccaccca ccttggcctc ctaaagtatt
gggattatag ttgtgagcca 69600ccatgcccgg ccctgccacc tgccatcttt
tgagttcttc cctggagacc tagacctgaa 69660ccctcctgct tgttctcttg
ttatctaata cccctattga cagcgcagct tagatcatta 69720atggagagct
tgacctcatc tgataccttc actgaaggaa acaacttagt gtcttttgtg
69780ttgaacactg aggtaaaaaa ttggaatagt tgattatatg aactctgcta
aaattgagtg 69840cattttacat tttttaaggc cttgttgggc cctggttaaa
taattatttt taaaaatcct 69900taaggagcct attataaaca gatctgtggt
cttaatgaaa tgtgattaat actgtgcatt 69960attttaagaa cttttgactt
ttcaaaaaac ttttacaaca tttcccattt gatagcggca 70020taggtttaag
cacttctcat ctctaagtta gtggacaaaa aaccctcatg gatagtctaa
70080taatgtttgc tacaagtcca tgttgagttt tatactccat tttattttca
gttttaaaaa 70140ctgtggttaa atatgtgtaa cataaaattt atgttcttaa
ccattttttg cgtatacagt 70200tcgctggtat taaatacatt taaataatgt
catggaatca ttgctaccac ccatctctgt 70260aaccttttga tcatgtaaca
ctgaagctct gttcccattg aactctattc ctcctttccc 70320gccaagtccc
tggcaaccac gattcttctt tctgtcttct gaatttgact actttgggtt
70380ctcatatact ttaggagtca cacagtattt gttttactta gcataatgtc
cccaaagctc 70440atgcatgttg tagcctatgt tagaacttcc taatgtttca
ggccaaatac tattccattg 70500tatggatagg ccacattttg cttttccatt
cctctgtcca tggacacttg tattgcttca 70560tgttttagcc attgtgaatc
atgctgttat gaacgtgggt gtacagatag ctcctggaga 70620ctctgctttc
catttttttg gctaaatacc cagaaatgga gttgctttta cattccaatt
70680ttaatttaaa acattcatat cattgagtgt tttacttaat agtatagtag
ttaacaaact 70740taataaaata gtattttggt aataatttgc tggtagtcca
ttgttcagtt tttttaggta 70800aattacacag gacatttcaa gtggacatga
aacatcttgt gatgtggaat catgccccaa 70860gctgatggct aaacatatga
aataccatac cctaaattta gtagatttag tctttgcaat 70920ttaggagata
acctgttata ttgttaggtt tttgtcgaaa agctttgtcc tcatatttcc
70980aacttgctgt aaaatttgtt tgtgaagaca aatatttttg tatgggtttt
ttctttttca 71040tattaaaaag aaatgtccac attggaattt ttttggagtt
tttagagcta atagagcttt 71100tcataatgta gtgggaatga gtgatcagta
agctcttagc agtttccatg cgtgcatttc 71160tgtgccttga aataaatgac
agatgagtac atttgtgttc tgtgtgtaaa atgtgctctt 71220tcctcattgc
acttccatgt tggagggctt gtctcttggt gatcacactt caaaattctc
71280acagcccccc ttgaaccgtt taggtgttag acggtaccga caaccagtat
ttgggcctgc 71340agattggaca gccccaggat gaagatgagg aagccacagg
tattcttcct gatgaagcct 71400cggaggcctt caggaactct tccatgggta
tgtggactac aggtgatgcg ctacaaagtg 71460gtttgtattc agacctggac
atcttaatta
tatctttgct tccaagaaga agtcctttga 71520tactgttttc tgagttctga
atagctgatg aaaatgacca attgaggaat aatcatactt 71580tttcttgatc
taaatcttat acttttgagt tatcttagca taaatgtata attgtatttt
71640aagtggaaat ttgtcactta atcttgattt ctctgttttt aaagcccttc
aacaggcaca 71700tttattgaaa aacatgagtc actgcaggca gccttctgac
agcagtgttg ataaatttgt 71760gttgagagat gaagctactg aaccgggtga
tcaagaaaac aaggtgaggg acataggctt 71820gagacgactt ggtgtttctg
agcttgtgtg aggatttaaa atcgccctgg ctactgtcta 71880ctttattgct
ttcccatccc tgggccttta aatttcccct ttaaatacca gctcttccca
71940ggcctgttgt tttctgcctt tccaggtact acccacagcc ttgagaattg
cctgagttct 72000gcctcctttg agagtgtgcc ccagacaaat ctattctgta
ctgaatgttt ccttgtctga 72060tttcttggat cattcatttg atggttgcgt
atggcctgca acgtttcttg ttttggttct 72120actgaactgt tctaaaagtc
tctcttcata ttatcttttt acatgtaaat gtaactgtct 72180tcacttttaa
ttcctcaagg acaaggaata gcgtttcaca gttcgtccca tcaatcagaa
72240ttatagcctt tggcatctcc ctatctacca ggcccacttc ctcttagatt
tgggcttccc 72300caggctgttg cctttcccca agtagcttct gcttgtcctg
tagaagacct ttcatgcttt 72360gcttctgcag cagccgttcc tgaatgccta
gtgtcaactg ccttcttacc acgcccaccc 72420tccctgcatg ctgcatttat
cccctgccac agccctgtga ccctgtgtcc tgctgcctct 72480gacttgtctg
tttctgcttg gccatggtct ctgtgaggtc aggtgtgcat atgggcacaa
72540accagggcat ctctttatcc ccagcacctg gcttaagtgc tgctctggaa
ctatctgttg 72600aatgaactaa tgcatgaatg tattgttgag tatgagacaa
acaagtgtca ttgtctcctt 72660tctagccttg ccgcatcaaa ggtgacattg
gacagtccac tgatgatgac tctgcacctc 72720ttgtccattg tgtccgcctt
ttatctgctt cgtttttgct aacaggggga aaaaatggtg 72780agtacaaaag
gggatgtgca cagttgaagg aaataactag gtttcagagg tcagcttggt
72840ggcctgtttt tgccttgcgt gcagcagagg aagtagaatc tgaggatgag
tttggttttc 72900actagccgag gggagggagg aaatgatggg agcaggtagg
ttattgggtc tggttttgtt 72960catttgaaaa caatctgttg tttgaggctg
aaggtggctt gggtgatttc ttggcagtgc 73020tggttccgga cagggatgtg
agggtcagcg tgaaggccct ggccctcagc tgtgtgggag 73080cagctgtggc
cctccacccg gaatctttct tcagcaaact ctataaagtt cctcttgaca
73140ccacggaata ccctggtatg ttaaaagttc acatcttatt ttctcagatt
taatcattat 73200tgtaaaaact atttcagtat tgactatttt agttttagag
cagtaagtgt tttgagttca 73260tttgggatat ttgacctgcg ttgtagctct
tcagaaaaca catgaatagt gaagttcttt 73320gtttcatggg ttccctttag
atgaaaccca tagaggagaa aagtagaaac ctcagcacgt 73380aagagccaac
atatatacac atcggattta aacctaaagc acaaattgtg cctggtcgca
73440gtggcgctga gtcgcactca gccaggccag gcattcacac tcagggtgag
tgggaaccag 73500gactggctga ggcagcagtg gacccaagtc tccatcgcgc
ccatgcttac tatggagcct 73560tctcgttctc tctttttctt tgggtgagag
ggtacacttg tgtttttgaa tttatatgag 73620gtaagtgtgt aatagggttt
tttctaatct tttttaagtg gaatctggaa ttttaatcag 73680atttattatc
tgacaaccta gaattataat ccagaaagtc tgtggtattg aggacatatt
73740ggcaatatga tgaatctcta attcttaaat cctgaaactt tttttttttt
aatcacttag 73800ggttattata gtgaagtcat ttctgaattt ggatcttctc
ttcacacctc tttttctctt 73860tcctgagaat taagcttttg tttcgagtta
gaaagttgat agtagggaat tgttccatgg 73920ctgagcaatt tatctccaca
gaggaacagt atgtctcaga catcttgaac tacatcgatc 73980atggagaccc
acaggttcga ggagccactg ccattctctg tgggaccctc atctgctcca
74040tcctcagcag gtcccgcttc cacgtgggag attggatggg caccattaga
accctcacag 74100gtaacggcca gtttttcagc tgtgtttttt ctagttatgc
ttactaaggt ttaagtttag 74160atgatgatgt ttgttgcttg ttcttctggt
taggaaatac attttctttg gcggattgca 74220ttcctttgct gcggaaaaca
ctgaaggatg agtcttctgt tacttgcaag ttagcttgta 74280cagctgtgag
ggtgagcata atcttctgtg gaaccatttc ttcacttagt ggacatttta
74340tcattgctac aattaaaatt ggagcttaat aggaaatatt tccatgcact
ctaaagctgt 74400aaccagtaat acccaccatg tatccatctc tcagctttag
aaagaaaacg ttgccagtaa 74460agttaatgct tcataaactt cagtttaagt
tctaattctc agaatatttg tttgaaatag 74520acctcttcct aaaggatata
tttagaaata acctatcatt aagtgtaaag tctgttgaat 74580atgctgggca
cggtgactca cacctgtaat ctgaccactt tgggaggcca aggtggaagg
74640attgcttgag cccaggagtt caagactatg ggcaacatag ttgaccctgt
ccctacagaa 74700aattaaaaaa aaaaaaaaaa aaagtagctg ggtatggtgg
tgcatacctg tagtctcagc 74760tactcgggaa gctgaggtgg aggggggatt
gcttgagccc cagagatcaa ggctgcagta 74820aggcgtggtt acaccactgc
cctctagcct gggcaacaga gtgagactgt ctcaaaaata 74880atagtaataa
taatcagttg aattaaaaaa aaaaaaaaaa aaaccactgt gctaggccca
74940tagtatggta agagttaaag tgagccttag ggattattta ctcaacctct
gtttctgtat 75000aaagtggaat aggctcaatt ctttaagtga tagcatgttg
aacctttcca taccaactgg 75060ctcataagtc acaactggcc agtcaacaag
agtaaaaatt aactggtaaa aatcaaagca 75120aaaaacctac aattgtcaaa
tttgtgggat aactccccct tttaaaatgt catgcctgac 75180agtaatttct
ctctagtttc caggttttca gtcagttgtg tcttttttga gcagaaggaa
75240gcatgctaag agctcaatct tgtggctagc tgggggtctt tgtgtcagcc
atgcatgtga 75300tggtgcccct gggtgcttgg ggctgcaggg gaggggtaca
gcagtagggg cctgttctgt 75360tctctcgtgc tgtggagtac atagtgacat
agtggggtgg tccttggtgt aggtcccttg 75420ttcctacccc tgggtctgag
atttatttag aagtggtgtt ggggctgtgc ggcaggcccc 75480tctgtaactg
atcaatgttt gtgaagttgc tgtttgagag ttgaaaccat gacataagca
75540gaaatggaag gaagaaagaa ccagttatgt gaaagggaca catttacttt
taagcttgta 75600tttactgaga taaagtattc ttaatcaatg ttcttgagag
gtgtgggaaa aatgcaacat 75660cctggttgca gttaaaccca gaacattgtg
tgttgaagag tgacggttct caaaccgtca 75720agacgcgggt actgagtggg
actaacctgc tgtcctcttg ccttggacct tgtgttccag 75780aactgtgtca
tgagtctctg cagcagcagc tacagtgagt taggactgca gctgatcatc
75840gatgtgctga ctctgaggaa cagttcctat tggctggtga ggacagagct
tctggaaacc 75900cttgcagaga ttgacttcag gtaagtgagt cacatccatt
agatttcatg aactaagctc 75960aattgaaagt tctgggatca cttgatgcaa
ggaatgatgt tatcaagtac cctgtccatc 76020agaaatccga gtggtttagg
tagatgacag tgattttctc ctcccagtgg ctttttgctg 76080aactttgccc
tatgcttgga attttatttt attttattat ttatttagag acaagatctt
76140gctctgtcgc ccaggcttga atgcagtagc acaatcatag ctcactgaag
ctttgaactc 76200taggactcaa gtggtcctcc tgcctcagcc tcccgattag
ctaggagaat aggtgtgtgc 76260cgtcacactg gctaatattt tttgtagaaa
tggggtcttg ctatgttgcc caggctggtc 76320tcaaactcct gggcttgatt
gatcctccat cttggcctcc caaagtgctg ggattacagg 76380catgagccac
tgtgcctggc ctagaatttt aaaatataag tagaagagta gatttttttt
76440tttggtagtc ctcgtcattt aagtattctg gatagtggga ataaaagagc
ttagaatttt 76500tcatctttgt cttaaacttt taaaaaaatg tagcttatat
taattctgct tgtttaaaaa 76560gaatatactc ttcattatac tgaacctagg
taagacagct ggtttatatt ttgttgcaat 76620taaaaaacgt gagctgtggt
tgcagtgagc caagattgtg gccattgcac ttcagcctgg 76680caacagagtg
agacttggcc tcaaaaaaaa aaaaataaca tgagctgtgt tggcactttc
76740attttctaag agtagttttg gctggagaag ttttctttca gtactttctt
ttagaaggga 76800aattttcctt tataatttag ggtttgtttt ttttttttcc
aagccacctt ttatagagcc 76860cttgtgggtt atttcattta atccttagaa
tgtttataaa tctgggcttg ttctcggctc 76920cacccacaga tagggacgct
gagcgtgcat gagtgggcag caagatagca ggttatggag 76980ggcccagctc
accccttctg tggcttgagc caattttata gggcacttac agagtctttt
77040gaaatagtat ttattttgaa gaaaaagaaa aacagtttac tgagtactgt
cttattgagt 77100ctggaattgt gagaggaatg ccacctctat ttatttaaag
ccattggcct tttttgttgt 77160tttgagtaag tgctgcccaa ggtccttcca
gggcacctgg atgagcctgc tctggagcaa 77220gctggcggta agtgtttact
gagtaactaa atgatttcat tgttaaatgt gctcttttgt 77280taggctggtg
agctttttgg aggcaaaagc agaaaactta cacagagggg ctcatcatta
77340tacaggggta agcggtttat ttttgtgaga tgctgtttta ccttcaagaa
ggtgaaagtg 77400aggctttcct tgtggaattt ctctaaatgc attcgtcatg
ttttagatgt ttatttcaca 77460gtttatatca tgaaagttat aatcttgtca
tatggattta agtctagtaa tgttgagttc 77520tttctcacta gctttccaaa
atatcttacc taaaatttag tcaaatacaa gattatgttt 77580atttttatta
tccttctctc taaagctttt aaaactgcaa gaacgagtgc tcaataatgt
77640tgtcatccat ttgcttggag atgaagaccc cagggtgcga catgttgccg
cagcatcact 77700aattaggtat ttaccaatat tttatctctt ttcctttttt
ggttgaagta ctaaaagata 77760cgagaatgga aagagaggga agaattcaaa
ggatgtagag cagtattcct gaatctgagc 77820tcatttcagc cattctattc
ttaaactata atgaaaaaaa aatccaaaaa agtctaaaat 77880tataattaaa
aaaacaacaa aatactaact gtccattgta aaaagtaatg cactttcatt
77940gtaaaaattt tggactatag agaatagtac taagaagaaa aaaaaaatca
ccttcaattc 78000tgctgccacc tggaggtaat cactgttaat attttgctat
atactctatg agtttcttgt 78060tcaaaatcag gtcaaaatta catgcaattt
tgtaatctga caatttccac ttaatatttt 78120attagcattt tcctgttatg
aaacagtaat tttagttatg ggtcgttgtt ttgctatgcg 78180gttgggataa
aattttatat actttttttg gcaattactt attatacata aatgtttgtg
78240tatagttttc tttttctgag aattcctgga agttgagtta ccaggcccgg
ctttgaattt 78300ttttttttat tttttttttg agacagagtc ctgctctatt
gtccaggtgc tatctcggct 78360cactgcaacc tctgtctccc tggttcaagc
gattctcctg cctcagcctc ccgagtagct 78420gggattacag gggcacacca
ccacgcccaa ttaatttttg tatttttagt agagacaggg 78480tttcacgata
ttggccaggc tggtctcgaa cttctgaccc cgtgatccac ctgcattggc
78540ctcccaaagt gctgggatta caggcgtgag ccatggcgcc tggccaggct
ttaaatttaa 78600aacaaatctt ctaatagctt tatggaggtt ataatttaca
tttcttgaaa tgtactcact 78660ttgagtgtat agtaaactcc aattttatca
catttctgtc accccaaatg tatccttgtg 78720cccatttgct gtaacctccg
gttcctgccc caactcctag gcagccactc atctattttc 78780tgtcccttaa
gatttgtgtt ttcgccaggc gctcatgcct gtaatcccag cactttggga
78840ggccgaggtt ggtggatcac ttgaggtcag gagttcgaga ccagcctggc
caacatggtg 78900aaaccttgtc tctactaaaa atacaaaaat tagtcggatg
tggtggcaca cgcctgtaat 78960cccagctact cgggaggctg aggcaggaga
atcacttgaa cctgggaggc ggaggttgca 79020gtgagcagag atcgcgccac
tgccttccaa cctgggcaac agagagagac tgtctcaaaa 79080caaacaaaga
tttgtatttt ctggacattt tatagtactg gggtcatagt atagatggac
79140ttttgcattt ggcttctttt acttaattgt gagattggtt cttgttgtag
catgtatcag 79200tagtttgttc atttttattg gcgaaagtat tctattatat
gaataatacc atattttatc 79260tatccatcag atggatatta tagagttcat
gttttggcta atttatgaat tatggtactg 79320tgaacatttg cctgcaagat
tttgtgtaga catgtcttca tttctcttga gtagatcacc 79380tagaagtgga
tttttaaata attttggtac ttactgtgaa actgctcttc aaaaacatac
79440cattgttcct tccttccttc cttccttcct tccttccttc tttccttcct
cccttcctcc 79500ctcccttccc tacttccctc tccctttccc tttcccttcc
ccttttccct tccccttccc 79560gcctgcctgc ctgcctgcct tccttccttc
cttccttcgt ttctttctac atatacacat 79620ttttttaaat ttcaatggtt
tttggggtac aagtggtttt tggttacatg gctgaatttt 79680ggttacatgg
tgaagtctga gattttagta cacctgtcac ccgagtagtg taccttgtac
79740ccaatatgta gttttttgtc cctcaccttc cagccttccg ccttgtgagt
ctccaatgtc 79800cattatacca cactgtatgc ccttgcgtac ccacagctca
gctcccactt ctgagaacat 79860atagcagaaa catgccaaag tatactccca
ctaccagaat gtgattgtgc ctgattcttc 79920tcaccagtac aaatatttca
aaaaaagtta aatatgtatc agttttttgg gcagaagttg 79980atacttctct
ttatttattt attttttttg agatagggtc tcattctatg atgcccaggc
80040tggagtgtgg tggtgcgatc tcggctcact gcagtctctg cctcccaggt
tcaagtgatt 80100cccacgtcag cctcccagga agctggaatt acaggcgagg
gccaccactg ccagctaatt 80160tttgtatttt ttggtagaga tggggtttca
ccatgttggc cagactggtc tcaagctcct 80220gacctcaagt gatccacctg
ccttggcctt ccaaagtgct gggattacag gcgtgagcta 80280ccacacccgg
ctgatatttc tttttaaaat aacttacctt cttttgaaag taatacatgt
80340ttaatgaaca gaatttaagg aaaatataaa aaaacgaaat aatctttgta
atcaaactac 80400tgaaaagaaa accaaagtta cattttggtg catattcttt
ttcattttca tcattgtaat 80460ttgcatttct ttgattactt gtgagacact
cctttcattt acttaatagg tttatatgac 80520ttgcctattc agagattttg
cagctttacc attttctgca aatgatagca acttcttttt 80580gtttgtttgt
ttgtggagac agagtctcgc tctgtcactc aggcaggaat gcagtggtgg
80640aatcttggct cattgcaact attgcctcct gggttcaagc gattttcctg
cctcagcctc 80700ccaagtagct gggattacag gagtgtgcca ccatgcccgg
ctaatttttg tatctttagt 80760agagatgggg ttttgccatg ttggccgggc
tgatcttgaa ctcctggcct caagcggtcc 80820ccctgtctcg gcctcccaaa
gtgctgggat tacaggcgtg agccaccgta cccagccagt 80880agttacttct
tatattctag aaaaaattct actcatgatc aagtctccat gaggaaagag
80940actttaattg aagatcatgg ggcttgcaga ccaatatgat aaaatagttc
attgtttcta 81000aaagtattac tgagtgttga tggcagatat gaaccctttt
gtttttgtag gaaaatgtta 81060cccgtattct ccatttgaat tcagtttaga
tttgttagga atcgcagctt aagctttgcc 81120atctgggagt gtttgggaca
gttttgcaga caaaattgca aaagtgccta aggaatgcag 81180ctggcattca
gacctgctct gtgctcagta ctctgtggac agacactgtt cagcacttgt
81240tgatcagaag gtttagaaag agaactttca aagttggttt ttaattaaag
catttaatag 81300tgtaaataga aagggattaa attttatgac agacaaaaga
aagtacagca cccagctggg 81360cgtgggggct cacgcctgta atccagcact
atggggggct gaggtgggtg gatcacgagg 81420tcaggagttc aagagttcaa
gaacagcctg gccaaggtga tgaaaccctg tctctactaa 81480aactacaaaa
attagccggg cgcggtggca ggcgcctgta atcccagcta ctcaggaggc
81540tgaggcagga gaatcacttg aacctggacg gcagaggttg cagtgagcca
agattgcacc 81600attgtactcc ggcctgggcc acagagtgac attctgtctc
aaaaaaaaaa aaaaaagaaa 81660aaaagaaagt acagcaccca gttatgtccg
agtgggtgca tgagagtgac cctgagattg 81720gagacaacgc tgtcacgtgc
ttgaagaacg ccacctgaga aagggggcga gaagtggtgt 81780ccgctggtaa
ccagaggtgt tggcttagcc atctgcaggg aggagggtgg tctatcacag
81840gtgagtttca tctactttct taagcaaatt aaccttactt ttgtgttagg
cttgtcccaa 81900agctgtttta taaatgtgac caaggacaag ctgatccagt
agtggccgtg gcaagagatc 81960aaagcagtgt ttacctgaaa cttctcatgc
atgagacgca gcctccatct catttctccg 82020tcagcacaat aaccaggtat
gctgacccag tggcatcttc acattgtcgg gaaaatgccc 82080tttcctgatg
cctttcttta ggctttaatt gaaaacattt tattttctag aaaaaagctt
82140cagctcagga tgtttgagtg taggtcagtc ctttgatagg atattatcat
tttgaggatt 82200gaccacacca cctctgtatt taagctctgc cacaatcact
cagctgtgac actgtaaatc 82260tcttaatagt ttattacatt ccatgtgctg
acagttgtat ttttgtttgt gacacttacg 82320tattatctgt taaaacattt
tcactttagt tgtgttacct ttaaagagga ttgtattcta 82380tcatgcctgt
tgattttttg gtgagcgggc tattaaagtc agtgttattt agggttatcc
82440actagttcag tgatttgcga gattatcatt cacatttatt gtggagcttt
tgaatatcgt 82500gtcaaatggc cacatatatc ccattcttat ctgcttctta
ggtgagtggg acacagtgct 82560ttaatgaagc tataatcttc agaattctag
cttgcagaga agattgcaga agtgataaga 82620cttgtgcttt ttaattttgt
cttttaaatg ttattttaaa aattggcttt atatgatact 82680ctttttttct
gctgagtaac agtgttttac aaaacttgga ctaaatgact tctaagctta
82740aatgatcact tgatgctttt tttctgaatt aggaactcag cttatcaaat
atcaaagtca 82800taattcctga ataaataacg tcttttttca tgtaaagact
gctttaaaaa acacatggaa 82860ggctgggtgc ggtggctcac gcctgtaatc
ctaacacttt gggaggccca ggtgggcagg 82920tcgcttgagc tcaggggttc
aagaccaccc agggcaacat ggcaaaaccc acctctactc 82980aaatacaaaa
aattagccag gcgtggtggc gggcccctgt aatcccagct actcgggagg
83040ctgagggatg agaatcactt gagccccgga ggcagaggtt gcagtgagcc
aagattgtgc 83100cattgcactc ccagcttggg ctacagagtg agactctgtc
tcaaaaaaag acacacacac 83160aaacaaaaaa aacatggaga catttttttg
gccaccttaa tatttcccct cagataattt 83220cctttgttta aactcagaac
tggcattttc tctcttggag aagattcagg acaaatactc 83280ctttaagata
agtagaagca gtgaaagagg atttgattat caggaatttg ataagcttag
83340aataaattgt tgcttcttaa tgtcatttca gaagatgaat atttattaat
agatgccaac 83400tgagatatca ttaaaattga ttactaacta ctacttggaa
aagtctccca gttccaaact 83460tcagcaggcc tcttgacaat tcagctgtgg
tcaattgggt cttgcgtgat agatacaatg 83520accaattgtg cagcagagtg
tgctgcttag ctgcctattc tgttagcatt catgtgttaa 83580cttaaaatca
taatctcctt agttttgttg agtgtctccg tggacaagac actgtgaggg
83640atacaaaatc agattggctt tattcaaacc actggggtat tataattcat
ttataattta 83700ttttattttt tgcctttttt ccatgtgttc taaaggaatt
agagtttgta tataactata 83760atgggggata gaaattgaca tgtgccatga
agggaatgca aaaaagtgcc gtgggagatg 83820agaagtggag aaaggaattt
cttttttctt ggaagcagga ataacttcat gaagcatgta 83880tttcaactta
aacagatagt aggcaacgct gtaaggggag tatggctgca gcaaaagtgt
83940tcggggcaga ctgggaggaa gggagggaat aaattcagcc attgttatgg
aataatgatc 84000aaaatttatt ttcagcccgt ttcacttaaa agttgagact
gcttaacttt ttttaatctt 84060taatcttaaa cttttaaatg ccatttgatc
tttaaaaata tatgttttaa tagtgtattt 84120taagtctcta tatttttgtt
attagaatat atagaggcta taacctacta ccaagcataa 84180cagacgtcac
tatggaaaat aacctttcaa gagttattgc agcagtttct catgaactaa
84240tcacatcaac caccagagca ctcacagtaa gtctctttct tgatcggtct
tactgacatt 84300gtaatagttt ttggtagctt gtatggccag ttagttgtat
ggtcatctta cggtgaggtg 84360cttgtcttac agctcttact tatccatgag
gcttgctaag aaattgtgct tctgtgaaaa 84420gaatctcagc ttactccagg
aatgtaaatg actatgtttt ttctgattat taaagtaata 84480cacgcccaaa
ataaaaaaat tcagccaatt taggaagaca caacaattaa aataagccag
84540gcatggtggc tcatgcctgt aatcccagca ctttgggagg ccaaggttgg
gggctcactt 84600gaggtcagga gtcggatacc agcctggcca acgtggtgaa
accccatctc tactaaaaat 84660acaaaaatta gctgggcgtg gtggcgggcg
cctgtaatcc cagctactca ggaggctgag 84720gcaggagaat cgcttgaacc
tgggaggtag aggttgcagt gagctgaggt caagccactg 84780cactccagcc
tgtgcaatag agcgagactc tgtctcaaaa aaaaaaaaaa aaaaagaaaa
84840gaaaaaagta aactactgtc acctgcattg gtaatgtatc agaagtttaa
aatgtctaga 84900ttataattaa ctcagtgacc tggtaatata tactaaggga
aaaatattta taatttacat 84960ttttacattt ttattttttt aattttatta
tttttttttt gagacagagt tttgctcttg 85020ttgcccaggc tggagtgcaa
tggcatgatc tcagctcacc acaacctcca cctcccgggt 85080tcaagcaatt
ctcctgcctc agcctcctga gtagctggga ttacaggcat gcaccaccat
85140gcccggctaa ttttgtattt ttagtagaga cagggtttct ccatgttggt
caggctggtc 85200tcaaactccc aacctcaggt gatccgccct cctcgacccc
ccaaagtgct gggattacag 85260gtgtgagcca ccatgcctgg ccttacattt
ttataataag aatttatgtt gctgacatta 85320gaaaagaacc ataatatcca
agaatccaag aataattaaa ttatgtacat atgctagtat 85380atagtgtgat
gctttggaga atttttaaca atatggagat gtataatctg gattgtaata
85440ttgagtgaaa aaaggcagaa tacaaacctg gtgggggtat agtcggattt
cagttaagaa 85500aaataatatt tacatatata catttctcac actggcagat
aatcaccaag ataaattttg 85560ggattgtgga tgattttttt cttctttata
tttttcagat attctcaaat tttctaaaat 85620gagcaagtat aacttttgtt
atcagaaaaa aataatatac aaaagtaatg ttaatttgct 85680ggtgaccagg
ttaaaccttt ttatttttat tttttgagat ggaatctcac tctgttgccc
85740aggctagagc acagtggcat gatcttggct cactgcagcc tccgcttcct
gggttcaaat 85800gattctctgg ccccagcctc ctgagtggct ggaattacag
gcgtgtggca ccacacctgg 85860ctaatttttg tatttttagt agaggtaggg
tttcaccagg ttggtcaggc tggtctcgaa 85920ctcctgacct cgtgatccac
ccacctcggc ctcccaaagt gctgggatta caggcgtgag 85980ctactgcgcc
cagccagacc tttttatttt atttgacaaa agaaatactt ccatgttata
86040gaagactaaa tattgtttgg gctgtctgca gtatggtctt cccttgattt
gttcaaaata 86100tcgtaaactt tgcttattta tttttattgt ggccgactgt
gtcgggcact gttgtaggct 86160tgggatggaa aaacaggatt cctgccctta
gggtttctgc aggctggtca gggagacgat 86220gtggtaagct ggagctcagc
tcctaaggat gtgcaggggc agttgagagg cggaagggtg 86280ggagatcatt
ccagggtgtg ggcagcacag gaacctctct tcattgggat ataattgcca
86340ttctgataac acgtgtttga ggtgtctaaa gtaggaagtt gtaccatggt
gggacagata 86400tcctgtggtt atcatacaca gatctcagtt ttcttctcat
tgtttgtact ttttataaag 86460ggtaacagga gatataattc aataaacctt
tgtggtgttt gggtgtgatt ttattgtttc 86520tttcttctca gtttggatgc
tgtgaagctt
tgtgtcttct ttccactgcc ttcccagttt 86580gcatttggag tttaggttgg
cactgtgggt atgtattttc ctcagtatat attaatagtt 86640gtctacaaca
gtatgacata aacatagtta ttaggatgcc ctttttcttt ctttttaagt
86700cttttatcaa tttggctttt tggaaaaata tctgatggaa tacttgtttc
tgctatatta 86760gctgtgtgag actagtgaca ggagctgtgg gaaatgaatg
ccaaatgttc ttaggcattg 86820atgggaattt cagggtgtgg tcttcaagtt
catttaaggg aattttcata tgctggcaaa 86880aggcttttct cattagcttg
actctttcca aaattatttg ctgtgaatta gaagtttagg 86940aacctttttt
cacttaattg tgacctagca tacgaaatgg tgatgattta ggaactactg
87000ttcttgtatt aacagctttt atttaaaaat gattttcctc cagtagatgg
ccctactagc 87060atctgggaaa taatttcaag tcttctccag cattcaggaa
taggctttca ttttgtgtat 87120caattactga gaatgatttt ggtgactcac
atcacatttg agaagtaaac ctgcagattt 87180cttgtgtgtg tcagcaaatg
accaactgat atttgcttga agtggattac attatctgct 87240ctagaatgat
tgctttccca ccttcctcac atacagactg agcagctacg gtttctaatc
87300ataggtctgg cactagactt cacttctggg caactttggc attggagtaa
aatgtattaa 87360tttaaagaaa gttaaaaatc cgttcaagta aacatacagt
tctaatactt tttacaattt 87420aaaatataga tttaaatgat aaaataaaaa
agaaaatatg ggtagacacc ataatcctcg 87480tttctgcatc tgttcacaag
gggttgatat ttatgagttc tattctccat atccattcta 87540tgttctctta
atgctcagtc agcacctcag gtggttggag ttcaatgctt ggtagtttga
87600cttacactgt cttttctagg ggattgagcc ctgggtagtc ctgcttattt
gaggttgcaa 87660tttgtctttc aataactttt actacaagat atggcgtgtt
aaaggatacc attggggaac 87720caacataata atatcaggaa aactaaccac
gtcagacctg ccccattgtg tatcaagtac 87780actatttttc catagtaata
aagagttcac cccagccaat tctcttttat tttgtgcctg 87840tttactcaat
ggcattaaca tgcccaaatg tctgggtagc tgtctcatct ccagttcagc
87900agaaccattg tcatatgccc tagtaaaagc attccttcat tggacactta
ggccccaata 87960ctttcattca gatctactac ctgatttcat ttctcaaatg
atttttatgg agctctgatt 88020tataggaaag atgttagttg attaaaaata
aaacaatttc tgagctggta taaaatgtat 88080tgtgacatgc cttcctcttg
gaattgcaag agaaaggaag actgttgttt gcttaaaaat 88140tgtctataat
ttgactttgc aaatgtctgc ttccagagtg cctccactga gtgcctcaga
88200tgagtctagg aagagctgta ccgttgggat ggccacaatg attctgaccc
tgctctcgtc 88260agcttggttc ccattggatc tctcagccca tcaagatgct
ttgattttgg ccggaaactt 88320gcttgcaggt actggtactg agttgaaaca
gggactccag gacttggatt ttgatttcct 88380tagggggaat gggggtggtg
agcatatgag gggaaaatac tataaggtca ttgccagtga 88440tggcttgtcc
ctttagtcaa atttcagatg ttacctatat gcataaacac atgcagttgg
88500cagctgttct gtgctgagta ttttaaagta gcctcttccc aatatagccc
ctcagttaac 88560tacaagtaaa ctcattttga atttcatttt aatgggcacc
atatgccagt actccctcgg 88620gcactgggat gttaagaaag tataatgtat
ggacttcatt ctcaagttag ttttagatta 88680gagggggata cacgtaaaca
aaagtgcagt ggtcacacag agtggcccta atcactctcc 88740ttgggcagat
ttatgggctg gtaggaaaga gcacaacacg gagagggtgt agcaccttgg
88800cgatgataat ggaggatgtg gccagcaagg aagacggagt ccattgaaat
tgattttggg 88860agaagttgcc aatctccatg aaagaattgg ggcctgtgct
atttgcttca gggggctata 88920ggagagtttc gtgaaaggga ctaaaagatg
agtattttaa taagatcatt catccaactt 88980gaacatgggc tggaggagaa
ggtagggaga ctcaggagat taatgttgat gctaaggcaa 89040gataatggct
ttgggactgt agggaagaca ctgattgtaa gagaatgaag gaggcagaat
89100tgccaggcct ggttcaccaa ctgaacttcg gttgtgaaga caaagaaacc
tgggatgact 89160tcacatcctg ggcaggtgtg tggtggtgac agtcatggaa
attgggaaca cagatttgtg 89220cgggaaacat cagtttcagt ttgagtttgg
cttatcagtt gaatatcagg cacagatgtc 89280tggccaactc tcaacatagg
gtcttaaatg acttcagttc cccaagcaat ttgtccttcc 89340catgctattg
gggtggagag gtaatgtctg tgcccatatc acagccagtg ctcccaaatc
89400tctgagaagt tcatgggcct ctgaagaaga agccaaccca gcagccacca
agcaagagga 89460ggtctggcca gccctggggg accgggccct ggtgcccatg
gtggagcagc tcttctctca 89520cctgctgaag gtgattaaca tttgtgccca
cgtcctggat gacgtggctc ctggacccgc 89580aataaaggta atgtcccact
tgggtgctgg attcatacag ccttaatgac tatgggtttc 89640cagactacct
ttgtttagta atctgtccct tctttattct ctttttgctt taaatgaaca
89700aaattgctca gattgtgaca ctaaatttaa catcaaaatg tgaccatgtg
gatgggtgca 89760gtggctcgtg cctgttattc cagcactttg ggagactgag
gcaagtggat cacttgaggc 89820caagagttcg agaccagcct gggcaacatc
acgaaacccc ctctctacta aaaatacaaa 89880aaattagatg ggttgggccg
ggcgtggtgg ctcaagcctg taatcccagc actttgggag 89940gccgaggtgg
gcggatcacg aggtcaagag atcaagacca tcctggctaa cacagtgaaa
90000ccccgtctct actaaaaata caaaaaaatt atctgagcat ggtggcgggc
gcctgtagtc 90060ccagctgctc gggaggctga ggcaggagaa tggcgtgaat
ccgggaggcg gagcttgcag 90120tgagccgaga tcgtgccact gcactccagc
ctgggtgaca gagcgagact ccgtctcaaa 90180aaaaaaatta gatgggcatg
gtggtgcgtg cctgtaatcc cagctacttg ggaggctgag 90240gcaagagagt
tgcttgaacc tgggaggcgg agtttgcagt aagccttgat tgtgccgctg
90300cactccagcc tgggtgacag agtcagactc tttccaaaag aagaaaaaaa
tgtgaccatg 90360tgttttatag ctcttttagt atcatcagtc actgttatcc
ctaagaggga aatacctagc 90420tttagtttta ggtttccagc attagccaag
aaagctcaga attgatgttc ctggccaagt 90480acctcattgc tgtctcctta
aatcttggtt aatggctact gtcctggcta gcatagttat 90540ggagcatttc
catggttgta gaatgttctg ccaatctcag ggacagtttt gcttttctgt
90600gaagcaataa aatcaacttc aaaacaaatg ttaactattt gtacaatgga
tttaagatag 90660accagttcac atactttttt tttttttttt ttttgagatg
gagtttcatt cttgttgcct 90720gggctggagt gcaatggtgt gatctcagct
cactgcaact tctgcctcct gggttcaaac 90780gattcttctg cctcagcctc
tcgaggcaga ttacagctgg gattacaggc atgcaccacc 90840acacccagct
aatttttttg tagttttagt agagacgggg tttcaccatg ttggtcaggt
90900tggtctcaaa ctcctgacct gaagtgatct atccgcttcg gcctcccaaa
gtgttgggat 90960tacgggcatg agccaccacg cccagcctaa gatagaccag
ttcacttact gtttatatct 91020gattactctc tctttgcctt gtcttctacc
tttaaaaatc tccctactaa cttcccattc 91080tcctttagct gccatcagtc
ttctcccttc tctgcaaaca tctctggaga gtcccagcct 91140cagcccacag
agcttcccac tgctctgagg tggaccttgt ttgcaaggct tctttggctc
91200tcttggcctg gaccctgtct actacttcag ccatccttcc ttaacccctg
ctggtggttt 91260ctgttgccac actccatagc agcgtttccc gcccagatca
tgtctttaca tctctgggca 91320ctgctctggt cctgcctgcc tttccctctt
tgtatcctgc aggctgctac ccccatcttg 91380agtgtcctct tcagttggct
ttcagagggc ctcctgggtg ttcccttacc cacttgccac 91440tccccagtca
ctgggttcag tccttcctgc ccaccagcac atgctttcta ggctctgtcc
91500taggccgtct tctctctttg tagtctctgg gccagtgctg ttctagagag
tggcagaatt 91560ttctataacc atggcagtgc tccatagcta tgccaggcaa
gacagtagcc actaaacaca 91620tatagctgtt gagcccttga aatgcagcta
gtgtgactga agaactgaac cccgattcgg 91680tttaattttc attaaattta
aatttaaata accttatgtg ggtagtggct ccagtattgg 91740gcagggcagc
ctgagagtcg gggctgttct cctgtcttca gtgtctagat gagggacctc
91800agaggacctg tctctggagc tgcagttcaa tgtagccagc tgccccgtga
cacttacata 91860tagctgattt gtggatatgt cagacacggt gtgatgagct
cagctttctg tcctcctccc 91920cacatctgcc cctgccccat ttaccccact
ttgtgtctta tcaagctaga aacaggtcac 91980cacaagtctt catttccact
caccaagtct tttgtttccc ctactaaata ttttgcgaga 92040agaaagtgtg
tacctttgta ttcacataca tgtacatgca catatacatg cacatatgca
92100ggggtcccca acctctgtta aaaaccggac tgcaggccgt gcgtggtggc
tcacgcctgt 92160aattccagaa ctttgggagg ccgagaccag tgcatcacaa
ggtcaggaga tcgagaccat 92220tccggctcac acggtgaaac cccgtctcta
ctaaaaatac aaaaaaaaat tagccgggtg 92280tggtggcggg cgcccatagt
cccagctacc tgggaggctg atgcaggaga acggcgtgaa 92340cctgggaggc
ggagcttgca gtgagccgag attgtgccat tgcactccag cctgggcgac
92400agagcgagac tctgtctcaa aaacaaaaca aaacaaaaaa aaaaaaaacc
aggctgcaca 92460ggaagaagtg agcaagcatt accatctgag ctctatctcc
tctcaggcca gtggtggcat 92520tagattctca taggagcgtg tatgagttcg
ttctcacact tctgtaaaga catacctgag 92580acatataaag aaaagaggtt
taattggctc acagttctgc aggctgtaca ggcttctgtt 92640tctgggaagg
cctcaggaaa cttgcagtca tggcagaagg tgaaggggaa gtaggcacat
92700cttcacatgg cccacaggaa aaagagagaa ggagagagag agagagacag
agagagagag 92760agaaaaagaa agattgagag ggagagagga gggagaaagg
agagtgcctg tagggggagt 92820tgctacacaa aggagcacca gggggatggt
gctcaaccat tagaaactac ccccatgatc 92880caatcacctc ccaccaggcc
ccacctccga cactggagat tacaattcag catgagattt 92940gggtggggac
acagagccaa accatatcag agcatgaacc ctattgtgaa ctgcacattt
93000gagggatcta ggttgcatgc tccttatgag aatctaatgc ctgatgatga
tttgaggtgg 93060aacagtttca tcccgaaacc atcccccgcc aaccctggtt
tgtggaaaaa ttgtcttcca 93120cagaaccggt ccctggtgcc aaaaagtttg
gggacctctg cacatatgca tgcacctgta 93180catggacaca taatacatgt
acatatgcat actttatatt ctctgccact tctggtccag 93240actgatatac
tatctcattt ggattactgc actagccttt tgttttggaa acagcatttt
93300ttaaaaaatt taatttaatt tttttgagat agggtgtcat tctgttgccc
agcttggagt 93360gcagtgtcat gatcatagct cactgcggcc tcgatctccc
aggctcaagt gatccttctg 93420cctcagcctt ctcagtagtt gggactacag
gcatacccac catgcccagc taattttttg 93480attttttttt ttttttgaga
cagagtctca gcctgtcgcc caggctggag tgggttggcg 93540cgatctcagc
tcactgcaac ttctgcctcc caggttcaag tgattctcct gcctcagcct
93600cccgagtagt tgggattaca ggcgcctgcc accacaccca gctaactttt
tgtattttta 93660gtagagacgg ggtttcacca tgttggccag gctggtctcg
aacttgtgac ctcgtgatta 93720gcccgcctcg gcctcccaaa gtgctgggat
tacaggcgtg agctaccgct cccagccagg 93780aaacagcatt cttgagataa
ttcatataat tcacccattt aaagtatata attcattctc 93840tttagtatgc
ccacagagtt gtacagccat caccagaatc agttttagaa cccataaagg
93900aactctgtac tctttaccca aaacctccat gcctccagct gcaggcagcc
actaacctgc 93960cttctgtctc tgtgactcta cgtcttctgg acattactgt
ggatgggctc atacagtcag 94020tgagcttgtg actggtgcct tctaccaagc
agggttttca gtgtagcagc ctctctgttt 94080ttcttttttt tttaaattgt
gacggaactt ctgcctcccg ggttcaagcg attctcctgc 94140ctcagcctcc
cgagtggctg ggactacagg cccatgtcac catgcctggc taattttttt
94200tttttttttt tttagtagag atgggtttca acatgttagc cagggtggtc
tcgatctcct 94260gacttcatga tccgcctgcc tcggcctccc aaagtgctgg
gattacaggc gtgagccacc 94320atgcccggct aacctttcat ttactgtctg
catttcttcc ctgatgcctt ccagtccatg 94380cacccgattg tagccattca
tcctattatg gtttaaggtg actgtcttag tcagcatggg 94440ttgccataac
aaaataccat agcctgggtg gcttcaacaa cagaatttac ttctcacact
94500tctggaggtt gggaagtcca agatccagga ctttcgcctt gccctcatgt
ggtgaggggg 94560tgaggaagct ctgtggggcc tcttatatat ggatgctaat
ctcattcatg aggggtctgc 94620cctcatgacc cagtcacctc ccaaaggccc
cacctcctaa taccatcacc ctggtaatta 94680agtttcagtg tataaatttg
ggggactata gacattgaaa ccataacaag cacttttcta 94740agatcaggga
gtgagtaagt agcagagcta ggacctcaat tccacatgtc agtcatcttg
94800ccttcactct gctccatgat ggctgcctcc tagagcattg ggagtctcga
tgttctatat 94860gctctcatgt gttgtgtatt ggagatagtt gaggctttat
gaatacatct ggatttgttg 94920acttctagct ttgctggtaa ccagctgtga
ccttgaataa gttacttcat ctctgagcct 94980gtttcctctt ttagaaacag
gagtttaaaa tgctgctttg ggttgggcac ggtggctcat 95040gcctgtaatt
ccagcacttt gggaggctga gatgggagga tcactggagc ttggagttcg
95100agaccagcct gggcatcata gtgtgagatc ctgtctcctc aagaaattaa
aaaattagct 95160gggtgatgtg gcgtgtgcct gtggtcccat ctactctgga
ggctgaggtg ggaggattgc 95220ttgagcccag gaggttgagg ctacaatgaa
atatgattgc accccatcct gggtgacgag 95280tgagaccctg tctcaaaaaa
gaaaaaaaaa atgctgcttt gtaccccttt catgtcatgg 95340cgtcatggcc
aacatagaat gccctggttg tttgctgttg gagggcatgg gcctgggggc
95400tccctgaggg ctccttccat cttcaactca ttctctgtgc acctgttagg
aagttgtggg 95460ccagtcccta ccatgtatca ttgtgtgggt aaaagtaaat
aaaatgtgta cagtgtctga 95520actgtacata tcagggtcca agaacaaaat
gagtgacatg ggttagctct ttttaataaa 95580tggtaaaacc aaatattcta
attttcagtt ttgttatact tccatcacat gtttttgttt 95640ttttgttttt
tgtttttgtt tttctatttt aggcagcctt gccttctcta acaaaccccc
95700cttctctaag tcccatccga cgaaagggga aggagaaaga accaggagaa
caagcatctg 95760taccgttgag tcccaagaaa ggcagtgagg ccagtgcagg
taggaaacag cgtggggaag 95820ggagggacat gagtgcagca tctgtcatgt
agaaacatag gatttaagta acttggtgtt 95880ttagagaaat aaatataata
cacatcagta aagtgagaga aagtttctcc aggtgcggtt 95940caagatatta
gaaactaatg actgatgtac acagaccacc ttttggtctg aagcatttct
96000aagtgccact ggctgacatg cagcccctac agcctccagg cttccagccc
tagcatggag 96060catcactctc ctatgcttcc ctggttgcag gtgatggctg
gagaggcctc ctgattttca 96120gtaagggaag tggtgtagat gcttaggaat
agatgtagtg agtgaaaaaa ctgattctga 96180tatgtcaaaa attctgattg
gaaatggaat atttacattt ggaagagcta aaggcgagag 96240aaagtgggga
taaagtcatc tgagttggag gagcttaaac cattcacaag tttggaggac
96300ctttttttac ccatgaaaag gtcagaacag aaggggctag gatttaggtg
tgactgcagt 96360ttattgaatt cccatccata ctgctctcgg tgggcagtgg
caggggcagg agaggagcct 96420ggcaaagcat gaagtgactg ctgctgcctc
tgctatctgg gacgcctggc cacctgtctg 96480tacagtctcc ctccagaccc
attctcacgc tgtctcttgg cacccagggg ccagtgatgg 96540ttctcccatt
tgttttgtgt atatagcatt tatatcaagg ctatttattt atttatttat
96600tttatttatt tatttttttg agacagagtc tcactctgtc acccaggctg
gagtgcagtg 96660gtgcaatctc ggctcagtgc aagctctgcc tcctgggttc
aagcaattct cctgcctcag 96720cctcctgagt agctgggact acaggtgtgc
accaccacac ctggctaatt ttttgtattt 96780tttattagtg gagacggggt
ttcaccttgt tggccaggat ggtcttgatc tcctgacctc 96840gtgatccgtc
cacctcagcc tctcaaagtg ctgggattac aggcatgagt cactgtaccc
96900ggcctattta tttattttta attgacaaaa ttgtatatat ctgtaatata
caacatgatg 96960tttgaaatat gtgtacattg gccaggcgtg gtggctcaca
cctgtaatcc cagcactttg 97020ggaggctgag gtgggcggat cacgaggtcg
ggagttcaag accaaactgg ccagcatggt 97080gaaatcctgt ctctactaaa
aataccacaa aaaaaaaaaa aaaaaaaaaa agccgggcat 97140ggtggctcgc
gccagtcgtc ccagctactt gggaggctga ggcaggagaa ttgcttgaat
97200ctggcaggtg gaggttgcag tgagctgagt tcatgccact gcactctagc
ctgggcgata 97260gagcgagact ccgtctcaaa aaaaaaaaaa aaagaagaaa
tacatatgca ttgtggaatg 97320gctaattaac ctgtgcatca cctcacgtat
cattgttttg tggtgagaac acttaaaatc 97380tactctttca gtgattttct
tgcatatggt acattgctat taactgcagt caccatgcta 97440tacagtagat
ctcttgaact cattcctcct gtctataaat gaaattttgt atccttgacc
97500aacacattca aggttttttt tgagatggag tcttcttcac ccaggctgga
gtaccatggc 97560acgatctcat ctcactgcaa cctccgcctc ccaggttcaa
gcaattctcc tgcctcagcc 97620tcctgagtag ctgggattac aggcacatgc
tactgcacct ggctaatttt tgtattttta 97680gtagaagtgg agtttcacca
tgttggccag gctggtctcg aactcctgac ctcaagtgat 97740ccgcctgcct
tggcctgcca aagtgctggg attacaggtg tgagccactg cacccggcct
97800caagcgtttt aaaagatgct cttttctaag gattgactgt agtacaggag
gaagattgac 97860ctgttgaaaa gcctcagcct ttacaagtgt aaaattatca
gtatattact atcatctttc 97920tgatgaatta aataaactaa ggactccaag
tcaaaagtct tcaaactgaa gtagaatagt 97980tgtatatagt gcttggcact
ttaatattta gtatcggttt aatgataatg tttgtgcctt 98040tgccgtcttt
aaaacatttt tacatcatcc ctgtttgatt acttggtgtg ctcatgaagt
98100tgttggccac taaggaatct taggctcaga gaggttctgg aattggccag
tggtccttga 98160atcagctgct cctatgattc tctaactgat ttctcacaaa
gcaaacaagc aatcataaca 98220aaacaactgt gcacactgct cttcttattt
tgttatttaa aaagtactta ggctctactt 98280atgtttgtta gtcaatttct
cattacttct agttaatcaa aaggtcagag gaaatacttg 98340aatattttca
tactagaata ctttaaaaaa tcatgatttc cagtaatctc tttaaaactt
98400ggcaagttat tttgatctaa aagtttatct tttgtgtgca tatttttaaa
gcttctagac 98460aatctgatac ctcaggtcct gttacaacaa gtaaatcctc
atcactgggg agtttctatc 98520atcttccttc atacctcaaa ctgcatgatg
tcctgaaagc tacacacgct aactacaagg 98580tatgggcctc tgcatctttt
aaaaatatat atgcacacat acttacgtct aatggatagt 98640tgatgttttt
cttatgattt gtaggatgta taagcccttt gagatatgag ttacatttag
98700ttttttcaag tttgtttgtc tttcagcttt gtttatgata gcttctatca
tacaggtgtt 98760ttggattttc atattgtttg tactcacagc taagattgat
tacagtgaca gagctaggat 98820gtgcagccag gttatagggg gaagtggccc
tggtggagtc tggagggatc cgtgtacagg 98880cttccttccc tcccgtgagg
ctcacacaaa aatacagcaa catgctggtc ctgcaggtac 98940cctctgccta
acatgagcca caattccaga ctcacagaag aaaagcaggt gttcggcata
99000aaccatgtgt ttcaaatagt ctgggcatgg tgagccactt gttatcagct
agggaaagtt 99060tatgtcagcg taagaaactg ttcaccagat acccccaaga
gccagccttt ctgtctaggg 99120atgttttagt tttttagttc attttttttt
ttaactttaa aattttctgt tcatctgcaa 99180tttgttagat atgaagtatg
tgtctaattt aatttttgtt tttggttgtc cccaataatg 99240tttacagaag
aatttttctg cactaattgg cttgagttac ttacattctc atagttctct
99300agtttcagta gtttcattta ttattttgtt atatcaatct atctgtctgc
tcatctatta 99360gaagcatcct tgtttttttt ttttcttttt tagacagagt
cttgctctgt ccccaggttg 99420gagtgcagtg gtgcaaccat gcctccctgc
agtctcaggg ctcaagtgat cctcccacct 99480cagctcctga gtacctggga
ctaccggcat gtgccaccac acccagctaa tttttacatt 99540ttttgtagag
acagggtctc cctaagttgc ctgggctggt ctcaagctcc tggcttaagt
99600aatcctccct ccttggcctc ccaaagtgct gggattacag gtgtgagcaa
ctgcacccgg 99660ctacaagtat acttcttaat tattgtagct taatggtatt
tatgagggga tcagttcccc 99720tgttgttctt tagaattttc tggatattct
tctttattga ttttgggatg tgaacaatag 99780aatcaacttc tacttgtaga
ttgatttagg gagaacttat acctcagatg ttaagtcacc 99840ctgtccagaa
tgtgggatgc tttcctattt gttcagaact ttttaaatta cctcagaagc
99900acatgaaatt taaaggattt taaaaaaaac ttaaagatta tttcacatag
ctcttgcaca 99960tttcttgata aatgaatcct caggtattcc tctgtttttg
ttactaatag ttacttctta 100020tgggtttttt ttcccctgaa aatcatttat
caaacgtatg tggcttattt tctgaaggat 100080gtttgataat tttggaagat
atgaaagtct tcatatttta caaggtttga ggtctcttta 100140agctgcatgg
ttctcatgtc agctcccaaa gcagaagacg gcatgttgaa aaatgccgta
100200gagaagatac ttcttttcca cctgttttca actcatatca tcttgaattt
cagggcacct 100260ttccatgctc ctagtgcttg ctatctgttt attattttcc
ttcctgaata ccctgaactc 100320cagcatgttc tgctgtaatt ctggcctccc
tggcatcttg gactcctgtt tcctttgctc 100380tgtcatcccc gcggtcagct
cctgctgcgc agcttctcag ctgaagtgcg tttggagtgc 100440ctggcgtgtc
ttgctggatc tttgagtatt gcctctggtt tccttggttc cttctgctga
100500gttgctcagc gtctccactc cccatttctt gtgtggccct tcctgcactc
ctctgattcc 100560ttttgtcttc cctggtttct tgctttggtt tcgagtctcc
acagaacttt tgcagctctt 100620ctgaagacct ggaagctttt tcatcttaat
tctcatctca tgacctcttt tcccttcttt 100680gagagctaga acttcccatg
gtgaacttct ctttccagaa ttccatgcct tcttttccct 100740cccacttacc
tgttgtccag gagaggtcag attgctgtgc atattggagg agaacccttt
100800cttccctggg ctcttcatct cacatgacat caccacatca cctcgttcct
tggaccctca 100860gtggtgtcac tgctggattt ttctttcctt tggctggcct
tagggcacac ccaggttgac 100920tagcgtagtc atggtattta gatccactca
cattttcagt ttctgtgtct gtctcttgcc 100980tgcttctgac ttcgcccaga
gaaagcttct ctttcacaag ggttcttaga tttatgttca 101040ctgagcacct
tcttttctga ggcagtgttt taccaatatt tattttccta gtcagtctcg
101100ccttaccttt cttgttatgc atgtctttgg tcctgaccca ttctctgagt
ctgtaaaata 101160gaattgctgt ataatttaat tacatgaaat cctttagaat
cttaacacat cttacacctg 101220atttaatatt ttattgtatc caaattgaac
caaccctatg tgaatttgac agtgatttct 101280cccagggatc ctagtgtata
aggaatagga cttagtattt tctatttttt gatataccac 101340ataccagata
ctgattatga tggacattta accctttttt ctcattatga aagaaagtta
101400ggaattattt cttccagtag cgccagtgta acctgaaagc ctttgaaaga
gtagtttttg 101460tatagctatc tgaaaggaat ttctttccaa aatatttttc
cagtgctgac aacaaacacg 101520cagacacacc ctgcaaggtg agtgtacggc
gccgcacagt ggaggcatct gctgcagccg 101580tcgatgtttg tgtctttggt
tgtacattat
gagatcgtga cagggccagt aaccgtgtgt 101640tctctccttc accttcccaa
ggtcacgctg gatcttcaga acagcacgga aaagtttgga 101700gggtttctcc
gctcagcctt ggatgttctt tctcagatac tagagctggc cacactgcag
101760gacattggga aggtttgtgt cttgtttttt ctccttgggt tgtggctggc
acacttgatg 101820tgcgtcttct gggctgagtt catctaggat ggagcctggt
tctccagggt gcctccggga 101880gactcctccc tgccccacgt gcttgcgtca
caggacccaa gtctgactct gccttagcca 101940tgaagtttag ggggaagttt
ctatttgtat tctatttttg tctgttatca tgtattagct 102000tagacccagt
ttagtttgga aaatcagtgg gtttcaaaat gtgtttgtag agtcctttat
102060ttcttaactt gaccttttca agtggaaagg ggcaaaacag acgggtaagg
gggcggggcg 102120ggaggtgtga cttgctcttt tgtgcctgag gaagtaacag
agctggggtt gacagtcata 102180ttctctgaca cagatagtct ctgacttatc
tcacagaaag tcagcggcag agcctgagtt 102240aaaagtctcg tagattttct
ttttcttttt tttggtggct aatttcagtt ttatttatat 102300ttgtttattt
atttattata ctttaagttc tgggttacat gtgcagaatg tgcagttttg
102360ttacataggt atacacgtgc catgatggtt tgctgcaccc atcaacccat
cacctacatt 102420aggtatttct cctaatgtta tccctccccc agtcccctca
ctccccatgg gccccggtgt 102480gtgatgttct cctccctgtg cccatgtgtt
ctcattgttc aatttccact tgtgagtgag 102540aacatgcggt gtttggtttt
ctgatcttgt gatagtttgc tgagaatgat ggtttccagc 102600atcatccatg
tgcctgcaaa ggacatgaac tcatcctttt ttatggctgt atagtattcc
102660atggtgtata tgtgccacat tttcttaatc cagtctatca ttgatggaca
ttcgggttgg 102720ttccaagtct ttgctattgt gactagtgcc acaataaaca
tacatgtgca tgtgtcttta 102780tcgtagaatg atttataatc ctttgggtat
atgcccagta atgggattgc tgggtcaaat 102840ggtatttcta gttctagacc
tttgaggaat cgccagactg tcttccacaa tagttgaact 102900aatttacact
cccaccaaca gtgtaaaagt gttcctattt ttccacaacc tctccagcat
102960ctgttgtttc gtgacttttt aacgatcgcc atcctaactg gcgtgagatg
gtatctcatt 103020gtgattttga tctgcatttc tctaatgacc agtggtgatg
agcatttttt cgtatgtctg 103080ttggctgcat aaatgtcttc ttttgcgaag
tgtctgttca tatcctttgt ccattttttg 103140atggggttgt ttgctttttt
ttcgtaaatt tgtttaagtt ctttgtagat tctggatgtt 103200aatcttttgt
cagatgggta gattgcaaaa attttatccc attctgtagg ttgcctgttc
103260actctgatga tagtttcttt tgctatgcag aagctcttta gtttaattag
atcccgtttg 103320tcaattttgg cttttgttgc cattgctttt ggtgttttag
acatgaagtc tttgcctatg 103380cctatgtcct gaatgttatg gcccaggttt
tcttctagga tttttatggt cctaggtctt 103440atgtttaagt ctttgatcca
tcttgagttg atttttgtgt aaggtataag gaaggggtcc 103500agtttcagtt
ttctgcatgt ggctagccag ttttcccaac accatttatt aaatagggaa
103560tcttttcccc attgcttatg tgtgtcaggt ttgtcaaaga tcagatgatt
gtagatgtgt 103620ggtggtattt ctgaggcctc tgttctgttc cattggtcta
tatatctgtt ttggtaccag 103680taccatgcag ttttggttac tgtagtgttg
tagtatagtt tgaagtcagg tagtgtgatg 103740cctccagctt tgttcttcta
gcccaggatt gtcttggcta tgcaggctct tttttggttc 103800catatgaagt
ttaaaatagt tttttccaat tctgtgaaga aagtcagtga tagcttgatg
103860gggggatagc attgaatcta taaattactt tgggcagcaa ggccattttc
acgatattga 103920ttcgtcctat ccatgaacat ggaatgtttt tctatttgtt
tgtgtcctct cttatttcct 103980tgagcagtgg tttgtagttc tccttgaaga
ggtccttcac atcccttgta agttgtcttc 104040ctaggtgttt cattccctta
gtagcatttg tgaatgggag ttcactcatg atttggctct 104100ctgtttgtct
gttattggtg tataggaatg cttgtgattt ttgcacattg attttgtatc
104160ctgagacttt gctgaagttg ctaatcagct taaggagatt ttgagctgaa
ccaatagggt 104220tttctaaata tacaatcatg tcatctgcaa acagggacag
ttttacttcc tctcttccta 104280tttgaatacc ctttattgct ttctcttgcc
tgattgcgct ggccagaact tccaatacta 104340tgttgaatag gagtggtgag
agagggcatc cttgtcttgt gccggttttc gaagggaatg 104400cttccagttt
ttgcccattc agtatgatat tagctgtggg tttgtcataa atagctctta
104460ctatgttgag atacgttcca tcgataccta gtttattgag agtttttagc
atgaaaggct 104520gttgaatttt gtcaaaggcc ttttctgcat ctgttgagat
aatcatatgg tttttgttgt 104580tggttctgtt tatgtgatgg attacgttta
ttgatttgcg tatgttgaac cagccttgca 104640ttccagggat gaagctgact
tgattgtggt ggataagctt tttgatgtgc tgctggattc 104700agtttgccag
tattttattg aggattttca catcgatgtt catcagggat attggcctaa
104760aattctcttt ttttgttgtg tctctgccag gctttggtat caggatgatg
ctggcctcat 104820aaaatgagtt agggaggatt ctctcttttt ctattgattg
gaatagtttc agaaggaatg 104880gtaccatctc ctctttgtac ctctggtaga
attcggctgt gaatccatcc tggacttttt 104940ttggttagta ggctattaac
tattgcctca agtttagaac ctgttatcag tctattcaga 105000gattcagctt
ttttctggtt tagtcttggg agggtgtatg tgtccaggaa tttatccatt
105060tcttctagat tttctagttt atttgggtag agatgtttat agtattctct
gatggtagtt 105120tgtatttctg tgggatcggt ggtgatatcc cctttatcgt
ttttattgag tctatttgat 105180tcttctctct tttcttcttt attagtcttg
ctagcggtct acctatttta ttgatctttt 105240caaaaaacca gcacctggat
tcattgattt tttttggagg gttttttttc gtgtctctat 105300ctccttcagt
tctgctctga tcttagttat tttttgtctt ctgctagctt ttgaatttgt
105360ttgctcttgc ttttctagtt cttttaattg tgatgttagg gtgttaattt
tagatctttt 105420ctgctttctc ttgtgggcat ttagtgctat aaatttccct
ctacacactg ctttaaatgt 105480gtcccagaga ttctggtatg ttgtgtcttc
gttctcattg gtttccaaga aaatttttat 105540ttctgccttc atttcgttat
ttacccagta gtcattcaag agcaggttgt tcagtttcca 105600tgtagttgtg
tggttttgag tgagattctc aatcctgagt tctaatttga ttgcactgtg
105660gtctgacaga cagtttgttg tgatttctgt tcttttacat ttgctgagga
gtgttttact 105720tccaactatg tggtcagttt tagaataagt gcaatgtggt
gctgagaaga atgtatgttc 105780tgttgatttg gggtgcagag ttctgtagat
gtctattagg tccgcttggt ccagtgctga 105840gttcaagtcc tggatatcct
tgttaatttt ctggctcatt gatctgccta atattgacag 105900tggggtgtta
aagtctccca ctattaccgg gtgggagtct ctttgtaggt ctctaagaac
105960ttgcttcatg aatctgggtg ctcctgtatt gggggcgtgt atatttagga
tagttagctc 106020ttcttgttga attgatccct ttaccattat gtaatggcct
tctttgtctc ctttgaactt 106080tgttgattta aagtctgttt tatcagagac
taggattgca atccctgctt tttttttgct 106140ttccatttgc ttgttagatc
ttcctccatc cctttatttt gagccaatga gtgtctttgc 106200atgtgagatg
ggtctcctga atacagcaca ccaatgggtc ttgactcttt atccaatttg
106260ccagtctgtg tcttttaatt ggggcattta gcccatttac atttaaggtt
aatattgcta 106320tgtgtgaatt tgatcctgtc attatgatcc tagttggtta
ttttgcccgt taactgatgc 106380agtttcttca tagcgtcagt agtctttaca
atttggcatg tttttgcagt ggctggtact 106440ggttgttcct ttccatgttt
agtgcttcct tcaggagctc ttgtaaggca ggcctggtgg 106500tgacaaaatc
tctgcatttg cttgtctgta aaggatttta tttctcgttc acttatgaag
106560cttagtttgg ctggatatga aattctgggt tgaaaatact ttttttaaag
aatgttgaat 106620attggctccc actcttttct ggcttgtagg atttctgcag
agagatctgc tgttagtctg 106680atgggcttcc ctttgtgggt aacccgacct
ttctctctgg ctgccctttc cttcatttca 106740atcttggtgg atctgatgat
tatgtgtctt ggggttgctc ttctcgagga gtatctttgt 106800ggtgttctct
gtatttcctg aatttgaatg ttggtctgcc ttgctaggtt ggggaagttc
106860tcctggataa tatcctgaag agtgttttct aacttggttc tattctcccc
atcactttca 106920ggtacaccaa tcaaacgtag atttggtctt ttcacatagt
cccatatttc ttggaggctt 106980ggttcatttc ttttcactct tttttctcta
atcttgtctt ctcgctttat ttcattaatt 107040tgatcttcaa tcactgatat
cctttcttct gcttgattga atcggctgtc gaagcttgtg 107100tatacttcac
aaaattctcg ttctgtggtt tttagctcca tcaggtcatt taagctcttc
107160tctacactgg ttattctagc cattagtcta acattttttt caaggttttt
agcttccttg 107220tgatgggtta gaacatgctc ctttagctcg gagaagtttg
ttattaccga ccttctgaag 107280cctacttctg tcaattcatc aaactcattc
tccatccagt tttgttccct tgctggtgag 107340gagttgtgat cctttggagg
agaagaggtg ttctggtttt tggaattttc agcctttctg 107400ctatggtttc
tccccatcat tgtggtttta tctacctttg gtctttgatg ttggtgacct
107460acggatgggg ttttggtgtg ggtgtccttt ttgttgatgt tgatgctatt
cctttctgtt 107520tgttagtttt ccttctaaca gacaggcccc tcagctgcag
gtctgttgga gtttgctgga 107580ggtccactcc aggccctgtt tgcctgggca
tcaccagcag aggctgcaga acagcaaata 107640ttgctgcctg atccttcctc
tggaaacatc gtcccagagc acgaaggtgt ctgcctgtat 107700gaggtgtttg
ttggccccta ctgggaggtg tctcccagtc aggctacatg ggggtcaggg
107760acccacttga ggcagtctgt tcattatcgg agcttgaatg ccgtaccggg
agaaccactg 107820ctctcttcag agctgtcagg cacgtatgtt taaatctgga
gaagctgtct gctgcctttt 107880gttcagatgt gcccttcccc cagaggtgga
atctagagag gcagtaggcc ttgctgagct 107940gcagtgggct ctgcccagtt
cgagcttccc tgctgctttg tttacactgt gagcatagaa 108000ccacctactc
tagcctcagc agtggtggac acccctcccc cagccaagct cctgcatccc
108060aggtcgattt cagagtgctg cgctagcagt gagcaaggcc ccatgggcgt
gggacccgct 108120gagccaggca caggagagaa tctcctggtc tgctggttgt
gaagactgtg ggaaaagtgc 108180agtatttggg caggagtgta ctgctccttc
aggtacagtc actcatggct tcctttggct 108240tggaaaggga agtcccccga
ccccttgtgc ttcccaggtg aggcaacacc ccgccctgct 108300tcggcttgcc
ctccgtgggc tgcacccact gtccagcaag tcccagtgag atgaactagg
108360tacctcagtt ggaaatgcag aaatcacctg tcttctgtgt cgatctcact
gggagctgta 108420gactggagct gttcctattc ggccattttg gaagcatccc
ttgttttttg aggtggagtc 108480ttgctctgtc gcccaggctg acgtgcatcg
gcacaatctc ggcccactgc aacctttgcc 108540tcctggtttc aagcgattct
cctacctcag cctccggagt agctgggatt acaggcacct 108600gccaccatgc
ctggctaatt ttttgtattt ttagtggaga tggggtttca ccacattggc
108660caggctagtc tcgaactcct gaccttgtga tccacccacc tcagcctcct
agagtgctgg 108720gatcacaggt gtcagccacc acgcccagcc atattttcag
atctccctct ctttgcccta 108780aaccactgtg cttaataagt agtttttagt
ggccagcagt ctccatgtat aacacatttt 108840agcaaaatgg aaaatactat
atgttttaaa tttgaacgtg agattatact gaaataaaaa 108900tcatctaact
gggattcttt aaatagtaag attttctttt ttgtatgtgg gttttttttt
108960aaccttatta ttatgactgt catatataga aatggctgtt tttcagttac
agtcagtgaa 109020tgtatcaaat gctgccttat ccaaataata aaagtaaatt
attaataagt cacaatttaa 109080tgaagattga tgttagttga tctttatatt
cttgaaatca gccatatggt tgtgtgtgta 109140tgtatatatt tttaaaggta
cataaagata ataagctcat ctctgaaaat ttttacattt 109200ggcataagaa
taactggata attaagcatc ttattctctg gcctgtgtct ttacagttaa
109260aggtagattt actcacctct ccttttttgt ttttctaagt tcatcttttt
tgctgtttca 109320agacagaggc ccattttagc tttctcgcat atccttttgt
ttgtactttg gaagcctcac 109380ctgcttaatt gttgagtttt tatccgtggt
cttttagagg gggatatgta gggtagaagc 109440tttcacaggt tcttgtttgc
acttggcccc tgactgtttt gaggaatctc cctcactgac 109500tcacagcatg
gcaaggtttc agatctcttt ctgccacaca gcagttctga ggcagctgga
109560aagatatcca gatgcttaga ttgtcaggcc aggcttgaga tatacaaact
attgagcctt 109620atctgtgacc ttgcttaggt gaaggcatca gagcccctgc
accaacatgc ataggcctct 109680gcatgtgtgc ggggctgggt gttgaggtct
gagcacaagt gtagctggag aggtgagctt 109740gatgtggcga cgggtatgag
caggttttct tcagacttct gtgagtttac ctagttccag 109800gatttaaagg
cacagagact ttagaattaa aatagaatca ttttcttttt ctaaatagca
109860acactaggaa taaaaaataa taattccaca ttcttgacag gtaatgtttt
ttcttgtctt 109920ctaatcctta tttattccat actcattttt atacataatt
gaaatgtatt atgcattgga 109980tttttctttt gcattatatt atagacgatt
tttcatgtaa ctccttactg ttccatttta 110040tatgttttgt ctggtttaag
actttatctg caaaccggga aactgtctct acaaaaagaa 110100aaacaaaaat
agttggccgc agtggcatgc gtctgtggtc ccagctactc ggggctgagg
110160tgggaggatt gcttgagcct tgggaggttg aggctgcaaa gagccatgat
catgccattg 110220cactccagca tgggtgacag actttatact gtctgttttg
ggtgatttga taatgatatg 110280ccctgatgta gtttttttat atcttgtgtt
tcttgtgcct gggtttattg aggttgggtc 110340tgtggcttca tagtattttt
aaagtttgga aaattttagg ccattctttc tttctttctt 110400tctttttttt
ttttttgaga cagtgtctcg ctctgtcgcc tgcgttggag tgcagtgaca
110460ctatcttggc tcactgcaag ctctgcctcc tgggttcacg ccattctcct
gcctcagcct 110520cctgagtagc tgggactaca ggcgcctgcc accacgcctg
gctaattttt tgtattttta 110580gtagagacga ggtttcactg tgttagccag
gatggtctca atctcctgac ctcgtgatct 110640gcccgcctgg gcctcccaaa
gtgctgggat tacaggcgtg agccactgca cccagctagg 110700ccattatttc
ttcaaagatt ttttttctgc cctgcctccc tccttttttc cctctcttaa
110760aggggctgtg atttcctgaa tgattgctta gtgttgtccc atagcttact
gatgctcttt 110820tcagtgtttg attgttttat gtgttttctg ttttgtatag
tttctattat tgtgttttca 110880agttctctga tcttttcttc tacagtgtct
actctgttgt taatctgtta atctgttgtt 110940aatcctgtcc agcgtatttt
tttttttgtt tttgaaacag tctcactctg ttgcccaggc 111000tggagtttag
tggtgcgata tcagctcact gcaacctcca cctcccaggc tcaagcaatt
111060cttctgcctc agcctcccga gtagctggga ctataggcac gtgccaccac
acctggctaa 111120tttgtgtatt tttattagag atggggtttc accatgttgg
ccaaactggc cttgaactcc 111180tgacctcagg tgattcatcc gcctcggtct
cccaaagtgt tgggattata ggcatgagcc 111240accgtgtctg gcccctgttc
agtgtatatc actaattttg tttttatctc tagaagtttg 111300atttaggtct
tttaaaaatg tctccctgtg tttctgttta gctttgtgaa cacaattgta
111360ataactgttt taatatcctt ctctgctagt tctaagatct tctaataact
tcccagttct 111420tggtgtttct cattggttga ttgatactcc tcgttttggg
ttgtattttc ctgcctcttt 111480gtatggctgc caatttttta ttggatgccc
aaccttgtga attttacttt gttggatgct 111540atatattttt gtgttcccat
agatcttctt gagctttgtt ctgaggttag ttgagttaca 111600tatagatggt
ttactctttt gggtcttgct ttataatttg tcagatgggt tggagcagtg
111660cttagtttag gactaatttt ttttttggac taattattcc tctttaggaa
taattaggta 111720ccatgcttag gaggcaagac catcctgagt actctaccta
atgaaccaga aagtttgggt 111780tttccagtcc gcctgctgag aacagtgact
ttctagccct gtgtgagcgc tgagctctgc 111840tccttctaat cctttccaat
gcttctttcc ctggcctcag ggagttttct cacacacata 111900tctctgctga
gtactcgaga gggaccttcc ccagatctcc agagctctct ctgtcttgtt
111960ttctcttctc tggtgctctg tcttatgaac tgtggctgtc ttggtctcct
tagattctca 112020gcacctcttc aattcagagg gttgcctgtc cctcctcctt
gtgccacagc ctaggaactc 112080tctcaaagca gcgagttggg gcagccatag
ggctgactta gtctctcgtc tcccagggat 112140cactgtcctt cattgctcat
gtccagtgtc ttgaggactc tgggttttgt ctgttttgtt 112200ttttggtttg
ctttggttgt ctcaggcagg agggtaaacc cagtccctca ccctcattgt
112260gctcagtagt ggaagtctca ctctattaca ttagatatta gtatttgtag
cagagccctg 112320gttccctggt acttggggag ctcttgaaag gccagaaaca
gcatgctttc tcaccttttc 112380cagggcttca gtttctggtg cacatcaagc
attccataca catttgttaa agtcctttgt 112440tagacaagta gtgattcaca
ggttctattt gtaatttttt cagttaacat gtattgggta 112500tctgctggga
gctagtaaaa acaaaaagtg gtgtgtgaca aattcaattc tgacaagaac
112560aaccttaaac acttagaata tactttgagc atatcagaat tttaaaaatg
tgtggccctt 112620gagtatttga aaccaacaag aatctattgc ttattagtag
aggatatttt gttaaacaag 112680tggagagaga ggcattttca gtctaattgg
tgttggcttt tagcagctga tggaaaccag 112740ttcgtgatta gccaggcagt
ggtgaaacag gctgtgcatt ctgaatgcct aggtatctag 112800gcattcagaa
tggtggcgct ctttgagtta gcatcttctt ctttcttgat tctttttttt
112860ttttttttga gatggacttt cgctcttgtt gcccaggtaa caactccagt
gcaatggcgc 112920catctcggct cactgtaacc tctgcctccc tggttcaagc
gattctcctg cctcagcctc 112980tcaagtagct gggattacag gtgtgcgcca
ccacgcctgg ctaattttgt atttttggta 113040gagatggggt ttcactatat
tggtcaggct ggtcttgaac tcctgacctc aagtgatgca 113100cctgcctcga
tctcccaaaa tgctgggatt acaggcgtga gccaccactc ccagcccctt
113160cttgattctt gaaaaggaca ttgggtgctg tacatctcgt tatagatgtt
gataaaaatg 113220cttgtgagaa gagtaacatt aaggtagtta tttggtcatt
tttgcagatt attttaagac 113280aattctagga ctgatttgtg gtaaatcaca
cattgctgta tcatagttgt gttcactgaa 113340catattcagg ggctctacag
atgcagggct cttagctgct ttgcacactt ctgaattcct 113400gccctgcgaa
caggactgga tacctaatag acaacaggta cttgataaca gtttattgaa
113460ttaatgagtg aatgaacaga tacataaatg catgaaagaa tggttgtaat
gtatataact 113520tggatttcaa gactttttac tgactgttca aaataagaaa
ttgaaaactt tcctctgatt 113580ttcctctact atttacacaa tttaaatgga
agttatcttg taccttcaat ttctgtctag 113640gattcgtaca ataacgggtc
atctctgagt cgcttaatgt ctcacttgtc tttctacagt 113700gtgttgaaga
gatcctagga tacctgaaat cctgctttag tcgagaacca atgatggcaa
113760ctgtttgtgt tcaacaagta agagcttcat tcttttcctc ttctgttaag
acgttcgggt 113820atgacagcaa aacgctgcta ctccttaaga ggcaggcgct
gttggcataa tcagctggga 113880ggattgtggg gtccagcgca gcactttttg
gctcagtcca tgattgagcc aagaggccat 113940ccttcccttc actccccagg
aggacgaggt ctgtcactgt ggagggcaga ggacaccaga 114000agctcctctg
caacctcgct agttaacttc cagtccctcg gagtttctgt ttagaatgct
114060caatctcatt tagaattgca aggaaaccca aaacgcctat ttaaggtaca
aacagcactt 114120catacaatat ctcatgaggt attaatagtg attcacagga
agaatttcac gctgtgagtc 114180tttgctaaca tatccagtta tttacagatg
gatttgatat ttgtgtggga gattcttaaa 114240agtgttgttc acgccacatt
gttgatgcct catttttttc actgtagttg ttgaagactc 114300tctttggcac
aaacttggcc tcccagtttg atggcttatc ttccaacccc agcaagtcac
114360aaggccgagc acagcgcctt ggctcctcca gtgtgaggcc aggcttgtac
cactactgct 114420tcatggcccc gtacacccac ttcacccagg ccctcgctga
cgccagcctg aggaacatgg 114480tgcaggcgga gcaggagaac gacacctcgg
ggtaacagtt gtggcaagaa tgctgtcgtt 114540ggtggaagca cgaaagagca
agcaggaaat actttgtaaa agaataaaaa cgaaaaatgt 114600tagcgaacat
cttctaatag tctgctgtat tcagagaact ctaggagata tatatggttg
114660atgcaaagat gatttaaggc atagcccggc cttccaagaa gtgtgtggcc
agtgagtgag 114720atgggcttgg gacttacaca tctcagaggt gggggtagag
gaggaggaac actgagtggg 114780ctgagaagca gccagctctc attgccaaag
tgtgtcagca aaccagaatg cagttcataa 114840tgtccccacc cattcaaagc
acaggacctg tagagtggtg tggcatgtgt tggtggcact 114900tttcaggcct
gtaacaagga tgaaagaaca gcttcatagc agcacagtag tgctggtgtt
114960cagaggtgtg tgaaggccat agaagcatct tggatatatt accttgtgtt
ttgtcagctt 115020tatgactaga agtctctttt cacttaaatt tgtttttttt
ttttttgaga cggagtcttg 115080ctctgtcgcc caggctggag tgcagtggtg
caatctcagc tcactgcaag ctctgcatcc 115140tgggttcatg ccattctcct
gcctcagcct cccgagtagc tgggactaca ggcgcctgcc 115200atcacgcctg
gctaactttt ttttgtattt ttagtagaga cggggtttca ccatgttagc
115260caggatggtc tcgatctcct gacctcgtga tctgcccgtc ccggcctccc
aaagtgctgg 115320gattacaggc gtgagccacc gcgcccggcc tcttttcact
taaatttatg tttgtgtttt 115380taatgcctag tatacaggac ttcttaaatt
gccttaagta tgaacaggta tttgagttgc 115440taatctgtat agtagcaata
atagaatccc ttgtttttcc ttttataaat ttagcgatta 115500aatagctaca
attaaaacac tagagtcagg agtcaaggaa aatacccatg ttccaggctg
115560tatgttagtg atgtacttac tatatattgg agtttcagga gtaagtctgt
ttcaatgctt 115620tctgtaacca tttggggtat taataagcat gtgagtgtgt
gcatgtttgg gttaatttca 115680tatatgtttc ttagaaggga tatcattgat
gtaaatattt taaaggcttg tcctccaaaa 115740aaatcatgta atttcttcta
aattactgat cttttaaatg accttcacct ttctctcaaa 115800tctcacttaa
gactgggctg agtagtcagt ttcctgtagc agaaaaaagc tcagacttga
115860gtagccttct gcgagtgagg agacttgatg gctgtcaggc agctgtaaac
tctaaataga 115920gtgtcattat ctgaagaggg cgatgctgcc acactgagtg
gcctttcaag ttgtttctca 115980atctgacacg ttctgatcgt gtgaatgtga
aattggtttg agcaggagta tatctgagtg 116040cagaggagat tatttaaaga
tattctcatt ctctgcttcc cttttattcc catttggcag 116100atggtttgat
gtcctccaga aagtgtctac ccagttgaag acaaacctca cgagtgtcac
116160aaagaaccgt gcagataagg taaatggtgc cgtttgtggc atgtgaactc
aggcgtgtca 116220gtgctagaga ggaaactgga gctgagactt tccaggtatt
ttgcttgaag cttttagttg 116280aaggcttact tatggattct ttctttcttt
ttttcttttt tatagaatgc tattcataat 116340cacattcgtt tgtttgaacc
tcttgttata aaagctttaa aacagtacac gactacaaca 116400tgtgtgcagt
tacagaagca ggttttagat ttgctggcgc agctggttca gttacgggtt
116460aattactgtc ttctggattc agatcaggtt tgtcactttt atctttcatc
catcatacct 116520gttcctaatt tagtacaaat taccctaaaa gacactgaaa
tctactttaa agaaatgtgg 116580tctgcatgtt tccctcatca gttgctgctg
cttatctttt tcatgcacct agctggtgca 116640gaaggcctgg ggcatagcca
gcctcagcaa
gtcagcatcc ttgccccagc tccctggact 116700caaggctaac ctggggttgg
ctgttaggga tttccaaagg tttgtcccat ccacttgcct 116760cccctccaaa
ataagtttga atttaaattg tgagatacaa ttaagattta ttgtttgggg
116820aacatttttg caaaatctag agttagttta aacagattat caattattac
cataattgat 116880catctgcagt ttcaagctat ctaacaggtt cacttacctc
tttaaaaagg aatggaattt 116940agcaggacag taactgagac ccgtgctcct
ggagtccatg tgggagctgt gtggctctgc 117000acaagcattt gcacgcttcc
cctcttgact gcattacctt cctcctatag ttgctgtggg 117060caccagattc
tggctagtcc tgtcccttca tgatgcacat tttcctcaag attcgtccca
117120gttaaatcac tgcagatgaa actgcctttt catcgtcaaa atttaactgt
catttttgag 117180ccgtgatctt gggctacttt cttatgtggg gtaggaatat
ttgtgagtta gaaatattac 117240acttctctat ttccttctag acgtaaatct
gttaatcctg tcagcactgt tactcacctg 117300aaagggtctg tttccctagg
agaactgagg gcactcggtc aacactgatt ttccacagtg 117360ggtattgggg
tggtatctgc ttgttttttt tgttgttgtt gtttgttttt ttttgttttt
117420tttttgagat ggagtctcgc tctgtcaccc aggctggagt gcaggggtgc
gatctcggct 117480cactgccagc tccgcctcag aggttcacgc cattctcctg
cctcagcctc ccgagtagct 117540gggactacag gcacccacca ctacgccagg
ctaatttttt gtatttttag tagagacgag 117600gtttcactgt gttagccagg
atggtctcca tctcctgacc tcgtgatctg cccgcctcgg 117660cctcccaaag
tgctgggatg acaggcgtga gccaccgcgc ccggcctggg gtctgctttt
117720aatgaaggag gcatcaaggg gtgggctttg cgttggcctg atgctttcat
ctttctttca 117780caaaacctgt ccgaagaaaa tccgtctaaa tgggccattg
ctctcctcag gaaatagtca 117840ttgggaactt cttttccttt cctttgacac
taggaggctg actggggaga agccctggtc 117900tatggctgtg ggcagcaggg
gctgagagga gcaggctctc aggggggcac gggtacccca 117960agggaagcca
gagccctgat ttgttccatt ctagtaagaa caaagactgc tctggtttca
118020tgtttgttct gattgccttt catcaaccgg tcccctttct cccagttctt
aagattcagt 118080acagtgacag ttttatgaac aagaatagaa cactagaaca
gacaaaccat tgaactctat 118140gctgataaag atttattgag ctcctgctgt
atgtttgcat tctgcccaga ggctctgaga 118200aaaccaggcc atatgctcca
tgctttatcc atggaagctc cccgtcaggt tgggaaagct 118260gacagctgca
gggaatacag tgtgacacaa aactggctcc catgcagccc ttacgtgtcg
118320cctctcagat ggttggggga cgaaggtcga ctcctttggg tatcttatta
ctaaaccagt 118380ttcagggaat ctgtgccacc ctatctgcca ttaacgtgaa
cagatgagtc cccaaggtgt 118440aattttgggt attgtctgat gtctcttgga
atttattatt tgtttttcca atgagatttc 118500acctcagggt atagtaaagt
tgttgagggg attcctggat gtgttctgca attatctagg 118560ctgatttcag
aatagagtta tgcttatagt caaatttatc agctgtcaag aattttattt
118620aaaatttatg cagataagca ggaggaaaag aagcctggtt tttacatttt
aatcctatta 118680ttgatgtgaa attttatttt ccttcctgta ggtgtttatt
ggctttgtat tgaaacagtt 118740tgaatacatt gaagtgggcc agttcaggta
atagcatttt attattttag atttttttct 118800tcttcttgtg tacttacatg
taatttaggt tattaagtga atgtttaaac tactgttagg 118860catttttgct
gttttcttta aatggaaatc tgactaacat actgtgcatt tttgcttctc
118920ttaaaaatta atgtatatct caagacttgt ttggaagtag ttatgtatct
gaaaattcca 118980tatgttgtca gtattcattg cacatttcaa agcatttaat
tgtgttgaca gatggtggaa 119040tgaaatcttg tggtggagca ctagttttta
aatcttctta gagaaagcag ttttatataa 119100tgttgtcttt agtaattatt
atgcatttgt attctctgca gctttttctt gctagatgtt 119160gaggttttaa
tacttcttgc tagtccatta caggtttata attattaaaa gttaaaattc
119220ttttagtacc taaaatgctt aataaacatt gtaattagga aaatttagtg
cagaaggaaa 119280gtgttcccag attccctggg gtctggaaac atagtgttta
ttctaattac atgacacctc 119340cactgtgttt tggggcaagt tactgtttct
cttttgagtt tcaatttctt caagagcaaa 119400gaggcagagg agagctagga
agatcgtagc tgctgtgccc ctgtgccgtc gggtgccttc 119460tacctgctgc
ctccgaacct ttacacatgt ccctgctctg cgcgagggca cagatgggat
119520gcactgtggc aggggtgggg ttagagtaga tcacggacac ctgttagctt
gatgtgtgct 119580tgctgtcaag gttgaatcat gaattatttt atgttgctta
tattgatatg tatcttaatt 119640ttaaaagaaa ggtctaaatg gatgtttttg
tttttaggga atcagaggca atcattccaa 119700acatcttttt cttcttggta
ttactatctt atgaacgcta tcattcaaaa cagatcattg 119760gaattcctaa
aatcattcag ctctgtgatg gcatcatggc cagtggaagg aaggctgtga
119820cacatggtaa cgggacacac ctttcactgt cgtcttcggt gtcgtgatgt
gcttggcagt 119880gttcgttttc atatacccac tttgaacgtt gtcagtggca
gccatgtgct tctcaggctc 119940tgcatgtgtg tctgtgtatg tgaaggtact
ggttagagac gtttcaaaag agaagagagc 120000atattcttta ctctcagcaa
tttgtaatct tctcagggaa aaaaattcaa gaaacagtaa 120060gataacctaa
ggtacagata gattctgaat ataaagttcc tgttcattca catgaaacgc
120120taaaagttct tcacttgatc ttagccaaaa ggccaagaag cgatgcaaca
ctaaaaattc 120180ttaaatcgaa cttgccgtga attaaatttt gatctctcat
ccagtggtat tggagatata 120240gtttgacttg ggttcagggc tttctgtttt
gcctgatgat tttgctggag cttaaataag 120300gaacccagga gatggccagc
tgtgcaagcc cccagcctgt ggaaggagct agtgtggttt 120360tatgaatgag
ttgcaaatct ttctttgagc tttttgaact gatcttccag cattgcccta
120420ttgacccctc cctgactcct ttgctggaat ctgtaggctt ttgaactttg
acagggacac 120480atcctaagac ccttgcaaac tcccagatgt gagaatggca
ctactactta gagtcttttc 120540gactcagcgt gtgtgcagaa gagcatcaac
cgggctgtgt tgcgaggcag ggccttggct 120600gacctctcag tgtttacata
gctaagccag ttagtgtttg ccacggcctc acaagggctt 120660cagattcaca
cagccaaagt atagattatt aaaggcatag gtgtttggtt tcctggactt
120720ggagggtctt tggacagaaa atcagtaggc aaccacaccc agtactttgt
gctgggaagc 120780ttggtcatct gtgagagggt cagagagtat acccatgcgt
gcatgccacc gaagggtcag 120840tgagtattcc tgtgtgtgca tgtctcaggg
ccggagagag tatgtgtcac tgagaggtca 120900gagtgtttgt gtgtgtgtca
aagagggttg cattgtgccc ttcactgagg ggtcagaggg 120960tgcctcgcgt
gtgtgtgtgt gtacgtgtgt gtgtgtcact gaggggtcag agtgtgcctg
121020tgtgtgtgct tgtgtgtgcg tacatgtcac tgaggggtca gagtgtgcct
ctgtgtgtgt 121080gctcatgtgt gtgcatacgt gtcactgagg ggtcagagtg
tgcctctgtg tgtgctcatt 121140tgtgagcgta tgtgtcactg agggggtcag
agtgtgcctc tgtgtgtgtg ctcatgtgtg 121200agcgtatgtg tcactgaggg
ggtcagagtg tgcctctgtg tgtgtgctca tgtgtgagcg 121260tatgtgtcac
tgaggggtca gtgttcctat gtgctcatga cattgagggt cagagtgtgc
121320ctgtgtgcca atgaaaggca tttcttatat ttttttatat gtggtcatag
tagaccagtt 121380aatttatttt gactcctgtg ttagaccaaa ataagacttg
ggggaaagtc ccttatctat 121440ctaatgacag agtgagttta cttaaaaaag
cataataatc cagtggcttt gactaaatgt 121500attatgtgga agtctttatt
gtcttttcag atgaatcaag tagattattc ttgagaccag 121560gaatgttgct
gttttggtta tttggaaagt tttatcattt tcaaattgac ttttgaattt
121620gagtcacctt ttttcagaag tggtgttaaa ttataggagc cctaggtttt
ttttcttttt 121680ttagaagtca tcacaaaatg atcagtgttc agaggaagag
ctttgacctt ccacatggta 121740taatgattga taaccttaat tcatctctta
ccataaacca agtatgtgta agggttttct 121800ttatttcttg aaagcatttt
gtagatgttg agagcagttt tccaaatgta atttccatga 121860aatgcctgat
aagggtaccc ttttgtcccc acagccatac cggctctgca gcccatagtc
121920cacgacctct ttgtattaag aggaacaaat aaagctgatg caggaaaaga
gcttgaaacc 121980caaaaagagg tggtggtgtc aatgttactg agactcatcc
agtaccatca ggtaagagga 122040atgtatgttg gaactgtcgt ggatacttta
ttgacccgtg cagatggaag gaagtgccat 122100gtggtaacgc tcactgttaa
ctgtgttact ttgaaccagg tttgggcttt ctggggcctg 122160ggtagatgcc
ggtgcagggg gatggggagg gaggcggggg gtgggggggt gtggtggagt
122220tggggaggtg cagtggcagg aggtgttgtt ggtgtgtatc cttttttttt
ttttgagatg 122280gagtctctct ccgtcgccca ggctggagtg tggtggcacg
atcttggctc attgcaagct 122340ccacctcccg ggtttaagca attctcctgc
ctccacctcc cgagtagctg ggattacagg 122400catgcaccac catgcccagc
aaattttttt ttttgtattt ttagtagaga tggggtttca 122460ccatgatggc
caagctgttt cgaactcctg acctcaagtg atcctcctgc cttggcctcc
122520caaagtgcta ggattacagg cgtgagccac catgcccagc ctggtgttta
tctttaaagt 122580gggcacagcc acaggagttc acctgactcc tggtctgaga
gtcacgagat cgttcaagat 122640agtgaggccc tcttttccaa aacgaggacc
aaaaatcaat tgacagtgtt ggtcaagatg 122700gtagaaacct taaaatgata
gaaatctcaa ctctgaaata aaaactttat ttgtatattt 122760atttaccact
attttgacat agggctaagg tctttttctt tgagctgatt tctggttttg
122820ttttcttaaa gtggcataag aattcaaaga cattttgagg aaggctgagt
gcagaaatct 122880ctctttttaa atgacttctc ctttctttta acttgcactg
ttgtctagcc ctcacttatt 122940ttgtcaattc tttttagctg tttgtctttg
aatcttcata aagccatagc ttttctcata 123000agaagcagca ctttctttgt
tcattcatat tttaatgaac ccctgtagta tttaattaaa 123060tacttaatgc
ctaattaaat cacataattg caatgcaaaa gtacatgtat cataaagagg
123120tctgaaaatg agcaactggc aagcaggtgg tggcaggcag agctgcttgg
gtgggtgggt 123180gtcatggaga ggagttcatc agccacatgt tcagtgagct
ctggatatgt ctgtttagaa 123240atgatcacta ataaacttgt gctcaaccat
gtatacctct gggaagcagg tgctcttcag 123300tagattgcct ctgcagagaa
cacagaattg aagtgaatgt ccacaaaggc aatgagccac 123360ctgcagaata
gtttagtcaa ggctgtgttt gaagtttgcc aaagattaat atacatttga
123420ttttcatgtt gtgccttttc tctgattgtg aaatattaca aattctatac
aaataacaat 123480gatggcaaat cctcctgagc aaagtgtgca ccttgtatgt
gccctagagg aacttgtgtt 123540tcgttctgat tcccctacat ttctcatgtc
atagagtggg ggttgcatta gtgtccccct 123600gtcctcgctg ggatcacatc
tgtttggatc ctagagtctt ccagctgaac tgggacaagt 123660ataacagacg
gacacgtagg ggtggaaagg cgtctcttgg cagcagactt tctaattgtg
123720cacgctctta taggtgttgg agatgttcat tcttgtcctg cagcagtgcc
acaaggagaa 123780tgaagacaag tggaagcgac tgtctcgaca gatagctgac
atcatcctcc caatgttagc 123840caaacagcag gtttgtcccc gcagccttgg
cttgttgttg catagtgatg gtagcttaag 123900gtccttgtga aaggtgggtg
gctggaatca gctcttcctt cagtcctaat ctgtgccttg 123960atagcagttc
tccgtgctag tcatgggaca gctgacttca tttcttctca caatgccatc
124020tcaggttggt attgcccacc tactttacag gggggatccc acagctccga
gaggttatgg 124080aggtgatcag gcagcacaca gctttagagt gctggggtga
gggcgggcca aggctaactc 124140taaagcccga acccttacct cctacactgc
ctcctgcatt ctggtcaacc cagtgtttta 124200tttggtggtt agatttttgt
ttttgttacc ttactgcttg taatttagca gttttccttt 124260cctttccctt
cctttccttt ccgacagggt ctcactctgt cacccaggct agagtgcagt
124320cgtgtaatct cactgcaaca acctctgcct cccaggttca accaattctc
ccacctcagc 124380ctcctgagta gcaaggacca caggtgtgca ccactacgcc
tggctagttt tttgtatttt 124440tagtagagat gaggtctcgc tgtgttgccc
aggctggttt taaactcctg ggcgcaagtg 124500atccaccaac cttggcctgc
caaagtgctg gcattacagg tgtgagccac ctcgcctggc 124560ctattcatca
ctaatcagaa tttctatgat caaatgacat gaatcattgt ttccacaact
124620gcagtggaag gaaatggcct ggcagtgcca gtttcagaag cagcctgccc
ccagtcaggc 124680acaggccact gtgcccccag tgtagcagca cctctgtagc
tcacagagaa gggtggtggg 124740gacctccttg aggcagctct gccagaaaat
ctcatgagct gcctggcaca gcttgaggtt 124800gccttttaag tggactcagc
aaatacatgt ttgttcatct tgattataca caataaacaa 124860ctactctgta
tagtacgagt agtccgtggt ttttggcatt tgatttaaac ttagaggcat
124920gtgatattga tgttactgcc ttcatgactg cacccccatt ctgatttcat
aatggaatgt 124980tatcttgaga ccagttagac aacaggacag ggatcttggc
ttctggtgag attgacagca 125040gttttagtgt ggtcagggtc tccctgccta
cagatggttt tagaatggtg ccctggaagc 125100tttatcccat tcttttctgt
gcgtaatctg agtagagtgg agatcgaagg cctgaataca 125160tagtaaatac
ctgacttaat atctgccgca atggaaattg tgtgatacaa catttatgaa
125220acgcttagtg cagcacctgc caggtagctc accacaggtg catgttgcat
tcagaagtag 125280tgctagatac tatcctgtta ctggcagtgc atacatcagt
gatcaaagca gattaaagaa 125340agaccccctg ccttcttgga gtgaagattt
tgttgggatg cgggtaaggg gacagacaat 125400agaaaagcaa gtgagtgaag
tctataccat ggcggctgat caggaacacc gtacagaaga 125460atccaggagg
gaagagagtt aggtggtgtc tgcggtggga gtggcattgt tcagctggtg
125520atgagaagaa gctttggtga tctggtgaca tttgagtgaa tttgcagaaa
ggaaagatac 125580aagcctagga gatacctggg gaaggaacat tccaggcaga
gcaaatagca gtgcaaaggc 125640cctggcgggg ggcggacatg ctgttagggt
acaagcaatg agggtggagg agtggggcag 125700ccatggggag ggaagggagt
gaggcctggt ggggtgaggc cagtgtggag gagccttgag 125760agggtttgcg
ctgatgtggt gtaggtttta gcaggatcat tcttattcct gagttgagaa
125820tagccttgag ggggaggtga gggcagagca gggccaccca tgtgagaccc
ggcactggag 125880tggaatggcc caagtcagca tcccttggca gcatgaaagc
aaaaccagca aggtttgctg 125940gtggcttaga tgtggcatgt gagagagagc
agggctttgg gggtgatttc agggtgagga 126000cagggtggct gtggacaagg
tagggcagac attgggggca gcaggaggtc agagcctgtc 126060tggatgtagc
agttgagacc ccataggtgc ctaatgaggt gaggccagca tcaggtgtat
126120gagcctggag ttgtcgagag actgtggggc agggggtcag catctgagat
gtccactcac 126180agtggaccca gactggctgg agaggaggag gagcttgaat
accgagcctg ctgagtccca 126240gctccaaggt caggtaggtg aggggagcca
gtgctggggc agggggagta ggcaggtgtg 126300gggttcctaa agccaagatt
ttttttaagg cattttgtgc aggagggcga catctgctgt 126360cagcaccttg
ggaacttggc ccaggtttgg cagcaccgag ggcactgatg agtgcttttg
126420gaggagcaaa gggagccaaa ccctaatggg aatgtgttcc tgaaaggaca
ggagagagac 126480ttgggaaaag gttttacttg aagagggaac ggagaaatag
ggcagtagcc agaggaggag 126540aggagtcggc aatgggttaa gttggcagaa
atgaaggcct gtttacgcac tgagggcaga 126600agcaacaggg aggatcagtt
catgacacag gagacacaaa tcgccgttgt ggtgttcaca 126660gacatgggtt
aggattggct gcatggatga cagagcactg tgggttctcc cagagttgct
126720ggggaggagg cagagttggt gagcacaggc gagggtccag gatgcaggaa
tcctggagct 126780caagtcagtt gttcccttgt tgtaagatgt ggccagtgtt
gtgagcttca catctgtgcc 126840ttgaaaaaca ccacatctgt ttgcagagtt
gtttactatg tatacacact cagtagaaac 126900aaaaattgga aacagtcagt
gcccaccatc aataagtaat ggttgaacac actgtggtat 126960aagcttagac
tattttagct tgggctattt tgcatgatta aaaatgttct ggccaggtgt
127020ggtggctcat gcctgtaatc ccagcacttt gggaggccaa ggcaggcaga
ttgcttgagc 127080tcaggagttt gagaccagcc tgggcaacat ggtgaaaccc
tgtctctact agaaatacaa 127140aaagtagctg ggtgtggtgg tgtgcgcctg
tagtcctggc taactcagga ggctgaggtg 127200ggaggatcac ttgagcccat
tcgtgcgcca ctgcactcct ggggcacaga gtgagactct 127260gttagaaaga
gagagagaga aagaagagag agggagggag gaaggaagga aggaaataaa
127320tggaagaaat ggaagggagg aaggggaggg aggaaggaag aaaggaagtt
cagccagttg 127380ccttgggagt tctccattgc actgggttaa gtgagaagag
cagagacgtt tatgattttt 127440caaaacaact aaaacaaaac ctctgtgggt
gagggggcaa ggatatggct ataggaacat 127500ggggcagatt aagaaaggga
tatacacaca ccacttagca tttgttacaa ctgttgtggg 127560agggatggag
tgcagaaaaa gaaaaaaaaa agtgcacacc atcccatgta tgtgtataca
127620aagggacgct tggaagactg gtccccaaaa tgttggtaat gattgtgtca
gggtgctgca 127680gtgctagttg attttttttc acacttttgt atatttgagt
cttttacaga aagcatttat 127740tatttatgta ataaaaatct aaatgacaag
atttctgtta tgggaaaaat gtagctatac 127800agtgttgttg taaaaatgtt
tgcttggttc accactgaac ttaaaatgct tttaaatgag 127860ggaaggtgac
gatgagatga ttatgatgat ttgcccttga gttacatagc tggtgtacag
127920gaagctgtcg tttcttttgg cttacgtaga aatgtttgtg gtgtctaatt
ccacagatgc 127980acattgactc tcatgaagcc cttggagtgt taaatacatt
atttgagatt ttggcccctt 128040cctccctccg tccggtagac atgcttttac
ggagtatgtt cgtcactcca aacacaatgg 128100tgagtctctc gcctggctca
gcagatgaat ctggacggct tgttcaggct ctgattactg 128160ggaccacccc
cagaatgtct gagtcagtca gtttgggtag ggcttcttga gagtttgctt
128220tttttttttt tttttttttt ggtgtggggg tggtgcggaa cagagtctca
ctctgtcgcc 128280caggctggag tacagtgtca tgatctcggc tcactgcaag
ctctgccttc cagcttcaca 128340ccattctcct gcctcagcct cccgagttgc
tgggactaca agcgcccacc accacgcccg 128400gctaattttt ttgtattttt
agtagagatg gggtttcacc gtgttagcca ggatggtctt 128460gatctcctga
cctcgtgacc cgcccatctc agcctcccaa agtgctggga ttacaggcgt
128520gagccaccgc acccggcctt tttatttttt ttggagatgg agccttgctc
tgtcacccag 128580gctggagtac agtggcgcta cctcgactca ctgcaacctc
cgcctcccgg gttcaagcaa 128640ttttcctgcc tcagcctccc gagtagctgg
gactacaggt gcgtgccact gtgcccggct 128700aattttttgt atttttagta
gagacggggt ttcactgtgt tagccaggat ggtcgcgatc 128760tcctgacctt
gtgatccgcc cgcctcggcc tcccaaagtg ttgggattac aggtggctct
128820cgcaccaagc caagagtttg catttttagc aaattcccag gtgaaactaa
tgcctgcttt 128880tctgggagca cactttggga ctcagtgata gagaggttta
ttggtaggat agtaaaatag 128940gagttatttt ctttcacaaa attggcaatt
gggggaaatt taatcttcct tttttcttca 129000gctgtgactt atgtattatg
tttattttag gcgtccgtga gcactgttca actgtggata 129060tcgggaattc
tggccatttt gagggttctg atttcccagt caactgaaga tattgttctt
129120tctcgtattc aggagctctc cttctctccg tatttaatct cctgtacagt
aattaatagg 129180ttaagagatg gggacagtac ttcaacgcta gaagaacaca
gtgaagggaa acaaataaag 129240aatttgccag aagaaacatt ttcaaggtat
gctttctatc tgagcctata actaacccat 129300gccttttggg aagtcacgtg
atgtttcaca gtcagtaagt ctggaataat acctggtctt 129360gcttcacttc
tgagttgggt aaagaagtct gtatcagtgt aattttctaa tccgtcctgc
129420attatctatg gctcttggtt catacctgtc ttgaagttct gtcatgttct
gtctcttgtc 129480ctcagtagag atgctacagc agtggctcgc ctcaggcagg
gcagggcagt ggggtggctg 129540tcctgggggc aggcagtagg ggcacgctga
cgtcagggaa gttgaaaccc aagagaagcc 129600agtaaaagtg agtctcagat
tgtcaccatg tgctggcagt tttacacgct gtcagtaata 129660aaagtcttct
ccctgcaggg cagcctgcct ccaataaata cgtgtagtat caaatcctgt
129720cttccctcat aaattgtttg gaagctcccc aaggacagtg atgaggcact
cgtaagtgct 129780tgctgcctag atgggtccct ctccaccttt gctagattct
gagcattcac tgagttagag 129840ctgcttctgc aaatgtgctg cttctgctaa
gtggctgtga cttcatgcag ccttcacttg 129900gtttgtcatc agtggagatg
ccctgtgttg tcgaaggaga taagcccagt aagcctgctg 129960ggcacctttt
ggtttgcagg ttcagcaggc agcccatggc tttccctgtg tcgcattgaa
130020gcagctggct aaaattgatg atacattaaa ttcctgtgac agatgatcag
cttgtatttg 130080tgtaatggtg tacagttcac aaagcttaaa aaaatgctac
ctgccatttc atcctcagtg 130140aggaaggtga tacacagaga gaccaagtga
ctgtgtccac ggcgacggcg ctctgcattt 130200cactttagcg gttaatgtac
tctacctata tttttacttt atatttacca tatatctttt 130260catgtatact
tggcgtaagt gctttatagt agtcacctaa ttcactgtca tcttttttgt
130320ttcttggaag gtttctatta caactggttg gtattctttt agaagacatt
gttacaaaac 130380agctgaaggt ggaaatgagt gagcagcaac atactttcta
ttgccaggaa ctaggcacac 130440tgctaatgtg tctgatccac atcttcaagt
ctggtaggtg aatcacatta gtcttcctgg 130500agtgtctcgt tccccattct
gcactataca ctctcagagt gtaggagctg tgctgcccgg 130560tagaaactct
gccttgccca gtgtgccagt tgaaaatatt tgttgctgta agagtacacc
130620tgataccatg tgacccagca gttccactct tgggtatata cccaaaagaa
tggaaagcag 130680ggtggtgaaa agatatttgc atgccagcat tcatagcagc
attattcacg atagctaaaa 130740tgtggaacca actgaagtgt ccctcgatgg
atgaatggat aagcaaaatc tggtgtatat 130800ttacagtgga atattattca
gccttaaaaa aaggacattc tgacacatgc tacaacatgg 130860gtgaccctta
aggacattat gctaaatgaa ataagccagt cacaaaagga caaatactat
130920gtgattccac ttacatgagg gacctggagt agttaattca tagatataga
aagtagaatg 130980gtggttgcca ggggctgcag gggaggggag ttatttttac
aagatgaaga gagttattct 131040agaaatgaat ggtggtgatg gttgtataac
attatgaatg tacttaatgc tactgaactg 131100tacagttaaa aatagttaag
aggaccaggt gtcatggctc atgcctgaaa tccaagcact 131160ttgagaggcc
aaggcaggag gattgcttga gccaaggagt ttgagaccag cctcagcaac
131220atggtaggac cccatctgta caaacaaact agccggggat agtggtgtgc
atgtggtccc 131280agctactcag gagactgagg ctggaggatc gcttgagccc
aggaggttaa gtctctagtg 131340agatgtgttc atgccactgc actccagcct
cggctataga gtaagaccct gcctcaaaaa 131400aacaaaacaa aacaagacaa
gagccaaaaa tggttaagat gggccaatca cagtggctta 131460tgcctgtaat
cccaacactt tgggaggtca aggtaaaagg atcacttgaa gccaggagct
131520tgggaccagc ctgagcaaca tatcgagacc cctatctcta caaagaaaat
caaaaactag 131580ctagatatgg tgggcacatg cctgtagtcc cagctacttg
ggaggctgag gtgggaggat 131640ctcttgagct caggagttcg aggctgcagg
gagctattat tgcactccag cctgggctac 131700agaatgatac cctgcctctt
attaaaaaaa
aatccaaaaa aaaaaaaaag taaacctgag 131760agcttcctcc tcctgtgtta
aatttggagg ccaagatgtt tttgttactt ttacaaatga 131820tcaaggacgg
tgaaggttgg gcatggtagc tcacacctga aatcccagca ctttgggagg
131880ctgaggcggg gtgatcgctt gagcttgaga ccagcctgga caacatagca
agagacccca 131940tctccacaaa aataaaaaaa taaaaaaaaa tagccaggag
tagtggcatg agcctgagcc 132000caggaggtca agctgtagtg agccatgatc
atgccactgc actccagcct gggcgagatc 132060gagaccatgt ctctagagaa
agaaaatgac aaggacagtg aacccaagaa agtcataaga 132120tgccagctgt
gcagcaagca tggaaagcag ccagtccaaa ttaggacagt gtgttttcca
132180agaagaacga tcgtttgtaa tgagaatgct ttgctttaaa taaatgacta
aatagctaga 132240agcctagttc taggggatag gcacgtcttt cttctctcaa
gaaaatagaa aggcaattct 132300aatttctagt aacagcaaac agcattaagt
catggtccaa atatgaggca aaccaaaatg 132360tggcttgatt gttcagcagt
tgatctgttg gaagcccttg atattaaaaa ggttctcctt 132420taagcggctt
aggagtcacg atcaaagacc tatagaaaga gatgccatcc ttctaggatc
132480cttggctctc ttgggaacta gattcagata gtcataatgt aaatactgct
tgagctttct 132540ttctttcttt ctttctttct tttttttttt gagacagagt
ttcactcttg ttgcccatcc 132600tggagtgcaa tggtgccatc tcggctcacc
gcaacctctg cctcccaggt tcaagcaatt 132660ctcctgcctc agcctcccga
gtagctggga ttacgggcat gcaccaccac gcctggctaa 132720ttttttgtat
ttttagtaga gacagggttt ctccatgttg aggctggtct cgaactcctg
132780acctcaggtg atccacccgc ctcggcctcc caaagtgctg ggattacagg
tgtgagccac 132840cgcacccggc ccgagctttc atttttgaaa tcaatgtatg
actgaaacac tgaagactta 132900ctgacttaat tatggtttca gaacagaatg
aaaatgtctt cggttctgat gaatataaaa 132960ggaaaactaa ccaagttaat
ttggcaagta gatggtagag atagaggtgg ggagtggaag 133020gggaactaaa
atcttcacct agcattgttg ggattatatg gttacatcat ctgaagttga
133080cagaccaaaa tatagaggct tcagaggtct ccaaatagaa ctaaacatgt
aattcagatt 133140gttaggaggt agtataaatg agctaaatct catctttatt
acggtagagt taatgggtga 133200tgtctaaagt tgtctgaagt ctataaatca
tgacaaatta tgatgtggtg attgtattca 133260acagtctttc agttgcaggg
ataaaacccc agtttaaact agagtaagag aaagaatgtg 133320ttggtttaag
ctcctggaaa gtgcaggcaa gggtagttgg taggactgca tctagtgttg
133380taattctgtg gtctgcattg tatatttatg catctcagct ctgctttctt
cttttcattt 133440atataatttt taaattttat tttaaagata gggtctcact
ttgtcgccta ggctgaagtg 133500cagtggcatg aagtgcagtg cgaggctcac
tctagcctcg aactcctggg ctctagagtt 133560cttcctgcct cagccttcta
agtagctgag acaataggca tgtaccaaca tgcctggata 133620ggttttaaaa
tttttttgta gaaatggaag tcttgctgtg ttgcccaggc gggtctttaa
133680ctcttagctt caggcgatcc tcctgcctct gcctcccaaa atgctgaggt
tataggtgtc 133740acccaccacg cccagtctca tctctgcttc ctgtgttagt
tttgttctct ggtgggctgt 133800tttcacatga ccgaagatga cctctagcag
gctgtgttct cagcccctca agtaggccta 133860tgtgattggc cttgcatgag
taatatgggt gaccataaac ccctgaatgc tctggtccac 133920atgggccaaa
tgggagactg gacagcattc cattgatgag gaggtggggc tggtctccgg
133980gagtaaggga gaggagcaca tgcagtaact gatggtctgc tgcaagggat
agcagcacag 134040cagttagaat tttggaggta actaccagaa ctgaaaacag
aaatgataac aagtagttgc 134100cttaaaaagg gatgggagca gggtgctttt
gtgatcaaag ctcctttctc ttactggatt 134160tttgtacaca ttttgcatac
atatcttaga gtaaaagata gcattttcag ccttggtcca 134220tttgaggata
ctcttggcgt ggcccgcctc catgctagca ggctctggtt gtgccaagtt
134280cagttgagca tcctggctct tgcctgcacg gaacttccag tcagtgcgtc
agtatcacaa 134340gtcttgatat ttcctatgaa gaagaacagt agtgcagtga
cagacgaaat gggtgggcag 134400gcagaggcag gatttctgag ggagagaagt
agctagcttt ttgcagagaa gagttccggc 134460acccaagaga gcagctgaga
gtacaggcag gcaggcagga tgccggtagg gcccggccgc 134520acggcgccac
agaatcctgg agaaaggggc ctcttcatgg cctctgcatt cagctgctgt
134580caccctccgc acaggccatg gccaaaattt aattttcata gtggactcta
gtttttgagc 134640cttacttgct attattgaaa taattttctt gtttcttttt
aaagatcttc ggattatgct 134700tcactgacca ctgtaataag tttaaagttg
agaaaatatg gcttgttaat gaatgatagg 134760tcaattttag tatgttggtc
attttaatat tttgccacca gttggtttgg atttgatgcc 134820aggaggagac
agcctcattt ctaaggacta gtcttgcctt tgtgggataa gggtggtgtg
134880ttctgtgtcc ttctacatgt ccgagcgatc tctgtgcagc tcaaatgtgg
tcactgtctt 134940attgcgctga tttcctctcc ttccatctca caattgaggc
aaaatattgt tactgttgaa 135000gtgttgtcca ataggacttc cagcagagac
aggatgtctg cactgtctaa tttagttgcc 135060tttagccaca tgtggtgttc
tgtacctgaa atgtggctgg tctgattgga tagcttaatt 135120tataatttta
tttaatttta attaacttaa atttaaacag ctctgtgtgg atagtggctc
135180ctgtatgaga cagtgcaggt ctgttgagaa gcagctttac tggtgggagt
ggagggcttg 135240gagagggcac gtgggtttcc tgctggtatc ttttgacctt
atttaatctg cccaacattt 135300gcaagtaagt tgtgtgtgtg tgtatatata
aatgtgtgtt tctgtcttct tgtttccttt 135360gactgcattt atttgaaaga
cactaggtgg cagaattact gtatttgatt ggtttcaaga 135420taagagttga
aataattcat ctcgtgtttt tatataagta aggtgtgttt agcatgtaaa
135480attggtaata tgtattcacg tactgcttaa acaaaggcta tgaattccac
ccataaaccg 135540aaaatgaaga cctttaaatt tgtccatttc aggcgtgggt
acttcttaaa taatacctgg 135600ttcaggaact agtcagaatg gcacccttga
ctttttgttt cctgcttttc ctcttgttgg 135660gagaggaggg tattcatccc
aaagtggttt gcctatttca cattccatct aggataagca 135720gaatagccaa
gaaagatagc tgtcctcctg tttacaacat ttggggtaac cagcatccct
135780ctcttttggt ccaagataga ctggtttaga aacagatgat ggcaccagag
gcccaggagg 135840tggaaacatc agctttgttt gttgtccatg tggctgaatt
agagctgtct ggccttgtag 135900cctcaacacg gccttccagc tttgctcacc
gtgattttca aggacacatc ttgtgctctt 135960ccctgcctgc catccagact
atacccagtc agggtggcag gagctgctgc cccttcctcc 136020ctgagtcctg
gtcgtgggtg gtggagatgt gccatgacgc tcacggaggc atgctcaccc
136080cttcctctgt ggcagagggg atggctgcac gacagctctt ccctgtcctt
tccaaagcgt 136140ctgtggttcc actttttggg gcaaagcagg aatactggaa
gagagagaaa gtggtccttt 136200ctatagtaat aaagttgaca ttgattcaag
ttcatgcttg gggaaaggac agggctacta 136260acaattataa tgctgggagc
aatggaattt tctcatgggt atgtggtagg tttaatttta 136320attatcccag
ttaattctta gaactgctct gtgaagtatt tcccgctttg tgcttaagtt
136380ctaaaagatc ctgtgccaaa accaagaatg aaaacccaag cattctttct
tgcccatcga 136440tctttctctc atcaggccac ttcttgggtt gatagtggtg
agtgtagccg ctgccacttt 136500cagaataccc accatgggcc ccagtcactg
tgtggcgtgg agaagagatg gttctctctg 136560tgtcatagct gaacaagccc
agcccagaga ggtttctgcc ctaggagctc tcgatggtgg 136620aattgggatg
cgatcccaca tcctgcctgt tttgaaaaca gcattcttta tttccaattc
136680ctgcttccat tgttcctttt aatatttctt tgtttagctc acaaaaacac
ggcttgcgga 136740gctgctgcgt gcagctgtag ctgtttctct gggtgcagcc
tgcatccgcc ttcctgcccg 136800cctcctttcc tgcactgcca tcgtggtctc
cgggcacttg gtccctttct cttcccctga 136860gtccctttgg ctcccctgtg
ccacccttgt gatccacagg ctctgccttc tttctgtctc 136920agactgctgc
tcatcactac tcgggaccct aggaagggag gttccaccga gaagcatctt
136980ctcatctcag ccacgttctc agtgccactg ttgtctttgt taggtaatgg
tagctactgt 137040aacaaataaa ccaacatttc catggcttca caccagagaa
ggttgtttct tggttttatg 137100acaatgtatt gagggtgttc ttggttcacg
gatggttttc ctccatgtgg gaattcgggg 137160acccaggctc ctttccttct
tttggttctg ttctccaggc cttcacatcc tctgtgtctg 137220gttggggaca
aggagaggga aggtaaagaa ggctttgtgg ccttggataa gtgacaggca
137280tgcctttgct ggtgttctct cgtggtgaca ggtcacagcc ccaccctgta
aaaggggact 137340gagagacgtc gtcctgctgc ttcccagcag cagcactgtg
gtctctgatg tgttttctgt 137400gaggataaaa acaggtgatt ccaggatgag
gaaagtcagg gaaacccttg gaaggagggg 137460accaggcggg tgtcaccatg
ggattagtgg tggcttcaga atgagctgca gcgagtgcca 137520tgccttctaa
agcttttgct attctgatat gcccacacca tgcccagcag gtgtctgcct
137580tgctctccgc agagagagtg atgaatcctt ctcatgagcc tctgtccagt
tgttcctccc 137640tccacctgga agggaccctg ggttcctcat aacatcccag
cggaacaggg gaccttctat 137700cctgtcccca agttcatcct catcctcctg
ccggcttcct ggcccctctt atgtctgctt 137760cctgacgcca catccttctg
gattctctgg aattgaattt tgcctttgat gcttatttaa 137820aaatatccat
tgcaggccag gtgtggtggc tcacacctgt aatcctgtgc actttgggaa
137880gccaaggtgg gcagattgct tgagcccagg agtttgagat tagcctgagc
aacatgttga 137940aatcctgttt ctatagaaaa tacaaaaatt agctgggcat
ggtggcgcac acctatactc 138000ccagctactc aggaacctga gacaggagga
tcaattgagc cccggaggcc aaagctacag 138060tgggctgtga tcgtgccact
gtactccagt ctggtcaaac agagtgagac cctgtctgaa 138120aaaaaaaaaa
aaatccattg catacttcac cgtagcgaaa catgtatgtc ttacctttcc
138180tttcctgcct gtagctgctc ttttacactt aacagccaca ctaagccagc
cttaaatgaa 138240aaacaaacca gcacttcctg tgccctcctg cttccttcat
gaggggtccc tccctctgtg 138300tacactccat tctcattgcc catggtggtt
tgtttccctc ttgtttctca agccatggca 138360gcctgcctct tgccctcttt
actaaaaagg cctttgcaga ggctgcctgt gttctttctt 138420tctaggtctc
tctcatccta ggccctccag cttgattctg tggagctgcc ctcttgtcac
138480tcagtagctt gtggggtctt ctctgtctag ccacttaatt gattgtgttc
ctcgagttgc 138540tgtccatggt ctctcgttac tgttttctct gtgtttctgc
ctctctcctt ggccttggta 138600ggtccatccc ctttgtgacc ttggctgttg
ctctcatgga caactttctc ttgctggtcc 138660ttgtagtcct ggcatccagc
ttctcgacac gggacttgtc ctgccagtac ctcagacttg 138720cacttaaaat
tgaactagca ccactgtcac tctccagggc ctcttcttgt taattagatc
138780attagggatg ttcagaatcc cagcatcata gtatgttcct cctcccgcta
ccccaggaac 138840cctaacctta cctcctcctc tctatctact aggaggtggc
cctcagagtc cgtctcatct 138900tccacctgaa cttccctaat aggctccagc
agctgccacc ccgggggctg agtacttcct 138960ccatgccttg tgcagtgctg
agccctttac ctgggttctc ctgtttgctc cttattacag 139020ccctgcgaac
agatactgct cttaattcca tcttacacct aaggaagctg aggccccagg
139080taaggtgcat ccaaggtcac ccaggtagta gacagtagag ccacgatctg
aaccaggcag 139140tctgattcag agcctgtgtt gacactcagc cacctagaac
acagcttgga ttgtgggttt 139200ctattacctg ttcaaaaccc ctacatcccg
ggtctgtccc tgcacgtgct ctgtggcctg 139260gctgcatctt ccttgaaggc
agtgcatgcc tcttcactca gggggcccat gcaggaacag 139320agggccccac
agaaggatga ggccagtgca gaatgggctg gaggggacaa tgctgaccag
139380gaagcaagtg tagagaaatc ccaggaaacc tggaggagcc agagacaagg
cattagaact 139440cctcgtcgtg acctggtctg cattctctga gtgtgctgct
tctgttagct cgcttccttg 139500gtctcaggtt atagtttaag gcattgtgga
gccctaaaaa gcctgtactc tgtttttacc 139560tgttttagga ccctttcact
ttggggatgt gttgattttt tttttttttt tttttttttt 139620tttgagatag
agtctcgctc cattgcccag gctagagtgc agtggcacga tcttggccac
139680tgctgcccct gcctcctggg ttcaagcaat tcttgtgctc ccgcctccca
aatacctggg 139740attacaggca cccgccacca cactcggcca atttttgtat
ttttagtgga gacagggttt 139800taccatgttg gtcaggctgg tctcgaactc
ctgacctcaa gtgatctgcc caccttggcc 139860tcccaaagtg ctgtgattat
aggcgtgagc caccacaccc ggcctgaaat ttaaatcaga 139920aataaaattt
tgatcccaac agtgatgcca ggcagcccag atctggggga gagggtggcc
139980ttggccagct gggcctttct ctgtttccca agtcttgctg cctctccctg
ctgggctttg 140040cagcctgtgc atgtctctgt gcctttgacc ttgtttatcc
aaaggagagg atagaatgaa 140100gtcatgattc ctggagccct gagaaggatg
ctgtggagaa atttgccggt agaatctagc 140160tgagtgtgtt gctgaggtgc
cagcattgtg tgtggggagg ctgaccgctt ggcctgccta 140220ggcccaggat
gctccatggc cgggcacaga ggccacttgg ctgtcaggtg tcaggagcct
140280gcagagggca cacagagcct ggaccgcagg ggggtcctgc tttctcacct
ggcctccttc 140340agcatttctg tccctcagtc cttagcaagc ccaggagctg
ttgagtttgg caggtgccga 140400gtgctgttcc tgcctgtgta gctgtggctc
agtcctgtgg gggccccgct gtggcccgag 140460tgcagtgatt cgaggcgctg
agtgttccct gactccttct ccaggagctg tgttcagact 140520ttcgcagctc
ttggcttgga gctcctggag ggcttggcat tgccgaccaa tgtggaggtc
140580gacagtgaga gaggaggaat gctagctttc ttgaccagtc cattaaataa
gtgggatatt 140640ggccaggcac ggcggctcac gccttaatcc cagcactttg
ggaggctgag gcgggtggat 140700cacgagctca ggagttcaag accagcctgg
ccaacatggt gaaaccccct ctatactaaa 140760aatacaaata ttagctgggc
gtggtggcag gcgcctgtaa tcctagctac ttgggaggct 140820gaggcaggag
aacagcttga aaccggaagg tggagtttgc agtgagccaa gattgcgcca
140880ctgcactcca acctgggcaa caagagcaaa actctatctc aaaaaaaaaa
aaaaaagtag 140940gatatctgtt tctgcttaga aaaatcagaa ttttctaaat
gccaggtgtt ctgaatacgt 141000aagtatggga gacgactcag cctgtttcat
ttttatgtaa aatcttcgcg tagccatgtg 141060gcactggacc gagatgaaag
caaagacatt tctccttaac tttgtttcta ggaatgttcc 141120ggagaatcac
agcagctgcc actaggctgt tccgcagtga tggctgtggc ggcagtttct
141180acaccctgga cagcttgaac ttgcgggctc gttccatgat caccacccac
ccggccctgg 141240tgctgctctg gtgtcagata ctgctgcttg tcaaccacac
cgactaccgc tggtgggcag 141300aagtgcagca gaccccgaag taggttcata
atgccccaca gcccagggcg ccagcccagc 141360accctgtcct gagactccca
gtaacctgag ctttggccac cgttaaagca ttttcatttt 141420ccattttttg
tgagggcttg tgaaatttct gctgcatatt aatattcctt tcatggacag
141480catattattg ggacaaacat gcggtccagc taaaggcatt caaaatagca
gttgctttct 141540aaatgcgatt ttctttggca ggttctttga caccattgca
tcttgtggga tatgcttgtc 141600atgctctgtg gctcctacta agttctagtc
cttaaattgg ttccatagcc agacatgttg 141660caatgtctta acctcattat
aaagtaaatg tggttctggt tatccttaga taatgaagta 141720acagtgtagc
aaatttcaaa acctcttgga aatgttattt taccattcaa aaaggcttac
141780taaggttctc gttatgggtg gccctctttt tgcaaaaggt tttcaggctt
aagctccatt 141840tctaggtgct ccaacactcc attatttgta tatgtatgga
aataaaagct gtgaccaccc 141900ccaaccctgg cccccgccca gctgaatcct
cagcacagta tttctggaag gctcaagatc 141960ccacgctggg gaaaagaagt
tctggagaca aaagagggca ggtgctgccg tgcctctctg 142020ctcagtatgg
atactggacc ttgtgctgcc agggctccca gtagggccag ttcatggcac
142080tcagctggaa agtccactgt tgggaggcat tcttaaccat ccactctgtg
ccgtatgtag 142140tggggtctgg tcattctgtt ggaggagaca gaccagtgac
gacatttgaa atgcttggtg 142200gatgtcttag gcctgttacg atgactgagc
actgtggggg caggagacag aaagtcagtg 142260tctcctagtt ctgtgctgct
ttaacgtgca tagaaatcag ctgcggattc agcagatcac 142320tccttttctg
acagatgggc ctgcttactc tgatgttata tcagaaagct ctgaatctgg
142380gaattgtgtc ccctgaattg gagtaacaga aatgcttaga tgatgagtgt
ttaaaagaaa 142440taaaccaaag gtaaatttag tttggaattc agcaagcgtc
ttcattcagc cctctgaggg 142500caaactacag ctttttgtaa atgtaggtaa
attctgtgac tgtttcgtga ccccctctga 142560tccagttttc ctttataacc
ttctgtattg ttccttctat tatcctgaaa taacattaat 142620agattaggct
gggcgtggtg gctcatgcct ataatcccag caccttggga agccaaggcg
142680ggcagatcac ctgaggccag gacttcgaga ccagcctggc caacatgatg
aaatgctgtc 142740tctactgaaa ataacaaaaa ttagccgagc atggtgacag
gtgcctgtag tccctgctac 142800tcagaaggct gaggcgggag aatcgcttga
acctaggagg aaaaggttgc agtgagctga 142860gatcgcgcca ctgcactcta
gcctgggtga cagagtgaga ctccatctca aaaaaaaaaa 142920aaaaaaaaaa
aaattaatgg atcaatggat ttttaaccta ataattaaat ttcaaaaaat
142980atcgttcttt aatggtaatg taaaggtaaa attaagataa tatgtaacaa
gcatgtgagt 143040gtctaaggtg tccccgtggt ggaaggaaaa aataaatccc
cataagtgtc caagatgccc 143100atagagagca gagctgttct ggtttaaacc
cctgctctta gcactgtgtt tttccagctg 143160tgggtggtgg gggatgagta
tctttttatt tccatgagat gagaaaaatg aattactaga 143220agtgtgaaat
acaaaacaca gctgctcttt ttttagccat agactcagca gccataaaat
143280tgctgtatcc agttgcagaa attcctgctg cttactcttg accctctctc
ggtttgtgtg 143340catctcctct caggctggct cccagatggg agctggctcc
aggcgacact gggtgctctg 143400ctccaggagg tccttatgtg ggtcctgccc
tagcctagcc cctctcttat ggactctgtc 143460actgtgggtt tatgattcac
tctcaatctg tcttacctct tggtgaactg ttagagtcct 143520gcctatactt
tggcgcttgt gggtgtgttg tggtacacat gatgtgttgg tcacttccca
143580gctcatcttg ttctgagtca ccctagattt gggacattca ttcgccacca
gtaccgggcg 143640gtgtatggcc tgagatttgg gggggcttgt gctgctacaa
attggggctg aatttgagtt 143700gacagtggac cttctttatg tctactgctc
atatttgaat tgcaaatact gcctcttctc 143760tttcagaggc tcattaccct
atagctgtat tattgcaaag tgcacaatta cagcttgagt 143820gtaagtcaca
ctgcgctggc aggacggccc actgagaaag ggcacgtttc ctgttcgtta
143880gttttcacat tgacacataa tttacaatac agtaaaatgt acttttctat
caactgtagt 143940cagtaacagc ccccctcccc caaccacatc aagatataga
ggagtgctgt cacttcaaac 144000agttccctct tcctctgcca catcctgccc
ctccccaggt ctaaccacca atccgtgctc 144060tgtccctctg ttcagcccat
tgcagaaggc catagaaata gaatctatag gctaggtgtg 144120gtggctcatg
cctgtaatcc cagtattttg agaggctgaa gtgggaggat gacttgaggc
144180tgggagttca agactagcct gggctgccta gcaagacccc atctccagaa
aaaaaaaatt 144240taaaaattac aatcacgtcc ctgtagttca gctgcttggg
aggctgaggc aggaggatca 144300cttgagctca ggagttagag gttacagtga
gctatgatcg tgccactgtg ctccagccta 144360ggtgacacag caagacgttg
tctctgggga aaaaagaaag aaacggaacc acgcggtgtg 144420cagccttctg
agtctggccc ctttcggtga gcagtgtcta aagttctgtc gcgtgttgcc
144480cacgcgtcgg tggctcgctc cttgcaactg ctgagcattg tatggctagg
ctgtagtttg 144540ttttcacttc accagttggg aaacagagaa aaggcacttt
ttaaaaagtt taaatctgta 144600gaattttggt ttttaccagt tctcttctaa
atcctgaggg attacaggaa aagttgttgt 144660atttcagaat attcttagct
tgatgtgacc tctgtccccg ttaaggccct ttgccgcaat 144720gggaaggacg
tcgctcggtc agaccctgaa ggtcagaggg gcagtttggg agtgtgtcaa
144780cattttaact gtatggacta gagccaagag tctcaaggtt tataattccc
acgtattcaa 144840aaagaaaaaa acaataaagt gagaagtcag tgtagagtga
aataacctgt gttagtgggg 144900aagaagtgtt tttaaacagg atttccataa
cgtataacat caacatgttt agagtggtga 144960tgtttcattg ggaaacgaac
agtaaaacat gaaagcaggg aggttttcat tctggcagtt 145020ggcaactttc
acggcagatg gagaatttca aaagcaattg ctcaattatc aaacatagcc
145080agtgtgagtt ctgaaataaa ggtgctgatt gaatgtgcag ctttatggtg
gattttgcta 145140ttcaggcaag cattttaatt ttctgcctgt taaattctgt
tttctttagt ttttcatatg 145200tggtttattg tagcttagga atagataact
gagagtatat attacacata caacattctg 145260atatggcaat atttaaaaca
acttgtctgt tttagaacta gaattaaaca taatcatctt 145320cagtattttg
caaataagct cactgccatc cagaaacatt gtcaatgcat ctgttgctcc
145380ttctagaaga cacagtctgt ccagcacaaa gttacttagt ccccagatgt
ctggagaaga 145440ggaggattct gacttggcag ccaaacttgg aatgtgcaat
agagaaatag tacgaagagg 145500ggctctcatt ctcttctgtg attatgtcgt
aagtttgaaa tgcctgtaaa cggggttgag 145560ggaggtgggg accaggagaa
catcctgtgt agatgacact tgcatggacc ctctggaacc 145620cagaccgccc
ggtgtcctgc caagctccat cgaaactaaa tctagaatga atgtttactt
145680ctgctgtgac atataattgg agaccaggcc tggccttcca gtcactggat
tctaagttgg 145740actgtgagag tttttgcagc tgactcattt atcaaatgcc
cggctattgg ctcacgccta 145800catgatgctg ggtatgtttg ttaatttgag
ggaagcaatg gaataataat aactaatgat 145860ttaaaaaaca aagtaagtgc
attgactgta gtggggttct gattttaaat ttttttaaaa 145920attaatacca
ggagcagtgg cttatgccta aattccagca actcgagagg ctgaggtagg
145980aagatcactt gagcccagga gtttgagaca agcctgggct atggtgtgag
acacccatct 146040ctaaaaaaat aaaaaataaa aaattatcca agtgtggtgg
ctcgtgcctg taatcacagc 146100tctttgagaa gctgagggcg gaggatggct
tgagcctggg agttcgagac cagcctggca 146160acacagagaa accctgcctc
taccaaaaaa agaaagagag gaagaaagaa aaattagcct 146220ggcgtggtgg
tgcatgcctg tggtcccagc cacctgagag actgagaagg gaggattgct
146280tgagcccaga agtttgaggc tgcagtgagc tgtgactgtg tcactgcact
ccggcctggg 146340tgacaaggcg agacccctgc tctaaaataa tttttttaag
ttaatttgta gaaaaggtgt 146400tagatgttct ttgtcacatt ttatgatgga
ttcctgttta aatgccgttc tctttaaaga 146460aaaaaaaata acttgtggga
gtttttaacc ataaaactag catcacatat ttaccatgga 146520gaatttacaa
aaaaacaaat aaacggagga aaataaaacc tcctgtaatc atactactca
146580gagataactt gctgttagat tttggtctag atttaatact ttttctatat
ttatattaaa 146640aatatttaaa acatatgcat ttctttgtca caaacatggt
atcttataga tactactgtc 146700acatagcaaa acagtgttaa atattctgaa
tcagaaaagg aagccgactc tccaactgaa 146760agaggtgtta tcctagagac
tttttctggt
gatgacaatt tattaatagt cactttttgc 146820tttactttct ctattgaagt
agtttttcta ttttgttcta cttttaagga taatataatt 146880tataatgctg
tttttcacag aaatataaga aaaaagatac taattttata agttaataaa
146940gtttgatcat cccaaatcca aaaatctgaa atccaaaatg ctccaaattc
tgaagctttt 147000tgagtgctga cattatgttc aaaggaaatg ttcattggaa
ggtttcagat tttcggattt 147060agggagctca acaaataagt ataatgcaca
tatttcaaaa cctgaaaaaa atcctaaatt 147120cagaatactt ctgatcccaa
acatttcaga taagggttat tcaacctgta ctgtcagatg 147180atcccaaatg
aaaaatatta atcgttaacc aaatatcaag gaattgatca cattttacag
147240tttctgccta ggattatgaa tcaagatgaa aaggctctgc atgtttaaaa
atatatattt 147300ttattttctt ataaatctta aatatctaca cttaagattt
atttgatatg tgggatccat 147360tcatattttg gattcaacag ttctgtcaaa
actgtggcag tgatagggga ttcttttttt 147420cccactgaac tatcacaaaa
ttggaaaaag agtaattgga gaaccccact ggcttagccg 147480gcccgaagcc
cgggagaggg caggcagtgc tgtggatggg gtcatcccag cgcaacgctg
147540cccctgctac ctgcggatct cgctgaggcc tgcctttgtc ctttgaccct
tggccatttg 147600ttagtgtctc tgagagctgg actgctgtac cctacttccc
cagggggcct aacttcacac 147660agcctctgcc gcagtgcgtg gttggaggtg
acggccttgg taaatcgagt ttcctacctc 147720ctcaattatt tgtgctcata
cactgtatat ttttagtgag gtttatattt gggatgtgtt 147780ttctccttct
taccctttct ggcctttcta tggcattaat acctggtctc ttcttgtgta
147840cttgaaaatg aatctctcat catatttttc cttagtgtca gaacctccat
gactccgagc 147900acttaacgtg gctcattgta aatcacattc aagatctgat
cagcctttcc cacgagcctc 147960cagtacagga cttcatcagt gccgttcatc
ggaactctgc tgccagcggc ctgttcatcc 148020aggcaattca gtctcgttgt
gaaaaccttt caactgtacg tcttcatcct gccgactatt 148080gccagttgca
gttttccctg ccttaaaaat ggagtattga aatttttaac tttaatttct
148140gatttgcaaa atagtcatct tttgttcttt tccttcttgc tgttagccaa
ccatgctgaa 148200gaaaactctt cagtgcttgg aggggatcca tctcagccag
tcgggagctg tgctcacgct 148260gtatgtggac aggcttctgt gcaccccttt
ccgtgtgctg gctcgcatgg tcgacatcct 148320tgcttgtcgc cgggtagaaa
tgcttctggc tgcaaattta caggtattgg gaagagaaac 148380cctgatattg
atttatattg aaaatttagc aggccaagca aaacaggtgg ctggcttttt
148440cctccgtaag tatggtcttg acatggtcac cgatagaaac atggaaacat
ctgcaaactt 148500gccgttactc gtgtgtccga tctgactgtt tcttgtattt
ttttctagtc tgcccttact 148560aggatgaact gtacacatca gttcatcctt
tttaaatgag catgaggtta ttttgggttg 148620ttaggtgtta caaacacact
aatgtgtttt tgtctattag agcagcatgg cccagttgcc 148680aatggaagaa
ctcaacagaa tccaggaata ccttcagagc agcgggctcg ctcagaggta
148740atgctggaaa cacaggtcgt ccttgtgtta ggacaaccca ggatataaag
gatatagatt 148800tgtacgggaa taaattcaca ggacaagaaa tcgatgtgcc
ttataggtgg gtttactgca 148860gaagtgccat aatagaacct tcctactttt
aaaacaacca gatctcactt tctaaagagt 148920aaaggatgac cggcaggatc
acgtctgtga cgtgagtgga ggcagtttgc actcctggtg 148980gctgtttgag
aggtagcatt tagaatgcct gtattcactg tcctgtgatg agtgggaaaa
149040taggttatca ggtttatctt agcaaaatca aagcatgtca tctaattgct
aaacaagagt 149100tggcaaatct gagagacatt actcaatcct tggcatgcag
gacttacatc tgcatcctgt 149160tgccatttta tgtcttcaaa gcatttaatc
atttagttgt gtttgcaaag tctttgagaa 149220gcctttgtca gaaatcccta
catctcctat gtgagtgtat ttccatgact gcagaataag 149280ttaaactttt
acctttttcc ttcccttgcg gggcggggtg gggggcaggg attgtgtgtg
149340tgagagggag agagagacag cagagaagga gaatataatt atcatgctgt
gtactttgag 149400ctgaaactgc aaaaaaggaa aaacacacaa aaattattat
gcttttcagt ctttagagta 149460ccttgtctat tatgcttttc agtctttaga
gtaccttgtt gatggtgttt ttaaatggga 149520ttgggcacaa ttaggtggac
agtttgggat gatttttcag tctgtagggc caagctcttt 149580tgtaatttgc
attatgaagt tgtcactctc atagcagatg gcgggagata aactattatt
149640actttttgac cctagactta gtcttcagtc cagatgaggg agattaaaag
attataaata 149700tcttgtgcca gatgaggtga ttttattttg aaatgaccat
gaattcctat cagttgtctt 149760actgggatat ttgatagtgg aatttgtgca
tttgagtctt agatgatctg ttttacattt 149820attaagaaag cctttattag
cttttatact gtgtattgcc tgttgcagtg tttgagtata 149880aatgaaattt
ctggaaaata ttaatggagt acaaactgtg atacttaaaa gtaaactagg
149940gcctgcattt gtatcatgac ctgtttgagt attgatgaga agatagctgt
gaagaaaaag 150000gtttaaacaa gtgtattttc ctttaagaag ccactaatag
tgcatctcct tagagtgtat 150060atttctagaa tcctagtgtg cagagtttag
actaagacta aaaaaaaaaa aaaacaaatt 150120atactgtaat ttcattttta
tttgtatttt agacaccaaa ggctctattc cctgctggac 150180aggtttcgtc
tctccaccat gcaagactca cttagtccct ctcctccagt ctcttcccac
150240ccgctggacg gggatgggca cgtgtcactg gaaacagtga gtccggacaa
agtaagtgtc 150300cagcgtgtct gcatgggagg cacagggcgc tgagtgcctc
tgtcacctgt ggcagataca 150360gagagtgcag aggaggtgcc gtggacccaa
ggagttctgg cgctcggctc ggctcagtga 150420agctgtggtt agagacgtgg
ggggccatca aggtctgagg gagccaagca gtgctgatgt 150480gggacccttt
tggtaggagt gtggggtgag tagttagtgg gtgaatcaag gaatagtcgg
150540ccgtggcctg caggcccctg actgcacagg ccttcaagca catgtcaatg
ccgttagcct 150600ccctccatct cctcatacct tctggccacc tgtgagttgc
actgccactg ccagccattc 150660tggtatgttg tcagcacctc cactgctcat
acctcatggt tagggaccac ctggagcctt 150720ggtagagcct tggtagagcc
ttggtactct actttcctgg acaaagttca gcttatgaat 150780atgaatttag
atttcaaaaa ccagcagccc aagtataaga aagcgaaggt tcagtcctgc
150840cttcttaggc tctattcgct aagcacctgc cctgccctgg ttgctgggga
gagatgagta 150900aagcagacaa cccaggagag gatggcaaag gggccgctaa
cccttagtgg tttagctata 150960tttggaaggc ctattggaag ttcaccaggt
gaagggggag gctgtgaggg tgcccaggca 151020ggtaacagaa gtccaaaggg
gaaaacctgt ggtgtggtga gccgtatagc cacagcctgc 151080cggccggcag
ccctctcagc ctagtgcggt gttcccaagc actggcctag gcctgtagct
151140ccagggatgt gaagtcccct tgaacgccgc ccatcatgtt ccccttatcc
atttttttct 151200tcccaggact ggtacgttca tcttgtcaaa tcccagtgtt
ggaccaggtc agattctgca 151260ctgctggaag gtgcagagct ggtgaatcgg
attcctgctg aagatatgaa tgccttcatg 151320atgaactcgg tacgggggga
gcagtggagg caaggaatcc tcagcttttc ttgtgacttc 151380caagtgggat
ttgtctcatc atcatgtgac ccacttgttg acaacacatg ttggggactc
151440cagtctgggc agggacggga tgtcggagag actccactct gaatggggcc
gggaagtggg 151500gaggactcca tttcagatgg ggtcgggaca tgggggttat
gctgatcgag acagaaaagc 151560acattgtttc agccacatta gaatccacgg
aggtgttgtt ttgaaatcca gctggcccca 151620aggctgggtg tatggtttgg
gatgagaact atctggcctc cactggagga acaaacacag 151680gatgttatca
tctaagctcc atggccaaga cagaatggaa gtcaaggttg cgtatttgcc
151740gtagacttca acacagtgtc gtaatgcgtg acgtcaataa cttgtttcta
gtgtcttgga 151800agttgatctt tagtcgtaaa agagaccctt ggatgcagcg
agatttcctc tactcacacc 151860tctgttagat gtagtgaggt tcttcacccc
ccaaccccag atgtcagagg gcaccctgcg 151920cagagctagg aggccatgca
aagccttggt gtccctgtcc ctcacccgtg ggcaggtcct 151980gtgagcagtg
ggggggccac ctcttgggta tggtgcagcc atggcccaag cagggcttct
152040tctcagacct actaggacgg gagaaacctc ctggtgcttt agccctgcgt
tgatatgcag 152100caaatgggag ggaagtgggc acctgggagg acaaatgcct
gtagaggccg ggagtgacgg 152160caggtgttca tgaaaagaga ccttgtgggg
agggcaacac aacagtgtgt tctgatgtac 152220tgaagagctc aactgaaaac
aacaggagaa ttagcccaaa atccatttac taaaattgtt 152280tatctttttt
tttttttttg agacaaagtc tcgctgttgt cccccaggct ggagtgcaat
152340ggcgctatct tggctcactg caacctccgc ctcctgggtt catacgattc
tcctgcctca 152400gcctcccaaa tagctggtat taacaggcat gcaccaccac
gcccggctaa tttttgtatt 152460tttagtagag acgggatttc accatgttgg
ccaggctggt ctcaaactcc tgacctcagg 152520tgatccgccc acctcggcct
cccaaagtgc tgggattata ggcctgagcc accacgcccg 152580gcctaaaatt
gtttatctta agattcatgc agtgaaagct aacttactga gtgataaatt
152640tgcttagtga tctgtttatt aggttttcca aatttgctaa ttgggctttg
aacagctgta 152700aaagttctga ctgtaaaaga aagcttcaac ttttggcatt
catgatgctt ttctgagtat 152760taaactaaga tagatgtttt acctgaagga
tcggccacca atctttaaat ggctaaacaa 152820aagggttgct aaaacataat
ccaaattgac ataagaaata ccatttttcc aaccaaaatt 152880ttggcattca
tatggctact tttacgtatt tcagctgcat ttgaacatct ttttcaaact
152940ttagggtggt tggtgtatca ctgaggtctt ggatgacact ttagctttga
ttttgttttt 153000atgaattaaa attgtcatac caaaattttt atttcaagca
aatccaagag cataaaaaat 153060taaaatatta cttaaaatac taagagagaa
cagatatata ttttactaag catatgttga 153120atgaaattgt tcaaatattt
ataacaggca tagagtagaa ttttcttaaa aatatttttg 153180atggtatacc
aatttgtatt ttctcagaaa catttgcctt attctttttt ctgttgtgtt
153240tttcttacct gattgaaagc tcataatctg ttgttattgt ttgttaacct
ttaatgctct 153300gatttcagga gttcaaccta agcctgctag ctccatgctt
aagcctaggg atgagtgaaa 153360tttctggtgg ccagaagagt gccctttttg
aagcagcccg tgaggtgact ctggcccgtg 153420tgagcggcac cgtgcagcag
ctccctgctg tccatcatgt cttccagccc gagctgcctg 153480cagagccggc
ggcctactgg agcaagttga atgatctgtt tggtaattaa aattaaaatt
153540tatcttattt ttaaaaagca ttccagggcc agtatagtac tttgcaccaa
gtaaatgtac 153600aataaaggca gtggatctaa tacattgaaa gcgtttacag
aggtagctaa agagcagcac 153660gggtgtcctc ggctcagaat ttcttcctgt
gtgtttgcca ctttgccatt cattgacatg 153720gtcatggaca tagggctcta
agcccttgag gaaggctggg ccagacctca ggggagatgc 153780agccccaaac
cacgtgcagt cctgtggacg gatgtgtaga tgtgccactg aggaacaatg
153840tcttgagctt tcatcagatt ctcagagaat tgcttgactg cctttcgaag
ttgatgcatc 153900tgtgctcacg tttgcaccca cccacgaggt ccttctgttt
caggggatgc tgcactgtat 153960cagtccctgc ccactctggc ccgggccctg
gcacagtacc tggtggtggt ctccaaactg 154020cccagtcatt tgcaccttcc
tcctgagaaa gagaaggaca ttgtgaaatt cgtggtggca 154080acccttgagg
taagaggcag ctcgggagct cagtgttgct gtggggaggg ggcatggggc
154140tgacactgaa gagggtaaag cagttttatt tgaaaagcaa gatctctgac
cagtccagtc 154200acttttccat ctcagcctgg cagtaagtct tgtcaccgtc
aagttattgt agccatcctt 154260caccctcacc tcgccactcc tcatggtggc
ctgtgaggtc agccaggtcc ccttctcatc 154320tgcacctacc atgttaggtg
gatcctaatt ttagagacat gaaaaataat catctggaag 154380tactttatgt
cttaagttgg cctggacatg tcagccaagg aatacttact tggtttgtgt
154440tagtgcttgt aattcgcccc cagaatgtgt acacgttctg gatgcattaa
agtctggcct 154500gtatccttaa agggccatcg ctgtgctgcc tgccctcagc
aaggacacac tttgcagacc 154560cacagaggct ccgcctccac ctcacaccaa
agaaagggag gagtccaaag ggcatcagtg 154620ccattactca caaaatgata
aatacaccct tattctgaac cacgtggagt catatggttt 154680gtgatccctg
tccttcaggt ttcagcttag tggggaagtg ggaaagtcag cgtgtgatca
154740cagcacaggg tgattgctgc tgattatatt atgtgcctgc tgtatgcagg
atgaaatact 154800ttatatgcgt catcttattt gactctcaca accccctgtg
agataggctc tgttactccc 154860atttgacagg tgaggaaagc aaggcttaga
gaatttcagt gacttgccca ggtcctctga 154920gctaggaagt agccattctg
gcatttgaac ccaaggcctg ctatccctag aacccacgct 154980ctcaaattca
acctatgaca gaggcaagcc ctggtgctgt gggagcccca aggaagagcc
155040tctggcctgg tggccacgta gcccaggaga gatttctaca ggagcccaca
gcgctgaagg 155100agagagaggc agcagagtaa gggggctttg tggcagagag
gggactggca ctttggggaa 155160taggtgggtc aggactgaat gtaatggagc
catgtcagag ctgtccttct ggaagggcaa 155220gggcacctgg acgcgctgcc
cctcagtgct ttggacggtt ccacaactgt gattcacacg 155280gcttccccaa
acgaaggtac acgagtgggc attctgtgac tcggtacttc cctttaggcc
155340ctgtcctggc atttgatcca tgagcagatc ccgctgagtc tggatctcca
ggcagggctg 155400gactgctgct gcctggccct gcagctgcct ggcctctgga
gcgtggtctc ctccacagag 155460tttgtgaccc acgcctgctc cctcatctac
tgtgtgcact tcatcctgga ggccggtgag 155520tccccgtcca tgaacggtgg
gttcctatca tagttcctgt ctgcttcacc atgtttttat 155580tttgtgctgc
ctgtttgcca ggtactaagc taggaattgg ggatggagag gtagataaaa
155640tatgcatcag gaagggctgg gccccatctc ttactctcca atatattgga
gtctacactg 155700gaatttaact ggaatttgct tttttagtca ttttatttag
attttgaagt ttcagctttc 155760atcaaaaata cctctaaact ttatgtctct
gtgatctttg gtcttagctg ttttatgtat 155820ttagtcttat atgatcataa
gattaataac attacattca gaagattatt tgttttctgt 155880cagagttaaa
atgtttgttt ttatactgca ttgtaatatt aacgtactgt aaaataaaag
155940tggcttgttc ttttcaagga acagtatcct caacaagggt cattagccac
aatttttaaa 156000aaattggacg tcatagttta catgttagag ggcgttttga
agctttgtat ttttaaatta 156060aatgttatag agtgatgttt tcatgtttca
taattgtttt catctgtgca tttgtagcca 156120acttgaaaac aaagatccag
ggattactac ttaaaagcca gacttcttgg aggttatagt 156180gatgattttg
atagtatctt gagccgtctc ataataacct cagggtgaga gatggccaac
156240aggagacagt cgagggactt agaaatctga atgaaatctg aagttcaaat
cttcagacat 156300ataccactaa ccaagagatt ggtacctcag tctagtattg
tctgtttgtc taaaattggt 156360tctaaggaat ctaggctagt ctgtctatcc
ctttcaactt ttgtgaggct gcacaaatgt 156420aaaatgttga ataaaaagca
ctgatggaag tgtgtagaaa ttcttctctt tgttctgttg 156480taattttagt
tgcagtgcag cctggagagc agcttcttag tccagaaaga aggacaaata
156540ccccaaaagc catcagcgag gaggaggagg aagtagatcc aaacacacag
agtaagtctc 156600aggacccatt tttttcttac atgttgttcc tccaggactt
aaaaatcatt cacagagacg 156660tgcaccgcgg tgagtgtgga ctcctggaag
cgcaccgtag ctccgctgtg tcctgctgct 156720cctccctagc tgtcagggag
gctgtagtcc attgctttgc cagctctttt gtttccgagt 156780gaacacctta
tccgtacaca tgcggctgtc tctgacccta cagaccagct gggatgccac
156840tgggggagcg ctcccttccc cccgcacttc ccacactctg cagttattct
gagatccttg 156900agggcaggga acaggtttgt cttctttgtg ttctcagaaa
ttaatgctcg gcctctggtc 156960agcaagcaac aaccttttgt tgagtgataa
tgaataaata aatgtttccc acatgagtat 157020tcagtaacct cagtgtcagg
ttcagccatc tgttttggtg gatatttaaa agaaaattcc 157080gcttttccta
cagaaaaaaa aaaaaatcca aatcccagtg atttaagcca gttatagact
157140tagacatata ctacggcttt tcatgcactt tcctcccaat tctagagtag
gtattttact 157200aggaaaatgg tggcagtgcc tgttgggagg aagattcttt
ggccaagtgt cttttgttct 157260tgccagggcc cctaggctgc tggggtgctt
cagcttcttt agcccagtgt ctggtgggga 157320atggcccctg ttgcctgtcc
cacagaggtg ggggtgcctc acctggagcc tgtccacaca 157380ttttacacag
cacgcttacc tggagcatca ggcatctttt ccatgctctg tggctcagga
157440aacacgcctt ttcaatcatg agtgcaccag tgcttttggg ctttttctcc
ccgcttttgt 157500gcaatcctgg ttgtggatgg agttttcctg tctttagtct
tctgcatagt acttttctct 157560tctggttccc ggttcaaggt tttgtaatta
gagaatgacc cagaagcaat ggcattttaa 157620tgcacagcca aggacttctc
tgaatttgta tctcaaacct ctgtgggtcc ttcaggcttc 157680agtttgtgat
ttcatgattt cttgttgcta cctaaggaat atgaaaacac ccacctccct
157740actctgcatc ttccagccga gtggcacctc aggctgtgga tcctgtgctt
ctgtggtgag 157800gataagaata gtgccaaccg tgtggattga aatcaatcag
ttaatccctc catgtaaagc 157860acctggaacg gatgacagtc ttgttatgaa
tactcaacaa atgctatcat gatttttagt 157920tagatttcca ttgctttaaa
acagttgaga catcttggcg gtttgagtta gagcaacggg 157980ccctgaagtg
ggttctgttt gggtgaagat gattatgctt attccccatg gccctcttta
158040ggcaagagtg ggaagctttc tttgtttttt taatcacctc gataggacgt
tacttcttaa 158100aggtcatcca ataaatatta ataggccggg cgcggtggct
cacgcctgta atcccagcac 158160tttgggaggc cgaggcgggc ggatcacgag
gtcaggagat cgagaccatc ccagctaaaa 158220cggtgaaacc ccgtctctac
taaaaataca aaaaattagc cgggcgtagt ggcgggcgcc 158280tgtagtccca
gctacttggg aggctgaggc aggagaatgg cgtgaacccg ggaggcggag
158340cttgcagtga gccgagatcc cgccactgca ctccagcctg ggcgacagag
caagactccg 158400tctcaaaaaa aaaaaaaaat attaataaag ccaactcgtt
agcgtggggc ttaattgctt 158460aagtccaatg agaagtcctt ctctatccta
ggaagttgcc caaactgtag aatctcgtgg 158520cctgtgggta atagccacgt
aatacacact cactgcctca acaaatcata ttttagtagg 158580tatgatattc
tagactcaag acaccattct gtggatcttc ccaagggtgt gaagtgtcca
158640cagcgtctgc cttgggagtt tccatgccca ccagaaccat gccccaagcc
cctcaagcac 158700tctgacctag gaaagccagt gaagcaagga tgacaacatg
gccctttgat actagctgag 158760ggacagacac aggtcctggg agaccagaga
aagacgaggg gcagaggagg tgtcctaaag 158820gaagtctgag gctgaggagc
cacaggatgg cttccagctg tcacaggctg ctgctggcct 158880tatcacagag
agtgggccag agggctggga accaaggcca gagctcaggt tcaggaccat
158940tccagcaatc ccagcagaaa atggggagaa ttgtatggta taggcggata
tgaaggtaga 159000atctgcaggc cttcagtggc caactcagag tctaagtgga
ttccacagtt acagcttgag 159060cagctggttg taggtcatgc tttctacact
gggcatatag gatgtgtttt ttaaaaagtc 159120ctctcttaac cgttgcttgt
ttagatccta agtatatcac tgcagcctgt gagatggtgg 159180cagaaatggt
ggagtctctg cagtcggtgt tggccttggg tcataaaagg aatagcggcg
159240tgccggcgtt tctcacgcca ttgctaagga acatcatcat cagcctggcc
cgcctgcccc 159300ttgtcaacag ctacacacgt gtgcccccac tggtgagtct
gctcgttcct tgcagaagac 159360caagtacggt gaaaggcacc ggtaggccct
gggctgggca cacgtgagag ggcgggacag 159420aatccccgca gcccagaggc
tgcctgctgt ggttctggtg cccactgtgg ttctggtgcc 159480aggctgcttt
cctcaggcac cacgtgtgga ggtcgctagt agaaatactg ggttttctaa
159540aatgaactga ggccctacat ccctaagaga ttagtgttag acctgattct
agagcaacta 159600gaccactttg cttaatagca gaccagaaac cacaccccct
cgagtgagtg agattttcct 159660ttggagataa ttcatgtttt tctacacagt
tttgcagttg tcttcagaat tggtttaaag 159720taggtgttat tgccaggcgc
agtagctcat gcctgtaatc ccagcacttt gggaagccaa 159780ggtgggcgga
tcacttgagg tcaggatttc gagaccagcc tggccaacat ggtgaaaccc
159840catctctact aaaaatataa aaattagcca ggtgtggtgg tgtacgcctg
taatcccagc 159900tactcaggag actgagacag gagaatcgct tgaacccagg
aggcgaaggt tgcagtaagc 159960cgagatcgcg ccactgcact ctagcctggg
caacagagca agactccgtc tcaaaaaaaa 160020aaaaggtagg tgttattgat
cagaaccctt gtttcagata acatgaggag cttagcttga 160080ggagagtgag
ggttgatgga gggggactga cttctgccca gtgaaatggc atcatctccc
160140accagcccgc tgaaataaga tgatggggcc tgttccttag ggcctgcagc
atcctcaggc 160200aggaaagaaa ggccgacctg gcagggtgtg agccagcagg
tgtaggtcag ggagaatgga 160260gccaggtccc agggaagagg cttgtggctg
cctgagaagg gtgcgtgcct gcctgtgtgt 160320gtgtgtgcac gtgtgtgtat
gtatgctgga gagtctaggg aggcttgctc caaggacgca 160380gtattgtttg
atcctgagag ataaggattc tgccgcaggg aatgaaggta ttccagatgg
160440cgggcttatt ccgaagaaga ggccagtgcc tggcggtgct ggaagcagtt
gcagaacagg 160500gagttgtagg ctttcctggg aagagagcag caggggtgct
ggagaagcag gccacacttg 160560ctgcatgggg ttgctctcgg ccccactctt
ggtgcacagc gagtcactgt gggttcatta 160620gcatctggtt atgagacagt
aactgctcct ttggaggggc tcgtggagac catgcaggag 160680ggcacggtct
tgaggtcatg ccgtccagag cacacctgag gataggccag gacgggctgc
160740acgctgtagg taaaattcct ccagcaagct cttcactggc attgaggagt
tccctgagtg 160800cggtcatctg gaaggcagct gtaacaggca ctgcagtctc
tccctgggtg ggtaccagag 160860aggagcatag gggagcataa ccgatttaaa
gagagggctt tcctgtggtg aggtaagaga 160920ttagctggtc attatcatag
agccccctct gcctttgtgc agatgggctg tgggaatcct 160980ggggttccgt
tgggtccttt gtcacctcac tgaaggcatg taagctgagc tggccagacc
161040gtgagctgat cctgccactt gaacagcatc aagcctgcct ctggattctt
ctgtgcatgg 161100cacttgtctg agcacctcac gcacagagaa ctggacttca
gagtttacag aaataagctg 161160tatggttcat tttcatgcct gcttgccaat
aaacatatct gagctgaacc tcattgaacg 161220cctgccttta ttctagcaca
gcacctgctg tttgtgggcg aggggtgctg tctctaactc 161280ctgcctgctt
ctcccagcac tccctgagtg gggtgtgcca gcagcctcag gatgaggaca
161340ggaagtggga gggcagagca gatttgggag ggccacttga tggggaagga
agtcccagga 161400agcagttgga gctgttttct gggggagaag gtgccagctc
tgggacagtg ttggggtagt 161460gaggagggag cccagtggag agaagtcggg
cttcctgctt cctcacagta tgtctgtcct 161520gactcaactc ggatgatgtc
acttcctttt catcttctca ggtgtggaag cttggatggt 161580cacccaaacc
gggaggggat tttggcacag cattccctga gatccccgtg gagttcctcc
161640aggaaaagga agtctttaag gagttcatct accgcatcaa cacactaggt
actcttgggg 161700cctctccttc aggtcaccat tgtcggacat ctaccgggag
gaaatccaga gcccccagta 161760ctgggatctt ctcatttgac tccagaaaag
atttaagcat gataataata caaacctatg 161820tgaatacatt ttgcagtgtt
ggcaaaactc
cttttatact gagaaaatag atcccagttc 161880ctgtgttttg tggcttgaat
cccagctttg tgtattccgg gcttgtttga agtcaggaaa 161940ggttcatgtg
tagtggacaa cgtgagacca aattctgcct tagattttgc atttaggcta
162000aacagtggca gcacttgtct cagaatgttt tcttgtgttc accagtctga
tcctgttgtg 162060tctcagtggt ccattttctc atatgggaac aagcagacgg
gagcagatgg agtcaggttt 162120cttggcactc gccttcccca gagcctagag
gcagcatggg gagaaagcag gcttggggct 162180cagacagtcc tggtctgctt
ccagccctcc tacctgagca gcgcagggca agtccgtcta 162240acctctagag
accctcagtt ttgtcatatg taaaatgggg gtcgtgtcta tttcatagaa
162300ttgttgcaga tttagaaatt acatttctaa acaaatgtta ccccttattt
ctaaataagt 162360gtctaaatga ataagtcacc acttttgccc ctatttgatg
gcaagaggtg tgatcttgtg 162420gtgggactgt aatcagtcag ttctcagtga
ctgtgccctg ctgtggtgtt tcctggaatg 162480ttcctgtctt gtcctagaaa
gtctggcagg ggcaccctga ctccactgtc cagtcctctc 162540cccagtccct
cgggcttctg cagatttgag gcttgtttgg atcccagaag gttgtggcag
162600gagacacctt gcctctactt tcccctttat aattcaatgt ccaaagagag
ccctgagcag 162660gtacctcacg ccagctgcct cacggagctc ctcctcttcc
tggctgtgag gatcggtatc 162720agtggcctcc tgctctctcc cccttgccta
acacgagcac ctttgcttac ttgggtgccc 162780ttgctcttga actgcccatc
ggacgtgcgt gacccaagac tgtgccgcag tccttgcctt 162840gtctgtgctc
attttctttg ttcatttttt tccctgtaac gtaaattgtt atatttgtct
162900gtatctgtgt ctgaatcagt cctgcacgct ctccttctct ctgtctcttg
ttctttcttt 162960accccgttta tcacggggac cccgatgtcc attgctctag
ttctcctgtc ctaagcaccc 163020catcccgtct ctctggcctt accacaagtg
gcgtggctgc ctcagacatc atgatgggga 163080catgaagcac agctgtcaga
aacaactgtt cgttagatac actcgaatgc agctcatcaa 163140tagggatgga
gggtctgtcg gatgtatttt cactgaatcc ccgttcctac cttgatacac
163200tctttttaat ctattcttct agacaggtca gaggaaccat tactttgact
tttaaatttt 163260tagcagcttt attgaggtag aattcacata ctacagattt
cacccactct aagcggacag 163320cttggtggcc attagtttta tccacagagt
tgtgcagcca gctgcacagt ctcagggctg 163380gactccaggg aagattttag
cccatttagt gagtggggca gaagtggccc tggccctgca 163440cgaggttgcc
tgcatgggcg tccctgccct gtccctgtgt ctgctccact gggggttgac
163500caggctgcca gggccgactt gggcctgtgc cacctgcctc tcatgtgtct
cggacagtgc 163560agccgatgtc tatacttcgg tttcctcaat gatgaaatgg
aggggatagt gttccccgca 163620tcatagaact gtgtgaggtt taagggactc
actgcccttg gcgtggagcc ttctccaggg 163680gccgtgctgt gtcggcgtag
ctgtcagctc tccgttacag gcttgagaag ggttgacact 163740ctctcatgta
acatttatat ttctaggctg gaccagtcgt actcagtttg aagaaacttg
163800ggccaccctc cttggtgtcc tggtgacgca gcccctcgtg atggagcagg
aggagagccc 163860accagaagta aggccacacc ctgtgctggt tggcacatgg
gcagttatgg ccgcttgcag 163920gcctttggtg gggaataaaa taaggcagca
agctggtgtt ctttttttct cttaccttat 163980ttttgaaaga gtagctgaat
ggtgtcttga ctgatattcc agagcaggga caaagcctgc 164040tgaggtctgg
gggctgcgat taccaatggc tggaatgcat tttattacgg tgcattccat
164100gttaaggatc aatacgattg tgccctttct ggaaaatatc ttttagttta
tcaatattca 164160gaggagtgta ggttgaatta aaatgaaaag gcactttata
aaggccatga gtagtacctg 164220gtttcatttt tctaatgtct tgcagagatt
ttatcaggct tcttgaagtg ttcacgtaca 164280ttacgctaac acgatattaa
taataactgt gctctggtac agcggagcca gcagaatggg 164340aagttgtgga
atgcaggccc ttgattctga tagaaggtgt ggtttgaact cacagaaatg
164400acagtttgga gggtagacat atgtcacaag tcatcaagat tgtctttaaa
ttcatgcata 164460gaagctaaca gggtgtcata agcaaggcct gtaaaatgta
tgagggaatt caaagataat 164520ttattaaaaa gtaattcatg tttggagttt
tgtgcccaaa ggagtccttg atttgaaaaa 164580tgggcttttg cccatcagat
tgtttcaggg cccgtgtgtg cggaggccct gccttgtgcc 164640ccgtgagctc
agcctgacag aaatcctttg gtagcactta aggctcctct tcctcccatt
164700gaggcaggga agactctggg ttctgcaggc agaggtggtt gtgggtgtct
tgctgctctt 164760gttgacatgt gggctctcct tccaggaaga cacagagagg
acccagatca acgtcctggc 164820cgtgcaggcc atcacctcac tggtgctcag
tgcaatgact gtgcctgtgg ccggcaaccc 164880agctgtaagc tgcttggagc
agcagccccg gaacaagcct ctgaaagctc tcgacaccag 164940gtttgcttga
gttcccacgt gtctctggga catagcaggt gctggggaca gtgggttccc
165000cgctgaagcg tccagcagct tcaaccaggc cgttttcctt cattgctaga
attgaaaaca 165060ccgtccgtgt ggcctgtgca ggagatgcag acccaaaggt
ggcctcctgg tcagtgagaa 165120gctggaaacg tgacaggaac tgacgtgggg
ttattgagca tttaggggaa gacgttagca 165180gagcaggaat gagcaggcaa
ctagtagaac acccacttaa gggctcacgg acaggtgctc 165240acttaggaag
tgagtttcat ttggtattac accaggttcc tttaggcaaa gcggagggaa
165300agttctggtg tttttcactt gtaagatttt gaaggaaaca aaacactctt
tacctttttt 165360ctaaaatgta ggtttgggag gaagctgagc attatcagag
ggattgtgga gcaagagatt 165420caagcaatgg tttcaaagag agagaatatt
gccacccatc atttatatca ggcatgggat 165480cctgtccctt ctctgtctcc
ggctactaca ggtacctgag ggaaagggtg cgggggagcg 165540gttgtacttg
ggctagaatg agagaagact ggcatgctca ccacaccagt gatgcgggaa
165600gacctgagtg tggtctgagt tggaggctgt ggtgctaaat acgctgcccc
tttcataagc 165660aggagtctta gtcaggccca gggaggaagt aaaatctgga
aatgaatgag aagcattctc 165720tcctgccagt caagaaatga gaagcgaaag
aattctcacg ggctgtaaga ccagcaggat 165780ttaaaagttg aattagttgc
ttatgttaag aactcaacca agttcatcta cacaagctga 165840atctccagct
tttcctaaga aaccatgtgt ggcagtggct gcagggcagg gcacagctgg
165900gcctgagcac cccgctccct gcacctctcc cctccctggg ccctgcctgt
cactgcccac 165960tctcccacca agccttccgg ttgtgtgcct gccctatcac
aggcatcgga gcttgtcacc 166020tggtttaaaa gaagagagtt gtgtggggat
ttgggatgca cgtttttcac tcaaaagtat 166080tttagcgtag agctctgtga
ttccgtagct atttaggagt ttaagcacct tgaaggcttt 166140aattgcagaa
agttctatgt ggacgtgcaa tgtgttatac gcagtgtcta tgagactcaa
166200atgtttatta gggcgttgaa gtaaactgag cacttggagg gccatggatc
cagccttcaa 166260ggagctcata agtcaggagg acccaggagc aatgacctgt
catagaaggc agaaaagagg 166320ggcacagagg tgggtgggag gcatacacag
gcagctcctg gagctccaag gggagcaagt 166380gcttccaggg aagggggcgt
ggaggcccct ttggaggagg caagttgatc tggggtctgg 166440cagagggtta
gctggggaca tttagcggga ggctggtgcc cgggaattgg ggggatgccc
166500agcagaaaga catgaggagg ctggcctggg gcgtgggggg gtgtgaaagg
ttaagtgggg 166560gcattatcct gctcccgctc ctgccggctg tatctggtca
gcctgggcac cgaggtgggg 166620ttctggaagg cactgttcac caaaatgctt
atctgggtcc cccagagagc ttgcctgcct 166680ggactgtcgg ctcgcctgca
actgctgact cctaagcttt tgcagctcag cccacaacca 166740gttcctattc
acagaggtgg gagctgaggg gtgacaagtg actgctgcag tcttatttgt
166800catagagaaa aagtgacaga gtccagcttg cccactggcc ctgccagctt
aactggttat 166860aaagtgacaa atccccaaga cccacagggc tctgcacaac
ctgggccctc ctgccagtgg 166920cggcgagggc aggtggctca cggctgggtg
cctgtctggg caggagctgg gctggtatgg 166980ggtgggcctg cggccctgcc
cccctgtgca gatcaagact cagggtgctg gtgttcacag 167040gtgccctcat
cagccacgag aagctgctgc tacagatcaa ccccgagcgg gagctgggga
167100gcatgagcta caaactcggc caggtcagtc tcgcgccccc gccgcctggc
ctctgtccgt 167160ttctgtcctc agactttggc gcttgacaca cccaggagaa
aagctcagtg cactttttaa 167220atgaaaggaa gttttccttt tttttaaaaa
aaaatttaat gttcattgtt tttatctgtt 167280ttattcctag gtcccgcaag
cagaggaagc attagttttg tttttattta tgttctgtat 167340tccagaaagt
agttaagaga cctcacatgt agcgatagag atgtgtgtaa gagacagtga
167400gagggcgtga cttggactta agcaaggacc gtgagacaca aaaagggggg
tgaggacaga 167460gtggagtcag ctgaaatgct caggaggaag tagacgccat
gaagggccat ggtatggggg 167520gccgcaggcg tggccgtgag tgtccctggg
gccagctctt ggggggctcc ctgagtgtcc 167580ctgtccctgt ggccagttct
gggtgggagc cccgtgtgca ggcagacagc tcggccactt 167640cctagcaggt
cacattggtc tgtgcttctg tttcctcctc agataagtga agggattcaa
167700gggtctgggt gtggtggcta acacctgtaa tctataacat tttaggaggc
tgaggcagga 167760ggcttacctg agctcaggag gttgaggctg cagtgagcca
tgattgcacc actgcactcc 167820agcctgggca acagaccagt actctgtccc
ttaaaaaaaa atgtaaacag aaacgtaggg 167880ccatttgcat atgatggcac
atggcgtgga gccctacagg tgtatgctgg gcggggcccg 167940gctgtgctgg
ccgacttgca cctttccctc caccccggtg ctgtgtcttt cgctcaccgg
168000gttcctgatt tagtgaaagc agttgtgcag gacagttctc tttgtagctt
ttgtttctgt 168060ggaaatgggt cagaatatgg tgtttagaaa cacttatgag
ctctgagagt ttcctcttct 168120gagttcctgg cctgcagcct tcacagcaga
aaccctgtga tgtcacaagc ctgtttctgt 168180tccctgctct ctgcctgtac
tgtcctgttt tgtgcctgcc ggtttcagtg acaggaagca 168240gggagctact
ggaccagcct gtatttttct agacatagtt ggaaaaagaa gtcccactct
168300tctgtccttt cacctttgac agatgtttcc accccaagat aagtgaaaat
gaccaatagg 168360atgcactgta tttttcatga aagtgtttct gaagggcagg
ctgagagtga gaggcctggg 168420gctcactggg tgcctctggc cttgtcctgg
gcccagggac actggtctgt gcccgaggta 168480ttccctatcc ccccaacccc
gctgcatttg gccacatcct tcaatgtttg cgttgtgtcc 168540agcgtccgca
aaccaactgt catgggatca tactggggct gaagtacggt cccacccctg
168600ccctgtctgg ggctgaagta cagtgccacc cctgccctgt ctggggctga
aggacagtgc 168660cacccctgcc ctgtctgggg ctgaagtaca gtgccacccc
tgccctgtct ggggctgaag 168720gacagtgcca ccccttccct gtctggggct
gaaggacagt gccacccctg ccctgtctgg 168780ggctgaagga cagtgccacc
cctgccctgt ctggggctga aggacagtgc cacccctgcc 168840ctgtctgggg
ctgaaggaca gtgccacccc tgccctgtct ggggctgaag gacagtgcca
168900cccctgccct gtctggggct gaaggacagt gccacccctg ccctgtctgg
ggctgaagga 168960cagtgccacc cctgccctgt ctggggctga aggacagtgc
cacccctgcc ctgtctgggg 169020ctgaaggaca gtgccacccc tgccctgtct
ggggctgaag gacagtgcca cccctgccct 169080gtctggggct gaaggacagt
gccacccctg ccctgtctgg ggctgaagga cagtgccacc 169140cctgccctgt
ctggggctga aggacagtgc cacccctgcc ctgtctgggg ctgaaggaca
169200gtgccacccc tgccctgtct gggatgttta gcccctagat gccactggac
tgagccgcta 169260cttgcttttg ggaaagaggg gtgggggtta ggggtctggg
cgaggggagt gcaggggctc 169320ctccttggcc tgagagctgt tcatacagac
tcctcgccca ctccctgcag ggtgctgggt 169380cccagggggg aaatggccct
tggtgccaag aacgtgagtt ggggctagtg ccagtgatga 169440tggagaacag
ctttttatgg gcacacagcc cacagcactg tgccaagtgc tcgaggcttc
169500ccgagaacca ggcagaaagg aggacagtcg aggtgtgctg actgcgtggt
ggctgcgtga 169560tctagagcgc gggtcacaaa ggcgcgaggg agctctggcc
ttgggtttac cgcaatgact 169620gccagtgcgg gagactggaa aaggaatctc
acgtattggt tccgtgtttt ggggactcca 169680ttcagatgtc acttaggagt
gaaagcatcc cttcgtagag cctctttctg tgtcaccctc 169740ctcagctgct
cctggggttg actggcccct gattcatgcc tttagcatgt gctggagctt
169800cccagcagct gtccagcccc tgccccaccc tctctgtggg ctcccttgcc
cgtaacctgg 169860ggtgtctgaa cgacccttgc taaggggcag actgttagac
ggtaggcatg tgctgagtcc 169920cagtggccac acccacccac caggagcctg
gcactgtggc cgcagcactg agcagtgccc 169980cgtttctgtg gcaggtgtcc
atacactccg tgtggctggg gaacagcatc acacccctga 170040gggaggagga
atgggacgag gaagaggagg aggaggccga cgcccctgca ccttcgtcac
170100cacccacgtc tccagtcaac tccaggtttt ccaatggcct ttttcttttt
aacagaaatt 170160tgaaatttct tatcagtcat ttgatttgtt tgaggtgctt
cttgaaatga gcctctcatc 170220tcatgtactt ggaaaatacc catctcgcat
attccacagg aaacaccggg ctggagttga 170280catccactcc tgttcgcagt
ttttgcttga gttgtacagc cgctggatcc tgccgtccag 170340ctcagccagg
aggaccccgg ccatcctgat cagtgaggtg gtcagatccg taagtgagcc
170400ttcccattcc cctcacacct gcacgtgcca cacgcaccac acacgccaca
caccccacac 170460acacacaccg cccacacaca tgccacttgc acacacaccc
ctcatgcatg caacacacac 170520acaggccaca cgcaccatag acaccacaca
cacatgccac atgcacacac atacacggca 170580tgcaccatac acacaacaca
cacagcacac atgccacaca cacacgccac accacatgca 170640ccacacacat
gccacatgca cacacactcc acatgcatgc accacacaca cacacacaca
170700ccacacacac cacatgcacc acaccacaca ggttacatgc acacaacaca
cacatgccac 170760gtgcacacac cccacacacc acatgtatgt gccacacaca
gcacacaacc acacacatgc 170820accacacaca tgccacatgt gcatgcacca
gacacatggc acacactaca cacacgccac 170880gtgcacacac cccacacaca
tgtacgcacc acacacatgc cacacacaca tgcaccacac 170940acatgccaca
tgtacacaca tgtatataca caccccacac cacacacaca ccacttgcac
171000accacgcaca cacaccacat gcgcacacac acaccacata cgccacatgt
acacaccata 171060cacacaccat acatgcacca cgtgtaccac gcacccacac
agacacagca cacgcataca 171120ccacacacac acgcacacat gcgtcccgca
cagtaatgtc tcttgggtgt aagaacacga 171180cttgccagta gtagcgttct
ggatgcgttg cctggattct aacagcgcga ttctcccctt 171240gccctcctgg
ttttccacat ctccagcttc tagtggtctc agacttgttc accgagcgca
171300accagtttga gctgatgtat gtgacgctga cagaactgcg aagggtgcac
ccttcagaag 171360acgagatcct cgctcagtac ctggtgcctg ccacctgcaa
ggcagctgcc gtccttggga 171420tggtaagtga caggtggcac agaggtttct
gtgctgaagc cacgggggcc catctgcctt 171480gggacctggt gttggccaga
ggtgccgggt gcggctgcct ccttccaaga gttgacccga 171540accggactcc
acggcccacg tgagctgcag tgcttctcag atggaggggg ttcagcgacg
171600gtcagtgcca ttcacaggtc actgtgatgt gggttgtggc ggccaagcca
tggtttgggg 171660tcccgtatcc ctgggcttat gacatcattg tagtagccca
tccccacaga accacggtgt 171720gtggtggcgc tgaggcatcg tagatggtgg
aaatgctact ggcttcccca tgctctgccc 171780tgaggcctga ctgcctcact
ccccttctca gttatgttcc aggccccccg agcttcctgg 171840ctggacagct
tctctcctgg gggccgtttt gtcacagtga ccctgtgttt ctagtcccaa
171900atctgggtgc tatagtctct ttttagcgtg gtggttgtct tagtcttttt
tggctgctac 171960cacaagttac cttagactgg gtaatttata aacagtggaa
atttacttct caccgttctg 172020ggggctggaa gttttcatgg tcaaggtgcc
agcagatttg gtgtgtgatg agggctgctc 172080tctgcttcat agatggcatc
ttctggctgg gtcctcacgg tggaaggagt gaacaagctc 172140cctcaggcct
tttagaaggg ccccaatcca caagggctct cccatcatga cctcatcacc
172200tcccaaggcc ccaccttctt gtactgtggc actgcaaatt aggtgtcagt
gtaggagttt 172260caggagggat agaaacattc agaccatccc agcggtcaag
tgttcatcct cttgagttcc 172320tccttattct gcttctggtt tatcaggatt
cagccagtgc agcatggtac ctgtattctg 172380tggcacatca ccacatggta
tttgccaagt atccatcacc tgcacacgtg aaatcattgc 172440ccgtgggtcc
cgacatctgg cgaagcatat tcaaggatgg cagaactgtc agagctggca
172500cctctggttc cttgtcatgt ggcattacct agtaatccat tttatgatag
caatggaaac 172560tcatttcttc aacaaacacc tgagtggctg ccgtgtgcca
gccgtctggg gcccttggtg 172620agaatggcat ggtggtgccc atcagggcct
gcctagcccg tgctctggac gggctcctgt 172680gtgtcaggaa cgacaatgct
gtcatgacgg tgaatgattt ttttttttgc catcactcca 172740gccgctaaca
tttgcggagc tcttcctccc gcacccccac ctgacaaggc caagggtgac
172800cttggcccca ccctaggcgg ccaaggtcag aggttagctg gcttgtctgg
gtcacacaaa 172860atgcagcaga ggttgaggtg agcacatgtc cgtgacctgg
agcctgactc cctctctgcg 172920agtcttgact gctcttgcct agactctgtc
ctccccgagc ccaaacgcca gtcatcttcc 172980cttgtgggtg tccttcagcc
tggtgccatg ctggtgactc agcagccgtc cagggagtgg 173040aaacaattga
gtgtgtgggt tccctgtgtg ggcatctctc ttcacggcga acaccctctg
173100ggtgttgccc acacgatgtc aaagcggctc ttggaagggg tccttctcct
ttgtgggaag 173160tttcagctgc tgggctaact tgaattgtaa ctgtggtttt
gtgctcaggc ccagatcccc 173220ctaggcaagt gttgtgccat cagtaatcaa
atgagaaata atcattttga aaagcagatc 173280ctaaggcagg atggtcatgg
acactcactc ccagctcttt gtgcactcat gctttctgga 173340agatggccat
cctctgtgaa ggttttcagc gcgtcatgct tggtacccac gtatccagag
173400catgtcgttt tgaggtattt gcccaccgtt gtgaaatccg tgccacccga
gagcaggtcc 173460tgatgtgggg ctttcagaag tgggacctgg ggccgtacgc
agtccttagg gaggggccgt 173520gtggcgttgt gcgtgtgagg ggatagcaca
gggtgaggtg ggggcccaag aaggaagtga 173580cccacaaaga acagcctcct
cttttggtcc ttgttcctgg gatggctggg agtggcttct 173640gtgtcgtccg
gccatttccc ctgcggagag gctcctacca ctgccgagaa cctcatcatt
173700ccacaaaaac aagaggccgc ctggccatcc agcgctccat gggaattctg
tgtccccata 173760gtcttgggct gaaggagggt gacattcctt gctgacttct
gcaggggtct cctcactgtt 173820aaagagcaga ttgaaagtga agaacgtggg
ctaagtgttt aggtcgatat ttaaccctgc 173880taggttttgg atactaagtg
aaattgaggc cattttggtt gaagttgaca gaaaccacta 173940tcagggatcc
ccaagactac cccaggcttt tctagaaaga ctctcagcta agatgtgtta
174000tggtaaaagc acacaaaaca aaatcagcaa agaaaattag caagggcaga
ggcccatggg 174060gcgatgtccc gaggacacca ggcttgagct tccagaatcc
tctcccagcg gggtcgtgca 174120ggacgcactt aactccccgc acagtgagcc
gtgacagcgc gtgtgcagtg tcgtcgccag 174180gaaagcacac tagagactcg
gtgccagggt ttttactggg ggctgggcac atgggcaccc 174240tctgcctgcc
tcgtgcccag actctggact cccggaggga aggcaagttc tcagcaccaa
174300ccctggtgcc cacacaagca gctgagcaca gggagcccct cctcagtgag
gatggtgggc 174360accgtcccaa caccagccag gggccagcct tgcacacagg
cctctcagga tggtctccgg 174420cctgctgtgt agtctcttct gcacacaagc
gtgagggcag cgcccccgcc tcggctgtgg 174480ggaggagcca ctgggacgtg
agctctggtg gcatgcagca gcttttgtct gtgtgtgcct 174540aggacaaggc
cgtggcggag cctgtcagcc gcctgctgga gagcacgctc aggagcagcc
174600acctgcccag cagggttgga gccctgcacg gcgtcctcta tgtgctggag
tgcgacctgc 174660tggacgacac tgccaagcag ctcatcccgg tcatcagcga
ctatctcctc tccaacctga 174720aagggatcgc ccagtgagtg ggagcctggc
tggggctggg gcgggggtct cagaatgagc 174780tgtgaaggaa gcagcatcac
cctctccaag tgcccaggct cctggccaga tggcaggcca 174840ggtatcagtg
ggaacccagg tgggtgccat ggctgaggtc agtgagacgc aagagcacag
174900gtgcgtccta gaggcttcct cgggcacctc cagcgagctg gagctctcgc
ctctgctgct 174960gtctcatgtg gcgcttagca cactctccca cgtgcccatt
cctgactctg ctctcgaggc 175020catcggctct cattctctgc tcccagaacc
ctgttattac ccaggctagc ctcctctctg 175080caccttcccc gccctggccc
agtacctccc tcttgtttcc actgtgattc cgacctcacc 175140ttatcttaaa
gctgctggac ggcaggttct gtacacacgt gtccttgaca aagcacggct
175200ggtgccgcaa cccctcagcg agcaagtcaa gctcttcaca gcgatgtctt
acaagcgcag 175260agggctctgt gacaccctgg tctcaccgcc actcttccaa
agtcgcagag gctttagcag 175320agatgggccc agcctctctg agtcataggc
ttctgcacac gggagctgtc tttagaggga 175380gggtggaatt tcatcagcca
cccacatggg ggagttgagg gcaagaatta ggagcaaaga 175440tgggaagggg
tctgggagga atggccagtg atcccctttg acaagtgggc aggaaacggg
175500ggctaggtca aagttgagtg gaagacctgg agggagacgg gaaggtctct
gtaggcacag 175560ttcagacagg agggaggtgt gagccagggc acatgccggt
ggccgtctgg caggatttgg 175620gacatgctgg agcagggaca gcggctcatc
aggggccatt gccctcatcc aggccagagt 175680gtcacaagcc cgtggggagg
cccttctcgc ctgtcatcct tgctgggcag tgggtgctgt 175740gctagcagga
caggcggacg gctggcaact gtctctgcat ccctggagcc tggcataggg
175800ccaagtcaca cggggcacag gcctgcaaat caggcacata tgttggtgca
gtgacgtgat 175860tttggggggc agccccagaa caggccccag acacaggcca
aagccctgcc tgtgctggtg 175920tgttgggctg ttctatggct cttgctgtgg
gcatggagga ctcagggaag gagagttgag 175980gtggtccagg agttgcgttt
gggatgcaga gagcttgtgg catccaggta gaaatggtgc 176040gtggggctga
cctcagcacc atgggcagag gggccgtgtc acgtgcctcc gaggtggagg
176100tgggaccacg tggtgacaga tatacgcatc actgggcacg tttttgtggg
tgttgggggg 176160catcgtattg gctcctctgt tcacagtggc cactcattca
gtccctggct accaggtcct 176220cactgtgcca tggggaaggc cggcgctgtc
gggggatcac agaaggcagc acgtcatgat 176280ggcatgtgcc atgaaggaaa
agcacagggc actcaggaag tagaggggac tggcctgggg 176340tgtgggaatc
tagggcctcg ttgagggaca gagagaggaa gtgtgtggtg gccagcatgg
176400aggtggccac aggggaggct gagttaggcc gagagggcag ggcgttgggg
aggtagacgg 176460gctcagccac tcagggagtg gtcaagcaga ggctgaaggg
tcaggccagg ttgcaggggc 176520ctgggggagc cactcagggt aggcgctccc
gggagcccgc ctggcccata gctctacact 176580cccgcgtggg gccggacatg
ctgtgaagcc ctctccacgt tggatggggg tggctgagcc 176640tggatgctgt
ctcccgtttt cagctgcgtg aacattcaca gccagcagca cgtactggtc
176700atgtgtgcca ctgcgtttta cctcattgag aactatcctc tggacgtagg
gccggaattt 176760tcagcatcaa taatacaggt gagtgggccc tggctgtctt
cctctgcaca cggggagtgg 176820gcttcccttc tcttttcctt gcaggatcat
accagtgggc cagttttgac ttggtcggga 176880ggaggcatga acacctgaga
ctgtgcagcg
attctttgac acagaggcct ttctccctgt 176940gcagatgtgt ggggtgatgc
tgtctggaag tgaggagtcc accccctcca tcatttacca 177000ctgtgccctc
agaggcctgg agcgcctcct gctctctgag cagctctccc gcctggatgc
177060agaatcgctg gtcaagctga gtgtggacag agtgaacgtg cacagcccgc
accgggccat 177120ggcggctctg ggcctgatgc tcacctgcat gtacacaggt
gagcatgtac acggtgccca 177180taaggccagc ccaagtcctg ttcaagggag
gcaggagcat gctcactcaa gggacctcga 177240ctaggtgccc tctgatttca
cacttctggt gttgccccaa gccggcccca tcaccttgca 177300agaaaggctc
tggagccccc agggctggag tacctggtca gggttgaccg tccctgtggt
177360cactcatccc atgtggctga gctgggctgg gtcctgggca agcaaggggc
tgatatcacc 177420tgctttcaga tctccaggga ctcactggac ccctgtgtac
aaagcactgt ctacagagcc 177480tattgggttg tatagaggta accttcgtac
tgaacacttt tgttacagga aaggagaaag 177540tcagtccggg tagaacttca
gaccctaatc ctgcagcccc cgacagcgag tcagtgattg 177600ttgctatgga
gcgggtatct gttctttttg ataggtaaga agcgaagccc catccctcag
177660ccgttagctt ccctagaact ttggcctgaa gctgtgcttt tgtgtgtgtc
tgctgatccc 177720ctggcgctgt tgctggagtc ctgccagtga ttccccacca
cagcctgacc atgggctgcc 177780ttggctcagg gttccactgg cgagctggtg
gtccttggac cccagcactc aggtgtagcg 177840ttgaccagtt ccaaggttgt
cccagtgcct gcccatctct cctgagggct cagggacagt 177900acctggcagt
tgggggtgtg gcagggggca ggaatgacca gcctctggga gggtggggca
177960gaagcctgta cagtgaggag gagctggctc agcctggctg cctatcgtga
gaggggagcc 178020cacggggctg tgggaggggg gccgtggtgc ctgtgagcag
ggtgaggagc agcggcagga 178080ggatgaaggt ggaacccaca catgcatctt
tgagacccgt gtggtcagtg gcttctgccc 178140cccaccaccc cccactgctg
tgcgtgcata gaattggctt ccctcacctg ctctggaagt 178200gggttaggag
cttggtaggg ctttttctca aggacaaggg cccctgattt gctctcaggc
178260ctcagtcctg gcgacatggt ggatctggag ccttgttgca ctgccttgcc
tgtgctctcc 178320aatcagggtg gccagtgggg agccatttgg cttttctcaa
gagcatactc aggtggacct 178380tgctccactg tttgaccaga tgaggcattc
tgaacagcca agcctgtgct ggtctgtttt 178440catgttgatt tttttttttc
ttttcttttt gagatggagt ttttcccttg tcacccaggc 178500tggagtgcaa
tggtgtgatc tcggctcact gcaacctccg cctcccgggt tcaagtgatt
178560ctcctgcctc agcctcccta gtagctggga ttacaggcac acaccaccat
gcccagctaa 178620tttttgtgtt tttagtagag acggggtttc accgtgttgg
ctgggctggt ctcgaactcc 178680tgaactcaag tgatccaccc tccttggcct
cccaaagtgc tgggattgca ggcgtgagcc 178740actgcgcccg gcccccatgt
cgatttttaa atgcacctct gcatcgttct tcagtcccca 178800tatgctcact
gagcaccact gcgactggca gacgggcaca gggaggcgcc acgaccagtc
178860ctggccttca aggggcttgt ggtctagtgg gcccaatgct aggtggcgag
tgctccaaag 178920agtgtggtgc acgccttccg cttgaccgct ctccagacgc
cacagggagg cacctcgcag 178980ctgaccacag atttctctct gtggagcagt
gtcttcagag cggctgccat gccactgctg 179040ggcgagggtc tgcgggcggg
tagagccagg agcacctgtg aggaagtgca ctgccatttt 179100cgtagctgct
tcccgtgtgt ctcagttaca cacggctggc atgtgtgcac tgatgagacg
179160ggaacgtgat ggttgctttt cagcactgaa agggatactg ctcagggggc
gtgtttcagg 179220atctggttag ggaagaagca gcgagagcac agatggggcc
ctgtgtggta acaagaaaaa 179280agtcctggtt gacaacagtg ccacgaagcg
ttagaacaca tagggatgtt tgtggagcat 179340ttgcatgtgg aaagcagcaa
aaacataatg ggaacgggtt cttttgttat gatttttaaa 179400aatctctttt
gtaacatcct tcccgctgcg ccgtttctgc atattccttt atgtagcttt
179460caaactcctc ttaggagttc tggtccctac agggcgtggg agcccaggct
ttacgtagct 179520ttcaaactcc tcttaggagt tctggtccct acagggtgtg
ggagcccagg gcctgtgccg 179580agcagcctgc ctccacgagc tagacagagg
aagggctggg gttttgcctt tttagtctca 179640aaattcgtac tccagttgct
taggctctga ctttccccac ttggaaagtc cctcacggcc 179700gagggtccct
cccagccctg atttcacatc ggcattttcc ccagtattag agccaaggcc
179760ctccgcgggc aggtggggca gctgtgggag ctggtgccag tctctgacct
gcgtccctcc 179820tcccaggatc aggaaaggct ttccttgtga agccagagtg
gtggccagga tcctgcccca 179880gtttctagac gacttcttcc caccccagga
catcatgaac aaagtcatcg gagagtttct 179940gtccaaccag cagccatacc
cccagttcat ggccaccgtg gtgtataagg tgaggttgca 180000tgtgggatgg
ggatggagtg ggaaagcctg gaggtggagt tgcctccgac ttcccagcag
180060attcgccagc agagcccagc tcctccgctt taaagcagca atgcctctgg
cccccacccc 180120acccccgcca cccaggcgca gcaggtgctt cccgtccccc
cagccctgac actcaggcac 180180ctgcttgctc cttgcaggtg tttcagactc
tgcacagcac cgggcagtcg tccatggtcc 180240gggactgggt catgctgtcc
ctctccaact tcacgcagag ggccccggtc gccatggcca 180300cgtggagcct
ctcctgcttc tttgtcagcg cgtccaccag cccgtgggtc gcggcgatgt
180360atcctctctg ggtccctggt gctggccccg tttcccttgt caacaccgag
gctcatgttt 180420catgataagg ttttgaaacc taacctttgc aaaaacccca
cagatgccag ggtgacaggc 180480cctcagcccc agggaagtaa aatgctgaca
ggggtacaga aaggagcacg tccagacatt 180540tgctgaccag ggcctctcag
aggggccggt gtatggcagg agggtcgcag ctgaggggcc 180600tttctgtgga
gggcctgggt gaggggagcg agggtgggcg gtggtctctg cagacgtccc
180660gcccactcgc gggctctgtg tggctgggct tctcctgaca ctgcttctca
ttagctttgg 180720tcattgtgcc tcgatcgccc tctcggggaa aggcttaagt
aaagatccag ttcccacccc 180780cagatgctgg ctgccaggag tttccctttc
cacagccctt ccccaagaca gaccacaaga 180840gcctccaagc agcacagttg
tcctggtgct gacagcacag ccttgcccgg cgtgcctggc 180900acggctctgc
cctcactgca ttggagcagg gctagtggag gccagcggaa gcaccggcca
180960ccagcgctgc acaggagcca ggccaggtga gtgctgccga gtgggtgccc
tgcctgcagg 181020gcatccagcc agccaagggt tgcaggaatg gaggtggagg
cgctgatgca gctggaggca 181080tccaggtggc ccttccgggg ctctgctcgc
tctccaggct ccctggaccc ctttgtagac 181140tgtttcagga gaggaactcc
caggtgagga cagggaggca gcattcccct catttgccgg 181200cctttttcct
taactcctgc accagcctcc cacatgtcat cagcaggatg ggcaagctgg
181260agcaggtgga cgtgaacctt ttctgcctgg tcgccacaga cttctacaga
caccagatag 181320aggaggagct cgaccgcagg gccttccagt ctgtgcttga
ggtggttgca gccccaggaa 181380gcccatatca ccggctgctg acttgtttac
gaaatgtcca caaggtcacc acctgctgag 181440cgccatggtg ggagagactg
tgaggcggca gctggggccg gagcctttgg aagtctgcgc 181500ccttgtgccc
tgcctccacc gagccagctt ggtccctatg ggcttccgca catgccgcgg
181560gcggccaggc aacgtgcgtg tctctgccat gtggcagaag tgctctttgt
ggcagtggcc 181620aggcagggag tgtctgcagt cctggtgggg ctgagcctga
ggccttccag aaagcaggag 181680cagctgtgct gcaccccatg tgggtgacca
ggtcctttct cctgatagtc acctgctggt 181740tgttgccagg ttgcagctgc
tcttgcatct gggccagaag tcctccctcc tgcaggctgg 181800ctgttggccc
ctctgctgtc ctgcagtaga aggtgccgtg agcaggcttt gggaacactg
181860gcctgggtct ccctggtggg gtgtgcatgc cacgccccgt gtctggatgc
acagatgcca 181920tggcctgtgc tgggccagtg gctgggggtg ctagacaccc
ggcaccattc tcccttctct 181980cttttcttct caggatttaa aatttaatta
tatcagtaaa gagattaatt ttaacgtaac 182040tctttctatg cccgtgtaaa
gtatgtgaat cgcaaggcct gtgctgcatg cgacagcgtc 182100cggggtggtg
gacagggccc ccggccacgc tccctctcct gtagccactg gcatagccct
182160cctgagcacc cgctgacatt tccgttgtac atgttcctgt ttatgcattc
acaaggtgac 182220tgggatgtag agaggcgtta gtgggcaggt ggccacagca
ggactgagga caggccccca 182280ttatcctagg ggtgcgctca cctgcagccc
ctcctcctcg ggcacagacg actgtcgttc 182340tccacccacc agtcagggac
agcagcctcc ctgtcactca gctgagaagg ccagccctcc 182400ctggctgtga
gcagcctcca ctgtgtccag agacatgggc ctcccactcc tgttccttgc
182460tagccctggg gtggcgtctg cctaggagct ggctggcagg tgttgggacc
tgctgctcca 182520tggatgcatg ccctaagagt gtcactgagc tgtgttttgt
ctgagcctct ctcggtcaac 182580agcaaagctt ggtgtcttgg cactgttagt
gacagagccc agcatccctt ctgcccccgt 182640tccagctgac atcttgcacg
gtgacccctt ttagtcagga gagtgcagat ctgtgctcat 182700cggagactgc
cccacggccc tgtcagagcc gccactccta tccccaggcc aggtccctgg
182760accagcctcc tgtttgcagg cccagaggag ccaagtcatt aaaatggaag
tggattctgg 182820atggccgggc tgctgctgat gtaggagctg gatttgggag
ctctgcttgc cgactggctg 182880tgagacgagg caggggctct gcttcctcag
ccctagaggc gagccaggca aggttggcga 182940ctgtcatgtg gcttggtttg
gtcatgcccg tcgatgtttt gggtattgaa tgtggtaagt 183000ggaggaaatg
ttggaactct gtgcaggtgc tgccttgaga cccccaagct tccacctgtc
183060cctctcctat gtggcagctg gggagcagct gagatgtgga cttgtatgct
gcccacatac 183120gtgaggggga gctgaaaggg agcccctcct ctgagcagcc
tctgccaggc ctgtatgagg 183180cttttcccac cagctcccaa cagaggcctc
ccccagccag gaccacctcg tcctcgtggc 183240ggggcagcag gagcggtaga
aaggggtccg atgtttgagg aggcccttaa gggaagctac 183300tgaattataa
cacgtaagaa aatcaccatt ccgtattggt tgggggctcc tgtttctcat
183360cctagctttt tcctggaaag cccgctagaa ggtttgggaa cgaggggaaa
gttctcagaa 183420ctgttggctg ctccccaccc gcctcccgcc tcccccgcag
gttatgtcag cagctctgag 183480acagcagtat cacaggccag atgttgttcc
tggctagatg tttacatttg taagaaataa 183540cactgtgaat gtaaaacaga
gccattccct tggaatgcat atcgctgggc tcaacataga 183600gtttgtcttc
ctcttgttta cgacgtgatc taaaccagtc cttagcaagg ggctcagaac
183660accccgctct ggcagtaggt gtcccccacc cccaaagacc tgcctgtgtg
ctccggagat 183720gaatatgagc tcattagtaa aaatgacttc acccacgcat
atacataaag tatccatgca 183780tgtgcatata gacacatcta taattttaca
cacacacctc tcaagacgga gatgcatggc 183840ctctaagagt gcccgtgtcg
gttcttcctg gaagttgact ttccttagac ccgccaggtc 183900aagttagccg
cgtgacggac atccaggcgt gggacgtggt cagggcaggg ctcattcatt
183960gcccactagg atcccactgg cgaagatggt ctccatatca gctctctgca
gaagggagga 184020agactttatc atgttcctaa aaatctgtgg caagcaccca
tcgtattatc caaattttgt 184080tgcaaatgtg attaatttgg ttgtcaagtt
ttgggggtgg gctgtgggga gattgctttt 184140gttttcctgc tggtaatatc
gggaaagatt ttaatgaaac cagggtagaa ttgtttggca 184200atgcactgaa
gcgtgtttct ttcccaaaat gtgcctccct tccgctgcgg gcccagctga
184260gtctatgtag gtgatgtttc cagctgccaa gtgctctttg ttactgtcca
ccctcatttc 184320tgccagcgca tgtgtccttt caaggggaaa atgtgaagct
gaaccccctc cagacaccca 184380gaatgtagca tctgagaagg ccctgtgccc
taaaggacac ccctcgcccc catcttcatg 184440gagggggtca tttcagagcc
ctcggagcca atgaacagct cctcctcttg gagctgagat 184500gagccccacg
tggagctcgg gacggatagt agacagcaat aactcggtgt gtggccgcct
184560ggcaggtgga acttcctccc gttgcggggt ggagtgaggt tagttctgtg
tgtctggtgg 184620gtggagtcag gcttctcttg ctacctgtga gcatccttcc
cagcagacat cctcatcggg 184680ctttgtccct cccccgcttc ctccctctgc
ggggaggacc cgggaccaca gctgctggcc 184740agggtagact tggagctgtc
ctccagaggg gtcacgtgta ggagtgagaa gaaggaagat 184800cttgagagct
gctgagggac cttggagagc tcaggatggc tcagacgagg acactcgctt
184860gccgggcctg ggcctcctgg gaaggaggga gctgctcaga atgccgcatg
acaactgaag 184920gcaacctgga aggttcaggg gccgctcttc ccccatgtgc
ctgtcacgct ctggtgcagt 184980caaaggaacg ccttcccctc agttgtttct
aagagcagag tctcccgctg caatctgggt 185040ggtaactgcc agccttggag
gatcgtggcc aacgtggacc tgcctacgga gggtgggctc 185100tgacccaagt
ggggcctcct tgtccaggtc tcactgcttt gcaccgtggt cagagggact
185160gtcagctgag cttgagctcc cctggagcca gcagggctgt gatgggcgag
tcccggagcc 185220ccacccagac ctgaatgctt ctgagagcaa agggaaggac
tgacgagaga tgtatattta 185280attttttaac tgctgcaaac attgtacatc
caaattaaag gaaaaaaatg gaaaccatca 185340gttgttgctg tgtgaggctt
gctttgcttc atgagaacct agaccttgct gagctggagt 185400cttaggaagc
agtctcctaa gtgcttctcc agcaggggca gaaactgtcc caccagctaa
185460catctggcat tatggagggt cccccaggca gctgccagca gggacaggcc
ccgtgttttc 185520tgtagccagg gatgaggaag tggccccagg gcatgggcct
ggctgggtgc ttctgcaagg 185580gccttcccaa accacagtac aggtggtctt
cctgccctgc agatgggagc tgtgggagct 185640gctggagctg ctggagcctt
catggtcaag tgacatcata agcttatatg acatacacaa 185700gcctcaggac
ttggcccatg gcactgaagc aggtcatcag gcccagcaca gagactagag
185760ctgtgttctc acagggccca ccacccttcc acctccttgg ccattgacac
ctgcgtccct 185820ggcccagctg ctcccaggta acccccaaag cagctggcac
atcccacctc tggtgtggcc 185880ggggctgctg tgtgtccgca gggcctgccc
cgtctattct agcttgtttg tcctgtctga 185940accagcgcct actccaagaa
gcctctgctc agcccagcgg ggatgcttct aagctccgga 186000cgagcctctc
ggaagccttg gtgattggtg gtgtagtcat cttgggatgc agatgtctta
186060ccaacctgca agaacaaaaa ccctgtggct tcctctggtg cagggtattt
agtcaatgtt 186120tgctgaggtc ccgtctggtt ctggctaatt ggcaggggtc
gtccacccat tctttccctg 186180ctctgctgtc tgtgccagga gagacggggg
ccagtcggcc aaggggccag ctcctgctgc 186240ctgctcctct tgggcacgtg
cgggggcccc ctttctctga gcagggatag ggatcagtct 186300gccggaggga
tgtggtggac aggcctaaag catttggggc ggggcatgcc acttgagctc
186360cctaaatctg tctcctcata ggtgacaccg ctccagggcc ccccagtggc
ctctcctttc 186420agagctacct aaattctggt cacttcagag aaatggagca
cccccttctc cctggtccag 186480gtgtggacag cctggcacac tgagcacacc
tggcatggct ggtaatttca gaaagaagag 186540gggccggggt ccagtgggaa
gcagcggtga acccctcgtg agtgggcttt gcagtccctc 186600cccatgccac
ggcagagctg ccctcaacac agccttcctc ttcctcatcg gagagcacac
186660cctgtcccct tgccgagctg tgccctgtgc cttcggtggt atttgatttt
ggctgctact 186720ggctttgttg ggatctggaa gtcgcttccc ctgcgtggtg
cgtggagcac tgtaagtcag 186780atgagggaag tagccagggt gaggtgagta
ccgggtggag ccgccactga agggactggg 186840taggggggcc ttgcctctac
atgatgtgac acagccaacc gaggacagag gaagccccgt 186900tcctgggggt
gtggggtgca cccctcaggg aagcctgcag tggggcctga ggaaaggcat
186960cctccgcgag cccacgagtc tggtccatga gcaccgtgac agtgtctgtg
ggtagaggtg 187020gacccggcct tgtgtcatca ccaggacctc ttttgggaaa
ccatgtggac atcgcttgcg 187080ggtcccccag gctctgcagc cccagcagcc
tggctgcctt ttgggcaagt ggcttgagcc 187140acagaggacc cagtcctgtt
gcagccacat cctctggggg ggcccgccag tgtggccggc 187200tttctccacc
ctacaccagg cctccaggtg tcctggtcgg gggtgtctgg gccctgggtg
187260ggccctgtgg acctgtgagg tcagggtcag ggcatcactg gaggcagagg
gctgaagttg 187320tgggtctggg ttccccttgt gtgcacaggc ccctgccctc
catgcttggt caggcagcta 187380cccccaaaac tgctaggaca ggctggtcct
gaggtggatc ctggcccctg taccctctgg 187440acagcccacc cgcccaacct
tctaccctgc cccagcggcg gcagtgttgg ccacatcctt 187500cccctcctgg
ccccaattgc tctggggaag tccaggctcc ggagcctgcc caggggcccc
187560ccgtgatttg ggcccaggac tccacgtggt tctctgcctt cacccaagcc
ctgaactcct 187620cagctgccaa atccccaccc atctgcacag gctgtgctca
ccactgctgc tcctggaagg 187680tgcccctcag tgggacgccc acctcctctc
tgggcttctg tgtttgggag ccctgctgcc 187740cccacccttg gtcagtcccc
atgtcctgct ggcctgtcag gcagggcaga aaatccaccc 187800agaaatgctg
agcaggatga gagtctagtt gggcccagcc tcattattta gaagggatgg
187860aggcctaggg agcatgcttc tagcctgagc ccagcagggc cccgcccatg
tcccaggtct 187920gcaccaggga cagctcctgc cgaggcctga cctgcccctt
ctccctcagg tgctgctggt 187980tgaccagcct ctggccctag gagaccccgt
agcgactgag ggtcccagca ggccatgcag 188040ctttgccaag gtacgagccc
ctccccagca ggggacagat gtggggaccc tcccaggcag 188100gagcagctgg
gtgcctggtg ctgccatctg ctgcctgcct ggttcttgtc ctcacattgg
188160aggtcagtgt gagggctctg cctcgggaaa ggccatggag cttgccctgt
ccagggcctc 188220ccatgtgcac tgagcctggg aagagagggt tggagttgag
ccttttaccc tgggaatgct 188280gcctggagga tggtgcgggt gtggggtggc
accctgccag gcagggccct gcctccctgc 188340gcccactgga actcgggcag
gcaggggtgt aggtgcctcc tctagagccg tccggtgggg 188400gcccccggca
gtggtggtgg tgtccactgg ccagcagctg ccccttcagc caggacagta
188460ggcctgacgc tgtccccagc agctccaagg tggatttgtg gaagggggta
gagggcacgt 188520agaggcccca tgacctcccc agggttctgg gagggctgtg
cccccttagc cagcaccatg 188580ctgggtgata tagtcagatc ctgttacccc
tgttgtggag gtgaggaaac aggttagtgg 188640ggaggacatg actaaggtcc
atgctgagtc gctagagctg cacccagaac cactgctggg 188700accccatgcc
tttctgctta ccccttgtgc cgggagatgc caagagatgc tgggagccag
188760ccccacctct gcccttggag tcatggctac ggaaagggca ttcggaccgg
tccctgacct 188820caccggggag ggccgaaccc tgttcctgag gagccagggc
ttcctagagg aggtaggcct 188880tctagtcact ccttcatctg caggcactcc
acagagctct ctgtgccagc ccccagcacg 188940gagggctgac cttagtcgag
tggagatgcc ccagtgccag gcagtaggga tgatgtctcc 189000tgaggcccag
atggaaggga ctggactagt ctcatggggc tgatggtggg gccaggcctt
189060gaccagggac ccagtgtagg gggtgcagag acccctctga gttcctcaca
catccctggg 189120gccctcccca tacacttcct atcctgactg cgggcaagag
ggagccccag ttcgccttcc 189180ctatgctggg cacccacagt ggggctgggc
acccccgcca tgcccctgcc ctgtccttcc 189240cctgagagcc tcggtcccac
ctccaaggtg cctcagagga cagcaggggc agcgggcaga 189300ggccgagatg
cctcctcatt ccaggctcag ctgcccttct tggggcagcc cacacctgag
189360agtctcctgc agttggtcag gcctgaggag ggcagggggg tgcctgctgt
ccctctgctg 189420accacagtgg catttagcct gggcaccgcg cccagcacag
tccatgctgc acaggtgccg 189480tgggctccac agagccctgc ctgacatgca
tgtgttacgt ttcgggtgcc gatgcccttg 189540ggcggcactt ctccgggcag
aacccccagg ccaccgctcc ggttccggtt ccgctgcatc 189600tggggctctc
ggcaggctgt ggtcctccgg ccagcctggg ggcatctcag tccctcagcc
189660ccacaggggc ctgccccgca gcctgggcct cgagccccgt ctccgcacgc
tgtgccgaat 189720ctggctgccc atcagctccc tgcgtaccca gactgtgccc
tgccatgccc gtggctcttc 189780ccaggagtgc cctgtggcct ccccctggct
tgctgggctg attccctcct gtgtctcaaa 189840cagagctcac ctttgccatc
actgctgtcc tcaccggccg gtgccagagg cccgtgtctg 189900tgtaccctgt
gtctgcacct ctgggcaggg cctggctctg accaacccgg gcttccagtg
189960tccacagacc taaggcccag ggcgcctggg ggctggagca agagaagcaa
aaggagccaa 190020gggtgggggt ttggggttct tgtgagggcc cagccccagg
accccaggac caggacaccc 190080aggagcccca gggcccagcc ccagttcaga
aggcaggggc cttctgaggg agcttaaggg 190140tcccacagcc caggaccccc
accagggcca gtggccagcg ttgggggact cagcctcctc 190200gtcgctcgtc
ctctctgttt ctcccacctt ttgccccctt tctccttgcc tgttcccacc
190260cgaggccccc tcttggcctg cgtgagccgg ggcggcactg aactgggggc
cgatccgcct 190320gggcggcggt gagaggcagg gccgggagcc gggccgctgg
gtttgggcct ggcccgctcg 190380ccgcaatatt gatggcccgt cagtgcagcc
ctgattcctg tgctttcagt taaaaggttt 190440ctgttgttgt agcttatgca
gttgctctgt tgctatggaa acgtgacatc aaaatgacgt 190500ttcccgttta
aaagctttta actaaattcc tgcctgtcag atgtaggccc cattttgagc
190560gtggagctgc cttcgagcga gcgtgagcgg cgcctcccgc ccatggtgcg
tggggccggg 190620ccggggccct cgctgagcgc gctctctcac cccacaggcg
cctccggcat ggcggcggcc 190680gaggggcccg gctacctcgt gtctccccag
gcggagaagc accggcgggc ccgcaactgg 190740acggacgccg agatgcgcgg
cctcatgctg gtctgggagg agttcttcga cgagctcaag 190800cagaccaagc
gcaacgccaa ggtgtacgag aagatggcca gcaagctctt cgagatgacc
190860ggcgagcgca ggctgggcga ggagatcaag atcaagatca ccaacatgac
cttccagtac 190920aggtgggcga gcgggcagtg tgggccccac caggacgggc
gggcccgggc gtggcgggcc 190980gctcctgact ttcttggagc tctgagtcgg
gacgatgtgt gggtcgtggc ctgcctgtcg 191040gtctcctctg gccgggtatg
ggcagaaccc cacggggtga gacggggccc acggaaaccg 191100tgtgtgcagc
cttccattgg ggaagtgggg aaactgaggc ccagcaaggg caggaaacca
191160gtctaagagc tgaggggtag caggggtggg gctggtgctg ggcagaggcc
aggatggctc 191220ccaggacgta tgggcggtct gggcactgtc cctcggaggc
agcaacactc atggtggtgc 191280ccactgacct cacaccctgc tcccccatag
ggaggcggcg gctgccagtg ccctccccac 191340caccaagctc ccaagctcag
caggggtttc aggggcctac tgcgtcattg gggaaattga 191400gactgcaagt
gagaaggagg ctcagtgctc tgcgacttgg agcatccact gagcctctgc
191460catgagccgg tgagccccac tggggctggc cctagggtca cggtggggta
tttccagaaa 191520tcaccaggtg aggtgcagga ccagccagcg catgggtggg
gcttacggtg cgaagaagaa 191580agaggtggag gcctgccctg gcccaggact
cccagcgtgg gggctcccgg cctggcccca 191640cctctgctcc tgctacatgg
caggtgggcc cttcctgccc tggcaacctg cagggaaggc 191700cggaggggac
cacccagcca gggagatgtt ggcgtctagg aggggacagg tgtggtccca
191760cacacccagc atcttaaagt gcgtgggtcc ccagcccatt aggacagggt
cccgggtggg 191820caggggtcat ggtggggtga aggtctcagg cacaggcaag
gtcacaggtg cggtgagggt 191880cttgcagggt gtgaaggtca taggtgtgcg
gtgaaggtca caggtgtggg gtgatggttt 191940tgggtgtggg gagggtcttg
cacggagcga
gggtggcagc aagagctgga agctgcaggg 192000ggagaatggc agcagagagc
acccggccct gtgggcggcc tggacagggc tgggcctggg 192060gctgccggag
agcctgtcag cttccaggat gggagtggcc tcactcagct gctccacctc
192120cgggtcaggc aggtgagcct ggggcagaga ggctgagagc acctgagcca
cttgtgggag 192180aggccacccc cactgccccc ctcaggcgag gagccggcct
ccagcacagc agaagggaac 192240ccccagtccc cagccctagt gggagtgggg
aagaggccca gcaaggcccc ggacagaccg 192300ccagcctgtg aggtctccgc
tttcagttgc gttgatttga ttttttctga gccttgaagg 192360aggggtccgg
ggcctggccc tgcccaaagg cccctaggca ggccccaaag ccgggaccta
192420gggtgctgag catgacggat gttgggtttg agcggctggc ttgcgacgtg
agggctgagg 192480tgtgagcctg ggtatcttca gaggttcggt ggacacaggc
agctgcccgc ggccccactg 192540ttcccgtggc ctcctagtcc tgctcaggca
cctggtgagg aagggacgca gagggcagtg 192600ggaggtggcc acgactgttc
cagcaggctc ccctctgact caggaattca cgggcaccac 192660ctccctggct
ggctctggtt ggtgtctggc caggttattc attatttatg ctgaaagcct
192720cttcagagtc ccaggggagg gtttctgtct ccattcctgg aggctgagag
atgagggtgc 192780agcagagtgg gggcctccac tccagaccct gcagtctggg
ctggccaagg gctgcaccgg 192840tgcactgcac gtcatggctg atgaagcact
tccacaccgc agcccctcag agctgccaca 192900gtcagcctta gttcaccgag
ggggaagctg aggcccagag catgagaggg acttgcccag 192960ggccacatag
tccttagcag aggaagctgt ggctgggtga ctcgatcttt gtcctttttc
193020tttatacccg cagtctcccc atagcagagg cttttctttt ttttttcttt
ttcttttttt 193080tttttttaca agaactcttt atatattaag gctgttgggc
tgaagaagcc tgagagggtg 193140gctggttctg tggagcatgg tttgttgaag
tacagtttgg gggcctccta cactgagaat 193200aggccttttc tcgtttctcc
aaagagtggg ctggctcaag tagggcagag agagaagcct 193260ggggcagagg
ttagggatgg gcacccagcg cctgccctca cacgctctgt gctggtgtct
193320tcacagccac gtgccaccct gggcagcatc ccctgctcac catctggctg
tgcctgtttg 193380ctgggggcac ctcattcaga atccagctta ttgtttccaa
cggccaatgg ccacaccctg 193440gcaggtagca agagtaggag agaggagaca
cccactccga gcacaggttg ggtttggagc 193500ccggccttgg ggcactctgt
cactcaaagg cagagtgggg agtgggcact gggccttagg 193560aggtactggg
tccagtgagg cagagatgcc cctgccccac ccccaccttg tggcttcttc
193620cctggcctgg ccagagctgt ctggccgcca tggggccctg tgtctcctgc
cttgacctcc 193680cagagggcag ccgaggccca ggggaggcct ggggacttag
cctctcaggg caggacctgt 193740ctgcaggagt aggtgggtgc tgggggtccc
agtggtaatg aggcatcagg cagtgtggga 193800aggggcccat ccggcccacc
ccagggcctc tgggcaggtt gcaggttgta gcgctggatc 193860taggctcctg
cccagactgt aggttcaacc aagaatggca tgggagccca gcctgctgtt
193920tgctttatta aatctgccct gtagctgggg gaggggctta ctttgatcat
cactatgtca 193980ttgatataaa aatagaggct cagagaggtg aatgaacctg
cccaaagtca cacagcaaag 194040tgtggagatg agatactgac tcagggctgt
ggacactgaa gcctgtgctc taacgccagt 194100ggctgtcgct ccctgaggca
ttctctcccg aacaacacag ttattatatt acaaaatatt 194160atcactatat
ttatatatct tataatacct tattattaca ataaaacctt attactctac
194220ctttcaaaat gaattattta aaaagcagta tttgctcatt gcagagagtc
tagaaactat 194280agaaaagcaa gggaaaagca ataggaccag ccccaaggtc
ccagcatgca cagataacct 194340tagtaatact gggacgtgtg cttccttttt
aacatctgag cccgtgtagg tcctgaagcc 194400cagcttcttt ctaagtccat
tgtcatcttg accctggagc ctggccgatt ttgctgggga 194460ggcccttgcc
agccgagagc ggctcctgcc tgtgccggcg tggcgcgccc ctctgctgag
194520gctgggcagg acaggggctg ggccagctct gtttctcacc cttggctctt
gtgtctctcg 194580tttcaggaaa ttaaaatgca tgacagatag cgagtccgcc
ccgcccgact ggccctatta 194640cctagccatt gatgggattc tggccaaggt
ccccgagtcc tgtgatggca aactgccgga 194700cagccagccg ccggggccct
ccacgtccca gaccgaggcg tccctgtcgc cgcccgctaa 194760gtccacccct
ctgtacttcc cgtataacca gtgctcctac gaaggccgct tcgaggatga
194820tcgctccgac agctcctcca gcttactgtc ccttaagttc aggtagtgtg
tctgcttgtc 194880cttcccctgc cctggggtat ctcagccccc accatttaga
gaaagggact gggagtggca 194940aggccggcgg cggcggccac agtggttgca
gaggccgtgg ctgcgggcag cgcctccagg 195000gacaggcggc ctcagaccag
ggagggcttt agtgtccaca ggcagaccga gtttgtctcc 195060cagctccatc
acttttgagc tgcacggaaa gttccttgac ttctctggcc tcagtctccc
195120tcctataaaa tgggggtaaa tcagtacctt tctcagaggg tggctgggag
catcacagga 195180gagaagacgc agcatggggc ccggcacacg gagggagacc
aagccccaga ccccagaatg 195240cgccccctgg cctcccttag cccacacaga
ccccaccctc acaggctagc tgccctctca 195300gcactgggga gggtgtcggg
ctgcacctca tcacgtgttg ccgtgggcat gacccgtccc 195360ctctgccatc
catcccacac ctcagacccg tcccgtgctg gccacgtgac tgtgcctgca
195420agatgctcac agggcagccg ggagccaggc agcatgcagg acagacacct
gcggggtggg 195480cctggggagc ccagagaagg tgcttttgag gaggggacat
ttggggtggg ctttcaaggt 195540aaaatagaag ttggccattt ggaggcaaga
acaggaagat tgtggatttg agtcacagct 195600tctcccctgc cctggtcttc
aagtctttct gacaggaggt gtcagaaaag tatctttagt 195660agagaaggcg
tctccgagga gggtccctct catgccgggg gccgctgctt gactcaggat
195720ttctcattga agacctgaga caaaaacgct tttgctggca gctagaagga
accagcagga 195780ggcctgagat ttgtggctgt tgttcccgtg gactgagccc
agttctcaga ctcagctgcc 195840tggggccttg cacaggactg gggcgtgggg
gctgccctcc ctgatcaggc ccaaagcgcg 195900gatctcacgc ccctgaggtt
ggctgtaccc tctcagctca gagcagagtg tgggccaggg 195960atgagcaggc
actggagcag ggccctgggg tctgtgggtt ttggcagctc cctgcccttc
196020agggaggtct gctgagacca cgggtggccc ctaccccagc agcagagctc
tcaggaggcg 196080cccacagggc tggactgcct ttactcacca cctctaccag
agctctgagg tcctggggag 196140agagcccagg cctcttgtgg gccccacacc
ctctaggtgc ctgtccttct gcctctctac 196200caaggtgtgc cggccccatt
tctaggccgc cgggagataa gggggctcac atctcaggcc 196260cttccttctg
ggacctcagt ttccccatct gcctaaggcc gggtggggct ggtggtcttg
196320gcttccctac aggggtcctg agtactctgc actacccagc accccccacc
cctgccttca 196380tctctccctg ggggtggtct ctccacccct ggcccccaac
tggggctgag cccccacctg 196440cccagtttgg tgggtgaagg gtgctccctg
gcaggatatg cccctctgca gcccagaaca 196500tcccaccctt tccagaccga
aggggtgtgg attgtcctgg gaccctggtc attggggtca 196560tccgctagtc
gcaaaggacg gcaatgcctg tggcctctct ttctttcttt ttcttttttt
196620ttttttttga gacggagtct cgctcttgtg cagagagcag tggcgcgatc
ttggctcact 196680gcaacctccg cctcgtgggt tcaagcgatt ctcctgcctc
agcctcccga gtagctggga 196740ttacaggcac ccgccacaac gcctggctaa
tttttgtatt tttagtagag atggggtttc 196800accatgttgg ccaggctggt
cttgaactcc tgacctcagg tgatccacct gcctctgcct 196860cccaaagtgc
tgggattaca ggcataagcc tccacacccg gccacccctg ttactttctg
196920tcaaaggcgg tgggttctgg cccctccttt gcacatggaa tatgagaccc
tgagtaagtg 196980acctgactcc ctggggcctc agtttcccca tttgcccagt
aggattgtcg ggagggtccg 197040gtgaggcccc tggtgtgccc aggctctgtg
gccagcacgt ccacagccgg cactgtcctt 197100ccaggtcgga ggagcggccg
gtgaagaagc gcaaggtgca gagctgccac ctgcagaaga 197160agcagctgcg
gctgctggag gccatggtgg aggagcagcg ccggctgagc cgcgccgtgg
197220aggagacctg ccgcgaggtg cgccgcgtgc tggaccagca gcacatcctg
caggtgcaga 197280gcctgcagct gcaggagcgc atgatgagtc tgctggagag
gatcatcacc aagtccagcg 197340tctaggccag caggcggcgg cggcggcggg
gccgggcggc tggtggtact gctcaggcca 197400cccagggcag gccactcagg
ccaggcgggc aagggggccg ccccgcgagc ggagaccgcc 197460ttccacctgg
cctctggcag gatgtccctt ctgaggggta ttttgaggaa cccccaggcc
197520ctggggaccg tgaggctcca gtctccagca tgaatgccct tcctcggaca
caggccaggg 197580cctctggggt tcactccgag taagaacgtc ctagagccac
tctccagtgt cgttactatc 197640aatgatactt gacgtggctt tgatattaaa
cgtatacttt ttcattcttg cctggaacgc 197700acagtttgct gttgctggct
tggtgaggat gccctgattg atggatcccg aaaatgaaag 197760cagatggaaa
cgggttgggg caggctggag ctgggggagc tctctcctga agggaaccct
197820gtgtcctccc tcaccaggac ctctgcgtct ctccttaaat ggcctctgac
gcctgatgaa 197880aaccccagcg accttccagg aggcttttat tcagctctgt
ttggagcatc aggtgtttcc 197940actgcctcct tagcaatgac actaataaaa
gtcgtaacac ctgttcacat gcacagccct 198000gttgagtgtt ctgggtgctg
gagatatcat ggtggatgac acaaaggccc tggcctcttg 198060gagcttatgc
tcccatgcgg ggaagacaca tgggtcagta gagaaatggt tgcaggttgt
198120gataagtgct ggaagggagg ggttggcctg aggacacgga ggcagacata
cgtggagctg 198180ggaacagtgg ccacacaggg aacggccagt gcgaaggccc
agaggcagag gacactggag 198240caagcccagg agcagctagg aggctggtgg
ccagcagcca ggccacggaa gcccgtgcag 198300cccgtgggga ggagtgttca
tgcttttcaa gcttagtggg agtcttttgg ccagtgcagc 198360tctgggtctg
acatcggtgg gggacagagg ggtggtggag cggccacagc tgcaagctca
198420cctcactgcc ggcccttcca ccagtttcaa actctttcta gaagctccag
ctttcccaaa 198480gctgaattct ctatgagcct ccttggccgg gactcgggcg
tctggttgcc ctggctgcaa 198540aggaggctgg ggccaggtgt gtttgagtca
cctcctggaa ttaggcaagt tgctgcccaa 198600atagaaggtt gttggcaggt
gggtcagcag gtgaacagca tggtttgact cagggttcag 198660aaaaatctcc
ctctggctgc caagcgagca ggccgtggag acaggtgcag aggcaggtgt
198720ggcagcaggc atcctgccag gcagtgctgc agtcatcctg cgacaagcag
cagcagctca 198780tcctaccctc tagggggtct tgaggtcagc caggcaagag
agcagcttgg actccactgg 198840gtgtgggacc agcctgtgga ccatggtggt
gtggagggtg ccctcggcct gcctgtgtga 198900aggagaggcc ggcgtgttct
gtggagccca aaggggagct gggcaagcag gattcacttc 198960actctgaggg
tcctggagct cccaccctcc tcagccatct ccccagagcc tgtgtgccga
199020ggactcggcc catgttgctg tgggatgaga ggcagagtgt cgtgagggtg
taaggagcgg 199080cggcagtggt gggaggaggg agcagcagcc agcgctacgg
tgccagtttc cagctgccag 199140atgacgccgc tgaccctgtg gttgagaaga
gatgcacaga gccagctctt gcaagccagt 199200gtggctgcca tagcacctgc
cgagaagcag aaggaagggt ggccccagga ggacagagga 199260tgcgggcaca
tctgatgcgg gcctgagttt tgggagcttt tgctctagcc agtttccagc
199320tccgggaccc acccgcctcg taggcaagac accacccaag aaatcatttg
cttaacaaac 199380acactgggct ccaactggac acctgtgcca ccctagatgc
tgggaaccca gccatgacac 199440aggcacctgc ccccagctgc tgaccactga
ggctggctag cagctcccat ggggccagtg 199500tggggttccc cagcctccta
acagggagcc agtcacaagc cctcgagagg gaagggtgcc 199560cgcggccctg
gcaggaaggt taggctggac gctcccacaa gacataacag atggaggttc
199620taaatgatgt agcaacttct tcaccctgaa actgctgtag agtcagccat
gacgcaccgg 199680tacttcagta actgccaggc atccgggaca gcacaccgcg
agtcgctgct gtgcttgggt 199740tagaagtggt ttggtctgtt ttcttctcgc
cctctctaat cagagtcagt gattcatgcc 199800cttccatcac cttagagaag
gggcaggcgc tgcccgacct tctccaggct ggagcagcat 199860cgcctcatgt
cagcagaact cagctgtaga atatcgtggg gttggtgcct ttcatcagca
199920gcatgtcctt aacaactttc tgatttcttc cttagttgtt ggtccattaa
ggagaaaaaa 199980aatgatctca gccattgcta aaatatttga taagattcag
caaagcagca tgttaacatt 200040gaaaactaga atcaggagcc aggcagatgt
gcttgctttt cacctgtagt atttcatgtt 200100gttttgacgt ttttagctaa
tgcattaaga taaataaaca aaagccgggc acggtggttc 200160acgcctgtaa
tcccagcact ttgggaggct gaggcgggag gatcctctga ggtcaggagt
200220tcaagaccag cctgaccaac atggagaaac ctcgtcatta ctaaaaatac
aaaattagct 200280gggcgtggtg gtgcatgcct gtaatcccag ctacttggga
ggctgaggca ggagaatcgc 200340ttgaacccgg gaggcggagg ttgcagtgag
ctgagattgc accactgcac tccagcctgg 200400gtgacagtga aactcggtct
caaaaaaaaa aaaaaattaa aaaaagataa ataaaataag 200460caggataaga
aatgaagaaa gtagagttac ctttgttttc agatttcatt tttgtatacc
200520cagaaagcca aatgtacaaa agactgggag ctctttaaac cagcttaaac
ttgttgaaaa 200580tgaggatgaa gaaatatccc attcagagtt ggaatgaatt
taacccagaa ggaacaggac 200640ctctactgaa gagaactatg cagtcttact
gaaaaatcta aataatacct gagcgctgga 200700gaaacttcgc acactcctga
aagctccaaa gtcaatgtca tcattttatt aatgtcattc 200760caaacatagt
ctcaataata tcacttcttg gttttgacat ggacgcgatg atgtttaaat
200820tcatatgaaa aaagaacggg gccaaaagtc caaggccagt cagcgtgaga
agaccgctcg 200880gcctccctcg gagtcgggga gttggaaccg cagactgaga
tcatgtggct gctggaggcc 200940aggacgaacg tcgggaaatg gagactcctg
cgttgctggt gggatgtggt gcagccgctt 201000ccaggagcaa tttggtgtcc
cgtcctaaag ctgaagaaac gcatttcctc tggtcagtgc 201060cactcctaga
caggccaccc tgcggcagcc gtcctcaaac tggtctgagg acccctcaac
201120gctcttaaaa atcattaaaa gtgggccagg tgcggtggct cacacctgta
atcccagcac 201180tttgggaggc caagacaggc ggatcacgag gtcaggacat
tgagatcatc ctggctaaca 201240cggtgaaacc ccgtctctac taaaaataca
aaaaattagc cgggcgtggt ggcgggcgcc 201300tgtagtccca gctacttggg
aggctgagcc aggagaatgg cgtgaaccca ggaggtggag 201360cttgcagtga
gctgagatca ctccactgca ctccagcctg ggcagcagag cgagactctg
201420tctcaaaaaa aaataataaa taaataaata aaaataaaat aaaataaaat
tcattaaaag 201480tgccaaagaa cttttgctta tgtgagttct aatgaccaat
attaatacac attagaatat 201540cttattagaa attaaacctg agacctttag
aaaacatgta ttcatttcaa aatagcaata 201600aacccatgac atattaacat
aaataacaat tgtatgaaaa atatattttc caaaacaaaa 201660agttttcggg
agaagtgtgg catagtttta catggtcgta aatctctggc ttaagagaag
201720cccactggcc tctcagcagg ctctgggtcc gtccactttg ggggtgtttt
ggttgtgaag 201780tataggagtg aatggagaag ctcattctta cccagatgtg
tatttgaaaa gaaaaggaac 201840attttaataa cctttgcaaa taatcggtat
attcttccgt gatcctattc caacactgga 201900caggtggtgg tttgtttttt
ttttttggag acggagtccc gctctgtcac tcaggctgga 201960gtgcagtggc
gcgatttcag ctcactgcaa gctccgcctc c 202001917DNAArtificial
sequenceSynthetic oligonucleotide 9ataaattgtc atcacca
171013481DNAHomo sapiens 10gctgccggga cgggtccaag atggacggcc
gctcaggttc tgcttttacc tgcggcccag 60agccccattc attgccccgg tgctgagcgg
cgccgcgagt cggcccgagg cctccgggga 120ctgccgtgcc gggcgggaga
ccgccatggc gaccctggaa aagctgatga aggccttcga 180gtccctcaag
tccttccagc agcagcagca gcagcagcag cagcagcagc agcagcagca
240gcagcagcag cagcagcagc aacagccgcc accgccgccg ccgccgccgc
cgcctcctca 300gcttcctcag ccgccgccgc aggcacagcc gctgctgcct
cagccgcagc cgcccccgcc 360gccgcccccg ccgccacccg gcccggctgt
ggctgaggag ccgctgcacc gaccaaagaa 420agaactttca gctaccaaga
aagaccgtgt gaatcattgt ctgacaatat gtgaaaacat 480agtggcacag
tctgtcagaa attctccaga atttcagaaa cttctgggca tcgctatgga
540actttttctg ctgtgcagtg atgacgcaga gtcagatgtc aggatggtgg
ctgacgaatg 600cctcaacaaa gttatcaaag ctttgatgga ttctaatctt
ccaaggttac agctcgagct 660ctataaggaa attaaaaaga atggtgcccc
tcggagtttg cgtgctgccc tgtggaggtt 720tgctgagctg gctcacctgg
ttcggcctca gaaatgcagg ccttacctgg tgaaccttct 780gccgtgcctg
actcgaacaa gcaagagacc cgaagaatca gtccaggaga ccttggctgc
840agctgttccc aaaattatgg cttcttttgg caattttgca aatgacaatg
aaattaaggt 900tttgttaaag gccttcatag cgaacctgaa gtcaagctcc
cccaccattc ggcggacagc 960ggctggatca gcagtgagca tctgccagca
ctcaagaagg acacaatatt tctatagttg 1020gctactaaat gtgctcttag
gcttactcgt tcctgtcgag gatgaacact ccactctgct 1080gattcttggc
gtgctgctca ccctgaggta tttggtgccc ttgctgcagc agcaggtcaa
1140ggacacaagc ctgaaaggca gcttcggagt gacaaggaaa gaaatggaag
tctctccttc 1200tgcagagcag cttgtccagg tttatgaact gacgttacat
catacacagc accaagacca 1260caatgttgtg accggagccc tggagctgtt
gcagcagctc ttcagaacgc ctccacccga 1320gcttctgcaa accctgaccg
cagtcggggg cattgggcag ctcaccgctg ctaaggagga 1380gtctggtggc
cgaagccgta gtgggagtat tgtggaactt atagctggag ggggttcctc
1440atgcagccct gtcctttcaa gaaaacaaaa aggcaaagtg ctcttaggag
aagaagaagc 1500cttggaggat gactctgaat cgagatcgga tgtcagcagc
tctgccttaa cagcctcagt 1560gaaggatgag atcagtggag agctggctgc
ttcttcaggg gtttccactc cagggtcagc 1620aggtcatgac atcatcacag
aacagccacg gtcacagcac acactgcagg cggactcagt 1680ggatctggcc
agctgtgact tgacaagctc tgccactgat ggggatgagg aggatatctt
1740gagccacagc tccagccagg tcagcgccgt cccatctgac cctgccatgg
acctgaatga 1800tgggacccag gcctcgtcgc ccatcagcga cagctcccag
accaccaccg aagggcctga 1860ttcagctgtt accccttcag acagttctga
aattgtgtta gacggtaccg acaaccagta 1920tttgggcctg cagattggac
agccccagga tgaagatgag gaagccacag gtattcttcc 1980tgatgaagcc
tcggaggcct tcaggaactc ttccatggcc cttcaacagg cacatttatt
2040gaaaaacatg agtcactgca ggcagccttc tgacagcagt gttgataaat
ttgtgttgag 2100agatgaagct actgaaccgg gtgatcaaga aaacaagcct
tgccgcatca aaggtgacat 2160tggacagtcc actgatgatg actctgcacc
tcttgtccat tgtgtccgcc ttttatctgc 2220ttcgtttttg ctaacagggg
gaaaaaatgt gctggttccg gacagggatg tgagggtcag 2280cgtgaaggcc
ctggccctca gctgtgtggg agcagctgtg gccctccacc cggaatcttt
2340cttcagcaaa ctctataaag ttcctcttga caccacggaa taccctgagg
aacagtatgt 2400ctcagacatc ttgaactaca tcgatcatgg agacccacag
gttcgaggag ccactgccat 2460tctctgtggg accctcatct gctccatcct
cagcaggtcc cgcttccacg tgggagattg 2520gatgggcacc attagaaccc
tcacaggaaa tacattttct ttggcggatt gcattccttt 2580gctgcggaaa
acactgaagg atgagtcttc tgttacttgc aagttagctt gtacagctgt
2640gaggaactgt gtcatgagtc tctgcagcag cagctacagt gagttaggac
tgcagctgat 2700catcgatgtg ctgactctga ggaacagttc ctattggctg
gtgaggacag agcttctgga 2760aacccttgca gagattgact tcaggctggt
gagctttttg gaggcaaaag cagaaaactt 2820acacagaggg gctcatcatt
atacagggct tttaaaactg caagaacgag tgctcaataa 2880tgttgtcatc
catttgcttg gagatgaaga ccccagggtg cgacatgttg ccgcagcatc
2940actaattagg cttgtcccaa agctgtttta taaatgtgac caaggacaag
ctgatccagt 3000agtggccgtg gcaagagatc aaagcagtgt ttacctgaaa
cttctcatgc atgagacgca 3060gcctccatct catttctccg tcagcacaat
aaccagaata tatagaggct ataacctact 3120accaagcata acagacgtca
ctatggaaaa taacctttca agagttattg cagcagtttc 3180tcatgaacta
atcacatcaa ccaccagagc actcacattt ggatgctgtg aagctttgtg
3240tcttctttcc actgccttcc cagtttgcat ttggagttta ggttggcact
gtggagtgcc 3300tccactgagt gcctcagatg agtctaggaa gagctgtacc
gttgggatgg ccacaatgat 3360tctgaccctg ctctcgtcag cttggttccc
attggatctc tcagcccatc aagatgcttt 3420gattttggcc ggaaacttgc
ttgcagccag tgctcccaaa tctctgagaa gttcatgggc 3480ctctgaagaa
gaagccaacc cagcagccac caagcaagag gaggtctggc cagccctggg
3540ggaccgggcc ctggtgccca tggtggagca gctcttctct cacctgctga
aggtgattaa 3600catttgtgcc cacgtcctgg atgacgtggc tcctggaccc
gcaataaagg cagccttgcc 3660ttctctaaca aacccccctt ctctaagtcc
catccgacga aaggggaagg agaaagaacc 3720aggagaacaa gcatctgtac
cgttgagtcc caagaaaggc agtgaggcca gtgcagcttc 3780tagacaatct
gatacctcag gtcctgttac aacaagtaaa tcctcatcac tggggagttt
3840ctatcatctt ccttcatacc tcaaactgca tgatgtcctg aaagctacac
acgctaacta 3900caaggtcacg ctggatcttc agaacagcac ggaaaagttt
ggagggtttc tccgctcagc 3960cttggatgtt ctttctcaga tactagagct
ggccacactg caggacattg ggaagtgtgt 4020tgaagagatc ctaggatacc
tgaaatcctg ctttagtcga gaaccaatga tggcaactgt 4080ttgtgttcaa
caattgttga agactctctt tggcacaaac ttggcctccc agtttgatgg
4140cttatcttcc aaccccagca agtcacaagg ccgagcacag cgccttggct
cctccagtgt 4200gaggccaggc ttgtaccact actgcttcat ggccccgtac
acccacttca cccaggccct 4260cgctgacgcc agcctgagga acatggtgca
ggcggagcag gagaacgaca cctcgggatg 4320gtttgatgtc ctccagaaag
tgtctaccca gttgaagaca aacctcacga gtgtcacaaa 4380gaaccgtgca
gataagaatg ctattcataa tcacattcgt ttgtttgaac ctcttgttat
4440aaaagcttta aaacagtaca cgactacaac atgtgtgcag ttacagaagc
aggttttaga 4500tttgctggcg cagctggttc agttacgggt taattactgt
cttctggatt cagatcaggt 4560gtttattggc tttgtattga aacagtttga
atacattgaa gtgggccagt tcagggaatc 4620agaggcaatc attccaaaca
tctttttctt cttggtatta ctatcttatg aacgctatca 4680ttcaaaacag
atcattggaa ttcctaaaat cattcagctc tgtgatggca tcatggccag
4740tggaaggaag gctgtgacac atgccatacc ggctctgcag cccatagtcc
acgacctctt 4800tgtattaaga ggaacaaata aagctgatgc aggaaaagag
cttgaaaccc aaaaagaggt 4860ggtggtgtca atgttactga gactcatcca
gtaccatcag gtgttggaga tgttcattct 4920tgtcctgcag cagtgccaca
aggagaatga agacaagtgg aagcgactgt ctcgacagat 4980agctgacatc
atcctcccaa tgttagccaa acagcagatg cacattgact ctcatgaagc
5040ccttggagtg ttaaatacat tatttgagat tttggcccct tcctccctcc
gtccggtaga 5100catgctttta cggagtatgt tcgtcactcc aaacacaatg
gcgtccgtga gcactgttca 5160actgtggata tcgggaattc tggccatttt
gagggttctg atttcccagt caactgaaga 5220tattgttctt tctcgtattc
aggagctctc cttctctccg tatttaatct cctgtacagt 5280aattaatagg
ttaagagatg gggacagtac ttcaacgcta gaagaacaca gtgaagggaa
5340acaaataaag aatttgccag aagaaacatt ttcaaggttt ctattacaac
tggttggtat 5400tcttttagaa gacattgtta caaaacagct gaaggtggaa
atgagtgagc agcaacatac 5460tttctattgc caggaactag gcacactgct
aatgtgtctg atccacatct tcaagtctgg 5520aatgttccgg agaatcacag
cagctgccac taggctgttc cgcagtgatg gctgtggcgg 5580cagtttctac
accctggaca gcttgaactt gcgggctcgt tccatgatca ccacccaccc
5640ggccctggtg ctgctctggt gtcagatact gctgcttgtc aaccacaccg
actaccgctg 5700gtgggcagaa gtgcagcaga ccccgaaaag acacagtctg
tccagcacaa agttacttag 5760tccccagatg tctggagaag aggaggattc
tgacttggca gccaaacttg gaatgtgcaa 5820tagagaaata gtacgaagag
gggctctcat tctcttctgt gattatgtct gtcagaacct 5880ccatgactcc
gagcacttaa cgtggctcat tgtaaatcac attcaagatc tgatcagcct
5940ttcccacgag cctccagtac aggacttcat cagtgccgtt catcggaact
ctgctgccag 6000cggcctgttc atccaggcaa ttcagtctcg ttgtgaaaac
ctttcaactc caaccatgct 6060gaagaaaact cttcagtgct tggaggggat
ccatctcagc cagtcgggag ctgtgctcac 6120gctgtatgtg gacaggcttc
tgtgcacccc tttccgtgtg ctggctcgca tggtcgacat 6180ccttgcttgt
cgccgggtag aaatgcttct ggctgcaaat ttacagagca gcatggccca
6240gttgccaatg gaagaactca acagaatcca ggaatacctt cagagcagcg
ggctcgctca 6300gagacaccaa aggctctatt ccctgctgga caggtttcgt
ctctccacca tgcaagactc 6360acttagtccc tctcctccag tctcttccca
cccgctggac ggggatgggc acgtgtcact 6420ggaaacagtg agtccggaca
aagactggta cgttcatctt gtcaaatccc agtgttggac 6480caggtcagat
tctgcactgc tggaaggtgc agagctggtg aatcggattc ctgctgaaga
6540tatgaatgcc ttcatgatga actcggagtt caacctaagc ctgctagctc
catgcttaag 6600cctagggatg agtgaaattt ctggtggcca gaagagtgcc
ctttttgaag cagcccgtga 6660ggtgactctg gcccgtgtga gcggcaccgt
gcagcagctc cctgctgtcc atcatgtctt 6720ccagcccgag ctgcctgcag
agccggcggc ctactggagc aagttgaatg atctgtttgg 6780ggatgctgca
ctgtatcagt ccctgcccac tctggcccgg gccctggcac agtacctggt
6840ggtggtctcc aaactgccca gtcatttgca ccttcctcct gagaaagaga
aggacattgt 6900gaaattcgtg gtggcaaccc ttgaggccct gtcctggcat
ttgatccatg agcagatccc 6960gctgagtctg gatctccagg cagggctgga
ctgctgctgc ctggccctgc agctgcctgg 7020cctctggagc gtggtctcct
ccacagagtt tgtgacccac gcctgctccc tcatctactg 7080tgtgcacttc
atcctggagg ccgttgcagt gcagcctgga gagcagcttc ttagtccaga
7140aagaaggaca aataccccaa aagccatcag cgaggaggag gaggaagtag
atccaaacac 7200acagaatcct aagtatatca ctgcagcctg tgagatggtg
gcagaaatgg tggagtctct 7260gcagtcggtg ttggccttgg gtcataaaag
gaatagcggc gtgccggcgt ttctcacgcc 7320attgctaagg aacatcatca
tcagcctggc ccgcctgccc cttgtcaaca gctacacacg 7380tgtgccccca
ctggtgtgga agcttggatg gtcacccaaa ccgggagggg attttggcac
7440agcattccct gagatccccg tggagttcct ccaggaaaag gaagtcttta
aggagttcat 7500ctaccgcatc aacacactag gctggaccag tcgtactcag
tttgaagaaa cttgggccac 7560cctccttggt gtcctggtga cgcagcccct
cgtgatggag caggaggaga gcccaccaga 7620agaagacaca gagaggaccc
agatcaacgt cctggccgtg caggccatca cctcactggt 7680gctcagtgca
atgactgtgc ctgtggccgg caacccagct gtaagctgct tggagcagca
7740gccccggaac aagcctctga aagctctcga caccaggttt gggaggaagc
tgagcattat 7800cagagggatt gtggagcaag agattcaagc aatggtttca
aagagagaga atattgccac 7860ccatcattta tatcaggcat gggatcctgt
cccttctctg tctccggcta ctacaggtgc 7920cctcatcagc cacgagaagc
tgctgctaca gatcaacccc gagcgggagc tggggagcat 7980gagctacaaa
ctcggccagg tgtccataca ctccgtgtgg ctggggaaca gcatcacacc
8040cctgagggag gaggaatggg acgaggaaga ggaggaggag gccgacgccc
ctgcaccttc 8100gtcaccaccc acgtctccag tcaactccag gaaacaccgg
gctggagttg acatccactc 8160ctgttcgcag tttttgcttg agttgtacag
ccgctggatc ctgccgtcca gctcagccag 8220gaggaccccg gccatcctga
tcagtgaggt ggtcagatcc cttctagtgg tctcagactt 8280gttcaccgag
cgcaaccagt ttgagctgat gtatgtgacg ctgacagaac tgcgaagggt
8340gcacccttca gaagacgaga tcctcgctca gtacctggtg cctgccacct
gcaaggcagc 8400tgccgtcctt gggatggaca aggccgtggc ggagcctgtc
agccgcctgc tggagagcac 8460gctcaggagc agccacctgc ccagcagggt
tggagccctg cacggcgtcc tctatgtgct 8520ggagtgcgac ctgctggacg
acactgccaa gcagctcatc ccggtcatca gcgactatct 8580cctctccaac
ctgaaaggga tcgcccactg cgtgaacatt cacagccagc agcacgtact
8640ggtcatgtgt gccactgcgt tttacctcat tgagaactat cctctggacg
tagggccgga 8700attttcagca tcaataatac agatgtgtgg ggtgatgctg
tctggaagtg aggagtccac 8760cccctccatc atttaccact gtgccctcag
aggcctggag cgcctcctgc tctctgagca 8820gctctcccgc ctggatgcag
aatcgctggt caagctgagt gtggacagag tgaacgtgca 8880cagcccgcac
cgggccatgg cggctctggg cctgatgctc acctgcatgt acacaggaaa
8940ggagaaagtc agtccgggta gaacttcaga ccctaatcct gcagcccccg
acagcgagtc 9000agtgattgtt gctatggagc gggtatctgt tctttttgat
aggatcagga aaggctttcc 9060ttgtgaagcc agagtggtgg ccaggatcct
gccccagttt ctagacgact tcttcccacc 9120ccaggacatc atgaacaaag
tcatcggaga gtttctgtcc aaccagcagc cataccccca 9180gttcatggcc
accgtggtgt ataaggtgtt tcagactctg cacagcaccg ggcagtcgtc
9240catggtccgg gactgggtca tgctgtccct ctccaacttc acgcagaggg
ccccggtcgc 9300catggccacg tggagcctct cctgcttctt tgtcagcgcg
tccaccagcc cgtgggtcgc 9360ggcgatcctc ccacatgtca tcagcaggat
gggcaagctg gagcaggtgg acgtgaacct 9420tttctgcctg gtcgccacag
acttctacag acaccagata gaggaggagc tcgaccgcag 9480ggccttccag
tctgtgcttg aggtggttgc agccccagga agcccatatc accggctgct
9540gacttgttta cgaaatgtcc acaaggtcac cacctgctga gcgccatggt
gggagagact 9600gtgaggcggc agctggggcc ggagcctttg gaagtctgcg
cccttgtgcc ctgcctccac 9660cgagccagct tggtccctat gggcttccgc
acatgccgcg ggcggccagg caacgtgcgt 9720gtctctgcca tgtggcagaa
gtgctctttg tggcagtggc caggcaggga gtgtctgcag 9780tcctggtggg
gctgagcctg aggccttcca gaaagcagga gcagctgtgc tgcaccccat
9840gtgggtgacc aggtcctttc tcctgatagt cacctgctgg ttgttgccag
gttgcagctg 9900ctcttgcatc tgggccagaa gtcctccctc ctgcaggctg
gctgttggcc cctctgctgt 9960cctgcagtag aaggtgccgt gagcaggctt
tgggaacact ggcctgggtc tccctggtgg 10020ggtgtgcatg ccacgccccg
tgtctggatg cacagatgcc atggcctgtg ctgggccagt 10080ggctgggggt
gctagacacc cggcaccatt ctcccttctc tcttttcttc tcaggattta
10140aaatttaatt atatcagtaa agagattaat tttaacgtaa ctctttctat
gcccgtgtaa 10200agtatgtgaa tcgcaaggcc tgtgctgcat gcgacagcgt
ccggggtggt ggacagggcc 10260cccggccacg ctccctctcc tgtagccact
ggcatagccc tcctgagcac ccgctgacat 10320ttccgttgta catgttcctg
tttatgcatt cacaaggtga ctgggatgta gagaggcgtt 10380agtgggcagg
tggccacagc aggactgagg acaggccccc attatcctag gggtgcgctc
10440acctgcagcc cctcctcctc gggcacagac gactgtcgtt ctccacccac
cagtcaggga 10500cagcagcctc cctgtcactc agctgagaag gccagccctc
cctggctgtg agcagcctcc 10560actgtgtcca gagacatggg cctcccactc
ctgttccttg ctagccctgg ggtggcgtct 10620gcctaggagc tggctggcag
gtgttgggac ctgctgctcc atggatgcat gccctaagag 10680tgtcactgag
ctgtgttttg tctgagcctc tctcggtcaa cagcaaagct tggtgtcttg
10740gcactgttag tgacagagcc cagcatccct tctgcccccg ttccagctga
catcttgcac 10800ggtgacccct tttagtcagg agagtgcaga tctgtgctca
tcggagactg ccccacggcc 10860ctgtcagagc cgccactcct atccccaggc
caggtccctg gaccagcctc ctgtttgcag 10920gcccagagga gccaagtcat
taaaatggaa gtggattctg gatggccggg ctgctgctga 10980tgtaggagct
ggatttggga gctctgcttg ccgactggct gtgagacgag gcaggggctc
11040tgcttcctca gccctagagg cgagccaggc aaggttggcg actgtcatgt
ggcttggttt 11100ggtcatgccc gtcgatgttt tgggtattga atgtggtaag
tggaggaaat gttggaactc 11160tgtgcaggtg ctgccttgag acccccaagc
ttccacctgt ccctctccta tgtggcagct 11220ggggagcagc tgagatgtgg
acttgtatgc tgcccacata cgtgaggggg agctgaaagg 11280gagcccctcc
tctgagcagc ctctgccagg cctgtatgag gcttttccca ccagctccca
11340acagaggcct cccccagcca ggaccacctc gtcctcgtgg cggggcagca
ggagcggtag 11400aaaggggtcc gatgtttgag gaggccctta agggaagcta
ctgaattata acacgtaaga 11460aaatcaccat tccgtattgg ttgggggctc
ctgtttctca tcctagcttt ttcctggaaa 11520gcccgctaga aggtttggga
acgaggggaa agttctcaga actgttggct gctccccacc 11580cgcctcccgc
ctcccccgca ggttatgtca gcagctctga gacagcagta tcacaggcca
11640gatgttgttc ctggctagat gtttacattt gtaagaaata acactgtgaa
tgtaaaacag 11700agccattccc ttggaatgca tatcgctggg ctcaacatag
agtttgtctt cctcttgttt 11760acgacgtgat ctaaaccagt ccttagcaag
gggctcagaa caccccgctc tggcagtagg 11820tgtcccccac ccccaaagac
ctgcctgtgt gctccggaga tgaatatgag ctcattagta 11880aaaatgactt
cacccacgca tatacataaa gtatccatgc atgtgcatat agacacatct
11940ataattttac acacacacct ctcaagacgg agatgcatgg cctctaagag
tgcccgtgtc 12000ggttcttcct ggaagttgac tttccttaga cccgccaggt
caagttagcc gcgtgacgga 12060catccaggcg tgggacgtgg tcagggcagg
gctcattcat tgcccactag gatcccactg 12120gcgaagatgg tctccatatc
agctctctgc agaagggagg aagactttat catgttccta 12180aaaatctgtg
gcaagcaccc atcgtattat ccaaattttg ttgcaaatgt gattaatttg
12240gttgtcaagt tttgggggtg ggctgtgggg agattgcttt tgttttcctg
ctggtaatat 12300cgggaaagat tttaatgaaa ccagggtaga attgtttggc
aatgcactga agcgtgtttc 12360tttcccaaaa tgtgcctccc ttccgctgcg
ggcccagctg agtctatgta ggtgatgttt 12420ccagctgcca agtgctcttt
gttactgtcc accctcattt ctgccagcgc atgtgtcctt 12480tcaaggggaa
aatgtgaagc tgaaccccct ccagacaccc agaatgtagc atctgagaag
12540gccctgtgcc ctaaaggaca cccctcgccc ccatcttcat ggagggggtc
atttcagagc 12600cctcggagcc aatgaacagc tcctcctctt ggagctgaga
tgagccccac gtggagctcg 12660ggacggatag tagacagcaa taactcggtg
tgtggccgcc tggcaggtgg aacttcctcc 12720cgttgcgggg tggagtgagg
ttagttctgt gtgtctggtg ggtggagtca ggcttctctt 12780gctacctgtg
agcatccttc ccagcagaca tcctcatcgg gctttgtccc tcccccgctt
12840cctccctctg cggggaggac ccgggaccac agctgctggc cagggtagac
ttggagctgt 12900cctccagagg ggtcacgtgt aggagtgaga agaaggaaga
tcttgagagc tgctgaggga 12960ccttggagag ctcaggatgg ctcagacgag
gacactcgct tgccgggcct gggcctcctg 13020ggaaggaggg agctgctcag
aatgccgcat gacaactgaa ggcaacctgg aaggttcagg 13080ggccgctctt
cccccatgtg cctgtcacgc tctggtgcag tcaaaggaac gccttcccct
13140cagttgtttc taagagcaga gtctcccgct gcaatctggg tggtaactgc
cagccttgga 13200ggatcgtggc caacgtggac ctgcctacgg agggtgggct
ctgacccaag tggggcctcc 13260ttgtccaggt ctcactgctt tgcaccgtgg
tcagagggac tgtcagctga gcttgagctc 13320ccctggagcc agcagggctg
tgatgggcga gtcccggagc cccacccaga cctgaatgct 13380tctgagagca
aagggaagga ctgacgagag atgtatattt aattttttaa ctgctgcaaa
13440cattgtacat ccaaattaaa ggaaaaaaat ggaaaccatc a
134811116DNAArtificial sequenceSynthetic oligonucleotide
11atcatggctg cagctt 161216DNAArtificial sequenceSynthetic
oligonucleotide 12ttcagtcatg acttcc 161316DNAArtificial
sequenceSynthetic oligonucleotide 13nnnnnnnnnn nnnnnn
161416DNAArtificial sequenceSynthetic oligonucleotide 14nnnnnnnnnn
nnnnnn 161516DNAArtificial sequenceSynthetic oligonucleotide
15nnnnnnnnnn nnnnnn 161616DNAArtificial sequenceSynthetic
oligonucleotide 16nnnnnnnnnn nnnnnn 16
* * * * *
References