| 质粒名称: | pdCas9-DNMT3A-EGFP |
|---|---|
| 出品公司： | addgene |
| 目录编号: | 71666 |
| 存储实验室: | Vlatka Zoldoš |
|
载体骨架基本信息 |
|
| 载体名称: | PX462 |
| 空载体大小: | 4246 bp |
| 完整质粒大小: | 10266bp |
| 载体修饰: | 人细胞色素c氧化酶亚基VIII（human cytochrome C oxidase subunit VIII） |
| 载体类型: | 哺乳细胞表达 |
| 筛选标记: | 新霉素（G418） |
| 载体抗性: | 氨苄霉素 |
| 生长温度: | 37 ℃ |
| 宿主菌: | DH5a |
| 拷贝数: | 未知 |
|
插入基因信息 |
|
| 插入基因名称: | S. pyogenes dCas9 fused with the catalytic domain of human DNMT3A (amino acids P602-V912) and T2A-EGFP/Cas9-DNMT3A-EGFP/SpdCas9-DNMT3A-T2A-EGFP/Cas9-DNMT3A |
| 物种来源: | H. sapiens (human), Synthetic; S. pyogenes |
| 插入大小: | 6020bp |
| 基因突变: | D10A and H840A in S.pyogenes Cas9 |
| GenBank编号 | AKA60242.1 NP_072046.2 |
| 启动子: | CBh |
| 标签/融合蛋白: |
3xFLAG (N terminal on insert)
SV40 NLS (N terminal on insert)
T2A-EGFP (C terminal on insert)
|
| 克隆方法: | 限制性内切酶 |
| 5’克隆位点: | BamHI(未破坏) |
| 3’克隆位点: | FseI(未破坏) |
| 5’测序引物 | rGS-linker For 5' GAAGAGGTACACCAGCACCAAAG 3' |
| 3’测序引物 | BGH Rev |
|
文献引用方法 |
|
| 材料和方法部分: | pdCas9-DNMT3A-EGFP was a gift from Vlatka Zoldoš (Addgene plasmid # 71666 ; http://n2t.net/addgene:71666 ; RRID:Addgene_71666) |
| 参考文献部分: | Repurposing the CRISPR-Cas9 system for targeted DNA methylation. Vojta A, Dobrinic P, Tadic V, Bockor L, Korac P, Julg B, Klasic M, Zoldos V. Nucleic Acids Res. 2016 Mar 11. pii: gkw159. 10.1093/nar/gkw159 PubMed 26969735 |
买家导航
载体基本信息
载体图谱质粒图谱和多克隆位点信息
载体简介
载体序列
LOCUS pdCas9-DNMT3A-EGFP 10266 bp ds-DNA circular SYN 12-MAY-2021
DEFINITION Expression of human codon-optimized dCas9-DNMT3A fusion with
T2A-EGFP for targeted DNA methylation in mammalian cells; cloning
backbone for sgRNA.
ACCESSION .
VERSION .
KEYWORDS pdCas9-DNMT3A-EGFP
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 10266)
AUTHORS Vojta A, Dobrinic P, Tadic V, Bockor L, Korac P, Julg B, Klasic M,
Zoldos V
TITLE Repurposing the CRISPR-Cas9 system for targeted DNA methylation.
JOURNAL Nucleic Acids Res. 2016 Mar 11. pii: gkw159.
PUBMED 26969735
REFERENCE 2 (bases 1 to 10266)
AUTHORS .
TITLE Direct Submission
JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58
http://www.biofeng.com
FEATURES Location/Qualifiers
source 1..10266
/organism="synthetic DNA construct"
/mol_type="other DNA"
promoter 1..241
/label=U6 promoter
/note="RNA polymerase III promoter for human U6 snRNA"
primer_bind 1..21
/label=hU6-F
/note="Human U6 promoter, forward primer"
primer_bind 172..191
/label=LKO.1 5'
/note="Human U6 promoter, forward primer"
misc_RNA 268..343
/label=gRNA scaffold
/note="guide RNA scaffold for the Streptococcus pyogenes
CRISPR/Cas9 system"
enhancer 440..725
/label=CMV enhancer
/note="human cytomegalovirus immediate early enhancer;
contains an 18-bp deletion relative to the standard CMV
enhancer"
promoter 727..1004
/label=chicken beta-actin promoter
intron 1005..1233
/label=hybrid intron
/note="hybrid between chicken beta-actin (CBA) and minute
virus of mice (MMV) introns (Gray et al., 2011)"
regulatory 1245..1254
/regulatory_class="other"
/note="vertebrate consensus sequence for strong initiation
of translation (Kozak, 1987)"
CDS 1254..1319
/codon_start=1
/product="three tandem FLAG(R) epitope tags, followed by an
enterokinase cleavage site"
/label=3xFLAG
/translation="DYKDHDGDYKDHDIDYKDDDDK"
CDS 1326..1346
/codon_start=1
/product="nuclear localization signal of SV40 (simian virus
40) large T antigen"
/label=SV40 NLS
/translation="PKKKRKV"
CDS 1371..5471
/codon_start=1
/product="catalytically dead mutant of the Cas9
endonuclease from the Streptococcus pyogenes Type II
CRISPR/Cas system"
/label=dCas9
/note="RNA-guided DNA-binding protein that lacks
endonuclease activity due to the D10A mutation in the RuvC
catalytic domain and the H840A mutation in the HNH
catalytic domain"
/translation="DKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN
LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF
LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF
RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE
NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI
GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF
TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS
VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL
KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ
LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH
KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL
YYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPS
EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV
AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE
ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE
SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT
IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL
ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA
NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD
ATLIHQSITGLYETRIDLSQLGGD"
CDS 5472..5519
/codon_start=1
/product="bipartite nuclear localization signal from
nucleoplasmin"
/label=nucleoplasmin NLS
/translation="KRPAATKKAGQAKKKK"
CDS 5547..6479
/codon_start=1
/product="catalytic domain of DNA methyltransferase 3A"
/label=DNMT3A catalytic domain
/translation="PSRLQMFFANNHDQEFDPPKVYPPVPAEKRKPIRVLSLFDGIATG
LLVLKDLGIQVDRYIASEVCEDSITVGMVRHQGKIMYVGDVRSVTQKHIQEWGPFDLVI
GGSPCNDLSIVNPARKGLYEGTGRLFFEFYRLLHDARPKEGDDRPFFWLFENVVAMGVS
DKRDISRFLESNPVMIDAKEVSAAHRARYFWGNLPGMNRPLASTVNDKLELQECLEHGR
IAKFSKVRTITTRSNSIKQGKDQHFPVFMNEKEDILWCTEMERVFGFPVHYTDVSNMSR
LARQRLLGRSWSVPVIRHLFAPLKEYFACV"
CDS 6507..6560
/codon_start=1
/product="2A peptide from Thosea asigna virus capsid
protein"
/label=T2A
/note="Eukaryotic ribosomes fail to insert a peptide bond
between the Gly and Pro residues, yielding separate
polypeptides."
/translation="EGRGSLLTCGDVEENPGP"
CDS 6561..7274
/codon_start=1
/product="enhanced GFP"
/label=EGFP
/translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
FVTAAGITLGMDELYK"
CDS 6561..7274
/codon_start=1
/product="the original enhanced GFP (Yang et al., 1996)"
/label=EGFP
/note="mammalian codon-optimized"
/translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
FVTAAGITLGMDELYK"
primer_bind complement(6603..6624)
/label=EGFP-N
/note="EGFP, reverse primer"
primer_bind complement(6864..6883)
/label=EXFP-R
/note="For distinguishing EGFP variants, reverse primer"
primer_bind 7211..7232
/label=EGFP-C
/note="EGFP, forward primer"
primer_bind complement(7307..7324)
/label=BGH-rev
/note="Bovine growth hormone terminator, reverse primer.
Also called BGH reverse"
polyA_signal 7313..7520
/label=bGH poly(A) signal
/note="bovine growth hormone polyadenylation signal"
repeat_region 7529..7669
/label=AAV2 ITR
/note="inverted terminal repeat of adeno-associated virus
serotype 2"
repeat_region 7529..7658
/label=AAV2 ITR
rep_origin 7744..8199
/direction=RIGHT
/label=f1 ori
/note="f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
primer_bind complement(7831..7850)
/label=F1ori-R
/note="F1 origin, reverse primer"
primer_bind 8041..8062
/label=F1ori-F
/note="F1 origin, forward primer"
primer_bind complement(8216..8235)
/label=pRS-marker
/note="pRS vectors, use to sequence yeast selectable
marker"
primer_bind 8335..8357
/label=pGEX 3'
/note="pGEX vectors, reverse primer"
primer_bind complement(8395..8413)
/label=pBRforEco
/note="pBR322 vectors, upsteam of EcoRI site, forward
primer"
promoter 8481..8585
/gene="bla"
/label=AmpR promoter
CDS 8586..9446
/codon_start=1
/gene="bla"
/product="beta-lactamase"
/label=AmpR
/note="confers resistance to ampicillin, carbenicillin, and
related antibiotics"
/translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
LIKHW"
primer_bind complement(8804..8823)
/label=Amp-R
/note="Ampicillin resistance gene, reverse primer"
rep_origin 9617..10205
/direction=RIGHT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
primer_bind 10106..10125
/label=pBR322ori-F
/note="pBR322 origin, forward primer"
ORIGIN
1 gagggcctat ttcccatgat tccttcatat ttgcatatac gatacaaggc tgttagagag
61 ataattggaa ttaatttgac tgtaaacaca aagatattag tacaaaatac gtgacgtaga
121 aagtaataat ttcttgggta gtttgcagtt ttaaaattat gttttaaaat ggactatcat
181 atgcttaccg taacttgaaa gtatttcgat ttcttggctt tatatatctt gtggaaagga
241 cgaaacaccg ggtcttcgag aagacctgtt ttagagctag aaatagcaag ttaaaataag
301 gctagtccgt tatcaacttg aaaaagtggc accgagtcgg tgcttttttg ttttagagct
361 agaaatagca agttaaaata aggctagtcc gtttttagcg cgtgcgccaa ttctgcagac
421 aaatggctct agaggtaccc gttacataac ttacggtaaa tggcccgcct ggctgaccgc
481 ccaacgaccc ccgcccattg acgtcaatag taacgccaat agggactttc cattgacgtc
541 aatgggtgga gtatttacgg taaactgccc acttggcagt acatcaagtg tatcatatgc
601 caagtacgcc ccctattgac gtcaatgacg gtaaatggcc cgcctggcat tgtgcccagt
661 acatgacctt atgggacttt cctacttggc agtacatcta cgtattagtc atcgctatta
721 ccatggtcga ggtgagcccc acgttctgct tcactctccc catctccccc ccctccccac
781 ccccaatttt gtatttattt attttttaat tattttgtgc agcgatgggg gcgggggggg
841 ggggggggcg cgcgccaggc ggggcggggc ggggcgaggg gcggggcggg gcgaggcgga
901 gaggtgcggc ggcagccaat cagagcggcg cgctccgaaa gtttcctttt atggcgaggc
961 ggcggcggcg gcggccctat aaaaagcgaa gcgcgcggcg ggcgggagtc gctgcgacgc
1021 tgccttcgcc ccgtgccccg ctccgccgcc gcctcgcgcc gcccgccccg gctctgactg
1081 accgcgttac tcccacaggt gagcgggcgg gacggccctt ctcctccggg ctgtaattag
1141 ctgagcaaga ggtaagggtt taagggatgg ttggttggtg gggtattaat gtttaattac
1201 ctggagcacc tgcctgaaat cacttttttt caggttggac cggtgccacc atggactata
1261 aggaccacga cggagactac aaggatcatg atattgatta caaagacgat gacgataaga
1321 tggccccaaa gaagaagcgg aaggtcggta tccacggagt cccagcagcc gacaagaagt
1381 acagcatcgg cctggccatc ggcaccaact ctgtgggctg ggccgtgatc accgacgagt
1441 acaaggtgcc cagcaagaaa ttcaaggtgc tgggcaacac cgaccggcac agcatcaaga
1501 agaacctgat cggagccctg ctgttcgaca gcggcgaaac agccgaggcc acccggctga
1561 agagaaccgc cagaagaaga tacaccagac ggaagaaccg gatctgctat ctgcaagaga
1621 tcttcagcaa cgagatggcc aaggtggacg acagcttctt ccacagactg gaagagtcct
1681 tcctggtgga agaggataag aagcacgagc ggcaccccat cttcggcaac atcgtggacg
1741 aggtggccta ccacgagaag taccccacca tctaccacct gagaaagaaa ctggtggaca
1801 gcaccgacaa ggccgacctg cggctgatct atctggccct ggcccacatg atcaagttcc
1861 ggggccactt cctgatcgag ggcgacctga accccgacaa cagcgacgtg gacaagctgt
1921 tcatccagct ggtgcagacc tacaaccagc tgttcgagga aaaccccatc aacgccagcg
1981 gcgtggacgc caaggccatc ctgtctgcca gactgagcaa gagcagacgg ctggaaaatc
2041 tgatcgccca gctgcccggc gagaagaaga atggcctgtt cggcaacctg attgccctga
2101 gcctgggcct gacccccaac ttcaagagca acttcgacct ggccgaggat gccaaactgc
2161 agctgagcaa ggacacctac gacgacgacc tggacaacct gctggcccag atcggcgacc
2221 agtacgccga cctgtttctg gccgccaaga acctgtccga cgccatcctg ctgagcgaca
2281 tcctgagagt gaacaccgag atcaccaagg cccccctgag cgcctctatg atcaagagat
2341 acgacgagca ccaccaggac ctgaccctgc tgaaagctct cgtgcggcag cagctgcctg
2401 agaagtacaa agagattttc ttcgaccaga gcaagaacgg ctacgccggc tacattgacg
2461 gcggagccag ccaggaagag ttctacaagt tcatcaagcc catcctggaa aagatggacg
2521 gcaccgagga actgctcgtg aagctgaaca gagaggacct gctgcggaag cagcggacct
2581 tcgacaacgg cagcatcccc caccagatcc acctgggaga gctgcacgcc attctgcggc
2641 ggcaggaaga tttttaccca ttcctgaagg acaaccggga aaagatcgag aagatcctga
2701 ccttccgcat cccctactac gtgggccctc tggccagggg aaacagcaga ttcgcctgga
2761 tgaccagaaa gagcgaggaa accatcaccc cctggaactt cgaggaagtg gtggacaagg
2821 gcgcttccgc ccagagcttc atcgagcgga tgaccaactt cgataagaac ctgcccaacg
2881 agaaggtgct gcccaagcac agcctgctgt acgagtactt caccgtgtat aacgagctga
2941 ccaaagtgaa atacgtgacc gagggaatga gaaagcccgc cttcctgagc ggcgagcaga
3001 aaaaggccat cgtggacctg ctgttcaaga ccaaccggaa agtgaccgtg aagcagctga
3061 aagaggacta cttcaagaaa atcgagtgct tcgactccgt ggaaatctcc ggcgtggaag
3121 atcggttcaa cgcctccctg ggcacatacc acgatctgct gaaaattatc aaggacaagg
3181 acttcctgga caatgaggaa aacgaggaca ttctggaaga tatcgtgctg accctgacac
3241 tgtttgagga cagagagatg atcgaggaac ggctgaaaac ctatgcccac ctgttcgacg
3301 acaaagtgat gaagcagctg aagcggcgga gatacaccgg ctggggcagg ctgagccgga
3361 agctgatcaa cggcatccgg gacaagcagt ccggcaagac aatcctggat ttcctgaagt
3421 ccgacggctt cgccaacaga aacttcatgc agctgatcca cgacgacagc ctgaccttta
3481 aagaggacat ccagaaagcc caggtgtccg gccagggcga tagcctgcac gagcacattg
3541 ccaatctggc cggcagcccc gccattaaga agggcatcct gcagacagtg aaggtggtgg
3601 acgagctcgt gaaagtgatg ggccggcaca agcccgagaa catcgtgatc gaaatggcca
3661 gagagaacca gaccacccag aagggacaga agaacagccg cgagagaatg aagcggatcg
3721 aagagggcat caaagagctg ggcagccaga tcctgaaaga acaccccgtg gaaaacaccc
3781 agctgcagaa cgagaagctg tacctgtact acctgcagaa tgggcgggat atgtacgtgg
3841 accaggaact ggacatcaac cggctgtccg actacgatgt ggacgctatc gtgcctcaga
3901 gctttctgaa ggacgactcc atcgacaaca aggtgctgac cagaagcgac aagaaccggg
3961 gcaagagcga caacgtgccc tccgaagagg tcgtgaagaa gatgaagaac tactggcggc
4021 agctgctgaa cgccaagctg attacccaga gaaagttcga caatctgacc aaggccgaga
4081 gaggcggcct gagcgaactg gataaggccg gcttcatcaa gagacagctg gtggaaaccc
4141 ggcagatcac aaagcacgtg gcacagatcc tggactcccg gatgaacact aagtacgacg
4201 agaatgacaa gctgatccgg gaagtgaaag tgatcaccct gaagtccaag ctggtgtccg
4261 atttccggaa ggatttccag ttttacaaag tgcgcgagat caacaactac caccacgccc
4321 acgacgccta cctgaacgcc gtcgtgggaa ccgccctgat caaaaagtac cctaagctgg
4381 aaagcgagtt cgtgtacggc gactacaagg tgtacgacgt gcggaagatg atcgccaaga
4441 gcgagcagga aatcggcaag gctaccgcca agtacttctt ctacagcaac atcatgaact
4501 ttttcaagac cgagattacc ctggccaacg gcgagatccg gaagcggcct ctgatcgaga
4561 caaacggcga aaccggggag atcgtgtggg ataagggccg ggattttgcc accgtgcgga
4621 aagtgctgag catgccccaa gtgaatatcg tgaaaaagac cgaggtgcag acaggcggct
4681 tcagcaaaga gtctatcctg cccaagagga acagcgataa gctgatcgcc agaaagaagg
4741 actgggaccc taagaagtac ggcggcttcg acagccccac cgtggcctat tctgtgctgg
4801 tggtggccaa agtggaaaag ggcaagtcca agaaactgaa gagtgtgaaa gagctgctgg
4861 ggatcaccat catggaaaga agcagcttcg agaagaatcc catcgacttt ctggaagcca
4921 agggctacaa agaagtgaaa aaggacctga tcatcaagct gcctaagtac tccctgttcg
4981 agctggaaaa cggccggaag agaatgctgg cctctgccgg cgaactgcag aagggaaacg
5041 aactggccct gccctccaaa tatgtgaact tcctgtacct ggccagccac tatgagaagc
5101 tgaagggctc ccccgaggat aatgagcaga aacagctgtt tgtggaacag cacaagcact
5161 acctggacga gatcatcgag cagatcagcg agttctccaa gagagtgatc ctggccgacg
5221 ctaatctgga caaagtgctg tccgcctaca acaagcaccg ggataagccc atcagagagc
5281 aggccgagaa tatcatccac ctgtttaccc tgaccaatct gggagcccct gccgccttca
5341 agtactttga caccaccatc gaccggaaga ggtacaccag caccaaagag gtgctggacg
5401 ccaccctgat ccaccagagc atcaccggcc tgtacgagac acggatcgac ctgtctcagc
5461 tgggaggcga caaaaggccg gcggccacga aaaaggccgg acaggccaaa aagaaaaagc
5521 tcgagggcgg aggcgggagc ggatccccct cccggctcca gatgttcttc gctaataacc
5581 acgaccagga atttgaccct ccaaaggttt acccacctgt cccagctgag aagaggaagc
5641 ccatccgggt gctgtctctc tttgatggaa tcgctacagg gctcctggtg ctgaaggact
5701 tgggcattca ggtggaccgc tacattgcct cggaggtgtg tgaggactcc atcacggtgg
5761 gcatggtgcg gcaccagggg aagatcatgt acgtcgggga cgtccgcagc gtcacacaga
5821 agcatatcca ggagtggggc ccattcgatc tggtgattgg gggcagtccc tgcaatgacc
5881 tctccatcgt caaccctgct cgcaagggcc tctacgaggg cactggccgg ctcttctttg
5941 agttctaccg cctcctgcat gatgcgcggc ccaaggaggg agatgatcgc cccttcttct
6001 ggctctttga gaatgtggtg gccatgggcg ttagtgacaa gagggacatc tcgcgatttc
6061 tcgagtccaa ccctgtgatg attgatgcca aagaagtgtc agctgcacac agggcccgct
6121 acttctgggg taaccttccc ggtatgaaca ggccgttggc atccactgtg aatgataagc
6181 tggagctgca ggagtgtctg gagcatggca ggatagccaa gttcagcaaa gtgaggacca
6241 ttactacgag gtcaaactcc ataaagcagg gcaaagacca gcattttcct gtgttcatga
6301 atgagaaaga ggacatctta tggtgcactg aaatggaaag ggtatttggt ttcccagtcc
6361 actatactga cgtctccaac atgagccgct tggcgaggca gagactgctg ggccggtcat
6421 ggagcgtgcc agtcatccgc cacctcttcg ctccgctgaa ggagtatttt gcgtgtgtgt
6481 ccggccggcc cgaattcggc agtggagagg gcagaggaag tctgctaaca tgcggtgacg
6541 tcgaggagaa tcctggccca gtgagcaagg gcgaggagct gttcaccggg gtggtgccca
6601 tcctggtcga gctggacggc gacgtaaacg gccacaagtt cagcgtgtcc ggcgagggcg
6661 agggcgatgc cacctacggc aagctgaccc tgaagttcat ctgcaccacc ggcaagctgc
6721 ccgtgccctg gcccaccctc gtgaccaccc tgacctacgg cgtgcagtgc ttcagccgct
6781 accccgacca catgaagcag cacgacttct tcaagtccgc catgcccgaa ggctacgtcc
6841 aggagcgcac catcttcttc aaggacgacg gcaactacaa gacccgcgcc gaggtgaagt
6901 tcgagggcga caccctggtg aaccgcatcg agctgaaggg catcgacttc aaggaggacg
6961 gcaacatcct ggggcacaag ctggagtaca actacaacag ccacaacgtc tatatcatgg
7021 ccgacaagca gaagaacggc atcaaggtga acttcaagat ccgccacaac atcgaggacg
7081 gcagcgtgca gctcgccgac cactaccagc agaacacccc catcggcgac ggccccgtgc
7141 tgctgcccga caaccactac ctgagcaccc agtccgccct gagcaaagac cccaacgaga
7201 agcgcgatca catggtcctg ctggagttcg tgaccgccgc cgggatcact ctcggcatgg
7261 acgagctgta caaggaattc aattctaact agagctcgct gatcagcctc gactgtgcct
7321 tctagttgcc agccatctgt tgtttgcccc tcccccgtgc cttccttgac cctggaaggt
7381 gccactccca ctgtcctttc ctaataaaat gaggaaattg catcgcattg tctgagtagg
7441 tgtcattcta ttctgggggg tggggtgggg caggacagca agggggagga ttgggaagag
7501 aatagcaggc atgctgggga gcggccgcag gaacccctag tgatggagtt ggccactccc
7561 tctctgcgcg ctcgctcgct cactgaggcc gggcgaccaa aggtcgcccg acgcccgggc
7621 tttgcccggg cggcctcagt gagcgagcga gcgcgcagct gcctgcaggg gcgcctgatg
7681 cggtattttc tccttacgca tctgtgcggt atttcacacc gcatacgtca aagcaaccat
7741 agtacgcgcc ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga
7801 ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct tcctttctcg
7861 ccacgttcgc cggctttccc cgtcaagctc taaatcgggg gctcccttta gggttccgat
7921 ttagtgcttt acggcacctc gaccccaaaa aacttgattt gggtgatggt tcacgtagtg
7981 ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg ttctttaata
8041 gtggactctt gttccaaact ggaacaacac tcaaccctat ctcgggctat tcttttgatt
8101 tataagggat tttgccgatt tcggcctatt ggttaaaaaa tgagctgatt taacaaaaat
8161 ttaacgcgaa ttttaacaaa atattaacgt ttacaatttt atggtgcact ctcagtacaa
8221 tctgctctga tgccgcatag ttaagccagc cccgacaccc gccaacaccc gctgacgcgc
8281 cctgacgggc ttgtctgctc ccggcatccg cttacagaca agctgtgacc gtctccggga
8341 gctgcatgtg tcagaggttt tcaccgtcat caccgaaacg cgcgagacga aagggcctcg
8401 tgatacgcct atttttatag gttaatgtca tgataataat ggtttcttag acgtcaggtg
8461 gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt atttttctaa atacattcaa
8521 atatgtatcc gctcatgaga caataaccct gataaatgct tcaataatat tgaaaaagga
8581 agagtatgag tattcaacat ttccgtgtcg cccttattcc cttttttgcg gcattttgcc
8641 ttcctgtttt tgctcaccca gaaacgctgg tgaaagtaaa agatgctgaa gatcagttgg
8701 gtgcacgagt gggttacatc gaactggatc tcaacagcgg taagatcctt gagagttttc
8761 gccccgaaga acgttttcca atgatgagca cttttaaagt tctgctatgt ggcgcggtat
8821 tatcccgtat tgacgccggg caagagcaac tcggtcgccg catacactat tctcagaatg
8881 acttggttga gtactcacca gtcacagaaa agcatcttac ggatggcatg acagtaagag
8941 aattatgcag tgctgccata accatgagtg ataacactgc ggccaactta cttctgacaa
9001 cgatcggagg accgaaggag ctaaccgctt ttttgcacaa catgggggat catgtaactc
9061 gccttgatcg ttgggaaccg gagctgaatg aagccatacc aaacgacgag cgtgacacca
9121 cgatgcctgt agcaatggca acaacgttgc gcaaactatt aactggcgaa ctacttactc
9181 tagcttcccg gcaacaatta atagactgga tggaggcgga taaagttgca ggaccacttc
9241 tgcgctcggc ccttccggct ggctggttta ttgctgataa atctggagcc ggtgagcgtg
9301 gaagccgcgg tatcattgca gcactggggc cagatggtaa gccctcccgt atcgtagtta
9361 tctacacgac ggggagtcag gcaactatgg atgaacgaaa tagacagatc gctgagatag
9421 gtgcctcact gattaagcat tggtaactgt cagaccaagt ttactcatat atactttaga
9481 ttgatttaaa acttcatttt taatttaaaa ggatctaggt gaagatcctt tttgataatc
9541 tcatgaccaa aatcccttaa cgtgagtttt cgttccactg agcgtcagac cccgtagaaa
9601 agatcaaagg atcttcttga gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa
9661 aaaaaccacc gctaccagcg gtggtttgtt tgccggatca agagctacca actctttttc
9721 cgaaggtaac tggcttcagc agagcgcaga taccaaatac tgtccttcta gtgtagccgt
9781 agttaggcca ccacttcaag aactctgtag caccgcctac atacctcgct ctgctaatcc
9841 tgttaccagt ggctgctgcc agtggcgata agtcgtgtct taccgggttg gactcaagac
9901 gatagttacc ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc acacagccca
9961 gcttggagcg aacgacctac accgaactga gatacctaca gcgtgagcta tgagaaagcg
10021 ccacgcttcc cgaagggaga aaggcggaca ggtatccggt aagcggcagg gtcggaacag
10081 gagagcgcac gagggagctt ccagggggaa acgcctggta tctttatagt cctgtcgggt
10141 ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg cggagcctat
10201 ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc cttttgctgg ccttttgctc
10261 acatgt
//
