质粒名称: | pTPGI_dCas9 |
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出品公司: | Addgene |
目录编号: | 64279 |
存储实验室: | Timothy Lu |
载体骨架基本信息 |
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载体名称: | pRS304 |
载体出品公司: | -- |
空载体大小: | -- |
完整质粒大小: | 10900 bp |
载体修饰: | -- |
载体类型: | 酵母表达 |
筛选标记: | TRP1 |
载体抗性: | 氨苄霉素 |
生长温度: | 37 ℃ |
宿主菌: | DH5a |
拷贝数: | 高拷贝 |
插入基因信息 |
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插入基因名称: | Yeast-optimized dCas9 |
物种来源: | S. cerevisiae (budding yeast) |
插入大小: | 4101 bp |
基因突变: | D10A和H840A与化脓链球菌Cas9的关系 |
GenBank编号 | -- |
启动子: | pTPGI |
标签/融合蛋白: | SV40 NLS(插入件上的N端子) |
克隆方法: | 限制性内切酶 |
5’克隆位点: | BamHI (未破坏) |
3’克隆位点: | SalI (未破坏) |
5’测序引物 | CAAATTAACAACCATAGGATGATAATGCG |
3’测序引物 | GTTACATGCGTACACGCGTCTGTACAG |
文献引用方法 |
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材料和方法部分: | pTPGI_dCas9 was a gift from Timothy Lu (Addgene plasmid # 64279 ; http://n2t.net/addgene:64279 ; RRID:Addgene_64279) |
参考文献部分: | Tunable and Multifunctional Eukaryotic Transcription Factors Based on CRISPR/Cas. Farzadfard F, Perli SD, Lu TK. ACS Synth Biol. 2013 Sep 11. 10.1021/sb400081r PubMed 23977949 |
买家导航
pTPGIdCas9载体基本信息
pTPGIdCas9载体图谱质粒图谱和多克隆位点信息
pTPGIdCas9载体简介
pTPGIdCas9载体序列
LOCUS pTPGI_dCas9 10815 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION encodes yeast-optimized dCas9 synthetic transcription factor. ACCESSION . VERSION . KEYWORDS pTPGI_dCas9 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 10815) AUTHORS Farzadfard F, Perli SD, Lu TK TITLE Tunable and Multifunctional Eukaryotic Transcription Factors Based on CRISPR/Cas. JOURNAL ACS Synth Biol. 2013 Sep 11. PUBMED 23977949 REFERENCE 2 (bases 1 to 10815) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..10815 /organism="synthetic DNA construct" /mol_type="other DNA" primer_bind complement(29..51) /label=pGEX 3' /note="pGEX vectors, reverse primer" primer_bind 151..170 /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" promoter 187..467 /gene="S. cerevisiae TRP1" /label=TRP1 promoter CDS 468..1142 /codon_start=1 /gene="S. cerevisiae TRP1" /product="phosphoribosylanthranilate isomerase, required for tryptophan biosynthesis" /label=TRP1 /note="yeast auxotrophic marker" /translation="MSVINFTGSSGPLVKVCGLQSTEAAECALDSDADLLGIICVPNRK RTIDPVIARKISSLVKAYKNSSGTPKYLVGVFRNQPKEDVLALVNDYGIDIVQLHGDES WQEYQEFLGLPVIKRLVFPKDCNILLSAASQKPHSFIPLFDSEAGGTGELLDWNSISDW VGRQESPESLHFMLAGGLTPENVGDALRLNGVIGVDVSGGVETNGVKDSNKIANFVKNA KK" rep_origin complement(1241..1696) /direction=LEFT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(1378..1399) /label=F1ori-F /note="F1 origin, forward primer" primer_bind 1590..1609 /label=F1ori-R /note="F1 origin, reverse primer" terminator 1779..1944 /gene="S. cerevisiae ADH1" /label=ADH1 terminator /note="transcription terminator for the S. cerevisiae alcohol dehydrogenase 1 (ADH1) gene" primer_bind 1865..1889 /label=pHybLex-R /note="ADH terminator, reverse primer" CDS complement(2088..2711) /codon_start=1 /gene="tetR from transposon Tn10" /product="tetracycline repressor TetR" /label=TetR /note="TetR binds to the tetracycline operator tetO to inhibit transcription. This inhibition can be relieved by adding tetracycline or doxycycline." /translation="MSRLDKSKVINSALELLNEVGIEGLTTRKLAQKLGVEQPTLYWHV KNKRALLDALAIEMLDRHHTHFCPLEGESWQDFLRNNAKSFRCALLSHRDGAKVHLGTR PTEKQYETLENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEDQEHQVAKEERETPTT DSMPPLLRQAIELFDHQGAEPAFLFGLELIICGLEKQLKCESGS" primer_bind 2619..2638 /label=Tet-R /note="Tetracycline resistance gene, reverse primer" promoter complement(2729..3127) /gene="S. cerevisiae TEF1" /label=TEF1 promoter /note="promoter for EF-1-alpha" promoter 3643..4084 /label=TPGI promoter /note="S. cerevisiae GAL1 promoter modified to contain two copies of the tet operator (Ellis et al., 2009)" protein_bind 3643..3760 /label=UAS /bound_moiety="Gal4" /note="upstream activating sequence mediating Gal4-dependent induction" protein_bind 3968..3986 /gene="tetO" /label=tet operator /bound_moiety="tetracycline repressor TetR" /note="bacterial operator O2 for the tetR and tetA genes" protein_bind 3989..4007 /gene="tetO" /label=tet operator /bound_moiety="tetracycline repressor TetR" /note="bacterial operator O2 for the tetR and tetA genes" primer_bind 4055..4078 /label=GAL1 /note="S. cerevisiae GAL1 promoter, forward primer" CDS 4124..4144 /codon_start=1 /product="nuclear localization signal of SV40 (simian virus 40) large T antigen" /label=SV40 NLS /translation="PKKKRKV" CDS 4154..8257 /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" terminator 8284..8531 /gene="S. cerevisiae CYC1" /label=CYC1 terminator /note="transcription terminator for CYC1" primer_bind complement(8300..8318) /label=CYC1 /note="CYC1 transcription termination signal, reverse primer" promoter complement(8555..8573) /label=T3 promoter /note="promoter for bacteriophage T3 RNA polymerase" primer_bind complement(8594..8610) /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind complement(8594..8610) /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" primer_bind complement(8607..8629) /label=M13/pUC Reverse /note="In lacZ gene" protein_bind 8618..8634 /label=lac operator /bound_moiety="lac repressor encoded by lacI" /note="The lac repressor binds to the lac operator to inhibit transcription in E. coli. This inhibition can be relieved by adding lactose or isopropyl-beta-D-thiogalactopyranoside (IPTG)." promoter complement(8642..8672) /label=lac promoter /note="promoter for the E. coli lac operon" primer_bind complement(8825..8842) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(8996..9584) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" CDS complement(9755..10615) /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 10378..10397 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(10616..10720) /gene="bla" /label=AmpR promoter primer_bind 10788..10806 /label=pBRforEco /note="pBR322 vectors, upsteam of EcoRI site, forward primer" ORIGIN 1 tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca 61 cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg 121 ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc 181 accataaacg acattactat atatataata taggaagcat ttaatagaca gcatcgtaat 241 atatgtgtac tttgcagtta tgacgccaga tggcagtagt ggaagatatt ctttattgaa 301 aaatagcttg tcaccttacg tacaatcttg atccggagct tttctttttt tgccgattaa 361 gaattaattc ggtcgaaaaa agaaaaggag agggccaaga gggagggcat tggtgactat 421 tgagcacgtg agtatacgtg attaagcaca caaaggcagc ttggagtatg tctgttatta 481 atttcacagg tagttctggt ccattggtga aagtttgcgg cttgcagagc acagaggccg 541 cagaatgtgc tctagattcc gatgctgact tgctgggtat tatatgtgtg cccaatagaa 601 agagaacaat tgacccggtt attgcaagga aaatttcaag tcttgtaaaa gcatataaaa 661 atagttcagg cactccgaaa tacttggttg gcgtgtttcg taatcaacct aaggaggatg 721 ttttggctct ggtcaatgat tacggcattg atatcgtcca actgcatgga gatgagtcgt 781 ggcaagaata ccaagagttc ctcggtttgc cagttattaa aagactcgta tttccaaaag 841 actgcaacat actactcagt gcagcttcac agaaacctca ttcgtttatt cccttgtttg 901 attcagaagc aggtgggaca ggtgaacttt tggattggaa ctcgatttct gactgggttg 961 gaaggcaaga gagccccgaa agcttacatt ttatgttagc tggtggactg acgccagaaa 1021 atgttggtga tgcgcttaga ttaaatggcg ttattggtgt tgatgtaagc ggaggtgtgg 1081 agacaaatgg tgtaaaagac tctaacaaaa tagcaaattt cgtcaaaaat gctaagaaat 1141 aggttattac tgagtagtat ttatttaagt attgtttgtg cacttgccta tgcggtgtga 1201 aataccgcac agatgcgtaa ggagaaaata ccgcatcagg aaattgtaaa cgttaatatt 1261 ttgttaaaat tcgcgttaaa tttttgttaa atcagctcat tttttaacca ataggccgaa 1321 atcggcaaaa tcccttataa atcaaaagaa tagaccgaga tagggttgag tgttgttcca 1381 gtttggaaca agagtccact attaaagaac gtggactcca acgtcaaagg gcgaaaaacc 1441 gtctatcagg gcgatggccc actacgtgaa ccatcaccct aatcaagttt tttggggtcg 1501 aggtgccgta aagcactaaa tcggaaccct aaagggagcc cccgatttag agcttgacgg 1561 ggaaagccgg cgaacgtggc gagaaaggaa gggaagaaag cgaaaggagc gggcgctagg 1621 gcgctggcaa gtgtagcggt cacgctgcgc gtaaccacca cacccgccgc gcttaatgcg 1681 ccgctacagg gcgcgtcgcg ccattcgcca ttcaggctgc gcaactgttg ggaagggcga 1741 tcggtgcggg cctcttcgct attacgccag ctgaattgga gcgacctcat gctatacctg 1801 agaaagcaac ctgacctaca ggaaagagtt actcaagaat aagaattttc gttttaaaac 1861 ctaagagtca ctttaaaatt tgtatacact tatttttttt ataacttatt taataataaa 1921 aatcataaat cataagaaat tcgcttattt agaagtgtca acaacgtatc taccaacgat 1981 ttgacccttt tccatctttt cgtaaatttc tggcaaggta gacaagccga caaccttgat 2041 tggagacttg accaaacctc tggcgaagaa ttgttaatta agagctctta agacccactt 2101 tcacatttaa gttgtttttc taatccgcat atgatcaatt caaggccgaa taagaaggct 2161 ggctctgcac cttggtgatc aaataattcg atagcttgtc gtaataatgg cggcatacta 2221 tcagtagtag gtgtttccct ttcttcttta gcgacttgat gctcttgatc ttccaatacg 2281 caacctaaag taaaatgccc cacagcgctg agtgcatata atgcattctc tagtgaaaaa 2341 ccttgttggc ataaaaaggc taattgattt tcgagagttt catactgttt ttctgtaggc 2401 cgtgtaccta aatgtacttt tgctccatcg cgatgactta gtaaagcaca tctaaaactt 2461 ttagcgttat tacgtaaaaa atcttgccag ctttcccctt ctaaagggca aaagtgagta 2521 tggtgcctat ctaacatctc aatggctaag gcgtcgagca aagcccgctt attttttaca 2581 tgccaataca atgtaggctg ctctacacct agcttctggg cgagtttacg ggttgttaaa 2641 ccttcgattc cgacctcatt aagcagctct aatgcgctgt taatcacttt acttttatct 2701 aatctagaca ttttaataac tagtaaaact tagattagat tgctatgctt tctttctaat 2761 gagcaagaag taaaaaaagt tgtaatagaa caagaaaaat gaaactgaaa cttgagaaat 2821 tgaagaccgt ttattaactt aaatatcaat gggaggtcat cgaaagagaa aaaaatcaaa 2881 aaaaaaattt tcaagaaaaa gaaacgtgat aaaaattttt attgcctttt tcgacgaaga 2941 aaaagaaacg aggcggtctc ttttttcttt tccaaacctt tagtacgggt aattaacgac 3001 accctagagg aagaaagagg ggaaatttag tatgctgtgc ttgggtgttt tgaagtggta 3061 cggcgatgcg cggagtccga gaaaatctgg aagagtaaaa aaggagtaga aacattttga 3121 agctatggtg tggcggccgc atcttgactg atttttccat ggagggcaca gttaagccgc 3181 taaaggcatt atccgccaag tacaattttt tactcttcga agacagaaaa tttgctgaca 3241 ttggtaatac agtcaaattg cagtactctg cgggtgtata cagaatagca gaatgggcag 3301 acattacgaa tgcacacggt gtggtgggcc caggtattgt tagcggtttg aagcaggcgg 3361 cagaagaagt aacaaaggaa cctagaggcc ttttgatgtt agcagaattg tcatgcaagg 3421 gctccctatc tactggagaa tatactaagg gtactgttga cattgcgaag agcgacaaag 3481 attttgttat cggctttatt gctcaaagag acatgggtgg aagagatgaa ggttacgatt 3541 ggttgattat gacacccggt gtgggtttag atgacaaggg agacgcattg ggtcaacagt 3601 atagaaccgt ggatgatgtg gtctctacag gaattcgaag tacggattag aagccgccga 3661 gcgggtgaca gccctccgaa ggaagactct cctccgtgcg tcctcgtctt caccggtcgc 3721 gttcctgaaa cgcagatgtg cctcgcgccg cactgctccg aacaataaag attctacaat 3781 actagctttt atggttatga agaggaaaaa ttggcagtaa cctggcccca caaaccttca 3841 aatgaacgaa tcaaattaac aaccatagga tgataatgcg attagttttt tagccttatt 3901 tctggggtac tgcagcagcg aagcgatgat ttttgatcta ttaacagata tataaatgca 3961 aaaactgtcc ctatcagtga tagagatctc cctatcagtg atagagatat tacttcttat 4021 tcaaatgtaa taaaagtatc aacaaaaaat tgttaatata cctctatact ttaacgtcaa 4081 ggagaaaaac cccggatcca cgattaaaag aatgtctaga gccccaaaga agaagagaaa 4141 agttagaccc ggggataaga aatactctat tggtttggct atcggtacaa actctgttgg 4201 ttgggctgtt attactgatg aatacaaggt tccatccaag aagttcaagg ttttgggtaa 4261 cactgataga cactccatca aaaagaactt gattggtgcc ttgttgttcg attctggtga 4321 aactgctgaa gctactagat tgaaaagaac cgctagaaga agatacacca gaagaaagaa 4381 cagaatctgc tacttgcaag aaatcttctc caacgaaatg gccaaggttg atgattcatt 4441 cttccacaga ttggaagaat ccttcttggt cgaagaagat aagaagcacg aaagacatcc 4501 aatcttcggt aacatcgttg atgaagttgc ttaccacgaa aagtacccaa ctatctacca 4561 tttgagaaag aagttggttg actctaccga taaggctgat ttgagattga tctatttggc 4621 tttggcccac atgattaagt tcagaggtca tttcttgatc gaaggtgatt tgaacccaga 4681 taactccgat gttgataagt tgttcatcca attagtccaa acctacaatc aattattcga 4741 agaaaaccca atcaacgcct ctggtgttga tgctaaagct attttgtctg ccagattgtc 4801 caagtccaga agattagaaa atttgatcgc ccaattacca ggtgaaaaga agaatggttt 4861 gttcggtaat ttgattgcct tgtctttggg tttgactcca aacttcaagt ccaatttcga 4921 tttggctgaa gatgccaagt tgcaattatc taaggatacc tacgatgacg atttggataa 4981 cttgttggct caaatcggtg atcaatacgc tgatttgttt ttggctgcta agaacttgtc 5041 cgatgccatt ttgttgtccg atattttgag agtcaacacc gaaattacta aggctccatt 5101 gtctgcctct atgatcaaaa gatacgatga acaccaccaa gacttgactt tgttgaaggc 5161 tttggtcaga caacaattac ctgaaaagta caaagaaatt ttcttcgatc aatccaagaa 5221 cggttacgcc ggttatattg atggtggtgc ttctcaagaa gaattttaca agttcatcaa 5281 gccaatcttg gaaaagatgg acggtactga agaattattg gtcaagttga acagagaaga 5341 tttgttgaga aagcaaagaa ccttcgacaa cggttctatt ccacatcaaa ttcacttggg 5401 tgaattgcac gcaattttga gaagacaaga agatttttat ccattcttga aggacaacag 5461 agaaaagatc gaaaagattc tgaccttcag aatcccttac tacgttggtc cattggctag 5521 aggtaattca agatttgcct ggatgactag aaagtccgaa gaaactatta ctccttggaa 5581 cttcgaagaa gttgtagata agggtgcttc tgcccaatcc tttattgaaa gaatgaccaa 5641 cttcgacaag aacttgccaa acgaaaaggt tttgccaaag cactctttgt tgtacgaata 5701 cttcaccgtc tacaacgaat tgactaaggt taagtacgtc accgaaggta tgagaaaacc 5761 agctttttta tccggtgaac aaaagaaggc tatcgtcgat ttgttgttca agaccaacag 5821 aaaggttact gtcaagcaat taaaagaaga ttacttcaag aaaatcgaat gcttcgactc 5881 cgttgaaatt tctggtgtcg aagatagatt caatgcctct ttaggtactt accatgactt 5941 gttgaaaatc atcaaggaca aggatttctt ggacaacgaa gaaaacgaag atattttgga 6001 agatattgtc ttgacattga ccttgtttga agatagagaa atgattgaag aaagattgaa 6061 aacctacgcc cacttgttcg atgataaggt tatgaagcaa ttaaagagaa gaagatacac 6121 tggttggggt agattgtcca gaaaattgat taacggtatc agagacaagc aatccggtaa 6181 gaccattttg gactttttga agtctgatgg tttcgctaac agaaacttca tgcaattaat 6241 ccacgacgat tccttgactt tcaaagaaga tatacaaaag gcccaagtct ctggtcaagg 6301 tgattcttta catgaacata tcgctaactt ggctggttct ccagctatta agaagggtat 6361 tttacaaacc gttaaggtcg ttgacgaatt ggtcaaagtt atgggtagac ataagccaga 6421 aaacatcgtt atcgaaatgg ctagagaaaa tcaaaccacc caaaagggtc aaaagaactc 6481 cagagaaaga atgaagagaa tcgaagaagg tatcaaagaa ttgggttccc aaattttgaa 6541 agaacaccca gttgaaaaca cccaattaca aaacgaaaag ttgtacttgt actacttgca 6601 aaacggtaga gatatgtacg ttgaccaaga attggacatc aacagattgt ctgattacga 6661 tgttgacgct atcgttccac aatctttttt gaaggatgac tccattgaca acaaggtctt 6721 gactagatcc gataagaata gaggtaagtc cgataacgtt ccatctgaag aagtcgttaa 6781 gaaaatgaag aactattgga gacaattatt gaacgccaag ttgatcaccc aaagaaagtt 6841 tgacaatttg accaaggctg aaagaggtgg tttgtctgaa ttggataagg caggtttcat 6901 caaaagacaa ttagtagaaa ccagacaaat caccaagcac gttgctcaaa ttttggatag 6961 tagaatgaac actaagtacg acgaaaacga caaattgatc agagaagtta aggtcattac 7021 cttgaagtcc aagttggttt ccgatttcag aaaggacttc caattctaca aggtcagaga 7081 aatcaacaac taccatcatg cacatgatgc ttacttgaat gctgttgttg gtactgcctt 7141 gattaagaag tatccaaagt tggaatccga atttgtctac ggtgattaca aggtttacga 7201 cgttagaaag atgatcgcca agtccgaaca agaaattggt aaagctactg ccaaatactt 7261 cttctactcc aatattatga atttctttaa gaccgaaatc actttggcca acggtgaaat 7321 tagaaaaaga ccattgattg aaactaatgg tgaaacaggt gaaatcgttt gggataaggg 7381 tagagatttt gccactgtta gaaaggtatt gtccatgcca caagtaaaca tcgtcaaaaa 7441 gaccgaagtt caaactggtg gtttctccaa agaatccatt ttgcctaaga gaaactccga 7501 taagttgatc gctagaaaaa aagactggga cccaaaaaag tacggtggtt ttgattctcc 7561 aactgttgct tactctgttt tggttgttgc taaggtcgaa aagggtaaga gtaagaagtt 7621 gaagtccgtc aaagaattat taggtatcac tatcatggaa agatcctcat tcgaaaagaa 7681 tcctatcgac tttttggaag ccaagggtta caaagaagtc aagaaggact tgatcattaa 7741 gttgccaaag tacagtttgt tcgaattgga aaatggtaga aagagaatgt tggcttctgc 7801 cggtgaatta caaaagggta atgaattggc tttgccatcc aagtacgtta atttcttata 7861 cttggcctcc cactacgaaa aattgaaagg ttctcctgaa gataacgaac aaaagcaatt 7921 atttgtcgaa caacacaagc actacttgga cgaaatcatt gaacaaattt ccgaattttc 7981 caaaagagtc attttggctg acgccaattt ggacaaagtt ttgtcagctt acaacaagca 8041 cagagataag ccaattagag aacaagctga aaacatcatt cacttgttca ctttgactaa 8101 cttgggtgct ccagctgctt ttaagtattt cgataccact atcgacagaa agagatacac 8161 ctctaccaaa gaagttttgg acgctacttt gatccaccaa tctattactg gtttgtacga 8221 aactagaatc gacttgtctc aattaggtgg tgattaatag gtcgacggtc ctaggaactc 8281 gagtcatgta attagttatg tcacgcttac attcacgccc tccccccaca tccgctctaa 8341 ccgaaaagga aggagttaga caacctgaag tctaggtccc tatttatttt tttatagtta 8401 tgttagtatt aagaacgtta tttatatttc aaatttttct tttttttctg tacagacgcg 8461 tgtacgcatg taacattata ctgaaaacct tgcttgagaa ggttttggga cgctcgaagg 8521 ctttaatttg cggcccggta cccagctttt gttcccttta gtgagggtta attccgagct 8581 tggcgtaatc atggtcatag ctgtttcctg tgtgaaattg ttatccgctc acaattccac 8641 acaacatagg agccggaagc ataaagtgta aagcctgggg tgcctaatga gtgaggtaac 8701 tcacattaat tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg tcgtgccagc 8761 tgcattaatg aatcggccaa cgcgcgggga gaggcggttt gcgtattggg cgctcttccg 8821 cttcctcgct cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc 8881 actcaaaggc ggtaatacgg ttatccacag aatcagggga taacgcagga aagaacatgt 8941 gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc 9001 ataggctcgg cccccctgac gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa 9061 acccgacagg actataaaga taccaggcgt tcccccctgg aagctccctc gtgcgctctc 9121 ctgttccgac cctgccgctt accggatacc tgtccgcctt tctcccttcg ggaagcgtgg 9181 cgctttctca atgctcacgc tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc 9241 tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc ggtaactatc 9301 gtcttgagtc caacccggta agacacgact tatcgccact ggcagcagcc actggtaaca 9361 ggattagcag agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg tggcctaact 9421 acggctacac tagaaggaca gtatttggta tctgcgctct gctgaagcca gttaccttcg 9481 gaaaaagagt tggtagctct tgatccggca aacaaaccac cgctggtagc ggtggttttt 9541 ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat cctttgatct 9601 tttctacggg gtctgacgct cagtggaacg aaaactcacg ttaagggatt ttggtcatga 9661 gattatcaaa aaggatcttc acctagatcc ttttaaatta aaaatgaagt tttaaatcaa 9721 tctaaagtat atatgagtaa acttggtctg acagttacca atgcttaatc agtgaggcac 9781 ctatctcagc gatctgtcta tttcgttcat ccatagttgc ctgactgccc gtcgtgtaga 9841 taactacgat acgggagggc ttaccatctg gccccagtgc tgcaatgata ccgcgagacc 9901 cacgctcacc ggctccagat ttatcagcaa taaaccagcc agccggaagg gccgagcgca 9961 gaagtggtcc tgcaacttta tccgcctcca tccagtctat taattgttgc cgggaagcta 10021 gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt tgccattgct acaggcatcg 10081 tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc cggttcccaa cgatcaaggc 10141 gagttacatg atcccccatg ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg 10201 ttgtcagaag taagttggcc gcagtgttat cactcatggt tatggcagca ctgcataatt 10261 ctcttactgt catgccatcc gtaagatgct tttctgtgac tggtgagtac tcaaccaagt 10321 cattctgaga atagtgtatg cggcgaccga gttgctcttg cccggcgtca atacgggata 10381 ataccgcgcc acatagcaga actttaaaag tgctcatcat tggaaaacgt tcttcggggc 10441 gaaaactctc aaggatctta ccgctgttga gatccagttc gatgtaaccc actcgtgcac 10501 ccaactgatc ttcagcatct tttactttca ccagcgtttc tgggtgagca aaaacaggaa 10561 ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa atgttgaata ctcatactct 10621 tcctttttca atattattga agcatttatc agggttattg tctcatgagc ggatacatat 10681 ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg cacatttccc cgaaaagtgc 10741 cacctgacgt ctaagaaacc attattatca tgacattaac ctataaaaat aggcgtatca 10801 cgaggccctt tcgtc //
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