LOCUS Exported 10757 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION encodes yeast-optimized dCas9_VP64 synthetic transcription factor. ACCESSION . VERSION . KEYWORDS pTPGI_dCas9_VP64 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 10757) 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 10757) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..10757 /organism="synthetic DNA construct" /mol_type="other DNA" promoter 235..676 /label=TPGI promoter /note="S. cerevisiae GAL1 promoter modified to contain two copies of the tet operator (Ellis et al., 2009)" protein_bind 235..352 /label=UAS /bound_moiety="Gal4" /note="upstream activating sequence mediating Gal4-dependent induction" protein_bind 560..578 /gene="tetO" /label=tet operator /bound_moiety="tetracycline repressor TetR" /note="bacterial operator O2 for the tetR and tetA genes" protein_bind 581..599 /gene="tetO" /label=tet operator /bound_moiety="tetracycline repressor TetR" /note="bacterial operator O2 for the tetR and tetA genes" primer_bind 647..670 /label=GAL1 /note="S. cerevisiae GAL1 promoter, forward primer" CDS 716..736 /codon_start=1 /product="nuclear localization signal of SV40 (simian virus 40) large T antigen" /label=SV40 NLS /translation="PKKKRKV" CDS 746..4846 /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 4883..5032 /codon_start=1 /product="tetrameric repeat of the minimal activation domain of herpes simplex virus VP16 (Beerli et al., 1998)" /label=VP64 /translation="DALDDFDLDMLGSDALDDFDLDMLGSDALDDFDLDMLGSDALDDF DLDML" primer_bind complement(5064..5082) /label=CYC1 /note="CYC1 transcription termination signal, reverse primer" terminator 5094..5283 /gene="S. cerevisiae CYC1" /label=CYC1 terminator /note="transcription terminator for CYC1" primer_bind complement(5355..5372) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(5526..6114) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(5606..5625) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS complement(6285..7145) /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 6908..6927 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(7146..7250) /gene="bla" /label=AmpR promoter primer_bind 7318..7336 /label=pBRforEco /note="pBR322 vectors, upsteam of EcoRI site, forward primer" primer_bind complement(7374..7396) /label=pGEX 3' /note="pGEX vectors, reverse primer" primer_bind 7496..7515 /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" promoter 7536..7817 /gene="S. cerevisiae TRP1" /label=TRP1 promoter CDS 7818..8492 /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(8595..9050) /direction=LEFT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(8732..8753) /label=F1ori-F /note="F1 origin, forward primer" primer_bind 8944..8963 /label=F1ori-R /note="F1 origin, reverse primer" terminator 9128..9293 /gene="S. cerevisiae ADH1" /label=ADH1 terminator /note="transcription terminator for the S. cerevisiae alcohol dehydrogenase 1 (ADH1) gene" primer_bind 9214..9238 /label=pHybLex-R /note="ADH terminator, reverse primer" CDS complement(9437..10060) /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 9968..9987 /label=Tet-R /note="Tetracycline resistance gene, reverse primer" promoter complement(10078..10476) /gene="S. cerevisiae TEF1" /label=TEF1 promoter /note="promoter for EF-1-alpha" ORIGIN 1 tgtcatgcaa gggctcccta tctactggag aatatactaa gggtactgtt gacgttgcga 61 agagcgacaa agattttgtt atcagcttta ttgctcaaag agacatgggt ggaagagatg 121 aaggttacga ttggttgatt atgacacccg gtgtgggttt agatgacaag ggagacgcat 181 tgggtcaaca gtatagaacc gtggatgatg tggtctctac aggaattcga agtacggatt 241 agaagccgcc gagcgggtga cagccctccg aaggaagact ctcctccgtg cgtcctcgtc 301 ttcaccggtc gcgttcctga aacgcagatg tgcctcgcgc cgcactgctc cgaacaataa 361 agattctaca atactagctt ttatggttat gaagaggaaa aattggcagt aacctggccc 421 cacaaacctt caaatgaacg aatcaaatta acaaccatag gatgataatg cgattacttt 481 tttagcctta tttctggggt actgcagcag cgaagcgatg atttttgatc tattaacaga 541 tatataaatg caaaaactgt ccctatcagt gatagagatc tccctatcag tgatagagat 601 attacttctt attcaaatgt aataaaagta tcaacaaaaa attgttaata tacctctata 661 ctttaacgtc aaggagaaaa accccggatc cacgattaaa agaatgtcta gagccccaaa 721 gaagaagaga aaagttagac ccggggataa gaaatactct attggtttgg ctatcggtac 781 aaactctgtt ggttgggctg ttattactga tgaatacaag gttccatcca agaagttcaa 841 ggttttgggt aacactgata gacactccat caaaaagaac ttgattggtg ccttgttgtt 901 cgattctggt gaaactgctg aagctactag attgaaaaga accgctagaa gaagatacac 961 cagaagaaag aacagaatct gctacttgca agaaatcttc tccaacgaaa tggccaaggt 1021 tgatgattca ttcttccaca gattggaaga atccttcttg gtcgaagaag ataagaagca 1081 cgaaagacat ccaatcttcg gtaacatcgt tgatgaagtt gcttaccacg aaaagtaccc 1141 aactatctac catttgagaa agaagttggt tgactctacc gataaggctg atttgagatt 1201 gatctatttg gctttggccc acatgattaa gttcagaggt catttcttga tcgaaggtga 1261 tttgaaccca gataactccg atgttgataa gttgttcatc caattagtcc aaacctacaa 1321 tcaattattc gaagaaaacc caatcaacgc ctctggtgtt gatgctaaag ctattttgtc 1381 tgccagattg tccaagtcca gaagattaga aaatttgatc gcccaattac caggtgaaaa 1441 gaagaatggt ttgttcggta atttgattgc cttgtctttg ggtttgactc caaacttcaa 1501 gtccaatttc gatttggctg aagatgccaa gttgcaatta tctaaggata cctacgatga 1561 cgatttggat aacttgttgg ctcaaatcgg tgatcaatac gctgatttgt ttttggctgc 1621 taagaacttg tccgatgcca ttttgttgtc cgatattttg agagtcaaca ccgaaattac 1681 taaggctcca ttgtctgcct ctatgatcaa aagatacgat gaacaccacc aagacttgac 1741 tttgttgaag gctttggtca gacaacaatt acctgaaaag tacaaagaaa ttttcttcga 1801 tcaatccaag aacggttacg ccggttatat tgatggtggt gcttctcaag aagaatttta 1861 caagttcatc aagccaatct tggaaaagat ggacggtact gaagaattat tggtcaagtt 1921 gaacagagaa gatttgttga gaaagcaaag aaccttcgac aacggttcta ttccacatca 1981 aattcacttg ggtgaattgc acgcaatttt gagaagacaa gaagattttt atccattctt 2041 gaaggacaac agagaaaaga tcgaaaagat tctgaccttc agaatccctt actacgttgg 2101 tccattggct agaggtaatt caagatttgc ctggatgact agaaagtccg aagaaactat 2161 tactccttgg aacttcgaag aagttgtaga taagggtgct tctgcccaat cctttattga 2221 aagaatgacc aacttcgaca agaacttgcc aaacgaaaag gttttgccaa agcactcttt 2281 gttgtacgaa tacttcaccg tctacaacga attgactaag gttaagtacg tcaccgaagg 2341 tatgagaaaa ccagcttttt tatccggtga acaaaagaag gctatcgtcg atttgttgtt 2401 caagaccaac agaaaggtta ctgtcaagca attaaaagaa gattacttca agaaaatcga 2461 atgcttcgac tccgttgaaa tttctggtgt cgaagataga ttcaatgcct ctttaggtac 2521 ttaccatgac ttgttgaaaa tcatcaagga caaggatttc ttggacaacg aagaaaacga 2581 agatattttg gaagatattg tcttgacatt gaccttgttt gaagatagag aaatgattga 2641 agaaagattg aaaacctacg cccacttgtt cgatgataag gttatgaagc aattaaagag 2701 aagaagatac actggttggg gtagattgtc cagaaaattg attaacggta tcagagacaa 2761 gcaatccggt aagaccattt tggacttttt gaagtctgat ggtttcgcta acagaaactt 2821 catgcaatta atccacgacg attccttgac tttcaaagaa gatatacaaa aggcccaagt 2881 ctctggtcaa ggtgattctt tacatgaaca tatcgctaac ttggctggtt ctccagctat 2941 taagaagggt attttacaaa ccgttaaggt cgttgacgaa ttggtcaaag ttatgggtag 3001 acataagcca gaaaacatcg ttatcgaaat ggctagagaa aatcaaacca cccaaaaggg 3061 tcaaaagaac tccagagaaa gaatgaagag aatcgaagaa ggtatcaaag aattgggttc 3121 ccaaattttg aaagaacacc cagttgaaaa cacccaatta caaaacgaaa agttgtactt 3181 gtactacttg caaaacggta gagatatgta cgttgaccaa gaattggaca tcaacagatt 3241 gtctgattac gatgttgacg ctatcgttcc acaatctttt ttgaaggatg actccattga 3301 caacaaggtc ttgactagat ccgataagaa tagaggtaag tccgataacg ttccatctga 3361 agaagtcgtt aagaaaatga agaactattg gagacaatta ttgaacgcca agttgatcac 3421 ccaaagaaag tttgacaatt tgaccaaggc tgaaagaggt ggtttgtctg aattggataa 3481 ggcaggtttc atcaaaagac aattagtaga aaccagacaa atcaccaagc acgttgctca 3541 aattttggat agtagaatga acactaagta cgacgaaaac gacaaattga tcagagaagt 3601 taaggtcatt accttgaagt ccaagttggt ttccgatttc agaaaggact tccaattcta 3661 caaggtcaga gaaatcaaca actaccatca tgcacatgat gcttacttga atgctgttgt 3721 tggtactgcc ttgattaaga agtatccaaa gttggaatcc gaatttgtct acggtgatta 3781 caaggtttac gacgttagaa agatgatcgc caagtccgaa caagaaattg gtaaagctac 3841 tgccaaatac ttcttctact ccaatattat gaatttcttt aagaccgaaa tcactttggc 3901 caacggtgaa attagaaaaa gaccattgat tgaaactaat ggtgaaacag gtgaaatcgt 3961 ttgggataag ggtagagatt ttgccactgt tagaaaggta ttgtccatgc cacaagtaaa 4021 catcgtcaaa aagaccgaag ttcaaactgg tggtttctcc aaagaatcca ttttgcctaa 4081 gagaaactcc gataagttga tcgctagaaa aaaagactgg gacccaaaaa agtacggtgg 4141 ttttgattct ccaactgttg cttactctgt tttggttgtt gctaaggtcg aaaagggtaa 4201 gagtaagaag ttgaagtccg tcaaagaatt attaggtatc actatcatgg aaagatcctc 4261 attcgaaaag aatcctatcg actttttgga agccaagggt tacaaagaag tcaagaagga 4321 cttgatcatt aagttgccaa agtacagttt gttcgaattg gaaaatggta gaaagagaat 4381 gttggcttct gccggtgaat tacaaaaggg taatgaattg gctttgccat ccaagtacgt 4441 taatttctta tacttggcct cccactacga aaaattgaaa ggttctcctg aagataacga 4501 acaaaagcaa ttatttgtcg aacaacacaa gcactacttg gacgaaatca ttgaacaaat 4561 ttccgaattt tccaaaagag tcattttggc tgacgccaat ttggacaaag ttttgtcagc 4621 ttacaacaag cacagagata agccaattag agaacaagct gaaaacatca ttcacttgtt 4681 cactttgact aacttgggtg ctccagctgc ttttaagtat ttcgatacca ctatcgacag 4741 aaagagatac acctctacca aagaagtttt ggacgctact ttgatccacc aatctattac 4801 tggtttgtac gaaactagaa tcgacttgtc tcaattaggt ggtgatggtt ctggtagatc 4861 tggagtcgac ggtggaggtt ctgacgcttt ggacgacttc gacttggata tgctgggttc 4921 tgatgcgcta gatgactttg acctcgacat gcttggaagt gacgccttag atgattttga 4981 cctggatatg cttggatcag acgctctgga cgatttcgac ttagacatgc tttcctagga 5041 actcgagtca tgtaattagt tatgtcacgc ttacattcac gcccttcccc cacatccgct 5101 ctaaccgaaa aggaaggagt tagacaacct gaagtctagg tccctattta tttttttata 5161 gttatgttag tattaagaac gttatttata tttcaaattt ttcttttttt tctgtacaga 5221 cgcgtgtacg catgtaacat tatactgaaa accttgcttg agaaggtttt gggacgctcg 5281 aagatccagc tgcattaatg aatcggccaa cgcgcgggga gaggcggttt gcgtattggg 5341 cgctcttccg cttcctcgct cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg 5401 gtatcagctc actcaaaggc ggtaatacgg ttatccacag aatcagggga taacgcagga 5461 aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg 5521 gcgtttttcc ataggctccg cccccctgac gagcatcaca aaaatcgacg ctcaagtcag 5581 aggtggcgaa acccgacagg actataaaga taccaggcgt ttccccctgg aagctccctc 5641 gtgcgctctc ctgttccgac cctgccgctt accggatacc tgtccgcctt tctcccttcg 5701 ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt 5761 cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc 5821 ggtaactatc gtcttgagtc caacccggta agacacgact tatcgccact ggcagcagcc 5881 actggtaaca ggattagcag agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg 5941 tggcctaact acggctacac tagaagaaca gtatttggta tctgcgctct gctgaagcca 6001 gttaccttcg gaaaaagagt tggtagctct tgatccggca aacaaaccac cgctggtagc 6061 ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat 6121 cctttgatct tttctacggg gtctgacgct cagtggaacg aaaactcacg ttaagggatt 6181 ttggtcatga gattatcaaa aaggatcttc acctagatcc ttttaaatta aaaatgaagt 6241 tttaaatcaa tctaaagtat atatgagtaa acttggtctg acagttacca atgcttaatc 6301 agtgaggcac ctatctcagc gatctgtcta tttcgttcat ccatagttgc ctgactcccc 6361 gtcgtgtaga taactacgat acgggagggc ttaccatctg gccccagtgc tgcaatgata 6421 ccgcgagacc cacgctcacc ggctccagat ttatcagcaa taaaccagcc agccggaagg 6481 gccgagcgca gaagtggtcc tgcaacttta tccgcctcca tccagtctat taattgttgc 6541 cgggaagcta gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt tgccattgct 6601 acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc cggttcccaa 6661 cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa aagcggttag ctccttcggt 6721 cctccgatcg ttgtcagaag taagttggcc gcagtgttat cactcatggt tatggcagca 6781 ctgcataatt ctcttactgt catgccatcc gtaagatgct tttctgtgac tggtgagtac 6841 tcaaccaagt cattctgaga atagtgtatg cggcgaccga gttgctcttg cccggcgtca 6901 atacgggata ataccgcgcc acatagcaga actttaaaag tgctcatcat tggaaaacgt 6961 tcttcggggc gaaaactctc aaggatctta ccgctgttga gatccagttc gatgtaaccc 7021 actcgtgcac ccaactgatc ttcagcatct tttactttca ccagcgtttc tgggtgagca 7081 aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa atgttgaata 7141 ctcatactct tcctttttca atattattga agcatttatc agggttattg tctcatgagc 7201 ggatacatat ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg cacatttccc 7261 cgaaaagtgc cacctgacgt ctaagaaacc attattatca tgacattaac ctataaaaat 7321 aggcgtatca cgaggccctt tcgtctcgcg cgtttcggtg atgacggtga aaacctctga 7381 cacatgcagc tcccggagac ggtcacagct tgtctgtaag cggatgccgg gagcagacaa 7441 gcccgtcagg gcgcgtcagc gggtgttggc gggtgtcggg gctggcttaa ctatgcggca 7501 tcagagcaga ttgtactgag agtgcaccat agatcaacga cattactata tatataatat 7561 aggaagcatt taatagaaca gcatcgtaat atatgtgtac tttgcagtta tgacgccaga 7621 tggcagtagt ggaagatatt ctttattgaa aaatagcttg tcaccttacg tacaatcttg 7681 atccggagct tttctttttt tgccgattaa gaattaattc ggtcgaaaaa agaaaaggag 7741 agggccaaga gggagggcat tggtgactat tgagcacgtg agtatacgtg attaagcaca 7801 caaaggcagc ttggagtatg tctgttatta atttcacagg tagttctggt ccattggtga 7861 aagtttgcgg cttgcagagc acagaggccg cagaatgtgc tctagattcc gatgctgact 7921 tgctgggtat tatatgtgtg cccaatagaa agagaacaat tgacccggtt attgcaagga 7981 aaatttcaag tcttgtaaaa gcatataaaa atagttcagg cactccgaaa tacttggttg 8041 gcgtgtttcg taatcaacct aaggaggatg ttttggctct ggtcaatgat tacggcattg 8101 atatcgtcca actgcatgga gatgagtcgt ggcaagaata ccaagagttc ctcggtttgc 8161 cagttattaa aagactcgta tttccaaaag actgcaacat actactcagt gcagcttcac 8221 agaaacctca ttcgtttatt cccttgtttg attcagaagc aggtgggaca ggtgaacttt 8281 tggattggaa ctcgatttct gactgggttg gaaggcaaga gagccccgaa agcttacatt 8341 ttatgttagc tggtggactg acgccagaaa atgttggtga tgcgcttaga ttaaatggcg 8401 ttattggtgt tgatgtaagc ggaggtgtgg agacaaatgg tgtaaaagac tctaacaaaa 8461 tagcaaattt cgtcaaaaat gctaagaaat aggttattac tgagtagtat ttatttaagt 8521 attgtttgtg cacttgccga tctatgcggt gtgaaatacc gcacagatgc gtaaggagaa 8581 aataccgcat caggaaattg taagcgttaa tattttgtta aaattcgcgt taaatttttg 8641 ttaaatcagc tcatttttta accaataggc cgaaatcggc aaaatccctt ataaatcaaa 8701 agaatagacc gagatagggt tgagtgttgt tccagtttgg aacaagagtc cactattaaa 8761 gaacgtggac tccaacgtca aagggcgaaa aaccgtctat cagggcgatg gcccactacg 8821 tgaaccatca ccctaatcaa gttttttggg gtcgaggtgc cgtaaagcac taaatcggaa 8881 ccctaaaggg agcccccgat ttagagcttg acggggaaag ccggcgaacg tggcgagaaa 8941 ggaagggaag aaagcgaaag gagcgggcgc tagggcgctg gcaagtgtag cggtcacgct 9001 gcgcgtaacc accacacccg ccgcgcttaa tgcgccgcta cagggcgcgt ccattcgcca 9061 ttcaggctgc gcaactgttg ggaagggcga tcggtgcggg cctcttcgct attacgccac 9121 tgaattggag cgacctcatg ctatacctga gaaagcaacc tgacctacag gaaagagtta 9181 ctcaagaata agaattttcg ttttaaaacc taagagtcac tttaaaattt gtatacactt 9241 atttttttta taacttattt aataataaaa atcataaatc ataagaaatt cgcttattta 9301 gaagtgtcaa caacgtatct accaacgatt tgaccctttt ccatcttttc gtaaatttct 9361 ggcaaggtag acaagccgac aaccttgatt ggagacttga ccaaacctct ggcgaagaat 9421 tgttaattaa gagctcttaa gacccacttt cacatttaag ttgtttttct aatccgcata 9481 tgatcaattc aaggccgaat aagaaggctg gctctgcacc ttggtgatca aataattcga 9541 tagcttgtcg taataatggc ggcatactat cagtagtagg tgtttccctt tcttctttag 9601 cgacttgatg ctcttgatct tccaatacgc aacctaaagt aaaatgcccc acagcgctga 9661 gtgcatataa tgcattctct agtgaaaaac cttgttggca taaaaaggct aattgatttt 9721 cgagagtttc atactgtttt tctgtaggcc gtgtacctaa atgtactttt gctccatcgc 9781 gatgacttag taaagcacat ctaaaacttt tagcgttatt acgtaaaaaa tcttgccagc 9841 tttccccttc taaagggcaa aagtgagtat ggtgcctatc taacatctca atggctaagg 9901 cgtcgagcaa agcccgctta ttttttacat gccaatacaa tgtaggctgc tctacaccta 9961 gcttctgggc gagtttacgg gttgttaaac cttcgattcc gacctcatta agcagctcta 10021 atgcgctgtt aatcacttta cttttatcta atctagacat tttaataact agtaaaactt 10081 agattagatt gctatgcttt ctttctaatg agcaagaagt aaaaaaagtt gtaatagaac 10141 aagaaaaatg aaactgaaac ttgagaaatt gaagaccgtt tattaactta aatatcaatg 10201 ggaggtcatc gaaagagaaa aaaatcaaaa aaaaaatttt caagaaaaag aaacgtgata 10261 aaaattttta ttgccttttt cgacgaagaa aaagaaacga ggcggtctct tttttctttt 10321 ccaaaccttt agtacgggta attaacgaca ccctagagga agaaagaggg gaaatttagt 10381 atgctgtgct tgggtgtttt gaagtggtac ggcgatgcgc ggagtccgag aaaatctgga 10441 agagtaaaaa aggagtagaa acattttgaa gctatggtgt ggcggccgca tcttgactga 10501 tttttccatg gagggcacag ttaagccgct aaaggcatta tccgccaagt acaatttttt 10561 actcttcgaa gacagaaaat ttgctgacat tggtaataca gtcaaattgc agtactctgc 10621 gggtgtatac agaatagcag aatgggcaga cattacgaat gcacacggtg tggtgggccc 10681 aggtattgtt agcggtttga agcaggcggc agaagaagta acaaaggaac ctagaggcct 10741 tttgatgtta gcagaat //