LOCUS Exported 11106 bp ds-DNA circular SYN 13-MAY-2021 DEFINITION All-in-one CRISPR plasmid for integrating, deleting, or replacing a gene in S. cerevisiae. High-copy, URA3 plasmid contains human codon-optimized Cas9 and a guide RNA targeting ARS1206a sequence CGAACATTTTTCCATGCGCT in yeast chromosome 12.. ACCESSION . VERSION . KEYWORDS p426_Cas9_gRNA-ARS1206a SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 11106) AUTHORS Reider Apel A, d'Espaux L, Wehrs M, Sachs D, Li RA, Tong GJ, Garber M, Nnadi O, Zhuang W, Hillson NJ, Keasling JD, Mukhopadhyay A TITLE A Cas9-based toolkit to program gene expression in Saccharomyces cerevisiae. JOURNAL Nucleic Acids Res. 2017 Jan 9;45(1):496-508. doi: 10.1093/nar/gkw1023. Epub 2016 Nov 28. PUBMED 27899650 REFERENCE 2 (bases 1 to 11106) AUTHORS . TITLE Direct Submission JOURNAL Exported May 13, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..11106 /organism="synthetic DNA construct" /mol_type="other DNA" promoter complement(111..326) /gene="S. cerevisiae URA3" /label=URA3 promoter primer_bind complement(350..369) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" primer_bind 469..491 /label=pGEX 3' /note="pGEX vectors, reverse primer" primer_bind complement(529..547) /label=pBRforEco /note="pBR322 vectors, upsteam of EcoRI site, forward primer" rep_origin 588..1930 /label=2u ori /note="yeast 2u plasmid origin of replication" protein_bind 920..967 /label=FRT /bound_moiety="FLP recombinase from the Saccharomyces cerevisiae 2u plasmid" /note="FLP-mediated recombination occurs in the 8-bp core sequence TCTAGAAA (Turan and Bode, 2011)." promoter 1957..2061 /gene="bla" /label=AmpR promoter CDS 2062..2922 /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(2280..2299) /label=Amp-R /note="Ampicillin resistance gene, reverse primer" rep_origin 3093..3681 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 3582..3601 /label=pBR322ori-F /note="pBR322 origin, forward primer" primer_bind 3835..3852 /label=L4440 /note="L4440 vector, forward primer" protein_bind 3969..3990 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." promoter 4005..4035 /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 4043..4059 /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)." primer_bind 4048..4070 /label=M13/pUC Reverse /note="In lacZ gene" primer_bind 4067..4083 /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind 4067..4083 /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" primer_bind 4102..4122 /label=T3 /note="T3 promoter, forward primer" promoter 4104..4122 /label=T3 promoter /note="promoter for bacteriophage T3 RNA polymerase" misc_RNA 4349..4424 /label=gRNA scaffold /note="guide RNA scaffold for the Streptococcus pyogenes CRISPR/Cas9 system" promoter 4510..5214 /gene="S. cerevisiae ADH1" /label=ADH1 promoter /note="promoter for alcohol dehydrogenase 1" CDS 5224..9327 /codon_start=1 /product="Cas9 (Csn1) endonuclease from the Streptococcus pyogenes Type II CRISPR/Cas system" /label=Cas9 /note="generates RNA-guided double strand breaks in DNA" /translation="MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK NLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES FLVEEDKKNERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER LKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFM QLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY LYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVP SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD ANLDKVLSAYNKHRDKPIREQAENIIYLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL DATLIHQSITGLYETRIDLSQLGGD" CDS 9340..9360 /codon_start=1 /product="nuclear localization signal of SV40 (simian virus 40) large T antigen" /label=SV40 NLS /translation="PKKKRKV" terminator 9376..9623 /gene="S. cerevisiae CYC1" /label=CYC1 terminator /note="transcription terminator for CYC1" primer_bind complement(9641..9660) /label=T7 /note="T7 promoter, forward primer" promoter complement(9642..9660) /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" primer_bind complement(9670..9687) /label=M13 Forward /note="In lacZ gene. Also called M13-F20 or M13 (-21) Forward" primer_bind complement(9670..9686) /label=M13 fwd /note="common sequencing primer, one of multiple similar variants" primer_bind complement(9679..9701) /label=M13/pUC Forward /note="In lacZ gene" rep_origin 9827..10282 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(9914..9933) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 10124..10145 /label=F1ori-F /note="F1 origin, forward primer" CDS complement(10413..110) /codon_start=1 /gene="S. cerevisiae URA3" /product="orotidine-5'-phosphate decarboxylase, required for uracil biosynthesis" /label=URA3 /note="yeast auxotrophic marker, counterselectable with 5-fluoroorotic acid (5-FOA)" /translation="MSKATYKERAATHPSPVAAKLFNIMHEKQTNLCASLDVRTTKELL ELVEALGPKICLLKTHVDILTDFSMEGTVKPLKALSAKYNFLLFEDRKFADIGNTVKLQ YSAGVYRIAEWADITNAHGVVGPGIVSGLKQAAEEVTKEPRGLLMLAELSCKGSLSTGE YTKGTVDIAKSDKDFVIGFIAQRDMGGRDEGYDWLIMTPGVGLDDKGDALGQQYRTVDD VVSTGSDIIIVGRGLFAKGRDAKVEGERYRKAGWEAYLRRCGQQN" ORIGIN 1 tccaatgaag cacacaagtt tgtttgcttt tcgtgcatga tattaaatag cttggcagca 61 acaggactag gatgagtagc agcacgttcc ttatatgtag ctttcgacat gatttatctt 121 cgtttcctgc aggtttttgt tctgtgcagt tgggttaaga atactgggca atttcatgtt 181 tcttcaacac tacatatgcg tatatatacc aatctaagtc tgtgctcctt ccttcgttct 241 tccttctgtt cggagattac cgaatcaaaa aaatttcaag gaaaccgaaa tcaaaaaaaa 301 gaataaaaaa aaaatgatga attgaaaagg tggtatggtg cactctcagt acaatctgct 361 ctgatgccgc atagttaagc cagccccgac acccgccaac acccgctgac gcgccctgac 421 gggcttgtct gctcccggca tccgcttaca gacaagctgt gaccgtctcc gggagctgca 481 tgtgtcagag gttttcaccg tcatcaccga aacgcgcgag acgaaagggc ctcgtgatac 541 gcctattttt ataggttaat gtcatgataa taatggtttc ttagtatgat ccaatatcaa 601 aggaaatgat agcattgaag gatgagacta atccaattga ggagtggcag catatagaac 661 agctaaaggg tagtgctgaa ggaagcatac gataccccgc atggaatggg ataatatcac 721 aggaggtact agactacctt tcatcctaca taaatagacg catataagta cgcatttaag 781 cataaacacg cactatgccg ttcttctcat gtatatatat atacaggcaa cacgcagata 841 taggtgcgac gtgaacagtg agctgtatgt gcgcagctcg cgttgcattt tcggaagcgc 901 tcgttttcgg aaacgctttg aagttcctat tccgaagttc ctattctcta gaaagtatag 961 gaacttcaga gcgcttttga aaaccaaaag cgctctgaag acgcactttc aaaaaaccaa 1021 aaacgcaccg gactgtaacg agctactaaa atattgcgaa taccgcttcc acaaacattg 1081 ctcaaaagta tctctttgct atatatctct gtgctatatc cctatataac ctacccatcc 1141 acctttcgct ccttgaactt gcatctaaac tcgacctcta cattttttat gtttatctct 1201 agtattactc tttagacaaa aaaattgtag taagaactat tcatagagtg aatcgaaaac 1261 aatacgaaaa tgtaaacatt tcctatacgt agtatataga gacaaaatag aagaaaccgt 1321 tcataatttt ctgaccaatg aagaatcatc aacgctatca ctttctgttc acaaagtatg 1381 cgcaatccac atcggtatag aatataatcg gggatgcctt tatcttgaaa aaatgcaccc 1441 gcagcttcgc tagtaatcag taaacgcggg aagtggagtc aggctttttt tatggaagag 1501 aaaatagaca ccaaagtagc cttcttctaa ccttaacgga cctacagtgc aaaaagttat 1561 caagagactg cattatagag cgcacaaagg agaaaaaaag taatctaaga tgctttgtta 1621 gaaaaatagc gctctcggga tgcatttttg tagaacaaaa aagaagtata gattctttgt 1681 tggtaaaata gcgctctcgc gttgcatttc tgttctgtaa aaatgcagct cagattcttt 1741 gtttgaaaaa ttagcgctct cgcgttgcat ttttgtttta caaaaatgaa gcacagattc 1801 ttcgttggta aaatagcgct ttcgcgttgc atttctgttc tgtaaaaatg cagctcagat 1861 tctttgtttg aaaaattagc gctctcgcgt tgcatttttg ttctacaaaa tgaagcacag 1921 atgcttcgtt caggtggcac ttttcgggga aatgtgcgcg gaacccctat ttgtttattt 1981 ttctaaatac attcaaatat gtatccgctc atgagacaat aaccctgata aatgcttcaa 2041 taatattgaa aaaggaagag tatgagtatt caacatttcc gtgtcgccct tattcccttt 2101 tttgcggcat tttgccttcc tgtttttgct cacccagaaa cgctggtgaa agtaaaagat 2161 gctgaagatc agttgggtgc acgagtgggt tacatcgaac tggatctcaa cagcggtaag 2221 atccttgaga gttttcgccc cgaagaacgt tttccaatga tgagcacttt taaagttctg 2281 ctatgtggcg cggtattatc ccgtattgac gccgggcaag agcaactcgg tcgccgcata 2341 cactattctc agaatgactt ggttgagtac tcaccagtca cagaaaagca tcttacggat 2401 ggcatgacag taagagaatt atgcagtgct gccataacca tgagtgataa cactgcggcc 2461 aacttacttc tgacaacgat cggaggaccg aaggagctaa ccgctttttt gcacaacatg 2521 ggggatcatg taactcgcct tgatcgttgg gaaccggagc tgaatgaagc cataccaaac 2581 gacgagcgtg acaccacgat gcctgtagca atggcaacaa cgttgcgcaa actattaact 2641 ggcgaactac ttactctagc ttcccggcaa caattaatag actggatgga ggcggataaa 2701 gttgcaggac cacttctgcg ctcggccctt ccggctggct ggtttattgc tgataaatct 2761 ggagccggtg agcgtgggtc tcgcggtatc attgcagcac tggggccaga tggtaagccc 2821 tcccgtatcg tagttatcta cacgacgggg agtcaggcaa ctatggatga acgaaataga 2881 cagatcgctg agataggtgc ctcactgatt aagcattggt aactgtcaga ccaagtttac 2941 tcatatatac tttagattga tttaaaactt catttttaat ttaaaaggat ctaggtgaag 3001 atcctttttg ataatctcat gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg 3061 tcagaccccg tagaaaagat caaaggatct tcttgagatc ctttttttct gcgcgtaatc 3121 tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc ggatcaagag 3181 ctaccaactc tttttccgaa ggtaactggc ttcagcagag cgcagatacc aaatactgtt 3241 cttctagtgt agccgtagtt aggccaccac ttcaagaact ctgtagcacc gcctacatac 3301 ctcgctctgc taatcctgtt accagtggct gctgccagtg gcgataagtc gtgtcttacc 3361 gggttggact caagacgata gttaccggat aaggcgcagc ggtcgggctg aacggggggt 3421 tcgtgcacac agcccagctt ggagcgaacg acctacaccg aactgagata cctacagcgt 3481 gagctatgag aaagcgccac gcttcccgaa gggagaaagg cggacaggta tccggtaagc 3541 ggcagggtcg gaacaggaga gcgcacgagg gagcttccag ggggaaacgc ctggtatctt 3601 tatagtcctg tcgggtttcg ccacctctga cttgagcgtc gatttttgtg atgctcgtca 3661 ggggggcgga gcctatggaa aaacgccagc aacgcggcct ttttacggtt cctggccttt 3721 tgctggcctt ttgctcacat gttctttcct gcgttatccc ctgattctgt ggataaccgt 3781 attaccgcct ttgagtgagc tgataccgct cgccgcagcc gaacgaccga gcgcagcgag 3841 tcagtgagcg aggaagcgga agagcgccca atacgcaaac cgcctctccc cgcgcgttgg 3901 ccgattcatt aatgcagctg gcacgacagg tttcccgact ggaaagcggg cagtgagcgc 3961 aacgcaatta atgtgagtta gctcactcat taggcacccc aggctttaca ctttatgctt 4021 ccggctcgta tgttgtgtgg aattgtgagc ggataacaat ttcacacagg aaacagctat 4081 gaccatgatt acgccaagcg cgcaattaac cctcactaaa gggaacaaaa gctggagcta 4141 gtatactctt tcttcaacaa ttaaatactc tcggtagcca agttggttta aggcgcaaga 4201 ctgtaattta tcactacgaa atcttgagat cgggcgttcg actcgccccc gggagagatg 4261 gccggcatgg tcccagcctc ctcgctggcg ccggctgggc aacaccttcg ggtggcgaat 4321 gggactttcg aacatttttc catgcgctgt tttagagcta gaaatagcaa gttaaaataa 4381 ggctagtccg ttatcaactt gaaaaagtgg caccgagtcg gtgctttttt tattttttgt 4441 cactattgtt atgtaaaatg ccacctctga cagtatggaa cgcaaacttc tgtctagtgg 4501 atagtcgaca tccttttgtt gtttccgggt gtacaatatg gacttcctct tttctggcaa 4561 ccaaacccat acatcgggat tcctataata ccttcgttgg tctccctaac atgtaggtgg 4621 cggaggggag atatacaata gaacagatac cagacaagac ataatgggct aaacaagact 4681 acaccaatta cactgcctca ttgatggtgg tacataacga actaatactg tagccctaga 4741 cttgatagcc atcatcatat cgaagtttca ctaccctttt tccatttgcc atctattgaa 4801 gtaataatag gcgcatgcaa cttcttttct ttttttttct tttctctctc ccccgttgtt 4861 gtctcaccat atccgcaatg acaaaaaaat gatggaagac actaaaggaa aaaattaacg 4921 acaaagacag caccaacaga tgtcgttgtt ccagagctga tgaggggtat ctcgaagcac 4981 acgaaacttt ttccttcctt cattcacgca cactactctc taatgagcaa cggtatacgg 5041 ccttccttcc agttacttga atttgaaata aaaaaaagtt tgctgtcttg ctatcaagta 5101 taaatagacc tgcaattatt aatcttttgt ttcctcgtca ttgttctcgt tccctttctt 5161 ccttgtttct ttttctgcac aatatttcaa gctataccaa gcatacaatc aacttggaaa 5221 aaaatggaca agaagtactc cattgggctc gatatcggca caaacagcgt cggttgggcc 5281 gtcattacgg acgagtacaa ggtgccgagc aaaaaattca aagttctggg caataccgat 5341 cgccacagca taaagaagaa cctcattggc gccctcctgt tcgactccgg ggagacggcc 5401 gaagccacgc ggctcaaaag aacagcacgg cgcagatata cccgcagaaa gaatcggatc 5461 tgctacctgc aggagatctt tagtaatgag atggctaagg tggatgactc tttcttccat 5521 aggctggagg agtccttttt ggtggaggag gataaaaaga acgagcgcca cccaatcttt 5581 ggcaatatcg tggacgaggt ggcgtaccat gaaaagtacc caaccatata tcatctgagg 5641 aagaagcttg tagacagtac tgataaggct gacttgcggt tgatctatct cgcgctggcg 5701 catatgatca aatttcgggg acacttcctc atcgaggggg acctgaaccc agacaacagc 5761 gatgtcgaca aactctttat ccaactggtt cagacttaca atcagctttt cgaagagaac 5821 ccgatcaacg catccggagt tgacgccaaa gcaatcctga gcgctaggct gtccaaatcc 5881 cggcggctcg aaaacctcat cgcacagctc cctggggaga agaagaacgg cctgtttggt 5941 aatcttatcg ccctgtcact cgggctgacc cccaacttta aatctaactt cgacctggcc 6001 gaagatgcca agcttcaact gagcaaagac acctacgatg atgatctcga caatctgctg 6061 gcccagatcg gcgaccagta cgcagacctt tttttggcgg caaagaacct gtcagacgcc 6121 attctgctga gtgatattct gcgagtgaac acggagatca ccaaagctcc gctgagcgct 6181 agtatgatca agcgctatga tgagcaccac caagacttga ctttgctgaa ggcccttgtc 6241 agacagcaac tgcctgagaa gtacaaggaa attttcttcg atcagtctaa aaatggctac 6301 gccggataca ttgacggcgg agcaagccag gaggaatttt acaaatttat taagcccatc 6361 ttggaaaaaa tggacggcac cgaggagctg ctggtaaagc ttaacagaga agatctgttg 6421 cgcaaacagc gcactttcga caatggaagc atcccccacc agattcacct gggcgaactg 6481 cacgctatcc tcaggcggca agaggatttc tacccctttt tgaaagataa cagggaaaag 6541 attgagaaaa tcctcacatt tcggataccc tactatgtag gccccctcgc ccggggaaat 6601 tccagattcg cgtggatgac tcgcaaatca gaagagacca tcactccctg gaacttcgag 6661 gaagtcgtgg ataagggggc ctctgcccag tccttcatcg aaaggatgac taactttgat 6721 aaaaatctgc ctaacgaaaa ggtgcttcct aaacactctc tgctgtacga gtacttcaca 6781 gtttataacg agctcaccaa ggtcaaatac gtcacagaag ggatgagaaa gccagcattc 6841 ctgtctggag agcagaagaa agctatcgtg gacctcctct tcaagacgaa ccggaaagtt 6901 accgtgaaac agctcaaaga agactatttc aaaaagattg aatgtttcga ctctgttgaa 6961 atcagcggag tggaggatcg cttcaacgca tccctgggaa cgtatcacga tctcctgaaa 7021 atcattaaag acaaggactt cctggacaat gaggagaacg aggacattct tgaggacatt 7081 gtcctcaccc ttacgttgtt tgaagatagg gagatgattg aagaacgctt gaaaacttac 7141 gctcatctct tcgacgacaa agtcatgaaa cagctcaaga ggcgccgata tacaggatgg 7201 gggcggctgt caagaaaact gatcaatggg atccgagaca agcagagtgg aaagacaatc 7261 ctggattttc ttaagtccga tggatttgcc aaccggaact tcatgcagtt gatccatgat 7321 gactctctca cctttaagga ggacatccag aaagcacaag tttctggcca gggggacagt 7381 cttcacgagc acatcgctaa tcttgcaggt agcccagcta tcaaaaaggg aatactgcag 7441 accgttaagg tcgtggatga actcgtcaaa gtaatgggaa ggcataagcc cgagaatatc 7501 gttatcgaga tggcccgaga gaaccaaact acccagaagg gacagaagaa cagtagggaa 7561 aggatgaaga ggattgaaga gggtataaaa gaactggggt cccaaatcct taaggaacac 7621 ccagttgaaa acacccagct tcagaatgag aagctctacc tgtactacct gcagaacggc 7681 agggacatgt acgtggatca ggaactggac atcaatcggc tctccgacta cgacgtggat 7741 catatcgtgc cccagtcttt tctcaaagat gattctattg ataataaagt gttgacaaga 7801 tccgataaaa atagagggaa gagtgataac gtcccctcag aagaagttgt caagaaaatg 7861 aaaaattatt ggcggcagct gctgaacgcc aaactgatca cacaacggaa gttcgataat 7921 ctgactaagg ctgaacgagg tggcctgtct gagttggata aagccggctt catcaaaagg 7981 cagcttgttg agacacgcca gatcaccaag cacgtggccc aaattctcga ttcacgcatg 8041 aacaccaagt acgatgaaaa tgacaaactg attcgagagg tgaaagttat tactctgaag 8101 tctaagctgg tctcagattt cagaaaggac tttcagtttt ataaggtgag agagatcaac 8161 aattaccacc atgcgcatga tgcctacctg aatgcagtgg taggcactgc acttatcaaa 8221 aaatatccca agcttgaatc tgaatttgtt tacggagact ataaagtgta cgatgttagg 8281 aaaatgatcg caaagtctga gcaggaaata ggcaaggcca ccgctaagta cttcttttac 8341 agcaatatta tgaatttttt caagaccgag attacactgg ccaatggaga gattcggaag 8401 cgaccactta tcgaaacaaa cggagaaaca ggagaaatcg tgtgggacaa gggtagggat 8461 ttcgcgacag tccggaaggt cctgtccatg ccgcaggtga acatcgttaa aaagaccgaa 8521 gtacagaccg gaggcttctc caaggaaagt atcctcccga aaaggaacag cgacaagctg 8581 atcgcacgca aaaaagattg ggaccccaag aaatacggcg gattcgattc tcctacagtc 8641 gcttacagtg tactggttgt ggccaaagtg gagaaaggga agtctaaaaa actcaaaagc 8701 gtcaaggaac tgctgggcat cacaatcatg gagcgatcaa gcttcgaaaa aaaccccatc 8761 gactttctcg aggcgaaagg atataaagag gtcaaaaaag acctcatcat taagcttccc 8821 aagtactctc tctttgagct tgaaaacggc cggaaacgaa tgctcgctag tgcgggcgag 8881 ctgcagaaag gtaacgagct ggcactgccc tctaaatacg ttaatttctt gtatctggcc 8941 agccactatg aaaagctcaa agggtctccc gaagataatg agcagaagca gctgttcgtg 9001 gaacaacaca aacactacct tgatgagatc atcgagcaaa taagcgaatt ctccaaaaga 9061 gtgatcctcg ccgacgctaa cctcgataag gtgctttctg cttacaataa gcacagggat 9121 aagcccatca gggagcaggc agaaaacatt atctacttgt ttactctgac caacttgggc 9181 gcgcctgcag ccttcaagta cttcgacacc accatagaca gaaagcggta cacctctaca 9241 aaggaggtcc tggacgccac actgattcat cagtcaatta cggggctcta tgaaacaaga 9301 atcgacctct ctcagctcgg tggagacagc agggctgacc ccaagaagaa gaggaaggtg 9361 tgatctcttc tcgagtcatg taattagtta tgtcacgctt acgttcacgc cctcccccca 9421 catccgctct aaccgaaaag gaaggagtta gacaacctga agtctaggtc cctatttatt 9481 tttttatagt tatgttagta ttaagaacgt tatttatatt tcaaattttt cttttttttc 9541 tgtacagacg cgtgtacgca tgtaacatta tactgaaaac cttgcttgag aaggttttgg 9601 gacgctcgaa ggctttaatt tgcggccggt acccaattcg ccctatagtg agtcgtatta 9661 cgcgcgctca ctggccgtcg ttttacaacg tcgtgactgg gaaaaccctg gcgttaccca 9721 acttaatcgc cttgcagcac atcccccttt cgccagctgg cgtaatagcg aagaggcccg 9781 caccgatcgc ccttcccaac agttgcgcag cctgaatggc gaatggacgc gccctgtagc 9841 ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac acttgccagc 9901 gccctagcgc ccgctccttt cgctttcttc ccttcctttc tcgccacgtt cgccggcttt 9961 ccccgtcaag ctctaaatcg ggggctccct ttagggttcc gatttagtgc tttacggcac 10021 ctcgacccca aaaaacttga ttagggtgat ggttcacgta gtgggccatc gccctgatag 10081 acggtttttc gccctttgac gttggagtcc acgttcttta atagtggact cttgttccaa 10141 actggaacaa cactcaaccc tatctcggtc tattcttttg atttataagg gattttgccg 10201 atttcggcct attggttaaa aaatgagctg atttaacaaa aatttaacgc gaattttaac 10261 aaaatattaa cgcttacaat ttcctgatgc ggtattttct ccttacgcat ctgtgcggta 10321 tttcacaccg catagggtaa taactgatat aattaaattg aagctctaat ttgtgagttt 10381 agtatacatg catttactta taatacagtt ttttagtttt gctggccgca tcttctcaaa 10441 tatgcttccc agcctgcttt tctgtaacgt tcaccctcta ccttagcatc ccttcccttt 10501 gcaaatagtc ctcttccaac aataataatg tcagatcctg tagagaccac atcatccacg 10561 gttctatact gttgacccaa tgcgtctccc ttgtcatcta aacccacacc gggtgtcata 10621 atcaaccaat cgtaaccttc atctcttcca cccatgtctc tttgagcaat aaagccgata 10681 acaaaatctt tgtcgctctt cgcaatgtca acagtaccct tagtatattc tccagtagat 10741 agggagccct tgcatgacaa ttctgctaac atcaaaaggc ctctaggttc ctttgttact 10801 tcttctgccg cctgcttcaa accgctaaca atacctgggc ccaccacacc gtgtgcattc 10861 gtaatgtctg cccattctgc tattctgtat acacccgcag agtactgcaa tttgactgta 10921 ttaccaatgt cagcaaattt tctgtcttcg aagagtaaaa aattgtactt ggcggataat 10981 gcctttagcg gcttaactgt gccctccatg gaaaaatcag tcaagatatc cacatgtgtt 11041 tttagtaaac aaattttggg acctaatgct tcaactaact ccagtaattc cttggtggta 11101 cgaaca //