LOCUS Exported 12349 bp ds-DNA circular SYN 13-MAY-2021 DEFINITION Pre-express Cas9 in yeast. ACCESSION . VERSION . KEYWORDS pCas9 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 12349) TITLE CRISPR course for high school and undergraduate students REFERENCE 2 (bases 1 to 12349) AUTHORS . TITLE Direct Submission JOURNAL Exported May 13, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..12349 /organism="synthetic DNA construct" /mol_type="other DNA" gene 1..1576 /label=hphMX6 /note="yeast selectable marker conferring hygromycin resistance" promoter 1..344 /label=TEF promoter /note="Ashbya gossypii TEF promoter" CDS 345..1373 /codon_start=1 /gene="aph(4)-Ia" /product="aminoglycoside phosphotransferase from E. coli" /label=HygR /note="confers resistance to hygromycin" /translation="MGKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGR GYVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDL PETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHW QTVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAM FGDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVD GNFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRA KE" terminator 1379..1576 /label=TEF terminator /note="Ashbya gossypii TEF terminator" promoter 1864..2271 /gene="S. cerevisiae LEU2" /label=LEU2 promoter CDS 2272..3366 /codon_start=1 /gene="S. cerevisiae LEU2" /product="3-isopropylmalate dehydrogenase, required for leucine biosynthesis" /label=LEU2 /note="yeast auxotrophic marker" /translation="MSAPKKIVVLPGDHVGQEITAEAIKVLKAISDVRSNVKFDFENHL IGGAAIDATGVPLPDEALEASKKVDAVLLGAVGGPKWGTGSVRPEQGLLKIRKELQLYA NLRPCNFASDSLLDLSPIKPQFAKGTDFVVVRELVGGIYFGKRKEDDGDGVAWDSEQYT VPEVQRITRMAAFMALQHEPPLPIWSLDKANVLASSRLWRKTVEETIKNEFPTLKVQHQ LIDSAAMILVKNPTHLNGIIITSNMFGDIISDEASVIPGSLGLLPSASLASLPDKNTAF GLYEPCHGSAPDLPKNKVDPIATILSAAMMLKLSLNLPEEGKAIEDAVKKVLDAGIRTG DLGGSNSTTEVGDAVAEEVKKILA" primer_bind complement(3859..3878) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" primer_bind 3978..4000 /label=pGEX 3' /note="pGEX vectors, reverse primer" primer_bind complement(4038..4056) /label=pBRforEco /note="pBR322 vectors, upsteam of EcoRI site, forward primer" misc_feature 4098..4601 /label=CEN/ARS /note="S. cerevisiae CEN6 centromere fused to an autonomously replicating sequence" promoter 4638..4742 /gene="bla" /label=AmpR promoter CDS 4743..5603 /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(4961..4980) /label=Amp-R /note="Ampicillin resistance gene, reverse primer" rep_origin 5774..6362 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 6263..6282 /label=pBR322ori-F /note="pBR322 origin, forward primer" primer_bind 6516..6533 /label=L4440 /note="L4440 vector, forward primer" promoter 6686..6716 /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 6724..6740 /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 6729..6751 /label=M13/pUC Reverse /note="In lacZ gene" primer_bind 6748..6764 /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind 6748..6764 /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" primer_bind 6783..6803 /label=T3 /note="T3 promoter, forward primer" promoter 6785..6803 /label=T3 promoter /note="promoter for bacteriophage T3 RNA polymerase" promoter 6821..7219 /gene="S. cerevisiae TEF1" /label=TEF1 promoter /note="promoter for EF-1-alpha" primer_bind 7223..7239 /label=SK primer /note="common sequencing primer, one of multiple similar variants" primer_bind 7223..7239 /label=pBluescriptSK /note="For pBluescript vector" CDS 7251..11354 /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 FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER LKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFM QLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY LYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVP SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL DATLIHQSITGLYETRIDLSQLGGD" CDS 11367..11387 /codon_start=1 /product="nuclear localization signal of SV40 (simian virus 40) large T antigen" /label=SV40 NLS /translation="PKKKRKV" terminator 11403..11650 /gene="S. cerevisiae CYC1" /label=CYC1 terminator /note="transcription terminator for CYC1" primer_bind complement(11419..11437) /label=CYC1 /note="CYC1 transcription termination signal, reverse primer" primer_bind complement(11668..11687) /label=T7 /note="T7 promoter, forward primer" promoter complement(11669..11687) /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" primer_bind complement(11697..11714) /label=M13 Forward /note="In lacZ gene. Also called M13-F20 or M13 (-21) Forward" primer_bind complement(11697..11713) /label=M13 fwd /note="common sequencing primer, one of multiple similar variants" primer_bind complement(11706..11728) /label=M13/pUC Forward /note="In lacZ gene" rep_origin 11858..12313 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(11945..11964) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 12155..12176 /label=F1ori-F /note="F1 origin, forward primer" ORIGIN 1 gacatggagg cccagaatac cctccttgac agtcttgacg tgcgcagctc aggggcatga 61 tgtgactgtc gcccgtacat ttagcccata catccccatg tataatcatt tgcatccata 121 cattttgatg gccgcacggc gcgaagcaaa aattacggct cctcgctgcg gacctgcgag 181 cagggaaacg ctcccctcac agacgcgttg aattgtcccc acgccgcgcc cctgtagaga 241 aatataaaag gttaggattt gccactgagg ttcttctttc atatacttcc ttttaaaatc 301 ttgctaggat acagttctca catcacatcc gaacataaac aaccatgggt aaaaagcctg 361 aactcaccgc gacgtctgtc gagaagtttc tgatcgaaaa gttcgacagc gtctccgacc 421 tgatgcagct ctcggagggc gaagaatctc gtgctttcag cttcgatgta ggagggcgtg 481 gatatgtcct gcgggtaaat agctgcgccg atggtttcta caaagatcgt tatgtttatc 541 ggcactttgc atcggccgcg ctcccgattc cggaagtgct tgacattggg gaattcagcg 601 agagcctgac ctattgcatc tcccgccgtg cacagggtgt cacgttgcaa gacctgcctg 661 aaaccgaact gcccgctgtt ctgcagccgg tcgcggaggc catggatgcg atcgctgcgg 721 ccgatcttag ccagacgagc gggttcggcc cattcggacc gcaaggaatc ggtcaataca 781 ctacatggcg tgatttcata tgcgcgattg ctgatcccca tgtgtatcac tggcaaactg 841 tgatggacga caccgtcagt gcgtccgtcg cgcaggctct cgatgagctg atgctttggg 901 ccgaggactg ccccgaagtc cggcacctcg tgcacgcgga tttcggctcc aacaatgtcc 961 tgacggacaa tggccgcata acagcggtca ttgactggag cgaggcgatg ttcggggatt 1021 cccaatacga ggtcgccaac atcttcttct ggaggccgtg gttggcttgt atggagcagc 1081 agacgcgcta cttcgagcgg aggcatccgg agcttgcagg atcgccgcgg ctccgggcgt 1141 atatgctccg cattggtctt gaccaactct atcagagctt ggttgacggc aatttcgatg 1201 atgcagcttg ggcgcagggt cgatgcgacg caatcgtccg atccggagcc gggactgtcg 1261 ggcgtacaca aatcgcccgc agaagcgcgg ccgtctggac cgatggctgt gtagaagtac 1321 tcgccgatag tggaaaccga cgccccagca ctcgtccgag ggcaaaggaa taatcagtac 1381 tgacaataaa aagattcttg ttttcaagaa cttgtcattt gtatagtttt tttatattgt 1441 agttgttcta ttttaatcaa atgttagcgt gatttatatt ttttttcgcc tcgacatcat 1501 ctgcccagat gcgaagttaa gtgcgcagaa agtaatatca tgcgtcaatc gtatgtgaat 1561 gctggtcgct atactgctgt cgattcgata ctaacgccgc catccagtgt taattaacga 1621 cggtcgagga gaacttctag tatatctaca tacctaatat tattgcctta ttaaaaatgg 1681 aatcccaaca attacatcaa aatccacatt ctcttcaaaa tcaattgtcc tgtacttcct 1741 tgttcatgtg tgttcaaaaa cgttatattt ataggataat tatactctat ttctcaacaa 1801 gtaattggtt gtttggccga gcggtctaag gcgcctgatt caagaaatat cttgaccgca 1861 gttaactgtg ggaatactca ggtatcgtaa gatgcaagag ttcgaatctc ttagcaacca 1921 ttattttttt cctcaacata acgagaacac acaggggcgc tatcgcacag aatcaaattc 1981 gatgactgga aattttttgt taatttcaga ggtcgcctga cgcatatacc tttttcaact 2041 gaaaaattgg gagaaaaagg aaaggtgaga gcgccggaac cggcttttca tatagaatag 2101 agaagcgttc atgactaaat gcttgcatca caatacttga agttgacaat attatttaag 2161 gacctattgt tttttccaat aggtggttag caatcgtctt actttctaac ttttcttacc 2221 ttttacattt cagcaatata tatatatata tttcaaggat ataccattct aatgtctgcc 2281 cctaagaaga tcgtcgtttt gccaggtgac cacgttggtc aagaaatcac agccgaagcc 2341 attaaggttc ttaaagctat ttctgatgtt cgttccaatg tcaagttcga tttcgaaaat 2401 catttaattg gtggtgctgc tatcgatgct acaggtgttc cacttccaga tgaggcgctg 2461 gaagcctcca agaaggttga tgccgttttg ttaggtgctg tgggtggtcc taaatggggt 2521 accggtagtg ttagacctga acaaggttta ctaaaaatcc gtaaagaact tcaattgtac 2581 gccaacttaa gaccatgtaa ctttgcatcc gactctcttt tagacttatc tccaatcaag 2641 ccacaatttg ctaaaggtac tgacttcgtt gttgtcagag aattagtggg aggtatttac 2701 tttggtaaga gaaaggaaga cgatggtgat ggtgtcgctt gggatagtga acaatacacc 2761 gttccagaag tgcaaagaat cacaagaatg gccgctttca tggccctaca acatgagcca 2821 ccattgccta tttggtcctt ggataaagct aatgttttgg cctcttcaag attatggaga 2881 aaaactgtgg aggaaaccat caagaacgaa ttccctacat tgaaggttca acatcaattg 2941 attgattctg ccgccatgat cctagttaag aacccaaccc acctaaatgg tattataatc 3001 accagcaata tgtttggtga tatcatctcc gatgaagcct ccgttatccc aggttccttg 3061 ggtttgttgc catctgcgtc cttggcctct ttgccagaca agaacaccgc atttggtttg 3121 tacgaaccat gccacggttc tgctccagat ttgccaaaga ataaggttga ccctatcgcc 3181 actatcttgt ctgctgcaat gatgttgaaa ttgtcattga acttgcctga agaaggtaag 3241 gccattgaag atgcagttaa aaaggttttg gatgcaggta tcagaactgg tgatttaggt 3301 ggttccaaca gtaccaccga agtcggtgat gctgtcgccg aagaagttaa gaaaatcctt 3361 gcttaaaaag attctctttt tttatgatat ttgtacataa actttataaa tgaaattcat 3421 aatagaaacg acacgaaatt acaaaatgga atatgttcat agggtagacg aaactatata 3481 cgcaatctac atacatttat caagaaggag aaaaaggagg atagtaaagg aatacaggta 3541 agcaaattga tactaatggc tcaacgtgat aaggaaaaag aattgcactt taacattaat 3601 attgacaagg aggagggcac cacacaaaaa gttaggtgta acagaaaatc atgaaactac 3661 gattcctaat ttgatattgg aggattttct ctaaaaaaaa aaaaatacaa caaataaaaa 3721 acactcaatg acctgaccat ttgatggagt ttaagtcaat accttcttga accatttccc 3781 ataatggtga aagttccctc aagaatttta ctctgtcaga aacggcctta cgacgtagtc 3841 gatatggtgc actctcagta caatctgctc tgatgccgca tagttaagcc agccccgaca 3901 cccgccaaca cccgctgacg cgccctgacg ggcttgtctg ctcccggcat ccgcttacag 3961 acaagctgtg accgtctccg ggagctgcat gtgtcagagg ttttcaccgt catcaccgaa 4021 acgcgcgaga cgaaagggcc tcgtgatacg cctattttta taggttaatg tcatgataat 4081 aatggtttct taggacggat cgcttgcctg taacttacac gcgcctcgta tcttttaatg 4141 atggaataat ttgggaattt actctgtgtt tatttatttt tatgttttgt atttggattt 4201 tagaaagtaa ataaagaagg tagaagagtt acggaatgaa gaaaaaaaaa taaacaaagg 4261 tttaaaaaat ttcaacaaaa agcgtacttt acatatatat ttattagaca agaaaagcag 4321 attaaataga tatacattcg attaacgata agtaaaatgt aaaatcacag gattttcgtg 4381 tgtggtcttc tacacagaca agatgaaaca attcggcatt aatacctgag agcaggaaga 4441 aaaagataaa aggtagtatt tgttggcgat ccccctagag tcttttacat cttcggaaaa 4501 caaaaactat tttttcttta atttcttttt ttactttcta tttttaattt atatatttat 4561 attaaaaaat ttaaattata attattttta tagcacgtga tgaaaaggac ccaggtggca 4621 cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt tttctaaata cattcaaata 4681 tgtatccgct catgagacaa taaccctgat aaatgcttca ataatattga aaaaggaaga 4741 gtatgagtat tcaacatttc cgtgtcgccc ttattccctt ttttgcggca ttttgccttc 4801 ctgtttttgc tcacccagaa acgctggtga aagtaaaaga tgctgaagat cagttgggtg 4861 cacgagtggg ttacatcgaa ctggatctca acagcggtaa gatccttgag agttttcgcc 4921 ccgaagaacg ttttccaatg atgagcactt ttaaagttct gctatgtggc gcggtattat 4981 cccgtattga cgccgggcaa gagcaactcg gtcgccgcat acactattct cagaatgact 5041 tggttgagta ctcaccagtc acagaaaagc atcttacgga tggcatgaca gtaagagaat 5101 tatgcagtgc tgccataacc atgagtgata acactgcggc caacttactt ctgacaacga 5161 tcggaggacc gaaggagcta accgcttttt tgcacaacat gggggatcat gtaactcgcc 5221 ttgatcgttg ggaaccggag ctgaatgaag ccataccaaa cgacgagcgt gacaccacga 5281 tgcctgtagc aatggcaaca acgttgcgca aactattaac tggcgaacta cttactctag 5341 cttcccggca acaattaata gactggatgg aggcggataa agttgcagga ccacttctgc 5401 gctcggccct tccggctggc tggtttattg ctgataaatc tggagccggt gagcgtgggt 5461 ctcgcggtat cattgcagca ctggggccag atggtaagcc ctcccgtatc gtagttatct 5521 acacgacggg gagtcaggca actatggatg aacgaaatag acagatcgct gagataggtg 5581 cctcactgat taagcattgg taactgtcag accaagttta ctcatatata ctttagattg 5641 atttaaaact tcatttttaa tttaaaagga tctaggtgaa gatccttttt gataatctca 5701 tgaccaaaat cccttaacgt gagttttcgt tccactgagc gtcagacccc gtagaaaaga 5761 tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa 5821 aaccaccgct accagcggtg gtttgtttgc cggatcaaga gctaccaact ctttttccga 5881 aggtaactgg cttcagcaga gcgcagatac caaatactgt ccttctagtg tagccgtagt 5941 taggccacca cttcaagaac tctgtagcac cgcctacata cctcgctctg ctaatcctgt 6001 taccagtggc tgctgccagt ggcgataagt cgtgtcttac cgggttggac tcaagacgat 6061 agttaccgga taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca cagcccagct 6121 tggagcgaac gacctacacc gaactgagat acctacagcg tgagctatga gaaagcgcca 6181 cgcttcccga agggagaaag gcggacaggt atccggtaag cggcagggtc ggaacaggag 6241 agcgcacgag ggagcttcca gggggaaacg cctggtatct ttatagtcct gtcgggtttc 6301 gccacctctg acttgagcgt cgatttttgt gatgctcgtc aggggggcgg agcctatgga 6361 aaaacgccag caacgcggcc tttttacggt tcctggcctt ttgctggcct tttgctcaca 6421 tgttctttcc tgcgttatcc cctgattctg tggataaccg tattaccgcc tttgagtgag 6481 ctgataccgc tcgccgcagc cgaacgaccg agcgcagcga gtcagtgagc gaggaagcgg 6541 aagagcgccc aatacgcaaa ccgcctctcc ccgcgcgttg gccgattcat taatgcagct 6601 ggcacgacag gtttcccgac tggaaagcgg gcagtgagcg caacgcaatt aatgtgagtt 6661 acctcactca ttaggcaccc caggctttac actttatgct tccggctcct atgttgtgtg 6721 gaattgtgag cggataacaa tttcacacag gaaacagcta tgaccatgat tacgccaagc 6781 gcgcaattaa ccctcactaa agggaacaaa agctggagct catagcttca aaatgtttct 6841 actccttttt tactcttcca gattttctcg gactccgcgc atcgccgtac cacttcaaaa 6901 cacccaagca cagcatacta aatttcccct ctttcttcct ctagggtgtc gttaattacc 6961 cgtactaaag gtttggaaaa gaaaaaagag accgcctcgt ttctttttct tcgtcgaaaa 7021 aggcaataaa aatttttatc acgtttcttt ttcttgaaaa tttttttttt gatttttttc 7081 tctttcgatg acctcccatt gatatttaag ttaataaacg gtcttcaatt tctcaagttt 7141 cagtttcatt tttcttgttc tattacaact ttttttactt cttgctcatt agaaagaaag 7201 catagcaatc taatctaagt tttctagaac tagtggatcc cccgggaaaa atggacaaga 7261 agtactccat tgggctcgat atcggcacaa acagcgtcgg ttgggccgtc attacggacg 7321 agtacaaggt gccgagcaaa aaattcaaag ttctgggcaa taccgatcgc cacagcataa 7381 agaagaacct cattggcgcc ctcctgttcg actccgggga gacggccgaa gccacgcggc 7441 tcaaaagaac agcacggcgc agatataccc gcagaaagaa tcggatctgc tacctgcagg 7501 agatctttag taatgagatg gctaaggtgg atgactcttt cttccatagg ctggaggagt 7561 cctttttggt ggaggaggat aaaaagcacg agcgccaccc aatctttggc aatatcgtgg 7621 acgaggtggc gtaccatgaa aagtacccaa ccatatatca tctgaggaag aagcttgtag 7681 acagtactga taaggctgac ttgcggttga tctatctcgc gctggcgcat atgatcaaat 7741 ttcggggaca cttcctcatc gagggggacc tgaacccaga caacagcgat gtcgacaaac 7801 tctttatcca actggttcag acttacaatc agcttttcga agagaacccg atcaacgcat 7861 ccggagttga cgccaaagca atcctgagcg ctaggctgtc caaatcccgg cggctcgaaa 7921 acctcatcgc acagctccct ggggagaaga agaacggcct gtttggtaat cttatcgccc 7981 tgtcactcgg gctgaccccc aactttaaat ctaacttcga cctggccgaa gatgccaagc 8041 ttcaactgag caaagacacc tacgatgatg atctcgacaa tctgctggcc cagatcggcg 8101 accagtacgc agaccttttt ttggcggcaa agaacctgtc agacgccatt ctgctgagtg 8161 atattctgcg agtgaacacg gagatcacca aagctccgct gagcgctagt atgatcaagc 8221 gctatgatga gcaccaccaa gacttgactt tgctgaaggc ccttgtcaga cagcaactgc 8281 ctgagaagta caaggaaatt ttcttcgatc agtctaaaaa tggctacgcc ggatacattg 8341 acggcggagc aagccaggag gaattttaca aatttattaa gcccatcttg gaaaaaatgg 8401 acggcaccga ggagctgctg gtaaagctta acagagaaga tctgttgcgc aaacagcgca 8461 ctttcgacaa tggaagcatc ccccaccaga ttcacctggg cgaactgcac gctatcctca 8521 ggcggcaaga ggatttctac ccctttttga aagataacag ggaaaagatt gagaaaatcc 8581 tcacatttcg gataccctac tatgtaggcc ccctcgcccg gggaaattcc agattcgcgt 8641 ggatgactcg caaatcagaa gagaccatca ctccctggaa cttcgaggaa gtcgtggata 8701 agggggcctc tgcccagtcc ttcatcgaaa ggatgactaa ctttgataaa aatctgccta 8761 acgaaaaggt gcttcctaaa cactctctgc tgtacgagta cttcacagtt tataacgagc 8821 tcaccaaggt caaatacgtc acagaaggga tgagaaagcc agcattcctg tctggagagc 8881 agaagaaagc tatcgtggac ctcctcttca agacgaaccg gaaagttacc gtgaaacagc 8941 tcaaagaaga ctatttcaaa aagattgaat gtttcgactc tgttgaaatc agcggagtgg 9001 aggatcgctt caacgcatcc ctgggaacgt atcacgatct cctgaaaatc attaaagaca 9061 aggacttcct ggacaatgag gagaacgagg acattcttga ggacattgtc ctcaccctta 9121 cgttgtttga agatagggag atgattgaag aacgcttgaa aacttacgct catctcttcg 9181 acgacaaagt catgaaacag ctcaagaggc gccgatatac aggatggggg cggctgtcaa 9241 gaaaactgat caatgggatc cgagacaagc agagtggaaa gacaatcctg gattttctta 9301 agtccgatgg atttgccaac cggaacttca tgcagttgat ccatgatgac tctctcacct 9361 ttaaggagga catccagaaa gcacaagttt ctggccaggg ggacagtctt cacgagcaca 9421 tcgctaatct tgcaggtagc ccagctatca aaaagggaat actgcagacc gttaaggtcg 9481 tggatgaact cgtcaaagta atgggaaggc ataagcccga gaatatcgtt atcgagatgg 9541 cccgagagaa ccaaactacc cagaagggac agaagaacag tagggaaagg atgaagagga 9601 ttgaagaggg tataaaagaa ctggggtccc aaatccttaa ggaacaccca gttgaaaaca 9661 cccagcttca gaatgagaag ctctacctgt actacctgca gaacggcagg gacatgtacg 9721 tggatcagga actggacatc aatcggctct ccgactacga cgtggatcat atcgtgcccc 9781 agtcttttct caaagatgat tctattgata ataaagtgtt gacaagatcc gataaaaata 9841 gagggaagag tgataacgtc ccctcagaag aagttgtcaa gaaaatgaaa aattattggc 9901 ggcagctgct gaacgccaaa ctgatcacac aacggaagtt cgataatctg actaaggctg 9961 aacgaggtgg cctgtctgag ttggataaag ccggcttcat caaaaggcag cttgttgaga 10021 cacgccagat caccaagcac gtggcccaaa ttctcgattc acgcatgaac accaagtacg 10081 atgaaaatga caaactgatt cgagaggtga aagttattac tctgaagtct aagctggtct 10141 cagatttcag aaaggacttt cagttttata aggtgagaga gatcaacaat taccaccatg 10201 cgcatgatgc ctacctgaat gcagtggtag gcactgcact tatcaaaaaa tatcccaagc 10261 ttgaatctga atttgtttac ggagactata aagtgtacga tgttaggaaa atgatcgcaa 10321 agtctgagca ggaaataggc aaggccaccg ctaagtactt cttttacagc aatattatga 10381 attttttcaa gaccgagatt acactggcca atggagagat tcggaagcga ccacttatcg 10441 aaacaaacgg agaaacagga gaaatcgtgt gggacaaggg tagggatttc gcgacagtcc 10501 ggaaggtcct gtccatgccg caggtgaaca tcgttaaaaa gaccgaagta cagaccggag 10561 gcttctccaa ggaaagtatc ctcccgaaaa ggaacagcga caagctgatc gcacgcaaaa 10621 aagattggga ccccaagaaa tacggcggat tcgattctcc tacagtcgct tacagtgtac 10681 tggttgtggc caaagtggag aaagggaagt ctaaaaaact caaaagcgtc aaggaactgc 10741 tgggcatcac aatcatggag cgatcaagct tcgaaaaaaa ccccatcgac tttctcgagg 10801 cgaaaggata taaagaggtc aaaaaagacc tcatcattaa gcttcccaag tactctctct 10861 ttgagcttga aaacggccgg aaacgaatgc tcgctagtgc gggcgagctg cagaaaggta 10921 acgagctggc actgccctct aaatacgtta atttcttgta tctggccagc cactatgaaa 10981 agctcaaagg gtctcccgaa gataatgagc agaagcagct gttcgtggaa caacacaaac 11041 actaccttga tgagatcatc gagcaaataa gcgaattctc caaaagagtg atcctcgccg 11101 acgctaacct cgataaggtg ctttctgctt acaataagca cagggataag cccatcaggg 11161 agcaggcaga aaacattatc cacttgttta ctctgaccaa cttgggcgcg cctgcagcct 11221 tcaagtactt cgacaccacc atagacagaa agcggtacac ctctacaaag gaggtcctgg 11281 acgccacact gattcatcag tcaattacgg ggctctatga aacaagaatc gacctctctc 11341 agctcggtgg agacagcagg gctgacccca agaagaagag gaaggtgtga tctcttctcg 11401 agtcatgtaa ttagttatgt cacgcttaca ttcacgccct ccccccacat ccgctctaac 11461 cgaaaaggaa ggagttagac aacctgaagt ctaggtccct atttattttt ttatagttat 11521 gttagtatta agaacgttat ttatatttca aatttttctt ttttttctgt acagacgcgt 11581 gtacgcatgt aacattatac tgaaaacctt gcttgagaag gttttgggac gctcgaaggc 11641 tttaatttgc ggccggtacc caattcgccc tatagtgagt cgtattacgc gcgctcactg 11701 gccgtcgttt tacaacgtcg tgactgggaa aaccctggcg ttacccaact taatcgcctt 11761 gcagcacatc cccctttcgc cagctggcgt aatagcgaag aggcccgcac cgatcgccct 11821 tcccaacagt tgcgcagcct gaatggcgaa tggcgcgacg cgccctgtag cggcgcatta 11881 agcgcggcgg gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag cgccctagcg 11941 cccgctcctt tcgctttctt cccttccttt ctcgccacgt tcgccggctt tccccgtcaa 12001 gctctaaatc gggggctccc tttagggttc cgatttagtg ctttacggca cctcgacccc 12061 aaaaaacttg attagggtga tggttcacgt agtgggccat cgccctgata gacggttttt 12121 cgccctttga cgttggagtc cacgttcttt aatagtggac tcttgttcca aactggaaca 12181 acactcaacc ctatctcggt ctattctttt gatttataag ggattttgcc gatttcggcc 12241 tattggttaa aaaatgagct gatttaacaa aaatttaacg cgaattttaa caaaatatta 12301 acgtttacaa tttcctgatg cggtattttc tccttacgca tctgtgcgg //