LOCUS Exported 11310 bp ds-DNA circular SYN 13-MAY-2021 DEFINITION Tunable and temporal expression control of Cas9. ACCESSION . VERSION . KEYWORDS XClone-Cas9-Cronus-T2A-BFP SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 11310) TITLE Tunable and temporal expression control of Cas9 REFERENCE 2 (bases 1 to 11310) AUTHORS . TITLE Direct Submission JOURNAL Exported May 13, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..11310 /organism="synthetic DNA construct" /mol_type="other DNA" promoter 9..376 /label=TRE3GS promoter /note="3rd-generation Tet-responsive promoter that can be activated by binding of Tet-On(R) 3G, modified to eliminate binding sites for endogenous mammalian transcription factors" protein_bind 17..35 /gene="tetO" /label=tet operator /bound_moiety="tetracycline repressor TetR" /note="bacterial operator O2 for the tetR and tetA genes" protein_bind 53..71 /gene="tetO" /label=tet operator /bound_moiety="tetracycline repressor TetR" /note="bacterial operator O2 for the tetR and tetA genes" protein_bind 89..107 /gene="tetO" /label=tet operator /bound_moiety="tetracycline repressor TetR" /note="bacterial operator O2 for the tetR and tetA genes" protein_bind 125..143 /gene="tetO" /label=tet operator /bound_moiety="tetracycline repressor TetR" /note="bacterial operator O2 for the tetR and tetA genes" protein_bind 161..179 /gene="tetO" /label=tet operator /bound_moiety="tetracycline repressor TetR" /note="bacterial operator O2 for the tetR and tetA genes" protein_bind 197..215 /gene="tetO" /label=tet operator /bound_moiety="tetracycline repressor TetR" /note="bacterial operator O2 for the tetR and tetA genes" protein_bind 233..251 /gene="tetO" /label=tet operator /bound_moiety="tetracycline repressor TetR" /note="bacterial operator O2 for the tetR and tetA genes" CDS 383..4486 /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" RBS 4399..4407 /label=Shine-Dalgarno sequence /note="full consensus sequence for ribosome-binding sites upstream of start codons in E. coli; complementary to a region in the 3' end of the 16S rRNA (Chen et al., 1994)" CDS 5315..5368 /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 5381..6079 /codon_start=1 /product="monomeric blue fluorescent protein" /label=TagBFP /note="mammalian codon-optimized" /translation="MSELIKENMHMKLYMEGTVDNHHFKCTSEGEGKPYEGTQTMRIKV VEGGPLPFAFDILATSFLYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLTA TQDTSLQDGCLIYNVKIRGVNFTSNGPVMQKKTLGWEAFTETLYPADGGLEGRNDMALK LVGGSHLIANIKTTYRSKKPAKNLKMPGVYYVDYRLERIKEANNETYVEQHEVAVARYC DLPSKLGHKLN" polyA_signal 6272..6406 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" repeat_region 6682..6716 /label=piggyBac left (5') inverted repeat /note="piggyBac transposon-specific inverted terminal repeat sequence (ITR)" primer_bind complement(6727..6744) /label=M13 Forward /note="In lacZ gene. Also called M13-F20 or M13 (-21) Forward" primer_bind complement(6727..6743) /label=M13 fwd /note="common sequencing primer, one of multiple similar variants" primer_bind complement(6736..6758) /label=M13/pUC Forward /note="In lacZ gene" primer_bind complement(6952..6971) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" primer_bind 7071..7093 /label=pGEX 3' /note="pGEX vectors, reverse primer" primer_bind complement(7131..7149) /label=pBRforEco /note="pBR322 vectors, upsteam of EcoRI site, forward primer" promoter 7217..7321 /gene="bla" /label=AmpR promoter CDS 7322..8182 /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(7540..7559) /label=Amp-R /note="Ampicillin resistance gene, reverse primer" rep_origin 8353..8941 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 8842..8861 /label=pBR322ori-F /note="pBR322 origin, forward primer" primer_bind 9095..9112 /label=L4440 /note="L4440 vector, forward primer" protein_bind 9229..9250 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." promoter 9265..9295 /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 9303..9319 /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 9308..9330 /label=M13/pUC Reverse /note="In lacZ gene" primer_bind 9327..9343 /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind 9327..9343 /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" repeat_region 9369..9431 /label=piggyBac right (3') inverted repeat /note="piggyBac transposon-specific inverted terminal repeat sequence (ITR)" polyA_signal 9607..9688 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" CDS complement(9805..10203) /codon_start=1 /gene="Aspergillus terreus BSD" /product="blasticidin S deaminase" /label=BSD /note="confers resistance to blasticidin" /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI KAIVKDSDGQPTAVGIRELLPSGYVWEG" CDS complement(10204..10260) /codon_start=1 /product="2A peptide from porcine teschovirus-1 polyprotein" /label=P2A /note="Eukaryotic ribosomes fail to insert a peptide bond between the Gly and Pro residues, yielding separate polypeptides." /translation="ATNFSLLKQAGDVEENPGP" CDS complement(10270..11013) /codon_start=1 /product="modified rtTA protein that binds tightly to promoters containing the tet operator in the presence of doxycycline" /label=Tet-On(R) 3G /translation="MSRLDKSKVINSALELLNGVGIEGLTTRKLAQKLGVEQPTLYWHV KNKRALLDALPIEMLDRHHTHSCPLEGESWQDFLRNNAKSYRCALLSHRDGAKVHLGTR PTEKQYETLENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEEQEHQVAKEERETPTT DSMPPLLKQAIELFDRQGAEPAFLFGLELIICGLEKQLKCESGGPTDALDDFDLDMLPA DALDDFDLDMLPADALDDFDLDMLPG" CDS complement(11014..11037) /codon_start=1 /product="FLAG(R) epitope tag, followed by an enterokinase cleavage site" /label=FLAG /translation="DYKDDDDK" regulatory 11037..11046 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" promoter complement(11066..11277) /label=EF-1-alpha core promoter /note="core promoter for human elongation factor EF-1-alpha" ORIGIN 1 atcacctcga gtttactccc tatcagtgat agagaacgta tgaagagttt actccctatc 61 agtgatagag aacgtatgca gactttactc cctatcagtg atagagaacg tataaggagt 121 ttactcccta tcagtgatag agaacgtatg accagtttac tccctatcag tgatagagaa 181 cgtatctaca gtttactccc tatcagtgat agagaacgta tatccagttt actccctatc 241 agtgatagag aacgtataag ctttgcttat gtaaaccagg gcgcctataa aagagtgctg 301 attttttgag taaacttcaa ttccacaaca cttttgtctt ataccaactt tccgtaccac 361 ttcctaccct cgtaaaggta ccatggataa gaaatacagc attggactgg acattgggac 421 aaactccgtg ggatgggccg tgattacaga cgaatacaaa gtgccttcaa agaagttcaa 481 ggtgctgggc aacaccgata gacacagcat caagaaaaat ctgattggag ccctgctgtt 541 cgactccggc gagacagctg aagcaactcg gctgaaaaga actgctcgga gaaggtatac 601 ccgccgaaag aataggatct gctacctgca ggagattttc agcaacgaaa tggccaaggt 661 ggacgatagt ttctttcacc gcctggagga atcattcctg gtcgaggaag ataagaaaca 721 cgagcggcat cccatctttg gcaacattgt ggacgaggtc gcttatcacg aaaagtaccc 781 taccatctat catctgagga agaaactggt ggactccaca gataaagcag acctgcgcct 841 gatctatctg gccctggctc acatgattaa gttccggggc cattttctga tcgaggggga 901 tctgaaccca gacaattctg atgtggacaa gctgttcatc cagctggtcc agacatacaa 961 tcagctgttt gaggaaaacc ccattaatgc atctggcgtg gacgcaaaag ccatcctgag 1021 tgccagactg tctaagagtc ggagactgga gaacctgatc gctcagctgc caggggaaaa 1081 gaaaaacggc ctgtttggga atctgattgc actgtcactg ggactgactc ccaacttcaa 1141 gagcaatttt gatctggccg aggacgctaa actgcagctg tccaaggaca cctatgacga 1201 tgacctggat aacctgctgg ctcagatcgg ggatcagtac gcagacctgt tcctggccgc 1261 taagaatctg tctgacgcca tcctgctgag tgatattctg cgcgtgaaca ccgagattac 1321 aaaagccccc ctgtcagcta gcatgatcaa gagatatgac gagcaccatc aggatctgac 1381 cctgctgaag gctctggtga ggcagcagct gcctgagaag tacaaggaaa tcttctttga 1441 tcagtctaag aacggatacg ccggctatat tgacggcggg gctagtcagg aggagttcta 1501 caagtttatc aaacccattc tggagaagat ggatggcaca gaggaactgc tggtgaaact 1561 gaatcgggaa gacctgctga ggaagcagcg cacttttgat aacggaagca tccctcacca 1621 gattcatctg ggagagctgc acgcaatcct gaggcgccag gaagacttct acccatttct 1681 gaaggataac agggagaaga tcgaaaaaat tctgacattc cgcatcccct actatgtggg 1741 ccctctggca agaggcaaca gccggtttgc ctggatgact cgcaaatctg aggaaacaat 1801 cactccctgg aacttcgagg aagtggtcga taagggcgct tccgcacagt ctttcattga 1861 gcggatgaca aacttcgaca agaacctgcc aaacgaaaaa gtgctgccca agcactctct 1921 gctgtacgag tatttcacag tctataacga actgactaag gtgaaatacg tcaccgaggg 1981 gatgagaaag cctgccttcc tgagtggaga acagaagaaa gctatcgtgg acctgctgtt 2041 taaaaccaat aggaaggtga cagtcaagca gctgaaagag gactatttca agaaaattga 2101 atgtttcgat tctgtggaga tcagtggcgt cgaagacagg tttaacgcct ccctggggac 2161 ctaccacgat ctgctgaaga tcattaagga taaagacttc ctggacaacg aggaaaatga 2221 ggatatcctg gaagacattg tgctgaccct gacactgttt gaggataggg aaatgatcga 2281 ggaacgcctg aagacctatg cccatctgtt cgatgacaaa gtgatgaaac agctgaagcg 2341 acggagatac acaggatggg gccgactgtc tcggaagctg atcaatggga ttcgcgacaa 2401 acagagtgga aagaccatcc tggactttct gaaatcagat ggcttcgcca accggaactt 2461 catgcagctg attcacgatg acagcctgac attcaaagag gatatccaga aggcacaggt 2521 gtccgggcag ggagactctc tgcacgagca tatcgcaaac ctggccggca gccctgccat 2581 caagaaaggg attctgcaga ccgtgaaggt ggtggacgag ctggtgaaag tcatgggaag 2641 acataagcca gaaaacatcg tgattgagat ggccagggaa aatcagacca cacagaaagg 2701 ccagaagaac tcaagggagc gcatgaaaag aatcgaggaa ggaattaagg aactgggcag 2761 ccagatcctg aaagagcacc ccgtggaaaa cacacagctg cagaatgaga agctgtatct 2821 gtactatctg cagaatggac gcgatatgta cgtggaccag gagctggata ttaaccgact 2881 gtccgattac gacgtggatc atatcgtccc acagtcattc ctgaaagatg acagcattga 2941 caataaggtg ctgacccgct ctgacaaaaa ccgaggcaag agtgataatg tcccctcaga 3001 ggaagtggtc aagaaaatga agaactactg gaggcagctg ctgaatgcca aactgatcac 3061 acagcgaaag tttgataacc tgactaaagc tgagcgggga ggcctgagtg aactggacaa 3121 agcaggcttc attaagcgac agctggtgga gacacggcag atcacaaagc acgtcgccca 3181 gattctggat tcaagaatga acactaagta cgatgagaat gacaaactga tcagagaagt 3241 gaaggtcatt accctgaagt caaaactggt gagcgacttt cggaaagatt tccagtttta 3301 taaggtcaga gagatcaaca actaccacca tgctcatgac gcatacctga acgcagtggt 3361 cggcacagcc ctgattaaga aataccctaa actggagtcc gagttcgtgt acggggacta 3421 taaggtgtac gatgtcagaa aaatgatcgc caagtctgag caggaaattg gcaaagccac 3481 tgctaagtat ttcttttaca gtaacatcat gaatttcttt aagactgaga tcaccctggc 3541 aaatggggaa atccgaaagc ggccactgat tgagactaac ggcgagacag gagaaatcgt 3601 gtgggacaaa ggaagagatt ttgctaccgt gaggaaggtc ctgagcatgc cccaagtgaa 3661 tattgtcaag aaaacagagg tgcagactgg gggattcagt aaggaatcaa ttctgcctaa 3721 acgcaactcc gataagctga tcgcccgaaa gaaagactgg gaccccaaga agtatggcgg 3781 gttcgactcc ccaactgtgg cttactctgt cctggtggtc gcaaaggtgg agaagggaaa 3841 aagcaagaaa ctgaaatccg tcaaggaact gctgggcatc accattatgg agcgcagctc 3901 cttcgaaaag aatcctatcg attttctgga ggccaaaggc tataaggaag tgaagaaaga 3961 cctgatcatc aagctgccaa agtactcact gtttgagctg gaaaacggga gaaagaggat 4021 gctggcaagc gccggggagc tgcagaaagg aaatgaactg gccctgccct ccaagtacgt 4081 gaacttcctg tatctggcta gccactacga gaagctgaaa gggtcccctg aggataacga 4141 acagaaacag ctgtttgtgg agcagcacaa gcattatctg gacgagatca ttgaacagat 4201 tagcgagttc tccaaaagag tgatcctggc tgacgcaaat ctggataagg tcctgagcgc 4261 atacaacaaa caccgggata agccaatcag agagcaggcc gaaaatatca ttcatctgtt 4321 cactctgacc aacctgggag cccccgcagc cttcaagtat tttgacacta ccatcgatcg 4381 caaacgatac acaagcacta aggaggtgct ggacgctacc ctgattcatc agagcattac 4441 tggcctgtat gaaacaagga ttgacctgtc tcagctgggc ggcgactccg gagctgacgg 4501 aagcgaaaat cctggtaaca aaacaatagt tcctgcaacg ttaccacaac tcacccctac 4561 cctggtgtca ctgttggagg ttattgaacc tgaagtgtta tatgcaggat atgatagctc 4621 tgttccagac tcaacttgga ggatcatgac tacgctcaac atgttaggag ggcggcaagt 4681 gattgcagca gtgaaatggg caaaggcaat accaggtttc aggaacttac acctggatga 4741 ccaaatgacc ctactgcagt actcctggat gtttcttatg gcatttgctc tggggtggag 4801 atcatataga caatcaagtg caaacctgct gtgttttgct cctgatctga ttattaatga 4861 gcagagaatg actctaccct gcatgtacga ccaatgtaaa cacatgctgt atgtttcctc 4921 tgagttacac aggcttcagg tatcttatga agagtatctc tgtatgaaaa ccttactgct 4981 tctctcttca gttcctaagg acggtctgaa gagccaagag ctatttgatg aaattagaat 5041 gacctacatc aaagagctag gaaaagccat tgtcaagagg gaaggaaact ccagccagaa 5101 ctggcagcgg ttttatcaac tgacaaaact cttggattct atgcatgaag tggttgaaaa 5161 tctccttaac tattgcttcc aaacattttt ggataagacc atgtccatcg agttccccga 5221 gatgttagct gaaatcatca ccaatcagat accaaaatat tcaaatggaa atatcaaaaa 5281 acttctgttt catcaaaagg gtaccggatc cggagagggc agaggaagtc tgctaacatg 5341 cggtgacgtc gaggagaatc ctggcccacc ggtcgccacc atgagcgagc tgattaagga 5401 gaacatgcac atgaagctgt acatggaggg caccgtggac aaccatcact tcaagtgcac 5461 atccgagggc gaaggcaagc cctacgaggg cacccagacc atgagaatca aggtggtcga 5521 gggcggccct ctccccttcg ccttcgacat cctggctact agcttcctct acggcagcaa 5581 gaccttcatc aaccacaccc agggcatccc cgacttcttc aagcagtcct tccctgaggg 5641 cttcacatgg gagagagtca ccacatacga agacgggggc gtgctgaccg ctacccagga 5701 caccagcctc caggacggct gcctcatcta caacgtcaag atcagagggg tgaacttcac 5761 atccaacggc cctgtgatgc agaagaaaac actcggctgg gaggccttca ccgagacgct 5821 gtaccccgct gacggcggcc tggaaggcag aaacgacatg gccctgaagc tcgtgggcgg 5881 gagccatctg atcgcaaaca tcaagaccac atatagatcc aagaaacccg ctaagaacct 5941 caagatgcct ggcgtctact atgtggacta cagactggaa agaatcaagg aggccaacaa 6001 cgagacctac gtcgagcagc acgaggtggc agtggccaga tactgcgacc tccctagcaa 6061 actggggcac aagcttaatg gatccggcta aactagtaga ccacctcccc tgcgagctaa 6121 gctggacagc caatgacggg taagagagtg acatttttca ctaacctaag acaggagggc 6181 cgtcagagct actgcctaat ccaaagacgg gtaaaagtga taaaaatgta tcactccaac 6241 ctaagacagg cgcagcttcc gagggatttg agatccagac atgataagat acattgatga 6301 gtttggacaa accaaaacta gaatgcagtg aaaaaaatgc cttatttgtg aaatttgtga 6361 tgctattgcc ttatttgtaa ccattataag ctgcaataaa caagtttgat atctataaca 6421 agaaaatata tatataataa gttatcacgt aagtagaaca tgaaataaca atataattat 6481 cgtatgagtt aaatcttaaa agtcacgtaa aagataatca tgcgtcattt tgactcacgc 6541 ggtcgttata gttcaaaatc agtgacactt accgcattga caagcacgcc tcacgggagc 6601 tccaagcggc gactgagatg tcctaaatgc acagcgacgg attcgcgcta tttagaaaga 6661 gagagcaata tttcaagaat gcatgcgtca attttacgca gactatcttt ctagggttaa 6721 gaattcactg gccgtcgttt tacaacgtcg tgactgggaa aaccctggcg ttacccaact 6781 taatcgcctt gcagcacatc cccctttcgc cagctggcgt aatagcgaag aggcccgcac 6841 cgatcgccct tcccaacagt tgcgcagcct gaatggcgaa tggcgcctga tgcggtattt 6901 tctccttacg catctgtgcg gtatttcaca ccgcatatgg tgcactctca gtacaatctg 6961 ctctgatgcc gcatagttaa gccagccccg acacccgcca acacccgctg acgcgccctg 7021 acgggcttgt ctgctcccgg catccgctta cagacaagct gtgaccgtct ccgggagctg 7081 catgtgtcag aggttttcac cgtcatcacc gaaacgcgcg agacgaaagg gcctcgtgat 7141 acgcctattt ttataggtta atgtcatgat aataatggtt tcttagacgt caggtggcac 7201 ttttcgggga aatgtgcgcg gaacccctat ttgtttattt ttctaaatac attcaaatat 7261 gtatccgctc atgagacaat aaccctgata aatgcttcaa taatattgaa aaaggaagag 7321 tatgagtatt caacatttcc gtgtcgccct tattcccttt tttgcggcat tttgccttcc 7381 tgtttttgct cacccagaaa cgctggtgaa agtaaaagat gctgaagatc agttgggtgc 7441 acgagtgggt tacatcgaac tggatctcaa cagcggtaag atccttgaga gttttcgccc 7501 cgaagaacgt tttccaatga tgagcacttt taaagttctg ctatgtggcg cggtattatc 7561 ccgtattgac gccgggcaag agcaactcgg tcgccgcata cactattctc agaatgactt 7621 ggttgagtac tcaccagtca cagaaaagca tcttacggat ggcatgacag taagagaatt 7681 atgcagtgct gccataacca tgagtgataa cactgcggcc aacttacttc tgacaacgat 7741 cggaggaccg aaggagctaa ccgctttttt gcacaacatg ggggatcatg taactcgcct 7801 tgatcgttgg gaaccggagc tgaatgaagc cataccaaac gacgagcgtg acaccacgat 7861 gcctgtagca atggcaacaa cgttgcgcaa actattaact ggcgaactac ttactctagc 7921 ttcccggcaa caattaatag actggatgga ggcggataaa gttgcaggac cacttctgcg 7981 ctcggccctt ccggctggct ggtttattgc tgataaatct ggagccggtg agcgtgggtc 8041 tcgcggtatc attgcagcac tggggccaga tggtaagccc tcccgtatcg tagttatcta 8101 cacgacgggg agtcaggcaa ctatggatga acgaaataga cagatcgctg agataggtgc 8161 ctcactgatt aagcattggt aactgtcaga ccaagtttac tcatatatac tttagattga 8221 tttaaaactt catttttaat ttaaaaggat ctaggtgaag atcctttttg ataatctcat 8281 gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg tcagaccccg tagaaaagat 8341 caaaggatct tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa 8401 accaccgcta ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc tttttccgaa 8461 ggtaactggc ttcagcagag cgcagatacc aaatactgtt cttctagtgt agccgtagtt 8521 aggccaccac ttcaagaact ctgtagcacc gcctacatac ctcgctctgc taatcctgtt 8581 accagtggct gctgccagtg gcgataagtc gtgtcttacc gggttggact caagacgata 8641 gttaccggat aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac agcccagctt 8701 ggagcgaacg acctacaccg aactgagata cctacagcgt gagctatgag aaagcgccac 8761 gcttcccgaa gggagaaagg cggacaggta tccggtaagc ggcagggtcg gaacaggaga 8821 gcgcacgagg gagcttccag ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg 8881 ccacctctga cttgagcgtc gatttttgtg atgctcgtca ggggggcgga gcctatggaa 8941 aaacgccagc aacgcggcct ttttacggtt cctggccttt tgctggcctt ttgctcacat 9001 gttctttcct gcgttatccc ctgattctgt ggataaccgt attaccgcct ttgagtgagc 9061 tgataccgct cgccgcagcc gaacgaccga gcgcagcgag tcagtgagcg aggaagcgga 9121 agagcgccca atacgcaaac cgcctctccc cgcgcgttgg ccgattcatt aatgcagctg 9181 gcacgacagg tttcccgact ggaaagcggg cagtgagcgc aacgcaatta atgtgagtta 9241 gctcactcat taggcacccc aggctttaca ctttatgctt ccggctcgta tgttgtgtgg 9301 aattgtgagc ggataacaat ttcacacagg aaacagctat gaccatgatt acgccaaggt 9361 cgacttaacc ctagaaagat aatcatattg tgacgtacgt taaagataat catgcgtaaa 9421 attgacgcat gtgttttatc ggtctgtata tcgaggttta tttattaatt tgaatagata 9481 ttaagtttta ttatatttac acttacatac taataataaa ttcaacaaac aatttattta 9541 tgtttattta tttattaaaa aaaaacaaaa actcaaaatt tcttctataa agtaacaaaa 9601 cttttagcag tgaaaaaaat gctttatttg tgaaatttgt gatgctattg ctttatttgt 9661 aaccattata agctgcaata aacaagttaa caacaacaat tgcattcatt ttatgtttca 9721 ggttcagggg gaggtgtggg aggtttttta aagcaagtaa aacctctaca aatgtggtat 9781 ggctgattat gatcctctgg agatttagcc ctcccacaca taaccagagg gcagcaattc 9841 acgaatccca actgccgtcg gctgtccatc actgtccttc actatggctt tgatcccagg 9901 atgcagatcg agaagcacct gtcggcaccg tccgcagggg ctcaagatgc ccctgttctc 9961 atttccgatc gcgacgatac aagtcaggtt gccagctgcc gcagcagcag cagtgcccag 10021 caccacgagt tctgcacaag gtcccccagt aaaatgatat acattgacac cagtgaagat 10081 gcggccgtcg ctagagagag ctgcgctggc gacgctgtag tcttcagaga tggggatgct 10141 gttgattgta gccgttgctc tttcaatgag ggtggattct tcttgagaca aaggcttggc 10201 catggggccg gggttctcct ccacgtcgcc ggcctgcttc agcaggctga agttggtggc 10261 gccgctgccc ccggggagca tgtcaaggtc aaaatcgtca agagcgtcag caggcagcat 10321 atcaaggtca aagtcgtcaa gggcatcggc tgggagcatg tctaagtcaa aatcgtcaag 10381 ggcgtcggtc ggcccgccgc tttcgcactt tagctgtttc tccaggccac atatgattag 10441 ttccaggccg aaaaggaagg caggttcggc tccctgccgg tcgaacagct caattgcttg 10501 tttcagaagt gggggcatag aatcggtggt aggtgtctct ctttcctctt ttgctacttg 10561 atgctcctgt tcctccaata cgcagcccag tgtaaagtgg cccacggcgg acagagcgta 10621 cagtgcgttc tccagggaga agccttgctg acacaggaac gcgagctgat tttccagggt 10681 ttcgtactgt ttctctgttg ggcgggtgcc gagatgcact ttagccccgt cgcgatgtga 10741 gaggagagca cagcggtatg acttggcgtt gttccgcaga aagtcttgcc atgactcgcc 10801 ttccaggggg caggagtggg tatgatgcct gtccagcatc tcgattggca gggcatcgag 10861 cagggcccgc ttgttcttca cgtgccagta cagggtaggc tgctcaactc ccagcttttg 10921 agcgagtttc cttgtcgtca ggccttcgat accgactcca ttgagtaatt ccagagcaga 10981 gtttatgact ttgctcttgt ccagtctaga catcttatcg tcatcgtctt tgtaatccat 11041 ggtggcggat cccgcgtcac gacacctgtg ttctggcggc aaacccgttg cgaaaaagaa 11101 cgttcacggc gactactgca cttatatacg gttctccccc accctcggga aaaaggcgga 11161 gccagtacac gacatcactt tcccagttta ccccgcgcca ccttctctag gcaccggttc 11221 aattgccgac ccctcccccc aacttctcgg ggactgtggg cgatgtgcgc tctgcccact 11281 gacgggcacc ggagccactc gagtggaatt //