LOCUS pX459HF1 9175 bp ds-DNA circular 11-SEP-2017 DEFINITION Cas9 from S. pyogenes with 2A-Puro, and cloning backbone for sgRNA (V2.0). ACCESSION . VERSION . KEYWORDS pSpCas9(BB)-2A-Puro (PX459) V2.0 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 9175) AUTHORS Ran FA, Hsu PD, Wright J, Agarwala V, Scott DA, Zhang F TITLE Genome engineering using the CRISPR-Cas9 system. JOURNAL Nat Protoc. 2013 Nov;8(11):2281-308. doi: 10.1038/nprot.2013.143. Epub 2013 Oct 24. PUBMED 24157548 REFERENCE 2 (bases 1 to 9175) AUTHORS . TITLE Direct Submission JOURNAL Exported Friday, Jun 16, 2017 from SnapGene Console 4.0.0a8 http://www.snapgene.com COMMENT COMMENT COMMENT ApEinfo:methylated:1 FEATURES Location/Qualifiers source 1..9175 /organism="synthetic DNA construct" /mol_type="other DNA" /label=source:synthetic DNA construct /ApEinfo_fwdcolor="pink" /ApEinfo_revcolor="pink" /ApEinfo_graphicformat="arrow_data {{0 1 2 0 0 -1} {} 0} width 5 offset 0" promoter 1..241 /note="RNA polymerase III promoter for human U6 snRNA" /label=U6 promoter /ApEinfo_fwdcolor="#346ee0" /ApEinfo_revcolor="#346ee0" /ApEinfo_graphicformat="arrow_data {{0 1 2 0 0 -1} {} 0} width 5 offset 0" primer_bind 1..21 /note="Human U6 promoter, forward primer" /label=hU6-F /ApEinfo_fwdcolor="#14c0bd" /ApEinfo_revcolor="#4ec02b" /ApEinfo_graphicformat="arrow_data {{0 1 2 0 0 -1} {} 0} width 5 offset 0" primer_bind 172..191 /note="Human U6 promoter, forward primer" /label=LKO.1 5' /ApEinfo_fwdcolor="#14c0bd" /ApEinfo_revcolor="#4ec02b" /ApEinfo_graphicformat="arrow_data {{0 1 2 0 0 -1} {} 0} width 5 offset 0" misc_RNA 268..343 /note="guide RNA scaffold for the Streptococcus pyogenes CRISPR/Cas9 system" /label=g RNA scaffold /ApEinfo_fwdcolor="pink" /ApEinfo_revcolor="pink" /ApEinfo_graphicformat="arrow_data {{0 1 2 0 0 -1} {} 0} width 5 offset 0" enhancer 440..725 /note="human cytomegalovirus immediate early enhancer; contains an 18-bp deletion relative to the standard CMV enhancer" /label=CMV enhancer /ApEinfo_fwdcolor="#5ac3fa" /ApEinfo_revcolor="#5ac3fa" /ApEinfo_graphicformat="arrow_data {{0 1 2 0 0 -1} {} 0} width 5 offset 0" promoter 727..1004 /label=chicken beta-actin promoter /ApEinfo_fwdcolor="#346ee0" /ApEinfo_revcolor="#346ee0" /ApEinfo_graphicformat="arrow_data {{0 1 2 0 0 -1} {} 0} width 5 offset 0" intron 1005..1233 /note="hybrid between chicken beta-actin (CBA) and minute virus of mice (MMV) introns (Gray et al., 2011)" /label=hybrid intron /ApEinfo_fwdcolor="#eb606c" /ApEinfo_revcolor="#eb606c" /ApEinfo_graphicformat="arrow_data {{0 1 2 0 0 -1} {} 0} width 5 offset 0" CDS 1254..1319 /codon_start=1 /product="three tandem FLAG(R) epitope tags, followed by an enterokinase cleavage site" /translation="DYKDHDGDYKDHDIDYKDDDDK" /label=3xFLAG /ApEinfo_fwdcolor="#e9d024" /ApEinfo_revcolor="#e9d024" /ApEinfo_graphicformat="arrow_data {{0 1 2 0 0 -1} {} 0} width 5 offset 0" CDS 1326..1346 /codon_start=1 /product="nuclear localization signal of SV40 large T antigen" /translation="PKKKRKV" /label=SV40 NLS /ApEinfo_fwdcolor="#e9d024" /ApEinfo_revcolor="#e9d024" /ApEinfo_graphicformat="arrow_data {{0 1 2 0 0 -1} {} 0} width 5 offset 0" CDS 1371..5471 /codon_start=1 /product="Cas9 (Csn1) endonuclease from the Streptococcus pyogenes Type II CRISPR/Cas system" /note="generates RNA-guided double strand breaks in DNA" /translation="DKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK N LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES F LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK F RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL E NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ I GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR Q QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL R KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG N SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY F TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD S VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER L KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFM Q LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR H KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY L YYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVP S EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH V AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL N AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT E ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK E SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI T IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE L ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD A NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL D ATLIHQSITGLYETRIDLSQLGGD" /label=Cas9 /ApEinfo_fwdcolor="#e9d024" /ApEinfo_revcolor="#e9d024" /ApEinfo_graphicformat="arrow_data {{0 1 2 0 0 -1} {} 0} width 5 offset 0" primer_bind complement(4974..4994) /note="EGFP, reverse primer" /label=EGFP-N /ApEinfo_fwdcolor="#14c0bd" /ApEinfo_revcolor="#4ec02b" /ApEinfo_graphicformat="arrow_data {{0 1 2 0 0 -1} {} 0} width 5 offset 0" CDS 5472..5519 /codon_start=1 /product="bipartite nuclear localization signal from nucleoplasmin" /translation="KRPAATKKAGQAKKKK" /label=nucleoplasmin NLS /ApEinfo_fwdcolor="#e9d024" /ApEinfo_revcolor="#e9d024" /ApEinfo_graphicformat="arrow_data {{0 1 2 0 0 -1} {} 0} width 5 offset 0" CDS 5535..5588 /codon_start=1 /product="2A peptide from Thosea asigna virus capsid protein" /note="Eukaryotic ribosomes fail to insert a peptide bond between the Gly and Pro residues, yielding separate polypeptides." /translation="EGRGSLLTCGDVEENPGP" /label=T2A /ApEinfo_fwdcolor="#e9d024" /ApEinfo_revcolor="#e9d024" /ApEinfo_graphicformat="arrow_data {{0 1 2 0 0 -1} {} 0} width 5 offset 0" CDS 5589..6188 /codon_start=1 /gene="pac from Streptomyces alboniger" /product="puromycin N-acetyltransferase" /note="confers resistance to puromycin" /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIE R VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRL A AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLET S APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA" /label=PuroR /ApEinfo_fwdcolor="#e9d024" /ApEinfo_revcolor="#e9d024" /ApEinfo_graphicformat="arrow_data {{0 1 2 0 0 -1} {} 0} width 5 offset 0" primer_bind complement(5589..5608) /note="Puromycin resistance gene, reverse primer. Also called puro-variant-R" /label=Puro-R /ApEinfo_fwdcolor="#14c0bd" /ApEinfo_revcolor="#4ec02b" /ApEinfo_graphicformat="arrow_data {{0 1 2 0 0 -1} {} 0} width 5 offset 0" primer_bind 6085..6105 /note="Puromycin resistance gene, forward primer" /label=Puro-F /ApEinfo_fwdcolor="#14c0bd" /ApEinfo_revcolor="#4ec02b" /ApEinfo_graphicformat="arrow_data {{0 1 2 0 0 -1} {} 0} width 5 offset 0" primer_bind complement(6216..6233) /note="Bovine growth hormone terminator, reverse primer. Also called BGH reverse" /label=BGH-rev /ApEinfo_fwdcolor="#14c0bd" /ApEinfo_revcolor="#4ec02b" /ApEinfo_graphicformat="arrow_data {{0 1 2 0 0 -1} {} 0} width 5 offset 0" polyA_signal 6222..6429 /note="bovine growth hormone polyadenylation signal" /label=bGH poly(A) signal /ApEinfo_fwdcolor="#ff3eee" /ApEinfo_revcolor="#ff3eee" /ApEinfo_graphicformat="arrow_data {{0 1 2 0 0 -1} {} 0} width 5 offset 0" repeat_region 6438..6578 /note="inverted terminal repeat of adeno-associated virus serotype 2" /label=AAV2 ITR /ApEinfo_fwdcolor="#0dfff7" /ApEinfo_revcolor="#0dfff7" /ApEinfo_graphicformat="arrow_data {{0 1 2 0 0 -1} {} 0} width 5 offset 0" repeat_region 6438..6567 /label=AAV2 ITR(1) /ApEinfo_label="AAV2 ITR" /ApEinfo_fwdcolor="#0dfff7" /ApEinfo_revcolor="#0dfff7" /ApEinfo_graphicformat="arrow_data {{0 1 2 0 0 -1} {} 0} width 5 offset 0" primer_bind complement(6484..6495) /note="L4440 vector, forward primer" /label=L4440 /ApEinfo_fwdcolor="#14c0bd" /ApEinfo_revcolor="#4ec02b" /ApEinfo_graphicformat="arrow_data {{0 1 2 0 0 -1} {} 0} width 5 offset 0" primer_bind 6545..6556 /note="L4440 vector, forward primer" /label=L4440(1) /ApEinfo_label="L4440" /ApEinfo_fwdcolor="#14c0bd" /ApEinfo_revcolor="#4ec02b" /ApEinfo_graphicformat="arrow_data {{0 1 2 0 0 -1} {} 0} width 5 offset 0" rep_origin 6653..7108 /direction=RIGHT /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" /label=f1 ori /ApEinfo_fwdcolor="#999999" /ApEinfo_revcolor="#999999" /ApEinfo_graphicformat="arrow_data {{0 1 2 0 0 -1} {} 0} width 5 offset 0" primer_bind complement(6740..6759) /note="F1 origin, reverse primer" /label=F1ori-R /ApEinfo_fwdcolor="#14c0bd" /ApEinfo_revcolor="#4ec02b" /ApEinfo_graphicformat="arrow_data {{0 1 2 0 0 -1} {} 0} width 5 offset 0" primer_bind 6950..6971 /note="F1 origin, forward primer" /label=F1ori-F /ApEinfo_fwdcolor="#14c0bd" /ApEinfo_revcolor="#4ec02b" /ApEinfo_graphicformat="arrow_data {{0 1 2 0 0 -1} {} 0} width 5 offset 0" primer_bind complement(7125..7144) /note="pRS vectors, use to sequence yeast selectable marker" /label=pRS-marker /ApEinfo_fwdcolor="#14c0bd" /ApEinfo_revcolor="#4ec02b" /ApEinfo_graphicformat="arrow_data {{0 1 2 0 0 -1} {} 0} width 5 offset 0" primer_bind 7244..7266 /note="pGEX vectors, reverse primer" /label=pGEX 3' /ApEinfo_fwdcolor="#14c0bd" /ApEinfo_revcolor="#4ec02b" /ApEinfo_graphicformat="arrow_data {{0 1 2 0 0 -1} {} 0} width 5 offset 0" primer_bind complement(7304..7322) /note="pBR322 vectors, upsteam of EcoRI site, forward primer" /label=pBRforEco /ApEinfo_fwdcolor="#14c0bd" /ApEinfo_revcolor="#4ec02b" /ApEinfo_graphicformat="arrow_data {{0 1 2 0 0 -1} {} 0} width 5 offset 0" promoter 7390..7494 /gene="bla" /label=AmpR promoter /ApEinfo_fwdcolor="#346ee0" /ApEinfo_revcolor="#346ee0" /ApEinfo_graphicformat="arrow_data {{0 1 2 0 0 -1} {} 0} width 5 offset 0" CDS 7495..8355 /codon_start=1 /gene="bla" /product="beta-lactamase" /note="confers resistance to ampicillin, carbenicillin, and related antibiotics" /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGY I ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEY S PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDR W EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRS A LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGA S LIKHW" /label=AmpR /ApEinfo_fwdcolor="#e9d024" /ApEinfo_revcolor="#e9d024" /ApEinfo_graphicformat="arrow_data {{0 1 2 0 0 -1} {} 0} width 5 offset 0" primer_bind complement(7713..7732) /note="Ampicillin resistance gene, reverse primer" /label=Amp-R /ApEinfo_fwdcolor="#14c0bd" /ApEinfo_revcolor="#4ec02b" /ApEinfo_graphicformat="arrow_data {{0 1 2 0 0 -1} {} 0} width 5 offset 0" rep_origin 8526..9114 /direction=RIGHT /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" /label=ori /ApEinfo_fwdcolor="#999999" /ApEinfo_revcolor="#999999" /ApEinfo_graphicformat="arrow_data {{0 1 2 0 0 -1} {} 0} width 5 offset 0" primer_bind 9015..9034 /note="pBR322 origin, forward primer" /label=pBR322ori-F /ApEinfo_fwdcolor="#14c0bd" /ApEinfo_revcolor="#4ec02b" /ApEinfo_graphicformat="arrow_data {{0 1 2 0 0 -1} {} 0} width 5 offset 0" misc_feature 2856..2858 /label=N497A /ApEinfo_fwdcolor="#ff0018" /ApEinfo_revcolor="green" /ApEinfo_graphicformat="arrow_data {{0 1 2 0 0 -1} {} 0} width 5 offset 0" misc_feature 3348..3350 /label=R661A /ApEinfo_fwdcolor="#ff0003" /ApEinfo_revcolor="green" /ApEinfo_graphicformat="arrow_data {{0 1 2 0 0 -1} {} 0} width 5 offset 0" misc_feature 3450..3452 /label=Q695A /ApEinfo_fwdcolor="#ff0011" /ApEinfo_revcolor="green" /ApEinfo_graphicformat="arrow_data {{0 1 2 0 0 -1} {} 0} width 5 offset 0" misc_feature 4143..4145 /label=Q926A /ApEinfo_fwdcolor="#ff0023" /ApEinfo_revcolor="green" /ApEinfo_graphicformat="arrow_data {{0 1 2 0 0 -1} {} 0} width 5 offset 0" misc_feature 2637..2654 /label=Cas9 FW1 /ApEinfo_fwdcolor="#ff00e4" /ApEinfo_revcolor="green" /ApEinfo_graphicformat="arrow_data {{0 1 2 0 0 -1} {} 0} width 5 offset 0" misc_feature complement(4319..4336) /label=Cas9 RV2 /ApEinfo_fwdcolor="cyan" /ApEinfo_revcolor="#0030ff" /ApEinfo_graphicformat="arrow_data {{0 1 2 0 0 -1} {} 0} width 5 offset 0" ORIGIN 1 gagggcctat ttcccatgat tccttcatat ttgcatatac gatacaaggc tgttagagag 61 ataattggaa ttaatttgac tgtaaacaca aagatattag tacaaaatac gtgacgtaga 121 aagtaataat ttcttgggta gtttgcagtt ttaaaattat gttttaaaat ggactatcat 181 atgcttaccg taacttgaaa gtatttcgat ttcttggctt tatatatctt gtggaaagga 241 cgaaacaccg ggtcttcgag aagacctgtt ttagagctag aaatagcaag ttaaaataag 301 gctagtccgt tatcaacttg aaaaagtggc accgagtcgg tgcttttttg ttttagagct 361 agaaatagca agttaaaata aggctagtcc gtttttagcg cgtgcgccaa ttctgcagac 421 aaatggctct agaggtaccc gttacataac ttacggtaaa tggcccgcct ggctgaccgc 481 ccaacgaccc ccgcccattg acgtcaatag taacgccaat agggactttc cattgacgtc 541 aatgggtgga gtatttacgg taaactgccc acttggcagt acatcaagtg tatcatatgc 601 caagtacgcc ccctattgac gtcaatgacg gtaaatggcc cgcctggcat tgtgcccagt 661 acatgacctt atgggacttt cctacttggc agtacatcta cgtattagtc atcgctatta 721 ccatggtcga ggtgagcccc acgttctgct tcactctccc catctccccc ccctccccac 781 ccccaatttt gtatttattt attttttaat tattttgtgc agcgatgggg gcgggggggg 841 ggggggggcg cgcgccaggc ggggcggggc ggggcgaggg gcggggcggg gcgaggcgga 901 gaggtgcggc ggcagccaat cagagcggcg cgctccgaaa gtttcctttt atggcgaggc 961 ggcggcggcg gcggccctat aaaaagcgaa gcgcgcggcg ggcgggagtc gctgcgacgc 1021 tgccttcgcc ccgtgccccg ctccgccgcc gcctcgcgcc gcccgccccg gctctgactg 1081 accgcgttac tcccacaggt gagcgggcgg gacggccctt ctcctccggg ctgtaattag 1141 ctgagcaaga ggtaagggtt taagggatgg ttggttggtg gggtattaat gtttaattac 1201 ctggagcacc tgcctgaaat cacttttttt caggttggac cggtgccacc atggactata 1261 aggaccacga cggagactac aaggatcatg atattgatta caaagacgat gacgataaga 1321 tggccccaaa gaagaagcgg aaggtcggta tccacggagt cccagcagcc gacaagaagt 1381 acagcatcgg cctggacatc ggcaccaact ctgtgggctg ggccgtgatc accgacgagt 1441 acaaggtgcc cagcaagaaa ttcaaggtgc tgggcaacac cgaccggcac agcatcaaga 1501 agaacctgat cggagccctg ctgttcgaca gcggcgaaac agccgaggcc acccggctga 1561 agagaaccgc cagaagaaga tacaccagac ggaagaaccg gatctgctat ctgcaagaga 1621 tcttcagcaa cgagatggcc aaggtggacg acagcttctt ccacagactg gaagagtcct 1681 tcctggtgga agaggataag aagcacgagc ggcaccccat cttcggcaac atcgtggacg 1741 aggtggccta ccacgagaag taccccacca tctaccacct gagaaagaaa ctggtggaca 1801 gcaccgacaa ggccgacctg cggctgatct atctggccct ggcccacatg atcaagttcc 1861 ggggccactt cctgatcgag ggcgacctga accccgacaa cagcgacgtg gacaagctgt 1921 tcatccagct ggtgcagacc tacaaccagc tgttcgagga aaaccccatc aacgccagcg 1981 gcgtggacgc caaggccatc ctgtctgcca gactgagcaa gagcagacgg ctggaaaatc 2041 tgatcgccca gctgcccggc gagaagaaga atggcctgtt cggaaacctg attgccctga 2101 gcctgggcct gacccccaac ttcaagagca acttcgacct ggccgaggat gccaaactgc 2161 agctgagcaa ggacacctac gacgacgacc tggacaacct gctggcccag atcggcgacc 2221 agtacgccga cctgtttctg gccgccaaga acctgtccga cgccatcctg ctgagcgaca 2281 tcctgagagt gaacaccgag atcaccaagg cccccctgag cgcctctatg atcaagagat 2341 acgacgagca ccaccaggac ctgaccctgc tgaaagctct cgtgcggcag cagctgcctg 2401 agaagtacaa agagattttc ttcgaccaga gcaagaacgg ctacgccggc tacattgacg 2461 gcggagccag ccaggaagag ttctacaagt tcatcaagcc catcctggaa aagatggacg 2521 gcaccgagga actgctcgtg aagctgaaca gagaggacct gctgcggaag cagcggacct 2581 tcgacaacgg cagcatcccc caccagatcc acctgggaga gctgcacgcc attctgcggc 2641 ggcaggaaga tttttaccca ttcctgaagg acaaccggga aaagatcgag aagatcctga 2701 ccttccgcat cccctactac gtgggccctc tggccagggg aaacagcaga ttcgcctgga 2761 tgaccagaaa gagcgaggaa accatcaccc cctggaactt cgaggaagtg gtggacaagg 2821 gcgcttccgc ccagagcttc atcgagcgga tgaccGCctt cgataagaac ctgcccaacg 2881 agaaggtgct gcccaagcac agcctgctgt acgagtactt caccgtgtat aacgagctga 2941 ccaaagtgaa atacgtgacc gagggaatga gaaagcccgc cttcctgagc ggcgagcaga 3001 aaaaggccat cgtggacctg ctgttcaaga ccaaccggaa agtgaccgtg aagcagctga 3061 aagaggacta cttcaagaaa atcgagtgct tcgactccgt ggaaatctcc ggcgtggaag 3121 atcggttcaa cgcctccctg ggcacatacc acgatctgct gaaaattatc aaggacaagg 3181 acttcctgga caatgaggaa aacgaggaca ttctggaaga tatcgtgctg accctgacac 3241 tgtttgagga cagagagatg atcgaggaac ggctgaaaac ctatgcccac ctgttcgacg 3301 acaaagtgat gaagcagctg aagcggcgga gatacaccgg ctggggcGCg ctgagccgga 3361 agctgatcaa cggcatccgg gacaagcagt ccggcaagac aatcctggat ttcctgaagt 3421 ccgacggctt cgccaacaga aacttcatgG Cgctgatcca cgacgacagc ctgaccttta 3481 aagaggacat ccagaaagcc caggtgtccg gccagggcga tagcctgcac gagcacattg 3541 ccaatctggc cggcagcccc gccattaaga agggcatcct gcagacagtg aaggtggtgg 3601 acgagctcgt gaaagtgatg ggccggcaca agcccgagaa catcgtgatc gaaatggcca 3661 gagagaacca gaccacccag aagggacaga agaacagccg cgagagaatg aagcggatcg 3721 aagagggcat caaagagctg ggcagccaga tcctgaaaga acaccccgtg gaaaacaccc 3781 agctgcagaa cgagaagctg tacctgtact acctgcagaa tgggcgggat atgtacgtgg 3841 accaggaact ggacatcaac cggctgtccg actacgatgt ggaccatatc gtgcctcaga 3901 gctttctgaa ggacgactcc atcgacaaca aggtgctgac cagaagcgac aagaaccggg 3961 gcaagagcga caacgtgccc tccgaagagg tcgtgaagaa gatgaagaac tactggcggc 4021 agctgctgaa cgccaagctg attacccaga gaaagttcga caatctgacc aaggccgaga 4081 gaggcggcct gagcgaactg gataaggccg gcttcatcaa gagacagctg gtggaaaccc 4141 ggGCgatcac aaagcacgtg gcacagatcc tggactcccg gatgaacact aagtacgacg 4201 agaatgacaa gctgatccgg gaagtgaaag tgatcaccct gaagtccaag ctggtgtccg 4261 atttccggaa ggatttccag ttttacaaag tgcgcgagat caacaactac caccacgccc 4321 acgacgccta cctgaacgcc gtcgtgggaa ccgccctgat caaaaagtac cctaagctgg 4381 aaagcgagtt cgtgtacggc gactacaagg tgtacgacgt gcggaagatg atcgccaaga 4441 gcgagcagga aatcggcaag gctaccgcca agtacttctt ctacagcaac atcatgaact 4501 ttttcaagac cgagattacc ctggccaacg gcgagatccg gaagcggcct ctgatcgaga 4561 caaacggcga aaccggggag atcgtgtggg ataagggccg ggattttgcc accgtgcgga 4621 aagtgctgag catgccccaa gtgaatatcg tgaaaaagac cgaggtgcag acaggcggct 4681 tcagcaaaga gtctatcctg cccaagagga acagcgataa gctgatcgcc agaaagaagg 4741 actgggaccc taagaagtac ggcggcttcg acagccccac cgtggcctat tctgtgctgg 4801 tggtggccaa agtggaaaag ggcaagtcca agaaactgaa gagtgtgaaa gagctgctgg 4861 ggatcaccat catggaaaga agcagcttcg agaagaatcc catcgacttt ctggaagcca 4921 agggctacaa agaagtgaaa aaggacctga tcatcaagct gcctaagtac tccctgttcg 4981 agctggaaaa cggccggaag agaatgctgg cctctgccgg cgaactgcag aagggaaacg 5041 aactggccct gccctccaaa tatgtgaact tcctgtacct ggccagccac tatgagaagc 5101 tgaagggctc ccccgaggat aatgagcaga aacagctgtt tgtggaacag cacaagcact 5161 acctggacga gatcatcgag cagatcagcg agttctccaa gagagtgatc ctggccgacg 5221 ctaatctgga caaagtgctg tccgcctaca acaagcaccg ggataagccc atcagagagc 5281 aggccgagaa tatcatccac ctgtttaccc tgaccaatct gggagcccct gccgccttca 5341 agtactttga caccaccatc gaccggaaga ggtacaccag caccaaagag gtgctggacg 5401 ccaccctgat ccaccagagc atcaccggcc tgtacgagac acggatcgac ctgtctcagc 5461 tgggaggcga caaaaggccg gcggccacga aaaaggccgg ccaggcaaaa aagaaaaagg 5521 aattcggcag tggagagggc agaggaagtc tgctaacatg cggtgacgtc gaggagaatc 5581 ctggcccaat gaccgagtac aagcccacgg tgcgcctcgc cacccgcgac gacgtcccca 5641 gggccgtacg caccctcgcc gccgcgttcg ccgactaccc cgccacgcgc cacaccgtcg 5701 atccggaccg ccacatcgag cgggtcaccg agctgcaaga actcttcctc acgcgcgtcg 5761 ggctcgacat cggcaaggtg tgggtcgcgg acgacggcgc cgcggtggcg gtctggacca 5821 cgccggagag cgtcgaagcg ggggcggtgt tcgccgagat cggcccgcgc atggccgagt 5881 tgagcggttc ccggctggcc gcgcagcaac agatggaagg cctcctggcg ccgcaccggc 5941 ccaaggagcc cgcgtggttc ctggccaccg tcggagtctc gcccgaccac cagggcaagg 6001 gtctgggcag cgccgtcgtg ctccccggag tggaggcggc cgagcgcgcc ggggtgcccg 6061 ccttcctgga gacctccgcg ccccgcaacc tccccttcta cgagcggctc ggcttcaccg 6121 tcaccgccga cgtcgaggtg cccgaaggac cgcgcacctg gtgcatgacc cgcaagcccg 6181 gtgcctgaga attctaacta gagctcgctg atcagcctcg actgtgcctt ctagttgcca 6241 gccatctgtt gtttgcccct cccccgtgcc ttccttgacc ctggaaggtg ccactcccac 6301 tgtcctttcc taataaaatg aggaaattgc atcgcattgt ctgagtaggt gtcattctat 6361 tctggggggt ggggtggggc aggacagcaa gggggaggat tgggaagaga atagcaggca 6421 tgctggggag cggccgcagg aacccctagt gatggagttg gccactccct ctctgcgcgc 6481 tcgctcgctc actgaggccg ggcgaccaaa ggtcgcccga cgcccgggct ttgcccgggc 6541 ggcctcagtg agcgagcgag cgcgcagctg cctgcagggg cgcctgatgc ggtattttct 6601 ccttacgcat ctgtgcggta tttcacaccg catacgtcaa agcaaccata gtacgcgccc 6661 tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt 6721 gccagcgccc tagcgcccgc tcctttcgct ttcttccctt cctttctcgc cacgttcgcc 6781 ggctttcccc gtcaagctct aaatcggggg ctccctttag ggttccgatt tagtgcttta 6841 cggcacctcg accccaaaaa acttgatttg ggtgatggtt cacgtagtgg gccatcgccc 6901 tgatagacgg tttttcgccc tttgacgttg gagtccacgt tctttaatag tggactcttg 6961 ttccaaactg gaacaacact caaccctatc tcgggctatt cttttgattt ataagggatt 7021 ttgccgattt cggcctattg gttaaaaaat gagctgattt aacaaaaatt taacgcgaat 7081 tttaacaaaa tattaacgtt tacaatttta tggtgcactc tcagtacaat ctgctctgat 7141 gccgcatagt taagccagcc ccgacacccg ccaacacccg ctgacgcgcc ctgacgggct 7201 tgtctgctcc cggcatccgc ttacagacaa gctgtgaccg tctccgggag ctgcatgtgt 7261 cagaggtttt caccgtcatc accgaaacgc gcgagacgaa agggcctcgt gatacgccta 7321 tttttatagg ttaatgtcat gataataatg gtttcttaga cgtcaggtgg cacttttcgg 7381 ggaaatgtgc gcggaacccc tatttgttta tttttctaaa tacattcaaa tatgtatccg 7441 ctcatgagac aataaccctg ataaatgctt caataatatt gaaaaaggaa gagtatgagt 7501 attcaacatt tccgtgtcgc ccttattccc ttttttgcgg cattttgcct tcctgttttt 7561 gctcacccag aaacgctggt gaaagtaaaa gatgctgaag atcagttggg tgcacgagtg 7621 ggttacatcg aactggatct caacagcggt aagatccttg agagttttcg ccccgaagaa 7681 cgttttccaa tgatgagcac ttttaaagtt ctgctatgtg gcgcggtatt atcccgtatt 7741 gacgccgggc aagagcaact cggtcgccgc atacactatt ctcagaatga cttggttgag 7801 tactcaccag tcacagaaaa gcatcttacg gatggcatga cagtaagaga attatgcagt 7861 gctgccataa ccatgagtga taacactgcg gccaacttac ttctgacaac gatcggagga 7921 ccgaaggagc taaccgcttt tttgcacaac atgggggatc atgtaactcg ccttgatcgt 7981 tgggaaccgg agctgaatga agccatacca aacgacgagc gtgacaccac gatgcctgta 8041 gcaatggcaa caacgttgcg caaactatta actggcgaac tacttactct agcttcccgg 8101 caacaattaa tagactggat ggaggcggat aaagttgcag gaccacttct gcgctcggcc 8161 cttccggctg gctggtttat tgctgataaa tctggagccg gtgagcgtgg aagccgcggt 8221 atcattgcag cactggggcc agatggtaag ccctcccgta tcgtagttat ctacacgacg 8281 gggagtcagg caactatgga tgaacgaaat agacagatcg ctgagatagg tgcctcactg 8341 attaagcatt ggtaactgtc agaccaagtt tactcatata tactttagat tgatttaaaa 8401 cttcattttt aatttaaaag gatctaggtg aagatccttt ttgataatct catgaccaaa 8461 atcccttaac gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga 8521 tcttcttgag atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg 8581 ctaccagcgg tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact 8641 ggcttcagca gagcgcagat accaaatact gtccttctag tgtagccgta gttaggccac 8701 cacttcaaga actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg 8761 gctgctgcca gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg 8821 gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga 8881 acgacctaca ccgaactgag atacctacag cgtgagctat gagaaagcgc cacgcttccc 8941 gaagggagaa aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg 9001 agggagcttc cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc 9061 tgacttgagc gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc 9121 agcaacgcgg cctttttacg gttcctggcc ttttgctggc cttttgctca catgt //