LOCUS Exported 10657 bp ds-DNA circular SYN 28-JUN-2021 DEFINITION CRISPR/Cas9 expressing plasmid containing two gRNAs targeting mouse Otx2.. ACCESSION . VERSION . KEYWORDS pX458-Ef1a-Cas9-H2B-mCherry (dual gRNA: Otx2 x2) SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 10657) TITLE Method: add second guide to any existing pX458-compatible plasmid REFERENCE 2 (bases 1 to 10657) AUTHORS . TITLE Direct Submission JOURNAL Exported Jun 28, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..10657 /organism="synthetic DNA construct" /mol_type="other DNA" promoter 208..1389 /label=EF-1-alpha promoter /note="strong constitutive promoter for human elongation factor EF-1-alpha" intron 438..1380 /label=EF-1-alpha intron A /note="intron upstream of the start codon of human EF-1-alpha" primer_bind 1337..1357 /label=EF1a-F /note="Human elongation factor-1a promoter, forward primer" regulatory 1395..1404 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" CDS 1404..1469 /codon_start=1 /product="three tandem FLAG(R) epitope tags, followed by an enterokinase cleavage site" /label=3xFLAG /translation="DYKDHDGDYKDHDIDYKDDDDK" CDS 1476..1496 /codon_start=1 /product="nuclear localization signal of SV40 (simian virus 40) large T antigen" /label=SV40 NLS /translation="PKKKRKV" CDS 1521..5621 /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="DKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL YYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPS EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD ATLIHQSITGLYETRIDLSQLGGD" CDS 5622..5669 /codon_start=1 /product="bipartite nuclear localization signal from nucleoplasmin" /label=nucleoplasmin NLS /translation="KRPAATKKAGQAKKKK" CDS 5685..5738 /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 5739..6113 /codon_start=1 /gene="HIST1H2BB" /product="human histone H2B" /label=H2B /translation="MPEPSKSAPAPKKGSKKAITKAQKKDGKKRKRSRKESYSIYVYKV LKQVHPDTGISSKAMGIMNSFVNDIFERIAGEASRLAHYNKRSTITSREIQTAVRLLLP GELAKHAVSEGTKAVTKYTSS" regulatory 6129..6138 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" CDS 6135..6842 /codon_start=1 /product="monomeric derivative of DsRed fluorescent protein (Shaner et al., 2004)" /label=mCherry /note="mammalian codon-optimized" /translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERA EGRHSTGGMDELYK" primer_bind complement(6280..6298) /label=mCherry-R /note="mCherry, reverse primer" primer_bind complement(6870..6887) /label=BGH-rev /note="Bovine growth hormone terminator, reverse primer. Also called BGH reverse" polyA_signal 6876..7083 /label=bGH poly(A) signal /note="bovine growth hormone polyadenylation signal" repeat_region 7092..7232 /label=AAV2 ITR /note="inverted terminal repeat of adeno-associated virus serotype 2" repeat_region 7092..7221 /label=AAV2 ITR rep_origin 7307..7762 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(7394..7413) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 7604..7625 /label=F1ori-F /note="F1 origin, forward primer" primer_bind complement(7779..7798) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" primer_bind 7898..7920 /label=pGEX 3' /note="pGEX vectors, reverse primer" primer_bind complement(7958..7976) /label=pBRforEco /note="pBR322 vectors, upsteam of EcoRI site, forward primer" promoter 8044..8148 /gene="bla" /label=AmpR promoter CDS 8149..9009 /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(8367..8386) /label=Amp-R /note="Ampicillin resistance gene, reverse primer" rep_origin 9180..9768 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 9669..9688 /label=pBR322ori-F /note="pBR322 origin, forward primer" promoter 9830..10070 /label=U6 promoter /note="RNA polymerase III promoter for human U6 snRNA" misc_RNA 10100..10175 /label=gRNA scaffold /note="guide RNA scaffold for the Streptococcus pyogenes CRISPR/Cas9 system" promoter 10271..10511 /label=U6 promoter /note="RNA polymerase III promoter for human U6 snRNA" misc_RNA 10541..10616 /label=gRNA scaffold /note="guide RNA scaffold for the Streptococcus pyogenes CRISPR/Cas9 system" ORIGIN 1 tagtccgttt ttagcgcgtg cgccaattct gcagacaaat ggctctacag gtaccagtaa 61 ttcatacaaa aggactcgcc cctgccttgg ggaatcccag ggaccgtcgt taaactccca 121 ctaacgtaga acccagagat cgctgcgttc ccgccccctc acccgcccgc tctcgtcatc 181 actgaggtgg agaagagcat gcgtgaggct ccggtgcccg tcagtgggca gagcgcacat 241 cgcccacagt ccccgagaag ttggggggag gggtcggcaa ttgaaccggt gcctagagaa 301 ggtggcgcgg ggtaaactgg gaaagtgatg tcgtgtactg gctccgcctt tttcccgagg 361 gtgggggaga accgtatata agtgcagtag tcgccgtgaa cgttcttttt cgcaacgggt 421 ttgccgccag aacacaggta agtgccgtgt gtggttcccg cgggcctggc ctctttacgg 481 gttatggccc ttgcgtgcct tgaattactt ccacgcccct ggctgcagta cgtgattctt 541 gatcccgagc ttcgggttgg aagtgggtgg gagagttcga ggccttgcgc ttaaggagcc 601 ccttcgcctc gtgcttgagt tgaggcctgg cttgggcgct ggggccgccg cgtgcgaatc 661 tggtggcacc ttcgcgcctg tctcgctgct ttcgataagt ctctagccat ttaaaatttt 721 tgatgacctg ctgcgacgct ttttttctgg caagatagtc ttgtaaatgc gggccaagat 781 ctgcacactg gtatttcggt ttttggggcc gcgggcggcg acggggcccg tgcgtcccag 841 cgcacatgtt cggcgaggcg gggcctgcga gcgcggccac cgagaatcgg acgggggtag 901 tctcaagctg gccggcctgc tctggtgcct ggcctcgcgc cgccgtgtat cgccccgccc 961 tgggcggcaa ggctggcccg gtcggcacca gttgcgtgag cggaaagatg gccgcttccc 1021 ggccctgctg cagggagctc aaaatggagg acgcggcgct cgggagagcg ggcgggtgag 1081 tcacccacac aaaggaaaag ggcctttccg tcctcagccg tcgcttcatg tgactccacg 1141 gagtaccggg cgccgtccag gcacctcgat tagttctcga gcttttggag tacgtcgtct 1201 ttaggttggg gggaggggtt ttatgcgatg gagtttcccc acactgagtg ggtggagact 1261 gaagttaggc cagcttggca cttgatgtaa ttctccttgg aatttgccct ttttgagttt 1321 ggatcttggt tcattctcaa gcctcagaca gtggttcaaa gtttttttct tccatttcag 1381 gtgtcgtgac cggtgccacc atggactata aggaccacga cggagactac aaggatcatg 1441 atattgatta caaagacgat gacgataaga tggccccaaa gaagaagcgg aaggtcggta 1501 tccacggagt cccagcagcc gacaagaagt acagcatcgg cctggacatc ggcaccaact 1561 ctgtgggctg ggccgtgatc accgacgagt acaaggtgcc cagcaagaaa ttcaaggtgc 1621 tgggcaacac cgaccggcac agcatcaaga agaacctgat cggagccctg ctgttcgaca 1681 gcggcgaaac agccgaggcc acccggctga agagaaccgc cagaagaaga tacaccagac 1741 ggaagaaccg gatctgctat ctgcaagaga tcttcagcaa cgagatggcc aaggtggacg 1801 acagcttctt ccacagactg gaagagtcct tcctggtgga agaggataag aagcacgagc 1861 ggcaccccat cttcggcaac atcgtggacg aggtggccta ccacgagaag taccccacca 1921 tctaccacct gagaaagaaa ctggtggaca gcaccgacaa ggccgacctg cggctgatct 1981 atctggccct ggcccacatg atcaagttcc ggggccactt cctgatcgag ggcgacctga 2041 accccgacaa cagcgacgtg gacaagctgt tcatccagct ggtgcagacc tacaaccagc 2101 tgttcgagga aaaccccatc aacgccagcg gcgtggacgc caaggccatc ctgtctgcca 2161 gactgagcaa gagcagacgg ctggaaaatc tgatcgccca gctgcccggc gagaagaaga 2221 atggcctgtt cggaaacctg attgccctga gcctgggcct gacccccaac ttcaagagca 2281 acttcgacct ggccgaggat gccaaactgc agctgagcaa ggacacctac gacgacgacc 2341 tggacaacct gctggcccag atcggcgacc agtacgccga cctgtttctg gccgccaaga 2401 acctgtccga cgccatcctg ctgagcgaca tcctgagagt gaacaccgag atcaccaagg 2461 cccccctgag cgcctctatg atcaagagat acgacgagca ccaccaggac ctgaccctgc 2521 tgaaagctct cgtgcggcag cagctgcctg agaagtacaa agagattttc ttcgaccaga 2581 gcaagaacgg ctacgccggc tacattgacg gcggagccag ccaggaagag ttctacaagt 2641 tcatcaagcc catcctggaa aagatggacg gcaccgagga actgctcgtg aagctgaaca 2701 gagaggacct gctgcggaag cagcggacct tcgacaacgg cagcatcccc caccagatcc 2761 acctgggaga gctgcacgcc attctgcggc ggcaggaaga tttttaccca ttcctgaagg 2821 acaaccggga aaagatcgag aagatcctga ccttccgcat cccctactac gtgggccctc 2881 tggccagggg aaacagcaga ttcgcctgga tgaccagaaa gagcgaggaa accatcaccc 2941 cctggaactt cgaggaagtg gtggacaagg gcgcttccgc ccagagcttc atcgagcgga 3001 tgaccaactt cgataagaac ctgcccaacg agaaggtgct gcccaagcac agcctgctgt 3061 acgagtactt caccgtgtat aacgagctga ccaaagtgaa atacgtgacc gagggaatga 3121 gaaagcccgc cttcctgagc ggcgagcaga aaaaggccat cgtggacctg ctgttcaaga 3181 ccaaccggaa agtgaccgtg aagcagctga aagaggacta cttcaagaaa atcgagtgct 3241 tcgactccgt ggaaatctcc ggcgtggaag atcggttcaa cgcctccctg ggcacatacc 3301 acgatctgct gaaaattatc aaggacaagg acttcctgga caatgaggaa aacgaggaca 3361 ttctggaaga tatcgtgctg accctgacac tgtttgagga cagagagatg atcgaggaac 3421 ggctgaaaac ctatgcccac ctgttcgacg acaaagtgat gaagcagctg aagcggcgga 3481 gatacaccgg ctggggcagg ctgagccgga agctgatcaa cggcatccgg gacaagcagt 3541 ccggcaagac aatcctggat ttcctgaagt ccgacggctt cgccaacaga aacttcatgc 3601 agctgatcca cgacgacagc ctgaccttta aagaggacat ccagaaagcc caggtgtccg 3661 gccagggcga tagcctgcac gagcacattg ccaatctggc cggcagcccc gccattaaga 3721 agggcatcct gcagacagtg aaggtggtgg acgagctcgt gaaagtgatg ggccggcaca 3781 agcccgagaa catcgtgatc gaaatggcca gagagaacca gaccacccag aagggacaga 3841 agaacagccg cgagagaatg aagcggatcg aagagggcat caaagagctg ggcagccaga 3901 tcctgaaaga acaccccgtg gaaaacaccc agctgcagaa cgagaagctg tacctgtact 3961 acctgcagaa tgggcgggat atgtacgtgg accaggaact ggacatcaac cggctgtccg 4021 actacgatgt ggaccatatc gtgcctcaga gctttctgaa ggacgactcc atcgacaaca 4081 aggtgctgac cagaagcgac aagaaccggg gcaagagcga caacgtgccc tccgaagagg 4141 tcgtgaagaa gatgaagaac tactggcggc agctgctgaa cgccaagctg attacccaga 4201 gaaagttcga caatctgacc aaggccgaga gaggcggcct gagcgaactg gataaggccg 4261 gcttcatcaa gagacagctg gtggaaaccc ggcagatcac aaagcacgtg gcacagatcc 4321 tggactcccg gatgaacact aagtacgacg agaatgacaa gctgatccgg gaagtgaaag 4381 tgatcaccct gaagtccaag ctggtgtccg atttccggaa ggatttccag ttttacaaag 4441 tgcgcgagat caacaactac caccacgccc acgacgccta cctgaacgcc gtcgtgggaa 4501 ccgccctgat caaaaagtac cctaagctgg aaagcgagtt cgtgtacggc gactacaagg 4561 tgtacgacgt gcggaagatg atcgccaaga gcgagcagga aatcggcaag gctaccgcca 4621 agtacttctt ctacagcaac atcatgaact ttttcaagac cgagattacc ctggccaacg 4681 gcgagatccg gaagcggcct ctgatcgaga caaacggcga aaccggggag atcgtgtggg 4741 ataagggccg ggattttgcc accgtgcgga aagtgctgag catgccccaa gtgaatatcg 4801 tgaaaaagac cgaggtgcag acaggcggct tcagcaaaga gtctatcctg cccaagagga 4861 acagcgataa gctgatcgcc agaaagaagg actgggaccc taagaagtac ggcggcttcg 4921 acagccccac cgtggcctat tctgtgctgg tggtggccaa agtggaaaag ggcaagtcca 4981 agaaactgaa gagtgtgaaa gagctgctgg ggatcaccat catggaaaga agcagcttcg 5041 agaagaatcc catcgacttt ctggaagcca agggctacaa agaagtgaaa aaggacctga 5101 tcatcaagct gcctaagtac tccctgttcg agctggaaaa cggccggaag agaatgctgg 5161 cctctgccgg cgaactgcag aagggaaacg aactggccct gccctccaaa tatgtgaact 5221 tcctgtacct ggccagccac tatgagaagc tgaagggctc ccccgaggat aatgagcaga 5281 aacagctgtt tgtggaacag cacaagcact acctggacga gatcatcgag cagatcagcg 5341 agttctccaa gagagtgatc ctggccgacg ctaatctgga caaagtgctg tccgcctaca 5401 acaagcaccg ggataagccc atcagagagc aggccgagaa tatcatccac ctgtttaccc 5461 tgaccaatct gggagcccct gccgccttca agtactttga caccaccatc gaccggaaga 5521 ggtacaccag caccaaagag gtgctggacg ccaccctgat ccaccagagc atcaccggcc 5581 tgtacgagac acggatcgac ctgtctcagc tgggaggcga caaaaggccg gcggccacga 5641 aaaaggccgg ccaggcaaaa aagaaaaagg aattcggcag tggagagggc agaggaagtc 5701 tgctaacatg cggtgacgtc gaggagaatc ctggcccaat gcctgaaccc tctaagtctg 5761 ctccagcccc taaaaagggt tctaagaagg ctatcactaa ggcgcagaag aaggatggta 5821 agaagcgtaa gcgcagccgc aaggagagct attctatcta tgtgtacaag gttctgaagc 5881 aggtccaccc cgacaccggc atctcatcca aggccatggg gatcatgaac tccttcgtca 5941 acgacatctt cgagcgcatc gcgggcgagg cttctcgcct ggctcactac aataagcgct 6001 cgaccatcac ctccagggag attcagacgg ctgtgcgcct gctgctgcct ggggagctgg 6061 ctaagcatgc tgtgtccgag ggcactaagg cagttaccaa gtacactagc tctaaggatc 6121 caccggtcgc caccatggtg agcaagggcg aggaggataa catggccatc atcaaggagt 6181 tcatgcgctt caaggtgcac atggagggct ccgtgaacgg ccacgagttc gagatcgagg 6241 gcgagggcga gggccgcccc tacgagggca cccagaccgc caagctgaag gtgaccaagg 6301 gtggccccct gcccttcgcc tgggacatcc tgtcccctca gttcatgtac ggctccaagg 6361 cctacgtgaa gcaccccgcc gacatccccg actacttgaa gctgtccttc cccgagggct 6421 tcaagtggga gcgcgtgatg aacttcgagg acggcggcgt ggtgaccgtg acccaggact 6481 cctccctgca ggacggcgag ttcatctaca aggtgaagct gcgcggcacc aacttcccct 6541 ccgacggccc cgtaatgcag aagaaaacca tgggctggga ggcctcctcc gagcggatgt 6601 accccgagga cggcgccctg aagggcgaga tcaagcagag gctgaagctg aaggacggcg 6661 gccactacga cgctgaggtc aagaccacct acaaggccaa gaagcccgtg cagctgcccg 6721 gcgcctacaa cgtcaacatc aagttggaca tcacctccca caacgaggac tacaccatcg 6781 tggaacagta cgaacgcgcc gagggccgcc actccaccgg cggcatggac gagctgtaca 6841 aggaattcta actagagctc gctgatcagc ctcgactgtg ccttctagtt gccagccatc 6901 tgttgtttgc ccctcccccg tgccttcctt gaccctggaa ggtgccactc ccactgtcct 6961 ttcctaataa aatgaggaaa ttgcatcgca ttgtctgagt aggtgtcatt ctattctggg 7021 gggtggggtg gggcaggaca gcaaggggga ggattgggaa gagaatagca ggcatgctgg 7081 ggagcggccg caggaacccc tagtgatgga gttggccact ccctctctgc gcgctcgctc 7141 gctcactgag gccgggcgac caaaggtcgc ccgacgcccg ggctttgccc gggcggcctc 7201 agtgagcgag cgagcgcgca gctgcctgca ggggcgcctg atgcggtatt ttctccttac 7261 gcatctgtgc ggtatttcac accgcatacg tcaaagcaac catagtacgc gccctgtagc 7321 ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac acttgccagc 7381 gccttagcgc ccgctccttt cgctttcttc ccttcctttc tcgccacgtt cgccggcttt 7441 ccccgtcaag ctctaaatcg ggggctccct ttagggttcc gatttagtgc tttacggcac 7501 ctcgacccca aaaaacttga tttgggtgat ggttcacgta gtgggccatc gccctgatag 7561 acggtttttc gccctttgac gttggagtcc acgttcttta atagtggact cttgttccaa 7621 actggaacaa cactcaactc tatctcgggc tattcttttg atttataagg gattttgccg 7681 atttcggtct attggttaaa aaatgagctg atttaacaaa aatttaacgc gaattttaac 7741 aaaatattaa cgtttacaat tttatggtgc actctcagta caatctgctc tgatgccgca 7801 tagttaagcc agccccgaca cccgccaaca cccgctgacg cgccctgacg ggcttgtctg 7861 ctcccggcat ccgcttacag acaagctgtg accgtctccg ggagctgcat gtgtcagagg 7921 ttttcaccgt catcaccgaa acgcgcgaga cgaaagggcc tcgtgatacg cctattttta 7981 taggttaatg tcatgataat aatggtttct tagacgtcag gtggcacttt tcggggaaat 8041 gtgcgcggaa cccctatttg tttatttttc taaatacatt caaatatgta tccgctcatg 8101 agacaataac cctgataaat gcttcaataa tattgaaaaa ggaagagtat gagtattcaa 8161 catttccgtg tcgcccttat tccctttttt gcggcatttt gccttcctgt ttttgctcac 8221 ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt tgggtgcacg agtgggttac 8281 atcgaactgg atctcaacag cggtaagatc cttgagagtt ttcgccccga agaacgtttt 8341 ccaatgatga gcacttttaa agttctgcta tgtggcgcgg tattatcccg tattgacgcc 8401 gggcaagagc aactcggtcg ccgcatacac tattctcaga atgacttggt tgagtactca 8461 ccagtcacag aaaagcatct tacggatggc atgacagtaa gagaattatg cagtgctgcc 8521 ataaccatga gtgataacac tgcggccaac ttacttctga caacgatcgg aggaccgaag 8581 gagctaaccg cttttttgca caacatgggg gatcatgtaa ctcgccttga tcgttgggaa 8641 ccggagctga atgaagccat accaaacgac gagcgtgaca ccacgatgcc tgtagcaatg 8701 gcaacaacgt tgcgcaaact attaactggc gaactactta ctctagcttc ccggcaacaa 8761 ttaatagact ggatggaggc ggataaagtt gcaggaccac ttctgcgctc ggcccttccg 8821 gctggctggt ttattgctga taaatctgga gccggtgagc gtggaagccg cggtatcatt 8881 gcagcactgg ggccagatgg taagccctcc cgtatcgtag ttatctacac gacggggagt 8941 caggcaacta tggatgaacg aaatagacag atcgctgaga taggtgcctc actgattaag 9001 cattggtaac tgtcagacca agtttactca tatatacttt agattgattt aaaacttcat 9061 ttttaattta aaaggatcta ggtgaagatc ctttttgata atctcatgac caaaatccct 9121 taacgtgagt tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa aggatcttct 9181 tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca 9241 gcggtggttt gtttgccgga tcaagagcta ccaactcttt ttccgaaggt aactggcttc 9301 agcagagcgc agataccaaa tactgttctt ctagtgtagc cgtagttagg ccaccacttc 9361 aagaactctg tagcaccgcc tacatacctc gctctgctaa tcctgttacc agtggctgct 9421 gccagtggcg ataagtcgtg tcttaccggg ttggactcaa gacgatagtt accggataag 9481 gcgcagcggt cgggctgaac ggggggttcg tgcacacagc ccagcttgga gcgaacgacc 9541 tacaccgaac tgagatacct acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg 9601 agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa caggagagcg cacgagggag 9661 cttccagggg gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca cctctgactt 9721 gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac 9781 gcggcctttt tacggttcct ggccttttgc tggccttttg ctcacatgtg agggcctatt 9841 tcccatgatt ccttcatatt tgcatatacg atacaaggct gttagagaga taattggaat 9901 taatttgact gtaaacacaa agatattagt acaaaatacg tgacgtagaa agtaataatt 9961 tcttgggtag tttgcagttt taaaattatg ttttaaaatg gactatcata tgcttaccgt 10021 aacttgaaag tatttcgatt tcttggcttt atatatcttg tggaaaggac gaaacaccgc 10081 ttaccggggt agcccacggg ttttagagct agaaatagca agttaaaata aggctagtcc 10141 gttatcaact tgaaaaagtg gcaccgagtc ggtgcttttt tgttttagag ctagaaatag 10201 caagttaaaa taaggctagt ccgtttttag cgcgtgcgcc aattctgcag acaaatggct 10261 ctagacatgt gagggcctat ttcccatgat tccttcatat ttgcatatac gatacaaggc 10321 tgttagagag ataattggaa ttaatttgac tgtaaacaca aagatattag tacaaaatac 10381 gtgacgtaga aagtaataat ttcttgggta gtttgcagtt ttaaaattat gttttaaaat 10441 ggactatcat atgcttaccg taacttgaaa gtatttcgat ttcttggctt tatatatctt 10501 gtggaaagga cgaaacaccg caccccccgg aaacagcgaa gttttagagc tagaaatagc 10561 aagttaaaat aaggctagtc cgttatcaac ttgaaaaagt ggcaccgagt cggtgctttt 10621 ttgttttaga gctagaaata gcaagttaaa ataaggc //