LOCUS Exported 15390 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION constitutive expression of dCas9-BFP-KRAB from the CLYBL locus (Ward lab). ACCESSION . VERSION . KEYWORDS pC13N-dCas9-BFP-KRAB SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 15390) AUTHORS Tian R, Gachechiladze MA, Ludwig CH, Laurie MT, Hong JY, Nathaniel D, Prabhu AV, Fernandopulle MS, Patel R, Abshari M, Ward ME, Kampmann M TITLE CRISPR Interference-Based Platform for Multimodal Genetic Screens in Human iPSC-Derived Neurons. JOURNAL Neuron. 2019 Aug 5. pii: S0896-6273(19)30640-3. doi: 10.1016/j.neuron.2019.07.014. PUBMED 31422865 REFERENCE 2 (bases 1 to 15390) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..15390 /organism="synthetic DNA construct" /mol_type="other DNA" protein_bind 9..42 /label=lox2272 /bound_moiety="Cre recombinase" /note="Cre-mediated recombination occurs in the 8-bp core sequence (GGATACTT). lox2272 sites are compatible with each other, but incompatible with loxP or loxN sites (Lee and Saito, 1988)." enhancer 113..416 /label=CMV enhancer /note="human cytomegalovirus immediate early enhancer" promoter 418..695 /label=chicken beta-actin promoter intron 696..1712 /label=chimeric intron /note="chimera between introns from chicken beta-actin and rabbit beta-globin" primer_bind 1636..1658 /label=pCAGGS-5 /note="Chimeric intron in CAG promoter, forward primer" primer_bind 1720..1739 /label=pCAG-F /note="Rabbit beta-globin intron, for pCAG plasmids, forward primer" regulatory 1779..1788 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" CDS 1785..5888 /codon_start=1 /product="catalytically dead mutant of the Cas9 endonuclease from the Streptococcus pyogenes Type II CRISPR/Cas system" /label=dCas9 /note="RNA-guided DNA-binding protein that lacks endonuclease activity due to the D10A mutation in the RuvC catalytic domain and the H840A mutation in the HNH catalytic domain" /translation="MDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK NLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER LKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFM QLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY LYYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVP SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL DATLIHQSITGLYETRIDLSQLGGD" CDS 5892..5918 /codon_start=1 /product="HA (human influenza hemagglutinin) epitope tag" /label=HA /translation="YPYDVPDYA" CDS 5937..5957 /codon_start=1 /product="nuclear localization signal of SV40 (simian virus 40) large T antigen" /label=SV40 NLS /translation="PKKKRKV" CDS 5964..5984 /codon_start=1 /product="nuclear localization signal of SV40 (simian virus 40) large T antigen" /label=SV40 NLS /translation="PKKKRKV" CDS 6024..6719 /codon_start=1 /product="monomeric blue fluorescent protein" /label=TagBFP /note="mammalian codon-optimized" /translation="SELIKENMHMKLYMEGTVDNHHFKCTSEGEGKPYEGTQTMRIKVV EGGPLPFAFDILATSFLYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLTAT QDTSLQDGCLIYNVKIRGVNFTSNGPVMQKKTLGWEAFTETLYPADGGLEGRNDMALKL VGGSHLIANIKTTYRSKKPAKNLKMPGVYYVDYRLERIKEANNETYVEQHEVAVARYCD LPSKLGHKLN" CDS 6765..6950 /codon_start=1 /product="Kruppel-associated box (KRAB) transcriptional repression domain from the human zinc finger protein ZNF10 (Margolin et al., 1994)" /label=KRAB /translation="RTLVTFKDVFVDFTREEWKLLDTAQQIVYRNVMLENYKNLVSLGY QLTKPDVILRLEKGEEP" primer_bind complement(7043..7062) /label=Bglob-pA-R /note="Rabbit beta-globin polyA region, reverse primer" polyA_signal 7108..7163 /label=-beta-globin poly(A) signal /note="rabbit beta-globin polyadenylation signal (Gil and Proudfoot, 1987)" primer_bind complement(7162..7181) /label=rbglobpA-R /note="Rabbit beta-globin polyA, reverse primer. Also called rb-glob-pA-term-R" protein_bind complement(7496..7529) /label=loxP511 /bound_moiety="Cre recombinase" /note="Cre-mediated recombination occurs in the 8-bp core sequence (GTATACAT). loxP511 sites are compatible with each other, but incompatible with wild-type loxP sites." primer_bind complement(9585..9602) /label=M13 Forward /note="In lacZ gene. Also called M13-F20 or M13 (-21) Forward" primer_bind complement(9585..9601) /label=M13 fwd /note="common sequencing primer, one of multiple similar variants" primer_bind complement(9594..9616) /label=M13/pUC Forward /note="In lacZ gene" primer_bind complement(9810..9829) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" primer_bind 9929..9951 /label=pGEX 3' /note="pGEX vectors, reverse primer" primer_bind complement(10082..10101) /label=pENTR-R /note="pENTR vectors, reverse primer" CDS 10093..10902 /codon_start=1 /gene="aph(3')-Ia" /product="aminoglycoside phosphotransferase" /label=KanR /note="confers resistance to kanamycin in bacteria or G418 (Geneticin(R)) in eukaryotes" /translation="MSHIQRETSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYGKP DAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGKTA FQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDASD FDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGIAD RYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF" primer_bind complement(10160..10179) /label=Kan-R /note="Kanamycin resistance gene, reverse primer" rep_origin 11080..11668 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 11569..11588 /label=pBR322ori-F /note="pBR322 origin, forward primer" primer_bind 11822..11839 /label=L4440 /note="L4440 vector, forward primer" protein_bind 11956..11977 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." promoter 11992..12022 /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 12030..12046 /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 12035..12057 /label=M13/pUC Reverse /note="In lacZ gene" primer_bind 12054..12070 /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind 12054..12070 /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" protein_bind 12931..12964 /label=loxP /bound_moiety="Cre recombinase" /note="Cre-mediated recombination occurs in the 8-bp core sequence (GCATACAT)." misc_feature 12965..12990 /label=SA /note="splice acceptor site" CDS 13014..13067 /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 13071..13862 /codon_start=1 /gene="aph(3')-II (or nptII)" /product="aminoglycoside phosphotransferase from Tn5" /label=NeoR/KanR /note="confers resistance to neomycin, kanamycin, and G418 (Geneticin(R))" /translation="IEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRPV LFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLSS HLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQG LAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIAL ATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF" primer_bind complement(13122..13141) /label=Neo-R /note="Neomycin resistance gene, reverse primer" primer_bind 13732..13751 /label=Neo-F /note="Neomycin resistance gene, forward primer" polyA_signal 13897..14121 /label=bGH poly(A) signal /note="bovine growth hormone polyadenylation signal" protein_bind 14122..14155 /label=loxP /bound_moiety="Cre recombinase" /note="Cre-mediated recombination occurs in the 8-bp core sequence (GCATACAT)." ORIGIN 1 cggtcgacat aacttcgtat aggatacttt atacgaagtt atcaattcag gctagttatt 61 aatagtaatc aattacgggg tcattagttc atagcccata tatggagttc cgcgttacat 121 aacttacggt aaatggcccg cctggctgac cgcccaacga cccccgccca ttgacgtcaa 181 taatgacgta tgttcccata gtaacgccaa tagggacttt ccattgacgt caatgggtgg 241 agtatttacg gtaaactgcc cacttggcag tacatcaagt gtatcatatg ccaagtacgc 301 cccctattga cgtcaatgac ggtaaatggc ccgcctggca ttatgcccag tacatgacct 361 tatgggactt tcctacttgg cagtacatct acgtattagt catcgctatt accatggtcg 421 aggtgagccc cacgttctgc ttcactctcc ccatctcccc cccctcccca cccccaattt 481 tgtatttatt tattttttaa ttattttgtg cagcgrkggg ggsggggggg gggggggggc 541 gcgcgccrgg sggggcgggg sggggcgagg ggcggggcgg ggcgaggcgg agaggtgcgg 601 cggcagccaa tcagagcggc gcgctccgaa agtttccttt tatggcgagg cggcggcggc 661 ggcggcccta taaaaagcga agcgcgcggc gggcgggagt cgctgcgcgc tgccttcgcc 721 ccgtgccccg ctccgccgcc gcctcgcgcc gcccgccccg gctctgactg accgcgttac 781 tcccacaggt gagcgggcgg gacggccctt ctcctccggg ctgtaattag cgcttggttt 841 aatgacggct tgtttctttt ctgtggctgc gtgaaagcct tgaggggctc cgggagggcc 901 ctttgtgcgg ggggagcggc tcggggggtg cgtgcgtgtg tgtgtgcgtg gggagcgccg 961 cgtgcggctc cgcgctgccc ggcggctgtg agcgctgcgg gcgcggcgcg gggctttgtg 1021 cgctccgcag tgtgcgcgag gggagcgcgg ccgggggcgg tgccccgcgg tgcggggggg 1081 gctgcgaggg gaacaaaggc tgcgtgcggg gtgtgtgcgt gggggggtga gcagggggtg 1141 tgggcgcgtc ggtcgggctg caaccccccc tgcacccccc tccccgagtt gctgagcacg 1201 gcccggcttc gggtgcgggg ctccgtacgg ggcgtggcgc ggggctcgcc gtgccgggcg 1261 gggggtggcg gcaggtgggg gtgccgggcg gggcggggcc gcctcgggcc ggggagggct 1321 cgggggaggg gcgcggcggc ccccggagcg ccggcggctg tcgaggcgcg gcgagccgca 1381 gccattgcct tttatggtaa tcgtgcgaga gggcgcaggg acttcctttg tcccaaatct 1441 gtgcggagcc gaaatctggg aggcgccgcc gcaccccctc tagcgggcgc ggggcgaagc 1501 ggtgcggcgc cggcaggaag gaaatgggcg gggagggcct tcgtgcgtcg ccgcgccgcc 1561 gtccccttct ccctctccag cctcggggct gtccgcgggg ggacggctgc cttcgggggg 1621 gacggggcag ggcggggttc ggcttctggc gtgtgaccgg cggctctaga gcctctgcta 1681 accatgttca tgccttcttc tttttcctac agctcctggg caacgtgctg gttattgtgc 1741 tgtctcatca ttttggcaaa gaattgtgta caactagtgc caccatggac aagaagtaca 1801 gcatcggcct ggccatcggc accaactctg tgggctgggc cgtgatcacc gacgagtaca 1861 aggtgcccag caagaaattc aaggtgctgg gcaacaccga ccggcacagc atcaagaaga 1921 acctgatcgg cgccctgctg ttcgacagcg gagaaacagc cgaggccacc cggctgaaga 1981 gaaccgccag aagaagatac accagacgga agaaccggat ctgctatctg caagagatct 2041 tcagcaacga gatggccaag gtggacgaca gcttcttcca cagactggaa gagtccttcc 2101 tggtggaaga ggataagaag cacgagcggc accccatctt cggcaacatc gtggacgagg 2161 tggcctacca cgagaagtac cccaccatct accacctgag aaagaaactg gtggacagca 2221 ccgacaaggc cgacctgcgg ctgatctatc tggccctggc ccacatgatc aagttccggg 2281 gccacttcct gatcgagggc gacctgaacc ccgacaacag cgacgtggac aagctgttca 2341 tccagctggt gcagacctac aaccagctgt tcgaggaaaa ccccatcaac gccagcggcg 2401 tggacgccaa ggccatcctg tctgccagac tgagcaagag cagacggctg gaaaatctga 2461 tcgcccagct gcccggcgag aagaagaatg gcctgttcgg caacctgatt gccctgagcc 2521 tgggcctgac ccccaacttc aagagcaact tcgacctggc cgaggatgcc aaactgcagc 2581 tgagcaagga cacctacgac gacgacctgg acaacctgct ggcccagatc ggcgaccagt 2641 acgccgacct gtttctggcc gccaagaacc tgtccgacgc catcctgctg agcgacatcc 2701 tgagagtgaa caccgagatc accaaggccc ccctgagcgc ctctatgatc aagagatacg 2761 acgagcacca ccaggacctg accctgctga aagctctcgt gcggcagcag ctgcctgaga 2821 agtacaaaga gattttcttc gaccagagca agaacggcta cgccggctac atcgatggcg 2881 gagccagcca ggaagagttc tacaagttca tcaagcccat cctggaaaag atggacggca 2941 ccgaggaact gctcgtgaag ctgaacagag aggacctgct gcggaagcag cggaccttcg 3001 acaacggcag catcccccac cagatccacc tgggagagct gcacgccatt ctgcggcggc 3061 aggaagattt ttacccattc ctgaaggaca accgggaaaa gatcgagaag atcctgacct 3121 tccgcatccc ctactacgtg ggccctctgg ccaggggaaa cagcagattc gcctggatga 3181 ccagaaagag cgaggaaacc atcaccccct ggaacttcga ggaagtggtg gacaagggcg 3241 ccagcgccca gagcttcatc gagcggatga ccaacttcga taagaacctg cccaacgaga 3301 aggtgctgcc caagcacagc ctgctgtacg agtacttcac cgtgtacaac gagctgacca 3361 aagtgaaata cgtgaccgag ggaatgagaa agcccgcctt cctgagcggc gagcagaaaa 3421 aagccatcgt ggacctgctg ttcaagacca accggaaagt gaccgtgaag cagctgaaag 3481 aggactactt caagaaaatc gagtgcttcg actccgtgga aatctccggc gtggaagatc 3541 ggttcaacgc ctccctgggc acataccacg atctgctgaa aattatcaag gacaaggact 3601 tcctggacaa tgaggaaaac gaggacattc tggaagatat cgtgctgacc ctgacactgt 3661 ttgaggacag agagatgatc gaggaacggc tgaaaaccta tgcccacctg ttcgacgaca 3721 aagtgatgaa gcagctgaag cggcggagat acaccggctg gggcaggctg agccggaagc 3781 tgatcaacgg catccgggac aagcagtccg gcaagacaat cctggatttc ctgaagtccg 3841 acggcttcgc caacagaaac ttcatgcagc tgatccacga cgacagcctg acctttaaag 3901 aggacatcca gaaagcccag gtgtccggcc agggcgatag cctgcacgag cacattgcca 3961 atctggccgg cagccccgcc attaagaagg gcatcctgca gacagtgaag gtggtggacg 4021 agctcgtgaa agtgatgggc cggcacaagc ccgagaacat cgtgatcgaa atggccagag 4081 agaaccagac cacccagaag ggacagaaga acagccgcga gagaatgaag cggatcgaag 4141 agggcatcaa agagctgggc agccagatcc tgaaagaaca ccccgtggaa aacacccagc 4201 tgcagaacga gaagctgtac ctgtactacc tgcagaatgg gcgggatatg tacgtggacc 4261 aggaactgga catcaaccgg ctgtccgact acgatgtgga cgctatcgtg cctcagagct 4321 ttctgaagga cgactccatc gataacaaag tgctgactcg gagcgacaag aaccggggca 4381 agagcgacaa cgtgccctcc gaagaggtcg tgaagaagat gaagaactac tggcgccagc 4441 tgctgaatgc caagctgatt acccagagga agttcgacaa tctgaccaag gccgagagag 4501 gcggcctgag cgaactggat aaggccggct tcatcaagag acagctggtg gaaacccggc 4561 agatcacaaa gcacgtggca cagatcctgg actcccggat gaacactaag tacgacgaga 4621 acgacaaact gatccgggaa gtgaaagtga tcaccctgaa gtccaagctg gtgtccgatt 4681 tccggaagga tttccagttt tacaaagtgc gcgagatcaa caactaccac cacgcccacg 4741 acgcctacct gaacgccgtc gtgggaaccg ccctgatcaa aaagtaccct aagctggaaa 4801 gcgagttcgt gtacggcgac tacaaggtgt acgacgtgcg gaagatgatc gccaagagcg 4861 agcaggaaat cggcaaggct accgccaagt acttcttcta cagcaacatc atgaactttt 4921 tcaagaccga gattaccctg gccaacggcg agatccggaa gcggcctctg atcgagacaa 4981 acggcgaaac aggcgagatc gtgtgggata agggccggga ctttgccacc gtgcggaaag 5041 tgctgtctat gccccaagtg aatatcgtga aaaagaccga ggtgcagaca ggcggcttca 5101 gcaaagagtc tatcctgccc aagaggaaca gcgacaagct gatcgccaga aagaaggact 5161 gggaccctaa gaagtacggc ggcttcgaca gccccaccgt ggcctattct gtgctggtgg 5221 tggccaaagt ggaaaagggc aagtccaaga aactgaagag tgtgaaagag ctgctgggga 5281 tcaccatcat ggaaagaagc agcttcgaga agaatcccat cgactttctg gaagccaagg 5341 gctacaaaga agtgaaaaag gacctgatca tcaagctgcc taagtactcc ctgttcgagc 5401 tggaaaacgg ccggaagaga atgctggcct ctgccggcga actgcagaag ggaaacgaac 5461 tggccctgcc ctccaaatat gtgaacttcc tgtacctggc cagccactat gagaagctga 5521 agggctcccc cgaggataat gagcagaaac agctgtttgt ggaacagcac aaacactacc 5581 tggacgagat catcgagcag atcagcgagt tctccaagag agtgatcctg gccgacgcta 5641 atctggacaa ggtgctgagc gcctacaaca agcacagaga caagcctatc agagagcagg 5701 ccgagaatat catccacctg tttaccctga ccaatctggg agcccctgcc gccttcaagt 5761 actttgacac caccatcgac cggaagaggt acaccagcac caaagaggtg ctggacgcca 5821 ccctgatcca ccagagcatc accggcctgt acgagacacg gatcgacctg tctcagctgg 5881 gaggcgacgc ctatccctat gacgtgcccg attatgccag cctgggcagc ggctccccca 5941 agaaaaaacg caaggtggaa gatcctaaga aaaagcggaa agtggacggc attggtagtg 6001 ggagcaacgg cagcagcgga tccagcgagc tgattaagga gaacatgcac atgaagctgt 6061 acatggaggg caccgtggac aaccatcact tcaagtgcac atccgagggc gaaggcaagc 6121 cctacgaggg cacccagacc atgagaatca aggtggtcga gggcggccct ctccccttcg 6181 ccttcgacat cctggctact agcttcctct acggcagcaa gaccttcatc aaccacaccc 6241 agggcatccc cgacttcttc aagcagtcct tccctgaggg cttcacatgg gagagagtca 6301 ccacatacga agacgggggc gtgctgaccg ctacccagga caccagcctc caggacggct 6361 gcctcatcta caacgtcaag atcagagggg tgaacttcac atccaacggc cctgtgatgc 6421 agaagaaaac actcggctgg gaggccttca ccgagacgct gtaccccgct gacggcggcc 6481 tggaaggcag aaacgacatg gccctgaagc tcgtgggcgg gagccatctg atcgcaaaca 6541 tcaagaccac atatagatcc aagaaacccg ctaagaacct caagatgcct ggcgtctact 6601 atgtggacta cagactggaa agaatcaagg aggccaacaa cgagacctac gtcgagcagc 6661 acgaggtggc agtggccaga tactgcgacc tccctagcaa actggggcac aagcttaatg 6721 gcggtggcgg agggatggat gctaagtcac taactgcctg gtcccggaca ctggtgacct 6781 tcaaggatgt atttgtggac ttcaccaggg aggagtggaa gctgctggac actgctcagc 6841 agatcgtgta cagaaatgtg atgctggaga actataagaa cctggtttcc ttgggttatc 6901 agcttactaa gccagatgtg atcctccggt tggagaaggg agaagagccc tagacgcgtg 6961 aattcactcc tcaggtgcag gctgcctatc agaaggtggt ggctggtgtg gccaatgccc 7021 tggctcacaa ataccactga gatctttttc cctctgccaa aaattatggg gacatcatga 7081 agccccttga gcatctgact tctggctaat aaaggaaatt tattttcatt gcaatagtgt 7141 gttggaattt tttgtgtctc tcactcggaa ggacatatgg gagggcaaat catttaaaac 7201 atcagaatga gtatttggtt tagagtttgg caacatatgc ccatatgctg gctgccatga 7261 acaaaggttg gctataaaga ggtcatcagt atatgaaaca gccccctgct gtccattcct 7321 tattccatag aaaagccttg acttgaggtt agattttttt tatattttgt tttgtgttat 7381 ttttttcttt aacatcccta aaattttcct tacatgtttt actagccaga tttttcctcc 7441 tctcctgact actcccagtc atagctgtcc ctcttctctt atggagatca ccggtataac 7501 ttcgtataat gtatactata cgaagttatg gtaccttaat taagttaaca tgcatagtac 7561 tgagctcacg gggacagccc ccccccaaag cccccaggga tgtaattacg tccctccccc 7621 gctagggggc agcagcgagc cgcccggggc tccgctccgg tccggcgctc cccccgcatc 7681 cccgagccgg cagcgtgcgg ggacagcccg ggcacgggga aggtggcacg ggatcgcttt 7741 cctctgaacg cttctcgctg ctctttgagc ctgcagacac ctggggggat acggggaaaa 7801 agctttaggc tgaaagagag atttagaatg acagaatcat agaacggcct gggttgcaaa 7861 ggagcacagt gctcatccag atccaacccc ctgctatgtg cagggtcatc aaccagcagc 7921 ccaggctgcc cagagccaca tccagcctgg ccttgaatgc ctgcagggat ggggcatcca 7981 cagcctcctt gggcaacctg ttcagtgcgt caccaccctc tgggggaaaa actgcctcct 8041 catatccaac ccaaacctcc cctgtctcag tgtaaagcca ttcccccttg tcctatcaag 8101 ggggagtttg ctgtgacatt gttggtctgg ggtgacacat gtttgccaat tcagtgcatc 8161 acggagaggc agatcttggg gataaggaag tgcaggacag catggacgtg ggacatgcag 8221 gtgttgaggg ctctgggaca ctctccaagt cacagcgttc agaacagcct taaggataag 8281 aagataggat agaaggacaa agagcaagtt aaaacccagc atggagagga gcacaaaaag 8341 gccacagaca ctgctggtcc ctgtgtctga gcctgcatgt ttgatggtgt ctggatgcaa 8401 gcagaagggg tggaagagct tgcctggaga gatacagctg ggtcagtagg actgggacag 8461 gcagctggag aattgccatg tagatgttca tacaatcgtc aaatcatgaa ggctggaaaa 8521 gccctccaag atccccaaga ccaaccccaa cccacccacc gtgcccactg gccatgtccc 8581 tcagtgccac atccccacag ttcttcatca cctccaggga cggtgacccc cccacctccg 8641 tgggcagctg tgccactgca gcaccgctct ttggagaagg taaatcttgc taaatccagc 8701 ccgaccctcc cctggcacaa cgtaaggcca ttatctctca tccaactcca ggacggagtc 8761 agtgagaata ttgtttaaac tcctcatcct tccaacataa atatattttg ggattatatc 8821 aacattcaat gttacttaaa gtgaccttgt aaatattttc acaactgagc catgtttgat 8881 ttgtatactt atgtttactt tactgttttt cctgaagtta ataattgcct tgaatttatt 8941 tatttcttta aaaatgtttc attactcagg actgtagttt acattacgat tctttgtgtt 9001 atacagttga tgggtttctt ttctttctta atttctttaa aaaatagaga tggggtctta 9061 ctatattacc caggctggtc ttgaagtcct gggctcaagt gatcttcctg tctcagccta 9121 ccaagtagct gagactatag gtgcaaaaaa gccactatac ctggctagtt tacaggtttt 9181 aacaaatgca ttatgccacg tatccattat tacaggatca cacaagatat tttcattacc 9241 ctgaaagcat ccctgtgttc caccaattca tcctgcctcc atgagccgct ggcaaccact 9301 gatctctata gttttgcctt ttctaaaatg tcatataatt ggaatcatac agtctgtagc 9361 attttcagac tagcttttaa aatttggcaa tatgcattta aggttcctcc ttaaatgtga 9421 agggcatagc caatgtggct tgatagctca tttcttttta ttggtgaata tttcattgtc 9481 tggatgttcc acagtttgtt tatccattca cctattcaat ttgctttttt tctgtgtatc 9541 tatcactaat tcaatactgg actctccaac agagccgttt aaacactggc cgtcgtttta 9601 caacgtcgtg actgggaaaa ccctggcgtt acccaactta atcgccttgc agcacatccc 9661 cctttcgcca gctggcgtaa tagcgaagag gcccgcaccg atcgcccttc ccaacagttg 9721 cgcagcctga atggcgaatg gcgcctgatg cggtattttc tccttacgca tctgtgcggt 9781 atttcacacc gcatatggtg cactctcagt acaatctgct ctgatgccgc atagttaagc 9841 cagccccgac acccgccaac acccgctgac gcgccctgac gggcttgtct gctcccggca 9901 tccgcttaca gacaagctgt gaccgtctcc gggagctgca tgtgtcagag gttttcaccg 9961 tcatcaccga aacgcgcgat gcagctctgg cccgtgtctc aaaatctctg atgttacatt 10021 gcacaagata aaaatatatc atcatgaaca ataaaactgt ctgcttacat aaacagtaat 10081 acaaggggtg ttatgagcca tattcaacgg gaaacgtcga ggccgcgatt aaattccaac 10141 atggatgctg atttatatgg gtataaatgg gctcgcgata atgtcgggca atcaggtgcg 10201 acaatctatc gcttgtatgg gaagcccgat gcgccagagt tgtttctgaa acatggcaaa 10261 ggtagcgttg ccaatgatgt tacagatgag atggtcagac taaactggct gacggaattt 10321 atgcctcttc cgaccatcaa gcattttatc cgtactcctg atgatgcatg gttactcacc 10381 actgcgatcc ccggaaaaac agcattccag gtattagaag aatatcctga ttcaggtgaa 10441 aatattgttg atgcgctggc agtgttcctg cgccggttgc attcgattcc tgtttgtaat 10501 tgtcctttta acagcgatcg cgtatttcgt ctcgctcagg cgcaatcacg aatgaataac 10561 ggtttggttg atgcgagtga ttttgatgac gagcgtaatg gctggcctgt tgaacaagtc 10621 tggaaagaaa tgcataaact tttgccattc tcaccggatt cagtcgtcac tcatggtgat 10681 ttctcacttg ataaccttat ttttgacgag gggaaattaa taggttgtat tgatgttgga 10741 cgagtcggaa tcgcagaccg ataccaggat cttgccatcc tatggaactg cctcggtgag 10801 ttttctcctt cattacagaa acggcttttt caaaaatatg gtattgataa tcctgatatg 10861 aataaattgc agtttcattt gatgctcgat gagtttttct aatcagaatt ggttaattgg 10921 ttgtaacatt attcagattg ggcttgattt aaaacttcat ttttaattta aaaggatcta 10981 ggtgaagatc ctttttgata atctcatgac caaaatccct taacgtgagt tttcgttcca 11041 ctgagcgtca gaccccgtag aaaagatcaa aggatcttct tgagatcctt tttttctgcg 11101 cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca gcggtggttt gtttgccgga 11161 tcaagagcta ccaactcttt ttccgaaggt aactggcttc agcagagcgc agataccaaa 11221 tactgttctt ctagtgtagc cgtagttagg ccaccacttc aagaactctg tagcaccgcc 11281 tacatacctc gctctgctaa tcctgttacc agtggctgct gccagtggcg ataagtcgtg 11341 tcttaccggg ttggactcaa gacgatagtt accggataag gcgcagcggt cgggctgaac 11401 ggggggttcg tgcacacagc ccagcttgga gcgaacgacc tacaccgaac tgagatacct 11461 acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc 11521 ggtaagcggc agggtcggaa caggagagcg cacgagggag cttccagggg gaaacgcctg 11581 gtatctttat agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg 11641 ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac gcggcctttt tacggttcct 11701 ggccttttgc tggccttttg ctcacatgtt ctttcctgcg ttatcccctg attctgtgga 11761 taaccgtatt accgcctttg agtgagctga taccgctcgc cgcagccgaa cgaccgagcg 11821 cagcgagtca gtgagcgagg aagcggaaga gcgcccaata cgcaaaccgc ctctccccgc 11881 gcgttggccg attcattaat gcagctggca cgacaggttt cccgactgga aagcgggcag 11941 tgagcgcaac gcaattaatg tgagttagct cactcattag gcaccccagg ctttacactt 12001 tatgcttccg gctcgtatgt tgtgtggaat tgtgagcgga taacaatttc acacaggaaa 12061 cagctatgac catgattacg ccgcggccgc agcaggagaa tcatgccaat gggccaatat 12121 acattctgac ccacagtttc ataataaaat aaaatggttg tggttgtaag ccactatgtt 12181 tcagagtggt ttgttacaca gcaataaata actaatatag taggcatacc atcaagtcca 12241 aagtaggtag agaagaatgt aaatagcaga gcaaaacagc atgactggtg gctgggaggc 12301 ttaaaactgg gacaggatca gagtcatgaa agaagtcaaa gaaatggttc agaagtaagg 12361 ctgagactga cttacaaaag ctgaaagtcc ctttaagttg gtgtttggtg cattggcagg 12421 ggcaggtatg gtgacttaaa agagccatgc tcaacaagat caagcacaac acaatcacgg 12481 gtcaccccag cagaccttag cgagtctagc catttctttg gtggtggtca cagtcatgct 12541 tcagcccagt ttccacttgg acaaatggta catattttca atgagatgaa aattaagata 12601 caatccatgt gctcagagag tgatcacagc tctgactaaa cactgtgccc caaagtgttg 12661 aggaattggg aaaacctagc tgagttagtg gtctcttttc tgttacaata aagctcataa 12721 tgaaaattag ccttctttgt tcttccccaa gtctttcttt ctagacgaaa ctactttcaa 12781 ctgttttaac ttccttactg ttaacttcca tatttctaga tagtatggtc agactgttat 12841 ttcttgactt ttaaatgtaa gatattatct actgacttcc ttctatgtaa gatgaggatt 12901 gagctctctt acccttctcc cagcggccgc ataacttcgt atagcataca ttatacgaag 12961 ttatctgacc tcttctcttc ctcccacagg gcctcgagag atctggcagc ggagagggca 13021 gaggaagtct tctaacatgc ggtgacgtgg aggagaatcc cggccctagg attgaacaag 13081 atggattgca cgcaggttct ccggccgctt gggtggagag gctattcggc tatgactggg 13141 cacaacagac aatcggctgc tctgatgccg ccgtgttccg gctgtcagcg caggggcgcc 13201 cggttctttt tgtcaagacc gacctgtccg gtgccctgaa tgaactgcag gacgaggcag 13261 cgcggctatc gtggctggcc acgacgggcg ttccttgcgc agctgtgctc gacgttgtca 13321 ctgaagcggg aagggactgg ctgctattgg gcgaagtgcc ggggcaggat ctcctgtcat 13381 ctcaccttgc tcctgccgag aaagtatcca tcatggctga tgcaatgcgg cggctgcata 13441 cgcttgatcc ggctacctgc ccattcgacc accaagcgaa acatcgcatc gagcgagcac 13501 gtactcggat ggaagccggt cttgtcgatc aggatgatct ggacgaagag catcaggggc 13561 tcgcgccagc cgaactgttc gccaggctca aggcgcgcat gcccgacggc gaggatctcg 13621 tcgtgaccca tggcgatgcc tgcttgccga atatcatggt ggaaaatggc cgcttttctg 13681 gattcatcga ctgtggccgg ctgggtgtgg cggaccgcta tcaggacata gcgttggcta 13741 cccgtgatat tgctgaagag cttggcggcg aatgggctga ccgcttcctc gtgctttacg 13801 gtatcgccgc tcccgattcg cagcgcatcg ccttctatcg ccttcttgac gagttcttct 13861 aacacctggt gcatgacccg caagcccggt gcctgactgt gccttctagt tgccagccat 13921 ctgttgtttg cccctccccc gtgccttcct tgaccctgga aggtgccact cccactgtcc 13981 tttcctaata aaatgaggaa attgcatcgc attgtctgag taggtgtcat tctattctgg 14041 ggggtggggt ggggcaggac agcaaggggg aggattggga agacaatagc aggcatgctg 14101 gggatgcggt gggctctatg gataacttcg tatagcatac attatacgaa gttatgagct 14161 cacggggaca gccccccccc aaagccccca gggatgtaat tacgtccctc ccccgctagg 14221 gggcagcagc gagccgcccg gggctccgct ccggtccggc gctccccccg catccccgag 14281 ccggcagcgt gcggggacag cccgggcacg gggaaggtgg cacgggatcg ctttcctctg 14341 aacgcttctc gctgctcttt gagcctgcag acacctgggg ggatacgggg aaaaagcttt 14401 aggctgaaag agagatttag aatgacagaa tcatagaacg gcctgggttg caaaggagca 14461 cagtgctcat ccagatccaa ccccctgcta tgtgcagggt catcaaccag cagcccaggc 14521 tgcccagagc cacatccagc ctggccttga atgcctgcag ggatggggca tccacagcct 14581 ccttgggcaa cctgttcagt gcgtcaccac cctctggggg aaaaactgcc tcctcatatc 14641 caacccaaac ctcccctgtc tcagtgtaaa gccattcccc cttgtcctat caagggggag 14701 tttgctgtga cattgttggt ctggggtgac acatgtttgc caattcagtg catcacggag 14761 aggcagatct tggggataag gaagtgcagg acagcatgga cgtgggacat gcaggtgttg 14821 agggctctgg gacactctcc aagtcacagc gttcagaaca gccttaagga taagaagata 14881 ggatagaagg acaaagagca agttaaaacc cagcatggag aggagcacaa aaaggccaca 14941 gacactgctg gtccctgtgt ctgagcctgc atgtttgatg gtgtctggat gcaagcagaa 15001 ggggtggaag agcttgcctg gagagataca gctgggtcag taggactggg acaggcagct 15061 ggagaattgc catgtagatg ttcatacaat cgtcaaatca tgaaggctgg aaaagccctc 15121 caagatcccc aagaccaacc ccaacccacc caccgtgccc actggccatg tccctcagtg 15181 ccacatcccc acagttcttc atcacctcca gggacggtga cccccccacc tccgtgggca 15241 gctgtgccac tgcagcaccg ctctttggag aaggtaaatc ttgctaaatc cagcccgacc 15301 ctcccctggc acaacgtaag gccattatct ctcatccaac tccaggacgg agtcagtgag 15361 aatattggcg cgccgaattc tcgcgacgat //