LOCUS Exported 14027 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION Expresses dCas9-p300 and MS2-p65-HSF1 fusion proteins. ACCESSION . VERSION . KEYWORDS pPB-R1R2_EF1adCas9p300_T2A_MS2p65HSF1-IRESbsdpA SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 14027) AUTHORS Chong ZS, Ohnishi S, Yusa K, Wright GJ TITLE Pooled extracellular receptor-ligand interaction screening using CRISPR activation. JOURNAL Genome Biol. 2018 Nov 26;19(1):205. doi: 10.1186/s13059-018-1581-3. PUBMED 30477585 REFERENCE 2 (bases 1 to 14027) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..14027 /organism="synthetic DNA construct" /mol_type="other DNA" rep_origin complement(3..458) /direction=LEFT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(140..161) /label=F1ori-F /note="F1 origin, forward primer" primer_bind 352..371 /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 585..607 /label=M13/pUC Forward /note="In lacZ gene" primer_bind 599..616 /label=M13 Forward /note="In lacZ gene. Also called M13-F20 or M13 (-21) Forward" primer_bind 600..616 /label=M13 fwd /note="common sequencing primer, one of multiple similar variants" primer_bind 626..645 /label=T7 /note="T7 promoter, forward primer" promoter 626..644 /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" repeat_region 674..708 /label=piggyBac left (5') inverted repeat /note="piggyBac transposon-specific inverted terminal repeat sequence (ITR)" protein_bind 998..1022 /gene="mutant version of attB" /label=attB1 /bound_moiety="BP Clonase(TM)" /note="recombination site for the Gateway(R) BP reaction" promoter 1492..1703 /label=EF-1-alpha core promoter /note="core promoter for human elongation factor EF-1-alpha" intron 2447..2650 /label=EF-1-alpha intron A /note="truncated intron upstream of the start codon of human EF-1-alpha" primer_bind 2607..2627 /label=EF1a-F /note="Human elongation factor-1a promoter, forward primer" CDS 2690..2737 /codon_start=1 /product="bipartite nuclear localization signal from nucleoplasmin" /label=nucleoplasmin NLS /translation="KRPAATKKAGQAKKKK" CDS 2738..6838 /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 N863A mutation in the HNH catalytic domain" /translation="DKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL YYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKARGKSDNVPS EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD ATLIHQSITGLYETRIDLSQLGGD" CDS 6893..6913 /codon_start=1 /product="nuclear localization signal of SV40 (simian virus 40) large T antigen" /label=SV40 NLS /translation="PKKKRKV" CDS 6935..8785 /codon_start=1 /gene="EP300" /product="catalytic histone acetyltransferase core of the human E1A-associated protein p300 (Delvecchio et al., 2013)" /label=p300 core /translation="IFKPEELRQALMPTLEALYRQDPESLPFRQPVDPQLLGIPDYFDI VKSPMDLSTIKRKLDTGQYQEPWQYVDDIWLMFNNAWLYNRKTSRVYKYCSKLSEVFEQ EIDPVMQSLGYCCGRKLEFSPQTLCCYGKQLCTIPRDATYYSYQNRYHFCEKCFNEIQG ESVSLGDDPSQPQTTINKEQFSKRKNDTLDPELFVECTECGRKMHQICVLHHEIIWPAG FVCDGCLKKSARTRKENKFSAKRLPSTRLGTFLENRVNDFLRRQNHPESGEVTVRVVHA SDKTVEVKPGMKARFVDSGEMAESFPYRTKALFAFEEIDGVDLCFFGMHVQEYGSDCPP PNQRRVYISYLDSVHFFRPKCLRTAVYHEILIGYLEYVKKLGYTTGHIWACPPSEGDDY IFHCHPPDQKIPKPKRLQEWYKKMLDKAVSERIVHDYKDIFKQATEDRLTSAKELPYFE GDFWPNVLEESIKELEQEEEERKREENTSNESTDVTKGDSKNAKKKNNKKTSKNKSSLS RGNKKKPGMPNVSNDLSQKLYATMEKHKEVFFVIRLIAGPAANSLPPIVDPDPLIPCDL MDGRDAFLTLARDKHLEFSSLRRAQWSTMCMLVELHTQSQD" regulatory 8549..8558 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" CDS 8786..8839 /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 8849..9235 /codon_start=1 /product="bacteriophage MS2 coat protein" /label=MS2-N55K /note="binds with high affinity to a specific stem-loop structure in the viral RNA (Lim et al., 1994)" /translation="ASNFTQFVLVDNGGTGDVTVAPSNFANGVAEWISSNSRSQAYKVT CSVRQSSAQKRKYTIKVEVPKVATQTVGGVELPVAAWRSYLNMELTIPIFATNSDCELI VKAMQGLLKDGNPIPSAIAANSGIY" CDS 9290..9310 /codon_start=1 /product="nuclear localization signal of SV40 (simian virus 40) large T antigen" /label=SV40 NLS /translation="PKKKRKV" CDS 9326..9868 /codon_start=1 /product="C-terminal portion of the p65 subunit of mouse NF-kappa-B" /label=p65 /translation="PSGQISNQALALAPSSAPVLAQTMVPSSAMVPLAQPPAPAPVLTP GPPQSLSAPVPKSTQAGEGTLSEALLHLQFDADEDLGALLGNSTDPGVFTDLASVDNSE FQQLLNQGVSMSHSTAEPMLMEYPEAITRLVTGSQRPPDPAPTPLGTSGLPNGLSGDED FSSIADMDFSALLSQISS" CDS 9893..10264 /codon_start=1 /product="C-terminal activation domain from the human heat shock transcription factor HSF1" /label=HSF1 /translation="GFSVDTSALLDLFSPSVTVPDMSLPDLDSSLASIQELLSPQEPPR PPEAENSSPDSGKQLVHYTAQPLFLLDPGSVDTGSNDLPVLFELGEGSYFSEGDGFAED PTISLLTGSEPPKAKDPTVS" misc_feature 10290..10875 /label=IRES2 /note="internal ribosome entry site (IRES) of the encephalomyocarditis virus (EMCV)" primer_bind complement(10457..10474) /label=IRES reverse /note="IRES internal ribosome entry site, reverse primer. Also called pCDH-rev" primer_bind 10684..10703 /label=IRES-F /note="IRES internal ribosome entry site, forward primer" CDS 10876..11271 /codon_start=1 /gene="Aspergillus terreus BSD" /product="blasticidin S deaminase" /label=BSD /note="confers resistance to blasticidin" /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI KAIVKDSDGQPTAVGIRELLPSGYVWEG" polyA_signal 11275..11499 /label=bGH poly(A) signal /note="bovine growth hormone polyadenylation signal" protein_bind complement(11520..11544) /gene="mutant version of attB" /label=attB2 /bound_moiety="BP Clonase(TM)" /note="recombination site for the Gateway(R) BP reaction" repeat_region 11726..11788 /label=piggyBac right (3') inverted repeat /note="piggyBac transposon-specific inverted terminal repeat sequence (ITR)" primer_bind complement(11839..11859) /label=T3 /note="T3 promoter, forward primer" promoter complement(11839..11857) /label=T3 promoter /note="promoter for bacteriophage T3 RNA polymerase" primer_bind complement(11878..11894) /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind complement(11878..11894) /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" primer_bind complement(11891..11913) /label=M13/pUC Reverse /note="In lacZ gene" protein_bind 11902..11918 /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)." promoter complement(11926..11956) /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 11971..11992 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." primer_bind complement(12109..12126) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(12280..12868) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(12360..12379) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS complement(13039..13899) /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 13662..13681 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(13900..14004) /gene="bla" /label=AmpR promoter ORIGIN 1 ctaaattgta agcgttaata ttttgttaaa attcgcgtta aatttttgtt aaatcagctc 61 attttttaac caataggccg aaatcggcaa aatcccttat aaatcaaaag aatagaccga 121 gatagggttg agtgttgttc cagtttggaa caagagtcca ctattaaaga acgtggactc 181 caacgtcaaa gggcgaaaaa ccgtctatca gggcgatggc ccactacgtg aaccatcacc 241 ctaatcaagt tttttggggt cgaggtgccg taaagcacta aatcggaacc ctaaagggag 301 cccccgattt agagcttgac ggggaaagcc ggcgaacgtg gcgagaaagg aagggaagaa 361 agcgaaagga gcgggcgcta gggcgctggc aagtgtagcg gtcacgctgc gcgtaaccac 421 cacacccgcc gcgcttaatg cgccgctaca gggcgcgtcc cattcgccat tcaggctgcg 481 caactgttgg gaagggcgat cggtgcgggc ctcttcgcta ttacgccagc tggcgaaagg 541 gggatgtgct gcaaggcgat taagttgggt aacgccaggg ttttcccagt cacgacgttg 601 taaaacgacg gccagtgagc gcgcgtaata cgactcacta tagggcgaat tggggcgcgc 661 cattctagat taaccctaga aagatagtct gcgtaaaatt gacgcatgca ttcttgaaat 721 attgctctct ctttctaaat agcgcgaatc cgtcgctgtg catttaggac atctcagtcg 781 ccgcttggag ctcccgtgag gcgtgcttgt caatgcggta agtgtcactg attttgaact 841 ataacgaccg cgtgagtcaa aatgacgcat gattatcttt tacgtgactt ttaagattta 901 actcatacga taattatatt gttatttcat gttctactta cgtgataact tattatatat 961 atattttctt gttatagata tcaactagaa tgctagcaca agtttgtaca aaaaagcagg 1021 ctggcgccgg aaccaattca gtcgactgga tccacgcgtg aggggcgcgg aagccggggc 1081 ggaggaggag ccaaccccgg gctgggctga gacccgcaga ggaagacgct ctagggattt 1141 gtcccggact agcgagatgg caaggctgag gacgggaggc tgattgagag gcgaaggtac 1201 accctaatct caatacaacc tttggagcta agccagcaat ggtagaggga agattctgca 1261 cgtcccttcc aggcggcctc cccgtcacca ccccccccaa cccgccccga ccggagctga 1321 gagtaattca tacaaaagga ctcgcccctg ccttggggaa tcccagggac cgtcgttaaa 1381 ctcccactaa cgtagaaccc agagatcgct gcgttcccgc cccctcaccc gcccgctctc 1441 gtcatcactg aggtggagaa gagcatgcgt gaggctccgg tgcccgtcag tgggcagagc 1501 gcacatcgcc cacagtcccc gagaagttgg ggggaggggt cggcaattga accggtgcct 1561 agagaaggtg gcgcggggta aactgggaaa gtgatgtcgt gtactggctc cgcctttttc 1621 ccgagggtgg gggagaaccg tatataagtg cagtagtcgc cgtgaacgtt ctttttcgca 1681 acgggtttgc cgccagaaca caggtaagtg ccgtgtgtgg ttcccgcggg cctggcctct 1741 ttacgggtta tggcccttgc gtgccttgaa ttacttccac gcccctggct gcagtacgtg 1801 attcttgatc ccgagcttcg ggttggaagt gggtgggaga gttcgaggcc ttgcgcttaa 1861 ggagcccctt cgcctcgtgc ttgagttgag gcctggcttg ggcgctgggg ccgccgcgtg 1921 cgaatctggt ggcaccttcg cgcctgtctc gctgctttcg ataagtctct agccatttaa 1981 aatttttgat gacctgctgc gacgcttttt ttctggcaag atagtcttgt aaatgcgggc 2041 caagatcgat ctgcacactg gtatttcggt ttttggggcc gcgggcggcg acggggcccg 2101 tgcgtcccag cgcacatgtt cggcgaggcg gggcctgcga gcgcggccac cgagaatcgg 2161 acgggggtag tctcaagctg gccggcctgc tctggtgcct ggcctcgcgc cgccgtgtat 2221 cgccccgccc tgggcggcaa ggctggcccg gtcggcacca gttgcgtgag cggaaagatg 2281 gccgcttccc ggccctgctg cagggagctc aaaatggagg acgcggcgct cgggagagcg 2341 ggcgggtgag tcacccacac aaaggaaaag ggcctttccg tcctcagccg tcgcttcatg 2401 tgactccacg gagtaccggg cgccgtccag gcacctcgat tagttctcga gcttttggag 2461 tacgtcgtct ttaggttggg gggaggggtt ttatgcgatg gagtttcccc acactgagtg 2521 ggtggagact gaagttaggc cagcttggca cttgatgtaa ttctccttgg aatttgccct 2581 ttttgagttt ggatcttggt tcattctcaa gcctcagaca gtggttcaaa gtttttttct 2641 tccatttcag gtgtcgtggg aagcttggcg cgcccgtacg gccaccatga aaaggccggc 2701 ggccacgaaa aaggccggcc aggcaaaaaa gaaaaaggac aagaagtaca gcatcggcct 2761 ggccatcggc accaactctg tgggctgggc cgtgatcacc gacgagtaca aggtgcccag 2821 caagaaattc aaggtgctgg gcaacaccga ccggcacagc atcaagaaga acctgatcgg 2881 agccctgctg ttcgacagcg gcgaaacagc cgaggccacc cggctgaaga gaaccgccag 2941 aagaagatac accagacgga agaaccggat ctgctatctg caagagatct tcagcaacga 3001 gatggccaag gtggacgaca gcttcttcca cagactggaa gagtccttcc tggtggaaga 3061 ggataagaag cacgagcggc accccatctt cggcaacatc gtggacgagg tggcctacca 3121 cgagaagtac cccaccatct accacctgag aaagaaactg gtggacagca ccgacaaggc 3181 cgacctgcgg ctgatctatc tggccctggc ccacatgatc aagttccggg gccacttcct 3241 gatcgagggc gacctgaacc ccgacaacag cgacgtggac aagctgttca tccagctggt 3301 gcagacctac aaccagctgt tcgaggaaaa ccccatcaac gccagcggcg tggacgccaa 3361 ggccatcctg tctgccagac tgagcaagag cagacggctg gaaaatctga tcgcccagct 3421 gcccggcgag aagaagaatg gcctgttcgg caacctgatt gccctgagcc tgggcctgac 3481 ccccaacttc aagagcaact tcgacctggc cgaggatgcc aaactgcagc tgagcaagga 3541 cacctacgac gacgacctgg acaacctgct ggcccagatc ggcgaccagt acgccgacct 3601 gtttctggcc gccaagaacc tgtccgacgc catcctgctg agcgacatcc tgagagtgaa 3661 caccgagatc accaaggccc ccctgagcgc ctctatgatc aagagatacg acgagcacca 3721 ccaggacctg accctgctga aagctctcgt gcggcagcag ctgcctgaga agtacaaaga 3781 gattttcttc gaccagagca agaacggcta cgccggctac attgacggcg gagccagcca 3841 ggaagagttc tacaagttca tcaagcccat cctggaaaag atggacggca ccgaggaact 3901 gctcgtgaag ctgaacagag aggacctgct gcggaagcag cggaccttcg acaacggcag 3961 catcccccac cagatccacc tgggagagct gcacgccatt ctgcggcggc aggaagattt 4021 ttacccattc ctgaaggaca accgggaaaa gatcgagaag atcctgacct tccgcatccc 4081 ctactacgtg ggccctctgg ccaggggaaa cagcagattc gcctggatga ccagaaagag 4141 cgaggaaacc atcaccccct ggaacttcga ggaagtggtg gacaagggcg cttccgccca 4201 gagcttcatc gagcggatga ccaacttcga taagaacctg cccaacgaga aggtgctgcc 4261 caagcacagc ctgctgtacg agtacttcac cgtgtataac gagctgacca aagtgaaata 4321 cgtgaccgag ggaatgagaa agcccgcctt cctgagcggc gagcagaaaa aggccatcgt 4381 ggacctgctg ttcaagacca accggaaagt gaccgtgaag cagctgaaag aggactactt 4441 caagaaaatc gagtgcttcg actccgtgga aatctccggc gtggaagatc ggttcaacgc 4501 ctccctgggc acataccacg atctgctgaa aattatcaag gacaaggact tcctggacaa 4561 tgaggaaaac gaggacattc tggaagatat cgtgctgacc ctgacactgt ttgaggacag 4621 agagatgatc gaggaacggc tgaaaaccta tgcccacctg ttcgacgaca aagtgatgaa 4681 gcagctgaag cggcggagat acaccggctg gggcaggctg agccggaagc tgatcaacgg 4741 catccgggac aagcagtccg gcaagacaat cctggatttc ctgaagtccg acggcttcgc 4801 caacagaaac ttcatgcagc tgatccacga cgacagcctg acctttaaag aggacatcca 4861 gaaagcccag gtgtccggcc agggcgatag cctgcacgag cacattgcca atctggccgg 4921 cagccccgcc attaagaagg gcatcctgca gacagtgaag gtggtggacg agctcgtgaa 4981 agtgatgggc cggcacaagc ccgagaacat cgtgatcgaa atggccagag agaaccagac 5041 cacccagaag ggacagaaga acagccgcga gagaatgaag cggatcgaag agggcatcaa 5101 agagctgggc agccagatcc tgaaagaaca ccccgtggaa aacacccagc tgcagaacga 5161 gaagctgtac ctgtactacc tgcagaatgg gcgggatatg tacgtggacc aggaactgga 5221 catcaaccgg ctgtccgact acgatgtgga ccacatcgtg cctcagagct ttctgaagga 5281 cgactccatc gacaacaagg tgctgaccag aagcgacaag gcccggggca agagcgacaa 5341 cgtgccctcc gaagaggtcg tgaagaagat gaagaactac tggcggcagc tgctgaacgc 5401 caagctgatt acccagagaa agttcgacaa tctgaccaag gccgagagag gcggcctgag 5461 cgaactggat aaggccggct tcatcaagag acagctggtg gaaacccggc agatcacaaa 5521 gcacgtggca cagatcctgg actcccggat gaacactaag tacgacgaga atgacaagct 5581 gatccgggaa gtgaaagtga tcaccctgaa gtccaagctg gtgtccgatt tccggaagga 5641 tttccagttt tacaaagtgc gcgagatcaa caactaccac cacgcccacg acgcctacct 5701 gaacgccgtc gtgggaaccg ccctgatcaa aaagtaccct aagctggaaa gcgagttcgt 5761 gtacggcgac tacaaggtgt acgacgtgcg gaagatgatc gccaagagcg agcaggaaat 5821 cggcaaggct accgccaagt acttcttcta cagcaacatc atgaactttt tcaagaccga 5881 gattaccctg gccaacggcg agatccggaa gcggcctctg atcgagacaa acggcgaaac 5941 cggggagatc gtgtgggata agggccggga ttttgccacc gtgcggaaag tgctgagcat 6001 gccccaagtg aatatcgtga aaaagaccga ggtgcagaca ggcggcttca gcaaagagtc 6061 tatcctgccc aagaggaaca gcgataagct gatcgccaga aagaaggact gggaccctaa 6121 gaagtacggc ggcttcgaca gccccaccgt ggcctattct gtgctggtgg tggccaaagt 6181 ggaaaagggc aagtccaaga aactgaagag tgtgaaagag ctgctgggga tcaccatcat 6241 ggaaagaagc agcttcgaga agaatcccat cgactttctg gaagccaagg gctacaaaga 6301 agtgaaaaag gacctgatca tcaagctgcc taagtactcc ctgttcgagc tggaaaacgg 6361 ccggaagaga atgctggcct ctgccggcga actgcagaag ggaaacgaac tggccctgcc 6421 ctccaaatat gtgaacttcc tgtacctggc cagccactat gagaagctga agggctcccc 6481 cgaggataat gagcagaaac agctgtttgt ggaacagcac aagcactacc tggacgagat 6541 catcgagcag atcagcgagt tctccaagag agtgatcctg gccgacgcta atctggacaa 6601 agtgctgtcc gcctacaaca agcaccggga taagcccatc agagagcagg ccgagaatat 6661 catccacctg tttaccctga ccaatctggg agcccctgcc gccttcaagt actttgacac 6721 caccatcgac cggaagaggt acaccagcac caaagaggtg ctggacgcca ccctgatcca 6781 ccagagcatc accggcctgt acgagacacg gatcgacctg tctcagctgg gaggcgacag 6841 cgctggagga ggtggaagcg gaggaggagg aagcggagga ggaggtagcg gacctaagaa 6901 aaagaggaag gtggcgggag gtggaagcgg aggaattttc aaaccagaag aactacgaca 6961 ggcactgatg ccaactttgg aggcacttta ccgtcaggat ccagaatccc ttccctttcg 7021 tcaacctgtg gaccctcagc ttttaggaat ccctgattac tttgatattg tgaagagccc 7081 catggatctt tctaccatta agaggaagtt agacactgga cagtatcagg agccctggca 7141 gtatgtcgat gatatttggc ttatgttcaa taatgcctgg ttatataacc ggaaaacatc 7201 acgggtatac aaatactgct ccaagctctc tgaggtcttt gaacaagaaa ttgacccagt 7261 gatgcaaagc cttggatact gttgtggcag aaagttggag ttctctccac agacactgtg 7321 ttgctacggc aaacagttgt gcacaatacc tcgtgatgcc acttattaca gttaccagaa 7381 caggtatcat ttctgtgaga agtgtttcaa tgagatccaa ggggagagcg tttctttggg 7441 ggatgaccct tcccagcctc aaactacaat aaataaagaa caattttcca agagaaaaaa 7501 tgacacactg gatcctgaac tgtttgttga atgtacagag tgcggaagaa agatgcatca 7561 gatctgtgtc cttcaccatg agatcatctg gcctgctgga ttcgtctgtg atggctgttt 7621 aaagaaaagt gcacgaacta ggaaagaaaa taagttttct gctaaaaggt tgccatctac 7681 cagacttggc acctttcttg agaatcgtgt gaatgacttt ctgaggcgac agaatcaccc 7741 tgagtcagga gaggtcactg ttagagtagt tcatgcttct gacaaaaccg tggaagtaaa 7801 accaggcatg aaagcaaggt ttgtggacag tggagagatg gcagaatcct ttccataccg 7861 aaccaaagcc ctctttgcct ttgaagaaat tgatggtgtt gacctgtgct tctttggcat 7921 gcatgttcaa gagtatggct ctgactgccc tccacccaac cagaggagag tatacatatc 7981 ttacctcgat agtgttcatt tcttccgtcc taaatgcttg aggactgcag tctatcatga 8041 aatcctaatt ggatatttag aatatgtcaa gaaattaggt tacacaacag ggcatatttg 8101 ggcatgtcca ccaagtgagg gagatgatta tatcttccat tgccatcctc ctgaccagaa 8161 gatacccaag cccaagcgac tgcaggaatg gtacaaaaaa atgcttgaca aggctgtatc 8221 agagcgtatt gtccatgact acaaggatat ttttaaacaa gctactgaag atagattaac 8281 aagtgcaaag gaattgcctt atttcgaggg tgatttctgg cccaatgttc tggaagaaag 8341 cattaaggaa ctggaacagg aggaagaaga gagaaaacga gaggaaaaca ccagcaatga 8401 aagcacagat gtgaccaagg gagacagcaa aaatgctaaa aagaagaata ataagaaaac 8461 cagcaaaaat aagagcagcc tgagtagggg caacaagaag aaacccggga tgcccaatgt 8521 atctaacgac ctctcacaga aactatatgc caccatggag aagcataaag aggtcttctt 8581 tgtgatccgc ctcattgctg gccctgctgc caactccctg cctcccattg ttgatcctga 8641 tcctctcatc ccctgcgatc tgatggatgg tcgggatgcg tttctcacgc tggcaaggga 8701 caagcacctg gagttctctt cactccgaag agcccagtgg tccaccatgt gcatgctggt 8761 ggagctgcac acgcagagcc aggacgaggg cagaggaagt ctcctaacat gcggtgacgt 8821 ggaggagaat cctggcccag gaggtatggc ttcaaacttt actcagttcg tgctcgtgga 8881 caatggtggg acaggggatg tgacagtggc tccttctaat ttcgctaatg gggtggcaga 8941 gtggatcagc tccaactcac ggagccaggc ctacaaggtg acatgcagcg tcaggcagtc 9001 tagtgcccag aagagaaagt ataccatcaa ggtggaggtc cccaaagtgg ctacccagac 9061 agtgggcgga gtcgaactgc ctgtcgccgc ttggaggtcc tacctgaaca tggagctcac 9121 tatcccaatt ttcgctacca attctgactg tgaactcatc gtgaaggcaa tgcaggggct 9181 cctcaaagac ggtaatccta tcccttccgc catcgccgct aactcaggta tctacagcgc 9241 tggaggaggt ggaagcggag gaggaggaag cggaggagga ggtagcggac ctaagaaaaa 9301 gaggaaggtg gcggccgctg gatccccttc agggcagatc agcaaccagg ccctggctct 9361 ggcccctagc tccgctccag tgctggccca gactatggtg ccctctagtg ctatggtgcc 9421 tctggcccag ccacctgctc cagcccctgt gctgacccca ggaccacccc agtcactgag 9481 cgctccagtg cccaagtcta cacaggccgg cgaggggact ctgagtgaag ctctgctgca 9541 cctgcagttc gacgctgatg aggacctggg agctctgctg gggaacagca ccgatcccgg 9601 agtgttcaca gatctggcct ccgtggacaa ctctgagttt cagcagctgc tgaatcaggg 9661 cgtgtccatg tctcatagta cagccgaacc aatgctgatg gagtaccccg aagccattac 9721 ccggctggtg accggcagcc agcggccccc cgaccccgct ccaactcccc tgggaaccag 9781 cggcctgcct aatgggctgt ccggagatga agacttctca agcatcgctg atatggactt 9841 tagtgccctg ctgtcacaga tttcctctag tgggcaggga ggaggtggaa gcggcttcag 9901 cgtggacacc agtgccctgc tggacctgtt cagcccctcg gtgaccgtgc ccgacatgag 9961 cctgcctgac cttgacagca gcctggccag tatccaagag ctcctgtctc cccaggagcc 10021 ccccaggcct cccgaggcag agaacagcag cccggattca gggaagcagc tggtgcacta 10081 cacagcgcag ccgctgttcc tgctggaccc cggctccgtg gacaccggga gcaacgacct 10141 gccggtgctg tttgagctgg gagagggctc ctacttctcc gaaggggacg gcttcgccga 10201 ggaccccacc atctccctgc tgacaggctc ggagcctccc aaagccaagg accccactgt 10261 ctcctgtaca tgaggaattc cgcccccccc ccctctccct cccccccccc taacgttact 10321 ggccgaagcc gcttggaata aggccggtgt gcgtttgtct atatgttatt ttccaccata 10381 ttgccgtctt ttggcaatgt gagggcccgg aaacctggcc ctgtcttctt gacgagcatt 10441 cctaggggtc tttcccctct cgccaaagga atgcaaggtc tgttgaatgt cgtgaaggaa 10501 gcagttcctc tggaagcttc ttgaagacaa acaacgtctg tagcgaccct ttgcaggcag 10561 cggaaccccc cacctggcga caggtgcctc tgcggccaaa agccacgtgt ataagataca 10621 cctgcaaagg cggcacaacc ccagtgccac gttgtgagtt ggatagttgt ggaaagagtc 10681 aaatggctct cctcaagcgt attcaacaag gggctgaagg atgcccagaa ggtaccccat 10741 tgtatgggat ctgatctggg gcctcggtgc acatgcttta catgtgttta gtcgaggtta 10801 aaaaacgtct aggccccccg aaccacgggg acgtggtttt cctttgaaaa acacgatgat 10861 aatatggcca caaccatggc caagcctttg tctcaagaag aatccaccct cattgaaaga 10921 gcaacggcta caatcaacag catccccatc tctgaagact acagcgtcgc cagcgcagct 10981 ctctctagcg acggccgcat cttcactggt gtcaatgtat atcattttac tgggggacct 11041 tgtgcagaac tcgtggtgct gggcactgct gctgctgcgg cagctggcaa cctgacttgt 11101 atcgtcgcga tcggaaatga gaacaggggc atcttgagcc cctgcggacg gtgccgacag 11161 gtgcttctcg atctgcatcc tgggatcaaa gccatagtga aggacagtga tggacagccg 11221 acggcagttg ggattcgtga attgctgccc tctggttatg tgtgggaggg ccgactgtgc 11281 cttctagttg ccagccatct gttgtttgcc cctcccccgt gccttccttg accctggaag 11341 gtgccactcc cactgtcctt tcctaataaa atgaggaaat tgcatcgcat tgtctgagta 11401 ggtgtcattc tattctgggg ggtggggtgg ggcaggacag caagggggag gattgggaag 11461 tcaatagcag gcatgctggg gatgcggtgg gctctatgga ctagtctcga gatatctaga 11521 cccagctttc ttgtacaaag tggtactagt taaaagtttt gttactttat agaagaaatt 11581 ttgagttttt gttttttttt aataaataaa taaacataaa taaattgttt gttgaattta 11641 ttattagtat gtaagtgtaa atataataaa acttaatatc tattcaaatt aataaataaa 11701 cctcgatata cagaccgata aaacacatgc gtcaatttta cgcatgatta tctttaacgt 11761 acgtcacaat atgattatct ttctagggtt aatctagtat acgcgtatgc ggccgcttaa 11821 ttaatccagc ttttgttccc tttagtgagg gttaattgcg cgcttggcgt aatcatggtc 11881 atagctgttt cctgtgtgaa attgttatcc gctcacaatt ccacacaaca tacgagccgg 11941 aagcataaag tgtaaagcct ggggtgccta atgagtgagc taactcacat taattgcgtt 12001 gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg 12061 ccaacgcgcg gggagaggcg gtttgcgtat tgggcgctct tccgcttcct cgctcactga 12121 ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca gctcactcaa aggcggtaat 12181 acggttatcc acagaatcag gggataacgc aggaaagaac atgtgagcaa aaggccagca 12241 aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc tccgcccccc 12301 tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga caggactata 12361 aagataccag gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc 12421 gcttaccgga tacctgtccg cctttctccc ttcgggaagc gtggcgcttt ctcatagctc 12481 acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga 12541 accccccgtt cagcccgacc gctgcgcctt atccggtaac tatcgtcttg agtccaaccc 12601 ggtaagacac gacttatcgc cactggcagc agccactggt aacaggatta gcagagcgag 12661 gtatgtaggc ggtgctacag agttcttgaa gtggtggcct aactacggct acactagaag 12721 gacagtattt ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa gagttggtag 12781 ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt gcaagcagca 12841 gattacgcgc agaaaaaaag gatctcaaga agatcctttg atcttttcta cggggtctga 12901 cgctcagtgg aacgaaaact cacgttaagg gattttggtc atgagattat caaaaaggat 12961 cttcacctag atccttttaa attaaaaatg aagttttaaa tcaatctaaa gtatatatga 13021 gtaaacttgg tctgacagtt accaatgctt aatcagtgag gcacctatct cagcgatctg 13081 tctatttcgt tcatccatag ttgcctgact ccccgtcgtg tagataacta cgatacggga 13141 gggcttacca tctggcccca gtgctgcaat gataccgcga gacccacgct caccggctcc 13201 agatttatca gcaataaacc agccagccgg aagggccgag cgcagaagtg gtcctgcaac 13261 tttatccgcc tccatccagt ctattaattg ttgccgggaa gctagagtaa gtagttcgcc 13321 agttaatagt ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc 13381 gtttggtatg gcttcattca gctccggttc ccaacgatca aggcgagtta catgatcccc 13441 catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca gaagtaagtt 13501 ggccgcagtg ttatcactca tggttatggc agcactgcat aattctctta ctgtcatgcc 13561 atccgtaaga tgcttttctg tgactggtga gtactcaacc aagtcattct gagaatagtg 13621 tatgcggcga ccgagttgct cttgcccggc gtcaatacgg gataataccg cgccacatag 13681 cagaacttta aaagtgctca tcattggaaa acgttcttcg gggcgaaaac tctcaaggat 13741 cttaccgctg ttgagatcca gttcgatgta acccactcgt gcacccaact gatcttcagc 13801 atcttttact ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa 13861 aaagggaata agggcgacac ggaaatgttg aatactcata ctcttccttt ttcaatatta 13921 ttgaagcatt tatcagggtt attgtctcat gagcggatac atatttgaat gtatttagaa 13981 aaataaacaa ataggggttc cgcgcacatt tccccgaaaa gtgccac //