LOCUS Exported 15702 bp ds-DNA circular SYN 29-MAR-2022 DEFINITION 3rd generation lenti vector encoding dCas9 (S. pyogenes) fused with VP64-p65-Rta (VPR) and 2A puromycin resistance marker; EF1a-dCas9-VPR-P2A-Puro-WPRE). ACCESSION . VERSION . KEYWORDS lenti-EF1a-dCas9-VPR-Puro SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 15702) AUTHORS Ho SM, Hartley BJ, Flaherty E, Rajarajan P, Abdelaal R, Obiorah I, Barretto N, Muhammad H, Phatnani HP, Akbarian S, Brennand KJ TITLE Evaluating Synthetic Activation and Repression of Neuropsychiatric-Related Genes in hiPSC-Derived NPCs, Neurons, and Astrocytes. JOURNAL Stem Cell Reports. 2017 Aug 8;9(2):615-628. doi: 10.1016/j.stemcr.2017.06.012. Epub 2017 Jul 27. PUBMED 28757163 REFERENCE 2 (bases 1 to 15702) AUTHORS . TITLE Direct Submission JOURNAL Exported Mar 29, 2022 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..15702 /organism="synthetic DNA construct" /mol_type="other DNA" primer_bind 9..30 /label=F1ori-F /note="F1 origin, forward primer" primer_bind complement(149..169) /label=pBABE 3' /note="SV40 enhancer, reverse primer for pBABE vectors" promoter 154..483 /label=SV40 promoter /note="SV40 enhancer and early promoter" rep_origin 334..469 /label=SV40 ori /note="SV40 origin of replication" primer_bind 396..415 /label=SV40pro-F /note="SV40 promoter/origin, forward primer" promoter 531..578 /label=EM7 promoter /note="synthetic bacterial promoter " CDS 597..971 /codon_start=1 /gene="Sh ble from Streptoalloteichus hindustanus" /product="antibiotic-binding protein" /label=BleoR /note="confers resistance to bleomycin, phleomycin, and Zeocin(TM)" /translation="MAKLTSAVPVLTARDVAGAVEFWTDRLGFSRDFVEDDFAGVVRDD VTLFISAVQDQVVPDNTLAWVWVRGLDELYAEWSEVVSTNFRDASGPAMTEIGEQPWGR EFALRDPAGNCVHFVAEEQD" polyA_signal 1101..1222 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(1138..1157) /label=SV40pA-R /note="SV40 polyA, reverse primer" primer_bind 1192..1211 /label=EBV-rev /note="SV40 polyA terminator, reverse primer" primer_bind complement(1271..1287) /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind complement(1271..1287) /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" primer_bind complement(1284..1306) /label=M13/pUC Reverse /note="In lacZ gene" protein_bind 1295..1311 /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(1319..1349) /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 1364..1385 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." primer_bind complement(1502..1519) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(1673..2261) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(1753..1772) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS complement(2432..3292) /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 3055..3074 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(3293..3397) /gene="bla" /label=AmpR promoter primer_bind complement(3472..3491) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" enhancer 3663..4042 /label=CMV enhancer /note="human cytomegalovirus immediate early enhancer" promoter 4044..4242 /label=CMV promoter /note="human cytomegalovirus (CMV) immediate early promoter" primer_bind 4197..4217 /label=CMV-F /note="Human CMV immediate early promoter, forward primer" LTR 4260..4440 /label=5' LTR (truncated) /note="truncated 5' long terminal repeat (LTR) from HIV-1" misc_feature 4487..4612 /label=HIV-1 Psi /note="packaging signal of human immunodeficiency virus type 1" misc_feature 5105..5338 /label=RRE /note="The Rev response element (RRE) of HIV-1 allows for Rev-dependent mRNA export from the nucleus to the cytoplasm." CDS 5523..5567 /codon_start=1 /product="antigenic peptide corresponding to amino acids 655 to 669 of the HIV envelope protein gp41 (Lutje Hulsik et al., 2013)" /label=gp41 peptide /note="recognized by the 2H10 single-chain llama nanobody" /translation="KNEQELLELDKWASL" misc_feature 5865..5982 /label=cPPT/CTS /note="central polypurine tract and central termination sequence of HIV-1" promoter 6112..7290 /label=EF-1-alpha promoter /note="strong constitutive promoter for human elongation factor EF-1-alpha" intron 6343..7281 /label=EF-1-alpha intron A /note="intron upstream of the start codon of human EF-1-alpha" primer_bind 7238..7258 /label=EF1a-F /note="Human elongation factor-1a promoter, forward primer" regulatory 7315..7324 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" CDS 7321..11424 /codon_start=1 /product="catalytically dead mutant of the Cas9 endonuclease from the Streptococcus pyogenes Type II CRISPR/Cas system" /label=Cas9m4 /note="RNA-guided DNA-binding protein that lacks endonuclease activity due to the mutations D10A, D839A, H840A, and N863A (Mali et al., 2013)" /translation="MDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK NLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER LKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFM QLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY LYYLQNGRDMYVDQELDINRLSDYDVAAIVPQSFLKDDSIDNKVLTRSDKARGKSDNVP SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL DATLIHQSITGLYETRIDLSQLGGD" CDS 11437..11457 /codon_start=1 /product="nuclear localization signal of SV40 (simian virus 40) large T antigen" /label=SV40 NLS /translation="PKKKRKV" protein_bind 11494..11518 /gene="mutant version of attB" /label=attB1 /bound_moiety="BP Clonase(TM)" /note="recombination site for the Gateway(R) BP reaction" CDS 11548..11697 /codon_start=1 /product="tetrameric repeat of the minimal activation domain of herpes simplex virus VP16 (Beerli et al., 1998)" /label=VP64 /translation="DALDDFDLDMLGSDALDDFDLDMLGSDALDDFDLDMLGSDALDDF DLDML" CDS 11722..11742 /codon_start=1 /product="nuclear localization signal of SV40 (simian virus 40) large T antigen" /label=SV40 NLS /translation="PKKKRKV" CDS 12172..12528 /codon_start=1 /product="transcriptional activation domain of human RelA, also known as p65 (O'Shea and Perkins, 2008)" /label=RelA (p65) AD /translation="PTQAGEGTLSEALLQLQFDDEDLGALLGNSTDPAVFTDLASVDNS EFQQLLNQGIPVAPHTTEPMLMEYPEAITRLVTGAQRPPDPAPAPLGAPGLPNGLLSGD EDFSSIADMDFSALL" CDS 12547..13116 /codon_start=1 /product="transcriptional activation domain from the human herpesvirus 4 (Epstein-Barr virus) replication and transcription activator Rta/BRLF1 (Hardwick et al., 1992; Chavez et al., 2015)" /label=Rta AD /translation="RDSREGMFLPKPEAGSAISDVFEGREVCQPKRIRPFHPPGSPWAN RPLPASLAPTPTGPVHEPVGSLTPAPVPQPLDPAPAVTPEASHLLEDPDEETSQAVKAL REMADTVIPQKEEAAICGQMDLSHPPPRGHLDELTTTLESMTEDLNLDSPLTPELNEIL DTFLNDECLLHAMHISTGLSIFDTSLF" CDS 13132..13188 /codon_start=1 /product="2A peptide from porcine teschovirus-1 polyprotein" /label=P2A /note="Eukaryotic ribosomes fail to insert a peptide bond between the Gly and Pro residues, yielding separate polypeptides." /translation="ATNFSLLKQAGDVEENPGP" CDS 13201..13794 /codon_start=1 /gene="pac from Streptomyces alboniger" /product="puromycin N-acetyltransferase" /label=PuroR /note="confers resistance to puromycin" /translation="EYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIERVT ELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLAAQ QQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETSAP RNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA" primer_bind 13691..13711 /label=Puro-F /note="Puromycin resistance gene, forward primer" misc_feature 13819..14407 /label=WPRE /note="woodchuck hepatitis virus posttranscriptional regulatory element" primer_bind complement(13872..13892) /label=WPRE-R /note="WPRE, reverse primer" CDS complement(14290..14301) /codon_start=1 /product="Factor Xa recognition and cleavage site" /label=Factor Xa site /translation="IEGR" primer_bind complement(14410..14426) /label=KS primer /note="common sequencing primer, one of multiple similar variants" primer_bind complement(14411..14427) /label=pBluescriptKS /note="For pBluescript vector" LTR 14932..15112 /label=5' LTR (truncated) /note="truncated 5' long terminal repeat (LTR) from HIV-1" primer_bind complement(15138..15155) /label=BGH-rev /note="Bovine growth hormone terminator, reverse primer. Also called BGH reverse" polyA_signal 15144..15368 /label=bGH poly(A) signal /note="bovine growth hormone polyadenylation signal" rep_origin 15414..140 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(15501..15520) /label=F1ori-R /note="F1 origin, reverse primer" ORIGIN 1 ctttaatagt ggactcttgt tccaaactgg aacaacactc aaccctatct cggtctattc 61 ttttgattta taagggattt tgccgatttc ggcctattgg ttaaaaaatg agctgattta 121 acaaaaattt aacgcgaatt aattctgtgg aatgtgtgtc agttagggtg tggaaagtcc 181 ccaggctccc cagcaggcag aagtatgcaa agcatgcatc tcaattagtc agcaaccagg 241 tgtggaaagt ccccaggctc cccagcaggc agaagtatgc aaagcatgca tctcaattag 301 tcagcaacca tagtcccgcc cctaactccg cccatcccgc ccctaactcc gcccagttcc 361 gcccattctc cgccccatgg ctgactaatt ttttttattt atgcagaggc cgaggccgcc 421 tctgcctctg agctattcca gaagtagtga ggaggctttt ttggaggcct aggcttttgc 481 aaaaagctcc cgggagcttg tatatccatt ttcggatctg atcagcacgt gttgacaatt 541 aatcatcggc atagtatatc ggcatagtat aatacgacaa ggtgaggaac taaaccatgg 601 ccaagttgac cagtgccgtt ccggtgctca ccgcgcgcga cgtcgccgga gcggtcgagt 661 tctggaccga ccggctcggg ttctcccggg acttcgtgga ggacgacttc gccggtgtgg 721 tccgggacga cgtgaccctg ttcatcagcg cggtccagga ccaggtggtg ccggacaaca 781 ccctggcctg ggtgtgggtg cgcggcctgg acgagctgta cgccgagtgg tcggaggtcg 841 tgtccacgaa cttccgggac gcctccgggc cggccatgac cgagatcggc gagcagccgt 901 gggggcggga gttcgccctg cgcgacccgg ccggcaactg cgtgcacttc gtggccgagg 961 agcaggactg acacgtgcta cgagatttcg attccaccgc cgccttctat gaaaggttgg 1021 gcttcggaat cgttttccgg gacgccggct ggatgatcct ccagcgcggg gatctcatgc 1081 tggagttctt cgcccacccc aacttgttta ttgcagctta taatggttac aaataaagca 1141 atagcatcac aaatttcaca aataaagcat ttttttcact gcattctagt tgtggtttgt 1201 ccaaactcat caatgtatct tatcatgtct gtataccgtc gacctctagc tagagcttgg 1261 cgtaatcatg gtcatagctg tttcctgtgt gaaattgtta tccgctcaca attccacaca 1321 acatacgagc cggaagcata aagtgtaaag cctggggtgc ctaatgagtg agctaactca 1381 cattaattgc gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc 1441 attaatgaat cggccaacgc gcggggagag gcggtttgcg tattgggcgc tcttccgctt 1501 cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta tcagctcact 1561 caaaggcggt aatacggtta tccacagaat caggggataa cgcaggaaag aacatgtgag 1621 caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg tttttccata 1681 ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg tggcgaaacc 1741 cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg cgctctcctg 1801 ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga agcgtggcgc 1861 tttctcatag ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc tccaagctgg 1921 gctgtgtgca cgaacccccc gttcagcccg accgctgcgc cttatccggt aactatcgtc 1981 ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact ggtaacagga 2041 ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg cctaactacg 2101 gctacactag aagaacagta tttggtatct gcgctctgct gaagccagtt accttcggaa 2161 aaagagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt ggtttttttg 2221 tttgcaagca gcagattacg cgcagaaaaa aaggatctca agaagatcct ttgatctttt 2281 ctacggggtc tgacgctcag tggaacgaaa actcacgtta agggattttg gtcatgagat 2341 tatcaaaaag gatcttcacc tagatccttt taaattaaaa atgaagtttt aaatcaatct 2401 aaagtatata tgagtaaact tggtctgaca gttaccaatg cttaatcagt gaggcaccta 2461 tctcagcgat ctgtctattt cgttcatcca tagttgcctg actccccgtc gtgtagataa 2521 ctacgatacg ggagggctta ccatctggcc ccagtgctgc aatgataccg cgagacccac 2581 gctcaccggc tccagattta tcagcaataa accagccagc cggaagggcc gagcgcagaa 2641 gtggtcctgc aactttatcc gcctccatcc agtctattaa ttgttgccgg gaagctagag 2701 taagtagttc gccagttaat agtttgcgca acgttgttgc cattgctaca ggcatcgtgg 2761 tgtcacgctc gtcgtttggt atggcttcat tcagctccgg ttcccaacga tcaaggcgag 2821 ttacatgatc ccccatgttg tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg 2881 tcagaagtaa gttggccgca gtgttatcac tcatggttat ggcagcactg cataattctc 2941 ttactgtcat gccatccgta agatgctttt ctgtgactgg tgagtactca accaagtcat 3001 tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata cgggataata 3061 ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa 3121 aactctcaag gatcttaccg ctgttgagat ccagttcgat gtaacccact cgtgcaccca 3181 actgatcttc agcatctttt actttcacca gcgtttctgg gtgagcaaaa acaggaaggc 3241 aaaatgccgc aaaaaaggga ataagggcga cacggaaatg ttgaatactc atactcttcc 3301 tttttcaata ttattgaagc atttatcagg gttattgtct catgagcgga tacatatttg 3361 aatgtattta gaaaaataaa caaatagggg ttccgcgcac atttccccga aaagtgccac 3421 ctgacgtcga cggatcggga gatctcccga tcccctatgg tgcactctca gtacaatctg 3481 ctctgatgcc gcatagttaa gccagtatct gctccctgct tgtgtgttgg aggtcgctga 3541 gtagtgcgcg agcaaaattt aagctacaac aaggcaaggc ttgaccgaca attgcatgaa 3601 gaatctgctt agggttaggc gttttgcgct gcttcgcgat gtacgggcca gatatacgcg 3661 ttgacattga ttattgacta gttattaata gtaatcaatt acggggtcat tagttcatag 3721 cccatatatg gagttccgcg ttacataact tacggtaaat ggcccgcctg gctgaccgcc 3781 caacgacccc cgcccattga cgtcaataat gacgtatgtt cccatagtaa cgccaatagg 3841 gactttccat tgacgtcaat gggtggagta tttacggtaa actgcccact tggcagtaca 3901 tcaagtgtat catatgccaa gtacgccccc tattgacgtc aatgacggta aatggcccgc 3961 ctggcattat gcccagtaca tgaccttatg ggactttcct acttggcagt acatctacgt 4021 attagtcatc gctattacca tggtgatgcg gttttggcag tacatcaatg ggcgtggata 4081 gcggtttgac tcacggggat ttccaagtct ccaccccatt gacgtcaatg ggagtttgtt 4141 ttggcaccaa aatcaacggg actttccaaa atgtcgtaac aactccgccc cattgacgca 4201 aatgggcggt aggcgtgtac ggtgggaggt ctatataagc agcgcgtttt gcctgtactg 4261 ggtctctctg gttagaccag atctgagcct gggagctctc tggctaacta gggaacccac 4321 tgcttaagcc tcaataaagc ttgccttgag tgcttcaagt agtgtgtgcc cgtctgttgt 4381 gtgactctgg taactagaga tccctcagac ccttttagtc agtgtggaaa atctctagca 4441 gtggcgcccg aacagggact tgaaagcgaa agggaaacca gaggagctct ctcgacgcag 4501 gactcggctt gctgaagcgc gcacggcaag aggcgagggg cggcgactgg tgagtacgcc 4561 aaaaattttg actagcggag gctagaagga gagagatggg tgcgagagcg tcagtattaa 4621 gcgggggaga attagatcgc gatgggaaaa aattcggtta aggccagggg gaaagaaaaa 4681 atataaatta aaacatatag tatgggcaag cagggagcta gaacgattcg cagttaatcc 4741 tggcctgtta gaaacatcag aaggctgtag acaaatactg ggacagctac aaccatccct 4801 tcagacagga tcagaagaac ttagatcatt atataataca gtagcaaccc tctattgtgt 4861 gcatcaaagg atagagataa aagacaccaa ggaagcttta gacaagatag aggaagagca 4921 aaacaaaagt aagaccaccg cacagcaagc ggccgctgat cttcagacct ggaggaggag 4981 atatgaggga caattggaga agtgaattat ataaatataa agtagtaaaa attgaaccat 5041 taggagtagc acccaccaag gcaaagagaa gagtggtgca gagagaaaaa agagcagtgg 5101 gaataggagc tttgttcctt gggttcttgg gagcagcagg aagcactatg ggcgcagcgt 5161 caatgacgct gacggtacag gccagacaat tattgtctgg tatagtgcag cagcagaaca 5221 atttgctgag ggctattgag gcgcaacagc atctgttgca actcacagtc tggggcatca 5281 agcagctcca ggcaagaatc ctggctgtgg aaagatacct aaaggatcaa cagctcctgg 5341 ggatttgggg ttgctctgga aaactcattt gcaccactgc tgtgccttgg aatgctagtt 5401 ggagtaataa atctctggaa cagatttgga atcacacgac ctggatggag tgggacagag 5461 aaattaacaa ttacacaagc ttaatacact ccttaattga agaatcgcaa aaccagcaag 5521 aaaagaatga acaagaatta ttggaattag ataaatgggc aagtttgtgg aattggttta 5581 acataacaaa ttggctgtgg tatataaaat tattcataat gatagtagga ggcttggtag 5641 gtttaagaat agtttttgct gtactttcta tagtgaatag agttaggcag ggatattcac 5701 cattatcgtt tcagacccac ctcccaaccc cgaggggacc cgacaggccc gaaggaatag 5761 aagaagaagg tggagagaga gacagagaca gatccattcg attagtgaac ggatcggcac 5821 tgcgtgcgcc aattctgcag acaaatggca gtattcatcc acaattttaa aagaaaaggg 5881 gggattgggg ggtacagtgc aggggaaaga atagtagaca taatagcaac agacatacaa 5941 actaaagaat tacaaaaaca aattacaaaa attcaaaatt ttcgggttta ttacagggac 6001 agcagagatc cagtttggtt aattagctag ctgcaaagat ggataaagtt ttaaacagag 6061 aggaatcttt gcagctaatg gaccttctag gtcttgaaag gagtgggaat tggctccggt 6121 gcccgtcagt gggcagagcg cacatcgccc acagtccccg agaagttggg gggaggggtc 6181 ggcaattgaa ccggtgccta gagaaggtgg cgcggggtaa actgggaaag tgatgtcgtg 6241 tactggctcc gcctttttcc cgagggtggg ggagaaccgt atataagtgc agtagtcgcc 6301 gtgaacgttc tttttcgcaa cgggtttgcc gccagaacac aggtaagtgc cgtgtgtggt 6361 tcccgcgggc ctggcctctt tacgggttat ggcccttgcg tgccttgaat tacttccacc 6421 tggctgcagt acgtgattct tgatcccgag cttcgggttg gaagtgggtg ggagagttcg 6481 aggccttgcg cttaaggagc cccttcgcct cgtgcttgag ttgaggcctg gcctgggcgc 6541 tggggccgcc gcgtgcgaat ctggtggcac cttcgcgcct gtctcgctgc tttcgataag 6601 tctctagcca tttaaaattt ttgatgacct gctgcgacgc tttttttctg gcaagatagt 6661 cttgtaaatg cgggccaaga tctgcacact ggtatttcgg tttttggggc cgcgggcggc 6721 gacggggccc gtgcgtccca gcgcacatgt tcggcgaggc ggggcctgcg agcgcggcca 6781 ccgagaatcg gacgggggta gtctcaagct ggccggcctg ctctggtgcc tggcctcgcg 6841 ccgccgtgta tcgccccgcc ctgggcggca aggctggccc ggtcggcacc agttgcgtga 6901 gcggaaagat ggccgcttcc cggccctgct gcagggagct caaaatggag gacgcggcgc 6961 tcgggagagc gggcgggtga gtcacccaca caaaggaaaa gggcctttcc gtcctcagcc 7021 gtcgcttcat gtgactccac ggagtaccgg gcgccgtcca ggcacctcga ttagttctcg 7081 agcttttgga gtacgtcgtc tttaggttgg ggggaggggt tttatgcgat ggagtttccc 7141 cacactgagt gggtggagac tgaagttagg ccagcttggc acttgatgta attctccttg 7201 gaatttgccc tttttgagtt tggatcttgg ttcattctca agcctcagac agtggttcaa 7261 agtttttttc ttccatttca ggtgtcgtga cgtacgctag aggatcgaac ccttgccacc 7321 atggacaaga agtactccat tgggctcgct atcggcacaa acagcgtcgg ctgggccgtc 7381 attacggacg agtacaaggt gccgagcaaa aaattcaaag ttctgggcaa taccgatcgc 7441 cacagcataa agaagaacct cattggcgcc ctcctgttcg actccgggga gacggccgaa 7501 gccacgcggc tcaaaagaac agcacggcgc agatataccc gcagaaagaa tcggatctgc 7561 tacctgcagg agatctttag taatgagatg gctaaggtgg atgactcttt cttccatagg 7621 ctggaggagt cctttttggt ggaggaggat aaaaagcacg agcgccaccc aatctttggc 7681 aatatcgtgg acgaggtggc gtaccatgaa aagtacccaa ccatatatca tctgaggaag 7741 aagcttgtag acagtactga taaggctgac ttgcggttga tctatctcgc gctggcgcat 7801 atgatcaaat ttcggggaca cttcctcatc gagggggacc tgaacccaga caacagcgat 7861 gtcgacaaac tctttatcca actggttcag acttacaatc agcttttcga agagaacccg 7921 atcaacgcat ccggagttga cgccaaagca atcctgagcg ctaggctgtc caaatcccgg 7981 cggctcgaaa acctcatcgc acagctccct ggggagaaga agaacggcct gtttggtaat 8041 cttatcgccc tgtcactcgg gctgaccccc aactttaaat ctaacttcga cctggccgaa 8101 gatgccaagc ttcaactgag caaagacacc tacgatgatg atctcgacaa tctgctggcc 8161 cagatcggcg accagtacgc agaccttttt ttggcggcaa agaacctgtc agacgccatt 8221 ctgctgagtg atattctgcg agtgaacacg gagatcacca aagctccgct gagcgctagt 8281 atgatcaagc gctatgatga gcaccaccaa gacttgactt tgctgaaggc ccttgtcaga 8341 cagcaactgc ctgagaagta caaggaaatt ttcttcgatc agtctaaaaa tggctacgcc 8401 ggatacattg acggcggagc aagccaggag gaattttaca aatttattaa gcccatcttg 8461 gaaaaaatgg acggcaccga ggagctgctg gtaaagctta acagagaaga tctgttgcgc 8521 aaacagcgca ctttcgacaa tggaagcatc ccccaccaga ttcacctggg cgaactgcac 8581 gctatcctca ggcggcaaga ggatttctac ccctttttga aagataacag ggaaaagatt 8641 gagaaaatcc tcacatttcg gataccctac tatgtaggcc ccctcgcccg gggaaattcc 8701 agattcgcgt ggatgactcg caaatcagaa gagaccatca ctccctggaa cttcgaggaa 8761 gtcgtggata agggggcctc tgcccagtcc ttcatcgaaa ggatgactaa ctttgataaa 8821 aatctgccta acgaaaaggt gcttcctaaa cactctctgc tgtacgagta cttcacagtt 8881 tataacgagc tcaccaaggt caaatacgtc acagaaggga tgagaaagcc agcattcctg 8941 tctggagagc agaagaaagc tatcgtggac ctcctcttca agacgaaccg gaaagttacc 9001 gtgaaacagc tcaaagaaga ctatttcaaa aagattgaat gtttcgactc tgttgaaatc 9061 agcggagtgg aggatcgctt caacgcatcc ctgggaacgt atcacgatct cctgaaaatc 9121 attaaagaca aggacttcct ggacaatgag gagaacgagg acattcttga ggacattgtc 9181 ctcaccctta cgttgtttga agatagggag atgattgaag aacgcttgaa aacttacgct 9241 catctcttcg acgacaaagt catgaaacag ctcaagaggc gccgatatac aggatggggg 9301 cggctgtcaa gaaaactgat caatgggatc cgagacaagc agagtggaaa gacaatcctg 9361 gattttctta agtccgatgg atttgccaac cggaacttca tgcagttgat ccatgatgac 9421 tctctcacct ttaaggagga catccagaaa gcacaagttt ctggccaggg ggacagtctt 9481 cacgagcaca tcgctaatct tgcaggtagc ccagctatca aaaagggaat actgcagacc 9541 gttaaggtcg tggatgaact cgtcaaagta atgggaaggc ataagcccga gaatatcgtt 9601 atcgagatgg cccgagagaa ccaaactacc cagaagggac agaagaacag tagggaaagg 9661 atgaagagga ttgaagaggg tataaaagaa ctggggtccc aaatccttaa ggaacaccca 9721 gttgaaaaca cccagcttca gaatgagaag ctctacctgt actacctgca gaacggcagg 9781 gacatgtacg tggatcagga actggacatc aatcggctct ccgactacga cgtggctgct 9841 atcgtgcccc agtcttttct caaagatgat tctattgata ataaagtgtt gacaagatcc 9901 gataaagcta gagggaagag tgataacgtc ccctcagaag aagttgtcaa gaaaatgaaa 9961 aattattggc ggcagctgct gaacgccaaa ctgatcacac aacggaagtt cgataatctg 10021 actaaggctg aacgaggtgg cctgtctgag ttggataaag ccggcttcat caaaaggcag 10081 cttgttgaga cacgccagat caccaagcac gtggcccaaa ttctcgattc acgcatgaac 10141 accaagtacg atgaaaatga caaactgatt cgagaggtga aagttattac tctgaagtct 10201 aagctggtct cagatttcag aaaggacttt cagttttata aggtgagaga gatcaacaat 10261 taccaccatg cgcatgatgc ctacctgaat gcagtggtag gcactgcact tatcaaaaaa 10321 tatcccaagc ttgaatctga atttgtttac ggagactata aagtgtacga tgttaggaaa 10381 atgatcgcaa agtctgagca ggaaataggc aaggccaccg ctaagtactt cttttacagc 10441 aatattatga attttttcaa gaccgagatt acactggcca atggagagat tcggaagcga 10501 ccacttatcg aaacaaacgg agaaacagga gaaatcgtgt gggacaaggg tagggatttc 10561 gcgacagtcc ggaaggtcct gtccatgccg caggtgaaca tcgttaaaaa gaccgaagta 10621 cagaccggag gcttctccaa ggaaagtatc ctcccgaaaa ggaacagcga caagctgatc 10681 gcacgcaaaa aagattggga ccccaagaaa tacggcggat tcgattctcc tacagtcgct 10741 tacagtgtac tggttgtggc caaagtggag aaagggaagt ctaaaaaact caaaagcgtc 10801 aaggaactgc tgggcatcac aatcatggag cgatcaagct tcgaaaaaaa ccccatcgac 10861 tttctcgagg cgaaaggata taaagaggtc aaaaaagacc tcatcattaa gcttcccaag 10921 tactctctct ttgagcttga aaacggccgg aaacgaatgc tcgctagtgc gggcgagctg 10981 cagaaaggta acgagctggc actgccctct aaatacgtta atttcttgta tctggccagc 11041 cactatgaaa agctcaaagg gtctcccgaa gataatgagc agaagcagct gttcgtggaa 11101 caacacaaac actaccttga tgagatcatc gagcaaataa gcgaattctc caaaagagtg 11161 atcctcgccg acgctaacct cgataaggtg ctttctgctt acaataagca cagggataag 11221 cccatcaggg agcaggcaga aaacattatc cacttgttta ctctgaccaa cttgggcgcg 11281 cctgcagcct tcaagtactt cgacaccacc atagacagaa agcggtacac ctctacaaag 11341 gaggtcctgg acgccacact gattcatcag tcaattacgg ggctctatga aacaagaatc 11401 gacctctctc agctcggtgg agacagcagg gctgacccca agaagaagag gaaggtgtcg 11461 ccagggatcc gtcgacttga cgcgttgata tcaacaagtt tgtacaaaaa agcaggctac 11521 aaagaggcca gcggttccgg acgggctgac gcattggacg attttgatct ggatatgctg 11581 ggaagtgacg ccctcgatga ttttgacctt gacatgcttg gttcggatgc ccttgatgac 11641 tttgacctcg acatgctcgg cagtgacgcc cttgatgatt tcgacctgga catgctgatt 11701 aactctagaa gttccggatc tccgaaaaag aaacgcaaag ttggtagcca gtacctgccc 11761 gacaccgacg accggcaccg gatcgaggaa aagcggaagc ggacctacga gacattcaag 11821 agcatcatga agaagtcccc cttcagcggc cccaccgacc ctagacctcc acctagaaga 11881 atcgccgtgc ccagcagatc cagcgccagc gtgccaaaac ctgcccccca gccttacccc 11941 ttcaccagca gcctgagcac catcaactac gacgagttcc ctaccatggt gttccccagc 12001 ggccagatct ctcaggcctc tgctctggct ccagcccctc ctcaggtgct gcctcaggct 12061 cctgctcctg caccagctcc agccatggtg tctgcactgg ctcaggcacc agcacccgtg 12121 cctgtgctgg ctcctggacc tccacaggct gtggctccac cagcccctaa acctacacag 12181 gccggcgagg gcacactgtc tgaagctctg ctgcagctgc agttcgacga cgaggatctg 12241 ggagccctgc tgggaaacag caccgatcct gccgtgttca ccgacctggc cagcgtggac 12301 aacagcgagt tccagcagct gctgaaccag ggcatccctg tggcccctca caccaccgag 12361 cccatgctga tggaataccc cgaggccatc acccggctcg tgacaggcgc tcagaggcct 12421 cctgatccag ctcctgcccc tctgggagca ccaggcctgc ctaatggact gctgtctggc 12481 gacgaggact tcagctctat cgccgatatg gatttctcag ccttgctggg ctctggcagc 12541 ggcagccggg attccaggga agggatgttt ttgccgaagc ctgaggccgg ctccgctatt 12601 agtgacgtgt ttgagggccg cgaggtgtgc cagccaaaac gaatccggcc atttcatcct 12661 ccaggaagtc catgggccaa ccgcccactc cccgccagcc tcgcaccaac accaaccggt 12721 ccagtacatg agccagtcgg gtcactgacc ccggcaccag tccctcagcc actggatcca 12781 gcgcccgcag tgactcccga ggccagtcac ctgttggagg atcccgatga agagacgagc 12841 caggctgtca aagcccttcg ggagatggcc gatactgtga ttccccagaa ggaagaggct 12901 gcaatctgtg gccaaatgga cctttcccat ccgcccccaa ggggccatct ggatgagctg 12961 acaaccacac ttgagtccat gaccgaggat ctgaacctgg actcacccct gaccccggaa 13021 ttgaacgaga ttctggatac cttcctgaac gacgagtgcc tcttgcatgc catgcatatc 13081 agcacaggac tgtccatctt cgacacatct ctctttgaat tcggatctgg tgccacgaat 13141 ttctcgctac tcaagcaggc cggtgatgtc gaggaaaacc ctggtcctat ggtgtctaga 13201 gagtacaagc ccacggtgcg cctcgccacc cgcgacgacg tccccagggc cgtacgcacc 13261 ctcgccgccg cgttcgccga ctaccccgcc acgcgccaca ccgtcgatcc ggaccgccac 13321 atcgagcggg tcaccgagct gcaagaactc ttcctcacgc gcgtcgggct cgacatcggc 13381 aaggtgtggg tcgcggacga cggcgccgcg gtggcggtct ggaccacgcc ggagagcgtc 13441 gaagcggggg cggtgttcgc cgagatcggc ccgcgcatgg ccgagttgag cggttcccgg 13501 ctggccgcgc agcaacagat ggaaggcctc ctggcgccgc accggcccaa ggagcccgcg 13561 tggttcctgg ccaccgtcgg cgtctcgccc gaccaccagg gcaagggtct gggcagcgcc 13621 gtcgtgctcc ccggagtgga ggcggccgag cgcgccgggg tgcccgcctt cctggagacc 13681 tccgcgcccc gcaacctccc cttctacgag cggctcggct tcaccgtcac cgccgacgtc 13741 gaggtgcccg aaggaccgcg cacctggtgc atgacccgca agcccggtgc ctgagaattc 13801 gatatcaagc ttatcggtaa tcaacctctg gattacaaaa tttgtgaaag attgactggt 13861 attcttaact atgttgctcc ttttacgcta tgtggatacg ctgctttaat gcctttgtat 13921 catgctattg cttcccgtat ggctttcatt ttctcctcct tgtataaatc ctggttgctg 13981 tctctttatg aggagttgtg gcccgttgtc aggcaacgtg gcgtggtgtg cactgtgttt 14041 gctgacgcaa cccccactgg ttggggcatt gccaccacct gtcagctcct ttccgggact 14101 ttcgctttcc ccctccctat tgccacggcg gaactcatcg ccgcctgcct tgcccgctgc 14161 tggacagggg ctcggctgtt gggcactgac aattccgtgg tgttgtcggg gaaatcatcg 14221 tcctttcctt ggctgctcgc ctgtgttgcc acctggattc tgcgcgggac gtccttctgc 14281 tacgtccctt cggccctcaa tccagcggac cttccttccc gcggcctgct gccggctctg 14341 cggcctcttc cgcgtcttcg ccttcgccct cagacgagtc ggatctccct ttgggccgcc 14401 tccccgcatc gataccgtcg acctcgagac ctagaaaaac atggagcaat cacaagtagc 14461 aatacagcag ctaccaatgc tgattgtgcc tggctagaag cacaagagga ggaggaggtg 14521 ggttttccag tcacacctca ggtaccttta agaccaatga cttacaaggc agctgtagat 14581 cttagccact ttttaaaaga aaagggggga ctggaagggc taattcactc ccaacgaaga 14641 caagatatcc ttgatctgtg gatctaccac acacaaggct acttccctga ttggcagaac 14701 tacacaccag ggccagggat cagatatcca ctgacctttg gatggtgcta caagctagta 14761 ccagttgagc aagagaaggt agaagaagcc aatgaaggag agaacacccg cttgttacac 14821 cctgtgagcc tgcatgggat ggatgacccg gagagagaag tattagagtg gaggtttgac 14881 agccgcctag catttcatca catggcccga gagctgcatc cggactgtac tgggtctctc 14941 tggttagacc agatctgagc ctgggagctc tctggctaac tagggaaccc actgcttaag 15001 cctcaataaa gcttgccttg agtgcttcaa gtagtgtgtg cccgtctgtt gtgtgactct 15061 ggtaactaga gatccctcag acccttttag tcagtgtgga aaatctctag cagggcccgt 15121 ttaaacccgc tgatcagcct cgactgtgcc ttctagttgc cagccatctg ttgtttgccc 15181 ctcccccgtg ccttccttga ccctggaagg tgccactccc actgtccttt cctaataaaa 15241 tgaggaaatt gcatcgcatt gtctgagtag gtgtcattct attctggggg gtggggtggg 15301 gcaggacagc aagggggagg attgggaaga caatagcagg catgctgggg atgcggtggg 15361 ctctatggct tctgaggcgg aaagaaccag ctggggctct agggggtatc cccacgcgcc 15421 ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga ccgctacact 15481 tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct tcctttctcg ccacgttcgc 15541 cggctttccc cgtcaagctc taaatcgggg gctcccttta gggttccgat ttagtgcttt 15601 acggcacctc gaccccaaaa aacttgatta gggtgatggt tcacgtagtg ggccatcgcc 15661 ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg tt //