LOCUS Exported 14982 bp ds-DNA circular SYN 29-JUL-2021 DEFINITION Express sgRNA and dCas9-KRAB from 3rd generation lentiviral vector. ACCESSION . VERSION . KEYWORDS pLV hU6-sgRNA hUbC-dCas9-KRAB-T2a-GFP SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 14982) AUTHORS Thakore PI, D'Ippolito AM, Song L, Safi A, Shivakumar NK, Kabadi AM, Reddy TE, Crawford GE, Gersbach CA TITLE Highly specific epigenome editing by CRISPR-Cas9 repressors for silencing of distal regulatory elements. JOURNAL Nat Methods. 2015 Dec;12(12):1143-9. doi: 10.1038/nmeth.3630. Epub 2015 Oct 26. PUBMED 26501517 REFERENCE 2 (bases 1 to 14982) AUTHORS . TITLE Direct Submission JOURNAL Exported Jul 29, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..14982 /organism="synthetic DNA construct" /mol_type="other DNA" enhancer 85..464 /label=CMV enhancer /note="human cytomegalovirus immediate early enhancer" promoter 466..664 /label=CMV promoter /note="human cytomegalovirus (CMV) immediate early promoter" primer_bind 619..639 /label=CMV-F /note="Human CMV immediate early promoter, forward primer" LTR 682..862 /label=5' LTR (truncated) /note="truncated 5' long terminal repeat (LTR) from HIV-1" misc_feature 909..1034 /label=HIV-1 Psi /note="packaging signal of human immunodeficiency virus type 1" misc_feature 1527..1760 /label=RRE /note="The Rev response element (RRE) of HIV-1 allows for Rev-dependent mRNA export from the nucleus to the cytoplasm." CDS 1945..1989 /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 2287..2404 /label=cPPT/CTS /note="central polypurine tract and central termination sequence of HIV-1" protein_bind 2448..2481 /label=loxP /bound_moiety="Cre recombinase" /note="Cre-mediated recombination occurs in the 8-bp core sequence (GCATACAT)." misc_RNA complement(2492..2567) /label=gRNA scaffold /note="guide RNA scaffold for the Streptococcus pyogenes CRISPR/Cas9 system" promoter complement(2592..2832) /label=U6 promoter /note="RNA polymerase III promoter for human U6 snRNA" primer_bind complement(2642..2661) /label=LKO.1 5' /note="Human U6 promoter, forward primer" primer_bind complement(2812..2832) /label=hU6-F /note="Human U6 promoter, forward primer" promoter 2865..4076 /label=UbC promoter /note="human ubiquitin C promoter" primer_bind 3913..3932 /label=hUBCpro-F /note="Human Ubiquitin C (UbC) promoter, forward primer" regulatory 4105..4114 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" CDS 4114..4179 /codon_start=1 /product="three tandem FLAG(R) epitope tags, followed by an enterokinase cleavage site" /label=3xFLAG /translation="DYKDHDGDYKDHDIDYKDDDDK" CDS 4186..4206 /codon_start=1 /product="nuclear localization signal of SV40 (simian virus 40) large T antigen" /label=SV40 NLS /translation="PKKKRKV" CDS 4216..8319 /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 8332..8352 /codon_start=1 /product="nuclear localization signal of SV40 (simian virus 40) large T antigen" /label=SV40 NLS /translation="PKKKRKV" CDS 8386..8580 /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="RTLVTFKDVFVDFTREEWKLLDTAQQILYRNVMLENYKNLVSLGY QLTKPDVILRLEKGEEPWLV" CDS 8647..8667 /codon_start=1 /product="nuclear localization signal of SV40 (simian virus 40) large T antigen" /label=SV40 NLS /translation="PKKKRKV" CDS 8674..8727 /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 8734..9447 /codon_start=1 /product="enhanced GFP" /label=EGFP /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE FVTAAGITLGMDELYK" CDS 8734..9447 /codon_start=1 /product="the original enhanced GFP (Yang et al., 1996)" /label=EGFP /note="mammalian codon-optimized" /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE FVTAAGITLGMDELYK" primer_bind complement(8776..8797) /label=EGFP-N /note="EGFP, reverse primer" primer_bind complement(9037..9056) /label=EXFP-R /note="For distinguishing EGFP variants, reverse primer" primer_bind 9384..9405 /label=EGFP-C /note="EGFP, forward primer" protein_bind 9463..9496 /label=loxP /bound_moiety="Cre recombinase" /note="Cre-mediated recombination occurs in the 8-bp core sequence (GCATACAT)." misc_feature 9521..10109 /label=WPRE /note="woodchuck hepatitis virus posttranscriptional regulatory element" primer_bind complement(9574..9594) /label=WPRE-R /note="WPRE, reverse primer" CDS complement(9992..10003) /codon_start=1 /product="Factor Xa recognition and cleavage site" /label=Factor Xa site /translation="IEGR" primer_bind complement(10112..10128) /label=KS primer /note="common sequencing primer, one of multiple similar variants" primer_bind complement(10113..10129) /label=pBluescriptKS /note="For pBluescript vector" LTR 10634..10814 /label=5' LTR (truncated) /note="truncated 5' long terminal repeat (LTR) from HIV-1" primer_bind complement(10840..10857) /label=BGH-rev /note="Bovine growth hormone terminator, reverse primer. Also called BGH reverse" polyA_signal 10846..11070 /label=bGH poly(A) signal /note="bovine growth hormone polyadenylation signal" rep_origin 11116..11544 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(11203..11222) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 11413..11434 /label=F1ori-F /note="F1 origin, forward primer" primer_bind complement(11553..11573) /label=pBABE 3' /note="SV40 enhancer, reverse primer for pBABE vectors" promoter 11558..11887 /label=SV40 promoter /note="SV40 enhancer and early promoter" rep_origin 11738..11873 /label=SV40 ori /note="SV40 origin of replication" primer_bind 11800..11819 /label=SV40pro-F /note="SV40 promoter/origin, forward primer" promoter 11935..11982 /label=EM7 promoter /note="synthetic bacterial promoter " CDS 12001..12375 /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 12505..12626 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(12542..12561) /label=SV40pA-R /note="SV40 polyA, reverse primer" primer_bind 12596..12615 /label=EBV-rev /note="SV40 polyA terminator, reverse primer" primer_bind complement(12675..12691) /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind complement(12675..12691) /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" primer_bind complement(12688..12710) /label=M13/pUC Reverse /note="In lacZ gene" protein_bind 12699..12715 /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(12723..12753) /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 12768..12789 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." primer_bind complement(12906..12923) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(13077..13665) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(13157..13176) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS complement(13836..14696) /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 14459..14478 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(14697..14801) /gene="bla" /label=AmpR promoter primer_bind complement(14876..14895) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" ORIGIN 1 gcttgaccga caattgcatg aagaatctgc ttagggttag gcgttttgcg ctgcttcgcg 61 atgtacgggc cagatatacg cgttgacatt gattattgac tagttattaa tagtaatcaa 121 ttacggggtc attagttcat agcccatata tggagttccg cgttacataa cttacggtaa 181 atggcccgcc tggctgaccg cccaacgacc cccgcccatt gacgtcaata atgacgtatg 241 ttcccatagt aacgccaata gggactttcc attgacgtca atgggtggag tatttacggt 301 aaactgccca cttggcagta catcaagtgt atcatatgcc aagtacgccc cctattgacg 361 tcaatgacgg taaatggccc gcctggcatt atgcccagta catgacctta tgggactttc 421 ctacttggca gtacatctac gtattagtca tcgctattac catggtgatg cggttttggc 481 agtacatcaa tgggcgtgga tagcggtttg actcacgggg atttccaagt ctccacccca 541 ttgacgtcaa tgggagtttg ttttggcacc aaaatcaacg ggactttcca aaatgtcgta 601 acaactccgc cccattgacg caaatgggcg gtaggcgtgt acggtgggag gtctatataa 661 gcagcgcgtt ttgcctgtac tgggtctctc tggttagacc agatctgagc ctgggagctc 721 tctggctaac tagggaaccc actgcttaag cctcaataaa gcttgccttg agtgcttcaa 781 gtagtgtgtg cccgtctgtt gtgtgactct ggtaactaga gatccctcag acccttttag 841 tcagtgtgga aaatctctag cagtggcgcc cgaacaggga cttgaaagcg aaagggaaac 901 cagaggagct ctctcgacgc aggactcggc ttgctgaagc gcgcacggca agaggcgagg 961 ggcggcgact ggtgagtacg ccaaaaattt tgactagcgg aggctagaag gagagagatg 1021 ggtgcgagag cgtcagtatt aagcggggga gaattagatc gcgatgggaa aaaattcggt 1081 taaggccagg gggaaagaaa aaatataaat taaaacatat agtatgggca agcagggagc 1141 tagaacgatt cgcagttaat cctggcctgt tagaaacatc agaaggctgt agacaaatac 1201 tgggacagct acaaccatcc cttcagacag gatcagaaga acttagatca ttatataata 1261 cagtagcaac cctctattgt gtgcatcaaa ggatagagat aaaagacacc aaggaagctt 1321 tagacaagat agaggaagag caaaacaaaa gtaagaccac cgcacagcaa gcggccgctg 1381 atcttcagac ctggaggagg agatatgagg gacaattgga gaagtgaatt atataaatat 1441 aaagtagtaa aaattgaacc attaggagta gcacccacca aggcaaagag aagagtggtg 1501 cagagagaaa aaagagcagt gggaatagga gctttgttcc ttgggttctt gggagcagca 1561 ggaagcacta tgggcgcagc gtcaatgacg ctgacggtac aggccagaca attattgtct 1621 ggtatagtgc agcagcagaa caatttgctg agggctattg aggcgcaaca gcatctgttg 1681 caactcacag tctggggcat caagcagctc caggcaagaa tcctggctgt ggaaagatac 1741 ctaaaggatc aacagctcct ggggatttgg ggttgctctg gaaaactcat ttgcaccact 1801 gctgtgcctt ggaatgctag ttggagtaat aaatctctgg aacagatttg gaatcacacg 1861 acctggatgg agtgggacag agaaattaac aattacacaa gcttaataca ctccttaatt 1921 gaagaatcgc aaaaccagca agaaaagaat gaacaagaat tattggaatt agataaatgg 1981 gcaagtttgt ggaattggtt taacataaca aattggctgt ggtatataaa attattcata 2041 atgatagtag gaggcttggt aggtttaaga atagtttttg ctgtactttc tatagtgaat 2101 agagttaggc agggatattc accattatcg tttcagaccc acctcccaac cccgagggga 2161 cccgacaggc ccgaaggaat agaagaagaa ggtggagaga gagacagaga cagatccatt 2221 cgattagtga acggatcggc actgcgtgcg ccaattctgc agacaaatgg cagtattcat 2281 ccacaatttt aaaagaaaag gggggattgg ggggtacagt gcaggggaaa gaatagtaga 2341 cataatagca acagacatac aaactaaaga attacaaaaa caaattacaa aaattcaaaa 2401 ttttcgggtt tattacaggg acagcagaga tccagtttgg ttaattaata acttcgtata 2461 gcatacatta tacgaagtta ttaaaaaaaa agcaccgact cggtgccact ttttcaagtt 2521 gataacggac tagccttatt ttaacttgct atttctagct ctaaaactga gacgtccgtc 2581 tccggtgttt cgtcctttcc acaagatata taaagccaag aaatcgaaat actttcaagt 2641 tacggtaagc atatgatagt ccattttaaa acataatttt aaaactgcaa actacccaag 2701 aaattattac tttctacgtc acgtattttg tactaatatc tttgtgttta cagtcaaatt 2761 aattccaatt atctctctaa cagccttgta tcgtatatgc aaatatgaag gaatcatggg 2821 aaataggccc tcttaattaa ttaacccgtg tcggctccag atctggcctc cgcgccgggt 2881 tttggcgcct cccgcgggcg cccccctcct cacggcgagc gctgccacgt cagacgaagg 2941 gcgcagcgag cgtcctgatc cttccgcccg gacgctcagg acagcggccc gctgctcata 3001 agactcggcc ttagaacccc agtatcagca gaaggacatt ttaggacggg acttgggtga 3061 ctctagggca ctggttttct ttccagagag cggaacaggc gaggaaaagt agtcccttct 3121 cggcgattct gcggagggat ctccgtgggg cggtgaacgc cgatgattat ataaggacgc 3181 gccgggtgtg gcacagctag ttccgtcgca gccgggattt gggtcgcggt tcttgtttgt 3241 ggatcgctgt gatcgtcact tggtgagtag cgggctgctg ggctggccgg ggctttcgtg 3301 gccgccgggc cgctcggtgg gacggaagcg tgtggagaga ccgccaaggg ctgtagtctg 3361 ggtccgcgag caaggttgcc ctgaactggg ggttgggggg agcgcagcaa aatggcggct 3421 gttcccgagt cttgaatgga agacgcttgt gaggcgggct gtgaggtcgt tgaaacaagg 3481 tggggggcat ggtgggcggc aagaacccaa ggtcttgagg ccttcgctaa tgcgggaaag 3541 ctcttattcg ggtgagatgg gctggggcac catctgggga ccctgacgtg aagtttgtca 3601 ctgactggag aactcggttt gtcgtctgtt gcgggggcgg cagttatggc ggtgccgttg 3661 ggcagtgcac ccgtaccttt gggagcgcgc gccctcgtcg tgtcgtgacg tcacccgttc 3721 tgttggctta taatgcaggg tggggccacc tgccggtagg tgtgcggtag gcttttctcc 3781 gtcgcaggac gcagggttcg ggcctagggt aggctctcct gaatcgacag gcgccggacc 3841 tctggtgagg ggagggataa gtgaggcgtc agtttctttg gtcggtttta tgtacctatc 3901 ttcttaagta gctgaagctc cggttttgaa ctatgcgctc ggggttggcg agtgtgtttt 3961 gtgaagtttt ttaggcacct tttgaaatgt aatcatttgg gtcaatatgt aattttcagt 4021 gttagactag taaattgtcc gctaaattct ggccgttttt ggcttttttg ttagacgaag 4081 cttgggctgc aggtcgactc tagagccacc atggactaca aagaccatga cggtgattat 4141 aaagatcatg acatcgatta caaggatgac gatgacaaga tggcccccaa gaagaagagg 4201 aaggtgggcc gcggaatgga caagaagtac tccattgggc tcgccatcgg cacaaacagc 4261 gtcggctggg ccgtcattac ggacgagtac aaggtgccga gcaaaaaatt caaagttctg 4321 ggcaataccg atcgccacag cataaagaag aacctcattg gcgccctcct gttcgactcc 4381 ggggaaaccg ccgaagccac gcggctcaaa agaacagcac ggcgcagata tacccgcaga 4441 aagaatcgga tctgctacct gcaggagatc tttagtaatg agatggctaa ggtggatgac 4501 tctttcttcc ataggctgga ggagtccttt ttggtggagg aggataaaaa gcacgagcgc 4561 cacccaatct ttggcaatat cgtggacgag gtggcgtacc atgaaaagta cccaaccata 4621 tatcatctga ggaagaagct tgtagacagt actgataagg ctgacttgcg gttgatctat 4681 ctcgcgctgg cgcatatgat caaatttcgg ggacacttcc tcatcgaggg ggacctgaac 4741 ccagacaaca gcgatgtcga caaactcttt atccaactgg ttcagactta caatcagctt 4801 ttcgaagaga acccgatcaa cgcatccgga gttgacgcca aagcaatcct gagcgctagg 4861 ctgtccaaat cccggcggct cgaaaacctc atcgcacagc tccctgggga gaagaagaac 4921 ggcctgtttg gtaatcttat cgccctgtca ctcgggctga cccccaactt taaatctaac 4981 ttcgacctgg ccgaagatgc caagcttcaa ctgagcaaag acacctacga tgatgatctc 5041 gacaatctgc tggcccagat cggcgaccag tacgcagacc tttttttggc ggcaaagaac 5101 ctgtcagacg ccattctgct gagtgatatt ctgcgagtga acacggagat caccaaagct 5161 ccgctgagcg ctagtatgat caagcgctat gatgagcacc accaagactt gactttgctg 5221 aaggcccttg tcagacagca actgcctgag aagtacaagg aaattttctt cgatcagtct 5281 aaaaatggct acgccggata cattgacggc ggagcaagcc aggaggaatt ttacaaattt 5341 attaagccca tcttggaaaa aatggacggc accgaggagc tgctggtaaa gcttaacaga 5401 gaagatctgt tgcgcaaaca gcgcactttc gacaatggaa gcatccccca ccagattcac 5461 ctgggcgaac tgcacgctat cctcaggcgg caagaggatt tctacccctt tttgaaagat 5521 aacagggaaa agattgagaa aatcctcaca tttcggatac cctactatgt aggccccctc 5581 gcccggggaa attccagatt cgcgtggatg actcgcaaat cagaagagac catcactccc 5641 tggaacttcg aggaagtcgt ggataagggg gcctctgccc agtccttcat cgaaaggatg 5701 actaactttg ataaaaatct gcctaacgaa aaggtgcttc ctaaacactc tctgctgtac 5761 gagtacttca cagtttataa cgagctcacc aaggtcaaat acgtcacaga agggatgaga 5821 aagccagcat tcctgtctgg agagcagaag aaagctatcg tggacctcct cttcaagacg 5881 aaccggaaag ttaccgtgaa acagctcaaa gaagactatt tcaaaaagat tgaatgtttc 5941 gactctgttg aaatcagcgg agtggaggat cgcttcaacg catccctggg aacgtatcac 6001 gatctcctga aaatcattaa agacaaggac ttcctggaca atgaggagaa cgaggacatt 6061 cttgaggaca ttgtcctcac ccttacgttg tttgaagata gggagatgat tgaagaacgc 6121 ttgaaaactt acgctcatct cttcgacgac aaagtcatga aacagctcaa gaggcgccga 6181 tatacaggat gggggcggct gtcaagaaaa ctgatcaatg ggatccgaga caagcagagt 6241 ggaaagacaa tcctggattt tcttaagtcc gatggatttg ccaaccggaa cttcatgcag 6301 ttgatccatg atgactctct cacctttaag gaggacatcc agaaagcaca agtttctggc 6361 cagggggaca gtcttcacga gcacatcgct aatcttgcag gtagcccagc tatcaaaaag 6421 ggaatactgc agaccgttaa ggtcgtggat gaactcgtca aagtaatggg aaggcataag 6481 cccgagaata tcgttatcga gatggcccga gagaaccaaa ctacccagaa gggacagaag 6541 aacagtaggg aaaggatgaa gaggattgaa gagggtataa aagaactggg gtcccaaatc 6601 cttaaggaac acccagttga aaacacccag cttcagaatg agaagctcta cctgtactac 6661 ctgcagaacg gcagggacat gtacgtggat caggaactgg acatcaatcg gctctccgac 6721 tacgacgtgg atgccatcgt gccccagtct tttctcaaag atgattctat tgataataaa 6781 gtgttgacaa gatccgataa aaatagaggg aagagtgata acgtcccctc agaagaagtt 6841 gtcaagaaaa tgaaaaatta ttggcggcag ctgctgaacg ccaaactgat cacacaacgg 6901 aagttcgata atctgactaa ggctgaacga ggtggcctgt ctgagttgga taaagccggc 6961 ttcatcaaaa ggcagcttgt tgagacacgc cagatcacca agcacgtggc ccaaattctc 7021 gattcacgca tgaacaccaa gtacgatgaa aatgacaaac tgattcgaga ggtgaaagtt 7081 attactctga agtctaagct ggtctcagat ttcagaaagg actttcagtt ttataaggtg 7141 agagagatca acaattacca ccatgcgcat gatgcctacc tgaatgcagt ggtaggcact 7201 gcacttatca aaaaatatcc caagcttgaa tctgaatttg tttacggaga ctataaagtg 7261 tacgatgtta ggaaaatgat cgcaaagtct gagcaggaaa taggcaaggc caccgctaag 7321 tacttctttt acagcaatat tatgaatttt ttcaagaccg agattacact ggccaatgga 7381 gagattcgga agcgaccact tatcgaaaca aacggagaaa caggagaaat cgtgtgggac 7441 aagggtaggg atttcgcgac agtccggaag gtcctgtcca tgccgcaggt gaacatcgtt 7501 aaaaagaccg aagtacagac cggaggcttc tccaaggaaa gtatcctccc gaaaaggaac 7561 agcgacaagc tgatcgcacg caaaaaagat tgggacccca agaaatacgg cggattcgat 7621 tctcctacag tcgcttacag tgtactggtt gtggccaaag tggagaaagg gaagtctaaa 7681 aaactcaaaa gcgtcaagga actgctgggc atcacaatca tggagcgatc aagcttcgaa 7741 aaaaacccca tcgactttct cgaggcgaaa ggatataaag aggtcaaaaa agacctcatc 7801 attaagcttc ccaagtactc tctctttgag cttgaaaacg gccggaaacg aatgctcgct 7861 agtgcgggcg agctgcagaa aggtaacgag ctggcactgc cctctaaata cgttaatttc 7921 ttgtatctgg ccagccacta tgaaaagctc aaagggtctc ccgaagataa tgagcagaag 7981 cagctgttcg tggaacaaca caaacactac cttgatgaga tcatcgagca aataagcgaa 8041 ttctccaaaa gagtgatcct cgccgacgct aacctcgata aggtgctttc tgcttacaat 8101 aagcacaggg ataagcccat cagggagcag gcagaaaaca ttatccactt gtttactctg 8161 accaacttgg gcgcgcctgc agccttcaag tacttcgaca ccaccataga cagaaagcgg 8221 tacacctcta caaaggaggt cctggacgcc acactgattc atcagtcaat tacggggctc 8281 tatgaaacaa gaatcgacct ctctcagctc ggtggagaca gcagggctga ccccaagaag 8341 aagaggaagg tggctagcga tgctaagtca ctgactgcct ggtcccggac actggtgacc 8401 ttcaaggatg tgtttgtgga cttcaccagg gaggagtgga agctgctgga cactgctcag 8461 cagatcctgt acagaaatgt gatgctggag aactataaga acctggtttc cttgggttat 8521 cagcttacta agccagatgt gatcctccgg ttggagaagg gagaagagcc ctggctggtg 8581 gagagagaaa ttcaccaaga gacccatcct gattcagaga ctgcatttga aatcaaatca 8641 tcagttccga aaaagaaacg caaagttgct agcgagggca gaggaagtct tctaacatgc 8701 ggtgacgtgg aggagaatcc cggccctggt accgtgagca agggcgagga gctgttcacc 8761 ggggtggtgc ccatcctggt cgagctggac ggcgacgtaa acggccacaa gttcagcgtg 8821 tccggcgagg gcgagggcga tgccacctac ggcaagctga ccctgaagtt catctgcacc 8881 accggcaagc tgcccgtgcc ctggcccacc ctcgtgacca ccctgaccta cggcgtgcag 8941 tgcttcagcc gctaccccga ccacatgaag cagcacgact tcttcaagtc cgccatgccc 9001 gaaggctacg tccaggagcg caccatcttc ttcaaggacg acggcaacta caagacccgc 9061 gccgaggtga agttcgaggg cgacaccctg gtgaaccgca tcgagctgaa gggcatcgac 9121 ttcaaggagg acggcaacat cctggggcac aagctggagt acaactacaa cagccacaac 9181 gtctatatca tggccgacaa gcagaagaac ggcatcaagg tgaacttcaa gatccgccac 9241 aacatcgagg acggcagcgt gcagctcgcc gaccactacc agcagaacac ccccatcggc 9301 gacggccccg tgctgctgcc cgacaaccac tacctgagca cccagtccgc cctgagcaaa 9361 gaccccaacg agaagcgcga tcacatggtc ctgctggagt tcgtgaccgc cgccgggatc 9421 actctcggca tggacgagct gtacaagacc ggttgataat agataacttc gtatagcata 9481 cattatacga agttatgaat tcgatatcaa gcttatcgat aatcaacctc tggattacaa 9541 aatttgtgaa agattgactg gtattcttaa ctatgttgct ccttttacgc tatgtggata 9601 cgctgcttta atgcctttgt atcatgctat tgcttcccgt atggctttca ttttctcctc 9661 cttgtataaa tcctggttgc tgtctcttta tgaggagttg tggcccgttg tcaggcaacg 9721 tggcgtggtg tgcactgtgt ttgctgacgc aacccccact ggttggggca ttgccaccac 9781 ctgtcagctc ctttccggga ctttcgcttt ccccctccct attgccacgg cggaactcat 9841 cgccgcctgc cttgcccgct gctggacagg ggctcggctg ttgggcactg acaattccgt 9901 ggtgttgtcg gggaaatcat cgtcctttcc ttggctgctc gcctgtgttg ccacctggat 9961 tctgcgcggg acgtccttct gctacgtccc ttcggccctc aatccagcgg accttccttc 10021 ccgcggcctg ctgccggctc tgcggcctct tccgcgtctt cgccttcgcc ctcagacgag 10081 tcggatctcc ctttgggccg cctccccgca tcgataccgt cgacctcgag acctagaaaa 10141 acatggagca atcacaagta gcaatacagc agctaccaat gctgattgtg cctggctaga 10201 agcacaagag gaggaggagg tgggttttcc agtcacacct caggtacctt taagaccaat 10261 gacttacaag gcagctgtag atcttagcca ctttttaaaa gaaaaggggg gactggaagg 10321 gctaattcac tcccaacgaa gacaagatat ccttgatctg tggatctacc acacacaagg 10381 ctacttccct gattggcaga actacacacc agggccaggg atcagatatc cactgacctt 10441 tggatggtgc tacaagctag taccagttga gcaagagaag gtagaagaag ccaatgaagg 10501 agagaacacc cgcttgttac accctgtgag cctgcatggg atggatgacc cggagagaga 10561 agtattagag tggaggtttg acagccgcct agcatttcat cacatggccc gagagctgca 10621 tccggactgt actgggtctc tctggttaga ccagatctga gcctgggagc tctctggcta 10681 actagggaac ccactgctta agcctcaata aagcttgcct tgagtgcttc aagtagtgtg 10741 tgcccgtctg ttgtgtgact ctggtaacta gagatccctc agaccctttt agtcagtgtg 10801 gaaaatctct agcagggccc gtttaaaccc gctgatcagc ctcgactgtg ccttctagtt 10861 gccagccatc tgttgtttgc ccctcccccg tgccttcctt gaccctggaa ggtgccactc 10921 ccactgtcct ttcctaataa aatgaggaaa ttgcatcgca ttgtctgagt aggtgtcatt 10981 ctattctggg gggtggggtg gggcaggaca gcaaggggga ggattgggaa gacaatagca 11041 ggcatgctgg ggatgcggtg ggctctatgg cttctgaggc ggaaagaacc agctggggct 11101 ctagggggta tccccacgcg ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta 11161 cgcgcagcgt gaccgctaca cttgccagcg ccctagcgcc cgctcctttc gctttcttcc 11221 cttcctttct cgccacgttc gccggctttc cccgtcaagc tctaaatcgg gggctccctt 11281 tagggttccg atttagtgct ttacggcacc tcgaccccaa aaaacttgat tagggtgatg 11341 gttcacgtag tgggccatcg ccctgataga cggtttttcg ccctttgacg ttggagtcca 11401 cgttctttaa tagtggactc ttgttccaaa ctggaacaac actcaaccct atctcggtct 11461 attcttttga tttataaggg attttgccga tttcggccta ttggttaaaa aatgagctga 11521 tttaacaaaa atttaacgcg aattaattct gtggaatgtg tgtcagttag ggtgtggaaa 11581 gtccccaggc tccccagcag gcagaagtat gcaaagcatg catctcaatt agtcagcaac 11641 caggtgtgga aagtccccag gctccccagc aggcagaagt atgcaaagca tgcatctcaa 11701 ttagtcagca accatagtcc cgcccctaac tccgcccatc ccgcccctaa ctccgcccag 11761 ttccgcccat tctccgcccc atggctgact aatttttttt atttatgcag aggccgaggc 11821 cgcctctgcc tctgagctat tccagaagta gtgaggaggc ttttttggag gcctaggctt 11881 ttgcaaaaag ctcccgggag cttgtatatc cattttcgga tctgatcagc acgtgttgac 11941 aattaatcat cggcatagta tatcggcata gtataatacg acaaggtgag gaactaaacc 12001 atggccaagt tgaccagtgc cgttccggtg ctcaccgcgc gcgacgtcgc cggagcggtc 12061 gagttctgga ccgaccggct cgggttctcc cgggacttcg tggaggacga cttcgccggt 12121 gtggtccggg acgacgtgac cctgttcatc agcgcggtcc aggaccaggt ggtgccggac 12181 aacaccctgg cctgggtgtg ggtgcgcggc ctggacgagc tgtacgccga gtggtcggag 12241 gtcgtgtcca cgaacttccg ggacgcctcc gggccggcca tgaccgagat cggcgagcag 12301 ccgtgggggc gggagttcgc cctgcgcgac ccggccggca actgcgtgca cttcgtggcc 12361 gaggagcagg actgacacgt gctacgagat ttcgattcca ccgccgcctt ctatgaaagg 12421 ttgggcttcg gaatcgtttt ccgggacgcc ggctggatga tcctccagcg cggggatctc 12481 atgctggagt tcttcgccca ccccaacttg tttattgcag cttataatgg ttacaaataa 12541 agcaatagca tcacaaattt cacaaataaa gcattttttt cactgcattc tagttgtggt 12601 ttgtccaaac tcatcaatgt atcttatcat gtctgtatac cgtcgacctc tagctagagc 12661 ttggcgtaat catggtcata gctgtttcct gtgtgaaatt gttatccgct cacaattcca 12721 cacaacatac gagccggaag cataaagtgt aaagcctggg gtgcctaatg agtgagctaa 12781 ctcacattaa ttgcgttgcg ctcactgccc gctttccagt cgggaaacct gtcgtgccag 12841 ctgcattaat gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg gcgctcttcc 12901 gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct 12961 cactcaaagg cggtaatacg gttatccaca gaatcagggg ataacgcagg aaagaacatg 13021 tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc 13081 cataggctcc gcccccctga cgagcatcac aaaaatcgac gctcaagtca gaggtggcga 13141 aacccgacag gactataaag ataccaggcg tttccccctg gaagctccct cgtgcgctct 13201 cctgttccga ccctgccgct taccggatac ctgtccgcct ttctcccttc gggaagcgtg 13261 gcgctttctc atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag 13321 ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct gcgccttatc cggtaactat 13381 cgtcttgagt ccaacccggt aagacacgac ttatcgccac tggcagcagc cactggtaac 13441 aggattagca gagcgaggta tgtaggcggt gctacagagt tcttgaagtg gtggcctaac 13501 tacggctaca ctagaagaac agtatttggt atctgcgctc tgctgaagcc agttaccttc 13561 ggaaaaagag ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggtggtttt 13621 tttgtttgca agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc 13681 ttttctacgg ggtctgacgc tcagtggaac gaaaactcac gttaagggat tttggtcatg 13741 agattatcaa aaaggatctt cacctagatc cttttaaatt aaaaatgaag ttttaaatca 13801 atctaaagta tatatgagta aacttggtct gacagttacc aatgcttaat cagtgaggca 13861 cctatctcag cgatctgtct atttcgttca tccatagttg cctgactccc cgtcgtgtag 13921 ataactacga tacgggaggg cttaccatct ggccccagtg ctgcaatgat accgcgagac 13981 ccacgctcac cggctccaga tttatcagca ataaaccagc cagccggaag ggccgagcgc 14041 agaagtggtc ctgcaacttt atccgcctcc atccagtcta ttaattgttg ccgggaagct 14101 agagtaagta gttcgccagt taatagtttg cgcaacgttg ttgccattgc tacaggcatc 14161 gtggtgtcac gctcgtcgtt tggtatggct tcattcagct ccggttccca acgatcaagg 14221 cgagttacat gatcccccat gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc 14281 gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg ttatggcagc actgcataat 14341 tctcttactg tcatgccatc cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag 14401 tcattctgag aatagtgtat gcggcgaccg agttgctctt gcccggcgtc aatacgggat 14461 aataccgcgc cacatagcag aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg 14521 cgaaaactct caaggatctt accgctgttg agatccagtt cgatgtaacc cactcgtgca 14581 cccaactgat cttcagcatc ttttactttc accagcgttt ctgggtgagc aaaaacagga 14641 aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat actcatactc 14701 ttcctttttc aatattattg aagcatttat cagggttatt gtctcatgag cggatacata 14761 tttgaatgta tttagaaaaa taaacaaata ggggttccgc gcacatttcc ccgaaaagtg 14821 ccacctgacg tcgacggatc gggagatctc ccgatcccct atggtgcact ctcagtacaa 14881 tctgctctga tgccgcatag ttaagccagt atctgctccc tgcttgtgtg ttggaggtcg 14941 ctgagtagtg cgcgagcaaa atttaagcta caacaaggca ag //