LOCUS Exported 15030 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION expresses VP64-dCas9-VP64, same plasmid as Addgene 59791 but with LoxP sites removed. ACCESSION . VERSION . KEYWORDS pLV hUbC- VP64-dCas9-VP64-T2A- GFP(No LoxP sites) SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 15030) AUTHORS Gersbach C TITLE Multiplex CRISPR/Cas9 Genome Engineering for Directing Myogenic Cellular Differentiation in Primary Human Cells JOURNAL Unpublished REFERENCE 2 (bases 1 to 15030) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..15030 /organism="synthetic DNA construct" /mol_type="other DNA" primer_bind complement(47..66) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" enhancer 238..617 /label=CMV enhancer /note="human cytomegalovirus immediate early enhancer" promoter 619..817 /label=CMV promoter /note="human cytomegalovirus (CMV) immediate early promoter" primer_bind 772..792 /label=CMV-F /note="Human CMV immediate early promoter, forward primer" LTR 835..1015 /label=5' LTR (truncated) /note="truncated 5' long terminal repeat (LTR) from HIV-1" misc_feature 1062..1187 /label=HIV-1 Psi /note="packaging signal of human immunodeficiency virus type 1" misc_feature 1680..1913 /label=RRE /note="The Rev response element (RRE) of HIV-1 allows for Rev-dependent mRNA export from the nucleus to the cytoplasm." CDS 2098..2142 /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 2440..2557 /label=cPPT/CTS /note="central polypurine tract and central termination sequence of HIV-1" CDS complement(2594..2914) /codon_start=1 /gene="lacZ fragment" /product="LacZ-alpha fragment of beta-galactosidase" /label=lacZ-alpha /translation="MTMITPSAQLTLTKGNKSWRTSRGGPVPNSPYSESYYARSLAVVL QRRDWENPGVTQLNRLAAHPPFASWRNSEEARTDRPSQQLRSLNGEWDAPCSGATTVSY LN" primer_bind 2764..2786 /label=M13/pUC Forward /note="In lacZ gene" primer_bind 2778..2795 /label=M13 Forward /note="In lacZ gene. Also called M13-F20 or M13 (-21) Forward" primer_bind 2779..2795 /label=M13 fwd /note="common sequencing primer, one of multiple similar variants" primer_bind 2805..2824 /label=T7 /note="T7 promoter, forward primer" promoter 2805..2823 /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" primer_bind complement(2871..2891) /label=T3 /note="T3 promoter, forward primer" promoter complement(2871..2889) /label=T3 promoter /note="promoter for bacteriophage T3 RNA polymerase" primer_bind complement(2910..2926) /label=M13 rev /note="common sequencing primer, one of multiple similar variants" protein_bind 2934..2950 /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(2958..2988) /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 3003..3024 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." promoter 3090..4300 /label=UbC promoter /note="human ubiquitin C promoter" primer_bind 4137..4156 /label=hUBCpro-F /note="Human Ubiquitin C (UbC) promoter, forward primer" regulatory 4329..4338 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" CDS 4338..4403 /codon_start=1 /product="three tandem FLAG(R) epitope tags, followed by an enterokinase cleavage site" /label=3xFLAG /translation="DYKDHDGDYKDHDIDYKDDDDK" CDS 4419..4565 /codon_start=1 /product="tetrameric repeat of the minimal activation domain of herpes simplex virus VP16 (Beerli et al., 1998)" /label=VP64 /translation="DALDDFDLDMLGSDALDDFDLDMLGSDALDDFDLDMLGSDALDDF DLDM" CDS 4572..4592 /codon_start=1 /product="nuclear localization signal of SV40 (simian virus 40) large T antigen" /label=SV40 NLS /translation="PKKKRKV" CDS 4602..8705 /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 8718..8738 /codon_start=1 /product="nuclear localization signal of SV40 (simian virus 40) large T antigen" /label=SV40 NLS /translation="PKKKRKV" CDS 8751..8900 /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 8910..8963 /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 8970..9683 /codon_start=1 /product="enhanced GFP" /label=EGFP /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE FVTAAGITLGMDELYK" CDS 8970..9683 /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(9012..9033) /label=EGFP-N /note="EGFP, reverse primer" primer_bind complement(9273..9292) /label=EXFP-R /note="For distinguishing EGFP variants, reverse primer" primer_bind 9620..9641 /label=EGFP-C /note="EGFP, forward primer" misc_feature 9722..10310 /label=WPRE /note="woodchuck hepatitis virus posttranscriptional regulatory element" primer_bind complement(9775..9795) /label=WPRE-R /note="WPRE, reverse primer" CDS complement(10193..10204) /codon_start=1 /product="Factor Xa recognition and cleavage site" /label=Factor Xa site /translation="IEGR" primer_bind complement(10313..10329) /label=KS primer /note="common sequencing primer, one of multiple similar variants" primer_bind complement(10314..10330) /label=pBluescriptKS /note="For pBluescript vector" LTR 10835..11015 /label=5' LTR (truncated) /note="truncated 5' long terminal repeat (LTR) from HIV-1" primer_bind complement(11041..11058) /label=BGH-rev /note="Bovine growth hormone terminator, reverse primer. Also called BGH reverse" polyA_signal 11047..11271 /label=bGH poly(A) signal /note="bovine growth hormone polyadenylation signal" rep_origin 11317..11745 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(11404..11423) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 11614..11635 /label=F1ori-F /note="F1 origin, forward primer" primer_bind complement(11754..11774) /label=pBABE 3' /note="SV40 enhancer, reverse primer for pBABE vectors" promoter 11759..12088 /label=SV40 promoter /note="SV40 enhancer and early promoter" rep_origin 11939..12074 /label=SV40 ori /note="SV40 origin of replication" primer_bind 12001..12020 /label=SV40pro-F /note="SV40 promoter/origin, forward primer" promoter 12136..12183 /label=EM7 promoter /note="synthetic bacterial promoter " CDS 12202..12576 /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 12706..12827 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(12743..12762) /label=SV40pA-R /note="SV40 polyA, reverse primer" primer_bind 12797..12816 /label=EBV-rev /note="SV40 polyA terminator, reverse primer" primer_bind complement(12876..12892) /label=M13 rev /note="common sequencing primer, one of multiple similar variants" protein_bind 12900..12916 /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(12924..12954) /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 12969..12990 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." primer_bind complement(13107..13124) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(13278..13866) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(13358..13377) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS complement(14037..14897) /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 14660..14679 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(14898..15002) /gene="bla" /label=AmpR promoter ORIGIN 1 gtcgacggat cgggagatct cccgatcccc tatggtgcac tctcagtaca atctgctctg 61 atgccgcata gttaagccag tatctgctcc ctgcttgtgt gttggaggtc gctgagtagt 121 gcgcgagcaa aatttaagct acaacaaggc aaggcttgac cgacaattgc atgaagaatc 181 tgcttagggt taggcgtttt gcgctgcttc gcgatgtacg ggccagatat acgcgttgac 241 attgattatt gactagttat taatagtaat caattacggg gtcattagtt catagcccat 301 atatggagtt ccgcgttaca taacttacgg taaatggccc gcctggctga ccgcccaacg 361 acccccgccc attgacgtca ataatgacgt atgttcccat agtaacgcca atagggactt 421 tccattgacg tcaatgggtg gagtatttac ggtaaactgc ccacttggca gtacatcaag 481 tgtatcatat gccaagtacg ccccctattg acgtcaatga cggtaaatgg cccgcctggc 541 attatgccca gtacatgacc ttatgggact ttcctacttg gcagtacatc tacgtattag 601 tcatcgctat taccatggtg atgcggtttt ggcagtacat caatgggcgt ggatagcggt 661 ttgactcacg gggatttcca agtctccacc ccattgacgt caatgggagt ttgttttggc 721 accaaaatca acgggacttt ccaaaatgtc gtaacaactc cgccccattg acgcaaatgg 781 gcggtaggcg tgtacggtgg gaggtctata taagcagcgc gttttgcctg tactgggtct 841 ctctggttag accagatctg agcctgggag ctctctggct aactagggaa cccactgctt 901 aagcctcaat aaagcttgcc ttgagtgctt caagtagtgt gtgcccgtct gttgtgtgac 961 tctggtaact agagatccct cagacccttt tagtcagtgt ggaaaatctc tagcagtggc 1021 gcccgaacag ggacttgaaa gcgaaaggga aaccagagga gctctctcga cgcaggactc 1081 ggcttgctga agcgcgcacg gcaagaggcg aggggcggcg actggtgagt acgccaaaaa 1141 ttttgactag cggaggctag aaggagagag atgggtgcga gagcgtcagt attaagcggg 1201 ggagaattag atcgcgatgg gaaaaaattc ggttaaggcc agggggaaag aaaaaatata 1261 aattaaaaca tatagtatgg gcaagcaggg agctagaacg attcgcagtt aatcctggcc 1321 tgttagaaac atcagaaggc tgtagacaaa tactgggaca gctacaacca tcccttcaga 1381 caggatcaga agaacttaga tcattatata atacagtagc aaccctctat tgtgtgcatc 1441 aaaggataga gataaaagac accaaggaag ctttagacaa gatagaggaa gagcaaaaca 1501 aaagtaagac caccgcacag caagcggccg ctgatcttca gacctggagg aggagatatg 1561 agggacaatt ggagaagtga attatataaa tataaagtag taaaaattga accattagga 1621 gtagcaccca ccaaggcaaa gagaagagtg gtgcagagag aaaaaagagc agtgggaata 1681 ggagctttgt tccttgggtt cttgggagca gcaggaagca ctatgggcgc agcgtcaatg 1741 acgctgacgg tacaggccag acaattattg tctggtatag tgcagcagca gaacaatttg 1801 ctgagggcta ttgaggcgca acagcatctg ttgcaactca cagtctgggg catcaagcag 1861 ctccaggcaa gaatcctggc tgtggaaaga tacctaaagg atcaacagct cctggggatt 1921 tggggttgct ctggaaaact catttgcacc actgctgtgc cttggaatgc tagttggagt 1981 aataaatctc tggaacagat ttggaatcac acgacctgga tggagtggga cagagaaatt 2041 aacaattaca caagcttaat acactcctta attgaagaat cgcaaaacca gcaagaaaag 2101 aatgaacaag aattattgga attagataaa tgggcaagtt tgtggaattg gtttaacata 2161 acaaattggc tgtggtatat aaaattattc ataatgatag taggaggctt ggtaggttta 2221 agaatagttt ttgctgtact ttctatagtg aatagagtta ggcagggata ttcaccatta 2281 tcgtttcaga cccacctccc aaccccgagg ggacccgaca ggcccgaagg aatagaagaa 2341 gaaggtggag agagagacag agacagatcc attcgattag tgaacggatc ggcactgcgt 2401 gcgccaattc tgcagacaaa tggcagtatt catccacaat tttaaaagaa aaggggggat 2461 tggggggtac agtgcagggg aaagaatagt agacataata gcaacagaca tacaaactaa 2521 agaattacaa aaacaaatta caaaaattca aaattttcgg gtttattaca gggacagcag 2581 agatccagtt tggttaatta agataagaga cggtggtggc gccgctacag ggcgcgtccc 2641 attcgccatt caggctgcgc aactgttggg aagggcgatc ggtgcgggcc tcttcgctat 2701 tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta acgccagggt 2761 tttcccagtc acgacgttgt aaaacgacgg ccagtgagcg cgcgtaatac gactcactat 2821 agggcgaatt gggtaccggg ccccccctcg aggtcctcca gcttttgttc cctttagtga 2881 gggttaattg cgcgcttggc gtaatcatgg tcatagctgt ttcctgtgtg aaattgttat 2941 ccgctcacaa ttccacacaa catacgagcc ggaagcataa agtgtaaagc ctggggtgcc 3001 taatgagtga gctaactcac attaattgcg ttgcgctcac tgcccgcttt ccactgcatg 3061 acgtctccac aattaattaa gggtgcagcg gcctccgcgc cgggttttgg cgcctcccgc 3121 gggcgccccc ctcctcacgg cgagcgctgc cacgtcagac gaagggcgca ggagcgttcc 3181 tgatccttcc gcccggacgc tcaggacagc ggcccgctgc tcataagact cggccttaga 3241 accccagtat cagcagaagg acattttagg acgggacttg ggtgactcta gggcactggt 3301 tttctttcca gagagcggaa caggcgagga aaagtagtcc cttctcggcg attctgcgga 3361 gggatctccg tggggcggtg aacgccgatg attatataag gacgcgccgg gtgtggcaca 3421 gctagttccg tcgcagccgg gatttgggtc gcggttcttg tttgtggatc gctgtgatcg 3481 tcacttggtg agttgcgggc tgctgggctg gccggggctt tcgtggccgc cgggccgctc 3541 ggtgggacgg aagcgtgtgg agagaccgcc aagggctgta gtctgggtcc gcgagcaagg 3601 ttgccctgaa ctgggggttg gggggagcgc acaaaatggc ggctgttccc gagtcttgaa 3661 tggaagacgc ttgtaaggcg ggctgtgagg tcgttgaaac aaggtggggg gcatggtggg 3721 cggcaagaac ccaaggtctt gaggccttcg ctaatgcggg aaagctctta ttcgggtgag 3781 atgggctggg gcaccatctg gggaccctga cgtgaagttt gtcactgact ggagaactcg 3841 ggtttgtcgt ctggttgcgg gggcggcagt tatgcggtgc cgttgggcag tgcacccgta 3901 cctttgggag cgcgcgcctc gtcgtgtcgt gacgtcaccc gttctgttgg cttataatgc 3961 agggtggggc cacctgccgg taggtgtgcg gtaggctttt ctccgtcgca ggacgcaggg 4021 ttcgggccta gggtaggctc tcctgaatcg acaggcgccg gacctctggt gaggggaggg 4081 ataagtgagg cgtcagtttc tttggtcggt tttatgtacc tatcttctta agtagctgaa 4141 gctccggttt tgaactatgc gctcggggtt ggcgagtgtg ttttgtgaag ttttttaggc 4201 accttttgaa atgtaatcat ttgggtcaat atgtaatttt cagtgttaga ctagtaaatt 4261 gtccgctaaa ttctggccgt ttttggcttt tttgttagac gaagcttggg ctgcaggtcg 4321 actctagagc caccatggac tacaaagacc atgacggtga ttataaagat catgacatcg 4381 attacaagga tgacgatgac aagcacggtc cgcgggctga cgcattggac gattttgatc 4441 tggatatgct gggaagtgac gccctcgatg attttgacct tgacatgctt ggttcggatg 4501 cccttgatga ctttgacctc gacatgctcg gcagtgacgc ccttgatgat ttcgacctgg 4561 acatggttaa ccccaagaag aagaggaagg tgggccgcgg aatggacaag aagtactcca 4621 ttgggctcgc catcggcaca aacagcgtcg gctgggccgt cattacggac gagtacaagg 4681 tgccgagcaa aaaattcaaa gttctgggca ataccgatcg ccacagcata aagaagaacc 4741 tcattggcgc cctcctgttc gactccgggg aaaccgccga agccacgcgg ctcaaaagaa 4801 cagcacggcg cagatatacc cgcagaaaga atcggatctg ctacctgcag gagatcttta 4861 gtaatgagat ggctaaggtg gatgactctt tcttccatag gctggaggag tcctttttgg 4921 tggaggagga taaaaagcac gagcgccacc caatctttgg caatatcgtg gacgaggtgg 4981 cgtaccatga aaagtaccca accatatatc atctgaggaa gaagcttgta gacagtactg 5041 ataaggctga cttgcggttg atctatctcg cgctggcgca tatgatcaaa tttcggggac 5101 acttcctcat cgagggggac ctgaacccag acaacagcga tgtcgacaaa ctctttatcc 5161 aactggttca gacttacaat cagcttttcg aagagaaccc gatcaacgca tccggagttg 5221 acgccaaagc aatcctgagc gctaggctgt ccaaatcccg gcggctcgaa aacctcatcg 5281 cacagctccc tggggagaag aagaacggcc tgtttggtaa tcttatcgcc ctgtcactcg 5341 ggctgacccc caactttaaa tctaacttcg acctggccga agatgccaag cttcaactga 5401 gcaaagacac ctacgatgat gatctcgaca atctgctggc ccagatcggc gaccagtacg 5461 cagacctttt tttggcggca aagaacctgt cagacgccat tctgctgagt gatattctgc 5521 gagtgaacac ggagatcacc aaagctccgc tgagcgctag tatgatcaag cgctatgatg 5581 agcaccacca agacttgact ttgctgaagg cccttgtcag acagcaactg cctgagaagt 5641 acaaggaaat tttcttcgat cagtctaaaa atggctacgc cggatacatt gacggcggag 5701 caagccagga ggaattttac aaatttatta agcccatctt ggaaaaaatg gacggcaccg 5761 aggagctgct ggtaaagctt aacagagaag atctgttgcg caaacagcgc actttcgaca 5821 atggaagcat cccccaccag attcacctgg gcgaactgca cgctatcctc aggcggcaag 5881 aggatttcta cccctttttg aaagataaca gggaaaagat tgagaaaatc ctcacatttc 5941 ggatacccta ctatgtaggc cccctcgccc ggggaaattc cagattcgcg tggatgactc 6001 gcaaatcaga agagaccatc actccctgga acttcgagga agtcgtggat aagggggcct 6061 ctgcccagtc cttcatcgaa aggatgacta actttgataa aaatctgcct aacgaaaagg 6121 tgcttcctaa acactctctg ctgtacgagt acttcacagt ttataacgag ctcaccaagg 6181 tcaaatacgt cacagaaggg atgagaaagc cagcattcct gtctggagag cagaagaaag 6241 ctatcgtgga cctcctcttc aagacgaacc ggaaagttac cgtgaaacag ctcaaagaag 6301 actatttcaa aaagattgaa tgtttcgact ctgttgaaat cagcggagtg gaggatcgct 6361 tcaacgcatc cctgggaacg tatcacgatc tcctgaaaat cattaaagac aaggacttcc 6421 tggacaatga ggagaacgag gacattcttg aggacattgt cctcaccctt acgttgtttg 6481 aagataggga gatgattgaa gaacgcttga aaacttacgc tcatctcttc gacgacaaag 6541 tcatgaaaca gctcaagagg cgccgatata caggatgggg gcggctgtca agaaaactga 6601 tcaatgggat ccgagacaag cagagtggaa agacaatcct ggattttctt aagtccgatg 6661 gatttgccaa ccggaacttc atgcagttga tccatgatga ctctctcacc tttaaggagg 6721 acatccagaa agcacaagtt tctggccagg gggacagtct tcacgagcac atcgctaatc 6781 ttgcaggtag cccagctatc aaaaagggaa tactgcagac cgttaaggtc gtggatgaac 6841 tcgtcaaagt aatgggaagg cataagcccg agaatatcgt tatcgagatg gcccgagaga 6901 accaaactac ccagaaggga cagaagaaca gtagggaaag gatgaagagg attgaagagg 6961 gtataaaaga actggggtcc caaatcctta aggaacaccc agttgaaaac acccagcttc 7021 agaatgagaa gctctacctg tactacctgc agaacggcag ggacatgtac gtggatcagg 7081 aactggacat caatcggctc tccgactacg acgtggatgc catcgtgccc cagtcttttc 7141 tcaaagatga ttctattgat aataaagtgt tgacaagatc cgataaaaat agagggaaga 7201 gtgataacgt cccctcagaa gaagttgtca agaaaatgaa aaattattgg cggcagctgc 7261 tgaacgccaa actgatcaca caacggaagt tcgataatct gactaaggct gaacgaggtg 7321 gcctgtctga gttggataaa gccggcttca tcaaaaggca gcttgttgag acacgccaga 7381 tcaccaagca cgtggcccaa attctcgatt cacgcatgaa caccaagtac gatgaaaatg 7441 acaaactgat tcgagaggtg aaagttatta ctctgaagtc taagctggtc tcagatttca 7501 gaaaggactt tcagttttat aaggtgagag agatcaacaa ttaccaccat gcgcatgatg 7561 cctacctgaa tgcagtggta ggcactgcac ttatcaaaaa atatcccaag cttgaatctg 7621 aatttgttta cggagactat aaagtgtacg atgttaggaa aatgatcgca aagtctgagc 7681 aggaaatagg caaggccacc gctaagtact tcttttacag caatattatg aattttttca 7741 agaccgagat tacactggcc aatggagaga ttcggaagcg accacttatc gaaacaaacg 7801 gagaaacagg agaaatcgtg tgggacaagg gtagggattt cgcgacagtc cggaaggtcc 7861 tgtccatgcc gcaggtgaac atcgttaaaa agaccgaagt acagaccgga ggcttctcca 7921 aggaaagtat cctcccgaaa aggaacagcg acaagctgat cgcacgcaaa aaagattggg 7981 accccaagaa atacggcgga ttcgattctc ctacagtcgc ttacagtgta ctggttgtgg 8041 ccaaagtgga gaaagggaag tctaaaaaac tcaaaagcgt caaggaactg ctgggcatca 8101 caatcatgga gcgatcaagc ttcgaaaaaa accccatcga ctttctcgag gcgaaaggat 8161 ataaagaggt caaaaaagac ctcatcatta agcttcccaa gtactctctc tttgagcttg 8221 aaaacggccg gaaacgaatg ctcgctagtg cgggcgagct gcagaaaggt aacgagctgg 8281 cactgccctc taaatacgtt aatttcttgt atctggccag ccactatgaa aagctcaaag 8341 ggtctcccga agataatgag cagaagcagc tgttcgtgga acaacacaaa cactaccttg 8401 atgagatcat cgagcaaata agcgaattct ccaaaagagt gatcctcgcc gacgctaacc 8461 tcgataaggt gctttctgct tacaataagc acagggataa gcccatcagg gagcaggcag 8521 aaaacattat ccacttgttt actctgacca acttgggcgc gcctgcagcc ttcaagtact 8581 tcgacaccac catagacaga aagcggtaca cctctacaaa ggaggtcctg gacgccacac 8641 tgattcatca gtcaattacg gggctctatg aaacaagaat cgacctctct cagctcggtg 8701 gagacagcag ggctgacccc aagaagaaga ggaaggtggc tagccgcgcc gacgcgctgg 8761 acgatttcga tctcgacatg ctgggttctg atgccctcga tgactttgac ctggatatgt 8821 tgggaagcga cgcattggat gactttgatc tggacatgct cggctccgat gctctggacg 8881 atttcgatct cgatatgtta atcgctagcg agggcagagg aagtcttcta acatgcggtg 8941 acgtggagga gaatcccggc cctggtaccg tgagcaaggg cgaggagctg ttcaccgggg 9001 tggtgcccat cctggtcgag ctggacggcg acgtaaacgg ccacaagttc agcgtgtccg 9061 gcgagggcga gggcgatgcc acctacggca agctgaccct gaagttcatc tgcaccaccg 9121 gcaagctgcc cgtgccctgg cccaccctcg tgaccaccct gacctacggc gtgcagtgct 9181 tcagccgcta ccccgaccac atgaagcagc acgacttctt caagtccgcc atgcccgaag 9241 gctacgtcca ggagcgcacc atcttcttca aggacgacgg caactacaag acccgcgccg 9301 aggtgaagtt cgagggcgac accctggtga accgcatcga gctgaagggc atcgacttca 9361 aggaggacgg caacatcctg gggcacaagc tggagtacaa ctacaacagc cacaacgtct 9421 atatcatggc cgacaagcag aagaacggca tcaaggtgaa cttcaagatc cgccacaaca 9481 tcgaggacgg cagcgtgcag ctcgccgacc actaccagca gaacaccccc atcggcgacg 9541 gccccgtgct gctgcccgac aaccactacc tgagcaccca gtccgccctg agcaaagacc 9601 ccaacgagaa gcgcgatcac atggtcctgc tggagttcgt gaccgccgcc gggatcactc 9661 tcggcatgga cgagctgtac aagaccggtt gataatagaa ttcgatatca agcttatcga 9721 taatcaacct ctggattaca aaatttgtga aagattgact ggtattctta actatgttgc 9781 tccttttacg ctatgtggat acgctgcttt aatgcctttg tatcatgcta ttgcttcccg 9841 tatggctttc attttctcct ccttgtataa atcctggttg ctgtctcttt atgaggagtt 9901 gtggcccgtt gtcaggcaac gtggcgtggt gtgcactgtg tttgctgacg caacccccac 9961 tggttggggc attgccacca cctgtcagct cctttccggg actttcgctt tccccctccc 10021 tattgccacg gcggaactca tcgccgcctg ccttgcccgc tgctggacag gggctcggct 10081 gttgggcact gacaattccg tggtgttgtc ggggaaatca tcgtcctttc cttggctgct 10141 cgcctgtgtt gccacctgga ttctgcgcgg gacgtccttc tgctacgtcc cttcggccct 10201 caatccagcg gaccttcctt cccgcggcct gctgccggct ctgcggcctc ttccgcgtct 10261 tcgccttcgc cctcagacga gtcggatctc cctttgggcc gcctccccgc atcgataccg 10321 tcgacctcga gacctagaaa aacatggagc aatcacaagt agcaatacag cagctaccaa 10381 tgctgattgt gcctggctag aagcacaaga ggaggaggag gtgggttttc cagtcacacc 10441 tcaggtacct ttaagaccaa tgacttacaa ggcagctgta gatcttagcc actttttaaa 10501 agaaaagggg ggactggaag ggctaattca ctcccaacga agacaagata tccttgatct 10561 gtggatctac cacacacaag gctacttccc tgattggcag aactacacac cagggccagg 10621 gatcagatat ccactgacct ttggatggtg ctacaagcta gtaccagttg agcaagagaa 10681 ggtagaagaa gccaatgaag gagagaacac ccgcttgtta caccctgtga gcctgcatgg 10741 gatggatgac ccggagagag aagtattaga gtggaggttt gacagccgcc tagcatttca 10801 tcacatggcc cgagagctgc atccggactg tactgggtct ctctggttag accagatctg 10861 agcctgggag ctctctggct aactagggaa cccactgctt aagcctcaat aaagcttgcc 10921 ttgagtgctt caagtagtgt gtgcccgtct gttgtgtgac tctggtaact agagatccct 10981 cagacccttt tagtcagtgt ggaaaatctc tagcagggcc cgtttaaacc cgctgatcag 11041 cctcgactgt gccttctagt tgccagccat ctgttgtttg cccctccccc gtgccttcct 11101 tgaccctgga aggtgccact cccactgtcc tttcctaata aaatgaggaa attgcatcgc 11161 attgtctgag taggtgtcat tctattctgg ggggtggggt ggggcaggac agcaaggggg 11221 aggattggga agacaatagc aggcatgctg gggatgcggt gggctctatg gcttctgagg 11281 cggaaagaac cagctggggc tctagggggt atccccacgc gccctgtagc ggcgcattaa 11341 gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac acttgccagc gccctagcgc 11401 ccgctccttt cgctttcttc ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag 11461 ctctaaatcg ggggctccct ttagggttcc gatttagtgc tttacggcac ctcgacccca 11521 aaaaacttga ttagggtgat ggttcacgta gtgggccatc gccctgatag acggtttttc 11581 gccctttgac gttggagtcc acgttcttta atagtggact cttgttccaa actggaacaa 11641 cactcaaccc tatctcggtc tattcttttg atttataagg gattttgccg atttcggcct 11701 attggttaaa aaatgagctg atttaacaaa aatttaacgc gaattaattc tgtggaatgt 11761 gtgtcagtta gggtgtggaa agtccccagg ctccccagca ggcagaagta tgcaaagcat 11821 gcatctcaat tagtcagcaa ccaggtgtgg aaagtcccca ggctccccag caggcagaag 11881 tatgcaaagc atgcatctca attagtcagc aaccatagtc ccgcccctaa ctccgcccat 11941 cccgccccta actccgccca gttccgccca ttctccgccc catggctgac taattttttt 12001 tatttatgca gaggccgagg ccgcctctgc ctctgagcta ttccagaagt agtgaggagg 12061 cttttttgga ggcctaggct tttgcaaaaa gctcccggga gcttgtatat ccattttcgg 12121 atctgatcag cacgtgttga caattaatca tcggcatagt atatcggcat agtataatac 12181 gacaaggtga ggaactaaac catggccaag ttgaccagtg ccgttccggt gctcaccgcg 12241 cgcgacgtcg ccggagcggt cgagttctgg accgaccggc tcgggttctc ccgggacttc 12301 gtggaggacg acttcgccgg tgtggtccgg gacgacgtga ccctgttcat cagcgcggtc 12361 caggaccagg tggtgccgga caacaccctg gcctgggtgt gggtgcgcgg cctggacgag 12421 ctgtacgccg agtggtcgga ggtcgtgtcc acgaacttcc gggacgcctc cgggccggcc 12481 atgaccgaga tcggcgagca gccgtggggg cgggagttcg ccctgcgcga cccggccggc 12541 aactgcgtgc acttcgtggc cgaggagcag gactgacacg tgctacgaga tttcgattcc 12601 accgccgcct tctatgaaag gttgggcttc ggaatcgttt tccgggacgc cggctggatg 12661 atcctccagc gcggggatct catgctggag ttcttcgccc accccaactt gtttattgca 12721 gcttataatg gttacaaata aagcaatagc atcacaaatt tcacaaataa agcatttttt 12781 tcactgcatt ctagttgtgg tttgtccaaa ctcatcaatg tatcttatca tgtctgtata 12841 ccgtcgacct ctagctagag cttggcgtaa tcatggtcat agctgtttcc tgtgtgaaat 12901 tgttatccgc tcacaattcc acacaacata cgagccggaa gcataaagtg taaagcctgg 12961 ggtgcctaat gagtgagcta actcacatta attgcgttgc gctcactgcc cgctttccag 13021 tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt 13081 ttgcgtattg ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg 13141 ctgcggcgag cggtatcagc tcactcaaag gcggtaatac ggttatccac agaatcaggg 13201 gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag 13261 gccgcgttgc tggcgttttt ccataggctc cgcccccctg acgagcatca caaaaatcga 13321 cgctcaagtc agaggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct 13381 ggaagctccc tcgtgcgctc tcctgttccg accctgccgc ttaccggata cctgtccgcc 13441 tttctccctt cgggaagcgt ggcgctttct catagctcac gctgtaggta tctcagttcg 13501 gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac cccccgttca gcccgaccgc 13561 tgcgccttat ccggtaacta tcgtcttgag tccaacccgg taagacacga cttatcgcca 13621 ctggcagcag ccactggtaa caggattagc agagcgaggt atgtaggcgg tgctacagag 13681 ttcttgaagt ggtggcctaa ctacggctac actagaagaa cagtatttgg tatctgcgct 13741 ctgctgaagc cagttacctt cggaaaaaga gttggtagct cttgatccgg caaacaaacc 13801 accgctggta gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga 13861 tctcaagaag atcctttgat cttttctacg gggtctgacg ctcagtggaa cgaaaactca 13921 cgttaaggga ttttggtcat gagattatca aaaaggatct tcacctagat ccttttaaat 13981 taaaaatgaa gttttaaatc aatctaaagt atatatgagt aaacttggtc tgacagttac 14041 caatgcttaa tcagtgaggc acctatctca gcgatctgtc tatttcgttc atccatagtt 14101 gcctgactcc ccgtcgtgta gataactacg atacgggagg gcttaccatc tggccccagt 14161 gctgcaatga taccgcgaga cccacgctca ccggctccag atttatcagc aataaaccag 14221 ccagccggaa gggccgagcg cagaagtggt cctgcaactt tatccgcctc catccagtct 14281 attaattgtt gccgggaagc tagagtaagt agttcgccag ttaatagttt gcgcaacgtt 14341 gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc 14401 tccggttccc aacgatcaag gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt 14461 agctccttcg gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg 14521 gttatggcag cactgcataa ttctcttact gtcatgccat ccgtaagatg cttttctgtg 14581 actggtgagt actcaaccaa gtcattctga gaatagtgta tgcggcgacc gagttgctct 14641 tgcccggcgt caatacggga taataccgcg ccacatagca gaactttaaa agtgctcatc 14701 attggaaaac gttcttcggg gcgaaaactc tcaaggatct taccgctgtt gagatccagt 14761 tcgatgtaac ccactcgtgc acccaactga tcttcagcat cttttacttt caccagcgtt 14821 tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg 14881 aaatgttgaa tactcatact cttccttttt caatattatt gaagcattta tcagggttat 14941 tgtctcatga gcggatacat atttgaatgt atttagaaaa ataaacaaat aggggttccg 15001 cgcacatttc cccgaaaagt gccacctgac //