LOCUS Exported 14993 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION expresses a single gRNA and VP64-dCas9-VP64. ACCESSION . VERSION . KEYWORDS pLV hU6-gRNA(anti-sense) hUbC-VP64-dCas9-VP64-T2A-GFP SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 14993) 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Multiplex CRISPR/Cas9 Genome Engineering for Directing Myogenic Cellular Differentiation in Primary Human Cells JOURNAL Unpublished REFERENCE 2 (bases 1 to 14993) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..14993 /organism="synthetic DNA construct" /mol_type="other DNA" enhancer 1..380 /label=CMV enhancer /note="human cytomegalovirus immediate early enhancer" promoter 382..580 /label=CMV promoter /note="human cytomegalovirus (CMV) immediate early promoter" primer_bind 535..555 /label=CMV-F /note="Human CMV immediate early promoter, forward primer" LTR 598..778 /label=5' LTR (truncated) /note="truncated 5' long terminal repeat (LTR) from HIV-1" misc_feature 825..950 /label=HIV-1 Psi /note="packaging signal of human immunodeficiency virus type 1" misc_feature 1443..1676 /label=RRE /note="The Rev response element (RRE) of HIV-1 allows for Rev-dependent mRNA export from the nucleus to the cytoplasm." CDS 1861..1905 /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 2203..2320 /label=cPPT/CTS /note="central polypurine tract and central termination sequence of HIV-1" protein_bind 2364..2397 /label=loxP /bound_moiety="Cre recombinase" /note="Cre-mediated recombination occurs in the 8-bp core sequence (GCATACAT)." misc_RNA complement(2408..2483) /label=gRNA scaffold /note="guide RNA scaffold for the Streptococcus pyogenes CRISPR/Cas9 system" promoter complement(2508..2748) /label=U6 promoter /note="RNA polymerase III promoter for human U6 snRNA" primer_bind complement(2558..2577) /label=LKO.1 5' /note="Human U6 promoter, forward primer" primer_bind complement(2728..2748) /label=hU6-F /note="Human U6 promoter, forward primer" promoter 2780..3991 /label=UbC promoter /note="human ubiquitin C promoter" primer_bind 3828..3847 /label=hUBCpro-F /note="Human Ubiquitin C (UbC) promoter, forward primer" regulatory 4020..4029 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" CDS 4029..4094 /codon_start=1 /product="three tandem FLAG(R) epitope tags, followed by an enterokinase cleavage site" /label=3xFLAG /translation="DYKDHDGDYKDHDIDYKDDDDK" CDS 4110..4256 /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 4263..4283 /codon_start=1 /product="nuclear localization signal of SV40 (simian virus 40) large T antigen" /label=SV40 NLS /translation="PKKKRKV" CDS 4293..8396 /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 8409..8429 /codon_start=1 /product="nuclear localization signal of SV40 (simian virus 40) large T antigen" /label=SV40 NLS /translation="PKKKRKV" CDS 8442..8591 /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 8601..8654 /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 8661..9374 /codon_start=1 /product="enhanced GFP" /label=EGFP /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE FVTAAGITLGMDELYK" CDS 8661..9374 /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(8703..8724) /label=EGFP-N /note="EGFP, reverse primer" primer_bind complement(8964..8983) /label=EXFP-R /note="For distinguishing EGFP variants, reverse primer" primer_bind 9311..9332 /label=EGFP-C /note="EGFP, forward primer" protein_bind 9390..9423 /label=loxP /bound_moiety="Cre recombinase" /note="Cre-mediated recombination occurs in the 8-bp core sequence (GCATACAT)." misc_feature 9448..10036 /label=WPRE /note="woodchuck hepatitis virus posttranscriptional regulatory element" primer_bind complement(9501..9521) /label=WPRE-R /note="WPRE, reverse primer" CDS complement(9919..9930) /codon_start=1 /product="Factor Xa recognition and cleavage site" /label=Factor Xa site /translation="IEGR" primer_bind complement(10039..10055) /label=KS primer /note="common sequencing primer, one of multiple similar variants" primer_bind complement(10040..10056) /label=pBluescriptKS /note="For pBluescript vector" LTR 10561..10741 /label=5' LTR (truncated) /note="truncated 5' long terminal repeat (LTR) from HIV-1" primer_bind complement(10767..10784) /label=BGH-rev /note="Bovine growth hormone terminator, reverse primer. Also called BGH reverse" polyA_signal 10773..10997 /label=bGH poly(A) signal /note="bovine growth hormone polyadenylation signal" rep_origin 11043..11471 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(11130..11149) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 11340..11361 /label=F1ori-F /note="F1 origin, forward primer" primer_bind complement(11480..11500) /label=pBABE 3' /note="SV40 enhancer, reverse primer for pBABE vectors" promoter 11485..11814 /label=SV40 promoter /note="SV40 enhancer and early promoter" rep_origin 11665..11800 /label=SV40 ori /note="SV40 origin of replication" primer_bind 11727..11746 /label=SV40pro-F /note="SV40 promoter/origin, forward primer" promoter 11862..11909 /label=EM7 promoter /note="synthetic bacterial promoter " CDS 11928..12302 /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 12432..12553 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(12469..12488) /label=SV40pA-R /note="SV40 polyA, reverse primer" primer_bind 12523..12542 /label=EBV-rev /note="SV40 polyA terminator, reverse primer" primer_bind complement(12602..12618) /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind complement(12602..12618) /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" primer_bind complement(12615..12637) /label=M13/pUC Reverse /note="In lacZ gene" protein_bind 12626..12642 /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(12650..12680) /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 12695..12716 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." primer_bind complement(12833..12850) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(13004..13592) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(13084..13103) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS complement(13763..14623) /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 14386..14405 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(14624..14728) /gene="bla" /label=AmpR promoter primer_bind complement(14803..14822) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" ORIGIN 1 gacattgatt attgactagt tattaatagt aatcaattac ggggtcatta gttcatagcc 61 catatatgga gttccgcgtt acataactta cggtaaatgg cccgcctggc tgaccgccca 121 acgacccccg cccattgacg tcaataatga cgtatgttcc catagtaacg ccaataggga 181 ctttccattg acgtcaatgg gtggagtatt tacggtaaac tgcccacttg gcagtacatc 241 aagtgtatca tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct 301 ggcattatgc ccagtacatg accttatggg actttcctac ttggcagtac atctacgtat 361 tagtcatcgc tattaccatg gtgatgcggt tttggcagta catcaatggg cgtggatagc 421 ggtttgactc acggggattt ccaagtctcc accccattga cgtcaatggg agtttgtttt 481 ggcaccaaaa tcaacgggac tttccaaaat gtcgtaacaa ctccgcccca ttgacgcaaa 541 tgggcggtag gcgtgtacgg tgggaggtct atataagcag cgcgttttgc ctgtactggg 601 tctctctggt tagaccagat ctgagcctgg gagctctctg gctaactagg gaacccactg 661 cttaagcctc aataaagctt gccttgagtg cttcaagtag tgtgtgcccg tctgttgtgt 721 gactctggta actagagatc cctcagaccc ttttagtcag tgtggaaaat ctctagcagt 781 ggcgcccgaa cagggacttg aaagcgaaag ggaaaccaga ggagctctct cgacgcagga 841 ctcggcttgc tgaagcgcgc acggcaagag gcgaggggcg gcgactggtg agtacgccaa 901 aaattttgac tagcggaggc tagaaggaga gagatgggtg cgagagcgtc agtattaagc 961 gggggagaat tagatcgcga tgggaaaaaa ttcggttaag gccaggggga aagaaaaaat 1021 ataaattaaa acatatagta tgggcaagca gggagctaga acgattcgca gttaatcctg 1081 gcctgttaga aacatcagaa ggctgtagac aaatactggg acagctacaa ccatcccttc 1141 agacaggatc agaagaactt agatcattat ataatacagt agcaaccctc tattgtgtgc 1201 atcaaaggat agagataaaa gacaccaagg aagctttaga caagatagag gaagagcaaa 1261 acaaaagtaa gaccaccgca cagcaagcgg ccgctgatct tcagacctgg aggaggagat 1321 atgagggaca attggagaag tgaattatat aaatataaag tagtaaaaat tgaaccatta 1381 ggagtagcac ccaccaaggc aaagagaaga gtggtgcaga gagaaaaaag agcagtggga 1441 ataggagctt tgttccttgg gttcttggga gcagcaggaa gcactatggg cgcagcgtca 1501 atgacgctga cggtacaggc cagacaatta ttgtctggta tagtgcagca gcagaacaat 1561 ttgctgaggg ctattgaggc gcaacagcat ctgttgcaac tcacagtctg gggcatcaag 1621 cagctccagg caagaatcct ggctgtggaa agatacctaa aggatcaaca gctcctgggg 1681 atttggggtt gctctggaaa actcatttgc accactgctg tgccttggaa tgctagttgg 1741 agtaataaat ctctggaaca gatttggaat cacacgacct ggatggagtg ggacagagaa 1801 attaacaatt acacaagctt aatacactcc ttaattgaag aatcgcaaaa ccagcaagaa 1861 aagaatgaac aagaattatt ggaattagat aaatgggcaa gtttgtggaa ttggtttaac 1921 ataacaaatt ggctgtggta tataaaatta ttcataatga tagtaggagg cttggtaggt 1981 ttaagaatag tttttgctgt actttctata gtgaatagag ttaggcaggg atattcacca 2041 ttatcgtttc agacccacct cccaaccccg aggggacccg acaggcccga aggaatagaa 2101 gaagaaggtg gagagagaga cagagacaga tccattcgat tagtgaacgg atcggcactg 2161 cgtgcgccaa ttctgcagac aaatggcagt attcatccac aattttaaaa gaaaaggggg 2221 gattgggggg tacagtgcag gggaaagaat agtagacata atagcaacag acatacaaac 2281 taaagaatta caaaaacaaa ttacaaaaat tcaaaatttt cgggtttatt acagggacag 2341 cagagatcca gtttggttaa ttaataactt cgtatagcat acattatacg aagttattaa 2401 aaaaaaagca ccgactcggt gccacttttt caagttgata acggactagc cttattttaa 2461 cttgctattt ctagctctaa aactgagacg tccgtctccg gtgtttcgtc ctttccacaa 2521 gatatataaa gccaagaaat cgaaatactt tcaagttacg gtaagcatat gatagtccat 2581 tttaaaacat aattttaaaa ctgcaaacta cccaagaaat tattactttc tacgtcacgt 2641 attttgtact aatatctttg tgtttacagt caaattaatt ccaattatct ctctaacagc 2701 cttgtatcgt atatgcaaat atgaaggaat catgggaaat aggccctctt attaattaac 2761 ccgtgtcggc tccagatctg gcctccgcgc cgggttttgg cgcctcccgc gggcgccccc 2821 ctcctcacgg cgagcgctgc cacgtcagac gaagggcgca gcgagcgtcc tgatccttcc 2881 gcccggacgc tcaggacagc ggcccgctgc tcataagact cggccttaga accccagtat 2941 cagcagaagg acattttagg acgggacttg ggtgactcta gggcactggt tttctttcca 3001 gagagcggaa caggcgagga aaagtagtcc cttctcggcg attctgcgga gggatctccg 3061 tggggcggtg aacgccgatg attatataag gacgcgccgg gtgtggcaca gctagttccg 3121 tcgcagccgg gatttgggtc gcggttcttg tttgtggatc gctgtgatcg tcacttggtg 3181 agtagcgggc tgctgggctg gccggggctt tcgtggccgc cgggccgctc ggtgggacgg 3241 aagcgtgtgg agagaccgcc aagggctgta gtctgggtcc gcgagcaagg ttgccctgaa 3301 ctgggggttg gggggagcgc agcaaaatgg cggctgttcc cgagtcttga atggaagacg 3361 cttgtgaggc gggctgtgag gtcgttgaaa caaggtgggg ggcatggtgg gcggcaagaa 3421 cccaaggtct tgaggccttc gctaatgcgg gaaagctctt attcgggtga gatgggctgg 3481 ggcaccatct ggggaccctg acgtgaagtt tgtcactgac tggagaactc ggtttgtcgt 3541 ctgttgcggg ggcggcagtt atggcggtgc cgttgggcag tgcacccgta cctttgggag 3601 cgcgcgccct cgtcgtgtcg tgacgtcacc cgttctgttg gcttataatg cagggtgggg 3661 ccacctgccg gtaggtgtgc ggtaggcttt tctccgtcgc aggacgcagg gttcgggcct 3721 agggtaggct ctcctgaatc gacaggcgcc ggacctctgg tgaggggagg gataagtgag 3781 gcgtcagttt ctttggtcgg ttttatgtac ctatcttctt aagtagctga agctccggtt 3841 ttgaactatg cgctcggggt tggcgagtgt gttttgtgaa gttttttagg caccttttga 3901 aatgtaatca tttgggtcaa tatgtaattt tcagtgttag actagtaaat tgtccgctaa 3961 attctggccg tttttggctt ttttgttaga cgaagcttgg gctgcaggtc gactctagag 4021 ccaccatgga ctacaaagac catgacggtg attataaaga tcatgacatc gattacaagg 4081 atgacgatga caagcacggt ccgcgggctg acgcattgga cgattttgat ctggatatgc 4141 tgggaagtga cgccctcgat gattttgacc ttgacatgct tggttcggat gcccttgatg 4201 actttgacct cgacatgctc ggcagtgacg cccttgatga tttcgacctg gacatggtta 4261 accccaagaa gaagaggaag gtgggccgcg gaatggacaa gaagtactcc attgggctcg 4321 ccatcggcac aaacagcgtc ggctgggccg tcattacgga cgagtacaag gtgccgagca 4381 aaaaattcaa agttctgggc aataccgatc gccacagcat aaagaagaac ctcattggcg 4441 ccctcctgtt cgactccggg gaaaccgccg aagccacgcg gctcaaaaga acagcacggc 4501 gcagatatac ccgcagaaag aatcggatct gctacctgca ggagatcttt agtaatgaga 4561 tggctaaggt ggatgactct ttcttccata ggctggagga gtcctttttg gtggaggagg 4621 ataaaaagca cgagcgccac ccaatctttg gcaatatcgt ggacgaggtg gcgtaccatg 4681 aaaagtaccc aaccatatat catctgagga agaagcttgt agacagtact gataaggctg 4741 acttgcggtt gatctatctc gcgctggcgc atatgatcaa atttcgggga cacttcctca 4801 tcgaggggga cctgaaccca gacaacagcg atgtcgacaa actctttatc caactggttc 4861 agacttacaa tcagcttttc gaagagaacc cgatcaacgc atccggagtt gacgccaaag 4921 caatcctgag cgctaggctg tccaaatccc ggcggctcga aaacctcatc gcacagctcc 4981 ctggggagaa gaagaacggc ctgtttggta atcttatcgc cctgtcactc gggctgaccc 5041 ccaactttaa atctaacttc gacctggccg aagatgccaa gcttcaactg agcaaagaca 5101 cctacgatga tgatctcgac aatctgctgg cccagatcgg cgaccagtac gcagaccttt 5161 ttttggcggc aaagaacctg tcagacgcca ttctgctgag tgatattctg cgagtgaaca 5221 cggagatcac caaagctccg ctgagcgcta gtatgatcaa gcgctatgat gagcaccacc 5281 aagacttgac tttgctgaag gcccttgtca gacagcaact gcctgagaag tacaaggaaa 5341 ttttcttcga tcagtctaaa aatggctacg ccggatacat tgacggcgga gcaagccagg 5401 aggaatttta caaatttatt aagcccatct tggaaaaaat ggacggcacc gaggagctgc 5461 tggtaaagct taacagagaa gatctgttgc gcaaacagcg cactttcgac aatggaagca 5521 tcccccacca gattcacctg ggcgaactgc acgctatcct caggcggcaa gaggatttct 5581 accccttttt gaaagataac agggaaaaga ttgagaaaat cctcacattt cggataccct 5641 actatgtagg ccccctcgcc cggggaaatt ccagattcgc gtggatgact cgcaaatcag 5701 aagagaccat cactccctgg aacttcgagg aagtcgtgga taagggggcc tctgcccagt 5761 ccttcatcga aaggatgact aactttgata aaaatctgcc taacgaaaag gtgcttccta 5821 aacactctct gctgtacgag tacttcacag tttataacga gctcaccaag gtcaaatacg 5881 tcacagaagg gatgagaaag ccagcattcc tgtctggaga gcagaagaaa gctatcgtgg 5941 acctcctctt caagacgaac cggaaagtta ccgtgaaaca gctcaaagaa gactatttca 6001 aaaagattga atgtttcgac tctgttgaaa tcagcggagt ggaggatcgc ttcaacgcat 6061 ccctgggaac gtatcacgat ctcctgaaaa tcattaaaga caaggacttc ctggacaatg 6121 aggagaacga ggacattctt gaggacattg tcctcaccct tacgttgttt gaagataggg 6181 agatgattga agaacgcttg aaaacttacg ctcatctctt cgacgacaaa gtcatgaaac 6241 agctcaagag gcgccgatat acaggatggg ggcggctgtc aagaaaactg atcaatggga 6301 tccgagacaa gcagagtgga aagacaatcc tggattttct taagtccgat ggatttgcca 6361 accggaactt catgcagttg atccatgatg actctctcac ctttaaggag gacatccaga 6421 aagcacaagt ttctggccag ggggacagtc ttcacgagca catcgctaat cttgcaggta 6481 gcccagctat caaaaaggga atactgcaga ccgttaaggt cgtggatgaa ctcgtcaaag 6541 taatgggaag gcataagccc gagaatatcg ttatcgagat ggcccgagag aaccaaacta 6601 cccagaaggg acagaagaac agtagggaaa ggatgaagag gattgaagag ggtataaaag 6661 aactggggtc ccaaatcctt aaggaacacc cagttgaaaa cacccagctt cagaatgaga 6721 agctctacct gtactacctg cagaacggca gggacatgta cgtggatcag gaactggaca 6781 tcaatcggct ctccgactac gacgtggatg ccatcgtgcc ccagtctttt ctcaaagatg 6841 attctattga taataaagtg ttgacaagat ccgataaaaa tagagggaag agtgataacg 6901 tcccctcaga agaagttgtc aagaaaatga aaaattattg gcggcagctg ctgaacgcca 6961 aactgatcac acaacggaag ttcgataatc tgactaaggc tgaacgaggt ggcctgtctg 7021 agttggataa agccggcttc atcaaaaggc agcttgttga gacacgccag atcaccaagc 7081 acgtggccca aattctcgat tcacgcatga acaccaagta cgatgaaaat gacaaactga 7141 ttcgagaggt gaaagttatt actctgaagt ctaagctggt ctcagatttc agaaaggact 7201 ttcagtttta taaggtgaga gagatcaaca attaccacca tgcgcatgat gcctacctga 7261 atgcagtggt aggcactgca cttatcaaaa aatatcccaa gcttgaatct gaatttgttt 7321 acggagacta taaagtgtac gatgttagga aaatgatcgc aaagtctgag caggaaatag 7381 gcaaggccac cgctaagtac ttcttttaca gcaatattat gaattttttc aagaccgaga 7441 ttacactggc caatggagag attcggaagc gaccacttat cgaaacaaac ggagaaacag 7501 gagaaatcgt gtgggacaag ggtagggatt tcgcgacagt ccggaaggtc ctgtccatgc 7561 cgcaggtgaa catcgttaaa aagaccgaag tacagaccgg aggcttctcc aaggaaagta 7621 tcctcccgaa aaggaacagc gacaagctga tcgcacgcaa aaaagattgg gaccccaaga 7681 aatacggcgg attcgattct cctacagtcg cttacagtgt actggttgtg gccaaagtgg 7741 agaaagggaa gtctaaaaaa ctcaaaagcg tcaaggaact gctgggcatc acaatcatgg 7801 agcgatcaag cttcgaaaaa aaccccatcg actttctcga ggcgaaagga tataaagagg 7861 tcaaaaaaga cctcatcatt aagcttccca agtactctct ctttgagctt gaaaacggcc 7921 ggaaacgaat gctcgctagt gcgggcgagc tgcagaaagg taacgagctg gcactgccct 7981 ctaaatacgt taatttcttg tatctggcca gccactatga aaagctcaaa gggtctcccg 8041 aagataatga gcagaagcag ctgttcgtgg aacaacacaa acactacctt gatgagatca 8101 tcgagcaaat aagcgaattc tccaaaagag tgatcctcgc cgacgctaac ctcgataagg 8161 tgctttctgc ttacaataag cacagggata agcccatcag ggagcaggca gaaaacatta 8221 tccacttgtt tactctgacc aacttgggcg cgcctgcagc cttcaagtac ttcgacacca 8281 ccatagacag aaagcggtac acctctacaa aggaggtcct ggacgccaca ctgattcatc 8341 agtcaattac ggggctctat gaaacaagaa tcgacctctc tcagctcggt ggagacagca 8401 gggctgaccc caagaagaag aggaaggtgg ctagccgcgc cgacgcgctg gacgatttcg 8461 atctcgacat gctgggttct gatgccctcg atgactttga cctggatatg ttgggaagcg 8521 acgcattgga tgactttgat ctggacatgc tcggctccga tgctctggac gatttcgatc 8581 tcgatatgtt aatcgctagc gagggcagag gaagtcttct aacatgcggt gacgtggagg 8641 agaatcccgg ccctggtacc gtgagcaagg gcgaggagct gttcaccggg gtggtgccca 8701 tcctggtcga gctggacggc gacgtaaacg gccacaagtt cagcgtgtcc ggcgagggcg 8761 agggcgatgc cacctacggc aagctgaccc tgaagttcat ctgcaccacc ggcaagctgc 8821 ccgtgccctg gcccaccctc gtgaccaccc tgacctacgg cgtgcagtgc ttcagccgct 8881 accccgacca catgaagcag cacgacttct tcaagtccgc catgcccgaa ggctacgtcc 8941 aggagcgcac catcttcttc aaggacgacg gcaactacaa gacccgcgcc gaggtgaagt 9001 tcgagggcga caccctggtg aaccgcatcg agctgaaggg catcgacttc aaggaggacg 9061 gcaacatcct ggggcacaag ctggagtaca actacaacag ccacaacgtc tatatcatgg 9121 ccgacaagca gaagaacggc atcaaggtga acttcaagat ccgccacaac atcgaggacg 9181 gcagcgtgca gctcgccgac cactaccagc agaacacccc catcggcgac ggccccgtgc 9241 tgctgcccga caaccactac ctgagcaccc agtccgccct gagcaaagac cccaacgaga 9301 agcgcgatca catggtcctg ctggagttcg tgaccgccgc cgggatcact ctcggcatgg 9361 acgagctgta caagaccggt tgataataga taacttcgta tagcatacat tatacgaagt 9421 tatgaattcg atatcaagct tatcgataat caacctctgg attacaaaat ttgtgaaaga 9481 ttgactggta ttcttaacta tgttgctcct tttacgctat gtggatacgc tgctttaatg 9541 cctttgtatc atgctattgc ttcccgtatg gctttcattt tctcctcctt gtataaatcc 9601 tggttgctgt ctctttatga ggagttgtgg cccgttgtca ggcaacgtgg cgtggtgtgc 9661 actgtgtttg ctgacgcaac ccccactggt tggggcattg ccaccacctg tcagctcctt 9721 tccgggactt tcgctttccc cctccctatt gccacggcgg aactcatcgc cgcctgcctt 9781 gcccgctgct ggacaggggc tcggctgttg ggcactgaca attccgtggt gttgtcgggg 9841 aaatcatcgt cctttccttg gctgctcgcc tgtgttgcca cctggattct gcgcgggacg 9901 tccttctgct acgtcccttc ggccctcaat ccagcggacc ttccttcccg cggcctgctg 9961 ccggctctgc ggcctcttcc gcgtcttcgc cttcgccctc agacgagtcg gatctccctt 10021 tgggccgcct ccccgcatcg ataccgtcga cctcgagacc tagaaaaaca tggagcaatc 10081 acaagtagca atacagcagc taccaatgct gattgtgcct ggctagaagc acaagaggag 10141 gaggaggtgg gttttccagt cacacctcag gtacctttaa gaccaatgac ttacaaggca 10201 gctgtagatc ttagccactt tttaaaagaa aaggggggac tggaagggct aattcactcc 10261 caacgaagac aagatatcct tgatctgtgg atctaccaca cacaaggcta cttccctgat 10321 tggcagaact acacaccagg gccagggatc agatatccac tgacctttgg atggtgctac 10381 aagctagtac cagttgagca agagaaggta gaagaagcca atgaaggaga gaacacccgc 10441 ttgttacacc ctgtgagcct gcatgggatg gatgacccgg agagagaagt attagagtgg 10501 aggtttgaca gccgcctagc atttcatcac atggcccgag agctgcatcc ggactgtact 10561 gggtctctct ggttagacca gatctgagcc tgggagctct ctggctaact agggaaccca 10621 ctgcttaagc ctcaataaag cttgccttga gtgcttcaag tagtgtgtgc ccgtctgttg 10681 tgtgactctg gtaactagag atccctcaga cccttttagt cagtgtggaa aatctctagc 10741 agggcccgtt taaacccgct gatcagcctc gactgtgcct tctagttgcc agccatctgt 10801 tgtttgcccc tcccccgtgc cttccttgac cctggaaggt gccactccca ctgtcctttc 10861 ctaataaaat gaggaaattg catcgcattg tctgagtagg tgtcattcta ttctgggggg 10921 tggggtgggg caggacagca agggggagga ttgggaagac aatagcaggc atgctgggga 10981 tgcggtgggc tctatggctt ctgaggcgga aagaaccagc tggggctcta gggggtatcc 11041 ccacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac 11101 cgctacactt gccagcgccc tagcgcccgc tcctttcgct ttcttccctt cctttctcgc 11161 cacgttcgcc ggctttcccc gtcaagctct aaatcggggg ctccctttag ggttccgatt 11221 tagtgcttta cggcacctcg accccaaaaa acttgattag ggtgatggtt cacgtagtgg 11281 gccatcgccc tgatagacgg tttttcgccc tttgacgttg gagtccacgt tctttaatag 11341 tggactcttg ttccaaactg gaacaacact caaccctatc tcggtctatt cttttgattt 11401 ataagggatt ttgccgattt cggcctattg gttaaaaaat gagctgattt aacaaaaatt 11461 taacgcgaat taattctgtg gaatgtgtgt cagttagggt gtggaaagtc cccaggctcc 11521 ccagcaggca gaagtatgca aagcatgcat ctcaattagt cagcaaccag gtgtggaaag 11581 tccccaggct ccccagcagg cagaagtatg caaagcatgc atctcaatta gtcagcaacc 11641 atagtcccgc ccctaactcc gcccatcccg cccctaactc cgcccagttc cgcccattct 11701 ccgccccatg gctgactaat tttttttatt tatgcagagg ccgaggccgc ctctgcctct 11761 gagctattcc agaagtagtg aggaggcttt tttggaggcc taggcttttg caaaaagctc 11821 ccgggagctt gtatatccat tttcggatct gatcagcacg tgttgacaat taatcatcgg 11881 catagtatat cggcatagta taatacgaca aggtgaggaa ctaaaccatg gccaagttga 11941 ccagtgccgt tccggtgctc accgcgcgcg acgtcgccgg agcggtcgag ttctggaccg 12001 accggctcgg gttctcccgg gacttcgtgg aggacgactt cgccggtgtg gtccgggacg 12061 acgtgaccct gttcatcagc gcggtccagg accaggtggt gccggacaac accctggcct 12121 gggtgtgggt gcgcggcctg gacgagctgt acgccgagtg gtcggaggtc gtgtccacga 12181 acttccggga cgcctccggg ccggccatga ccgagatcgg cgagcagccg tgggggcggg 12241 agttcgccct gcgcgacccg gccggcaact gcgtgcactt cgtggccgag gagcaggact 12301 gacacgtgct acgagatttc gattccaccg ccgccttcta tgaaaggttg ggcttcggaa 12361 tcgttttccg ggacgccggc tggatgatcc tccagcgcgg ggatctcatg ctggagttct 12421 tcgcccaccc caacttgttt attgcagctt ataatggtta caaataaagc aatagcatca 12481 caaatttcac aaataaagca tttttttcac tgcattctag ttgtggtttg tccaaactca 12541 tcaatgtatc ttatcatgtc tgtataccgt cgacctctag ctagagcttg gcgtaatcat 12601 ggtcatagct gtttcctgtg tgaaattgtt atccgctcac aattccacac aacatacgag 12661 ccggaagcat aaagtgtaaa gcctggggtg cctaatgagt gagctaactc acattaattg 12721 cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa 12781 tcggccaacg cgcggggaga ggcggtttgc gtattgggcg ctcttccgct tcctcgctca 12841 ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg 12901 taatacggtt atccacagaa tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc 12961 agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc gtttttccat aggctccgcc 13021 cccctgacga gcatcacaaa aatcgacgct caagtcagag gtggcgaaac ccgacaggac 13081 tataaagata ccaggcgttt ccccctggaa gctccctcgt gcgctctcct gttccgaccc 13141 tgccgcttac cggatacctg tccgcctttc tcccttcggg aagcgtggcg ctttctcata 13201 gctcacgctg taggtatctc agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc 13261 acgaaccccc cgttcagccc gaccgctgcg ccttatccgg taactatcgt cttgagtcca 13321 acccggtaag acacgactta tcgccactgg cagcagccac tggtaacagg attagcagag 13381 cgaggtatgt aggcggtgct acagagttct tgaagtggtg gcctaactac ggctacacta 13441 gaagaacagt atttggtatc tgcgctctgc tgaagccagt taccttcgga aaaagagttg 13501 gtagctcttg atccggcaaa caaaccaccg ctggtagcgg tggttttttt gtttgcaagc 13561 agcagattac gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt tctacggggt 13621 ctgacgctca gtggaacgaa aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa 13681 ggatcttcac ctagatcctt ttaaattaaa aatgaagttt taaatcaatc taaagtatat 13741 atgagtaaac ttggtctgac agttaccaat gcttaatcag tgaggcacct atctcagcga 13801 tctgtctatt tcgttcatcc atagttgcct gactccccgt cgtgtagata actacgatac 13861 gggagggctt accatctggc cccagtgctg caatgatacc gcgagaccca cgctcaccgg 13921 ctccagattt atcagcaata aaccagccag ccggaagggc cgagcgcaga agtggtcctg 13981 caactttatc cgcctccatc cagtctatta attgttgccg ggaagctaga gtaagtagtt 14041 cgccagttaa tagtttgcgc aacgttgttg ccattgctac aggcatcgtg gtgtcacgct 14101 cgtcgtttgg tatggcttca ttcagctccg gttcccaacg atcaaggcga gttacatgat 14161 cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt gtcagaagta 14221 agttggccgc agtgttatca ctcatggtta tggcagcact gcataattct cttactgtca 14281 tgccatccgt aagatgcttt tctgtgactg gtgagtactc aaccaagtca ttctgagaat 14341 agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat acgggataat accgcgccac 14401 atagcagaac tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga aaactctcaa 14461 ggatcttacc gctgttgaga tccagttcga tgtaacccac tcgtgcaccc aactgatctt 14521 cagcatcttt tactttcacc agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg 14581 caaaaaaggg aataagggcg acacggaaat gttgaatact catactcttc ctttttcaat 14641 attattgaag catttatcag ggttattgtc tcatgagcgg atacatattt gaatgtattt 14701 agaaaaataa acaaataggg gttccgcgca catttccccg aaaagtgcca cctgacgtcg 14761 acggatcggg agatctcccg atcccctatg gtgcactctc agtacaatct gctctgatgc 14821 cgcatagtta agccagtatc tgctccctgc ttgtgtgttg gaggtcgctg agtagtgcgc 14881 gagcaaaatt taagctacaa caaggcaagg cttgaccgac aattgcatga agaatctgct 14941 tagggttagg cgttttgcgc tgcttcgcga tgtacgggcc agatatacgc gtt //