LOCUS Exported 15087 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION Co-expressed human optimized S. pyogenese dCas9 fused to KRAB repressor domain and GFP. ACCESSION . VERSION . KEYWORDS pLV hUbC-dCas9 KRAB-T2A-GFP SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 15087) AUTHORS Kabadi AM, Ousterout DG, Hilton IB, Gersbach CA TITLE Multiplex CRISPR/Cas9-based genome engineering from a single lentiviral vector. JOURNAL Nucleic Acids Res. 2014 Aug 13. pii: gku749. PUBMED 25122746 REFERENCE 2 (bases 1 to 15087) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..15087 /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" protein_bind 2602..2635 /label=loxP /bound_moiety="Cre recombinase" /note="Cre-mediated recombination occurs in the 8-bp core sequence (GCATACAT)." CDS complement(2622..2948) /codon_start=1 /gene="lacZ fragment" /product="LacZ-alpha fragment of beta-galactosidase" /label=lacZ-alpha /translation="MTMITPSAQLTLTKGNKSWRTSRGGPVPNSPYSESYYARSLAVVL QRRDWENPGVTQLNRLAAHPPFASWRNSEEARTDRPSQQLRSLNGEWDAPCSGATTVSY HNFV" primer_bind 2798..2820 /label=M13/pUC Forward /note="In lacZ gene" primer_bind 2812..2829 /label=M13 Forward /note="In lacZ gene. Also called M13-F20 or M13 (-21) Forward" primer_bind 2813..2829 /label=M13 fwd /note="common sequencing primer, one of multiple similar variants" primer_bind 2839..2858 /label=T7 /note="T7 promoter, forward primer" promoter 2839..2857 /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" primer_bind complement(2905..2925) /label=T3 /note="T3 promoter, forward primer" promoter complement(2905..2923) /label=T3 promoter /note="promoter for bacteriophage T3 RNA polymerase" primer_bind complement(2944..2960) /label=M13 rev /note="common sequencing primer, one of multiple similar variants" protein_bind 2968..2984 /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(2992..3022) /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 3037..3058 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." promoter 3124..4334 /label=UbC promoter /note="human ubiquitin C promoter" primer_bind 4171..4190 /label=hUBCpro-F /note="Human Ubiquitin C (UbC) promoter, forward primer" regulatory 4363..4372 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" CDS 4372..4437 /codon_start=1 /product="three tandem FLAG(R) epitope tags, followed by an enterokinase cleavage site" /label=3xFLAG /translation="DYKDHDGDYKDHDIDYKDDDDK" CDS 4444..4464 /codon_start=1 /product="nuclear localization signal of SV40 (simian virus 40) large T antigen" /label=SV40 NLS /translation="PKKKRKV" CDS 4474..8577 /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 8590..8610 /codon_start=1 /product="nuclear localization signal of SV40 (simian virus 40) large T antigen" /label=SV40 NLS /translation="PKKKRKV" CDS 8644..8838 /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 8905..8925 /codon_start=1 /product="nuclear localization signal of SV40 (simian virus 40) large T antigen" /label=SV40 NLS /translation="PKKKRKV" CDS 8932..8985 /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 8992..9705 /codon_start=1 /product="enhanced GFP" /label=EGFP /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE FVTAAGITLGMDELYK" CDS 8992..9705 /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(9034..9055) /label=EGFP-N /note="EGFP, reverse primer" primer_bind complement(9295..9314) /label=EXFP-R /note="For distinguishing EGFP variants, reverse primer" primer_bind 9642..9663 /label=EGFP-C /note="EGFP, forward primer" protein_bind 9721..9754 /label=loxP /bound_moiety="Cre recombinase" /note="Cre-mediated recombination occurs in the 8-bp core sequence (GCATACAT)." misc_feature 9779..10367 /label=WPRE /note="woodchuck hepatitis virus posttranscriptional regulatory element" primer_bind complement(9832..9852) /label=WPRE-R /note="WPRE, reverse primer" CDS complement(10250..10261) /codon_start=1 /product="Factor Xa recognition and cleavage site" /label=Factor Xa site /translation="IEGR" primer_bind complement(10370..10386) /label=KS primer /note="common sequencing primer, one of multiple similar variants" primer_bind complement(10371..10387) /label=pBluescriptKS /note="For pBluescript vector" LTR 10892..11072 /label=5' LTR (truncated) /note="truncated 5' long terminal repeat (LTR) from HIV-1" primer_bind complement(11098..11115) /label=BGH-rev /note="Bovine growth hormone terminator, reverse primer. Also called BGH reverse" polyA_signal 11104..11328 /label=bGH poly(A) signal /note="bovine growth hormone polyadenylation signal" rep_origin 11374..11802 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(11461..11480) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 11671..11692 /label=F1ori-F /note="F1 origin, forward primer" primer_bind complement(11811..11831) /label=pBABE 3' /note="SV40 enhancer, reverse primer for pBABE vectors" promoter 11816..12145 /label=SV40 promoter /note="SV40 enhancer and early promoter" rep_origin 11996..12131 /label=SV40 ori /note="SV40 origin of replication" primer_bind 12058..12077 /label=SV40pro-F /note="SV40 promoter/origin, forward primer" promoter 12193..12240 /label=EM7 promoter /note="synthetic bacterial promoter " CDS 12259..12633 /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 12763..12884 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(12800..12819) /label=SV40pA-R /note="SV40 polyA, reverse primer" primer_bind 12854..12873 /label=EBV-rev /note="SV40 polyA terminator, reverse primer" primer_bind complement(12933..12949) /label=M13 rev /note="common sequencing primer, one of multiple similar variants" protein_bind 12957..12973 /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(12981..13011) /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 13026..13047 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." primer_bind complement(13164..13181) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(13335..13923) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(13415..13434) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS complement(14094..14954) /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 14717..14736 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(14955..15059) /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 aataacttcg tatagcatac attatacgaa gttatgataa 2641 gagacggtgg tggcgccgct acagggcgcg tcccattcgc cattcaggct gcgcaactgt 2701 tgggaagggc gatcggtgcg ggcctcttcg ctattacgcc agctggcgaa agggggatgt 2761 gctgcaaggc gattaagttg ggtaacgcca gggttttccc agtcacgacg ttgtaaaacg 2821 acggccagtg agcgcgcgta atacgactca ctatagggcg aattgggtac cgggcccccc 2881 ctcgaggtcc tccagctttt gttcccttta gtgagggtta attgcgcgct tggcgtaatc 2941 atggtcatag ctgtttcctg tgtgaaattg ttatccgctc acaattccac acaacatacg 3001 agccggaagc ataaagtgta aagcctgggg tgcctaatga gtgagctaac tcacattaat 3061 tgcgttgcgc tcactgcccg ctttccactg catgacgtct ccacaattaa ttaagggtgc 3121 agcggcctcc gcgccgggtt ttggcgcctc ccgcgggcgc ccccctcctc acggcgagcg 3181 ctgccacgtc agacgaaggg cgcaggagcg ttcctgatcc ttccgcccgg acgctcagga 3241 cagcggcccg ctgctcataa gactcggcct tagaacccca gtatcagcag aaggacattt 3301 taggacggga cttgggtgac tctagggcac tggttttctt tccagagagc ggaacaggcg 3361 aggaaaagta gtcccttctc ggcgattctg cggagggatc tccgtggggc ggtgaacgcc 3421 gatgattata taaggacgcg ccgggtgtgg cacagctagt tccgtcgcag ccgggatttg 3481 ggtcgcggtt cttgtttgtg gatcgctgtg atcgtcactt ggtgagttgc gggctgctgg 3541 gctggccggg gctttcgtgg ccgccgggcc gctcggtggg acggaagcgt gtggagagac 3601 cgccaagggc tgtagtctgg gtccgcgagc aaggttgccc tgaactgggg gttgggggga 3661 gcgcacaaaa tggcggctgt tcccgagtct tgaatggaag acgcttgtaa ggcgggctgt 3721 gaggtcgttg aaacaaggtg gggggcatgg tgggcggcaa gaacccaagg tcttgaggcc 3781 ttcgctaatg cgggaaagct cttattcggg tgagatgggc tggggcacca tctggggacc 3841 ctgacgtgaa gtttgtcact gactggagaa ctcgggtttg tcgtctggtt gcgggggcgg 3901 cagttatgcg gtgccgttgg gcagtgcacc cgtacctttg ggagcgcgcg cctcgtcgtg 3961 tcgtgacgtc acccgttctg ttggcttata atgcagggtg gggccacctg ccggtaggtg 4021 tgcggtaggc ttttctccgt cgcaggacgc agggttcggg cctagggtag gctctcctga 4081 atcgacaggc gccggacctc tggtgagggg agggataagt gaggcgtcag tttctttggt 4141 cggttttatg tacctatctt cttaagtagc tgaagctccg gttttgaact atgcgctcgg 4201 ggttggcgag tgtgttttgt gaagtttttt aggcaccttt tgaaatgtaa tcatttgggt 4261 caatatgtaa ttttcagtgt tagactagta aattgtccgc taaattctgg ccgtttttgg 4321 cttttttgtt agacgaagct tgggctgcag gtcgactcta gagccaccat ggactacaaa 4381 gaccatgacg gtgattataa agatcatgac atcgattaca aggatgacga tgacaagatg 4441 gcccccaaga agaagaggaa ggtgggccgc ggaatggaca agaagtactc cattgggctc 4501 gccatcggca caaacagcgt cggctgggcc gtcattacgg acgagtacaa ggtgccgagc 4561 aaaaaattca aagttctggg caataccgat cgccacagca taaagaagaa cctcattggc 4621 gccctcctgt tcgactccgg ggaaaccgcc gaagccacgc ggctcaaaag aacagcacgg 4681 cgcagatata cccgcagaaa gaatcggatc tgctacctgc aggagatctt tagtaatgag 4741 atggctaagg tggatgactc tttcttccat aggctggagg agtccttttt ggtggaggag 4801 gataaaaagc acgagcgcca cccaatcttt ggcaatatcg tggacgaggt ggcgtaccat 4861 gaaaagtacc caaccatata tcatctgagg aagaagcttg tagacagtac tgataaggct 4921 gacttgcggt tgatctatct cgcgctggcg catatgatca aatttcgggg acacttcctc 4981 atcgaggggg acctgaaccc agacaacagc gatgtcgaca aactctttat ccaactggtt 5041 cagacttaca atcagctttt cgaagagaac ccgatcaacg catccggagt tgacgccaaa 5101 gcaatcctga gcgctaggct gtccaaatcc cggcggctcg aaaacctcat cgcacagctc 5161 cctggggaga agaagaacgg cctgtttggt aatcttatcg ccctgtcact cgggctgacc 5221 cccaacttta aatctaactt cgacctggcc gaagatgcca agcttcaact gagcaaagac 5281 acctacgatg atgatctcga caatctgctg gcccagatcg gcgaccagta cgcagacctt 5341 tttttggcgg caaagaacct gtcagacgcc attctgctga gtgatattct gcgagtgaac 5401 acggagatca ccaaagctcc gctgagcgct agtatgatca agcgctatga tgagcaccac 5461 caagacttga ctttgctgaa ggcccttgtc agacagcaac tgcctgagaa gtacaaggaa 5521 attttcttcg atcagtctaa aaatggctac gccggataca ttgacggcgg agcaagccag 5581 gaggaatttt acaaatttat taagcccatc ttggaaaaaa tggacggcac cgaggagctg 5641 ctggtaaagc ttaacagaga agatctgttg cgcaaacagc gcactttcga caatggaagc 5701 atcccccacc agattcacct gggcgaactg cacgctatcc tcaggcggca agaggatttc 5761 tacccctttt tgaaagataa cagggaaaag attgagaaaa tcctcacatt tcggataccc 5821 tactatgtag gccccctcgc ccggggaaat tccagattcg cgtggatgac tcgcaaatca 5881 gaagagacca tcactccctg gaacttcgag gaagtcgtgg ataagggggc ctctgcccag 5941 tccttcatcg aaaggatgac taactttgat aaaaatctgc ctaacgaaaa ggtgcttcct 6001 aaacactctc tgctgtacga gtacttcaca gtttataacg agctcaccaa ggtcaaatac 6061 gtcacagaag ggatgagaaa gccagcattc ctgtctggag agcagaagaa agctatcgtg 6121 gacctcctct tcaagacgaa ccggaaagtt accgtgaaac agctcaaaga agactatttc 6181 aaaaagattg aatgtttcga ctctgttgaa atcagcggag tggaggatcg cttcaacgca 6241 tccctgggaa cgtatcacga tctcctgaaa atcattaaag acaaggactt cctggacaat 6301 gaggagaacg aggacattct tgaggacatt gtcctcaccc ttacgttgtt tgaagatagg 6361 gagatgattg aagaacgctt gaaaacttac gctcatctct tcgacgacaa agtcatgaaa 6421 cagctcaaga ggcgccgata tacaggatgg gggcggctgt caagaaaact gatcaatggg 6481 atccgagaca agcagagtgg aaagacaatc ctggattttc ttaagtccga tggatttgcc 6541 aaccggaact tcatgcagtt gatccatgat gactctctca cctttaagga ggacatccag 6601 aaagcacaag tttctggcca gggggacagt cttcacgagc acatcgctaa tcttgcaggt 6661 agcccagcta tcaaaaaggg aatactgcag accgttaagg tcgtggatga actcgtcaaa 6721 gtaatgggaa ggcataagcc cgagaatatc gttatcgaga tggcccgaga gaaccaaact 6781 acccagaagg gacagaagaa cagtagggaa aggatgaaga ggattgaaga gggtataaaa 6841 gaactggggt cccaaatcct taaggaacac ccagttgaaa acacccagct tcagaatgag 6901 aagctctacc tgtactacct gcagaacggc agggacatgt acgtggatca ggaactggac 6961 atcaatcggc tctccgacta cgacgtggat gccatcgtgc cccagtcttt tctcaaagat 7021 gattctattg ataataaagt gttgacaaga tccgataaaa atagagggaa gagtgataac 7081 gtcccctcag aagaagttgt caagaaaatg aaaaattatt ggcggcagct gctgaacgcc 7141 aaactgatca cacaacggaa gttcgataat ctgactaagg ctgaacgagg tggcctgtct 7201 gagttggata aagccggctt catcaaaagg cagcttgttg agacacgcca gatcaccaag 7261 cacgtggccc aaattctcga ttcacgcatg aacaccaagt acgatgaaaa tgacaaactg 7321 attcgagagg tgaaagttat tactctgaag tctaagctgg tctcagattt cagaaaggac 7381 tttcagtttt ataaggtgag agagatcaac aattaccacc atgcgcatga tgcctacctg 7441 aatgcagtgg taggcactgc acttatcaaa aaatatccca agcttgaatc tgaatttgtt 7501 tacggagact ataaagtgta cgatgttagg aaaatgatcg caaagtctga gcaggaaata 7561 ggcaaggcca ccgctaagta cttcttttac agcaatatta tgaatttttt caagaccgag 7621 attacactgg ccaatggaga gattcggaag cgaccactta tcgaaacaaa cggagaaaca 7681 ggagaaatcg tgtgggacaa gggtagggat ttcgcgacag tccggaaggt cctgtccatg 7741 ccgcaggtga acatcgttaa aaagaccgaa gtacagaccg gaggcttctc caaggaaagt 7801 atcctcccga aaaggaacag cgacaagctg atcgcacgca aaaaagattg ggaccccaag 7861 aaatacggcg gattcgattc tcctacagtc gcttacagtg tactggttgt ggccaaagtg 7921 gagaaaggga agtctaaaaa actcaaaagc gtcaaggaac tgctgggcat cacaatcatg 7981 gagcgatcaa gcttcgaaaa aaaccccatc gactttctcg aggcgaaagg atataaagag 8041 gtcaaaaaag acctcatcat taagcttccc aagtactctc tctttgagct tgaaaacggc 8101 cggaaacgaa tgctcgctag tgcgggcgag ctgcagaaag gtaacgagct ggcactgccc 8161 tctaaatacg ttaatttctt gtatctggcc agccactatg aaaagctcaa agggtctccc 8221 gaagataatg agcagaagca gctgttcgtg gaacaacaca aacactacct tgatgagatc 8281 atcgagcaaa taagcgaatt ctccaaaaga gtgatcctcg ccgacgctaa cctcgataag 8341 gtgctttctg cttacaataa gcacagggat aagcccatca gggagcaggc agaaaacatt 8401 atccacttgt ttactctgac caacttgggc gcgcctgcag ccttcaagta cttcgacacc 8461 accatagaca gaaagcggta cacctctaca aaggaggtcc tggacgccac actgattcat 8521 cagtcaatta cggggctcta tgaaacaaga atcgacctct ctcagctcgg tggagacagc 8581 agggctgacc ccaagaagaa gaggaaggtg gctagcgatg ctaagtcact gactgcctgg 8641 tcccggacac tggtgacctt caaggatgtg tttgtggact tcaccaggga ggagtggaag 8701 ctgctggaca ctgctcagca gatcctgtac agaaatgtga tgctggagaa ctataagaac 8761 ctggtttcct tgggttatca gcttactaag ccagatgtga tcctccggtt ggagaaggga 8821 gaagagccct ggctggtgga gagagaaatt caccaagaga cccatcctga ttcagagact 8881 gcatttgaaa tcaaatcatc agttccgaaa aagaaacgca aagttgctag cgagggcaga 8941 ggaagtcttc taacatgcgg tgacgtggag gagaatcccg gccctggtac cgtgagcaag 9001 ggcgaggagc tgttcaccgg ggtggtgccc atcctggtcg agctggacgg cgacgtaaac 9061 ggccacaagt tcagcgtgtc cggcgagggc gagggcgatg ccacctacgg caagctgacc 9121 ctgaagttca tctgcaccac cggcaagctg cccgtgccct ggcccaccct cgtgaccacc 9181 ctgacctacg gcgtgcagtg cttcagccgc taccccgacc acatgaagca gcacgacttc 9241 ttcaagtccg ccatgcccga aggctacgtc caggagcgca ccatcttctt caaggacgac 9301 ggcaactaca agacccgcgc cgaggtgaag ttcgagggcg acaccctggt gaaccgcatc 9361 gagctgaagg gcatcgactt caaggaggac ggcaacatcc tggggcacaa gctggagtac 9421 aactacaaca gccacaacgt ctatatcatg gccgacaagc agaagaacgg catcaaggtg 9481 aacttcaaga tccgccacaa catcgaggac ggcagcgtgc agctcgccga ccactaccag 9541 cagaacaccc ccatcggcga cggccccgtg ctgctgcccg acaaccacta cctgagcacc 9601 cagtccgccc tgagcaaaga ccccaacgag aagcgcgatc acatggtcct gctggagttc 9661 gtgaccgccg ccgggatcac tctcggcatg gacgagctgt acaagaccgg ttgataatag 9721 ataacttcgt atagcataca ttatacgaag ttatgaattc gatatcaagc ttatcgataa 9781 tcaacctctg gattacaaaa tttgtgaaag attgactggt attcttaact atgttgctcc 9841 ttttacgcta tgtggatacg ctgctttaat gcctttgtat catgctattg cttcccgtat 9901 ggctttcatt ttctcctcct tgtataaatc ctggttgctg tctctttatg aggagttgtg 9961 gcccgttgtc aggcaacgtg gcgtggtgtg cactgtgttt gctgacgcaa cccccactgg 10021 ttggggcatt gccaccacct gtcagctcct ttccgggact ttcgctttcc ccctccctat 10081 tgccacggcg gaactcatcg ccgcctgcct tgcccgctgc tggacagggg ctcggctgtt 10141 gggcactgac aattccgtgg tgttgtcggg gaaatcatcg tcctttcctt ggctgctcgc 10201 ctgtgttgcc acctggattc tgcgcgggac gtccttctgc tacgtccctt cggccctcaa 10261 tccagcggac cttccttccc gcggcctgct gccggctctg cggcctcttc cgcgtcttcg 10321 ccttcgccct cagacgagtc ggatctccct ttgggccgcc tccccgcatc gataccgtcg 10381 acctcgagac ctagaaaaac atggagcaat cacaagtagc aatacagcag ctaccaatgc 10441 tgattgtgcc tggctagaag cacaagagga ggaggaggtg ggttttccag tcacacctca 10501 ggtaccttta agaccaatga cttacaaggc agctgtagat cttagccact ttttaaaaga 10561 aaagggggga ctggaagggc taattcactc ccaacgaaga caagatatcc ttgatctgtg 10621 gatctaccac acacaaggct acttccctga ttggcagaac tacacaccag ggccagggat 10681 cagatatcca ctgacctttg gatggtgcta caagctagta ccagttgagc aagagaaggt 10741 agaagaagcc aatgaaggag agaacacccg cttgttacac cctgtgagcc tgcatgggat 10801 ggatgacccg gagagagaag tattagagtg gaggtttgac agccgcctag catttcatca 10861 catggcccga gagctgcatc cggactgtac tgggtctctc tggttagacc agatctgagc 10921 ctgggagctc tctggctaac tagggaaccc actgcttaag cctcaataaa gcttgccttg 10981 agtgcttcaa gtagtgtgtg cccgtctgtt gtgtgactct ggtaactaga gatccctcag 11041 acccttttag tcagtgtgga aaatctctag cagggcccgt ttaaacccgc tgatcagcct 11101 cgactgtgcc ttctagttgc cagccatctg ttgtttgccc ctcccccgtg ccttccttga 11161 ccctggaagg tgccactccc actgtccttt cctaataaaa tgaggaaatt gcatcgcatt 11221 gtctgagtag gtgtcattct attctggggg gtggggtggg gcaggacagc aagggggagg 11281 attgggaaga caatagcagg catgctgggg atgcggtggg ctctatggct tctgaggcgg 11341 aaagaaccag ctggggctct agggggtatc cccacgcgcc ctgtagcggc gcattaagcg 11401 cggcgggtgt ggtggttacg cgcagcgtga ccgctacact tgccagcgcc ctagcgcccg 11461 ctcctttcgc tttcttccct tcctttctcg ccacgttcgc cggctttccc cgtcaagctc 11521 taaatcgggg gctcccttta gggttccgat ttagtgcttt acggcacctc gaccccaaaa 11581 aacttgatta gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc 11641 ctttgacgtt ggagtccacg ttctttaata gtggactctt gttccaaact ggaacaacac 11701 tcaaccctat ctcggtctat tcttttgatt tataagggat tttgccgatt tcggcctatt 11761 ggttaaaaaa tgagctgatt taacaaaaat ttaacgcgaa ttaattctgt ggaatgtgtg 11821 tcagttaggg tgtggaaagt ccccaggctc cccagcaggc agaagtatgc aaagcatgca 11881 tctcaattag tcagcaacca ggtgtggaaa gtccccaggc tccccagcag gcagaagtat 11941 gcaaagcatg catctcaatt agtcagcaac catagtcccg cccctaactc cgcccatccc 12001 gcccctaact ccgcccagtt ccgcccattc tccgccccat ggctgactaa ttttttttat 12061 ttatgcagag gccgaggccg cctctgcctc tgagctattc cagaagtagt gaggaggctt 12121 ttttggaggc ctaggctttt gcaaaaagct cccgggagct tgtatatcca ttttcggatc 12181 tgatcagcac gtgttgacaa ttaatcatcg gcatagtata tcggcatagt ataatacgac 12241 aaggtgagga actaaaccat ggccaagttg accagtgccg ttccggtgct caccgcgcgc 12301 gacgtcgccg gagcggtcga gttctggacc gaccggctcg ggttctcccg ggacttcgtg 12361 gaggacgact tcgccggtgt ggtccgggac gacgtgaccc tgttcatcag cgcggtccag 12421 gaccaggtgg tgccggacaa caccctggcc tgggtgtggg tgcgcggcct ggacgagctg 12481 tacgccgagt ggtcggaggt cgtgtccacg aacttccggg acgcctccgg gccggccatg 12541 accgagatcg gcgagcagcc gtgggggcgg gagttcgccc tgcgcgaccc ggccggcaac 12601 tgcgtgcact tcgtggccga ggagcaggac tgacacgtgc tacgagattt cgattccacc 12661 gccgccttct atgaaaggtt gggcttcgga atcgttttcc gggacgccgg ctggatgatc 12721 ctccagcgcg gggatctcat gctggagttc ttcgcccacc ccaacttgtt tattgcagct 12781 tataatggtt acaaataaag caatagcatc acaaatttca caaataaagc atttttttca 12841 ctgcattcta gttgtggttt gtccaaactc atcaatgtat cttatcatgt ctgtataccg 12901 tcgacctcta gctagagctt ggcgtaatca tggtcatagc tgtttcctgt gtgaaattgt 12961 tatccgctca caattccaca caacatacga gccggaagca taaagtgtaa agcctggggt 13021 gcctaatgag tgagctaact cacattaatt gcgttgcgct cactgcccgc tttccagtcg 13081 ggaaacctgt cgtgccagct gcattaatga atcggccaac gcgcggggag aggcggtttg 13141 cgtattgggc gctcttccgc ttcctcgctc actgactcgc tgcgctcggt cgttcggctg 13201 cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt tatccacaga atcaggggat 13261 aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc 13321 gcgttgctgg cgtttttcca taggctccgc ccccctgacg agcatcacaa aaatcgacgc 13381 tcaagtcaga ggtggcgaaa cccgacagga ctataaagat accaggcgtt tccccctgga 13441 agctccctcg tgcgctctcc tgttccgacc ctgccgctta ccggatacct gtccgccttt 13501 ctcccttcgg gaagcgtggc gctttctcat agctcacgct gtaggtatct cagttcggtg 13561 taggtcgttc gctccaagct gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc 13621 gccttatccg gtaactatcg tcttgagtcc aacccggtaa gacacgactt atcgccactg 13681 gcagcagcca ctggtaacag gattagcaga gcgaggtatg taggcggtgc tacagagttc 13741 ttgaagtggt ggcctaacta cggctacact agaagaacag tatttggtat ctgcgctctg 13801 ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa acaaaccacc 13861 gctggtagcg gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct 13921 caagaagatc ctttgatctt ttctacgggg tctgacgctc agtggaacga aaactcacgt 13981 taagggattt tggtcatgag attatcaaaa aggatcttca cctagatcct tttaaattaa 14041 aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa cttggtctga cagttaccaa 14101 tgcttaatca gtgaggcacc tatctcagcg atctgtctat ttcgttcatc catagttgcc 14161 tgactccccg tcgtgtagat aactacgata cgggagggct taccatctgg ccccagtgct 14221 gcaatgatac cgcgagaccc acgctcaccg gctccagatt tatcagcaat aaaccagcca 14281 gccggaaggg ccgagcgcag aagtggtcct gcaactttat ccgcctccat ccagtctatt 14341 aattgttgcc gggaagctag agtaagtagt tcgccagtta atagtttgcg caacgttgtt 14401 gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc 14461 ggttcccaac gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa agcggttagc 14521 tccttcggtc ctccgatcgt tgtcagaagt aagttggccg cagtgttatc actcatggtt 14581 atggcagcac tgcataattc tcttactgtc atgccatccg taagatgctt ttctgtgact 14641 ggtgagtact caaccaagtc attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc 14701 ccggcgtcaa tacgggataa taccgcgcca catagcagaa ctttaaaagt gctcatcatt 14761 ggaaaacgtt cttcggggcg aaaactctca aggatcttac cgctgttgag atccagttcg 14821 atgtaaccca ctcgtgcacc caactgatct tcagcatctt ttactttcac cagcgtttct 14881 gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa 14941 tgttgaatac tcatactctt cctttttcaa tattattgaa gcatttatca gggttattgt 15001 ctcatgagcg gatacatatt tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc 15061 acatttcccc gaaaagtgcc acctgac //