LOCUS Exported 13500 bp ds-DNA circular SYN 29-JUL-2021 DEFINITION lenti sgRNA cloning backbone with MS2 loops at tetraloop and stemloop 2, dCas9-VP64, and blast resistance marker. Contains BsmBI sites for insertion of spacer sequences.. ACCESSION . VERSION . KEYWORDS lentiSAMv2 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 13500) AUTHORS Joung J, Konermann S, Gootenberg JS, Abudayyeh OO, Platt RJ, Brigham MD, Sanjana NE, Zhang F TITLE Genome-scale CRISPR-Cas9 knockout and transcriptional activation screening. JOURNAL Nat Protoc. 2017 Apr;12(4):828-863. doi: 10.1038/nprot.2017.016. Epub 2017 Mar 23. PUBMED 28333914 REFERENCE 2 (bases 1 to 13500) AUTHORS . TITLE Direct Submission JOURNAL Exported Jul 29, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..13500 /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" promoter 2607..2847 /label=U6 promoter /note="RNA polymerase III promoter for human U6 snRNA" primer_bind 2607..2627 /label=hU6-F /note="Human U6 promoter, forward primer" primer_bind 2778..2797 /label=LKO.1 5' /note="Human U6 promoter, forward primer" misc_RNA 2893..2911 /label=MS2 stem loop /note="stem loop that binds the bacteriophage MS2 coat protein" misc_RNA 2963..2981 /label=MS2 stem loop /note="stem loop that binds the bacteriophage MS2 coat protein" promoter 3068..3279 /label=EF-1-alpha core promoter /note="core promoter for human elongation factor EF-1-alpha" CDS 3296..3343 /codon_start=1 /product="bipartite nuclear localization signal from nucleoplasmin" /label=nucleoplasmin NLS /translation="KRPAATKKAGQAKKKK" CDS 3344..7444 /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 N863A mutation in the HNH catalytic domain" /translation="DKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL YYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKARGKSDNVPS EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD ATLIHQSITGLYETRIDLSQLGGD" CDS 7499..7519 /codon_start=1 /product="nuclear localization signal of SV40 (simian virus 40) large T antigen" /label=SV40 NLS /translation="PKKKRKV" CDS 7544..7693 /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 7715..7768 /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 7769..8167 /codon_start=1 /gene="Aspergillus terreus BSD" /product="blasticidin S deaminase" /label=BSD /note="confers resistance to blasticidin" /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI KAIVKDSDGQPTAVGIRELLPSGYVWEG" misc_feature 8192..8780 /label=WPRE /note="woodchuck hepatitis virus posttranscriptional regulatory element" primer_bind complement(8245..8265) /label=WPRE-R /note="WPRE, reverse primer" CDS complement(8663..8674) /codon_start=1 /product="Factor Xa recognition and cleavage site" /label=Factor Xa site /translation="IEGR" primer_bind complement(8783..8799) /label=KS primer /note="common sequencing primer, one of multiple similar variants" primer_bind complement(8784..8800) /label=pBluescriptKS /note="For pBluescript vector" LTR 9305..9485 /label=5' LTR (truncated) /note="truncated 5' long terminal repeat (LTR) from HIV-1" primer_bind complement(9511..9528) /label=BGH-rev /note="Bovine growth hormone terminator, reverse primer. Also called BGH reverse" polyA_signal 9517..9741 /label=bGH poly(A) signal /note="bovine growth hormone polyadenylation signal" rep_origin 9787..10215 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(9874..9893) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 10084..10105 /label=F1ori-F /note="F1 origin, forward primer" primer_bind complement(10224..10244) /label=pBABE 3' /note="SV40 enhancer, reverse primer for pBABE vectors" promoter 10229..10558 /label=SV40 promoter /note="SV40 enhancer and early promoter" rep_origin 10409..10544 /label=SV40 ori /note="SV40 origin of replication" primer_bind 10471..10490 /label=SV40pro-F /note="SV40 promoter/origin, forward primer" promoter 10606..10653 /label=EM7 promoter /note="synthetic bacterial promoter " CDS 10672..11046 /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 11176..11297 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(11213..11232) /label=SV40pA-R /note="SV40 polyA, reverse primer" primer_bind 11267..11286 /label=EBV-rev /note="SV40 polyA terminator, reverse primer" primer_bind complement(11346..11362) /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind complement(11346..11362) /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" primer_bind complement(11359..11381) /label=M13/pUC Reverse /note="In lacZ gene" protein_bind 11370..11386 /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(11394..11424) /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 11439..11460 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." primer_bind complement(11577..11594) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(11748..12336) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(11828..11847) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS complement(12507..13367) /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 13130..13149 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(13368..13472) /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 gctagcgagg gcctatttcc catgattcct tcatatttgc 2641 atatacgata caaggctgtt agagagataa ttggaattaa tttgactgta aacacaaaga 2701 tattagtaca aaatacgtga cgtagaaagt aataatttct tgggtagttt gcagttttaa 2761 aattatgttt taaaatggac tatcatatgc ttaccgtaac ttgaaagtat ttcgatttct 2821 tggctttata tatcttgtgg aaaggacgaa acaccggaga cgggataccg tctctgtttt 2881 agagctaggc caacatgagg atcacccatg tctgcagggc ctagcaagtt aaaataaggc 2941 tagtccgtta tcaacttggc caacatgagg atcacccatg tctgcagggc caagtggcac 3001 cgagtcggtg ctttttttgg atcctaggtc ttgaaaggag tgggaattgg ctccggtgcc 3061 cgtcagtggg cagagcgcac atcgcccaca gtccccgaga agttgggggg aggggtcggc 3121 aattgatccg gtgcctagag aaggtggcgc ggggtaaact gggaaagtga tgtcgtgtac 3181 tggctccgcc tttttcccga gggtggggga gaaccgtata taagtgcagt agtcgccgtg 3241 aacgttcttt ttcgcaacgg gtttgccgcc agaacacagg cgtacggcca ccatgaaaag 3301 gccggcggcc acgaaaaagg ccggccaggc aaaaaagaaa aaggacaaga agtacagcat 3361 cggcctggcc atcggcacca actctgtggg ctgggccgtg atcaccgacg agtacaaggt 3421 gcccagcaag aaattcaagg tgctgggcaa caccgaccgg cacagcatca agaagaacct 3481 gatcggagcc ctgctgttcg acagcggcga aacagccgag gccacccggc tgaagagaac 3541 cgccagaaga agatacacca gacggaagaa ccggatctgc tatctgcaag agatcttcag 3601 caacgagatg gccaaggtgg acgacagctt cttccacaga ctggaagagt ccttcctggt 3661 ggaagaggat aagaagcacg agcggcaccc catcttcggc aacatcgtgg acgaggtggc 3721 ctaccacgag aagtacccca ccatctacca cctgagaaag aaactggtgg acagcaccga 3781 caaggccgac ctgcggctga tctatctggc cctggcccac atgatcaagt tccggggcca 3841 cttcctgatc gagggcgacc tgaaccccga caacagcgac gtggacaagc tgttcatcca 3901 gctggtgcag acctacaacc agctgttcga ggaaaacccc atcaacgcca gcggcgtgga 3961 cgccaaggcc atcctgtctg ccagactgag caagagcaga cggctggaaa atctgatcgc 4021 ccagctgccc ggcgagaaga agaatggcct gttcggcaac ctgattgccc tgagcctggg 4081 cctgaccccc aacttcaaga gcaacttcga cctggccgag gatgccaaac tgcagctgag 4141 caaggacacc tacgacgacg acctggacaa cctgctggcc cagatcggcg accagtacgc 4201 cgacctgttt ctggccgcca agaacctgtc cgacgccatc ctgctgagcg acatcctgag 4261 agtgaacacc gagatcacca aggcccccct gagcgcctct atgatcaaga gatacgacga 4321 gcaccaccag gacctgaccc tgctgaaagc tctcgtgcgg cagcagctgc ctgagaagta 4381 caaagagatt ttcttcgacc agagcaagaa cggctacgcc ggctacattg acggcggagc 4441 cagccaggaa gagttctaca agttcatcaa gcccatcctg gaaaagatgg acggcaccga 4501 ggaactgctc gtgaagctga acagagagga cctgctgcgg aagcagcgga ccttcgacaa 4561 cggcagcatc ccccaccaga tccacctggg agagctgcac gccattctgc ggcggcagga 4621 agatttttac ccattcctga aggacaaccg ggaaaagatc gagaagatcc tgaccttccg 4681 catcccctac tacgtgggcc ctctggccag gggaaacagc agattcgcct ggatgaccag 4741 aaagagcgag gaaaccatca ccccctggaa cttcgaggaa gtggtggaca agggcgcttc 4801 cgcccagagc ttcatcgagc ggatgaccaa cttcgataag aacctgccca acgagaaggt 4861 gctgcccaag cacagcctgc tgtacgagta cttcaccgtg tataacgagc tgaccaaagt 4921 gaaatacgtg accgagggaa tgagaaagcc cgccttcctg agcggcgagc agaaaaaggc 4981 catcgtggac ctgctgttca agaccaaccg gaaagtgacc gtgaagcagc tgaaagagga 5041 ctacttcaag aaaatcgagt gcttcgactc cgtggaaatc tccggcgtgg aagatcggtt 5101 caacgcctcc ctgggcacat accacgatct gctgaaaatt atcaaggaca aggacttcct 5161 ggacaatgag gaaaacgagg acattctgga agatatcgtg ctgaccctga cactgtttga 5221 ggacagagag atgatcgagg aacggctgaa aacctatgcc cacctgttcg acgacaaagt 5281 gatgaagcag ctgaagcggc ggagatacac cggctggggc aggctgagcc ggaagctgat 5341 caacggcatc cgggacaagc agtccggcaa gacaatcctg gatttcctga agtccgacgg 5401 cttcgccaac agaaacttca tgcagctgat ccacgacgac agcctgacct ttaaagagga 5461 catccagaaa gcccaggtgt ccggccaggg cgatagcctg cacgagcaca ttgccaatct 5521 ggccggcagc cccgccatta agaagggcat cctgcagaca gtgaaggtgg tggacgagct 5581 cgtgaaagtg atgggccggc acaagcccga gaacatcgtg atcgaaatgg ccagagagaa 5641 ccagaccacc cagaagggac agaagaacag ccgcgagaga atgaagcgga tcgaagaggg 5701 catcaaagag ctgggcagcc agatcctgaa agaacacccc gtggaaaaca cccagctgca 5761 gaacgagaag ctgtacctgt actacctgca gaatgggcgg gatatgtacg tggaccagga 5821 actggacatc aaccggctgt ccgactacga tgtggaccac atcgtgcctc agagctttct 5881 gaaggacgac tccatcgaca acaaggtgct gaccagaagc gacaaggccc ggggcaagag 5941 cgacaacgtg ccctccgaag aggtcgtgaa gaagatgaag aactactggc ggcagctgct 6001 gaacgccaag ctgattaccc agagaaagtt cgacaatctg accaaggccg agagaggcgg 6061 cctgagcgaa ctggataagg ccggcttcat caagagacag ctggtggaaa cccggcagat 6121 cacaaagcac gtggcacaga tcctggactc ccggatgaac actaagtacg acgagaatga 6181 caagctgatc cgggaagtga aagtgatcac cctgaagtcc aagctggtgt ccgatttccg 6241 gaaggatttc cagttttaca aagtgcgcga gatcaacaac taccaccacg cccacgacgc 6301 ctacctgaac gccgtcgtgg gaaccgccct gatcaaaaag taccctaagc tggaaagcga 6361 gttcgtgtac ggcgactaca aggtgtacga cgtgcggaag atgatcgcca agagcgagca 6421 ggaaatcggc aaggctaccg ccaagtactt cttctacagc aacatcatga actttttcaa 6481 gaccgagatt accctggcca acggcgagat ccggaagcgg cctctgatcg agacaaacgg 6541 cgaaaccggg gagatcgtgt gggataaggg ccgggatttt gccaccgtgc ggaaagtgct 6601 gagcatgccc caagtgaata tcgtgaaaaa gaccgaggtg cagacaggcg gcttcagcaa 6661 agagtctatc ctgcccaaga ggaacagcga taagctgatc gccagaaaga aggactggga 6721 ccctaagaag tacggcggct tcgacagccc caccgtggcc tattctgtgc tggtggtggc 6781 caaagtggaa aagggcaagt ccaagaaact gaagagtgtg aaagagctgc tggggatcac 6841 catcatggaa agaagcagct tcgagaagaa tcccatcgac tttctggaag ccaagggcta 6901 caaagaagtg aaaaaggacc tgatcatcaa gctgcctaag tactccctgt tcgagctgga 6961 aaacggccgg aagagaatgc tggcctctgc cggcgaactg cagaagggaa acgaactggc 7021 cctgccctcc aaatatgtga acttcctgta cctggccagc cactatgaga agctgaaggg 7081 ctcccccgag gataatgagc agaaacagct gtttgtggaa cagcacaagc actacctgga 7141 cgagatcatc gagcagatca gcgagttctc caagagagtg atcctggccg acgctaatct 7201 ggacaaagtg ctgtccgcct acaacaagca ccgggataag cccatcagag agcaggccga 7261 gaatatcatc cacctgttta ccctgaccaa tctgggagcc cctgccgcct tcaagtactt 7321 tgacaccacc atcgaccgga agaggtacac cagcaccaaa gaggtgctgg acgccaccct 7381 gatccaccag agcatcaccg gcctgtacga gacacggatc gacctgtctc agctgggagg 7441 cgacagcgct ggaggaggtg gaagcggagg aggaggaagc ggaggaggag gtagcggacc 7501 taagaaaaag aggaaggtgg cggccgctgg atccggacgg gctgacgcat tggacgattt 7561 tgatctggat atgctgggaa gtgacgccct cgatgatttt gaccttgaca tgcttggttc 7621 ggatgccctt gatgactttg acctcgacat gctcggcagt gacgcccttg atgatttcga 7681 cctggacatg ctgattaact gtacaggcag tggagagggc agaggaagtc tgctaacatg 7741 cggtgacgtc gaggagaatc ctggcccaat ggccaagcct ttgtctcaag aagaatccac 7801 cctcattgaa agagcaacgg ctacaatcaa cagcatcccc atctctgaag actacagcgt 7861 cgccagcgca gctctctcta gcgacggccg catcttcact ggtgtcaatg tatatcattt 7921 tactggggga ccttgtgcag aactcgtggt gctgggcact gctgctgctg cggcagctgg 7981 caacctgact tgtatcgtcg cgatcggaaa tgagaacagg ggcatcttga gcccctgcgg 8041 acggtgccga caggtgcttc tcgatctgca tcctgggatc aaagccatag tgaaggacag 8101 tgatggacag ccgacggcag ttgggattcg tgaattgctg ccctctggtt atgtgtggga 8161 gggctaagaa ttcgatatca agcttatcgg taatcaacct ctggattaca aaatttgtga 8221 aagattgact ggtattctta actatgttgc tccttttacg ctatgtggat acgctgcttt 8281 aatgcctttg tatcatgcta ttgcttcccg tatggctttc attttctcct ccttgtataa 8341 atcctggttg ctgtctcttt atgaggagtt gtggcccgtt gtcaggcaac gtggcgtggt 8401 gtgcactgtg tttgctgacg caacccccac tggttggggc attgccacca cctgtcagct 8461 cctttccggg actttcgctt tccccctccc tattgccacg gcggaactca tcgccgcctg 8521 ccttgcccgc tgctggacag gggctcggct gttgggcact gacaattccg tggtgttgtc 8581 ggggaaatca tcgtcctttc cttggctgct cgcctgtgtt gccacctgga ttctgcgcgg 8641 gacgtccttc tgctacgtcc cttcggccct caatccagcg gaccttcctt cccgcggcct 8701 gctgccggct ctgcggcctc ttccgcgtct tcgccttcgc cctcagacga gtcggatctc 8761 cctttgggcc gcctccccgc atcgataccg tcgacctcga gacctagaaa aacatggagc 8821 aatcacaagt agcaatacag cagctaccaa tgctgattgt gcctggctag aagcacaaga 8881 ggaggaggag gtgggttttc cagtcacacc tcaggtacct ttaagaccaa tgacttacaa 8941 ggcagctgta gatcttagcc actttttaaa agaaaagggg ggactggaag ggctaattca 9001 ctcccaacga agacaagata tccttgatct gtggatctac cacacacaag gctacttccc 9061 tgattggcag aactacacac cagggccagg gatcagatat ccactgacct ttggatggtg 9121 ctacaagcta gtaccagttg agcaagagaa ggtagaagaa gccaatgaag gagagaacac 9181 ccgcttgtta caccctgtga gcctgcatgg gatggatgac ccggagagag aagtattaga 9241 gtggaggttt gacagccgcc tagcatttca tcacatggcc cgagagctgc atccggactg 9301 tactgggtct ctctggttag accagatctg agcctgggag ctctctggct aactagggaa 9361 cccactgctt aagcctcaat aaagcttgcc ttgagtgctt caagtagtgt gtgcccgtct 9421 gttgtgtgac tctggtaact agagatccct cagacccttt tagtcagtgt ggaaaatctc 9481 tagcagggcc cgtttaaacc cgctgatcag cctcgactgt gccttctagt tgccagccat 9541 ctgttgtttg cccctccccc gtgccttcct tgaccctgga aggtgccact cccactgtcc 9601 tttcctaata aaatgaggaa attgcatcgc attgtctgag taggtgtcat tctattctgg 9661 ggggtggggt ggggcaggac agcaaggggg aggattggga agacaatagc aggcatgctg 9721 gggatgcggt gggctctatg gcttctgagg cggaaagaac cagctggggc tctagggggt 9781 atccccacgc gccctgtagc ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg 9841 tgaccgctac acttgccagc gccctagcgc ccgctccttt cgctttcttc ccttcctttc 9901 tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg ggggctccct ttagggttcc 9961 gatttagtgc tttacggcac ctcgacccca aaaaacttga ttagggtgat ggttcacgta 10021 gtgggccatc gccctgatag acggtttttc gccctttgac gttggagtcc acgttcttta 10081 atagtggact cttgttccaa actggaacaa cactcaaccc tatctcggtc tattcttttg 10141 atttataagg gattttgccg atttcggcct attggttaaa aaatgagctg atttaacaaa 10201 aatttaacgc gaattaattc tgtggaatgt gtgtcagtta gggtgtggaa agtccccagg 10261 ctccccagca ggcagaagta tgcaaagcat gcatctcaat tagtcagcaa ccaggtgtgg 10321 aaagtcccca ggctccccag caggcagaag tatgcaaagc atgcatctca attagtcagc 10381 aaccatagtc ccgcccctaa ctccgcccat cccgccccta actccgccca gttccgccca 10441 ttctccgccc catggctgac taattttttt tatttatgca gaggccgagg ccgcctctgc 10501 ctctgagcta ttccagaagt agtgaggagg cttttttgga ggcctaggct tttgcaaaaa 10561 gctcccggga gcttgtatat ccattttcgg atctgatcag cacgtgttga caattaatca 10621 tcggcatagt atatcggcat agtataatac gacaaggtga ggaactaaac catggccaag 10681 ttgaccagtg ccgttccggt gctcaccgcg cgcgacgtcg ccggagcggt cgagttctgg 10741 accgaccggc tcgggttctc ccgggacttc gtggaggacg acttcgccgg tgtggtccgg 10801 gacgacgtga ccctgttcat cagcgcggtc caggaccagg tggtgccgga caacaccctg 10861 gcctgggtgt gggtgcgcgg cctggacgag ctgtacgccg agtggtcgga ggtcgtgtcc 10921 acgaacttcc gggacgcctc cgggccggcc atgaccgaga tcggcgagca gccgtggggg 10981 cgggagttcg ccctgcgcga cccggccggc aactgcgtgc acttcgtggc cgaggagcag 11041 gactgacacg tgctacgaga tttcgattcc accgccgcct tctatgaaag gttgggcttc 11101 ggaatcgttt tccgggacgc cggctggatg atcctccagc gcggggatct catgctggag 11161 ttcttcgccc accccaactt gtttattgca gcttataatg gttacaaata aagcaatagc 11221 atcacaaatt tcacaaataa agcatttttt tcactgcatt ctagttgtgg tttgtccaaa 11281 ctcatcaatg tatcttatca tgtctgtata ccgtcgacct ctagctagag cttggcgtaa 11341 tcatggtcat agctgtttcc tgtgtgaaat tgttatccgc tcacaattcc acacaacata 11401 cgagccggaa gcataaagtg taaagcctgg ggtgcctaat gagtgagcta actcacatta 11461 attgcgttgc gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa 11521 tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg ggcgctcttc cgcttcctcg 11581 ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag 11641 gcggtaatac ggttatccac agaatcaggg gataacgcag gaaagaacat gtgagcaaaa 11701 ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc 11761 cgcccccctg acgagcatca caaaaatcga cgctcaagtc agaggtggcg aaacccgaca 11821 ggactataaa gataccaggc gtttccccct ggaagctccc tcgtgcgctc tcctgttccg 11881 accctgccgc ttaccggata cctgtccgcc tttctccctt cgggaagcgt ggcgctttct 11941 catagctcac gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt 12001 gtgcacgaac cccccgttca gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag 12061 tccaacccgg taagacacga cttatcgcca ctggcagcag ccactggtaa caggattagc 12121 agagcgaggt atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa ctacggctac 12181 actagaagaa cagtatttgg tatctgcgct ctgctgaagc cagttacctt cggaaaaaga 12241 gttggtagct cttgatccgg caaacaaacc accgctggta gcggtggttt ttttgtttgc 12301 aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag atcctttgat cttttctacg 12361 gggtctgacg ctcagtggaa cgaaaactca cgttaaggga ttttggtcat gagattatca 12421 aaaaggatct tcacctagat ccttttaaat taaaaatgaa gttttaaatc aatctaaagt 12481 atatatgagt aaacttggtc tgacagttac caatgcttaa tcagtgaggc acctatctca 12541 gcgatctgtc tatttcgttc atccatagtt gcctgactcc ccgtcgtgta gataactacg 12601 atacgggagg gcttaccatc tggccccagt gctgcaatga taccgcgaga cccacgctca 12661 ccggctccag atttatcagc aataaaccag ccagccggaa gggccgagcg cagaagtggt 12721 cctgcaactt tatccgcctc catccagtct attaattgtt gccgggaagc tagagtaagt 12781 agttcgccag ttaatagttt gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca 12841 cgctcgtcgt ttggtatggc ttcattcagc tccggttccc aacgatcaag gcgagttaca 12901 tgatccccca tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat cgttgtcaga 12961 agtaagttgg ccgcagtgtt atcactcatg gttatggcag cactgcataa ttctcttact 13021 gtcatgccat ccgtaagatg cttttctgtg actggtgagt actcaaccaa gtcattctga 13081 gaatagtgta tgcggcgacc gagttgctct tgcccggcgt caatacggga taataccgcg 13141 ccacatagca gaactttaaa agtgctcatc attggaaaac gttcttcggg gcgaaaactc 13201 tcaaggatct taccgctgtt gagatccagt tcgatgtaac ccactcgtgc acccaactga 13261 tcttcagcat cttttacttt caccagcgtt tctgggtgag caaaaacagg aaggcaaaat 13321 gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa tactcatact cttccttttt 13381 caatattatt gaagcattta tcagggttat tgtctcatga gcggatacat atttgaatgt 13441 atttagaaaa ataaacaaat aggggttccg cgcacatttc cccgaaaagt gccacctgac //