LOCUS Exported 15200 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION Lentiviral vector encoding a doxycycline inducible EGFP reporter downstream of FLAG-tagged spCas9, separated by a P2A self-cleavage sequence. ACCESSION . VERSION . KEYWORDS Lenti-iCas9-neo SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 15200) AUTHORS Cao J, Wu L, Zhang SM, Lu M, Cheung WK, Cai W, Gale M, Xu Q, Yan Q TITLE An easy and efficient inducible CRISPR/Cas9 platform with improved specificity for multiple gene targeting. JOURNAL Nucleic Acids Res. 2016 Nov 2;44(19):e149. Epub 2016 Jul 25. PUBMED 27458201 REFERENCE 2 (bases 1 to 15200) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..15200 /organism="synthetic DNA construct" /mol_type="other DNA" LTR 1..634 /label=3' LTR /note="3' long terminal repeat (LTR) from HIV-1" misc_feature 681..806 /label=HIV-1 Psi /note="packaging signal of human immunodeficiency virus type 1" misc_feature 1303..1536 /label=RRE /note="The Rev response element (RRE) of HIV-1 allows for Rev-dependent mRNA export from the nucleus to the cytoplasm." CDS 1721..1765 /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 2058..2175 /label=cPPT/CTS /note="central polypurine tract and central termination sequence of HIV-1" protein_bind 2230..2500 /label=tetracycline response element /bound_moiety="tetracycline repressor TetR" /note="contains seven copies of the tetracycline operator tetO" promoter 2533..2571 /label=minimal CMV promoter /note="human cytomegalovirus (CMV) immediate early promoter" primer_bind 2568..2592 /label=LNCX /note="Human CMV promoter, forward primer" protein_bind 2701..2725 /gene="mutant version of attB" /label=attB1 /bound_moiety="BP Clonase(TM)" /note="recombination site for the Gateway(R) BP reaction" CDS 2746..2769 /codon_start=1 /product="FLAG(R) epitope tag, followed by an enterokinase cleavage site" /label=FLAG /translation="DYKDDDDK" CDS 2776..2796 /codon_start=1 /product="nuclear localization signal of SV40 (simian virus 40) large T antigen" /label=SV40 NLS /translation="PKKKRKV" CDS 2821..6921 /codon_start=1 /product="Cas9 (Csn1) endonuclease from the Streptococcus pyogenes Type II CRISPR/Cas system" /label=Cas9 /note="generates RNA-guided double strand breaks in DNA" /translation="DKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF LAEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL YYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPS EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD ATLIHQSITGLYETRIDLSQLGGD" CDS 6922..6969 /codon_start=1 /product="bipartite nuclear localization signal from nucleoplasmin" /label=nucleoplasmin NLS /translation="KRPAATKKAGQAKKKK" CDS 6985..7041 /codon_start=1 /product="2A peptide from porcine teschovirus-1 polyprotein" /label=P2A /note="Eukaryotic ribosomes fail to insert a peptide bond between the Gly and Pro residues, yielding separate polypeptides." /translation="ATNFSLLKQAGDVEENPGP" CDS 7048..7764 /codon_start=1 /product="enhanced GFP" /label=EGFP /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL EFVTAAGITLGMDELYK" primer_bind complement(7093..7114) /label=EGFP-N /note="EGFP, reverse primer" primer_bind complement(7354..7373) /label=EXFP-R /note="For distinguishing EGFP variants, reverse primer" primer_bind 7701..7722 /label=EGFP-C /note="EGFP, forward primer" protein_bind complement(7772..7796) /gene="mutant version of attB" /label=attB2 /bound_moiety="BP Clonase(TM)" /note="recombination site for the Gateway(R) BP reaction" CDS 7849..7875 /codon_start=1 /product="HA (human influenza hemagglutinin) epitope tag" /label=HA /translation="YPYDVPDYA" promoter 7961..9172 /label=UbC promoter /note="human ubiquitin C promoter" primer_bind 9009..9028 /label=hUBCpro-F /note="Human Ubiquitin C (UbC) promoter, forward primer" CDS 9231..9977 /codon_start=1 /product="improved tetracycline-controlled transactivator" /label=rtTA-Advanced /note="In the Tet-On(R) system, rtTA-Advanced binds to the Tet-responsive element and stimulates transcription only in the presence of tetracycline or doxycycline." /translation="MSRLDKSKVINGALELLNGVGIEGLTTRKLAQKLGVEQPTLYWHV KNKRALLDALPIEMLDRHHTHFCPLEGESWQDFLRNNAKSYRCALLSHRDGAKVHLGTR PTEKQYETLENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEEQEHQVAKEERETPTT DSMPPLLRQAIELFDRQGAEPAFLFGLELIICGLEKQLKCESGGPTDALDDFDLDMLPA DALDDFDLDMLPADALDDFDLDMLPG" misc_feature 10030..10603 /label=IRES /note="internal ribosome entry site (IRES) of the encephalomyocarditis virus (EMCV)" primer_bind complement(10197..10214) /label=IRES reverse /note="IRES internal ribosome entry site, reverse primer. Also called pCDH-rev" primer_bind 10424..10443 /label=IRES-F /note="IRES internal ribosome entry site, forward primer" CDS 10619..11413 /codon_start=1 /gene="aph(3')-II (or nptII)" /product="aminoglycoside phosphotransferase from Tn5" /label=NeoR/KanR /note="confers resistance to neomycin, kanamycin, and G418 (Geneticin(R))" /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF" primer_bind complement(10673..10692) /label=Neo-R /note="Neomycin resistance gene, reverse primer" primer_bind 11283..11302 /label=Neo-F /note="Neomycin resistance gene, forward primer" misc_feature 11590..12178 /label=WPRE /note="woodchuck hepatitis virus posttranscriptional regulatory element" primer_bind complement(11643..11663) /label=WPRE-R /note="WPRE, reverse primer" CDS complement(12061..12072) /codon_start=1 /product="Factor Xa recognition and cleavage site" /label=Factor Xa site /translation="IEGR" LTR 12253..12486 /label=3' LTR (Delta-U3) /note="self-inactivating 3' long terminal repeat (LTR) from HIV-1" primer_bind complement(12605..12623) /label=pBRforEco /note="pBR322 vectors, upsteam of EcoRI site, forward primer" promoter 12691..12795 /gene="bla" /label=AmpR promoter CDS 12796..13656 /codon_start=1 /gene="bla" /product="beta-lactamase" /label=AmpR /note="confers resistance to ampicillin, carbenicillin, and related antibiotics" /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS LIKHW" primer_bind complement(13014..13033) /label=Amp-R /note="Ampicillin resistance gene, reverse primer" rep_origin 13827..14415 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 14316..14335 /label=pBR322ori-F /note="pBR322 origin, forward primer" primer_bind 14569..14586 /label=L4440 /note="L4440 vector, forward primer" primer_bind 14678..14697 /label=SV40pro-F /note="SV40 promoter/origin, forward primer" protein_bind 14826..14847 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." promoter 14862..14892 /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 14900..14916 /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)." primer_bind 14905..14927 /label=M13/pUC Reverse /note="In lacZ gene" primer_bind 14924..14940 /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind 14924..14940 /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" primer_bind 14990..15009 /label=EBV-rev /note="SV40 polyA terminator, reverse primer" ORIGIN 1 tggaagggct aattcactcc caaagaagac aagatatcct tgatctgtgg atctaccaca 61 cacaaggcta cttccctgat tagcagaact acacaccagg gccaggggtc agatatccac 121 tgacctttgg atggtgctac aagctagtac cagttgagcc agataaggta gaagaggcca 181 ataaaggaga gaacaccagc ttgttacacc ctgtgagcct gcatgggatg gatgacccgg 241 agagagaagt gttagagtgg aggtttgaca gccgcctagc atttcatcac gtggcccgag 301 agctgcatcc ggagtacttc aagaactgct gatatcgagc ttgctacaag ggactttccg 361 ctggggactt tccagggagg cgtggcctgg gcgggactgg ggagtggcga gccctcagat 421 cctgcatata agcagctgct ttttgcctgt actgggtctc tctggttaga ccagatctga 481 gcctgggagc tctctggcta actagggaac ccactgctta agcctcaata aagcttgcct 541 tgagtgcttc aagtagtgtg tgcccgtctg ttgtgtgact ctggtaacta gagatccctc 601 agaccctttt agtcagtgtg gaaaatctct agcagtggcg cccgaacagg gacttgaaag 661 cgaaagggaa accagaggag ctctctcgac gcaggactcg gcttgctgaa gcgcgcacgg 721 caagaggcga ggggcggcga ctggtgagta cgccaaaaat tttgactagc ggaggctaga 781 aggagagaga tgggtgcgag agcgtcagta ttaagcgggg gagaattaga tcgcgatggg 841 aaaaaattcg gttaaggcca gggggaaaga aaaaatataa attaaaacat atagtatggg 901 caagcaggga gctagaacga ttcgcagtta atcctggcct gttagaaaca tcagaaggct 961 gtagacaaat actgggacag ctacaaccat cccttcagac aggatcagaa gaacttagat 1021 cattatataa tacagtagca accctctatt gtgtgcatca aaggatagag ataaaagaca 1081 ccaaggaagc tttagacaag atagaggaag agcaaaacaa aagtaagacc accgcacagc 1141 aagcggccgg ccgctgatct tcagacctgg aggaggagat atgagggaca attggagaag 1201 tgaattatat aaatataaag tagtaaaaat tgaaccatta ggagtagcac ccaccaaggc 1261 aaagagaaga gtggtgcaga gagaaaaaag agcagtggga ataggagctt tgttccttgg 1321 gttcttggga gcagcaggaa gcactatggg cgcagcgtca atgacgctga cggtacaggc 1381 cagacaatta ttgtctggta tagtgcagca gcagaacaat ttgctgaggg ctattgaggc 1441 gcaacagcat ctgttgcaac tcacagtctg gggcatcaag cagctccagg caagaatcct 1501 ggctgtggaa agatacctaa aggatcaaca gctcctgggg atttggggtt gctctggaaa 1561 actcatttgc accactgctg tgccttggaa tgctagttgg agtaataaat ctctggaaca 1621 gatttggaat cacacgacct ggatggagtg ggacagagaa attaacaatt acacaagctt 1681 aatacactcc ttaattgaag aatcgcaaaa ccagcaagaa aagaatgaac aagaattatt 1741 ggaattagat aaatgggcaa gtttgtggaa ttggtttaac ataacaaatt ggctgtggta 1801 tataaaatta ttcataatga tagtaggagg cttggtaggt ttaagaatag tttttgctgt 1861 actttctata gtgaatagag ttaggcaggg atattcacca ttatcgtttc agacccacct 1921 cccaaccccg aggggacccg acaggcccga aggaatagaa gaagaaggtg gagagagaga 1981 cagagacaga tccattcgat tagtgaacgg atctcgacgg tatcgccgaa ttcacaaatg 2041 gcagtattca tccacaattt taaaagaaaa ggggggattg gggggtacag tgcaggggaa 2101 agaatagtag acataatagc aacagacata caaactaaag aattacaaaa acaaattaca 2161 aaaattcaaa attttcgggt ttattacagg gacagcagag atccagtttg gactaggatc 2221 ctttaccact ccctatcagt gatagagaaa agtgaaagtc gagtttacca ctccctatca 2281 gtgatagaga aaagtgaaag tcgagtttac cactccctat cagtgataga gaaaagtgaa 2341 agtcgagttt accactccct atcagtgata gagaaaagtg aaagtcgagt ttaccactcc 2401 ctatcagtga tagagaaaag tgaaagtcga gtttaccact ccctatcagt gatagagaaa 2461 agtgaaagtc gagtttacca ctccctatca gtgatagaga aaagtgaaag tcgagctcgg 2521 tacccgggtc gaggtaggcg tgtacggtgg gaggcctata taagcagagc tcgtttagtg 2581 aaccgtcaga tcgcctggag acgccatcca cgctgttttg acctccatag aagacaccgg 2641 gaccgatcca gcctccgcgg ccccgaacta gtccagtgtg gtggaattct gcagatatca 2701 acaagtttgt acaaaaaagc aggcttcgaa ggagatagaa ccatggatta caaagacgat 2761 gacgataaga tggccccaaa gaagaagcgg aaggtcggta tccacggagt cccagcagcc 2821 gacaagaagt acagcatcgg cctggacatc ggcaccaact ctgtgggctg ggccgtgatc 2881 accgacgagt acaaggtgcc cagcaagaaa ttcaaggtgc tgggcaacac cgaccggcac 2941 agcatcaaga agaacctgat cggagccctg ctgttcgaca gcggcgaaac agccgaggcc 3001 acccggctga agagaaccgc cagaagaaga tacaccagac ggaagaaccg gatctgctat 3061 ctgcaagaga tcttcagcaa cgagatggcc aaggtggacg acagcttctt ccacagactg 3121 gaagagtcct tcctggcgga agaggataag aagcacgagc ggcaccccat cttcggcaac 3181 atcgtggacg aggtggccta ccacgagaag taccccacca tctaccacct gagaaagaaa 3241 ctggtggaca gcaccgacaa ggccgacctg cggctgatct atctggccct ggcccacatg 3301 atcaagttcc ggggccactt cctgatcgag ggcgacctga accccgacaa cagcgacgtg 3361 gacaagctgt tcatccagct ggtgcagacc tacaaccagc tgttcgagga aaaccccatc 3421 aacgccagcg gcgtggacgc caaggccatc ctgtctgcca gactgagcaa gagcagacgg 3481 ctggaaaatc tgatcgccca gctgcccggc gagaagaaga atggcctgtt cggaaacctg 3541 attgccctga gcctgggcct gacccccaac ttcaagagca acttcgacct ggccgaggat 3601 gccaaactgc agctgagcaa ggacacctac gacgacgacc tggacaacct gctggcccag 3661 atcggcgacc agtacgccga cctgtttctg gccgccaaga acctgtccga cgccatcctg 3721 ctgagcgaca tcctgagagt gaacaccgag atcaccaagg cccccctgag cgcctctatg 3781 atcaagagat acgacgagca ccaccaggac ctgaccctgc tgaaagctct cgtgcggcag 3841 cagctgcctg agaagtacaa agagattttc ttcgaccaga gcaagaacgg ctacgccggc 3901 tacattgacg gcggagccag ccaggaagag ttctacaagt tcatcaagcc catcctggaa 3961 aagatggacg gcaccgagga actgctcgtg aagctgaaca gagaggacct gctgcggaag 4021 cagcggacct tcgacaacgg cagcatcccc caccagatcc acctgggaga gctgcacgcc 4081 attctgcggc ggcaggaaga tttttaccca ttcctgaagg acaaccggga aaagatcgag 4141 aagatcctga ccttccgcat cccctactac gtgggccctc tggccagggg aaacagcaga 4201 ttcgcctgga tgaccagaaa gagcgaggaa accatcaccc cctggaactt cgaggaagtg 4261 gtggacaagg gcgcttccgc ccagagcttc atcgagcgga tgaccaactt cgataagaac 4321 ctgcccaacg agaaggtgct gcccaagcac agcctgctgt acgagtactt caccgtgtat 4381 aacgagctga ccaaagtgaa atacgtgacc gagggaatga gaaagcccgc cttcctgagc 4441 ggcgagcaga aaaaggccat cgtggacctg ctgttcaaga ccaaccggaa agtgaccgtg 4501 aagcagctga aagaggacta cttcaagaaa atcgagtgct tcgactccgt ggaaatctcc 4561 ggcgtggaag atcggttcaa cgcctccctg ggcacatacc acgatctgct gaaaattatc 4621 aaggacaagg acttcctgga caatgaggaa aacgaggaca ttctggaaga tatcgtgctg 4681 accctgacac tgtttgagga cagagagatg atcgaggaac ggctgaaaac ctatgcccac 4741 ctgttcgacg acaaagtgat gaagcagctg aagcggcgga gatacaccgg ctggggcagg 4801 ctgagccgga agctgatcaa cggcatccgg gacaagcagt ccggcaagac aatcctggat 4861 ttcctgaagt ccgacggctt cgccaacaga aacttcatgc agctgatcca cgacgacagc 4921 ctgaccttta aagaggacat ccagaaagcc caggtgtccg gccagggcga tagcctgcac 4981 gagcacattg ccaatctggc cggcagcccc gccattaaga agggcatcct gcagacagtg 5041 aaggtggtgg acgagctcgt gaaagtgatg ggccggcaca agcccgagaa catcgtgatc 5101 gaaatggcca gagagaacca gaccacccag aagggacaga agaacagccg cgagagaatg 5161 aagcggatcg aagagggcat caaagagctg ggcagccaga tcctgaaaga acaccccgtg 5221 gaaaacaccc agctgcagaa cgagaagctg tacctgtact acctgcagaa tgggcgggat 5281 atgtacgtgg accaggaact ggacatcaac cggctgtccg actacgatgt ggaccatatc 5341 gtgcctcaga gctttctgaa ggacgactcc atcgacaaca aggtgctgac cagaagcgac 5401 aagaaccggg gcaagagcga caacgtgccc tccgaagagg tcgtgaagaa gatgaagaac 5461 tactggcggc agctgctgaa cgccaagctg attacccaga gaaagttcga caatctgacc 5521 aaggccgaga gaggcggcct gagcgaactg gataaggccg gcttcatcaa gagacagctg 5581 gtggaaaccc ggcagatcac aaagcacgtg gcacagatcc tggactcccg gatgaacact 5641 aagtacgacg agaatgacaa gctgatccgg gaagtgaaag tgatcaccct gaagtccaag 5701 ctggtgtccg atttccggaa ggatttccag ttttacaaag tgcgcgagat caacaactac 5761 caccacgccc acgacgccta cctgaacgcc gtcgtgggaa ccgccctgat caaaaagtac 5821 cctaagctgg aaagcgagtt cgtgtacggc gactacaagg tgtacgacgt gcggaagatg 5881 atcgccaaga gcgagcagga aatcggcaag gctaccgcca agtacttctt ctacagcaac 5941 atcatgaact ttttcaagac cgagattacc ctggccaacg gcgagatccg gaagcggcct 6001 ctgatcgaga caaacggcga aaccggggag atcgtgtggg ataagggccg ggattttgcc 6061 accgtgcgga aagtgctgag catgccccaa gtgaatatcg tgaaaaagac cgaggtgcag 6121 acaggcggct tcagcaaaga gtctatcctg cccaagagga acagcgataa gctgatcgcc 6181 agaaagaagg actgggaccc taagaagtac ggcggcttcg acagccccac cgtggcctat 6241 tctgtgctgg tggtggccaa agtggaaaag ggcaagtcca agaaactgaa gagtgtgaaa 6301 gagctgctgg ggatcaccat catggaaaga agcagcttcg agaagaatcc catcgacttt 6361 ctggaagcca agggctacaa agaagtgaaa aaggacctga tcatcaagct gcctaagtac 6421 tccctgttcg agctggaaaa cggccggaag agaatgctgg cctctgccgg cgaactgcag 6481 aagggaaacg aactggccct gccctccaaa tatgtgaact tcctgtacct ggccagccac 6541 tatgagaagc tgaagggctc ccccgaggat aatgagcaga aacagctgtt tgtggaacag 6601 cacaagcact acctggacga gatcatcgag cagatcagcg agttctccaa gagagtgatc 6661 ctggccgacg ctaatctgga caaagtgctg tccgcctaca acaagcaccg ggataagccc 6721 atcagagagc aggccgagaa tatcatccac ctgtttaccc tgaccaatct gggagcccct 6781 gccgccttca agtactttga caccaccatc gaccggaaga ggtacaccag caccaaagag 6841 gtgctggacg ccaccctgat ccaccagagc atcaccggcc tgtacgagac acggatcgac 6901 ctgtctcagc tgggaggcga caagcgtcct gctgctacta agaaagctgg tcaagctaag 6961 aaaaagaaag ctagcggcag cggcgccacc aacttcagcc tgctgaagca ggccggcgac 7021 gtggaggaga accccggccc cgaattcatg gtgagcaagg gcgaggagct gttcaccggg 7081 gtggtgccca tcctggtcga gctggacggc gacgtaaacg gccacaagtt cagcgtgtcc 7141 ggcgagggcg agggcgatgc cacctacggc aagctgaccc tgaagttcat ctgcaccacc 7201 ggcaagctgc ccgtgccctg gcccaccctc gtgaccaccc tgacctacgg cgtgcagtgc 7261 ttcagccgct accccgacca catgaagcag cacgacttct tcaagtccgc catgcccgaa 7321 ggctacgtcc aggagcgcac catcttcttc aaggacgacg gcaactacaa gacccgcgcc 7381 gaggtgaagt tcgagggcga caccctggtg aaccgcatcg agctgaaggg catcgacttc 7441 aaggaggacg gcaacatcct ggggcacaag ctggagtaca actacaacag ccacaacgtc 7501 tatatcatgg ccgacaagca gaagaacggc atcaaggtga acttcaagat ccgccacaac 7561 atcgaggacg gcagcgtgca gctcgccgac cactaccagc agaacacccc catcggcgac 7621 ggccccgtgc tgctgcccga caaccactac ctgagcaccc agtccgccct gagcaaagac 7681 cccaacgaga agcgcgatca catggtcctg ctggagttcg tgaccgccgc cgggatcact 7741 ctcggcatgg acgagctgta caagtgatag gacccagctt tcttgtacaa agtggttgat 7801 atccagcaca gtggcggccg ctcgagtcta gagggcccgc ggttcgaata cccatacgac 7861 gtcccagact acgcttagta atgattaatt aaactagaaa ttctaccggg taggggaggc 7921 gcttttccca aggcagtctg gagtaaccca cccaagatct ggcctccgcg ccgggttttg 7981 gcgcctcccg cgggcgcccc cctcctcacg gcgagcgctg ccacgtcaga cgaagggcgc 8041 agcgagcgtc ctgatccttc cgcccggacg ctcaggacag cggcccgctg ctcataagac 8101 tcggccttag aaccccagta tcagcagaag gacattttag gacgggactt gggtgactct 8161 agggcactgg ttttctttcc agagagcgga acaggcgagg aaaagtagtc ccttctcggc 8221 gattctgcgg agggatctcc gtggggcggt gaacgccgat gattatataa ggacgcgccg 8281 ggtgtggcac agctagttcc gtcgcagccg ggatttgggt cgcggttctt gtttgtggat 8341 cgctgtgatc gtcacttggt gagtagcggg ctgctgggct ggccggggct ttcgtggccg 8401 ccgggccgct cggtgggacg gaagcgtgtg gagagaccgc caagggctgt agtctgggtc 8461 cgcgagcaag gttgccctga actgggggtt ggggggagcg cagcaaaatg gcggctgttc 8521 ccgagtcttg aatggaagac gcttgtgagg cgggctgtga ggtcgttgaa acaaggtggg 8581 gggcatggtg ggcggcaaga acccaaggtc ttgaggcctt cgctaatgcg ggaaagctct 8641 tattcgggtg agatgggctg gggcaccatc tggggaccct gacgtgaagt ttgtcactga 8701 ctggagaact cggtttgtcg tctgttgcgg gggcggcagt tatggcggtg ccgttgggca 8761 gtgcacccgt acctttggga gcgcgcgccc tcgtcgtgtc gtgacgtcac ccgttctgtt 8821 ggcttataat gcagggtggg gccacctgcc ggtaggtgtg cggtaggctt ttctccgtcg 8881 caggacgcag ggttcgggcc tagggtaggc tctcctgaat cgacaggcgc cggacctctg 8941 gtgaggggag ggataagtga ggcgtcagtt tctttggtcg gttttatgta cctatcttct 9001 taagtagctg aagctccggt tttgaactat gcgctcgggg ttggcgagtg tgttttgtga 9061 agttttttag gcaccttttg aaatgtaatc atttgggtca atatgtaatt ttcagtgtta 9121 gactagtaaa ttgtccgcta aattctggcc gtttttggct tttttgttag acgaagcttg 9181 gtaccgagct cggatctcca ccccgtaccg gtcctgcagt cgaattcacc atgtctagac 9241 tggacaagag caaagtcata aacggagctc tggaattact caatggtgtc ggtatcgaag 9301 gcctgacgac aaggaaactc gctcaaaagc tgggagttga gcagcctacc ctgtactggc 9361 acgtgaagaa caagcgggcc ctgctcgatg ccctgccaat cgagatgctg gacaggcatc 9421 atacccactt ctgccccctg gaaggcgagt catggcaaga ctttctgcgg aacaacgcca 9481 agtcataccg ctgtgctctc ctctcacatc gcgacggggc taaagtgcat ctcggcaccc 9541 gcccaacaga gaaacagtac gaaaccctgg aaaatcagct cgcgttcctg tgtcagcaag 9601 gcttctccct ggagaacgca ctgtacgctc tgtccgccgt gggccacttt acactgggct 9661 gcgtattgga ggaacaggag catcaagtag caaaagagga aagagagaca cctaccaccg 9721 attctatgcc cccacttctg agacaagcaa ttgagctgtt cgaccggcag ggagccgaac 9781 ctgccttcct tttcggcctg gaactaatca tatgtggcct ggagaaacag ctaaagtgcg 9841 aaagcggcgg gccgaccgac gcccttgacg attttgactt agacatgctc ccagccgatg 9901 cccttgacga ctttgacctt gatatgctgc ctgctgacgc tcttgacgat tttgaccttg 9961 acatgctccc cgggtaacta agtaaggatc cgcggccgca ctagaggaat tccgcccctc 10021 tccccccccc ccctctccct cccccccccc taacgttact ggccgaagcc gcttggaata 10081 aggccggtgt gcgtttgtct atatgttatt ttccaccata ttgccgtctt ttggcaatgt 10141 gagggcccgg aaacctggcc ctgtcttctt gacgagcatt cctaggggtc tttcccctct 10201 cgccaaagga atgcaaggtc tgttgaatgt cgtgaaggaa gcagttcctc tggaagcttc 10261 ttgaagacaa acaacgtctg tagcgaccct ttgcaggcag cggaaccccc cacctggcga 10321 caggtgcctc tgcggccaaa agccacgtgt ataagataca cctgcaaagg cggcacaacc 10381 ccagtgccac gttgtgagtt ggatagttgt ggaaagagtc aaatggctct cctcaagcgt 10441 attcaacaag gggctgaagg atgcccagaa ggtaccccat tgtatgggat ctgatctggg 10501 gcctcggtgc acatgcttta catgtgttta gtcgaggtta aaaaaacgtc taggcccccc 10561 gaaccacggg gacgtggttt tcctttgaaa aacacgatga taagcttacc ggtccaccat 10621 gattgaacaa gatggattgc acgcaggttc tccggccgct tgggtggaga ggctattcgg 10681 ctatgactgg gcacaacaga caatcggctg ctctgatgcc gccgtgttcc ggctgtcagc 10741 gcaggggcgc ccggttcttt ttgtcaagac cgacctgtcc ggtgccctga atgaactgca 10801 agacgaggca gcgcggctat cgtggctggc cacgacgggc gttccttgcg cagctgtgct 10861 cgacgttgtc actgaagcgg gaagggactg gctgctattg ggcgaagtgc cggggcagga 10921 tctcctgtca tctcaccttg ctcctgccga gaaagtatcc atcatggctg atgcaatgcg 10981 gcggctgcat acgcttgatc cggctacctg cccattcgac caccaagcga aacatcgcat 11041 cgagcgagca cgtactcgga tggaagccgg tcttgtcgat caggatgatc tggacgaaga 11101 gcatcagggg ctcgcgccag ccgaactgtt cgccaggctc aaggcgagca tgcccgacgg 11161 cgaggatctc gtcgtgaccc atggcgatgc ctgcttgccg aatatcatgg tggaaaatgg 11221 ccgcttttct ggattcatcg actgtggccg gctgggtgtg gcggaccgct atcaggacat 11281 agcgttggct acccgtgata ttgctgaaga gcttggcggc gaatgggctg accgcttcct 11341 cgtgctttac ggtatcgccg ctcccgattc gcagcgcatc gccttctatc gccttcttga 11401 cgagttcttc tgatgtacaa gtaaagcggc cgcgactcta gatcataatc agccatacca 11461 catttgtaga ggttttactt gctttaaaaa acctcccaca cctccccctg aacctgaaac 11521 ataaaatgaa tgcaattgtt gttgtttagt ccctcccaat tcgatatcaa gcttatcgcg 11581 atagatccta atcaacctct ggattacaaa atttgtgaaa gattgactgg tattcttaac 11641 tatgttgctc cttttacgct atgtggatac gctgctttaa tgcctttgta tcatgctatt 11701 gcttcccgta tggctttcat tttctcctcc ttgtataaat cctggttgct gtctctttat 11761 gaggagttgt ggcccgttgt caggcaacgt ggcgtggtgt gcactgtgtt tgctgacgca 11821 acccccactg gttggggcat tgccaccacc tgtcagctcc tttccgggac tttcgctttc 11881 cccctcccta ttgccacggc ggaactcatc gccgcctgcc ttgcccgctg ctggacaggg 11941 gctcggctgt tgggcactga caattccgtg gtgttgtcgg ggaaatcatc gtcctttcct 12001 tggctgctcg cctgtgttgc cacctggatt ctgcgcggga cgtccttctg ctacgtccct 12061 tcggccctca atccagcgga ccttccttcc cgcggcctgc tgccggctct gcggcctctt 12121 ccgcgtcttc gccttcgccc tcagacgagt cggatctccc tttgggccgc ctccccgcct 12181 gagatccttt aagaccaatg acttacaagg cagctgtaga tcttagccac tttttaaaag 12241 aaaagggggg actggaaggg ctaattcact cccaacgaag acaagatctg ctttttgctt 12301 gtactgggtc tctctggtta gaccagatct gagcctggga gctctctggc taactaggga 12361 acccactgct taagcctcaa taaagcttgc cttgagtgct tcaagtagtg tgtgcccgtc 12421 tgttgtgtga ctctggtaac tagagatccc tcagaccctt ttagtcagtg tggaaaatct 12481 ctagcagtag tagttcatgt catcttatta ttcagtattt ataacttgca aagaaatgaa 12541 tatcagagag tgagaggccc gggttaatta aggaaagggc tagatcattc ttgaagacga 12601 aagggcctcg tgatacgcct atttttatag gttaatgtca tgataataat ggtttcttag 12661 acgtcaggtg gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt atttttctaa 12721 atacattcaa atatgtatcc gctcatgaga caataaccct gataaatgct tcaataatat 12781 tgaaaaagga agagtatgag tattcaacat ttccgtgtcg cccttattcc cttttttgcg 12841 gcattttgcc ttcctgtttt tgctcaccca gaaacgctgg tgaaagtaaa agatgctgaa 12901 gatcagttgg gtgcacgagt gggttacatc gaactggatc tcaacagcgg taagatcctt 12961 gagagttttc gccccgaaga acgttttcca atgatgagca cttttaaagt tctgctatgt 13021 ggcgcggtat tatcccgtgt tgacgccggg caagagcaac tcggtcgccg catacactat 13081 tctcagaatg acttggttga gtactcacca gtcacagaaa agcatcttac ggatggcatg 13141 acagtaagag aattatgcag tgctgccata accatgagtg ataacactgc ggccaactta 13201 cttctgacaa cgatcggagg accgaaggag ctaaccgctt ttttgcacaa catgggggat 13261 catgtaactc gccttgatcg ttgggaaccg gagctgaatg aagccatacc aaacgacgag 13321 cgtgacacca cgatgcctgt agcaatggca acaacgttgc gcaaactatt aactggcgaa 13381 ctacttactc tagcttcccg gcaacaatta atagactgga tggaggcgga taaagttgca 13441 ggaccacttc tgcgctcggc ccttccggct ggctggttta ttgctgataa atctggagcc 13501 ggtgagcgtg ggtctcgcgg tatcattgca gcactggggc cagatggtaa gccctcccgt 13561 atcgtagtta tctacacgac ggggagtcag gcaactatgg atgaacgaaa tagacagatc 13621 gctgagatag gtgcctcact gattaagcat tggtaactgt cagaccaagt ttactcatat 13681 atactttaga ttgatttaaa acttcatttt taatttaaaa ggatctaggt gaagatcctt 13741 tttgataatc tcatgaccaa aatcccttaa cgtgagtttt cgttccactg agcgtcagac 13801 cccgtagaaa agatcaaagg atcttcttga gatccttttt ttctgcgcgt aatctgctgc 13861 ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt tgccggatca agagctacca 13921 actctttttc cgaaggtaac tggcttcagc agagcgcaga taccaaatac tgttcttcta 13981 gtgtagccgt agttaggcca ccacttcaag aactctgtag caccgcctac atacctcgct 14041 ctgctaatcc tgttaccagt ggctgctgcc agtggcgata agtcgtgtct taccgggttg 14101 gactcaagac gatagttacc ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc 14161 acacagccca gcttggagcg aacgacctac accgaactga gatacctaca gcgtgagcta 14221 tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca ggtatccggt aagcggcagg 14281 gtcggaacag gagagcgcac gagggagctt ccagggggaa acgcctggta tctttatagt 14341 cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg 14401 cggagcctat ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc cttttgctgg 14461 ccttttgctc acatgttctt tcctgcgtta tcccctgatt ctgtggataa ccgtattacc 14521 gcctttgagt gagctgatac cgctcgccgc agccgaacga ccgagcgcag cgagtcagtg 14581 agcgaggaag cggaagagcg cccaatacgc aaaccgcctc tccccgcgcg ttggccgatt 14641 cattaatgca gcaagctcat ggctgactaa ttttttttat ttatgcagag gccgaggccg 14701 cctcggcctc tgagctattc cagaagtagt gaggaggctt ttttggaggc ctaggctttt 14761 gcaaaaagct ccccgtggca cgacaggttt cccgactgga aagcgggcag tgagcgcaac 14821 gcaattaatg tgagttagct cactcattag gcaccccagg ctttacactt tatgcttccg 14881 gctcgtatgt tgtgtggaat tgtgagcgga taacaatttc acacaggaaa cagctatgac 14941 atgattacga atttcacaaa taaagcattt ttttcactgc attctagttg tggtttgtcc 15001 aaactcatca atgtatctta tcatgtctgg atcaactgga taactcaagc taaccaaaat 15061 catcccaaac ttcccacccc ataccctatt accactgcca attacctgtg gtttcattta 15121 ctctaaacct gtgattcctc tgaattattt tcattttaaa gaaattgtat ttgttaaata 15181 tgtactacaa acttagtagt //