LOCUS Exported 16811 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION LentiCRISPR v2 was modified into an all-in-one dox inducible system.. ACCESSION . VERSION . KEYWORDS TLCV2-LoxP SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 16811) TITLE Karpf Lab plasmids JOURNAL Unpublished REFERENCE 2 (bases 1 to 16811) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..16811 /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 2599..2632 /label=loxP /bound_moiety="Cre recombinase" /note="Cre-mediated recombination occurs in the 8-bp core sequence (GCATACAT)." promoter 2647..2887 /label=U6 promoter /note="RNA polymerase III promoter for human U6 snRNA" primer_bind 2647..2667 /label=hU6-F /note="Human U6 promoter, forward primer" primer_bind 2818..2837 /label=LKO.1 5' /note="Human U6 promoter, forward primer" misc_RNA 4777..4852 /label=gRNA scaffold /note="guide RNA scaffold for the Streptococcus pyogenes CRISPR/Cas9 system" promoter 4889..5203 /label=tight TRE promoter /note="Tet-responsive promoter PTight, consisting of seven tet operator sequences followed by the minimal CMV promoter" protein_bind 4897..4915 /gene="tetO" /label=tet operator /bound_moiety="tetracycline repressor TetR" /note="bacterial operator O2 for the tetR and tetA genes" protein_bind 4933..4951 /gene="tetO" /label=tet operator /bound_moiety="tetracycline repressor TetR" /note="bacterial operator O2 for the tetR and tetA genes" protein_bind 4968..4986 /gene="tetO" /label=tet operator /bound_moiety="tetracycline repressor TetR" /note="bacterial operator O2 for the tetR and tetA genes" protein_bind 5004..5022 /gene="tetO" /label=tet operator /bound_moiety="tetracycline repressor TetR" /note="bacterial operator O2 for the tetR and tetA genes" protein_bind 5040..5058 /gene="tetO" /label=tet operator /bound_moiety="tetracycline repressor TetR" /note="bacterial operator O2 for the tetR and tetA genes" protein_bind 5075..5093 /gene="tetO" /label=tet operator /bound_moiety="tetracycline repressor TetR" /note="bacterial operator O2 for the tetR and tetA genes" protein_bind 5111..5129 /gene="tetO" /label=tet operator /bound_moiety="tetracycline repressor TetR" /note="bacterial operator O2 for the tetR and tetA genes" primer_bind 5176..5200 /label=LNCX /note="Human CMV promoter, forward primer" regulatory 5229..5238 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" CDS 5235..9338 /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="MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK NLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER LKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFM QLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY LYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVP SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL DATLIHQSITGLYETRIDLSQLGGD" CDS 9339..9386 /codon_start=1 /product="bipartite nuclear localization signal from nucleoplasmin" /label=nucleoplasmin NLS /translation="KRPAATKKAGQAKKKK" CDS 9387..9410 /codon_start=1 /product="FLAG(R) epitope tag, followed by an enterokinase cleavage site" /label=FLAG /translation="DYKDDDDK" CDS 9420..9473 /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 9474..10190 /codon_start=1 /product="the original enhanced GFP (Yang et al., 1996)" /label=EGFP /note="mammalian codon-optimized" /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE FVTAAGITLGMDELYK" CDS 9474..10187 /codon_start=1 /product="enhanced GFP" /label=EGFP /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE FVTAAGITLGMDELYK" primer_bind complement(9516..9537) /label=EGFP-N /note="EGFP, reverse primer" primer_bind complement(9777..9796) /label=EXFP-R /note="For distinguishing EGFP variants, reverse primer" primer_bind 10124..10145 /label=EGFP-C /note="EGFP, forward primer" promoter 10201..10412 /label=EF-1-alpha core promoter /note="core promoter for human elongation factor EF-1-alpha" CDS 10434..11030 /codon_start=1 /gene="pac from Streptomyces alboniger" /product="puromycin N-acetyltransferase" /label=PuroR /note="confers resistance to puromycin" /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA" primer_bind complement(10434..10453) /label=Puro-R /note="Puromycin resistance gene, reverse primer. Also called puro-variant-R" primer_bind 10930..10950 /label=Puro-F /note="Puromycin resistance gene, forward primer" CDS 11040..11096 /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 11097..11843 /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" protein_bind 11850..11883 /label=loxP /bound_moiety="Cre recombinase" /note="Cre-mediated recombination occurs in the 8-bp core sequence (GCATACAT)." misc_feature 11903..12491 /label=WPRE /note="woodchuck hepatitis virus posttranscriptional regulatory element" primer_bind complement(11956..11976) /label=WPRE-R /note="WPRE, reverse primer" CDS complement(12374..12385) /codon_start=1 /product="Factor Xa recognition and cleavage site" /label=Factor Xa site /translation="IEGR" LTR 12563..12796 /label=3' LTR (Delta-U3) /note="self-inactivating 3' long terminal repeat (LTR) from HIV-1" primer_bind complement(12822..12839) /label=BGH-rev /note="Bovine growth hormone terminator, reverse primer. Also called BGH reverse" polyA_signal 12828..13052 /label=bGH poly(A) signal /note="bovine growth hormone polyadenylation signal" rep_origin 13098..13526 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(13185..13204) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 13395..13416 /label=F1ori-F /note="F1 origin, forward primer" primer_bind complement(13535..13555) /label=pBABE 3' /note="SV40 enhancer, reverse primer for pBABE vectors" promoter 13540..13869 /label=SV40 promoter /note="SV40 enhancer and early promoter" rep_origin 13720..13855 /label=SV40 ori /note="SV40 origin of replication" primer_bind 13782..13801 /label=SV40pro-F /note="SV40 promoter/origin, forward primer" promoter 13917..13964 /label=EM7 promoter /note="synthetic bacterial promoter " CDS 13983..14357 /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 14487..14608 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(14524..14543) /label=SV40pA-R /note="SV40 polyA, reverse primer" primer_bind 14578..14597 /label=EBV-rev /note="SV40 polyA terminator, reverse primer" primer_bind complement(14657..14673) /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind complement(14657..14673) /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" primer_bind complement(14670..14692) /label=M13/pUC Reverse /note="In lacZ gene" protein_bind 14681..14697 /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(14705..14735) /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 14750..14771 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." primer_bind complement(14888..14905) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(15059..15647) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(15139..15158) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS complement(15818..16678) /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 16441..16460 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(16679..16783) /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 tggttaatat aacttcgtat agcatacatt atacgaagtt atttaattaa 2641 ggtaccgagg gcctatttcc catgattcct tcatatttgc atatacgata caaggctgtt 2701 agagagataa ttagaattaa tttgactgta aacacaaaga tattagtaca aaatacgtga 2761 cgtagaaagt aataatttct tgggtagttt gcagttttaa aattatgttt taaaatggac 2821 tatcatatgc ttaccgtaac ttgaaagtat ttcgatttct tggctttata tatcttgtgg 2881 aaaggacgaa acaccggaga cggttgtaaa tgagcacaca aaatacacat gctaaaatat 2941 tatattctat gacctttata aaatcaacca aaatcttctt tttaataact ttagtatcaa 3001 taattagaat ttttatgttc ctttttgcaa acttttaata aaaatgagca aaataaaaaa 3061 acgctagttt tagtaactcg cgttgttttc ttcaccttta ataatagcta ctccaccact 3121 tgttcctaag cggtcagctc ctgcttcaat cattttttga gcatcttcaa atgttctaac 3181 tccaccagct gctttaacta aagcattgtc tttaacaact gacttcatta gtttaacatc 3241 ttcaaatgtt gcacctgatt ttgaaaatcc tgttgatgtt ttaacaaatt ctaatccagc 3301 ttcaacagct atttcacaag ctttcatgat ttcttctttt gttaataaac aattttccat 3361 aatacattta acaacatgtg atccagctgc tttttttaca gctttcatgt cttctaaaac 3421 taattcataa tttttgtctt ttaatgcacc aatatttaat accatatcaa tttctgttgc 3481 accatcttta attgcttcag aaacttcgaa tgcttttgta gctgttgtgc atgcacctag 3541 aggaaaacct acaacatttg ttattcctac atttgtgcct tttaataatt ctttacaata 3601 gcttgttcaa tatgaattaa cacaaactgt tgcaaaatca aattcaattg cttcatcaca 3661 taattgttta atttcagctt tcgtagcatc ttgttttaat aatgtgtgat ctatatattt 3721 gtttagtttc attttttctc ctatatattc atttttaatt ttaattcttt aataatttcg 3781 tctactttaa ctttagcgtt ttgaacagat tcaccaacac ctataaaata aatttttagt 3841 ttaggttcag ttccacttgg gcgaacagca aatcatgact tatcttctaa ataaaatttt 3901 agtaagtctt gtcctggcat attatacatt ccatcgatgt agtcttcaac attaacaact 3961 ttaagtccag caatttgagt taagggtgtt gctctcaatg atttcattaa tggttcaatt 4021 tttaatttct tttcttctgg tttaaaattc aagtttaaag tgaaagtgta atatgcaccc 4081 atttctttaa ataaatcttc taaatagtct actaatgttt tattttgttt tttataaaat 4141 caagcagcct ctgctattaa tatagaagct tgtattccat ctttatctct agctgagtca 4201 tcaattacat atccataact ttcttcataa gcaaaaacaa aatttaatcc gttatcttct 4261 tctttagcaa tttctctacc cattcattta aatccagtta aagtttttac aatattaact 4321 ccatattttt catgagcgat tctatcaccc aaatcacttg ttacaaaact tgaatataga 4381 gccggatttt ttggaatgct atttaagcgt tttagatttg ataattttca atcaattaaa 4441 attggtcctg tttgatttcc atctaatctt acaaaatgac catcatgttt tattgccatt 4501 ccaaatctgt cagcatctgg gtcattcata ataataatat ctgcatcatg tttaatacca 4561 tattcaagcg gtatttttca tgcaggatca aattctggat ttggatttac aacattttta 4621 aatgtttcat cttcaaatgc atgctcttca acctcaataa cgttatatcc tgattcacgt 4681 aatatttttg gggtaaattt agttcctgtt ccattaactg cgctaaaaat aatttttaaa 4741 tcttttttag cttcttgctc ttttttgtac gtctctgttt tagagctaga aatagcaagt 4801 taaaataagg ctagtccgtt atcaacttga aaaagtggca ccgagtcggt gcttttttga 4861 attcactttg gccgcgaatc gatatgtcga gtttactccc tatcagtgat agagaacgta 4921 tgtcgagttt actccctatc agtgatagag aacgatgtcg agtttactcc ctatcagtga 4981 tagagaacgt atgtcgagtt tactccctat cagtgataga gaacgtatgt cgagtttact 5041 ccctatcagt gatagagaac gtatgtcgag tttatcccta tcagtgatag agaacgtatg 5101 tcgagtttac tccctatcag tgatagagaa cgtatgtcga ggtaggcgtg tacggtggga 5161 ggcctatata agcagagctc gtttagtgaa ccgtcagatc gcctggagaa taccggttct 5221 agagcgctgc caccatggac aagaagtaca gcatcggcct ggacatcggc accaactctg 5281 tgggctgggc cgtgatcacc gacgagtaca aggtgcccag caagaaattc aaggtgctgg 5341 gcaacaccga ccggcacagc atcaagaaga acctgatcgg agccctgctg ttcgacagcg 5401 gcgaaacagc cgaggccacc cggctgaaga gaaccgccag aagaagatac accagacgga 5461 agaaccggat ctgctatctg caagagatct tcagcaacga gatggccaag gtggacgaca 5521 gcttcttcca cagactggaa gagtccttcc tggtggaaga ggataagaag cacgagcggc 5581 accccatctt cggcaacatc gtggacgagg tggcctacca cgagaagtac cccaccatct 5641 accacctgag aaagaaactg gtggacagca ccgacaaggc cgacctgcgg ctgatctatc 5701 tggccctggc ccacatgatc aagttccggg gccacttcct gatcgagggc gacctgaacc 5761 ccgacaacag cgacgtggac aagctgttca tccagctggt gcagacctac aaccagctgt 5821 tcgaggaaaa ccccatcaac gccagcggcg tggacgccaa ggccatcctg tctgccagac 5881 tgagcaagag cagacggctg gaaaatctga tcgcccagct gcccggcgag aagaagaatg 5941 gcctgttcgg aaacctgatt gccctgagcc tgggcctgac ccccaacttc aagagcaact 6001 tcgacctggc cgaggatgcc aaactgcagc tgagcaagga cacctacgac gacgacctgg 6061 acaacctgct ggcccagatc ggcgaccagt acgccgacct gtttctggcc gccaagaacc 6121 tgtccgacgc catcctgctg agcgacatcc tgagagtgaa caccgagatc accaaggccc 6181 ccctgagcgc ctctatgatc aagagatacg acgagcacca ccaggacctg accctgctga 6241 aagctctcgt gcggcagcag ctgcctgaga agtacaaaga gattttcttc gaccagagca 6301 agaacggcta cgccggctac attgacggcg gagccagcca ggaagagttc tacaagttca 6361 tcaagcccat cctggaaaag atggacggca ccgaggaact gctcgtgaag ctgaacagag 6421 aggacctgct gcggaagcag cggaccttcg acaacggcag catcccccac cagatccacc 6481 tgggagagct gcacgccatt ctgcggcggc aggaagattt ttacccattc ctgaaggaca 6541 accgggaaaa gatcgagaag atcctgacct tccgcatccc ctactacgtg ggccctctgg 6601 ccaggggaaa cagcagattc gcctggatga ccagaaagag cgaggaaacc atcaccccct 6661 ggaacttcga ggaagtggtg gacaagggcg cttccgccca gagcttcatc gagcggatga 6721 ccaacttcga taagaacctg cccaacgaga aggtgctgcc caagcacagc ctgctgtacg 6781 agtacttcac cgtgtataac gagctgacca aagtgaaata cgtgaccgag ggaatgagaa 6841 agcccgcctt cctgagcggc gagcagaaaa aggccatcgt ggacctgctg ttcaagacca 6901 accggaaagt gaccgtgaag cagctgaaag aggactactt caagaaaatc gagtgcttcg 6961 actccgtgga aatctccggc gtggaagatc ggttcaacgc ctccctgggc acataccacg 7021 atctgctgaa aattatcaag gacaaggact tcctggacaa tgaggaaaac gaggacattc 7081 tggaagatat cgtgctgacc ctgacactgt ttgaggacag agagatgatc gaggaacggc 7141 tgaaaaccta tgcccacctg ttcgacgaca aagtgatgaa gcagctgaag cggcggagat 7201 acaccggctg gggcaggctg agccggaagc tgatcaacgg catccgggac aagcagtccg 7261 gcaagacaat cctggatttc ctgaagtccg acggcttcgc caacagaaac ttcatgcagc 7321 tgatccacga cgacagcctg acctttaaag aggacatcca gaaagcccag gtgtccggcc 7381 agggcgatag cctgcacgag cacattgcca atctggccgg cagccccgcc attaagaagg 7441 gcatcctgca gacagtgaag gtggtggacg agctcgtgaa agtgatgggc cggcacaagc 7501 ccgagaacat cgtgatcgaa atggccagag agaaccagac cacccagaag ggacagaaga 7561 acagccgcga gagaatgaag cggatcgaag agggcatcaa agagctgggc agccagatcc 7621 tgaaagaaca ccccgtggaa aacacccagc tgcagaacga gaagctgtac ctgtactacc 7681 tgcagaatgg gcgggatatg tacgtggacc aggaactgga catcaaccgg ctgtccgact 7741 acgatgtgga ccatatcgtg cctcagagct ttctgaagga cgactccatc gacaacaagg 7801 tgctgaccag aagcgacaag aaccggggca agagcgacaa cgtgccctcc gaagaggtcg 7861 tgaagaagat gaagaactac tggcggcagc tgctgaacgc caagctgatt acccagagaa 7921 agttcgacaa tctgaccaag gccgagagag gcggcctgag cgaactggat aaggccggct 7981 tcatcaagag acagctggtg gaaacccggc agatcacaaa gcacgtggca cagatcctgg 8041 actcccggat gaacactaag tacgacgaga atgacaagct gatccgggaa gtgaaagtga 8101 tcaccctgaa gtccaagctg gtgtccgatt tccggaagga tttccagttt tacaaagtgc 8161 gcgagatcaa caactaccac cacgcccacg acgcctacct gaacgccgtc gtgggaaccg 8221 ccctgatcaa aaagtaccct aagctggaaa gcgagttcgt gtacggcgac tacaaggtgt 8281 acgacgtgcg gaagatgatc gccaagagcg agcaggaaat cggcaaggct accgccaagt 8341 acttcttcta cagcaacatc atgaactttt tcaagaccga gattaccctg gccaacggcg 8401 agatccggaa gcggcctctg atcgagacaa acggcgaaac cggggagatc gtgtgggata 8461 agggccggga ttttgccacc gtgcggaaag tgctgagcat gccccaagtg aatatcgtga 8521 aaaagaccga ggtgcagaca ggcggcttca gcaaagagtc tatcctgccc aagaggaaca 8581 gcgataagct gatcgccaga aagaaggact gggaccctaa gaagtacggc ggcttcgaca 8641 gccccaccgt ggcctattct gtgctggtgg tggccaaagt ggaaaagggc aagtccaaga 8701 aactgaagag tgtgaaagag ctgctgggga tcaccatcat ggaaagaagc agcttcgaga 8761 agaatcccat cgactttctg gaagccaagg gctacaaaga agtgaaaaag gacctgatca 8821 tcaagctgcc taagtactcc ctgttcgagc tggaaaacgg ccggaagaga atgctggcct 8881 ctgccggcga actgcagaag ggaaacgaac tggccctgcc ctccaaatat gtgaacttcc 8941 tgtacctggc cagccactat gagaagctga agggctcccc cgaggataat gagcagaaac 9001 agctgtttgt ggaacagcac aagcactacc tggacgagat catcgagcag atcagcgagt 9061 tctccaagag agtgatcctg gccgacgcta atctggacaa agtgctgtcc gcctacaaca 9121 agcaccggga taagcccatc agagagcagg ccgagaatat catccacctg tttaccctga 9181 ccaatctggg agcccctgcc gccttcaagt actttgacac caccatcgac cggaagaggt 9241 acaccagcac caaagaggtg ctggacgcca ccctgatcca ccagagcatc accggcctgt 9301 acgagacacg gatcgacctg tctcagctgg gaggcgacaa gcgacctgcc gccacaaaga 9361 aggctggaca ggctaagaag aagaaagatt acaaagacga tgacgataag ggatccggag 9421 agggcagagg aagtctgcta acatgcggtg acgtcgagga gaatcctggc ccagtgagca 9481 agggcgagga gctgttcacc ggggtggtgc ccatcctggt cgagctggac ggcgacgtaa 9541 acggccacaa gttcagcgtg tccggcgagg gcgagggcga tgccacctac ggcaagctga 9601 ccctgaagtt catctgcacc accggcaagc tgcccgtgcc ctggcccacc ctcgtgacca 9661 ccctgaccta cggcgtgcag tgcttcagcc gctaccccga ccacatgaag cagcacgact 9721 tcttcaagtc cgccatgccc gaaggctacg tccaggagcg caccatcttc ttcaaggacg 9781 acggcaacta caagacccgc gccgaggtga agttcgaggg cgacaccctg gtgaaccgca 9841 tcgagctgaa gggcatcgac ttcaaggagg acggcaacat cctggggcac aagctggagt 9901 acaactacaa cagccacaac gtctatatca tggccgacaa gcagaagaac ggcatcaagg 9961 tgaacttcaa gatccgccac aacatcgagg acggcagcgt gcagctcgcc gaccactacc 10021 agcagaacac ccccatcggc gacggccccg tgctgctgcc cgacaaccac tacctgagca 10081 cccagtccgc cctgagcaaa gaccccaacg agaagcgcga tcacatggtc ctgctggagt 10141 tcgtgaccgc cgccgggatc actctcggca tggacgagct gtacaagtaa gctagcatcc 10201 gggcagagcg cacatcgccc acagtccccg agaagttggg gggaggggtc ggcaattgat 10261 ccggtgccta gagaaggtgg cgcggggtaa actgggaaag tgatgtcgtg tactggctcc 10321 gcctttttcc cgagggtggg ggagaaccgt atataagtgc agtagtcgcc gtgaacgttc 10381 tttttcgcaa cgggtttgcc gccagaacac agactgcgat cgcaatgtac agtatgaccg 10441 agtacaagcc cacggtgcgc ctcgccaccc gcgacgacgt ccccagggcc gtacgcaccc 10501 tcgccgccgc gttcgccgac taccccgcca cgcgccacac cgtcgatccg gaccgccaca 10561 tcgagcgggt caccgagctg caagaactct tcctcacgcg cgtcgggctc gacatcggca 10621 aggtgtgggt cgcggacgac ggcgccgcgg tggcggtctg gaccacgccg gagagcgtcg 10681 aagcgggggc ggtgttcgcc gagatcggcc cgcgcatggc cgagttgagc ggttcccggc 10741 tggccgcgca gcaacagatg gaaggcctcc tggcgccgca ccggcccaag gagcccgcgt 10801 ggttcctggc caccgtcgga gtctcgcccg accaccaggg caagggtctg ggcagcgccg 10861 tcgtgctccc cggagtggag gcggccgagc gcgccggggt gcccgccttc ctggagacct 10921 ccgcgccccg caacctcccc ttctacgagc ggctcggctt caccgtcacc gccgacgtcg 10981 aggtgcccga aggaccgcgc acctggtgca tgacccgcaa gcccggtgcc ggttccggcg 11041 caacaaactt ctctctgctg aaacaagccg gagatgtcga agagaatcct ggaccgatgt 11101 ctagactgga caagagcaaa gtcataaacg gagctctgga attactcaat ggtgtcggta 11161 tcgaaggcct gacgacaagg aaactcgctc aaaagctggg agttgagcag cctaccctgt 11221 actggcacgt gaagaacaag cgggccctgc tcgatgccct gccaatcgag atgctggaca 11281 ggcatcatac ccacttctgc cccctggaag gcgagtcatg gcaagacttt ctgcggaaca 11341 acgccaagtc ataccgctgt gctctcctct cacatcgcga cggggctaaa gtgcatctcg 11401 gcacccgccc aacagagaaa cagtacgaaa ccctggaaaa tcagctcgcg ttcctgtgtc 11461 agcaaggctt ctccctggag aacgcactgt acgctctgtc cgccgtgggc cactttacac 11521 tgggctgcgt attggaggaa caggagcatc aagtagcaaa agaggaaaga gagacaccta 11581 ccaccgattc tatgccccca cttctgagac aagcaattga gctgttcgac cggcagggag 11641 ccgaacctgc cttccttttc ggcctggaac taatcatatg tggcctggag aaacagctaa 11701 agtgcgaaag cggcgggccg accgacgccc ttgacgattt tgacttagac atgctcccag 11761 ccgatgccct tgacgacttt gaccttgata tgctgcctgc tgacgctctt gacgattttg 11821 accttgacat gctccccggg taactcgaga taacttcgta tagcatacat tatacgaagt 11881 tattcgagcg cgttaagtcg acaatcaacc tctggattac aaaatttgtg aaagattgac 11941 tggtattctt aactatgttg ctccttttac gctatgtgga tacgctgctt taatgccttt 12001 gtatcatgct attgcttccc gtatggcttt cattttctcc tccttgtata aatcctggtt 12061 gctgtctctt tatgaggagt tgtggcccgt tgtcaggcaa cgtggcgtgg tgtgcactgt 12121 gtttgctgac gcaaccccca ctggttgggg cattgccacc acctgtcagc tcctttccgg 12181 gactttcgct ttccccctcc ctattgccac ggcggaactc atcgccgcct gccttgcccg 12241 ctgctggaca ggggctcggc tgttgggcac tgacaattcc gtggtgttgt cggggaaatc 12301 atcgtccttt ccttggctgc tcgcctgtgt tgccacctgg attctgcgcg ggacgtcctt 12361 ctgctacgtc ccttcggccc tcaatccagc ggaccttcct tcccgcggcc tgctgccggc 12421 tctgcggcct cttccgcgtc ttcgccttcg ccctcagacg agtcggatct ccctttgggc 12481 cgcctccccg cgtcgacttt aagaccaatg acttacaagg cagctgtaga tcttagccac 12541 tttttaaaag aaaagggggg actggaaggg ctaattcact cccaacgaag acaagatctg 12601 ctttttgctt gtactgggtc tctctggtta gaccagatct gagcctggga gctctctggc 12661 taactaggga acccactgct taagcctcaa taaagcttgc cttgagtgct tcaagtagtg 12721 tgtgcccgtc tgttgtgtga ctctggtaac tagagatccc tcagaccctt ttagtcagtg 12781 tggaaaatct ctagcagggc ccgtttaaac ccgctgatca gcctcgactg tgccttctag 12841 ttgccagcca tctgttgttt gcccctcccc cgtgccttcc ttgaccctgg aaggtgccac 12901 tcccactgtc ctttcctaat aaaatgagga aattgcatcg cattgtctga gtaggtgtca 12961 ttctattctg gggggtgggg tggggcagga cagcaagggg gaggattggg aagacaatag 13021 caggcatgct ggggatgcgg tgggctctat ggcttctgag gcggaaagaa ccagctgggg 13081 ctctaggggg tatccccacg cgccctgtag cggcgcatta agcgcggcgg gtgtggtggt 13141 tacgcgcagc gtgaccgcta cacttgccag cgccctagcg cccgctcctt tcgctttctt 13201 cccttccttt ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc gggggctccc 13261 tttagggttc cgatttagtg ctttacggca cctcgacccc aaaaaacttg attagggtga 13321 tggttcacgt agtgggccat cgccctgata gacggttttt cgccctttga cgttggagtc 13381 cacgttcttt aatagtggac tcttgttcca aactggaaca acactcaacc ctatctcggt 13441 ctattctttt gatttataag ggattttgcc gatttcggcc tattggttaa aaaatgagct 13501 gatttaacaa aaatttaacg cgaattaatt ctgtggaatg tgtgtcagtt agggtgtgga 13561 aagtccccag gctccccagc aggcagaagt atgcaaagca tgcatctcaa ttagtcagca 13621 accaggtgtg gaaagtcccc aggctcccca gcaggcagaa gtatgcaaag catgcatctc 13681 aattagtcag caaccatagt cccgccccta actccgccca tcccgcccct aactccgccc 13741 agttccgccc attctccgcc ccatggctga ctaatttttt ttatttatgc agaggccgag 13801 gccgcctctg cctctgagct attccagaag tagtgaggag gcttttttgg aggcctaggc 13861 ttttgcaaaa agctcccggg agcttgtata tccattttcg gatctgatca gcacgtgttg 13921 acaattaatc atcggcatag tatatcggca tagtataata cgacaaggtg aggaactaaa 13981 ccatggccaa gttgaccagt gccgttccgg tgctcaccgc gcgcgacgtc gccggagcgg 14041 tcgagttctg gaccgaccgg ctcgggttct cccgggactt cgtggaggac gacttcgccg 14101 gtgtggtccg ggacgacgtg accctgttca tcagcgcggt ccaggaccag gtggtgccgg 14161 acaacaccct ggcctgggtg tgggtgcgcg gcctggacga gctgtacgcc gagtggtcgg 14221 aggtcgtgtc cacgaacttc cgggacgcct ccgggccggc catgaccgag atcggcgagc 14281 agccgtgggg gcgggagttc gccctgcgcg acccggccgg caactgcgtg cacttcgtgg 14341 ccgaggagca ggactgacac gtgctacgag atttcgattc caccgccgcc ttctatgaaa 14401 ggttgggctt cggaatcgtt ttccgggacg ccggctggat gatcctccag cgcggggatc 14461 tcatgctgga gttcttcgcc caccccaact tgtttattgc agcttataat ggttacaaat 14521 aaagcaatag catcacaaat ttcacaaata aagcattttt ttcactgcat tctagttgtg 14581 gtttgtccaa actcatcaat gtatcttatc atgtctgtat accgtcgacc tctagctaga 14641 gcttggcgta atcatggtca tagctgtttc ctgtgtgaaa ttgttatccg ctcacaattc 14701 cacacaacat acgagccgga agcataaagt gtaaagcctg gggtgcctaa tgagtgagct 14761 aactcacatt aattgcgttg cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc 14821 agctgcatta atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt gggcgctctt 14881 ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag 14941 ctcactcaaa ggcggtaata cggttatcca cagaatcagg ggataacgca ggaaagaaca 15001 tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt 15061 tccataggct ccgcccccct gacgagcatc acaaaaatcg acgctcaagt cagaggtggc 15121 gaaacccgac aggactataa agataccagg cgtttccccc tggaagctcc ctcgtgcgct 15181 ctcctgttcc gaccctgccg cttaccggat acctgtccgc ctttctccct tcgggaagcg 15241 tggcgctttc tcatagctca cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca 15301 agctgggctg tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta tccggtaact 15361 atcgtcttga gtccaacccg gtaagacacg acttatcgcc actggcagca gccactggta 15421 acaggattag cagagcgagg tatgtaggcg gtgctacaga gttcttgaag tggtggccta 15481 actacggcta cactagaaga acagtatttg gtatctgcgc tctgctgaag ccagttacct 15541 tcggaaaaag agttggtagc tcttgatccg gcaaacaaac caccgctggt agcggtggtt 15601 tttttgtttg caagcagcag attacgcgca gaaaaaaagg atctcaagaa gatcctttga 15661 tcttttctac ggggtctgac gctcagtgga acgaaaactc acgttaaggg attttggtca 15721 tgagattatc aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga agttttaaat 15781 caatctaaag tatatatgag taaacttggt ctgacagtta ccaatgctta atcagtgagg 15841 cacctatctc agcgatctgt ctatttcgtt catccatagt tgcctgactc cccgtcgtgt 15901 agataactac gatacgggag ggcttaccat ctggccccag tgctgcaatg ataccgcgag 15961 acccacgctc accggctcca gatttatcag caataaacca gccagccgga agggccgagc 16021 gcagaagtgg tcctgcaact ttatccgcct ccatccagtc tattaattgt tgccgggaag 16081 ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt gctacaggca 16141 tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag ctccggttcc caacgatcaa 16201 ggcgagttac atgatccccc atgttgtgca aaaaagcggt tagctccttc ggtcctccga 16261 tcgttgtcag aagtaagttg gccgcagtgt tatcactcat ggttatggca gcactgcata 16321 attctcttac tgtcatgcca tccgtaagat gcttttctgt gactggtgag tactcaacca 16381 agtcattctg agaatagtgt atgcggcgac cgagttgctc ttgcccggcg tcaatacggg 16441 ataataccgc gccacatagc agaactttaa aagtgctcat cattggaaaa cgttcttcgg 16501 ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg 16561 cacccaactg atcttcagca tcttttactt tcaccagcgt ttctgggtga gcaaaaacag 16621 gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg gaaatgttga atactcatac 16681 tcttcctttt tcaatattat tgaagcattt atcagggtta ttgtctcatg agcggataca 16741 tatttgaatg tatttagaaa aataaacaaa taggggttcc gcgcacattt ccccgaaaag 16801 tgccacctga c //