LOCUS Exported 13364 bp ds-DNA circular SYN 13-MAY-2021 DEFINITION Multiplex Guide RNAs to generate Nr4a1 knockout by CRISPR. ACCESSION . VERSION . KEYWORDS lentiCRISPR v2-Nr4a1 double gRNAs SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 13364) AUTHORS Zhang Y, Federation AJ, Kim S, O'Keefe JP, Lun M, Xiang D, Brown JD, Steinhauser ML TITLE Targeting nuclear receptor NR4A1-dependent adipocyte progenitor quiescence promotes metabolic adaptation to obesity. JOURNAL J Clin Invest. 2018 Nov 1;128(11):4898-4911. doi: 10.1172/JCI98353. Epub 2018 Oct 2. PUBMED 30277475 REFERENCE 2 (bases 1 to 13364) AUTHORS . TITLE Direct Submission JOURNAL Exported May 13, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..13364 /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 2608..2848 /label=U6 promoter /note="RNA polymerase III promoter for human U6 snRNA" misc_RNA 2878..2953 /label=gRNA scaffold /note="guide RNA scaffold for the Streptococcus pyogenes CRISPR/Cas9 system" promoter 2966..3206 /label=U6 promoter /note="RNA polymerase III promoter for human U6 snRNA" misc_RNA 3235..3310 /label=gRNA scaffold /note="guide RNA scaffold for the Streptococcus pyogenes CRISPR/Cas9 system" promoter 3366..3577 /label=EF-1-alpha core promoter /note="core promoter for human elongation factor EF-1-alpha" regulatory 3596..3605 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" CDS 3602..7705 /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 7706..7753 /codon_start=1 /product="bipartite nuclear localization signal from nucleoplasmin" /label=nucleoplasmin NLS /translation="KRPAATKKAGQAKKKK" CDS 7754..7777 /codon_start=1 /product="FLAG(R) epitope tag, followed by an enterokinase cleavage site" /label=FLAG /translation="DYKDDDDK" CDS 7787..7843 /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 7844..8440 /codon_start=1 /gene="pac from Streptomyces alboniger" /product="puromycin N-acetyltransferase" /label=PuroR /note="confers resistance to puromycin" /translation="TEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIERV TELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLAA QQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETSA PRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA" primer_bind 8337..8357 /label=Puro-F /note="Puromycin resistance gene, forward primer" misc_feature 8456..9044 /label=WPRE /note="woodchuck hepatitis virus posttranscriptional regulatory element" primer_bind complement(8509..8529) /label=WPRE-R /note="WPRE, reverse primer" CDS complement(8927..8938) /codon_start=1 /product="Factor Xa recognition and cleavage site" /label=Factor Xa site /translation="IEGR" LTR 9116..9349 /label=3' LTR (Delta-U3) /note="self-inactivating 3' long terminal repeat (LTR) from HIV-1" primer_bind complement(9375..9392) /label=BGH-rev /note="Bovine growth hormone terminator, reverse primer. Also called BGH reverse" polyA_signal 9381..9605 /label=bGH poly(A) signal /note="bovine growth hormone polyadenylation signal" rep_origin 9651..10079 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(9738..9757) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 9948..9969 /label=F1ori-F /note="F1 origin, forward primer" primer_bind complement(10088..10108) /label=pBABE 3' /note="SV40 enhancer, reverse primer for pBABE vectors" promoter 10093..10422 /label=SV40 promoter /note="SV40 enhancer and early promoter" rep_origin 10273..10408 /label=SV40 ori /note="SV40 origin of replication" primer_bind 10335..10354 /label=SV40pro-F /note="SV40 promoter/origin, forward primer" promoter 10470..10517 /label=EM7 promoter /note="synthetic bacterial promoter " CDS 10536..10910 /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 11040..11161 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(11077..11096) /label=SV40pA-R /note="SV40 polyA, reverse primer" primer_bind 11131..11150 /label=EBV-rev /note="SV40 polyA terminator, reverse primer" primer_bind complement(11210..11226) /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind complement(11210..11226) /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" primer_bind complement(11223..11245) /label=M13/pUC Reverse /note="In lacZ gene" protein_bind 11234..11250 /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(11258..11288) /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 11303..11324 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." primer_bind complement(11441..11458) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(11612..12200) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(11692..11711) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS complement(12371..13231) /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 12994..13013 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(13232..13336) /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 aggtaccgag ggcctatttc ccatgattcc ttcatatttg 2641 catatacgat acaaggctgt tagagagata attagaatta atttgactgt aaacacaaag 2701 atattagtac aaaatacgtg acgtagaaag taataatttc ttgggtagtt tgcagtttta 2761 aaattatgtt ttaaaatgga ctatcatatg cttaccgtaa cttgaaagta tttcgatttc 2821 ttggctttat atatcttgtg gaaaggacga aacaccgccg ggtagcagcc gtacaccgtt 2881 ttagagctag aaatagcaag ttaaaataag gctagtccgt tatcaacttg aaaaagtggc 2941 accgagtcgg tgcttttttg aattcgaggg cctatttccc atgattcctt catatttgca 3001 tatacgatac aaggctgtta gagagataat tagaattaat ttgactgtaa acacaaagat 3061 attagtacaa aatacgtgac gtagaaagta ataatttctt gggtagtttg cagttttaaa 3121 attatgtttt aaaatggact atcatatgct taccgtaact tgaaagtatt tcgatttctt 3181 ggctttatat atcttgtgga aaggacgaaa caccgtccaa gtgtgcccgg atgagtttta 3241 gagctagaaa tagcaagtta aaataaggct agtccgttat caacttgaaa aagtggcacc 3301 gagtcggtgc ttttttgcta gctaggtctt gaaaggagtg ggaattggct ccggtgcccg 3361 tcagtgggca gagcgcacat cgcccacagt ccccgagaag ttggggggag gggtcggcaa 3421 ttgatccggt gcctagagaa ggtggcgcgg ggtaaactgg gaaagtgatg tcgtgtactg 3481 gctccgcctt tttcccgagg gtgggggaga accgtatata agtgcagtag tcgccgtgaa 3541 cgttcttttt cgcaacgggt ttgccgccag aacacaggac cggttctaga gcgctgccac 3601 catggacaag aagtacagca tcggcctgga catcggcacc aactctgtgg gctgggccgt 3661 gatcaccgac gagtacaagg tgcccagcaa gaaattcaag gtgctgggca acaccgaccg 3721 gcacagcatc aagaagaacc tgatcggagc cctgctgttc gacagcggcg aaacagccga 3781 ggccacccgg ctgaagagaa ccgccagaag aagatacacc agacggaaga accggatctg 3841 ctatctgcaa gagatcttca gcaacgagat ggccaaggtg gacgacagct tcttccacag 3901 actggaagag tccttcctgg tggaagagga taagaagcac gagcggcacc ccatcttcgg 3961 caacatcgtg gacgaggtgg cctaccacga gaagtacccc accatctacc acctgagaaa 4021 gaaactggtg gacagcaccg acaaggccga cctgcggctg atctatctgg ccctggccca 4081 catgatcaag ttccggggcc acttcctgat cgagggcgac ctgaaccccg acaacagcga 4141 cgtggacaag ctgttcatcc agctggtgca gacctacaac cagctgttcg aggaaaaccc 4201 catcaacgcc agcggcgtgg acgccaaggc catcctgtct gccagactga gcaagagcag 4261 acggctggaa aatctgatcg cccagctgcc cggcgagaag aagaatggcc tgttcggaaa 4321 cctgattgcc ctgagcctgg gcctgacccc caacttcaag agcaacttcg acctggccga 4381 ggatgccaaa ctgcagctga gcaaggacac ctacgacgac gacctggaca acctgctggc 4441 ccagatcggc gaccagtacg ccgacctgtt tctggccgcc aagaacctgt ccgacgccat 4501 cctgctgagc gacatcctga gagtgaacac cgagatcacc aaggcccccc tgagcgcctc 4561 tatgatcaag agatacgacg agcaccacca ggacctgacc ctgctgaaag ctctcgtgcg 4621 gcagcagctg cctgagaagt acaaagagat tttcttcgac cagagcaaga acggctacgc 4681 cggctacatt gacggcggag ccagccagga agagttctac aagttcatca agcccatcct 4741 ggaaaagatg gacggcaccg aggaactgct cgtgaagctg aacagagagg acctgctgcg 4801 gaagcagcgg accttcgaca acggcagcat cccccaccag atccacctgg gagagctgca 4861 cgccattctg cggcggcagg aagattttta cccattcctg aaggacaacc gggaaaagat 4921 cgagaagatc ctgaccttcc gcatccccta ctacgtgggc cctctggcca ggggaaacag 4981 cagattcgcc tggatgacca gaaagagcga ggaaaccatc accccctgga acttcgagga 5041 agtggtggac aagggcgctt ccgcccagag cttcatcgag cggatgacca acttcgataa 5101 gaacctgccc aacgagaagg tgctgcccaa gcacagcctg ctgtacgagt acttcaccgt 5161 gtataacgag ctgaccaaag tgaaatacgt gaccgaggga atgagaaagc ccgccttcct 5221 gagcggcgag cagaaaaagg ccatcgtgga cctgctgttc aagaccaacc ggaaagtgac 5281 cgtgaagcag ctgaaagagg actacttcaa gaaaatcgag tgcttcgact ccgtggaaat 5341 ctccggcgtg gaagatcggt tcaacgcctc cctgggcaca taccacgatc tgctgaaaat 5401 tatcaaggac aaggacttcc tggacaatga ggaaaacgag gacattctgg aagatatcgt 5461 gctgaccctg acactgtttg aggacagaga gatgatcgag gaacggctga aaacctatgc 5521 ccacctgttc gacgacaaag tgatgaagca gctgaagcgg cggagataca ccggctgggg 5581 caggctgagc cggaagctga tcaacggcat ccgggacaag cagtccggca agacaatcct 5641 ggatttcctg aagtccgacg gcttcgccaa cagaaacttc atgcagctga tccacgacga 5701 cagcctgacc tttaaagagg acatccagaa agcccaggtg tccggccagg gcgatagcct 5761 gcacgagcac attgccaatc tggccggcag ccccgccatt aagaagggca tcctgcagac 5821 agtgaaggtg gtggacgagc tcgtgaaagt gatgggccgg cacaagcccg agaacatcgt 5881 gatcgaaatg gccagagaga accagaccac ccagaaggga cagaagaaca gccgcgagag 5941 aatgaagcgg atcgaagagg gcatcaaaga gctgggcagc cagatcctga aagaacaccc 6001 cgtggaaaac acccagctgc agaacgagaa gctgtacctg tactacctgc agaatgggcg 6061 ggatatgtac gtggaccagg aactggacat caaccggctg tccgactacg atgtggacca 6121 tatcgtgcct cagagctttc tgaaggacga ctccatcgac aacaaggtgc tgaccagaag 6181 cgacaagaac cggggcaaga gcgacaacgt gccctccgaa gaggtcgtga agaagatgaa 6241 gaactactgg cggcagctgc tgaacgccaa gctgattacc cagagaaagt tcgacaatct 6301 gaccaaggcc gagagaggcg gcctgagcga actggataag gccggcttca tcaagagaca 6361 gctggtggaa acccggcaga tcacaaagca cgtggcacag atcctggact cccggatgaa 6421 cactaagtac gacgagaatg acaagctgat ccgggaagtg aaagtgatca ccctgaagtc 6481 caagctggtg tccgatttcc ggaaggattt ccagttttac aaagtgcgcg agatcaacaa 6541 ctaccaccac gcccacgacg cctacctgaa cgccgtcgtg ggaaccgccc tgatcaaaaa 6601 gtaccctaag ctggaaagcg agttcgtgta cggcgactac aaggtgtacg acgtgcggaa 6661 gatgatcgcc aagagcgagc aggaaatcgg caaggctacc gccaagtact tcttctacag 6721 caacatcatg aactttttca agaccgagat taccctggcc aacggcgaga tccggaagcg 6781 gcctctgatc gagacaaacg gcgaaaccgg ggagatcgtg tgggataagg gccgggattt 6841 tgccaccgtg cggaaagtgc tgagcatgcc ccaagtgaat atcgtgaaaa agaccgaggt 6901 gcagacaggc ggcttcagca aagagtctat cctgcccaag aggaacagcg ataagctgat 6961 cgccagaaag aaggactggg accctaagaa gtacggcggc ttcgacagcc ccaccgtggc 7021 ctattctgtg ctggtggtgg ccaaagtgga aaagggcaag tccaagaaac tgaagagtgt 7081 gaaagagctg ctggggatca ccatcatgga aagaagcagc ttcgagaaga atcccatcga 7141 ctttctggaa gccaagggct acaaagaagt gaaaaaggac ctgatcatca agctgcctaa 7201 gtactccctg ttcgagctgg aaaacggccg gaagagaatg ctggcctctg ccggcgaact 7261 gcagaaggga aacgaactgg ccctgccctc caaatatgtg aacttcctgt acctggccag 7321 ccactatgag aagctgaagg gctcccccga ggataatgag cagaaacagc tgtttgtgga 7381 acagcacaag cactacctgg acgagatcat cgagcagatc agcgagttct ccaagagagt 7441 gatcctggcc gacgctaatc tggacaaagt gctgtccgcc tacaacaagc accgggataa 7501 gcccatcaga gagcaggccg agaatatcat ccacctgttt accctgacca atctgggagc 7561 ccctgccgcc ttcaagtact ttgacaccac catcgaccgg aagaggtaca ccagcaccaa 7621 agaggtgctg gacgccaccc tgatccacca gagcatcacc ggcctgtacg agacacggat 7681 cgacctgtct cagctgggag gcgacaagcg acctgccgcc acaaagaagg ctggacaggc 7741 taagaagaag aaagattaca aagacgatga cgataaggga tccggcgcaa caaacttctc 7801 tctgctgaaa caagccggag atgtcgaaga gaatcctgga ccgaccgagt acaagcccac 7861 ggtgcgcctc gccacccgcg acgacgtccc cagggccgta cgcaccctcg ccgccgcgtt 7921 cgccgactac cccgccacgc gccacaccgt cgatccggac cgccacatcg agcgggtcac 7981 cgagctgcaa gaactcttcc tcacgcgcgt cgggctcgac atcggcaagg tgtgggtcgc 8041 ggacgacggc gccgcggtgg cggtctggac cacgccggag agcgtcgaag cgggggcggt 8101 gttcgccgag atcggcccgc gcatggccga gttgagcggt tcccggctgg ccgcgcagca 8161 acagatggaa ggcctcctgg cgccgcaccg gcccaaggag cccgcgtggt tcctggccac 8221 cgtcggagtc tcgcccgacc accagggcaa gggtctgggc agcgccgtcg tgctccccgg 8281 agtggaggcg gccgagcgcg ccggggtgcc cgccttcctg gagacctccg cgccccgcaa 8341 cctccccttc tacgagcggc tcggcttcac cgtcaccgcc gacgtcgagg tgcccgaagg 8401 accgcgcacc tggtgcatga cccgcaagcc cggtgcctga acgcgttaag tcgacaatca 8461 acctctggat tacaaaattt gtgaaagatt gactggtatt cttaactatg ttgctccttt 8521 tacgctatgt ggatacgctg ctttaatgcc tttgtatcat gctattgctt cccgtatggc 8581 tttcattttc tcctccttgt ataaatcctg gttgctgtct ctttatgagg agttgtggcc 8641 cgttgtcagg caacgtggcg tggtgtgcac tgtgtttgct gacgcaaccc ccactggttg 8701 gggcattgcc accacctgtc agctcctttc cgggactttc gctttccccc tccctattgc 8761 cacggcggaa ctcatcgccg cctgccttgc ccgctgctgg acaggggctc ggctgttggg 8821 cactgacaat tccgtggtgt tgtcggggaa atcatcgtcc tttccttggc tgctcgcctg 8881 tgttgccacc tggattctgc gcgggacgtc cttctgctac gtcccttcgg ccctcaatcc 8941 agcggacctt ccttcccgcg gcctgctgcc ggctctgcgg cctcttccgc gtcttcgcct 9001 tcgccctcag acgagtcgga tctccctttg ggccgcctcc ccgcgtcgac tttaagacca 9061 atgacttaca aggcagctgt agatcttagc cactttttaa aagaaaaggg gggactggaa 9121 gggctaattc actcccaacg aagacaagat ctgctttttg cttgtactgg gtctctctgg 9181 ttagaccaga tctgagcctg ggagctctct ggctaactag ggaacccact gcttaagcct 9241 caataaagct tgccttgagt gcttcaagta gtgtgtgccc gtctgttgtg tgactctggt 9301 aactagagat ccctcagacc cttttagtca gtgtggaaaa tctctagcag ggcccgttta 9361 aacccgctga tcagcctcga ctgtgccttc tagttgccag ccatctgttg tttgcccctc 9421 ccccgtgcct tccttgaccc tggaaggtgc cactcccact gtcctttcct aataaaatga 9481 ggaaattgca tcgcattgtc tgagtaggtg tcattctatt ctggggggtg gggtggggca 9541 ggacagcaag ggggaggatt gggaagacaa tagcaggcat gctggggatg cggtgggctc 9601 tatggcttct gaggcggaaa gaaccagctg gggctctagg gggtatcccc acgcgccctg 9661 tagcggcgca ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg ctacacttgc 9721 cagcgcccta gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca cgttcgccgg 9781 ctttccccgt caagctctaa atcgggggct ccctttaggg ttccgattta gtgctttacg 9841 gcacctcgac cccaaaaaac ttgattaggg tgatggttca cgtagtgggc catcgccctg 9901 atagacggtt tttcgccctt tgacgttgga gtccacgttc tttaatagtg gactcttgtt 9961 ccaaactgga acaacactca accctatctc ggtctattct tttgatttat aagggatttt 10021 gccgatttcg gcctattggt taaaaaatga gctgatttaa caaaaattta acgcgaatta 10081 attctgtgga atgtgtgtca gttagggtgt ggaaagtccc caggctcccc agcaggcaga 10141 agtatgcaaa gcatgcatct caattagtca gcaaccaggt gtggaaagtc cccaggctcc 10201 ccagcaggca gaagtatgca aagcatgcat ctcaattagt cagcaaccat agtcccgccc 10261 ctaactccgc ccatcccgcc cctaactccg cccagttccg cccattctcc gccccatggc 10321 tgactaattt tttttattta tgcagaggcc gaggccgcct ctgcctctga gctattccag 10381 aagtagtgag gaggcttttt tggaggccta ggcttttgca aaaagctccc gggagcttgt 10441 atatccattt tcggatctga tcagcacgtg ttgacaatta atcatcggca tagtatatcg 10501 gcatagtata atacgacaag gtgaggaact aaaccatggc caagttgacc agtgccgttc 10561 cggtgctcac cgcgcgcgac gtcgccggag cggtcgagtt ctggaccgac cggctcgggt 10621 tctcccggga cttcgtggag gacgacttcg ccggtgtggt ccgggacgac gtgaccctgt 10681 tcatcagcgc ggtccaggac caggtggtgc cggacaacac cctggcctgg gtgtgggtgc 10741 gcggcctgga cgagctgtac gccgagtggt cggaggtcgt gtccacgaac ttccgggacg 10801 cctccgggcc ggccatgacc gagatcggcg agcagccgtg ggggcgggag ttcgccctgc 10861 gcgacccggc cggcaactgc gtgcacttcg tggccgagga gcaggactga cacgtgctac 10921 gagatttcga ttccaccgcc gccttctatg aaaggttggg cttcggaatc gttttccggg 10981 acgccggctg gatgatcctc cagcgcgggg atctcatgct ggagttcttc gcccacccca 11041 acttgtttat tgcagcttat aatggttaca aataaagcaa tagcatcaca aatttcacaa 11101 ataaagcatt tttttcactg cattctagtt gtggtttgtc caaactcatc aatgtatctt 11161 atcatgtctg tataccgtcg acctctagct agagcttggc gtaatcatgg tcatagctgt 11221 ttcctgtgtg aaattgttat ccgctcacaa ttccacacaa catacgagcc ggaagcataa 11281 agtgtaaagc ctggggtgcc taatgagtga gctaactcac attaattgcg ttgcgctcac 11341 tgcccgcttt ccagtcggga aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg 11401 cggggagagg cggtttgcgt attgggcgct cttccgcttc ctcgctcact gactcgctgc 11461 gctcggtcgt tcggctgcgg cgagcggtat cagctcactc aaaggcggta atacggttat 11521 ccacagaatc aggggataac gcaggaaaga acatgtgagc aaaaggccag caaaaggcca 11581 ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag gctccgcccc cctgacgagc 11641 atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc gacaggacta taaagatacc 11701 aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg 11761 gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta 11821 ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg 11881 ttcagcccga ccgctgcgcc ttatccggta actatcgtct tgagtccaac ccggtaagac 11941 acgacttatc gccactggca gcagccactg gtaacaggat tagcagagcg aggtatgtag 12001 gcggtgctac agagttcttg aagtggtggc ctaactacgg ctacactaga agaacagtat 12061 ttggtatctg cgctctgctg aagccagtta ccttcggaaa aagagttggt agctcttgat 12121 ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag cagattacgc 12181 gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc tacggggtct gacgctcagt 12241 ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt atcaaaaagg atcttcacct 12301 agatcctttt aaattaaaaa tgaagtttta aatcaatcta aagtatatat gagtaaactt 12361 ggtctgacag ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc tgtctatttc 12421 gttcatccat agttgcctga ctccccgtcg tgtagataac tacgatacgg gagggcttac 12481 catctggccc cagtgctgca atgataccgc gagacccacg ctcaccggct ccagatttat 12541 cagcaataaa ccagccagcc ggaagggccg agcgcagaag tggtcctgca actttatccg 12601 cctccatcca gtctattaat tgttgccggg aagctagagt aagtagttcg ccagttaata 12661 gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt gtcacgctcg tcgtttggta 12721 tggcttcatt cagctccggt tcccaacgat caaggcgagt tacatgatcc cccatgttgt 12781 gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag 12841 tgttatcact catggttatg gcagcactgc ataattctct tactgtcatg ccatccgtaa 12901 gatgcttttc tgtgactggt gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc 12961 gaccgagttg ctcttgcccg gcgtcaatac gggataatac cgcgccacat agcagaactt 13021 taaaagtgct catcattgga aaacgttctt cggggcgaaa actctcaagg atcttaccgc 13081 tgttgagatc cagttcgatg taacccactc gtgcacccaa ctgatcttca gcatctttta 13141 ctttcaccag cgtttctggg tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa 13201 taagggcgac acggaaatgt tgaatactca tactcttcct ttttcaatat tattgaagca 13261 tttatcaggg ttattgtctc atgagcggat acatatttga atgtatttag aaaaataaac 13321 aaataggggt tccgcgcaca tttccccgaa aagtgccacc tgac //