pCas

LOCUS       pCas       12542 bp DNA     circular SYN 25-AUG-2024
DEFINITION  Constitutive expression of cas9 and inducible expression of lambda 
            RED and sgR.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 12542)
  AUTHORS   Jiang Y, Chen B, Duan C, Sun B, Yang J, Yang S
  TITLE     Multigene editing in the Escherichia coli genome using the 
            CRISPR-Cas9 system.
  JOURNAL   Appl Environ Microbiol. 2015 Jan 30. pii: AEM.04023-14.
  PUBMED    25636838
REFERENCE   2  (bases 1 to 12542)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Aug 25, 2024 from SnapGene Server 7.0.3
            https://www.biofeng.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Appl 
            Environ Microbiol. 2015 Jan 30. pii: AEM.04023-14."
FEATURES             Location/Qualifiers
     source          1..12542
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             286..4392
                     /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             286..4389
                     /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             complement(4522..5400)
                     /codon_start=1
                     /gene="araC"
                     /product="L-arabinose regulatory protein"
                     /label=araC
                     /translation="MAEAQNDPLLPGYSFNAHLVAGLTPIEANGYLDFFIDRPLGMKGY
                     ILNLTIRGQGVVKNQGREFVCRPGDILLFPPGEIHHYGRHPEAREWYHQWVYFRPRAYW
                     HEWLNWPSIFANTGFFRPDEAHQPHFSDLFGQIINAGQGEGRYSELLAINLLEQLLLRH
                     MEAINESLHPPMDNRVREACQYISDHLADSNFDIASVAQHVCLSPSRLSHLFRQQLGIS
                     VLSWREDQRISQAKLLLSTTRMPIATVGRNVGFDDQLYFSRVFKKCTGASPSEFRAGCE
                     EKVNDVAVKLS"
     promoter        5427..5711
                     /gene="araBAD"
                     /label=araBAD promoter
                     /note="promoter of the L-arabinose operon of E. coli; the 
                     araC regulatory gene is transcribed in the opposite 
                     direction (Guzman et al., 1995)"
     primer_bind     5634..5653
                     /label=pBAD Forward
                     /note="For vectors with E. coli araBAD promoter, forward 
                     primer"
     RBS             5738..5746
                     /label=Shine-Dalgarno sequence
                     /note="full consensus sequence for ribosome-binding sites 
                     upstream of start codons in E. coli; complementary to a 
                     region in the 3' end of the 16S rRNA (Chen et al., 1994)"
     CDS             5755..6171
                     /codon_start=1
                     /gene="gam"
                     /product="inhibitor of the host RecBCD nuclease in the 
                     lambda Red system"
                     /label=Gam
                     /note="protects the double-stranded DNA substrate for 
                     recombination"
                     /translation="MDINTETEIKQKHSLTPFPVFLISPAFRGRYFHSYFRSSAMNAYY
                     IQDRLEAQSWARHYQQLAREEKEAELADDMEKGLPQHLFESLCIDHLQRHGASKKSITR
                     AFDDDVEFQERMAEHIRYMVETIAHHQVDIDSEV"
     CDS             6177..6962
                     /codon_start=1
                     /gene="bet"
                     /product="single-stranded DNA binding recombinase in the 
                     lambda Red system"
                     /label=Beta
                     /note="mediates pairing of a single-stranded DNA with a 
                     complementary target"
                     /translation="MSTALATLAGKLAERVGMDSVDPQELITTLRQTAFKGDASDAQFI
                     ALLIVANQYGLNPWTKEIYAFPDKQNGIVPVVGVDGWSRIINENQQFDGMDFEQDNESC
                     TCRIYRKDRNHPICVTEWMDECRREPFKTREGREITGPWQSHPKRMLRHKAMIQCARLA
                     FGFAGIYDKDEAERIVENTAYTAERQPERDITPVNDETMQEINTLLIALDKTWDDDLLP
                     LCSQIFRRDIRASSELTQAEAVKALGFLKQKAAEQKVAA"
     CDS             6959..7639
                     /codon_start=1
                     /gene="exo"
                     /product="5' to 3' double-stranded DNA exonuclease in the 
                     lambda Red system"
                     /label=Exo
                     /note="creates 3' single-stranded DNA tails"
                     /translation="MTPDIILQRTGIDVRAVEQGDDAWHKLRLGVITASEVHNVIAKPR
                     SGKKWPDMKMSYFHTLLAEVCTGVAPEVNAKALAWGKQYENDARTLFEFTSGVNVTESP
                     IIYRDESMRTACSPDGLCSDGNGLELKCPFTSRDFMKFRLGGFEAIKSAYMAQVQYSMW
                     VTRKNAWYFANYDPRMKREGLHYVVIERDEKYMASFDEIVPEFIEKMDEALAEIGFVFG
                     EQWR"
     terminator      7640..7884
                     /label=lambda tL3 terminator
                     /note="transcription terminator tL3 from phage lambda"
     misc_RNA        complement(7928..8003)
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     protein_bind    8033..8049
                     /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)."
     CDS             complement(8318..9400)
                     /codon_start=1
                     /gene="lacI"
                     /product="lac repressor"
                     /label=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)."
                     /translation="MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     primer_bind     9362..9381
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(9401..9478)
                     /gene="lacI (mutant)"
                     /label=lacIq promoter
                     /note="In the lacIq allele, a single base change in the 
                     promoter boosts expression of the lacI gene about 10-fold."
     CDS             complement(9611..10561)
                     /codon_start=1
                     /gene="rep101 encoding an A56V mutation"
                     /product="temperature-sensitive version of the RepA protein
                     needed for replication with the pSC101 origin (Armstrong et
                     al., 1984)"
                     /label=Rep101(Ts)
                     /translation="MSELVVFKANELAISRYDLTEHETKLILCCVALLNPTIENPTRKE
                     RTVSFTYNQYVQMMNISRENAYGVLAKATRELMTRTVEIRNPLVKGFEIFQWTNYAKFS
                     SEKLELVFSEEILPYLFQLKKFIKYNLEHVKSFENKYSMRIYEWLLKELTQKKTHKANI
                     EISLDEFKFMLMLENNYHEFKRLNQWVLKPISKDLNTYSNMKLVVDKRGRPTDTLIFQV
                     ELDRQMDLVTELENNQIKMNGDKIPTTITSDSYLHNGLRKTLHDALTAKIQLTSFEAKF
                     LSDMQSKYDLNGSFSWLTQKQRTTLENILAKYGRI"
     rep_origin      10609..10831
                     /label=pSC101 ori
                     /note="low-copy replication origin that requires the Rep101
                     protein"
     CDS             complement(11483..12298)
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     CDS             complement(11483..12298)
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     12206..12225
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     12290..12309
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     misc_RNA        complement(12454..12532)
                     /label=tracrRNA
                     /note="trans-activating CRISPR RNA for the Streptococcus 
                     pyogenes CRISPR/Cas9 system"
ORIGIN
        1 ttttataact tttataacaa ataatcaagg agaaattcaa agaaatttat cagccataaa
       61 acaatactta atactataga atgataacaa aataaactac tttttaaaag aattttgtgt
      121 tataatctat ttattattaa gtattgggta atattttttg aagagatatt ttgaaaaaga
      181 aaaattaaag catattaaac taatttcgga ggtcattaaa actattattg aaatcatcaa
      241 actcattatg gatttaattt aaacttttta ttttaggagg caaaaatgga taagaaatac
      301 tcaataggct tagatatcgg cacaaatagc gtcggatggg cggtgatcac tgatgaatat
      361 aaggttccgt ctaaaaagtt caaggttctg ggaaatacag accgccacag tatcaaaaaa
      421 aatcttatag gggctctttt atttgacagt ggagagacag cggaagcgac tcgtctcaaa
      481 cggacagctc gtagaaggta tacacgtcgg aagaatcgta tttgttatct acaggagatt
      541 ttttcaaatg agatggcgaa agtagatgat agtttctttc atcgacttga agagtctttt
      601 ttggtggaag aagacaagaa gcatgaacgt catcctattt ttggaaatat agtagatgaa
      661 gttgcttatc atgagaaata tccaactatc tatcatctgc gaaaaaaatt ggtagattct
      721 actgataaag cggatttgcg cttaatctat ttggccttag cgcatatgat taagtttcgt
      781 ggtcattttt tgattgaggg agatttaaat cctgataata gtgatgtgga caaactattt
      841 atccagttgg tacaaaccta caatcaatta tttgaagaaa accctattaa cgcaagtgga
      901 gtagatgcta aagcgattct ttctgcacga ttgagtaaat caagacgatt agaaaatctc
      961 attgctcagc tccccggtga gaagaaaaat ggcttatttg ggaatctcat tgctttgtca
     1021 ttgggtttga cccctaattt taaatcaaat tttgatttgg cagaagatgc taaattacag
     1081 ctttcaaaag atacttacga tgatgattta gataatttat tggcgcaaat tggagatcaa
     1141 tatgctgatt tgtttttggc agctaagaat ttatcagatg ctattttact ttcagatatc
     1201 ctaagagtaa atactgaaat aactaaggct cccctatcag cttcaatgat taaacgctac
     1261 gatgaacatc atcaagactt gactctttta aaagctttag ttcgacaaca acttccagaa
     1321 aagtataaag aaatcttttt tgatcaatca aaaaacggat atgcaggtta tattgatggg
     1381 ggagctagcc aagaagaatt ttataaattt atcaaaccaa ttttagaaaa aatggatggt
     1441 actgaggaat tattggtgaa actaaatcgt gaagatttgc tgcgcaagca acggaccttt
     1501 gacaacggct ctattcccca tcaaattcac ttgggtgagc tgcatgctat tttgagaaga
     1561 caagaagact tttatccatt tttaaaagac aatcgtgaga agattgaaaa aatcttgact
     1621 tttcgaattc cttattatgt tggtccattg gcgcgtggca atagtcgttt tgcatggatg
     1681 actcggaagt ctgaagaaac aattacccca tggaattttg aagaagttgt cgataaaggt
     1741 gcttcagctc aatcatttat tgaacgcatg acaaactttg ataaaaatct tccaaatgaa
     1801 aaagtactac caaaacatag tttgctttat gagtatttta cggtttataa cgaattgaca
     1861 aaggtcaaat atgttactga aggaatgcga aaaccagcat ttctttcagg tgaacagaag
     1921 aaagccattg ttgatttact cttcaaaaca aatcgaaaag taaccgttaa gcaattaaaa
     1981 gaagattatt tcaaaaaaat agaatgtttt gatagtgttg aaatttcagg agttgaagat
     2041 agatttaatg cttcattagg tacctaccat gatttgctaa aaattattaa agataaagat
     2101 tttttggata atgaagaaaa tgaagatatc ttagaggata ttgttttaac attgacctta
     2161 tttgaagata gggagatgat tgaggaaaga cttaaaacat atgctcacct ctttgatgat
     2221 aaggtgatga aacagcttaa acgtcgccgt tatactggtt ggggacgttt gtctcgaaaa
     2281 ttgattaatg gtattaggga taagcaatct ggcaaaacaa tattagattt tttgaaatca
     2341 gatggttttg ccaatcgcaa ttttatgcag ctgatccatg atgatagttt gacatttaaa
     2401 gaagacattc aaaaagcaca agtgtctgga caaggcgata gtttacatga acatattgca
     2461 aatttagctg gtagccctgc tattaaaaaa ggtattttac agactgtaaa agttgttgat
     2521 gaattggtca aagtaatggg gcggcataag ccagaaaata tcgttattga aatggcacgt
     2581 gaaaatcaga caactcaaaa gggccagaaa aattcgcgag agcgtatgaa acgaatcgaa
     2641 gaaggtatca aagaattagg aagtcagatt cttaaagagc atcctgttga aaatactcaa
     2701 ttgcaaaatg aaaagctcta tctctattat ctccaaaatg gaagagacat gtatgtggac
     2761 caagaattag atattaatcg tttaagtgat tatgatgtcg atcacattgt tccacaaagt
     2821 ttccttaaag acgattcaat agacaataag gtcttaacgc gttctgataa aaatcgtggt
     2881 aaatcggata acgttccaag tgaagaagta gtcaaaaaga tgaaaaacta ttggagacaa
     2941 cttctaaacg ccaagttaat cactcaacgt aagtttgata atttaacgaa agctgaacgt
     3001 ggaggtttga gtgaacttga taaagctggt tttatcaaac gccaattggt tgaaactcgc
     3061 caaatcacta agcatgtggc acaaattttg gatagtcgca tgaatactaa atacgatgaa
     3121 aatgataaac ttattcgaga ggttaaagtg attaccttaa aatctaaatt agtttctgac
     3181 ttccgaaaag atttccaatt ctataaagta cgtgagatta acaattacca tcatgcccat
     3241 gatgcgtatc taaatgccgt cgttggaact gctttgatta agaaatatcc aaaacttgaa
     3301 tcggagtttg tctatggtga ttataaagtt tatgatgttc gtaaaatgat tgctaagtct
     3361 gagcaagaaa taggcaaagc aaccgcaaaa tatttctttt actctaatat catgaacttc
     3421 ttcaaaacag aaattacact tgcaaatgga gagattcgca aacgccctct aatcgaaact
     3481 aatggggaaa ctggagaaat tgtctgggat aaagggcgag attttgccac agtgcgcaaa
     3541 gtattgtcca tgccccaagt caatattgtc aagaaaacag aagtacagac aggcggattc
     3601 tccaaggagt caattttacc aaaaagaaat tcggacaagc ttattgctcg taaaaaagac
     3661 tgggatccaa aaaaatatgg tggttttgat agtccaacgg tagcttattc agtcctagtg
     3721 gttgctaagg tggaaaaagg gaaatcgaag aagttaaaat ccgttaaaga gttactaggg
     3781 atcacaatta tggaaagaag ttcctttgaa aaaaatccga ttgacttttt agaagctaaa
     3841 ggatataagg aagttaaaaa agacttaatc attaaactac ctaaatatag tctttttgag
     3901 ttagaaaacg gtcgtaaacg gatgctggct agtgccggag aattacaaaa aggaaatgag
     3961 ctggctctgc caagcaaata tgtgaatttt ttatatttag ctagtcatta tgaaaagttg
     4021 aagggtagtc cagaagataa cgaacaaaaa caattgtttg tggagcagca taagcattat
     4081 ttagatgaga ttattgagca aatcagtgaa ttttctaagc gtgttatttt agcagatgcc
     4141 aatttagata aagttcttag tgcatataac aaacatagag acaaaccaat acgtgaacaa
     4201 gcagaaaata ttattcattt atttacgttg acgaatcttg gagctcccgc tgcttttaaa
     4261 tattttgata caacaattga tcgtaaacga tatacgtcta caaaagaagt tttagatgcc
     4321 actcttatcc atcaatccat cactggtctt tatgaaacac gcattgattt gagtcagcta
     4381 ggaggtgact gaagtatatt ttagatgaag attatttctt aatctagaca tgagcggata
     4441 catatttgaa tgtatttaga aaaataaaca aataggggtt ccgcgcacat ttccccgaaa
     4501 agtgccacct gcatcgattt attatgacaa cttgacggct acatcattca ctttttcttc
     4561 acaaccggca cggaactcgc tcgggctggc cccggtgcat tttttaaata cccgcgagaa
     4621 gtagagttga tcgtcaaaac caacattgcg accgacggtg gcgataggca tccgggtggt
     4681 gctcaaaagc agcttcgcct ggctgatacg ttggtcctcg cgccagctta agacgctaat
     4741 ccctaactgc tggcggaaaa gatgtgacag acgcgacggc gacaagcaaa catgctgtgc
     4801 gacgctggcg atatcaaaat tgctgtctgc caggtgatcg ctgatgtact gacaagcctc
     4861 gcgtacccga ttatccatcg gtggatggag cgactcgtta atcgcttcca tgtgccgcag
     4921 taacaattgc tcaagcagat ttatcgccag cagctccgaa tagcgccctt ccccttgccc
     4981 ggcgttaatg atttgcccaa acaggtcgct gaaatgcggc tggtgcgctt catccgggcg
     5041 aaagaacccc gtattggcaa atattgacgg ccagttaagc cattcatgcc agtaggcgcg
     5101 cggacgaaag taaacccact ggtgatacca ttcgcgagcc tccggatgac gaccgtagtg
     5161 atgaatctct cctggcggga acagcaaaat atcacccggt cggcaaacaa attctcgtcc
     5221 ctgatttttc accaccccct gaccgcgaat ggtgagattg agaatataac ctttcattcc
     5281 cagcggtcgg tcgataaaaa aatcgagata accgttggcc tcaatcggcg ttaaacccgc
     5341 caccagatgg gcattaaacg agtatcccgg cagcagggga tcattttgcg cttcagccat
     5401 acttttcata ctcccgccat tcagagaaga aaccaattgt ccatattgca tcagacattg
     5461 ccgtcactgc gtcttttact ggctcttctc gctaaccaaa ccggtaaccc cgcttattaa
     5521 aagcattctg taacaaagcg ggaccaaagc catgacaaaa acgcgtaaca aaagtgtcta
     5581 taatcacggc agaaaagtcc acattgatta tttgcacggc gtcacacttt gctatgccat
     5641 agcattttta tccataagat tagcggatcc tacctgacgc tttttatcgc aactctctac
     5701 tgtttctcca tacccgtttt tttgggaatt cgagctctaa ggaggttata aaaaatggat
     5761 attaatactg aaactgagat caagcaaaag cattcactaa ccccctttcc tgttttccta
     5821 atcagcccgg catttcgcgg gcgatatttt cacagctatt tcaggagttc agccatgaac
     5881 gcttattaca ttcaggatcg tcttgaggct cagagctggg cgcgtcacta ccagcagctc
     5941 gcccgtgaag agaaagaggc agaactggca gacgacatgg aaaaaggcct gccccagcac
     6001 ctgtttgaat cgctatgcat cgatcatttg caacgccacg gggccagcaa aaaatccatt
     6061 acccgtgcgt ttgatgacga tgttgagttt caggagcgca tggcagaaca catccggtac
     6121 atggttgaaa ccattgctca ccaccaggtt gatattgatt cagaggtata aaacgaatga
     6181 gtactgcact cgcaacgctg gctgggaagc tggctgaacg tgtcggcatg gattctgtcg
     6241 acccacagga actgatcacc actcttcgcc agacggcatt taaaggtgat gccagcgatg
     6301 cgcagttcat cgcattactg atcgttgcca accagtacgg ccttaatccg tggacgaaag
     6361 aaatttacgc ctttcctgat aagcagaatg gcatcgttcc ggtggtgggc gttgatggct
     6421 ggtcccgcat catcaatgaa aaccagcagt ttgatggcat ggactttgag caggacaatg
     6481 aatcctgtac atgccggatt taccgcaagg accgtaatca tccgatctgc gttaccgaat
     6541 ggatggatga atgccgccgc gaaccattca aaactcgcga aggcagagaa atcacggggc
     6601 cgtggcagtc gcatcccaaa cggatgttac gtcataaagc catgattcag tgtgcccgtc
     6661 tggccttcgg atttgctggt atctatgaca aggatgaagc cgagcgcatt gtcgaaaata
     6721 ctgcatacac tgcagaacgt cagccggaac gcgacatcac tccggttaac gatgaaacca
     6781 tgcaggagat taacactctg ctgatcgccc tggataaaac atgggatgac gacttattgc
     6841 cgctctgttc ccagatattt cgccgcgaca ttcgtgcatc gtcagaactg acacaggccg
     6901 aagcagtaaa agctcttgga ttcctgaaac agaaagccgc agagcagaag gtggcagcat
     6961 gacaccggac attatcctgc agcgtaccgg gatcgatgtg agagctgtcg aacaggggga
     7021 tgatgcgtgg cacaaattac ggctcggcgt catcaccgct tcagaagttc acaacgtgat
     7081 agcaaaaccc cgctccggaa agaagtggcc tgacatgaaa atgtcctact tccacaccct
     7141 gcttgctgag gtttgcaccg gtgtggctcc ggaagttaac gctaaagcac tggcctgggg
     7201 aaaacagtac gagaacgacg ccagaaccct gtttgaattc acttccggcg tgaatgttac
     7261 tgaatccccg atcatctatc gcgacgaaag tatgcgtacc gcctgctctc ccgatggttt
     7321 atgcagtgac ggcaacggcc ttgaactgaa atgcccgttt acctcccggg atttcatgaa
     7381 gttccggctc ggtggtttcg aggccataaa gtcagcttac atggcccagg tgcagtacag
     7441 catgtgggtg acgcgaaaaa atgcctggta ctttgccaac tatgacccgc gtatgaagcg
     7501 tgaaggcctg cattatgtcg tgattgagcg ggatgaaaag tacatggcga gttttgacga
     7561 gatcgtgccg gagttcatcg aaaaaatgga cgaggcactg gctgaaattg gttttgtatt
     7621 tggggagcaa tggcgatgac gcatcctcac gataatatcc gggtaggcgc aatcactttc
     7681 gtctactccg ttacaaagcg aggctgggta tttcccggcc tttctgttat ccgaaatcca
     7741 ctgaaagcac agcggctggc tgaggagata aataataaac gaggggctgt atgcacaaag
     7801 catcttctgt tgagttaaga acgagtatcg agatggcaca tagccttgct caaattggaa
     7861 tcaggtttgt gccaatacca gtagaaacag acgaagaatc catgggtatg gacagatctc
     7921 aaaaaaagca ccgactcggt gccacttttt caagttgata acggactagc cttattttaa
     7981 cttgctattt ctagctctaa aacctggtaa caggattagc agatgtgtga aattgttatc
     8041 cgctcacaat tccacacaac atacgagccg gatgattaat tgtcaacagc tcatttcaga
     8101 atatttgcca gaaccgttat gatgtcggcg caaaaaacat tatccagaac gggagtgcgc
     8161 cttgagcgac acgaattatg cagtgattta cgacctgcac agccatacca cagcttccga
     8221 tggctgcctg acgccagaag cattggtgca ccgtgcagtc gatgataagc tgtcaaacca
     8281 gatcaattcg cgctaactta cattaattgc gttgcgctca ctgcccgctt tccagtcggg
     8341 aaacctgtcg tgccagctgc attaatgaat cggccaacgc gcggggagag gcggtttgcg
     8401 tattgggcgc cagggtggtt tttcttttca ccagtgagac gggcaacagc tgattgccct
     8461 tcaccgcctg gccctgagag agttgcagca agcggtccac gctggtttgc cccagcaggc
     8521 gaaaatcctg tttgatggtg gttaacggcg ggatataaca tgagctgtct tcggtatcgt
     8581 cgtatcccac taccgagata tccgcaccaa cgcgcagccc ggactcggta atggcgcgca
     8641 ttgcgcccag cgccatctga tcgttggcaa ccagcatcgc agtgggaacg atgccctcat
     8701 tcagcatttg catggtttgt tgaaaaccgg acatggcact ccagtcgcct tcccgttccg
     8761 ctatcggctg aatttgattg cgagtgagat atttatgcca gccagccaga cgcagacgcg
     8821 ccgagacaga acttaatggg cccgctaaca gcgcgatttg ctggtgaccc aatgcgacca
     8881 gatgctccac gcccagtcgc gtaccgtctt catgggagaa aataatactg ttgatgggtg
     8941 tctggtcaga gacatcaaga aataacgccg gaacattagt gcaggcagct tccacagcaa
     9001 tggcatcctg gtcatccagc ggatagttaa tgatcagccc actgacgcgt tgcgcgagaa
     9061 gattgtgcac cgccgcttta caggcttcga cgccgcttcg ttctaccatc gacaccacca
     9121 cgctggcacc cagttgatcg gcgcgagatt taatcgccgc gacaatttgc gacggcgcgt
     9181 gcagggccag actggaggtg gcaacgccaa tcagcaacga ctgtttgccc gccagttgtt
     9241 gtgccacgcg gttgggaatg taattcagct ccgccatcgc cgcttccact ttttcccgcg
     9301 ttttcgcaga aacgtggctg gcctggttca ccacgcggga aacggtctga taagagacac
     9361 cggcatactc tgcgacatcg tataacgtta ctggtttcac attcaccacc ctgaattgac
     9421 tctcttccgg gcgctatcat gccataccgc gaaaggtttt gcaccattcg atggtgtcaa
     9481 cgtaaatgca tgccgctcgc cttccatggg tatggacagt tttccctttg atatgtaacg
     9541 gtgaacagtt gttctacttt tgtttgttag tcttgatgct tcactgatag atacaagagc
     9601 cataagaacc tcagatcctt ccgtatttag ccagtatgtt ctctagtgtg gttcgttgtt
     9661 tttgcgtgag ccatgagaac gaaccattga gatcatactt actttgcatg tcactcaaaa
     9721 attttgcctc aaaactggtg agctgaattt ttgcagttaa agcatcgtgt agtgtttttc
     9781 ttagtccgtt atgtaggtag gaatctgatg taatggttgt tggtattttg tcaccattca
     9841 tttttatctg gttgttctca agttcggtta cgagatccat ttgtctatct agttcaactt
     9901 ggaaaatcaa cgtatcagtc gggcggcctc gcttatcaac caccaatttc atattgctgt
     9961 aagtgtttaa atctttactt attggtttca aaacccattg gttaagcctt ttaaactcat
    10021 ggtagttatt ttcaagcatt aacatgaact taaattcatc aaggctaatc tctatatttg
    10081 ccttgtgagt tttcttttgt gttagttctt ttaataacca ctcataaatc ctcatagagt
    10141 atttgttttc aaaagactta acatgttcca gattatattt tatgaatttt tttaactgga
    10201 aaagataagg caatatctct tcactaaaaa ctaattctaa tttttcgctt gagaacttgg
    10261 catagtttgt ccactggaaa atctcaaagc ctttaaccaa aggattcctg atttccacag
    10321 ttctcgtcat cagctctctg gttgctttag ctaatacacc ataagcattt tccctactga
    10381 tgttcatcat ctgaacgtat tggttataag tgaacgatac cgtccgttct ttccttgtag
    10441 ggttttcaat cgtggggttg agtagtgcca cacagcataa aattagcttg gtttcatgct
    10501 ccgttaagtc atagcgacta atcgctagtt catttgcttt gaaaacaact aattcagaca
    10561 tacatctcaa ttggtctagg tgattttaat cactatacca attgagatgg gctagtcaat
    10621 gataattact agtccttttc ctttgagttg tgggtatctg taaattctgc tagacctttg
    10681 ctggaaaact tgtaaattct gctagaccct ctgtaaattc cgctagacct ttgtgtgttt
    10741 tttttgttta tattcaagtg gttataattt atagaataaa gaaagaataa aaaaagataa
    10801 aaagaataga tcccagccct gtgtataact cactacttta gtcagttccg cagtattaca
    10861 aaaggatgtc gcaaacgctg tttgctcctc tacaaaacag accttaaaac cctaaaggct
    10921 taagtagcac cctcgcaagc tcggttgcgg ccgcaatcgg gcaaatcgct gaatattcct
    10981 tttgtctccg accatcaggc acctgagtcg ctgtcttttt cgtgacattc agttcgctgc
    11041 gctcacggct ctggcagtga atgggggtaa atggcactac aggcgccttt tatggattca
    11101 tgcaaggaaa ctacccataa tacaagaaaa gcccgtcacg ggcttctcag ggcgttttat
    11161 ggcgggtctg ctatgtggtg ctatctgact ttttgctgtt cagcagttcc tgccctctga
    11221 ttttccagtc tgaccacttc ggattatccc gtgacaggtc attcagactg gctaatgcac
    11281 ccagtaaggc agcggtatca tcaacggggt ctgacgctca gtggaacgaa aactcacgtt
    11341 aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa
    11401 aatgaagttt taaatcaatc taaagtatat atgagtaaac ttggtctgac agttacgttt
    11461 ccaaccaatt aaccaattct gattagaaaa actcatcgag catcaaatga aactgcaatt
    11521 tattcatatc aggattatca ataccatatt tttgaaaaag ccgtttctgt aatgaaggag
    11581 aaaactcacc gaggcagttc cataggatgg caagatcctg gtatcggtct gcgattccga
    11641 ctcgtccaac atcaatacaa cctattaatt tcccctcgtc aaaaataagg ttatcaagtg
    11701 agaaatcacc atgagtgacg actgaatccg gtgagaatgg caaaagctta tgcatttctt
    11761 tccagacttg ttcaacaggc cagccattac gctcgtcatc aaaatcactc gcatcaacca
    11821 aaccgttatt cattcgtgat tgcgcctgag cgagacgaaa tacgcgatcg ctgttaaaag
    11881 gacaattaca aacaggaatc gaatgcaacc ggcgcaggaa cactgccagc gcatcaacaa
    11941 tattttcacc tgaatcagga tattcttcta atacctggaa tgctgttttc ccggggatcg
    12001 cagtggtgag taaccatgca tcatcaggag tacggataaa atgcttgatg gtcggaagag
    12061 gcataaattc cgtcagccag tttagtctga ccatctcatc tgtaacatca ttggcaacgc
    12121 tacctttgcc atgtttcaga aacaactctg gcgcatcggg cttcccatac aatcgataga
    12181 ttgtcgcacc tgattgcccg acattatcgc gagcccattt atacccatat aaatcagcat
    12241 ccatgttgga atttaatcgc ggcctcgagc aagacgtttc ccgttgaata tggctcataa
    12301 caccccttgt attactgttt atgtaagcag acagttttat tgttcatgat gatatatttt
    12361 tatcttgtgc aatgtaacat cagagatttt gagacacaac gtggctttcc ctgcagggtt
    12421 tgcagtcaga gtagaataga agtatcaaaa aaagcaccga ctcggtgcca ctttttcaag
    12481 ttgataacgg actagcctta ttttaacttg ctatgctgtt ttgaatggtt ccaacaagat
    12541 ta
//