LOCUS       sequence-419024-       14589 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Constitutive expression of cas9 and inducible expression of lambda 
            RED and sgRNA.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 14589)
  AUTHORS   Li Q, Sun B, Chen J, Zhang Y, Jiang Y, Yang S
  TITLE     A modified pCas/pTargetF system for CRISPR-Cas9-assisted genome 
            editing in Escherichia coli.
  JOURNAL   Acta Biochim Biophys Sin (Shanghai). 2021 Apr 15;53(5):620-627. doi:
            10.1093/abbs/gmab036.
  PUBMED    33764372
REFERENCE   2  (bases 1 to 14589)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; doi: "10"; journalName: 
            "Acta Biochim Biophys Sin (Shanghai)"; date: "2021-04-15- 15"; 
            volume: "53"; issue: "5"; pages: "620-627"
COMMENT     Sequence Label: pEcCas
FEATURES             Location/Qualifiers
     source          1..14589
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      245..467
                     /label=pSC101 ori
                     /note="low-copy replication origin that requires the Rep101
                     protein"
     CDS             complement(956..1771)
                     /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
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFL"
     CDS             complement(956..1771)
                     /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
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFL"
     primer_bind     1679..1698
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     1763..1782
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             2119..6225
                     /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="MDKKYSIGLDIGTNSVGWAVITDDYKVPSKKFKVLGNTDRHSIKK
                     NLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES
                     FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK
                     FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL
                     ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ
                     IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR
                     QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL
                     RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG
                     NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY
                     FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD
                     SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER
                     LKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFM
                     QLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR
                     HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY
                     LYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVP
                     SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH
                     VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL
                     NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT
                     EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK
                     ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI
                     TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE
                     LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD
                     ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL
                     DATLIHQSITGLYETRIDLSQLGGD"
     CDS             2119..6222
                     /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="MDKKYSIGLDIGTNSVGWAVITDDYKVPSKKFKVLGNTDRHSIKK
                     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(6355..7233)
                     /codon_start=1
                     /gene="araC"
                     /product="L-arabinose regulatory protein"
                     /label=araC
                     /translation="MAEAQNDPLLPGYSFNAHLVAGLTPIEANGYLDFFIDRPLGMKGY
                     ILNLTIRGQGVVKNQGREFVCRPGDILLFPPGEIHHYGRHPEAREWYHQWVYFRPRAYW
                     HEWLNWPSIFANTGFFRPDEAHQPHFSDLFGQIINAGQGEGRYSELLAINLLEQLLLRR
                     MEAINESLHPPMDNRVREACQYISDHLADSNFDIASVAQHVCLSPSRLSHLFRQQLGIS
                     VLSWREDQRISQAKLLLSTTRMPIATVGRNVGFDDQLYFSRVFKKCTGASPSEFRAGCE
                     EKVNDVAVKLS"
     promoter        7260..7544
                     /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     7467..7486
                     /label=pBAD Forward
                     /note="For vectors with E. coli araBAD promoter, forward 
                     primer"
     RBS             7571..7579
                     /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             7588..8004
                     /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             8010..8795
                     /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             8792..9472
                     /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      9473..9717
                     /label=lambda tL3 terminator
                     /note="transcription terminator tL3 from phage lambda"
     CDS             join(10054..10140,10141..11475)
                     /codon_start=1
                     /gene="Bacillus subtilis sacB"
                     /product="secreted levansucrase that renders bacterial 
                     growth sensitive to sucrose"
                     /label=SacB
                     /note="negative selection marker"
                     /translation="MNIKKFAKQATVLTFTTALLAGGATQAFAKETNQKPYKETYGISH
                     ITRHDMLQIPEQQKNEKYQVPEFDSSTIKNISSAKGLDVWDSWPLQNADGTVANYHGYH
                     IVFALAGDPKNADDTSIYMFYQKVGETSIDSWKNAGRVFKDSDKFDANDSILKDQTQEW
                     SGSATFTSDGKIRLFYTDFSGKHYGKQTLTTAQVNVSASDSSLNINGVEDYKSIFDGDG
                     KTYQNVQQFIDEGNYSSGDNHTLRDPHYVEDKGHKYLVFEANTGTEDGYQGEESLFNKA
                     YYGKSTSFFRQESQKLLQSDKKRTAELANGALGMIELNDDYTLKKVMKPLIASNTVTDE
                     IERANVFKMNGKWYLFTDSRGSKMTIDGITSNDIYMLGYVSNSLTGPYKPLNKTGLVLK
                     MDLDPNDVTFTYSHFAVPQAKGNNVVITSYMTNRGFYADKQSTFAPSFLLNIKGKKTSV
                     VKDSILEQGQLTVNK"
     misc_RNA        complement(11598..11673)
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        complement(11695..11813)
                     /label=rhaB promoter
                     /note="promoter of the E. coli rhaBAD operon, conferring 
                     tight induction with L-rhamnose and repression with 
                     D-glucose in the presence of RhaR and RhaS (Giacalone et 
                     al., 2006)"
     CDS             11959..12795
                     /codon_start=1
                     /gene="E. coli rhaS"
                     /product="positive regulator of the rhaB promoter
                     "
                     /label=rhaS
                     /note="activated by L-rhamnose"
                     /translation="MTVLHSVDFFPSGNASVAIEPRLPQADFPEHHHDFHEIVIVEHGT
                     GIHVFNGQPYTITGGTVCFVRDHDRHLYEHTDNLCLTNVLYRSPDRFQFLAGLNQLLPQ
                     ELDGQYPSHWRVNHSVLQQVRQLVAQMEQQEGENDLPSTASREILFMQLLLLLRKSSLQ
                     ENLENSASRLNLLLAWLEDHFADEVNWDAVADQFSLSLRTLHRQLKQQTGLTPQRYLNR
                     LRLMKARHLLRHSEASVTDIAYRCGFSDSNHFSTLFRREFNWSPRDIRQGRDGFLQ"
     CDS             12869..13717
                     /codon_start=1
                     /gene="E. coli rhaR"
                     /product="transcriptional activator of rhaR and rhaS"
                     /label=rhaR
                     /note="needed for rhamnose-dependent activation of the rhaB
                     promoter"
                     /translation="MAHQLKLLKDDFFASDQQAVAVADRYPQDVFAEHTHDFCELVIVW
                     RGNGLHVLNDRPYRITRGDLFYIHADDKHSYASVNDLVLQNIIYCPERLKLNLDWQGAI
                     PGFNASAGQPHWRLGSMGMAQARQVIGQLEHESSQHVPFANEMAELLFGQLVMLLNRHR
                     YTSDSLPPTSSETLLDKLITRLAASLKSPFALDKFCDEASCSERVLRQQFRQQTGMTIN
                     QYLRQVRVCHAQYLLQHSRLLISDISTECGFEDSNYFSVVFTRETGMTPSQWRHLNSQK
                     D"
     CDS             complement(join(13836..14589,1..197))
                     /codon_start=1
                     /gene="rep101"
                     /product="RepA protein needed for replication with the 
                     pSC101 origin"
                     /label=Rep101
                     /translation="MSELVVFKANELAISRYDLTEHETKLILCCVALLNPTIENPTRKE
                     RTVSFTYNQYAQMMNISRENAYGVLAKATRELMTRTVEIRNPLVKGFEIFQWTNYAKFS
                     SEKLELVFSEEILPYLFQLKKFIKYNLEHVKSFENKYSMRIYEWLLKELTQKKTHKANI
                     EISLDEFKFMLMLENNYHEFKRLNQWVLKPISKDLNTYSNMKLVVDKRGRPTDTLIFQV
                     ELDRQMDLVTELENNQIKMNGDKIPTTITSDSYLHNGLRKTLHDALTAKIQLTSFEAKF
                     LSDMQSKHDLNGSFSWLTQKQRTTLENILAKYGRI"
ORIGIN
        1 gcattttccc tactgatgtt catcatctga gcgtattggt tataagtgaa cgataccgtc
       61 cgttctttcc ttgtagggtt ttcaatcgtg gggttgagta gtgccacaca gcataaaatt
      121 agcttggttt catgctccgt taagtcatag cgactaatcg ctagttcatt tgctttgaaa
      181 acaactaatt cagacataca tctcaattgg tctaggtgat tttaatcact ataccaattg
      241 agatgggcta gtcaatgata attactagtc cttttccttt gagttgtggg tatctgtaaa
      301 ttctgctaga cctttgctgg aaaacttgta aattctgcta gaccctctgt aaattccgct
      361 agacctttgt gtgttttttt tgtttatatt caagtggtta taatttatag aataaagaaa
      421 gaataaaaaa agataaaaag aatagatccc agccctgtgt ataactcact actttagtca
      481 gttccgcagt attacaaaag gatgtcgcaa acgctgtttg ctcctctaca aaacagacct
      541 taaaacccta aaggcttaag tagcaccctc gcaagctcgg gcaaatcgct gaatattcct
      601 tttgtctccg accatcaggc acctgagtcg ctgtcttttt cgtgacattc agttcgctgc
      661 gctcacggct ctggcagtga atgggggtaa atggcactac aggcgccttt tatggattca
      721 tgcaaggaaa ctacccataa tacaagaaaa gcccgtcacg ggcttctcag ggcgttttat
      781 ggcgggtctg ctatgtggtg ctatctgact ttttgctgtt cagcagttcc tgccctctga
      841 ttttccagtc tgaccacttc ggattatccc gtgacaggtc attcagactg gctaatgcac
      901 ccagtaaggc agcggtatca tcaacccact gcagcaacca attaaccaat tctgattata
      961 aaaactcatc gagcatcaaa tgaaactgca atttattcat atcaggatta tcaataccat
     1021 atttttgaaa aagccgtttc tgtaatgaag gagaaaactc accgaggcag ttccatagga
     1081 tggcaagatc ctggtatcgg tctgcgattc cgactcgtcc aacatcaata caacctatta
     1141 atttcccctc gtcaaaaata aggttatcaa gtgagaaatc accatgagtg acgactgaat
     1201 ccggtgagaa tggcaaaagc ttatgcattt ctttccagac ttgttcaaca ggccagccat
     1261 tacgctcgtc atcaaaatca ctcgcatcaa ccaaaccgtt attcattcgt gattgcgcct
     1321 gagcgagacg aaatacgcga tcgctgttaa aaggacaatt acaaacagga atcgaatgca
     1381 accggcgcag gaacactgcc agcgcatcaa caatattttc acctgaatca ggatattctt
     1441 ctaatacctg gaatgctgtt ttcccgggga tcgcagtggt gagtaaccat gcatcatcag
     1501 gagtacggat aaaatgcttg atggtcggaa gaggcataaa ttccgtcagc cagtttagtc
     1561 tgaccatctc atctgtaaca tcattggcaa cgctaccttt gccatgtttc agaaacaact
     1621 ctggcgcatc gggcttccca tacaatcgat agattgtcgc acctgattgc ccgacattat
     1681 cgcgagccca tttataccca tataaatcag catccatgtt ggaatttaat cgcggcctcg
     1741 agcaagacgt ttcccgttga atatggctca taacacccct tgtattactg tttatgtaag
     1801 cagacagttt tattgttcat gatgatatat ttttatcttg tgcaatgtaa catcagagat
     1861 tttgagacac aacgtggctt tccctgcagc tgaacaatac ttaatactat agaatgataa
     1921 caaaataaac tactttttaa aagaattttg tgttataatc tatttattat taagtattgg
     1981 gtaatatttt ttgaagagat attttgaaaa agaaaaatta aagcatatta aactaatttc
     2041 ggaggtcatt aaaactatta ttgaaatcat caaactcatt atggatttaa tttaaacttt
     2101 ttattttagg aggcaaaaat ggataagaaa tactcaatag gcttagatat cggcacaaat
     2161 agcgtcggat gggcggtgat cactgatgat tataaggttc cgtctaaaaa gttcaaggtt
     2221 ctgggaaata cagaccgcca cagtatcaaa aaaaatctta taggggctct tttatttgac
     2281 agtggagaga cagcggaagc gactcgtctc aaacggacag ctcgtagaag gtatacacgt
     2341 cggaagaatc gtatttgtta tctacaggag attttttcaa atgagatggc gaaagtagat
     2401 gatagtttct ttcatcgact tgaagagtct tttttggtgg aagaagacaa gaagcatgaa
     2461 cgtcatccta tttttggaaa tatagtagat gaagttgctt atcatgagaa atatccaact
     2521 atctatcatc tgcgaaaaaa attggtagat tctactgata aagcggattt gcgcttaatc
     2581 tatttggcct tagcgcatat gattaagttt cgtggtcatt ttttgattga gggagattta
     2641 aatcctgata atagtgatgt ggacaaacta tttatccagt tggtacaaac ctacaatcaa
     2701 ttatttgaag aaaaccctat taacgcaagt ggagtagatg ctaaagcgat tctttctgca
     2761 cgattgagta aatcaagacg attagaaaat ctcattgctc agctccccgg tgagaagaaa
     2821 aatggcttat ttgggaatct cattgctttg tcattgggtt tgacccctaa ttttaaatca
     2881 aattttgatt tggcagaaga tgctaaatta cagctttcaa aagatactta cgatgatgat
     2941 ttagataatt tattggcgca aattggagat caatatgctg atttgttttt ggcagctaag
     3001 aatttatcag atgctatttt actttcagat atcctaagag taaatactga aataactaag
     3061 gctcccctat cagcttcaat gattaaacgc tacgatgaac atcatcaaga cttgactctt
     3121 ttaaaagctt tagttcgaca acaacttcca gaaaagtata aagaaatctt ttttgatcaa
     3181 tcaaaaaacg gatatgcagg ttatattgat gggggagcta gccaagaaga attttataaa
     3241 tttatcaaac caattttaga aaaaatggat ggtactgagg aattattggt gaaactaaat
     3301 cgtgaagatt tgctgcgcaa gcaacggacc tttgacaacg gctctattcc ccatcaaatt
     3361 cacttgggtg agctgcatgc tattttgaga agacaagaag acttttatcc atttttaaaa
     3421 gacaatcgtg agaagattga aaaaatcttg acttttcgaa ttccttatta tgttggtcca
     3481 ttggcgcgtg gcaatagtcg ttttgcatgg atgactcgga agtctgaaga aacaattacc
     3541 ccatggaatt ttgaagaagt tgtcgataaa ggtgcttcag ctcaatcatt tattgaacgc
     3601 atgacaaact ttgataaaaa tcttccaaat gaaaaagtac taccaaaaca tagtttgctt
     3661 tatgagtatt ttacggttta taacgaattg acaaaggtca aatatgttac tgaaggaatg
     3721 cgaaaaccag catttctttc aggtgaacag aagaaagcca ttgttgattt actcttcaaa
     3781 acaaatcgaa aagtaaccgt taagcaatta aaagaagatt atttcaaaaa aatagaatgt
     3841 tttgatagtg ttgaaatttc aggagttgaa gatagattta atgcttcatt aggtacctac
     3901 catgatttgc taaaaattat taaagataaa gattttttgg ataatgaaga aaatgaagat
     3961 atcttagagg atattgtttt aacattgacc ttatttgaag atagggagat gattgaggaa
     4021 agacttaaaa catatgctca cctctttgat gataaggtga tgaaacagct taaacgtcgc
     4081 cgttatactg gttggggacg tttgtctcga aaattgatta atggtattag ggataagcaa
     4141 tctggcaaaa caatattaga ttttttgaaa tcagatggtt ttgccaatcg caattttatg
     4201 cagctgatcc atgatgatag tttgacattt aaagaagaca ttcaaaaagc acaagtgtct
     4261 ggacaaggcg atagtttaca tgaacatatt gcaaatttag ctggtagccc tgctattaaa
     4321 aaaggtattt tacagactgt aaaagttgtt gatgaattgg tcaaagtaat ggggcggcat
     4381 aagccagaaa atatcgttat tgaaatggca cgtgaaaatc agacaactca aaagggccag
     4441 aaaaattcgc gagagcgtat gaaacgaatc gaagaaggta tcaaagaatt aggaagtcag
     4501 attcttaaag agcatcctgt tgaaaatact caattgcaaa atgaaaagct ctatctctat
     4561 tatctccaaa atggaagaga catgtatgtg gaccaagaat tagatattaa tcgtttaagt
     4621 gattatgatg tcgatcacat tgttccacaa agtttcctta aagacgattc aatagacaat
     4681 aaggtcttaa cgcgttctga taaaaatcgt ggtaaatcgg ataacgttcc aagtgaagaa
     4741 gtagtcaaaa agatgaaaaa ctattggaga caacttctaa acgccaagtt aatcactcaa
     4801 cgtaagtttg ataatttaac gaaagctgaa cgtggaggtt tgagtgaact tgataaagct
     4861 ggttttatca aacgccaatt ggttgaaact cgccaaatca ctaagcatgt ggcacaaatt
     4921 ttggatagtc gcatgaatac taaatacgat gaaaatgata aacttattcg agaggttaaa
     4981 gtgattacct taaaatctaa attagtttct gacttccgaa aagatttcca attctataaa
     5041 gtacgtgaga ttaacaatta ccatcatgcc catgatgcgt atctaaatgc cgtcgttgga
     5101 actgctttga ttaagaaata tccaaaactt gaatcggagt ttgtctatgg tgattataaa
     5161 gtttatgatg ttcgtaaaat gattgctaag tctgagcaag aaataggcaa agcaaccgca
     5221 aaatatttct tttactctaa tatcatgaac ttcttcaaaa cagaaattac acttgcaaat
     5281 ggagagattc gcaaacgccc tctaatcgaa actaatgggg aaactggaga aattgtctgg
     5341 gataaagggc gagattttgc cacagtgcgc aaagtattgt ccatgcccca agtcaatatt
     5401 gtcaagaaaa cagaagtaca gacaggcgga ttctccaagg agtcaatttt accaaaaaga
     5461 aattcggaca agcttattgc tcgtaaaaaa gactgggatc caaaaaaata tggtggtttt
     5521 gatagtccaa cggtagctta ttcagtccta gtggttgcta aggtggaaaa agggaaatcg
     5581 aagaagttaa aatccgttaa agagttacta gggatcacaa ttatggaaag aagttccttt
     5641 gaaaaaaatc cgattgactt tttagaagct aaaggatata aggaagttaa aaaagactta
     5701 atcattaaac tacctaaata tagtcttttt gagttagaaa acggtcgtaa acggatgctg
     5761 gctagtgccg gagaattaca aaaaggaaat gagctggctc tgccaagcaa atatgtgaat
     5821 tttttatatt tagctagtca ttatgaaaag ttgaagggta gtccagaaga taacgaacaa
     5881 aaacaattgt ttgtggagca gcataagcat tatttagatg agattattga gcaaatcagt
     5941 gaattttcta agcgtgttat tttagcagat gccaatttag ataaagttct tagtgcatat
     6001 aacaaacata gagacaaacc aatacgtgaa caagcagaaa atattattca tttatttacg
     6061 ttgacgaatc ttggagctcc cgctgctttt aaatattttg atacaacaat tgatcgtaaa
     6121 cgatatacgt ctacaaaaga agttttagat gccactctta tccatcaatc catcactggt
     6181 ctttatgaaa cacgcattga tttgagtcag ctaggaggtg actgaagtat attttagatg
     6241 aagattattt cttaatctag acatgagcgg atacatattt gaatgtattt agaaaaataa
     6301 acaaataggg gttccgcgca catttccccg aaaagtgcca cctgcatcga tttattatga
     6361 caacttgacg gctacatcat tcactttttc ttcacaaccg gcacggaact cgctcgggct
     6421 ggccccggtg cattttttaa atacccgcga gaagtagagt tgatcgtcaa aaccaacatt
     6481 gcgaccgacg gtggcgatag gcatccgggt ggtgctcaaa agcagcttcg cctggctgat
     6541 acgttggtcc tcgcgccagc ttaagacgct aatccctaac tgctggcgga aaagatgtga
     6601 cagacgcgac ggcgacaagc aaacatgctg tgcgacgctg gcgatatcaa aattgctgtc
     6661 tgccaggtga tcgctgatgt actgacaagc ctcgcgtacc cgattatcca tcggtggatg
     6721 gagcgactcg ttaatcgctt ccatgcgccg cagtaacaat tgctcaagca gatttatcgc
     6781 cagcagctcc gaatagcgcc cttccccttg cccggcgtta atgatttgcc caaacaggtc
     6841 gctgaaatgc ggctggtgcg cttcatccgg gcgaaagaac cccgtattgg caaatattga
     6901 cggccagtta agccattcat gccagtaggc gcgcggacga aagtaaaccc actggtgata
     6961 ccattcgcga gcctccggat gacgaccgta gtgatgaatc tctcctggcg ggaacagcaa
     7021 aatatcaccc ggtcggcaaa caaattctcg tccctgattt ttcaccaccc cctgaccgcg
     7081 aatggtgaga ttgagaatat aacctttcat tcccagcggt cggtcgataa aaaaatcgag
     7141 ataaccgttg gcctcaatcg gcgttaaacc cgccaccaga tgggcattaa acgagtatcc
     7201 cggcagcagg ggatcatttt gcgcttcagc catacttttc atactcccgc cattcagaga
     7261 agaaaccaat tgtccatatt gcatcagaca ttgccgtcac tgcgtctttt actggctctt
     7321 ctcgctaacc aaaccggtaa ccccgcttat taaaagcatt ctgtaacaaa gcgggaccaa
     7381 agccatgaca aaaacgcgta acaaaagtgt ctataatcac ggcagaaaag tccacattga
     7441 ttatttgcac ggcgtcacac tttgctatgc catagcattt ttatccataa gattagcgga
     7501 tcctacctga cgctttttat cgcaactctc tactgtttct ccatacccgt ttttttggga
     7561 attcgagctc taaggaggtt ataaaaaatg gatattaata ctgaaactga gatcaagcaa
     7621 aagcattcac taaccccctt tcctgttttc ctaatcagcc cggcatttcg cgggcgatat
     7681 tttcacagct atttcaggag ttcagccatg aacgcttatt acattcagga tcgtcttgag
     7741 gctcagagct gggcgcgtca ctaccagcag ctcgcccgtg aagagaaaga ggcagaactg
     7801 gcagacgaca tggaaaaagg cctgccccag cacctgtttg aatcgctatg catcgatcat
     7861 ttgcaacgcc acggggccag caaaaaatcc attacccgtg cgtttgatga cgatgttgag
     7921 tttcaggagc gcatggcaga acacatccgg tacatggttg aaaccattgc tcaccaccag
     7981 gttgatattg attcagaggt ataaaacgaa tgagtactgc actcgcaacg ctggctggga
     8041 agctggctga acgtgtcggc atggattctg tcgacccaca ggaactgatc accactcttc
     8101 gccagacggc atttaaaggt gatgccagcg atgcgcagtt catcgcatta ctgatcgttg
     8161 ccaaccagta cggccttaat ccgtggacga aagaaattta cgcctttcct gataagcaga
     8221 atggcatcgt tccggtggtg ggcgttgatg gctggtcccg catcatcaat gaaaaccagc
     8281 agtttgatgg catggacttt gagcaggaca atgaatcctg tacatgccgg atttaccgca
     8341 aggaccgtaa tcatccgatc tgcgttaccg aatggatgga tgaatgccgc cgcgaaccat
     8401 tcaaaactcg cgaaggcaga gaaatcacgg ggccgtggca gtcgcatccc aaacggatgt
     8461 tacgtcataa agccatgatt cagtgtgccc gtctggcctt cggatttgct ggtatctatg
     8521 acaaggatga agccgagcgc attgtcgaaa atactgcata cactgcagaa cgtcagccgg
     8581 aacgcgacat cactccggtt aacgatgaaa ccatgcagga gattaacact ctgctgatcg
     8641 ccctggataa aacatgggat gacgacttat tgccgctctg ttcccagata tttcgccgcg
     8701 acattcgtgc atcgtcagaa ctgacacagg ccgaagcagt aaaagctctt ggattcctga
     8761 aacagaaagc cgcagagcag aaggtggcag catgacaccg gacattatcc tgcagcgtac
     8821 cgggatcgat gtgagagctg tcgaacaggg ggatgatgcg tggcacaaat tacggctcgg
     8881 cgtcatcacc gcttcagaag ttcacaacgt gatagcaaaa ccccgctccg gaaagaagtg
     8941 gcctgacatg aaaatgtcct acttccacac cctgcttgct gaggtttgca ccggtgtggc
     9001 tccggaagtt aacgctaaag cactggcctg gggaaaacag tacgagaacg acgccagaac
     9061 cctgtttgaa ttcacttccg gcgtgaatgt tactgaatcc ccgatcatct atcgcgacga
     9121 aagtatgcgt accgcctgct ctcccgatgg tttatgcagt gacggcaacg gccttgaact
     9181 gaaatgcccg tttacctccc gggatttcat gaagttccgg ctcggtggtt tcgaggccat
     9241 aaagtcagct tacatggccc aggtgcagta cagcatgtgg gtgacgcgaa aaaatgcctg
     9301 gtactttgcc aactatgacc cgcgtatgaa gcgtgaaggc ctgcattatg tcgtgattga
     9361 gcgggatgaa aagtacatgg cgagttttga cgagatcgtg ccggagttca tcgaaaaaat
     9421 ggacgaggca ctggctgaaa ttggttttgt atttggggag caatggcgat gacgcatcct
     9481 cacgataata tccgggtagg cgcaatcact ttcgtctact ccgttacaaa gcgaggctgg
     9541 gtatttcccg gcctttctgt tatccgaaat ccactgaaag cacagcggct ggctgaggag
     9601 ataaataata aacgaggggc tgtatgcaca aagcatcttc tgttgagtta agaacgagta
     9661 tcgagatggc acatagcctt gctcaaattg gaatcaggtt tgtgccaata ccagtagaaa
     9721 cagacgaaga atccatgggt atggacagat cctctttagg cccgtagtct gcaaatcctt
     9781 ttatgatttt ctatcaaaca aaagaggaaa atagaccagt tgcaatccaa acgagagtct
     9841 aatagaatga ggtcgaaaag taaatcgcgc gggtttgtta ctgataaagc aggcaagacc
     9901 taaaatgtgt aaagggcaaa gtgtatactt tggcgtcacc ccttacatat tttaggtctt
     9961 tttttattgt gcgtaactaa cttgccatct tcaaacagga gggctggaag aagcagaccg
    10021 ctaacacagt acataaaaaa ggagacatga acgatgaaca tcaaaaagtt tgcaaaacaa
    10081 gcaacagtat taacctttac taccgcactg ctggcaggag gcgcaactca agcgtttgcg
    10141 aaagaaacga accaaaagcc atataaggaa acatacggca tttcccatat tacacgccat
    10201 gatatgctgc aaatccctga acagcaaaaa aatgaaaaat atcaagttcc tgaattcgat
    10261 tcgtccacaa ttaaaaatat ctcttctgca aaaggcctgg acgtttggga cagctggcca
    10321 ttacaaaacg ctgacggcac tgtcgcaaac tatcacggct accacatcgt ctttgcatta
    10381 gccggagatc ctaaaaatgc ggatgacaca tcgatttaca tgttctatca aaaagtcggc
    10441 gaaacttcta ttgacagctg gaaaaacgct ggccgcgtct ttaaagacag cgacaaattc
    10501 gatgcaaatg attctatcct aaaagaccaa acacaagaat ggtcaggttc agccacattt
    10561 acatctgacg gaaaaatccg tttattctac actgatttct ccggtaaaca ttacggcaaa
    10621 caaacactga caactgcaca agttaacgta tcagcatcag acagctcttt gaacatcaac
    10681 ggtgtagagg attataaatc aatctttgac ggtgacggaa aaacgtatca aaatgtacag
    10741 cagttcatcg atgaaggcaa ctacagctca ggcgacaacc atacgctgag agatcctcac
    10801 tacgtagaag ataaaggcca caaatactta gtatttgaag caaacactgg aactgaagat
    10861 ggctaccaag gcgaagaatc tttatttaac aaagcatact atggcaaaag cacatcattc
    10921 ttccgtcaag aaagtcaaaa acttctgcaa agcgataaaa aacgcacggc tgagttagca
    10981 aacggcgctc tcggtatgat tgagctaaac gatgattaca cactgaaaaa agtgatgaaa
    11041 ccgctgattg catctaacac agtaacagat gaaattgaac gcgcgaacgt ctttaaaatg
    11101 aacggcaaat ggtacctgtt cactgactcc cgcggatcaa aaatgacgat tgacggcatt
    11161 acgtctaacg atatttacat gcttggttat gtttctaatt ctttaactgg cccatacaag
    11221 ccgctgaaca aaactggcct tgtgttaaaa atggatcttg atcctaacga tgtaaccttt
    11281 acttactcac acttcgctgt acctcaagcg aaaggaaaca atgtcgtgat tacaagctat
    11341 atgacaaaca gaggattcta cgcagacaaa caatcaacgt ttgcgccaag cttcctgctg
    11401 aacatcaaag gcaagaaaac atctgttgtc aaagacagca tccttgaaca aggacaatta
    11461 acagttaaca aataaaaacg caaaagaaaa tgccgatatc ctattggcat tttcttttat
    11521 ttcttatcaa cataaaggtg aatcccatat gaactatata aaagcaggca aatggctaac
    11581 cgtattcctc aaaaaaagca ccgactcggt gccacttttt caagttgata acggactagc
    11641 cttattttaa cttgctattt ctagctctaa aacggttgga ctcaagacga tagttacgac
    11701 cagtctaaaa agcgcctgaa ttcgcgacct tctcgttact gacaggaaaa tgggccattg
    11761 gcaaccaggg aaagatgaac gtgatgatgt tcacaatttg ctgaattgtg gtgatgtgat
    11821 gctcaccgca tttcctgaaa attcacgctg tatcttgaaa aatcgacgtt ttttacgtgg
    11881 ttttccgtcg aaaatttaag gtaagaacct gacctcgtga ttactatttc gccgtgttga
    11941 cgacatcagg aggccagtat gaccgtatta catagtgtgg atttttttcc gtctggtaac
    12001 gcgtccgtgg cgatagaacc ccggctcccg caggcggatt ttcctgaaca tcatcatgat
    12061 tttcatgaaa ttgtgattgt cgaacatggc acgggtattc atgtgtttaa tgggcagccc
    12121 tataccatca ccggtggcac ggtctgtttc gtacgcgatc atgatcggca tctgtatgaa
    12181 cataccgata atctgtgtct gaccaatgtg ctgtatcgct cgccggatcg atttcagttt
    12241 ctcgccgggc tgaatcagtt gctgccacaa gagctggatg ggcagtatcc gtctcactgg
    12301 cgcgttaacc acagcgtatt gcagcaggtg cgacagctgg ttgcacagat ggaacagcag
    12361 gaaggggaaa atgatttacc ctcgaccgcc agtcgcgaga tcttgtttat gcaattactg
    12421 ctcttgctgc gtaaaagcag tttgcaggag aacctggaaa acagcgcatc acgtctcaac
    12481 ttgcttctgg cctggctgga ggaccatttt gccgatgagg tgaattggga tgccgtggcg
    12541 gatcaatttt ctctttcact gcgtacgcta catcggcagc ttaagcagca aacgggactg
    12601 acgcctcagc gatacctgaa ccgcctgcga ctgatgaaag cccgacatct gctacgccac
    12661 agcgaggcca gcgttactga catcgcctat cgctgtggat tcagcgacag taaccacttt
    12721 tcgacgcttt ttcgccgaga gtttaactgg tcaccgcgtg atattcgcca gggacgggat
    12781 ggctttctgc aataacgcga atcttctcaa cgtatttgta cgccatattg cgaataatca
    12841 acttcgttct ctggccgagg tagccacggt ggcgcatcag ttaaaacttc tcaaagatga
    12901 tttttttgcc agcgaccagc aggcagtcgc tgtggctgac cgttatccgc aagatgtctt
    12961 tgctgaacat acacatgatt tttgtgagct ggtgattgtc tggcgcggta atggcctgca
    13021 tgtactcaac gatcgccctt atcgcattac ccgtggcgat ctcttttaca ttcatgctga
    13081 cgataaacac tcctacgctt ccgttaacga tctggttttg cagaatatta tttattgccc
    13141 ggagcgtctg aagctgaatc ttgactggca gggggcgatt ccgggattta acgccagcgc
    13201 agggcaacca cactggcgct taggtagcat ggggatggcg caggcgcggc aggttatcgg
    13261 tcagcttgag catgaaagta gtcagcatgt gccgtttgct aacgaaatgg ctgagttgct
    13321 gttcgggcag ttggtgatgt tgctgaatcg ccatcgttac accagtgatt cgttgccgcc
    13381 aacatccagc gaaacgttgc tggataagct gattacccgg ctggcggcta gcctgaaaag
    13441 tccctttgcg ctggataaat tttgtgatga ggcatcgtgc agtgagcgcg ttttgcgtca
    13501 gcaatttcgc cagcagactg gaatgaccat caatcaatat ctgcgacagg tcagagtgtg
    13561 tcatgcgcaa tatcttctcc agcatagccg cctgttaatc agtgatattt cgaccgaatg
    13621 tggctttgaa gatagtaact atttttcggt ggtgtttacc cgggaaaccg ggatgacgcc
    13681 cagccagtgg cgtcatctca attcgcagaa agattaattc gccatgcccc atgggtatgg
    13741 acagttttcc ctttgatatg taacggtgaa cagttgttct acttttgttt gttagtcttg
    13801 atgcttcact gatagataca agagccataa gaacctcaga tccttccgta tttagccagt
    13861 atgttctcta gtgtggttcg ttgtttttgc gtgagccatg agaacgaacc attgagatca
    13921 tgcttacttt gcatgtcact caaaaatttt gcctcaaaac tggtgagctg aatttttgca
    13981 gttaaagcat cgtgtagtgt ttttcttagt ccgttatgta ggtaggaatc tgatgtaatg
    14041 gttgttggta ttttgtcacc attcattttt atctggttgt tctcaagttc ggttacgaga
    14101 tccatttgtc tatctagttc aacttggaaa atcaacgtat cagtcgggcg gcctcgctta
    14161 tcaaccacca atttcatatt gctgtaagtg tttaaatctt tacttattgg tttcaaaacc
    14221 cattggttaa gccttttaaa ctcatggtag ttattttcaa gcattaacat gaacttaaat
    14281 tcatcaaggc taatctctat atttgccttg tgagttttct tttgtgttag ttcttttaat
    14341 aaccactcat aaatcctcat agagtatttg ttttcaaaag acttaacatg ttccagatta
    14401 tattttatga atttttttaa ctggaaaaga taaggcaata tctcttcact aaaaactaat
    14461 tctaattttt cgcttgagaa cttggcatag tttgtccact ggaaaatctc aaagccttta
    14521 accaaaggat tcctgatttc cacagttctc gtcatcagct ctctggttgc tttagctaat
    14581 acaccataa
//
