LOCUS       sequence-333602-       15461 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Constitutive expression of Cas9, inducible expression of the Red 
            recombineering system, and inducible expression of the plasmid 
            curing system for CRISPR mediated genome editing of E. coli..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 15461)
  AUTHORS   Li Y, Lin Z, Huang C, Zhang Y, Wang Z, Tang YJ, Chen T, Zhao X
  TITLE     Metabolic engineering of Escherichia coli using CRISPR-Cas9 
            meditated genome editing.
  JOURNAL   Metab Eng. 2015 Sep;31:13-21. doi: 10.1016/j.ymben.2015.06.006. Epub
            2015 Jun 30.
  PUBMED    26141150
REFERENCE   2  (bases 1 to 15461)
  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"; journalName: "Metab 
            Eng."; date: "2015-09"; volume: "31:13-21. doi"; pages: " 
            10.1016/j.ymben.2015.06.006. Epub 2015 Jun 30"
COMMENT     Sequence Label: pREDCas9
FEATURES             Location/Qualifiers
     source          1..15461
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_RNA        complement(370..448)
                     /label=tracrRNA
                     /note="trans-activating CRISPR RNA for the Streptococcus 
                     pyogenes CRISPR/Cas9 system"
     CDS             744..4850
                     /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             744..4847
                     /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"
     misc_feature    4871..5002
                     /label=crRNA leader
                     /note="crRNA leader sequence for the Streptococcus pyogenes
                     CRISPR/Cas system"
     repeat_region   5003..5038
                     /label=DR
                     /note="direct repeat for the Streptococcus pyogenes 
                     CRISPR/Cas system"
     CDS             complement(5248..6126)
                     /codon_start=1
                     /gene="araC"
                     /product="L-arabinose regulatory protein"
                     /label=araC
                     /translation="MAEAQNDPLLPGYSFNAHLVAGLTPIEANGYLDFFIDRPLGMKGY
                     ILNLTIRGQGVVKNQGREFVCRPGDILLFPPGEIHHYGRHPEAREWYHQWVYFRPRAYW
                     HEWLNWPSIFANTGFFRPDEAHQPHFSDLFGQIINAGQGEGRYSELLAINLLEQLLLRR
                     MEAINESLHPPMDNRVREACQYISDHLADSNFDIASVAQHVCLSPSRLSHLFRQQLGIS
                     VLSWREDQRISQAKLLLSTTRMPIATVGRNVGFDDQLYFSRVFKKCTGASPSEFRAGCE
                     EKVNDVAVKLS"
     promoter        6153..6437
                     /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     6360..6379
                     /label=pBAD Forward
                     /note="For vectors with E. coli araBAD promoter, forward 
                     primer"
     repeat_region   6584..6619
                     /label=DR
                     /note="direct repeat for the Streptococcus pyogenes 
                     CRISPR/Cas system"
     CDS             7397..8188
                     /codon_start=1
                     /gene="aadA"
                     /product="aminoglycoside adenylyltransferase (Murphy, 
                     1985)"
                     /label=SmR
                     /note="confers resistance to spectinomycin and 
                     streptomycin"
                     /translation="MREAVIAEVSTQLSEVVGVIERHLEPTLLAVHLYGSAVDGGLKPH
                     SDIDLLVTVTVRLDETTRRALINDLLETSASPGESEILRAVEVTIVVHDDIIPWRYPAK
                     RELQFGEWQRNDILAGIFEPATIDIDLAILLTKAREHSVALVGPAAEELFDPVPEQDLF
                     EALNETLTLWNSPPDWAGDERNVVLTLSRIWYSAVTGKIAPKDVAAYWAMERLPAQYQP
                     VILEARQAYLGQEEDRLASRADQLEEFVHYVKGEITKVVGK"
     CDS             complement(8512..9192)
                     /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="MTPDIILQRTGIDVRAVEQGEDAWHKLRLGVITASEVHNVIAKPR
                     SGKKWPDMKMSYFHTLLAEVCTGVAPEVNAKALAWGKQYENDARTLFEFTSGVNVTESP
                     IIYRDESMRTACSPDGLCSDGNGLELKCPFTSRDFMKFRLGGFEAIKSAYMAQVQYSMW
                     VTRKNAWYFANYDPRMKREGLHYVVIERDEKYMASFDEIVPEFIEKMDEALAEIGFVFG
                     EQWR"
     CDS             complement(9189..9974)
                     /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             complement(9980..10396)
                     /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"
     primer_bind     complement(10419..10441)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    10430..10446
                     /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(join(10454..10460,10461..10478,10479..10484))
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an ""up"" mutation"
     CDS             complement(10610..11671)
                     /codon_start=1
                     /gene="recA"
                     /product="DNA repair protein from E. coli"
                     /label=RecA
                     /translation="MAIDENKQKALAAALGQIEKQFGKGSIMRLGEDRSMDVETISTGS
                     LSLDIALGAGGLPMGRIVEIYGPESSGKTTLTLQVIAAAQREGKTCAFIDAEHALDPIY
                     ARKLGVDIDNLLCSQPDTGEQALEICDALARSGAVDVIVVDSVAALTPKAEIEGEIGDS
                     HMGLAARMMSQAMRKLAGNLKQSNTLLIFINQIRMKIGVMFGNPETTTGGNALKFYASV
                     RLDIRRIGAVKEGENVVGSETRVKVVKNKIAAPFKQAEFQILYGEGINFYGELVDLGVK
                     EKLIEKAGAWYSYKGEKIGQGKANATAWLKDNPETAKEIEKKVRELLLSNPNSTPDFSV
                     DDSEGVAETNEDF"
     primer_bind     13205..13224
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(13244..13321)
                     /gene="lacI"
                     /label=lacI promoter
     CDS             complement(13429..14379)
                     /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      14427..14649
                     /label=pSC101 ori
                     /note="low-copy replication origin that requires the Rep101
                     protein"
ORIGIN
        1 ctgctcgcgc aggctgggtg ccaagctctc gggtaacatc aaggcccgat ccttggagcc
       61 cttgccctcc cgcacgatga tcgtgccgtg atcgaaatcc agatccttga cccgcagttg
      121 caaaccctca ctgatccgca tgcggatctt gctgtaggca taggcttggt tatgccggta
      181 ctgccgggcc tcttgcggga ttacgaaatc atcctgtgga gcttagtagg tttagcaaga
      241 tggcagcgcc taaatgtaga atgataaaag gattaagaga ttaatttccc taaaaatgat
      301 aaaacaagcg ttttgaaagc gcttgttttt ttggtttgca gtcagagtag aatagaagta
      361 tcaaaaaaag caccgactcg gtgccacttt ttcaagttga taacggacta gccttatttt
      421 aacttgctat gctgttttga atggttccaa caagattatt ttataacttt tataacaaat
      481 aatcaaggag aaattcaaag aaatttatca gccataaaac aatacttaat actatagaat
      541 gataacaaaa taaactactt tttaaaagaa ttttgtgtta taatctattt attattaagt
      601 attgggtaat attttttgaa gagatatttt gaaaaagaaa aattaaagca tattaaacta
      661 atttcggagg tcattaaaac tattattgaa atcatcaaac tcattatgga tttaatttaa
      721 actttttatt ttaggaggca aaaatggata agaaatactc aataggctta gatatcggca
      781 caaatagcgt cggatgggcg gtgatcactg atgaatataa ggttccgtct aaaaagttca
      841 aggttctggg aaatacagac cgccacagta tcaaaaaaaa tcttataggg gctcttttat
      901 ttgacagtgg agagacagcg gaagcgactc gtctcaaacg gacagctcgt agaaggtata
      961 cacgtcggaa gaatcgtatt tgttatctac aggagatttt ttcaaatgag atggcgaaag
     1021 tagatgatag tttctttcat cgacttgaag agtctttttt ggtggaagaa gacaagaagc
     1081 atgaacgtca tcctattttt ggaaatatag tagatgaagt tgcttatcat gagaaatatc
     1141 caactatcta tcatctgcga aaaaaattgg tagattctac tgataaagcg gatttgcgct
     1201 taatctattt ggccttagcg catatgatta agtttcgtgg tcattttttg attgagggag
     1261 atttaaatcc tgataatagt gatgtggaca aactatttat ccagttggta caaacctaca
     1321 atcaattatt tgaagaaaac cctattaacg caagtggagt agatgctaaa gcgattcttt
     1381 ctgcacgatt gagtaaatca agacgattag aaaatctcat tgctcagctc cccggtgaga
     1441 agaaaaatgg cttatttggg aatctcattg ctttgtcatt gggtttgacc cctaatttta
     1501 aatcaaattt tgatttggca gaagatgcta aattacagct ttcaaaagat acttacgatg
     1561 atgatttaga taatttattg gcgcaaattg gagatcaata tgctgatttg tttttggcag
     1621 ctaagaattt atcagatgct attttacttt cagatatcct aagagtaaat actgaaataa
     1681 ctaaggctcc cctatcagct tcaatgatta aacgctacga tgaacatcat caagacttga
     1741 ctcttttaaa agctttagtt cgacaacaac ttccagaaaa gtataaagaa atcttttttg
     1801 atcaatcaaa aaacggatat gcaggttata ttgatggggg agctagccaa gaagaatttt
     1861 ataaatttat caaaccaatt ttagaaaaaa tggatggtac tgaggaatta ttggtgaaac
     1921 taaatcgtga agatttgctg cgcaagcaac ggacctttga caacggctct attccccatc
     1981 aaattcactt gggtgagctg catgctattt tgagaagaca agaagacttt tatccatttt
     2041 taaaagacaa tcgtgagaag attgaaaaaa tcttgacttt tcgaattcct tattatgttg
     2101 gtccattggc gcgtggcaat agtcgttttg catggatgac tcggaagtct gaagaaacaa
     2161 ttaccccatg gaattttgaa gaagttgtcg ataaaggtgc ttcagctcaa tcatttattg
     2221 aacgcatgac aaactttgat aaaaatcttc caaatgaaaa agtactacca aaacatagtt
     2281 tgctttatga gtattttacg gtttataacg aattgacaaa ggtcaaatat gttactgaag
     2341 gaatgcgaaa accagcattt ctttcaggtg aacagaagaa agccattgtt gatttactct
     2401 tcaaaacaaa tcgaaaagta accgttaagc aattaaaaga agattatttc aaaaaaatag
     2461 aatgttttga tagtgttgaa atttcaggag ttgaagatag atttaatgct tcattaggta
     2521 cctaccatga tttgctaaaa attattaaag ataaagattt tttggataat gaagaaaatg
     2581 aagatatctt agaggatatt gttttaacat tgaccttatt tgaagatagg gagatgattg
     2641 aggaaagact taaaacatat gctcacctct ttgatgataa ggtgatgaaa cagcttaaac
     2701 gtcgccgtta tactggttgg ggacgtttgt ctcgaaaatt gattaatggt attagggata
     2761 agcaatctgg caaaacaata ttagattttt tgaaatcaga tggttttgcc aatcgcaatt
     2821 ttatgcagct gatccatgat gatagtttga catttaaaga agacattcaa aaagcacaag
     2881 tgtctggaca aggcgatagt ttacatgaac atattgcaaa tttagctggt agccctgcta
     2941 ttaaaaaagg tattttacag actgtaaaag ttgttgatga attggtcaaa gtaatggggc
     3001 ggcataagcc agaaaatatc gttattgaaa tggcacgtga aaatcagaca actcaaaagg
     3061 gccagaaaaa ttcgcgagag cgtatgaaac gaatcgaaga aggtatcaaa gaattaggaa
     3121 gtcagattct taaagagcat cctgttgaaa atactcaatt gcaaaatgaa aagctctatc
     3181 tctattatct ccaaaatgga agagacatgt atgtggacca agaattagat attaatcgtt
     3241 taagtgatta tgatgtcgat cacattgttc cacaaagttt ccttaaagac gattcaatag
     3301 acaataaggt cttaacgcgt tctgataaaa atcgtggtaa atcggataac gttccaagtg
     3361 aagaagtagt caaaaagatg aaaaactatt ggagacaact tctaaacgcc aagttaatca
     3421 ctcaacgtaa gtttgataat ttaacgaaag ctgaacgtgg aggtttgagt gaacttgata
     3481 aagctggttt tatcaaacgc caattggttg aaactcgcca aatcactaag catgtggcac
     3541 aaattttgga tagtcgcatg aatactaaat acgatgaaaa tgataaactt attcgagagg
     3601 ttaaagtgat taccttaaaa tctaaattag tttctgactt ccgaaaagat ttccaattct
     3661 ataaagtacg tgagattaac aattaccatc atgcccatga tgcgtatcta aatgccgtcg
     3721 ttggaactgc tttgattaag aaatatccaa aacttgaatc ggagtttgtc tatggtgatt
     3781 ataaagttta tgatgttcgt aaaatgattg ctaagtctga gcaagaaata ggcaaagcaa
     3841 ccgcaaaata tttcttttac tctaatatca tgaacttctt caaaacagaa attacacttg
     3901 caaatggaga gattcgcaaa cgccctctaa tcgaaactaa tggggaaact ggagaaattg
     3961 tctgggataa agggcgagat tttgccacag tgcgcaaagt attgtccatg ccccaagtca
     4021 atattgtcaa gaaaacagaa gtacagacag gcggattctc caaggagtca attttaccaa
     4081 aaagaaattc ggacaagctt attgctcgta aaaaagactg ggatccaaaa aaatatggtg
     4141 gttttgatag tccaacggta gcttattcag tcctagtggt tgctaaggtg gaaaaaggga
     4201 aatcgaagaa gttaaaatcc gttaaagagt tactagggat cacaattatg gaaagaagtt
     4261 cctttgaaaa aaatccgatt gactttttag aagctaaagg atataaggaa gttaaaaaag
     4321 acttaatcat taaactacct aaatatagtc tttttgagtt agaaaacggt cgtaaacgga
     4381 tgctggctag tgccggagaa ttacaaaaag gaaatgagct ggctctgcca agcaaatatg
     4441 tgaatttttt atatttagct agtcattatg aaaagttgaa gggtagtcca gaagataacg
     4501 aacaaaaaca attgtttgtg gagcagcata agcattattt agatgagatt attgagcaaa
     4561 tcagtgaatt ttctaagcgt gttattttag cagatgccaa tttagataaa gttcttagtg
     4621 catataacaa acatagagac aaaccaatac gtgaacaagc agaaaatatt attcatttat
     4681 ttacgttgac gaatcttgga gctcccgctg cttttaaata ttttgataca acaattgatc
     4741 gtaaacgata tacgtctaca aaagaagttt tagatgccac tcttatccat caatccatca
     4801 ctggtcttta tgaaacacgc attgatttga gtcagctagg aggtgactga agtatatttt
     4861 agatgaagat tatttcttaa taactaaaaa tatggtataa tactcttaat aaatgcagta
     4921 atacaggggc ttttcaagac tgaagtctag ctgagacaaa tagtgcgatt acgaaatttt
     4981 ttagacaaaa atagtctacg aggttttaga gctatgctgt tttgaatggt cccaaaactg
     5041 cagcgcaata gttggcgaag taatcgcaac atccgcatta aaatctagcg agggctttac
     5101 taagctccgc aaaaaacccc gcccctgaca gggcggggtt ttttcgcgcc cgatccttgg
     5161 agcccttgcc ctcccgcacg atgatcgtgc cgtgatcgaa atccagatcc ttgacccgca
     5221 gttgcaaacc ctcactgatc cgcatgctta tgacaacttg acggctacat cattcacttt
     5281 ttcttcacaa ccggcacgga actcgctcgg gctggccccg gtgcattttt taaatacccg
     5341 cgagaaatag agttgatcgt caaaaccaac attgcgaccg acggtggcga taggcatccg
     5401 ggtggtgctc aaaagcagct tcgcctggct gatacgttgg tcctcgcgcc agcttaagac
     5461 gctaatccct aactgctggc ggaaaagatg tgacagacgc gacggcgaca agcaaacatg
     5521 ctgtgcgacg ctggcgatat caaaattgct gtctgccagg tgatcgctga tgtactgaca
     5581 agcctcgcgt acccgattat ccatcggtgg atggagcgac tcgttaatcg cttccatgcg
     5641 ccgcagtaac aattgctcaa gcagatttat cgccagcagc tccgaatagc gcccttcccc
     5701 ttgcccggcg ttaatgattt gcccaaacag gtcgctgaaa tgcggctggt gcgcttcatc
     5761 cgggcgaaag aaccccgtat tggcaaatat tgacggccag ttaagccatt catgccagta
     5821 ggcgcgcgga cgaaagtaaa cccactggtg ataccattcg cgagcctccg gatgacgacc
     5881 gtagtgatga atctctcctg gcgggaacag caaaatatca cccggtcggc aaacaaattc
     5941 tcgtccctga tttttcacca ccccctgacc gcgaatggtg agattgagaa tataaccttt
     6001 cattcccagc ggtcggtcga taaaaaaatc gagataaccg ttggcctcaa tcggcgttaa
     6061 acccgccacc agatgggcat taaacgagta tcccggcagc aggggatcat tttgcgcttc
     6121 agccatactt ttcatactcc cgccattcag agaagaaacc aattgtccat attgcatcag
     6181 acattgccgt cactgcgtct tttactggct cttctcgcta accaaaccgg taaccccgct
     6241 tattaaaagc attctgtaac aaagcgggac caaagccatg acaaaaacgc gtaacaaaag
     6301 tgtctataat cacggcagaa aagtccacat tgattatttg cacggcgtca cactttgcta
     6361 tgccatagca tttttatcca taagattagc ggatcctacc tgacgctttt tatcgcaact
     6421 ctctactgtt tctccatgta ggtcgttcgc tccaagcgtt ttagagctag aaatagcaag
     6481 ttaaaataag gctagtccgt tatcaacttg aaaaagtggc accagacccc gttgatgata
     6541 ccgctgcctt actgggtgca ttagccagtc tgaatgacct cgagttttag agctatgctg
     6601 ttttgaatgg tcccaaaact tcagcacact gagacttgtt gagttccatg ttttagagct
     6661 atgctgtttt gaatggactc cattcaacat tgccgatgat aacttgagaa agagggttaa
     6721 taccagcagt cggatacctt cctattcttt ctgttaaagc gttttcatgt tataataggc
     6781 aaaagaagag tagtgtgatc gtccattccg acgtcgagcc gttccataca gaagctgggc
     6841 gaacaaacga tgctcgcctt ccagaaaacc gaggatgcga accacttcat ccggggtcag
     6901 caccaccggc aagcgccgcg acggccgagg tcttccgatc tcctgaagcc agggcagatc
     6961 cgtgcacagc accttgccgt agaagaacag caaggccgcc aatgcctgac gatgcgtgga
     7021 gaccgaaacc ttgcgctcgt tcgccagcca ggacagaaat gcctcgactt cgctgctgcc
     7081 caaggttgcc gggtgacgca caccgtggaa acggatgaag gcacgaaccc agtggacata
     7141 agcctgttcg gttcgtaagc tgtaatgcaa gtagcgtatg cgctcacgca actggtccag
     7201 aaccttgacc gaacgcagcg gtggtaacgg cgcagtggcg gttttcatgg cttgttatga
     7261 ctgttttttt ggggtacagt ctatgcctcg ggcatccaag cagcaagcgc gttacgccgt
     7321 gggtcgatgt ttgatgttat ggagcagcaa cgatgttacg cagcagggca gtcgccctaa
     7381 aacaaagtta aacatcatga gggaagcggt gatcgccgaa gtatcgactc aactatcaga
     7441 ggtagttggc gtcatcgagc gccatctcga accgacgttg ctggccgtac atttgtacgg
     7501 ctccgcagtg gatggcggcc tgaagccaca cagtgatatt gatttgctgg ttacggtgac
     7561 cgtaaggctt gatgaaacaa cgcggcgagc tttgatcaac gaccttttgg aaacttcggc
     7621 ttcccctgga gagagcgaga ttctccgcgc tgtagaagtc accattgttg tgcacgacga
     7681 catcattccg tggcgttatc cagctaagcg cgaactgcaa tttggagaat ggcagcgcaa
     7741 tgacattctt gcaggtatct tcgagccagc cacgatcgac attgatctgg ctatcttgct
     7801 gacaaaagca agagaacata gcgttgcctt ggtaggtcca gcggcggagg aactctttga
     7861 tccggttcct gaacaggatc tatttgaggc gctaaatgaa accttaacgc tatggaactc
     7921 gccgcccgac tgggctggcg atgagcgaaa tgtagtgctt acgttgtccc gcatttggta
     7981 cagcgcagta accggcaaaa tcgcgccgaa ggatgtcgct gcctactggg caatggagcg
     8041 cctgccggcc cagtatcagc ccgtcatact tgaagctaga caggcttatc ttggacaaga
     8101 agaagatcgc ttggcctcgc gcgcagatca gttggaagaa tttgtccact acgtgaaagg
     8161 cgagatcacc aaggtagtcg gcaaataatg tctaacaatt cgttcaagcc gacgccgctt
     8221 cgcggcgcgg cttaactcaa gcgttagatg cactaagcac ataattgctc acagccaaac
     8281 tatcaggtca agtctgcttt tattattttt aagcgtgcat aataagccct acacaaattg
     8341 ggagatatat catgaaaggc tggctttttc ttgttatcgc aatagttggc gaagtaatcg
     8401 caacatccgc attaaaatct agcgagggct ttactaagct cctgcagaga tctgaattcc
     8461 ctagagagac gaaagtgatt gcgcctaccc ggatattatc gtgaggatgc gtcatcgcca
     8521 ttgctcccca aatacaaaac caatttcagc cagtgcctcg tccatttttt cgatgaactc
     8581 cggcacgatc tcgtcaaaac tcgccatgta cttttcatcc cgctcaatca cgacataatg
     8641 caggccttca cgcttcatac gcgggtcata gttggcaaag taccaggcat tttttcgcgt
     8701 cacccacatg ctgtactgca cctgggccat gtaagctgac tttatggcct cgaaaccacc
     8761 gagccggaac ttcatgaaat cccgggaggt aaacgggcat ttcagttcaa ggccgttgcc
     8821 gtcactgcat aaaccatcgg gagagcaggc ggtacgcata ctttcgtcgc gatagatgat
     8881 cggggattca gtaacattca cgccggaagt gaattcaaac agggttctgg cgtcgttctc
     8941 gtactgtttt ccccaggcca gtgctttagc gttaacttcc ggagccacac cggtgcaaac
     9001 ctcagcaagc agggtgtgga agtaggacat tttcatgtca ggccacttct ttccggagcg
     9061 gggttttgct atcacgttgt gaacttctga agcggtgatg acgccgagcc gtaatttgtg
     9121 ccacgcatct tccccctgtt cgacagctct cacatcgatc ccggtacgct gcaggataat
     9181 gtccggtgtc atgctgccac cttctgctct gcggctttct gtttcaggaa tccaagagct
     9241 tttactgctt cggcctgtgt cagttctgac gatgcacgaa tgtcgcggcg aaatatctgg
     9301 gaacagagcg gcaataagtc gtcatcccat gttttatcca gggcgatcag cagagtgtta
     9361 atctcctgca ttgtttcatc gttaaccgga gtgatgtcgc gttccggctg acgttctgca
     9421 gtgtatgcag tattttcgac aatgcgctcg gcttcatcct tgtcatagat accagcaaat
     9481 ccgaaggcca gacgggcaca ctgaatcatg gctttatgac gtaacatccg tttgggatgc
     9541 gactgccacg gccccgtgat ttctctgcct tcgcgagttt tgaatggttc gcggcggcat
     9601 tcatccatcc attcggtaac gcagatcgga tgattacggt ccttgcggta aatccggcat
     9661 gtacaggatt cattgtcctg ctcaaagtcc atgccatcaa actgctggtt ttcattgatg
     9721 atgcgggacc agccatcaac gcccaccacc ggaacgatgc cattctgctt atcaggaaag
     9781 gcgtaaattt ctttcgtcca cggattaagg ccgtactggt tggcaacgat cagtaatgcg
     9841 atgaactgcg catcgctggc atcaccttta aatgccgtct ggcgaagagt ggtgatcagt
     9901 tcctgtgggt cgacagaatc catgccgaca cgttcagcca gcttcccagc cagcgttgcg
     9961 agtgcagtac tcattcgttt tatacctctg aatcaatatc aacctggtgg tgagcaatgg
    10021 tttcaaccat gtaccggatg tgttctgcca tgcgctcctg aaactcaaca tcgtcatcaa
    10081 acgcacgggt aatggatttt ttgctggccc cgtggcgttg caaatgatcg atgcatagcg
    10141 attcaaacag gtgctggggc aggccttttt ccatgtcgtc tgccagttct gcctctttct
    10201 cttcacgggc gagctgctgg tagtgacgcg cccagctctg agcctcaaga cgatcctgaa
    10261 tgtaataagc gttcatggct gaactcctga aatagctgtg aaaatatcgc ccgcgaaatg
    10321 ccgggctgat taggaaaaca ggaaaggggg ttagtgaatg cttttgcttg atctcagttt
    10381 cagtattaat atccattttt tataagcgtc gactgtttcc tgtgtgaaat tgttatccgc
    10441 tcacaattcc acacattata cgagccggaa gcataaagtg taaagcctgg ggtgcctaat
    10501 gagtgagaat tcggatctcg acgggatgtt gattctgtca tggcatatcc ttacaactta
    10561 aaaaagcaaa agggccgcag atgcgaccct tgtgtatcaa acaagacgat taaaaatctt
    10621 cgttagtttc tgctacgcct tcgctatcat ctacagagaa atccggcgtt gagttcgggt
    10681 tgctcagcag caactcacgt actttcttct cgatctcttt cgcggtttcc gggttatctt
    10741 tcagccaggc agtcgcattc gctttaccct gaccgatctt ctcacctttg tagctgtacc
    10801 acgcgcctgc tttctcgatc agcttctctt ttacgcccag gtcaaccagt tcgccgtaga
    10861 agttgatacc ttcgccgtag aggatctgga attcagcctg tttaaacggc gcagcgattt
    10921 tgttcttcac cactttcacg cgggtttcgc tacccaccac gttttcgccc tctttcaccg
    10981 cgccgatacg acggatgtcg agacgaacag aggcgtagaa tttcagcgcg ttaccaccgg
    11041 tagtggtttc cgggttaccg aacatcacac caattttcat acggatctgg ttgatgaaga
    11101 tcagcagcgt gttggactgc ttcaggttac ccgccagctt acgcatcgcc tggctcatca
    11161 tacgtgccgc aaggcccatg tgagagtcgc cgatttcgcc ttcgatttcc gctttcggcg
    11221 tcagtgccgc cacggagtca acgacgataa cgtctactgc gccagaacgc gccagggcgt
    11281 cacagatttc cagtgcctgc tcgccggtgt ccggctggga gcacagcagg ttgtcgatat
    11341 cgacgcccag tttacgtgcg tagattgggt ccagcgcgtg ttcagcatcg ataaacgcac
    11401 aggttttacc ttcacgctgc gctgcggcga tcacctgcag cgtcagcgtg gttttaccgg
    11461 aagattccgg tccgtagatt tcgacgatac ggcccatcgg cagaccacct gccccaagcg
    11521 cgatatccag tgaaagcgaa ccggtagaga tggtttccac atccatggaa cggtcttcac
    11581 ccaggcgcat gatggagcct ttaccaaatt gtttctcaat ctggcccagt gctgccgcca
    11641 acgctttctg tttgttttcg tcgatagcca tttttactcc tgtcatgccg ggtaataccg
    11701 gatagtcaat atgttctgtt gaagcaatta tactgtatgc tcatacagta tcaagtgttt
    11761 tgtagaaatt gttgccacaa ggtctgcaat gcatacgcag tagcctgacg acgcaccgca
    11821 tcacggtcgc cgctgaagca ttcccgccgg gtaatgcctt caccgcgggc agtggcaaaa
    11881 gcaaaccaga cggtgccgac aggcttctct agatccgcct acctttcacg agttgcgcag
    11941 tttgtctgca agactctatg agaagcagat aagcgataag tttgctcaac atcttctcgg
    12001 gcataagtcg gacaccatgg catcacagta tcgtgatgac agaggcaggg agtgggacaa
    12061 aattgaaatc aaataatgat tttattttga ctgatagtga cctgttcgtt gcaacaaatt
    12121 gataagcaat gccaagcttg gcactggctg atcagctagc atcgaatggc gcaaaacctt
    12181 ksgcrrymtg kcrtgmyrgc tgcattaatg aatcggccaa cgcgcgggga gaggcggttt
    12241 gcgtattggg cgccagggtg gtttttcttt tcaccagtga gacgggcaac agctgattgc
    12301 ccttcaccgc ctggccctga gagagttgca gcaagcggtc cacgctggtt tgccccagca
    12361 ggcgaaaatc ctgtttgatg gtggttaacg gcgggatata acatgagctg tcttcggtat
    12421 cgtcgtatcc cactaccgag atatccgcac caacgcgcag cccggactcg gtaatggcgc
    12481 gcattgcgcc cagcgccatc tgatcgttgg caaccagcat cgcagtggga acgatgccct
    12541 cattcagcat ttgcatggtt tgttgaaaac cggacatggc actccagtcg ccttcccgtt
    12601 ccgctatcgg ctgaatttga ttgcgagtga gatatttatg ccagccagcc agacgcagac
    12661 gcgccgagac agaacttaat gggcccgcta acagcgcgat ttgctggtga cccaatgcga
    12721 ccagatgctc cacgcccagt cgcgtaccgt cttcatggga gaaaataata ctgttgatgg
    12781 gtgtctggtc agagacatca agaaataacg ccggaacatt agtgcaggca gcttccacag
    12841 caatggcatc ctggtcatcc agcggatagt taatgatcag cccactgacg cgttgcgcga
    12901 gaagattgtg caccgccgct ttacaggctt cgacgccgct tcgttctacc atcgacacca
    12961 ccacgctggc acccagttga tcggcgcgag atttaatcgc cgcgacaatt tgcgacggcg
    13021 cgtgcagggc cagactggag gtggcaacgc caatcagcaa cgactgtttg cccgccagtt
    13081 gttgtgccac gcggttggga atgtaattca gctccgccat cgccgcttcc actttttccc
    13141 gcgttttcgc agaaacgtgg ctggcctggt tcaccacgcg ggaaacggtc tgataagaga
    13201 caccggcata ctctgcgaca tcgtataacg ttactggttt cacattcacc accctgaatt
    13261 gactctcttc cgggcgctat catgccatac cgcgaaaggt tttgcgccat tcgatgctag
    13321 cccatgggta tggacagttt tccctttgat atgtaacggt gaacagttgt tctacttttg
    13381 tttgttagtc ttgatgcttc actgatagat acaagagcca taagaacctc agatccttcc
    13441 gtatttagcc agtatgttct ctagtgtggt tcgttgtttt tgcgtgagcc atgagaacga
    13501 accattgaga tcatacttac tttgcatgtc actcaaaaat tttgcctcaa aactggtgag
    13561 ctgaattttt gcagttaaag catcgtgtag tgtttttctt agtccgttat gtaggtagga
    13621 atctgatgta atggttgttg gtattttgtc accattcatt tttatctggt tgttctcaag
    13681 ttcggttacg agatccattt gtctatctag ttcaacttgg aaaatcaacg tatcagtcgg
    13741 gcggcctcgc ttatcaacca ccaatttcat attgctgtaa gtgtttaaat ctttacttat
    13801 tggtttcaaa acccattggt taagcctttt aaactcatgg tagttatttt caagcattaa
    13861 catgaactta aattcatcaa ggctaatctc tatatttgcc ttgtgagttt tcttttgtgt
    13921 tagttctttt aataaccact cataaatcct catagagtat ttgttttcaa aagacttaac
    13981 atgttccaga ttatatttta tgaatttttt taactggaaa agataaggca atatctcttc
    14041 actaaaaact aattctaatt tttcgcttga gaacttggca tagtttgtcc actggaaaat
    14101 ctcaaagcct ttaaccaaag gattcctgat ttccacagtt ctcgtcatca gctctctggt
    14161 tgctttagct aatacaccat aagcattttc cctactgatg ttcatcatct gaacgtattg
    14221 gttataagtg aacgataccg tccgttcttt ccttgtaggg ttttcaatcg tggggttgag
    14281 tagtgccaca cagcataaaa ttagcttggt ttcatgctcc gttaagtcat agcgactaat
    14341 cgctagttca tttgctttga aaacaactaa ttcagacata catctcaatt ggtctaggtg
    14401 attttaatca ctataccaat tgagatgggc tagtcaatga taattactag tccttttcct
    14461 ttgagttgtg ggtatctgta aattctgcta gacctttgct ggaaaacttg taaattctgc
    14521 tagaccctct gtaaattccg ctagaccttt gtgtgttttt tttgtttata ttcaagtggt
    14581 tataatttat agaataaaga aagaataaaa aaagataaaa agaatagatc ccagccctgt
    14641 gtataactca ctactttagt cagttccgca gtattacaaa aggatgtcgc aaacgctgtt
    14701 tgctcctcta caaaacagac cttaaaaccc taaaggctta agtagcaccc tcgcaagctc
    14761 ggttgcggcc gcaatcgggc aaatcgctga atattccttt tgtctccgac catcaggcac
    14821 ctgagtcgct gtctttttcg tgacattcag ttcgctgcgc tcacggctct ggcagtgaat
    14881 gggggtaaat ggcactacag gcgcctttta tggattcatg caaggaaact acccataata
    14941 caagaaaagc ccgtcacggg cttctcaggg cgttttatgg cgggtctgct atgtggtgct
    15001 atctgacttt ttgctgttca gcagttcctg ccctctgatt ttccagtctg accacttcgg
    15061 attatcccgt gacaggtcat tcagactggc taatgcaccc agtaaggcag cggtatcatc
    15121 aacggggtct gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt
    15181 atcaaaaagg atcttcacct agatcctttt aaattaaaaa tgaagtttta aatcaatcta
    15241 aagtatatat gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg aggcacctat
    15301 ctcagcgatc tgtctatttc gttcatccat agttgcctga ctccccgtcg tgtagataac
    15361 tacgatacgg gagggcttac catctggccc cagtgctgca atgataccgc gagacccacg
    15421 ctcaccggct ccagatttat cagcaataaa ccagccagcc g
//
