LOCUS       sequence-180994-       11507 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Allows inducible expression of gRNAs and Cas9 for plasmid curing as 
            well as self-curing via a temperature sensitive RK2 replicon..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 10980 to 11014)
  AUTHORS   Lauritsen I, Porse A, Sommer MOA, Norholm MHH
  TITLE     A versatile one-step CRISPR-Cas9 based approach to plasmid-curing.
  JOURNAL   Microb Cell Fact. 2017 Aug 2;16(1):135. doi: 
            10.1186/s12934-017-0748-z.
  PUBMED    28764701
REFERENCE   2  (bases 1 to 11507)
  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: "Microb Cell
            Fact."; date: "2017-08-2"; volume: "16(1):135. doi"; pages: " 
            10.1186/s12934-017-0748-z"
COMMENT     Sequence Label: pFREE-RK2
FEATURES             Location/Qualifiers
     source          1..11507
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    17..35
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    42..60
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     CDS             146..4252
                     /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             146..4249
                     /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"
     primer_bind     complement(4287..4305)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      4301..4348
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     CDS             4482..5105
                     /codon_start=1
                     /gene="tetR from transposon Tn10"
                     /product="tetracycline repressor TetR"
                     /label=TetR
                     /note="TetR binds to the tetracycline operator tetO to 
                     inhibit transcription. This inhibition can be relieved by 
                     adding tetracycline or doxycycline."
                     /translation="MSRLDKSKVINSALELLNEVGIEGLTTRKLAQKLGVEQPTLYWHV
                     KNKRALLDALAIEMLDRHHTHFCPLEGESWQDFLRNNAKSFRCALLSHRDGAKVHLGTR
                     PTEKQYETLENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEDQEHQVAKEERETPTT
                     DSMPPLLRQAIELFDHQGAEPAFLFGLELIICGLEKQLKCESGS"
     primer_bind     complement(4555..4574)
                     /label=Tet-R
                     /note="Tetracycline resistance gene, reverse primer"
     CDS             complement(5267..6082)
                     /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(5267..6082)
                     /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     5990..6009
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     promoter        complement(6083..6174)
                     /gene="bla"
                     /label=AmpR promoter
     RBS             6257..6265
                     /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)"
     misc_feature    6280..6298
                     /label=Tn5 ME
                     /note="hyperactive mosaic end for Tn5 transposase 
                     recognition (Reznikoff et al., 2004)"
     RBS             6802..6810
                     /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)"
     misc_feature    6825..6843
                     /label=Tn5 ME
                     /note="hyperactive mosaic end for Tn5 transposase 
                     recognition (Reznikoff et al., 2004)"
     rep_origin      8217..8748
                     /label=oriV
                     /note="incP origin of replication"
     CDS             9477..10622
                     /codon_start=1
                     /product="trans-acting replication protein that binds to 
                     and activates oriV"
                     /label=trfA
                     /translation="MNRTFDRKAYRQELIDAGFSAEDAETIASRTVMRAPRETFQSVGS
                     MVQQATAKIERDSVQLAPPALPAPSAAVERSRRLEQEAAGLAKSMTIDTRGTMTTKKRK
                     TAGEDLAKQVSEAKQAALLKHTKQQIKEMQLSLFDIAPWPDTMRAMPNDTARSALFTTR
                     NKKIPREALQNKVIFHVNKDVKITYTGVELRADDDELVWQQVLEYAKRTPIGEPITFTF
                     YELCQDLGWSINGRYYTKAEECLSRLQATAMGFTSDRVGHLESVSLLHRFRVLDRGKKT
                     SRCQVLIDEEIVVLFAGDHYTKFIWEKYRKLSPTARRMFDYFSSHREPYPLKLETFRLM
                     CGSDSTRVKKWREQVGEACEELRGSGLVEHAWVNDLVHCKR"
     misc_feature    10662..10680
                     /label=Tn5 ME
                     /note="hyperactive mosaic end for Tn5 transposase 
                     recognition (Reznikoff et al., 2004)"
     misc_RNA        complement(10901..10979)
                     /label=tracrRNA
                     /note="trans-activating CRISPR RNA for the Streptococcus 
                     pyogenes CRISPR/Cas9 system"
     promoter        complement(10980..11014)
                     /label=﻿BBa_J23100
                     /note="Anderson constitutive promoter 
                     (https://parts.igem.org/Promoters/Catalog/Anderson). 
                     Measured strength relative to BBa_J223100: 1.0
                     "
                     /citation=[1]
     repeat_region   11151..11186
                     /label=DR
                     /note="direct repeat for the Streptococcus pyogenes 
                     CRISPR/Cas system"
     repeat_region   11217..11252
                     /label=DR
                     /note="direct repeat for the Streptococcus pyogenes 
                     CRISPR/Cas system"
     repeat_region   11283..11318
                     /label=DR
                     /note="direct repeat for the Streptococcus pyogenes 
                     CRISPR/Cas system"
     repeat_region   11349..11384
                     /label=DR
                     /note="direct repeat for the Streptococcus pyogenes 
                     CRISPR/Cas system"
     repeat_region   11415..11450
                     /label=DR
                     /note="direct repeat for the Streptococcus pyogenes 
                     CRISPR/Cas system"
ORIGIN
        1 tcgagtctgg ttataatccc tatcagtgat agagattgac atccctatca gtgatagaga
       61 tactgagcac agaaggagat atacatggat aagaaatact gagcactatc agtgatagag
      121 atactgagca cagaaggaga tatacatgga taagaaatac tcaataggct tagatatcgg
      181 cacaaatagc gtcggatggg cggtgatcac tgatgaatat aaggttccgt ctaaaaagtt
      241 caaggttctg ggaaatacag accgccacag tatcaaaaaa aatcttatag gggctctttt
      301 atttgacagt ggagagacag cggaagcgac tcgtctcaaa cggacagctc gtagaaggta
      361 tacacgtcgg aagaatcgta tttgttatct acaggagatt ttttcaaatg agatggcgaa
      421 agtagatgat agtttctttc atcgacttga agagtctttt ttggtggaag aagacaagaa
      481 gcatgaacgt catcctattt ttggaaatat agtagatgaa gttgcttatc atgagaaata
      541 tccaactatc tatcatctgc gaaaaaaatt ggtagattct actgataaag cggatttgcg
      601 cttaatctat ttggccttag cgcatatgat taagtttcgt ggtcattttt tgattgaggg
      661 agatttaaat cctgataata gtgatgtgga caaactattt atccagttgg tacaaaccta
      721 caatcaatta tttgaagaaa accctattaa cgcaagtgga gtagatgcta aagcgattct
      781 ttctgcacga ttgagtaaat caagacgatt agaaaatctc attgctcagc tccccggtga
      841 gaagaaaaat ggcttatttg ggaatctcat tgctttgtca ttgggtttga cccctaattt
      901 taaatcaaat tttgatttgg cagaagatgc taaattacag ctttcaaaag atacttacga
      961 tgatgattta gataatttat tggcgcaaat tggagatcaa tatgctgatt tgtttttggc
     1021 agctaagaat ttatcagatg ctattttact ttcagatatc ctaagagtaa atactgaaat
     1081 aactaaggct cccctatcag cttcaatgat taaacgctac gatgaacatc atcaagactt
     1141 gactctttta aaagctttag ttcgacaaca acttccagaa aagtataaag aaatcttttt
     1201 tgatcaatca aaaaacggat atgcaggtta tattgatggg ggagctagcc aagaagaatt
     1261 ttataaattt atcaaaccaa ttttagaaaa aatggatggt actgaggaat tattggtgaa
     1321 actaaatcgt gaagatttgc tgcgcaagca acggaccttt gacaacggct ctattcccca
     1381 tcaaattcac ttgggtgagc tgcatgctat tttgagaaga caagaagact tttatccatt
     1441 tttaaaagac aatcgtgaga agattgaaaa aatcttgact tttcgaattc cttattatgt
     1501 tggtccattg gcgcgtggca atagtcgttt tgcatggatg actcggaagt ctgaagaaac
     1561 aattacccca tggaattttg aagaagttgt cgataaaggt gcttcagctc aatcatttat
     1621 tgaacgcatg acaaactttg ataaaaatct tccaaatgaa aaagtactac caaaacatag
     1681 tttgctttat gagtatttta cggtttataa cgaattgaca aaggtcaaat atgttactga
     1741 aggaatgcga aaaccagcat ttctttcagg tgaacagaag aaagccattg ttgatttact
     1801 cttcaaaaca aatcgaaaag taaccgttaa gcaattaaaa gaagattatt tcaaaaaaat
     1861 agaatgtttt gatagtgttg aaatttcagg agttgaagat agatttaatg cttcattagg
     1921 tacctaccat gatttgctaa aaattattaa agataaagat tttttggata atgaagaaaa
     1981 tgaagatatc ttagaggata ttgttttaac attgacctta tttgaagata gggagatgat
     2041 tgaggaaaga cttaaaacat atgctcacct ctttgatgat aaggtgatga aacagcttaa
     2101 acgtcgccgt tatactggtt ggggacgttt gtctcgaaaa ttgattaatg gtattaggga
     2161 taagcaatct ggcaaaacaa tattagattt tttgaaatca gatggttttg ccaatcgcaa
     2221 ttttatgcag ctgatccatg atgatagttt gacatttaaa gaagacattc aaaaagcaca
     2281 agtgtctgga caaggcgata gtttacatga acatattgca aatttagctg gtagccctgc
     2341 tattaaaaaa ggtattttac agactgtaaa agttgttgat gaattggtca aagtaatggg
     2401 gcggcataag ccagaaaata tcgttattga aatggcacgt gaaaatcaga caactcaaaa
     2461 gggccagaaa aattcgcgag agcgtatgaa acgaatcgaa gaaggtatca aagaattagg
     2521 aagtcagatt cttaaagagc atcctgttga aaatactcaa ttgcaaaatg aaaagctcta
     2581 tctctattat ctccaaaatg gaagagacat gtatgtggac caagaattag atattaatcg
     2641 tttaagtgat tatgatgtcg atcacattgt tccacaaagt ttccttaaag acgattcaat
     2701 agacaataag gtcttaacgc gttctgataa aaatcgtggt aaatcggata acgttccaag
     2761 tgaagaagta gtcaaaaaga tgaaaaacta ttggagacaa cttctaaacg ccaagttaat
     2821 cactcaacgt aagtttgata atttaacgaa agctgaacgt ggaggtttga gtgaacttga
     2881 taaagctggt tttatcaaac gccaattggt tgaaactcgc caaatcacta agcatgtggc
     2941 acaaattttg gatagtcgca tgaatactaa atacgatgaa aatgataaac ttattcgaga
     3001 ggttaaagtg attaccttaa aatctaaatt agtttctgac ttccgaaaag atttccaatt
     3061 ctataaagta cgtgagatta acaattacca tcatgcccat gatgcgtatc taaatgccgt
     3121 cgttggaact gctttgatta agaaatatcc aaaacttgaa tcggagtttg tctatggtga
     3181 ttataaagtt tatgatgttc gtaaaatgat tgctaagtct gagcaagaaa taggcaaagc
     3241 aaccgcaaaa tatttctttt actctaatat catgaacttc ttcaaaacag aaattacact
     3301 tgcaaatgga gagattcgca aacgccctct aatcgaaact aatggggaaa ctggagaaat
     3361 tgtctgggat aaagggcgag attttgccac agtgcgcaaa gtattgtcca tgccccaagt
     3421 caatattgtc aagaaaacag aagtacagac aggcggattc tccaaggagt caattttacc
     3481 aaaaagaaat tcggacaagc ttattgctcg taaaaaagac tgggatccaa aaaaatatgg
     3541 tggttttgat agtccaacgg tagcttattc agtcctagtg gttgctaagg tggaaaaagg
     3601 gaaatcgaag aagttaaaat ccgttaaaga gttactaggg atcacaatta tggaaagaag
     3661 ttcctttgaa aaaaatccga ttgacttttt agaagctaaa ggatataagg aagttaaaaa
     3721 agacttaatc attaaactac ctaaatatag tctttttgag ttagaaaacg gtcgtaaacg
     3781 gatgctggct agtgccggag aattacaaaa aggaaatgag ctggctctgc caagcaaata
     3841 tgtgaatttt ttatatttag ctagtcatta tgaaaagttg aagggtagtc cagaagataa
     3901 cgaacaaaaa caattgtttg tggagcagca taagcattat ttagatgaga ttattgagca
     3961 aatcagtgaa ttttctaagc gtgttatttt agcagatgcc aatttagata aagttcttag
     4021 tgcatataac aaacatagag acaaaccaat acgtgaacaa gcagaaaata ttattcattt
     4081 atttacgttg acgaatcttg gagctcccgc tgcttttaaa tattttgata caacaattga
     4141 tcgtaaacga tatacgtcta caaaagaagt tttagatgcc actcttatcc atcaatccat
     4201 cactggtctt tatgaaacac gcattgattt gagtcagcta ggaggtgact gagatcccat
     4261 gttaccggta tcctaggctg ctgccaccgc tgagcaataa ctagcataac cccttggggc
     4321 ctctaaacgg gtcttgaggg gttttttgct gaaacctcag gcatttgaga agcacacggt
     4381 cacactgctt ccggtagtca ataaaccggt attgacgcca acgggtcaca cgggtacagt
     4441 gctagcagaa acatatccat gaaatcccgc cccgaattca tatgtctaga ttagataaaa
     4501 gtaaagtgat taacagcgca ttagagctgc ttaatgaggt cggaatcgaa ggtttaacaa
     4561 cccgtaaact cgcccagaag ctaggtgtag agcagcctac attgtattgg catgtaaaaa
     4621 ataagcgggc tttgctcgac gccttagcca ttgagatgtt agataggcac catactcact
     4681 tttgcccttt agaaggggaa agctggcaag attttttacg taataacgct aaaagtttta
     4741 gatgtgcttt actaagtcat cgcgatggag caaaagtaca tttaggtaca cggcctacag
     4801 aaaaacagta tgaaactctc gaaaatcaat tagccttttt atgccaacaa ggtttttcac
     4861 tagagaatgc attatatgca ctcagcgctg tggggcattt tactttaggt tgcgtattgg
     4921 aagatcaaga gcatcaagtc gctaaagaag aaagggaaac acctactact gatagtatgc
     4981 cgccattatt acgacaagct atcgaattat ttgatcacca aggtgcagag ccagccttct
     5041 tattcggcct tgaattgatc atatgcggat tagaaaaaca acttaaatgt gaaagtgggt
     5101 cttaagcaat agacataagc ggctatttaa cgaccctgcc ctgaaccgac gacaagctga
     5161 cgaccgggtc tccgcaagtg gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt
     5221 atttttctaa atacattcaa atatgtatcc gctcatgaat taattcttag aaaaactcat
     5281 cgagcatcaa atgaaactgc aatttattca tatcaggatt atcaatacca tatttttgaa
     5341 aaagccgttt ctgtaatgaa ggagaaaact caccgaggca gttccatagg atggcaagat
     5401 cctggtatcg gtctgcgatt ccgactcgtc caacatcaat acaacctatt aatttcccct
     5461 cgtcaaaaat aaggttatca agtgagaaat caccatgagt gacgactgaa tccggtgaga
     5521 atggcaaaag tttatgcatt tctttccaga cttgttcaac aggccagcca ttacgctcgt
     5581 catcaaaatc actcgcatca accaaaccgt tattcattcg tgattgcgcc tgagcgagac
     5641 gaaatacgcg gtcgctgtta aaaggacaat tacaaacagg aatcgaatgc aaccggcgca
     5701 ggaacactgc cagcgcatca acaatatttt cacctgaatc aggatattct tctaatacct
     5761 ggaatgctgt tttcccgggg atcgcagtgg tgagtaacca tgcatcatca ggagtacgga
     5821 taaaatgctt gatggtcgga agaggcataa attccgtcag ccagtttagt ctgaccatct
     5881 catctgtaac atcattggca acgctacctt tgccatgttt cagaaacaac tctggcgcat
     5941 cgggcttccc atacaatcga tagattgtcg cacctgattg cccgacatta tcgcgagccc
     6001 atttataccc atataaatca gcatccatgt tggaatttaa tcgcggccta gagcaagacg
     6061 tttcccgttg aatatggctc atactcttcc tttttcaata ttattgaagc atttatcagg
     6121 gttattgtct catgagcgga tacatatttg aatgtattta gaaaaataaa caaataggca
     6181 tgctagcgca gaataaatat ctaacaccgt gcgtgttgac tattttacct ctggcggtga
     6241 taatggttgc atgtactaag gaggttgtat ggaacaacga gatgtgtata agagacagct
     6301 gaaacgccgt cgaagccgtg tgcgagacac cgcggccgcc ggcgttgtgg atacctcgcg
     6361 gaaaacttgg ccctcactga cagatgaggg gcggacgttg acacttgagg ggccgactca
     6421 cccggcgcgg cgttgacaga tgaggggcag gctcgatttc ggccggcgac gtggagctgg
     6481 ccagcctcgc aaatcggcga aaacgcctga ttttacgcga gtttcccaca gatgatgtgg
     6541 acaagcctgg ggataagtgc cctgcggtat tgacacttga ggggcgcgac tactgacaga
     6601 tgaggggcgc gatccttgac acttgagggg cagagtgctg acagatgagg ggcgcaccta
     6661 ttgacatttg aggggctgtc cacaggcaga aaatccagca tttgcaaggg tttccgcccg
     6721 tttttcggcc accgctaacc tgtcttttaa cctgctttta aaccaatatt tataaacctt
     6781 gtttttaatg gttgcatgta ctaaggaggt tgtatggaac aacgagatgt gtataagaga
     6841 cagctgaaac gccgtcgaag ccgtgtgctg agagatcccc tcataatttc cccaaagcgt
     6901 aaccatgtgt gaataaattt tgagctagta gggttgcagc cacgagtaag tcttcccttg
     6961 ttattgtgta gccagaatgc cgcaaaactt ccatgcctaa gcgaactgtt gagagtacgt
     7021 ttcgatttct gactgtgtta gcctggaagt gcttgtccca accttgtttc tgagcatgaa
     7081 cgcccgcaag ccaacatgtt agttgaagca tcagggcgat tagcagcatg atatcaaaac
     7141 gctctgagct gctcgttcgg ctatggcgta ggcctagtcc gtaggcagga cttttcaagt
     7201 ctcggaaggt ttcttcaatc tgcattcgct tcgaatagat attaacaagt tgtttgggtg
     7261 ttcgaatttc aacaggtaag ttagttgcta gaacccatgg ctcctttgcc gacgctgagt
     7321 agattttagg tgacgggtgg tgacaatgag tccgtgtcga gcgctgattt tttcggcctt
     7381 tagagcgaga tttatacaat agaatttggc atgagattgg attgctttta gtcagcctct
     7441 tatagcctaa agtctttgag tgactagatg acatatcatg taagttgctg ataggtttcc
     7501 agttttccgc tcctaggtct gcatattgta cttttcctct tactcgactt aaccagtacc
     7561 aacccagctt ctcaacggat ttataccatg gcactttaaa gccagcatca ctgacaatga
     7621 gcggtgtggt gttactcggt agaatgctcg caaggtcggc tagaaattgg tcatgagctt
     7681 tctttgaaca ttgctctgaa agcgggaacg ctttctcata aagagtaaca gaacgaccgt
     7741 gtagtgcgac tgaagctcgc aataccataa gtcgtttttg ctcacgaata tcagaccagt
     7801 caacaagtac aatgggcatc gtattgcccg aacagataaa gctagcatgc caacggtata
     7861 cagcgagtcg ctctttgtgg aggtgacgat tacctaacaa tcggtcgatt cgtttgatgt
     7921 tatgttttgt tctcgctttg gttggcaggt tacggccaag ttcggtaaga gtgagagttt
     7981 tacagtcaag taatgcgtgg caagccaacg ttaagctgtt gagtcgtttt aagtgtaatt
     8041 cggggcagaa ttggtaaaga gagtcgtgta aaatatcgag ttcgcacatc ttgttgtctg
     8101 attattgatt tttcgcgaaa ccatttgatc atatgacaag atgtgtatcc accttaactt
     8161 aatgattttt accaaaatca ttaggggatt catcagagcc gtgtgcgaga caccgcggcc
     8221 gccggcgttg tggatacctc gcggaaaact tggccctcac tgacagatga ggggcggacg
     8281 ttgacacttg aggggccgac tcacccggcg cggcgttgac agatgagggg caggctcgat
     8341 ttcggccggc gacgtggagc tggccagcct cgcaaatcgg cgaaaacgcc tgattttacg
     8401 cgagtttccc acagatgatg tggacaagcc tggggataag tgccctgcgg tattgacact
     8461 tgaggggcgc gactactgac agatgagggg cgcgatcctt gacacttgag gggcagagtg
     8521 ctgacagatg aggggcgcac ctattgacat ttgaggggct gtccacaggc agaaaatcca
     8581 gcatttgcaa gggtttccgc ccgtttttcg gccaccgcta acctgtcttt taacctgctt
     8641 ttaaaccaat atttataaac cttgttttta accagggctg cgccctgtgc gcgtgaccgc
     8701 gcacgccgaa ggggggtgcc cccccttctc gaaccctccc ggcccgctaa cgcgggcctc
     8761 ccatcccccc aggggctgcg cccctcggcc gcgaacggcc tcaccccaaa aatggcagcg
     8821 ctggcagtcc ttgccattgc cgggatcggg gcagtaacgg gatgggcgat cagcccgagc
     8881 gcgacgcccg gaagcattga cgtgccgcag gtgctggcat cgacattcag cgaccaggtg
     8941 ccgggcagtg agggcggcgg cctgggtggc ggcctgccct tcacttcggc cgtcggggca
     9001 ttcacggact tcatggcggg gccggcaatt tttaccttgg gcattcttgg catagtggtc
     9061 gcgggtgccg tgctcgtgtt cgggggtgaa ttaattcccc ggatccgtca gcttcacgct
     9121 gccgcaagca ctcagggcgc aagggctgct aaaggaagcg gaacacgtag aaagccagtc
     9181 cgcagaaacg gtgctgaccc cggatgaatg tcagctactg ggctatctgg acaagggaaa
     9241 acgcaagcgc aaagagaaag caggtagctt gcagtgggct tacatggcga tagctagact
     9301 gggcggtttt atggacagca agcgaaccgg aattgccagc tggggcgccc tctggtaagg
     9361 ttgggaagcc ctgcaaagta aactggatgg ctttcttgcc gccaaggatc tgatggcgca
     9421 ggggatcaag atcgacggat ccggggaatt ccggggcaat cccgcaagga gggtgaatga
     9481 atcggacgtt tgaccggaag gcatacaggc aagaactgat cgacgcgggg ttttccgccg
     9541 aggatgccga aaccatcgca agccgcaccg tcatgcgtgc gccccgcgaa accttccagt
     9601 ccgtcggctc gatggtccag caagctacgg ccaagatcga gcgcgacagc gtgcaactgg
     9661 ctccccctgc cctgcccgcg ccatcggccg ccgtggagcg ttcgcgtcgt ctcgaacagg
     9721 aggcggcagg tttggcgaag tcgatgacca tcgacacgcg aggaactatg acgaccaaga
     9781 agcgaaaaac cgccggcgag gacctggcaa aacaggtcag cgaggccaag caggccgcgt
     9841 tgctgaaaca cacgaagcag cagatcaagg aaatgcagct ttccttgttc gatattgcgc
     9901 cgtggccgga cacgatgcga gcgatgccaa acgacacggc ccgctctgcc ctgttcacca
     9961 cgcgcaacaa gaaaatcccg cgcgaggcgc tgcaaaacaa ggtcattttc cacgtcaaca
    10021 aggacgtgaa gatcacctac accggcgtcg agctgcgggc cgacgatgac gaactggtgt
    10081 ggcagcaggt gttggagtac gcgaagcgca cccctatcgg cgagccgatc accttcacgt
    10141 tctacgagct ttgccaggac ctgggctggt cgatcaatgg ccggtattac acgaaggccg
    10201 aggaatgcct gtcgcgccta caggcgacgg cgatgggctt cacgtccgac cgcgttgggc
    10261 acctggaatc ggtgtcgctg ctgcaccgct tccgcgtcct ggaccgtggc aagaaaacgt
    10321 cccgttgcca ggtcctgatc gacgaggaaa tcgtcgtgct gtttgctggc gaccactaca
    10381 cgaaattcat atgggagaag taccgcaagc tgtcgccgac ggcccgacgg atgttcgact
    10441 atttcagctc gcaccgggag ccgtacccgc tcaagctgga aaccttccgc ctcatgtgcg
    10501 gatcggattc cacccgcgtg aagaagtggc gcgagcaggt cggcgaagcc tgcgaagagt
    10561 tgcgaggcag cggcctggtg gaacacgcct gggtcaatga cctggtgcat tgcaaacgct
    10621 agggccttgt ggggtcagtt ccggctgggg gttcagcaca gctgtctctt atacacatct
    10681 cttcaaatgt agcacctgaa gtcagcccca tacgatataa gttgtaattc tcatgttagt
    10741 catgccccgc gcccaccgga aggagctgac tgggttgaag gctctcaagg gcatcggtcg
    10801 agatcccggt gcctaatgag tgagctaact tacattaatt gcgttgcgca caccgactag
    10861 cgaaaaaacc ccgccgaagc ggggtttttt gcgaaaaaaa gcaccgactc ggtgccactt
    10921 tttcaagttg ataacggact agccttattt taacttgcta tgctgttttg aatggttccg
    10981 ctagcactgt acctaggact gagctagccg tcaaaagcag cgtatatacc atggccacaa
    11041 ttcagcaaat tgtgaacatc atcacgttca tctttccctg gttgccaatg gcccattttc
    11101 ctgtcagtaa cgagaaggtc gcgaattcag gcgcttttta gactggtcgt gttttagagc
    11161 tatgctgttt tgaatggtcc caaaacatga actagcgatt agtcgctatg acttaagttt
    11221 tagagctatg ctgttttgaa tggtcccaaa acaaccacac tagagaacat actggctaaa
    11281 tagttttaga gctatgctgt tttgaatggt cccaaaacgg ttggactcaa gacgatagtt
    11341 accggatagt tttagagcta tgctgttttg aatggtccca aaacggcgaa acccgacagg
    11401 actataaaga taccgtttta gagctatgct gttttgaatg gtcccaaaac aaaaaaaaac
    11461 cccgcccctg acagggcggg gttttttttt taattaaagt ctggtcc
//
