LOCUS       sequence-393255-        9944 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses Streptococcus pyogenes' cas9 and a tailored sgRNA for 
            counterselection in gram-negative bacteria.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 9944)
  AUTHORS   Pankratz D, Gomez NO, Nielsen A, Mustafayeva A, Gur M, 
            Arce-Rodriguez F, Nikel PI, Haussler S, Arce-Rodriguez A
  TITLE     An expanded CRISPR-Cas9-assisted recombineering toolkit for 
            engineering genetically intractable Pseudomonas aeruginosa isolates.
  JOURNAL   Nat Protoc. 2023 Oct 5. doi: 10.1038/s41596-023-00882-z.
  PUBMED    37798358
REFERENCE   2  (bases 1 to 9944)
  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: "Nat Protoc.
            2023 Oct 5. doi: 10.1038/s41596-023-00882-z."
COMMENT     Sequence Label: pS148∙CsR
FEATURES             Location/Qualifiers
     source          1..9944
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     191..210
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(344..1177)
                     /codon_start=1
                     /product="replication protein for the broad-host-range 
                     plasmid pRO1600 from Pseudomonas aeruginosa "
                     /label=pRO1600 Rep
                     /translation="MASPPMVYKSNALVEAAYRLSVQEQRIVLACISQVKRSEPVTDEV
                     MYSVTAEDIATMAGVPIESSYNQLKEAALRLKRREVRLTQEPNGKGKRPSVMITGWVQT
                     IIYREGEGRVELRFTKDMLPYLTELTKQFTKYALADVAKMDSTHAIRLYELLMQWDSIG
                     QREIEIDQLRKWFQLEGRYPSIKDFKLRVLDPAVTQINEHSPLQVEWAQRKTGRKVTHL
                     LFSFGPKKPAKAVGKAPAKRKAGKISDAEIAKQARPGETWEAARARLTQMPLDLA"
     rep_origin      1191..1542
                     /label=pRO1600 oriV
                     /note="broad-host-range origin of replication from 
                     Pseudomonas aeruginosa plasmid pRO1600; requires the 
                     pRO1600 Rep protein for replication (West et al., 1994)"
     terminator      complement(1615..1701)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     CDS             complement(1726..2691)
                     /codon_start=1
                     /gene="xylS"
                     /product="XylS regulator encoded by the Pseudomonas putida 
                     TOL plasmid pWWO"
                     /label=XylS
                     /note="stimulates transcription from the Pm promoter in the
                     presence of aromatic compounds such as m-toluate or 
                     benzoate"
                     /translation="MDFCLLNEKSQIFVHAEPYAVSDYVNQYVGTHSIRLPKGGRPAGR
                     LHHRIFGCLDLCRISYGGSVRVISPGLETCYHLQIILKGHCLWRGHGQEHYFAPGELLL
                     LNPDDQADLTYSEDCEKFIVKLPSVVLDRACSDNNWHKPREGIRFAARHNLQQLDGFIN
                     LLGLVCDEAEHTKSMPRVQEHYAGIIASKLLEMLGSNVSREIFSKGNPSFERVVQFIEE
                     NLKRNISLERLAELAMMSPRSLYNLFEKHAGTTPKNYIRNRKLESIRACLNDPSANVRS
                     ITEIALDYGFLHLGRFAENYRSAFGELPSDTLRQCKKEVA"
     promoter        3591..3671
                     /label=Pm promoter
                     /note="The bacterial Pm promoter is activated by XylS in 
                     the presence of benzoate or m-toluate (Marques et al., 
                     1999)."
     CDS             3731..7837
                     /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             3731..7834
                     /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"
     terminator      7910..8004
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     promoter        8027..8074
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     misc_RNA        8132..8207
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        8303..8394
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(8395..8463,8464..9255)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     oriT            9444..9552
                     /note="incP origin of transfer"
     rep_origin      join(9646..9944,1..290)
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
ORIGIN
        1 aggcgcagcg gtcgggctga acggggggtt cgtgcacaca gcccagcttg gagcgaacga
       61 cctacaccga actgagatac ctacagcgtg agctatgaga aagcgccacg cttcccgaag
      121 ggagaaaggc ggacaggcat ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg
      181 agcttccagg gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc cacctctgac
      241 ttgagcgtcg atttttgtga tgctcgtcag gggggcggag cctatggaaa aacgccagca
      301 acgcggccgt gaaaggcagg ccggtccgtg gtggccacgg cctctaggcc agatccagcg
      361 gcatctgggt tagtcgagcg cgggccgctt cccatgtctc accagggcga gcctgtttcg
      421 cgatctcagc atctgaaatc ttcccggcct tgcgcttcgc tggggcctta cccaccgcct
      481 tggcgggctt cttcggtcca aaactgaaca acagatgtgt gaccttgcgc ccggtctttc
      541 gctgcgccca ctccacctgt agcgggctgt gctcgttgat ctgcgtcacg gctggatcaa
      601 gcactcgcaa cttgaagtcc ttgatcgagg gataccggcc ttccagttga aaccactttc
      661 gcagctggtc aatttctatt tcgcgctggc cgatgctgtc ccattgcatg agcagctcgt
      721 aaagcctgat cgcgtgggtg ctgtccatct tggccacgtc agccaaggcg tatttggtga
      781 actgtttggt gagttccgtc aggtacggca gcatgtcttt ggtgaacctg agttctacac
      841 ggccctcacc ctcccggtag atgattgttt gcacccagcc ggtaatcatc acactcggtc
      901 ttttcccctt gccattgggc tcttgggtta accggacttc ccgccgtttc aggcgcaggg
      961 ccgcttcttt gagctggttg taggaagatt cgatagggac acccgccatc gtcgctatgt
     1021 cctccgccgt cactgaatac atcacttcat cggtgacagg ctcgctcctc ttcacctggc
     1081 taatacaggc cagaacgatc cgctgttcct gaacactgag gcgatacgcg gcctcgacca
     1141 gggcattgct tttgtaaacc attgggggtg aggccacgtt cgacattcct tgtgtataag
     1201 gggacactgt atctgcgtcc cacaatacaa caaatccgtc cctttacaac aacaaatccg
     1261 tcccttctta acaacaaatc cgtcccttaa tggcaacaaa tccgtccctt tttaaactct
     1321 acaggccacg gattacgtgg cctgtagacg tcctaaaagg tttaaaaggg aaaaggaaga
     1381 aaagggtgga aacgcaaaaa acgcaccact acgtggcccc gttggggccg catttgtgcc
     1441 cctgaagggg cgggggaggc gtctgggcaa tccccgtttt accagtcccc tatcgccgcc
     1501 tgagagggcg caggaagcga gtaatcaggg tatcgaggcg gattcaccct tggcgtccaa
     1561 ccagcggcac cagcggcgcc tgagaggggc gcgcccagct gtctagggcg gcggatttgt
     1621 cctactcagg agagcgttca ccgacaaaca acagataaaa cgaaaggccc agtctttcga
     1681 ctgagctttt cgttttattt gatgccttta attaaacgtt cgtaatcaag ccacttcctt
     1741 tttgcattga cgcagggtgt cggaaggcaa ctcgccgaac gcgctcctat agttttcagc
     1801 gaagcgtccc aaatgtaaga agccgtagtc tagggctatc tcagttatac tacgcacatt
     1861 ggcactggga tcgttcaagc aggcgcggat gctttcgagc ttgcggttgc ggatgtagtt
     1921 cttcggcgtg gtgccggcgt gcttctcgaa caaattgtag agcgagcgtg gactcatcat
     1981 cgccagctcc gctaaccgct caaggctgat attccgtttg agattctcct caatgaattg
     2041 aacgactcgc tcgaaagacg ggttaccttt gctgaaaatt tcacggctga cattgctgcc
     2101 cagcatttcg agcagcttgg aagcgatgat ccccgcatag tgctcttgga cccgaggcat
     2161 cgactttgta tgttccgctt cgtcacaaac taacccgagt agattgataa agccatcgag
     2221 ttgctggaga ttgtgtcgcg cggcgaaacg gataccctcc ctcggcttgt gccaattgtt
     2281 gtcactgcac gcccgatcaa ggaccactga gggcaattta acgataaatt tctcgcaatc
     2341 ttctgaatag gtcaggtcgg cttggtcatc cggattgagc agcaatagtt cgcccggcgc
     2401 aaaatagtgc tcctggccat ggccacgcca caggcaatgg cctttgagta ttatttgcag
     2461 atgataacag gtttctaatc caggcgagat taccctcacg ctaccgccgt agctgattcg
     2521 acacagatcg aggcatccga agattctgtg gtgcagcctg cctgccgggc gcccgccctt
     2581 gggcaggcga atagagtgcg taccgacata ctggttaaca taatcggaga ctgcataggg
     2641 ctcggcgtgg acgaagatct gacttttctc gttcaataag caaaaatcca tagttcacgg
     2701 ttctcttatt ttaatgtggg ctgcttggtg tgatgtagaa aggcgccaag tcgatgaaaa
     2761 tgcatctcga cgtgatgcgt atacgggtta cccccattgc cacgttgcgc catccttttt
     2821 gcaatcagtg accacttttc caagcaaaaa taacgccaag cagaacgaag acgttctttt
     2881 taagaagcga gaacaccaga agttcgtgct gtcggggcat ggggcgacga attggcggat
     2941 aaaggggatc tgctggatat tacggccttt ttaaagaccg taaagaaaaa taagcacaag
     3001 ttttatccgg cctttattca cattcttgcc cgcctgatga atgctcatcc gtaattacgt
     3061 atggcaatga aagacggtga gctggtgata tgggatagtg ttcacccttg ttacaccgtt
     3121 ttccatgagc aaactgaaac gttttcatcg ctctggagtg aataccacga cgatttccgg
     3181 cagtttctac acatatattc gcaagatgtg gcgtgttacg gtgaaaacct ggcctatttc
     3241 cctaaagggt ttattgagaa tatgtttttc gtctcagcca atccctgggt gagtttcacc
     3301 agttttgatt taaacgtggc caatatggac aacttcttcg cccccgtttt caccatgcat
     3361 gggaattagc ttgatctgac caacgaccgg tagcggagct atccaacggc ggtataccag
     3421 gaaaacacac agcaggtaca tcagaacagt accatgactg aagaacaaat agttttttcc
     3481 tgatccataa agcagaacgg cctgctccat gacaaatctg gctccccaac taatgcccca
     3541 tgcagccagc ataaccagca taaagtgcag tgtccggttt gatagggata agtccagcct
     3601 tgcaagaagc ggatacagga gtgcaaaaaa tggctatctc tagtaaggcc taccccttag
     3661 gctttatgca acagaaacaa taataatgga gtcatgacca tgcctaggcc gcggttagga
     3721 ggaaaaacat atggataaga aatactcaat aggcttagat atcggcacaa atagcgtcgg
     3781 atgggcggtg atcactgatg aatataaggt tccgtctaaa aagttcaagg ttctgggaaa
     3841 tacagaccgc cacagtatca aaaaaaatct tataggggct cttttatttg acagtggaga
     3901 gacagcggaa gcgactcgtc tcaaacggac agctcgtaga aggtatacac gtcggaagaa
     3961 tcgtatttgt tatctacagg agattttttc aaatgagatg gcgaaagtag atgatagttt
     4021 ctttcatcga cttgaagagt cttttttggt ggaagaagac aagaagcatg aacgtcatcc
     4081 tatttttgga aatatagtag atgaagttgc ttatcatgag aaatatccaa ctatctatca
     4141 tctgcgaaaa aaattggtag attctactga taaagcggat ttgcgcttaa tctatttggc
     4201 cttagcgcat atgattaagt ttcgtggtca ttttttgatt gagggagatt taaatcctga
     4261 taatagtgat gtggacaaac tatttatcca gttggtacaa acctacaatc aattatttga
     4321 agaaaaccct attaacgcaa gtggagtaga tgctaaagcg attctttctg cacgattgag
     4381 taaatcaaga cgattagaaa atctcattgc tcagctcccc ggtgagaaga aaaatggctt
     4441 atttgggaat ctcattgctt tgtcattggg tttgacccct aattttaaat caaattttga
     4501 tttggcagaa gatgctaaat tacagctttc aaaagatact tacgatgatg atttagataa
     4561 tttattggcg caaattggag atcaatatgc tgatttgttt ttggcagcta agaatttatc
     4621 agatgctatt ttactttcag atatcctaag agtaaatact gaaataacta aggctcccct
     4681 atcagcttca atgattaaac gctacgatga acatcatcaa gacttgactc ttttaaaagc
     4741 tttagttcga caacaacttc cagaaaagta taaagaaatc ttttttgatc aatcaaaaaa
     4801 cggatatgca ggttatattg atgggggagc tagccaagaa gaattttata aatttatcaa
     4861 accaatttta gaaaaaatgg atggtactga ggaattattg gtgaaactaa atcgtgaaga
     4921 tttgctgcgc aagcaacgga cctttgacaa cggctctatt ccccatcaaa ttcacttggg
     4981 tgagctgcat gctattttga gaagacaaga agacttttat ccatttttaa aagacaatcg
     5041 tgagaagatt gaaaaaatct tgacttttcg aattccttat tatgttggtc cattggcgcg
     5101 tggcaatagt cgttttgcat ggatgactcg gaagtctgaa gaaacaatta ccccatggaa
     5161 ttttgaagaa gttgtcgata aaggtgcttc agctcaatca tttattgaac gcatgacaaa
     5221 ctttgataaa aatcttccaa atgaaaaagt actaccaaaa catagtttgc tttatgagta
     5281 ttttacggtt tataacgaat tgacaaaggt caaatatgtt actgaaggaa tgcgaaaacc
     5341 agcatttctt tcaggtgaac agaagaaagc cattgttgat ttactcttca aaacaaatcg
     5401 aaaagtaacc gttaagcaat taaaagaaga ttatttcaaa aaaatagaat gttttgatag
     5461 tgttgaaatt tcaggagttg aagatagatt taatgcttca ttaggtacct accatgattt
     5521 gctaaaaatt attaaagata aagatttttt ggataatgaa gaaaatgaag atatcttaga
     5581 ggatattgtt ttaacattga ccttatttga agatagggag atgattgagg aaagacttaa
     5641 aacatatgct cacctctttg atgataaggt gatgaaacag cttaaacgtc gccgttatac
     5701 tggttgggga cgtttgtctc gaaaattgat taatggtatt agggataagc aatctggcaa
     5761 aacaatatta gattttttga aatcagatgg ttttgccaat cgcaatttta tgcagctgat
     5821 ccatgatgat agtttgacat ttaaagaaga cattcaaaaa gcacaagtgt ctggacaagg
     5881 cgatagttta catgaacata ttgcaaattt agctggtagc cctgctatta aaaaaggtat
     5941 tttacagact gtaaaagttg ttgatgaatt ggtcaaagta atggggcggc ataagccaga
     6001 aaatatcgtt attgaaatgg cacgtgaaaa tcagacaact caaaagggcc agaaaaattc
     6061 gcgagagcgt atgaaacgaa tcgaagaagg tatcaaagaa ttaggaagtc agattcttaa
     6121 agagcatcct gttgaaaata ctcaattgca aaatgaaaag ctctatctct attatctcca
     6181 aaatggaaga gacatgtatg tggaccaaga attagatatt aatcgtttaa gtgattatga
     6241 tgtcgatcac attgttccac aaagtttcct taaagacgat tcaatagaca ataaggtctt
     6301 aacgcgttct gataaaaatc gtggtaaatc ggataacgtt ccaagtgaag aagtagtcaa
     6361 aaagatgaaa aactattgga gacaacttct aaacgccaag ttaatcactc aacgtaagtt
     6421 tgataattta acgaaagctg aacgtggagg tttgagtgaa cttgataaag ctggttttat
     6481 caaacgccaa ttggttgaaa ctcgccaaat cactaagcat gtggcacaaa ttttggatag
     6541 tcgcatgaat actaaatacg atgaaaatga taaacttatt cgagaggtta aagtgattac
     6601 cttaaaatct aaattagttt ctgacttccg aaaagatttc caattctata aagtacgtga
     6661 gattaacaat taccatcatg cccatgatgc gtatctaaat gccgtcgttg gaactgcttt
     6721 gattaagaaa tatccaaaac ttgaatcgga gtttgtctat ggtgattata aagtttatga
     6781 tgttcgtaaa atgattgcta agtctgagca agaaataggc aaagcaaccg caaaatattt
     6841 cttttactct aatatcatga acttcttcaa aacagaaatt acacttgcaa atggagagat
     6901 tcgcaaacgc cctctaatcg aaactaatgg ggaaactgga gaaattgtct gggataaagg
     6961 gcgagatttt gccacagtgc gcaaagtatt gtccatgccc caagtcaata ttgtcaagaa
     7021 aacagaagta cagacaggcg gattctccaa ggagtcaatt ttaccaaaaa gaaattcgga
     7081 caagcttatt gctcgtaaaa aagactggga tccaaaaaaa tatggtggtt ttgatagtcc
     7141 aacggtagct tattcagtcc tagtggttgc taaggtggaa aaagggaaat cgaagaagtt
     7201 aaaatccgtt aaagagttac tagggatcac aattatggaa agaagttcct ttgaaaaaaa
     7261 tccgattgac tttttagaag ctaaaggata taaggaagtt aaaaaagact taatcattaa
     7321 actacctaaa tatagtcttt ttgagttaga aaacggtcgt aaacggatgc tggctagtgc
     7381 cggagaatta caaaaaggaa atgagctggc tctgccaagc aaatatgtga attttttata
     7441 tttagctagt cattatgaaa agttgaaggg tagtccagaa gataacgaac aaaaacaatt
     7501 gtttgtggag cagcataagc attatttaga tgagattatt gagcaaatca gtgaattttc
     7561 taagcgtgtt attttagcag atgccaattt agataaagtt cttagtgcat ataacaaaca
     7621 tagagacaaa ccaatacgtg aacaagcaga aaatattatt catttattta cgttgacgaa
     7681 tcttggagct cccgctgctt ttaaatattt tgatacaaca attgatcgta aacgatatac
     7741 gtctacaaaa gaagttttag atgccactct tatccatcaa tccatcactg gtctttatga
     7801 aacacgcatt gatttgagtc agctaggagg tgactgatct agagtcgacc tgcaggcatg
     7861 caagcttgcg gccgcgtcgt gactgggaaa accctggcga ctagtcttgg actcctgttg
     7921 atagatccag taatgacctc agaactccat ctggatttgt tcagaacgct cggttgccgc
     7981 cgggcgtttt ttattggtga gaatccaggg gtccctttaa ttaattgttg acaattaatc
     8041 atcggcatag tatatcggca tagtataata cgacaaggtg aggaactaaa cccctaggcc
     8101 gcggccgcgc ggagacccga gactggtctc agttttagag ctagaaatag caagttaaaa
     8161 taaggctagt ccgttatcaa cttgaaaaag tggcaccgag tcggtgcaat tcgagctcgg
     8221 tacccgggga tcccaataat tacgatttac gtatttaaat tagtagcccg cctaatgagc
     8281 gggctttttt ttaattcccc tatttgttta tttttctaaa tacattcaaa tatgtatccg
     8341 ctcatgagac aataaccctg ataaatgctt caataatatt gaaaaaggaa gagtatgagc
     8401 attcagcatt ttcgtgtggc gctgattccg ttttttgcgg cgttttgcct gccggtgttt
     8461 gcgcatccgg aaaccctggt gaaagtgaaa gatgcggaag atcaactggg tgcgcgcgtg
     8521 ggctatattg aactggatct gaacagcggc aaaattctgg aatcttttcg tccggaagaa
     8581 cgttttccga tgatgagcac ctttaaagtg ctgctgtgcg gtgcggttct gagccgtgtg
     8641 gatgcgggcc aggaacaact gggccgtcgt attcattata gccagaacga tctggtggaa
     8701 tatagcccgg tgaccgaaaa acatctgacc gatggcatga ccgtgcgtga actgtgcagc
     8761 gcggcgatta ccatgagcga taacaccgcg gcgaacctgc tgctgacgac cattggcggt
     8821 ccgaaagaac tgaccgcgtt tctgcataac atgggcgatc atgtgacccg tctggatcgt
     8881 tgggaaccgg aactgaacga agcgattccg aacgatgaac gtgataccac catgccggca
     8941 gcaatggcga ccaccctgcg taaactgctg acgggtgagc tgctgaccct ggcaagccgc
     9001 cagcaactga ttgattggat ggaagcggat aaagtggcgg gtccgctgct gcgtagcgcg
     9061 ctgccggctg gctggtttat tgcggataaa agcggtgcgg gcgaacgtgg cagccgtggc
     9121 attattgcgg cgctgggccc ggatggtaaa ccgagccgta ttgtggtgat ttataccacc
     9181 ggcagccagg cgacgatgga tgaacgtaac cgtcagattg cggaaattgg cgcgagcctg
     9241 attaaacatt ggtaaaccga tacaattaaa ggctcctttt ggagcctttt ttttggacga
     9301 cccttgtcct tttccgctgc ataaccctgc ttcggggtca ttatagcgat tttttcggta
     9361 tatccatcct ttttcgcacg atatacagga ttttgccaaa gggttcgtgt agactttcct
     9421 tggtgtatcc aacggcgtca gccgggcagg ataggtgaag taggcccacc cgcgagcggg
     9481 tgttccttct tcactgtccc ttattcgcac ctggcggtgc tcaacgggaa tcctgctctg
     9541 cgaggctggc cgtaggccgg ccgataatct catgaccaaa atcccttaac gtgagttttc
     9601 gttccactga gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag atcctttttt
     9661 tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt
     9721 gccggatcaa gagctaccaa ctctttttcc gaaggtaact ggcttcagca gagcgcagat
     9781 accaaatact gttcttctag tgtagccgta gttaggccac cacttcaaga actctgtagc
     9841 accgcctaca tacctcgctc tgctaatcct gttaccagtg gctgctgcca gtggcgataa
     9901 gtcgtgtctt accgggttgg actcaagacg atagttaccg gata
//
