LOCUS       sequence-350007-       10874 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  CRISPR gene editing in Streptomyces, codon-optimized Cas9 behind 
            theophylline riboswitch and D4 promoter, with custom gRNA.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 10874)
  AUTHORS   Ye S, Enghiad B, Zhao H, Takano E
  TITLE     Fine-tuning the regulation of Cas9 expression levels for efficient 
            CRISPR-Cas9 mediated recombination in Streptomyces.
  JOURNAL   J Ind Microbiol Biotechnol. 2020 May;47(4-5):413-423. doi: 
            10.1007/s10295-020-02277-5. Epub 2020 May 4.
  PUBMED    32367443
REFERENCE   2  (bases 1 to 10874)
  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: "J Ind 
            Microbiol Biotechnol."; date: "2020-05"; volume: "47(4-5):413-423. 
            doi"; pages: " 10.1007/s10295-020-02277-5. Epub 2020 May 4"
COMMENT     Sequence Label: pCMU-4
FEATURES             Location/Qualifiers
     source          1..10874
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(407..1849)
                     /codon_start=1
                     /gene="rep"
                     /product="replication initiator protein from the 
                     Streptomyces ghanaensis plasmid pSG5"
                     /label=pSG5 Rep
                     /translation="MWKPGEATWGNTCRCNNVHTCPWCMSRILAVRGSNVQLAADGLAD
                     AGYGLHLGTNTLRHFERMAFGTVRKGMRHGLVAVLHDGWKGAYGSSGRRWRTMRDDFGI
                     IGYERAFEDTFGWGSGWHLHWHTLWVTREVLGPDAQAAFRDALAGAWAAGVESAGGYTV
                     SETCDRPGCSCEGKGHGTDVRPLNGADAADGDAGKQARYLYKDGDKTKGGVAKIGLELA
                     GQNFKAGRGDDRMGPLDLGDAAAAELQRLRRPGPFVEKYREREFGVFQVRKHYRSQNLN
                     RLIKELGIQQDVRTEEEITDDTEGLVAIAVIPAYIWYRYIARVAGRRLDLIKVAETYGL
                     PGVRRLVESWGLVWGKDVLDPPAPEAPAAPGDLDADQMRFEVMSEEEAAFREARRKANE
                     ARTEELAASLDRVRQPKKEAIRPTISLRKRLKPKPVTVDVKTPPPGAASPVCRRCKGKL
                     APVLQPWGQHPGDCLRVDTAVA"
     oriT            2889..2998
                     /note="incP origin of transfer"
     CDS             3031..3402
                     /codon_start=1
                     /gene="traJ"
                     /product="oriT-recognizing protein"
                     /label=traJ
                     /translation="MADETKPTRKGSPPIKVYCLPDERRAIEEKAAAAGMSLSAYLLAV
                     GQGYKITGVVDYEHVRELARINGDLGRLGGLLKLWLTDDPRTARFGDATILALLAKIEE
                     KQDELGKVMMGVVRPRAEP"
     CDS             3529..7635
                     /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             3529..7632
                     /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"
     promoter        7835..8119
                     /label=gapdh promoter
                     /note="glyceraldehyde-3-phosphate dehydrogenase promoter 
                     from Eggerthella lenta"
     CDS             complement(8128..8424)
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITPSAQLTLTKGNKSWRTSRGGPVPNSPYSESYYARSLAVVL
                     QRRDWENPGVTQLNRLAAHPPFASWRNSEEARTDRPSQQLRSLNGEWDAPCSG"
     primer_bind     8274..8296
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     8288..8305
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     8289..8305
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     8315..8334
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        8315..8333
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(8381..8401)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(8381..8399)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(8420..8436)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(8420..8436)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(8433..8455)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    8444..8460
                     /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(8468..8474,8475..8492,8493..8498))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    8513..8534
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     misc_RNA        8577..8652
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     terminator      8674..8708
                     /label=lambda t0 terminator
                     /note="minimal transcription terminator from phage lambda 
                     (Scholtissek and Grosse, 1987)"
     rep_origin      complement(8750..9338)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(8830..8849)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             9542..10342
                     /codon_start=1
                     /gene="aac(3)IV"
                     /product="aminoglycoside 3-N-acetyltransferase type IV     
                               "
                     /label=ApmR
                     /note="confers resistance to apramycin (Paget and Davies, 
                     1996)"
                     /translation="MSSAVECNVVQYEWRKAELIGQLLNLGVTPGGVLLVHSSFRSVRP
                     LEDGPLGLIEALRAALGPGGTLVMPSWSGLDDEPFDPATSPVTPDLGVVSDTFWRLPNV
                     KRSAHPFAFAAAGPQAEQIISDPLPLPPHSPASPVARVHELDGQVLLLGVGHDANTTLH
                     LAELMAKVPYGVPRHCTILQDGKLVRVDYLENDHCCERFALADRWLKEKSLQKEGPVGH
                     AFARLIRSRDIVATALGQLGRDPLIFLHPPEAGCEECDAARQSIG"
ORIGIN
        1 gggtaaagga ccagcctcgc caactaggag cgctaccaag gcgaacacaa cccaggactt
       61 ggtagaaccc ttcgccggag aagttcagac tcatcggcat gagcacccct cgccgcgcac
      121 tcggcaccgg tccgtcgacc accaccagga cgtcgttgtc gacgtcggcc ccgcggctcc
      181 tgcccgccga acgcgtcgtc gtcgacggcc tggtgctcat cgacgagcac ccggagccag
      241 gtgaaaagcg ccggcggacg ctcggactgg gcgcgggatt ccagcagtaa cccaggtccg
      301 ccggccacct cacggcaggc agaccctcgg ctttcgcgcc gggaccgcca tgagaccgcc
      361 acccggatgt ccggggtggc ggtctcatgg cggtccctca gcggccctac gcgaccgctg
      421 tgtcgacgcg gaggcagtct ccggggtgct ggccccaggg ctggagtacc ggggcgagct
      481 tgcccttgca gcggcggcac acgggcgacg cggcgccggg cgggggagtc ttgacgtcga
      541 cggtgaccgg cttcggcttc aggcgcttcc gaaggctgat cgtcgggcgg atcgcctcct
      601 tcttcggctg acgcactcgg tcgagcgaag cggccagctc ctccgtgcgg gcctcgttgg
      661 ccttccggcg cgcctcgcgg aaagcagctt cctcctcgga catgacctcg aacctcatct
      721 ggtcagcgtc aaggtcgccc ggcgcggccg gcgcttccgg ggcgggcggg tccaggacgt
      781 ccttgcccca caccaggccc caggactcga cgagccgccg gacgcccggt aggccgtacg
      841 tctcggcgac cttgatgagg tcgaggcgac gtccggcgac gcgggcgatg tatcggtacc
      901 agatgtaggc cgggatgacc gcgatggcga ccaggccctc ggtgtcgtcg gtgatctcct
      961 cctcggtgcg gacgtcctgc tggatgccga gttccttgat cagccggttc aggttctgcg
     1021 accggtagtg cttgcggacc tggaacacgc cgaactcgcg ctcgcggtac ttctcgacga
     1081 acgggccggg ccgacgaagc cgctgcagct cggcggccgc cgcgtcgccc aggtcgagcg
     1141 gtcccatgcg gtcgtcgccg cgaccggcct tgaagttctg tccggccagc tccaggccga
     1201 tcttggcgac gccgcccttg gtcttgtcgc cgtccttgta gaggtagcgg gcctgcttgc
     1261 ccgcatcgcc gtcagcggcg tccgcgccgt tgagtgggcg cacgtcggtg ccgtggccct
     1321 tgccctcaca ggagcaaccg ggccggtcgc acgtctcgct gacggtgtag ccgcccgcgg
     1381 attcgacccc ggcggcccag gctccggcga gtgcgtcgcg gaacgcggcc tgggcgtccg
     1441 ggccgagcac ctcgcgggtg acccagagcg tgtgccagtg caggtgccag ccggagcccc
     1501 agccgaaggt gtcctcgaag gcccgctcgt agccgatgat cccgaagtcg tcgcgcatcg
     1561 tgcgccagcg gcggccggac gagccgtacg cgcccttcca gccgtcgtgc aagaccgcga
     1621 ccaggccgtg ccgcattccc ttgcggacgg tgccgaacgc catgcgctcg aagtggcgca
     1681 acgtgttcgt gccaaggtgc agcccgtacc cggcgtccgc gagaccgtcg gcggcgagct
     1741 gcacgttcga gccccgtacg gccaggatgc ggctcatgca ccacgggcag gtgtggacgt
     1801 tgttgcagcg gcacgtgttg ccccacgtcg cctcgcccgg cttccacatc agctcggccg
     1861 tcccggcagt gagccgggtc ccgcagccct tgaacgcctc gttcagcgac accgtctggt
     1921 gccggtcccg ccgggcgaac cgctcgtcgc gcgggtcctc ccgcccggct gtcgcggcac
     1981 cctcgtttgg ggtagaaccc gttccagtta cagcgctctg acctgcagtg gacggagatt
     2041 ttcccttact actaaagccc gcgtccggat tacccgctgt agtcgtgctt gctacgctgc
     2101 gtgactggtc cgcaatgaga cgctttgcgc gctttcggca ggcgtccgag cagtagattt
     2161 tggggcgctt cccggggatg tggacgatcg gggtgccgca gtggcacttc ggtccggcgg
     2221 ggcgcggtgg tgtcgacgcg ctgttctctc gtacgctcgt cacagagcaa acgtcctcac
     2281 tcggcatgct gcgccggttc gggggcggcg agcccgggag gccaatcccg ggctcgtgcc
     2341 atttctgggt cctgttgatc ctggcattgg tgtggccgtt cattgcccct gctcgctcct
     2401 gacgcgccga tagacgtccg atacgcccgg tgctggtggg atttgatagg tcggaagaag
     2461 ccccgcccgg gcctgggcgg ggcttcctgt gcgtcaggac ctcctcgtcg tgagcctctt
     2521 cggcctatgg acggagtgac ctcgtgatcc gttacagccg cgcgcgctcg cgtagagcgg
     2581 tctcatcagt tccacgaacg gtcctcttcg cagatcaggg cgttggggcg gagtctcacc
     2641 aaggactacg tctgctggcg atttccgtta caccccgggc ggtggccggc gcacacgcgc
     2701 gcccgcgttg ggcagtgcag aaagtgcaga aacctaggtg caggtcgacg gatcttttcc
     2761 gctgcataac cctgcttcgg ggtcattata gcgatttttt cggtatatcc atcctttttc
     2821 gcacgatata caggattttg ccaaagggtt cgtgtagact ttccttggtg tatccaacgg
     2881 cgtcagccgg gcaggatagg tgaagtaggc ccacccgcga gcgggtgttc cttcttcact
     2941 gtcccttatt cgcacctggc ggtgctcaac gggaatcctg ctctgcgagg ctggccggct
     3001 accgccggcg taacagatga gggcaagcgg atggctgatg aaaccaagcc aaccaggaag
     3061 ggcagcccac ctatcaaggt gtactgcctt ccagacgaac gaagagcgat tgaggaaaag
     3121 gcggcggcgg ccggcatgag cctgtcggcc tacctgctgg ccgtcggcca gggctacaaa
     3181 atcacgggcg tcgtggacta tgagcacgtc cgcgagctgg cccgcatcaa tggcgacctg
     3241 ggccgcctgg gcggcctgct gaaactctgg ctcaccgacg acccgcgcac ggcgcggttc
     3301 ggtgatgcca cgatcctcgc cctgctggcg aagatcgaag agaagcagga cgagcttggc
     3361 aaggtcatga tgggcgtggt ccgcccgagg gcagagccat gacccgtcgg ctggaaacac
     3421 ttgatccaac ttaggatact tccggtacca atacgactca ctataggttc cggtgatacc
     3481 agcatcgtct tgatgccctt ggcagcaccc tgctaaggag gcaacaagat ggacaagaag
     3541 tacagcatcg gcctggacat cggcaccaac agcgtgggct gggcggtcat caccgacgag
     3601 tacaaggtcc cctccaagaa gttcaaggtc ctgggcaaca ccgaccggca ctcgatcaag
     3661 aagaacctga tcggcgccct gctcttcgac agcggcgaaa ccgccgaggc gacccgcctg
     3721 aagcggaccg cccgtcgccg ctacacccgg cgcaagaacc gcatctgcta cctgcaggag
     3781 atcttctcca acgagatggc caaggtcgac gactcgttct tccaccggct cgaggagagc
     3841 ttcctggtgg aggaggacaa gaagcacgag cgccacccga tcttcggcaa catcgtcgac
     3901 gaggtggcct accacgagaa gtaccccacc atctaccacc tccgcaagaa gctggtcgac
     3961 tcgaccgaca aggcggacct gcggctcatc tacctggccc tcgcgcacat gatcaagttc
     4021 cgcggccact tcctcatcga gggcgacctg aacccggaca actccgacgt cgacaagctc
     4081 ttcatccagc tggtgcagac ctacaaccag ctgttcgagg agaaccccat caacgccagc
     4141 ggcgtcgacg ccaaggcgat cctctccgcg cgcctgagca agtcccggcg cctggagaac
     4201 ctcatcgccc agctgccggg cgagaagaag aacggcctct tcggcaacct gatcgcgctg
     4261 tcgctcggcc tgacccccaa cttcaagagc aacttcgacc tggccgagga cgcgaagctc
     4321 cagctgtcca aggacaccta cgacgacgac ctggacaacc tgctcgccca gatcggcgac
     4381 cagtacgcgg acctcttcct ggccgcgaag aacctctcgg acgccatcct gctcagcgac
     4441 atcctgcggg tcaacaccga gatcaccaag gccccgctgt cggcgagcat gatcaagcgg
     4501 tacgacgagc accaccagga cctgaccctg ctcaaggccc tcgtgcgcca gcagctgccc
     4561 gagaagtaca aggagatctt cttcgaccag tccaagaacg gctacgccgg ctacatcgac
     4621 ggcggcgcgt cgcaggagga gttctacaag ttcatcaagc cgatcctgga gaagatggac
     4681 ggcaccgagg agctgctcgt caagctgaac cgcgaggacc tgctccgcaa gcagcggacc
     4741 ttcgacaacg gctccatccc gcaccagatc cacctgggcg agctccacgc catcctccgg
     4801 cgccaggagg acttctaccc cttcctgaag gacaaccgcg agaagatcga gaagatcctg
     4861 accttccgga tcccgtacta cgtcggcccc ctggcccgcg gcaactcccg gttcgcgtgg
     4921 atgacccgga agtcggagga aaccatcacc ccgtggaact tcgaggaggt cgtggacaag
     4981 ggcgcctccg cgcagtcgtt catcgagcgc atgaccaact tcgacaagaa cctcccgaac
     5041 gagaaggtcc tgcccaagca cagcctgctc tacgagtact tcaccgtgta caacgagctg
     5101 accaaggtca agtacgtgac cgagggcatg cggaagccgg ccttcctgtc cggcgagcag
     5161 aagaaggcga tcgtcgacct gctcttcaag accaaccgca aggtcaccgt gaagcagctg
     5221 aaggaggact acttcaagaa gatcgagtgc ttcgactccg tcgagatctc gggcgtggag
     5281 gaccgcttca acgcctccct gggcacctac cacgacctgc tcaagatcat caaggacaag
     5341 gacttcctcg acaacgagga gaacgaggac atcctggagg acatcgtcct caccctgacc
     5401 ctcttcgagg accgcgagat gatcgaggag cggctcaaga cctacgccca cctgttcgac
     5461 gacaaggtga tgaagcagct gaagcggcgc cggtacaccg gctggggccg cctctcccgg
     5521 aagctgatca acggcatccg ggacaagcag agcggcaaga ccatcctgga cttcctcaag
     5581 tccgacggct tcgccaaccg caacttcatg cagctcatcc acgacgactc gctgaccttc
     5641 aaggaggaca tccagaaggc ccaggtgtcc ggccagggcg acagcctcca cgagcacatc
     5701 gccaacctgg cgggctcccc ggcgatcaag aagggcatcc tccagaccgt caaggtcgtg
     5761 gacgagctgg tcaaggtgat gggccgccac aagcccgaga acatcgtgat cgagatggcc
     5821 cgggagaacc agaccaccca gaagggccag aagaactccc gcgagcggat gaagcgcatc
     5881 gaggagggca tcaaggagct cggctcgcag atcctgaagg agcacccggt cgagaacacc
     5941 cagctccaga acgagaagct gtacctctac tacctgcaga acggccgcga catgtacgtg
     6001 gaccaggagc tcgacatcaa ccggctgagc gactacgacg tcgaccacat cgtgccgcag
     6061 tccttcctga aggacgactc gatcgacaac aaggtcctga cccgctccga caagaaccgg
     6121 ggcaagtccg acaacgtgcc ctcggaggag gtcgtgaaga agatgaagaa ctactggcgc
     6181 cagctgctca acgccaagct catcacccag cgcaagttcg acaacctgac caaggccgag
     6241 cggggcggcc tgtcggagct cgacaaggcg ggcttcatca agcgccagct cgtcgaaacc
     6301 cggcagatca ccaagcacgt ggcccagatc ctggacagcc ggatgaacac caagtacgac
     6361 gagaacgaca agctgatccg cgaggtcaag gtgatcaccc tcaagagcaa gctggtgtcc
     6421 gacttccgca aggacttcca gttctacaag gtccgggaga tcaacaacta ccaccacgcc
     6481 cacgacgcgt acctgaacgc cgtcgtgggc accgcgctga tcaagaagta cccgaagctg
     6541 gagtccgagt tcgtctacgg cgactacaag gtctacgacg tgcgcaagat gatcgccaag
     6601 tcggagcagg agatcggcaa ggccaccgcg aagtacttct tctacagcaa catcatgaac
     6661 ttcttcaaga ccgagatcac cctggccaac ggcgagatcc gcaagcggcc cctgatcgaa
     6721 accaacggcg aaaccggcga gatcgtctgg gacaagggcc gcgacttcgc caccgtccgg
     6781 aaggtgctgt ccatgccgca ggtcaacatc gtcaagaaaa ccgaggtgca gaccggcggc
     6841 ttcagcaagg agtccatcct ccccaagcgc aactcggaca agctgatcgc ccggaagaag
     6901 gactgggacc cgaagaagta cggcggcttc gacagcccca ccgtcgccta ctccgtgctg
     6961 gtcgtggcga aggtcgagaa gggcaagagc aagaagctga agtccgtgaa ggagctgctc
     7021 ggcatcacca tcatggagcg ctcctcgttc gagaagaacc cgatcgactt cctggaggcc
     7081 aagggctaca aggaggtcaa gaaggacctc atcatcaagc tgcccaagta ctcgctgttc
     7141 gagctcgaga acggccgcaa gcggatgctc gccagcgcgg gcgagctgca gaagggcaac
     7201 gagctggccc tcccgtccaa gtacgtcaac ttcctgtacc tcgcgtccca ctacgagaag
     7261 ctgaagggct cgcccgagga caacgagcag aagcagctct tcgtggagca gcacaagcac
     7321 tacctggacg agatcatcga gcagatctcg gagttcagca agcgggtcat cctggccgac
     7381 gcgaacctcg acaaggtgct gtccgcctac aacaagcacc gcgacaagcc gatccgggag
     7441 caggcggaga acatcatcca cctgttcacc ctcaccaacc tgggcgcccc cgccgcgttc
     7501 aagtacttcg acaccaccat cgaccgcaag cggtacacca gcaccaagga ggtcctcgac
     7561 gcgaccctga tccaccagtc catcaccggc ctgtacgaaa cccgcatcga cctctcccag
     7621 ctcggcggcg actgagaatt cagatctacg cgttccccgc aaaagcggcc tttgactccc
     7681 tgcaagcctc agcgaccgaa tatatcggtt atgcgtgggc gatggttgtt gtcattgtcg
     7741 gcgcaactat cggtatcaag ctgtttaaga aattcacctc gaaagcaagc tgataaaccg
     7801 atacaattaa aggctccttt tggagccttt ttttgctgct ccttcggtcg gacgtgcgtc
     7861 tacgggcacc ttaccgcagc cgtcggctgt gcgacacgga cggatcgggc gaactggccg
     7921 atgctgggag aagcgcgctg ctgtacggcg cgcaccgggt gcggagcccc tcggcgagcg
     7981 gtgtgaaact tctgtgaatg gcctgttcgg ttgctttttt tatacggctg ccagataagg
     8041 cttgcagcat ctgggcggct accgctatga tcggggcgtt cctgcaattc ttagtgcgag
     8101 tatctgaaag gggatacgca agtcttctca gccgctacag ggcgcgtccc attcgccatt
     8161 caggctgcgc aactgttggg aagggcgatc ggtgcgggcc tcttcgctat tacgccagct
     8221 ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta acgccagggt tttcccagtc
     8281 acgacgttgt aaaacgacgg ccagtgagcg cgcgtaatac gactcactat agggcgaatt
     8341 gggtaccggg ccccccctcg aggtcctcca gcttttgttc cctttagtga gggttaattg
     8401 cgcgcttggc gtaatcatgg tcatagctgt ttcctgtgtg aaattgttat ccgctcacaa
     8461 ttccacacaa catacgagcc ggaagcataa agtgtaaagc ctggggtgcc taatgagtga
     8521 gctaactcac attaattgcg ttgcgctcac tgcccgcttt ccaccggtga agacatgttt
     8581 tagagctaga aatagcaagt taaaataagg ctagtccgtt atcaacttga aaaagtggca
     8641 ccgagtcggt gctttttact ccatctggat ttgttcagaa cgctcggttg ccgccgggcg
     8701 ttttttatct agaggccagg aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc
     8761 tccgcccccc tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga
     8821 caggactata aagataccag gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc
     8881 cgaccctgcc gcttaccgga tacctgtccg cctttctccc ttcgggaagc gtggcgcttt
     8941 ctcatagctc acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc aagctgggct
     9001 gtgtgcacga cccccccgtt cagcccgacc gctgcgcctt atccggtaac tatcgtcttg
     9061 agtccaaccc ggtaagacac gacttatcgc cactggcagc agccactggt aacaggatta
     9121 gcagagcgag gtatgtaggc ggtgctacag agttcttgaa gtggtggcct aactacggct
     9181 acactagaag aacagtattt ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa
     9241 gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt
     9301 gcaagcagca gattacgcgc agaaaaaaag gatctcaaga agatcctttg atcttttcta
     9361 cggggtctga cgctcagtgg aacgaaaact cacgttaagg gattttggtc atgagattat
     9421 caaaaaggat cttcacctag atccttttgg ttcatgtgca gctccatcag caaaagggga
     9481 tgataagttt atcaccaccg actatttgca acagtgccgt tgatcgtgct atgatcgact
     9541 gatgtcatca gcggtggagt gcaatgtcgt gcaatacgaa tggcgaaaag ccgagctcat
     9601 cggtcagctt ctcaaccttg gggttacccc cggcggtgtg ctgctggtcc acagctcctt
     9661 ccgtagcgtc cggcccctcg aagatgggcc acttggactg atcgaggccc tgcgtgctgc
     9721 gctgggtccg ggagggacgc tcgtcatgcc ctcgtggtca ggtctggacg acgagccgtt
     9781 cgatcctgcc acgtcgcccg ttacaccgga ccttggagtt gtctctgaca cattctggcg
     9841 cctgccaaat gtaaagcgca gcgcccatcc atttgccttt gcggcagcgg ggccacaggc
     9901 agagcagatc atctctgatc cattgcccct gccacctcac tcgcctgcaa gcccggtcgc
     9961 ccgtgtccat gaactcgatg ggcaggtact tctcctcggc gtgggacacg atgccaacac
    10021 gacgctgcat cttgccgagt tgatggcaaa ggttccctat ggggtgccga gacactgcac
    10081 cattcttcag gatggcaagt tggtacgcgt cgattatctc gagaatgacc actgctgtga
    10141 gcgctttgcc ttggcggaca ggtggctcaa ggagaagagc cttcagaagg aaggtccagt
    10201 cggtcatgcc tttgctcggt tgatccgctc ccgcgacatt gtggcgacag ccctgggtca
    10261 actgggccga gatccgttga tcttcctgca tccgccagag gcgggatgcg aagaatgcga
    10321 tgccgctcgc cagtcgattg gctgagctca tgagcggaga acgagatgac gttggagggg
    10381 caaggtcgcg ctgattgctg gggcaacacg tggagcggat cggggattgt ctttcttcag
    10441 ctcgctgatg atatgctgac gctcaatgcc gtttggcctc cgactaacga aaatcccgca
    10501 tttggacggc tgatccgatt ggcacggcgg acggcgaatg gcggagcaga cgctcgtccg
    10561 ggggcaatga gatatgaaaa agcctgaact caccgcgacg tatcgatgtc gaggttcctc
    10621 aggggagcca ccccagagaa gccctcggag ctgagcggag ctatttccaa agccatgcca
    10681 gctagagaca gtgcacaccg ccaagcatct gcaaaaccct cgcctggagg gaaagtgcaa
    10741 tgtacgtacg ctggtttccc tccagaaagg gattctgcgg cttctcaact ttggaagaag
    10801 aggcggaacg aactgctgtt tcagcctact catgtgagga ggctggtcct ttaccctgat
    10861 gcccgagggc atca
//
