LOCUS       sequence-418474-       11711 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  All-in-one cumate-based inducible CRISPRi plasmid for use in 
            Streptomyces.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 11711)
  AUTHORS   Bai C, van Wezel GP
  TITLE     CUBIC: A Versatile Cumate-Based Inducible CRISPRi System in 
            Streptomyces.
  JOURNAL   ACS Synth Biol. 2023 Oct 6. doi: 10.1021/acssynbio.3c00464.
  PUBMED    37801665
REFERENCE   2  (bases 1 to 11711)
  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: "ACS Synth 
            Biol. 2023 Oct 6. doi: 10.1021/acssynbio.3c00464."
COMMENT     Sequence Label: pCB-2
FEATURES             Location/Qualifiers
     source          1..11711
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             147..758
                     /codon_start=1
                     /gene="cymR from Pseudomonas putida"
                     /product="cumate repressor (Mullick et al., 2006)"
                     /label=CymR
                     /translation="MSPKRRTQAERAMETQGKLIAAALGVLREKGYAGFRIADVPGAAG
                     VSRGAQSHHFPTKLELLLATFEWLYEQITERSRARLAKLKPEDDVIQQMLDDAAEFFLD
                     DDFSISLDLIVAADRDPALREGIQRTVERNRFVVEDMWLGVLVSRGLSRDDAEDILWLI
                     FNSVRGLAVRSLWQKDKERFERVRNSTLEIARERYAKFKR"
     protein_bind    887..914
                     /label=CuO
                     /bound_moiety="CymR"
                     /note="CymR-binding P2 operator sequence from the p-cmt 
                     operon of Pseudomonas putida (Mullick et al., 2006)"
     misc_feature    930..951
                     /label=BioBrick prefix
                     /note="BioBrick prefix for parts that do not start with 
                     ""ATG"""
     protein_bind    1052..1073
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(1088..1093,1094..1111,1112..1118)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1126..1142
                     /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)."
     RBS             1160..1171
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             1178..1855
                     /codon_start=1
                     /product="monomeric derivative of DsRed (Campbell et al., 
                     2002)"
                     /label=mRFP1
                     /translation="MASSEDVIKEFMRFKVRMEGSVNGHEFEIEGEGEGRPYEGTQTAK
                     LKVTKGGPLPFAWDILSPQFQYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGV
                     VTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASTERMYPEDGALKGEIKM
                     RLKLKDGGHYDAEVKTTYMAKKPVQLPGAYKTDIKLDITSHNEDYTIVEQYERAEGRHS
                     TGA"
     terminator      1900..1971
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      1987..2014
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     misc_feature    2021..2041
                     /label=BioBrick suffix
                     /note="universal suffix for all parts"
     misc_RNA        2049..2124
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     protein_bind    2269..2296
                     /label=CuO
                     /bound_moiety="CymR"
                     /note="CymR-binding P2 operator sequence from the p-cmt 
                     operon of Pseudomonas putida (Mullick et al., 2006)"
     CDS             join(2321..2347,2348..2350,2351..4837,4838..4840,
                     4841..6427)
                     /codon_start=1
                     /product="catalytically dead mutant of the Cas9 
                     endonuclease from the Streptococcus pyogenes Type II 
                     CRISPR/Cas system"
                     /label=dCas9
                     /note="RNA-guided DNA-binding protein that lacks 
                     endonuclease activity due to the D10A mutation in the RuvC 
                     catalytic domain and the H840A mutation in the HNH 
                     catalytic domain"
                     /translation="MDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK
                     NLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES
                     FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK
                     FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL
                     ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ
                     IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR
                     QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL
                     RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG
                     NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY
                     FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD
                     SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER
                     LKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFM
                     QLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR
                     HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY
                     LYYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVP
                     SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH
                     VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL
                     NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT
                     EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK
                     ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI
                     TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE
                     LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD
                     ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL
                     DATLIHQSITGLYETRIDLSQLGGD"
     terminator      6579..6627
                     /label=fd terminator
                     /note="central terminator from bacteriophage fd (Otsuka and
                     Kunisawa, 1982)"
     CDS             complement(6840..6851)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     protein_bind    6922..7021
                     /label=phage phi-C31 attP
                     /bound_moiety="phage phi-C31 integrase"
                     /note="attachment site of phage phi-C31"
     CDS             7031..8848
                     /codon_start=1
                     /product="integrase from phage phi-C31"
                     /label=phage phi-C31 integrase
                     /translation="MDTYAGAYDRQSRERENSSAASPATQRSANEDKAADLQREVERDG
                     GRFRFVGHFSEAPGTSAFGTAERPEFERILNECRAGRLNMIIVYDVSRFSRLKVMDAIP
                     IVSELLALGVTIVSTQEGVFRQGNVMDLIHLIMRLDASHKESSLKSAKILDTKNLQREL
                     GGYVGGKAPYGFELVSETKEITRNGRMVNVVINKLAHSTTPLTGPFEFEPDVIRWWWRE
                     IKTHKHLPFKPGSQAAIHPGSITGLCKRMDADAVPTRGETIGKKTASSAWDPATVMRIL
                     RDPRIAGFAAEVIYKKKPDGTPTTKIEGYRIQRDPITLRPVELDCGPIIEPAEWYELQA
                     WLDGRGRGKGLSRGQAILSAMDKLYCECGAVMTSKRGEESIKDSYRCRRRKVVDPSAPG
                     QHEGTCNVSMAALDKFVAERIFNKIRHAEGDEETLALLWEAARRFGKLTEAPEKSGERA
                     NLVAERADALNALEELYEDRAAGAYDGPVGRKHFRKQQAALTLRQQGAEERLAELEAAE
                     APKLPLDQWFPEDADADPTGPKSWCGRASVDDKRVFVGLFVDKIVVTKSTTGRGQGTPI
                     EKRASITWAKPPTDDDEDDAQDGTEDVAA"
     oriT            8997..9106
                     /note="incP origin of transfer"
     CDS             9139..9510
                     /codon_start=1
                     /gene="traJ"
                     /product="oriT-recognizing protein"
                     /label=traJ
                     /translation="MADETKPTRKGSPPIKVYCLPDERRAIEEKAAAAGMSLSAYLLAV
                     GQGYKITGVVDYEHVRELARINGDLGRLGGLLKLWLTDDPRTARFGDATILALLAKIEE
                     KQDELGKVMMGVVRPRAEP"
     CDS             complement(9942..10742)
                     /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"
     rep_origin      10901..11445
                     /direction=RIGHT
                     /label=p15A ori
                     /note="Plasmids containing the medium-copy-number p15A 
                     origin of replication can be propagated in E. coli cells 
                     that contain a second plasmid with the ColE1 origin.
                     "
     primer_bind     11347..11366
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     11562..11579
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 cagaatcaac aataaggcct ccctaacggg gggccttttt tattgataac aaaagtatgt
       61 tcacattcga accgtctctg ctttgacaat ggcttgaatt ggggtgtaaa gtcgtggcca
      121 cctaacgagg agatcggttc atgatcatgt cccccaagcg gcggacccag gcggagcggg
      181 cgatggaaac ccagggcaag ctgatcgcgg cggccctcgg cgtcctgcgc gagaagggct
      241 acgccggctt ccggatcgcg gacgtcccgg gcgccgcggg cgtgtcgcgc ggcgcccaga
      301 gccaccactt ccccaccaag ctcgagctgc tcctggcgac cttcgagtgg ctgtacgagc
      361 agatcaccga gcgctcccgg gcccgcctcg cgaagctgaa gccggaggac gacgtcatcc
      421 agcagatgct cgacgacgcc gcggagttct tcctggacga cgacttctcc atctcgctcg
      481 acctgatcgt cgccgcggac cgggaccccg ccctgcgcga gggcatccag cggaccgtgg
      541 agcggaaccg cttcgtcgtg gaggacatgt ggctcggcgt cctggtgagc cggggcctgt
      601 cccgcgacga cgccgaggac atcctctggc tgatcttcaa ctccgtccgc ggcctcgccg
      661 tgcggtcgct gtggcagaag gacaaggagc ggttcgagcg cgtgcggaac agcaccctgg
      721 agatcgcccg ggagcggtac gccaagttca agcggtgata gactaatctt ttcgaaaaaa
      781 ggcctcccaa atcggggggc ctttttttat agcaacaaaa gtatgttcac attcgaaccg
      841 tctctgcttt gacatcgtgt ggcgcttggg tgtaaagtcg tggccaaaca aacagacaat
      901 ctggtctgtt tgtattatgc caagagaccg aattcgcggc cgcttctaga gcaatacgca
      961 aaccgcctct ccccgcgcgt tggccgattc attaatgcag ctggcacgac aggtttcccg
     1021 actggaaagc gggcagtgag cgcaacgcaa ttaatgtgag ttagctcact cattaggcac
     1081 cccaggcttt acactttatg cttccggctc gtatgttgtg tggaattgtg agcggataac
     1141 aatttcacac atactagaga aagaggagaa atactagatg gcttcctccg aagacgttat
     1201 caaagagttc atgcgtttca aagttcgtat ggaaggttcc gttaacggtc acgagttcga
     1261 aatcgaaggt gaaggtgaag gtcgtccgta cgaaggtacc cagaccgcta aactgaaagt
     1321 taccaaaggt ggtccgctgc cgttcgcttg ggacatcctg tccccgcagt tccagtacgg
     1381 ttccaaagct tacgttaaac acccggctga catcccggac tacctgaaac tgtccttccc
     1441 ggaaggtttc aaatgggaac gtgttatgaa cttcgaagac ggtggtgttg ttaccgttac
     1501 ccaggactcc tccctgcaag acggtgagtt catctacaaa gttaaactgc gtggtaccaa
     1561 cttcccgtcc gacggtccgg ttatgcagaa aaaaaccatg ggttgggaag cttccaccga
     1621 acgtatgtac ccggaagacg gtgctctgaa aggtgaaatc aaaatgcgtc tgaaactgaa
     1681 agacggtggt cactacgacg ctgaagttaa aaccacctac atggctaaaa aaccggttca
     1741 gctgccgggt gcttacaaaa ccgacatcaa actggacatc acctcccaca acgaagacta
     1801 caccatcgtt gaacagtacg aacgtgctga aggtcgtcac tccaccggtg cttaataacg
     1861 ctgatagtgc tagtgtagat cgctactaga gccaggcatc aaataaaacg aaaggctcag
     1921 tcgaaagact gggcctttcg ttttatctgt tgtttgtcgg tgaacgctct ctactagagt
     1981 cacactggct caccttcggg tgggcctttc tgcgtttata tactagtagc ggccgctgca
     2041 gggtctctgt tttagagcta gaaatagcaa gttaaaataa ggctagtccg ttatcaactt
     2101 gaaaaagtgg caccgagtcg gtgctttttt ttagtgccat cctcggtacc aaattccaga
     2161 aaagaggcct cccgaaaggg gggccttttt tcgttttggt ccgtatgttc acattcgaac
     2221 cgtctctgct ttgacatcgt gtggcgcttg ggtgtaaagt cgtggccaaa caaacagaca
     2281 atctggtctg tttgtattat tctaagtaag gagtgtccat atggacaaga agtactccat
     2341 cggcctcgcg atcggcacca actccgtggg ctgggcggtc atcaccgacg agtacaaggt
     2401 cccctccaag aagttcaagg tcctgggcaa caccgaccgg cactcgatca agaagaacct
     2461 gatcggcgcc ctgctgttcg acagcggcga gacggccgag gcgacccgcc tgaagcggac
     2521 cgcgcgtcgc cgctacaccc ggcgcaagaa ccgcatctgc tacctgcagg aaatcttctc
     2581 caacgagatg gccaaggtgg acgactcgtt cttccaccgc ctggaggaga gcttcctggt
     2641 ggaggaggac aagaagcacg agcgccaccc gatcttcggc aacatcgtgg acgaggtggc
     2701 ctaccacgag aagtacccca ccatctacca cctccgcaag aagctggtgg actcgaccga
     2761 caaggcggac ctgcggctca tctacctggc cctcgcgcac atgatcaagt tccgcggcca
     2821 cttcctcatc gagggcgacc tgaacccgga caactccgac gtggacaagc tgttcatcca
     2881 gctggtgcag acctacaacc agctgttcga ggagaacccc atcaacgcca gcggcgtgga
     2941 cgccaaggcg atcctctccg cgcgcctgag caagtcccgg cgcctggaga acctcatcgc
     3001 ccagctgccg ggcgagaaga agaacggcct cttcggcaac ctgatcgcgc tgtcgctcgg
     3061 cctgaccccc aacttcaaga gcaacttcga cctggccgag gacgcgaagc tccagctgtc
     3121 caaggacacc tacgacgacg acctggacaa cctgctcgcc cagatcggcg accagtacgc
     3181 ggacctcttc ctggccgcga agaacctctc ggacgccatc ctgctcagcg acatcctgcg
     3241 ggtcaacacc gagatcacca aggccccgct gtcggcgagc atgatcaagc ggtacgacga
     3301 gcaccaccag gacctgaccc tgctcaaggc cctcgtgcgc cagcagctgc ccgagaagta
     3361 caaggaaatc ttcttcgacc agtccaagaa cggctacgcc ggctacatcg acggcggcgc
     3421 gtcgcaggag gagttctaca agttcatcaa gccgatcctg gagaagatgg acggcaccga
     3481 ggagctgctc gtcaagctga accgcgagga cctgctccgc aagcagcgga ccttcgacaa
     3541 cggctccatc ccgcaccaga tccacctggg cgagctccac gccatcctcc ggcgccagga
     3601 ggacttctac cccttcctga aggacaaccg cgagaagatc gagaagatcc tgaccttccg
     3661 catcccgtac tacgtcggcc ccctggcccg cggcaactcc cggttcgcgt ggatgacccg
     3721 gaagtcggag gagacgatca ccccgtggaa cttcgaggag gtcgtggaca agggcgcgtc
     3781 cgcgcagtcg ttcatcgagc gcatgaccaa cttcgacaag aacctcccga acgagaaggt
     3841 cctgcccaag cactccctgc tctacgagta cttcaccgtg tacaacgagc tgaccaaggt
     3901 caagtacgtg accgagggca tgcggaagcc ggccttcctg tcgggcgagc agaagaaggc
     3961 gatcgtggac ctgctgttca agaccaaccg caaggtcacc gtgaagcagc tgaaggagga
     4021 ctacttcaag aagatcgagt gcttcgactc cgtcgagatc agcggcgtgg aggaccgctt
     4081 caacgcctcc ctgggcacct accacgacct gctcaagatc atcaaggaca aggacttcct
     4141 cgacaacgag gagaacgagg acatcctgga ggacatcgtc ctcaccctga ccctcttcga
     4201 ggaccgcgag atgatcgagg agcggctcaa gacctacgcc cacctgttcg acgacaaggt
     4261 gatgaagcag ctgaagcgtc gccgctacac cggctggggc cgcctctccc ggaagctgat
     4321 caacggcatc cgggacaagc agagcggcaa gaccatcctg gacttcctca agtccgacgg
     4381 cttcgccaac cgcaacttca tgcagctcat ccacgacgac agcctgacct tcaaggagga
     4441 catccagaag gcccaggtgt cgggccaggg cgacagcctc cacgagcaca tcgccaacct
     4501 ggcgggctcc ccggcgatca agaagggcat cctccagacc gtcaaggtcg tggacgagct
     4561 ggtcaaggtg atgggccgcc acaagcccga gaacatcgtg atcgagatgg cccgggagaa
     4621 ccagaccacc cagaagggcc agaagaactc gcgcgagcgg atgaagcgga tcgaggaggg
     4681 catcaaggag ctcggcagcc agatcctgaa ggagcacccg gtcgagaaca cccagctgca
     4741 gaacgagaag ctgtacctct actacctgca gaacggccgc gacatgtacg tggaccagga
     4801 gctcgacatc aaccggctgt ccgactacga cgtggacgcg atcgtgccgc agtccttcct
     4861 gaaggacgac tcgatcgaca acaaggtcct gacccgctcg gacaagaacc ggggcaagtc
     4921 cgacaacgtg ccctcggagg aggtcgtgaa gaagatgaag aactactggc gccagctgct
     4981 caacgccaag ctcatcaccc agcgcaagtt cgacaacctg accaaggccg agcggggcgg
     5041 cctgagcgag ctcgacaagg cgggcttcat caagcgccag ctggtcgaga cgcggcagat
     5101 caccaagcac gtggcccaga tcctggactc ccggatgaac accaagtacg acgagaacga
     5161 caagctgatc cgcgaggtca aggtgatcac cctcaagagc aagctggtgt ccgacttccg
     5221 caaggacttc cagttctaca aggtccggga gatcaacaac taccaccacg cccacgacgc
     5281 gtacctgaac gccgtcgtgg gcaccgcgct gatcaagaag tacccgaagc tggagtccga
     5341 gttcgtctac ggcgactaca aggtctacga cgtgcgcaag atgatcgcca agagcgagca
     5401 ggagatcggc aaggccaccg cgaagtactt cttctactcc aacatcatga acttcttcaa
     5461 gaccgagatc accctggcca acggcgagat ccgcaagcgg cccctgatcg agacgaacgg
     5521 cgagacgggc gagatcgtct gggacaaggg ccgcgacttc gccaccgtcc ggaaggtgct
     5581 gtcgatgccg caggtcaaca tcgtgaagaa gaccgaggtg cagaccggcg gcttcagcaa
     5641 ggagtccatc ctccccaagc gcaacagcga caagctgatc gcccggaaga aggactggga
     5701 cccgaagaag tacggcggct tcgacagccc caccgtcgcc tactccgtgc tggtcgtggc
     5761 gaaggtcgag aagggcaaga gcaagaagct gaagtccgtg aaggagctgc tcggcatcac
     5821 catcatggag cgctcctcgt tcgagaagaa cccgatcgac ttcctggagg ccaagggcta
     5881 caaggaggtc aagaaggacc tcatcatcaa gctgcccaag tacagcctgt tcgagctgga
     5941 gaacggccgc aagcggatgc tcgcctccgc gggcgagctg cagaagggca acgagctggc
     6001 cctcccgtcg aagtacgtca acttcctgta cctcgcgtcc cactacgaga agctgaaggg
     6061 ctcgcccgag gacaacgagc agaagcagct gttcgtggag cagcacaagc actacctgga
     6121 cgagatcatc gagcagatca gcgagttcag caagcgcgtc atcctggccg acgcgaacct
     6181 cgacaaggtg ctgtccgcct acaacaagca ccgcgacaag ccgatccggg agcaggcgga
     6241 gaacatcatc cacctgttca ccctcaccaa cctgggcgcc cccgccgcgt tcaagtactt
     6301 cgacaccacc atcgaccgca agcggtacac ctccaccaag gaggtcctcg acgcgaccct
     6361 gatccaccag agcatcaccg gcctgtacga gacgcgcatc gacctgtccc agctcggcgg
     6421 cgactgatag gagcgatccc gcaaaagcgg cctttgactc cctgcaagcc tcagcgaccg
     6481 aatatatcgg ttatgcgtgg gcgatggttg ttgtcattgt cggcgcaact atcggtatca
     6541 agctgtttaa gaaattcacc tcgaaagcaa gctgataaac cgatacaatt aaaggctcct
     6601 tttggagcct ttttttttgg agattttcaa cgtgaaaaaa ttattattcg caattccttt
     6661 agttgttcct ttctattctc actccgctga aactgttgaa agttgtttag caaaacctca
     6721 tacagaaaat tcattcttag acgtcaggtg gcagcatgca agctctagcg attccagacg
     6781 tcccgaaggc gtggcgcggc ttccccgtgc cggagcaatc gccctgggtg ggttacacga
     6841 cgcccctcta tggcccgtac tgacggacac accgaagccc cggcggcaac cctcagcgga
     6901 tgccccgggg cttcacgttt tcccaggtca gaagcggttt tcgggagtag tgccccaact
     6961 ggggtaacct ttgagttctc tcagttgggg gcgtagggtc gccgacatga cacaaggggt
     7021 tgtgaccggg gtggacacgt acgcgggtgc ttacgaccgt cagtcgcgcg agcgcgaaaa
     7081 ttcgagcgca gcaagcccag cgacacagcg tagcgccaac gaagacaagg cggccgacct
     7141 tcagcgcgaa gtcgagcgcg acgggggccg gttcaggttc gtcgggcatt tcagcgaagc
     7201 gccgggcacg tcggcgttcg ggacggcgga gcgcccggag ttcgaacgca tcctgaacga
     7261 atgccgcgcc gggcggctca acatgatcat tgtctatgac gtgtcgcgct tctcgcgcct
     7321 gaaggtcatg gacgcgattc cgattgtctc ggaattgctc gccctgggcg tgacgattgt
     7381 ttccactcag gaaggcgtct tccggcaggg aaacgtcatg gacctgattc acctgattat
     7441 gcggctcgac gcgtcgcaca aagaatcttc gctgaagtcg gcgaagattc tcgacacgaa
     7501 gaaccttcag cgcgaattgg gcgggtacgt cggcgggaag gcgccttacg gcttcgagct
     7561 tgtttcggag acgaaggaga tcacgcgcaa cggccgaatg gtcaatgtcg tcatcaacaa
     7621 gcttgcgcac tcgaccactc cccttaccgg acccttcgag ttcgagcccg acgtaatccg
     7681 gtggtggtgg cgtgagatca agacgcacaa acaccttccc ttcaagccgg gcagtcaagc
     7741 cgccattcac ccgggcagca tcacggggct ttgtaagcgc atggacgctg acgccgtgcc
     7801 gacccggggc gagacgattg ggaagaagac cgcttcaagc gcctgggacc cggcaaccgt
     7861 tatgcgaatc cttcgggacc cgcgtattgc gggcttcgcc gctgaggtga tctacaagaa
     7921 gaagccggac ggcacgccga ccacgaagat tgagggttac cgcattcagc gcgacccgat
     7981 cacgctccgg ccggtcgagc ttgattgcgg accgatcatc gagcccgctg agtggtatga
     8041 gcttcaggcg tggttggacg gcagggggcg cggcaagggg ctttcccggg ggcaagccat
     8101 tctgtccgcc atggacaagc tgtactgcga gtgtggcgcc gtcatgactt cgaagcgcgg
     8161 ggaagaatcg atcaaggact cttaccgctg ccgtcgccgg aaggtggtcg acccgtccgc
     8221 acctgggcag cacgaaggca cgtgcaacgt cagcatggcg gcactcgaca agttcgttgc
     8281 ggaacgcatc ttcaacaaga tcaggcacgc cgaaggcgac gaagagacgt tggcgcttct
     8341 gtgggaagcc gcccgacgct tcggcaagct cactgaggcg cctgagaaga gcggcgaacg
     8401 ggcgaacctt gttgcggagc gcgccgacgc cctgaacgcc cttgaagagc tgtacgaaga
     8461 ccgcgcggca ggcgcgtacg acggacccgt tggcaggaag cacttccgga agcaacaggc
     8521 agcgctgacg ctccggcagc aaggggcgga agagcggctt gccgaacttg aagccgccga
     8581 agccccgaag cttccccttg accaatggtt ccccgaagac gccgacgctg acccgaccgg
     8641 ccctaagtcg tggtgtgggc gcgcgtcagt agacgacaag cgcgtgttcg tcgggctctt
     8701 cgtagacaag atcgttgtca cgaagtcgac tacgggcagg gggcagggaa cgcccatcga
     8761 gaagcgcgct tcgatcacgt gggcgaagcc gccgaccgac gacgacgaag acgacgccca
     8821 ggacggcacg gaagacgtag cggcgtagca ggtcgacgga tcttttccgc tgcataaccc
     8881 tgcttcgggg tcattatagc gattttttcg gtatatccat cctttttcgc acgatataca
     8941 ggattttgcc aaagggttcg tgtagacttt ccttggtgta tccaacggcg tcagccgggc
     9001 aggataggtg aagtaggccc acccgcgagc gggtgttcct tcttcactgt cccttattcg
     9061 cacctggcgg tgctcaacgg gaatcctgct ctgcgaggct ggccggctac cgccggcgta
     9121 acagatgagg gcaagcggat ggctgatgaa accaagccaa ccaggaaggg cagcccacct
     9181 atcaaggtgt actgccttcc agacgaacga agagcgattg aggaaaaggc ggcggcggcc
     9241 ggcatgagcc tgtcggccta cctgctggcc gtcggccagg gctacaaaat cacgggcgtc
     9301 gtggactatg agcacgtccg cgagctggcc cgcatcaatg gcgacctggg ccgcctgggc
     9361 ggcctgctga aactctggct caccgacgac ccgcgcacgg cgcggttcgg tgatgccacg
     9421 atcctcgccc tgctggcgaa gatcgaagag aagcaggacg agcttggcaa ggtcatgatg
     9481 ggcgtggtcc gcccgagggc agagccatga cttttttagc cgctaaaacg gccggggggt
     9541 gcgcgtgatt gccaagcacg tccccatgcg ctccatcaag aagagcgact tcgcggagct
     9601 ggtgaagtac atcaccgacg agcaaggcaa gaccgatccc cggggaccca ggtcgactct
     9661 agctagctgg ccagggcccg atacgtcgcg gtgagttcag gctttttcat atctcattgc
     9721 ccccggacga gcgtctgctc cgccattcgc cgtccgccgt gccaatcgga tcagccgtcc
     9781 aaatgcggga ttttcgttag tcggaggcca aacggcattg agcgtcagca tatcatcagc
     9841 gagctgaaga aagacaatcc ccgatccgct ccacgtgttg ccccagcaat cagcgcgacc
     9901 ttgcccctcc aacgtcatct cgttctccgc tcatgagctc agccaatcga ctggcgagcg
     9961 gcatcgcatt cttcgcatcc cgcctctggc ggatgcagga agatcaacgg atctcggccc
    10021 agttgaccca gggctgtcgc cacaatgtcg cgggagcgga tcaaccgagc aaaggcatga
    10081 ccgactggac cttccttctg aaggctcttc tccttgagcc acctgtccgc caaggcaaag
    10141 cgctcacagc agtggtcatt ctcgagataa tcgacgcgta ccaacttgcc atcctgaaga
    10201 atggtgcagt gtctcggcac cccataggga acctttgcca tcaactcggc aagatgcagc
    10261 gtcgtgttgg catcgtgtcc cacgccgagg agaagtacct gcccatcgag ttcatggaca
    10321 cgggcgaccg ggcttgcagg cgagtgaggt ggcaggggca atggatcaga gatgatctgc
    10381 tctgcctgtg gccccgctgc cgcaaaggca aatggatggg cgctgcgctt tacatttggc
    10441 aggcgccaga atgtgtcaga gacaactcca aggtccggtg taacgggcga cgtggcagga
    10501 tcgaacggct cgtcgtccag acctgaccac gagggcatga cgagcgtccc tcccggaccc
    10561 agcgcagcac gcagggcctc gatcagtcca agtggcccat cttcgagggg ccggacgcta
    10621 cggaaggagc tgtggaccag cagcacaccg ccgggggtaa ccccaaggtt gagaagctga
    10681 ccgatgagct cggcttttcg ccattcgtat tgcacgacat tgcactccac cgctgatgac
    10741 atcagtcgat catagcacga tcaacggcac tgttgcaaat agtcggtggt gataaactta
    10801 tcatcccctt ttgctgatgg agctgcacat gaaccaaaag gatctaggtg aagatccttt
    10861 ttgataatct catgaccaaa atcttaataa gatgatcttc ttgagatcgt tttggtctgc
    10921 gcgtaatctc ttgctctgaa aacgaaaaaa ccgccttgca gggcggtttt ttgaaggttc
    10981 tctgagctac caactctttg aaccgaggta actggcttgg aggagcgcag tcaccaaaac
    11041 ttgtcctttc agtttagcct taaccggcgc atgacttcaa gactaactcc tctaaatcaa
    11101 ttaccagtgg ctgctgccag tggtgctttt gcatgtcttt ccgggttgga ctcaagacga
    11161 tagttaccgg ataaggcgca gcggtcggac tgaacggggg gttcgtgcat acagtccagc
    11221 ttggagcgaa ctgcctaccc ggaactgagt gtcaggcgtg gaatgagaca aacgcggcca
    11281 taacagcgga atgacaccgg taaaccgaaa ggcaggaaca ggagagcgca cgagggagcc
    11341 gccaggggga aacgcctggt atctttatag tcctgtcggg tttcgccacc actgatttga
    11401 gcgtcagatt tcgtgatgct tgtcaggggg cggagcctat ggaaaaacgg ctttgccgcg
    11461 gccctctcac ttccctgtta agtatcttcc tggcatcttc caggaaatct ccgccccgtt
    11521 cgtaagccat ttccgctcgc cgcagtcgaa cgaccgagcg tagcgagtca gtgagcgagg
    11581 aagcggaata tatcctgtat cacatattct gctgacgcac cggtgcagcc ttttttctcc
    11641 tgccacatga agcacttcac tgacaccctc atcagtgcca acatagtaag ccagtataca
    11701 ctccgctagc g
//
