pCutamp

LOCUS       pCutamp              14764 bp ds-DNA     circular SYN 12-MAY-2021
DEFINITION  Plasmid-curing in Escherichia coli by targeting the AmpR promoter.
ACCESSION   .
VERSION     .
KEYWORDS    pCutamp
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 14764)
  AUTHORS   Zhang Y, Sun X, Wang Q, Xu J, Dong F, Yang S, Yang J, Zhang Z, Qian 
            Y, Chen J, Zhang J, Liu Y, Tao R, Jiang Y, Yang J, Yang S
  TITLE     Multicopy chromosomal integration using CRISPR-associated 
            transposases.
  JOURNAL   ACS Synth Biol. 2020 Jun 17. doi: 10.1021/acssynbio.0c00073.
  PUBMED    32551502
REFERENCE   2  (bases 1 to 14764)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 12, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..14764
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     CDS             complement(530..1408)
                     /codon_start=1
                     /gene="araC"
                     /product="L-arabinose regulatory protein"
                     /label=araC
                     /translation="MAEAQNDPLLPGYSFNAHLVAGLTPIEANGYLDFFIDRPLGMKGY
                     ILNLTIRGQGVVKNQGREFVCRPGDILLFPPGEIHHYGRHPEAREWYHQWVYFRPRAYW
                     HEWLNWPSIFANTGFFRPDEAHQPHFSDLFGQIINAGQGEGRYSELLAINLLEQLLLRR
                     MEAINESLHPPMDNRVREACQYISDHLADSNFDIASVAQHVCLSPSRLSHLFRQQLGIS
                     VLSWREDQRISQAKLLLSTTRMPIATVGRNVGFDDQLYFSRVFKKCTGASPSEFRAGCE
                     EKVNDVAVKLS"
     promoter        1435..1719
                     /gene="araBAD"
                     /label=araBAD promoter
                     /note="promoter of the L-arabinose operon of E. coli; the 
                     araC regulatory gene is transcribed in the opposite 
                     direction (Guzman et al., 1995)"
     primer_bind     1642..1661
                     /label=pBAD Forward
                     /note="For vectors with E. coli araBAD promoter, forward 
                     primer"
     RBS             1746..1754
                     /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)"
     CDS             1763..2179
                     /codon_start=1
                     /gene="gam"
                     /product="inhibitor of the host RecBCD nuclease in the 
                     lambda Red system"
                     /label=Gam
                     /note="protects the double-stranded DNA substrate for 
                     recombination"
                     /translation="MDINTETEIKQKHSLTPFPVFLISPAFRGRYFHSYFRSSAMNAYY
                     IQDRLEAQSWARHYQQLAREEKEAELADDMEKGLPQHLFESLCIDHLQRHGASKKSITR
                     AFDDDVEFQERMAEHIRYMVETIAHHQVDIDSEV"
     CDS             2185..2970
                     /codon_start=1
                     /gene="bet"
                     /product="single-stranded DNA binding recombinase in the 
                     lambda Red system"
                     /label=Beta
                     /note="mediates pairing of a single-stranded DNA with a 
                     complementary target"
                     /translation="MSTALATLAGKLAERVGMDSVDPQELITTLRQTAFKGDASDAQFI
                     ALLIVANQYGLNPWTKEIYAFPDKQNGIVPVVGVDGWSRIINENQQFDGMDFEQDNESC
                     TCRIYRKDRNHPICVTEWMDECRREPFKTREGREITGPWQSHPKRMLRHKAMIQCARLA
                     FGFAGIYDKDEAERIVENTAYTAERQPERDITPVNDETMQEINTLLIALDKTWDDDLLP
                     LCSQIFRRDIRASSELTQAEAVKALGFLKQKAAEQKVAA"
     CDS             2967..3647
                     /codon_start=1
                     /gene="exo"
                     /product="5' to 3' double-stranded DNA exonuclease in the 
                     lambda Red system"
                     /label=Exo
                     /note="creates 3' single-stranded DNA tails"
                     /translation="MTPDIILQRTGIDVRAVEQGDDAWHKLRLGVITASEVHNVIAKPR
                     SGKKWPDMKMSYFHTLLAEVCTGVAPEVNAKALAWGKQYENDARTLFEFTSGVNVTESP
                     IIYRDESMRTACSPDGLCSDGNGLELKCPFTSRDFMKFRLGGFEAIKSAYMAQVQYSMW
                     VTRKNAWYFANYDPRMKREGLHYVVIERDEKYMASFDEIVPEFIEKMDEALAEIGFVFG
                     EQWR"
     terminator      3648..3892
                     /label=-lambda tL3 terminator
                     /note="transcription terminator tL3 from phage lambda"
     CDS             4229..5650
                     /codon_start=1
                     /gene="Bacillus subtilis sacB"
                     /product="secreted levansucrase that renders bacterial 
                     growth sensitive to sucrose"
                     /label=SacB
                     /note="negative selection marker"
                     /translation="MNIKKFAKQATVLTFTTALLAGGATQAFAKETNQKPYKETYGISH
                     ITRHDMLQIPEQQKNEKYQVPEFDSSTIKNISSAKGLDVWDSWPLQNADGTVANYHGYH
                     IVFALAGDPKNADDTSIYMFYQKVGETSIDSWKNAGRVFKDSDKFDANDSILKDQTQEW
                     SGSATFTSDGKIRLFYTDFSGKHYGKQTLTTAQVNVSASDSSLNINGVEDYKSIFDGDG
                     KTYQNVQQFIDEGNYSSGDNHTLRDPHYVEDKGHKYLVFEANTGTEDGYQGEESLFNKA
                     YYGKSTSFFRQESQKLLQSDKKRTAELANGALGMIELNDDYTLKKVMKPLIASNTVTDE
                     IERANVFKMNGKWYLFTDSRGSKMTIDGITSNDIYMLGYVSNSLTGPYKPLNKTGLVLK
                     MDLDPNDVTFTYSHFAVPQAKGNNVVITSYMTNRGFYADKQSTFAPSFLLNIKGKKTSV
                     VKDSILEQGQLTVNK"
     misc_RNA        complement(5773..5848)
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        complement(5870..5988)
                     /label=rhaB promoter
                     /note="promoter of the E. coli rhaBAD operon, conferring 
                     tight induction with L-rhamnose and repression with 
                     D-glucose in the presence of RhaR and RhaS (Giacalone et 
                     al., 2006)"
     CDS             6134..6970
                     /codon_start=1
                     /gene="E. coli rhaS"
                     /product="positive regulator of the rhaB promoter
                     "
                     /label=rhaS
                     /note="activated by L-rhamnose"
                     /translation="MTVLHSVDFFPSGNASVAIEPRLPQADFPEHHHDFHEIVIVEHGT
                     GIHVFNGQPYTITGGTVCFVRDHDRHLYEHTDNLCLTNVLYRSPDRFQFLAGLNQLLPQ
                     ELDGQYPSHWRVNHSVLQQVRQLVAQMEQQEGENDLPSTASREILFMQLLLLLRKSSLQ
                     ENLENSASRLNLLLAWLEDHFADEVNWDAVADQFSLSLRTLHRQLKQQTGLTPQRYLNR
                     LRLMKARHLLRHSEASVTDIAYRCGFSDSNHFSTLFRREFNWSPRDIRQGRDGFLQ"
     CDS             7044..7892
                     /codon_start=1
                     /gene="E. coli rhaR"
                     /product="transcriptional activator of rhaR and rhaS"
                     /label=rhaR
                     /note="needed for rhamnose-dependent activation of the rhaB
                     promoter"
                     /translation="MAHQLKLLKDDFFASDQQAVAVADRYPQDVFAEHTHDFCELVIVW
                     RGNGLHVLNDRPYRITRGDLFYIHADDKHSYASVNDLVLQNIIYCPERLKLNLDWQGAI
                     PGFNASAGQPHWRLGSMGMAQARQVIGQLEHESSQHVPFANEMAELLFGQLVMLLNRHR
                     YTSDSLPPTSSETLLDKLITRLAASLKSPFALDKFCDEASCSERVLRQQFRQQTGMTIN
                     QYLRQVRVCHAQYLLQHSRLLISDISTECGFEDSNYFSVVFTRETGMTPSQWRHLNSQK
                     D"
     CDS             complement(8011..8961)
                     /codon_start=1
                     /gene="rep101"
                     /product="RepA protein needed for replication with the 
                     pSC101 origin"
                     /label=Rep101
                     /translation="MSELVVFKANELAISRYDLTEHETKLILCCVALLNPTIENPTRKE
                     RTVSFTYNQYAQMMNISRENAYGVLAKATRELMTRTVEIRNPLVKGFEIFQWTNYAKFS
                     SEKLELVFSEEILPYLFQLKKFIKYNLEHVKSFENKYSMRIYEWLLKELTQKKTHKANI
                     EISLDEFKFMLMLENNYHEFKRLNQWVLKPISKDLNTYSNMKLVVDKRGRPTDTLIFQV
                     ELDRQMDLVTELENNQIKMNGDKIPTTITSDSYLHNGLRKTLHDALTAKIQLTSFEAKF
                     LSDMQSKHDLNGSFSWLTQKQRTTLENILAKYGRI"
     rep_origin      9009..9231
                     /label=pSC101 ori
                     /note="low-copy replication origin that requires the Rep101
                     protein"
     CDS             complement(9708..10508)
                     /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"
     CDS             11058..400
                     /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="MDKKYSIGLDIGTNSVGWAVITDDYKVPSKKFKVLGNTDRHSIKK
                     NLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES
                     FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK
                     FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL
                     ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ
                     IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR
                     QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL
                     RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG
                     NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY
                     FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD
                     SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER
                     LKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFM
                     QLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR
                     HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY
                     LYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVP
                     SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH
                     VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL
                     NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT
                     EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK
                     ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI
                     TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE
                     LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD
                     ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL
                     DATLIHQSITGLYETRIDLSQLGGD"
ORIGIN
        1 atatttagct agtcattatg aaaagttgaa gggtagtcca gaagataacg aacaaaaaca
       61 attgtttgtg gagcagcata agcattattt agatgagatt attgagcaaa tcagtgaatt
      121 ttctaagcgt gttattttag cagatgccaa tttagataaa gttcttagtg catataacaa
      181 acatagagac aaaccaatac gtgaacaagc agaaaatatt attcatttat ttacgttgac
      241 gaatcttgga gctcccgctg cttttaaata ttttgataca acaattgatc gtaaacgata
      301 tacgtctaca aaagaagttt tagatgccac tcttatccat caatccatca ctggtcttta
      361 tgaaacacgc attgatttga gtcagctagg aggtgactga agtatatttt agatgaagat
      421 tatttcttaa tctagacatg agcggataca tatttgaatg tatttagaaa aataaacaaa
      481 taggggttcc gcgcacattt ccccgaaaag tgccacctgc atcgatttat tatgacaact
      541 tgacggctac atcattcact ttttcttcac aaccggcacg gaactcgctc gggctggccc
      601 cggtgcattt tttaaatacc cgcgagaagt agagttgatc gtcaaaacca acattgcgac
      661 cgacggtggc gataggcatc cgggtggtgc tcaaaagcag cttcgcctgg ctgatacgtt
      721 ggtcctcgcg ccagcttaag acgctaatcc ctaactgctg gcggaaaaga tgtgacagac
      781 gcgacggcga caagcaaaca tgctgtgcga cgctggcgat atcaaaattg ctgtctgcca
      841 ggtgatcgct gatgtactga caagcctcgc gtacccgatt atccatcggt ggatggagcg
      901 actcgttaat cgcttccatg cgccgcagta acaattgctc aagcagattt atcgccagca
      961 gctccgaata gcgcccttcc ccttgcccgg cgttaatgat ttgcccaaac aggtcgctga
     1021 aatgcggctg gtgcgcttca tccgggcgaa agaaccccgt attggcaaat attgacggcc
     1081 agttaagcca ttcatgccag taggcgcgcg gacgaaagta aacccactgg tgataccatt
     1141 cgcgagcctc cggatgacga ccgtagtgat gaatctctcc tggcgggaac agcaaaatat
     1201 cacccggtcg gcaaacaaat tctcgtccct gatttttcac caccccctga ccgcgaatgg
     1261 tgagattgag aatataacct ttcattccca gcggtcggtc gataaaaaaa tcgagataac
     1321 cgttggcctc aatcggcgtt aaacccgcca ccagatgggc attaaacgag tatcccggca
     1381 gcaggggatc attttgcgct tcagccatac ttttcatact cccgccattc agagaagaaa
     1441 ccaattgtcc atattgcatc agacattgcc gtcactgcgt cttttactgg ctcttctcgc
     1501 taaccaaacc ggtaaccccg cttattaaaa gcattctgta acaaagcggg accaaagcca
     1561 tgacaaaaac gcgtaacaaa agtgtctata atcacggcag aaaagtccac attgattatt
     1621 tgcacggcgt cacactttgc tatgccatag catttttatc cataagatta gcggatccta
     1681 cctgacgctt tttatcgcaa ctctctactg tttctccata cccgtttttt tgggaattcg
     1741 agctctaagg aggttataaa aaatggatat taatactgaa actgagatca agcaaaagca
     1801 ttcactaacc ccctttcctg ttttcctaat cagcccggca tttcgcgggc gatattttca
     1861 cagctatttc aggagttcag ccatgaacgc ttattacatt caggatcgtc ttgaggctca
     1921 gagctgggcg cgtcactacc agcagctcgc ccgtgaagag aaagaggcag aactggcaga
     1981 cgacatggaa aaaggcctgc cccagcacct gtttgaatcg ctatgcatcg atcatttgca
     2041 acgccacggg gccagcaaaa aatccattac ccgtgcgttt gatgacgatg ttgagtttca
     2101 ggagcgcatg gcagaacaca tccggtacat ggttgaaacc attgctcacc accaggttga
     2161 tattgattca gaggtataaa acgaatgagt actgcactcg caacgctggc tgggaagctg
     2221 gctgaacgtg tcggcatgga ttctgtcgac ccacaggaac tgatcaccac tcttcgccag
     2281 acggcattta aaggtgatgc cagcgatgcg cagttcatcg cattactgat cgttgccaac
     2341 cagtacggcc ttaatccgtg gacgaaagaa atttacgcct ttcctgataa gcagaatggc
     2401 atcgttccgg tggtgggcgt tgatggctgg tcccgcatca tcaatgaaaa ccagcagttt
     2461 gatggcatgg actttgagca ggacaatgaa tcctgtacat gccggattta ccgcaaggac
     2521 cgtaatcatc cgatctgcgt taccgaatgg atggatgaat gccgccgcga accattcaaa
     2581 actcgcgaag gcagagaaat cacggggccg tggcagtcgc atcccaaacg gatgttacgt
     2641 cataaagcca tgattcagtg tgcccgtctg gccttcggat ttgctggtat ctatgacaag
     2701 gatgaagccg agcgcattgt cgaaaatact gcatacactg cagaacgtca gccggaacgc
     2761 gacatcactc cggttaacga tgaaaccatg caggagatta acactctgct gatcgccctg
     2821 gataaaacat gggatgacga cttattgccg ctctgttccc agatatttcg ccgcgacatt
     2881 cgtgcatcgt cagaactgac acaggccgaa gcagtaaaag ctcttggatt cctgaaacag
     2941 aaagccgcag agcagaaggt ggcagcatga caccggacat tatcctgcag cgtaccggga
     3001 tcgatgtgag agctgtcgaa cagggggatg atgcgtggca caaattacgg ctcggcgtca
     3061 tcaccgcttc agaagttcac aacgtgatag caaaaccccg ctccggaaag aagtggcctg
     3121 acatgaaaat gtcctacttc cacaccctgc ttgctgaggt ttgcaccggt gtggctccgg
     3181 aagttaacgc taaagcactg gcctggggaa aacagtacga gaacgacgcc agaaccctgt
     3241 ttgaattcac ttccggcgtg aatgttactg aatccccgat catctatcgc gacgaaagta
     3301 tgcgtaccgc ctgctctccc gatggtttat gcagtgacgg caacggcctt gaactgaaat
     3361 gcccgtttac ctcccgggat ttcatgaagt tccggctcgg tggtttcgag gccataaagt
     3421 cagcttacat ggcccaggtg cagtacagca tgtgggtgac gcgaaaaaat gcctggtact
     3481 ttgccaacta tgacccgcgt atgaagcgtg aaggcctgca ttatgtcgtg attgagcggg
     3541 atgaaaagta catggcgagt tttgacgaga tcgtgccgga gttcatcgaa aaaatggacg
     3601 aggcactggc tgaaattggt tttgtatttg gggagcaatg gcgatgacgc atcctcacga
     3661 taatatccgg gtaggcgcaa tcactttcgt ctactccgtt acaaagcgag gctgggtatt
     3721 tcccggcctt tctgttatcc gaaatccact gaaagcacag cggctggctg aggagataaa
     3781 taataaacga ggggctgtat gcacaaagca tcttctgttg agttaagaac gagtatcgag
     3841 atggcacata gccttgctca aattggaatc aggtttgtgc caataccagt agaaacagac
     3901 gaagaatcca tgggtatgga cagatcctct ttaggcccgt agtctgcaaa tccttttatg
     3961 attttctatc aaacaaaaga ggaaaataga ccagttgcaa tccaaacgag agtctaatag
     4021 aatgaggtcg aaaagtaaat cgcgcgggtt tgttactgat aaagcaggca agacctaaaa
     4081 tgtgtaaagg gcaaagtgta tactttggcg tcacccctta catattttag gtcttttttt
     4141 attgtgcgta actaacttgc catcttcaaa caggagggct ggaagaagca gaccgctaac
     4201 acagtacata aaaaaggaga catgaacgat gaacatcaaa aagtttgcaa aacaagcaac
     4261 agtattaacc tttactaccg cactgctggc aggaggcgca actcaagcgt ttgcgaaaga
     4321 aacgaaccaa aagccatata aggaaacata cggcatttcc catattacac gccatgatat
     4381 gctgcaaatc cctgaacagc aaaaaaatga aaaatatcaa gttcctgaat tcgattcgtc
     4441 cacaattaaa aatatctctt ctgcaaaagg cctggacgtt tgggacagct ggccattaca
     4501 aaacgctgac ggcactgtcg caaactatca cggctaccac atcgtctttg cattagccgg
     4561 agatcctaaa aatgcggatg acacatcgat ttacatgttc tatcaaaaag tcggcgaaac
     4621 ttctattgac agctggaaaa acgctggccg cgtctttaaa gacagcgaca aattcgatgc
     4681 aaatgattct atcctaaaag accaaacaca agaatggtca ggttcagcca catttacatc
     4741 tgacggaaaa atccgtttat tctacactga tttctccggt aaacattacg gcaaacaaac
     4801 actgacaact gcacaagtta acgtatcagc atcagacagc tctttgaaca tcaacggtgt
     4861 agaggattat aaatcaatct ttgacggtga cggaaaaacg tatcaaaatg tacagcagtt
     4921 catcgatgaa ggcaactaca gctcaggcga caaccatacg ctgagagatc ctcactacgt
     4981 agaagataaa ggccacaaat acttagtatt tgaagcaaac actggaactg aagatggcta
     5041 ccaaggcgaa gaatctttat ttaacaaagc atactatggc aaaagcacat cattcttccg
     5101 tcaagaaagt caaaaacttc tgcaaagcga taaaaaacgc acggctgagt tagcaaacgg
     5161 cgctctcggt atgattgagc taaacgatga ttacacactg aaaaaagtga tgaaaccgct
     5221 gattgcatct aacacagtaa cagatgaaat tgaacgcgcg aacgtcttta aaatgaacgg
     5281 caaatggtac ctgttcactg actcccgcgg atcaaaaatg acgattgacg gcattacgtc
     5341 taacgatatt tacatgcttg gttatgtttc taattcttta actggcccat acaagccgct
     5401 gaacaaaact ggccttgtgt taaaaatgga tcttgatcct aacgatgtaa cctttactta
     5461 ctcacacttc gctgtacctc aagcgaaagg aaacaatgtc gtgattacaa gctatatgac
     5521 aaacagagga ttctacgcag acaaacaatc aacgtttgcg ccaagcttcc tgctgaacat
     5581 caaaggcaag aaaacatctg ttgtcaaaga cagcatcctt gaacaaggac aattaacagt
     5641 taacaaataa aaacgcaaaa gaaaatgccg atatcctatt ggcattttct tttatttctt
     5701 atcaacataa aggtgaatcc catatgaact atataaaagc aggcaaatgg ctaaccgtat
     5761 tcctcaaaaa aagcaccgac tcggtgccac tttttcaagt tgataacgga ctagccttat
     5821 tttaacttgc tatttctagc tctaaaactg ataaatgctt caataatatt acgaccagtc
     5881 taaaaagcgc ctgaattcgc gaccttctcg ttactgacag gaaaatgggc cattggcaac
     5941 cagggaaaga tgaacgtgat gatgttcaca atttgctgaa ttgtggtgat gtgatgctca
     6001 ccgcatttcc tgaaaattca cgctgtatct tgaaaaatcg acgtttttta cgtggttttc
     6061 cgtcgaaaat ttaaggtaag aacctgacct cgtgattact atttcgccgt gttgacgaca
     6121 tcaggaggcc agtatgaccg tattacatag tgtggatttt tttccgtctg gtaacgcgtc
     6181 cgtggcgata gaaccccggc tcccgcaggc ggattttcct gaacatcatc atgattttca
     6241 tgaaattgtg attgtcgaac atggcacggg tattcatgtg tttaatgggc agccctatac
     6301 catcaccggt ggcacggtct gtttcgtacg cgatcatgat cggcatctgt atgaacatac
     6361 cgataatctg tgtctgacca atgtgctgta tcgctcgccg gatcgatttc agtttctcgc
     6421 cgggctgaat cagttgctgc cacaagagct ggatgggcag tatccgtctc actggcgcgt
     6481 taaccacagc gtattgcagc aggtgcgaca gctggttgca cagatggaac agcaggaagg
     6541 ggaaaatgat ttaccctcga ccgccagtcg cgagatcttg tttatgcaat tactgctctt
     6601 gctgcgtaaa agcagtttgc aggagaacct ggaaaacagc gcatcacgtc tcaacttgct
     6661 tctggcctgg ctggaggacc attttgccga tgaggtgaat tgggatgccg tggcggatca
     6721 attttctctt tcactgcgta cgctacatcg gcagcttaag cagcaaacgg gactgacgcc
     6781 tcagcgatac ctgaaccgcc tgcgactgat gaaagcccga catctgctac gccacagcga
     6841 ggccagcgtt actgacatcg cctatcgctg tggattcagc gacagtaacc acttttcgac
     6901 gctttttcgc cgagagttta actggtcacc gcgtgatatt cgccagggac gggatggctt
     6961 tctgcaataa cgcgaatctt ctcaacgtat ttgtacgcca tattgcgaat aatcaacttc
     7021 gttctctggc cgaggtagcc acggtggcgc atcagttaaa acttctcaaa gatgattttt
     7081 ttgccagcga ccagcaggca gtcgctgtgg ctgaccgtta tccgcaagat gtctttgctg
     7141 aacatacaca tgatttttgt gagctggtga ttgtctggcg cggtaatggc ctgcatgtac
     7201 tcaacgatcg cccttatcgc attacccgtg gcgatctctt ttacattcat gctgacgata
     7261 aacactccta cgcttccgtt aacgatctgg ttttgcagaa tattatttat tgcccggagc
     7321 gtctgaagct gaatcttgac tggcaggggg cgattccggg atttaacgcc agcgcagggc
     7381 aaccacactg gcgcttaggt agcatgggga tggcgcaggc gcggcaggtt atcggtcagc
     7441 ttgagcatga aagtagtcag catgtgccgt ttgctaacga aatggctgag ttgctgttcg
     7501 ggcagttggt gatgttgctg aatcgccatc gttacaccag tgattcgttg ccgccaacat
     7561 ccagcgaaac gttgctggat aagctgatta cccggctggc ggctagcctg aaaagtccct
     7621 ttgcgctgga taaattttgt gatgaggcat cgtgcagtga gcgcgttttg cgtcagcaat
     7681 ttcgccagca gactggaatg accatcaatc aatatctgcg acaggtcaga gtgtgtcatg
     7741 cgcaatatct tctccagcat agccgcctgt taatcagtga tatttcgacc gaatgtggct
     7801 ttgaagatag taactatttt tcggtggtgt ttacccggga aaccgggatg acgcccagcc
     7861 agtggcgtca tctcaattcg cagaaagatt aattcgccat gccccatggg tatggacagt
     7921 tttccctttg atatgtaacg gtgaacagtt gttctacttt tgtttgttag tcttgatgct
     7981 tcactgatag atacaagagc cataagaacc tcagatcctt ccgtatttag ccagtatgtt
     8041 ctctagtgtg gttcgttgtt tttgcgtgag ccatgagaac gaaccattga gatcatgctt
     8101 actttgcatg tcactcaaaa attttgcctc aaaactggtg agctgaattt ttgcagttaa
     8161 agcatcgtgt agtgtttttc ttagtccgtt atgtaggtag gaatctgatg taatggttgt
     8221 tggtattttg tcaccattca tttttatctg gttgttctca agttcggtta cgagatccat
     8281 ttgtctatct agttcaactt ggaaaatcaa cgtatcagtc gggcggcctc gcttatcaac
     8341 caccaatttc atattgctgt aagtgtttaa atctttactt attggtttca aaacccattg
     8401 gttaagcctt ttaaactcat ggtagttatt ttcaagcatt aacatgaact taaattcatc
     8461 aaggctaatc tctatatttg ccttgtgagt tttcttttgt gttagttctt ttaataacca
     8521 ctcataaatc ctcatagagt atttgttttc aaaagactta acatgttcca gattatattt
     8581 tatgaatttt tttaactgga aaagataagg caatatctct tcactaaaaa ctaattctaa
     8641 tttttcgctt gagaacttgg catagtttgt ccactggaaa atctcaaagc ctttaaccaa
     8701 aggattcctg atttccacag ttctcgtcat cagctctctg gttgctttag ctaatacacc
     8761 ataagcattt tccctactga tgttcatcat ctgagcgtat tggttataag tgaacgatac
     8821 cgtccgttct ttccttgtag ggttttcaat cgtggggttg agtagtgcca cacagcataa
     8881 aattagcttg gtttcatgct ccgttaagtc atagcgacta atcgctagtt catttgcttt
     8941 gaaaacaact aattcagaca tacatctcaa ttggtctagg tgattttaat cactatacca
     9001 attgagatgg gctagtcaat gataattact agtccttttc ctttgagttg tgggtatctg
     9061 taaattctgc tagacctttg ctggaaaact tgtaaattct gctagaccct ctgtaaattc
     9121 cgctagacct ttgtgtgttt tttttgttta tattcaagtg gttataattt atagaataaa
     9181 gaaagaataa aaaaagataa aaagaataga tcccagccct gtgtataact cactacttta
     9241 gtcagttccg cagtattaca aaaggatgtc gcaaacgctg tttgctcctc tacaaaacag
     9301 accttaaaac cctaaaggct taagtagcac cctcgcaagc tcgggcaaat cgctgaatat
     9361 tccttttgtc tccgaccatc aggcacctga gtcgctgtct ttttcgtgac attcagttcg
     9421 ctgcgctcac ggctctggca gtgaatgggg gtaaatggca ctacaggcgc cttttatgga
     9481 ttcatgcaag gaaactaccc ataatacaag aaaagcccgt cacgggcttc tcagggcgtt
     9541 ttatggcggg tctgctatgt ggtgctatct gactttttgc tgttcagcag ttcctgccct
     9601 ctgattttcc agtctgacca cttcggatta tcccgtgaca ggtcattcag actggctaat
     9661 gcacccagta aggcagcggt atcatcaacc cactgcagat gagctcagcc aatcgactgg
     9721 cgagcggcat cgcattcttc gcatcccgcc tctggcggat gcaggaagat caacggatct
     9781 cggcccagtt gacccagggc tgtcgccaca atgtcgcggg agcggatcaa ccgagcaaag
     9841 gcatgaccga ctggaccttc cttctgaagg ctcttctcct tgagccacct gtccgccaag
     9901 gcaaagcgct cacagcagtg gtcattctcg agataatcga cgcgtaccaa cttgccatcc
     9961 tgaagaatgg tgcagtgtct cggcacccca tagggaacct ttgccatcaa ctcggcaaga
    10021 tgcagcgtcg tgttggcatc gtgtcccacg ccgaggagaa gtacctgccc atcgagttca
    10081 tggacacggg cgaccgggct tgcaggcgag tgaggtggca ggggcaatgg atcagagatg
    10141 atctgctctg cctgtggccc cgctgccgca aaggcaaatg gatgggcgct gcgctttaca
    10201 tttggcaggc gccagaatgt gtcagagaca actccaaggt ccggtgtaac gggcgacgtg
    10261 gcaggatcga acggctcgtc gtccagacct gaccacgagg gcatgacgag cgtccctccc
    10321 ggacccagcg cagcacgcag ggcctcgatc agtccaagtg gcccatcttc gaggggccgg
    10381 acgctacgga aggagctgtg gaccagcagc acaccgccgg gggtaacccc aaggttgaga
    10441 agctgaccga tgagctcggc ttttcgccat tcgtattgca cgacattgca ctccaccgct
    10501 gatgacatca gtcgatcata gcacgatcaa cggcactgtt gcaaatagtc ggtggtgata
    10561 aacttatcat ccccttttgc ttatggagct gcacatgaac ccattcaaag gccggcattt
    10621 tcagcgtgac atcattctgt gggccgtacg ctggtactgc aaatacggca tcagttaccg
    10681 tgagctgcat tttccgctgc ataaccctgc ttcggggtca ttatagcgat tttttcggta
    10741 tatccatcct ttttcgcacg atatacagga ttttgccaaa gggttcgtgt agactttcct
    10801 tggtgtatcc aacggcgtca gcctgcagct gaacaatact taatactata gaatgataac
    10861 aaaataaact actttttaaa agaattttgt gttataatct atttattatt aagtattggg
    10921 taatattttt tgaagagata ttttgaaaaa gaaaaattaa agcatattaa actaatttcg
    10981 gaggtcatta aaactattat tgaaatcatc aaactcatta tggatttaat ttaaactttt
    11041 tattttagga ggcaaaaatg gataagaaat actcaatagg cttagatatc ggcacaaata
    11101 gcgtcggatg ggcggtgatc actgatgatt ataaggttcc gtctaaaaag ttcaaggttc
    11161 tgggaaatac agaccgccac agtatcaaaa aaaatcttat aggggctctt ttatttgaca
    11221 gtggagagac agcggaagcg actcgtctca aacggacagc tcgtagaagg tatacacgtc
    11281 ggaagaatcg tatttgttat ctacaggaga ttttttcaaa tgagatggcg aaagtagatg
    11341 atagtttctt tcatcgactt gaagagtctt ttttggtgga agaagacaag aagcatgaac
    11401 gtcatcctat ttttggaaat atagtagatg aagttgctta tcatgagaaa tatccaacta
    11461 tctatcatct gcgaaaaaaa ttggtagatt ctactgataa agcggatttg cgcttaatct
    11521 atttggcctt agcgcatatg attaagtttc gtggtcattt tttgattgag ggagatttaa
    11581 atcctgataa tagtgatgtg gacaaactat ttatccagtt ggtacaaacc tacaatcaat
    11641 tatttgaaga aaaccctatt aacgcaagtg gagtagatgc taaagcgatt ctttctgcac
    11701 gattgagtaa atcaagacga ttagaaaatc tcattgctca gctccccggt gagaagaaaa
    11761 atggcttatt tgggaatctc attgctttgt cattgggttt gacccctaat tttaaatcaa
    11821 attttgattt ggcagaagat gctaaattac agctttcaaa agatacttac gatgatgatt
    11881 tagataattt attggcgcaa attggagatc aatatgctga tttgtttttg gcagctaaga
    11941 atttatcaga tgctatttta ctttcagata tcctaagagt aaatactgaa ataactaagg
    12001 ctcccctatc agcttcaatg attaaacgct acgatgaaca tcatcaagac ttgactcttt
    12061 taaaagcttt agttcgacaa caacttccag aaaagtataa agaaatcttt tttgatcaat
    12121 caaaaaacgg atatgcaggt tatattgatg ggggagctag ccaagaagaa ttttataaat
    12181 ttatcaaacc aattttagaa aaaatggatg gtactgagga attattggtg aaactaaatc
    12241 gtgaagattt gctgcgcaag caacggacct ttgacaacgg ctctattccc catcaaattc
    12301 acttgggtga gctgcatgct attttgagaa gacaagaaga cttttatcca tttttaaaag
    12361 acaatcgtga gaagattgaa aaaatcttga cttttcgaat tccttattat gttggtccat
    12421 tggcgcgtgg caatagtcgt tttgcatgga tgactcggaa gtctgaagaa acaattaccc
    12481 catggaattt tgaagaagtt gtcgataaag gtgcttcagc tcaatcattt attgaacgca
    12541 tgacaaactt tgataaaaat cttccaaatg aaaaagtact accaaaacat agtttgcttt
    12601 atgagtattt tacggtttat aacgaattga caaaggtcaa atatgttact gaaggaatgc
    12661 gaaaaccagc atttctttca ggtgaacaga agaaagccat tgttgattta ctcttcaaaa
    12721 caaatcgaaa agtaaccgtt aagcaattaa aagaagatta tttcaaaaaa atagaatgtt
    12781 ttgatagtgt tgaaatttca ggagttgaag atagatttaa tgcttcatta ggtacctacc
    12841 atgatttgct aaaaattatt aaagataaag attttttgga taatgaagaa aatgaagata
    12901 tcttagagga tattgtttta acattgacct tatttgaaga tagggagatg attgaggaaa
    12961 gacttaaaac atatgctcac ctctttgatg ataaggtgat gaaacagctt aaacgtcgcc
    13021 gttatactgg ttggggacgt ttgtctcgaa aattgattaa tggtattagg gataagcaat
    13081 ctggcaaaac aatattagat tttttgaaat cagatggttt tgccaatcgc aattttatgc
    13141 agctgatcca tgatgatagt ttgacattta aagaagacat tcaaaaagca caagtgtctg
    13201 gacaaggcga tagtttacat gaacatattg caaatttagc tggtagccct gctattaaaa
    13261 aaggtatttt acagactgta aaagttgttg atgaattggt caaagtaatg gggcggcata
    13321 agccagaaaa tatcgttatt gaaatggcac gtgaaaatca gacaactcaa aagggccaga
    13381 aaaattcgcg agagcgtatg aaacgaatcg aagaaggtat caaagaatta ggaagtcaga
    13441 ttcttaaaga gcatcctgtt gaaaatactc aattgcaaaa tgaaaagctc tatctctatt
    13501 atctccaaaa tggaagagac atgtatgtgg accaagaatt agatattaat cgtttaagtg
    13561 attatgatgt cgatcacatt gttccacaaa gtttccttaa agacgattca atagacaata
    13621 aggtcttaac gcgttctgat aaaaatcgtg gtaaatcgga taacgttcca agtgaagaag
    13681 tagtcaaaaa gatgaaaaac tattggagac aacttctaaa cgccaagtta atcactcaac
    13741 gtaagtttga taatttaacg aaagctgaac gtggaggttt gagtgaactt gataaagctg
    13801 gttttatcaa acgccaattg gttgaaactc gccaaatcac taagcatgtg gcacaaattt
    13861 tggatagtcg catgaatact aaatacgatg aaaatgataa acttattcga gaggttaaag
    13921 tgattacctt aaaatctaaa ttagtttctg acttccgaaa agatttccaa ttctataaag
    13981 tacgtgagat taacaattac catcatgccc atgatgcgta tctaaatgcc gtcgttggaa
    14041 ctgctttgat taagaaatat ccaaaacttg aatcggagtt tgtctatggt gattataaag
    14101 tttatgatgt tcgtaaaatg attgctaagt ctgagcaaga aataggcaaa gcaaccgcaa
    14161 aatatttctt ttactctaat atcatgaact tcttcaaaac agaaattaca cttgcaaatg
    14221 gagagattcg caaacgccct ctaatcgaaa ctaatgggga aactggagaa attgtctggg
    14281 ataaagggcg agattttgcc acagtgcgca aagtattgtc catgccccaa gtcaatattg
    14341 tcaagaaaac agaagtacag acaggcggat tctccaagga gtcaatttta ccaaaaagaa
    14401 attcggacaa gcttattgct cgtaaaaaag actgggatcc aaaaaaatat ggtggttttg
    14461 atagtccaac ggtagcttat tcagtcctag tggttgctaa ggtggaaaaa gggaaatcga
    14521 agaagttaaa atccgttaaa gagttactag ggatcacaat tatggaaaga agttcctttg
    14581 aaaaaaatcc gattgacttt ttagaagcta aaggatataa ggaagttaaa aaagacttaa
    14641 tcattaaact acctaaatat agtctttttg agttagaaaa cggtcgtaaa cggatgctgg
    14701 ctagtgccgg agaattacaa aaaggaaatg agctggctct gccaagcaaa tatgtgaatt
    14761 tttt
//