pCasPA

LOCUS       pCasPA              17571 bp ds-DNA     circular SYN 12-MAY-2021
DEFINITION  Bacterial expression of Cas9 nuclease and lambda-Red system in 
            Pseudomonas aeruginosa.
ACCESSION   .
VERSION     .
KEYWORDS    pCasPA
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 17571)
  AUTHORS   Chen W, Zhang Y, Zhang Y, Pi Y, Gu T, Song L, Wang Y, Ji Q
  TITLE     CRISPR/Cas9-based Genome Editing in Pseudomonas aeruginosa and 
            Cytidine Deaminase-Mediated Base Editing in Pseudomonas Species.
  JOURNAL   iScience. 2018 Aug 31;6:222-231. doi: 10.1016/j.isci.2018.07.024. 
            Epub 2018 Aug 1.
  PUBMED    30240613
REFERENCE   2  (bases 1 to 17571)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 12, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..17571
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     rep_origin      11..642
                     /label=oriV
                     /note="incP origin of replication"
     primer_bind     complement(2109..2128)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2319..2340
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2603..2625
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     2617..2634
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     2618..2634
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     CDS             complement(2742..3620)
                     /codon_start=1
                     /gene="araC"
                     /product="L-arabinose regulatory protein"
                     /label=araC
                     /translation="MAEAQNDPLLPGYSFNAHLVAGLTPIEANGYLDFFIDRPLGMKGY
                     ILNLTIRGQGVVKNQGREFVCRPGDILLFPPGEIHHYGRHPEAREWYHQWVYFRPRAYW
                     HEWLNWPSIFANTGFFRPDEAHQPHFSDLFGQIINAGQGEGRYSELLAINLLEQLLLRR
                     MEAINESLHPPMDNRVREACQYISDHLADSNFDIASVAQHVCLSPSRLSHLFRQQLGIS
                     VLSWREDQRISQAKLLLSTTRMPIATVGRNVGFDDQLYFSRVFKKCTGASPSEFRAGCE
                     EKVNDVAVKLS"
     promoter        3647..3931
                     /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)"
     RBS             3958..3966
                     /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             3975..4391
                     /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             4397..5182
                     /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             5179..5859
                     /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      5860..6104
                     /label=-lambda tL3 terminator
                     /note="transcription terminator tL3 from phage lambda"
     regulatory      6120..6129
                     /regulatory_class="other"
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
     primer_bind     6145..6164
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     CDS             complement(6203..7624)
                     /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"
     promoter        8030..8314
                     /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)"
     RBS             8341..8349
                     /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             8358..12464
                     /codon_start=1
                     /product="Cas9 (Csn1) endonuclease from the Streptococcus 
                     pyogenes Type II CRISPR/Cas system"
                     /label=Cas9
                     /note="generates RNA-guided double strand breaks in DNA"
                     /translation="MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK
                     NLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES
                     FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK
                     FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL
                     ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ
                     IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR
                     QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL
                     RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG
                     NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY
                     FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD
                     SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER
                     LKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFM
                     QLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR
                     HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY
                     LYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVP
                     SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH
                     VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL
                     NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT
                     EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK
                     ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI
                     TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE
                     LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD
                     ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL
                     DATLIHQSITGLYETRIDLSQLGGD"
     primer_bind     complement(12519..12538)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(12520..12538)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(12552..12568)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(12552..12568)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(12565..12587)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    12576..12592
                     /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(12600..12630)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    12645..12666
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     CDS             13024..14223
                     /codon_start=1
                     /gene="tetA"
                     /product="tetracycline efflux protein"
                     /label=TcR
                     /note="confers resistance to tetracycline"
                     /translation="MKPNIPLIVILSTVALDAVGIGLIMPVLPGLLRDLVHSNDVTAHY
                     GILLALYALVQFACAPVLGALSDRFGRRPILLVSLAGATVDYAIMATAPFLWVLYIGRI
                     VAGITGATGAVAGAYIADITDGDERARHFGFMSACFGFGMVAGPVLGGLMGGFSPHAPF
                     FAAAALNGLNFLTGCFLLPESHKGERRPLRREALNPLASFRWARGMTVVAALMAVFFIM
                     QLVGQVPAALWVIFGEDRFHWDATTIGISLAAFGILHSLAQAMITGPVAARLGERRALM
                     LGMIADGTGYILLAFATRGWMAFPIMVLLASGGIGMPALQAMLSRQVDEERQGQLQGSL
                     AALTSLTSIVGPLLFTAIYAASITTWNGWAWIAGAALYLLCLPALRRGLWSGAGQRADR
                     "
     CDS             complement(15496..16644)
                     /codon_start=1
                     /product="trans-acting replication protein that binds to 
                     and activates oriV"
                     /label=trfA
                     /translation="MNRTFDRKAYRQELIDAGFSAEDAETIASRTVMRAPRETFQSVGS
                     MVQQATAKIERDSVQLAPPALPAPSAAVERSRRLEQEAAGLAKSMTIDTRGTMTTKKRK
                     TAGEDLAKQVSEAKQAALLKHTKQQIKEMQLSLFDIAPWPDTMRAMPNDTARSALFTTR
                     NKKIPREALQNKVIFHVNKDVKITYTGVELRADDDELVWQQVLEYAKRTPIGEPITFTF
                     YELCQDLGWSINGRYYTKAEECLSRLQATAMGFTSDRVGHLESVSLLHRFRVLDRGKKT
                     SRCQVLIDEEIVVLFAGDHYTKFIWEKYRKLSPTARRMFDYFSSHREPYPLKLETFRLM
                     CGSDSTRVKKWREQVGEACEELRGSGLVEHAWVNDDLVHCKR"
     CDS             complement(16916..17287)
                     /codon_start=1
                     /gene="traJ"
                     /product="oriT-recognizing protein"
                     /label=traJ
                     /translation="MADETKPTRKGSPPIKVYCLPDERRAIEEKAAAAGMSLSAYLLAV
                     GQGYKITGVVDYEHVRELARINGDLGRLGGLLKLWLTDDPRTARFGDATILALLAKIEE
                     KQDELGKVMMGVVRPRAEP"
     oriT            17320..17429
                     /note="incP origin of transfer"
ORIGIN
        1 gaccctttcc gacgctcacc gggctggttg ccctcgccgc tgggctggcg gccgtctatg
       61 gccctgcaaa cgcgccagaa acgccgtcga agccgtgtgc gagacaccgc ggccgccggc
      121 gttgtggata cctcgcggaa aacttggccc tcactgacag atgaggggcg gacgttgaca
      181 cttgaggggc cgactcaccc ggcgcggcgt tgacagatga ggggcaggct cgatttcggc
      241 cggcgacgtg gagctggcca gcctcgcaaa tcggcgaaaa cgcctgattt tacgcgagtt
      301 tcccacagat gatgtggaca agcctgggga taagtgccct gcggtattga cacttgaggg
      361 gcgcgactac tgacagatga ggggcgcgat ccttgacact tgaggggcag agtgctgaca
      421 gatgaggggc gcacctattg acatttgagg ggctgtccac aggcagaaaa tccagcattt
      481 gcaagggttt ccgcccgttt ttcggccacc gctaacctgt cttttaacct gcttttaaac
      541 caatatttat aaaccttgtt tttaaccagg gctgcgccct gtgcgcgtga ccgcgcacgc
      601 cgaagggggg tgccccccct tctcgaaccc tcccggcccg ctaacgcggg cctcccatcc
      661 ccccaggggc tgcgcccctc ggccgcgaac ggcctcaccc caaaaatggc aggacaactg
      721 gtgagtctgg atctgaagtt cattgttgtc atggatgatc tggaccgact ggagccatcc
      781 caggtggcgg aagtgttcag gcttgtgcgt gcagtagccg atctgccccg ctttacccat
      841 attctctgtt atgacaggca gattatcact catgccgttg aacatgcgct gaatatcgaa
      901 gatggcagcc gttatctcca gaaaatcatt cagcttagtt ttaaattacc ccgacctgaa
      961 gcctttgatt tacgtaatga atttcgccag cgggctgagg ctctatatca gcaaattaat
     1021 aatcaaccgc cagactctgg aatggtaagg gatctcatcg cggtgactga tacctatggt
     1081 gccgcacttt cgacgccacg ggaaatccat caggccatta attccctgat ttttctttat
     1141 ccggggatgc gggattttgt ttatttccct gatttgtgcc tgcttcagct tatacgggtg
     1201 acaaacccgg ctctgtatga ctggacagag cattacctga cagaacggtc cgtgattgaa
     1261 accggtcagg gtatgctttc tgacggagag aaagcagact tccgggaggg gcttatcaga
     1321 tgtatgaaga cgttcagggc atcaaatgca gactcgtttc tgacacttgc agactggatc
     1381 tatctcatct gcgcaaggca gaacgtgaag acggccgccc tggacctcgc ccgcgagcgc
     1441 caggcgcacg aggccggcgc gcggacccgc gccacggccc acgagcggac gccgcagcag
     1501 gagcgccaga aggccgccag agaggccgag cgcggccgtg aggcttggac gctagggcag
     1561 ggcatgaaaa agcccgtagc gggctgctac gggcgtctga cgcggtggaa agggggaggg
     1621 gatgttgtct acatggctct gctgtagtga gtgggttgcg ctccggcagc ggtcctgatc
     1681 aatcgtcacc ctttctcggt ccttcaacgt tcctgacaac gagcctcctt ttcgccaatc
     1741 catcgacaat caccgcgagt ccctgctcga acgctgcgtc cggaccggct tcgtcgaagg
     1801 cgtctatcgc ggcccgcaac agcggcgaga gcggagcctg ttcaacggtg ccgccgcgct
     1861 cgccggcatc gctgtcgccg gcctgctcct caagcacggc cccaacagtg aagtagctga
     1921 ttgtcatcag cgcattgacg gcgtccccgg ccgaaaaacc cgcctcgcag aggaagcgaa
     1981 gctgcgcgtc ggccgtttcc atctgcggtg cgcccggtcg cgtgccggca tggatgcgcg
     2041 cgccatcgcg gtaggcgagc agcgcctgcc tgaagctgcg ggcattcccg atcagaaatg
     2101 agcgcccgct cctttcgctt tcttcccttc ctttctcgcc acgttcgccg gctttccccg
     2161 tcaagctcta aatcgggggc tccctttagg gttccgattt agtgctttac ggcacctcga
     2221 ccccaaaaaa cttgattagg gtgatggttc acgtagtggg ccatcgccct gatagacggt
     2281 ttttcgccct ttgacgttgg agtccacgtt ctttaatagt ggactcttgt tccaaactgg
     2341 aacaacactc aaccctatct cggtctattc ttttgattta taagggattt tgccgatttc
     2401 ggcctattgg ttaaaaaatg agctgattta acaaaaattt aacgcgaatt ttaacaaaat
     2461 attaacgctt acaatttcca ttcgccattc aggctgcgca actgttggga agggcgatcg
     2521 gtgcgggcct cttcgctatt acgccagctg gcgaaagggg gatgtgctgc aaggcgatta
     2581 agttgggtaa cgccagggtt ttcccagtca cgacgttgta aaacgacggc cagtgaattc
     2641 gagctcggta ccctttcctg cgttgtcgac tgtatttaga aaaataaaca aataggggtt
     2701 ccgcgcacat ttccccgaaa agtgccacct gcatcgattt attatgacaa cttgacggct
     2761 acatcattca ctttttcttc acaaccggca cggaactcgc tcgggctggc cccggtgcat
     2821 tttttaaata cccgcgagaa gtagagttga tcgtcaaaac caacattgcg accgacggtg
     2881 gcgataggca tccgggtggt gctcaaaagc agcttcgcct ggctgatacg ttggtcctcg
     2941 cgccagctta agacgctaat ccctaactgc tggcggaaaa gatgtgacag acgcgacggc
     3001 gacaagcaaa catgctgtgc gacgctggcg atatcaaaat tgctgtctgc caggtgatcg
     3061 ctgatgtact gacaagcctc gcgtacccga ttatccatcg gtggatggag cgactcgtta
     3121 atcgcttcca tgcgccgcag taacaattgc tcaagcagat ttatcgccag cagctccgaa
     3181 tagcgccctt ccccttgccc ggcgttaatg atttgcccaa acaggtcgct gaaatgcggc
     3241 tggtgcgctt catccgggcg aaagaacccc gtattggcaa atattgacgg ccagttaagc
     3301 cattcatgcc agtaggcgcg cggacgaaag taaacccact ggtgatacca ttcgcgagcc
     3361 tccggatgac gaccgtagtg atgaatctct cctggcggga acagcaaaat atcacccggt
     3421 cggcaaacaa attctcgtcc ctgatttttc accaccccct gaccgcgaat ggtgagattg
     3481 agaatataac ctttcattcc cagcggtcgg tcgataaaaa aatcgagata accgttggcc
     3541 tcaatcggcg ttaaacccgc caccagatgg gcattaaacg agtatcccgg cagcagggga
     3601 tcattttgcg cttcagccat acttttcata ctcccgccat tcagagaaga aaccaattgt
     3661 ccatattgca tcagacattg ccgtcactgc gtcttttact ggctcttctc gctaaccaaa
     3721 ccggtaaccc cgcttattaa aagcattctg taacaaagcg ggaccaaagc catgacaaaa
     3781 acgcgtaaca aaagtgtcta taatcacggc agaaaagtcc acattgatta tttgcacggc
     3841 gtcacacttt gctatgccat agcattttta tccataagat tagcggatcc tacctgacgc
     3901 tttttatcgc aactctctac tgtttctcca tacccgtttt tttgggaatt cgagctctaa
     3961 ggaggttata aaaaatggat attaatactg aaactgagat caagcaaaag cattcactaa
     4021 ccccctttcc tgttttccta atcagcccgg catttcgcgg gcgatatttt cacagctatt
     4081 tcaggagttc agccatgaac gcttattaca ttcaggatcg tcttgaggct cagagctggg
     4141 cgcgtcacta ccagcagctc gcccgtgaag agaaagaggc agaactggca gacgacatgg
     4201 aaaaaggcct gccccagcac ctgtttgaat cgctatgcat cgatcatttg caacgccacg
     4261 gggccagcaa aaaatccatt acccgtgcgt ttgatgacga tgttgagttt caggagcgca
     4321 tggcagaaca catccggtac atggttgaaa ccattgctca ccaccaggtt gatattgatt
     4381 cagaggtata aaacgaatga gtactgcact cgcaacgctg gctgggaagc tggctgaacg
     4441 tgtcggcatg gattctgtcg acccacagga actgatcacc actcttcgcc agacggcatt
     4501 taaaggtgat gccagcgatg cgcagttcat cgcattactg atcgttgcca accagtacgg
     4561 ccttaatccg tggacgaaag aaatttacgc ctttcctgat aagcagaatg gcatcgttcc
     4621 ggtggtgggc gttgatggct ggtcccgcat catcaatgaa aaccagcagt ttgatggcat
     4681 ggactttgag caggacaatg aatcctgtac atgccggatt taccgcaagg accgtaatca
     4741 tccgatctgc gttaccgaat ggatggatga atgccgccgc gaaccattca aaactcgcga
     4801 aggcagagaa atcacggggc cgtggcagtc gcatcccaaa cggatgttac gtcataaagc
     4861 catgattcag tgtgcccgtc tggccttcgg atttgctggt atctatgaca aggatgaagc
     4921 cgagcgcatt gtcgaaaata ctgcatacac tgcagaacgt cagccggaac gcgacatcac
     4981 tccggttaac gatgaaacca tgcaggagat taacactctg ctgatcgccc tggataaaac
     5041 atgggatgac gacttattgc cgctctgttc ccagatattt cgccgcgaca ttcgtgcatc
     5101 gtcagaactg acacaggccg aagcagtaaa agctcttgga ttcctgaaac agaaagccgc
     5161 agagcagaag gtggcagcat gacaccggac attatcctgc agcgtaccgg gatcgatgtg
     5221 agagctgtcg aacaggggga tgatgcgtgg cacaaattac ggctcggcgt catcaccgct
     5281 tcagaagttc acaacgtgat agcaaaaccc cgctccggaa agaagtggcc tgacatgaaa
     5341 atgtcctact tccacaccct gcttgctgag gtttgcaccg gtgtggctcc ggaagttaac
     5401 gctaaagcac tggcctgggg aaaacagtac gagaacgacg ccagaaccct gtttgaattc
     5461 acttccggcg tgaatgttac tgaatccccg atcatctatc gcgacgaaag tatgcgtacc
     5521 gcctgctctc ccgatggttt atgcagtgac ggcaacggcc ttgaactgaa atgcccgttt
     5581 acctcccggg atttcatgaa gttccggctc ggtggtttcg aggccataaa gtcagcttac
     5641 atggcccagg tgcagtacag catgtgggtg acgcgaaaaa atgcctggta ctttgccaac
     5701 tatgacccgc gtatgaagcg tgaaggcctg cattatgtcg tgattgagcg ggatgaaaag
     5761 tacatggcga gttttgacga gatcgtgccg gagttcatcg aaaaaatgga cgaggcactg
     5821 gctgaaattg gttttgtatt tggggagcaa tggcgatgac gcatcctcac gataatatcc
     5881 gggtaggcgc aatcactttc gtctactccg ttacaaagcg aggctgggta tttcccggcc
     5941 tttctgttat ccgaaatcca ctgaaagcac agcggctggc tgaggagata aataataaac
     6001 gaggggctgt atgcacaaag catcttctgt tgagttaaga acgagtatcg agatggcaca
     6061 tagccttgct caaattggaa tcaggtttgt gccaatacca gtagaaacag acgaagaatc
     6121 catggcggcc gctgtggatc tagacggcat cagagcagat tgtactgaga gtgcaccata
     6181 atcggcattt tcttttgcgt ttttatttgt taactgttaa ttgtccttgt tcaaggatgc
     6241 tgtctttgac aacagatgtt ttcttgcctt tgatgttcag caggaagcta ggcgcaaacg
     6301 ttgattgttt gtctgcgtag aatcctctgt ttgtcatata gcttgtaatc acgacattgt
     6361 ttcctttcgc ttgaggtaca gcgaagtgtg agtaagtaaa ggttacatcg ttaggatcaa
     6421 gatccatttt taacacaagg ccagttttgt tcagcggctt gtatgggcca gttaaagaat
     6481 tagaaacata accaagcatg taaatatcgt tagacgtaat gccgtcaatc gtcatttttg
     6541 atccgcggga gtcagtgaac agataccatt tgccgttcat tttaaagacg ttcgcgcgtt
     6601 caatttcatc tgttactgtg ttagatgcaa tcagcggttt catcactttt ttcagtgtgt
     6661 aatcatcgtt tagctcaatc ataccgagag cgccgtttgc taactcagcc gtgcgttttt
     6721 tatcgctttg cagaagtttt tgactttctt gacggaagaa tgatgtgctt ttgccatagt
     6781 atgctttgtt aaataaagat tcttcgcctt ggtagccatc ttcagttcca gtgtttgctt
     6841 caaatactaa gtatttgtgg cctttatctt ctacgtagtg aggatctctc agcgtatggt
     6901 tgtcgcctga gctgtagttg ccttcatcga tgaactgctg tacattttga tacgtttttc
     6961 cgtcaccgtc aaagattgat ttataatcct ctacaccgtt gatgttcaaa gagctgtctg
     7021 atgctgatac gttaacttgt gcagttgtca gtgtttgttt gccgtaatgt ttaccggaga
     7081 aatcagtgta gaataaacgg atttttccgt cagatgtaaa tgtggctgaa cctgaccatt
     7141 cttgtgtttg gtcttttagg atagaatcat ttgcatcgaa tttgtcgctg tctttaaaga
     7201 cgcggccagc gtttttccag ctgtcaatag aagtttcgcc gactttttga tagaacatgt
     7261 aaatcgatgt gtcatccgca tttttaggat ctccggctaa tgcaaagacg atgtggtagc
     7321 cgtgatagtt tgcgacagtg ccgtcagcgt tttgtaatgg ccagctgtcc caaacgtcca
     7381 ggccttttgc agaagagata tttttaattg tggacgaatc gaactcagga acttgatatt
     7441 tttcattttt ttgctgttca gggatttgca gcatatcatg gcgtgtaata tgggaaatgc
     7501 cgtatgtttc cttatatggc ttttggttcg tttctttcgc aaacgcttga gttgcgcctc
     7561 ctgccagcag tgcggtagta aaggttaata ctgttgcttg ttttgcaaac tttttgatgt
     7621 tcatcgttca tgtctccttt tttatgtact gtgttagcgg tctgcttctt ccagccctcc
     7681 tgtttgaaga tggcaagtta gttacgcaca ataaaaaaag acctaaaata tgtaaggggt
     7741 gacgccaaag tatacacttt gccctttaca cattttaggt cttgcctgct ttatcagtaa
     7801 caaacccgcg cgatttactt ttcgacctca ttctattaga ctctcgtttg gattgcaact
     7861 ggtctatttt cctcttttgt ttgatagaaa atcataaaag gatttgcaga ctacgggcct
     7921 aaagaactaa aaaatctatc tgtttctttt cattctctgt attttttata gtttctgttg
     7981 catgggcata aagttgcctc gagacttttc atactcccgc cattcagaga agaaaccaat
     8041 tgtccatatt gcatcagaca ttgccgtcac tgcgtctttt actggctctt ctcgctaacc
     8101 aaaccggtaa ccccgcttat taaaagcatt ctgtaacaaa gcgggaccaa agccatgaca
     8161 aaaacgcgta acaaaagtgt ctataatcac ggcagaaaag tccacattga ttatttgcac
     8221 ggcgtcacac tttgctatgc catagcattt ttatccataa gattagcgga tcctacctga
     8281 cgctttttat cgcaactctc tactgtttct ccatacccgt ttttttggga attcgagctc
     8341 taaggaggtt ataaaaaatg gataagaaat actcaatagg cttagatatc ggcacaaata
     8401 gcgtcggatg ggcggtgatc actgatgaat ataaggttcc gtctaaaaag ttcaaggttc
     8461 tgggaaatac agaccgccac agtatcaaaa aaaatcttat aggggctctt ttatttgaca
     8521 gtggagagac agcggaagcg actcgtctca aacggacagc tcgtagaagg tatacacgtc
     8581 ggaagaatcg tatttgttat ctacaggaga ttttttcaaa tgagatggcg aaagtagatg
     8641 atagtttctt tcatcgactt gaagagtctt ttttggtgga agaagacaag aagcatgaac
     8701 gtcatcctat ttttggaaat atagtagatg aagttgctta tcatgagaaa tatccaacta
     8761 tctatcatct gcgaaaaaaa ttggtagatt ctactgataa agcggatttg cgcttaatct
     8821 atttggcctt agcgcatatg attaagtttc gtggtcattt tttgattgag ggagatttaa
     8881 atcctgataa tagtgatgtg gacaaactat ttatccagtt ggtacaaacc tacaatcaat
     8941 tatttgaaga aaaccctatt aacgcaagtg gagtagatgc taaagcgatt ctttctgcac
     9001 gattgagtaa atcaagacga ttagaaaatc tcattgctca gctccccggt gagaagaaaa
     9061 atggcttatt tgggaatctc attgctttgt cattgggttt gacccctaat tttaaatcaa
     9121 attttgattt ggcagaagat gctaaattac agctttcaaa agatacttac gatgatgatt
     9181 tagataattt attggcgcaa attggagatc aatatgctga tttgtttttg gcagctaaga
     9241 atttatcaga tgctatttta ctttcagata tcctaagagt aaatactgaa ataactaagg
     9301 ctcccctatc agcttcaatg attaaacgct acgatgaaca tcatcaagac ttgactcttt
     9361 taaaagcttt agttcgacaa caacttccag aaaagtataa agaaatcttt tttgatcaat
     9421 caaaaaacgg atatgcaggt tatattgatg ggggagctag ccaagaagaa ttttataaat
     9481 ttatcaaacc aattttagaa aaaatggatg gtactgagga attattggtg aaactaaatc
     9541 gtgaagattt gctgcgcaag caacggacct ttgacaacgg ctctattccc catcaaattc
     9601 acttgggtga gctgcatgct attttgagaa gacaagaaga cttttatcca tttttaaaag
     9661 acaatcgtga gaagattgaa aaaatcttga cttttcgaat tccttattat gttggtccat
     9721 tggcgcgtgg caatagtcgt tttgcatgga tgactcggaa gtctgaagaa acaattaccc
     9781 catggaattt tgaagaagtt gtcgataaag gtgcttcagc tcaatcattt attgaacgca
     9841 tgacaaactt tgataaaaat cttccaaatg aaaaagtact accaaaacat agtttgcttt
     9901 atgagtattt tacggtttat aacgaattga caaaggtcaa atatgttact gaaggaatgc
     9961 gaaaaccagc atttctttca ggtgaacaga agaaagccat tgttgattta ctcttcaaaa
    10021 caaatcgaaa agtaaccgtt aagcaattaa aagaagatta tttcaaaaaa atagaatgtt
    10081 ttgatagtgt tgaaatttca ggagttgaag atagatttaa tgcttcatta ggtacctacc
    10141 atgatttgct aaaaattatt aaagataaag attttttgga taatgaagaa aatgaagata
    10201 tcttagagga tattgtttta acattgacct tatttgaaga tagggagatg attgaggaaa
    10261 gacttaaaac atatgctcac ctctttgatg ataaggtgat gaaacagctt aaacgtcgcc
    10321 gttatactgg ttggggacgt ttgtctcgaa aattgattaa tggtattagg gataagcaat
    10381 ctggcaaaac aatattagat tttttgaaat cagatggttt tgccaatcgc aattttatgc
    10441 agctgatcca tgatgatagt ttgacattta aagaagacat tcaaaaagca caagtgtctg
    10501 gacaaggcga tagtttacat gaacatattg caaatttagc tggtagccct gctattaaaa
    10561 aaggtatttt acagactgta aaagttgttg atgaattggt caaagtaatg gggcggcata
    10621 agccagaaaa tatcgttatt gaaatggcac gtgaaaatca gacaactcaa aagggccaga
    10681 aaaattcgcg agagcgtatg aaacgaatcg aagaaggtat caaagaatta ggaagtcaga
    10741 ttcttaaaga gcatcctgtt gaaaatactc aattgcaaaa tgaaaagctc tatctctatt
    10801 atctccaaaa tggaagagac atgtatgtgg accaagaatt agatattaat cgtttaagtg
    10861 attatgatgt cgatcacatt gttccacaaa gtttccttaa agacgattca atagacaata
    10921 aggtcttaac gcgttctgat aaaaatcgtg gtaaatcgga taacgttcca agtgaagaag
    10981 tagtcaaaaa gatgaaaaac tattggagac aacttctaaa cgccaagtta atcactcaac
    11041 gtaagtttga taatttaacg aaagctgaac gtggaggttt gagtgaactt gataaagctg
    11101 gttttatcaa acgccaattg gttgaaactc gccaaatcac taagcatgtg gcacaaattt
    11161 tggatagtcg catgaatact aaatacgatg aaaatgataa acttattcga gaggttaaag
    11221 tgattacctt aaaatctaaa ttagtttctg acttccgaaa agatttccaa ttctataaag
    11281 tacgtgagat taacaattac catcatgccc atgatgcgta tctaaatgcc gtcgttggaa
    11341 ctgctttgat taagaaatat ccaaaacttg aatcggagtt tgtctatggt gattataaag
    11401 tttatgatgt tcgtaaaatg attgctaagt ctgagcaaga aataggcaaa gcaaccgcaa
    11461 aatatttctt ttactctaat atcatgaact tcttcaaaac agaaattaca cttgcaaatg
    11521 gagagattcg caaacgccct ctaatcgaaa ctaatgggga aactggagaa attgtctggg
    11581 ataaagggcg agattttgcc acagtgcgca aagtattgtc catgccccaa gtcaatattg
    11641 tcaagaaaac agaagtacag acaggcggat tctccaagga gtcaatttta ccaaaaagaa
    11701 attcggacaa gcttattgct cgtaaaaaag actgggatcc aaaaaaatat ggtggttttg
    11761 atagtccaac ggtagcttat tcagtcctag tggttgctaa ggtggaaaaa gggaaatcga
    11821 agaagttaaa atccgttaaa gagttactag ggatcacaat tatggaaaga agttcctttg
    11881 aaaaaaatcc gattgacttt ttagaagcta aaggatataa ggaagttaaa aaagacttaa
    11941 tcattaaact acctaaatat agtctttttg agttagaaaa cggtcgtaaa cggatgctgg
    12001 ctagtgccgg agaattacaa aaaggaaatg agctggctct gccaagcaaa tatgtgaatt
    12061 ttttatattt agctagtcat tatgaaaagt tgaagggtag tccagaagat aacgaacaaa
    12121 aacaattgtt tgtggagcag cataagcatt atttagatga gattattgag caaatcagtg
    12181 aattttctaa gcgtgttatt ttagcagatg ccaatttaga taaagttctt agtgcatata
    12241 acaaacatag agacaaacca atacgtgaac aagcagaaaa tattattcat ttatttacgt
    12301 tgacgaatct tggagctccc gctgctttta aatattttga tacaacaatt gatcgtaaac
    12361 gatatacgtc tacaaaagaa gttttagatg ccactcttat ccatcaatcc atcactggtc
    12421 tttatgaaac acgcattgat ttgagtcagc taggaggtga ctgatggctg gttggcgtac
    12481 tgttgtggta aatacccatg ggtatggaca gaagctttcc ctatagtgag tcgtattaaa
    12541 ttcgtaatca tgtcatagct gtttcctgtg tgaaattgtt atccgctcac aattccacac
    12601 aacatacgag ccggaagcat aaagtgtaaa gcctggggtg cctaatgagt gagctaactc
    12661 acattaattg cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc gtgccagctg
    12721 cattaatgaa tcggccaacg cgcggggaga ggcggtttgc gtattgggcg cccgcttgtt
    12781 cctgaagtgc cagtaaagcg ccggctgctg aacccccaac cgttccgcca gtttgcgtgt
    12841 cgtcagaccg tctacgccga cctcgttcaa caggtccagg gcggcacgga tcactgtatt
    12901 cggctgcaac tttgtcatgc ttgacgcttt atcactgata aacataatat gtccaccaac
    12961 ttatcagtga taaagaatcc gcgcgttcaa tcggaccagc ggaggctggt ccggaggcca
    13021 gacgtgaaac ccaacatacc cctgatcgta attctgagca ctgtcgcgct cgacgctgtc
    13081 ggcatcggcc tgattatgcc ggtgctgccg ggcctcctgc gcgatctggt tcactcgaac
    13141 gacgtcaccg cccactatgg cattctgctg gcgctgtatg cgttggtgca atttgcctgc
    13201 gcacctgtgc tgggcgcgct gtcggatcgt ttcgggcggc ggccaatctt gctcgtctcg
    13261 ctggccggcg ccactgtcga ctacgccatc atggcgacag cgcctttcct ttgggttctc
    13321 tatatcgggc ggatcgtggc cggcatcacc ggggcgactg gggcggtagc cggcgcttat
    13381 attgccgata tcactgatgg cgatgagcgc gcgcggcact tcggcttcat gagcgcctgt
    13441 ttcgggttcg ggatggtcgc gggacctgtg ctcggtgggc tgatgggcgg tttctccccc
    13501 cacgctccgt tcttcgccgc ggcagccttg aacggcctca atttcctgac gggctgtttc
    13561 cttttgccgg agtcgcacaa aggcgaacgc cggccgttac gccgggaggc tctcaacccg
    13621 ctcgcttcgt tccggtgggc ccggggcatg accgtcgtcg ccgccctgat ggcggtcttc
    13681 ttcatcatgc aacttgtcgg acaggtgccg gccgcgcttt gggtcatttt cggcgaggat
    13741 cgctttcact gggacgcgac cacgatcggc atttcgcttg ccgcatttgg cattctgcat
    13801 tcactcgccc aggcaatgat caccggccct gtagccgccc ggctcggcga aaggcgggca
    13861 ctcatgctcg gaatgattgc cgacggcaca ggctacatcc tgcttgcctt cgcgacacgg
    13921 ggatggatgg cgttcccgat catggtcctg cttgcttcgg gtggcatcgg aatgccggcg
    13981 ctgcaagcaa tgttgtccag gcaggtggat gaggaacgtc aggggcagct gcaaggctca
    14041 ctggcggcgc tcaccagcct gacctcgatc gtcggacccc tcctcttcac ggcgatctat
    14101 gcggcttcta taacaacgtg gaacgggtgg gcatggattg caggcgctgc cctctacttg
    14161 ctctgcctgc cggcgctgcg tcgcgggctt tggagcggcg cagggcaacg agccgatcgc
    14221 tgatcgtgga aacgataggc ctatgccatg cgggtcaagg cgacttccgg caagctatac
    14281 gcgccctagg agtgcggttg gaacgttggc ccagccagat actcccgatc acgagcagga
    14341 cgccgatgat ttgaagcgca ctcagcgtct gatccaagaa caaccatcct agcaacacgg
    14401 cggtccccgg gctgagaaag cccagtaagg aaacaactgt aggttcgagt cgcgagatcc
    14461 cccggaacca aaggaagtag gttaaacccg ctccgatcag gccgagccac gccaggccga
    14521 gaacattggt tcctgtaggc atcgggattg gcggatcaaa cactaaagct actggaacga
    14581 gcagaagtcc tccggccgcc agttgccagg cggtaaaggt gagcagaggc acgggaggtt
    14641 gccacttgcg ggtcagcacg gttccgaacg ccatggaaac cgcccccgcc aggcccgctg
    14701 cgacgccgac aggatctagc gctgcgtttg gtgtcaacac caacagcgcc acgcccgcag
    14761 ttccgcaaat agcccccagg accgccatca atcgtatcgg gctacctagc agagcggcag
    14821 agatgaacac gaccatcagc ggctgcacag cgcctaccgt cgccgcgacc ccgcccggca
    14881 ggcggtagac cgaaataaac aacaagctcc agaatagcga aatattaagt gcgccgagga
    14941 tgaagatgcg catccaccag attcccgttg gaatctgtcg gacgatcatc acgagcaata
    15001 aacccgccgg caacgcccgc agcagcatac cggcgacccc tcggcctcgc tgttcgggct
    15061 ccacgaaaac gccggacaga tgcgccttgt gagcgtcctt ggggccgtcc tcctgtttga
    15121 agaccgacag cccaatgatc tcgccgtcga tgtaggcgcc gaatgccacg gcatctcgca
    15181 accgttcagc gaacgcctcc atgggctttt tctcctcgtg ctcgtaaacg gacccgaaca
    15241 tctctggagc tttcttcagg gccgacaatc ggatctcgcg gaaatcctgc acgtcggccg
    15301 ctccaagccg tcgaatctga gccttaatca caattgtcaa ttttaatcct ctgtttatcg
    15361 gcagttcgta gagcgcgccg tgcgtcccga gcgatactga gcgaagcaag tgcgtcgagc
    15421 agtgcccgct tgttcctgaa atgccagtaa agcgctggct gctgaacccc cagccggaac
    15481 tgaccccaca aggccctagc gtttgcaatg caccaggtca tcattgaccc aggcgtgttc
    15541 caccaggccg ctgcctcgca actcttcgca ggcttcgccg acctgctcgc gccacttctt
    15601 cacgcgggtg gaatccgatc cgcacatgag gcggaaggtt tccagcttga gcgggtacgg
    15661 ctcccggtgc gagctgaaat agtcgaacat ccgtcgggcc gtcggcgaca gcttgcggta
    15721 cttctcccat atgaatttcg tgtagtggtc gccagcaaac agcacgacga tttcctcgtc
    15781 gatcaggacc tggcaacggg acgttttctt gccacggtcc aggacgcgga agcggtgcag
    15841 cagcgacacc gattccaggt gcccaacgcg gtcggacgtg aagcccatcg ccgtcgcctg
    15901 taggcgcgac aggcattcct cggccttcgt gtaataccgg ccattgatcg accagcccag
    15961 gtcctggcaa agctcgtaga acgtgaaggt gatcggctcg ccgatagggg tgcgcttcgc
    16021 gtactccaac acctgctgcc acaccagttc gtcatcgtcg gcccgcagct cgacgccggt
    16081 gtaggtgatc ttcacgtcct tgttgacgtg gaaaatgacc ttgttttgca gcgcctcgcg
    16141 cgggattttc ttgttgcgcg tggtgaacag ggcagagcgg gccgtgtcgt ttggcatcgc
    16201 tcgcatcgtg tccggccacg gcgcaatatc gaacaaggaa agctgcattt ccttgatctg
    16261 ctgcttcgtg tgtttcagca acgcggcctg cttggcctcg ctgacctgtt ttgccaggtc
    16321 ctcgccggcg gtttttcgct tcttggtcgt catagttcct cgcgtgtcga tggtcatcga
    16381 cttcgccaaa cctgccgcct cctgttcgag acgacgcgaa cgctccacgg cggccgatgg
    16441 cgcgggcagg gcagggggag ccagttgcac gctgtcgcgc tcgatcttgg ccgtagcttg
    16501 ctggaccatc gagccgacgg actggaaggt ttcgcggggc gcacgcatga cggtgcggct
    16561 tgcgatggtt tcggcatcct cggcggaaaa ccccgcgtcg atcagttctt gcctgtatgc
    16621 cttccggtca aacgtccgat tcattcaccc tccttgcggg attgccccga ctcacgccgg
    16681 ggcaatgtgc ccttattcct gatttgaccc gcctggtgcc ttggtgtcca gataatccac
    16741 cttatcggca atgaagtcgg tcccgtagac cgtctggccg tccttctcgt acttggtatt
    16801 ccgaatcttg ccctgcacga ataccagctc cgcgaagtcg ctcttcttga tggagcgcat
    16861 ggggacgtgc ttggcaatca cgcgcacccc ccggccgttt tagcggctaa aaaagtcatg
    16921 gctctgccct cgggcggacc acgcccatca tgaccttgcc aagctcgtcc tgcttctctt
    16981 cgatcttcgc cagcagggcg aggatcgtgg catcaccgaa ccgcgccgtg cgcgggtcgt
    17041 cggtgagcca gagtttcagc aggccgccca ggcggcccag gtcgccattg atgcgggcca
    17101 gctcgcggac gtgctcatag tccacgacgc ccgtgatttt gtagccctgg ccgacggcca
    17161 gcaggtaggc cgacaggctc atgccggccg ccgccgcctt ttcctcaatc gctcttcgtt
    17221 cgtctggaag gcagtacacc ttgataggtg ggctgccctt cctggttggc ttggtttcat
    17281 cagccatccg cttgccctca tctgttacgc cggcggtagc cggccagcct cgcagagcag
    17341 gattcccgtt gagcaccgcc aggtgcgaat aagggacagt gaagaaggaa cacccgctcg
    17401 cgggtgggcc tacttcacct atcctgcccg gctgacgccg ttggatacac caaggaaagt
    17461 ctacacgaac cctttggcaa aatcctgtat atcgtgcgaa aaaggatgga tataccgaaa
    17521 aaatcgctat aatgaccccg aagcagggtt atgcagcgga aaagatccgt c
//