LOCUS       sequence-351579-       15392 bp    DNA     circular SYN 15-APR-2025
DEFINITION  Binary expression vector with A.th. codon-optimized Cas9 and sgRNA 
            for miRNA160a.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 15392)
  AUTHORS   Lukan T, Veillet F, Kriznik M, Coll A, Mahkovec Povalej T, Pogacar 
            K, Stare K, Chauvin L, Chauvin JE, Gruden K
  TITLE     CRISPR/Cas9-mediated fine-tuning of miRNA expression in tetraploid 
            potato.
  JOURNAL   Hortic Res. 2022 Jun 30;9:uhac147. doi: 10.1093/hr/uhac147. 
            eCollection 2022.
  PUBMED    36072839
REFERENCE   2  (bases 1 to 15392)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Hortic 
            Res."; date: "2022-06-30"; volume: "9:uhac147. doi"; pages: " 
            10.1093/hr/uhac147. eCollection 2022"
COMMENT     Sequence Label: pDe_Cas9_KANA_R160a
FEATURES             Location/Qualifiers
     source          1..15392
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(108..899)
                     /codon_start=1
                     /gene="aadA"
                     /product="aminoglycoside adenylyltransferase (Murphy, 
                     1985)"
                     /label=SmR
                     /note="confers resistance to spectinomycin and 
                     streptomycin"
                     /translation="MGEAVIAEVSTQLSEVVGVIERHLEPTLLAVHLYGSAVDGGLKPH
                     SDIDLLVTVTVRLDETTRRALINDLLETSASPGESEILRAVEVTIVVHDDIIPWRYPAK
                     RELQFGEWQRNDILAGIFEPATIDIDLAILLTKAREHSVALVGPAAEELFDPVPEQDLF
                     EALNETLTLWNSPPDWAGDERNVVLTLSRIWYSAVTGKIAPKDVAADWAMERLPAQYQP
                     VILEARQAYLGQEEDRLASRADQLEEFVHYVKGEITKVVGK"
     misc_feature    1420..1444
                     /label=LB T-DNA repeat
                     /note="left border repeat from nopaline C58 T-DNA"
     protein_bind    1621..1642
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(1657..1662,1663..1680,1681..1687)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1695..1711
                     /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)."
     primer_bind     1700..1722
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     1719..1735
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     1719..1735
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             2736..6839
                     /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             2736..6839
                     /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             6852..6872
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     protein_bind    7384..7408
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     terminator      7921..7940
                     /label=SUP4 terminator
                     /note="transcription terminator for the S. cerevisiae SUP4 
                     tRNA gene"
     terminator      8381..8400
                     /label=SUP4 terminator
                     /note="transcription terminator for the S. cerevisiae SUP4 
                     tRNA gene"
     protein_bind    complement(8495..8519)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     terminator      complement(8649..8901)
                     /label=NOS terminator
                     /note="nopaline synthase terminator and poly(A) signal"
     CDS             complement(8941..9735)
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(9052..9071)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     9662..9681
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     promoter        complement(9769..9948)
                     /label=NOS promoter
                     /note="nopaline synthase promoter"
     primer_bind     complement(10038..10055)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(10038..10054)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(10047..10069)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     misc_feature    10250..10274
                     /label=RB T-DNA repeat
                     /note="right border repeat from nopaline C58 T-DNA"
     CDS             11605..12234
                     /codon_start=1
                     /product="stability protein from the Pseudomonas plasmid 
                     pVS1 (Heeb et al., 2000)"
                     /label=pVS1 StaA
                     /translation="MKVIAVLNQKGGSGKTTIATHLARALQLAGADVLLVDSDPQGSAR
                     DWAAVREDQPLTVVGIDRPTIDRDVKAIGRRDFVVIDGAPQAADLAVSAIKAADFVLIP
                     VQPSPYDIWATADLVELVKQRIEVTDGRLQAAFVVSRAIKGTRIGGEVAEALAGYELPI
                     LESRITQRVSYPGTAAAGTTVLESEPEGDAAREVQALAAEIKSKLI"
     CDS             12663..13736
                     /codon_start=1
                     /product="replication protein from the Pseudomonas plasmid 
                     pVS1 (Heeb et al., 2000)"
                     /label=pVS1 RepA
                     /translation="MSGRKPSGPVQIGAALGDDLVEKLKAAQAAQRQRIEAEARPGESW
                     QAAADRIRKESRQPPAAGAPSIRKPPKGDEQPDFFVPMLYDVGTRDSRSIMDVAVFRLS
                     KRDRRAGEVIRYELPDGHVEVSAGPAGMASVWDYDLVLMAVSHLTESMNRYREGKGDKP
                     GRVFRPHVADVLKFCRRADGGKQKDDLVETCIRLNTTHVAMQRTKKAKNGRLVTVSEGE
                     ALISRYKIVKSETGRPEYIEIELADWMYREITEGKNPDVLTVHPDYFLIDPGIGRFLYR
                     LARRAAGKAEARWLFKTIYERSGSAGEFKKFCFTVRKLIGSNDLPEYDLKEEAGQAGPI
                     LVMRYRNLIEGEASAGS"
     rep_origin      13802..13996
                     /label=pVS1 oriV
                     /note="origin of replication for the Pseudomonas plasmid 
                     pVS1 (Heeb et al., 2000)"
     primer_bind     complement(14232..14254)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    14340..14480
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     14395..14414
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(14495..14512)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(14666..15254)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(14746..14765)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 ttgacctgat agtttggctg tgagcaatta tgtgcttagt gcatctaacg cttgagttaa
       61 gccgcgccgc gaagcggcgt cggcttgaac gaatttctag ctagacatta tttgccgact
      121 accttggtga tctcgccttt cacgtagtgg acaaattctt ccaactgatc tgcgcgcgag
      181 gccaagcgat cttcttcttg tccaagataa gcctgtctag cttcaagtat gacgggctga
      241 tactgggccg gcaggcgctc cattgcccag tcggcagcga catccttcgg cgcgattttg
      301 ccggttactg cgctgtacca aatgcgggac aacgtaagca ctacatttcg ctcatcgcca
      361 gcccagtcgg gcggcgagtt ccatagcgtt aaggtttcat ttagcgcctc aaatagatcc
      421 tgttcaggaa ccggatcaaa gagttcctcc gccgctggac ctaccaaggc aacgctatgt
      481 tctcttgctt ttgtcagcaa gatagccaga tcaatgtcga tcgtggctgg ctcgaagata
      541 cctgcaagaa tgtcattgcg ctgccattct ccaaattgca gttcgcgctt agctggataa
      601 cgccacggaa tgatgtcgtc gtgcacaaca atggtgactt ctacagcgcg gagaatctcg
      661 ctctctccag gggaagccga agtttccaaa aggtcgttga tcaaagctcg ccgcgttgtt
      721 tcatcaagcc ttacggtcac cgtaaccagc aaatcaatat cactgtgtgg cttcaggccg
      781 ccatccactg cggagccgta caaatgtacg gccagcaacg tcggttcgag atggcgctcg
      841 atgacgccaa ctacctctga tagttgagtc gatacttcgg cgatcaccgc ttcccccatg
      901 atgtttaact ttgttttagg gcgactgccc tgctgcgtaa catcgttgct gctccataac
      961 atcaaacatc gacccacggc gtaacgcgct tgctgcttgg atgcccgagg catagactgt
     1021 accccaaaaa aacagtcata acaagccatg aaaaccgcca ctgcgccgtt accaccgctg
     1081 cgttcggtca aggttctgga ccagttgcgt gagcgcatac gctacttgca ttacagctta
     1141 cgaaccgaac aggcttatgt ccactgggtt cgtgcccgaa ttgatcacag gcagcaacgc
     1201 tctgtcatcg ttacaatcaa catgctaccc tccgcgagat catccgtgtt tcaaacccgg
     1261 cagcttagtt gccgttcttc cgaatagcat cggtaacatg agcaaagtct gccgccttac
     1321 aacggctctc ccgctgacgc cgtcccggac tgatgggctg cctgtatcga gtggtgattt
     1381 tgtgccgagc tgccggtcgg ggagctgttg gctggctggt ggcaggatat attgtggtgt
     1441 aaacaaattg acgcttagac aacttaataa cacattgcgg acgtttttaa tgtactgaat
     1501 taacgccgaa ttgctctagc caatacgcaa accgcctctc cccgcgcgtt ggccgattca
     1561 ttaatgcagc tggcacgaca ggtttcccga ctggaaagcg ggcagtgagc gcaacgcaat
     1621 taatgtgagt tagctcactc attaggcacc ccaggcttta cactttatgc ttccggctcg
     1681 tatgttgtgt ggaattgtga gcggataaca atttcacaca ggaaacagct atgacatgat
     1741 tacgaattca aaaattacgg atatgaatat aggcatatcc gtatccgaat tatccgtttg
     1801 acagctagca acgattgtac aattgcttct ttaaaaaagg aagaaagaaa gaaagaaaag
     1861 aatcaacatc agcgttaaca aacggccccg ttacggccca aacggtcata tagagtaacg
     1921 gcgttaagcg ttgaaagact cctatcgaaa tacgtaaccg caaacgtgtc atagtcagat
     1981 cccctcttcc ttcaccgcct caaacacaaa aataatcttc tacagcctat atatacaacc
     2041 cccccttcta tctctccttt ctcacaattc atcatctttc tttctctacc cccaatttta
     2101 agaaatcctc tcttctcctc ttcattttca aggtaaatct ctctctctct ctctctctct
     2161 gttattcctt gttttaatta ggtatgtatt attgctagtt tgttaatctg cttatcttat
     2221 gtatgcctta tgtgaatatc tttatcttgt tcatctcatc cgtttagaag ctataaattt
     2281 gttgatttga ctgtgtatct acacgtggtt atgtttatat ctaatcagat atgaatttct
     2341 tcatattgtt gcgtttgtgt gtaccaatcc gaaatcgttg atttttttca tttaatcgtg
     2401 tagctaattg tacgtataca tatggatcta cgtatcaatt gttcatctgt ttgtgtttgt
     2461 atgtatacag atctgaaaac atcacttctc tcatctgatt gtgttgttac atacatagat
     2521 atagatctgt tatatcattt tttttattaa ttgtgtatat atatatgtgc atagatctgg
     2581 attacatgat tgtgattatt tacatgattt tgttatttac gtatgtatat atgtagatct
     2641 ggactttttg gagttgttga cttgattgta tttgtgtgtg tatatgtgtg ttctgatctt
     2701 gatatgttat gtatgtgcag cgaattcggc gcgccatgga taagaagtac tctatcggac
     2761 tcgatatcgg aactaactct gtgggatggg ctgtgatcac cgatgagtac aaggtgccat
     2821 ctaagaagtt caaggttctc ggaaacaccg ataggcactc tatcaagaaa aaccttatcg
     2881 gtgctctcct cttcgattct ggtgaaactg ctgaggctac cagactcaag agaaccgcta
     2941 gaagaaggta caccagaaga aagaacagga tctgctacct ccaagagatc ttctctaacg
     3001 agatggctaa agtggatgat tcattcttcc acaggctcga agagtcattc ctcgtggaag
     3061 aagataagaa gcacgagagg caccctatct tcggaaacat cgttgatgag gtggcatacc
     3121 acgagaagta ccctactatc taccacctca gaaagaagct cgttgattct actgataagg
     3181 ctgatctcag gctcatctac ctcgctctcg ctcacatgat caagttcaga ggacacttcc
     3241 tcatcgaggg tgatctcaac cctgataact ctgatgtgga taagttgttc atccagctcg
     3301 tgcagaccta caaccagctt ttcgaagaga accctatcaa cgcttcaggt gtggatgcta
     3361 aggctatcct ctctgctagg ctctctaagt caagaaggct tgagaacctc attgctcagc
     3421 tccctggtga gaagaagaac ggacttttcg gaaacttgat cgctctctct ctcggactca
     3481 cccctaactt caagtctaac ttcgatctcg ctgaggatgc aaagctccag ctctcaaagg
     3541 atacctacga tgatgatctc gataacctcc tcgctcagat cggagatcag tacgctgatt
     3601 tgttcctcgc tgctaagaac ctctctgatg ctatcctcct cagtgatatc ctcagagtga
     3661 acaccgagat caccaaggct ccactctcag cttctatgat caagagatac gatgagcacc
     3721 accaggatct cacacttctc aaggctcttg ttagacagca gctcccagag aagtacaaag
     3781 agattttctt cgatcagtct aagaacggat acgctggtta catcgatggt ggtgcatctc
     3841 aagaagagtt ctacaagttc atcaagccta tcctcgagaa gatggatgga accgaggaac
     3901 tcctcgtgaa gctcaataga gaggatcttc tcagaaagca gaggaccttc gataacggat
     3961 ctatccctca tcagatccac ctcggagagt tgcacgctat ccttagaagg caagaggatt
     4021 tctacccatt cctcaaggat aacagggaaa agattgagaa gattctcacc ttcagaatcc
     4081 cttactacgt gggacctctc gctagaggaa actcaagatt cgcttggatg accagaaagt
     4141 ctgaggaaac catcacccct tggaacttcg aagaggtggt ggataagggt gctagtgctc
     4201 agtctttcat cgagaggatg accaacttcg ataagaacct tccaaacgag aaggtgctcc
     4261 ctaagcactc tttgctctac gagtacttca ccgtgtacaa cgagttgacc aaggttaagt
     4321 acgtgaccga gggaatgagg aagcctgctt ttttgtcagg tgagcaaaag aaggctatcg
     4381 ttgatctctt gttcaagacc aacagaaagg tgaccgtgaa gcagctcaaa gaggattact
     4441 tcaagaaaat cgagtgcttc gattcagttg agatttctgg tgttgaggat aggttcaacg
     4501 catctctcgg aacctaccac gatctcctca agatcattaa ggataaggat ttcttggata
     4561 acgaggaaaa cgaggatatc ttggaggata tcgttcttac cctcaccctc tttgaagata
     4621 gagagatgat tgaagaaagg ctcaagacct acgctcatct cttcgatgat aaggtgatga
     4681 agcagttgaa gagaagaaga tacactggtt ggggaaggct ctcaagaaag ctcattaacg
     4741 gaatcaggga taagcagtct ggaaagacaa tccttgattt cctcaagtct gatggattcg
     4801 ctaacagaaa cttcatgcag ctcatccacg atgattctct cacctttaaa gaggatatcc
     4861 agaaggctca ggtttcagga cagggtgata gtctccatga gcatatcgct aacctcgctg
     4921 gatctcctgc aatcaagaag ggaatcctcc agactgtgaa ggttgtggat gagttggtga
     4981 aggtgatggg aaggcataag cctgagaaca tcgtgatcga aatggctaga gagaaccaga
     5041 ccactcagaa gggacagaag aactctaggg aaaggatgaa gaggatcgag gaaggtatca
     5101 aagagcttgg atctcagatc ctcaaagagc accctgttga gaacactcag ctccagaatg
     5161 agaagctcta cctctactac ctccagaacg gaagggatat gtatgtggat caagagttgg
     5221 atatcaacag gctctctgat tacgatgttg atcatatcgt gccacagtca ttcttgaagg
     5281 atgattctat cgataacaag gtgctcacca ggtctgataa gaacaggggt aagagtgata
     5341 acgtgccaag tgaagaggtt gtgaagaaaa tgaagaacta ttggaggcag ctcctcaacg
     5401 ctaagctcat cactcagaga aagttcgata acttgactaa ggctgagagg ggaggactct
     5461 ctgaattgga taaggcagga ttcatcaaga ggcagcttgt ggaaaccagg cagatcacta
     5521 agcacgttgc acagatcctc gattctagga tgaacaccaa gtacgatgag aacgataagt
     5581 tgatcaggga agtgaaggtt atcaccctca agtcaaagct cgtgtctgat ttcagaaagg
     5641 atttccaatt ctacaaggtg agggaaatca acaactacca ccacgctcac gatgcttacc
     5701 ttaacgctgt tgttggaacc gctctcatca agaagtatcc taagctcgag tcagagttcg
     5761 tgtacggtga ttacaaggtg tacgatgtga ggaagatgat cgctaagtct gagcaagaga
     5821 tcggaaaggc taccgctaag tatttcttct actctaacat catgaatttc ttcaagaccg
     5881 agattaccct cgctaacggt gagatcagaa agaggccact catcgagaca aacggtgaaa
     5941 caggtgagat cgtgtgggat aagggaaggg atttcgctac cgttagaaag gtgctctcta
     6001 tgccacaggt gaacatcgtt aagaaaaccg aggtgcagac cggtggattc tctaaagagt
     6061 ctatcctccc taagaggaac tctgataagc tcattgctag gaagaaggat tgggacccta
     6121 agaaatacgg tggtttcgat tctcctaccg tggcttactc tgttctcgtt gtggctaagg
     6181 ttgagaaggg aaagagtaag aagctcaagt ctgttaagga acttctcgga atcactatca
     6241 tggaaaggtc atctttcgag aagaacccaa tcgatttcct cgaggctaag ggatacaaag
     6301 aggttaagaa ggatctcatc atcaagctcc caaagtactc actcttcgaa ctcgagaacg
     6361 gtagaaagag gatgctcgct tctgctggtg agcttcaaaa gggaaacgag cttgctctcc
     6421 catctaagta cgttaacttt ctttacctcg cttctcacta cgagaagttg aagggatctc
     6481 cagaagataa cgagcagaag caacttttcg ttgagcagca caagcactac ttggatgaga
     6541 tcatcgagca gatctctgag ttctctaaaa gggtgatcct cgctgatgca aacctcgata
     6601 aggtgttgtc tgcttacaac aagcacagag ataagcctat cagggaacag gcagagaaca
     6661 tcatccatct cttcaccctt accaacctcg gtgctcctgc tgctttcaag tacttcgata
     6721 caaccatcga taggaagaga tacacctcta ccaaagaagt gctcgatgct accctcatcc
     6781 atcagtctat cactggactc tacgagacta ggatcgatct ctcacagctc ggtggtgatt
     6841 caagggctga tcctaagaag aagaggaagg tttgaggcgc gccgagctcc aggcctccca
     6901 gctttcgtcc gtatcatcgg tttcgacaac gttcgtcaag ttcaatgcat cagtttcatt
     6961 gcccacacac cagaatccta ctaagtttga gtattatggc attggaaaag ctgttttctt
     7021 ctatcatttg ttctgcttgt aatttactgt gttctttcag tttttgtttt cggacatcaa
     7081 aatgcaaatg gatggataag agttaataaa tgatatggtc cttttgttca ttctcaaatt
     7141 attattatct gttgttttta ctttaatggg ttgaatttaa gtaagaaagg aactaacagt
     7201 gtgatattaa ggtgcaatgt tagacatata aaacagtctt tcacctctct ttggttatgt
     7261 cttgaattgg tttgtttctt cacttatctg tgtaatcaag tttactatga gtctatgatc
     7321 aagtaattat gcaatcaagt taagtacagt ataggcttga gctccctagg cctgttatcc
     7381 ctaacaagtt tgtacaaaaa agcaggctgg atccactagt gagctccttt ttttcttctt
     7441 cttcgttcat acagtttttt tttgtttatc agcttacatt ttcttgaacc gtagctttcg
     7501 ttttcttctt tttaactttc cattcggagt ttttgtatct tgtttcatag tttgtcccag
     7561 gattagaatg attaggcatc gaaccttcaa gaatttgatt gaataaaaca tcttcattct
     7621 taagatatga agataatctt caaaaggccc ctgggaatct gaaagaagag aagcaggccc
     7681 atttatatgg gaaagaacaa tagtatttct tatataggcc catttaagtt gaaaacaatc
     7741 ttcaaaagtc ccacatcgct tagataagaa aacgaagctg agtttatata cagctagagt
     7801 cgaagtagtg attggtcgtg tacacgtata tgccgtttaa gagctatgct ggaaacagca
     7861 tagcaagttt aaataaggct agtccgttat caacttgaaa aagtggcacc gagtcggtgc
     7921 tttttttgtt ttttatgtct agctctagct aacgccggcg gttcagttgc attatgtctt
     7981 tatacacccc tacctagatg attaagtttt actttagttg gtgtgaaatg gataaattct
     8041 aaaatatgag gtgtggaatg aaggattgtc atcaattagt tggccccaac caagtaaaat
     8101 aagaaggccg gcccattaca attaagtcgt cacacaagtg ggcttcattg aaacaagcgc
     8161 aaaaaggagt ccaggcccgt gttagcgtga agactcaacc agcgatttct ccctcatcgg
     8221 ttgcacagaa aagctgtgtg ttgtttatat ggcgaaacct aacagtctga cttggagctt
     8281 tgcaaatggc atacgtttaa gagctatgct ggaaacagca tagcaagttt aaataaggct
     8341 agtccgttat caacttgaaa aagtggcacc gagtcggtgc tttttttgtt ttttatgtct
     8401 ggtgaccata ggacatcggc cgagctgaat agtacgcgtt acgacttatc ggcgcgccga
     8461 attagatcac ctgagggagc tcactagtgg atccacccag ctttcttgta caaagtggtt
     8521 cgataattcc gatccagcct aggcccgggc ctgaggacgc gtccatggtt aattaagacg
     8581 tccggaccga ctagtggatc ctctagagtc gacctgcagg catgcaagct tcagcttgca
     8641 tgccggtcga tctagtaaca tagatgacac cgcgcgcgat aatttatcct agtttgcgcg
     8701 ctatattttg ttttctatcg cgtattaaat gtataattgc gggactctaa tcataaaaac
     8761 ccatctcata aataacgtca tgcattacat gttaattatt acatgcttaa cgtaattcaa
     8821 cagaaattat atgataatca tcgcaagacc ggcaacagga ttcaatctta agaagcttta
     8881 ttgccaaatg tttgaacgat ctgcttgact ctagctagag tccgaacccc agagtcccgc
     8941 tcagaagaac tcgtcaagaa ggcgatagaa ggcgatgcgc tgcgaatcgg gagcggcgat
     9001 accgtaaagc acgaggaagc ggtcagccca ttcgccgcca agctcttcag caatatcacg
     9061 ggtagccaac gctatgtcct gatagcggtc cgccacaccc agccggccac agtcgatgaa
     9121 tccagaaaag cggccatttt ccaccatgat attcggcaag caggcatcgc cgtgggtcac
     9181 gacgagatcc tcgccgtcgg gcatccgcgc cttgagcctg gcgaacagtt cggctggcgc
     9241 gagcccctga tgctcttcgt ccagatcatc ctgatcgaca agaccggctt ccatccgagt
     9301 acgtgctcgc tcgatgcgat gtttcgcttg gtggtcgaat gggcaggtag ccggatcaag
     9361 cgtatgcagc cgccgcattg catcagccat gatggatact ttctcggcag gagcaaggtg
     9421 agatgacagg agatcctgcc ccggcacttc gcccaatagc agccagtccc ttcccgcttc
     9481 agtgacaacg tcgagcacag ctgcgcaagg aacgcccgtc gtggccagcc acgatagccg
     9541 cgctgcctcg tcttggagtt cattcagggc accggacagg tcggtcttga caaaaagaac
     9601 cgggcgcccc tgcgctgaca gccggaacac ggcggcatca gagcagccga ttgtctgttg
     9661 tgcccagtca tagccgaata gcctctccac ccaagcggcc ggagaacctg cgtgcaatcc
     9721 atcttgttca atcatgcctc gatcgagttg agggtgaata tgagactcta attggatacc
     9781 gaggggaatt tatggaacgt cagtggagca tttttgacaa gaaatatttg ctagctgata
     9841 gtgaccttag gcgacttttg aacgcgcaat aatggtttct gacgtatgtg cttagctcat
     9901 taaactccag aaacccgcgg ctcagtggct ccttcaacgt tgcggttctg tcagttccaa
     9961 acgtaaaacg gcttgtcccg cgtcatcggc gggggtcata acgtgactcc cttaattctc
    10021 atgtatgaaa gcttggcact ggccgtcgtt ttacaacgtc gtgactggga aaaccctggc
    10081 gttacccaac ttaatcgcct tgcagcacat ccccctttcg ccagctggcg taatagcgaa
    10141 gaggcccgca ccgatcgccc ttcccaacag ttgcgcagcc tgaatggcga atgagcttga
    10201 gcttggatca gattgtcgtt tcccgccttc agtttaaact atcagtgttt gacaggatat
    10261 attggcgggt aaacctaaga gaaaagagcg tttattagaa taatcggata tttaaaaggg
    10321 cgtgaaaagg tttatccgtt cgtccatttg tatgtgcatg ccaaccacag ggttcccctc
    10381 gggatcaaag tactttaaag tactttaaag tactttaaag tactttgatc caacccctcc
    10441 gctgctatag tgcagtcggc ttctgacgtt cagtgcagcc gtcttctgaa aacgacatgt
    10501 cgcacaagtc ctaagttacg cgacaggctg ccgccctgcc cttttcctgg cgttttcttg
    10561 tcgcgtgttt tagtcgcata aagtagaata cttgcgacta gaaccggaga cattacgcca
    10621 tgaacaagag cgccgccgct ggcctgctgg gctatgcccg cgtcagcacc gacgaccagg
    10681 acttgaccaa ccaacgggcc gaactgcacg cggccggctg caccaagctg ttttccgaga
    10741 agatcaccgg caccaggcgc gaccgcccgg agctggccag gatgcttgac cacctacgcc
    10801 ctggcgacgt tgtgacagtg accaggctag accgcctggc ccgcagcacc cgcgacctac
    10861 tggacattgc cgagcgcatc caggaggccg gcgcgggcct gcgtagcctg gcagagccgt
    10921 gggccgacac caccacgccg gccggccgca tggtgttgac cgtgttcgcc ggcattgccg
    10981 agttcgagcg ttccctaatc atcgaccgca cccggagcgg gcgcgaggcc gccaaggccc
    11041 gaggcgtgaa gtttggcccc cgccctaccc tcaccccggc acagatcgcg cacgcccgcg
    11101 agctgatcga ccaggaaggc cgcaccgtga aagaggcggc tgcactgctt ggcgtgcatc
    11161 gctcgaccct gtaccgcgca cttgagcgca gcgaggaagt gacgcccacc gaggccaggc
    11221 ggcgcggtgc cttccgtgag gacgcattga ccgaggccga cgccctggcg gccgccgaga
    11281 atgaacgcca agaggaacaa gcatgaaacc gcaccaggac ggccaggacg aaccgttttt
    11341 cattaccgaa gagatcgagg cggagatgat cgcggccggg tacgtgttcg agccgcccgc
    11401 gcacgtctca accgtgcggc tgcatgaaat cctggccggt ttgtctgatg ccaagctggc
    11461 ggcctggccg gccagcttgg ccgctgaaga aaccgagcgc cgccgtctaa aaaggtgatg
    11521 tgtatttgag taaaacagct tgcgtcatgc ggtcgctgcg tatatgatgc gatgagtaaa
    11581 taaacaaata cgcaagggga acgcatgaag gttatcgctg tacttaacca gaaaggcggg
    11641 tcaggcaaga cgaccatcgc aacccatcta gcccgcgccc tgcaactcgc cggggccgat
    11701 gttctgttag tcgattccga tccccagggc agtgcccgcg attgggcggc cgtgcgggaa
    11761 gatcaaccgc taaccgttgt cggcatcgac cgcccgacga ttgaccgcga cgtgaaggcc
    11821 atcggccggc gcgacttcgt agtgatcgac ggagcgcccc aggcggcgga cttggctgtg
    11881 tccgcgatca aggcagccga cttcgtgctg attccggtgc agccaagccc ttacgacata
    11941 tgggccaccg ccgacctggt ggagctggtt aagcagcgca ttgaggtcac ggatggaagg
    12001 ctacaagcgg cctttgtcgt gtcgcgggcg atcaaaggca cgcgcatcgg cggtgaggtt
    12061 gccgaggcgc tggccgggta cgagctgccc attcttgagt cccgtatcac gcagcgcgtg
    12121 agctacccag gcactgccgc cgccggcaca accgttcttg aatcagaacc cgagggcgac
    12181 gctgcccgcg aggtccaggc gctggccgct gaaattaaat caaaactcat ttgagttaat
    12241 gaggtaaaga gaaaatgagc aaaagcacaa acacgctaag tgccggccgt ccgagcgcac
    12301 gcagcagcaa ggctgcaacg ttggccagcc tggcagacac gccagccatg aagcgggtca
    12361 actttcagtt gccggcggag gatcacacca agctgaagat gtacgcggta cgccaaggca
    12421 agaccattac cgagctgcta tctgaataca tcgcgcagct accagagtaa atgagcaaat
    12481 gaataaatga gtagatgaat tttagcggct aaaggaggcg gcatggaaaa tcaagaacaa
    12541 ccaggcaccg acgccgtgga atgccccatg tgtggaggaa cgggcggttg gccaggcgta
    12601 agcggctggg ttgtctgccg gccctgcaat ggcactggaa cccccaagcc cgaggaatcg
    12661 gcgtgagcgg tcgcaaacca tccggcccgg tacaaatcgg cgcggcgctg ggtgatgacc
    12721 tggtggagaa gttgaaggcc gcgcaggccg cccagcggca acgcatcgag gcagaagcac
    12781 gccccggtga atcgtggcaa gcggccgctg atcgaatccg caaagaatcc cggcaaccgc
    12841 cggcagccgg tgcgccgtcg attaggaagc cgcccaaggg cgacgagcaa ccagattttt
    12901 tcgttccgat gctctatgac gtgggcaccc gcgatagtcg cagcatcatg gacgtggccg
    12961 ttttccgtct gtcgaagcgt gaccgacgag ctggcgaggt gatccgctac gagcttccag
    13021 acgggcacgt agaggtttcc gcagggccgg ccggcatggc cagtgtgtgg gattacgacc
    13081 tggtactgat ggcggtttcc catctaaccg aatccatgaa ccgataccgg gaagggaagg
    13141 gagacaagcc cggccgcgtg ttccgtccac acgttgcgga cgtactcaag ttctgccggc
    13201 gagccgatgg cggaaagcag aaagacgacc tggtagaaac ctgcattcgg ttaaacacca
    13261 cgcacgttgc catgcagcgt acgaagaagg ccaagaacgg ccgcctggtg acggtatccg
    13321 agggtgaagc cttgattagc cgctacaaga tcgtaaagag cgaaaccggg cggccggagt
    13381 acatcgagat cgagctagct gattggatgt accgcgagat cacagaaggc aagaacccgg
    13441 acgtgctgac ggttcacccc gattactttt tgatcgatcc cggcatcggc cgttttctct
    13501 accgcctggc acgccgcgcc gcaggcaagg cagaagccag atggttgttc aagacgatct
    13561 acgaacgcag tggcagcgcc ggagagttca agaagttctg tttcaccgtg cgcaagctga
    13621 tcgggtcaaa tgacctgccg gagtacgatt tgaaggagga ggcggggcag gctggcccga
    13681 tcctagtcat gcgctaccgc aacctgatcg agggcgaagc atccgccggt tcctaatgta
    13741 cggagcagat gctagggcaa attgccctag caggggaaaa aggtcgaaaa ggtctctttc
    13801 ctgtggatag cacgtacatt gggaacccaa agccgtacat tgggaaccgg aacccgtaca
    13861 ttgggaaccc aaagccgtac attgggaacc ggtcacacat gtaagtgact gatataaaag
    13921 agaaaaaagg cgatttttcc gcctaaaact ctttaaaact tattaaaact cttaaaaccc
    13981 gcctggcctg tgcataactg tctggccagc gcacagccga agagctgcaa aaagcgccta
    14041 cccttcggtc gctgcgctcc ctacgccccg ccgcttcgcg tcggcctatc gcggccgctg
    14101 gccgctcaaa aatggctggc ctacggccag gcaatctacc agggcgcgga caagccgcgc
    14161 cgtcgccact cgaccgccgg cgcccacatc aaggcaccct gcctcgcgcg tttcggtgat
    14221 gacggtgaaa acctctgaca catgcagctc ccggagacgg tcacagcttg tctgtaagcg
    14281 gatgccggga gcagacaagc ccgtcagggc gcgtcagcgg gtgttggcgg gtgtcggggc
    14341 gcagccatga cccagtcacg tagcgatagc ggagtgtata ctggcttaac tatgcggcat
    14401 cagagcagat tgtactgaga gtgcaccata tgcggtgtga aataccgcac agatgcgtaa
    14461 ggagaaaata ccgcatcagg cgctcttccg cttcctcgct cactgactcg ctgcgctcgg
    14521 tcgttcggct gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg ttatccacag
    14581 aatcagggga taacgcagga aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc
    14641 gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg cccccctgac gagcatcaca
    14701 aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg actataaaga taccaggcgt
    14761 ttccccctgg aagctccctc gtgcgctctc ctgttccgac cctgccgctt accggatacc
    14821 tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc
    14881 tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc
    14941 ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc caacccggta agacacgact
    15001 tatcgccact ggcagcagcc actggtaaca ggattagcag agcgaggtat gtaggcggtg
    15061 ctacagagtt cttgaagtgg tggcctaact acggctacac tagaaggaca gtatttggta
    15121 tctgcgctct gctgaagcca gttaccttcg gaaaaagagt tggtagctct tgatccggca
    15181 aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa
    15241 aaaaaggatc tcaagaagat cctttgatct tttctacggg gtctgacgct cagtggaacg
    15301 aaaactcacg ttaagggatt ttggtcatgc atgatatatc tcccaatttg tgtagggctt
    15361 attatgcacg cttaaaaata ataaaagcag ac
//
