LOCUS       sequence-169789-       14421 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Golden Gate Level 2 construct encoding Cas9-YFP and 2 
            urease-targeting gRNAs.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 14421)
  AUTHORS   Hopes A, Nekrasov V, Kamoun S, Mock T
  TITLE     Editing of the urease gene by CRISPR-Cas in the diatom Thalassiosira
            pseudonana.
  JOURNAL   Plant Methods. 2016 Nov 24;12:49. doi: 10.1186/s13007-016-0148-0. 
            eCollection 2016.
  PUBMED    27904648
REFERENCE   2  (bases 1 to 14421)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Plant 
            Methods."; date: "2016-11-24"; volume: "12:49. doi"; pages: " 
            10.1186/s13007-016-0148-0. eCollection 2016"
COMMENT     Sequence Label: pAGM4723:TpCC_Urease
FEATURES             Location/Qualifiers
     source          1..14421
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_feature    36..60
                     /label=RB T-DNA repeat
                     /note="right border repeat from nopaline C58 T-DNA"
     CDS             340..969
                     /codon_start=1
                     /product="stability protein from the Pseudomonas plasmid 
                     pVS1 (Heeb et al., 2000)"
                     /label=pVS1 StaA
                     /translation="MKVIAVLNQKGGSGKTTIATHLARALQLAGADVLLVDSDPQGSAR
                     DWAAVREDQPLTVVGIDRPTIDRDVKAIGRRDFVVIDGAPQAADLAVSAIKAADFVLIP
                     VQPSPYDIWATADLVELVKQRIEVTDGRLQAAFVVSRAIKGTRIGGEVAEALAGYELPI
                     LESRITQRVSYPGTAAAGTTVLESEPEGDAAREVQALAAEIKSKLI"
     CDS             1398..2471
                     /codon_start=1
                     /product="replication protein from the Pseudomonas plasmid 
                     pVS1 (Heeb et al., 2000)"
                     /label=pVS1 RepA
                     /translation="MSGRKPSGPVQIGAALGDDLVEKLKAAQAAQRQRIEAEARPGESW
                     QAAADRIRKESRQPPAAGASSIRKPPKGDEQPDFFVPMLYDVGTRDSRSIMDVAVFRLS
                     KRDRRAGEVIRYELPDGHVEVSAGPAGMASVWDYDLVLMAVSHLTESMNRYREGKGDKP
                     GRVFRPHVADVLKFCRRADGGKQKDDLVETCIRLNTTHVAMQRTKKAKNGRLVTVSEGE
                     ALISRYKIVKSETGRPEYIEIELADWMYREITEGKNPDVLTVHPDYFLIDPGIGRFLYR
                     LARRAAGKAEARWLFKTIYERSGSAGEFKKFCFTVRKLIGSNDLPEYDLKEEAGQAGPI
                     LVMRYRNLIEGEASAGS"
     rep_origin      2537..2731
                     /label=pVS1 oriV
                     /note="origin of replication for the Pseudomonas plasmid 
                     pVS1 (Heeb et al., 2000)"
     promoter        2793..2897
                     /gene="bla"
                     /label=AmpR promoter
     CDS             2898..3692
                     /codon_start=1
                     /gene="aphA-3"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin"
                     /translation="MAKMRISPELKKLIEKYRCVKDTEGMSPAKVYKLVGENENLYLKM
                     TDSRYKGTTYDVEREKDMMLWLEGKLPVPKVLHFERHDGWSNLLMSEADGVLCSEEYED
                     EQSPEKIIELYAECIRLFHSIDISDCPYTNSLDSRLAELDYLLNNDLADVDCENWEEDT
                     PFKDPRELYDFLKTEKPEEELVFSHGDLGDSNIFVKDGKVSGFIDLGRSGRADKWYDIA
                     FCVRSLREDIGEEQYVELFFDLLGIKPDWEKIKYYILLDELF"
     rep_origin      3863..4451
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4352..4371
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     misc_feature    4587..4611
                     /label=LB T-DNA repeat
                     /note="left border repeat from nopaline C58 T-DNA"
     primer_bind     4701..4717
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     primer_bind     4702..4718
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     CDS             5701..6270
                     /codon_start=1
                     /gene="Streptomyces noursei nat1"
                     /product="nourseothricin acetyltransferase"
                     /label=NrsR
                     /note="confers resistance to nourseothricin"
                     /translation="MTTLDDTAYRYRTSVPGDAEAIEALDGSFTTDTVFRVTATGDGFT
                     LREVPVDPPLTKVFPDDESDDESDDGEDGDPDSRTFVAYGDDGDLAGFVVVSYSGWNRR
                     LTVEDIEVAPEHRGHGVGRALMGLATEFARERGAGHLWLEVTNVNAPAIHAYRRMGFTL
                     CGLDTALYDGTASDGEQALYMSMPCP"
     primer_bind     complement(6786..6802)
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6786..6802)
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     primer_bind     complement(6843..6863)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(6843..6861)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     CDS             7869..11972
                     /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             7869..11972
                     /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             7869..11972
                     /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             11985..12005
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             join(12012..12014,12015..12017,12018..12731)
                     /codon_start=1
                     /product="enhanced YFP"
                     /label=EYFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTFGYGLQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSYQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(12057..12078)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(12318..12337)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     12665..12686
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     misc_RNA        13730..13805
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     protein_bind    complement(13817..13841)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     misc_RNA        14340..14415
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
ORIGIN
        1 gggacacgaa gtgatccgtt taaactatca gtgtttgaca ggatatattg gcgggtaaac
       61 ctaagagaaa agagcgttta ttagaataat cggatattta aaagggcgtg aaaaggttta
      121 tccgttcgtc catttgtatg tgccagccgt gcggctgcat gaaatcctgg ccggtttgtc
      181 tgatgccaag ctggcggcct ggccggccag cttggccgct gaagaaaccg agcgccgccg
      241 tctaaaaagg tgatgtgtat ttgagtaaaa cagcttgcgt catgcggtcg ctgcgtatat
      301 gatgcgatga gtaaataaac aaatacgcaa ggggaacgca tgaaggttat cgctgtactt
      361 aaccagaaag gcgggtcagg caagacgacc atcgcaaccc atctagcccg cgccctgcaa
      421 ctcgccgggg ccgatgttct gttagtcgat tccgatcccc agggcagtgc ccgcgattgg
      481 gcggccgtgc gggaagatca accgctaacc gttgtcggca tcgaccgccc gacgattgac
      541 cgcgacgtga aggccatcgg ccggcgcgac ttcgtagtga tcgacggagc gccccaggcg
      601 gcggacttgg ctgtgtccgc gatcaaggca gccgacttcg tgctgattcc ggtgcagcca
      661 agcccttacg acatatgggc caccgccgac ctggtggagc tggttaagca gcgcattgag
      721 gtcacggatg gaaggctaca agcggccttt gtcgtgtcgc gggcgatcaa aggcacgcgc
      781 atcggcggtg aggttgccga ggcgctggcc gggtacgagc tgcccattct tgagtcccgt
      841 atcacgcagc gcgtgagcta cccaggcact gccgccgccg gcacaaccgt tcttgaatca
      901 gaacccgagg gcgacgctgc ccgcgaggtc caggcgctgg ccgctgaaat taaatcaaaa
      961 ctcatttgag ttaatgaggt aaagagaaaa tgagcaaaag cacaaacacg ctaagtgccg
     1021 gccgtccgag cgcacgcagc agcaaggctg caacgttggc cagcctggca gacacgccag
     1081 ccatgaagcg ggtcaacttt cagttgccgg cggaggatca caccaagctg aagatgtacg
     1141 cggtacgcca aggcaagacc attaccgagc tgctatctga atacatcgcg cagctaccag
     1201 agtaaatgag caaatgaata aatgagtaga tgaattttag cggctaaagg aggcggcatg
     1261 gaaaatcaag aacaaccagg caccgacgcc gtggaatgcc ccatgtgtgg aggaacgggc
     1321 ggttggccag gcgtaagcgg ctgggttgtc tgccggccct gcaatggcac tggaaccccc
     1381 aagcccgagg aatcggcgtg agcggtcgca aaccatccgg cccggtacaa atcggcgcgg
     1441 cgctgggtga tgacctggtg gagaagttga aggccgcgca ggccgcccag cggcaacgca
     1501 tcgaggcaga agcacgcccc ggtgaatcgt ggcaagcggc cgctgatcga atccgcaaag
     1561 aatcccggca accgccggca gccggtgcgt cgtcgattag gaagccgccc aagggcgacg
     1621 agcaaccaga ttttttcgtt ccgatgctct atgacgtggg cacccgcgat agtcgcagca
     1681 tcatggacgt ggccgttttc cgtctgtcga agcgtgaccg acgagctggc gaggtgatcc
     1741 gctacgagct tccagacggg cacgtagagg tttccgcagg gccggccggc atggccagtg
     1801 tgtgggatta cgacctggta ctgatggcgg tttcccatct aaccgaatcc atgaaccgat
     1861 accgggaagg gaagggagac aagcccggcc gcgtgttccg tccacacgtt gcggacgtac
     1921 tcaagttctg ccggcgagcc gatggcggaa agcagaaaga cgacctggta gaaacctgca
     1981 ttcggttaaa caccacgcac gttgccatgc agcgtacgaa gaaggccaag aacggccgcc
     2041 tggtgacggt atccgagggt gaagccttga ttagccgcta caagatcgta aagagcgaaa
     2101 ccgggcggcc ggagtacatc gagatcgagc tagctgattg gatgtaccgc gagatcacag
     2161 aaggcaagaa cccggacgtg ctgacggttc accccgatta ctttttgatc gatcccggca
     2221 tcggccgttt tctctaccgc ctggcacgcc gcgccgcagg caaggcagaa gccagatggt
     2281 tgttcaagac gatctacgaa cgcagtggca gcgccggaga gttcaagaag ttctgtttca
     2341 ccgtgcgcaa gctgatcggg tcaaatgacc tgccggagta cgatttgaag gaggaggcgg
     2401 ggcaggctgg cccgatccta gtcatgcgct accgcaacct gatcgagggc gaagcatccg
     2461 ccggttccta atgtacggag cagatgctag ggcaaattgc cctagcaggg gaaaaaggtc
     2521 gaaaaagctt ctttcctgtg gatagcacgt acattgggaa cccaaagccg tacattggga
     2581 accggaaccc gtacattggg aacccaaagc cgtacattgg gaaccggtca cacatgtaag
     2641 tgactgatat aaaagagaaa aaaggcgatt tttccgccta aaactcttta aaacttatta
     2701 aaactcttaa aacccgcctg gcctgtgcat aactgtctgg ccagcgcaca gccgaacagc
     2761 tgcaaaaagc gcccacatca aggctccgag tgcgcggaac ccctatttgt ttatttttct
     2821 aaatacattc aaatatgtat ccgctcatga gacaataacc ctgataaatg cttcaataat
     2881 attgaaaaag gaagagtatg gctaaaatga gaatatcacc ggaattgaaa aaactgatcg
     2941 aaaaataccg ctgcgtaaaa gatacggaag gaatgtctcc tgctaaggta tataagctgg
     3001 tgggagaaaa tgaaaaccta tatttaaaaa tgacggacag ccggtataaa gggaccacct
     3061 atgatgtgga acgggaaaag gacatgatgc tatggctgga aggaaagctg cctgttccaa
     3121 aggtcctgca ctttgaacgg catgatggct ggagcaatct gctcatgagt gaggccgatg
     3181 gcgtcctttg ctcggaagag tatgaagatg aacaaagccc tgaaaagatt atcgagctgt
     3241 atgcggagtg catcaggctc tttcactcca tcgacatatc ggattgtccc tatacgaata
     3301 gcttagacag ccgcttagcc gaattggatt acttactgaa taacgatctg gccgatgtgg
     3361 attgcgaaaa ctgggaagag gacactccat ttaaagatcc gcgcgagctg tatgattttt
     3421 taaagacgga aaagcccgaa gaggaacttg tcttttccca cggcgacctg ggagacagca
     3481 acatctttgt gaaagatggc aaagtaagtg gctttattga tcttgggaga agcggcaggg
     3541 cggacaagtg gtatgacatt gccttctgcg tccggtcgct cagggaggat atcggggaag
     3601 aacagtatgt cgagctattt tttgacttac tggggatcaa gcctgattgg gagaaaataa
     3661 aatattatat tttactggat gaattgtttt agctgtcaga ccaagtttac tcatatatac
     3721 tttagattga tttaaaactt catttttaat ttaaaaggat ctaggtgaag atcctttttg
     3781 ataatctcat gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg tcagaccccg
     3841 tagaaaagat caaaggatct tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc
     3901 aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc
     3961 tttttccgaa ggtaactggc ttcagcagag cgcagatacc aaatactgtt cttctagtgt
     4021 agccgtagtt aggccaccac ttcaagaact ctgtagcacc gcctacatac ctcgctctgc
     4081 taatcctgtt accagtggct gctgccagtg gcgataagtc gtgtcttacc gggttggact
     4141 caagacgata gttaccggat aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac
     4201 agcccagctt ggagcgaacg acctacaccg aactgagata cctacagcgt gagctatgag
     4261 aaagcgccac gcttcccgaa gggagaaagg cggacaggta tccggtaagc ggcagggtcg
     4321 gaacaggaga gcgcacgagg gagcttccag ggggaaacgc ctggtatctt tatagtcctg
     4381 tcgggtttcg ccacctctga cttgagcgtc gatttttgtg atgctcgtca ggggggcgga
     4441 gcctatggaa aaacgccagc aacgcggcct ttttacggtt cctgctcgga tctgttggac
     4501 cggacagtag tcatggttga tgggctgcct gtatcgagtg gtgattttgt gccgagctgc
     4561 cggtcgggga gctgttggct ggctggtggc aggatatatt gtggtgtaaa caaattgacg
     4621 cttagacaac ttaataacac attgcggacg tttttaatgt actggggttg aacactctgt
     4681 gccgaattcg gatccggagc tcgaggtcga cggtatcgat aagcttgcgc tttttccgag
     4741 aactccccat aagtcaacgg ctccaatcaa gaatgtatcc gacaacggcg agcatagcaa
     4801 cacgtccgtc tttggagtag aatcatcatg ttgtggatga atacacagat gaatgacatt
     4861 agaagcatga acatgttaga gagtaggagg tagagattga tatggtagca ttgcgatgtt
     4921 tgtttttggt cagcatatga tgagtggata ccaatatgat gaaagttgaa tctcgcgttt
     4981 gagctcagcg gtacgttatt gatcgaaagt agcctgatca aaatccttgg agagtacaag
     5041 aggatcaaag aatccagtgg gggcgataac tccaagctcg ttctcaaaga ggcaatggag
     5101 gtagaaactc atcccagttg agaagaagtg aaggcagtgg cggtggcgaa agcagaggca
     5161 acgaggacag acttcctgtg ggttgatgca acgaatattt ccagaaggag aagtttagag
     5221 agttgaaccg ctacctacaa tgacaaagta tcgtatcgat tttgatgttg gttggttatg
     5281 aattcaaact gtaagttgga ttgtgagaag atcagaagtt gaacgaacac atctttccga
     5341 tcattcacct ccacactgca acaacacggt acttcttccg cggcagatct ctgtcgccat
     5401 tctcttgtcc tgttgttggc tgtgagacga ggaaagcaac gacaagtttc acaaaaggga
     5461 gttcctttaa cgagatatgt tttttataaa gagtcccaat agaaagacaa attgattcct
     5521 ccgtgcaaac gcgcaaataa acaccacgtc cattatatcc atatctttca gagtatccaa
     5581 caagtgttga aggacaggta gttgaagtaa cgtatcttcc ccctcgactg gatccatcaa
     5641 caaggcgaac aaatccattc aacctctcat aaattatctg atttaccaaa ccgatccaaa
     5701 atgaccactc ttgacgacac ggcttaccgg taccgcacca gtgtcccggg ggacgccgag
     5761 gccatcgagg cactggatgg gtccttcacc accgacaccg tattccgcgt caccgccacc
     5821 ggggacggct tcaccctgcg ggaggtgccg gtggacccgc ccctgaccaa ggtgttcccc
     5881 gacgacgaat cggacgacga atcggacgac ggggaggacg gcgacccgga ctcccggacg
     5941 ttcgtcgcgt acggggacga cggcgacctg gcgggcttcg tggtcgtctc gtactccggc
     6001 tggaaccgcc ggctgaccgt cgaggacatc gaggtcgccc cggagcaccg ggggcacggg
     6061 gtcgggcgcg cgttgatggg gctcgcgacg gagttcgccc gcgagcgggg cgccgggcac
     6121 ctctggctgg aggtcaccaa cgtcaacgca ccggcgatcc acgcgtaccg gcggatgggg
     6181 ttcaccctct gcggcctgga caccgccctg tacgacggca ccgcctcgga cggcgagcag
     6241 gcgctctaca tgagcatgcc ctgcccctga gcggccgcat actggattgg tgaatcaatg
     6301 agccgtagca caatggttac attcggctag ctaagatcca atggcaagga ccaagtgctg
     6361 gaacttgttt tgctttagca gatcttagcg tgagaggtat ttgtcctctg tcaggagtag
     6421 atagtagatg ttctttttaa actaaaatgc taactgttcc gaattcctca tcgcagctaa
     6481 tccgtacatc aaaagacaaa atgctaggta tgtgtactac atctcctgtt gctagataag
     6541 acatatgata ggaaacacac catcaatagt cattgtagct ttacttatac tacgcatttg
     6601 cactttcccc tgagtggcag aggcgcattg agaaaatcga tctcaacata gtttatgtag
     6661 catcccctag atccattact ttaagtctcc ttcgtctttg gtgtaggcat gttggacaca
     6721 acgaggtaaa acacaacaca aacaatgtgt ccagcaaagt agtagctgct ccagttctcc
     6781 cgggggatcc actagttcta gagcggccgg ccgccaccgc ggtggagctc cagcttttgt
     6841 tccctttagt gagggttaat tgcgcgctgc aagaattcaa gcttggagag cttgcgcttt
     6901 ttccgagaac tccccataag tcaacggctc caatcaagaa tgtatccgac aacggcgagc
     6961 atagcaacac gtccgtcttt ggagtagaat catcatgttg tggatgaata cacagatgaa
     7021 tgacattaga agcatgaacg tgttagagag taggaggtag agattgatat ggtagcattg
     7081 cgatgtttgt ttttggtcag catatgatga gtggatacca atatgatgaa agttgaatct
     7141 cgcgtttgag ctcagcggta cgttattgat cgaaagtagc ctgatcaaaa tccttggaga
     7201 gtacaagagg atcaaagaat ccagtggggg cgataactcc aagctcgttc tcaaagaggc
     7261 aatggaggta gaaactcatc ccagttgaga agaagtgaag gcagtggcgg tggcgaaagc
     7321 agaggcaacg aggtcagact tcctgtaggt tgatgcaacg aatatttcca gaaggagaag
     7381 tttagagagt tgaaccgcta cctacaatga caaagtatcg tatcgatttt gatgttggtt
     7441 ggttatgaat tcaaactgta agttggattg tgagaagatc agaagttgaa cgaacacatc
     7501 tttccgatca ttcacctcca cactgcaaca acacggtact tcttccgcgg cagatctctg
     7561 tcgccattct cttgtcctgt tgttggctgt gagacgagga aagcaacgac aagtttcaca
     7621 aaagggagtt cctttaacga gatatgtttt ttataaagag tcccaataga aagacaaatt
     7681 gattcctccg tgcaaacgcg caaataaaca ccacgtccat tatatccata tctttcagag
     7741 tatccaacaa gtgttgaagg acaggtagtt gaagtaacgt atcttccccc tcgactggat
     7801 ccatcaacaa ggcgaacaaa tccattcaac ctctcataaa ttatctgatt taccaaacca
     7861 ataccaaaat ggacaagaag tactccattg ggctcgatat cggcacaaac agcgtcggct
     7921 gggccgtcat tacggacgag tacaaggtgc cgagcaaaaa attcaaagtt ctgggcaata
     7981 ccgatcgcca cagcataaag aagaacctca ttggcgccct cctgttcgac tccggggaga
     8041 cggccgaagc cacgcggctc aaaagaacag cacggcgcag atatacccgc agaaagaatc
     8101 ggatctgcta cctgcaggag atctttagta atgagatggc taaggtggat gactctttct
     8161 tccataggct ggaggagtcc tttttggtgg aggaggataa aaagcacgag cgccacccaa
     8221 tctttggcaa tatcgtggac gaggtggcgt accatgaaaa gtacccaacc atatatcatc
     8281 tgaggaagaa gcttgtagac agtactgata aggctgactt gcggttgatc tatctcgcgc
     8341 tggcgcatat gatcaaattt cggggacact tcctcatcga gggggacctg aacccagaca
     8401 acagcgatgt cgacaaactc tttatccaac tggttcagac ttacaatcag cttttcgaag
     8461 agaacccgat caacgcatcc ggagttgacg ccaaagcaat cctgagcgct aggctgtcca
     8521 aatcccggcg gctcgaaaac ctcatcgcac agctccctgg ggagaagaag aacggcctgt
     8581 ttggtaatct tatcgccctg tcactcgggc tgacccccaa ctttaaatct aacttcgacc
     8641 tggccgaaga tgccaagctt caactgagca aagacaccta cgatgatgat ctcgacaatc
     8701 tgctggccca gatcggcgac cagtacgcag accttttttt ggcggcaaag aacctgtcag
     8761 acgccattct gctgagtgat attctgcgag tgaacacgga gatcaccaaa gctccgctga
     8821 gcgctagtat gatcaagcgc tatgatgagc accaccaaga cttgactttg ctgaaggccc
     8881 ttgtcagaca gcaactgcct gagaagtaca aggaaatttt cttcgatcag tctaaaaatg
     8941 gctacgccgg atacattgac ggcggagcaa gccaggagga attttacaaa tttattaagc
     9001 ccatcttgga aaaaatggac ggcaccgagg agctgctggt aaagcttaac agagaagatc
     9061 tgttgcgcaa acagcgcact ttcgacaatg gaagcatccc ccaccagatt cacctgggcg
     9121 aactgcacgc tatcctcagg cggcaagagg atttctaccc ctttttgaaa gataacaggg
     9181 aaaagattga gaaaatcctc acatttcgga taccctacta tgtaggcccc ctcgcccggg
     9241 gaaattccag attcgcgtgg atgactcgca aatcagaaga gactatcact ccctggaact
     9301 tcgaggaagt cgtggataag ggggcctctg cccagtcctt catcgaaagg atgactaact
     9361 ttgataaaaa tctgcctaac gaaaaggtgc ttcctaaaca ctctctgctg tacgagtact
     9421 tcacagttta taacgagctc accaaggtca aatacgtcac agaagggatg agaaagccag
     9481 cattcctgtc tggagagcag aagaaagcta tcgtggacct cctcttcaag acgaaccgga
     9541 aagttaccgt gaaacagctc aaagaagatt atttcaaaaa gattgaatgt ttcgactctg
     9601 ttgaaatcag cggagtggag gatcgcttca acgcatccct gggaacgtat cacgatctcc
     9661 tgaaaatcat taaagacaag gacttcctgg acaatgagga gaacgaggac attcttgagg
     9721 acattgtcct cacccttacg ttgtttgaag atagggagat gattgaagaa cgcttgaaaa
     9781 cttacgctca tctcttcgac gacaaagtca tgaaacagct caagaggcgc cgatatacag
     9841 gatgggggcg gctgtcaaga aaactgatca atgggatccg agacaagcag agtggaaaga
     9901 caatcctgga ttttcttaag tccgatggat ttgccaaccg gaacttcatg cagttgatcc
     9961 atgatgactc tctcaccttt aaggaggaca tccagaaagc acaagtttct ggccaggggg
    10021 acagtctcca cgagcacatc gctaatcttg caggtagccc agctatcaaa aagggaatac
    10081 tgcagaccgt taaggtcgtg gatgaactcg tcaaagtaat gggaaggcat aagcccgaga
    10141 atatcgttat cgagatggcc cgagagaacc aaactaccca gaagggacag aagaacagta
    10201 gggaaaggat gaagaggatt gaagagggta taaaagaact ggggtcccaa atccttaagg
    10261 aacacccagt tgaaaacacc cagcttcaga atgagaagct ctacctgtac tacctgcaga
    10321 acggcaggga catgtacgtg gatcaggaac tggacatcaa tcggctctcc gactacgacg
    10381 tggatcatat cgtgccccag tcttttctca aagatgattc tattgataat aaagtgttga
    10441 caagatccga taaaaataga gggaagagtg ataacgtccc ctcagaagaa gttgtcaaga
    10501 aaatgaaaaa ttattggcgg cagctgctga acgccaaact gatcacacaa cggaagttcg
    10561 ataatctgac taaggctgaa cgaggtggcc tgtctgagtt ggataaagcc ggcttcatca
    10621 aaaggcagct tgttgagaca cgccagatca ccaagcacgt ggcccaaatt ctcgattcac
    10681 gcatgaacac caagtacgat gaaaatgaca aactgattcg agaggtgaaa gttattactc
    10741 tgaagtctaa gctggtttca gatttcagaa aggactttca gttttataag gtgagagaga
    10801 tcaacaatta ccaccatgcg catgatgcct acctgaatgc agtggtaggc actgcactta
    10861 tcaaaaaata tcccaagctt gaatctgaat ttgtttacgg agactataaa gtgtacgatg
    10921 ttaggaaaat gatcgcaaag tctgagcagg aaataggcaa ggccaccgct aagtacttct
    10981 tttacagcaa tattatgaat tttttcaaga ccgagattac actggccaat ggagagattc
    11041 ggaagcgacc acttatcgaa acaaacggag aaacaggaga aatcgtgtgg gacaagggta
    11101 gggatttcgc gacagtccgg aaggtcctgt ccatgccgca ggtgaacatc gttaaaaaga
    11161 ccgaagtaca gaccggaggc ttctccaagg aaagtatcct cccgaaaagg aacagcgaca
    11221 agctgatcgc acgcaaaaaa gattgggacc ccaagaaata cggcggattc gattctccta
    11281 cagtcgctta cagtgtactg gttgtggcca aagtggagaa agggaagtct aaaaaactca
    11341 aaagcgtcaa ggaactgctg ggcatcacaa tcatggagcg atcaagcttc gaaaaaaacc
    11401 ccatcgactt tctcgaggcg aaaggatata aagaggtcaa aaaagacctc atcattaagc
    11461 ttcccaagta ctctctcttt gagcttgaaa acggccggaa acgaatgctc gctagtgcgg
    11521 gcgagctgca gaaaggtaac gagctggcac tgccctctaa atacgttaat ttcttgtatc
    11581 tggccagcca ctatgaaaag ctcaaaggat ctcccgaaga taatgagcag aagcagctgt
    11641 tcgtggaaca acacaaacac taccttgatg agatcatcga gcaaataagc gaattctcca
    11701 aaagagtgat cctcgccgac gctaacctcg ataaggtgct ttctgcttac aataagcaca
    11761 gggataagcc catcagggag caggcagaaa acattatcca cttgtttact ctgaccaact
    11821 tgggcgcgcc tgcagccttc aagtacttcg acaccaccat agacagaaag cggtacacct
    11881 ctacaaagga ggtcctggac gccacactga ttcatcagtc aattacgggg ctctatgaaa
    11941 caagaatcga cctctctcag ctcggtggag acagcagggc tgaccccaag aagaagagga
    12001 aggtgggttc gatggtgagc aagggcgagg agctgttcac cggggtggtg cccatcctgg
    12061 tcgagctgga cggcgacgta aacggccaca agttcagcgt gtccggcgag ggcgagggcg
    12121 atgccaccta cggcaagctg accctgaagt tcatctgcac caccggcaag ctgcccgtgc
    12181 cctggcccac cctcgtgacc accttcggct acggcctgca gtgcttcgcc cgctaccccg
    12241 accacatgaa gcagcacgac ttcttcaagt ccgccatgcc cgaaggctac gtccaggagc
    12301 gcaccatctt cttcaaggac gacggcaact acaagacccg cgccgaggtg aagttcgagg
    12361 gcgacaccct ggtgaaccgc atcgagctga agggcatcga cttcaaggag gacggcaaca
    12421 tcctggggca caagctggag tacaactaca acagccacaa cgtctatatc atggccgaca
    12481 agcagaagaa cggcatcaag gtgaacttca agatccgcca caacatcgag gacggcagcg
    12541 tgcagctcgc cgaccactac cagcagaaca cccccatcgg cgacggcccc gtgctgctgc
    12601 ccgacaacca ctacctgagc taccagtccg ccctgagcaa agaccccaac gagaagcgcg
    12661 atcacatggt cctgctggag ttcgtgaccg ccgccgggat cactctcggc atggacgagc
    12721 tgtacaagtg agcttatact ggattggtga atcaatgagc cgtagcacaa tggttacatt
    12781 cggctagcta agatccaatg gcaaggacca agtgctggaa cttgttttgc tttagcagat
    12841 cttagcgtga gaggtatttg tcctctgtca ggagtagata gtagatgttc tttttaaact
    12901 aaaatgctaa ctgttccgaa ttcctcatcg cagctaatcc gtacatcaaa agacaaaatg
    12961 ctaggtatgt gtactacatc tcctgttgct agataagaca tatgatagga aacacaccat
    13021 caatagtcat tgtagcttta cttatactac gcatttgcac tttcccctga gtggcagagg
    13081 cgcattgaga aaatcgatct caacatagtt tatgtagcat cccctagatc cattacttta
    13141 agtctccttc gtctttggtg taggcatgtt ggacacaacg aggtaaaaca caacacaaac
    13201 aatgtgtcca gcaaagtagt agctgctcca gttctccgct actagaattc gagctcggag
    13261 cttcatcaag agagcaacca acaaagagga ggcgaacacg gtgcacagca ccgtctgcat
    13321 acttccgatt tgcaattcgc aacatatgat acatcatcca tctctctgag aaaactgggc
    13381 actgttagaa tctgggattt atcaacagcc accaactatc tactcaacgc aattggggca
    13441 cttgataatg tgacaatcct ccttgctcct ttccttgtcg cctcaaacaa cgtcgggaac
    13501 tgcacagaaa catcgctact tattcacgcc tcacctctct ttcctccttt actcacttat
    13561 tcgcgcctca cctctctttc ctcctttact caagtcctct gcttgccggg tttattcttc
    13621 cgtttgtgtt gttgcggtct gataggattt gtttggagac gaaccttttt ttttgattgc
    13681 tttataaatt aacgttttgc cgaaattgtg tcgtaatcaa gtattgccgg ttttagagct
    13741 agaaatagca agttaaaata aggctagtcc gttatcaact tgaaaaagtg gcaccgagtc
    13801 ggtgcttttt ttctagaccc agctttcttg tacaaagttg gcattacgct ttacgaattc
    13861 ccatggggag cttcatcaag agagcaacca acaaagaaga ggcgaacacg gtgcacagca
    13921 ccgtctgcat acttccgatt tgcaattcac aacatatgat acatcatcca tctctctgag
    13981 aaaactgggc actgttagaa tctgggattt atcaacagcc accaactatc tactcaacgc
    14041 aattggggca cttgataatg tgacaatcct ccttgctcct ttccttgtcg cctcaaacaa
    14101 cgtcgggaac tgcacagaaa catcgctact tattcacgcc tcacctctct ttcctccttt
    14161 actcacttat tcgcgcctca cctctctttc ctcctttact caagtcctct gcttgccggg
    14221 tttattcttc cgtttgtgtt gttgcggtct gataggattt gtttggagac gaaccttttt
    14281 ttttgattgc tttataaatt aacgttttgc cgaaattgtg tttccgatct aatgtccatg
    14341 ttttagagct agaaatagca agttaaaata aggctagtcc gttatcaact tgaaaaagtg
    14401 gcaccgagtc ggtgcttttt t
//
