LOCUS       sequence-209307-       15560 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  N. castellii Solo CRISPR vector, marked with URA3 and NatR.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 15560)
  AUTHORS   Vyas VK, Bushkin GG, Bernstein DA, Getz MA, Sewastianik M, Barrasa 
            MI, Bartel DP, Fink GR
  TITLE     New CRISPR Mutagenesis Strategies Reveal Variation in Repair 
            Mechanisms among Fungi.
  JOURNAL   mSphere. 2018 Apr 25;3(2). pii: 3/2/e00154-18. doi: 
            10.1128/mSphere.00154-18. Print 2018 Apr 25.
  PUBMED    29695624
REFERENCE   2  (bases 1 to 15560)
  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: "mSphere."; 
            date: "2018-04-25"; volume: "3(2). pii: 3/2/e00154-18. doi"; pages: 
            " 10.1128/mSphere.00154-18. Print 2018 Apr 25"
COMMENT     Sequence Label: pV1464
FEATURES             Location/Qualifiers
     source          1..15560
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(33..55)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     158..177
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        203..423
                     /gene="S. cerevisiae URA3"
                     /label=URA3 promoter
     CDS             424..1227
                     /codon_start=1
                     /gene="S. cerevisiae URA3"
                     /product="orotidine-5'-phosphate decarboxylase, required 
                     for uracil biosynthesis"
                     /label=URA3
                     /note="yeast auxotrophic marker, counterselectable with 
                     5-fluoroorotic acid (5-FOA)"
                     /translation="MSKATYKERAATHPSPVAAKLFNIMHEKQTNLCASLDVRTTKELL
                     ELVEALGPKICLLKTHVDILTDFSMEGTVKPLKALSAKYNFLLFEDRKFADIGNTVKLQ
                     YSAGVYRIAEWADITNAHGVVGPGIVSGLKQAAEEVTKEPRGLLMLAELSCKGSLSTGE
                     YTKGTVDIAKSDKDFVIGFIAQRDMGGRDEGYDWLIMTPGVGLDDKGDALGQQYRTVDD
                     VVSTGSDIIIVGRGLFAKGRDAKVEGERYRKAGWEAYLRRCGQQN"
     rep_origin      complement(1358..1813)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1495..1516)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     1707..1726
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1943..1965
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     1957..1974
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     1958..1974
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     1984..2003
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1984..2002
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             4548..8651
                     /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             4548..8651
                     /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             8664..8684
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             8688..8708
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             8712..8732
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             8733..8798
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     primer_bind     complement(8879..8897)
                     /label=CYC1
                     /note="CYC1 transcription termination signal, reverse 
                     primer"
     terminator      complement(9055..9252)
                     /label=TEF terminator
                     /note="Ashbya gossypii TEF terminator"
     promoter        complement(9853..10196)
                     /label=TEF promoter
                     /note="Ashbya gossypii TEF promoter"
     misc_RNA        10613..10688
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     primer_bind     complement(12786..12806)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(12786..12804)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(12825..12841)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(12825..12841)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(12838..12860)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    12849..12865
                     /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(join(12873..12879,12880..12897,12898..12903))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     primer_bind     complement(13056..13073)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(13227..13815)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(13307..13326)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(13986..14777,14778..14846))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     14609..14628
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(14847..14951)
                     /gene="bla"
                     /label=AmpR promoter
     misc_feature    14988..15491
                     /label=CEN/ARS
                     /note="S. cerevisiae CEN6 centromere fused to an 
                     autonomously replicating sequence"
     primer_bind     15533..15551
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
ORIGIN
        1 gaattcgcgc gtttcggtga tgacggtgaa aacctctgac acatgcagct cccggagatc
       61 tcggtcacag cttgtctgta agcggatgcc gggagcagac aagcccgtca gggcgcgtca
      121 gcgggtgttg gcgggtgtcg gggctggctt aactatgcgg catcagagca gattgtactg
      181 agagtgcacc ataccacagc ttttcaattc aattcatcat ttttttttta ttcttttttt
      241 tgatttcggt ttctttgaaa tttttttgat tcggtaatct ccgaacagaa ggaagaacga
      301 aggaaggagc acagacttag attggtatat atacgcatat gtagtgttga agaaacatga
      361 aattgcccag tattcttaac ccaactgcac agaacaaaaa cctgcaggaa acgaagataa
      421 atcatgtcga aagctacata taaggaacgt gctgctactc atcctagtcc tgttgctgcc
      481 aagctattta atatcatgca cgaaaagcaa acaaacttgt gtgcttcatt ggatgttcgt
      541 accaccaagg aattactgga gttagttgaa gcattaggtc ccaaaatttg tttactaaaa
      601 acacatgtgg atatcttgac tgatttttcc atggagggca cagttaagcc gctaaaggca
      661 ttatccgcca agtacaattt tttactcttc gaagacagaa aatttgctga cattggtaat
      721 acagtcaaat tgcagtactc tgcgggtgta tacagaatag cagaatgggc agacattacg
      781 aatgcacacg gtgtggtggg cccaggtatt gttagcggtt tgaagcaggc ggcagaagaa
      841 gtaacaaagg aacctagagg ccttttgatg ttagcagaat tgtcatgcaa gggctcccta
      901 tctactggag aatatactaa gggtactgtt gacattgcga agagcgacaa agattttgtt
      961 atcggcttta ttgctcaaag agacatgggt ggaagagatg aaggttacga ttggttgatt
     1021 atgacacccg gtgtgggttt agatgacaag ggtgatgcat tgggtcaaca gtatagaacc
     1081 gtggatgatg tggtctctac aggatctgac attattattg ttggaagagg actatttgca
     1141 aagggaaggg atgctaaggt agagggtgaa cgttacagaa aagcaggctg ggaagcatat
     1201 ttgagaagat gcggccagca aaactaaaaa actgtattat aagtaaatgc atgtatacta
     1261 aactcacaaa ttagagcttc aatttaatta tatcagttat taccctatgc ggtgtgaaat
     1321 accgcacaga tgcgtaagga gaaaataccg catcaggaaa ttgtaaacgt taatattttg
     1381 ttaaaattcg cgttaaattt ttgttaaatc agctcatttt ttaaccaata ggccgaaatc
     1441 ggcaaaatcc cttataaatc aaaagaatag accgagatag ggttgagtgt tgttccagtt
     1501 tggaacaaga gtccactatt aaagaacgtg gactccaacg tcaaagggcg aaaaaccgtc
     1561 tatcagggcg atggcccact acgtgaacca tcaccctaat caagtttttt ggggtcgagg
     1621 tgccgtaaag cactaaatcg gaaccctaaa gggagccccc gatttagagc ttgacgggga
     1681 aagccggcga acgtggcgag aaaggaaggg aagaaagcga aaggagcggg cgctagggcg
     1741 ctggcaagtg tagcggtcac gctgcgcgta accaccacac ccgccgcgct taatgcgccg
     1801 ctacagggcg cgtcgcgcca ttcgccattc aggctgcgca actgttggga agggcgatcg
     1861 gtgcgggcct cttcgctatt acgccagctg gcgaaagggg gatgtgctgc aaggcgatta
     1921 agttgggtaa cgccagggtt ttcccagtca cgacgttgta aaacgacggc cagtgagcgc
     1981 gcgtaatacg actcactata gggcgaattg ggtacctgcc actactacca ctgggagttt
     2041 cgttcttctc gatactatta gctttacttc ctgcactagc agtggttgga tcaacagaat
     2101 cttcataatc atcaaaatcg tcttttgaag accccccgtt tgatgtatgg ccctgtcttt
     2161 tcatcaaact ttttatatag ttgactgaac tgaggctaaa tatgtgatca tcttcactat
     2221 agacaatctt tctcttattt gcaccaccgc caccactagt ctttgagaaa ttctcaaaac
     2281 cttttacgat attaccaagc gggctctctt cgaaataatc tatctctttt tgatatatcg
     2341 aatcctctag cgtggttagc tttctagtta gttcttgctt cttaagaatt tgctggatta
     2401 gtttattttt caattcaacg tatttctcag agtcatcttt agattttgat gaagatgtgc
     2461 gttcattcgc tatatccttc ttggtcgtgt cttttcgatc ctccttggct ggcactgaac
     2521 tcgtcttttt tggcgttgct gttccagaca gacttatctc attagatttg gaacttgtgg
     2581 gtttaacatc atttgtatct ttagtagaca tgattgtgca ataccgtgat tatttgtttt
     2641 gaaaggtctg tcatatttct atcaatttca aaacaaaatg ttcatcagaa aaaagccaaa
     2701 aatgtctctt ctagtttctt agtggtgtcg cataatacac aatgtcgctc aacaatccac
     2761 attcccggcg catagctcaa atcacatgac tacagctaac aattacacaa aaaaaattct
     2821 ctttttgatg tagcaactat cttcaactaa aacattttct ccttcggccc atgattgtcc
     2881 tccgggtcga cagcaagccg ttacaattga gatggaaagc gacctacctt cactcgataa
     2941 ggtgcttaat tgtacttcat ataaatctgg cccggatcta aacaaatgag ttccattaag
     3001 ccgtgggttc tcaattaggg tttttgtttt tgatttagaa aaaagagatc aagatttgtt
     3061 tacaggtgat gccttttttt agaacttatg cgttgcaaaa gttgactaac gatttctata
     3121 aggtgatcca cactaattat acaaacgtac aaacagacat acttttcctg cgttcacctg
     3181 atgttggcca gatttctctc ttcattgcat agaacataac cacactaggg caacagaaaa
     3241 aaaaaaaaaa agtgcatcgg gaagttgtgt tccattcatt atatgtctac tactgcatat
     3301 gagtagccca cccaccacca ccatagtaag tttttgtgta tgcgcgccgt caggttattt
     3361 catttctgaa tttttcaacc accttactcc ctttattgtt gattgacaat tttgctcaca
     3421 gtaagatctt ttagactcca attaatataa aataagtctg attttccaat tcctgttttt
     3481 tctttttttt tctgtttcta tttctttcct tttctccctt ttttttaatt cttcattcaa
     3541 tcatcaattg ataattcagg aatattacaa caaccatggt ctatcgcaat tcttattagt
     3601 tgtagtattt tacttatcaa aatatctctg gttgtttcca gtctccaaca caaagtaaag
     3661 tcacttcaat ctgtagttgt agttaaatat agactctgtt ctgtgatttt gtagttttgc
     3721 gtaaagtgtg gcacacaatg tgtcaatgct acttgtgaag acaaaacacc cagcaaaagt
     3781 tgaaattaat aaccaagaaa acacgtttac cataacctca agatcaaaaa aattcttttt
     3841 aacgttacct agtgacgtcc ctatattttt acatccacat tgctcattaa aagaatgttg
     3901 gtgttaaatt catatctgac atctgttatc agtagtgctc acactcacaa aaagtttgac
     3961 tgtttgtcaa caaacacccg ttcatctttc agagtgtttt agccacccag acaccgcata
     4021 actaacaagg gaggcaagat gtattgaatg tcccccatca gagacattcc ctcctaagaa
     4081 atggccgcac ggttggaacg tccgccactt tgccaccaac aaaagtttac taagaaaaaa
     4141 ccgaagaaaa aatttgaaaa gacaaaccgc atgtgggcta cggcattttc actgtaccca
     4201 gcagggtata tttttacagt tttggtttgg caagcgcccc gtaggcactt tccccagaaa
     4261 taaccgcaca acgctcgaga ggattaggag ccagaaacga aagaaaaaag aaatgagcga
     4321 ggggcatctc ttcctttcct ttttcttttt ctcttttttt ttcactaaaa ttttcattcc
     4381 catccttaga aaattttttt cttacccatt aaacatttat atttaagatc atctcgtctc
     4441 aactttcagt gaaaaccctt ttttcttctg ttatttctta ttacaatctt gttcttagtt
     4501 taaaacctca tagcaatcca aactaattca catacataaa acccgggatg gataaaaagt
     4561 atagtattgg tttagatatt ggtactaact ctgtgggttg ggcagttatc accgacgaat
     4621 ataaagttcc atcaaagaaa tttaaggtgt taggtaacac tgacagacac tcaataaaaa
     4681 agaatcttat cggtgctctt ttgttcgact ccggtgaaac tgccgaggct acacgtttaa
     4741 aaagaacagc aagaagaaga tatacccgta gaaaaaatag aatatgttat ttacaagaaa
     4801 tcttttctaa tgaaatggct aaagttgatg attccttttt ccatagattg gaagagtcat
     4861 ttttggttga agaagacaaa aagcatgaga gacatccaat ctttgggaat atagttgatg
     4921 aagtggctta ccatgaaaaa tatcctacca tttatcattt aagaaagaaa ttggtagatt
     4981 caactgataa agctgacctt agattaatct atttagcact tgcccatatg attaaattta
     5041 gaggtcattt tttgattgaa ggtgatttga acccagataa ttctgacgtg gataaattat
     5101 ttattcaatt agtccaaacc tacaaccaat tatttgagga aaatccaatt aatgctagtg
     5161 gtgtcgatgc caaagctata ttatcagcca gattatcaaa atctagacgt ttggaaaatt
     5221 tgattgccca attgccagga gaaaaaaaga atggattatt tggaaacttg atcgcattat
     5281 cattgggttt gacaccaaat tttaaatcta attttgattt agctgaagat gctaaattac
     5341 aattatcaaa agacacctat gacgacgatt tggacaattt acttgctcaa attggtgatc
     5401 aatatgcaga tttgttctta gctgctaaaa acttatctga tgctattttg ttgtctgata
     5461 ttttgagagt gaacacagaa ataaccaaag ctccattatc agcatctatg atcaaacgtt
     5521 atgatgaaca ccatcaggat ttgactttat tgaaagcttt ggtgagacaa caattgccag
     5581 agaagtataa agaaatcttt ttcgatcaat ctaaaaacgg gtatgcaggt tatattgatg
     5641 ggggtgcctc ccaagaggaa ttttacaaat ttataaaacc tattttagaa aagatggatg
     5701 ggactgagga acttttggtc aaattgaaca gagaagattt gttacgtaaa cagagaactt
     5761 ttgataatgg tagtatacct caccaaattc atttgggtga gttgcatgca attttaagaa
     5821 gacaagaaga tttttatcca tttttaaaag ataatagaga aaaaatcgag aaaattttaa
     5881 cctttagaat tccatactat gttgggcctt tggctagagg taattcaaga tttgcctgga
     5941 tgacacgtaa atcagaagaa actattaccc cttggaattt tgaagaggtt gttgataaag
     6001 gagcatcagc acagagtttt attgaaagaa tgaccaattt cgataaaaac ttaccaaatg
     6061 aaaaagtttt accaaaacat tccttgttat acgaatattt tactgtttac aatgaactta
     6121 caaaggttaa atatgttact gaaggtatgc gtaagccagc ctttttatct ggagaacaga
     6181 aaaaggcaat agttgattta ttgtttaaaa caaatagaaa agttactgtt aaacaattaa
     6241 aagaagatta ctttaagaaa attgaatgtt ttgattcagt tgaaatcagt ggtgttgaag
     6301 acagatttaa tgctagttta ggaacttacc atgatttact taaaattatc aaagataaag
     6361 atttcttgga taacgaagaa aatgaagaca ttttagaaga cattgtttta accttaactt
     6421 tattcgaaga tagagagatg attgaagaac gtttgaagac ttatgcacat ttgtttgacg
     6481 ataaagtgat gaaacagttg aaaagaagac gttatactgg atggggtaga ttgtctcgta
     6541 aattgatcaa tggaattaga gataaacaaa gtggtaaaac tatcttggac tttttgaaat
     6601 ctgacggatt tgctaataga aatttcatgc aattgatcca cgacgatagt ttgacattta
     6661 aagaagacat ccaaaaggcc caagtgagtg ggcaaggtga ttcattacat gaacatattg
     6721 caaatttagc cggatctcct gctattaaga aagggatatt acaaactgtt aaagttgtgg
     6781 atgaattagt gaaagtaatg ggaagacata aacctgaaaa cattgtcatt gagatggcaa
     6841 gagaaaatca aactacacaa aaaggacaga aaaatagtag agaacgtatg aaaagaatag
     6901 aagagggtat taaagaattg ggtagtcaaa tattgaaaga acacccagtg gaaaataccc
     6961 agttgcaaaa tgaaaaatta tatctttact accttcaaaa tggacgtgat atgtatgttg
     7021 atcaggaatt agatataaat agactttcag attatgatgt agatcatata gttccacaat
     7081 ctttcttgaa agatgattcc atagacaata aagtattaac tagaagtgat aaaaatagag
     7141 gtaaaagtga taatgtccca agtgaggaag tcgtcaaaaa gatgaaaaat tactggcgtc
     7201 aacttttgaa tgctaaatta attactcaaa gaaaatttga taatttgact aaagcagaaa
     7261 gaggtgggct ttctgaatta gataaagccg ggttcattaa aagacaattg gtcgaaacta
     7321 gacaaattac taaacatgtt gcccaaattt tagattcccg tatgaacact aagtatgacg
     7381 aaaatgataa gttaatacgt gaggttaaag tcattacttt aaaatcaaaa cttgtctctg
     7441 atttcagaaa ggatttccaa ttctataaag ttagagaaat taataattat catcatgctc
     7501 atgatgcata tttgaatgct gtagttggaa ctgctttaat caagaaatac cctaaattag
     7561 aatctgaatt tgtatatggt gattacaaag tctatgatgt tagaaagatg attgctaaat
     7621 cagaacaaga aattggtaaa gctacagcta aatacttctt ttactctaac attatgaatt
     7681 tctttaaaac agaaattact ttggcaaacg gtgaaattag aaaaagacct cttattgaaa
     7741 caaatggtga gactggagag atagtttggg acaaagggcg tgatttcgct actgttagaa
     7801 aagttttatc aatgccacaa gttaacattg taaagaaaac agaggttcaa actggtggtt
     7861 tctcaaaaga aagtattttg cctaaaagaa atagtgataa attgattgcc agaaaaaagg
     7921 attgggatcc aaagaaatat ggtggtttcg actcaccaac cgtagcctat tctgttttgg
     7981 ttgtggcaaa ggttgaaaag ggtaaaagta aaaagcttaa atcagtaaaa gaacttttgg
     8041 gtattacaat aatggaaaga agttcctttg aaaagaaccc tattgatttt ttggaagcta
     8101 aaggttataa ggaagtaaag aaggacttaa taatcaaatt gcctaaatat tctttatttg
     8161 aattagaaaa tgggagaaaa agaatgttgg cttctgctgg agaattgcaa aagggtaatg
     8221 aattagcatt gccttccaaa tatgttaact tcttgtattt agcttcacac tatgaaaagt
     8281 tgaaagggtc accagaagat aacgagcaaa aacaattatt tgttgaacaa cacaaacact
     8341 acttagatga gattatagaa caaattagtg aattcagtaa aagagtgata ttagctgatg
     8401 caaatttaga taaagttttg tcagcctata acaaacatag agataagcca attagagaac
     8461 aagcagaaaa cattattcac ttatttaccc ttaccaattt aggagcacct gctgctttca
     8521 agtattttga tacaacaatt gatcgtaaaa gatatacctc aacaaaagaa gtcttagacg
     8581 ccaccttaat tcatcaatca atcactggat tgtatgagac aagaattgat ttgtctcaat
     8641 tgggtggtga tgaaggggct gatcctaaga agaaaagaaa agttgatcca aagaaaaagc
     8701 gtaaggtgga tcctaagaaa aagagaaagg ttgactacaa agaccatgac ggtgattata
     8761 aagatcatga catcgactac aaggatgacg atgacaagtg ataatgactg cagagatcca
     8821 tcgacctgcc gccaagctaa ttccgggcga atttctgtcg agtcatgtaa ttagttatgt
     8881 cacgcttaca ttcacgccct ccccccacat ccgctctaac cgaaaaggaa ggagttagac
     8941 aacctgaagt ctaggtccct atttattttt ttatagttat gttagtatta agaacgttat
     9001 ttatatttca aatttttctt ttttttcttg gcggcgttag tatcgaatcg acagcagtat
     9061 agcgaccagc attcacatac gattgacgca tgatattact ttctgcgcac ttaacttcgc
     9121 atctgggcag atgatgtcga ggcgaaaaaa aatataaatc acgctaacat ttgattaaaa
     9181 tagaacaact acaatataaa aaaactatac aaatgacaag ttcttgaaaa caagaatctt
     9241 tttattgtca gtactgactc gagttattat ggacatggca tagacatata caaagcttgt
     9301 tcaccatcgg aagcagtacc atcgtataaa gcagtatcca aaccacacaa agtgaaaccc
     9361 attcttctat aagcatgaat agctggagca ttaacattgg taacttccaa ccacaaatga
     9421 ccagcacctc tttctctggc gaattcagta gccaaaccca tcaaagctct accaacacca
     9481 tgacctctat gttctggagc aacttcaata tcttcaacag tcaatcttct gttccaacca
     9541 gaataagaaa caacaacgaa accagccaaa tcaccatcat caccataagc aacgaaagtt
     9601 ctagaatctg gatcaccatc ttcaccagca tcggattcat catcggattc atcatctggg
     9661 aaaaccttag tcaatggtgg atcaactgga acttctctca aagtgaaacc atcaccagta
     9721 gcagtaactc taaaaacagt atcggtagtg aaagaaccat ccaaagcttc aatagcttca
     9781 gcatcacctg gaacagaagt tctgtatcta taagcagtat catccaaagt agtagacata
     9841 attgtaggat ccggttgttt atgttcggat gtgatgtgag aactgtatcc tagcaagatt
     9901 ttaaaaggaa gtatatgaaa gaagaacctc agtggcaaat cctaaccttt tatatttctc
     9961 tacaggggcg cggcgtgggg acaattcaac gcgtctgtga ggggagcgtt tccctgctcg
    10021 caggtctgca gcgaggagcc gtaatttttg cttcgcgccg tgcggccatc aaaatgtatg
    10081 gatgcaaatg attatacatg gggatgtatg ggctaaatgt acgggcgaca gtcacatcat
    10141 gcccctgagc tgcgcacgtc aagactgtca aggagggtat tctgggcctc catgtcgctg
    10201 gccgggtgac ccggcgggga cgaggcaagc ttgatgcggc cgcgttgagg aggtagtagc
    10261 agctaataaa tgagattaaa gagggactaa ctaagatcaa gttgaataaa aacactgatg
    10321 tactaatttt tgaggataat aacgaaagag atgaatggga tggaaaggaa caacgacaac
    10381 aaactgaact gaaactggtg gtgttttttt tttccagaac gaaataggaa aaaaatcgta
    10441 agaagcgatc attacccgga ttaatcttgt gaccttaagt tttgcttatc aattcaaagt
    10501 ttctccgaaa ttttattgtt tcttgatttg gtcaagtggt aatttccttc ctaaaagggt
    10561 ttgggttcga aactaccttt gtgaacagta ataggagacg gaattccgtc tcgttttaga
    10621 gctagaaata gcaagttaaa ataaggctag tccgttatca acttgaaaaa gtggcaccga
    10681 gtcggtgctt tttttctcga gtttttttat cgagtgttta aggataatga taactgaaga
    10741 gaagaattag ttttgccgcc accgcgggtt tgcctctgat taaataaaaa aaagctggtg
    10801 cttttttttt cttttatagg aacatcttga atatatgaac taattaaatg ataatttttt
    10861 acccatcttt actcttaatc actgagctgc agtcaaagaa aaagggatac agcacctggt
    10921 gaagagatga acggagacta acttagacgc gttgattctt tttaattgca cattttatta
    10981 atcgatgcta acgtctattt acatatattc tttagagata ttatctaggg cttcaaataa
    11041 tctctggaca gcaataaaag tctcttcaaa agtattgtat aacggcaatg gggctaatct
    11101 gattacatct ggtcttcttt cgtcacagat tatagcatga tcatgcaagt acgcattaac
    11161 tcgttccatg acgttcttgt ccttttcatc gaaatgcggt tgaaacataa tggacaattg
    11221 acatcctctt tcagctggat tcaaaggagt taaaatttta aacccaaatt tggagtttga
    11281 tgtactggat tgtggtatgt aatacttgga attcgtcaat agatcctgta aaaattgagt
    11341 caaagcaaca cttttttcac gaagtttaga tactccaccc actttagcat acacttccaa
    11401 tgacgacttc acagcaacaa catcaagaac agaaggattt gactgtctgt aagaaagagc
    11461 cgagtttatt ggatcaaact cttctaacat tttgaatcgt tcttgggagt tattgcccca
    11521 ccaaccagct agtctaggaa cgaaactgct tttcttgttc tctatggtgt atttttcatg
    11581 cacaaaaatc ccacctatgg ctccaggtcc cgagtttaaa tatttgtagg aacaccaagc
    11641 agcaaaatct actccccaat catgtaaatt taatgggaca ttcccaactg catgggcaag
    11701 atcccaccca actttaattt gttggctctt ttccttagcg tatttagtta tttcctctat
    11761 cttgaaaaat tgaccagtgt agtattggat accaggaaaa cacactagag ccaattcatc
    11821 caggttctca tctatagcct tgattattct ttctgtttta atataagttt caccaggttg
    11881 aacttccaat tgaatcaaat gtttctcgtc gtatccgaac aatttaacaa tgttcaaaaa
    11941 tgcatagtag tcagaaggaa atgcttgttt ttcaaataaa attttggttc ttttcccctc
    12001 aggtttgtaa aaatggatca acaatgcatt caagtttgct gttaaagaac ccataactgc
    12061 aacttcgttt tcctttgcac caacaatggg ggctattaat ggtaataagg gtaaatcgat
    12121 gtctacccac ggtgttaaca gtttgtcagg atgattgaaa tgagactcaa cccctcgttc
    12181 aacccatgca tttaattcat cattgatagc tttctttgta ttcttaggca tcaacccaag
    12241 agagtttcca cataaataaa tagactcagt tgatgactca tatttattat ttttgatacc
    12301 taatgatcca aaagttggta tggcaaactc atttttaaaa gttgggaact ttttgtccaa
    12361 tttctttgcc tcggctaatg acatctgata ataaaatggg gttggagtag ttggtggtat
    12421 aaccggagag atagaattga agaaaaaaat cggaaacaac aaaaaaagtt gataccctgt
    12481 attatgtggg agataattgc gaatggtgga aaaaaaaaag acgccattga gtctcaacaa
    12541 caattctgtc agctgaagag ctttacaatc gagaaactat gattcattcc gttttaatat
    12601 gtatgtgttt agtaaactca tgaattttat ttgtggtcta ctttagtact aacataatca
    12661 ttggatagtc aataatgatg gtcttccgag actaatgaaa ttctatacca aagtcgatat
    12721 tccaacacag aaattgctct tgcaacaagt gcacctgttg atatctagag ctccagcttt
    12781 tgttcccttt agtgagggtt aattgcgcgc ttggcgtaat catggtcata gctgtttcct
    12841 gtgtgaaatt gttatccgct cacaattcca cacaacatag gagccggaag cataaagtgt
    12901 aaagcctggg gtgcctaatg agtgaggtaa ctcacattaa ttgcgttgcg ctcactgccc
    12961 gctttccagt cgggaaacct gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg
    13021 agaggcggtt tgcgtattgg gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg
    13081 gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg cggtaatacg gttatccaca
    13141 gaatcagggg ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac
    13201 cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc gcccccctga cgagcatcac
    13261 aaaaatcgac gctcaagtca gaggtggcga aacccgacag gactataaag ataccaggcg
    13321 tttccccctg gaagctccct cgtgcgctct cctgttccga ccctgccgct taccggatac
    13381 ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc atagctcacg ctgtaggtat
    13441 ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag
    13501 cccgaccgct gcgccttatc cggtaactat cgtcttgagt ccaacccggt aagacacgac
    13561 ttatcgccac tggcagcagc cactggtaac aggattagca gagcgaggta tgtaggcggt
    13621 gctacagagt tcttgaagtg gtggcctaac tacggctaca ctagaaggac agtatttggt
    13681 atctgcgctc tgctgaagcc agttaccttc ggaaaaagag ttggtagctc ttgatccggc
    13741 aaacaaacca ccgctggtag cggtggtttt tttgtttgca agcagcagat tacgcgcaga
    13801 aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc tcagtggaac
    13861 gaaaactcac gttaagggat tttggtcatg agattatcaa aaaggatctt cacctagatc
    13921 cttttaaatt aaaaatgaag ttttaaatca atctaaagta tatatgagta aacttggtct
    13981 gacagttacc aatgcttaat cagtgaggca cctatctcag cgatctgtct atttcgttca
    14041 tccatagttg cctgactccc cgtcgtgtag ataactacga tacgggaggg cttaccatct
    14101 ggccccagtg ctgcaatgat accgcgagac ccacgctcac cggctccaga tttatcagca
    14161 ataaaccagc cagccggaag ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc
    14221 atccagtcta ttaattgttg ccgggaagct agagtaagta gttcgccagt taatagtttg
    14281 cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct
    14341 tcattcagct ccggttccca acgatcaagg cgagttacat gatcccccat gttgtgcaaa
    14401 aaagcggtta gctccttcgg tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta
    14461 tcactcatgg ttatggcagc actgcataat tctcttactg tcatgccatc cgtaagatgc
    14521 ttttctgtga ctggtgagta ctcaaccaag tcattctgag aatagtgtat gcggcgaccg
    14581 agttgctctt gcccggcgtc aatacgggat aataccgcgc cacatagcag aactttaaaa
    14641 gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct caaggatctt accgctgttg
    14701 agatccagtt cgatgtaacc cactcgtgca cccaactgat cttcagcatc ttttactttc
    14761 accagcgttt ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg
    14821 gcgacacgga aatgttgaat actcatactc ttcctttttc aatattattg aagcatttat
    14881 cagggttatt gtctcatgag cggatacata tttgaatgta tttagaaaaa taaacaaata
    14941 ggggttccgc gcacatttcc ccgaaaagtg ccacctgggt ccttttcatc acgtgctata
    15001 aaaataatta taatttaaat tttttaatat aaatatataa attaaaaata gaaagtaaaa
    15061 aaagaaatta aagaaaaaat agtttttgtt ttccgaagat gtaaaagact ctagggggat
    15121 cgccaacaaa tactaccttt tatcttgctc ttcctgctct caggtattaa tgccgaattg
    15181 tttcatcttg tctgtgtaga agaccacaca cgaaaatcct gtgattttac attttactta
    15241 tcgttaatcg aatgtatatc tatttaatct gcttttcttg tctaataaat atatatgtaa
    15301 agtacgcttt ttgttgaaat tttttaaacc tttgtttatt tttttttctt cattccgtaa
    15361 ctcttctacc ttctttattt actttctaaa atccaaatac aaaacataaa aataaataaa
    15421 cacagagtaa attcccaaat tattccatca ttaaaagata cgaggcgcgt gtaagttaca
    15481 ggcaagcgat ccgtcctaag aaaccattat tatcatgaca ttaacctata aaaataggcg
    15541 tatcacgagg ccctttcgtc
//
