LOCUS       sequence-272086-       19102 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses R. toruloides codon-optimized SpCas9 under PGK1 promoter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 19102)
  AUTHORS   Schultz JC, Cao M, Zhao H
  TITLE     Development of a CRISPR/Cas9 system for high efficiency multiplexed 
            gene deletion in Rhodosporidium toruloides.
  JOURNAL   Biotechnol Bioeng. 2019 Apr 30. doi: 10.1002/bit.27001.
  PUBMED    31038202
REFERENCE   2  (bases 1 to 19102)
  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: "Biotechnol 
            Bioeng. 2019 Apr 30. doi: 10.1002/bit.27001."
COMMENT     Sequence Label: NM9-SpCas9-NLS3
FEATURES             Location/Qualifiers
     source          1..19102
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_feature    939..963
                     /label=LB T-DNA repeat
                     /note="left border repeat from nopaline C58 T-DNA"
     CDS             complement(4092..6476)
                     /codon_start=1
                     /product="C-terminal portion of Streptococcus pyogenes Cas9
                     (Zetsche et al., 2015)"
                     /label=Cas9(C)
                     /translation="CFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILED
                     IVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGK
                     TILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKG
                     ILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQI
                     LKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDN
                     KVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDK
                     AGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQF
                     YKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGK
                     ATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMP
                     QVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKV
                     EKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELEN
                     GRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLD
                     EIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKY
                     FDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGD"
     misc_feature    9930..9954
                     /label=RB T-DNA repeat
                     /note="right border repeat from nopaline C58 T-DNA"
     CDS             11255..11884
                     /codon_start=1
                     /product="stability protein from the Pseudomonas plasmid 
                     pVS1 (Heeb et al., 2000)"
                     /label=pVS1 StaA
                     /translation="MKVIAVLNQKGGSGKTTIATHLARALQLAGADVLLVDSDPQGSAR
                     DWAAVREDQPLTVVGIDRPTIDRDVKAIGRRDFVVIDGAPQAADLAVSAIKAADFVLIP
                     VQPSPYDIWATADLVELVKQRIEVTDGRLQAAFVVSRAIKGTRIGGEVAEALAGYELPI
                     LESRITQRVSYPGTAAAGTTVLESEPEGDAAREVQALAAEIKSKLI"
     CDS             12313..13386
                     /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      13452..13646
                     /label=pVS1 oriV
                     /note="origin of replication for the Pseudomonas plasmid 
                     pVS1 (Heeb et al., 2000)"
     primer_bind     complement(13882..13904)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    13990..14130
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     14045..14064
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(14145..14162)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(14316..14904)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(14396..14415)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(16329..17123)
                     /codon_start=1
                     /gene="aphA-3"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin"
                     /translation="MAKMRISPELKKLIEKYRCVKDTEGMSPAKVYKLVGENENLYLKM
                     TDSRYKGTTYDVEREKDMMLWLEGKLPVPKVLHFERHDGWSNLLMSEADGVLCSEEYED
                     EQSPEKIIELYAECIRLFHSIDISDCPYTNSLDSRLAELDYLLNNDLADVDCENWEEDT
                     PFKDPRELYDFLKTEKPEEELVFSHGDLGDSNIFVKDGKVSGFIDLGRSGRADKWYDIA
                     FCVRSIREDIGEEQYVELFFDLLGIKPDWEKIKYYILLDELF"
     CDS             complement(17310..18113)
                     /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
                     YTKGTVDIAKSDKDFVIGFIAQRDMGGGDEGYDWLIMTPGVGLDDKGDALGQQYRTVDD
                     VVSTGSDIIIVGRGLFAKGRDAKVEGERYRKAGWEAYLRRCGQQN"
     promoter        complement(18114..18329)
                     /gene="S. cerevisiae URA3"
                     /label=URA3 promoter
ORIGIN
        1 gcgctaattt ttcaaacaaa gaatctgagc tgcattttta cagaacagaa atgcaacgcg
       61 agagcgctat tttaccaaca aagaatctat acttcttttt tgttctacaa aaatgcatcc
      121 cgagagcgct atttttctaa caaagcatct tagattactt tttttctcct ttgtgcgctc
      181 tataatgcag tctcttgata actttttgca ctgtaggtcc gttaaggtta gaagaaggct
      241 actttggtgt ctattttctc ttccataaaa aaagcctgac tccacttccc gcgtttactg
      301 attactagcg aagctgcggg tgcatttttt caagataaag gcatccccga ttatattcta
      361 taccgatgtg gattgcgcat actttgtgaa cagaaagtga tagcgttgat gattcttcat
      421 tggtcagaaa attatgaacg gtttcttcta ttttgtctct atatactacg tataggaaat
      481 gtttacattt tcgtattgtt ttcgattcac tctatgaata gttcttacta caattttttt
      541 gtctaaagag taatactaga gataaacata aaaaatgtag aggtcgagtt tagatgcaag
      601 ttcaaggagc gaaaggtgga tgggtaggtt atatagggat atagcacaga gatatatagc
      661 aaagagatac ttttgagcaa tgtttgtgga agcgccgcgg tgatcacagg cagcaacgct
      721 ctgtcatcgt tacaatcaac atgctaccct ccgcgagatc atccgtgttt caaacccggc
      781 agcttagttg ccgttcttcc gaatagcatc ggtaacatga gcaaagtctg ccgccttaca
      841 acggctctcc cgctgacgcc gtcccggact gatgggctgc ctgtatcgag tggtgatttt
      901 gtgccgagct gccggtcggg gagctgttgg ctggctggtg gcaggatata ttgtggtgta
      961 aacaaattga cgcttagaca acttaataac acattgcgga cgtttttaat gtactgaatt
     1021 aacgccgaat taattcctag gccaccatgt tgggcccggc gcgccgaatt cgagctcggt
     1081 acccaactgc agaactacgc cctcgcgcat ccgacttcgg actcgaccgg cgaaacgact
     1141 gcgacctatt tctactgctt gccttcgacg tccgggcgag tcgccgctta ccccgtgcgc
     1201 tctcctcccc ctctggcccc tcaaactgca actcctgacc gcggcgatgc gcgcagtacc
     1261 ccgtcaaaat gcaactctac gcgactttcg gcttagaagt cgccaagctc cgtgcttcgc
     1321 cgcctcgagc tcttgcgttg cctgcagacg tcgtctatca cgaggcggag aagctcgagt
     1381 tgccggcttt gccgagggcg gtcaaggttg agtcggagac tgtcaaggta gaagttcgag
     1441 aagaggagga tgaggttaag ggtgaggtgc tgttagtgaa gacggactcg gttgtgcagg
     1501 aggacactga agcgggctcg gaaggagtgc aggttgagga aacgatgctg gtcaagctgg
     1561 aggcgagcag ttgattgccc atccttgcaa ctcacagctg cttcacgttc tctgcgcctc
     1621 gttccatgca ctgcgtcgca cttgtcggcg ggcgaagaca cgaggagagg acaggctgtg
     1681 tcctcgagcg agagtagatg ggaaggttac tggaagtcgt gctgttttct atcggcttgc
     1741 acgcatctgc tctggttcac tcgctgtgag gcccagcttc catgctgctg cgatctggga
     1801 gtgcaagccc gcggtcagca gccattgccc gcctacaatg tcttcccaaa gccgcccttg
     1861 ctcgctcgct gcgtggagtt cgacgttctc ctcgctccgc aagcattgga atgaaccttg
     1921 ctctctagtt ccctcctccg tgacctcgtt tcgtccttta gacggcacga tggaaggaag
     1981 aaatctctgc ggacaagcaa atctgctggc tcgccttgta ggtcgcctac cggagcaagc
     2041 cttgtgccgc cgggatgcca acgtcgtttt ttgacgtttg caagacgtag aggacgcttc
     2101 ggacgacgaa acaagctgtg aggacatgga agtcgtggga ggaacggcgc agagcggcgc
     2161 cgcgggagca taaggcaagc gagatagtcc agaaatcgcg gcgccaagta cagtaattta
     2221 ttggagcagg caccagaagc gggcagcagt atgcgcaggc ttggggtcga cgagagacga
     2281 ctccctcata ctcggttacc tcgagcaata caatcaatcg aagctgcgcg aatctcggct
     2341 tgtaagggtc ggaaaggaac ctcggagatg gccacgtcac atcaccaact tatcgatctc
     2401 agccgacgtc gcagagaggg cgagcgaagc ggtgaaggag ggaaacaatc cctcgagagc
     2461 atgatccgtc tgaatctgca gcgcaggaag ccgtcacacg cccgcctcga gcgcaggtcg
     2521 ggtccagccg ggggacgaaa cgcgcgaggg ctgatttcgt gagcgaagga agccgcatcg
     2581 acaagttcgc gtccctttgc cctctttccc atcacccgct ctcgctctac ccgctcagaa
     2641 caacaccaga tcagtcacaa tgggcaagga gaagacgcac gtctcgcgcc cgcgcctcaa
     2701 ctcgaacatg gacgccgacc tctacggcta caagtggtcc cgcgacaacg tcggccagtc
     2761 gggcgccacg atctaccgcc tctacggcaa gccggacgcc ccggagctct tcctcaagca
     2821 cggcaagggc tcggtcgcca acgacgtcac ggacgagatg gtccgcctca actggctcac
     2881 ggagttcatg ccgctcccga cgatcaagca cttcatccgc acgccggacg acgcctggct
     2941 cctcacgacg gccatcccgg gcaagacggc cttccaggtc ctcgaggagt acccggactc
     3001 gggcgagaac atcgtcgacg ccctcgccgt cttcctccgc cgcctccact cgatcccggt
     3061 ctgcaactgc ccgttcaact cggaccgcgt cttccgcctc gcccaggccc agtcgcgcat
     3121 gaacaacggc ctcgtcgacg cctcggactt cgacgacgag cgcaacggct ggccggtcga
     3181 gcaggtctgg aaggagatgc acaagctcct cccgttctcg ccggactcgg tcgtcacgca
     3241 cggcgacttc tcgctcgaca acctcatctt cgacgagggc aagctcatcg gctgcatcga
     3301 cgtcggccgc gtcggcatcg ccgaccgcta ccaggacctc gccatcctct ggaactgcct
     3361 cggcgagttc tcgccgtcgc tccagaagcg cctcttccag aagtacggca tcgacaaccc
     3421 ggacatgaac aagctccagt tccacctcat gctcgacgag ttcttctaga tcattccact
     3481 caacattcag gctcctctgc gcacgtaaag tgccaaaggc aataccctgc tcggtggaat
     3541 gccgccgggc ttgtcgattt tacgcacata tgcgcattct tgacttgaag cggaggagtt
     3601 cttcgttgcg ggttacagtg ttttaataaa agaatggtca aatcaaactg ctagatatac
     3661 ctgtcagaca ctctagttgt tgacccctat actcttaata catcagacag tacatgcatg
     3721 ttgcatgatg atgataatgt ctgtttagat tccaagtgtc tactgctggc gtcgacctag
     3781 ggggatcctc tagagtcgac ctgcaggcat gcaagcttta agaggagtcc accatggtag
     3841 atctactgga ttttggtttt aggaattaga aattttattg atagaagtat tttacaaata
     3901 caaatacata ctaagggttt cttatatgct caacacatga gcgaaaccct ataggaaccc
     3961 taattccctt atctgggaac tactcacaca ttattatgga gaaaactagt ctactcgtac
     4021 ttgacgcgct tgcgggtgcc cgcgtcgccc gcctcgcact cgccgaggcg gcgcttgagt
     4081 tcgcggatct cgtcgccgcc gagctgcgag aggtcgatgc gcgtctcgta gaggccggtg
     4141 atcgactggt ggatgagggt cgcgtcgagg acttccttgg tcgaggtgta gcgcttgcgg
     4201 tcgatggtgg tgtcgaagta cttgaacgcg gcaggggcgc cgaggttggt gagggtgaag
     4261 aggtggatga tgttctccgc ctgctcgcgg atgggcttgt cgcggtgctt gttgtaggcc
     4321 gagaggacct tgtcgaggtt cgcgtcggcg aggatgacgc gcttcgagaa ctcgctgatc
     4381 tgctcgatga tctcgtcgag gtagtgcttg tgctgctcga cgaagagctg cttctgctcg
     4441 ttgtcctcgg gcgagccctt gagcttctcg tagtgcgacg cgaggtagag gaagttgacg
     4501 tacttcgacg ggagggcgag ttcgttgccc ttctggagtt cgccggccga ggcgagcatg
     4561 cgcttgcggc cgttctccaa ctcgaagagc gagtacttgg ggagcttgat gatgaggtcc
     4621 ttcttgactt ccttgtagcc cttcgcttcg aggaagtcga tcgggttctt ctcgaacgac
     4681 gagcgctcca tgatggtgat gccgaggagt tccttgaccg acttgagctt cttcgacttg
     4741 cccttctcga ccttcgcgac gacgaggacc gagtaggcga ccgtcggcga gtcgaatccg
     4801 ccgtacttct tcgggtccca gtccttcttg cgggcgatga gcttgtccga gttgcgcttg
     4861 gggaggatcg actccttcga gaagccgccg gtctggacct cggtcttctt gacgatgttg
     4921 acctgcggca tcgagaggac cttgcggacg gtggcgaagt cgcggccctt gtcccagacg
     4981 atctcgcccg tctcgccgtt ggtctcgatg agggggcgct tgcggatctc gccgttggcg
     5041 agggtgatct cggtcttgaa gaagttcatg atgttcgagt agaagaagta cttcgcggtg
     5101 gccttgccga tctcctgctc cgacttggcg atcatcttgc ggacgtcgta gaccttgtag
     5161 tcgccgtaga cgaactccga ttcgagcttc gggtacttct tgatgagcgc ggtgccgacg
     5221 accgcgttga ggtaggcgtc gtgggcgtgg tggtagttgt tgatctcgcg gaccttgtag
     5281 aactggaagt ccttgcggaa gtccgagacg agcttcgact tgagggtgat gaccttgacc
     5341 tcgcggatga gcttgtcgtt ctcgtcgtac ttggtgttca tgcgcgagtc gaggatctgc
     5401 gcgacgtgct tggtgatctg gcgggtctcg acgagctggc gcttgatgaa gcccgccttg
     5461 tcgagttccg agagtccgcc gcgctccgcc ttcgtgaggt tgtcgaactt gcgctgggtg
     5521 atgagcttgg cgttgaggag ctggcgccag tagttcttca tcttcttgac gacttcctcc
     5581 gaggggacgt tgtccgactt gccgcggttc ttgtccgagc gggtgaggac cttgttgtcg
     5641 atcgagtcgt ccttgaggaa cgactgcggg acgatgtggt ccacgtcgta gtccgagagg
     5701 cggttgatgt cgagttcctg gtccacgtac atgtcgcggc cgttctggag gtagtagagg
     5761 tagagcttct cgttctggag ctgggtgttc tcgacggggt gctccttgag gatctgcgag
     5821 ccgagttcct tgatgccttc ctcgatgcgc ttcatgcgct cgcgcgagtt cttctggccc
     5881 ttctgggtgg tctggttctc gcgggccatc tcgatgacga tgttctcggg cttgtggcgg
     5941 cccatgacct tgacgagttc gtccacgacc ttgacggtct ggaggatgcc cttcttgatc
     6001 gcgggcgagc cggcgaggtt ggcgatgtgc tcgtggagcg agtcgccctg gcccgagacc
     6061 tgggccttct ggatgtcttc cttgaaggtg agcgagtcgt cgtggatgag ctgcatgaag
     6121 ttgcggttgg cgaagccgtc cgacttgagg aagtcgagga tggtcttgcc cgactgcttg
     6181 tcgcggatgc cgttgatgag cttgcgcgag aggcggcccc agcccgtgta tcggcggcgc
     6241 ttgagctgct tcatgacctt gtcgtcgaag aggtgggcgt aggtcttgag gcgctcctcg
     6301 atcatctcgc ggtcctcgaa gagggtgagg gtgaggacga tgtcttcgag gatgtcctcg
     6361 ttctcctcgt tgtcgaggaa gtccttgtcc ttgatgatct tgaggaggtc gtggtaggtg
     6421 ccgagcgacg cgttgaagcg gtcctcgacg ccgctgatct cgaccgagtc gaagcactcg
     6481 atcttcttga agtagtcttc cttgagctgc ttgacggtga ccttgcggtt ggtcttgaag
     6541 aggaggtcca cgatcgcctt cttctgctcg cccgagagga aggccggctt gcgcatgccc
     6601 tcggtgacgt acttgacctt ggtgagttcg ttgtagacgg tgaagtactc gtagaggagc
     6661 gagtgcttgg ggaggacctt ctcgttcggg aggttcttgt cgaagttggt catgcgctcg
     6721 atgaacgact gggccgaggc gcccttgtcc acgacttcct cgaagttcca gggggtgatg
     6781 gtctcctccg acttgcgggt catccacgcg aagcgcgagt tgccgcgggc gagggggccg
     6841 acgtagtacg ggatgcggaa ggtgaggatc ttctcgatct tctcgcggtt gtccttgagg
     6901 aacgggtaga agtcttcctg gcggcggagg atggcgtgga gttcgccgag gtggatctgg
     6961 tgggtgatcg agccgttgtc gaaggtgcgc tgcttgcgga ggaggtcctc gcggttgagc
     7021 ttgacgagga gttcctcggt gccgtccatc ttttcgagga tcggcttgat gaacttgtag
     7081 aactcttcct gcgacgcgcc gccgtcgatg tagcccgcgt agccgttctt cgactggtcg
     7141 aagaagattt ccttgtactt ctcggggagc tgctggcgga cgagcgcctt gaggagcgtg
     7201 aggtcctggt ggtgctcgtc gtagcgcttg atcatcgacg ccgagagcgg ggccttggtg
     7261 atctcggtgt tgacgcggag gatgtccgag aggaggatgg cgtccgagag gttcttcgcg
     7321 gcgaggaaga ggtccgcgta ctggtcgccg atctgggcga ggaggttgtc gaggtcgtcg
     7381 tcgtaggtgt ccttcgagag ctggagcttc gcgtcctcgg cgaggtcgaa gttcgacttg
     7441 aagttggggg tgaggccgag cgagagggcg atgaggttgc cgaagaggcc gttcttcttc
     7501 tcgcccggga gctgggcgat gaggttttcg aggcggcgcg acttcgagag gcgggccgag
     7561 aggatggcct tggcgtccac gcccgaggcg ttgatggggt tctcctcgaa gagctggttg
     7621 taggtctgga cgagctggat gaagagcttg tccacgtccg agttgtccgg gttgaggtcg
     7681 ccctcgatga ggaagtggcc gcggaacttg atcatgtgcg cgagggcgag gtagatgagg
     7741 cggaggtccg ccttgtaggt cgagtccacg agcttcttgc ggaggtggta gatggtgggg
     7801 tacttctcgt ggtaggcgac ctcgtccacg atgttgccga agatcgggtg gcgctcgtgc
     7861 ttcttgtctt cctcgacgag gaacgactct tcgaggcggt ggaagaacga gtcgtccacc
     7921 ttcgccatct cgttcgagaa gatttcctgg aggtagcaga tgcggttctt gcggcgggtg
     7981 tatcggcggc gggccgtgcg cttgaggcgg gtggcctccg ccgtctcgcc cgagtcgaag
     8041 aggagggcgc cgatgaggtt cttcttgatc gagtggcggt cggtgttgcc gaggaccttg
     8101 aacttcttcg acgggacctt gtactcgtcg gtgatgacgg cccagccgac cgagttggtg
     8161 ccgatgtcga ggccgatcga gtacttcttg tccatgtggg agggtgggag ttggacgggt
     8221 gagttgctgt gaacctgcag agtagtaagc gagtgactgt agctgtacca gagcgagtgc
     8281 gagtgagaag tgcaagttca acgatgcggc cgcacccgac gagcgagtga cgagcagggt
     8341 cgacctcgct cacccgcttg cactcgtctg gcgcattcgc tgacaggcta gacagactca
     8401 cacaggcttg ttctcccagg gccaagtcga gcggcgcact ccgagcgggt caggcgacga
     8461 ccacctcctc cgcttggact cgccagggaa gagtcgaccg actcgaggga tacaagggag
     8521 tgggacatga tacgagcaac agagtgcact gcagggatgg acaacacagg cgcagggatc
     8581 cgcccgccgt cgctcaccca gcccctcgtc tcgctcgcca gcatctcgac ggagcacttt
     8641 cggccctccc ctctcacgct cgactggctg tactcgcttc cattgccgcc aggatgtccc
     8701 gtccaacctt tcccacagac gagcgaggac tgggccctgg tcgagcagta ccgcctgctc
     8761 tgccaggggt tgacggtcat ggcggagtac gagcgggagc gggcgaggcg ccagcagcgc
     8821 ggggaggatc cggatgcgca gccgtgggat cctaggagtg cgtcgcagcg ttctgcctct
     8881 tctgccgcgc ttgcttacag ccacttgcct cgacagcctc ggacgacccg gccgtcgtct
     8941 tcgccgccga atgcctcttc aacgggccag actgggtcga gtcggaggac cacgcgtcgt
     9001 cgccgtccga gcacgacgac gccgcctacg aggtgatcga ctcgccctcg caagcggccg
     9061 acgcagcctt cctgggcgtc ctctccgccg tccagcaaca acaacagcgg aacgcctctt
     9121 cctcgtcctc gacatccttc acgacctcga tccccctctc cctctcggac ttgacagccc
     9181 tctcctccgc cctccccctc tcgtcctcct cctctcaacc gcctcaaacc cacctctcac
     9241 cctcccaaat ccccgcagac ggcatcctct ccgactcggc tctctcaccc gcctccctcc
     9301 caagcttgat gagcgccatc gccgagttgg agcagtgcgt cgcgcgattg agtttggagg
     9361 cgagcgaggc gaggaggttg cagaggcgga tgaggggggc cgtggtcggt gcttcggcgg
     9421 cgaatgtggg ggcggtgggt gatggagaag cagaggcggc gaagaagaag ggcaagaaca
     9481 ggaagaagaa gaaaaaggtc gccccagccc ctcctcctcc tccccctctc cctccggctg
     9541 tccctgcgcc acccgcgccg aaacagcgcg aaggagcagg aggagacgac tcggatttgt
     9601 tgaacgcgct gcactcggtt tcgatgctgg atcgcagggc ggacgagtac cgcgagcgat
     9661 tgagggttct caaggtccgt ctggcttaag attgaatcct gttgccggtc ttgcgatgat
     9721 tatcatataa tttctgttga attacgttaa gcatgtaata attaacatgt aatgcatgac
     9781 gttatttatg agatgggttt ttatgattag agtcccgcaa ttatacattt aatacgcgat
     9841 agaaaacaaa atatagcgcg caaactagga taaattatcg cgcgcggtgt catctatgtt
     9901 actagatcgg gaattaaact atcagtgttt gacaggatat attggcgggt aaacctaaga
     9961 gaaaagagcg tttattagaa taatcggata tttaaaaggg cgtgaaaagg tttatccgtt
    10021 cgtccatttg tatgtgcatg ccaaccacag ggttcccctc gggatcaaag tactttgatc
    10081 caacccctcc gctgctatag tgcagtcggc ttctgacgtt cagtgcagcc gtcttctgaa
    10141 aacgacatgt cgcacaagtc ctaagttacg cgacaggctg ccgccctgcc cttttcctgg
    10201 cgttttcttg tcgcgtgttt tagtcgcata aagtagaata cttgcgacta gaaccggaga
    10261 cattacgcca tgaacaagag cgccgccgct ggcctgctgg gctatgcccg cgtcagcacc
    10321 gacgaccagg acttgaccaa ccaacgggcc gaactgcacg cggccggctg caccaagctg
    10381 ttttccgaga agatcaccgg caccaggcgc gaccgcccgg agctggccag gatgcttgac
    10441 cacctacgcc ctggcgacgt tgtgacagtg accaggctag accgcctggc ccgcagcacc
    10501 cgcgacctac tggacattgc cgagcgcatc caggaggccg gcgcgggcct gcgtagcctg
    10561 gcagagccgt gggccgacac caccacgccg gccggccgca tggtgttgac cgtgttcgcc
    10621 ggcattgccg agttcgagcg ttccctaatc atcgaccgca cccggagcgg gcgcgaggcc
    10681 gccaaggccc gaggcgtgaa gtttggcccc cgccctaccc tcaccccggc acagatcgcg
    10741 cacgcccgcg agctgatcga ccaggaaggc cgcaccgtga aagaggcggc tgcactgctt
    10801 ggcgtgcatc gctcgaccct gtaccgcgca cttgagcgca gcgaggaagt gacgcccacc
    10861 gaggccaggc ggcgcggtgc cttccgtgag gacgcattga ccgaggccga cgccctggcg
    10921 gccgccgaga atgaacgcca agaggaacaa gcatgaaacc gcaccaggac ggccaggacg
    10981 aaccgttttt cattaccgaa gagatcgagg cggagatgat cgcggccggg tacgtgttcg
    11041 agccgcccgc gcacgtctca accgtgcggc tgcatgaaat cctggccggt ttgtctgatg
    11101 ccaagctggc ggcctggccg gccagcttgg ccgctgaaga aaccgagcgc cgccgtctaa
    11161 aaaggtgatg tgtatttgag taaaacagct tgcgtcatgc ggtcgctgcg tatatgatgc
    11221 gatgagtaaa taaacaaata cgcaagggga acgcatgaag gttatcgctg tacttaacca
    11281 gaaaggcggg tcaggcaaga cgaccatcgc aacccatcta gcccgcgccc tgcaactcgc
    11341 cggggccgat gttctgttag tcgattccga tccccagggc agtgcccgcg attgggcggc
    11401 cgtgcgggaa gatcaaccgc taaccgttgt cggcatcgac cgcccgacga ttgaccgcga
    11461 cgtgaaggcc atcggccggc gcgacttcgt agtgatcgac ggagcgcccc aggcggcgga
    11521 cttggctgtg tccgcgatca aggcagccga cttcgtgctg attccggtgc agccaagccc
    11581 ttacgacata tgggccaccg ccgacctggt ggagctggtt aagcagcgca ttgaggtcac
    11641 ggatggaagg ctacaagcgg cctttgtcgt gtcgcgggcg atcaaaggca cgcgcatcgg
    11701 cggtgaggtt gccgaggcgc tggccgggta cgagctgccc attcttgagt cccgtatcac
    11761 gcagcgcgtg agctacccag gcactgccgc cgccggcaca accgttcttg aatcagaacc
    11821 cgagggcgac gctgcccgcg aggtccaggc gctggccgct gaaattaaat caaaactcat
    11881 ttgagttaat gaggtaaaga gaaaatgagc aaaagcacaa acacgctaag tgccggccgt
    11941 ccgagcgcac gcagcagcaa ggctgcaacg ttggccagcc tggcagacac gccagccatg
    12001 aagcgggtca actttcagtt gccggcggag gatcacacca agctgaagat gtacgcggta
    12061 cgccaaggca agaccattac cgagctgcta tctgaataca tcgcgcagct accagagtaa
    12121 atgagcaaat gaataaatga gtagatgaat tttagcggct aaaggaggcg gcatggaaaa
    12181 tcaagaacaa ccaggcaccg acgccgtgga atgccccatg tgtggaggaa cgggcggttg
    12241 gccaggcgta agcggctggg ttgcctgccg gccctgcaat ggcactggaa cccccaagcc
    12301 cgaggaatcg gcgtgagcgg tcgcaaacca tccggcccgg tacaaatcgg cgcggcgctg
    12361 ggtgatgacc tggtggagaa gttgaaggcc gcgcaggccg cccagcggca acgcatcgag
    12421 gcagaagcac gccccggtga atcgtggcaa gcggccgctg atcgaatccg caaagaatcc
    12481 cggcaaccgc cggcagccgg tgcgccgtcg attaggaagc cgcccaaggg cgacgagcaa
    12541 ccagattttt tcgttccgat gctctatgac gtgggcaccc gcgatagtcg cagcatcatg
    12601 gacgtggccg ttttccgtct gtcgaagcgt gaccgacgag ctggcgaggt gatccgctac
    12661 gagcttccag acgggcacgt agaggtttcc gcagggccgg ccggcatggc cagtgtgtgg
    12721 gattacgacc tggtactgat ggcggtttcc catctaaccg aatccatgaa ccgataccgg
    12781 gaagggaagg gagacaagcc cggccgcgtg ttccgtccac acgttgcgga cgtactcaag
    12841 ttctgccggc gagccgatgg cggaaagcag aaagacgacc tggtagaaac ctgcattcgg
    12901 ttaaacacca cgcacgttgc catgcagcgt acgaagaagg ccaagaacgg ccgcctggtg
    12961 acggtatccg agggtgaagc cttgattagc cgctacaaga tcgtaaagag cgaaaccggg
    13021 cggccggagt acatcgagat cgagctagct gattggatgt accgcgagat cacagaaggc
    13081 aagaacccgg acgtgctgac ggttcacccc gattactttt tgatcgatcc cggcatcggc
    13141 cgttttctct accgcctggc acgccgcgcc gcaggcaagg cagaagccag atggttgttc
    13201 aagacgatct acgaacgcag tggcagcgcc ggagagttca agaagttctg tttcaccgtg
    13261 cgcaagctga tcgggtcaaa tgacctgccg gagtacgatt tgaaggagga ggcggggcag
    13321 gctggcccga tcctagtcat gcgctaccgc aacctgatcg agggcgaagc atccgccggt
    13381 tcctaatgta cggagcagat gctagggcaa attgccctag caggggaaaa aggtcgaaaa
    13441 ggactctttc ctgtggatag cacgtacatt gggaacccaa agccgtacat tgggaaccgg
    13501 aacccgtaca ttgggaaccc aaagccgtac attgggaacc ggtcacacat gtaagtgact
    13561 gatataaaag agaaaaaagg cgatttttcc gcctaaaact ctttaaaact tattaaaact
    13621 cttaaaaccc gcctggcctg tgcataactg tctggccagc gcacagccga agagctgcaa
    13681 aaagcgccta cccttcggtc gctgcgctcc ctacgccccg ccgcttcgcg tcggcctatc
    13741 gcggccgctg gccgctcaaa aatggctggc ctacggccag gcaatctacc agggcgcgga
    13801 caagccgcgc cgtcgccact cgaccgccgg cgcccacatc aaggcaccct gcctcgcgcg
    13861 tttcggtgat gacggtgaaa acctctgaca catgcagctc ccggagacgg tcacagcttg
    13921 tctgtaagcg gatgccggga gcagacaagc ccgtcagggc gcgtcagcgg gtgttggcgg
    13981 gtgtcggggc gcagccatga cccagtcacg tagcgatagc ggagtgtata ctggcttaac
    14041 tatgcggcat cagagcagat tgtactgaga gtgcaccata tgcggtgtga aataccgcac
    14101 agatgcgtaa ggagaaaata ccgcatcagg cgctcttccg cttcctcgct cactgactcg
    14161 ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg
    14221 ttatccacag aatcagggga taacgcagga aagaacatgt gagcaaaagg ccagcaaaag
    14281 gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg cccccctgac
    14341 gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg actataaaga
    14401 taccaggcgt ttccccctgg aagctccctc gtgcgctctc ctgttccgac cctgccgctt
    14461 accggatacc tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca tagctcacgc
    14521 tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc
    14581 cccgttcagc ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc caacccggta
    14641 agacacgact tatcgccact ggcagcagcc actggtaaca ggattagcag agcgaggtat
    14701 gtaggcggtg ctacagagtt cttgaagtgg tggcctaact acggctacac tagaaggaca
    14761 gtatttggta tctgcgctct gctgaagcca gttaccttcg gaaaaagagt tggtagctct
    14821 tgatccggca aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt
    14881 acgcgcagaa aaaaaggatc tcaagaagat cctttgatct tttctacggg gtctgacgct
    14941 cagtggaacg aaaactcacg ttaagggctg atgaatcccc taatgatttt tatcaaaatc
    15001 attaagttaa ggtagataca catcttgtca tatgatcaaa tggtttcgcc aaaaatcaat
    15061 aatcagacaa caaaatgtgc gaactcgata ttttacacga ctctctttac caattctgcc
    15121 ccgaattaca cttaaaacga ctcaacagct taacgttggc ttgccacgcc ttacttgact
    15181 gtaaaactct cactcttacc gaacttggcc gtaacctgcc aaccaaagcg agaacaaaac
    15241 ataacatcaa acgaatcgac cgattgttag gtaatcgtca cctccacaaa gagcgactcg
    15301 ctgtataccg ttggcatgct agctttatct gttcgggcaa tacgatgccc attgtacttg
    15361 ttgactggtc tgatatccgt gagcaaaaac ggcttatggt attgcgagct tcagtcgcac
    15421 tacacggtcg ttctgttact ctttatgaga aagcgttccc gctttcagag caatgttcaa
    15481 agaaagctca tgaccaattt ctagccgacc ttgcgagcat tctaccgagt aacaccacac
    15541 cgctcattgt cagtgatgct ggctttaaag tgccatggta taaatccgtt gagaagctgg
    15601 gttggtactg gttaagtcga gtaagaggaa aagtacaata tgcagaccta ggagcggaaa
    15661 actggaaacc tatcagcaac ttacatgata tgtcatctag tcactcaaag actttaggct
    15721 ataagaggct gactaaaagc aatccaatct catgccaaat tctattgtat aaatctcgct
    15781 ctaaaggccg aaaaaatcag cgctcgacac ggactcattg tcaccacccg tcacctaaaa
    15841 tctactcagc gtcggcaaag gagccatgga ttctagcaac taacttacct gttgaaattc
    15901 gaacacccaa acaacttgtt aatatctatt cgaagcgaat gcagattgaa gaaaccttcc
    15961 gagacttgaa aagtcctgcc tacggactag gcctacgcca tagccgaacg agcagctcag
    16021 agcgttttga tatcatgctg ctaatcgccc tgatgcttca actaacatgt tggcttgcgg
    16081 gcgttcatgc tcagaaacaa ggttgggaca agcacttcca ggctaacaca gtcagaaatc
    16141 gaaacgtact ctcaacagtt cgcttaggca tggaagtttt gcggcattct ggctacacaa
    16201 taacaaggga agacttactc gtggctgcaa ccctactagc tcaaaattta ttcacacatg
    16261 gttacgcttt ggggaaatta tgaggggatc tctcagcgtt aagggatttt ggtcatgcat
    16321 tctaggtact aaaacaattc atccagtaaa atataatatt ttattttctc ccaatcaggc
    16381 ttgatcccca gtaagtcaaa aaatagctcg acatactgtt cttccccgat atcctccctg
    16441 atcgaccgga cgcagaaggc aatgtcatac cacttgtccg ccctgccgct tctcccaaga
    16501 tcaataaagc cacttacttt gccatctttc acaaagatgt tgctgtctcc caggtcgccg
    16561 tgggaaaaga caagttcctc ttcgggcttt tccgtcttta aaaaatcata cagctcgcgc
    16621 ggatctttaa atggagtgtc ttcttcccag ttttcgcaat ccacatcggc cagatcgtta
    16681 ttcagtaagt aatccaattc ggctaagcgg ctgtctaagc tattcgtata gggacaatcc
    16741 gatatgtcga tggagtgaaa gagcctgatg cactccgcat acagctcgat aatcttttca
    16801 gggctttgtt catcttcata ctcttccgag caaaggacgc catcggcctc actcatgagc
    16861 agattgctcc agccatcatg ccgttcaaag tgcaggacct ttggaacagg cagctttcct
    16921 tccagccata gcatcatgtc cttttcccgt tccacatcat aggtggtccc tttataccgg
    16981 ctgtccgtca tttttaaata taggttttca ttttctccca ccagcttata taccttagca
    17041 ggagacattc cttccgtatc ttttacgcag cggtattttt cgatcagttt tttcaattcc
    17101 ggtgatattc tcattttagc catttattat ttccttcctc ttttctacag tatttaaaga
    17161 taccccaaga agctaattat aacaagacga actccaattc actgttcctt gcattctaaa
    17221 accttaaata ccagaaaaca gctttttcaa agttgttttc aaagttggcg tataacatag
    17281 tatcgacgga gccgattttg aaaccgcggt tagttttgct ggccgcatct tctcaaatat
    17341 gcttcccagc ctgcttttct gtaacgttca ccctctacct tagcatccct tccctttgca
    17401 aatagtcctc ttccaacaat aataatgtca gatcctgtag acaccacatc atccacggtt
    17461 ctatactgtt gacccaatgc gtctcccttg tcatctaaac ccacaccggg tgtcataatc
    17521 aaccaatcgt aaccttcatc tcctccaccc atgtctcttt gagcaataaa gccgataaca
    17581 aaatctttgt cgctcttcgc aatgtcaaca gtacccttag tatattctcc agtagatagg
    17641 gagcccttgc atgacaattc tgctaacatc aaaaggcctc taggttcctt tgttacttct
    17701 tctgccgcct gcttcaaacc gctaacaata cctgggccca ccacaccgtg tgcattcgta
    17761 atgtctgccc attctgctat tctgtataca cccgcagagt actgcaattt gactgtatta
    17821 ccaatgtcag caaattttct gtcttcgaag agtaaaaaat tgtacttggc ggataatgcc
    17881 tttagcggct taactgtgcc ctccatggaa aaatcagtca agatatccac atgtgttttt
    17941 agtaaacaaa ttttgggacc taatgcttca actaactcca gtaattcctt ggtggtacga
    18001 acatccaatg aagcacacaa gtttgtttgc ttttcgtgca tgatattaaa tagcttggca
    18061 gcaacaggac taggatgagt agcagcacgt tccttatatg tagctttcga catgatttat
    18121 cttcgtttcc tgcaggtttt tgttctgtgc agttgggtta agaatactgg gcaatttcat
    18181 gtttcttcaa cactacatat gcgtatatat accaatctaa gtctgtgctc cttccttcgt
    18241 tcttccttct gttcggagat taccgaatca aaaaaatttc aaggaaaccg aaatcaaaaa
    18301 aaagaataaa aaaaaaatga tgaattgaaa agctagctta tcgatgataa gctgtcaaac
    18361 atgagaatta attccacgga ctatagacta tacctagtat actccgtcta ctgtacgata
    18421 cacttccgct caggtccttg tcctttaacg aggccttacc actcttttgt tactctattg
    18481 atccagctca gcaaaggcag tgtgatctaa gattctatct tcgcgatgta gtaaaactag
    18541 ctagaccgag aaagagacta gaaatgcaaa aggcacttct acaatggctg ccatcattat
    18601 tatccgatgt gacgctgcag cttctcaatg atattcgaat acgctttgag gagatacagc
    18661 ctaatatccg acaaactgtt ttacagattt acgatcgtac ttgttaccca tcattgaatt
    18721 ttgaacatcc gaacctggga gttttccctg aaacagatag tatatttgaa cctgtataat
    18781 aatatatagt ctagcgcttt acggaagaca atgtatgtat ttcggttcct ggagaaacta
    18841 ttgcatctat tgcataggta atcttgcacg tcgcatcccc ggttcatttt ctgcgtttcc
    18901 atcttgcact tcaatagcat atctttgtta acgaagcatc tgtgcttcat tttgtagaac
    18961 aaaaatgcaa cgcgagagcg ctaatttttc aaacaaagaa tctgagctgc atttttacag
    19021 aacagaaatg caacgcgaaa gcgctatttt accaacgaag aatctgtgct tcatttttgt
    19081 aaaacaaaaa tgcaacgcga ga
//
