LOCUS       sequence-158700-       10018 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of Cas9 and gRNA cassette in S. cerevisiae; 
            Nourseothricin Resistance.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 10018)
  AUTHORS   Generoso WC, Gottardi M, Oreb M, Boles E
  TITLE     Simplified CRISPR-Cas genome editing for Saccharomyces cerevisiae.
  JOURNAL   J Microbiol Methods. 2016 Aug;127:203-5. doi: 
            10.1016/j.mimet.2016.06.020. Epub 2016 Jun 17.
  PUBMED    27327211
REFERENCE   2  (bases 1 to 10018)
  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: "J Microbiol
            Methods."; date: "2016-08"; volume: "127:203-5. doi"; pages: " 
            10.1016/j.mimet.2016.06.020. Epub 2016 Jun 17"
COMMENT     Sequence Label: pRCC-N
FEATURES             Location/Qualifiers
     source          1..10018
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(10..29)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     129..151
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(189..207)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     rep_origin      248..1590
                     /label=2u ori
                     /note="yeast 2u plasmid origin of replication"
     protein_bind    580..627
                     /label=FRT
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="FLP-mediated recombination occurs in the 8-bp core 
                     sequence TCTAGAAA (Turan and Bode, 2011)."
     promoter        1617..1721
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1722..1790,1791..2582)
                     /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     complement(1940..1959)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2753..3341
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3242..3261
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     promoter        3378..3646
                     /label=SNR52 promoter
                     /note="promoter for the S. cerevisiae small nucleolar RNA 
                     gene SNR52"
     misc_RNA        3670..3744
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     terminator      3749..3768
                     /label=SUP4 terminator
                     /note="transcription terminator for the S. cerevisiae SUP4 
                     tRNA gene"
     terminator      complement(3789..4036)
                     /gene="S. cerevisiae CYC1"
                     /label=CYC1 terminator
                     /note="transcription terminator for CYC1"
     primer_bind     4002..4020
                     /label=CYC1
                     /note="CYC1 transcription termination signal, reverse 
                     primer"
     CDS             complement(4052..4072)
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             complement(4085..8188)
                     /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             complement(4085..8188)
                     /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"
     gene            complement(8877..9996)
                     /label=natMX6
                     /note="yeast selectable marker conferring nourseothricin 
                     resistance"
     terminator      complement(8877..9074)
                     /label=TEF terminator
                     /note="Ashbya gossypii TEF terminator"
     CDS             complement(9080..9652)
                     /codon_start=1
                     /gene="Streptomyces noursei nat1"
                     /product="nourseothricin acetyltransferase"
                     /label=NrsR
                     /note="confers resistance to nourseothricin"
                     /translation="MGTTLDDTAYRYRTSVPGDAEAIEALDGSFTTDTVFRVTATGDGF
                     TLREVPVDPPLTKVFPDDESDDESDDGEDGDPDSRTFVAYGDDGDLAGFVVVSYSGWNR
                     RLTVEDIEVAPEHRGHGVGRALMGLATEFARERGAGHLWLEVTNVNAPAIHAYRRMGFT
                     LCGLDTALYDGTASDGEQALYMSMPCP"
     promoter        complement(9653..9996)
                     /label=TEF promoter
                     /note="Ashbya gossypii TEF promoter"
ORIGIN
        1 cactctcagt acaatctgct ctgatgccgc atagttaagc cagccccgac acccgccaac
       61 acccgctgac gcgccctgac gggcttgtct gctcccggca tccgcttaca gacaagctgt
      121 gaccgtctcc gggagctgca tgtgtcagag gttttcaccg tcatcaccga aacgcgcgag
      181 acgaaagggc ctcgtgatac gcctattttt ataggttaat gtcatgataa taatggtttc
      241 ttagtatgat ccaatatcaa aggaaatgat agcattgaag gatgagacta atccaattga
      301 ggagtggcag catatagaac agctaaaggg tagtgctgaa ggaagcatac gataccccgc
      361 atggaatggg ataatatcac aggaggtact agactacctt tcatcctaca taaatagacg
      421 catataagta cgcatttaag cataaacacg cactatgccg ttcttctcat gtatatatat
      481 atacaggcaa cacgcagata taggtgcgac gtgaacagtg agctgtatgt gcgcagctcg
      541 cgttgcattt tcggaagcgc tcgttttcgg aaacgctttg aagttcctat tccgaagttc
      601 ctattctcta gaaagtatag gaacttcaga gcgcttttga aaaccaaaag cgctctgaag
      661 acgcactttc aaaaaaccaa aaacgcaccg gactgtaacg agctactaaa atattgcgaa
      721 taccgcttcc acaaacattg ctcaaaagta tctctttgct atatatctct gtgctatatc
      781 cctatataac ctacccatcc acctttcgct ccttgaactt gcatctaaac tcgacctcta
      841 cattttttat gtttatctct agtattactc tttagacaaa aaaattgtag taagaactat
      901 tcatagagtg aatcgaaaac aatacgaaaa tgtaaacatt tcctatacgt agtatataga
      961 gacaaaatag aagaaaccgt tcataatttt ctgaccaatg aagaatcatc aacgctatca
     1021 ctttctgttc acaaagtatg cgcaatccac atcggtatag aatataatcg gggatgcctt
     1081 tatcttgaaa aaatgcaccc gcagcttcgc tagtaatcag taaacgcggg aagtggagtc
     1141 aggctttttt tatggaagag aaaatagaca ccaaagtagc cttcttctaa ccttaacgga
     1201 cctacagtgc aaaaagttat caagagactg cattatagag cgcacaaagg agaaaaaaag
     1261 taatctaaga tgctttgtta gaaaaatagc gctctcggga tgcatttttg tagaacaaaa
     1321 aagaagtata gattctttgt tggtaaaata gcgctctcgc gttgcatttc tgttctgtaa
     1381 aaatgcagct cagattcttt gtttgaaaaa ttagcgctct cgcgttgcat ttttgtttta
     1441 caaaaatgaa gcacagattc ttcgttggta aaatagcgct ttcgcgttgc atttctgttc
     1501 tgtaaaaatg cagctcagat tctttgtttg aaaaattagc gctctcgcgt tgcatttttg
     1561 ttctacaaaa tgaagcacag atgcttcgtt caggtggcac ttttcgggga aatgtgcgcg
     1621 gaacccctat ttgtttattt ttctaaatac attcaaatat gtatccgctc atgagacaat
     1681 aaccctgata aatgcttcaa taatattgaa aaaggaagag tatgagtatt caacatttcc
     1741 gtgtcgccct tattcccttt tttgcggcat tttgccttcc tgtttttgct cacccagaaa
     1801 cgctggtgaa agtaaaagat gctgaagatc agttgggtgc acgagtgggt tacatcgaac
     1861 tggatctcaa cagcggtaag atccttgaga gttttcgccc cgaagaacgt tttccaatga
     1921 tgagcacttt taaagttctg ctatgtggcg cggtattatc ccgtattgac gccgggcaag
     1981 agcaactcgg tcgccgcata cactattctc agaatgactt ggttgagtac tcaccagtca
     2041 cagaaaagca tcttacggat ggcatgacag taagagaatt atgcagtgct gccataacca
     2101 tgagtgataa cactgcggcc aacttacttc tgacaacgat cggaggaccg aaggagctaa
     2161 ccgctttttt gcacaacatg ggggatcatg taactcgcct tgatcgttgg gaaccggagc
     2221 tgaatgaagc cataccaaac gacgagcgtg acaccacgat gcctgtagca atggcaacaa
     2281 cgttgcgcaa actattaact ggcgaactac ttactctagc ttcccggcaa caattaatag
     2341 actggatgga ggcggataaa gttgcaggac cacttctgcg ctcggccctt ccggctggct
     2401 ggtttattgc tgataaatct ggagccggtg agcgtgggtc tcgcggtatc attgcagcac
     2461 tggggccaga tggtaagccc tcccgtatcg tagttatcta cacgacgggg agtcaggcaa
     2521 ctatggatga acgaaataga cagatcgctg agataggtgc ctcactgatt aagcattggt
     2581 aactgtcaga ccaagtttac tcatatatac tttagattga tttaaaactt catttttaat
     2641 ttaaaaggat ctaggtgaag atcctttttg ataatctcat gaccaaaatc ccttaacgtg
     2701 agttttcgtt ccactgagcg tcagaccccg tagaaaagat caaaggatct tcttgagatc
     2761 ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg
     2821 tttgtttgcc ggatcaagag ctaccaactc tttttccgaa ggtaactggc ttcagcagag
     2881 cgcagatacc aaatactgtc cttctagtgt agccgtagtt aggccaccac ttcaagaact
     2941 ctgtagcacc gcctacatac ctcgctctgc taatcctgtt accagtggct gctgccagtg
     3001 gcgataagtc gtgtcttacc gggttggact caagacgata gttaccggat aaggcgcagc
     3061 ggtcgggctg aacggggggt tcgtgcacac agcccagctt ggagcgaacg acctacaccg
     3121 aactgagata cctacagcgt gagctatgag aaagcgccac gcttcccgaa gggagaaagg
     3181 cggacaggta tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg gagcttccag
     3241 ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg ccacctctga cttgagcgtc
     3301 gatttttgtg atgctcgtca ggggggcgga gcctatggaa aaacgccagc aacgcgggct
     3361 agggataaca gggtaattct ttgaaaagat aatgtatgat tatgctttca ctcatattta
     3421 tacagaaact tgatgttttc tttcgagtat atacaaggtg attacatgta cgtttgaagt
     3481 acaactctag attttgtagt gccctcttgg gctagcggta aaggtgcgca ttttttcaca
     3541 ccctacaatg ttctgttcaa aagattttgg tcaaacgctg tagaagtgaa agttggtgcg
     3601 catgtttcgg cgttcgaaac ttctccgcag tgaaagataa atgatcggat ccacgttctc
     3661 tgcggccgcg ttttagagct agaaatagca agttaaaata aggctagtcc gttatcaact
     3721 tgaaaaagtg gcaccgagtc ggtggtgctt tttttgtttt ttatgtctcg gcgaattggg
     3781 taccggccgc aaattaaagc cttcgagcgt cccaaaacct tctcaagcaa ggttttcagt
     3841 ataatgttac atgcgtacac gcgtctgtac agaaaaaaaa gaaaaatttg aaatataaat
     3901 aacgttctta atactaacat aactataaaa aaataaatag ggacctagac ttcaggttgt
     3961 ctaactcctt ccttttcggt tagagcggat gtggggggag ggcgtgaatg taagcgtgac
     4021 ataactaatt acatgactcg aggtcgactt aaacctttct cttcttcttt gggtcagctc
     4081 tagagtcacc acccaattga gacaagtcaa ttctggtttc gtacaaaccg gtaatagatt
     4141 ggtgaatcaa ggtagcgtcc aaaacttcct tggtagaggt gtatctcttt ctgtcaatgg
     4201 tggtgtcgaa gtacttgaaa gcagctggag cacccaagtt ggtcaaggtg aacaagtgaa
     4261 taatgttttc agcttgttct ctaattggct tgtctctgtg cttgttgtaa gcagacaaaa
     4321 ccttgtccaa gttagcgtca gccaaaataa ctctcttaga gaattcagaa atttgttcaa
     4381 taatttcgtc caagtagtgc ttgtgttgtt caacgaacaa ttgcttttgt tcgttgtctt
     4441 ctggagaacc cttcaacttt tcgtagtgag aagccaagta caagaagtta acgtacttag
     4501 atggcaaagc caattcgtta cccttttgca attcaccagc agaagccaac attctctttc
     4561 taccgttttc caattcgaac aaagagtact ttggcaactt aataatcaag tccttcttaa
     4621 cttccttgta acccttagct tccaagaagt caattgggtt cttttcgaaa gaagatcttt
     4681 ccataatggt aatacccaac aattccttaa cagacttcaa cttcttagac ttaccctttt
     4741 caaccttagc aacaaccaaa acagagtaag caacggttgg agagtcgaaa ccaccgtact
     4801 tctttgggtc ccagtccttc tttctagcaa tcaacttgtc agagtttctc tttggcaaaa
     4861 tagattcctt agagaaacca ccggtttgaa cttcggtctt cttaacaatg ttaacttgtg
     4921 gcatagacaa aacctttcta acggtagcga agtctctacc cttgtcccaa acaatttcac
     4981 cggtttcacc gttggtttca atcaatggtc tctttctaat ttcaccgtta gccaaggtaa
     5041 tttcggtctt gaagaagttc ataatgttag agtagaagaa gtacttagcg gtagccttac
     5101 caatttcttg ttcagactta gcaatcatct ttctaacgtc gtaaaccttg tagtcaccgt
     5161 aaacgaattc agattccaac tttgggtact tcttaatcaa agcggtacca acaacagcgt
     5221 tcaagtaagc gtcgtgagcg tggtggtagt tgttaatttc tctaaccttg tagaattgga
     5281 agtcctttct gaagtcagaa accaacttag acttcaaggt aataacctta acttctctaa
     5341 tcaacttgtc gttttcgtcg tacttggtgt tcattctaga gtccaaaatt tgagcaacgt
     5401 gcttggtaat ttgtctggtt tcaaccaatt gtctcttaat gaaaccagcc ttgtccaatt
     5461 cagacaaacc acctctttca gccttggtca agttgtcgaa ctttctttgg gtaatcaact
     5521 tagcgttcaa caattgtctc cagtagttct tcatcttctt aacaacttct tcagatggaa
     5581 cgttgtcaga cttacctctg ttcttgtcag atctggtcaa aaccttgttg tcaatagagt
     5641 cgtccttcaa gaaagattgt ggaacaatgt ggtcaacgtc gtagtcagac aatctgttaa
     5701 tgtccaattc ttggtcaacg tacatgtctc taccgttttg caagtagtac aagtacaact
     5761 tttcgttttg caattgggtg ttttcaactg ggtgttcctt caaaatttga gaacccaatt
     5821 ccttaatacc ttcttcaatt ctcttcattc tttctctaga gttcttttga cccttttggg
     5881 tggtttggtt ttctctagcc atttcaataa caatgttttc tggcttgtgt ctacccataa
     5941 ccttaaccaa ttcgtcaaca accttaacgg tttgcaaaat acccttctta atagctggag
     6001 aaccagccaa gttagcaatg tgttcgtgca aagagtcacc ttgaccagaa acttgagcct
     6061 tttgaatgtc ttccttgaag gtcaaagagt cgtcgtgaat caattgcatg aagtttctgt
     6121 tagcgaaacc gtcagacttc aagaagtcca aaatggtctt accagattgc ttgtctctaa
     6181 taccgttaat caactttcta gacaatctac cccaaccggt gtatcttctt ctcttcaatt
     6241 gcttcataac cttgtcgtcg aacaagtgag cgtaggtctt caatctttct tcaatcattt
     6301 ctctgtcttc gaacaaggtc aaggtcaaaa caatgtcttc caaaatgtct tcgttttctt
     6361 cgttgtccaa gaagtccttg tccttaataa tcttcaacaa gtcgtggtag gtacccaaag
     6421 aagcgttgaa tctgtcttca acaccagaaa tttcaacaga gtcgaaacat tcaatcttct
     6481 tgaagtagtc ttccttcaat tgcttaacgg taacctttct gttggtcttg aacaacaagt
     6541 caacaatagc cttcttttgt tcaccagaca agaaagctgg ctttctcata ccttcggtaa
     6601 cgtacttaac cttggtcaat tcgttgtaaa cggtgaagta ttcgtacaac aaagagtgct
     6661 ttggcaaaac cttttcgttt ggcaagttct tgtcgaagtt ggtcattctt tcaatgaaag
     6721 attgagcaga agcacccttg tcaacaactt cttcgaagtt ccatggggta atggtttctt
     6781 cagactttct ggtcatccaa gcgaatctag agttacctct agccaatgga ccaacgtagt
     6841 atggaattct gaaggtcaaa atcttttcaa tcttttctct gttgtccttc aagaatgggt
     6901 agaagtcttc ttgtcttctc aaaatagcgt gcaattcacc caagtgaatt tggtgtggaa
     6961 tagaaccgtt gtcgaaggtt ctttgctttc tcaacaagtc ttctctgttc aacttaacca
     7021 acaattcttc ggtaccgtcc atcttttcca aaattggctt aatgaacttg tagaattctt
     7081 cttgagaagc accaccgtca atgtaaccag cgtaaccgtt cttagattgg tcgaagaaaa
     7141 tttccttgta cttttctggc aattgttgtc taaccaaagc cttcaacaag gtcaagtctt
     7201 ggtggtgttc gtcgtatctc ttaatcatag aagcagacaa tggagccttg gtaatttcgg
     7261 tgttaactct caaaatgtca gacaacaaaa tagcgtcaga caagttctta gcagccaaga
     7321 acaagtcagc gtattggtca ccaatttgag ccaacaagtt gtccaagtcg tcgtcgtagg
     7381 tgtccttaga caattgcaac ttagcgtctt cagccaagtc gaagttagac ttgaagtttg
     7441 gggtcaaacc caaagacaaa gcaatcaagt taccgaacaa accgttcttc ttttcacctg
     7501 gcaattgagc aatcaagttt tccaatcttc tagacttaga caatctagca gacaaaatag
     7561 ccttagcgtc aacaccagaa gcgttaattg ggttttcttc gaacaattgg ttgtaggttt
     7621 gaaccaattg aatgaacaac ttgtcaacgt cagagttgtc tgggttcaag tcaccttcaa
     7681 tcaagaagtg acctctgaac ttaatcatgt gagccaaagc caagtaaatc aatctcaagt
     7741 cagccttgtc ggtagagtca accaacttct ttctcaagtg gtaaatggtt gggtactttt
     7801 cgtggtaagc aacttcgtca acaatgttac cgaaaattgg gtgtctttcg tgcttcttgt
     7861 cttcttcaac caagaaagat tcttccaatc tgtggaagaa agagtcgtca accttagcca
     7921 tttcgttaga gaaaatttct tgcaagtaac aaattctgtt ctttcttctg gtgtatcttc
     7981 ttctagcggt tctcttcaat ctggtagctt cagcggtttc accagagtcg aacaacaaag
     8041 caccaatcaa gttcttctta atagagtgtc tgtcggtgtt acccaaaacc ttgaacttct
     8101 tagatggaac cttgtattcg tcggtaataa cagcccaacc aacagagttg gtaccaatgt
     8161 ccaaaccaat agagtacttc ttgtccatat gtattatcgt attccttttt cgttcactta
     8221 ctattgctat tcgcactcac tacagatcta tattttatgt tccaccctgt tgtcagatca
     8281 ggaaaggaat gaagaggctc tatttaaacc aaatacacct cttcgtttct caatattgtt
     8341 gggctttcct tttttttttc cttccacgtc tatctattgt ctctatcaaa attaggaaaa
     8401 agaaatgaag aacgatgagc caaacgaggc tgagacaatc atgcatccgt acattctatg
     8461 tatgtttggg tgtatctttc cagattcggt tctttttttt tttcagtcca attgaaaaaa
     8521 aaaaggaaca ggaacaacgg gtcgaaattt gccgtcacat tcagagcgaa gagcgacagg
     8581 ggaattggtg ggctaaccac ctgttgagcc cagctagcag ctgtcggtaa accgtcgttg
     8641 ggcggggcgc gacaatgcag ccagcggagg cattgtatgg tttccgtctg gagagtgggc
     8701 ttagccggcc tggcactcct tctctaagca gagtgagaag ggaatctttt tactggagaa
     8761 gagtgtttga atccagcaga aggtaatacg ctgcggtatt ttctccttac gcatctgtgc
     8821 ggtatttcac accgcgtttt cgacactgga tggcggcgtt agtatcgaat cgacagcagt
     8881 atagcgacca gcattcacat acgattgacg catgatatta ctttctgcgc acttaacttc
     8941 gcatctgggc agatgatgtc gaggcgaaaa aaaatataaa tcacgctaac atttgattaa
     9001 aatagaacaa ctacaatata aaaaaactat acaaatgaca agttcttgaa aacaagaatc
     9061 tttttattgt cagtactgat taggggcagg gcatgctcat gtagagcgcc tgctcgccgt
     9121 ccgaggcggt gccgtcgtac agggcggtgt ccaggccgca gagggtgaac cccatccgcc
     9181 ggtacgcgtg gatcgccggt gcgttgacgt tggtgacctc cagccagagg tgcccggcgc
     9241 cccgctcgcg ggcgaactcc gtcgcgagcc ccatcaacgc gcgcccgacc ccgtgccccc
     9301 ggtgctccgg ggcgacctcg atgtcctcga cggtcagccg gcggttccag ccggagtacg
     9361 agacgaccac gaagcccgcc aggtcgccgt cgtccccgta cgcgacgaac gtccgggagt
     9421 ccgggtcgcc gtcctccccg tcgtccgatt cgtcgtccga ttcgtcgtcg gggaacacct
     9481 tggtcagggg cgggtccacc ggcacctccc gcagggtgaa gccgtccccg gtggcggtga
     9541 cgcggaagac ggtgtcggtg gtgaaggacc catccagtgc ctcgatggcc tcggcgtccc
     9601 ccgggacact ggtgcggtac cggtaagccg tgtcgtcaag agtggtaccc atggttgttt
     9661 atgttcggat gtgatgtgag aactgtatcc tagcaagatt ttaaaaggaa gtatatgaaa
     9721 gaagaacctc agtggcaaat cctaaccttt tatatttctc tacaggggcg cggcgtgggg
     9781 acaattcaac gcgtctgtga ggggagcgtt tccctgctcg caggtctgca gcgaggagcc
     9841 gtaatttttg cttcgcgccg tgcggccatc aaaatgtatg gatgcaaatg attatacatg
     9901 gggatgtatg ggctaaatgt acgggcgaca gtcacatcat gcccctgagc tgcgcacgtc
     9961 aagactgtca aggagggtat tctgggcctc catgtcgctg attaccctgt tatcccta
//
