LOCUS       sequence-330352-       11755 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Plasmid for constituive spCas9 and tet-inducible CUP1-1 targeting 
            sgRNA expression for double stranded break formation in yeast.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 11755)
  TITLE     Burgess Lab Plasmids
REFERENCE   2  (bases 1 to 11755)
  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"
COMMENT     Sequence Label: pNSU299_CUP1-1
FEATURES             Location/Qualifiers
     source          1..11755
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    410..428
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     misc_RNA        514..589
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     terminator      602..664
                     /label=RPR1 terminator
                     /note="transcription terminator for the S. cerevisiae RNase
                     P RNA gene"
     CDS             1799..2422
                     /codon_start=1
                     /gene="tetR from transposon Tn10"
                     /product="tetracycline repressor TetR"
                     /label=TetR
                     /note="TetR binds to the tetracycline operator tetO to 
                     inhibit transcription. This inhibition can be relieved by 
                     adding tetracycline or doxycycline."
                     /translation="MSRLDKSKVINSALELLNEVGIEGLTTRKLAQKLGVEQPTLYWHV
                     KNKRALLDALAIEMLDRHHTHFCPLEGESWQDFLRNNAKSFRCALLSHRDGAKVHLGTR
                     PTEKQYETLENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEDQEHQVAKEERETPTT
                     DSMPPLLRQAIELFDHQGAEPAFLFGLELIICGLEKQLKCESGS"
     primer_bind     complement(1872..1891)
                     /label=Tet-R
                     /note="Tetracycline resistance gene, reverse primer"
     primer_bind     2919..2936
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    3053..3074
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(3089..3094,3095..3112,3113..3119)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3127..3143
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     primer_bind     3132..3154
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     3151..3167
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     3151..3167
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     3186..3206
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        3188..3206
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        3224..3622
                     /gene="S. cerevisiae TEF1"
                     /label=TEF1 promoter
                     /note="promoter for EF-1-alpha"
     primer_bind     3626..3642
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     3626..3642
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     CDS             3654..7757
                     /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             3654..7757
                     /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             3654..7757
                     /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             7770..7790
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     terminator      7806..8053
                     /gene="S. cerevisiae CYC1"
                     /label=CYC1 terminator
                     /note="transcription terminator for CYC1"
     primer_bind     complement(7822..7840)
                     /label=CYC1
                     /note="CYC1 transcription termination signal, reverse 
                     primer"
     primer_bind     complement(8071..8090)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(8072..8090)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(8100..8117)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(8100..8116)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(8109..8131)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      8257..8712
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(8344..8363)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     8554..8575
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(8815..9489)
                     /codon_start=1
                     /gene="S. cerevisiae TRP1"
                     /product="phosphoribosylanthranilate isomerase, required 
                     for tryptophan biosynthesis"
                     /label=TRP1
                     /note="yeast auxotrophic marker"
                     /translation="MSVINFTGSSGPLVKVCGLQSTEAAECALDSDADLLGIICVPNRK
                     RTIDPVIARKISSLVKAYKNSSGTPKYLVGVFRNQPKEDVLALVNDYGIDIVQLHGDES
                     WQEYQEFLGLPVIKRLVFPKDCNILLSAASQKPHSFIPLFDSEAGGTGELLDWNSISDW
                     VGRQESPESLHFMLAGGLTPENVGDALRLNGVIGVDVSGGVETNGVKDSNKIANFVKNA
                     KK"
     promoter        complement(9490..9771)
                     /gene="S. cerevisiae TRP1"
                     /label=TRP1 promoter
     primer_bind     complement(9792..9811)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     9911..9933
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(9971..9989)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        10057..10161
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(10162..10230,10231..11022)
                     /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(10380..10399)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      join(11193..11755,1..26)
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     11682..11701
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc ggccttttta cggttcctgg
       61 ccttttgctg gccttttgct cgggggatct gccaattgaa cataacatgg tagttacata
      121 tactagtaat atggttcggc acacattaaa agtataaaaa ctatctgaat tacgaattac
      181 atatattggt cataaaaatc aatcaatcat cgtgtgtttt atatgtctct tatctaagta
      241 taagaatatc catagttaat attcacttac gctacctttt aacctgtaat cattgtcaac
      301 aggatatgtt aacgacccac attgataaac gctagtattt ctttttcctc ttcttattgg
      361 ccggctgtct ctatactccc ctatagtctg tttcttttcg tttcgattgt ccctatcagt
      421 gatagagatg gcgcacatgg tacgctgtgg tgctcgcggc tgggaacgaa actctgggag
      481 ctgcgattgg cagaatcatg tagctgccca acggttttag agctagaaat agcaagttaa
      541 aataaggcta gtccgttatc aacttgaaaa agtggcaccg agtcggtgct tttttctcga
      601 gccatatcca acttccaatt taatctttct tttttaattt tcacttattt gcgatacaga
      661 aagaaaaaag cgatagtaac tattgaattt tgtttggatt tggttagatt agatatggtt
      721 tctctttata tttacatgct aaaaatgggc tacaccagag atacataatt agatatatat
      781 acgccagtac accttatcgg cccaagcctt gtcccaaggc agcgttttgt tcttggaaac
      841 gctgccctac acgttcgcta tgcttcaaga acttttctga gcacttcatg atgcatgttt
      901 gttccttatt ggttagcttt gatgttgtga agtcattgac acagtctgtg aaacatcttt
      961 ctaccagatt agagtacaaa cgcatgaaat ccttcatttg cttttgttcc actacttttt
     1021 ggaactcttg ttgttctttc ctgcaggtag tcgtgcaatg tatgacttta agatttgtga
     1081 gcaggaagaa aagggagaat cttctaacga taaacccttg aaaaactggg tagactacgc
     1141 tatgttgagt tgctacgcag gctgcacaat tacacgagaa tgctcccgcc taggatttaa
     1201 ggctaaggga cgtgcaatgc agacgacaga tctaaatgac cgtgtcggtg aagtgttcgc
     1261 caaacttttc ggttaacaca tgcagtgatg cacgcgcgat ggtgctaagt tacatatata
     1321 tatatatgta tagccatagt gatgtctaag taacctttat ggtatatttc ttaatgtgga
     1381 aagatactag cgcgcgcacc cacacacaag cttcgtcttt tcttgaagaa aagaggaagc
     1441 tcgctaaatg ggattccact ttccgttccc tgccagctga tggaaaaagg ttagtggaac
     1501 gatgaagaat aaaaagagag atccactgag gtgaaatttc agctgacagc gagtttcatg
     1561 atcgtgatga acaatggtaa cgagttgtgg ctgttgccag ggagggtggt tctcaacttt
     1621 taatgtatgg ccaaatcgct acttgggttt gttatataac aaagaagaaa taatgaactg
     1681 attctcttcc tccttcttgt cctttcttaa ttctgttgta attaccttcc tttgtaattt
     1741 tttttgtaat tattcttctt aataatccaa acaaacacac atattacaat atattaaaat
     1801 gtctagatta gataaaagta aagtgattaa cagcgcatta gagctgctta atgaggtcgg
     1861 aatcgaaggt ttaacaaccc gtaaactcgc ccagaagcta ggtgtagagc agcctacatt
     1921 gtattggcat gtaaaaaata agcgggcttt gctcgacgcc ttagccattg agatgttaga
     1981 taggcaccat actcactttt gccctttaga aggggaaagc tggcaagatt ttttacgtaa
     2041 taacgctaaa agttttagat gtgctttact aagtcatcgc gatggagcaa aagtacattt
     2101 aggtacacgg cctacagaaa aacagtatga aactctcgaa aatcaattag cctttttatg
     2161 ccaacaaggt ttttcactag agaatgcatt atatgcactc agcgctgtgg ggcattttac
     2221 tttaggttgc gtattggaag atcaagagca tcaagtcgct aaagaagaaa gggaaacacc
     2281 tactactgat agtatgccgc cattattacg acaagctatc gaattatttg atcaccaagg
     2341 tgcagagcca gccttcttat tcggccttga attgatcata tgcggattag aaaaacaact
     2401 taaatgtgaa agtgggtctt aagtctgaag aatgaatgat ttgatgattt ctttttccct
     2461 ccatttttct tactgaatat atcaatgata tagacttgta tagtttatta tttcaaatta
     2521 agtagctata tatagtcaag ataacgtttg tttgacacga ttacattatt cgtcgacatc
     2581 ttttttcagc ctgtcgtggt agcaatttga ggagtattat taattgaata ggttcatttt
     2641 gcgctcgcat aaacagtttt cgtcagggac agtatgttgg aatgagtggt aattaatggt
     2701 gacatgacat gttatagcaa taaccttgat gtttacatcg tagtttaatg tacaccccgc
     2761 gaattcgttc aagtaggagt gcaccaattg caaagggaaa agctgaatgg gcagttcgaa
     2821 ccgcggtctt tcctgcgtta tcccctgatt ctgtggataa ccgtattacc gcctttgagt
     2881 gagctgatac cgctcgccgc agccgaacga ccgagcgcag cgagtcagtg agcgaggaag
     2941 cggaagagcg cccaatacgc aaaccgcctc tccccgcgcg ttggccgatt cattaatgca
     3001 gctggcacga caggtttccc gactggaaag cgggcagtga gcgcaacgca attaatgtga
     3061 gttagctcac tcattaggca ccccaggctt tacactttat gcttccggct cgtatgttgt
     3121 gtggaattgt gagcggataa caatttcaca caggaaacag ctatgaccat gattacgcca
     3181 agcgcgcaat taaccctcac taaagggaac aaaagctgga gctcatagct tcaaaatgtt
     3241 tctactcctt ttttactctt ccagattttc tcggactccg cgcatcgccg taccacttca
     3301 aaacacccaa gcacagcata ctaaatttcc cctctttctt cctctagggt gtcgttaatt
     3361 acccgtacta aaggtttgga aaagaaaaaa gagaccgcct cgtttctttt tcttcgtcga
     3421 aaaaggcaat aaaaattttt atcacgtttc tttttcttga aaattttttt tttgattttt
     3481 ttctctttcg atgacctccc attgatattt aagttaataa acggtcttca atttctcaag
     3541 tttcagtttc atttttcttg ttctattaca acttttttta cttcttgctc attagaaaga
     3601 aagcatagca atctaatcta agttttctag aactagtgga tcccccggga aaaatggaca
     3661 agaagtactc cattgggctc gatatcggca caaacagcgt cggttgggcc gtcattacgg
     3721 acgagtacaa ggtgccgagc aaaaaattca aagttctggg caataccgat cgccacagca
     3781 taaagaagaa cctcattggc gccctcctgt tcgactccgg ggagacggcc gaagccacgc
     3841 ggctcaaaag aacagcacgg cgcagatata cccgcagaaa gaatcggatc tgctacctgc
     3901 aggagatctt tagtaatgag atggctaagg tggatgactc tttcttccat aggctggagg
     3961 agtccttttt ggtggaggag gataaaaagc acgagcgcca cccaatcttt ggcaatatcg
     4021 tggacgaggt ggcgtaccat gaaaagtacc caaccatata tcatctgagg aagaagcttg
     4081 tagacagtac tgataaggct gacttgcggt tgatctatct cgcgctggcg catatgatca
     4141 aatttcgggg acacttcctc atcgaggggg acctgaaccc agacaacagc gatgtcgaca
     4201 aactctttat ccaactggtt cagacttaca atcagctttt cgaagagaac ccgatcaacg
     4261 catccggagt tgacgccaaa gcaatcctga gcgctaggct gtccaaatcc cggcggctcg
     4321 aaaacctcat cgcacagctc cctggggaga agaagaacgg cctgtttggt aatcttatcg
     4381 ccctgtcact cgggctgacc cccaacttta aatctaactt cgacctggcc gaagatgcca
     4441 agcttcaact gagcaaagac acctacgatg atgatctcga caatctgctg gcccagatcg
     4501 gcgaccagta cgcagacctt tttttggcgg caaagaacct gtcagacgcc attctgctga
     4561 gtgatattct gcgagtgaac acggagatca ccaaagctcc gctgagcgct agtatgatca
     4621 agcgctatga tgagcaccac caagacttga ctttgctgaa ggcccttgtc agacagcaac
     4681 tgcctgagaa gtacaaggaa attttcttcg atcagtctaa aaatggctac gccggataca
     4741 ttgacggcgg agcaagccag gaggaatttt acaaatttat taagcccatc ttggaaaaaa
     4801 tggacggcac cgaggagctg ctggtaaagc ttaacagaga agatctgttg cgcaaacagc
     4861 gcactttcga caatggaagc atcccccacc agattcacct gggcgaactg cacgctatcc
     4921 tcaggcggca agaggatttc tacccctttt tgaaagataa cagggaaaag attgagaaaa
     4981 tcctcacatt tcggataccc tactatgtag gccccctcgc ccggggaaat tccagattcg
     5041 cgtggatgac tcgcaaatca gaagagacca tcactccctg gaacttcgag gaagtcgtgg
     5101 ataagggggc ctctgcccag tccttcatcg aaaggatgac taactttgat aaaaatctgc
     5161 ctaacgaaaa ggtgcttcct aaacactctc tgctgtacga gtacttcaca gtttataacg
     5221 agctcaccaa ggtcaaatac gtcacagaag ggatgagaaa gccagcattc ctgtctggag
     5281 agcagaagaa agctatcgtg gacctcctct tcaagacgaa ccggaaagtt accgtgaaac
     5341 agctcaaaga agactatttc aaaaagattg aatgtttcga ctctgttgaa atcagcggag
     5401 tggaggatcg cttcaacgca tccctgggaa cgtatcacga tctcctgaaa atcattaaag
     5461 acaaggactt cctggacaat gaggagaacg aggacattct tgaggacatt gtcctcaccc
     5521 ttacgttgtt tgaagatagg gagatgattg aagaacgctt gaaaacttac gctcatctct
     5581 tcgacgacaa agtcatgaaa cagctcaaga ggcgccgata tacaggatgg gggcggctgt
     5641 caagaaaact gatcaatggg atccgagaca agcagagtgg aaagacaatc ctggattttc
     5701 ttaagtccga tggatttgcc aaccggaact tcatgcagtt gatccatgat gactctctca
     5761 cctttaagga ggacatccag aaagcacaag tttctggcca gggggacagt cttcacgagc
     5821 acatcgctaa tcttgcaggt agcccagcta tcaaaaaggg aatactgcag accgttaagg
     5881 tcgtggatga actcgtcaaa gtaatgggaa ggcataagcc cgagaatatc gttatcgaga
     5941 tggcccgaga gaaccaaact acccagaagg gacagaagaa cagtagggaa aggatgaaga
     6001 ggattgaaga gggtataaaa gaactggggt cccaaatcct taaggaacac ccagttgaaa
     6061 acacccagct tcagaatgag aagctctacc tgtactacct gcagaacggc agggacatgt
     6121 acgtggatca ggaactggac atcaatcggc tctccgacta cgacgtggat catatcgtgc
     6181 cccagtcttt tctcaaagat gattctattg ataataaagt gttgacaaga tccgataaaa
     6241 atagagggaa gagtgataac gtcccctcag aagaagttgt caagaaaatg aaaaattatt
     6301 ggcggcagct gctgaacgcc aaactgatca cacaacggaa gttcgataat ctgactaagg
     6361 ctgaacgagg tggcctgtct gagttggata aagccggctt catcaaaagg cagcttgttg
     6421 agacacgcca gatcaccaag cacgtggccc aaattctcga ttcacgcatg aacaccaagt
     6481 acgatgaaaa tgacaaactg attcgagagg tgaaagttat tactctgaag tctaagctgg
     6541 tctcagattt cagaaaggac tttcagtttt ataaggtgag agagatcaac aattaccacc
     6601 atgcgcatga tgcctacctg aatgcagtgg taggcactgc acttatcaaa aaatatccca
     6661 agcttgaatc tgaatttgtt tacggagact ataaagtgta cgatgttagg aaaatgatcg
     6721 caaagtctga gcaggaaata ggcaaggcca ccgctaagta cttcttttac agcaatatta
     6781 tgaatttttt caagaccgag attacactgg ccaatggaga gattcggaag cgaccactta
     6841 tcgaaacaaa cggagaaaca ggagaaatcg tgtgggacaa gggtagggat ttcgcgacag
     6901 tccggaaggt cctgtccatg ccgcaggtga acatcgttaa aaagaccgaa gtacagaccg
     6961 gaggcttctc caaggaaagt atcctcccga aaaggaacag cgacaagctg atcgcacgca
     7021 aaaaagattg ggaccccaag aaatacggcg gattcgattc tcctacagtc gcttacagtg
     7081 tactggttgt ggccaaagtg gagaaaggga agtctaaaaa actcaaaagc gtcaaggaac
     7141 tgctgggcat cacaatcatg gagcgatcaa gcttcgaaaa aaaccccatc gactttctcg
     7201 aggcgaaagg atataaagag gtcaaaaaag acctcatcat taagcttccc aagtactctc
     7261 tctttgagct tgaaaacggc cggaaacgaa tgctcgctag tgcgggcgag ctgcagaaag
     7321 gtaacgagct ggcactgccc tctaaatacg ttaatttctt gtatctggcc agccactatg
     7381 aaaagctcaa agggtctccc gaagataatg agcagaagca gctgttcgtg gaacaacaca
     7441 aacactacct tgatgagatc atcgagcaaa taagcgaatt ctccaaaaga gtgatcctcg
     7501 ccgacgctaa cctcgataag gtgctttctg cttacaataa gcacagggat aagcccatca
     7561 gggagcaggc agaaaacatt atccacttgt ttactctgac caacttgggc gcgcctgcag
     7621 ccttcaagta cttcgacacc accatagaca gaaagcggta cacctctaca aaggaggtcc
     7681 tggacgccac actgattcat cagtcaatta cggggctcta tgaaacaaga atcgacctct
     7741 ctcagctcgg tggagacagc agggctgacc ccaagaagaa gaggaaggtg tgatctcttc
     7801 tcgagtcatg taattagtta tgtcacgctt acattcacgc cctcccccca catccgctct
     7861 aaccgaaaag gaaggagtta gacaacctga agtctaggtc cctatttatt tttttatagt
     7921 tatgttagta ttaagaacgt tatttatatt tcaaattttt cttttttttc tgtacagacg
     7981 cgtgtacgca tgtaacatta tactgaaaac cttgcttgag aaggttttgg gacgctcgaa
     8041 ggctttaatt tgcggccggt acccaattcg ccctatagtg agtcgtatta cgcgcgctca
     8101 ctggccgtcg ttttacaacg tcgtgactgg gaaaaccctg gcgttaccca acttaatcgc
     8161 cttgcagcac atcccccttt cgccagctgg cgtaatagcg aagaggcccg caccgatcgc
     8221 ccttcccaac agttgcgcag cctgaatggc gaatggacgc gccctgtagc ggcgcattaa
     8281 gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac acttgccagc gccctagcgc
     8341 ccgctccttt cgctttcttc ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag
     8401 ctctaaatcg ggggctccct ttagggttcc gatttagtgc tttacggcac ctcgacccca
     8461 aaaaacttga ttagggtgat ggttcacgta gtgggccatc gccctgatag acggtttttc
     8521 gccctttgac gttggagtcc acgttcttta atagtggact cttgttccaa actggaacaa
     8581 cactcaaccc tatctcggtc tattcttttg atttataagg gattttgccg atttcggcct
     8641 attggttaaa aaatgagctg atttaacaaa aatttaacgc gaattttaac aaaatattaa
     8701 cgcttacaat ttcctgatgc ggtattttct ccttacgcat ctgtgcggta tttcacaccg
     8761 catagatcgg caagtgcaca aacaatactt aaataaatac tactcagtaa taacctattt
     8821 cttagcattt ttgacgaaat ttgctatttt gttagagtct tttacaccat ttgtctccac
     8881 acctccgctt acatcaacac caataacgcc atttaatcta agcgcatcac caacattttc
     8941 tggcgtcagt ccaccagcta acataaaatg taagctttcg gggctctctt gccttccaac
     9001 ccagtcagaa atcgagttcc aatccaaaag ttcacctgtc ccacctgctt ctgaatcaaa
     9061 caagggaata aacgaatgag gtttctgtga agctgcactg agtagtatgt tgcagtcttt
     9121 tggaaatacg agtcttttaa taactggcaa accgaggaac tcttggtatt cttgccacga
     9181 ctcatctcca tgcagttgga cgatatcaat gccgtaatca ttgaccagag ccaaaacatc
     9241 ctccttaggt tgattacgaa acacgccaac caagtatttc ggagtgcctg aactattttt
     9301 atatgctttt acaagacttg aaattttcct tgcaataacc gggtcaattg ttctctttct
     9361 attgggcaca catataatac ccagcaagtc agcatcggaa tctagagcac attctgcggc
     9421 ctctgtgctc tgcaagccgc aaactttcac caatggacca gaactacctg tgaaattaat
     9481 aacagacata ctccaagctg cctttgtgtg cttaatcacg tatactcacg tgctcaatag
     9541 tcaccaatgc cctccctctt ggccctctcc ttttcttttt tcgaccgaat taattcttaa
     9601 tcggcaaaaa aagaaaagct ccggatcaag attgtacgta aggtgacaag ctatttttca
     9661 ataaagaata tcttccacta ctgccatctg gcgtcataac tgcaaagtac acatatatta
     9721 cgatgctgtt ctattaaatg cttcctatat tatatatata gtaatgtcgt tgatctatgg
     9781 tgcactctca gtacaatctg ctctgatgcc gcatagttaa gccagccccg acacccgcca
     9841 acacccgctg acgcgccctg acgggcttgt ctgctcccgg catccgctta cagacaagct
     9901 gtgaccgtct ccgggagctg catgtgtcag aggttttcac cgtcatcacc gaaacgcgcg
     9961 agacgaaagg gcctcgtgat acgcctattt ttataggtta atgtcatgat aataatggtt
    10021 tcttagacgt caggtggcac ttttcgggga aatgtgcgcg gaacccctat ttgtttattt
    10081 ttctaaatac attcaaatat gtatccgctc atgagacaat aaccctgata aatgcttcaa
    10141 taatattgaa aaaggaagag tatgagtatt caacatttcc gtgtcgccct tattcccttt
    10201 tttgcggcat tttgccttcc tgtttttgct cacccagaaa cgctggtgaa agtaaaagat
    10261 gctgaagatc agttgggtgc acgagtgggt tacatcgaac tggatctcaa cagcggtaag
    10321 atccttgaga gttttcgccc cgaagaacgt tttccaatga tgagcacttt taaagttctg
    10381 ctatgtggcg cggtattatc ccgtattgac gccgggcaag agcaactcgg tcgccgcata
    10441 cactattctc agaatgactt ggttgagtac tcaccagtca cagaaaagca tcttacggat
    10501 ggcatgacag taagagaatt atgcagtgct gccataacca tgagtgataa cactgcggcc
    10561 aacttacttc tgacaacgat cggaggaccg aaggagctaa ccgctttttt gcacaacatg
    10621 ggggatcatg taactcgcct tgatcgttgg gaaccggagc tgaatgaagc cataccaaac
    10681 gacgagcgtg acaccacgat gcctgtagca atggcaacaa cgttgcgcaa actattaact
    10741 ggcgaactac ttactctagc ttcccggcaa caattaatag actggatgga ggcggataaa
    10801 gttgcaggac cacttctgcg ctcggccctt ccggctggct ggtttattgc tgataaatct
    10861 ggagccggtg agcgtgggtc tcgcggtatc attgcagcac tggggccaga tggtaagccc
    10921 tcccgtatcg tagttatcta cacgacgggg agtcaggcaa ctatggatga acgaaataga
    10981 cagatcgctg agataggtgc ctcactgatt aagcattggt aactgtcaga ccaagtttac
    11041 tcatatatac tttagattga tttaaaactt catttttaat ttaaaaggat ctaggtgaag
    11101 atcctttttg ataatctcat gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg
    11161 tcagaccccg tagaaaagat caaaggatct tcttgagatc ctttttttct gcgcgtaatc
    11221 tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc ggatcaagag
    11281 ctaccaactc tttttccgaa ggtaactggc ttcagcagag cgcagatacc aaatactgtt
    11341 cttctagtgt agccgtagtt aggccaccac ttcaagaact ctgtagcacc gcctacatac
    11401 ctcgctctgc taatcctgtt accagtggct gctgccagtg gcgataagtc gtgtcttacc
    11461 gggttggact caagacgata gttaccggat aaggcgcagc ggtcgggctg aacggggggt
    11521 tcgtgcacac agcccagctt ggagcgaacg acctacaccg aactgagata cctacagcgt
    11581 gagctatgag aaagcgccac gcttcccgaa gggagaaagg cggacaggta tccggtaagc
    11641 ggcagggtcg gaacaggaga gcgcacgagg gagcttccag ggggaaacgc ctggtatctt
    11701 tatagtcctg tcgggtttcg ccacctctga cttgagcgtc gatttttgtg atgct
//
