LOCUS       sequence-329117-       11755 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Plasmid for constituive spCas9 and tet-inducible TDH2 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_TDH2
FEATURES             Location/Qualifiers
     source          1..11755
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    330..348
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     misc_RNA        434..509
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     terminator      522..584
                     /label=RPR1 terminator
                     /note="transcription terminator for the S. cerevisiae RNase
                     P RNA gene"
     CDS             1719..2342
                     /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(1792..1811)
                     /label=Tet-R
                     /note="Tetracycline resistance gene, reverse primer"
     primer_bind     2839..2856
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    2973..2994
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(3009..3014,3015..3032,3033..3039)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3047..3063
                     /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     3052..3074
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     3071..3087
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     3071..3087
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     3106..3126
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        3108..3126
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        3144..3542
                     /gene="S. cerevisiae TEF1"
                     /label=TEF1 promoter
                     /note="promoter for EF-1-alpha"
     primer_bind     3546..3562
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     3546..3562
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     CDS             3574..7677
                     /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             3574..7677
                     /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             3574..7677
                     /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             7690..7710
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     terminator      7726..7973
                     /gene="S. cerevisiae CYC1"
                     /label=CYC1 terminator
                     /note="transcription terminator for CYC1"
     primer_bind     complement(7742..7760)
                     /label=CYC1
                     /note="CYC1 transcription termination signal, reverse 
                     primer"
     primer_bind     complement(7991..8010)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(7992..8010)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(8020..8037)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(8020..8036)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(8029..8051)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      8177..8632
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(8264..8283)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     8474..8495
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(8735..9409)
                     /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(9410..9691)
                     /gene="S. cerevisiae TRP1"
                     /label=TRP1 promoter
     primer_bind     complement(9712..9731)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     9831..9853
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(9891..9909)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        9977..10081
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(10082..10150,10151..10942)
                     /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(10300..10319)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      11113..11701
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     11602..11621
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 cgggggatct gccaattgaa cataacatgg tagttacata tactagtaat atggttcggc
       61 acacattaaa agtataaaaa ctatctgaat tacgaattac atatattggt cataaaaatc
      121 aatcaatcat cgtgtgtttt atatgtctct tatctaagta taagaatatc catagttaat
      181 attcacttac gctacctttt aacctgtaat cattgtcaac aggatatgtt aacgacccac
      241 attgataaac gctagtattt ctttttcctc ttcttattgg ccggctgtct ctatactccc
      301 ctatagtctg tttcttttcg tttcgattgt ccctatcagt gatagagatg gcgcacatgg
      361 tacgctgtgg tgctcgcggc tgggaacgaa actctgggag ctgcgattgg cagttcaagt
      421 acgactctac tcagttttag agctagaaat agcaagttaa aataaggcta gtccgttatc
      481 aacttgaaaa agtggcaccg agtcggtgct tttttctcga gccatatcca acttccaatt
      541 taatctttct tttttaattt tcacttattt gcgatacaga aagaaaaaag cgatagtaac
      601 tattgaattt tgtttggatt tggttagatt agatatggtt tctctttata tttacatgct
      661 aaaaatgggc tacaccagag atacataatt agatatatat acgccagtac accttatcgg
      721 cccaagcctt gtcccaaggc agcgttttgt tcttggaaac gctgccctac acgttcgcta
      781 tgcttcaaga acttttctga gcacttcatg atgcatgttt gttccttatt ggttagcttt
      841 gatgttgtga agtcattgac acagtctgtg aaacatcttt ctaccagatt agagtacaaa
      901 cgcatgaaat ccttcatttg cttttgttcc actacttttt ggaactcttg ttgttctttc
      961 ctgcaggtag tcgtgcaatg tatgacttta agatttgtga gcaggaagaa aagggagaat
     1021 cttctaacga taaacccttg aaaaactggg tagactacgc tatgttgagt tgctacgcag
     1081 gctgcacaat tacacgagaa tgctcccgcc taggatttaa ggctaaggga cgtgcaatgc
     1141 agacgacaga tctaaatgac cgtgtcggtg aagtgttcgc caaacttttc ggttaacaca
     1201 tgcagtgatg cacgcgcgat ggtgctaagt tacatatata tatatatgta tagccatagt
     1261 gatgtctaag taacctttat ggtatatttc ttaatgtgga aagatactag cgcgcgcacc
     1321 cacacacaag cttcgtcttt tcttgaagaa aagaggaagc tcgctaaatg ggattccact
     1381 ttccgttccc tgccagctga tggaaaaagg ttagtggaac gatgaagaat aaaaagagag
     1441 atccactgag gtgaaatttc agctgacagc gagtttcatg atcgtgatga acaatggtaa
     1501 cgagttgtgg ctgttgccag ggagggtggt tctcaacttt taatgtatgg ccaaatcgct
     1561 acttgggttt gttatataac aaagaagaaa taatgaactg attctcttcc tccttcttgt
     1621 cctttcttaa ttctgttgta attaccttcc tttgtaattt tttttgtaat tattcttctt
     1681 aataatccaa acaaacacac atattacaat atattaaaat gtctagatta gataaaagta
     1741 aagtgattaa cagcgcatta gagctgctta atgaggtcgg aatcgaaggt ttaacaaccc
     1801 gtaaactcgc ccagaagcta ggtgtagagc agcctacatt gtattggcat gtaaaaaata
     1861 agcgggcttt gctcgacgcc ttagccattg agatgttaga taggcaccat actcactttt
     1921 gccctttaga aggggaaagc tggcaagatt ttttacgtaa taacgctaaa agttttagat
     1981 gtgctttact aagtcatcgc gatggagcaa aagtacattt aggtacacgg cctacagaaa
     2041 aacagtatga aactctcgaa aatcaattag cctttttatg ccaacaaggt ttttcactag
     2101 agaatgcatt atatgcactc agcgctgtgg ggcattttac tttaggttgc gtattggaag
     2161 atcaagagca tcaagtcgct aaagaagaaa gggaaacacc tactactgat agtatgccgc
     2221 cattattacg acaagctatc gaattatttg atcaccaagg tgcagagcca gccttcttat
     2281 tcggccttga attgatcata tgcggattag aaaaacaact taaatgtgaa agtgggtctt
     2341 aagtctgaag aatgaatgat ttgatgattt ctttttccct ccatttttct tactgaatat
     2401 atcaatgata tagacttgta tagtttatta tttcaaatta agtagctata tatagtcaag
     2461 ataacgtttg tttgacacga ttacattatt cgtcgacatc ttttttcagc ctgtcgtggt
     2521 agcaatttga ggagtattat taattgaata ggttcatttt gcgctcgcat aaacagtttt
     2581 cgtcagggac agtatgttgg aatgagtggt aattaatggt gacatgacat gttatagcaa
     2641 taaccttgat gtttacatcg tagtttaatg tacaccccgc gaattcgttc aagtaggagt
     2701 gcaccaattg caaagggaaa agctgaatgg gcagttcgaa ccgcggtctt tcctgcgtta
     2761 tcccctgatt ctgtggataa ccgtattacc gcctttgagt gagctgatac cgctcgccgc
     2821 agccgaacga ccgagcgcag cgagtcagtg agcgaggaag cggaagagcg cccaatacgc
     2881 aaaccgcctc tccccgcgcg ttggccgatt cattaatgca gctggcacga caggtttccc
     2941 gactggaaag cgggcagtga gcgcaacgca attaatgtga gttagctcac tcattaggca
     3001 ccccaggctt tacactttat gcttccggct cgtatgttgt gtggaattgt gagcggataa
     3061 caatttcaca caggaaacag ctatgaccat gattacgcca agcgcgcaat taaccctcac
     3121 taaagggaac aaaagctgga gctcatagct tcaaaatgtt tctactcctt ttttactctt
     3181 ccagattttc tcggactccg cgcatcgccg taccacttca aaacacccaa gcacagcata
     3241 ctaaatttcc cctctttctt cctctagggt gtcgttaatt acccgtacta aaggtttgga
     3301 aaagaaaaaa gagaccgcct cgtttctttt tcttcgtcga aaaaggcaat aaaaattttt
     3361 atcacgtttc tttttcttga aaattttttt tttgattttt ttctctttcg atgacctccc
     3421 attgatattt aagttaataa acggtcttca atttctcaag tttcagtttc atttttcttg
     3481 ttctattaca acttttttta cttcttgctc attagaaaga aagcatagca atctaatcta
     3541 agttttctag aactagtgga tcccccggga aaaatggaca agaagtactc cattgggctc
     3601 gatatcggca caaacagcgt cggttgggcc gtcattacgg acgagtacaa ggtgccgagc
     3661 aaaaaattca aagttctggg caataccgat cgccacagca taaagaagaa cctcattggc
     3721 gccctcctgt tcgactccgg ggagacggcc gaagccacgc ggctcaaaag aacagcacgg
     3781 cgcagatata cccgcagaaa gaatcggatc tgctacctgc aggagatctt tagtaatgag
     3841 atggctaagg tggatgactc tttcttccat aggctggagg agtccttttt ggtggaggag
     3901 gataaaaagc acgagcgcca cccaatcttt ggcaatatcg tggacgaggt ggcgtaccat
     3961 gaaaagtacc caaccatata tcatctgagg aagaagcttg tagacagtac tgataaggct
     4021 gacttgcggt tgatctatct cgcgctggcg catatgatca aatttcgggg acacttcctc
     4081 atcgaggggg acctgaaccc agacaacagc gatgtcgaca aactctttat ccaactggtt
     4141 cagacttaca atcagctttt cgaagagaac ccgatcaacg catccggagt tgacgccaaa
     4201 gcaatcctga gcgctaggct gtccaaatcc cggcggctcg aaaacctcat cgcacagctc
     4261 cctggggaga agaagaacgg cctgtttggt aatcttatcg ccctgtcact cgggctgacc
     4321 cccaacttta aatctaactt cgacctggcc gaagatgcca agcttcaact gagcaaagac
     4381 acctacgatg atgatctcga caatctgctg gcccagatcg gcgaccagta cgcagacctt
     4441 tttttggcgg caaagaacct gtcagacgcc attctgctga gtgatattct gcgagtgaac
     4501 acggagatca ccaaagctcc gctgagcgct agtatgatca agcgctatga tgagcaccac
     4561 caagacttga ctttgctgaa ggcccttgtc agacagcaac tgcctgagaa gtacaaggaa
     4621 attttcttcg atcagtctaa aaatggctac gccggataca ttgacggcgg agcaagccag
     4681 gaggaatttt acaaatttat taagcccatc ttggaaaaaa tggacggcac cgaggagctg
     4741 ctggtaaagc ttaacagaga agatctgttg cgcaaacagc gcactttcga caatggaagc
     4801 atcccccacc agattcacct gggcgaactg cacgctatcc tcaggcggca agaggatttc
     4861 tacccctttt tgaaagataa cagggaaaag attgagaaaa tcctcacatt tcggataccc
     4921 tactatgtag gccccctcgc ccggggaaat tccagattcg cgtggatgac tcgcaaatca
     4981 gaagagacca tcactccctg gaacttcgag gaagtcgtgg ataagggggc ctctgcccag
     5041 tccttcatcg aaaggatgac taactttgat aaaaatctgc ctaacgaaaa ggtgcttcct
     5101 aaacactctc tgctgtacga gtacttcaca gtttataacg agctcaccaa ggtcaaatac
     5161 gtcacagaag ggatgagaaa gccagcattc ctgtctggag agcagaagaa agctatcgtg
     5221 gacctcctct tcaagacgaa ccggaaagtt accgtgaaac agctcaaaga agactatttc
     5281 aaaaagattg aatgtttcga ctctgttgaa atcagcggag tggaggatcg cttcaacgca
     5341 tccctgggaa cgtatcacga tctcctgaaa atcattaaag acaaggactt cctggacaat
     5401 gaggagaacg aggacattct tgaggacatt gtcctcaccc ttacgttgtt tgaagatagg
     5461 gagatgattg aagaacgctt gaaaacttac gctcatctct tcgacgacaa agtcatgaaa
     5521 cagctcaaga ggcgccgata tacaggatgg gggcggctgt caagaaaact gatcaatggg
     5581 atccgagaca agcagagtgg aaagacaatc ctggattttc ttaagtccga tggatttgcc
     5641 aaccggaact tcatgcagtt gatccatgat gactctctca cctttaagga ggacatccag
     5701 aaagcacaag tttctggcca gggggacagt cttcacgagc acatcgctaa tcttgcaggt
     5761 agcccagcta tcaaaaaggg aatactgcag accgttaagg tcgtggatga actcgtcaaa
     5821 gtaatgggaa ggcataagcc cgagaatatc gttatcgaga tggcccgaga gaaccaaact
     5881 acccagaagg gacagaagaa cagtagggaa aggatgaaga ggattgaaga gggtataaaa
     5941 gaactggggt cccaaatcct taaggaacac ccagttgaaa acacccagct tcagaatgag
     6001 aagctctacc tgtactacct gcagaacggc agggacatgt acgtggatca ggaactggac
     6061 atcaatcggc tctccgacta cgacgtggat catatcgtgc cccagtcttt tctcaaagat
     6121 gattctattg ataataaagt gttgacaaga tccgataaaa atagagggaa gagtgataac
     6181 gtcccctcag aagaagttgt caagaaaatg aaaaattatt ggcggcagct gctgaacgcc
     6241 aaactgatca cacaacggaa gttcgataat ctgactaagg ctgaacgagg tggcctgtct
     6301 gagttggata aagccggctt catcaaaagg cagcttgttg agacacgcca gatcaccaag
     6361 cacgtggccc aaattctcga ttcacgcatg aacaccaagt acgatgaaaa tgacaaactg
     6421 attcgagagg tgaaagttat tactctgaag tctaagctgg tctcagattt cagaaaggac
     6481 tttcagtttt ataaggtgag agagatcaac aattaccacc atgcgcatga tgcctacctg
     6541 aatgcagtgg taggcactgc acttatcaaa aaatatccca agcttgaatc tgaatttgtt
     6601 tacggagact ataaagtgta cgatgttagg aaaatgatcg caaagtctga gcaggaaata
     6661 ggcaaggcca ccgctaagta cttcttttac agcaatatta tgaatttttt caagaccgag
     6721 attacactgg ccaatggaga gattcggaag cgaccactta tcgaaacaaa cggagaaaca
     6781 ggagaaatcg tgtgggacaa gggtagggat ttcgcgacag tccggaaggt cctgtccatg
     6841 ccgcaggtga acatcgttaa aaagaccgaa gtacagaccg gaggcttctc caaggaaagt
     6901 atcctcccga aaaggaacag cgacaagctg atcgcacgca aaaaagattg ggaccccaag
     6961 aaatacggcg gattcgattc tcctacagtc gcttacagtg tactggttgt ggccaaagtg
     7021 gagaaaggga agtctaaaaa actcaaaagc gtcaaggaac tgctgggcat cacaatcatg
     7081 gagcgatcaa gcttcgaaaa aaaccccatc gactttctcg aggcgaaagg atataaagag
     7141 gtcaaaaaag acctcatcat taagcttccc aagtactctc tctttgagct tgaaaacggc
     7201 cggaaacgaa tgctcgctag tgcgggcgag ctgcagaaag gtaacgagct ggcactgccc
     7261 tctaaatacg ttaatttctt gtatctggcc agccactatg aaaagctcaa agggtctccc
     7321 gaagataatg agcagaagca gctgttcgtg gaacaacaca aacactacct tgatgagatc
     7381 atcgagcaaa taagcgaatt ctccaaaaga gtgatcctcg ccgacgctaa cctcgataag
     7441 gtgctttctg cttacaataa gcacagggat aagcccatca gggagcaggc agaaaacatt
     7501 atccacttgt ttactctgac caacttgggc gcgcctgcag ccttcaagta cttcgacacc
     7561 accatagaca gaaagcggta cacctctaca aaggaggtcc tggacgccac actgattcat
     7621 cagtcaatta cggggctcta tgaaacaaga atcgacctct ctcagctcgg tggagacagc
     7681 agggctgacc ccaagaagaa gaggaaggtg tgatctcttc tcgagtcatg taattagtta
     7741 tgtcacgctt acattcacgc cctcccccca catccgctct aaccgaaaag gaaggagtta
     7801 gacaacctga agtctaggtc cctatttatt tttttatagt tatgttagta ttaagaacgt
     7861 tatttatatt tcaaattttt cttttttttc tgtacagacg cgtgtacgca tgtaacatta
     7921 tactgaaaac cttgcttgag aaggttttgg gacgctcgaa ggctttaatt tgcggccggt
     7981 acccaattcg ccctatagtg agtcgtatta cgcgcgctca ctggccgtcg ttttacaacg
     8041 tcgtgactgg gaaaaccctg gcgttaccca acttaatcgc cttgcagcac atcccccttt
     8101 cgccagctgg cgtaatagcg aagaggcccg caccgatcgc ccttcccaac agttgcgcag
     8161 cctgaatggc gaatggacgc gccctgtagc ggcgcattaa gcgcggcggg tgtggtggtt
     8221 acgcgcagcg tgaccgctac acttgccagc gccctagcgc ccgctccttt cgctttcttc
     8281 ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg ggggctccct
     8341 ttagggttcc gatttagtgc tttacggcac ctcgacccca aaaaacttga ttagggtgat
     8401 ggttcacgta gtgggccatc gccctgatag acggtttttc gccctttgac gttggagtcc
     8461 acgttcttta atagtggact cttgttccaa actggaacaa cactcaaccc tatctcggtc
     8521 tattcttttg atttataagg gattttgccg atttcggcct attggttaaa aaatgagctg
     8581 atttaacaaa aatttaacgc gaattttaac aaaatattaa cgcttacaat ttcctgatgc
     8641 ggtattttct ccttacgcat ctgtgcggta tttcacaccg catagatcgg caagtgcaca
     8701 aacaatactt aaataaatac tactcagtaa taacctattt cttagcattt ttgacgaaat
     8761 ttgctatttt gttagagtct tttacaccat ttgtctccac acctccgctt acatcaacac
     8821 caataacgcc atttaatcta agcgcatcac caacattttc tggcgtcagt ccaccagcta
     8881 acataaaatg taagctttcg gggctctctt gccttccaac ccagtcagaa atcgagttcc
     8941 aatccaaaag ttcacctgtc ccacctgctt ctgaatcaaa caagggaata aacgaatgag
     9001 gtttctgtga agctgcactg agtagtatgt tgcagtcttt tggaaatacg agtcttttaa
     9061 taactggcaa accgaggaac tcttggtatt cttgccacga ctcatctcca tgcagttgga
     9121 cgatatcaat gccgtaatca ttgaccagag ccaaaacatc ctccttaggt tgattacgaa
     9181 acacgccaac caagtatttc ggagtgcctg aactattttt atatgctttt acaagacttg
     9241 aaattttcct tgcaataacc gggtcaattg ttctctttct attgggcaca catataatac
     9301 ccagcaagtc agcatcggaa tctagagcac attctgcggc ctctgtgctc tgcaagccgc
     9361 aaactttcac caatggacca gaactacctg tgaaattaat aacagacata ctccaagctg
     9421 cctttgtgtg cttaatcacg tatactcacg tgctcaatag tcaccaatgc cctccctctt
     9481 ggccctctcc ttttcttttt tcgaccgaat taattcttaa tcggcaaaaa aagaaaagct
     9541 ccggatcaag attgtacgta aggtgacaag ctatttttca ataaagaata tcttccacta
     9601 ctgccatctg gcgtcataac tgcaaagtac acatatatta cgatgctgtt ctattaaatg
     9661 cttcctatat tatatatata gtaatgtcgt tgatctatgg tgcactctca gtacaatctg
     9721 ctctgatgcc gcatagttaa gccagccccg acacccgcca acacccgctg acgcgccctg
     9781 acgggcttgt ctgctcccgg catccgctta cagacaagct gtgaccgtct ccgggagctg
     9841 catgtgtcag aggttttcac cgtcatcacc gaaacgcgcg agacgaaagg gcctcgtgat
     9901 acgcctattt ttataggtta atgtcatgat aataatggtt tcttagacgt caggtggcac
     9961 ttttcgggga aatgtgcgcg gaacccctat ttgtttattt ttctaaatac attcaaatat
    10021 gtatccgctc atgagacaat aaccctgata aatgcttcaa taatattgaa aaaggaagag
    10081 tatgagtatt caacatttcc gtgtcgccct tattcccttt tttgcggcat tttgccttcc
    10141 tgtttttgct cacccagaaa cgctggtgaa agtaaaagat gctgaagatc agttgggtgc
    10201 acgagtgggt tacatcgaac tggatctcaa cagcggtaag atccttgaga gttttcgccc
    10261 cgaagaacgt tttccaatga tgagcacttt taaagttctg ctatgtggcg cggtattatc
    10321 ccgtattgac gccgggcaag agcaactcgg tcgccgcata cactattctc agaatgactt
    10381 ggttgagtac tcaccagtca cagaaaagca tcttacggat ggcatgacag taagagaatt
    10441 atgcagtgct gccataacca tgagtgataa cactgcggcc aacttacttc tgacaacgat
    10501 cggaggaccg aaggagctaa ccgctttttt gcacaacatg ggggatcatg taactcgcct
    10561 tgatcgttgg gaaccggagc tgaatgaagc cataccaaac gacgagcgtg acaccacgat
    10621 gcctgtagca atggcaacaa cgttgcgcaa actattaact ggcgaactac ttactctagc
    10681 ttcccggcaa caattaatag actggatgga ggcggataaa gttgcaggac cacttctgcg
    10741 ctcggccctt ccggctggct ggtttattgc tgataaatct ggagccggtg agcgtgggtc
    10801 tcgcggtatc attgcagcac tggggccaga tggtaagccc tcccgtatcg tagttatcta
    10861 cacgacgggg agtcaggcaa ctatggatga acgaaataga cagatcgctg agataggtgc
    10921 ctcactgatt aagcattggt aactgtcaga ccaagtttac tcatatatac tttagattga
    10981 tttaaaactt catttttaat ttaaaaggat ctaggtgaag atcctttttg ataatctcat
    11041 gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg tcagaccccg tagaaaagat
    11101 caaaggatct tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa
    11161 accaccgcta ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc tttttccgaa
    11221 ggtaactggc ttcagcagag cgcagatacc aaatactgtt cttctagtgt agccgtagtt
    11281 aggccaccac ttcaagaact ctgtagcacc gcctacatac ctcgctctgc taatcctgtt
    11341 accagtggct gctgccagtg gcgataagtc gtgtcttacc gggttggact caagacgata
    11401 gttaccggat aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac agcccagctt
    11461 ggagcgaacg acctacaccg aactgagata cctacagcgt gagctatgag aaagcgccac
    11521 gcttcccgaa gggagaaagg cggacaggta tccggtaagc ggcagggtcg gaacaggaga
    11581 gcgcacgagg gagcttccag ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg
    11641 ccacctctga cttgagcgtc gatttttgtg atgctcgtca ggggggcgga gcctatggaa
    11701 aaacgccagc aacgcggcct ttttacggtt cctggccttt tgctggcctt ttgct
//
