LOCUS       sequence-329123-       11755 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Plasmid for constituive spCas9 expression and tet-inducible 
            expression of sgRNA binding to the promoter of TDH3 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_TDH3
FEATURES             Location/Qualifiers
     source          1..11755
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    337..355
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     misc_RNA        441..516
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     terminator      529..591
                     /label=RPR1 terminator
                     /note="transcription terminator for the S. cerevisiae RNase
                     P RNA gene"
     CDS             1726..2349
                     /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(1799..1818)
                     /label=Tet-R
                     /note="Tetracycline resistance gene, reverse primer"
     primer_bind     2846..2863
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    2980..3001
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(3016..3021,3022..3039,3040..3046)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3054..3070
                     /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     3059..3081
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     3078..3094
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     3078..3094
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     3113..3133
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        3115..3133
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        3151..3549
                     /gene="S. cerevisiae TEF1"
                     /label=TEF1 promoter
                     /note="promoter for EF-1-alpha"
     primer_bind     3553..3569
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     3553..3569
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     CDS             3581..7684
                     /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             3581..7684
                     /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             3581..7684
                     /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             7697..7717
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     terminator      7733..7980
                     /gene="S. cerevisiae CYC1"
                     /label=CYC1 terminator
                     /note="transcription terminator for CYC1"
     primer_bind     complement(7749..7767)
                     /label=CYC1
                     /note="CYC1 transcription termination signal, reverse 
                     primer"
     primer_bind     complement(7998..8017)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(7999..8017)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(8027..8044)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(8027..8043)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(8036..8058)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      8184..8639
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(8271..8290)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     8481..8502
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(8742..9416)
                     /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(9417..9698)
                     /gene="S. cerevisiae TRP1"
                     /label=TRP1 promoter
     primer_bind     complement(9719..9738)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     9838..9860
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(9898..9916)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        9984..10088
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(10089..10157,10158..10949)
                     /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(10307..10326)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      11120..11708
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     11609..11628
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 ttttgctcgg gggatctgcc aattgaacat aacatggtag ttacatatac tagtaatatg
       61 gttcggcaca cattaaaagt ataaaaacta tctgaattac gaattacata tattggtcat
      121 aaaaatcaat caatcatcgt gtgttttata tgtctcttat ctaagtataa gaatatccat
      181 agttaatatt cacttacgct accttttaac ctgtaatcat tgtcaacagg atatgttaac
      241 gacccacatt gataaacgct agtatttctt tttcctcttc ttattggccg gctgtctcta
      301 tactccccta tagtctgttt cttttcgttt cgattgtccc tatcagtgat agagatggcg
      361 cacatggtac gctgtggtgc tcgcggctgg gaacgaaact ctgggagctg cgattggcag
      421 aataagtata taaagacggt gttttagagc tagaaatagc aagttaaaat aaggctagtc
      481 cgttatcaac ttgaaaaagt ggcaccgagt cggtgctttt ttctcgagcc atatccaact
      541 tccaatttaa tctttctttt ttaattttca cttatttgcg atacagaaag aaaaaagcga
      601 tagtaactat tgaattttgt ttggatttgg ttagattaga tatggtttct ctttatattt
      661 acatgctaaa aatgggctac accagagata cataattaga tatatatacg ccagtacacc
      721 ttatcggccc aagccttgtc ccaaggcagc gttttgttct tggaaacgct gccctacacg
      781 ttcgctatgc ttcaagaact tttctgagca cttcatgatg catgtttgtt ccttattggt
      841 tagctttgat gttgtgaagt cattgacaca gtctgtgaaa catctttcta ccagattaga
      901 gtacaaacgc atgaaatcct tcatttgctt ttgttccact actttttgga actcttgttg
      961 ttctttcctg caggtagtcg tgcaatgtat gactttaaga tttgtgagca ggaagaaaag
     1021 ggagaatctt ctaacgataa acccttgaaa aactgggtag actacgctat gttgagttgc
     1081 tacgcaggct gcacaattac acgagaatgc tcccgcctag gatttaaggc taagggacgt
     1141 gcaatgcaga cgacagatct aaatgaccgt gtcggtgaag tgttcgccaa acttttcggt
     1201 taacacatgc agtgatgcac gcgcgatggt gctaagttac atatatatat atatgtatag
     1261 ccatagtgat gtctaagtaa cctttatggt atatttctta atgtggaaag atactagcgc
     1321 gcgcacccac acacaagctt cgtcttttct tgaagaaaag aggaagctcg ctaaatggga
     1381 ttccactttc cgttccctgc cagctgatgg aaaaaggtta gtggaacgat gaagaataaa
     1441 aagagagatc cactgaggtg aaatttcagc tgacagcgag tttcatgatc gtgatgaaca
     1501 atggtaacga gttgtggctg ttgccaggga gggtggttct caacttttaa tgtatggcca
     1561 aatcgctact tgggtttgtt atataacaaa gaagaaataa tgaactgatt ctcttcctcc
     1621 ttcttgtcct ttcttaattc tgttgtaatt accttccttt gtaatttttt ttgtaattat
     1681 tcttcttaat aatccaaaca aacacacata ttacaatata ttaaaatgtc tagattagat
     1741 aaaagtaaag tgattaacag cgcattagag ctgcttaatg aggtcggaat cgaaggttta
     1801 acaacccgta aactcgccca gaagctaggt gtagagcagc ctacattgta ttggcatgta
     1861 aaaaataagc gggctttgct cgacgcctta gccattgaga tgttagatag gcaccatact
     1921 cacttttgcc ctttagaagg ggaaagctgg caagattttt tacgtaataa cgctaaaagt
     1981 tttagatgtg ctttactaag tcatcgcgat ggagcaaaag tacatttagg tacacggcct
     2041 acagaaaaac agtatgaaac tctcgaaaat caattagcct ttttatgcca acaaggtttt
     2101 tcactagaga atgcattata tgcactcagc gctgtggggc attttacttt aggttgcgta
     2161 ttggaagatc aagagcatca agtcgctaaa gaagaaaggg aaacacctac tactgatagt
     2221 atgccgccat tattacgaca agctatcgaa ttatttgatc accaaggtgc agagccagcc
     2281 ttcttattcg gccttgaatt gatcatatgc ggattagaaa aacaacttaa atgtgaaagt
     2341 gggtcttaag tctgaagaat gaatgatttg atgatttctt tttccctcca tttttcttac
     2401 tgaatatatc aatgatatag acttgtatag tttattattt caaattaagt agctatatat
     2461 agtcaagata acgtttgttt gacacgatta cattattcgt cgacatcttt tttcagcctg
     2521 tcgtggtagc aatttgagga gtattattaa ttgaataggt tcattttgcg ctcgcataaa
     2581 cagttttcgt cagggacagt atgttggaat gagtggtaat taatggtgac atgacatgtt
     2641 atagcaataa ccttgatgtt tacatcgtag tttaatgtac accccgcgaa ttcgttcaag
     2701 taggagtgca ccaattgcaa agggaaaagc tgaatgggca gttcgaaccg cggtctttcc
     2761 tgcgttatcc cctgattctg tggataaccg tattaccgcc tttgagtgag ctgataccgc
     2821 tcgccgcagc cgaacgaccg agcgcagcga gtcagtgagc gaggaagcgg aagagcgccc
     2881 aatacgcaaa ccgcctctcc ccgcgcgttg gccgattcat taatgcagct ggcacgacag
     2941 gtttcccgac tggaaagcgg gcagtgagcg caacgcaatt aatgtgagtt agctcactca
     3001 ttaggcaccc caggctttac actttatgct tccggctcgt atgttgtgtg gaattgtgag
     3061 cggataacaa tttcacacag gaaacagcta tgaccatgat tacgccaagc gcgcaattaa
     3121 ccctcactaa agggaacaaa agctggagct catagcttca aaatgtttct actccttttt
     3181 tactcttcca gattttctcg gactccgcgc atcgccgtac cacttcaaaa cacccaagca
     3241 cagcatacta aatttcccct ctttcttcct ctagggtgtc gttaattacc cgtactaaag
     3301 gtttggaaaa gaaaaaagag accgcctcgt ttctttttct tcgtcgaaaa aggcaataaa
     3361 aatttttatc acgtttcttt ttcttgaaaa tttttttttt gatttttttc tctttcgatg
     3421 acctcccatt gatatttaag ttaataaacg gtcttcaatt tctcaagttt cagtttcatt
     3481 tttcttgttc tattacaact ttttttactt cttgctcatt agaaagaaag catagcaatc
     3541 taatctaagt tttctagaac tagtggatcc cccgggaaaa atggacaaga agtactccat
     3601 tgggctcgat atcggcacaa acagcgtcgg ttgggccgtc attacggacg agtacaaggt
     3661 gccgagcaaa aaattcaaag ttctgggcaa taccgatcgc cacagcataa agaagaacct
     3721 cattggcgcc ctcctgttcg actccgggga gacggccgaa gccacgcggc tcaaaagaac
     3781 agcacggcgc agatataccc gcagaaagaa tcggatctgc tacctgcagg agatctttag
     3841 taatgagatg gctaaggtgg atgactcttt cttccatagg ctggaggagt cctttttggt
     3901 ggaggaggat aaaaagcacg agcgccaccc aatctttggc aatatcgtgg acgaggtggc
     3961 gtaccatgaa aagtacccaa ccatatatca tctgaggaag aagcttgtag acagtactga
     4021 taaggctgac ttgcggttga tctatctcgc gctggcgcat atgatcaaat ttcggggaca
     4081 cttcctcatc gagggggacc tgaacccaga caacagcgat gtcgacaaac tctttatcca
     4141 actggttcag acttacaatc agcttttcga agagaacccg atcaacgcat ccggagttga
     4201 cgccaaagca atcctgagcg ctaggctgtc caaatcccgg cggctcgaaa acctcatcgc
     4261 acagctccct ggggagaaga agaacggcct gtttggtaat cttatcgccc tgtcactcgg
     4321 gctgaccccc aactttaaat ctaacttcga cctggccgaa gatgccaagc ttcaactgag
     4381 caaagacacc tacgatgatg atctcgacaa tctgctggcc cagatcggcg accagtacgc
     4441 agaccttttt ttggcggcaa agaacctgtc agacgccatt ctgctgagtg atattctgcg
     4501 agtgaacacg gagatcacca aagctccgct gagcgctagt atgatcaagc gctatgatga
     4561 gcaccaccaa gacttgactt tgctgaaggc ccttgtcaga cagcaactgc ctgagaagta
     4621 caaggaaatt ttcttcgatc agtctaaaaa tggctacgcc ggatacattg acggcggagc
     4681 aagccaggag gaattttaca aatttattaa gcccatcttg gaaaaaatgg acggcaccga
     4741 ggagctgctg gtaaagctta acagagaaga tctgttgcgc aaacagcgca ctttcgacaa
     4801 tggaagcatc ccccaccaga ttcacctggg cgaactgcac gctatcctca ggcggcaaga
     4861 ggatttctac ccctttttga aagataacag ggaaaagatt gagaaaatcc tcacatttcg
     4921 gataccctac tatgtaggcc ccctcgcccg gggaaattcc agattcgcgt ggatgactcg
     4981 caaatcagaa gagaccatca ctccctggaa cttcgaggaa gtcgtggata agggggcctc
     5041 tgcccagtcc ttcatcgaaa ggatgactaa ctttgataaa aatctgccta acgaaaaggt
     5101 gcttcctaaa cactctctgc tgtacgagta cttcacagtt tataacgagc tcaccaaggt
     5161 caaatacgtc acagaaggga tgagaaagcc agcattcctg tctggagagc agaagaaagc
     5221 tatcgtggac ctcctcttca agacgaaccg gaaagttacc gtgaaacagc tcaaagaaga
     5281 ctatttcaaa aagattgaat gtttcgactc tgttgaaatc agcggagtgg aggatcgctt
     5341 caacgcatcc ctgggaacgt atcacgatct cctgaaaatc attaaagaca aggacttcct
     5401 ggacaatgag gagaacgagg acattcttga ggacattgtc ctcaccctta cgttgtttga
     5461 agatagggag atgattgaag aacgcttgaa aacttacgct catctcttcg acgacaaagt
     5521 catgaaacag ctcaagaggc gccgatatac aggatggggg cggctgtcaa gaaaactgat
     5581 caatgggatc cgagacaagc agagtggaaa gacaatcctg gattttctta agtccgatgg
     5641 atttgccaac cggaacttca tgcagttgat ccatgatgac tctctcacct ttaaggagga
     5701 catccagaaa gcacaagttt ctggccaggg ggacagtctt cacgagcaca tcgctaatct
     5761 tgcaggtagc ccagctatca aaaagggaat actgcagacc gttaaggtcg tggatgaact
     5821 cgtcaaagta atgggaaggc ataagcccga gaatatcgtt atcgagatgg cccgagagaa
     5881 ccaaactacc cagaagggac agaagaacag tagggaaagg atgaagagga ttgaagaggg
     5941 tataaaagaa ctggggtccc aaatccttaa ggaacaccca gttgaaaaca cccagcttca
     6001 gaatgagaag ctctacctgt actacctgca gaacggcagg gacatgtacg tggatcagga
     6061 actggacatc aatcggctct ccgactacga cgtggatcat atcgtgcccc agtcttttct
     6121 caaagatgat tctattgata ataaagtgtt gacaagatcc gataaaaata gagggaagag
     6181 tgataacgtc ccctcagaag aagttgtcaa gaaaatgaaa aattattggc ggcagctgct
     6241 gaacgccaaa ctgatcacac aacggaagtt cgataatctg actaaggctg aacgaggtgg
     6301 cctgtctgag ttggataaag ccggcttcat caaaaggcag cttgttgaga cacgccagat
     6361 caccaagcac gtggcccaaa ttctcgattc acgcatgaac accaagtacg atgaaaatga
     6421 caaactgatt cgagaggtga aagttattac tctgaagtct aagctggtct cagatttcag
     6481 aaaggacttt cagttttata aggtgagaga gatcaacaat taccaccatg cgcatgatgc
     6541 ctacctgaat gcagtggtag gcactgcact tatcaaaaaa tatcccaagc ttgaatctga
     6601 atttgtttac ggagactata aagtgtacga tgttaggaaa atgatcgcaa agtctgagca
     6661 ggaaataggc aaggccaccg ctaagtactt cttttacagc aatattatga attttttcaa
     6721 gaccgagatt acactggcca atggagagat tcggaagcga ccacttatcg aaacaaacgg
     6781 agaaacagga gaaatcgtgt gggacaaggg tagggatttc gcgacagtcc ggaaggtcct
     6841 gtccatgccg caggtgaaca tcgttaaaaa gaccgaagta cagaccggag gcttctccaa
     6901 ggaaagtatc ctcccgaaaa ggaacagcga caagctgatc gcacgcaaaa aagattggga
     6961 ccccaagaaa tacggcggat tcgattctcc tacagtcgct tacagtgtac tggttgtggc
     7021 caaagtggag aaagggaagt ctaaaaaact caaaagcgtc aaggaactgc tgggcatcac
     7081 aatcatggag cgatcaagct tcgaaaaaaa ccccatcgac tttctcgagg cgaaaggata
     7141 taaagaggtc aaaaaagacc tcatcattaa gcttcccaag tactctctct ttgagcttga
     7201 aaacggccgg aaacgaatgc tcgctagtgc gggcgagctg cagaaaggta acgagctggc
     7261 actgccctct aaatacgtta atttcttgta tctggccagc cactatgaaa agctcaaagg
     7321 gtctcccgaa gataatgagc agaagcagct gttcgtggaa caacacaaac actaccttga
     7381 tgagatcatc gagcaaataa gcgaattctc caaaagagtg atcctcgccg acgctaacct
     7441 cgataaggtg ctttctgctt acaataagca cagggataag cccatcaggg agcaggcaga
     7501 aaacattatc cacttgttta ctctgaccaa cttgggcgcg cctgcagcct tcaagtactt
     7561 cgacaccacc atagacagaa agcggtacac ctctacaaag gaggtcctgg acgccacact
     7621 gattcatcag tcaattacgg ggctctatga aacaagaatc gacctctctc agctcggtgg
     7681 agacagcagg gctgacccca agaagaagag gaaggtgtga tctcttctcg agtcatgtaa
     7741 ttagttatgt cacgcttaca ttcacgccct ccccccacat ccgctctaac cgaaaaggaa
     7801 ggagttagac aacctgaagt ctaggtccct atttattttt ttatagttat gttagtatta
     7861 agaacgttat ttatatttca aatttttctt ttttttctgt acagacgcgt gtacgcatgt
     7921 aacattatac tgaaaacctt gcttgagaag gttttgggac gctcgaaggc tttaatttgc
     7981 ggccggtacc caattcgccc tatagtgagt cgtattacgc gcgctcactg gccgtcgttt
     8041 tacaacgtcg tgactgggaa aaccctggcg ttacccaact taatcgcctt gcagcacatc
     8101 cccctttcgc cagctggcgt aatagcgaag aggcccgcac cgatcgccct tcccaacagt
     8161 tgcgcagcct gaatggcgaa tggacgcgcc ctgtagcggc gcattaagcg cggcgggtgt
     8221 ggtggttacg cgcagcgtga ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc
     8281 tttcttccct tcctttctcg ccacgttcgc cggctttccc cgtcaagctc taaatcgggg
     8341 gctcccttta gggttccgat ttagtgcttt acggcacctc gaccccaaaa aacttgatta
     8401 gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt
     8461 ggagtccacg ttctttaata gtggactctt gttccaaact ggaacaacac tcaaccctat
     8521 ctcggtctat tcttttgatt tataagggat tttgccgatt tcggcctatt ggttaaaaaa
     8581 tgagctgatt taacaaaaat ttaacgcgaa ttttaacaaa atattaacgc ttacaatttc
     8641 ctgatgcggt attttctcct tacgcatctg tgcggtattt cacaccgcat agatcggcaa
     8701 gtgcacaaac aatacttaaa taaatactac tcagtaataa cctatttctt agcatttttg
     8761 acgaaatttg ctattttgtt agagtctttt acaccatttg tctccacacc tccgcttaca
     8821 tcaacaccaa taacgccatt taatctaagc gcatcaccaa cattttctgg cgtcagtcca
     8881 ccagctaaca taaaatgtaa gctttcgggg ctctcttgcc ttccaaccca gtcagaaatc
     8941 gagttccaat ccaaaagttc acctgtccca cctgcttctg aatcaaacaa gggaataaac
     9001 gaatgaggtt tctgtgaagc tgcactgagt agtatgttgc agtcttttgg aaatacgagt
     9061 cttttaataa ctggcaaacc gaggaactct tggtattctt gccacgactc atctccatgc
     9121 agttggacga tatcaatgcc gtaatcattg accagagcca aaacatcctc cttaggttga
     9181 ttacgaaaca cgccaaccaa gtatttcgga gtgcctgaac tatttttata tgcttttaca
     9241 agacttgaaa ttttccttgc aataaccggg tcaattgttc tctttctatt gggcacacat
     9301 ataataccca gcaagtcagc atcggaatct agagcacatt ctgcggcctc tgtgctctgc
     9361 aagccgcaaa ctttcaccaa tggaccagaa ctacctgtga aattaataac agacatactc
     9421 caagctgcct ttgtgtgctt aatcacgtat actcacgtgc tcaatagtca ccaatgccct
     9481 ccctcttggc cctctccttt tcttttttcg accgaattaa ttcttaatcg gcaaaaaaag
     9541 aaaagctccg gatcaagatt gtacgtaagg tgacaagcta tttttcaata aagaatatct
     9601 tccactactg ccatctggcg tcataactgc aaagtacaca tatattacga tgctgttcta
     9661 ttaaatgctt cctatattat atatatagta atgtcgttga tctatggtgc actctcagta
     9721 caatctgctc tgatgccgca tagttaagcc agccccgaca cccgccaaca cccgctgacg
     9781 cgccctgacg ggcttgtctg ctcccggcat ccgcttacag acaagctgtg accgtctccg
     9841 ggagctgcat gtgtcagagg ttttcaccgt catcaccgaa acgcgcgaga cgaaagggcc
     9901 tcgtgatacg cctattttta taggttaatg tcatgataat aatggtttct tagacgtcag
     9961 gtggcacttt tcggggaaat gtgcgcggaa cccctatttg tttatttttc taaatacatt
    10021 caaatatgta tccgctcatg agacaataac cctgataaat gcttcaataa tattgaaaaa
    10081 ggaagagtat gagtattcaa catttccgtg tcgcccttat tccctttttt gcggcatttt
    10141 gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt
    10201 tgggtgcacg agtgggttac atcgaactgg atctcaacag cggtaagatc cttgagagtt
    10261 ttcgccccga agaacgtttt ccaatgatga gcacttttaa agttctgcta tgtggcgcgg
    10321 tattatcccg tattgacgcc gggcaagagc aactcggtcg ccgcatacac tattctcaga
    10381 atgacttggt tgagtactca ccagtcacag aaaagcatct tacggatggc atgacagtaa
    10441 gagaattatg cagtgctgcc ataaccatga gtgataacac tgcggccaac ttacttctga
    10501 caacgatcgg aggaccgaag gagctaaccg cttttttgca caacatgggg gatcatgtaa
    10561 ctcgccttga tcgttgggaa ccggagctga atgaagccat accaaacgac gagcgtgaca
    10621 ccacgatgcc tgtagcaatg gcaacaacgt tgcgcaaact attaactggc gaactactta
    10681 ctctagcttc ccggcaacaa ttaatagact ggatggaggc ggataaagtt gcaggaccac
    10741 ttctgcgctc ggcccttccg gctggctggt ttattgctga taaatctgga gccggtgagc
    10801 gtgggtctcg cggtatcatt gcagcactgg ggccagatgg taagccctcc cgtatcgtag
    10861 ttatctacac gacggggagt caggcaacta tggatgaacg aaatagacag atcgctgaga
    10921 taggtgcctc actgattaag cattggtaac tgtcagacca agtttactca tatatacttt
    10981 agattgattt aaaacttcat ttttaattta aaaggatcta ggtgaagatc ctttttgata
    11041 atctcatgac caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag
    11101 aaaagatcaa aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa
    11161 caaaaaaacc accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt
    11221 ttccgaaggt aactggcttc agcagagcgc agataccaaa tactgttctt ctagtgtagc
    11281 cgtagttagg ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa
    11341 tcctgttacc agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa
    11401 gacgatagtt accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc
    11461 ccagcttgga gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa
    11521 gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa
    11581 caggagagcg cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg
    11641 ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc
    11701 tatggaaaaa cgccagcaac gcggcctttt tacggttcct ggccttttgc tggcc
//
