LOCUS       sequence-329121-       11755 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Plasmid for constituive spCas9 and tet-inducible PDR12 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_PDR12
FEATURES             Location/Qualifiers
     source          1..11755
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        complement(80..361)
                     /gene="S. cerevisiae TRP1"
                     /label=TRP1 promoter
     primer_bind     complement(382..401)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     501..523
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(561..579)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        647..751
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(752..820,821..1612)
                     /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(970..989)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      1783..2371
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2272..2291
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     protein_bind    2755..2773
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     misc_RNA        2859..2934
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     terminator      2947..3009
                     /label=RPR1 terminator
                     /note="transcription terminator for the S. cerevisiae RNase
                     P RNA gene"
     CDS             4144..4767
                     /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(4217..4236)
                     /label=Tet-R
                     /note="Tetracycline resistance gene, reverse primer"
     primer_bind     5264..5281
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    5398..5419
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(5434..5439,5440..5457,5458..5464)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5472..5488
                     /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     5477..5499
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     5496..5512
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     5496..5512
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     5531..5551
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        5533..5551
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        5569..5967
                     /gene="S. cerevisiae TEF1"
                     /label=TEF1 promoter
                     /note="promoter for EF-1-alpha"
     primer_bind     5971..5987
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     5971..5987
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     CDS             5999..10102
                     /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             5999..10102
                     /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             5999..10102
                     /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             10115..10135
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     terminator      10151..10398
                     /gene="S. cerevisiae CYC1"
                     /label=CYC1 terminator
                     /note="transcription terminator for CYC1"
     primer_bind     complement(10167..10185)
                     /label=CYC1
                     /note="CYC1 transcription termination signal, reverse 
                     primer"
     primer_bind     complement(10416..10435)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(10417..10435)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(10445..10462)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(10445..10461)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(10454..10476)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      10602..11057
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(10689..10708)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     10899..10920
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(join(11160..11755,1..79))
                     /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"
ORIGIN
        1 attctgcggc ctctgtgctc tgcaagccgc aaactttcac caatggacca gaactacctg
       61 tgaaattaat aacagacata ctccaagctg cctttgtgtg cttaatcacg tatactcacg
      121 tgctcaatag tcaccaatgc cctccctctt ggccctctcc ttttcttttt tcgaccgaat
      181 taattcttaa tcggcaaaaa aagaaaagct ccggatcaag attgtacgta aggtgacaag
      241 ctatttttca ataaagaata tcttccacta ctgccatctg gcgtcataac tgcaaagtac
      301 acatatatta cgatgctgtt ctattaaatg cttcctatat tatatatata gtaatgtcgt
      361 tgatctatgg tgcactctca gtacaatctg ctctgatgcc gcatagttaa gccagccccg
      421 acacccgcca acacccgctg acgcgccctg acgggcttgt ctgctcccgg catccgctta
      481 cagacaagct gtgaccgtct ccgggagctg catgtgtcag aggttttcac cgtcatcacc
      541 gaaacgcgcg agacgaaagg gcctcgtgat acgcctattt ttataggtta atgtcatgat
      601 aataatggtt tcttagacgt caggtggcac ttttcgggga aatgtgcgcg gaacccctat
      661 ttgtttattt ttctaaatac attcaaatat gtatccgctc atgagacaat aaccctgata
      721 aatgcttcaa taatattgaa aaaggaagag tatgagtatt caacatttcc gtgtcgccct
      781 tattcccttt tttgcggcat tttgccttcc tgtttttgct cacccagaaa cgctggtgaa
      841 agtaaaagat gctgaagatc agttgggtgc acgagtgggt tacatcgaac tggatctcaa
      901 cagcggtaag atccttgaga gttttcgccc cgaagaacgt tttccaatga tgagcacttt
      961 taaagttctg ctatgtggcg cggtattatc ccgtattgac gccgggcaag agcaactcgg
     1021 tcgccgcata cactattctc agaatgactt ggttgagtac tcaccagtca cagaaaagca
     1081 tcttacggat ggcatgacag taagagaatt atgcagtgct gccataacca tgagtgataa
     1141 cactgcggcc aacttacttc tgacaacgat cggaggaccg aaggagctaa ccgctttttt
     1201 gcacaacatg ggggatcatg taactcgcct tgatcgttgg gaaccggagc tgaatgaagc
     1261 cataccaaac gacgagcgtg acaccacgat gcctgtagca atggcaacaa cgttgcgcaa
     1321 actattaact ggcgaactac ttactctagc ttcccggcaa caattaatag actggatgga
     1381 ggcggataaa gttgcaggac cacttctgcg ctcggccctt ccggctggct ggtttattgc
     1441 tgataaatct ggagccggtg agcgtgggtc tcgcggtatc attgcagcac tggggccaga
     1501 tggtaagccc tcccgtatcg tagttatcta cacgacgggg agtcaggcaa ctatggatga
     1561 acgaaataga cagatcgctg agataggtgc ctcactgatt aagcattggt aactgtcaga
     1621 ccaagtttac tcatatatac tttagattga tttaaaactt catttttaat ttaaaaggat
     1681 ctaggtgaag atcctttttg ataatctcat gaccaaaatc ccttaacgtg agttttcgtt
     1741 ccactgagcg tcagaccccg tagaaaagat caaaggatct tcttgagatc ctttttttct
     1801 gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc
     1861 ggatcaagag ctaccaactc tttttccgaa ggtaactggc ttcagcagag cgcagatacc
     1921 aaatactgtt cttctagtgt agccgtagtt aggccaccac ttcaagaact ctgtagcacc
     1981 gcctacatac ctcgctctgc taatcctgtt accagtggct gctgccagtg gcgataagtc
     2041 gtgtcttacc gggttggact caagacgata gttaccggat aaggcgcagc ggtcgggctg
     2101 aacggggggt tcgtgcacac agcccagctt ggagcgaacg acctacaccg aactgagata
     2161 cctacagcgt gagctatgag aaagcgccac gcttcccgaa gggagaaagg cggacaggta
     2221 tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg gagcttccag ggggaaacgc
     2281 ctggtatctt tatagtcctg tcgggtttcg ccacctctga cttgagcgtc gatttttgtg
     2341 atgctcgtca ggggggcgga gcctatggaa aaacgccagc aacgcggcct ttttacggtt
     2401 cctggccttt tgctggcctt ttgctcgggg gatctgccaa ttgaacataa catggtagtt
     2461 acatatacta gtaatatggt tcggcacaca ttaaaagtat aaaaactatc tgaattacga
     2521 attacatata ttggtcataa aaatcaatca atcatcgtgt gttttatatg tctcttatct
     2581 aagtataaga atatccatag ttaatattca cttacgctac cttttaacct gtaatcattg
     2641 tcaacaggat atgttaacga cccacattga taaacgctag tatttctttt tcctcttctt
     2701 attggccggc tgtctctata ctcccctata gtctgtttct tttcgtttcg attgtcccta
     2761 tcagtgatag agatggcgca catggtacgc tgtggtgctc gcggctggga acgaaactct
     2821 gggagctgcg attggcaggt tatcccaaga gtagatgggt tttagagcta gaaatagcaa
     2881 gttaaaataa ggctagtccg ttatcaactt gaaaaagtgg caccgagtcg gtgctttttt
     2941 ctcgagccat atccaacttc caatttaatc tttctttttt aattttcact tatttgcgat
     3001 acagaaagaa aaaagcgata gtaactattg aattttgttt ggatttggtt agattagata
     3061 tggtttctct ttatatttac atgctaaaaa tgggctacac cagagataca taattagata
     3121 tatatacgcc agtacacctt atcggcccaa gccttgtccc aaggcagcgt tttgttcttg
     3181 gaaacgctgc cctacacgtt cgctatgctt caagaacttt tctgagcact tcatgatgca
     3241 tgtttgttcc ttattggtta gctttgatgt tgtgaagtca ttgacacagt ctgtgaaaca
     3301 tctttctacc agattagagt acaaacgcat gaaatccttc atttgctttt gttccactac
     3361 tttttggaac tcttgttgtt ctttcctgca ggtagtcgtg caatgtatga ctttaagatt
     3421 tgtgagcagg aagaaaaggg agaatcttct aacgataaac ccttgaaaaa ctgggtagac
     3481 tacgctatgt tgagttgcta cgcaggctgc acaattacac gagaatgctc ccgcctagga
     3541 tttaaggcta agggacgtgc aatgcagacg acagatctaa atgaccgtgt cggtgaagtg
     3601 ttcgccaaac ttttcggtta acacatgcag tgatgcacgc gcgatggtgc taagttacat
     3661 atatatatat atgtatagcc atagtgatgt ctaagtaacc tttatggtat atttcttaat
     3721 gtggaaagat actagcgcgc gcacccacac acaagcttcg tcttttcttg aagaaaagag
     3781 gaagctcgct aaatgggatt ccactttccg ttccctgcca gctgatggaa aaaggttagt
     3841 ggaacgatga agaataaaaa gagagatcca ctgaggtgaa atttcagctg acagcgagtt
     3901 tcatgatcgt gatgaacaat ggtaacgagt tgtggctgtt gccagggagg gtggttctca
     3961 acttttaatg tatggccaaa tcgctacttg ggtttgttat ataacaaaga agaaataatg
     4021 aactgattct cttcctcctt cttgtccttt cttaattctg ttgtaattac cttcctttgt
     4081 aatttttttt gtaattattc ttcttaataa tccaaacaaa cacacatatt acaatatatt
     4141 aaaatgtcta gattagataa aagtaaagtg attaacagcg cattagagct gcttaatgag
     4201 gtcggaatcg aaggtttaac aacccgtaaa ctcgcccaga agctaggtgt agagcagcct
     4261 acattgtatt ggcatgtaaa aaataagcgg gctttgctcg acgccttagc cattgagatg
     4321 ttagataggc accatactca cttttgccct ttagaagggg aaagctggca agatttttta
     4381 cgtaataacg ctaaaagttt tagatgtgct ttactaagtc atcgcgatgg agcaaaagta
     4441 catttaggta cacggcctac agaaaaacag tatgaaactc tcgaaaatca attagccttt
     4501 ttatgccaac aaggtttttc actagagaat gcattatatg cactcagcgc tgtggggcat
     4561 tttactttag gttgcgtatt ggaagatcaa gagcatcaag tcgctaaaga agaaagggaa
     4621 acacctacta ctgatagtat gccgccatta ttacgacaag ctatcgaatt atttgatcac
     4681 caaggtgcag agccagcctt cttattcggc cttgaattga tcatatgcgg attagaaaaa
     4741 caacttaaat gtgaaagtgg gtcttaagtc tgaagaatga atgatttgat gatttctttt
     4801 tccctccatt tttcttactg aatatatcaa tgatatagac ttgtatagtt tattatttca
     4861 aattaagtag ctatatatag tcaagataac gtttgtttga cacgattaca ttattcgtcg
     4921 acatcttttt tcagcctgtc gtggtagcaa tttgaggagt attattaatt gaataggttc
     4981 attttgcgct cgcataaaca gttttcgtca gggacagtat gttggaatga gtggtaatta
     5041 atggtgacat gacatgttat agcaataacc ttgatgttta catcgtagtt taatgtacac
     5101 cccgcgaatt cgttcaagta ggagtgcacc aattgcaaag ggaaaagctg aatgggcagt
     5161 tcgaaccgcg gtctttcctg cgttatcccc tgattctgtg gataaccgta ttaccgcctt
     5221 tgagtgagct gataccgctc gccgcagccg aacgaccgag cgcagcgagt cagtgagcga
     5281 ggaagcggaa gagcgcccaa tacgcaaacc gcctctcccc gcgcgttggc cgattcatta
     5341 atgcagctgg cacgacaggt ttcccgactg gaaagcgggc agtgagcgca acgcaattaa
     5401 tgtgagttag ctcactcatt aggcacccca ggctttacac tttatgcttc cggctcgtat
     5461 gttgtgtgga attgtgagcg gataacaatt tcacacagga aacagctatg accatgatta
     5521 cgccaagcgc gcaattaacc ctcactaaag ggaacaaaag ctggagctca tagcttcaaa
     5581 atgtttctac tcctttttta ctcttccaga ttttctcgga ctccgcgcat cgccgtacca
     5641 cttcaaaaca cccaagcaca gcatactaaa tttcccctct ttcttcctct agggtgtcgt
     5701 taattacccg tactaaaggt ttggaaaaga aaaaagagac cgcctcgttt ctttttcttc
     5761 gtcgaaaaag gcaataaaaa tttttatcac gtttcttttt cttgaaaatt ttttttttga
     5821 tttttttctc tttcgatgac ctcccattga tatttaagtt aataaacggt cttcaatttc
     5881 tcaagtttca gtttcatttt tcttgttcta ttacaacttt ttttacttct tgctcattag
     5941 aaagaaagca tagcaatcta atctaagttt tctagaacta gtggatcccc cgggaaaaat
     6001 ggacaagaag tactccattg ggctcgatat cggcacaaac agcgtcggtt gggccgtcat
     6061 tacggacgag tacaaggtgc cgagcaaaaa attcaaagtt ctgggcaata ccgatcgcca
     6121 cagcataaag aagaacctca ttggcgccct cctgttcgac tccggggaga cggccgaagc
     6181 cacgcggctc aaaagaacag cacggcgcag atatacccgc agaaagaatc ggatctgcta
     6241 cctgcaggag atctttagta atgagatggc taaggtggat gactctttct tccataggct
     6301 ggaggagtcc tttttggtgg aggaggataa aaagcacgag cgccacccaa tctttggcaa
     6361 tatcgtggac gaggtggcgt accatgaaaa gtacccaacc atatatcatc tgaggaagaa
     6421 gcttgtagac agtactgata aggctgactt gcggttgatc tatctcgcgc tggcgcatat
     6481 gatcaaattt cggggacact tcctcatcga gggggacctg aacccagaca acagcgatgt
     6541 cgacaaactc tttatccaac tggttcagac ttacaatcag cttttcgaag agaacccgat
     6601 caacgcatcc ggagttgacg ccaaagcaat cctgagcgct aggctgtcca aatcccggcg
     6661 gctcgaaaac ctcatcgcac agctccctgg ggagaagaag aacggcctgt ttggtaatct
     6721 tatcgccctg tcactcgggc tgacccccaa ctttaaatct aacttcgacc tggccgaaga
     6781 tgccaagctt caactgagca aagacaccta cgatgatgat ctcgacaatc tgctggccca
     6841 gatcggcgac cagtacgcag accttttttt ggcggcaaag aacctgtcag acgccattct
     6901 gctgagtgat attctgcgag tgaacacgga gatcaccaaa gctccgctga gcgctagtat
     6961 gatcaagcgc tatgatgagc accaccaaga cttgactttg ctgaaggccc ttgtcagaca
     7021 gcaactgcct gagaagtaca aggaaatttt cttcgatcag tctaaaaatg gctacgccgg
     7081 atacattgac ggcggagcaa gccaggagga attttacaaa tttattaagc ccatcttgga
     7141 aaaaatggac ggcaccgagg agctgctggt aaagcttaac agagaagatc tgttgcgcaa
     7201 acagcgcact ttcgacaatg gaagcatccc ccaccagatt cacctgggcg aactgcacgc
     7261 tatcctcagg cggcaagagg atttctaccc ctttttgaaa gataacaggg aaaagattga
     7321 gaaaatcctc acatttcgga taccctacta tgtaggcccc ctcgcccggg gaaattccag
     7381 attcgcgtgg atgactcgca aatcagaaga gaccatcact ccctggaact tcgaggaagt
     7441 cgtggataag ggggcctctg cccagtcctt catcgaaagg atgactaact ttgataaaaa
     7501 tctgcctaac gaaaaggtgc ttcctaaaca ctctctgctg tacgagtact tcacagttta
     7561 taacgagctc accaaggtca aatacgtcac agaagggatg agaaagccag cattcctgtc
     7621 tggagagcag aagaaagcta tcgtggacct cctcttcaag acgaaccgga aagttaccgt
     7681 gaaacagctc aaagaagact atttcaaaaa gattgaatgt ttcgactctg ttgaaatcag
     7741 cggagtggag gatcgcttca acgcatccct gggaacgtat cacgatctcc tgaaaatcat
     7801 taaagacaag gacttcctgg acaatgagga gaacgaggac attcttgagg acattgtcct
     7861 cacccttacg ttgtttgaag atagggagat gattgaagaa cgcttgaaaa cttacgctca
     7921 tctcttcgac gacaaagtca tgaaacagct caagaggcgc cgatatacag gatgggggcg
     7981 gctgtcaaga aaactgatca atgggatccg agacaagcag agtggaaaga caatcctgga
     8041 ttttcttaag tccgatggat ttgccaaccg gaacttcatg cagttgatcc atgatgactc
     8101 tctcaccttt aaggaggaca tccagaaagc acaagtttct ggccaggggg acagtcttca
     8161 cgagcacatc gctaatcttg caggtagccc agctatcaaa aagggaatac tgcagaccgt
     8221 taaggtcgtg gatgaactcg tcaaagtaat gggaaggcat aagcccgaga atatcgttat
     8281 cgagatggcc cgagagaacc aaactaccca gaagggacag aagaacagta gggaaaggat
     8341 gaagaggatt gaagagggta taaaagaact ggggtcccaa atccttaagg aacacccagt
     8401 tgaaaacacc cagcttcaga atgagaagct ctacctgtac tacctgcaga acggcaggga
     8461 catgtacgtg gatcaggaac tggacatcaa tcggctctcc gactacgacg tggatcatat
     8521 cgtgccccag tcttttctca aagatgattc tattgataat aaagtgttga caagatccga
     8581 taaaaataga gggaagagtg ataacgtccc ctcagaagaa gttgtcaaga aaatgaaaaa
     8641 ttattggcgg cagctgctga acgccaaact gatcacacaa cggaagttcg ataatctgac
     8701 taaggctgaa cgaggtggcc tgtctgagtt ggataaagcc ggcttcatca aaaggcagct
     8761 tgttgagaca cgccagatca ccaagcacgt ggcccaaatt ctcgattcac gcatgaacac
     8821 caagtacgat gaaaatgaca aactgattcg agaggtgaaa gttattactc tgaagtctaa
     8881 gctggtctca gatttcagaa aggactttca gttttataag gtgagagaga tcaacaatta
     8941 ccaccatgcg catgatgcct acctgaatgc agtggtaggc actgcactta tcaaaaaata
     9001 tcccaagctt gaatctgaat ttgtttacgg agactataaa gtgtacgatg ttaggaaaat
     9061 gatcgcaaag tctgagcagg aaataggcaa ggccaccgct aagtacttct tttacagcaa
     9121 tattatgaat tttttcaaga ccgagattac actggccaat ggagagattc ggaagcgacc
     9181 acttatcgaa acaaacggag aaacaggaga aatcgtgtgg gacaagggta gggatttcgc
     9241 gacagtccgg aaggtcctgt ccatgccgca ggtgaacatc gttaaaaaga ccgaagtaca
     9301 gaccggaggc ttctccaagg aaagtatcct cccgaaaagg aacagcgaca agctgatcgc
     9361 acgcaaaaaa gattgggacc ccaagaaata cggcggattc gattctccta cagtcgctta
     9421 cagtgtactg gttgtggcca aagtggagaa agggaagtct aaaaaactca aaagcgtcaa
     9481 ggaactgctg ggcatcacaa tcatggagcg atcaagcttc gaaaaaaacc ccatcgactt
     9541 tctcgaggcg aaaggatata aagaggtcaa aaaagacctc atcattaagc ttcccaagta
     9601 ctctctcttt gagcttgaaa acggccggaa acgaatgctc gctagtgcgg gcgagctgca
     9661 gaaaggtaac gagctggcac tgccctctaa atacgttaat ttcttgtatc tggccagcca
     9721 ctatgaaaag ctcaaagggt ctcccgaaga taatgagcag aagcagctgt tcgtggaaca
     9781 acacaaacac taccttgatg agatcatcga gcaaataagc gaattctcca aaagagtgat
     9841 cctcgccgac gctaacctcg ataaggtgct ttctgcttac aataagcaca gggataagcc
     9901 catcagggag caggcagaaa acattatcca cttgtttact ctgaccaact tgggcgcgcc
     9961 tgcagccttc aagtacttcg acaccaccat agacagaaag cggtacacct ctacaaagga
    10021 ggtcctggac gccacactga ttcatcagtc aattacgggg ctctatgaaa caagaatcga
    10081 cctctctcag ctcggtggag acagcagggc tgaccccaag aagaagagga aggtgtgatc
    10141 tcttctcgag tcatgtaatt agttatgtca cgcttacatt cacgccctcc ccccacatcc
    10201 gctctaaccg aaaaggaagg agttagacaa cctgaagtct aggtccctat ttattttttt
    10261 atagttatgt tagtattaag aacgttattt atatttcaaa tttttctttt ttttctgtac
    10321 agacgcgtgt acgcatgtaa cattatactg aaaaccttgc ttgagaaggt tttgggacgc
    10381 tcgaaggctt taatttgcgg ccggtaccca attcgcccta tagtgagtcg tattacgcgc
    10441 gctcactggc cgtcgtttta caacgtcgtg actgggaaaa ccctggcgtt acccaactta
    10501 atcgccttgc agcacatccc cctttcgcca gctggcgtaa tagcgaagag gcccgcaccg
    10561 atcgcccttc ccaacagttg cgcagcctga atggcgaatg gacgcgccct gtagcggcgc
    10621 attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg ccagcgccct
    10681 agcgcccgct cctttcgctt tcttcccttc ctttctcgcc acgttcgccg gctttccccg
    10741 tcaagctcta aatcgggggc tccctttagg gttccgattt agtgctttac ggcacctcga
    10801 ccccaaaaaa cttgattagg gtgatggttc acgtagtggg ccatcgccct gatagacggt
    10861 ttttcgccct ttgacgttgg agtccacgtt ctttaatagt ggactcttgt tccaaactgg
    10921 aacaacactc aaccctatct cggtctattc ttttgattta taagggattt tgccgatttc
    10981 ggcctattgg ttaaaaaatg agctgattta acaaaaattt aacgcgaatt ttaacaaaat
    11041 attaacgctt acaatttcct gatgcggtat tttctcctta cgcatctgtg cggtatttca
    11101 caccgcatag atcggcaagt gcacaaacaa tacttaaata aatactactc agtaataacc
    11161 tatttcttag catttttgac gaaatttgct attttgttag agtcttttac accatttgtc
    11221 tccacacctc cgcttacatc aacaccaata acgccattta atctaagcgc atcaccaaca
    11281 ttttctggcg tcagtccacc agctaacata aaatgtaagc tttcggggct ctcttgcctt
    11341 ccaacccagt cagaaatcga gttccaatcc aaaagttcac ctgtcccacc tgcttctgaa
    11401 tcaaacaagg gaataaacga atgaggtttc tgtgaagctg cactgagtag tatgttgcag
    11461 tcttttggaa atacgagtct tttaataact ggcaaaccga ggaactcttg gtattcttgc
    11521 cacgactcat ctccatgcag ttggacgata tcaatgccgt aatcattgac cagagccaaa
    11581 acatcctcct taggttgatt acgaaacacg ccaaccaagt atttcggagt gcctgaacta
    11641 tttttatatg cttttacaag acttgaaatt ttccttgcaa taaccgggtc aattgttctc
    11701 tttctattgg gcacacatat aatacccagc aagtcagcat cggaatctag agcac
//
