LOCUS       sequence-329122-       11755 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Plasmid for constitutive spCas9 and tet-inducible PPE1 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_PPE1
FEATURES             Location/Qualifiers
     source          1..11755
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      1..589
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     490..509
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     protein_bind    973..991
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     misc_RNA        1077..1152
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     terminator      1165..1227
                     /label=RPR1 terminator
                     /note="transcription terminator for the S. cerevisiae RNase
                     P RNA gene"
     CDS             2362..2985
                     /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(2435..2454)
                     /label=Tet-R
                     /note="Tetracycline resistance gene, reverse primer"
     primer_bind     3482..3499
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    3616..3637
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(3652..3657,3658..3675,3676..3682)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3690..3706
                     /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     3695..3717
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     3714..3730
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     3714..3730
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     3749..3769
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        3751..3769
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        3787..4185
                     /gene="S. cerevisiae TEF1"
                     /label=TEF1 promoter
                     /note="promoter for EF-1-alpha"
     primer_bind     4189..4205
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     4189..4205
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     CDS             4217..8320
                     /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             4217..8320
                     /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             4217..8320
                     /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             8333..8353
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     terminator      8369..8616
                     /gene="S. cerevisiae CYC1"
                     /label=CYC1 terminator
                     /note="transcription terminator for CYC1"
     primer_bind     complement(8385..8403)
                     /label=CYC1
                     /note="CYC1 transcription termination signal, reverse 
                     primer"
     primer_bind     complement(8634..8653)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(8635..8653)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(8663..8680)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(8663..8679)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(8672..8694)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      8820..9275
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(8907..8926)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     9117..9138
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(9378..10052)
                     /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(10053..10334)
                     /gene="S. cerevisiae TRP1"
                     /label=TRP1 promoter
     primer_bind     complement(10355..10374)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     10474..10496
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(10534..10552)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        10620..10724
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(10725..10793,10794..11585)
                     /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(10943..10962)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc
       61 agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt
      121 cagcagagcg cagataccaa atactgttct tctagtgtag ccgtagttag gccaccactt
      181 caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc
      241 tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa
      301 ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac
      361 ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg
      421 gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga
      481 gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact
      541 tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa
      601 cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcggggga tctgccaatt
      661 gaacataaca tggtagttac atatactagt aatatggttc ggcacacatt aaaagtataa
      721 aaactatctg aattacgaat tacatatatt ggtcataaaa atcaatcaat catcgtgtgt
      781 tttatatgtc tcttatctaa gtataagaat atccatagtt aatattcact tacgctacct
      841 tttaacctgt aatcattgtc aacaggatat gttaacgacc cacattgata aacgctagta
      901 tttctttttc ctcttcttat tggccggctg tctctatact cccctatagt ctgtttcttt
      961 tcgtttcgat tgtccctatc agtgatagag atggcgcaca tggtacgctg tggtgctcgc
     1021 ggctgggaac gaaactctgg gagctgcgat tggcaggaca ttccaagaag catacggttt
     1081 tagagctaga aatagcaagt taaaataagg ctagtccgtt atcaacttga aaaagtggca
     1141 ccgagtcggt gcttttttct cgagccatat ccaacttcca atttaatctt tcttttttaa
     1201 ttttcactta tttgcgatac agaaagaaaa aagcgatagt aactattgaa ttttgtttgg
     1261 atttggttag attagatatg gtttctcttt atatttacat gctaaaaatg ggctacacca
     1321 gagatacata attagatata tatacgccag tacaccttat cggcccaagc cttgtcccaa
     1381 ggcagcgttt tgttcttgga aacgctgccc tacacgttcg ctatgcttca agaacttttc
     1441 tgagcacttc atgatgcatg tttgttcctt attggttagc tttgatgttg tgaagtcatt
     1501 gacacagtct gtgaaacatc tttctaccag attagagtac aaacgcatga aatccttcat
     1561 ttgcttttgt tccactactt tttggaactc ttgttgttct ttcctgcagg tagtcgtgca
     1621 atgtatgact ttaagatttg tgagcaggaa gaaaagggag aatcttctaa cgataaaccc
     1681 ttgaaaaact gggtagacta cgctatgttg agttgctacg caggctgcac aattacacga
     1741 gaatgctccc gcctaggatt taaggctaag ggacgtgcaa tgcagacgac agatctaaat
     1801 gaccgtgtcg gtgaagtgtt cgccaaactt ttcggttaac acatgcagtg atgcacgcgc
     1861 gatggtgcta agttacatat atatatatat gtatagccat agtgatgtct aagtaacctt
     1921 tatggtatat ttcttaatgt ggaaagatac tagcgcgcgc acccacacac aagcttcgtc
     1981 ttttcttgaa gaaaagagga agctcgctaa atgggattcc actttccgtt ccctgccagc
     2041 tgatggaaaa aggttagtgg aacgatgaag aataaaaaga gagatccact gaggtgaaat
     2101 ttcagctgac agcgagtttc atgatcgtga tgaacaatgg taacgagttg tggctgttgc
     2161 cagggagggt ggttctcaac ttttaatgta tggccaaatc gctacttggg tttgttatat
     2221 aacaaagaag aaataatgaa ctgattctct tcctccttct tgtcctttct taattctgtt
     2281 gtaattacct tcctttgtaa ttttttttgt aattattctt cttaataatc caaacaaaca
     2341 cacatattac aatatattaa aatgtctaga ttagataaaa gtaaagtgat taacagcgca
     2401 ttagagctgc ttaatgaggt cggaatcgaa ggtttaacaa cccgtaaact cgcccagaag
     2461 ctaggtgtag agcagcctac attgtattgg catgtaaaaa ataagcgggc tttgctcgac
     2521 gccttagcca ttgagatgtt agataggcac catactcact tttgcccttt agaaggggaa
     2581 agctggcaag attttttacg taataacgct aaaagtttta gatgtgcttt actaagtcat
     2641 cgcgatggag caaaagtaca tttaggtaca cggcctacag aaaaacagta tgaaactctc
     2701 gaaaatcaat tagccttttt atgccaacaa ggtttttcac tagagaatgc attatatgca
     2761 ctcagcgctg tggggcattt tactttaggt tgcgtattgg aagatcaaga gcatcaagtc
     2821 gctaaagaag aaagggaaac acctactact gatagtatgc cgccattatt acgacaagct
     2881 atcgaattat ttgatcacca aggtgcagag ccagccttct tattcggcct tgaattgatc
     2941 atatgcggat tagaaaaaca acttaaatgt gaaagtgggt cttaagtctg aagaatgaat
     3001 gatttgatga tttctttttc cctccatttt tcttactgaa tatatcaatg atatagactt
     3061 gtatagttta ttatttcaaa ttaagtagct atatatagtc aagataacgt ttgtttgaca
     3121 cgattacatt attcgtcgac atcttttttc agcctgtcgt ggtagcaatt tgaggagtat
     3181 tattaattga ataggttcat tttgcgctcg cataaacagt tttcgtcagg gacagtatgt
     3241 tggaatgagt ggtaattaat ggtgacatga catgttatag caataacctt gatgtttaca
     3301 tcgtagttta atgtacaccc cgcgaattcg ttcaagtagg agtgcaccaa ttgcaaaggg
     3361 aaaagctgaa tgggcagttc gaaccgcggt ctttcctgcg ttatcccctg attctgtgga
     3421 taaccgtatt accgcctttg agtgagctga taccgctcgc cgcagccgaa cgaccgagcg
     3481 cagcgagtca gtgagcgagg aagcggaaga gcgcccaata cgcaaaccgc ctctccccgc
     3541 gcgttggccg attcattaat gcagctggca cgacaggttt cccgactgga aagcgggcag
     3601 tgagcgcaac gcaattaatg tgagttagct cactcattag gcaccccagg ctttacactt
     3661 tatgcttccg gctcgtatgt tgtgtggaat tgtgagcgga taacaatttc acacaggaaa
     3721 cagctatgac catgattacg ccaagcgcgc aattaaccct cactaaaggg aacaaaagct
     3781 ggagctcata gcttcaaaat gtttctactc cttttttact cttccagatt ttctcggact
     3841 ccgcgcatcg ccgtaccact tcaaaacacc caagcacagc atactaaatt tcccctcttt
     3901 cttcctctag ggtgtcgtta attacccgta ctaaaggttt ggaaaagaaa aaagagaccg
     3961 cctcgtttct ttttcttcgt cgaaaaaggc aataaaaatt tttatcacgt ttctttttct
     4021 tgaaaatttt ttttttgatt tttttctctt tcgatgacct cccattgata tttaagttaa
     4081 taaacggtct tcaatttctc aagtttcagt ttcatttttc ttgttctatt acaacttttt
     4141 ttacttcttg ctcattagaa agaaagcata gcaatctaat ctaagttttc tagaactagt
     4201 ggatcccccg ggaaaaatgg acaagaagta ctccattggg ctcgatatcg gcacaaacag
     4261 cgtcggttgg gccgtcatta cggacgagta caaggtgccg agcaaaaaat tcaaagttct
     4321 gggcaatacc gatcgccaca gcataaagaa gaacctcatt ggcgccctcc tgttcgactc
     4381 cggggagacg gccgaagcca cgcggctcaa aagaacagca cggcgcagat atacccgcag
     4441 aaagaatcgg atctgctacc tgcaggagat ctttagtaat gagatggcta aggtggatga
     4501 ctctttcttc cataggctgg aggagtcctt tttggtggag gaggataaaa agcacgagcg
     4561 ccacccaatc tttggcaata tcgtggacga ggtggcgtac catgaaaagt acccaaccat
     4621 atatcatctg aggaagaagc ttgtagacag tactgataag gctgacttgc ggttgatcta
     4681 tctcgcgctg gcgcatatga tcaaatttcg gggacacttc ctcatcgagg gggacctgaa
     4741 cccagacaac agcgatgtcg acaaactctt tatccaactg gttcagactt acaatcagct
     4801 tttcgaagag aacccgatca acgcatccgg agttgacgcc aaagcaatcc tgagcgctag
     4861 gctgtccaaa tcccggcggc tcgaaaacct catcgcacag ctccctgggg agaagaagaa
     4921 cggcctgttt ggtaatctta tcgccctgtc actcgggctg acccccaact ttaaatctaa
     4981 cttcgacctg gccgaagatg ccaagcttca actgagcaaa gacacctacg atgatgatct
     5041 cgacaatctg ctggcccaga tcggcgacca gtacgcagac ctttttttgg cggcaaagaa
     5101 cctgtcagac gccattctgc tgagtgatat tctgcgagtg aacacggaga tcaccaaagc
     5161 tccgctgagc gctagtatga tcaagcgcta tgatgagcac caccaagact tgactttgct
     5221 gaaggccctt gtcagacagc aactgcctga gaagtacaag gaaattttct tcgatcagtc
     5281 taaaaatggc tacgccggat acattgacgg cggagcaagc caggaggaat tttacaaatt
     5341 tattaagccc atcttggaaa aaatggacgg caccgaggag ctgctggtaa agcttaacag
     5401 agaagatctg ttgcgcaaac agcgcacttt cgacaatgga agcatccccc accagattca
     5461 cctgggcgaa ctgcacgcta tcctcaggcg gcaagaggat ttctacccct ttttgaaaga
     5521 taacagggaa aagattgaga aaatcctcac atttcggata ccctactatg taggccccct
     5581 cgcccgggga aattccagat tcgcgtggat gactcgcaaa tcagaagaga ccatcactcc
     5641 ctggaacttc gaggaagtcg tggataaggg ggcctctgcc cagtccttca tcgaaaggat
     5701 gactaacttt gataaaaatc tgcctaacga aaaggtgctt cctaaacact ctctgctgta
     5761 cgagtacttc acagtttata acgagctcac caaggtcaaa tacgtcacag aagggatgag
     5821 aaagccagca ttcctgtctg gagagcagaa gaaagctatc gtggacctcc tcttcaagac
     5881 gaaccggaaa gttaccgtga aacagctcaa agaagactat ttcaaaaaga ttgaatgttt
     5941 cgactctgtt gaaatcagcg gagtggagga tcgcttcaac gcatccctgg gaacgtatca
     6001 cgatctcctg aaaatcatta aagacaagga cttcctggac aatgaggaga acgaggacat
     6061 tcttgaggac attgtcctca cccttacgtt gtttgaagat agggagatga ttgaagaacg
     6121 cttgaaaact tacgctcatc tcttcgacga caaagtcatg aaacagctca agaggcgccg
     6181 atatacagga tgggggcggc tgtcaagaaa actgatcaat gggatccgag acaagcagag
     6241 tggaaagaca atcctggatt ttcttaagtc cgatggattt gccaaccgga acttcatgca
     6301 gttgatccat gatgactctc tcacctttaa ggaggacatc cagaaagcac aagtttctgg
     6361 ccagggggac agtcttcacg agcacatcgc taatcttgca ggtagcccag ctatcaaaaa
     6421 gggaatactg cagaccgtta aggtcgtgga tgaactcgtc aaagtaatgg gaaggcataa
     6481 gcccgagaat atcgttatcg agatggcccg agagaaccaa actacccaga agggacagaa
     6541 gaacagtagg gaaaggatga agaggattga agagggtata aaagaactgg ggtcccaaat
     6601 ccttaaggaa cacccagttg aaaacaccca gcttcagaat gagaagctct acctgtacta
     6661 cctgcagaac ggcagggaca tgtacgtgga tcaggaactg gacatcaatc ggctctccga
     6721 ctacgacgtg gatcatatcg tgccccagtc ttttctcaaa gatgattcta ttgataataa
     6781 agtgttgaca agatccgata aaaatagagg gaagagtgat aacgtcccct cagaagaagt
     6841 tgtcaagaaa atgaaaaatt attggcggca gctgctgaac gccaaactga tcacacaacg
     6901 gaagttcgat aatctgacta aggctgaacg aggtggcctg tctgagttgg ataaagccgg
     6961 cttcatcaaa aggcagcttg ttgagacacg ccagatcacc aagcacgtgg cccaaattct
     7021 cgattcacgc atgaacacca agtacgatga aaatgacaaa ctgattcgag aggtgaaagt
     7081 tattactctg aagtctaagc tggtctcaga tttcagaaag gactttcagt tttataaggt
     7141 gagagagatc aacaattacc accatgcgca tgatgcctac ctgaatgcag tggtaggcac
     7201 tgcacttatc aaaaaatatc ccaagcttga atctgaattt gtttacggag actataaagt
     7261 gtacgatgtt aggaaaatga tcgcaaagtc tgagcaggaa ataggcaagg ccaccgctaa
     7321 gtacttcttt tacagcaata ttatgaattt tttcaagacc gagattacac tggccaatgg
     7381 agagattcgg aagcgaccac ttatcgaaac aaacggagaa acaggagaaa tcgtgtggga
     7441 caagggtagg gatttcgcga cagtccggaa ggtcctgtcc atgccgcagg tgaacatcgt
     7501 taaaaagacc gaagtacaga ccggaggctt ctccaaggaa agtatcctcc cgaaaaggaa
     7561 cagcgacaag ctgatcgcac gcaaaaaaga ttgggacccc aagaaatacg gcggattcga
     7621 ttctcctaca gtcgcttaca gtgtactggt tgtggccaaa gtggagaaag ggaagtctaa
     7681 aaaactcaaa agcgtcaagg aactgctggg catcacaatc atggagcgat caagcttcga
     7741 aaaaaacccc atcgactttc tcgaggcgaa aggatataaa gaggtcaaaa aagacctcat
     7801 cattaagctt cccaagtact ctctctttga gcttgaaaac ggccggaaac gaatgctcgc
     7861 tagtgcgggc gagctgcaga aaggtaacga gctggcactg ccctctaaat acgttaattt
     7921 cttgtatctg gccagccact atgaaaagct caaagggtct cccgaagata atgagcagaa
     7981 gcagctgttc gtggaacaac acaaacacta ccttgatgag atcatcgagc aaataagcga
     8041 attctccaaa agagtgatcc tcgccgacgc taacctcgat aaggtgcttt ctgcttacaa
     8101 taagcacagg gataagccca tcagggagca ggcagaaaac attatccact tgtttactct
     8161 gaccaacttg ggcgcgcctg cagccttcaa gtacttcgac accaccatag acagaaagcg
     8221 gtacacctct acaaaggagg tcctggacgc cacactgatt catcagtcaa ttacggggct
     8281 ctatgaaaca agaatcgacc tctctcagct cggtggagac agcagggctg accccaagaa
     8341 gaagaggaag gtgtgatctc ttctcgagtc atgtaattag ttatgtcacg cttacattca
     8401 cgccctcccc ccacatccgc tctaaccgaa aaggaaggag ttagacaacc tgaagtctag
     8461 gtccctattt atttttttat agttatgtta gtattaagaa cgttatttat atttcaaatt
     8521 tttctttttt ttctgtacag acgcgtgtac gcatgtaaca ttatactgaa aaccttgctt
     8581 gagaaggttt tgggacgctc gaaggcttta atttgcggcc ggtacccaat tcgccctata
     8641 gtgagtcgta ttacgcgcgc tcactggccg tcgttttaca acgtcgtgac tgggaaaacc
     8701 ctggcgttac ccaacttaat cgccttgcag cacatccccc tttcgccagc tggcgtaata
     8761 gcgaagaggc ccgcaccgat cgcccttccc aacagttgcg cagcctgaat ggcgaatgga
     8821 cgcgccctgt agcggcgcat taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc
     8881 tacacttgcc agcgccctag cgcccgctcc tttcgctttc ttcccttcct ttctcgccac
     8941 gttcgccggc tttccccgtc aagctctaaa tcgggggctc cctttagggt tccgatttag
     9001 tgctttacgg cacctcgacc ccaaaaaact tgattagggt gatggttcac gtagtgggcc
     9061 atcgccctga tagacggttt ttcgcccttt gacgttggag tccacgttct ttaatagtgg
     9121 actcttgttc caaactggaa caacactcaa ccctatctcg gtctattctt ttgatttata
     9181 agggattttg ccgatttcgg cctattggtt aaaaaatgag ctgatttaac aaaaatttaa
     9241 cgcgaatttt aacaaaatat taacgcttac aatttcctga tgcggtattt tctccttacg
     9301 catctgtgcg gtatttcaca ccgcatagat cggcaagtgc acaaacaata cttaaataaa
     9361 tactactcag taataaccta tttcttagca tttttgacga aatttgctat tttgttagag
     9421 tcttttacac catttgtctc cacacctccg cttacatcaa caccaataac gccatttaat
     9481 ctaagcgcat caccaacatt ttctggcgtc agtccaccag ctaacataaa atgtaagctt
     9541 tcggggctct cttgccttcc aacccagtca gaaatcgagt tccaatccaa aagttcacct
     9601 gtcccacctg cttctgaatc aaacaaggga ataaacgaat gaggtttctg tgaagctgca
     9661 ctgagtagta tgttgcagtc ttttggaaat acgagtcttt taataactgg caaaccgagg
     9721 aactcttggt attcttgcca cgactcatct ccatgcagtt ggacgatatc aatgccgtaa
     9781 tcattgacca gagccaaaac atcctcctta ggttgattac gaaacacgcc aaccaagtat
     9841 ttcggagtgc ctgaactatt tttatatgct tttacaagac ttgaaatttt ccttgcaata
     9901 accgggtcaa ttgttctctt tctattgggc acacatataa tacccagcaa gtcagcatcg
     9961 gaatctagag cacattctgc ggcctctgtg ctctgcaagc cgcaaacttt caccaatgga
    10021 ccagaactac ctgtgaaatt aataacagac atactccaag ctgcctttgt gtgcttaatc
    10081 acgtatactc acgtgctcaa tagtcaccaa tgccctccct cttggccctc tccttttctt
    10141 ttttcgaccg aattaattct taatcggcaa aaaaagaaaa gctccggatc aagattgtac
    10201 gtaaggtgac aagctatttt tcaataaaga atatcttcca ctactgccat ctggcgtcat
    10261 aactgcaaag tacacatata ttacgatgct gttctattaa atgcttccta tattatatat
    10321 atagtaatgt cgttgatcta tggtgcactc tcagtacaat ctgctctgat gccgcatagt
    10381 taagccagcc ccgacacccg ccaacacccg ctgacgcgcc ctgacgggct tgtctgctcc
    10441 cggcatccgc ttacagacaa gctgtgaccg tctccgggag ctgcatgtgt cagaggtttt
    10501 caccgtcatc accgaaacgc gcgagacgaa agggcctcgt gatacgccta tttttatagg
    10561 ttaatgtcat gataataatg gtttcttaga cgtcaggtgg cacttttcgg ggaaatgtgc
    10621 gcggaacccc tatttgttta tttttctaaa tacattcaaa tatgtatccg ctcatgagac
    10681 aataaccctg ataaatgctt caataatatt gaaaaaggaa gagtatgagt attcaacatt
    10741 tccgtgtcgc ccttattccc ttttttgcgg cattttgcct tcctgttttt gctcacccag
    10801 aaacgctggt gaaagtaaaa gatgctgaag atcagttggg tgcacgagtg ggttacatcg
    10861 aactggatct caacagcggt aagatccttg agagttttcg ccccgaagaa cgttttccaa
    10921 tgatgagcac ttttaaagtt ctgctatgtg gcgcggtatt atcccgtatt gacgccgggc
    10981 aagagcaact cggtcgccgc atacactatt ctcagaatga cttggttgag tactcaccag
    11041 tcacagaaaa gcatcttacg gatggcatga cagtaagaga attatgcagt gctgccataa
    11101 ccatgagtga taacactgcg gccaacttac ttctgacaac gatcggagga ccgaaggagc
    11161 taaccgcttt tttgcacaac atgggggatc atgtaactcg ccttgatcgt tgggaaccgg
    11221 agctgaatga agccatacca aacgacgagc gtgacaccac gatgcctgta gcaatggcaa
    11281 caacgttgcg caaactatta actggcgaac tacttactct agcttcccgg caacaattaa
    11341 tagactggat ggaggcggat aaagttgcag gaccacttct gcgctcggcc cttccggctg
    11401 gctggtttat tgctgataaa tctggagccg gtgagcgtgg gtctcgcggt atcattgcag
    11461 cactggggcc agatggtaag ccctcccgta tcgtagttat ctacacgacg gggagtcagg
    11521 caactatgga tgaacgaaat agacagatcg ctgagatagg tgcctcactg attaagcatt
    11581 ggtaactgtc agaccaagtt tactcatata tactttagat tgatttaaaa cttcattttt
    11641 aatttaaaag gatctaggtg aagatccttt ttgataatct catgaccaaa atcccttaac
    11701 gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga tcttc
//
