LOCUS       sequence-329605-       11853 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Plasmid for constituive spCas9 expression and tet-inducible 
            expression of sgRNA binding to the promoter of RPL22A for double 
            stranded break formation in yeast. .
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 11853)
  TITLE     Burgess Lab Plasmids
REFERENCE   2  (bases 1 to 11853)
  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_RPL22A
FEATURES             Location/Qualifiers
     source          1..11853
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     1..17
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     1..17
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     36..56
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        38..56
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        74..472
                     /gene="S. cerevisiae TEF1"
                     /label=TEF1 promoter
                     /note="promoter for EF-1-alpha"
     primer_bind     476..492
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     476..492
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     CDS             504..4607
                     /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             504..4607
                     /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             504..4607
                     /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             4620..4640
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     terminator      4656..4903
                     /gene="S. cerevisiae CYC1"
                     /label=CYC1 terminator
                     /note="transcription terminator for CYC1"
     primer_bind     complement(4672..4690)
                     /label=CYC1
                     /note="CYC1 transcription termination signal, reverse 
                     primer"
     primer_bind     complement(4921..4940)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4922..4940)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4950..4967)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4950..4966)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4959..4981)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      5107..5562
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5194..5213)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5404..5425
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(5665..6339)
                     /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(6340..6621)
                     /gene="S. cerevisiae TRP1"
                     /label=TRP1 promoter
     primer_bind     complement(6642..6661)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     6761..6783
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(6821..6839)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        6907..7011
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(7012..7080,7081..7872)
                     /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(7230..7249)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      8043..8631
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     8532..8551
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     protein_bind    9015..9033
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     misc_RNA        9217..9292
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     terminator      9305..9367
                     /label=RPR1 terminator
                     /note="transcription terminator for the S. cerevisiae RNase
                     P RNA gene"
     CDS             10502..11125
                     /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(10575..10594)
                     /label=Tet-R
                     /note="Tetracycline resistance gene, reverse primer"
     primer_bind     11622..11639
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    11756..11777
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(11792..11797,11798..11815,11816..11822)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    11830..11846
                     /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     join(11835..11853,1..4)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
ORIGIN
        1 caggaaacag ctatgaccat gattacgcca agcgcgcaat taaccctcac taaagggaac
       61 aaaagctgga gctcatagct tcaaaatgtt tctactcctt ttttactctt ccagattttc
      121 tcggactccg cgcatcgccg taccacttca aaacacccaa gcacagcata ctaaatttcc
      181 cctctttctt cctctagggt gtcgttaatt acccgtacta aaggtttgga aaagaaaaaa
      241 gagaccgcct cgtttctttt tcttcgtcga aaaaggcaat aaaaattttt atcacgtttc
      301 tttttcttga aaattttttt tttgattttt ttctctttcg atgacctccc attgatattt
      361 aagttaataa acggtcttca atttctcaag tttcagtttc atttttcttg ttctattaca
      421 acttttttta cttcttgctc attagaaaga aagcatagca atctaatcta agttttctag
      481 aactagtgga tcccccggga aaaatggaca agaagtactc cattgggctc gatatcggca
      541 caaacagcgt cggttgggcc gtcattacgg acgagtacaa ggtgccgagc aaaaaattca
      601 aagttctggg caataccgat cgccacagca taaagaagaa cctcattggc gccctcctgt
      661 tcgactccgg ggagacggcc gaagccacgc ggctcaaaag aacagcacgg cgcagatata
      721 cccgcagaaa gaatcggatc tgctacctgc aggagatctt tagtaatgag atggctaagg
      781 tggatgactc tttcttccat aggctggagg agtccttttt ggtggaggag gataaaaagc
      841 acgagcgcca cccaatcttt ggcaatatcg tggacgaggt ggcgtaccat gaaaagtacc
      901 caaccatata tcatctgagg aagaagcttg tagacagtac tgataaggct gacttgcggt
      961 tgatctatct cgcgctggcg catatgatca aatttcgggg acacttcctc atcgaggggg
     1021 acctgaaccc agacaacagc gatgtcgaca aactctttat ccaactggtt cagacttaca
     1081 atcagctttt cgaagagaac ccgatcaacg catccggagt tgacgccaaa gcaatcctga
     1141 gcgctaggct gtccaaatcc cggcggctcg aaaacctcat cgcacagctc cctggggaga
     1201 agaagaacgg cctgtttggt aatcttatcg ccctgtcact cgggctgacc cccaacttta
     1261 aatctaactt cgacctggcc gaagatgcca agcttcaact gagcaaagac acctacgatg
     1321 atgatctcga caatctgctg gcccagatcg gcgaccagta cgcagacctt tttttggcgg
     1381 caaagaacct gtcagacgcc attctgctga gtgatattct gcgagtgaac acggagatca
     1441 ccaaagctcc gctgagcgct agtatgatca agcgctatga tgagcaccac caagacttga
     1501 ctttgctgaa ggcccttgtc agacagcaac tgcctgagaa gtacaaggaa attttcttcg
     1561 atcagtctaa aaatggctac gccggataca ttgacggcgg agcaagccag gaggaatttt
     1621 acaaatttat taagcccatc ttggaaaaaa tggacggcac cgaggagctg ctggtaaagc
     1681 ttaacagaga agatctgttg cgcaaacagc gcactttcga caatggaagc atcccccacc
     1741 agattcacct gggcgaactg cacgctatcc tcaggcggca agaggatttc tacccctttt
     1801 tgaaagataa cagggaaaag attgagaaaa tcctcacatt tcggataccc tactatgtag
     1861 gccccctcgc ccggggaaat tccagattcg cgtggatgac tcgcaaatca gaagagacca
     1921 tcactccctg gaacttcgag gaagtcgtgg ataagggggc ctctgcccag tccttcatcg
     1981 aaaggatgac taactttgat aaaaatctgc ctaacgaaaa ggtgcttcct aaacactctc
     2041 tgctgtacga gtacttcaca gtttataacg agctcaccaa ggtcaaatac gtcacagaag
     2101 ggatgagaaa gccagcattc ctgtctggag agcagaagaa agctatcgtg gacctcctct
     2161 tcaagacgaa ccggaaagtt accgtgaaac agctcaaaga agactatttc aaaaagattg
     2221 aatgtttcga ctctgttgaa atcagcggag tggaggatcg cttcaacgca tccctgggaa
     2281 cgtatcacga tctcctgaaa atcattaaag acaaggactt cctggacaat gaggagaacg
     2341 aggacattct tgaggacatt gtcctcaccc ttacgttgtt tgaagatagg gagatgattg
     2401 aagaacgctt gaaaacttac gctcatctct tcgacgacaa agtcatgaaa cagctcaaga
     2461 ggcgccgata tacaggatgg gggcggctgt caagaaaact gatcaatggg atccgagaca
     2521 agcagagtgg aaagacaatc ctggattttc ttaagtccga tggatttgcc aaccggaact
     2581 tcatgcagtt gatccatgat gactctctca cctttaagga ggacatccag aaagcacaag
     2641 tttctggcca gggggacagt cttcacgagc acatcgctaa tcttgcaggt agcccagcta
     2701 tcaaaaaggg aatactgcag accgttaagg tcgtggatga actcgtcaaa gtaatgggaa
     2761 ggcataagcc cgagaatatc gttatcgaga tggcccgaga gaaccaaact acccagaagg
     2821 gacagaagaa cagtagggaa aggatgaaga ggattgaaga gggtataaaa gaactggggt
     2881 cccaaatcct taaggaacac ccagttgaaa acacccagct tcagaatgag aagctctacc
     2941 tgtactacct gcagaacggc agggacatgt acgtggatca ggaactggac atcaatcggc
     3001 tctccgacta cgacgtggat catatcgtgc cccagtcttt tctcaaagat gattctattg
     3061 ataataaagt gttgacaaga tccgataaaa atagagggaa gagtgataac gtcccctcag
     3121 aagaagttgt caagaaaatg aaaaattatt ggcggcagct gctgaacgcc aaactgatca
     3181 cacaacggaa gttcgataat ctgactaagg ctgaacgagg tggcctgtct gagttggata
     3241 aagccggctt catcaaaagg cagcttgttg agacacgcca gatcaccaag cacgtggccc
     3301 aaattctcga ttcacgcatg aacaccaagt acgatgaaaa tgacaaactg attcgagagg
     3361 tgaaagttat tactctgaag tctaagctgg tctcagattt cagaaaggac tttcagtttt
     3421 ataaggtgag agagatcaac aattaccacc atgcgcatga tgcctacctg aatgcagtgg
     3481 taggcactgc acttatcaaa aaatatccca agcttgaatc tgaatttgtt tacggagact
     3541 ataaagtgta cgatgttagg aaaatgatcg caaagtctga gcaggaaata ggcaaggcca
     3601 ccgctaagta cttcttttac agcaatatta tgaatttttt caagaccgag attacactgg
     3661 ccaatggaga gattcggaag cgaccactta tcgaaacaaa cggagaaaca ggagaaatcg
     3721 tgtgggacaa gggtagggat ttcgcgacag tccggaaggt cctgtccatg ccgcaggtga
     3781 acatcgttaa aaagaccgaa gtacagaccg gaggcttctc caaggaaagt atcctcccga
     3841 aaaggaacag cgacaagctg atcgcacgca aaaaagattg ggaccccaag aaatacggcg
     3901 gattcgattc tcctacagtc gcttacagtg tactggttgt ggccaaagtg gagaaaggga
     3961 agtctaaaaa actcaaaagc gtcaaggaac tgctgggcat cacaatcatg gagcgatcaa
     4021 gcttcgaaaa aaaccccatc gactttctcg aggcgaaagg atataaagag gtcaaaaaag
     4081 acctcatcat taagcttccc aagtactctc tctttgagct tgaaaacggc cggaaacgaa
     4141 tgctcgctag tgcgggcgag ctgcagaaag gtaacgagct ggcactgccc tctaaatacg
     4201 ttaatttctt gtatctggcc agccactatg aaaagctcaa agggtctccc gaagataatg
     4261 agcagaagca gctgttcgtg gaacaacaca aacactacct tgatgagatc atcgagcaaa
     4321 taagcgaatt ctccaaaaga gtgatcctcg ccgacgctaa cctcgataag gtgctttctg
     4381 cttacaataa gcacagggat aagcccatca gggagcaggc agaaaacatt atccacttgt
     4441 ttactctgac caacttgggc gcgcctgcag ccttcaagta cttcgacacc accatagaca
     4501 gaaagcggta cacctctaca aaggaggtcc tggacgccac actgattcat cagtcaatta
     4561 cggggctcta tgaaacaaga atcgacctct ctcagctcgg tggagacagc agggctgacc
     4621 ccaagaagaa gaggaaggtg tgatctcttc tcgagtcatg taattagtta tgtcacgctt
     4681 acattcacgc cctcccccca catccgctct aaccgaaaag gaaggagtta gacaacctga
     4741 agtctaggtc cctatttatt tttttatagt tatgttagta ttaagaacgt tatttatatt
     4801 tcaaattttt cttttttttc tgtacagacg cgtgtacgca tgtaacatta tactgaaaac
     4861 cttgcttgag aaggttttgg gacgctcgaa ggctttaatt tgcggccggt acccaattcg
     4921 ccctatagtg agtcgtatta cgcgcgctca ctggccgtcg ttttacaacg tcgtgactgg
     4981 gaaaaccctg gcgttaccca acttaatcgc cttgcagcac atcccccttt cgccagctgg
     5041 cgtaatagcg aagaggcccg caccgatcgc ccttcccaac agttgcgcag cctgaatggc
     5101 gaatggacgc gccctgtagc ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg
     5161 tgaccgctac acttgccagc gccctagcgc ccgctccttt cgctttcttc ccttcctttc
     5221 tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg ggggctccct ttagggttcc
     5281 gatttagtgc tttacggcac ctcgacccca aaaaacttga ttagggtgat ggttcacgta
     5341 gtgggccatc gccctgatag acggtttttc gccctttgac gttggagtcc acgttcttta
     5401 atagtggact cttgttccaa actggaacaa cactcaaccc tatctcggtc tattcttttg
     5461 atttataagg gattttgccg atttcggcct attggttaaa aaatgagctg atttaacaaa
     5521 aatttaacgc gaattttaac aaaatattaa cgcttacaat ttcctgatgc ggtattttct
     5581 ccttacgcat ctgtgcggta tttcacaccg catagatcgg caagtgcaca aacaatactt
     5641 aaataaatac tactcagtaa taacctattt cttagcattt ttgacgaaat ttgctatttt
     5701 gttagagtct tttacaccat ttgtctccac acctccgctt acatcaacac caataacgcc
     5761 atttaatcta agcgcatcac caacattttc tggcgtcagt ccaccagcta acataaaatg
     5821 taagctttcg gggctctctt gccttccaac ccagtcagaa atcgagttcc aatccaaaag
     5881 ttcacctgtc ccacctgctt ctgaatcaaa caagggaata aacgaatgag gtttctgtga
     5941 agctgcactg agtagtatgt tgcagtcttt tggaaatacg agtcttttaa taactggcaa
     6001 accgaggaac tcttggtatt cttgccacga ctcatctcca tgcagttgga cgatatcaat
     6061 gccgtaatca ttgaccagag ccaaaacatc ctccttaggt tgattacgaa acacgccaac
     6121 caagtatttc ggagtgcctg aactattttt atatgctttt acaagacttg aaattttcct
     6181 tgcaataacc gggtcaattg ttctctttct attgggcaca catataatac ccagcaagtc
     6241 agcatcggaa tctagagcac attctgcggc ctctgtgctc tgcaagccgc aaactttcac
     6301 caatggacca gaactacctg tgaaattaat aacagacata ctccaagctg cctttgtgtg
     6361 cttaatcacg tatactcacg tgctcaatag tcaccaatgc cctccctctt ggccctctcc
     6421 ttttcttttt tcgaccgaat taattcttaa tcggcaaaaa aagaaaagct ccggatcaag
     6481 attgtacgta aggtgacaag ctatttttca ataaagaata tcttccacta ctgccatctg
     6541 gcgtcataac tgcaaagtac acatatatta cgatgctgtt ctattaaatg cttcctatat
     6601 tatatatata gtaatgtcgt tgatctatgg tgcactctca gtacaatctg ctctgatgcc
     6661 gcatagttaa gccagccccg acacccgcca acacccgctg acgcgccctg acgggcttgt
     6721 ctgctcccgg catccgctta cagacaagct gtgaccgtct ccgggagctg catgtgtcag
     6781 aggttttcac cgtcatcacc gaaacgcgcg agacgaaagg gcctcgtgat acgcctattt
     6841 ttataggtta atgtcatgat aataatggtt tcttagacgt caggtggcac ttttcgggga
     6901 aatgtgcgcg gaacccctat ttgtttattt ttctaaatac attcaaatat gtatccgctc
     6961 atgagacaat aaccctgata aatgcttcaa taatattgaa aaaggaagag tatgagtatt
     7021 caacatttcc gtgtcgccct tattcccttt tttgcggcat tttgccttcc tgtttttgct
     7081 cacccagaaa cgctggtgaa agtaaaagat gctgaagatc agttgggtgc acgagtgggt
     7141 tacatcgaac tggatctcaa cagcggtaag atccttgaga gttttcgccc cgaagaacgt
     7201 tttccaatga tgagcacttt taaagttctg ctatgtggcg cggtattatc ccgtattgac
     7261 gccgggcaag agcaactcgg tcgccgcata cactattctc agaatgactt ggttgagtac
     7321 tcaccagtca cagaaaagca tcttacggat ggcatgacag taagagaatt atgcagtgct
     7381 gccataacca tgagtgataa cactgcggcc aacttacttc tgacaacgat cggaggaccg
     7441 aaggagctaa ccgctttttt gcacaacatg ggggatcatg taactcgcct tgatcgttgg
     7501 gaaccggagc tgaatgaagc cataccaaac gacgagcgtg acaccacgat gcctgtagca
     7561 atggcaacaa cgttgcgcaa actattaact ggcgaactac ttactctagc ttcccggcaa
     7621 caattaatag actggatgga ggcggataaa gttgcaggac cacttctgcg ctcggccctt
     7681 ccggctggct ggtttattgc tgataaatct ggagccggtg agcgtgggtc tcgcggtatc
     7741 attgcagcac tggggccaga tggtaagccc tcccgtatcg tagttatcta cacgacgggg
     7801 agtcaggcaa ctatggatga acgaaataga cagatcgctg agataggtgc ctcactgatt
     7861 aagcattggt aactgtcaga ccaagtttac tcatatatac tttagattga tttaaaactt
     7921 catttttaat ttaaaaggat ctaggtgaag atcctttttg ataatctcat gaccaaaatc
     7981 ccttaacgtg agttttcgtt ccactgagcg tcagaccccg tagaaaagat caaaggatct
     8041 tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta
     8101 ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc tttttccgaa ggtaactggc
     8161 ttcagcagag cgcagatacc aaatactgtt cttctagtgt agccgtagtt aggccaccac
     8221 ttcaagaact ctgtagcacc gcctacatac ctcgctctgc taatcctgtt accagtggct
     8281 gctgccagtg gcgataagtc gtgtcttacc gggttggact caagacgata gttaccggat
     8341 aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac agcccagctt ggagcgaacg
     8401 acctacaccg aactgagata cctacagcgt gagctatgag aaagcgccac gcttcccgaa
     8461 gggagaaagg cggacaggta tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg
     8521 gagcttccag ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg ccacctctga
     8581 cttgagcgtc gatttttgtg atgctcgtca ggggggcgga gcctatggaa aaacgccagc
     8641 aacgcggcct ttttacggtt cctggccttt tgctggcctt ttgctcgggg gatctgccaa
     8701 ttgaacataa catggtagtt acatatacta gtaatatggt tcggcacaca ttaaaagtat
     8761 aaaaactatc tgaattacga attacatata ttggtcataa aaatcaatca atcatcgtgt
     8821 gttttatatg tctcttatct aagtataaga atatccatag ttaatattca cttacgctac
     8881 cttttaacct gtaatcattg tcaacaggat atgttaacga cccacattga taaacgctag
     8941 tatttctttt tcctcttctt attggccggc tgtctctata ctcccctata gtctgtttct
     9001 tttcgtttcg attgtcccta tcagtgatag agatggcgca catggtacgc tgtggtgctc
     9061 gcggctggga acgaaactct gggagctgcg attggcagtt caaaagagaa gcgcactggt
     9121 tttagagcta gaaatagcaa gttaaaataa ggctagtcgc ggctgggaac gaaactctgg
     9181 gagctgcgat tggcagttca aaagagaagc gcactggttt tagagctaga aatagcaagt
     9241 taaaataagg ctagtccgtt atcaacttga aaaagtggca ccgagtcggt gcttttttct
     9301 cgagccatat ccaacttcca atttaatctt tcttttttaa ttttcactta tttgcgatac
     9361 agaaagaaaa aagcgatagt aactattgaa ttttgtttgg atttggttag attagatatg
     9421 gtttctcttt atatttacat gctaaaaatg ggctacacca gagatacata attagatata
     9481 tatacgccag tacaccttat cggcccaagc cttgtcccaa ggcagcgttt tgttcttgga
     9541 aacgctgccc tacacgttcg ctatgcttca agaacttttc tgagcacttc atgatgcatg
     9601 tttgttcctt attggttagc tttgatgttg tgaagtcatt gacacagtct gtgaaacatc
     9661 tttctaccag attagagtac aaacgcatga aatccttcat ttgcttttgt tccactactt
     9721 tttggaactc ttgttgttct ttcctgcagg tagtcgtgca atgtatgact ttaagatttg
     9781 tgagcaggaa gaaaagggag aatcttctaa cgataaaccc ttgaaaaact gggtagacta
     9841 cgctatgttg agttgctacg caggctgcac aattacacga gaatgctccc gcctaggatt
     9901 taaggctaag ggacgtgcaa tgcagacgac agatctaaat gaccgtgtcg gtgaagtgtt
     9961 cgccaaactt ttcggttaac acatgcagtg atgcacgcgc gatggtgcta agttacatat
    10021 atatatatat gtatagccat agtgatgtct aagtaacctt tatggtatat ttcttaatgt
    10081 ggaaagatac tagcgcgcgc acccacacac aagcttcgtc ttttcttgaa gaaaagagga
    10141 agctcgctaa atgggattcc actttccgtt ccctgccagc tgatggaaaa aggttagtgg
    10201 aacgatgaag aataaaaaga gagatccact gaggtgaaat ttcagctgac agcgagtttc
    10261 atgatcgtga tgaacaatgg taacgagttg tggctgttgc cagggagggt ggttctcaac
    10321 ttttaatgta tggccaaatc gctacttggg tttgttatat aacaaagaag aaataatgaa
    10381 ctgattctct tcctccttct tgtcctttct taattctgtt gtaattacct tcctttgtaa
    10441 ttttttttgt aattattctt cttaataatc caaacaaaca cacatattac aatatattaa
    10501 aatgtctaga ttagataaaa gtaaagtgat taacagcgca ttagagctgc ttaatgaggt
    10561 cggaatcgaa ggtttaacaa cccgtaaact cgcccagaag ctaggtgtag agcagcctac
    10621 attgtattgg catgtaaaaa ataagcgggc tttgctcgac gccttagcca ttgagatgtt
    10681 agataggcac catactcact tttgcccttt agaaggggaa agctggcaag attttttacg
    10741 taataacgct aaaagtttta gatgtgcttt actaagtcat cgcgatggag caaaagtaca
    10801 tttaggtaca cggcctacag aaaaacagta tgaaactctc gaaaatcaat tagccttttt
    10861 atgccaacaa ggtttttcac tagagaatgc attatatgca ctcagcgctg tggggcattt
    10921 tactttaggt tgcgtattgg aagatcaaga gcatcaagtc gctaaagaag aaagggaaac
    10981 acctactact gatagtatgc cgccattatt acgacaagct atcgaattat ttgatcacca
    11041 aggtgcagag ccagccttct tattcggcct tgaattgatc atatgcggat tagaaaaaca
    11101 acttaaatgt gaaagtgggt cttaagtctg aagaatgaat gatttgatga tttctttttc
    11161 cctccatttt tcttactgaa tatatcaatg atatagactt gtatagttta ttatttcaaa
    11221 ttaagtagct atatatagtc aagataacgt ttgtttgaca cgattacatt attcgtcgac
    11281 atcttttttc agcctgtcgt ggtagcaatt tgaggagtat tattaattga ataggttcat
    11341 tttgcgctcg cataaacagt tttcgtcagg gacagtatgt tggaatgagt ggtaattaat
    11401 ggtgacatga catgttatag caataacctt gatgtttaca tcgtagttta atgtacaccc
    11461 cgcgaattcg ttcaagtagg agtgcaccaa ttgcaaaggg aaaagctgaa tgggcagttc
    11521 gaaccgcggt ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg
    11581 agtgagctga taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg
    11641 aagcggaaga gcgcccaata cgcaaaccgc ctctccccgc gcgttggccg attcattaat
    11701 gcagctggca cgacaggttt cccgactgga aagcgggcag tgagcgcaac gcaattaatg
    11761 tgagttagct cactcattag gcaccccagg ctttacactt tatgcttccg gctcgtatgt
    11821 tgtgtggaat tgtgagcgga taacaatttc aca
//
