LOCUS      pJH2971       11162 bp    DNA     circular SYN 14-APR-2025
DEFINITION  Cas9 driven by PGK1 promoter. gRNA can be cloned into the BplI 
            sites. KANMX marker.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 11162)
  AUTHORS   
  TITLE     Cas9-mediated gene editing in Saccharomyces cerevisiae
  JOURNAL   Protocol Exchange (2017)
REFERENCE   2  (bases 1 to 11162)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 14, 2025 from SnapGene Server 8.0.1
            //gt12365.cn
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Protocol 
            Exchange (2017)"
COMMENT     Sequence Label: pJH2971
FEATURES             Location/Qualifiers
     source          1..11162
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(29..51)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     151..170
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     gene            complement(284..1640)
                     /label=kanMX
                     /note="yeast selectable marker conferring kanamycin 
                     resistance (Wach et al., 1994)"
     terminator      complement(284..481)
                     /label=TEF terminator
                     /note="Ashbya gossypii TEF terminator"
     CDS             complement(487..1296)
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin"
                     /translation="MGKEKTHVSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYGKP
                     DAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGKTA
                     FQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDASD
                     FDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGIAD
                     RYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     1210..1229
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     promoter        complement(1297..1640)
                     /label=TEF promoter
                     /note="Ashbya gossypii TEF promoter"
     CDS             2658..6761
                     /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             2658..6761
                     /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             6774..6794
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             6798..6818
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             6822..6842
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             6843..6908
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     terminator      6927..7174
                     /gene="S. cerevisiae CYC1"
                     /label=CYC1 terminator
                     /note="transcription terminator for CYC1"
     primer_bind     complement(6943..6961)
                     /label=CYC1
                     /note="CYC1 transcription termination signal, reverse 
                     primer"
     rep_origin      complement(7234..7689)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(7371..7392)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     7583..7602
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     7815..7837
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     7829..7846
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     7830..7846
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     7853..7872
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        7853..7871
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     7904..7920
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     7904..7920
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     promoter        7952..8220
                     /label=SNR52 promoter
                     /note="promoter for the S. cerevisiae small nucleolar RNA 
                     gene SNR52"
     misc_RNA        8248..8322
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     terminator      8327..8346
                     /label=SUP4 terminator
                     /note="transcription terminator for the S. cerevisiae SUP4 
                     tRNA gene"
     promoter        complement(8389..8407)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(8428..8444)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(8428..8444)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(8441..8463)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    8452..8468
                     /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)."
     promoter        complement(join(8476..8482,8483..8500,8501..8506))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    8521..8542
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(8659..8676)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(8830..9418)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(8910..8929)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(9589..10380,10381..10449))
                     /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     10212..10231
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(10450..10554)
                     /gene="bla"
                     /label=AmpR promoter
     misc_feature    10591..11094
                     /label=CEN/ARS
                     /note="S. cerevisiae CEN6 centromere fused to an 
                     autonomously replicating sequence"
     primer_bind     11135..11153
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
ORIGIN
        1 tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca
       61 cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg
      121 ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc
      181 accataccac cttttcaatt catcattttt tttttattct tttttttgat ttcgaattcg
      241 agctcgttta aactggatgg cggcgttagt atcgaatcga cagcagtata gcgaccagca
      301 ttcacatacg attgacgcat gatattactt tctgcgcact taacttcgca tctgggcaga
      361 tgatgtcgag gcgaaaaaaa atataaatca cgctaacatt tgattaaaat agaacaacta
      421 caatataaaa aaactataca aatgacaagt tcttgaaaac aagaatcttt ttattgtcag
      481 tactgattag aaaaactcat cgagcatcaa atgaaactgc aatttattca tatcaggatt
      541 atcaatacca tatttttgaa aaagccgttt ctgtaatgaa ggagaaaact caccgaggca
      601 gttccatagg atggcaagat cctggtatcg gtctgcgatt ccgactcgtc caacatcaat
      661 acaacctatt aatttcccct cgtcaaaaat aaggttatca agtgagaaat caccatgagt
      721 gacgactgaa tccggtgaga atggcaaaag cttatgcatt tctttccaga cttgttcaac
      781 aggccagcca ttacgctcgt catcaaaatc actcgcatca accaaaccgt tattcattcg
      841 tgattgcgcc tgagcgagac gaaatacgcg atcgctgtta aaaggacaat tacaaacagg
      901 aatcgaatgc aaccggcgca ggaacactgc cagcgcatca acaatatttt cacctgaatc
      961 aggatattct tctaatacct ggaatgctgt tttgccgggg atcgcagtgg tgagtaacca
     1021 tgcatcatca ggagtacgga taaaatgctt gatggtcgga agaggcataa attccgtcag
     1081 ccagtttagt ctgaccatct catctgtaac atcattggca acgctacctt tgccatgttt
     1141 cagaaacaac tctggcgcat cgggcttccc atacaatcga tagattgtcg cacctgattg
     1201 cccgacatta tcgcgagccc atttataccc atataaatca gcatccatgt tggaatttaa
     1261 tcgcggcctc gaaacgtgag tcttttcctt acccatggtt gtttatgttc ggatgtgatg
     1321 tgagaactgt atcctagcaa gattttaaaa ggaagtatat gaaagaagaa cctcagtggc
     1381 aaatcctaac cttttatatt tctctacagg ggcgcggcgt ggggacaatt caacgcgtct
     1441 gtgaggggag cgtttccctg ctcgcaggtc cgcagcgagg agccgtaatt tttgcttcgc
     1501 gccgtgcggc catcaaaatg tatggatgca aatgattata catggggatg tatgggctaa
     1561 atgtacgggc gacagtcaca tcatgcccct gagctgcgca cgtcaagact gtcaaggagg
     1621 gtattctggg cctccatgtc gctggccggg tgacccggcg gggaaagtac cttcaaagaa
     1681 tggggtctta tcttgttttg caagtaccac tgagcaggat aataatagaa atgataatat
     1741 actatagtag agataacgtc gatgacttcc catactgtaa ttgcttttag ttgtgtattt
     1801 ttagtgtgca agtttctgta aatcgattaa tttttttttc tttcctcttt ttattaacct
     1861 taatttttat tttagattcc tgacttcaac tcaagacgca cagatattat aacatctgca
     1921 taataggcat ttgcaagaat tactcgtgag taaggaaaga gtgaggaact atcgcatacc
     1981 tgcatttaaa gatgccgatt tgggcgcgaa tcctttattt tggcttcacc ctcatactat
     2041 tatcagggcc agaaaaagga agtgtttccc tccttcttga attgatgtta ccctcataaa
     2101 gcacgtggcc tcttatcgag aaagaaatta ccgtcgctcg tgatttgttt gcaaaaagaa
     2161 caaaactgaa aaaacccaga cacgctcgac ttcctgtctt cctattgatt gcagcttcca
     2221 atttcgtcac acaacaaggt cctagcgacg gctcacaggt tttgtaacaa gcaatcgaag
     2281 gttctggaat ggcgggaaag ggtttagtac cacatgctat gatgcccact gtgatctcca
     2341 gagcaaagtt cgttcgatcg tactgttact ctctctcttt caaacagaat tgtccgaatc
     2401 gtgtgacaac aacagcctgt tctcacacac tcttttcttc taaccaaggg ggtggtttag
     2461 tttagtagaa cctcgtgaaa cttacattta catatatata aacttgcata aattggtcaa
     2521 tgcaagaaat acatatttgg tcttttctaa ttcgtagttt ttcaagttct tagatgcttt
     2581 ctttttctct tttttacaga tcatcaagga agtaattatc tactttttac aacaaatata
     2641 aaacaccgga tctcaaaatg gataaaaagt atagtattgg tttagatatt ggtactaact
     2701 ctgtgggttg ggcagttatc actgacgaat ataaagttcc atcaaagaaa tttaaggtgt
     2761 taggtaacac tgacagacac tcaataaaaa agaatcttat cggtgctctt ttgttcgact
     2821 ccggtgaaac tgccgaggct acacgtttaa aaagaacagc aagaagaaga tatacccgta
     2881 gaaaaaatag aatatgttat ttacaagaaa tcttttctaa tgaaatggct aaagttgatg
     2941 attccttttt ccatagattg gaagagtcat ttttggttga agaagacaaa aagcatgaga
     3001 gacatccaat ctttgggaat atagttgatg aagtggctta ccatgaaaaa tatcctacca
     3061 tttatcattt aagaaagaaa ttggtagatt caactgataa agctgacctt agattaatct
     3121 atttagcact tgcccatatg attaaattta gaggtcattt tttgattgaa ggtgatttga
     3181 acccagataa ttctgacgtg gataaattat ttattcaatt agtccaaacc tacaaccaat
     3241 tatttgagga aaatccaatt aatgctagtg gtgtcgatgc caaagctata ttatcagcca
     3301 gattatcaaa atctagacgt ttggaaaatt tgattgccca attgccagga gaaaaaaaga
     3361 atggattatt tggaaacttg atcgcattat cattgggttt gacaccaaat tttaaatcta
     3421 attttgattt agctgaagat gctaaattac aattatcaaa agacacctat gacgacgatt
     3481 tggacaattt acttgctcaa attggtgatc aatatgcaga tttgttctta gctgctaaaa
     3541 acttatctga tgctattttg ttgtctgata ttttgagagt gaacacagaa ataaccaaag
     3601 ctccattatc agcatctatg atcaaacgtt atgatgaaca ccatcaggat ttgactttat
     3661 tgaaagcttt ggtgagacaa caattgccag agaagtataa agaaatcttt ttcgatcaat
     3721 ctaaaaacgg gtatgcaggt tatattgatg ggggtgcctc ccaagaggaa ttttacaaat
     3781 ttataaaacc tattttagaa aagatggatg ggactgagga acttttggtc aaattgaaca
     3841 gagaagattt gttacgtaaa cagagaactt ttgataatgg tagtatacct caccaaattc
     3901 atttgggtga gttgcatgca attttaagaa gacaagaaga tttttatcca tttttaaaag
     3961 ataatagaga aaaaatcgag aaaattttaa cctttagaat tccatactat gttgggcctt
     4021 tggctagagg taattcaaga tttgcctgga tgacacgtaa atcagaagaa actattaccc
     4081 cttggaattt tgaagaggtt gttgataaag gagcatcagc acagagtttt attgaaagaa
     4141 tgaccaattt cgataaaaac ttaccaaatg aaaaagtttt accaaaacat tccttgttat
     4201 acgaatattt tactgtttac aatgaactta caaaggttaa atatgttact gaaggtatgc
     4261 gtaagccagc ctttttatct ggagaacaga aaaaggcaat agttgattta ttgtttaaaa
     4321 caaatagaaa agttactgtt aaacaattaa aagaagatta ctttaagaaa attgaatgtt
     4381 ttgattcagt tgaaatcagt ggtgttgaag acagatttaa tgctagttta ggaacttacc
     4441 atgatttact taaaattatc aaagataaag atttcttgga taacgaagaa aatgaagaca
     4501 ttttagaaga cattgtttta accttaactt tattcgaaga tagagagatg attgaagaac
     4561 gtttgaagac ttatgcacat ttgtttgacg ataaagtgat gaaacagttg aaaagaagac
     4621 gttatactgg atggggtaga ttgtctcgta aattgatcaa tggaattaga gataaacaaa
     4681 gtggtaaaac tatcttggac tttttgaaat ctgacggatt tgctaataga aatttcatgc
     4741 aattgatcca cgacgatagt ttgacattta aagaagacat ccaaaaggcc caagtgagtg
     4801 ggcaaggtga ttcattacat gaacatattg caaatttagc cggatctcct gctattaaga
     4861 aagggatatt acaaactgtt aaagttgtgg atgaattagt gaaagtaatg ggaagacata
     4921 aacctgaaaa cattgtcatt gagatggcaa gagaaaatca aactacacaa aaaggacaga
     4981 aaaatagtag agaacgtatg aaaagaatag aagagggtat taaagaattg ggtagtcaaa
     5041 tattgaaaga acacccagtg gaaaataccc agttgcaaaa tgaaaaatta tatctttact
     5101 accttcaaaa tggacgtgat atgtatgttg atcaggaatt agatataaat agactttcag
     5161 attatgatgt agatcatata gttccacaat ctttcttgaa agatgattcc atagacaata
     5221 aagtattaac tagaagtgat aaaaatagag gtaaaagtga taatgtccca agtgaggaag
     5281 tcgtcaaaaa gatgaaaaat tactggcgtc aacttttgaa tgctaaatta attactcaaa
     5341 gaaaatttga taatttgact aaagcagaaa gaggtgggct ttctgaatta gataaagccg
     5401 ggttcattaa aagacaattg gtcgaaacta gacaaattac taaacatgtt gcccaaattt
     5461 tagattcccg tatgaacact aagtatgacg aaaatgataa gttaatacgt gaggttaaag
     5521 tcattacttt aaaatcaaaa cttgtctctg atttcagaaa ggatttccaa ttctataaag
     5581 ttagagaaat taataattat catcatgctc atgatgcata tttgaatgct gtagttggaa
     5641 ctgctttaat caagaaatac cctaaattag aatctgaatt tgtatatggt gattacaaag
     5701 tctatgatgt tagaaagatg attgctaaat cagaacaaga aattggtaaa gctacagcta
     5761 aatacttctt ttactctaac attatgaatt tctttaaaac agaaattact ttggcaaacg
     5821 gtgaaattag aaaaagacct cttattgaaa caaatggtga gactggagag atagtttggg
     5881 acaaagggcg tgatttcgct actgttagaa aagttttatc aatgccacaa gttaacattg
     5941 taaagaaaac agaggttcaa actggtggtt tctcaaaaga aagtattttg cctaaaagaa
     6001 atagtgataa attgattgcc agaaaaaagg attgggatcc aaagaaatat ggtggtttcg
     6061 actcaccaac cgtagcctat tctgttttgg ttgtggcaaa ggttgaaaag ggtaaaagta
     6121 aaaagcttaa atcagtaaaa gaacttttgg gtattacaat aatggaaaga agttcctttg
     6181 aaaagaaccc tattgatttt ttggaagcta aaggttataa ggaagtaaag aaggacttaa
     6241 taatcaaatt gcctaaatat tctttatttg aattagaaaa tgggagaaaa agaatgttgg
     6301 cttctgctgg agaattgcaa aagggtaatg aattagcatt gccttccaaa tatgttaact
     6361 tcttgtattt agcttcacac tatgaaaagt tgaaagggtc accagaagat aacgagcaaa
     6421 aacaattatt tgttgaacaa cacaaacact acttagatga gattatagaa caaattagtg
     6481 aattcagtaa aagagtgata ttagctgatg caaatttaga taaagttttg tcagcctata
     6541 acaaacatag agataagcca attagagaac aagcagaaaa cattattcac ttatttaccc
     6601 ttaccaattt aggagcacct gctgctttca agtattttga tacaacaatt gatcgtaaaa
     6661 gatatacctc aacaaaagaa gtcttagacg ccaccttaat tcatcaatca atcactggat
     6721 tgtatgagac aagaattgat ttgtctcaat tgggtggtga tgaaggggct gatcctaaga
     6781 agaaaagaaa agttgatcca aagaaaaagc gtaaggtgga tcctaagaaa aagagaaagg
     6841 ttgactacaa agaccatgac ggtgattata aagatcatga catcgactac aaggatgacg
     6901 atgacaagtg ataatctctt ctcgagtcat gtaattagtt atgtcacgct tacattcacg
     6961 ccctcccccc acatccgctc taaccgaaaa ggaaggagtt agacaacctg aagtctaggt
     7021 ccctatttat ttttttatag ttatgttagt attaagaacg ttatttatat ttcaaatttt
     7081 tctttttttt ctgtacagac gcgtgtacgc atgtaacatt atactgaaaa ccttgcttga
     7141 gaaggttttg ggacgctcga aggctttaat ttgcggccgg tactgcggtg tgaaataccg
     7201 cacagatgcg taaggagaaa ataccgcatc aggaaattgt aagcgttaat attttgttaa
     7261 aattcgcgtt aaatttttgt taaatcagct cattttttaa ccaataggcc gaaatcggca
     7321 aaatccctta taaatcaaaa gaatagaccg agatagggtt gagtgttgtt ccagtttgga
     7381 acaagagtcc actattaaag aacgtggact ccaacgtcaa agggcgaaaa accgtctatc
     7441 agggcgatgg cccactacgt gaaccatcac cctaatcaag ttttttgggg tcgaggtgcc
     7501 gtaaagcact aaatcggaac cctaaaggga gcccccgatt tagagcttga cggggaaagc
     7561 cggcgaacgt ggcgagaaag gaagggaaga aagcgaaagg agcgggcgct agggcgctgg
     7621 caagtgtagc ggtcacgctg cgcgtaacca ccacacccgc cgcgcttaat gcgccgctac
     7681 agggcgcgtc cattcgccat tcaggctgcg caactgttgg gaagggcgat cggtgcgggc
     7741 ctcttcgcta ttacgccagc tggcgaaagg gggatgtgct gcaaggcgat taagttgggt
     7801 aacgccaggg ttttcccagt cacgacgttg taaaacgacg gccagtgaat tgtaatacga
     7861 ctcactatag ggcgaattgg agctccaccg cggtggcggc cgctctagaa ctagtggatc
     7921 ccccgggctg caggaattcg atatcaagct ttctttgaaa agataatgta tgattatgct
     7981 ttcactcata tttatacaga aacttgatgt tttctttcga gtatatacaa ggtgattaca
     8041 tgtacgtttg aagtacaact ctagattttg tagtgccctc ttgggctagc ggtaaaggtg
     8101 cgcatttttt cacaccctac aatgttctgt tcaaaagatt ttggtcaaac gctgtagaag
     8161 tgaaagttgg tgcgcatgtt tcggcgttcg aaacttctcc gcagtgaaag ataaatgatc
     8221 ctagatgcga gatatcctca tgcctaggtt ttagagctag aaatagcaag ttaaaataag
     8281 gctagtccgt tatcaacttg aaaaagtggc accgagtcgg tggtgctttt tttgtttttt
     8341 atgtctaccg tcgacctcga gggggggccc ggtacccagc ttttgttccc tttagtgagg
     8401 gttaatttcg agcttggcgt aatcatggtc atagctgttt cctgtgtgaa attgttatcc
     8461 gctcacaatt ccacacaaca tacgagccgg aagcataaag tgtaaagcct ggggtgccta
     8521 atgagtgagc taactcacat taattgcgtt gcgctcactg cccgctttcc agtcgggaaa
     8581 cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat
     8641 tgggcgctct tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg
     8701 agcggtatca gctcactcaa aggcggtaat acggttatcc acagaatcag gggataacgc
     8761 aggaaagaac atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt
     8821 gctggcgttt ttccataggc tccgcccccc tgacgagcat cacaaaaatc gacgctcaag
     8881 tcagaggtgg cgaaacccga caggactata aagataccag gcgtttcccc ctggaagctc
     8941 cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga tacctgtccg cctttctccc
     9001 ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg tatctcagtt cggtgtaggt
     9061 cgttcgctcc aagctgggct gtgtgcacga accccccgtt cagcccgacc gctgcgcctt
     9121 atccggtaac tatcgtcttg agtccaaccc ggtaagacac gacttatcgc cactggcagc
     9181 agccactggt aacaggatta gcagagcgag gtatgtaggc ggtgctacag agttcttgaa
     9241 gtggtggcct aactacggct acactagaag aacagtattt ggtatctgcg ctctgctgaa
     9301 gccagttacc ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg
     9361 tagcggtggt ttttttgttt gcaagcagca gattacgcgc agaaaaaaag gatctcaaga
     9421 agatcctttg atcttttcta cggggtctga cgctcagtgg aacgaaaact cacgttaagg
     9481 gattttggtc atgagattat caaaaaggat cttcacctag atccttttaa attaaaaatg
     9541 aagttttaaa tcaatctaaa gtatatatga gtaaacttgg tctgacagtt accaatgctt
     9601 aatcagtgag gcacctatct cagcgatctg tctatttcgt tcatccatag ttgcctgact
     9661 ccccgtcgtg tagataacta cgatacggga gggcttacca tctggcccca gtgctgcaat
     9721 gataccgcga gacccacgct caccggctcc agatttatca gcaataaacc agccagccgg
     9781 aagggccgag cgcagaagtg gtcctgcaac tttatccgcc tccatccagt ctattaattg
     9841 ttgccgggaa gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat
     9901 tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg gcttcattca gctccggttc
     9961 ccaacgatca aggcgagtta catgatcccc catgttgtgc aaaaaagcgg ttagctcctt
    10021 cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg ttatcactca tggttatggc
    10081 agcactgcat aattctctta ctgtcatgcc atccgtaaga tgcttttctg tgactggtga
    10141 gtactcaacc aagtcattct gagaatagtg tatgcggcga ccgagttgct cttgcccggc
    10201 gtcaatacgg gataataccg cgccacatag cagaacttta aaagtgctca tcattggaaa
    10261 acgttcttcg gggcgaaaac tctcaaggat cttaccgctg ttgagatcca gttcgatgta
    10321 acccactcgt gcacccaact gatcttcagc atcttttact ttcaccagcg tttctgggtg
    10381 agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata agggcgacac ggaaatgttg
    10441 aatactcata ctcttccttt ttcaatatta ttgaagcatt tatcagggtt attgtctcat
    10501 gagcggatac atatttgaat gtatttagaa aaataaacaa ataggggttc cgcgcacatt
    10561 tccccgaaaa gtgccacctg ggtccttttc atcacgtgct ataaaaataa ttataattta
    10621 aattttttaa tataaatata taaattaaaa atagaaagta aaaaaagaaa ttaaagaaaa
    10681 aatagttttt gttttccgaa gatgtaaaag actctagggg gatcgccaac aaatactacc
    10741 ttttatcttg ctcttcctgc tctcaggtat taatgccgaa ttgtttcatc ttgtctgtgt
    10801 agaagaccac acacgaaaat cctgtgattt tacattttac ttatcgttaa tcgaatgtat
    10861 atctatttaa tctgcttttc ttgtctaata aatatatatg taaagtacgc tttttgttga
    10921 aattttttaa acctttgttt attttttttt cttcattccg taactcttct accttcttta
    10981 tttactttct aaaatccaaa tacaaaacat aaaaataaat aaacacagag taaattccca
    11041 aattattcca tcattaaaag atacgaggcg cgtgtaagtt acaggcaagc gatccgtcta
    11101 agaaaccatt attatcatga cattaaccta taaaaatagg cgtatcacga ggccctttcg
    11161 tc
//