LOCUS       sequence-358848-       10169 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Express SpCas9mSA in bacteria.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 10169)
  AUTHORS   Liang SQ, Liu P, Smith JL, Mintzer E, Maitland S, Dong X, Yang Q, 
            Lee J, Haynes CM, Zhu LJ, Watts JK, Sontheimer EJ, Wolfe SA, Xue W
  TITLE     Genome-wide detection of CRISPR editing in vivo using GUIDE-tag.
  JOURNAL   Nat Commun. 2022 Jan 21;13(1):437. doi: 10.1038/s41467-022-28135-9.
  PUBMED    35064134
REFERENCE   2  (bases 1 to 10169)
  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"; journalName: "Nat 
            Commun."; date: "2022-01-21"; volume: "13(1):437. doi"; pages: " 
            10.1038/s41467-022-28135-9"
COMMENT     Sequence Label: NLS-SpCas9mSA-His
FEATURES             Location/Qualifiers
     source          1..10169
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     660..679
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(699..776)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     982..1001
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     1085..1104
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1085..1103
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    1104..1128
                     /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)."
     RBS             1143..1165
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             1173..1205
                     /codon_start=1
                     /product="leader peptide from bacteriophage T7 gene 10"
                     /label=T7 tag (gene 10 leader)
                     /note="promotes efficient translation in E. coli"
                     /translation="MASMTGGQQMG"
     CDS             1215..1241
                     /codon_start=1
                     /product="nuclear localization signal (Makkerh et al., 
                     1996)"
                     /label=NLS
                     /translation="PAAKKKKLD"
     CDS             1254..5357
                     /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             1254..5357
                     /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             5364..5453
                     /codon_start=1
                     /product="three tandem HA epitope tags"
                     /label=3xHA
                     /translation="YPYDVPDYAGYPYDVPDYAGSYPYDVPDYA"
     CDS             5466..5492
                     /codon_start=1
                     /product="nuclear localization signal (Makkerh et al., 
                     1996)"
                     /label=NLS
                     /translation="PAAKKKKLD"
     CDS             5535..5876
                     /codon_start=1
                     /product="monomeric streptavidin/rhizavidin hybrid (Lim et 
                     al., 2013)"
                     /label=mSA
                     /translation="AEAGITGTWYNQSGSTFTVTAGADGNLTGQYENRAQGTGCQNSPY
                     TLTGRYNGTKLEWRVEWNNSTENCHSRTEWRGQYQGGAEARINTQWNLTYEGGSGPATE
                     QGQDTFTKVK"
     CDS             5898..5918
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             5931..5978
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             5985..6002
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(6055..6073)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      6069..6116
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      6153..6608
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6240..6259)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     6450..6471
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        6634..6738
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(6739..6807,6808..7599)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(6957..6976)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      7770..8358
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     8259..8278
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     8512..8529
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    8544..8683
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(8609..8628)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     8769..8791
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(8785..8976)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(join(9785..10169,1..698))
                     /codon_start=1
                     /gene="lacI"
                     /product="lac repressor"
                     /label=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)."
                     /translation="MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
ORIGIN
        1 agcatttgca tggtttgttg aaaaccggac atggcactcc agtcgccttc ccgttccgct
       61 atcggctgaa tttgattgcg agtgagatat ttatgccagc cagccagacg cagacgcgcc
      121 gagacagaac ttaatgggcc cgctaacagc gcgatttgct ggtgacccaa tgcgaccaga
      181 tgctccacgc ccagtcgcgt accgtcttca tgggagaaaa taatactgtt gatgggtgtc
      241 tggtcagaga catcaagaaa taacgccgga acattagtgc aggcagcttc cacagcaatg
      301 gcatcctggt catccagcgg atagttaatg atcagcccac tgacgcgttg cgcgagaaga
      361 ttgtgcaccg ccgctttaca ggcttcgacg ccgcttcgtt ctaccatcga caccaccacg
      421 ctggcaccca gttgatcggc gcgagattta atcgccgcga caatttgcga cggcgcgtgc
      481 agggccagac tggaggtggc aacgccaatc agcaacgact gtttgcccgc cagttgttgt
      541 gccacgcggt tgggaatgta attcagctcc gccatcgccg cttccacttt ttcccgcgtt
      601 ttcgcagaaa cgtggctggc ctggttcacc acgcgggaaa cggtctgata agagacaccg
      661 gcatactctg cgacatcgta taacgttact ggtttcacat tcaccaccct gaattgactc
      721 tcttccgggc gctatcatgc cataccgcga aaggttttgc gccattcgat ggtgtccggg
      781 atctcgacgc tctcccttat gcgactcctg cattaggaag cagcccagta gtaggttgag
      841 gccgttgagc accgccgccg caaggaatgg tgcatgcaag gagatggcgc ccaacagtcc
      901 cccggccacg gggcctgcca ccatacccac gccgaaacaa gcgctcatga gcccgaagtg
      961 gcgagcccga tcttccccat cggtgatgtc ggcgatatag gcgccagcaa ccgcacctgt
     1021 ggcgccggtg atgccggcca cgatgcgtcc ggcgtagagg atcgagatct cgatcccgcg
     1081 aaattaatac gactcactat aggggaattg tgagcggata acaattcccc tctagaaata
     1141 attttgttta actttaagaa ggagatatac atatggctag catgactggt ggacagcaaa
     1201 tgggtcgcgc cgctccggca gctaagaaaa agaaactgga tggcagcgtc gacatggata
     1261 agaaatactc aataggctta gatatcggca caaatagcgt cggatgggcg gtgatcactg
     1321 atgaatataa ggttccgtct aaaaagttca aggttctggg aaatacagac cgccacagta
     1381 tcaaaaaaaa tcttataggg gctcttttat ttgacagtgg agagacagcg gaagcgactc
     1441 gtctcaaacg gacagctcgt agaaggtata cacgtcggaa gaatcgtatt tgttatctac
     1501 aggagatttt ttcaaatgag atggcgaaag tagatgatag tttctttcat cgacttgaag
     1561 agtctttttt ggtggaagaa gacaagaagc atgaacgtca tcctattttt ggaaatatag
     1621 tagatgaagt tgcttatcat gagaaatatc caactatcta tcatctgcga aaaaaattgg
     1681 tagattctac tgataaagcg gatttgcgct taatctattt ggccttagcg catatgatta
     1741 agtttcgtgg tcattttttg attgagggag atttaaatcc tgataatagt gatgtggaca
     1801 aactatttat ccagttggta caaacctaca atcaattatt tgaagaaaac cctattaacg
     1861 caagtggagt agatgctaaa gcgattcttt ctgcacgatt gagtaaatca agacgattag
     1921 aaaatctcat tgctcagctc cccggtgaga agaaaaatgg cttatttggg aatctcattg
     1981 ctttgtcatt gggtttgacc cctaatttta aatcaaattt tgatttggca gaagatgcta
     2041 aattacagct ttcaaaagat acttacgatg atgatttaga taatttattg gcgcaaattg
     2101 gagatcaata tgctgatttg tttttggcag ctaagaattt atcagatgct attttacttt
     2161 cagatatcct aagagtaaat actgaaataa ctaaggctcc cctatcagct tcaatgatta
     2221 aacgctacga tgaacatcat caagacttga ctcttttaaa agctttagtt cgacaacaac
     2281 ttccagaaaa gtataaagaa atcttttttg atcaatcaaa aaacggatat gcaggttata
     2341 ttgatggggg agctagccaa gaagaatttt ataaatttat caaaccaatt ttagaaaaaa
     2401 tggatggtac tgaggaatta ttggtgaaac taaatcgtga agatttgctg cgcaagcaac
     2461 ggacctttga caacggctct attccccatc aaattcactt gggtgagctg catgctattt
     2521 tgagaagaca agaagacttt tatccatttt taaaagacaa tcgtgagaag attgaaaaaa
     2581 tcttgacttt tcgaattcct tattatgttg gtccattggc gcgtggcaat agtcgttttg
     2641 catggatgac tcggaagtct gaagaaacaa ttaccccatg gaattttgaa gaagttgtcg
     2701 ataaaggtgc ttcagctcaa tcatttattg aacgcatgac aaactttgat aaaaatcttc
     2761 caaatgaaaa agtactacca aaacatagtt tgctttatga gtattttacg gtttataacg
     2821 aattgacaaa ggtcaaatat gttactgaag gaatgcgaaa accagcattt ctttcaggtg
     2881 aacagaagaa agccattgtt gatttactct tcaaaacaaa tcgaaaagta accgttaagc
     2941 aattaaaaga agattatttc aaaaaaatag aatgttttga tagtgttgaa atttcaggag
     3001 ttgaagatag atttaatgct tcattaggta cctaccatga tttgctaaaa attattaaag
     3061 ataaagattt tttggataat gaagaaaatg aagatatctt agaggatatt gttttaacat
     3121 tgaccttatt tgaagatagg gagatgattg aggaaagact taaaacatat gctcacctct
     3181 ttgatgataa ggtgatgaaa cagcttaaac gtcgccgtta tactggttgg ggacgtttgt
     3241 ctcgaaaatt gattaatggt attagggata agcaatctgg caaaacaata ttagattttt
     3301 tgaaatcaga tggttttgcc aatcgcaatt ttatgcagct gatccatgat gatagtttga
     3361 catttaaaga agacattcaa aaagcacaag tgtctggaca aggcgatagt ttacatgaac
     3421 atattgcaaa tttagctggt agccctgcta ttaaaaaagg tattttacag actgtaaaag
     3481 ttgttgatga attggtcaaa gtaatggggc ggcataagcc agaaaatatc gttattgaaa
     3541 tggcacgtga aaatcagaca actcaaaagg gccagaaaaa ttcgcgagag cgtatgaaac
     3601 gaatcgaaga aggtatcaaa gaattaggaa gtcagattct taaagagcat cctgttgaaa
     3661 atactcaatt gcaaaatgaa aagctctatc tctattatct ccaaaatgga agagacatgt
     3721 atgtggacca agaattagat attaatcgtt taagtgatta tgatgtcgat cacattgttc
     3781 cacaaagttt ccttaaagac gattcaatag acaataaggt cttaacgcgt tctgataaaa
     3841 atcgtggtaa atcggataac gttccaagtg aagaagtagt caaaaagatg aaaaactatt
     3901 ggagacaact tctaaacgcc aagttaatca ctcaacgtaa gtttgataat ttaacgaaag
     3961 ctgaacgtgg aggtttgagt gaacttgata aagctggttt tatcaaacgc caattggttg
     4021 aaactcgcca aatcactaag catgtggcac aaattttgga tagtcgcatg aatactaaat
     4081 acgatgaaaa tgataaactt attcgagagg ttaaagtgat taccttaaaa tctaaattag
     4141 tttctgactt ccgaaaagat ttccaattct ataaagtacg tgagattaac aattaccatc
     4201 atgcccatga tgcgtatcta aatgccgtcg ttggaactgc tttgattaag aaatatccaa
     4261 aacttgaatc ggagtttgtc tatggtgatt ataaagttta tgatgttcgt aaaatgattg
     4321 ctaagtctga gcaagaaata ggcaaagcaa ccgcaaaata tttcttttac tctaatatca
     4381 tgaacttctt caaaacagaa attacacttg caaatggaga gattcgcaaa cgccctctaa
     4441 tcgaaactaa tggggaaact ggagaaattg tctgggataa agggcgagat tttgccacag
     4501 tgcgcaaagt attgtccatg ccccaagtca atattgtcaa gaaaacagaa gtacagacag
     4561 gcggattctc caaggagtca attttaccaa aaagaaattc ggacaagctt attgctcgta
     4621 aaaaagactg ggatccaaaa aaatatggtg gttttgatag tccaacggta gcttattcag
     4681 tcctagtggt tgctaaggtg gaaaaaggga aatcgaagaa gttaaaatcc gttaaagagt
     4741 tactagggat cacaattatg gaaagaagtt cctttgaaaa aaatccgatt gactttttag
     4801 aagctaaagg atataaggaa gttaaaaaag acttaatcat taaactacct aaatatagtc
     4861 tttttgagtt agaaaacggt cgtaaacgga tgctggctag tgccggagaa ttacaaaaag
     4921 gaaatgagct ggctctgcca agcaaatatg tgaatttttt atatttagct agtcattatg
     4981 aaaagttgaa gggtagtcca gaagataacg aacaaaaaca attgtttgtg gagcagcata
     5041 agcattattt agatgagatt attgagcaaa tcagtgaatt ttctaagcgt gttattttag
     5101 cagatgccaa tttagataaa gttcttagtg catataacaa acatagagac aaaccaatac
     5161 gtgaacaagc agaaaatatt attcatttat ttacgttgac gaatcttgga gctcccgctg
     5221 cttttaaata ttttgataca acaattgatc gtaaacgata tacgtctaca aaagaagttt
     5281 tagatgccac tcttatccat caatccatca ctggtcttta tgaaacacgc attgatttga
     5341 gtcagctagg aggtgacacc ggttacccat acgatgttcc tgactatgcg ggctatccct
     5401 atgacgtccc ggactatgca ggatcgtatc cttatgacgt tccagattac gctggatccg
     5461 ccgctccggc agctaagaaa aagaaactgg atttcgaatc cggagaattc ggggcccagc
     5521 cggccagatc tatggcggaa gcgggtatca ccggcacgtg gtacaaccag tctggttcta
     5581 ccttcaccgt taccgcgggt gcggacggta acctgaccgg tcagtacgaa aaccgtgcgc
     5641 agggcactgg ttgccagaac tctccgtaca ccctgaccgg tcgttacaac ggtaccaaac
     5701 tggaatggcg tgttgaatgg aacaactcta ccgaaaactg ccactctcgt accgaatggc
     5761 gtggtcagta ccagggtggt gcggaagcgc gtatcaacac ccagtggaac ctgacctacg
     5821 aaggtggttc tggtccggcg accgaacagg gtcaggacac cttcaccaaa gttaaagcgg
     5881 ccgcaggcag cggaagccct aagaaaaaac gaaaagttgg cagcggaagc aaaaggccgg
     5941 cggccacgaa aaaggccggc caggcaaaaa agaaaaagct cgagcaccac caccaccacc
     6001 actgagatcc ggctgctaac aaagcccgaa aggaagctga gttggctgct gccaccgctg
     6061 agcaataact agcataaccc cttggggcct ctaaacgggt cttgaggggt tttttgctga
     6121 aaggaggaac tatatccgga ttggcgaatg ggacgcgccc tgtagcggcg cattaagcgc
     6181 ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt gccagcgccc tagcgcccgc
     6241 tcctttcgct ttcttccctt cctttctcgc cacgttcgcc ggctttcccc gtcaagctct
     6301 aaatcggggg ctccctttag ggttccgatt tagtgcttta cggcacctcg accccaaaaa
     6361 acttgattag ggtgatggtt cacgtagtgg gccatcgccc tgatagacgg tttttcgccc
     6421 tttgacgttg gagtccacgt tctttaatag tggactcttg ttccaaactg gaacaacact
     6481 caaccctatc tcggtctatt cttttgattt ataagggatt ttgccgattt cggcctattg
     6541 gttaaaaaat gagctgattt aacaaaaatt taacgcgaat tttaacaaaa tattaacgct
     6601 tacaatttag gtggcacttt tcggggaaat gtgcgcggaa cccctatttg tttatttttc
     6661 taaatacatt caaatatgta tccgctcatg agacaataac cctgataaat gcttcaataa
     6721 tattgaaaaa ggaagagtat gagtattcaa catttccgtg tcgcccttat tccctttttt
     6781 gcggcatttt gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct
     6841 gaagatcagt tgggtgcacg agtgggttac atcgaactgg atctcaacag cggtaagatc
     6901 cttgagagtt ttcgccccga agaacgtttt ccaatgatga gcacttttaa agttctgcta
     6961 tgtggcgcgg tattatcccg tattgacgcc gggcaagagc aactcggtcg ccgcatacac
     7021 tattctcaga atgacttggt tgagtactca ccagtcacag aaaagcatct tacggatggc
     7081 atgacagtaa gagaattatg cagtgctgcc ataaccatga gtgataacac tgcggccaac
     7141 ttacttctga caacgatcgg aggaccgaag gagctaaccg cttttttgca caacatgggg
     7201 gatcatgtaa ctcgccttga tcgttgggaa ccggagctga atgaagccat accaaacgac
     7261 gagcgtgaca ccacgatgcc tgcagcaatg gcaacaacgt tgcgcaaact attaactggc
     7321 gaactactta ctctagcttc ccggcaacaa ttaatagact ggatggaggc ggataaagtt
     7381 gcaggaccac ttctgcgctc ggcccttccg gctggctggt ttattgctga taaatctgga
     7441 gccggtgagc gtgggtctcg cggtatcatt gcagcactgg ggccagatgg taagccctcc
     7501 cgtatcgtag ttatctacac gacggggagt caggcaacta tggatgaacg aaatagacag
     7561 atcgctgaga taggtgcctc actgattaag cattggtaac tgtcagacca agtttactca
     7621 tatatacttt agattgattt aaaacttcat ttttaattta aaaggatcta ggtgaagatc
     7681 ctttttgata atctcatgac caaaatccct taacgtgagt tttcgttcca ctgagcgtca
     7741 gaccccgtag aaaagatcaa aggatcttct tgagatcctt tttttctgcg cgtaatctgc
     7801 tgcttgcaaa caaaaaaacc accgctacca gcggtggttt gtttgccgga tcaagagcta
     7861 ccaactcttt ttccgaaggt aactggcttc agcagagcgc agataccaaa tactgtcctt
     7921 ctagtgtagc cgtagttagg ccaccacttc aagaactctg tagcaccgcc tacatacctc
     7981 gctctgctaa tcctgttacc agtggctgct gccagtggcg ataagtcgtg tcttaccggg
     8041 ttggactcaa gacgatagtt accggataag gcgcagcggt cgggctgaac ggggggttcg
     8101 tgcacacagc ccagcttgga gcgaacgacc tacaccgaac tgagatacct acagcgtgag
     8161 ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc
     8221 agggtcggaa caggagagcg cacgagggag cttccagggg gaaacgcctg gtatctttat
     8281 agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg
     8341 gggcggagcc tatggaaaaa cgccagcaac gcggcctttt tacggttcct ggccttttgc
     8401 tggccttttg ctcacatgtt ctttcctgcg ttatcccctg attctgtgga taaccgtatt
     8461 accgcctttg agtgagctga taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca
     8521 gtgagcgagg aagcggaaga gcgcctgatg cggtattttc tccttacgca tctgtgcggt
     8581 atttcacacc gcaatggtgc actctcagta caatctgctc tgatgccgca tagttaagcc
     8641 agtatacact ccgctatcgc tacgtgactg ggtcatggct gcgccccgac acccgccaac
     8701 acccgctgac gcgccctgac gggcttgtct gctcccggca tccgcttaca gacaagctgt
     8761 gaccgtctcc gggagctgca tgtgtcagag gttttcaccg tcatcaccga aacgcgcgag
     8821 gcagctgcgg taaagctcat cagcgtggtc gtgaagcgat tcacagatgt ctgcctgttc
     8881 atccgcgtcc agctcgttga gtttctccag aagcgttaat gtctggcttc tgataaagcg
     8941 ggccatgtta agggcggttt tttcctgttt ggtcactgat gcctccgtgt aagggggatt
     9001 tctgttcatg ggggtaatga taccgatgaa acgagagagg atgctcacga tacgggttac
     9061 tgatgatgaa catgcccggt tactggaacg ttgtgagggt aaacaactgg cggtatggat
     9121 gcggcgggac cagagaaaaa tcactcaggg tcaatgccag cgcttcgtta atacagatgt
     9181 aggtgttcca cagggtagcc agcagcatcc tgcgatgcag atccggaaca taatggtgca
     9241 gggcgctgac ttccgcgttt ccagacttta cgaaacacgg aaaccgaaga ccattcatgt
     9301 tgttgctcag gtcgcagacg ttttgcagca gcagtcgctt cacgttcgct cgcgtatcgg
     9361 tgattcattc tgctaaccag taaggcaacc ccgccagcct agccgggtcc tcaacgacag
     9421 gagcacgatc atgcgcaccc gtggggccgc catgccggcg ataatggcct gcttctcgcc
     9481 gaaacgtttg gtggcgggac cagtgacgaa ggcttgagcg agggcgtgca agattccgaa
     9541 taccgcaagc gacaggccga tcatcgtcgc gctccagcga aagcggtcct cgccgaaaat
     9601 gacccagagc gctgccggca cctgtcctac gagttgcatg ataaagaaga cagtcataag
     9661 tgcggcgacg atagtcatgc cccgcgccca ccggaaggag ctgactgggt tgaaggctct
     9721 caagggcatc ggtcgagatc ccggtgccta atgagtgagc taacttacat taattgcgtt
     9781 gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg
     9841 ccaacgcgcg gggagaggcg gtttgcgtat tgggcgccag ggtggttttt cttttcacca
     9901 gtgagacggg caacagctga ttgcccttca ccgcctggcc ctgagagagt tgcagcaagc
     9961 ggtccacgct ggtttgcccc agcaggcgaa aatcctgttt gatggtggtt aacggcggga
    10021 tataacatga gctgtcttcg gtatcgtcgt atcccactac cgagatatcc gcaccaacgc
    10081 gcagcccgga ctcggtaatg gcgcgcattg cgcccagcgc catctgatcg ttggcaacca
    10141 gcatcgcagt gggaacgatg ccctcattc
//
