LOCUS       sequence-410180-        9240 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of increased-fidelity (i.e. high-fidelity) SpCas9 variant
            in bacterial cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 9240)
  AUTHORS   Kulcsar PI, Talas A, Ligeti Z, Toth E, Rakvacs Z, Bartos Z, Krausz 
            SL, Welker A, Vegi VL, Huszar K, Welker E
  TITLE     A cleavage rule for selection of increased-fidelity SpCas9 variants 
            with high efficiency and no detectable off-targets.
  JOURNAL   Nat Commun. 2023 Sep 16;14(1):5746. doi: 10.1038/s41467-023-41393-5.
  PUBMED    37717069
REFERENCE   2  (bases 1 to 9240)
  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: "2023-09-16"; volume: "14(1):5746. doi"; pages: " 
            10.1038/s41467-023-41393-5"
COMMENT     Sequence Label: PET-HypaSpCas9-NLS-6xHis
FEATURES             Location/Qualifiers
     source          1..9240
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     1..20
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1..19
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    20..44
                     /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             59..81
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             join(88..2160,2161..2163,2164..2166,2167..2172,2173..2178,
                     2179..2181,2182..4191)
                     /codon_start=1
                     /product="Cas9 endonuclease from the Streptococcus pyogenes
                     Type II CRISPR/Cas system, mutated to improve targeting 
                     specificity without compromising on-target activity (Chen 
                     et al., 2017)"
                     /label=HypaCas9
                     /note="carries the mutations N692A, M694A, Q695A, and 
                     H698A"
                     /translation="MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK
                     NLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES
                     FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK
                     FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL
                     ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ
                     IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR
                     QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL
                     RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG
                     NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY
                     FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD
                     SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER
                     LKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRAFA
                     ALIADDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR
                     HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY
                     LYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVP
                     SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH
                     VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL
                     NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT
                     EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK
                     ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI
                     TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE
                     LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD
                     ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL
                     DATLIHQSITGLYETRIDLSQLGGD"
     CDS             88..4191
                     /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
                     LKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRAFA
                     ALIADDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR
                     HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY
                     LYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVP
                     SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH
                     VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL
                     NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT
                     EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK
                     ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI
                     TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE
                     LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD
                     ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL
                     DATLIHQSITGLYETRIDLSQLGGD"
     CDS             4192..4239
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             4249..4266
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(4288..4306)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      4302..4349
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      4386..4841
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4473..4492)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4683..4704
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(join(4956..5747,5748..5816))
                     /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     5579..5598
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5817..5909)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      5990..6578
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6479..6498
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6732..6749
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    6764..6903
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(6829..6848)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     6989..7011
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(7005..7196)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(7768..8850)
                     /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"
     primer_bind     8812..8831
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(8851..8928)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     9134..9153
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
ORIGIN
        1 taatacgact cactataggg gaattgtgag cggataacaa ttcccctcta gaaataattt
       61 tgtttaactt taagaaggag atataccatg gacaagaagt acagcatcgg cctggacatc
      121 ggcaccaact ctgtgggctg ggccgtgatc accgacgagt acaaggtgcc cagcaagaaa
      181 ttcaaggtgc tgggcaacac cgaccggcac agcatcaaga agaacctgat cggagccctg
      241 ctgttcgaca gcggcgaaac agccgaggcc acccggctga agagaaccgc cagaagaaga
      301 tacaccagac ggaagaaccg gatctgctat ctgcaagaga tcttcagcaa cgagatggcc
      361 aaggtggacg acagcttctt ccacagactg gaagagtcct tcctggtgga agaggataag
      421 aagcacgagc ggcaccccat cttcggcaac atcgtggacg aggtggccta ccacgagaag
      481 taccccacca tctaccacct gagaaagaaa ctggtggaca gcaccgacaa ggccgacctg
      541 cggctgatct atctggccct ggcccacatg atcaagttcc ggggccactt cctgatcgag
      601 ggcgacctga accccgacaa cagcgacgtg gacaagctgt tcatccagct ggtgcagacc
      661 tacaaccagc tgttcgagga aaaccccatc aacgccagcg gcgtggacgc caaggccatc
      721 ctgtctgcca gactgagcaa gagcagacgg ctggaaaatc tgatcgccca gctgcccggc
      781 gagaagaaga atggcctgtt cggaaacctg attgccctga gcctgggcct gacccccaac
      841 ttcaagagca acttcgacct ggccgaggat gccaaactgc agctgagcaa ggacacctac
      901 gacgacgacc tggacaacct gctggcccag atcggcgacc agtacgccga cctgtttctg
      961 gccgccaaga acctgtccga cgccatcctg ctgagcgaca tcctgagagt gaacaccgag
     1021 atcaccaagg cccccctgag cgcctctatg atcaagagat acgacgagca ccaccaggac
     1081 ctgaccctgc tgaaagctct cgtgcggcag cagctgcctg agaagtacaa agagattttc
     1141 ttcgaccaga gcaagaacgg ctacgccggc tacattgacg gcggagccag ccaggaagag
     1201 ttctacaagt tcatcaagcc catcctggaa aagatggacg gcaccgagga actgctcgtg
     1261 aagctgaaca gagaggacct gctgcggaag cagcggacct tcgacaacgg cagcatcccc
     1321 caccagatcc acctgggaga gctgcacgcc attctgcggc ggcaggaaga tttttaccca
     1381 ttcctgaagg acaaccggga aaagatcgag aagatcctga ccttccgcat cccctactac
     1441 gtgggccctc tggccagggg aaacagcaga ttcgcctgga tgaccagaaa gagcgaggaa
     1501 accatcaccc cctggaactt cgaggaagtg gtggacaagg gcgcttccgc ccagagcttc
     1561 atcgagcgga tgaccaactt cgataagaac ctgcccaacg agaaggtgct gcccaagcac
     1621 agcctgctgt acgagtactt caccgtgtat aacgagctga ccaaagtgaa atacgtgacc
     1681 gagggaatga gaaagcccgc cttcctgagc ggcgagcaga aaaaggccat cgtggacctg
     1741 ctgttcaaga ccaaccggaa agtgaccgtg aagcagctga aagaggacta cttcaagaaa
     1801 atcgagtgct tcgactccgt ggaaatctcc ggcgtggaag atcggttcaa cgcctccctg
     1861 ggcacatacc acgatctgct gaaaattatc aaggacaagg acttcctgga caatgaggaa
     1921 aacgaggaca ttctggaaga tatcgtgctg accctgacac tgtttgagga cagagagatg
     1981 atcgaggaac ggctgaaaac ctatgcccac ctgttcgacg acaaagtgat gaagcagctg
     2041 aagcggcgga gatacaccgg ctggggcagg ctgagccgga agctgatcaa cggcatccgc
     2101 gacaagcagt ccggcaagac aatcctggat ttcctgaagt ccgacggctt cgccaacaga
     2161 gcctttgcag ccctgatcgc tgacgacagc ctgaccttta aagaggacat ccagaaagcc
     2221 caggtgtccg gccagggcga tagcctgcac gagcacattg ccaatctggc cggcagcccc
     2281 gccattaaga agggcatcct gcagacagtg aaggtggtgg acgagctcgt gaaagtgatg
     2341 ggccggcaca agcccgagaa catcgtgatc gaaatggcca gagagaacca gaccacccag
     2401 aagggacaga agaacagccg cgagagaatg aagcggatcg aagagggcat caaagagctg
     2461 ggcagccaga tcctgaaaga acaccccgtg gaaaacaccc agctgcagaa cgagaagctg
     2521 tacctgtact acctgcagaa tgggcgggat atgtacgtgg accaggaact ggacatcaac
     2581 cggctgtccg actacgatgt ggaccatatc gtgcctcaga gctttctgaa ggacgactcc
     2641 atcgacaaca aggtgctgac cagaagcgac aagaaccggg gcaagagcga caacgtgccc
     2701 tccgaagagg tcgtgaagaa gatgaagaac tactggcggc agctgctgaa cgccaagctg
     2761 attacccaga gaaagttcga caatctgacc aaggccgaga gaggcggcct gagcgaactg
     2821 gataaggccg gcttcatcaa gagacagctg gtggaaaccc ggcagatcac aaagcacgtg
     2881 gcacagatcc tggactcccg gatgaacact aagtacgacg agaatgacaa gctgatccgg
     2941 gaagtgaaag tgatcaccct gaagtccaag ctggtgtccg atttccggaa ggatttccag
     3001 ttttacaaag tgcgcgagat caacaactac caccacgccc acgacgcgta cctgaacgcc
     3061 gtcgtgggaa ccgccctgat caaaaagtac cctaagctgg aaagcgagtt cgtgtacggc
     3121 gactacaagg tgtacgacgt acggaagatg atcgccaaga gcgagcagga aatcggcaag
     3181 gctaccgcca agtacttctt ctacagcaac atcatgaact ttttcaagac cgagattacc
     3241 ctggccaacg gcgagatccg gaagcggcct ctgatcgaga caaacggcga aaccggggag
     3301 atcgtgtggg ataagggccg ggattttgcc accgtgcgga aagtgctgag catgccccaa
     3361 gtgaatatcg tgaaaaagac cgaggtgcag acaggcggct tcagcaaaga gtctatcctg
     3421 cccaagagga acagcgataa gctgatcgcc agaaagaagg actgggaccc taagaagtac
     3481 ggcggcttcg acagccccac cgtggcctat tctgtgctgg tggtggccaa agtggaaaag
     3541 ggcaagtcca agaaactgaa gagtgtgaaa gagctgctgg ggatcaccat catggaaaga
     3601 agcagcttcg agaagaatcc catcgacttt ctggaagcca agggctacaa agaagtgaaa
     3661 aaggacctga tcatcaagct gcctaagtac tccctgttcg agctggaaaa cggccggaag
     3721 agaatgctgg cctctgccgg cgaactgcag aagggaaacg aactggccct gccctccaaa
     3781 tatgtgaact tcctgtacct ggccagccac tatgagaagc tgaagggctc ccccgaggat
     3841 aatgagcaga aacagctgtt tgtggaacag cacaagcact acctggacga gatcatcgag
     3901 cagatcagcg agttctccaa gagagtgatc ctggccgacg ctaatctgga caaagtgctg
     3961 tccgcctaca acaagcaccg ggataagccc atcagagagc aggccgagaa tatcatccac
     4021 ctgtttaccc tgaccaatct gggagcccct gccgccttca agtactttga caccaccatc
     4081 gaccggaaga ggtacaccag caccaaagag gtgctggacg ccaccctgat ccaccagagc
     4141 atcaccggcc tgtacgagac acggatcgac ctgtctcagc tgggaggcga caaaaggccg
     4201 gcggccacga aaaaggccgg ccaggcaaaa aagaaaaaga tggataagca tcaccaccac
     4261 catcactaat aacctaggct gctgccaccg ctgagcaata actagcataa ccccttgggg
     4321 cctctaaacg ggtcttgagg ggttttttgc tgaaaggagg aactatatcc ggattggcga
     4381 atgggacgcg ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta cgcgcagcgt
     4441 gaccgctaca cttgccagcg ccctagcgcc cgctcctttc gctttcttcc cttcctttct
     4501 cgccacgttc gccggctttc cccgtcaagc tctaaatcgg gggctccctt tagggttccg
     4561 atttagtgct ttacggcacc tcgaccccaa aaaacttgat tagggtgatg gttcacgtag
     4621 tgggccatcg ccctgataga cggtttttcg ccctttgacg ttggagtcca cgttctttaa
     4681 tagtggactc ttgttccaaa ctggaacaac actcaaccct atctcggtct attcttttga
     4741 tttataaggg attttgccga tttcggccta ttggttaaaa aatgagctga tttaacaaaa
     4801 atttaacgcg aattttaaca aaatattaac gtttacaatt tctggcggca cgatggcatg
     4861 agattatcaa aaaggatctt cacctagatc cttttaaatt aaaaatgaag ttttaaatca
     4921 atctaaagta tatatgagta aacttggtct gacagttacc aatgcttaat cagtgaggca
     4981 cctatctcag cgatctgtct atttcgttca tccatagttg cctgactccc cgtcgtgtag
     5041 ataactacga tacgggaggg cttaccatct ggccccagtg ctgcaatgat accgcgagac
     5101 ccacgctcac cggctccaga tttatcagca ataaaccagc cagccggaag ggccgagcgc
     5161 agaagtggtc ctgcaacttt atccgcctcc atccagtcta ttaattgttg ccgggaagct
     5221 agagtaagta gttcgccagt taatagtttg cgcaacgttg ttgccattgc tacaggcatc
     5281 gtggtgtcac gctcgtcgtt tggtatggct tcattcagct ccggttccca acgatcaagg
     5341 cgagttacat gatcccccat gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc
     5401 gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg ttatggcagc actgcataat
     5461 tctcttactg tcatgccatc cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag
     5521 tcattctgag aatagtgtat gcggcgaccg agttgctctt gcccggcgtc aatacgggat
     5581 aataccgcgc cacatagcag aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg
     5641 cgaaaactct caaggatctt accgctgttg agatccagtt cgatgtaacc cactcgtgca
     5701 cccaactgat cttcagcatc ttttactttc accagcgttt ctgggtgagc aaaaacagga
     5761 aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat actcatactc
     5821 ttcctttttc aatcatgatt gaagcattta tcagggttat tgtctcatga gcggatacat
     5881 atttgaatgt atttagaaaa ataaacaaat aggtcatgac caaaatccct taacgtgagt
     5941 tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa aggatcttct tgagatcctt
     6001 tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca gcggtggttt
     6061 gtttgccgga tcaagagcta ccaactcttt ttccgaaggt aactggcttc agcagagcgc
     6121 agataccaaa tactgtcctt ctagtgtagc cgtagttagg ccaccacttc aagaactctg
     6181 tagcaccgcc tacatacctc gctctgctaa tcctgttacc agtggctgct gccagtggcg
     6241 ataagtcgtg tcttaccggg ttggactcaa gacgatagtt accggataag gcgcagcggt
     6301 cgggctgaac ggggggttcg tgcacacagc ccagcttgga gcgaacgacc tacaccgaac
     6361 tgagatacct acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg
     6421 acaggtatcc ggtaagcggc agggtcggaa caggagagcg cacgagggag ctcccagggg
     6481 gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat
     6541 ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac gcggcctttt
     6601 tacggttcct ggccttttgc tggccttttg ctcacatgtt ctttcctgcg ttatcccctg
     6661 attctgtgga taaccgtatt accgcctttg agtgagctga taccgctcgc cgcagccgaa
     6721 cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga gcgcctgatg cggtattttc
     6781 tccttacgca tctgtgcggt atttcacacc gcaatggtgc actctcagta caatctgctc
     6841 tgatgccgca tagttaagcc agtatacact ccgctatcgc tacgtgactg ggtcatggct
     6901 gcgccccgac acccgccaac acccgctgac gcgccctgac gggcttgtct gctcccggca
     6961 tccgcttaca gacaagctgt gaccgtctcc gggagctgca tgtgtcagag gttttcaccg
     7021 tcatcaccga aacgcgcgag gcagctgcgg taaagctcat cagcgtggtc gtgaagcgat
     7081 tcacagatgt ctgcctgttc atccgcgtcc agctcgttga gtttctccag aagcgttaat
     7141 gtctggcttc tgataaagcg ggccatgtta agggcggttt tttcctgttt ggtcactgat
     7201 gcctccgtgt aagggggatt tctgttcatg ggggtaatga taccgatgaa acgagagagg
     7261 atgctcacga tacgggttac tgatgatgaa catgcccggt tactggaacg ttgtgagggt
     7321 aaacaactgg cggtatggat gcggcgggac cagagaaaaa tcactcaggg tcaatgccag
     7381 cgcttcgtta atacagatgt aggtgttcca cagggtagcc agcagcatcc tgcgatgcag
     7441 atccggaaca taatggtgca gggcgctgac ttccgcgttt ccagacttta cgaaacacgg
     7501 aaaccgaaga ccattcatgt tgttgctcag gtcgcagacg ttttgcagca gcagtcgctt
     7561 cacgttcgct cgcgtatcgg tgattcattc tgctaaccag taaggcaacc ccgccagcct
     7621 agccgggtcc tcaacgacag gagcacgatc atgctagtca tgccccgcgc ccaccggaag
     7681 gagctgactg ggttgaaggc tctcaagggc atcggtcgag atcccggtgc ctaatgagtg
     7741 agctaactta cattaattgc gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg
     7801 tgccagctgc attaatgaat cggccaacgc gcggggagag gcggtttgcg tattgggcgc
     7861 cagggtggtt tttcttttca ccagtgagac gggcaacagc tgattgccct tcaccgcctg
     7921 gccctgagag agttgcagca agcggtccac gctggtttgc cccagcaggc gaaaatcctg
     7981 tttgatggtg gttaacggcg ggatataaca tgagctgtct tcggtatcgt cgtatcccac
     8041 taccgagatg tccgcaccaa cgcgcagccc ggactcggta atggcgcgca ttgcgcccag
     8101 cgccatctga tcgttggcaa ccagcatcgc agtgggaacg atgccctcat tcagcatttg
     8161 catggtttgt tgaaaaccgg acatggcact ccagtcgcct tcccgttccg ctatcggctg
     8221 aatttgattg cgagtgagat atttatgcca gccagccaga cgcagacgcg ccgagacaga
     8281 acttaatggg cccgctaaca gcgcgatttg ctggtgaccc aatgcgacca gatgctccac
     8341 gcccagtcgc gtaccgtctt catgggagaa aataatactg ttgatgggtg tctggtcaga
     8401 gacatcaaga aataacgccg gaacattagt gcaggcagct tccacagcaa tggcatcctg
     8461 gtcatccagc ggatagttaa tgatcagccc actgacgcgt tgcgcgagaa gattgtgcac
     8521 cgccgcttta caggcttcga cgccgcttcg ttctaccatc gacaccacca cgctggcacc
     8581 cagttgatcg gcgcgagatt taatcgccgc gacaatttgc gacggcgcgt gcagggccag
     8641 actggaggtg gcaacgccaa tcagcaacga ctgtttgccc gccagttgtt gtgccacgcg
     8701 gttgggaatg taattcagct ccgccatcgc cgcttccact ttttcccgcg ttttcgcaga
     8761 aacgtggctg gcctggttca ccacgcggga aacggtctga taagagacac cggcatactc
     8821 tgcgacatcg tataacgtta ctggtttcac attcaccacc ctgaattgac tctcttccgg
     8881 gcgctatcat gccataccgc gaaaggtttt gcgccattcg atggtgtccg ggatctcgac
     8941 gctctccctt atgcgactcc tgcattagga agcagcccag tagtaggttg aggccgttga
     9001 gcaccgccgc cgcaaggaat ggtgcatgca aggagatggc gcccaacagt cccccggcca
     9061 cggggcctgc caccataccc acgccgaaac aagcgctcat gagcccgaag tggcgagccc
     9121 gatcttcccc atcggtgatg tcggcgatat aggcgccagc aaccgcacct gtggcgccgg
     9181 tgatgccggc cacgatgcgt ccggcgtaga ggatcgagat cgatctcgat cccgcgaaat
//
