LOCUS       sequence-410179-        9219 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 9219)
  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 9219)
  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-B-eSpCas9-NLS-6xHis
FEATURES             Location/Qualifiers
     source          1..9219
                     /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..2628,2629..2631,2632..3093,3094..3096,3097..3243,
                     3244..3246,3247..4170)
                     /codon_start=1
                     /product="Cas9 endonuclease from the Streptococcus pyogenes
                     Type II CRISPR/Cas system, mutated to improve targeting 
                     specificity (Slaymaker et al., 2016)"
                     /label=eSpCas9(1.1)
                     /note="carries the mutations K848A, K1003A, and R1060A"
                     /translation="MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK
                     NLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES
                     FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK
                     FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL
                     ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ
                     IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR
                     QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL
                     RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG
                     NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY
                     FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD
                     SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER
                     LKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFM
                     QLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR
                     HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY
                     LYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLADDSIDNKVLTRSDKNRGKSDNVP
                     SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH
                     VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL
                     NAVVGTALIKKYPALGGKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANG
                     EIRKAPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKR
                     NSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSS
                     FEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKY
                     VNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVL
                     SAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQ
                     SITGLYETRIDLSQLGGD"
     CDS             4171..4218
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             4228..4245
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(4267..4285)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      4281..4328
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      4365..4820
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4452..4471)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4662..4683
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(join(4935..5726,5727..5795))
                     /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     5558..5577
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5796..5888)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      5969..6557
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6458..6477
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6711..6728
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    6743..6882
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(6808..6827)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     6968..6990
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(6984..7175)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(7747..8829)
                     /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     8791..8810
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(8830..8907)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     9113..9132
                     /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 cggcatccgg
     2101 gacaagcagt ccggcaagac aatcctggat ttcctgaagt ccgacggctt cgccaacaga
     2161 aacttcatgc agctgatcca cgacgacagc 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 gctttctggc cgacgactcc
     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 cctgcgctgg gtggcaaggt gtacgatgta
     3121 cggaagatga tcgccaagag cgagcaggaa atcggcaagg ctaccgccaa gtacttcttc
     3181 tacagcaaca tcatgaactt tttcaagacc gagattaccc tggccaacgg cgagatccgg
     3241 aaggcgcctc tgatcgagac aaacggcgaa accggggaga tcgtgtggga taagggccgg
     3301 gattttgcca ccgtgcggaa agtgctgagc atgccccaag tgaatatcgt gaaaaagacc
     3361 gaggtgcaga caggcggctt cagcaaagag tctatcctgc ccaagaggaa cagcgataag
     3421 ctgatcgcca gaaagaagga ctgggaccct aagaagtacg gcggcttcga cagccccacc
     3481 gtggcctatt ctgtgctggt ggtggccaaa gtggaaaagg gcaagtccaa gaaactgaag
     3541 agtgtgaaag agctgctggg gatcaccatc atggaaagaa gcagcttcga gaagaatccc
     3601 atcgactttc tggaagccaa gggctacaaa gaagtgaaaa aggacctgat catcaagctg
     3661 cctaagtact ccctgttcga gctggaaaac ggccggaaga gaatgctggc ctctgccggc
     3721 gaactgcaga agggaaacga actggccctg ccctccaaat atgtgaactt cctgtacctg
     3781 gccagccact atgagaagct gaagggctcc cccgaggata atgagcagaa acagctgttt
     3841 gtggaacagc acaagcacta cctggacgag atcatcgagc agatcagcga gttctccaag
     3901 agagtgatcc tggccgacgc taatctggac aaagtgctgt ccgcctacaa caagcaccgg
     3961 gataagccca tcagagagca ggccgagaat atcatccacc tgtttaccct gaccaatctg
     4021 ggagcccctg ccgccttcaa gtactttgac accaccatcg accggaagag gtacaccagc
     4081 accaaagagg tgctggacgc caccctgatc caccagagca tcaccggcct gtacgagaca
     4141 cggatcgacc tgtctcagct gggaggcgac aaaaggccgg cggccacgaa aaaggccggc
     4201 caggcaaaaa agaaaaagat ggataagcat caccaccacc atcactaata acctaggctg
     4261 ctgccaccgc tgagcaataa ctagcataac cccttggggc ctctaaacgg gtcttgaggg
     4321 gttttttgct gaaaggagga actatatccg gattggcgaa tgggacgcgc cctgtagcgg
     4381 cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg accgctacac ttgccagcgc
     4441 cctagcgccc gctcctttcg ctttcttccc ttcctttctc gccacgttcg ccggctttcc
     4501 ccgtcaagct ctaaatcggg ggctcccttt agggttccga tttagtgctt tacggcacct
     4561 cgaccccaaa aaacttgatt agggtgatgg ttcacgtagt gggccatcgc cctgatagac
     4621 ggtttttcgc cctttgacgt tggagtccac gttctttaat agtggactct tgttccaaac
     4681 tggaacaaca ctcaacccta tctcggtcta ttcttttgat ttataaggga ttttgccgat
     4741 ttcggcctat tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga attttaacaa
     4801 aatattaacg tttacaattt ctggcggcac gatggcatga gattatcaaa aaggatcttc
     4861 acctagatcc ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa
     4921 acttggtctg acagttacca atgcttaatc agtgaggcac ctatctcagc gatctgtcta
     4981 tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga taactacgat acgggagggc
     5041 ttaccatctg gccccagtgc tgcaatgata ccgcgagacc cacgctcacc ggctccagat
     5101 ttatcagcaa taaaccagcc agccggaagg gccgagcgca gaagtggtcc tgcaacttta
     5161 tccgcctcca tccagtctat taattgttgc cgggaagcta gagtaagtag ttcgccagtt
     5221 aatagtttgc gcaacgttgt tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt
     5281 ggtatggctt cattcagctc cggttcccaa cgatcaaggc gagttacatg atcccccatg
     5341 ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg ttgtcagaag taagttggcc
     5401 gcagtgttat cactcatggt tatggcagca ctgcataatt ctcttactgt catgccatcc
     5461 gtaagatgct tttctgtgac tggtgagtac tcaaccaagt cattctgaga atagtgtatg
     5521 cggcgaccga gttgctcttg cccggcgtca atacgggata ataccgcgcc acatagcaga
     5581 actttaaaag tgctcatcat tggaaaacgt tcttcggggc gaaaactctc aaggatctta
     5641 ccgctgttga gatccagttc gatgtaaccc actcgtgcac ccaactgatc ttcagcatct
     5701 tttactttca ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag
     5761 ggaataaggg cgacacggaa atgttgaata ctcatactct tcctttttca atcatgattg
     5821 aagcatttat cagggttatt gtctcatgag cggatacata tttgaatgta tttagaaaaa
     5881 taaacaaata ggtcatgacc aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag
     5941 accccgtaga aaagatcaaa ggatcttctt gagatccttt ttttctgcgc gtaatctgct
     6001 gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg tttgccggat caagagctac
     6061 caactctttt tccgaaggta actggcttca gcagagcgca gataccaaat actgtccttc
     6121 tagtgtagcc gtagttaggc caccacttca agaactctgt agcaccgcct acatacctcg
     6181 ctctgctaat cctgttacca gtggctgctg ccagtggcga taagtcgtgt cttaccgggt
     6241 tggactcaag acgatagtta ccggataagg cgcagcggtc gggctgaacg gggggttcgt
     6301 gcacacagcc cagcttggag cgaacgacct acaccgaact gagataccta cagcgtgagc
     6361 tatgagaaag cgccacgctt cccgaaggga gaaaggcgga caggtatccg gtaagcggca
     6421 gggtcggaac aggagagcgc acgagggagc tcccaggggg aaacgcctgg tatctttata
     6481 gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg
     6541 ggcggagcct atggaaaaac gccagcaacg cggccttttt acggttcctg gccttttgct
     6601 ggccttttgc tcacatgttc tttcctgcgt tatcccctga ttctgtggat aaccgtatta
     6661 ccgcctttga gtgagctgat accgctcgcc gcagccgaac gaccgagcgc agcgagtcag
     6721 tgagcgagga agcggaagag cgcctgatgc ggtattttct ccttacgcat ctgtgcggta
     6781 tttcacaccg caatggtgca ctctcagtac aatctgctct gatgccgcat agttaagcca
     6841 gtatacactc cgctatcgct acgtgactgg gtcatggctg cgccccgaca cccgccaaca
     6901 cccgctgacg cgccctgacg ggcttgtctg ctcccggcat ccgcttacag acaagctgtg
     6961 accgtctccg ggagctgcat gtgtcagagg ttttcaccgt catcaccgaa acgcgcgagg
     7021 cagctgcggt aaagctcatc agcgtggtcg tgaagcgatt cacagatgtc tgcctgttca
     7081 tccgcgtcca gctcgttgag tttctccaga agcgttaatg tctggcttct gataaagcgg
     7141 gccatgttaa gggcggtttt ttcctgtttg gtcactgatg cctccgtgta agggggattt
     7201 ctgttcatgg gggtaatgat accgatgaaa cgagagagga tgctcacgat acgggttact
     7261 gatgatgaac atgcccggtt actggaacgt tgtgagggta aacaactggc ggtatggatg
     7321 cggcgggacc agagaaaaat cactcagggt caatgccagc gcttcgttaa tacagatgta
     7381 ggtgttccac agggtagcca gcagcatcct gcgatgcaga tccggaacat aatggtgcag
     7441 ggcgctgact tccgcgtttc cagactttac gaaacacgga aaccgaagac cattcatgtt
     7501 gttgctcagg tcgcagacgt tttgcagcag cagtcgcttc acgttcgctc gcgtatcggt
     7561 gattcattct gctaaccagt aaggcaaccc cgccagccta gccgggtcct caacgacagg
     7621 agcacgatca tgctagtcat gccccgcgcc caccggaagg agctgactgg gttgaaggct
     7681 ctcaagggca tcggtcgaga tcccggtgcc taatgagtga gctaacttac attaattgcg
     7741 ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt gccagctgca ttaatgaatc
     7801 ggccaacgcg cggggagagg cggtttgcgt attgggcgcc agggtggttt ttcttttcac
     7861 cagtgagacg ggcaacagct gattgccctt caccgcctgg ccctgagaga gttgcagcaa
     7921 gcggtccacg ctggtttgcc ccagcaggcg aaaatcctgt ttgatggtgg ttaacggcgg
     7981 gatataacat gagctgtctt cggtatcgtc gtatcccact accgagatgt ccgcaccaac
     8041 gcgcagcccg gactcggtaa tggcgcgcat tgcgcccagc gccatctgat cgttggcaac
     8101 cagcatcgca gtgggaacga tgccctcatt cagcatttgc atggtttgtt gaaaaccgga
     8161 catggcactc cagtcgcctt cccgttccgc tatcggctga atttgattgc gagtgagata
     8221 tttatgccag ccagccagac gcagacgcgc cgagacagaa cttaatgggc ccgctaacag
     8281 cgcgatttgc tggtgaccca atgcgaccag atgctccacg cccagtcgcg taccgtcttc
     8341 atgggagaaa ataatactgt tgatgggtgt ctggtcagag acatcaagaa ataacgccgg
     8401 aacattagtg caggcagctt ccacagcaat ggcatcctgg tcatccagcg gatagttaat
     8461 gatcagccca ctgacgcgtt gcgcgagaag attgtgcacc gccgctttac aggcttcgac
     8521 gccgcttcgt tctaccatcg acaccaccac gctggcaccc agttgatcgg cgcgagattt
     8581 aatcgccgcg acaatttgcg acggcgcgtg cagggccaga ctggaggtgg caacgccaat
     8641 cagcaacgac tgtttgcccg ccagttgttg tgccacgcgg ttgggaatgt aattcagctc
     8701 cgccatcgcc gcttccactt tttcccgcgt tttcgcagaa acgtggctgg cctggttcac
     8761 cacgcgggaa acggtctgat aagagacacc ggcatactct gcgacatcgt ataacgttac
     8821 tggtttcaca ttcaccaccc tgaattgact ctcttccggg cgctatcatg ccataccgcg
     8881 aaaggttttg cgccattcga tggtgtccgg gatctcgacg ctctccctta tgcgactcct
     8941 gcattaggaa gcagcccagt agtaggttga ggccgttgag caccgccgcc gcaaggaatg
     9001 gtgcatgcaa ggagatggcg cccaacagtc ccccggccac ggggcctgcc accataccca
     9061 cgccgaaaca agcgctcatg agcccgaagt ggcgagcccg atcttcccca tcggtgatgt
     9121 cggcgatata ggcgccagca accgcacctg tggcgccggt gatgccggcc acgatgcgtc
     9181 cggcgtagag gatcgagatc gatctcgatc ccgcgaaat
//
