LOCUS       sequence-410601-       10752 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of increased fidelity eSpCas9 in bacterial cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 10752)
  AUTHORS   Kulcsar PI, Talas A, Toth E, Nyeste A, Ligeti Z, Welker Z, Welker E
  TITLE     Blackjack mutations improve the on-target activities of increased 
            fidelity variants of SpCas9 with 5'G-extended sgRNAs.
  JOURNAL   Nat Commun. 2020 Mar 6;11(1):1223. doi: 10.1038/s41467-020-15021-5.
  PUBMED    32144253
REFERENCE   2  (bases 1 to 10752)
  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: "2020-03-6"; volume: "11(1):1223. doi"; pages: " 
            10.1038/s41467-020-15021-5"
COMMENT     Sequence Label: pET-FLAG-eSpCas9
FEATURES             Location/Qualifiers
     source          1..10752
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             1299..1490
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(1484..1506)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    1592..1731
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     1647..1666
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(1746..1763)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1917..2505)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1997..2016)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     complement(2616..2635)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             2627..3442
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     CDS             2627..3442
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     complement(2700..2719)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      complement(3534..3989)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3671..3692)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3883..3902
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     terminator      complement(4026..4073)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     4069..4087
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     CDS             complement(4140..4157)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     4190..4207
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     CDS             complement(4235..4282)
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             complement(join(4283..5206,5207..5209,5210..5377,5378..5380,
                     5381..5842,5843..5845,5846..8383))
                     /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="DKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN
                     LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF
                     LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF
                     RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE
                     NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI
                     GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
                     QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
                     KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
                     SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF
                     TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS
                     VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL
                     KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ
                     LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH
                     KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL
                     YYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLADDSIDNKVLTRSDKNRGKSDNVPS
                     EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV
                     AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
                     AVVGTALIKKYPALESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE
                     ITLANGEIRKAPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE
                     SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT
                     IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL
                     ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA
                     NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD
                     ATLIHQSITGLYETRIDLSQLGGD"
     CDS             complement(8408..8428)
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             complement(8435..8500)
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             complement(8513..8533)
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             complement(8564..9661)
                     /codon_start=1
                     /gene="malE (mutated)"
                     /product="maltose binding protein from E. coli"
                     /label=MBP
                     /note="This version of the gene does not encode a signal 
                     sequence, so MBP will remain in the cytosol."
                     /translation="KIEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLEE
                     KFPQVAATGDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDAVRYNGKLI
                     AYPIAVEALSLIYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWPLIAA
                     DGGYAFKYENGKYDIKDVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNKGET
                     AMTINGPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFLE
                     NYLLTDEGLEAVNKDKPLGAVALKSYEEELAKDPRIAATMENAQKGEIMPNIPQMSAFW
                     YAVRTAVINAASGRQTVDEALKDAQT"
     primer_bind     complement(8567..8590)
                     /label=MBP-F
                     /note="Maltose binding protein, forward primer"
     CDS             complement(9665..9682)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     RBS             9693..9715
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    9730..9754
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     primer_bind     complement(9754..9773)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(9755..9773)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(9857..9876)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        10082..10159
                     /gene="lacI"
                     /label=lacI promoter
     CDS             join(10160..10752,1..490)
                     /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     complement(10179..10198)
                     /label=LacI-R
                     /note="LacI, reverse primer"
ORIGIN
        1 ggctggctgg cataaatatc tcactcgcaa tcaaattcag ccgatagcgg aacgggaagg
       61 cgactggagt gccatgtccg gttttcaaca aaccatgcaa atgctgaatg agggcatcgt
      121 tcccactgcg atgctggttg ccaacgatca gatggcgctg ggcgcaatgc gcgccattac
      181 cgagtccggg ctgcgcgttg gtgcggatat ctcggtagtg ggatacgacg ataccgaaga
      241 cagctcatgt tatatcccgc cgttaaccac catcaaacag gattttcgcc tgctggggca
      301 aaccagcgtg gaccgcttgc tgcaactctc tcagggccag gcggtgaagg gcaatcagct
      361 gttgcccgtc tcactggtga aaagaaaaac caccctggcg cccaatacgc aaaccgcctc
      421 tccccgcgcg ttggccgatt cattaatgca gctggcacga caggtttccc gactggaaag
      481 cgggcagtga gcgcaacgca attaatgtaa gttagctcac tcattaggca ccgggatctc
      541 gaccgatgcc cttgagagcc ttcaacccag tcagctcctt ccggtgggcg cggggcatga
      601 ctatcgtcgc cgcacttatg actgtcttct ttatcatgca actcgtagga caggtgccgg
      661 cagcgctctg ggtcattttc ggcgaggacc gctttcgctg gagcgcgacg atgatcggcc
      721 tgtcgcttgc ggtattcgga atcttgcacg ccctcgctca agccttcgtc actggtcccg
      781 ccaccaaacg tttcggcgag aagcaggcca ttatcgccgg catggcggcc ccacgggtgc
      841 gcatgatcgt gctcctgtcg ttgaggaccc ggctaggctg gcggggttgc cttactggtt
      901 agcagaatga atcaccgata cgcgagcgaa cgtgaagcga ctgctgctgc aaaacgtctg
      961 cgacctgagc aacaacatga atggtcttcg gtttccgtgt ttcgtaaagt ctggaaacgc
     1021 ggaagtcagc gccctgcacc attatgttcc ggatctgcat cgcaggatgc tgctggctac
     1081 cctgtggaac acctacatct gtattaacga agcgctggca ttgaccctga gtgatttttc
     1141 tctggtcccg ccgcatccat accgccagtt gtttaccctc acaacgttcc agtaaccggg
     1201 catgttcatc atcagtaacc cgtatcgtga gcatcctctc tcgtttcatc ggtatcatta
     1261 cccccatgaa cagaaatccc ccttacacgg aggcatcagt gaccaaacag gaaaaaaccg
     1321 cccttaacat ggcccgcttt atcagaagcc agacattaac gcttctggag aaactcaacg
     1381 agctggacgc ggatgaacag gcagacatct gtgaatcgct tcacgaccac gctgatgagc
     1441 tttaccgcag ctgcctcgcg cgtttcggtg atgacggtga aaacctctga cacatgcagc
     1501 tcccggagac ggtcacagct tgtctgtaag cggatgccgg gagcagacaa gcccgtcagg
     1561 gcgcgtcagc gggtgttggc gggtgtcggg gcgcagccat gacccagtca cgtagcgata
     1621 gcggagtgta tactggctta actatgcggc atcagagcag attgtactga gagtgcacca
     1681 ttgcggtgtg aaataccgca cagatgcgta aggagaaaat accgcatcag gcgctcttcc
     1741 gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct
     1801 cactcaaagg cggtaatacg gttatccaca gaatcagggg ataacgcagg aaagaacatg
     1861 tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc
     1921 cataggctcc gcccccctga cgagcatcac aaaaatcgac gctcaagtca gaggtggcga
     1981 aacccgacag gactataaag ataccaggcg tttccccctg gaagctccct cgtgcgctct
     2041 cctgttccga ccctgccgct taccggatac ctgtccgcct ttctcccttc gggaagcgtg
     2101 gcgctttctc atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag
     2161 ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct gcgccttatc cggtaactat
     2221 cgtcttgagt ccaacccggt aagacacgac ttatcgccac tggcagcagc cactggtaac
     2281 aggattagca gagcgaggta tgtaggcggt gctacagagt tcttgaagtg gtggcctaac
     2341 tacggctaca ctagaaggac agtatttggt atctgcgctc tgctgaagcc agttaccttc
     2401 ggaaaaagag ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggtggtttt
     2461 tttgtttgca agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc
     2521 ttttctacgg ggtctgacgc tcagtggaac gaaaactcac gttaagggat tttggtcatg
     2581 aacaataaaa ctgtctgctt acataaacag taatacaagg ggtgttatga gccatattca
     2641 acgggaaacg tcttgctcta ggccgcgatt aaattccaac atggatgctg atttatatgg
     2701 gtataaatgg gctcgcgata atgtcgggca atcaggtgcg acaatctatc gattgtatgg
     2761 gaagcccgat gcgccagagt tgtttctgaa acatggcaaa ggtagcgttg ccaatgatgt
     2821 tacagatgag atggtcagac taaactggct gacggaattt atgcctcttc cgaccatcaa
     2881 gcattttatc cgtactcctg atgatgcatg gttactcacc actgcgatcc ccgggaaaac
     2941 agcattccag gtattagaag aatatcctga ttcaggtgaa aatattgttg atgcgctggc
     3001 agtgttcctg cgccggttgc attcgattcc tgtttgtaat tgtcctttta acagcgatcg
     3061 cgtatttcgt ctcgctcagg cgcaatcacg aatgaataac ggtttggttg atgcgagtga
     3121 ttttgatgac gagcgtaatg gctggcctgt tgaacaagtc tggaaagaaa tgcataaact
     3181 tttgccattc tcaccggatt cagtcgtcac tcatggtgat ttctcacttg ataaccttat
     3241 ttttgacgag gggaaattaa taggttgtat tgatgttgga cgagtcggaa tcgcagaccg
     3301 ataccaggat cttgccatcc tatggaactg cctcggtgag ttttctcctt cattacagaa
     3361 acggcttttt caaaaatatg gtattgataa tcctgatatg aataaattgc agtttcattt
     3421 gatgctcgat gagtttttct aagaattaat tcatgagcgg atacatattt gaatgtattt
     3481 agaaaaataa acaaataggg gttccgcgca catttccccg aaaagtgcca cctaaattgt
     3541 aagcgttaat attttgttaa aattcgcgtt aaatttttgt taaatcagct cattttttaa
     3601 ccaataggcc gaaatcggca aaatccctta taaatcaaaa gaatagaccg agatagggtt
     3661 gagtgttgtt ccagtttgga acaagagtcc actattaaag aacgtggact ccaacgtcaa
     3721 agggcgaaaa accgtctatc agggcgatgg cccactacgt gaaccatcac cctaatcaag
     3781 ttttttgggg tcgaggtgcc gtaaagcact aaatcggaac cctaaaggga gcccccgatt
     3841 tagagcttga cggggaaagc cggcgaacgt ggcgagaaag gaagggaaga aagcgaaagg
     3901 agcgggcgct agggcgctgg caagtgtagc ggtcacgctg cgcgtaacca ccacacccgc
     3961 cgcgcttaat gcgccgctac agggcgcgtc ccattcgcca atccggatat agttcctcct
     4021 ttcagcaaaa aacccctcaa gacccgttta gaggccccaa ggggttatgc tagttattgc
     4081 tcagcggtgg cagcagccaa ctcagcttcc tttcgggctt tgttagcagc cggatctcag
     4141 tggtggtggt ggtggtgctc gagtgcggcc gcaacagatg gctggcaact agaaggcaca
     4201 gtcgaggctg atcagcgagc tctaggaatt cttacttttt cttttttgcc tggccggcct
     4261 ttttcgtggc cgccggcctt ttgtcgcctc ccagctgaga caggtcgatc cgtgtctcgt
     4321 acaggccggt gatgctctgg tggatcaggg tggcgtccag cacctctttg gtgctggtgt
     4381 acctcttccg gtcgatggtg gtgtcaaagt acttgaaggc ggcaggggct cccagattgg
     4441 tcagggtaaa caggtggatg atattctcgg cctgctctct gatgggctta tcccggtgct
     4501 tgttgtaggc ggacagcact ttgtccagat tagcgtcggc caggatcact ctcttggaga
     4561 actcgctgat ctgctcgatg atctcgtcca ggtagtgctt gtgctgttcc acaaacagct
     4621 gtttctgctc attatcctcg ggggagccct tcagcttctc atagtggctg gccaggtaca
     4681 ggaagttcac atatttggag ggcagggcca gttcgtttcc cttctgcagt tcgccggcag
     4741 aggccagcat tctcttccgg ccgttttcca gctcgaacag ggagtactta ggcagcttga
     4801 tgatcaggtc ctttttcact tctttgtagc ccttggcttc cagaaagtcg atgggattct
     4861 tctcgaagct gcttctttcc atgatggtga tccccagcag ctctttcaca ctcttcagtt
     4921 tcttggactt gcccttttcc actttggcca ccaccagcac agaataggcc acggtggggc
     4981 tgtcgaagcc gccgtacttc ttagggtccc agtccttctt tctggcgatc agcttatcgc
     5041 tgttcctctt gggcaggata gactctttgc tgaagccgcc tgtctgcacc tcggtctttt
     5101 tcacgatatt cacttggggc atgctcagca ctttccgcac ggtggcaaaa tcccggccct
     5161 tatcccacac gatctccccg gtttcgccgt ttgtctcgat cagaggcgcc ttccggatct
     5221 cgccgttggc cagggtaatc tcggtcttga aaaagttcat gatgttgctg tagaagaagt
     5281 acttggcggt agccttgccg atttcctgct cgctcttggc gatcatcttc cgcacgtcgt
     5341 acaccttgta gtcgccgtac acgaactcgc tttccagcgc agggtacttt ttgatcaggg
     5401 cggttcccac gacggcgttc aggtaggcgt cgtgggcgtg gtggtagttg ttgatctcgc
     5461 gcactttgta aaactggaaa tccttccgga aatcggacac cagcttggac ttcagggtga
     5521 tcactttcac ttcccggatc agcttgtcat tctcgtcgta cttagtgttc atccgggagt
     5581 ccaggatctg tgccacgtgc tttgtgatct gccgggtttc caccagctgt ctcttgatga
     5641 agccggcctt atccagttcg ctcaggccgc ctctctcggc cttggtcaga ttgtcgaact
     5701 ttctctgggt aatcagcttg gcgttcagca gctgccgcca gtagttcttc atcttcttca
     5761 cgacctcttc ggagggcacg ttgtcgctct tgccccggtt cttgtcgctt ctggtcagca
     5821 ccttgttgtc gatggagtcg tcggccagaa agctctgagg cacgatatgg tccacatcgt
     5881 agtcggacag ccggttgatg tccagttcct ggtccacgta catatcccgc ccattctgca
     5941 ggtagtacag gtacagcttc tcgttctgca gctgggtgtt ttccacgggg tgttctttca
     6001 ggatctggct gcccagctct ttgatgccct cttcgatccg cttcattctc tcgcggctgt
     6061 tcttctgtcc cttctgggtg gtctggttct ctctggccat ttcgatcacg atgttctcgg
     6121 gcttgtgccg gcccatcact ttcacgagct cgtccaccac cttcactgtc tgcaggatgc
     6181 ccttcttaat ggcggggctg ccggccagat tggcaatgtg ctcgtgcagg ctatcgccct
     6241 ggccggacac ctgggctttc tggatgtcct ctttaaaggt caggctgtcg tcgtggatca
     6301 gctgcatgaa gtttctgttg gcgaagccgt cggacttcag gaaatccagg attgtcttgc
     6361 cggactgctt gtcccggatg ccgttgatca gcttccggct cagcctgccc cagccggtgt
     6421 atctccgccg cttcagctgc ttcatcactt tgtcgtcgaa caggtgggca taggttttca
     6481 gccgttcctc gatcatctct ctgtcctcaa acagtgtcag ggtcagcacg atatcttcca
     6541 gaatgtcctc gttttcctca ttgtccagga agtccttgtc cttgataatt ttcagcagat
     6601 cgtggtatgt gcccagggag gcgttgaacc gatcttccac gccggagatt tccacggagt
     6661 cgaagcactc gattttcttg aagtagtcct ctttcagctg cttcacggtc actttccggt
     6721 tggtcttgaa cagcaggtcc acgatggcct ttttctgctc gccgctcagg aaggcgggct
     6781 ttctcattcc ctcggtcacg tatttcactt tggtcagctc gttatacacg gtgaagtact
     6841 cgtacagcag gctgtgcttg ggcagcacct tctcgttggg caggttctta tcgaagttgg
     6901 tcatccgctc gatgaagctc tgggcggaag cgcccttgtc caccacttcc tcgaagttcc
     6961 agggggtgat ggtttcctcg ctctttctgg tcatccaggc gaatctgctg tttcccctgg
     7021 ccagagggcc cacgtagtag gggatgcgga aggtcaggat cttctcgatc ttttcccggt
     7081 tgtccttcag gaatgggtaa aaatcttcct gccgccgcag aatggcgtgc agctctccca
     7141 ggtggatctg gtgggggatg ctgccgttgt cgaaggtccg ctgcttccgc agcaggtcct
     7201 ctctgttcag cttcacgagc agttcctcgg tgccgtccat cttttccagg atgggcttga
     7261 tgaacttgta gaactcttcc tggctggctc cgccgtcaat gtagccggcg tagccgttct
     7321 tgctctggtc gaagaaaatc tctttgtact tctcaggcag ctgctgccgc acgagagctt
     7381 tcagcagggt caggtcctgg tggtgctcgt cgtatctctt gatcatagag gcgctcaggg
     7441 gggccttggt gatctcggtg ttcactctca ggatgtcgct cagcaggatg gcgtcggaca
     7501 ggttcttggc ggccagaaac aggtcggcgt actggtcgcc gatctgggcc agcaggttgt
     7561 ccaggtcgtc gtcgtaggtg tccttgctca gctgcagttt ggcatcctcg gccaggtcga
     7621 agttgctctt gaagttgggg gtcaggccca ggctcagggc aatcaggttt ccgaacaggc
     7681 cattcttctt ctcgccgggc agctgggcga tcagattttc cagccgtctg ctcttgctca
     7741 gtctggcaga caggatggcc ttggcgtcca cgccgctggc gttgatgggg ttttcctcga
     7801 acagctggtt gtaggtctgc accagctgga tgaacagctt gtccacgtcg ctgttgtcgg
     7861 ggttcaggtc gccctcgatc aggaagtggc cccggaactt gatcatgtgg gccagggcca
     7921 gatagatcag ccgcaggtcg gccttgtcgg tgctgtccac cagtttcttt ctcaggtggt
     7981 agatggtggg gtacttctcg tggtaggcca cctcgtccac gatgttgccg aagatggggt
     8041 gccgctcgtg cttcttatcc tcttccacca ggaaggactc ttccagtctg tggaagaagc
     8101 tgtcgtccac cttggccatc tcgttgctga agatctcttg cagatagcag atccggttct
     8161 tccgtctggt gtatcttctt ctggcggttc tcttcagccg ggtggcctcg gctgtttcgc
     8221 cgctgtcgaa cagcagggct ccgatcaggt tcttcttgat gctgtgccgg tcggtgttgc
     8281 ccagcacctt gaatttcttg ctgggcacct tgtactcgtc ggtgatcacg gcccagccca
     8341 cagagttggt gccgatgtcc aggccgatgc tgtacttctt gtcggctgct gggactccgt
     8401 ggataccgac cttccgcttc ttctttgggg ccatcttatc gtcatcgtct ttgtaatcaa
     8461 tatcatgatc cttgtagtct ccgtcgtggt ccttatagtc catcgaggct gctccctgga
     8521 aatacaggtt ttcactagtg ttgttgttgt tcgagctcga attagtctgc gcgtctttca
     8581 gggcttcatc gacagtctga cgaccgctgg cggcgttgat caccgcagta cgcacggcat
     8641 accagaaagc ggacatctgc gggatgttcg gcatgatttc acctttctgg gcgttttcca
     8701 tagtggcggc aatacgtgga tctttcgcca actcttcctc gtaagacttc agcgctacgg
     8761 cacccagcgg tttgtcttta ttaaccgctt ccagaccttc atcagtcagc agatagtttt
     8821 cgaggaactc ttttgccagc tctttgttcg gactggcggc gttaatacct gcgctcagca
     8881 cgccaacgaa cggtttggat ggttgaccct tgaaggtcgg cagtaccgtt acaccataat
     8941 tcactttgct ggtgtcgatg ttggaccatg cccacgggcc gttgatggtc atcgctgttt
     9001 cgcctttatt aaaggcagct tctgcgatgg agtaatcggt gtctgcattc atgtgtttgt
     9061 ttttaatcag gtcaaccagg aaggtcagac ccgctttcgc gccagcgtta tccacgccca
     9121 cgtctttaat gtcgtacttg ccgttttcat acttgaacgc ataacccccg tcagcagcaa
     9181 tcagcggcca ggtgaagtac ggttcttgca ggttgaacat cagcgcgctc ttacctttcg
     9241 ctttcagttc tttatccagc gccgggatct cttcccaggt ttttggcggg ttcggcagca
     9301 gatctttgtt ataaatcagc gataacgctt caacagcgat cgggtaagca atcagcttgc
     9361 cgttgtaacg tacggcatcc caggtaaacg gatacagctt gtcctggaac gctttgtccg
     9421 gggtgatttc agccaacagg ccagattgag cgtagccacc aaagcggtcg tgtgcccaga
     9481 agataatgtc agggccatcg ccagttgccg caacctgtgg gaatttctct tccagtttat
     9541 ccggatgctc aacggtgact ttaattccgg tatctttctc gaatttctta ccgacttcag
     9601 cgagaccgtt atagccttta tcgccgttaa tccagattac cagtttacct tcttcgattt
     9661 ttccatggtg atggtgatgg tgcatatgta tatctccttc ttaaagttaa acaaaattat
     9721 ttctagaggg gaattgttat ccgctcacaa ttcccctata gtgagtcgta ttaatttcgc
     9781 gggatcgaga tctcgatcct ctacgccgga cgcatcgtgg ccggcatcac cggcgccaca
     9841 ggtgcggttg ctggcgccta tatcgccgac atcaccgatg gggaagatcg ggctcgccac
     9901 ttcgggctca tgagcgcttg tttcggcgtg ggtatggtgg caggccccgt ggccggggga
     9961 ctgttgggcg ccatctcctt gcatgcacca ttccttgcgg cggcggtgct caacggcctc
    10021 aacctactac tgggctgctt cctaatgcag gagtcgcata agggagagcg tcgagatccc
    10081 ggacaccatc gaatggcgca aaacctttcg cggtatggca tgatagcgcc cggaagagag
    10141 tcaattcagg gtggtgaatg tgaaaccagt aacgttatac gatgtcgcag agtatgccgg
    10201 tgtctcttat cagaccgttt cccgcgtggt gaaccaggcc agccacgttt ctgcgaaaac
    10261 gcgggaaaaa gtggaagcgg cgatggcgga gctgaattac attcccaacc gcgtggcaca
    10321 acaactggcg ggcaaacagt cgttgctgat tggcgttgcc acctccagtc tggccctgca
    10381 cgcgccgtcg caaattgtcg cggcgattaa atctcgcgcc gatcaactgg gtgccagcgt
    10441 ggtggtgtcg atggtagaac gaagcggcgt cgaagcctgt aaagcggcgg tgcacaatct
    10501 tctcgcgcaa cgcgtcagtg ggctgatcat taactatccg ctggatgacc aggatgccat
    10561 tgctgtggaa gctgcctgca ctaatgttcc ggcgttattt cttgatgtct ctgaccagac
    10621 acccatcaac agtattattt tctcccatga agacggtacg cgactgggcg tggagcatct
    10681 ggtcgcattg ggtcaccagc aaatcgcgct gttagcgggc ccattaagtt ctgtctcggc
    10741 gcgtctgcgt ct
//
