LOCUS       sequence-410600-       10731 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of increased fidelity Blackjack-eSpCas9 in bacterial 
            cells. Cleaves both 20nt and 5'-extended 21nt spacer containing 
            sgRNAs with higher fidelity than that of eSpCas9.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 10731)
  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 10731)
  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-B-eSpCas9
FEATURES             Location/Qualifiers
     source          1..10731
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             1301..1492
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(1486..1508)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    1594..1733
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     1649..1668
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(1748..1765)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1919..2507)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1999..2018)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     complement(2618..2637)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             2629..3444
                     /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             2629..3444
                     /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(2702..2721)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      complement(3536..3991)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3673..3694)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3885..3904
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     terminator      complement(4028..4075)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     4071..4089
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     CDS             complement(4142..4159)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     4192..4209
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     CDS             complement(4237..4284)
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             complement(join(4285..5208,5209..5211,5212..5358,5359..5361,
                     5362..5823,5824..5826,5827..8364))
                     /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
                     AVVGTALIKKYPALGGKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGE
                     IRKAPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRN
                     SDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSF
                     EKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYV
                     NFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLS
                     AYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQS
                     ITGLYETRIDLSQLGGD"
     CDS             complement(8389..8409)
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             complement(8416..8481)
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             complement(8494..8514)
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             complement(8545..9642)
                     /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(8548..8571)
                     /label=MBP-F
                     /note="Maltose binding protein, forward primer"
     CDS             complement(9646..9663)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     RBS             9674..9696
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    9711..9735
                     /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(9735..9754)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(9736..9754)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(9838..9857)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        10063..10140
                     /gene="lacI"
                     /label=lacI promoter
     CDS             join(10141..10731,1..492)
                     /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(10160..10179)
                     /label=LacI-R
                     /note="LacI, reverse primer"
ORIGIN
        1 ctggctggct ggcataaata tctcactcgc aatcaaattc agccgatagc ggaacgggaa
       61 ggcgactgga gtgccatgtc cggttttcaa caaaccatgc aaatgctgaa tgagggcatc
      121 gttcccactg cgatgctggt tgccaacgat cagatggcgc tgggcgcaat gcgcgccatt
      181 accgagtccg ggctgcgcgt tggtgcggat atctcggtag tgggatacga cgataccgaa
      241 gacagctcat gttatatccc gccgttaacc accatcaaac aggattttcg cctgctgggg
      301 caaaccagcg tggaccgctt gctgcaactc tctcagggcc aggcggtgaa gggcaatcag
      361 ctgttgcccg tctcactggt gaaaagaaaa accaccctgg cgcccaatac gcaaaccgcc
      421 tctccccgcg cgttggccga ttcattaatg cagctggcac gacaggtttc ccgactggaa
      481 agcgggcagt gagcgcaacg caattaatgt aagttagctc actcattagg caccgggatc
      541 tcgaccgatg cccttgagag ccttcaaccc agtcagctcc ttccggtggg cgcggggcat
      601 gactatcgtc gccgcactta tgactgtctt ctttatcatg caactcgtag gacaggtgcc
      661 ggcagcgctc tgggtcattt tcggcgagga ccgctttcgc tggagcgcga cgatgatcgg
      721 cctgtcgctt gcggtattcg gaatcttgca cgccctcgct caagccttcg tcactggtcc
      781 cgccaccaaa cgtttcggcg agaagcaggc cattatcgcc ggcatggcgg ccccacgggt
      841 gcgcatgatc gtgctcctgt cgttgaggac ccggctaggc tggcggggtt gccttactgg
      901 ttagcagaat gaatcaccga tacgcgagcg aacgtgaagc gactgctgct gcaaaacgtc
      961 tgcgacctga gcaacaacat gaatggtctt cggtttccgt gtttcgtaaa gtctggaaac
     1021 gcggaagtca gcgccctgca ccattatgtt ccggatctgc atcgcaggat gctgctggct
     1081 accctgtgga acacctacat ctgtattaac gaagcgctgg cattgaccct gagtgatttt
     1141 tctctggtcc cgccgcatcc ataccgccag ttgtttaccc tcacaacgtt ccagtaaccg
     1201 ggcatgttca tcatcagtaa cccgtatcgt gagcatcctc tctcgtttca tcggtatcat
     1261 tacccccatg aacagaaatc ccccttacac ggaggcatca gtgaccaaac aggaaaaaac
     1321 cgcccttaac atggcccgct ttatcagaag ccagacatta acgcttctgg agaaactcaa
     1381 cgagctggac gcggatgaac aggcagacat ctgtgaatcg cttcacgacc acgctgatga
     1441 gctttaccgc agctgcctcg cgcgtttcgg tgatgacggt gaaaacctct gacacatgca
     1501 gctcccggag acggtcacag cttgtctgta agcggatgcc gggagcagac aagcccgtca
     1561 gggcgcgtca gcgggtgttg gcgggtgtcg gggcgcagcc atgacccagt cacgtagcga
     1621 tagcggagtg tatactggct taactatgcg gcatcagagc agattgtact gagagtgcac
     1681 cattgcggtg tgaaataccg cacagatgcg taaggagaaa ataccgcatc aggcgctctt
     1741 ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag
     1801 ctcactcaaa ggcggtaata cggttatcca cagaatcagg ggataacgca ggaaagaaca
     1861 tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt
     1921 tccataggct ccgcccccct gacgagcatc acaaaaatcg acgctcaagt cagaggtggc
     1981 gaaacccgac aggactataa agataccagg cgtttccccc tggaagctcc ctcgtgcgct
     2041 ctcctgttcc gaccctgccg cttaccggat acctgtccgc ctttctccct tcgggaagcg
     2101 tggcgctttc tcatagctca cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca
     2161 agctgggctg tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta tccggtaact
     2221 atcgtcttga gtccaacccg gtaagacacg acttatcgcc actggcagca gccactggta
     2281 acaggattag cagagcgagg tatgtaggcg gtgctacaga gttcttgaag tggtggccta
     2341 actacggcta cactagaagg acagtatttg gtatctgcgc tctgctgaag ccagttacct
     2401 tcggaaaaag agttggtagc tcttgatccg gcaaacaaac caccgctggt agcggtggtt
     2461 tttttgtttg caagcagcag attacgcgca gaaaaaaagg atctcaagaa gatcctttga
     2521 tcttttctac ggggtctgac gctcagtgga acgaaaactc acgttaaggg attttggtca
     2581 tgaacaataa aactgtctgc ttacataaac agtaatacaa ggggtgttat gagccatatt
     2641 caacgggaaa cgtcttgctc taggccgcga ttaaattcca acatggatgc tgatttatat
     2701 gggtataaat gggctcgcga taatgtcggg caatcaggtg cgacaatcta tcgattgtat
     2761 gggaagcccg atgcgccaga gttgtttctg aaacatggca aaggtagcgt tgccaatgat
     2821 gttacagatg agatggtcag actaaactgg ctgacggaat ttatgcctct tccgaccatc
     2881 aagcatttta tccgtactcc tgatgatgca tggttactca ccactgcgat ccccgggaaa
     2941 acagcattcc aggtattaga agaatatcct gattcaggtg aaaatattgt tgatgcgctg
     3001 gcagtgttcc tgcgccggtt gcattcgatt cctgtttgta attgtccttt taacagcgat
     3061 cgcgtatttc gtctcgctca ggcgcaatca cgaatgaata acggtttggt tgatgcgagt
     3121 gattttgatg acgagcgtaa tggctggcct gttgaacaag tctggaaaga aatgcataaa
     3181 cttttgccat tctcaccgga ttcagtcgtc actcatggtg atttctcact tgataacctt
     3241 atttttgacg aggggaaatt aataggttgt attgatgttg gacgagtcgg aatcgcagac
     3301 cgataccagg atcttgccat cctatggaac tgcctcggtg agttttctcc ttcattacag
     3361 aaacggcttt ttcaaaaata tggtattgat aatcctgata tgaataaatt gcagtttcat
     3421 ttgatgctcg atgagttttt ctaagaatta attcatgagc ggatacatat ttgaatgtat
     3481 ttagaaaaat aaacaaatag gggttccgcg cacatttccc cgaaaagtgc cacctaaatt
     3541 gtaagcgtta atattttgtt aaaattcgcg ttaaattttt gttaaatcag ctcatttttt
     3601 aaccaatagg ccgaaatcgg caaaatccct tataaatcaa aagaatagac cgagataggg
     3661 ttgagtgttg ttccagtttg gaacaagagt ccactattaa agaacgtgga ctccaacgtc
     3721 aaagggcgaa aaaccgtcta tcagggcgat ggcccactac gtgaaccatc accctaatca
     3781 agttttttgg ggtcgaggtg ccgtaaagca ctaaatcgga accctaaagg gagcccccga
     3841 tttagagctt gacggggaaa gccggcgaac gtggcgagaa aggaagggaa gaaagcgaaa
     3901 ggagcgggcg ctagggcgct ggcaagtgta gcggtcacgc tgcgcgtaac caccacaccc
     3961 gccgcgctta atgcgccgct acagggcgcg tcccattcgc caatccggat atagttcctc
     4021 ctttcagcaa aaaacccctc aagacccgtt tagaggcccc aaggggttat gctagttatt
     4081 gctcagcggt ggcagcagcc aactcagctt cctttcgggc tttgttagca gccggatctc
     4141 agtggtggtg gtggtggtgc tcgagtgcgg ccgcaacaga tggctggcaa ctagaaggca
     4201 cagtcgaggc tgatcagcga gctctaggaa ttcttacttt ttcttttttg cctggccggc
     4261 ctttttcgtg gccgccggcc ttttgtcgcc tcccagctga gacaggtcga tccgtgtctc
     4321 gtacaggccg gtgatgctct ggtggatcag ggtggcgtcc agcacctctt tggtgctggt
     4381 gtacctcttc cggtcgatgg tggtgtcaaa gtacttgaag gcggcagggg ctcccagatt
     4441 ggtcagggta aacaggtgga tgatattctc ggcctgctct ctgatgggct tatcccggtg
     4501 cttgttgtag gcggacagca ctttgtccag attagcgtcg gccaggatca ctctcttgga
     4561 gaactcgctg atctgctcga tgatctcgtc caggtagtgc ttgtgctgtt ccacaaacag
     4621 ctgtttctgc tcattatcct cgggggagcc cttcagcttc tcatagtggc tggccaggta
     4681 caggaagttc acatatttgg agggcagggc cagttcgttt cccttctgca gttcgccggc
     4741 agaggccagc attctcttcc ggccgttttc cagctcgaac agggagtact taggcagctt
     4801 gatgatcagg tcctttttca cttctttgta gcccttggct tccagaaagt cgatgggatt
     4861 cttctcgaag ctgcttcttt ccatgatggt gatccccagc agctctttca cactcttcag
     4921 tttcttggac ttgccctttt ccactttggc caccaccagc acagaatagg ccacggtggg
     4981 gctgtcgaag ccgccgtact tcttagggtc ccagtccttc tttctggcga tcagcttatc
     5041 gctgttcctc ttgggcagga tagactcttt gctgaagccg cctgtctgca cctcggtctt
     5101 tttcacgata ttcacttggg gcatgctcag cactttccgc acggtggcaa aatcccggcc
     5161 cttatcccac acgatctccc cggtttcgcc gtttgtctcg atcagaggcg ccttccggat
     5221 ctcgccgttg gccagggtaa tctcggtctt gaaaaagttc atgatgttgc tgtagaagaa
     5281 gtacttggcg gtagccttgc cgatttcctg ctcgctcttg gcgatcatct tccgtacatc
     5341 gtacaccttg ccacccagcg cagggtactt tttgatcagg gcggttccca cgacggcgtt
     5401 caggtacgcg tcgtgggcgt ggtggtagtt gttgatctcg cgcactttgt aaaactggaa
     5461 atccttccgg aaatcggaca ccagcttgga cttcagggtg atcactttca cttcccggat
     5521 cagcttgtca ttctcgtcgt acttagtgtt catccgggag tccaggatct gtgccacgtg
     5581 ctttgtgatc tgccgggttt ccaccagctg tctcttgatg aagccggcct tatccagttc
     5641 gctcaggccg cctctctcgg ccttggtcag attgtcgaac tttctctggg taatcagctt
     5701 ggcgttcagc agctgccgcc agtagttctt catcttcttc acgacctctt cggagggcac
     5761 gttgtcgctc ttgccccggt tcttgtcgct tctggtcagc accttgttgt cgatggagtc
     5821 gtcggccaga aagctctgag gcacgatatg gtccacatcg tagtcggaca gccggttgat
     5881 gtccagttcc tggtccacgt acatatcccg cccattctgc aggtagtaca ggtacagctt
     5941 ctcgttctgc agctgggtgt tttccacggg gtgttctttc aggatctggc tgcccagctc
     6001 tttgatgccc tcttcgatcc gcttcattct ctcgcggctg ttcttctgtc ccttctgggt
     6061 ggtctggttc tctctggcca tttcgatcac gatgttctcg ggcttgtgcc ggcccatcac
     6121 tttcacgagc tcgtccacca ccttcactgt ctgcaggatg cccttcttaa tggcggggct
     6181 gccggccaga ttggcaatgt gctcgtgcag gctatcgccc tggccggaca cctgggcttt
     6241 ctggatgtcc tctttaaagg tcaggctgtc gtcgtggatc agctgcatga agtttctgtt
     6301 ggcgaagccg tcggacttca ggaaatccag gattgtcttg ccggactgct tgtcccggat
     6361 gccgttgatc agcttccggc tcagcctgcc ccagccggtg tatctccgcc gcttcagctg
     6421 cttcatcact ttgtcgtcga acaggtgggc ataggttttc agccgttcct cgatcatctc
     6481 tctgtcctca aacagtgtca gggtcagcac gatatcttcc agaatgtcct cgttttcctc
     6541 attgtccagg aagtccttgt ccttgataat tttcagcaga tcgtggtatg tgcccaggga
     6601 ggcgttgaac cgatcttcca cgccggagat ttccacggag tcgaagcact cgattttctt
     6661 gaagtagtcc tctttcagct gcttcacggt cactttccgg ttggtcttga acagcaggtc
     6721 cacgatggcc tttttctgct cgccgctcag gaaggcgggc tttctcattc cctcggtcac
     6781 gtatttcact ttggtcagct cgttatacac ggtgaagtac tcgtacagca ggctgtgctt
     6841 gggcagcacc ttctcgttgg gcaggttctt atcgaagttg gtcatccgct cgatgaagct
     6901 ctgggcggaa gcgcccttgt ccaccacttc ctcgaagttc cagggggtga tggtttcctc
     6961 gctctttctg gtcatccagg cgaatctgct gtttcccctg gccagagggc ccacgtagta
     7021 ggggatgcgg aaggtcagga tcttctcgat cttttcccgg ttgtccttca ggaatgggta
     7081 aaaatcttcc tgccgccgca gaatggcgtg cagctctccc aggtggatct ggtgggggat
     7141 gctgccgttg tcgaaggtcc gctgcttccg cagcaggtcc tctctgttca gcttcacgag
     7201 cagttcctcg gtgccgtcca tcttttccag gatgggcttg atgaacttgt agaactcttc
     7261 ctggctggct ccgccgtcaa tgtagccggc gtagccgttc ttgctctggt cgaagaaaat
     7321 ctctttgtac ttctcaggca gctgctgccg cacgagagct ttcagcaggg tcaggtcctg
     7381 gtggtgctcg tcgtatctct tgatcataga ggcgctcagg ggggccttgg tgatctcggt
     7441 gttcactctc aggatgtcgc tcagcaggat ggcgtcggac aggttcttgg cggccagaaa
     7501 caggtcggcg tactggtcgc cgatctgggc cagcaggttg tccaggtcgt cgtcgtaggt
     7561 gtccttgctc agctgcagtt tggcatcctc ggccaggtcg aagttgctct tgaagttggg
     7621 ggtcaggccc aggctcaggg caatcaggtt tccgaacagg ccattcttct tctcgccggg
     7681 cagctgggcg atcagatttt ccagccgtct gctcttgctc agtctggcag acaggatggc
     7741 cttggcgtcc acgccgctgg cgttgatggg gttttcctcg aacagctggt tgtaggtctg
     7801 caccagctgg atgaacagct tgtccacgtc gctgttgtcg gggttcaggt cgccctcgat
     7861 caggaagtgg ccccggaact tgatcatgtg ggccagggcc agatagatca gccgcaggtc
     7921 ggccttgtcg gtgctgtcca ccagtttctt tctcaggtgg tagatggtgg ggtacttctc
     7981 gtggtaggcc acctcgtcca cgatgttgcc gaagatgggg tgccgctcgt gcttcttatc
     8041 ctcttccacc aggaaggact cttccagtct gtggaagaag ctgtcgtcca ccttggccat
     8101 ctcgttgctg aagatctctt gcagatagca gatccggttc ttccgtctgg tgtatcttct
     8161 tctggcggtt ctcttcagcc gggtggcctc ggctgtttcg ccgctgtcga acagcagggc
     8221 tccgatcagg ttcttcttga tgctgtgccg gtcggtgttg cccagcacct tgaatttctt
     8281 gctgggcacc ttgtactcgt cggtgatcac ggcccagccc acagagttgg tgccgatgtc
     8341 caggccgatg ctgtacttct tgtcggctgc tgggactccg tggataccga ccttccgctt
     8401 cttctttggg gccatcttat cgtcatcgtc tttgtaatca atatcatgat ccttgtagtc
     8461 tccgtcgtgg tccttatagt ccatcgaggc tgctccctgg aaatacaggt tttcactagt
     8521 gttgttgttg ttcgagctcg aattagtctg cgcgtctttc agggcttcat cgacagtctg
     8581 acgaccgctg gcggcgttga tcaccgcagt acgcacggca taccagaaag cggacatctg
     8641 cgggatgttc ggcatgattt cacctttctg ggcgttttcc atagtggcgg caatacgtgg
     8701 atctttcgcc aactcttcct cgtaagactt cagcgctacg gcacccagcg gtttgtcttt
     8761 attaaccgct tccagacctt catcagtcag cagatagttt tcgaggaact cttttgccag
     8821 ctctttgttc ggactggcgg cgttaatacc tgcgctcagc acgccaacga acggtttgga
     8881 tggttgaccc ttgaaggtcg gcagtaccgt tacaccataa ttcactttgc tggtgtcgat
     8941 gttggaccat gcccacgggc cgttgatggt catcgctgtt tcgcctttat taaaggcagc
     9001 ttctgcgatg gagtaatcgg tgtctgcatt catgtgtttg tttttaatca ggtcaaccag
     9061 gaaggtcaga cccgctttcg cgccagcgtt atccacgccc acgtctttaa tgtcgtactt
     9121 gccgttttca tacttgaacg cataaccccc gtcagcagca atcagcggcc aggtgaagta
     9181 cggttcttgc aggttgaaca tcagcgcgct cttacctttc gctttcagtt ctttatccag
     9241 cgccgggatc tcttcccagg tttttggcgg gttcggcagc agatctttgt tataaatcag
     9301 cgataacgct tcaacagcga tcgggtaagc aatcagcttg ccgttgtaac gtacggcatc
     9361 ccaggtaaac ggatacagct tgtcctggaa cgctttgtcc ggggtgattt cagccaacag
     9421 gccagattga gcgtagccac caaagcggtc gtgtgcccag aagataatgt cagggccatc
     9481 gccagttgcc gcaacctgtg ggaatttctc ttccagttta tccggatgct caacggtgac
     9541 tttaattccg gtatctttct cgaatttctt accgacttca gcgagaccgt tatagccttt
     9601 atcgccgtta atccagatta ccagtttacc ttcttcgatt tttccatggt gatggtgatg
     9661 gtgcatatgt atatctcctt cttaaagtta aacaaaatta tttctagagg ggaattgtta
     9721 tccgctcaca attcccctat agtgagtcgt attaatttcg cgggatcgag atctcgatcc
     9781 tctacgccgg acgcatcgtg gccggcatca ccggcgccac aggtgcggtt gctggcgcct
     9841 atatcgccga catcaccgat ggggaagatc gggctcgcca cttcgggctc atgagcgctt
     9901 gtttcggcgt gggtatggtg gcaggccccg tggccggggg actgttgggc gccatctcct
     9961 tgcatgcacc attccttgcg gcggcggtgc tcaacggcct caacctacta ctgggctgct
    10021 tcctaatgca ggagtcgcat aagggagagc gtcgagatcc cggacaccat cgaatggcgc
    10081 aaaacctttc gcggtatggc atgatagcgc ccggaagaga gtcaattcag ggtggtgaat
    10141 gtgaaaccag taacgttata cgatgtcgca gagtatgccg gtgtctctta tcagaccgtt
    10201 tcccgcgtgg tgaaccaggc cagccacgtt tctgcgaaaa cgcgggaaaa agtggaagcg
    10261 gcgatggcgg agctgaatta cattcccaac cgcgtggcac aacaactggc gggcaaacag
    10321 tcgttgctga ttggcgttgc cacctccagt ctggccctgc acgcgccgtc gcaaattgtc
    10381 gcggcgatta aatctcgcgc cgatcaactg ggtgccagcg tggtggtgtc gatggtagaa
    10441 cgaagcggcg tcgaagcctg taaagcggcg gtgcacaatc ttctcgcgca acgcgtcagt
    10501 gggctgatca ttaactatcc gctggatgac caggatgcca ttgctgtgga agctgcctgc
    10561 actaatgttc cggcgttatt tcttgatgtc tctgaccaga cacccatcaa cagtattatt
    10621 ttctcccatg aagacggtac gcgactgggc gtggagcatc tggtcgcatt gggtcaccag
    10681 caaatcgcgc tgttagcggg cccattaagt tctgtctcgg cgcgtctgcg t
//
