LOCUS       sequence-270836-       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"
     primer_bind     118..135
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    150..289
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(215..234)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     375..397
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(391..582)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(1391..2473)
                     /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     2435..2454
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(2474..2551)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     2757..2776
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     2860..2879
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        2860..2878
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    2879..2903
                     /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             2918..2940
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             2951..2968
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             2972..4069
                     /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     4043..4066
                     /label=MBP-F
                     /note="Maltose binding protein, forward primer"
     CDS             4100..4120
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             4133..4198
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             4205..4225
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             join(4250..6787,6788..6790,6791..7252,7253..7255,7256..7423,
                     7424..7426,7427..8350)
                     /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             8351..8398
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     primer_bind     complement(8426..8443)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     CDS             8476..8493
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(8546..8564)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      8560..8607
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      8644..9099
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(8731..8750)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     8941..8962
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(9191..10006)
                     /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             complement(9191..10006)
                     /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     9914..9933
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     9998..10017
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      10128..10716
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     10617..10636
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 ttttgctggc cttttgctca catgttcttt cctgcgttat cccctgattc tgtggataac
       61 cgtattaccg cctttgagtg agctgatacc gctcgccgca gccgaacgac cgagcgcagc
      121 gagtcagtga gcgaggaagc ggaagagcgc ctgatgcggt attttctcct tacgcatctg
      181 tgcggtattt cacaccgcaa tggtgcactc tcagtacaat ctgctctgat gccgcatagt
      241 taagccagta tacactccgc tatcgctacg tgactgggtc atggctgcgc cccgacaccc
      301 gccaacaccc gctgacgcgc cctgacgggc ttgtctgctc ccggcatccg cttacagaca
      361 agctgtgacc gtctccggga gctgcatgtg tcagaggttt tcaccgtcat caccgaaacg
      421 cgcgaggcag ctgcggtaaa gctcatcagc gtggtcgtga agcgattcac agatgtctgc
      481 ctgttcatcc gcgtccagct cgttgagttt ctccagaagc gttaatgtct ggcttctgat
      541 aaagcgggcc atgttaaggg cggttttttc ctgtttggtc actgatgcct ccgtgtaagg
      601 gggatttctg ttcatggggg taatgatacc gatgaaacga gagaggatgc tcacgatacg
      661 ggttactgat gatgaacatg cccggttact ggaacgttgt gagggtaaac aactggcggt
      721 atggatgcgg cgggaccaga gaaaaatcac tcagggtcaa tgccagcgct tcgttaatac
      781 agatgtaggt gttccacagg gtagccagca gcatcctgcg atgcagatcc ggaacataat
      841 ggtgcagggc gctgacttcc gcgtttccag actttacgaa acacggaaac cgaagaccat
      901 tcatgttgtt gctcaggtcg cagacgtttt gcagcagcag tcgcttcacg ttcgctcgcg
      961 tatcggtgat tcattctgct aaccagtaag gcaaccccgc cagcctagcc gggtcctcaa
     1021 cgacaggagc acgatcatgc gcacccgtgg ggccgccatg ccggcgataa tggcctgctt
     1081 ctcgccgaaa cgtttggtgg cgggaccagt gacgaaggct tgagcgaggg cgtgcaagat
     1141 tccgaatacc gcaagcgaca ggccgatcat cgtcgcgctc cagcgaaagc ggtcctcgcc
     1201 gaaaatgacc cagagcgctg ccggcacctg tcctacgagt tgcatgataa agaagacagt
     1261 cataagtgcg gcgacgatag tcatgccccg cgcccaccgg aaggagctga ctgggttgaa
     1321 ggctctcaag ggcatcggtc gagatcccgg tgcctaatga gtgagctaac ttacattaat
     1381 tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg tcgtgccagc tgcattaatg
     1441 aatcggccaa cgcgcgggga gaggcggttt gcgtattggg cgccagggtg gtttttcttt
     1501 tcaccagtga gacgggcaac agctgattgc ccttcaccgc ctggccctga gagagttgca
     1561 gcaagcggtc cacgctggtt tgccccagca ggcgaaaatc ctgtttgatg gtggttaacg
     1621 gcgggatata acatgagctg tcttcggtat cgtcgtatcc cactaccgag atatccgcac
     1681 caacgcgcag cccggactcg gtaatggcgc gcattgcgcc cagcgccatc tgatcgttgg
     1741 caaccagcat cgcagtggga acgatgccct cattcagcat ttgcatggtt tgttgaaaac
     1801 cggacatggc actccagtcg ccttcccgtt ccgctatcgg ctgaatttga ttgcgagtga
     1861 gatatttatg ccagccagcc agacgcagac gcgccgagac agaacttaat gggcccgcta
     1921 acagcgcgat ttgctggtga cccaatgcga ccagatgctc cacgcccagt cgcgtaccgt
     1981 cttcatggga gaaaataata ctgttgatgg gtgtctggtc agagacatca agaaataacg
     2041 ccggaacatt agtgcaggca gcttccacag caatggcatc ctggtcatcc agcggatagt
     2101 taatgatcag cccactgacg cgttgcgcga gaagattgtg caccgccgct ttacaggctt
     2161 cgacgccgct tcgttctacc atcgacacca ccacgctggc acccagttga tcggcgcgag
     2221 atttaatcgc cgcgacaatt tgcgacggcg cgtgcagggc cagactggag gtggcaacgc
     2281 caatcagcaa cgactgtttg cccgccagtt gttgtgccac gcggttggga atgtaattca
     2341 gctccgccat cgccgcttcc actttttccc gcgttttcgc agaaacgtgg ctggcctggt
     2401 tcaccacgcg ggaaacggtc tgataagaga caccggcata ctctgcgaca tcgtataacg
     2461 ttactggttt cacattcacc accctgaatt gactctcttc cgggcgctat catgccatac
     2521 cgcgaaaggt tttgcgccat tcgatggtgt ccgggatctc gacgctctcc cttatgcgac
     2581 tcctgcatta ggaagcagcc cagtagtagg ttgaggccgt tgagcaccgc cgccgcaagg
     2641 aatggtgcat gcaaggagat ggcgcccaac agtcccccgg ccacggggcc tgccaccata
     2701 cccacgccga aacaagcgct catgagcccg aagtggcgag cccgatcttc cccatcggtg
     2761 atgtcggcga tataggcgcc agcaaccgca cctgtggcgc cggtgatgcc ggccacgatg
     2821 cgtccggcgt agaggatcga gatctcgatc ccgcgaaatt aatacgactc actatagggg
     2881 aattgtgagc ggataacaat tcccctctag aaataatttt gtttaacttt aagaaggaga
     2941 tatacatatg caccatcacc atcaccatgg aaaaatcgaa gaaggtaaac tggtaatctg
     3001 gattaacggc gataaaggct ataacggtct cgctgaagtc ggtaagaaat tcgagaaaga
     3061 taccggaatt aaagtcaccg ttgagcatcc ggataaactg gaagagaaat tcccacaggt
     3121 tgcggcaact ggcgatggcc ctgacattat cttctgggca cacgaccgct ttggtggcta
     3181 cgctcaatct ggcctgttgg ctgaaatcac cccggacaaa gcgttccagg acaagctgta
     3241 tccgtttacc tgggatgccg tacgttacaa cggcaagctg attgcttacc cgatcgctgt
     3301 tgaagcgtta tcgctgattt ataacaaaga tctgctgccg aacccgccaa aaacctggga
     3361 agagatcccg gcgctggata aagaactgaa agcgaaaggt aagagcgcgc tgatgttcaa
     3421 cctgcaagaa ccgtacttca cctggccgct gattgctgct gacgggggtt atgcgttcaa
     3481 gtatgaaaac ggcaagtacg acattaaaga cgtgggcgtg gataacgctg gcgcgaaagc
     3541 gggtctgacc ttcctggttg acctgattaa aaacaaacac atgaatgcag acaccgatta
     3601 ctccatcgca gaagctgcct ttaataaagg cgaaacagcg atgaccatca acggcccgtg
     3661 ggcatggtcc aacatcgaca ccagcaaagt gaattatggt gtaacggtac tgccgacctt
     3721 caagggtcaa ccatccaaac cgttcgttgg cgtgctgagc gcaggtatta acgccgccag
     3781 tccgaacaaa gagctggcaa aagagttcct cgaaaactat ctgctgactg atgaaggtct
     3841 ggaagcggtt aataaagaca aaccgctggg tgccgtagcg ctgaagtctt acgaggaaga
     3901 gttggcgaaa gatccacgta ttgccgccac tatggaaaac gcccagaaag gtgaaatcat
     3961 gccgaacatc ccgcagatgt ccgctttctg gtatgccgtg cgtactgcgg tgatcaacgc
     4021 cgccagcggt cgtcagactg tcgatgaagc cctgaaagac gcgcagacta attcgagctc
     4081 gaacaacaac aacactagtg aaaacctgta tttccaggga gcagcctcga tggactataa
     4141 ggaccacgac ggagactaca aggatcatga tattgattac aaagacgatg acgataagat
     4201 ggccccaaag aagaagcgga aggtcggtat ccacggagtc ccagcagccg acaagaagta
     4261 cagcatcggc ctggacatcg gcaccaactc tgtgggctgg gccgtgatca ccgacgagta
     4321 caaggtgccc agcaagaaat tcaaggtgct gggcaacacc gaccggcaca gcatcaagaa
     4381 gaacctgatc ggagccctgc tgttcgacag cggcgaaaca gccgaggcca cccggctgaa
     4441 gagaaccgcc agaagaagat acaccagacg gaagaaccgg atctgctatc tgcaagagat
     4501 cttcagcaac gagatggcca aggtggacga cagcttcttc cacagactgg aagagtcctt
     4561 cctggtggaa gaggataaga agcacgagcg gcaccccatc ttcggcaaca tcgtggacga
     4621 ggtggcctac cacgagaagt accccaccat ctaccacctg agaaagaaac tggtggacag
     4681 caccgacaag gccgacctgc ggctgatcta tctggccctg gcccacatga tcaagttccg
     4741 gggccacttc ctgatcgagg gcgacctgaa ccccgacaac agcgacgtgg acaagctgtt
     4801 catccagctg gtgcagacct acaaccagct gttcgaggaa aaccccatca acgccagcgg
     4861 cgtggacgcc aaggccatcc tgtctgccag actgagcaag agcagacggc tggaaaatct
     4921 gatcgcccag ctgcccggcg agaagaagaa tggcctgttc ggaaacctga ttgccctgag
     4981 cctgggcctg acccccaact tcaagagcaa cttcgacctg gccgaggatg ccaaactgca
     5041 gctgagcaag gacacctacg acgacgacct ggacaacctg ctggcccaga tcggcgacca
     5101 gtacgccgac ctgtttctgg ccgccaagaa cctgtccgac gccatcctgc tgagcgacat
     5161 cctgagagtg aacaccgaga tcaccaaggc ccccctgagc gcctctatga tcaagagata
     5221 cgacgagcac caccaggacc tgaccctgct gaaagctctc gtgcggcagc agctgcctga
     5281 gaagtacaaa gagattttct tcgaccagag caagaacggc tacgccggct acattgacgg
     5341 cggagccagc caggaagagt tctacaagtt catcaagccc atcctggaaa agatggacgg
     5401 caccgaggaa ctgctcgtga agctgaacag agaggacctg ctgcggaagc agcggacctt
     5461 cgacaacggc agcatccccc accagatcca cctgggagag ctgcacgcca ttctgcggcg
     5521 gcaggaagat ttttacccat tcctgaagga caaccgggaa aagatcgaga agatcctgac
     5581 cttccgcatc ccctactacg tgggccctct ggccagggga aacagcagat tcgcctggat
     5641 gaccagaaag agcgaggaaa ccatcacccc ctggaacttc gaggaagtgg tggacaaggg
     5701 cgcttccgcc cagagcttca tcgagcggat gaccaacttc gataagaacc tgcccaacga
     5761 gaaggtgctg cccaagcaca gcctgctgta cgagtacttc accgtgtata acgagctgac
     5821 caaagtgaaa tacgtgaccg agggaatgag aaagcccgcc ttcctgagcg gcgagcagaa
     5881 aaaggccatc gtggacctgc tgttcaagac caaccggaaa gtgaccgtga agcagctgaa
     5941 agaggactac ttcaagaaaa tcgagtgctt cgactccgtg gaaatctccg gcgtggaaga
     6001 tcggttcaac gcctccctgg gcacatacca cgatctgctg aaaattatca aggacaagga
     6061 cttcctggac aatgaggaaa acgaggacat tctggaagat atcgtgctga ccctgacact
     6121 gtttgaggac agagagatga tcgaggaacg gctgaaaacc tatgcccacc tgttcgacga
     6181 caaagtgatg aagcagctga agcggcggag atacaccggc tggggcaggc tgagccggaa
     6241 gctgatcaac ggcatccggg acaagcagtc cggcaagaca atcctggatt tcctgaagtc
     6301 cgacggcttc gccaacagaa acttcatgca gctgatccac gacgacagcc tgacctttaa
     6361 agaggacatc cagaaagccc aggtgtccgg ccagggcgat agcctgcacg agcacattgc
     6421 caatctggcc ggcagccccg ccattaagaa gggcatcctg cagacagtga aggtggtgga
     6481 cgagctcgtg aaagtgatgg gccggcacaa gcccgagaac atcgtgatcg aaatggccag
     6541 agagaaccag accacccaga agggacagaa gaacagccgc gagagaatga agcggatcga
     6601 agagggcatc aaagagctgg gcagccagat cctgaaagaa caccccgtgg aaaacaccca
     6661 gctgcagaac gagaagctgt acctgtacta cctgcagaat gggcgggata tgtacgtgga
     6721 ccaggaactg gacatcaacc ggctgtccga ctacgatgtg gaccatatcg tgcctcagag
     6781 ctttctggcc gacgactcca tcgacaacaa ggtgctgacc agaagcgaca agaaccgggg
     6841 caagagcgac aacgtgccct ccgaagaggt cgtgaagaag atgaagaact actggcggca
     6901 gctgctgaac gccaagctga ttacccagag aaagttcgac aatctgacca aggccgagag
     6961 aggcggcctg agcgaactgg ataaggccgg cttcatcaag agacagctgg tggaaacccg
     7021 gcagatcaca aagcacgtgg cacagatcct ggactcccgg atgaacacta agtacgacga
     7081 gaatgacaag ctgatccggg aagtgaaagt gatcaccctg aagtccaagc tggtgtccga
     7141 tttccggaag gatttccagt tttacaaagt gcgcgagatc aacaactacc accacgccca
     7201 cgacgcctac ctgaacgccg tcgtgggaac cgccctgatc aaaaagtacc ctgcgctgga
     7261 aagcgagttc gtgtacggcg actacaaggt gtacgacgtg cggaagatga tcgccaagag
     7321 cgagcaggaa atcggcaagg ctaccgccaa gtacttcttc tacagcaaca tcatgaactt
     7381 tttcaagacc gagattaccc tggccaacgg cgagatccgg aaggcgcctc tgatcgagac
     7441 aaacggcgaa accggggaga tcgtgtggga taagggccgg gattttgcca ccgtgcggaa
     7501 agtgctgagc atgccccaag tgaatatcgt gaaaaagacc gaggtgcaga caggcggctt
     7561 cagcaaagag tctatcctgc ccaagaggaa cagcgataag ctgatcgcca gaaagaagga
     7621 ctgggaccct aagaagtacg gcggcttcga cagccccacc gtggcctatt ctgtgctggt
     7681 ggtggccaaa gtggaaaagg gcaagtccaa gaaactgaag agtgtgaaag agctgctggg
     7741 gatcaccatc atggaaagaa gcagcttcga gaagaatccc atcgactttc tggaagccaa
     7801 gggctacaaa gaagtgaaaa aggacctgat catcaagctg cctaagtact ccctgttcga
     7861 gctggaaaac ggccggaaga gaatgctggc ctctgccggc gaactgcaga agggaaacga
     7921 actggccctg ccctccaaat atgtgaactt cctgtacctg gccagccact atgagaagct
     7981 gaagggctcc cccgaggata atgagcagaa acagctgttt gtggaacagc acaagcacta
     8041 cctggacgag atcatcgagc agatcagcga gttctccaag agagtgatcc tggccgacgc
     8101 taatctggac aaagtgctgt ccgcctacaa caagcaccgg gataagccca tcagagagca
     8161 ggccgagaat atcatccacc tgtttaccct gaccaatctg ggagcccctg ccgccttcaa
     8221 gtactttgac accaccatcg accggaagag gtacaccagc accaaagagg tgctggacgc
     8281 caccctgatc caccagagca tcaccggcct gtacgagaca cggatcgacc tgtctcagct
     8341 gggaggcgac aaaaggccgg cggccacgaa aaaggccggc caggcaaaaa agaaaaagta
     8401 agaattccta gagctcgctg atcagcctcg actgtgcctt ctagttgcca gccatctgtt
     8461 gcggccgcac tcgagcacca ccaccaccac cactgagatc cggctgctaa caaagcccga
     8521 aaggaagctg agttggctgc tgccaccgct gagcaataac tagcataacc ccttggggcc
     8581 tctaaacggg tcttgagggg ttttttgctg aaaggaggaa ctatatccgg attggcgaat
     8641 gggacgcgcc ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga
     8701 ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct tcctttctcg
     8761 ccacgttcgc cggctttccc cgtcaagctc taaatcgggg gctcccttta gggttccgat
     8821 ttagtgcttt acggcacctc gaccccaaaa aacttgatta gggtgatggt tcacgtagtg
     8881 ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg ttctttaata
     8941 gtggactctt gttccaaact ggaacaacac tcaaccctat ctcggtctat tcttttgatt
     9001 tataagggat tttgccgatt tcggcctatt ggttaaaaaa tgagctgatt taacaaaaat
     9061 ttaacgcgaa ttttaacaaa atattaacgc ttacaattta ggtggcactt ttcggggaaa
     9121 tgtgcgcgga acccctattt gtttattttt ctaaatacat tcaaatatgt atccgctcat
     9181 gaattaattc ttagaaaaac tcatcgagca tcaaatgaaa ctgcaattta ttcatatcag
     9241 gattatcaat accatatttt tgaaaaagcc gtttctgtaa tgaaggagaa aactcaccga
     9301 ggcagttcca taggatggca agatcctggt atcggtctgc gattccgact cgtccaacat
     9361 caatacaacc tattaatttc ccctcgtcaa aaataaggtt atcaagtgag aaatcaccat
     9421 gagtgacgac tgaatccggt gagaatggca aaagtttatg catttctttc cagacttgtt
     9481 caacaggcca gccattacgc tcgtcatcaa aatcactcgc atcaaccaaa ccgttattca
     9541 ttcgtgattg cgcctgagcg agacgaaata cgcgatcgct gttaaaagga caattacaaa
     9601 caggaatcga atgcaaccgg cgcaggaaca ctgccagcgc atcaacaata ttttcacctg
     9661 aatcaggata ttcttctaat acctggaatg ctgttttccc ggggatcgca gtggtgagta
     9721 accatgcatc atcaggagta cggataaaat gcttgatggt cggaagaggc ataaattccg
     9781 tcagccagtt tagtctgacc atctcatctg taacatcatt ggcaacgcta cctttgccat
     9841 gtttcagaaa caactctggc gcatcgggct tcccatacaa tcgatagatt gtcgcacctg
     9901 attgcccgac attatcgcga gcccatttat acccatataa atcagcatcc atgttggaat
     9961 ttaatcgcgg cctagagcaa gacgtttccc gttgaatatg gctcataaca ccccttgtat
    10021 tactgtttat gtaagcagac agttttattg ttcatgacca aaatccctta acgtgagttt
    10081 tcgttccact gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg agatcctttt
    10141 tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt
    10201 ttgccggatc aagagctacc aactcttttt ccgaaggtaa ctggcttcag cagagcgcag
    10261 ataccaaata ctgtccttct agtgtagccg tagttaggcc accacttcaa gaactctgta
    10321 gcaccgccta catacctcgc tctgctaatc ctgttaccag tggctgctgc cagtggcgat
    10381 aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac cggataaggc gcagcggtcg
    10441 ggctgaacgg ggggttcgtg cacacagccc agcttggagc gaacgaccta caccgaactg
    10501 agatacctac agcgtgagct atgagaaagc gccacgcttc ccgaagggag aaaggcggac
    10561 aggtatccgg taagcggcag ggtcggaaca ggagagcgca cgagggagct tccaggggga
    10621 aacgcctggt atctttatag tcctgtcggg tttcgccacc tctgacttga gcgtcgattt
    10681 ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc ggccttttta
    10741 cggttcctgg cc
//
