LOCUS       sequence-264542-       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"
     primer_bind     538..557
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(577..654)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     860..879
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     963..982
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        963..981
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    982..1006
                     /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             1021..1043
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             1054..1071
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             1075..2172
                     /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     2146..2169
                     /label=MBP-F
                     /note="Maltose binding protein, forward primer"
     CDS             2203..2223
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             2236..2301
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             2308..2328
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             join(2353..4890,4891..4893,4894..5355,5356..5358,5359..5505,
                     5506..5508,5509..6432)
                     /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             6433..6480
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     primer_bind     complement(6508..6525)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     CDS             6558..6575
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(6628..6646)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      6642..6689
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      6726..7181
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6813..6832)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     7023..7044
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(7273..8088)
                     /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(7273..8088)
                     /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     7996..8015
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     8080..8099
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      8210..8798
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     8699..8718
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     8952..8969
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    8984..9123
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(9049..9068)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     9209..9231
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(9225..9416)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(join(10225..10731,1..576))
                     /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"
ORIGIN
        1 gacagaactt aatgggcccg ctaacagcgc gatttgctgg tgacccaatg cgaccagatg
       61 ctccacgccc agtcgcgtac cgtcttcatg ggagaaaata atactgttga tgggtgtctg
      121 gtcagagaca tcaagaaata acgccggaac attagtgcag gcagcttcca cagcaatggc
      181 atcctggtca tccagcggat agttaatgat cagcccactg acgcgttgcg cgagaagatt
      241 gtgcaccgcc gctttacagg cttcgacgcc gcttcgttct accatcgaca ccaccacgct
      301 ggcacccagt tgatcggcgc gagatttaat cgccgcgaca atttgcgacg gcgcgtgcag
      361 ggccagactg gaggtggcaa cgccaatcag caacgactgt ttgcccgcca gttgttgtgc
      421 cacgcggttg ggaatgtaat tcagctccgc catcgccgct tccacttttt cccgcgtttt
      481 cgcagaaacg tggctggcct ggttcaccac gcgggaaacg gtctgataag agacaccggc
      541 atactctgcg acatcgtata acgttactgg tttcacattc accaccctga attgactctc
      601 ttccgggcgc tatcatgcca taccgcgaaa ggttttgcgc cattcgatgg tgtccgggat
      661 ctcgacgctc tcccttatgc gactcctgca ttaggaagca gcccagtagt aggttgaggc
      721 cgttgagcac cgccgccgca aggaatggtg catgcaagga gatggcgccc aacagtcccc
      781 cggccacggg gcctgccacc atacccacgc cgaaacaagc gctcatgagc ccgaagtggc
      841 gagcccgatc ttccccatcg gtgatgtcgg cgatataggc gccagcaacc gcacctgtgg
      901 cgccggtgat gccggccacg atgcgtccgg cgtagaggat cgagatctcg atcccgcgaa
      961 attaatacga ctcactatag gggaattgtg agcggataac aattcccctc tagaaataat
     1021 tttgtttaac tttaagaagg agatatacat atgcaccatc accatcacca tggaaaaatc
     1081 gaagaaggta aactggtaat ctggattaac ggcgataaag gctataacgg tctcgctgaa
     1141 gtcggtaaga aattcgagaa agataccgga attaaagtca ccgttgagca tccggataaa
     1201 ctggaagaga aattcccaca ggttgcggca actggcgatg gccctgacat tatcttctgg
     1261 gcacacgacc gctttggtgg ctacgctcaa tctggcctgt tggctgaaat caccccggac
     1321 aaagcgttcc aggacaagct gtatccgttt acctgggatg ccgtacgtta caacggcaag
     1381 ctgattgctt acccgatcgc tgttgaagcg ttatcgctga tttataacaa agatctgctg
     1441 ccgaacccgc caaaaacctg ggaagagatc ccggcgctgg ataaagaact gaaagcgaaa
     1501 ggtaagagcg cgctgatgtt caacctgcaa gaaccgtact tcacctggcc gctgattgct
     1561 gctgacgggg gttatgcgtt caagtatgaa aacggcaagt acgacattaa agacgtgggc
     1621 gtggataacg ctggcgcgaa agcgggtctg accttcctgg ttgacctgat taaaaacaaa
     1681 cacatgaatg cagacaccga ttactccatc gcagaagctg cctttaataa aggcgaaaca
     1741 gcgatgacca tcaacggccc gtgggcatgg tccaacatcg acaccagcaa agtgaattat
     1801 ggtgtaacgg tactgccgac cttcaagggt caaccatcca aaccgttcgt tggcgtgctg
     1861 agcgcaggta ttaacgccgc cagtccgaac aaagagctgg caaaagagtt cctcgaaaac
     1921 tatctgctga ctgatgaagg tctggaagcg gttaataaag acaaaccgct gggtgccgta
     1981 gcgctgaagt cttacgagga agagttggcg aaagatccac gtattgccgc cactatggaa
     2041 aacgcccaga aaggtgaaat catgccgaac atcccgcaga tgtccgcttt ctggtatgcc
     2101 gtgcgtactg cggtgatcaa cgccgccagc ggtcgtcaga ctgtcgatga agccctgaaa
     2161 gacgcgcaga ctaattcgag ctcgaacaac aacaacacta gtgaaaacct gtatttccag
     2221 ggagcagcct cgatggacta taaggaccac gacggagact acaaggatca tgatattgat
     2281 tacaaagacg atgacgataa gatggcccca aagaagaagc ggaaggtcgg tatccacgga
     2341 gtcccagcag ccgacaagaa gtacagcatc ggcctggaca tcggcaccaa ctctgtgggc
     2401 tgggccgtga tcaccgacga gtacaaggtg cccagcaaga aattcaaggt gctgggcaac
     2461 accgaccggc acagcatcaa gaagaacctg atcggagccc tgctgttcga cagcggcgaa
     2521 acagccgagg ccacccggct gaagagaacc gccagaagaa gatacaccag acggaagaac
     2581 cggatctgct atctgcaaga gatcttcagc aacgagatgg ccaaggtgga cgacagcttc
     2641 ttccacagac tggaagagtc cttcctggtg gaagaggata agaagcacga gcggcacccc
     2701 atcttcggca acatcgtgga cgaggtggcc taccacgaga agtaccccac catctaccac
     2761 ctgagaaaga aactggtgga cagcaccgac aaggccgacc tgcggctgat ctatctggcc
     2821 ctggcccaca tgatcaagtt ccggggccac ttcctgatcg agggcgacct gaaccccgac
     2881 aacagcgacg tggacaagct gttcatccag ctggtgcaga cctacaacca gctgttcgag
     2941 gaaaacccca tcaacgccag cggcgtggac gccaaggcca tcctgtctgc cagactgagc
     3001 aagagcagac ggctggaaaa tctgatcgcc cagctgcccg gcgagaagaa gaatggcctg
     3061 ttcggaaacc tgattgccct gagcctgggc ctgaccccca acttcaagag caacttcgac
     3121 ctggccgagg atgccaaact gcagctgagc aaggacacct acgacgacga cctggacaac
     3181 ctgctggccc agatcggcga ccagtacgcc gacctgtttc tggccgccaa gaacctgtcc
     3241 gacgccatcc tgctgagcga catcctgaga gtgaacaccg agatcaccaa ggcccccctg
     3301 agcgcctcta tgatcaagag atacgacgag caccaccagg acctgaccct gctgaaagct
     3361 ctcgtgcggc agcagctgcc tgagaagtac aaagagattt tcttcgacca gagcaagaac
     3421 ggctacgccg gctacattga cggcggagcc agccaggaag agttctacaa gttcatcaag
     3481 cccatcctgg aaaagatgga cggcaccgag gaactgctcg tgaagctgaa cagagaggac
     3541 ctgctgcgga agcagcggac cttcgacaac ggcagcatcc cccaccagat ccacctggga
     3601 gagctgcacg ccattctgcg gcggcaggaa gatttttacc cattcctgaa ggacaaccgg
     3661 gaaaagatcg agaagatcct gaccttccgc atcccctact acgtgggccc tctggccagg
     3721 ggaaacagca gattcgcctg gatgaccaga aagagcgagg aaaccatcac cccctggaac
     3781 ttcgaggaag tggtggacaa gggcgcttcc gcccagagct tcatcgagcg gatgaccaac
     3841 ttcgataaga acctgcccaa cgagaaggtg ctgcccaagc acagcctgct gtacgagtac
     3901 ttcaccgtgt ataacgagct gaccaaagtg aaatacgtga ccgagggaat gagaaagccc
     3961 gccttcctga gcggcgagca gaaaaaggcc atcgtggacc tgctgttcaa gaccaaccgg
     4021 aaagtgaccg tgaagcagct gaaagaggac tacttcaaga aaatcgagtg cttcgactcc
     4081 gtggaaatct ccggcgtgga agatcggttc aacgcctccc tgggcacata ccacgatctg
     4141 ctgaaaatta tcaaggacaa ggacttcctg gacaatgagg aaaacgagga cattctggaa
     4201 gatatcgtgc tgaccctgac actgtttgag gacagagaga tgatcgagga acggctgaaa
     4261 acctatgccc acctgttcga cgacaaagtg atgaagcagc tgaagcggcg gagatacacc
     4321 ggctggggca ggctgagccg gaagctgatc aacggcatcc gggacaagca gtccggcaag
     4381 acaatcctgg atttcctgaa gtccgacggc ttcgccaaca gaaacttcat gcagctgatc
     4441 cacgacgaca gcctgacctt taaagaggac atccagaaag cccaggtgtc cggccagggc
     4501 gatagcctgc acgagcacat tgccaatctg gccggcagcc ccgccattaa gaagggcatc
     4561 ctgcagacag tgaaggtggt ggacgagctc gtgaaagtga tgggccggca caagcccgag
     4621 aacatcgtga tcgaaatggc cagagagaac cagaccaccc agaagggaca gaagaacagc
     4681 cgcgagagaa tgaagcggat cgaagagggc atcaaagagc tgggcagcca gatcctgaaa
     4741 gaacaccccg tggaaaacac ccagctgcag aacgagaagc tgtacctgta ctacctgcag
     4801 aatgggcggg atatgtacgt ggaccaggaa ctggacatca accggctgtc cgactacgat
     4861 gtggaccata tcgtgcctca gagctttctg gccgacgact ccatcgacaa caaggtgctg
     4921 accagaagcg acaagaaccg gggcaagagc gacaacgtgc cctccgaaga ggtcgtgaag
     4981 aagatgaaga actactggcg gcagctgctg aacgccaagc tgattaccca gagaaagttc
     5041 gacaatctga ccaaggccga gagaggcggc ctgagcgaac tggataaggc cggcttcatc
     5101 aagagacagc tggtggaaac ccggcagatc acaaagcacg tggcacagat cctggactcc
     5161 cggatgaaca ctaagtacga cgagaatgac aagctgatcc gggaagtgaa agtgatcacc
     5221 ctgaagtcca agctggtgtc cgatttccgg aaggatttcc agttttacaa agtgcgcgag
     5281 atcaacaact accaccacgc ccacgacgcg tacctgaacg ccgtcgtggg aaccgccctg
     5341 atcaaaaagt accctgcgct gggtggcaag gtgtacgatg tacggaagat gatcgccaag
     5401 agcgagcagg aaatcggcaa ggctaccgcc aagtacttct tctacagcaa catcatgaac
     5461 tttttcaaga ccgagattac cctggccaac ggcgagatcc ggaaggcgcc tctgatcgag
     5521 acaaacggcg aaaccgggga gatcgtgtgg gataagggcc gggattttgc caccgtgcgg
     5581 aaagtgctga gcatgcccca agtgaatatc gtgaaaaaga ccgaggtgca gacaggcggc
     5641 ttcagcaaag agtctatcct gcccaagagg aacagcgata agctgatcgc cagaaagaag
     5701 gactgggacc ctaagaagta cggcggcttc gacagcccca ccgtggccta ttctgtgctg
     5761 gtggtggcca aagtggaaaa gggcaagtcc aagaaactga agagtgtgaa agagctgctg
     5821 gggatcacca tcatggaaag aagcagcttc gagaagaatc ccatcgactt tctggaagcc
     5881 aagggctaca aagaagtgaa aaaggacctg atcatcaagc tgcctaagta ctccctgttc
     5941 gagctggaaa acggccggaa gagaatgctg gcctctgccg gcgaactgca gaagggaaac
     6001 gaactggccc tgccctccaa atatgtgaac ttcctgtacc tggccagcca ctatgagaag
     6061 ctgaagggct cccccgagga taatgagcag aaacagctgt ttgtggaaca gcacaagcac
     6121 tacctggacg agatcatcga gcagatcagc gagttctcca agagagtgat cctggccgac
     6181 gctaatctgg acaaagtgct gtccgcctac aacaagcacc gggataagcc catcagagag
     6241 caggccgaga atatcatcca cctgtttacc ctgaccaatc tgggagcccc tgccgccttc
     6301 aagtactttg acaccaccat cgaccggaag aggtacacca gcaccaaaga ggtgctggac
     6361 gccaccctga tccaccagag catcaccggc ctgtacgaga cacggatcga cctgtctcag
     6421 ctgggaggcg acaaaaggcc ggcggccacg aaaaaggccg gccaggcaaa aaagaaaaag
     6481 taagaattcc tagagctcgc tgatcagcct cgactgtgcc ttctagttgc cagccatctg
     6541 ttgcggccgc actcgagcac caccaccacc accactgaga tccggctgct aacaaagccc
     6601 gaaaggaagc tgagttggct gctgccaccg ctgagcaata actagcataa ccccttgggg
     6661 cctctaaacg ggtcttgagg ggttttttgc tgaaaggagg aactatatcc ggattggcga
     6721 atgggacgcg ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta cgcgcagcgt
     6781 gaccgctaca cttgccagcg ccctagcgcc cgctcctttc gctttcttcc cttcctttct
     6841 cgccacgttc gccggctttc cccgtcaagc tctaaatcgg gggctccctt tagggttccg
     6901 atttagtgct ttacggcacc tcgaccccaa aaaacttgat tagggtgatg gttcacgtag
     6961 tgggccatcg ccctgataga cggtttttcg ccctttgacg ttggagtcca cgttctttaa
     7021 tagtggactc ttgttccaaa ctggaacaac actcaaccct atctcggtct attcttttga
     7081 tttataaggg attttgccga tttcggccta ttggttaaaa aatgagctga tttaacaaaa
     7141 atttaacgcg aattttaaca aaatattaac gcttacaatt taggtggcac ttttcgggga
     7201 aatgtgcgcg gaacccctat ttgtttattt ttctaaatac attcaaatat gtatccgctc
     7261 atgaattaat tcttagaaaa actcatcgag catcaaatga aactgcaatt tattcatatc
     7321 aggattatca ataccatatt tttgaaaaag ccgtttctgt aatgaaggag aaaactcacc
     7381 gaggcagttc cataggatgg caagatcctg gtatcggtct gcgattccga ctcgtccaac
     7441 atcaatacaa cctattaatt tcccctcgtc aaaaataagg ttatcaagtg agaaatcacc
     7501 atgagtgacg actgaatccg gtgagaatgg caaaagttta tgcatttctt tccagacttg
     7561 ttcaacaggc cagccattac gctcgtcatc aaaatcactc gcatcaacca aaccgttatt
     7621 cattcgtgat tgcgcctgag cgagacgaaa tacgcgatcg ctgttaaaag gacaattaca
     7681 aacaggaatc gaatgcaacc ggcgcaggaa cactgccagc gcatcaacaa tattttcacc
     7741 tgaatcagga tattcttcta atacctggaa tgctgttttc ccggggatcg cagtggtgag
     7801 taaccatgca tcatcaggag tacggataaa atgcttgatg gtcggaagag gcataaattc
     7861 cgtcagccag tttagtctga ccatctcatc tgtaacatca ttggcaacgc tacctttgcc
     7921 atgtttcaga aacaactctg gcgcatcggg cttcccatac aatcgataga ttgtcgcacc
     7981 tgattgcccg acattatcgc gagcccattt atacccatat aaatcagcat ccatgttgga
     8041 atttaatcgc ggcctagagc aagacgtttc ccgttgaata tggctcataa caccccttgt
     8101 attactgttt atgtaagcag acagttttat tgttcatgac caaaatccct taacgtgagt
     8161 tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa aggatcttct tgagatcctt
     8221 tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca gcggtggttt
     8281 gtttgccgga tcaagagcta ccaactcttt ttccgaaggt aactggcttc agcagagcgc
     8341 agataccaaa tactgtcctt ctagtgtagc cgtagttagg ccaccacttc aagaactctg
     8401 tagcaccgcc tacatacctc gctctgctaa tcctgttacc agtggctgct gccagtggcg
     8461 ataagtcgtg tcttaccggg ttggactcaa gacgatagtt accggataag gcgcagcggt
     8521 cgggctgaac ggggggttcg tgcacacagc ccagcttgga gcgaacgacc tacaccgaac
     8581 tgagatacct acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg
     8641 acaggtatcc ggtaagcggc agggtcggaa caggagagcg cacgagggag cttccagggg
     8701 gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat
     8761 ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac gcggcctttt
     8821 tacggttcct ggccttttgc tggccttttg ctcacatgtt ctttcctgcg ttatcccctg
     8881 attctgtgga taaccgtatt accgcctttg agtgagctga taccgctcgc cgcagccgaa
     8941 cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga gcgcctgatg cggtattttc
     9001 tccttacgca tctgtgcggt atttcacacc gcaatggtgc actctcagta caatctgctc
     9061 tgatgccgca tagttaagcc agtatacact ccgctatcgc tacgtgactg ggtcatggct
     9121 gcgccccgac acccgccaac acccgctgac gcgccctgac gggcttgtct gctcccggca
     9181 tccgcttaca gacaagctgt gaccgtctcc gggagctgca tgtgtcagag gttttcaccg
     9241 tcatcaccga aacgcgcgag gcagctgcgg taaagctcat cagcgtggtc gtgaagcgat
     9301 tcacagatgt ctgcctgttc atccgcgtcc agctcgttga gtttctccag aagcgttaat
     9361 gtctggcttc tgataaagcg ggccatgtta agggcggttt tttcctgttt ggtcactgat
     9421 gcctccgtgt aagggggatt tctgttcatg ggggtaatga taccgatgaa acgagagagg
     9481 atgctcacga tacgggttac tgatgatgaa catgcccggt tactggaacg ttgtgagggt
     9541 aaacaactgg cggtatggat gcggcgggac cagagaaaaa tcactcaggg tcaatgccag
     9601 cgcttcgtta atacagatgt aggtgttcca cagggtagcc agcagcatcc tgcgatgcag
     9661 atccggaaca taatggtgca gggcgctgac ttccgcgttt ccagacttta cgaaacacgg
     9721 aaaccgaaga ccattcatgt tgttgctcag gtcgcagacg ttttgcagca gcagtcgctt
     9781 cacgttcgct cgcgtatcgg tgattcattc tgctaaccag taaggcaacc ccgccagcct
     9841 agccgggtcc tcaacgacag gagcacgatc atgcgcaccc gtggggccgc catgccggcg
     9901 ataatggcct gcttctcgcc gaaacgtttg gtggcgggac cagtgacgaa ggcttgagcg
     9961 agggcgtgca agattccgaa taccgcaagc gacaggccga tcatcgtcgc gctccagcga
    10021 aagcggtcct cgccgaaaat gacccagagc gctgccggca cctgtcctac gagttgcatg
    10081 ataaagaaga cagtcataag tgcggcgacg atagtcatgc cccgcgccca ccggaaggag
    10141 ctgactgggt tgaaggctct caagggcatc ggtcgagatc ccggtgccta atgagtgagc
    10201 taacttacat taattgcgtt gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc
    10261 cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat tgggcgccag
    10321 ggtggttttt cttttcacca gtgagacggg caacagctga ttgcccttca ccgcctggcc
    10381 ctgagagagt tgcagcaagc ggtccacgct ggtttgcccc agcaggcgaa aatcctgttt
    10441 gatggtggtt aacggcggga tataacatga gctgtcttcg gtatcgtcgt atcccactac
    10501 cgagatatcc gcaccaacgc gcagcccgga ctcggtaatg gcgcgcattg cgcccagcgc
    10561 catctgatcg ttggcaacca gcatcgcagt gggaacgatg ccctcattca gcatttgcat
    10621 ggtttgttga aaaccggaca tggcactcca gtcgccttcc cgttccgcta tcggctgaat
    10681 ttgattgcga gtgagatatt tatgccagcc agccagacgc agacgcgccg a
//
