LOCUS       Exported               10752 bp ds-DNA     circular SYN 16-OCT-2023
DEFINITION  Expression of increased fidelity eSpCas9 in bacterial cells.
ACCESSION   .
VERSION     .
KEYWORDS    pET-FLAG-eSpCas9
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 Oct 16, 2023 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..10752
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     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             2353..6453
                     /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             6454..6501
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     primer_bind     complement(6529..6546)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     CDS             6579..6596
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(6649..6667)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      6663..6710
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      6747..7202
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6834..6853)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     7044..7065
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(7294..8109)
                     /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     8017..8036
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     8101..8120
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      8231..8819
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     8720..8739
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     8973..8990
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    9005..9144
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(9070..9089)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     9230..9252
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(9246..9437)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(10246..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 gcggacgact 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 ccacgacgcc tacctgaacg ccgtcgtggg aaccgccctg
     5341 atcaaaaagt accctgcgct ggaaagcgag ttcgtgtacg gcgactacaa ggtgtacgac
     5401 gtgcggaaga tgatcgccaa gagcgagcag gaaatcggca aggctaccgc caagtacttc
     5461 ttctacagca acatcatgaa ctttttcaag accgagatta ccctggccaa cggcgagatc
     5521 cggaaggcgc ctctgatcga gacaaacggc gaaaccgggg agatcgtgtg ggataagggc
     5581 cgggattttg ccaccgtgcg gaaagtgctg agcatgcccc aagtgaatat cgtgaaaaag
     5641 accgaggtgc agacaggcgg cttcagcaaa gagtctatcc tgcccaagag gaacagcgat
     5701 aagctgatcg ccagaaagaa ggactgggac cctaagaagt acggcggctt cgacagcccc
     5761 accgtggcct attctgtgct ggtggtggcc aaagtggaaa agggcaagtc caagaaactg
     5821 aagagtgtga aagagctgct ggggatcacc atcatggaaa gaagcagctt cgagaagaat
     5881 cccatcgact ttctggaagc caagggctac aaagaagtga aaaaggacct gatcatcaag
     5941 ctgcctaagt actccctgtt cgagctggaa aacggccgga agagaatgct ggcctctgcc
     6001 ggcgaactgc agaagggaaa cgaactggcc ctgccctcca aatatgtgaa cttcctgtac
     6061 ctggccagcc actatgagaa gctgaagggc tcccccgagg ataatgagca gaaacagctg
     6121 tttgtggaac agcacaagca ctacctggac gagatcatcg agcagatcag cgagttctcc
     6181 aagagagtga tcctggccga cgctaatctg gacaaagtgc tgtccgccta caacaagcac
     6241 cgggataagc ccatcagaga gcaggccgag aatatcatcc acctgtttac cctgaccaat
     6301 ctgggagccc ctgccgcctt caagtacttt gacaccacca tcgaccggaa gaggtacacc
     6361 agcaccaaag aggtgctgga cgccaccctg atccaccaga gcatcaccgg cctgtacgag
     6421 acacggatcg acctgtctca gctgggaggc gacaaaaggc cggcggccac gaaaaaggcc
     6481 ggccaggcaa aaaagaaaaa gtaagaattc ctagagctcg ctgatcagcc tcgactgtgc
     6541 cttctagttg ccagccatct gttgcggccg cactcgagca ccaccaccac caccactgag
     6601 atccggctgc taacaaagcc cgaaaggaag ctgagttggc tgctgccacc gctgagcaat
     6661 aactagcata accccttggg gcctctaaac gggtcttgag gggttttttg ctgaaaggag
     6721 gaactatatc cggattggcg aatgggacgc gccctgtagc ggcgcattaa gcgcggcggg
     6781 tgtggtggtt acgcgcagcg tgaccgctac acttgccagc gccctagcgc ccgctccttt
     6841 cgctttcttc ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg
     6901 ggggctccct ttagggttcc gatttagtgc tttacggcac ctcgacccca aaaaacttga
     6961 ttagggtgat ggttcacgta gtgggccatc gccctgatag acggtttttc gccctttgac
     7021 gttggagtcc acgttcttta atagtggact cttgttccaa actggaacaa cactcaaccc
     7081 tatctcggtc tattcttttg atttataagg gattttgccg atttcggcct attggttaaa
     7141 aaatgagctg atttaacaaa aatttaacgc gaattttaac aaaatattaa cgcttacaat
     7201 ttaggtggca cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt tttctaaata
     7261 cattcaaata tgtatccgct catgaattaa ttcttagaaa aactcatcga gcatcaaatg
     7321 aaactgcaat ttattcatat caggattatc aataccatat ttttgaaaaa gccgtttctg
     7381 taatgaagga gaaaactcac cgaggcagtt ccataggatg gcaagatcct ggtatcggtc
     7441 tgcgattccg actcgtccaa catcaataca acctattaat ttcccctcgt caaaaataag
     7501 gttatcaagt gagaaatcac catgagtgac gactgaatcc ggtgagaatg gcaaaagttt
     7561 atgcatttct ttccagactt gttcaacagg ccagccatta cgctcgtcat caaaatcact
     7621 cgcatcaacc aaaccgttat tcattcgtga ttgcgcctga gcgagacgaa atacgcgatc
     7681 gctgttaaaa ggacaattac aaacaggaat cgaatgcaac cggcgcagga acactgccag
     7741 cgcatcaaca atattttcac ctgaatcagg atattcttct aatacctgga atgctgtttt
     7801 cccggggatc gcagtggtga gtaaccatgc atcatcagga gtacggataa aatgcttgat
     7861 ggtcggaaga ggcataaatt ccgtcagcca gtttagtctg accatctcat ctgtaacatc
     7921 attggcaacg ctacctttgc catgtttcag aaacaactct ggcgcatcgg gcttcccata
     7981 caatcgatag attgtcgcac ctgattgccc gacattatcg cgagcccatt tatacccata
     8041 taaatcagca tccatgttgg aatttaatcg cggcctagag caagacgttt cccgttgaat
     8101 atggctcata acaccccttg tattactgtt tatgtaagca gacagtttta ttgttcatga
     8161 ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc agaccccgta gaaaagatca
     8221 aaggatcttc ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac
     8281 caccgctacc agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg
     8341 taactggctt cagcagagcg cagataccaa atactgtcct tctagtgtag ccgtagttag
     8401 gccaccactt caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac
     8461 cagtggctgc tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt
     8521 taccggataa ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg
     8581 agcgaacgac ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc
     8641 ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc
     8701 gcacgaggga gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc
     8761 acctctgact tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa
     8821 acgccagcaa cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt
     8881 tctttcctgc gttatcccct gattctgtgg ataaccgtat taccgccttt gagtgagctg
     8941 ataccgctcg ccgcagccga acgaccgagc gcagcgagtc agtgagcgag gaagcggaag
     9001 agcgcctgat gcggtatttt ctccttacgc atctgtgcgg tatttcacac cgcaatggtg
     9061 cactctcagt acaatctgct ctgatgccgc atagttaagc cagtatacac tccgctatcg
     9121 ctacgtgact gggtcatggc tgcgccccga cacccgccaa cacccgctga cgcgccctga
     9181 cgggcttgtc tgctcccggc atccgcttac agacaagctg tgaccgtctc cgggagctgc
     9241 atgtgtcaga ggttttcacc gtcatcaccg aaacgcgcga ggcagctgcg gtaaagctca
     9301 tcagcgtggt cgtgaagcga ttcacagatg tctgcctgtt catccgcgtc cagctcgttg
     9361 agtttctcca gaagcgttaa tgtctggctt ctgataaagc gggccatgtt aagggcggtt
     9421 ttttcctgtt tggtcactga tgcctccgtg taagggggat ttctgttcat gggggtaatg
     9481 ataccgatga aacgagagag gatgctcacg atacgggtta ctgatgatga acatgcccgg
     9541 ttactggaac gttgtgaggg taaacaactg gcggtatgga tgcggcggga ccagagaaaa
     9601 atcactcagg gtcaatgcca gcgcttcgtt aatacagatg taggtgttcc acagggtagc
     9661 cagcagcatc ctgcgatgca gatccggaac ataatggtgc agggcgctga cttccgcgtt
     9721 tccagacttt acgaaacacg gaaaccgaag accattcatg ttgttgctca ggtcgcagac
     9781 gttttgcagc agcagtcgct tcacgttcgc tcgcgtatcg gtgattcatt ctgctaacca
     9841 gtaaggcaac cccgccagcc tagccgggtc ctcaacgaca ggagcacgat catgcgcacc
     9901 cgtggggccg ccatgccggc gataatggcc tgcttctcgc cgaaacgttt ggtggcggga
     9961 ccagtgacga aggcttgagc gagggcgtgc aagattccga ataccgcaag cgacaggccg
    10021 atcatcgtcg cgctccagcg aaagcggtcc tcgccgaaaa tgacccagag cgctgccggc
    10081 acctgtccta cgagttgcat gataaagaag acagtcataa gtgcggcgac gatagtcatg
    10141 ccccgcgccc accggaagga gctgactggg ttgaaggctc tcaagggcat cggtcgagat
    10201 cccggtgcct aatgagtgag ctaacttaca ttaattgcgt tgcgctcact gcccgctttc
    10261 cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc ggggagaggc
    10321 ggtttgcgta ttgggcgcca gggtggtttt tcttttcacc agtgagacgg gcaacagctg
    10381 attgcccttc accgcctggc cctgagagag ttgcagcaag cggtccacgc tggtttgccc
    10441 cagcaggcga aaatcctgtt tgatggtggt taacggcggg atataacatg agctgtcttc
    10501 ggtatcgtcg tatcccacta ccgagatatc cgcaccaacg cgcagcccgg actcggtaat
    10561 ggcgcgcatt gcgcccagcg ccatctgatc gttggcaacc agcatcgcag tgggaacgat
    10621 gccctcattc agcatttgca tggtttgttg aaaaccggac atggcactcc agtcgccttc
    10681 ccgttccgct atcggctgaa tttgattgcg agtgagatat ttatgccagc cagccagacg
    10741 cagacgcgcc ga
//
