pJSC281 - Bacterial expression plasmid for SpCas9 Cluster 1

LOCUS       pJSC281 - Bacterial expression plasmid for SpCas9 Cluster 1 (HypaCas9), REC2 FRET variant               10073 bp ds-DNA     circular SYN 13-MAY-2021
DEFINITION  Bacterial expression plasmid for SpCas9 Cluster 1 (HypaCas9), REC2 
            FRET variant.
ACCESSION   .
VERSION     .
KEYWORDS    pJSC281 - Bacterial expression plasmid for SpCas9 Cluster 1 (HypaCas9), REC2 FRET variant
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 10073)
  AUTHORS   Chen JS, Dagdas YS, Kleinstiver BP, Welch MM, Sousa AA, Harrington 
            LB, Sternberg SH, Joung JK, Yildiz A, Doudna JA
  TITLE     Enhanced proofreading governs CRISPR-Cas9 targeting accuracy.
  JOURNAL   Nature. 2017 Sep 20. doi: 10.1038/nature24268.
  PUBMED    28931002
REFERENCE   2  (bases 1 to 10073)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 13, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..10073
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     primer_bind     515..534
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     618..637
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        618..636
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     RBS             725..747
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             767..796
                     /codon_start=1
                     /product="10xHis affinity tag"
                     /label=10xHis
                     /translation="HHHHHHHHHH"
     CDS             806..1906
                     /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="MKIEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLE
                     EKFPQVAATGDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDAVRYNGKL
                     IAYPIAVEALSLIYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWPLIA
                     ADGGYAFKYENGKYDIKDVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNKGE
                     TAMTINGPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFL
                     ENYLLTDEGLEAVNKDKPLGAVALKSYEEELAKDPRIAATMENAQKGEIMPNIPQMSAF
                     WYAVRTAVINAASGRQTVDEALKDAQT"
     primer_bind     1880..1903
                     /label=MBP-F
                     /note="Maltose binding protein, forward primer"
     CDS             1961..1981
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQS"
     CDS             1988..6094
                     /codon_start=1
                     /product="Cas9 endonuclease from the Streptococcus pyogenes
                     Type II CRISPR/Cas system, mutated to improve targeting 
                     specificity without compromising on-target activity (Chen 
                     et al., 2017)"
                     /label=HypaCas9
                     /note="carries the mutations N692A, M694A, Q695A, and 
                     H698A"
                     /translation="MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK
                     NLIGALLFDSGETACATRLKRTARRRYTRRKNRISYLQEIFSNEMAKVDDSFFHRLEES
                     FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK
                     FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL
                     ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDCDLDNLLAQ
                     IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR
                     QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL
                     RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG
                     NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY
                     FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIESFD
                     SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER
                     LKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRAFA
                     ALIADDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR
                     HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY
                     LYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVP
                     SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH
                     VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL
                     NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT
                     EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK
                     ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI
                     TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE
                     LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD
                     ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL
                     DATLIHQSITGLYETRIDLSQLGGD"
     primer_bind     complement(6220..6238)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      6234..6281
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     promoter        complement(6647..6675)
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
     primer_bind     complement(6702..6720)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        6788..6892
                     /gene="bla"
                     /label=AmpR promoter
     CDS             6893..7753
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(7111..7130)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      7924..8512
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     8413..8432
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     8666..8683
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    8698..8837
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(8763..8782)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     8923..8945
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(8939..9130)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
ORIGIN
        1 acgccagcaa gacgtagccc agcgcgtcgg ccgccatgcc ggcgataatg gcctgcttct
       61 cgccgaaacg tttggtggcg ggaccagtga cgaaggcttg agcgagggcg tgcaagattc
      121 cgaataccgc aagcgacagg ccgatcatcg tcgcgctcca gcgaaagcgg tcctcgccga
      181 aaatgaccca gagcgctgcc ggcacctgtc ctacgagttg catgataaag aagacagtca
      241 taagtgcggc gacgatagtc atgccccgcg cccaccggaa ggagctgact gggttgaagg
      301 ctctcaaggg catcggtcga cgctctccct tatgcgactc ctgcattagg aagcagccca
      361 gtagtaggtt gaggccgttg agcaccgccg ccgcaaggaa tggtgcatgc aaggagatgg
      421 cgcccaacag tcccccggcc acggggcctg ccaccatacc cacgccgaaa caagcgctca
      481 tgagcccgaa gtggcgagcc cgatcttccc catcggtgat gtcggcgata taggcgccag
      541 caaccgcacc tgtggcgccg gtgatgccgg ccacgatgcg tccggcgtag aggatcgaga
      601 tctcgatccc gcgaaattaa tacgactcac tatagggaga ccacaacggt ttccctctag
      661 tgccggctcc ggagagctct ttaattaagc ggccgccctg caggactcga gttctagaaa
      721 taattttgtt taactttaag aaggagatat acatatgaaa tcttctcacc atcaccatca
      781 ccatcaccat caccatggtt cttctatgaa aatcgaagaa ggtaaactgg taatctggat
      841 taacggcgat aaaggctata acggtctcgc tgaagtcggt aagaaattcg agaaagatac
      901 cggaattaaa gtcaccgttg agcatccgga taaactggaa gagaaattcc cacaggttgc
      961 ggcaactggc gatggccctg acattatctt ctgggcacac gaccgctttg gtggctacgc
     1021 tcaatctggc ctgttggctg aaatcacccc ggacaaagcg ttccaggaca agctgtatcc
     1081 gtttacctgg gatgccgtac gttacaacgg caagctgatt gcttacccga tcgctgttga
     1141 agcgttatcg ctgatttata acaaagatct gctgccgaac ccgccaaaaa cctgggaaga
     1201 gatcccggcg ctggataaag aactgaaagc gaaaggtaag agcgcgctga tgttcaacct
     1261 gcaagaaccg tacttcacct ggccgctgat tgctgctgac gggggttatg cgttcaagta
     1321 tgaaaacggc aagtacgaca ttaaagacgt gggcgtggat aacgctggcg caaaagcggg
     1381 tctgaccttc ctggttgacc tgattaaaaa caaacacatg aatgcagaca ccgattactc
     1441 catcgcagaa gctgccttta ataaaggcga aacagcgatg accatcaacg gcccgtgggc
     1501 atggtccaac atcgacacca gcaaagtgaa ttatggtgta acggtactgc cgaccttcaa
     1561 gggtcaacca tccaaaccgt tcgttggcgt gctgagcgca ggtattaacg ccgccagtcc
     1621 gaacaaagag ctggcaaaag agttcctcga aaactatctg ctgactgatg aaggtctgga
     1681 agcggttaat aaagacaaac cgctgggtgc cgtagcgctg aagtcttacg aggaagagtt
     1741 ggcgaaagat ccacgtattg ccgccactat ggaaaacgcc cagaaaggtg aaatcatgcc
     1801 gaacatcccg cagatgtccg ctttctggta tgccgtgcgt actgcggtga tcaacgccgc
     1861 cagcggtcgt cagactgtcg atgaagccct gaaagacgcg cagactaatt cgagctcgaa
     1921 caacaacaac aataacaata acaacaacct cgggatcgag gaaaacctgt acttccaatc
     1981 caatgcaatg gataagaaat actcaatagg cttagatatc ggcacaaata gcgtcggatg
     2041 ggcggtgatc actgatgaat ataaggttcc gtctaaaaag ttcaaggttc tgggaaatac
     2101 agaccgccac agtatcaaaa aaaatcttat aggggctctt ttatttgaca gtggagagac
     2161 agcgtgtgcg actcgtctca aacggacagc tcgtagaagg tatacacgtc ggaagaatcg
     2221 tatttcttat ctacaggaga ttttttcaaa tgagatggcg aaagtagatg atagtttctt
     2281 tcatcgactt gaagagtctt ttttggtgga agaagacaag aagcatgaac gtcatcctat
     2341 ttttggaaat atagtagatg aagttgctta tcatgagaaa tatccaacta tctatcatct
     2401 gcgaaaaaaa ttggtagatt ctactgataa agcggatttg cgcttaatct atttggcctt
     2461 agcgcatatg attaagtttc gtggtcattt tttgattgag ggagatttaa atcctgataa
     2521 tagtgatgtg gacaaactat ttatccagtt ggtacaaacc tacaatcaat tatttgaaga
     2581 aaaccctatt aacgcaagtg gagtagatgc taaagcgatt ctttctgcac gattgagtaa
     2641 atcaagacga ttagaaaatc tcattgctca gctccccggt gagaagaaaa atggcttatt
     2701 tgggaatctc attgctttgt cattgggttt gacccctaat tttaaatcaa attttgattt
     2761 ggcagaagat gctaaattac agctttcaaa agatacttac gattgtgatt tagataattt
     2821 attggcgcaa attggagatc aatatgctga tttgtttttg gcagctaaga atttatcaga
     2881 tgctatttta ctttcagata tcctaagagt aaatactgaa ataactaagg ctcccctatc
     2941 agcttcaatg attaaacgct acgatgaaca tcatcaagac ttgactcttt taaaagcttt
     3001 agttcgacaa caacttccag aaaagtataa agaaatcttt tttgatcaat caaaaaacgg
     3061 atatgcaggt tatattgatg ggggagctag ccaagaagaa ttttataaat ttatcaaacc
     3121 aattttagaa aaaatggatg gtactgagga attattggtg aaactaaatc gtgaagattt
     3181 gctgcgcaag caacggacct ttgacaacgg ctctattccc catcaaattc acttgggtga
     3241 gctgcatgct attttgagaa gacaagaaga cttttatcca tttttaaaag acaatcgtga
     3301 gaagattgaa aaaatcttga cttttcgaat tccttattat gttggtccat tggcgcgtgg
     3361 caatagtcgt tttgcatgga tgactcggaa gtctgaagaa acaattaccc catggaattt
     3421 tgaagaagtt gtcgataaag gtgcttcagc tcaatcattt attgaacgca tgacaaactt
     3481 tgataaaaat cttccaaatg aaaaagtact accaaaacat agtttgcttt atgagtattt
     3541 tacggtttat aacgaattga caaaggtcaa atatgttact gaaggaatgc gaaaaccagc
     3601 atttctttca ggtgaacaga agaaagccat tgttgattta ctcttcaaaa caaatcgaaa
     3661 agtaaccgtt aagcaattaa aagaagatta tttcaaaaaa atagaatctt ttgatagtgt
     3721 tgaaatttca ggagttgaag atagatttaa tgcttcatta ggtacctacc atgatttgct
     3781 aaaaattatt aaagataaag attttttgga taatgaagaa aatgaagata tcttagagga
     3841 tattgtttta acattgacct tatttgaaga tagggagatg attgaggaaa gacttaaaac
     3901 atatgctcac ctctttgatg ataaggtgat gaaacagctt aaacgtcgcc gttatactgg
     3961 ttggggacgt ttgtctcgaa aattgattaa tggtattagg gataagcaat ctggcaaaac
     4021 aatattagat tttttgaaat cagatggttt tgccaatcgc gcttttgctg ctctgatcgc
     4081 tgatgatagt ttgacattta aagaagacat tcaaaaagca caagtgtctg gacaaggcga
     4141 tagtttacat gaacatattg caaatttagc tggtagccct gctattaaaa aaggtatttt
     4201 acagactgta aaagttgttg atgaattggt caaagtaatg gggcggcata agccagaaaa
     4261 tatcgttatt gaaatggcac gtgaaaatca gacaactcaa aagggccaga aaaattcgcg
     4321 agagcgtatg aaacgaatcg aagaaggtat caaagaatta ggaagtcaga ttcttaaaga
     4381 gcatcctgtt gaaaatactc aattgcaaaa tgaaaagctc tatctctatt atctccaaaa
     4441 tggaagagac atgtatgtgg accaagaatt agatattaat cgtttaagtg attatgatgt
     4501 cgatcacatt gttccacaaa gtttccttaa agacgattca atagacaata aggtcttaac
     4561 gcgttctgat aaaaatcgtg gtaaatcgga taacgttcca agtgaagaag tagtcaaaaa
     4621 gatgaaaaac tattggagac aacttctaaa cgccaagtta atcactcaac gtaagtttga
     4681 taatttaacg aaagctgaac gtggaggttt gagtgaactt gataaagctg gttttatcaa
     4741 acgccaattg gttgaaactc gccaaatcac taagcatgtg gcacaaattt tggatagtcg
     4801 catgaatact aaatacgatg aaaatgataa acttattcga gaggttaaag tgattacctt
     4861 aaaatctaaa ttagtttctg acttccgaaa agatttccaa ttctataaag tacgtgagat
     4921 taacaattac catcatgccc atgatgcgta tctaaatgcc gtcgttggaa ctgctttgat
     4981 taagaaatat ccaaaacttg aatcggagtt tgtctatggt gattataaag tttatgatgt
     5041 tcgtaaaatg attgctaagt ctgagcaaga aataggcaaa gcaaccgcaa aatatttctt
     5101 ttactctaat atcatgaact tcttcaaaac agaaattaca cttgcaaatg gagagattcg
     5161 caaacgccct ctaatcgaaa ctaatgggga aactggagaa attgtctggg ataaagggcg
     5221 agattttgcc acagtgcgca aagtattgtc catgccccaa gtcaatattg tcaagaaaac
     5281 agaagtacag acaggcggat tctccaagga gtcaatttta ccaaaaagaa attcggacaa
     5341 gcttattgct cgtaaaaaag actgggatcc aaaaaaatat ggtggttttg atagtccaac
     5401 ggtagcttat tcagtcctag tggttgctaa ggtggaaaaa gggaaatcga agaagttaaa
     5461 atccgttaaa gagttactag ggatcacaat tatggaaaga agttcctttg aaaaaaatcc
     5521 gattgacttt ttagaagcta aaggatataa ggaagttaaa aaagacttaa tcattaaact
     5581 acctaaatat agtctttttg agttagaaaa cggtcgtaaa cggatgctgg ctagtgccgg
     5641 agaattacaa aaaggaaatg agctggctct gccaagcaaa tatgtgaatt ttttatattt
     5701 agctagtcat tatgaaaagt tgaagggtag tccagaagat aacgaacaaa aacaattgtt
     5761 tgtggagcag cataagcatt atttagatga gattattgag caaatcagtg aattttctaa
     5821 gcgtgttatt ttagcagatg ccaatttaga taaagttctt agtgcatata acaaacatag
     5881 agacaaacca atacgtgaac aagcagaaaa tattattcat ttatttacgt tgacgaatct
     5941 tggagctccc gctgctttta aatattttga tacaacaatt gatcgtaaac gatatacgtc
     6001 tacaaaagaa gttttagatg ccactcttat ccatcaatcc atcactggtc tttatgaaac
     6061 acgcattgat ttgagtcagc taggaggtga ctaataacat tggaagtgga taacggatcc
     6121 gcgatcgcgg cgcgccacct ggtggccggc cggtaccacg cgtgcgcgct gatccggctg
     6181 ctaacaaagc ccgaaaggaa gctgagttgg ctgctgccac cgctgagcaa taactagcat
     6241 aaccccttgg ggcctctaaa cgggtcttga ggggtttttt gctgaaagga ggaactatat
     6301 ccggatatcc acaggacggg tgtggtcgcc atgatcgcgt agtcgatagt ggctccaagt
     6361 agcgaagcga gcaggactgg gcggcggcca aagcggtcgg acagtgctcc gagaacgggt
     6421 gcgcatagaa attgcatcaa cgcatatagc gctagcagca cgccatagtg actggcgatg
     6481 ctgtcggaat ggacgatatc ccgcaagagg cccggcagta ccggcataac caagcctatg
     6541 cctacagcat ccagggtgac ggtgccgagg atgacgatga gcgcattgtt agatttcata
     6601 cacggtgcct gactgcgtta gcaatttaac tgtgataaac taccgcatta aagcttatcg
     6661 atgataagct gtcaaacatg agaattcttg aagacgaaag ggcctcgtga tacgcctatt
     6721 tttataggtt aatgtcatga taataatggt ttcttagacg tcaggtggca cttttcgggg
     6781 aaatgtgcgc ggaaccccta tttgtttatt tttctaaata cattcaaata tgtatccgct
     6841 catgagacaa taaccctgat aaatgcttca ataacattga aaaaggaaga gtatgagtat
     6901 tcaacatttc cgtgtcgccc ttattccctt ttttgcggca ttttgccttc ctgtttttgc
     6961 tcacccagaa acgctggtga aagtaaaaga tgctgaagat cagttgggtg cacgagtggg
     7021 ttacatcgaa ctggatctca acagcggtaa gatccttgag agttttcgcc ccgaagaacg
     7081 ttttccaatg atgagcactt ttaaagttct gctatgtggc gcggtattat cccgtgttga
     7141 cgccgggcaa gagcaactcg gtcgccgcat acactattct cagaatgact tggttgagta
     7201 ctcaccagtc acagaaaagc atcttacgga tggcatgaca gtaagagaat tatgcagtgc
     7261 tgccataacc atgagtgata acactgcggc caacttactt ctgacaacga tcggaggacc
     7321 gaaggagcta accgcttttt tgcacaacat gggggatcat gtaactcgcc ttgatcgttg
     7381 ggaaccggag ctgaatgaag ccataccaaa cgacgagcgt gacaccacga tgcctgcagc
     7441 aatggcaaca acgttgcgca aactattaac tggcgaacta cttactctag cttcccggca
     7501 acaattaata gactggatgg aggcggataa agttgcagga ccacttctgc gctcggccct
     7561 tccggctggc tggtttattg ctgataaatc tggagccggt gagcgtgggt ctcgcggtat
     7621 cattgcagca ctggggccag atggtaagcc ctcccgtatc gtagttatct acacgacggg
     7681 gagtcaggca actatggatg aacgaaatag acagatcgct gagataggtg cctcactgat
     7741 taagcattgg taactgtcag accaagttta ctcatatata ctttagattg atttaaaact
     7801 tcatttttaa tttaaaagga tctaggtgaa gatccttttt gataatctca tgaccaaaat
     7861 cccttaacgt gagttttcgt tccactgagc gtcagacccc gtagaaaaga tcaaaggatc
     7921 ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct
     7981 accagcggtg gtttgtttgc cggatcaaga gctaccaact ctttttccga aggtaactgg
     8041 cttcagcaga gcgcagatac caaatactgt ccttctagtg tagccgtagt taggccacca
     8101 cttcaagaac tctgtagcac cgcctacata cctcgctctg ctaatcctgt taccagtggc
     8161 tgctgccagt ggcgataagt cgtgtcttac cgggttggac tcaagacgat agttaccgga
     8221 taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca cagcccagct tggagcgaac
     8281 gacctacacc gaactgagat acctacagcg tgagctatga gaaagcgcca cgcttcccga
     8341 agggagaaag gcggacaggt atccggtaag cggcagggtc ggaacaggag agcgcacgag
     8401 ggagcttcca gggggaaacg cctggtatct ttatagtcct gtcgggtttc gccacctctg
     8461 acttgagcgt cgatttttgt gatgctcgtc aggggggcgg agcctatgga aaaacgccag
     8521 caacgcggcc tttttacggt tcctggcctt ttgctggcct tttgctcaca tgttctttcc
     8581 tgcgttatcc cctgattctg tggataaccg tattaccgcc tttgagtgag ctgataccgc
     8641 tcgccgcagc cgaacgaccg agcgcagcga gtcagtgagc gaggaagcgg aagagcgcct
     8701 gatgcggtat tttctcctta cgcatctgtg cggtatttca caccgcaatg gtgcactctc
     8761 agtacaatct gctctgatgc cgcatagtta agccagtata cactccgcta tcgctacgtg
     8821 actgggtcat ggctgcgccc cgacacccgc caacacccgc tgacgcgccc tgacgggctt
     8881 gtctgctccc ggcatccgct tacagacaag ctgtgaccgt ctccgggagc tgcatgtgtc
     8941 agaggttttc accgtcatca ccgaaacgcg cgaggcagct gcggtaaagc tcatcagcgt
     9001 ggtcgtgaag cgattcacag atgtctgcct gttcatccgc gtccagctcg ttgagtttct
     9061 ccagaagcgt taatgtctgg cttctgataa agcgggccat gttaagggcg gttttttcct
     9121 gtttggtcac tgatgcctcc gtgtaagggg gatttctgtt catgggggta atgataccga
     9181 tgaaacgaga gaggatgctc acgatacggg ttactgatga tgaacatgcc cggttactgg
     9241 aacgttgtga gggtaaacaa ctggcggtat ggatgcggcg ggaccagaga aaaatcactc
     9301 agggtcaatg ccagcgcttc gttaatacag atgtaggtgt tccacagggt agccagcagc
     9361 atcctgcgat gcagatccgg aacataatgg tgcagggcgc tgacttccgc gtttccagac
     9421 tttacgaaac acggaaaccg aagaccattc atgttgttgc tcaggtcgca gacgttttgc
     9481 agcagcagtc gcttcacgtt cgctcgcgta tcggtgattc attctgctaa ccagtaaggc
     9541 aaccccgcca gcctagccgg gtcctcaacg acaggagcac gatcatgcgc acccgtggcc
     9601 aggacccaac gctgcccgag atgcgccgcg tgcggctgct ggagatggcg gacgcgatgg
     9661 atatgttctg ccaagggttg gtttgcgcat tcacagttct ccgcaagaat tgattggctc
     9721 caattcttgg agtggtgaat ccgttagcga ggtgccgccg gcttccattc aggtcgaggt
     9781 ggcccggctc catgcaccgc gacgcaacgc ggggaggcag acaaggtata gggcggcgcc
     9841 tacaatccat gccaacccgt tccatgtgct cgccgaggcg gcataaatcg ccgtgacgat
     9901 cagcggtcca atgatcgaag ttaggctggt aagagccgcg agcgatcctt gaagctgtcc
     9961 ctgatggtcg tcatctacct gcctggacag catggcctgc aacgcgggca tcccgatgcc
    10021 gccggaagcg agaagaatca taatggggaa ggccatccag cctcgcgtcg cga
//