LOCUS       sequence-198127-       10073 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial expression plasmid for SpCas9-HF1 variant.
ACCESSION   .
VERSION     .
KEYWORDS    .
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 Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Nature. 
            2017 Sep 20. doi: 10.1038/nature24268."
COMMENT     Sequence Label: pJSC111 - Bacterial expression plasmid for
            SpC...
FEATURES             Location/Qualifiers
     source          1..10073
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     167..186
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     270..289
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        270..288
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     RBS             377..399
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             419..448
                     /codon_start=1
                     /product="10xHis affinity tag"
                     /label=10xHis
                     /translation="HHHHHHHHHH"
     CDS             458..1558
                     /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     1532..1555
                     /label=MBP-F
                     /note="Maltose binding protein, forward primer"
     CDS             1613..1633
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQS"
     CDS             1640..5746
                     /codon_start=1
                     /product="Cas9 (Csn1) endonuclease from the Streptococcus 
                     pyogenes Type II CRISPR/Cas system"
                     /label=Cas9
                     /note="generates RNA-guided double strand breaks in DNA"
                     /translation="MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK
                     NLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES
                     FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK
                     FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL
                     ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ
                     IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR
                     QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL
                     RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG
                     NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTAFDKNLPNEKVLPKHSLLYEY
                     FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD
                     SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER
                     LKTYAHLFDDKVMKQLKRRRYTGWGALSRKLINGIRDKQSGKTILDFLKSDGFANRNFM
                     ALIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR
                     HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY
                     LYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVP
                     SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRAITKH
                     VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL
                     NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT
                     EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK
                     ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI
                     TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE
                     LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD
                     ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL
                     DATLIHQSITGLYETRIDLSQLGGD"
     CDS             join(1640..3127,3128..3130,3131..3619,3620..3622,3623..3721,
                     3722..3724,3725..4414,4415..4417,4418..5743)
                     /codon_start=1
                     /product="Cas9 endonuclease from the Streptococcus pyogenes
                     Type II CRISPR/Cas system, mutated to improve targeting 
                     specificity (Kleinstiver et al., 2016)"
                     /label=SpCas9-HF1
                     /note="carries the mutations N497A, R661A, Q695A, and 
                     Q926A"
                     /translation="MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK
                     NLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES
                     FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK
                     FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL
                     ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ
                     IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR
                     QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL
                     RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG
                     NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTAFDKNLPNEKVLPKHSLLYEY
                     FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD
                     SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER
                     LKTYAHLFDDKVMKQLKRRRYTGWGALSRKLINGIRDKQSGKTILDFLKSDGFANRNFM
                     ALIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR
                     HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY
                     LYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVP
                     SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRAITKH
                     VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL
                     NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT
                     EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK
                     ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI
                     TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE
                     LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD
                     ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL
                     DATLIHQSITGLYETRIDLSQLGGD"
     primer_bind     complement(5872..5890)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      5886..5933
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     promoter        complement(join(6299..6304,6305..6321,6322..6327))
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
     primer_bind     complement(6354..6372)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        6440..6544
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(6545..6613,6614..7405)
                     /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(6763..6782)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      7576..8164
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     8065..8084
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     8318..8335
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    8350..8489
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(8415..8434)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     8575..8597
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(8591..8782)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
ORIGIN
        1 ggaagcagcc cagtagtagg ttgaggccgt tgagcaccgc cgccgcaagg aatggtgcat
       61 gcaaggagat ggcgcccaac agtcccccgg ccacggggcc tgccaccata cccacgccga
      121 aacaagcgct catgagcccg aagtggcgag cccgatcttc cccatcggtg atgtcggcga
      181 tataggcgcc agcaaccgca cctgtggcgc cggtgatgcc ggccacgatg cgtccggcgt
      241 agaggatcga gatctcgatc ccgcgaaatt aatacgactc actataggga gaccacaacg
      301 gtttccctct agtgccggct ccggagagct ctttaattaa gcggccgccc tgcaggactc
      361 gagttctaga aataattttg tttaacttta agaaggagat atacatatga aatcttctca
      421 ccatcaccat caccatcacc atcaccatgg ttcttctatg aaaatcgaag aaggtaaact
      481 ggtaatctgg attaacggcg ataaaggcta taacggtctc gctgaagtcg gtaagaaatt
      541 cgagaaagat accggaatta aagtcaccgt tgagcatccg gataaactgg aagagaaatt
      601 cccacaggtt gcggcaactg gcgatggccc tgacattatc ttctgggcac acgaccgctt
      661 tggtggctac gctcaatctg gcctgttggc tgaaatcacc ccggacaaag cgttccagga
      721 caagctgtat ccgtttacct gggatgccgt acgttacaac ggcaagctga ttgcttaccc
      781 gatcgctgtt gaagcgttat cgctgattta taacaaagat ctgctgccga acccgccaaa
      841 aacctgggaa gagatcccgg cgctggataa agaactgaaa gcgaaaggta agagcgcgct
      901 gatgttcaac ctgcaagaac cgtacttcac ctggccgctg attgctgctg acgggggtta
      961 tgcgttcaag tatgaaaacg gcaagtacga cattaaagac gtgggcgtgg ataacgctgg
     1021 cgcgaaagcg ggtctgacct tcctggttga cctgattaaa aacaaacaca tgaatgcaga
     1081 caccgattac tccatcgcag aagctgcctt taataaaggc gaaacagcga tgaccatcaa
     1141 cggcccgtgg gcatggtcca acatcgacac cagcaaagtg aattatggtg taacggtact
     1201 gccgaccttc aagggtcaac catccaaacc gttcgttggc gtgctgagcg caggtattaa
     1261 cgccgccagt ccgaacaaag agctggcaaa agagttcctc gaaaactatc tgctgactga
     1321 tgaaggtctg gaagcggtta ataaagacaa accgctgggt gccgtagcgc tgaagtctta
     1381 cgaggaagag ttggcgaaag atccacgtat tgccgccact atggaaaacg cccagaaagg
     1441 tgaaatcatg ccgaacatcc cgcagatgtc cgctttctgg tatgccgtgc gtactgcggt
     1501 gatcaacgcc gccagcggtc gtcagactgt cgatgaagcc ctgaaagacg cgcagactaa
     1561 ttcgagctcg aacaacaaca acaataacaa taacaacaac ctcgggatcg aggaaaacct
     1621 gtacttccaa tccaatgcaa tggataagaa atactcaata ggcttagata tcggcacaaa
     1681 tagcgtcgga tgggcggtga tcactgatga atataaggtt ccgtctaaaa agttcaaggt
     1741 tctgggaaat acagaccgcc acagtatcaa aaaaaatctt ataggggctc ttttatttga
     1801 cagtggagag acagcggaag cgactcgtct caaacggaca gctcgtagaa ggtatacacg
     1861 tcggaagaat cgtatttgtt atctacagga gattttttca aatgagatgg cgaaagtaga
     1921 tgatagtttc tttcatcgac ttgaagagtc ttttttggtg gaagaagaca agaagcatga
     1981 acgtcatcct atttttggaa atatagtaga tgaagttgct tatcatgaga aatatccaac
     2041 tatctatcat ctgcgaaaaa aattggtaga ttctactgat aaagcggatt tgcgcttaat
     2101 ctatttggcc ttagcgcata tgattaagtt tcgtggtcat tttttgattg agggagattt
     2161 aaatcctgat aatagtgatg tggacaaact atttatccag ttggtacaaa cctacaatca
     2221 attatttgaa gaaaacccta ttaacgcaag tggagtagat gctaaagcga ttctttctgc
     2281 acgattgagt aaatcaagac gattagaaaa tctcattgct cagctccccg gtgagaagaa
     2341 aaatggctta tttgggaatc tcattgcttt gtcattgggt ttgaccccta attttaaatc
     2401 aaattttgat ttggcagaag atgctaaatt acagctttca aaagatactt acgatgatga
     2461 tttagataat ttattggcgc aaattggaga tcaatatgct gatttgtttt tggcagctaa
     2521 gaatttatca gatgctattt tactttcaga tatcctaaga gtaaatactg aaataactaa
     2581 ggctccccta tcagcttcaa tgattaaacg ctacgatgaa catcatcaag acttgactct
     2641 tttaaaagct ttagttcgac aacaacttcc agaaaagtat aaagaaatct tttttgatca
     2701 atcaaaaaac ggatatgcag gttatattga tgggggagct agccaagaag aattttataa
     2761 atttatcaaa ccaattttag aaaaaatgga tggtactgag gaattattgg tgaaactaaa
     2821 tcgtgaagat ttgctgcgca agcaacggac ctttgacaac ggctctattc cccatcaaat
     2881 tcacttgggt gagctgcatg ctattttgag aagacaagaa gacttttatc catttttaaa
     2941 agacaatcgt gagaagattg aaaaaatctt gacttttcga attccttatt atgttggtcc
     3001 attggcgcgt ggcaatagtc gttttgcatg gatgactcgg aagtctgaag aaacaattac
     3061 cccatggaat tttgaagaag ttgtcgataa aggtgcttca gctcaatcat ttattgaacg
     3121 catgacagcg tttgataaaa atcttccaaa tgaaaaagta ctaccaaaac atagtttgct
     3181 ttatgagtat tttacggttt ataacgaatt gacaaaggtc aaatatgtta ctgaaggaat
     3241 gcgaaaacca gcatttcttt caggtgaaca gaagaaagcc attgttgatt tactcttcaa
     3301 aacaaatcga aaagtaaccg ttaagcaatt aaaagaagat tatttcaaaa aaatagaatg
     3361 ttttgatagt gttgaaattt caggagttga agatagattt aatgcttcat taggtaccta
     3421 ccatgatttg ctaaaaatta ttaaagataa agattttttg gataatgaag aaaatgaaga
     3481 tatcttagag gatattgttt taacattgac cttatttgaa gatagggaga tgattgagga
     3541 aagacttaaa acatatgctc acctctttga tgataaggtg atgaaacagc ttaaacgtcg
     3601 ccgttatact ggttggggag cgttgtctcg aaaattgatt aatggtatta gggataagca
     3661 atctggcaaa acaatattag attttttgaa atcagatggt tttgccaatc gcaattttat
     3721 ggccctgatc catgatgata gtttgacatt taaagaagac attcaaaaag cacaagtgtc
     3781 tggacaaggc gatagtttac atgaacatat tgcaaattta gctggtagcc ctgctattaa
     3841 aaaaggtatt ttacagactg taaaagttgt tgatgaattg gtcaaagtaa tggggcggca
     3901 taagccagaa aatatcgtta ttgaaatggc acgtgaaaat cagacaactc aaaagggcca
     3961 gaaaaattcg cgagagcgta tgaaacgaat cgaagaaggt atcaaagaat taggaagtca
     4021 gattcttaaa gagcatcctg ttgaaaatac tcaattgcaa aatgaaaagc tctatctcta
     4081 ttatctccaa aatggaagag acatgtatgt ggaccaagaa ttagatatta atcgtttaag
     4141 tgattatgat gtcgatcaca ttgttccaca aagtttcctt aaagacgatt caatagacaa
     4201 taaggtctta acgcgttctg ataaaaatcg tggtaaatcg gataacgttc caagtgaaga
     4261 agtagtcaaa aagatgaaaa actattggag acaacttcta aacgccaagt taatcactca
     4321 acgtaagttt gataatttaa cgaaagctga acgtggaggt ttgagtgaac ttgataaagc
     4381 tggttttatc aaacgccaat tggttgaaac tcgcgccatc actaagcatg tggcacaaat
     4441 tttggatagt cgcatgaata ctaaatacga tgaaaatgat aaacttattc gagaggttaa
     4501 agtgattacc ttaaaatcta aattagtttc tgacttccga aaagatttcc aattctataa
     4561 agtacgtgag attaacaatt accatcatgc ccatgatgcg tatctaaatg ccgtcgttgg
     4621 aactgctttg attaagaaat atccaaaact tgaatcggag tttgtctatg gtgattataa
     4681 agtttatgat gttcgtaaaa tgattgctaa gtctgagcaa gaaataggca aagcaaccgc
     4741 aaaatatttc ttttactcta atatcatgaa cttcttcaaa acagaaatta cacttgcaaa
     4801 tggagagatt cgcaaacgcc ctctaatcga aactaatggg gaaactggag aaattgtctg
     4861 ggataaaggg cgagattttg ccacagtgcg caaagtattg tccatgcccc aagtcaatat
     4921 tgtcaagaaa acagaagtac agacaggcgg attctccaag gagtcaattt taccaaaaag
     4981 aaattcggac aagcttattg ctcgtaaaaa agactgggat ccaaaaaaat atggtggttt
     5041 tgatagtcca acggtagctt attcagtcct agtggttgct aaggtggaaa aagggaaatc
     5101 gaagaagtta aaatccgtta aagagttact agggatcaca attatggaaa gaagttcctt
     5161 tgaaaaaaat ccgattgact ttttagaagc taaaggatat aaggaagtta aaaaagactt
     5221 aatcattaaa ctacctaaat atagtctttt tgagttagaa aacggtcgta aacggatgct
     5281 ggctagtgcc ggagaattac aaaaaggaaa tgagctggct ctgccaagca aatatgtgaa
     5341 ttttttatat ttagctagtc attatgaaaa gttgaagggt agtccagaag ataacgaaca
     5401 aaaacaattg tttgtggagc agcataagca ttatttagat gagattattg agcaaatcag
     5461 tgaattttct aagcgtgtta ttttagcaga tgccaattta gataaagttc ttagtgcata
     5521 taacaaacat agagacaaac caatacgtga acaagcagaa aatattattc atttatttac
     5581 gttgacgaat cttggagctc ccgctgcttt taaatatttt gatacaacaa ttgatcgtaa
     5641 acgatatacg tctacaaaag aagttttaga tgccactctt atccatcaat ccatcactgg
     5701 tctttatgaa acacgcattg atttgagtca gctaggaggt gactaataac attggaagtg
     5761 gataacggat ccgcgatcgc ggcgcgccac ctggtggccg gccggtacca cgcgtgcgcg
     5821 ctgatccggc tgctaacaaa gcccgaaagg aagctgagtt ggctgctgcc accgctgagc
     5881 aataactagc ataacccctt ggggcctcta aacgggtctt gaggggtttt ttgctgaaag
     5941 gaggaactat atccggatat ccacaggacg ggtgtggtcg ccatgatcgc gtagtcgata
     6001 gtggctccaa gtagcgaagc gagcaggact gggcggcggc caaagcggtc ggacagtgct
     6061 ccgagaacgg gtgcgcatag aaattgcatc aacgcatata gcgctagcag cacgccatag
     6121 tgactggcga tgctgtcgga atggacgata tcccgcaaga ggcccggcag taccggcata
     6181 accaagccta tgcctacagc atccagggtg acggtgccga ggatgacgat gagcgcattg
     6241 ttagatttca tacacggtgc ctgactgcgt tagcaattta actgtgataa actaccgcat
     6301 taaagcttat cgatgataag ctgtcaaaca tgagaattct tgaagacgaa agggcctcgt
     6361 gatacgccta tttttatagg ttaatgtcat gataataatg gtttcttaga cgtcaggtgg
     6421 cacttttcgg ggaaatgtgc gcggaacccc tatttgttta tttttctaaa tacattcaaa
     6481 tatgtatccg ctcatgagac aataaccctg ataaatgctt caataacatt gaaaaaggaa
     6541 gagtatgagt attcaacatt tccgtgtcgc ccttattccc ttttttgcgg cattttgcct
     6601 tcctgttttt gctcacccag aaacgctggt gaaagtaaaa gatgctgaag atcagttggg
     6661 tgcacgagtg ggttacatcg aactggatct caacagcggt aagatccttg agagttttcg
     6721 ccccgaagaa cgttttccaa tgatgagcac ttttaaagtt ctgctatgtg gcgcggtatt
     6781 atcccgtgtt gacgccgggc aagagcaact cggtcgccgc atacactatt ctcagaatga
     6841 cttggttgag tactcaccag tcacagaaaa gcatcttacg gatggcatga cagtaagaga
     6901 attatgcagt gctgccataa ccatgagtga taacactgcg gccaacttac ttctgacaac
     6961 gatcggagga ccgaaggagc taaccgcttt tttgcacaac atgggggatc atgtaactcg
     7021 ccttgatcgt tgggaaccgg agctgaatga agccatacca aacgacgagc gtgacaccac
     7081 gatgcctgca gcaatggcaa caacgttgcg caaactatta actggcgaac tacttactct
     7141 agcttcccgg caacaattaa tagactggat ggaggcggat aaagttgcag gaccacttct
     7201 gcgctcggcc cttccggctg gctggtttat tgctgataaa tctggagccg gtgagcgtgg
     7261 gtctcgcggt atcattgcag cactggggcc agatggtaag ccctcccgta tcgtagttat
     7321 ctacacgacg gggagtcagg caactatgga tgaacgaaat agacagatcg ctgagatagg
     7381 tgcctcactg attaagcatt ggtaactgtc agaccaagtt tactcatata tactttagat
     7441 tgatttaaaa cttcattttt aatttaaaag gatctaggtg aagatccttt ttgataatct
     7501 catgaccaaa atcccttaac gtgagttttc gttccactga gcgtcagacc ccgtagaaaa
     7561 gatcaaagga tcttcttgag atcctttttt tctgcgcgta atctgctgct tgcaaacaaa
     7621 aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa gagctaccaa ctctttttcc
     7681 gaaggtaact ggcttcagca gagcgcagat accaaatact gtccttctag tgtagccgta
     7741 gttaggccac cacttcaaga actctgtagc accgcctaca tacctcgctc tgctaatcct
     7801 gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt accgggttgg actcaagacg
     7861 atagttaccg gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca cacagcccag
     7921 cttggagcga acgacctaca ccgaactgag atacctacag cgtgagctat gagaaagcgc
     7981 cacgcttccc gaagggagaa aggcggacag gtatccggta agcggcaggg tcggaacagg
     8041 agagcgcacg agggagcttc cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt
     8101 tcgccacctc tgacttgagc gtcgattttt gtgatgctcg tcaggggggc ggagcctatg
     8161 gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc ttttgctggc cttttgctca
     8221 catgttcttt cctgcgttat cccctgattc tgtggataac cgtattaccg cctttgagtg
     8281 agctgatacc gctcgccgca gccgaacgac cgagcgcagc gagtcagtga gcgaggaagc
     8341 ggaagagcgc ctgatgcggt attttctcct tacgcatctg tgcggtattt cacaccgcaa
     8401 tggtgcactc tcagtacaat ctgctctgat gccgcatagt taagccagta tacactccgc
     8461 tatcgctacg tgactgggtc atggctgcgc cccgacaccc gccaacaccc gctgacgcgc
     8521 cctgacgggc ttgtctgctc ccggcatccg cttacagaca agctgtgacc gtctccggga
     8581 gctgcatgtg tcagaggttt tcaccgtcat caccgaaacg cgcgaggcag ctgcggtaaa
     8641 gctcatcagc gtggtcgtga agcgattcac agatgtctgc ctgttcatcc gcgtccagct
     8701 cgttgagttt ctccagaagc gttaatgtct ggcttctgat aaagcgggcc atgttaaggg
     8761 cggttttttc ctgtttggtc actgatgcct ccgtgtaagg gggatttctg ttcatggggg
     8821 taatgatacc gatgaaacga gagaggatgc tcacgatacg ggttactgat gatgaacatg
     8881 cccggttact ggaacgttgt gagggtaaac aactggcggt atggatgcgg cgggaccaga
     8941 gaaaaatcac tcagggtcaa tgccagcgct tcgttaatac agatgtaggt gttccacagg
     9001 gtagccagca gcatcctgcg atgcagatcc ggaacataat ggtgcagggc gctgacttcc
     9061 gcgtttccag actttacgaa acacggaaac cgaagaccat tcatgttgtt gctcaggtcg
     9121 cagacgtttt gcagcagcag tcgcttcacg ttcgctcgcg tatcggtgat tcattctgct
     9181 aaccagtaag gcaaccccgc cagcctagcc gggtcctcaa cgacaggagc acgatcatgc
     9241 gcacccgtgg ccaggaccca acgctgcccg agatgcgccg cgtgcggctg ctggagatgg
     9301 cggacgcgat ggatatgttc tgccaagggt tggtttgcgc attcacagtt ctccgcaaga
     9361 attgattggc tccaattctt ggagtggtga atccgttagc gaggtgccgc cggcttccat
     9421 tcaggtcgag gtggcccggc tccatgcacc gcgacgcaac gcggggaggc agacaaggta
     9481 tagggcggcg cctacaatcc atgccaaccc gttccatgtg ctcgccgagg cggcataaat
     9541 cgccgtgacg atcagcggtc caatgatcga agttaggctg gtaagagccg cgagcgatcc
     9601 ttgaagctgt ccctgatggt cgtcatctac ctgcctggac agcatggcct gcaacgcggg
     9661 catcccgatg ccgccggaag cgagaagaat cataatgggg aaggccatcc agcctcgcgt
     9721 cgcgaacgcc agcaagacgt agcccagcgc gtcggccgcc atgccggcga taatggcctg
     9781 cttctcgccg aaacgtttgg tggcgggacc agtgacgaag gcttgagcga gggcgtgcaa
     9841 gattccgaat accgcaagcg acaggccgat catcgtcgcg ctccagcgaa agcggtcctc
     9901 gccgaaaatg acccagagcg ctgccggcac ctgtcctacg agttgcatga taaagaagac
     9961 agtcataagt gcggcgacga tagtcatgcc ccgcgcccac cggaaggagc tgactgggtt
    10021 gaaggctctc aagggcatcg gtcgacgctc tcccttatgc gactcctgca tta
//
