LOCUS       sequence-116220-        5292 bp    DNA     linear   UNA 15-DEC-2025
DEFINITION  natural linear DNA
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      natural DNA sequence
  ORGANISM  unspecified
REFERENCE   1  (bases 1 to 5292)
  AUTHORS   Jinek M, Chylinski K, Fonfara I, Hauer M, Doudna JA, Charpentier E
  TITLE     A Programmable Dual-RNA-Guided DNA Endonuclease in Adaptive 
            Bacterial Immunity.
  JOURNAL   Science. 2012 Aug 17;337(6096):816-21. doi: 10.1126/science.1225829.
            Epub 2012 Jun 28.
  PUBMED    22745249
REFERENCE   2  (bases 1 to 5292)
  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"; doi: 
            "10.1126/science.1225829"; journalName: "Science"; date: 
            "2012-08-17- 17"; volume: "337"; issue: "6096"; pages: "816-21"
COMMENT     Sequence Label: pMJ826
FEATURES             Location/Qualifiers
     source          1..5292
                     /mol_type="genomic DNA"
                     /organism="unspecified"
     CDS             4..24
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=7xHis
                     /translation="HHHHHHH"
     CDS             28..1125
                     /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     1099..1122
                     /label=MBP-F
                     /note="Maltose binding protein, forward primer"
     CDS             1156..1176
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             join(1186..3702,3703..3705,3706..5292)
                     /codon_start=1
                     /product="nickase mutant of the Cas9 endonuclease from the 
                     Streptococcus pyogenes Type II CRISPR/Cas system"
                     /label=Cas9(H840A)
                     /note="generates RNA-guided single strand nicks in DNA due 
                     to the H840A mutation in the HNH catalytic domain"
                     /translation="MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK
                     NLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES
                     FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK
                     FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL
                     ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ
                     IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR
                     QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL
                     RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG
                     NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY
                     FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD
                     SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER
                     LKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFM
                     QLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR
                     HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY
                     LYYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVP
                     SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH
                     VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL
                     NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT
                     EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK
                     ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI
                     TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE
                     LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD
                     ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL
                     DATLIHQSITGLYETRIDLSQLGGD"
ORIGIN
        1 atgcatcacc atcaccatca ccatggaaaa atcgaagaag gtaaactggt aatctggatt
       61 aacggcgata aaggctataa cggtctcgct gaagtcggta agaaattcga gaaagatacc
      121 ggaattaaag tcaccgttga gcatccggat aaactggaag agaaattccc acaggttgcg
      181 gcaactggcg atggccctga cattatcttc tgggcacacg accgctttgg tggctacgct
      241 caatctggcc tgttggctga aatcaccccg gacaaagcgt tccaggacaa gctgtatccg
      301 tttacctggg atgccgtacg ttacaacggc aagctgattg cttacccgat cgctgttgaa
      361 gcgttatcgc tgatttataa caaagatctg ctgccgaacc cgccaaaaac ctgggaagag
      421 atcccggcgc tggataaaga actgaaagcg aaaggtaaga gcgcgctgat gttcaacctg
      481 caagaaccgt acttcacctg gccgctgatt gctgctgacg ggggttatgc gttcaagtat
      541 gaaaacggca agtacgacat taaagacgtg ggcgtggata acgctggcgc gaaagcgggt
      601 ctgaccttcc tggttgacct gattaaaaac aaacacatga atgcagacac cgattactcc
      661 atcgcagaag ctgcctttaa taaaggcgaa acagcgatga ccatcaacgg cccgtgggca
      721 tggtccaaca tcgacaccag caaagtgaat tatggtgtaa cggtactgcc gaccttcaag
      781 ggtcaaccat ccaaaccgtt cgttggcgtg ctgagcgcag gtattaacgc cgccagtccg
      841 aacaaagagc tggcaaaaga gttcctcgaa aactatctgc tgactgatga aggtctggaa
      901 gcggttaata aagacaaacc gctgggtgcc gtagcgctga agtcttacga ggaagagttg
      961 gcgaaagatc cacgtattgc cgccactatg gaaaacgccc agaaaggtga aatcatgccg
     1021 aacatcccgc agatgtccgc tttctggtat gccgtgcgta ctgcggtgat caacgccgcc
     1081 agcggtcgtc agactgtcga tgaagccctg aaagacgcgc agactaattc gagctcgaac
     1141 aacaacaaca ctagtgaaaa cctgtatttc cagggagcag cctcgatgga taagaaatac
     1201 tcaataggct tagatatcgg cacaaatagc gtcggatggg cggtgatcac tgatgaatat
     1261 aaggttccgt ctaaaaagtt caaggttctg ggaaatacag accgccacag tatcaaaaaa
     1321 aatcttatag gggctctttt atttgacagt ggagagacag cggaagcgac tcgtctcaaa
     1381 cggacagctc gtagaaggta tacacgtcgg aagaatcgta tttgttatct acaggagatt
     1441 ttttcaaatg agatggcgaa agtagatgat agtttctttc atcgacttga agagtctttt
     1501 ttggtggaag aagacaagaa gcatgaacgt catcctattt ttggaaatat agtagatgaa
     1561 gttgcttatc atgagaaata tccaactatc tatcatctgc gaaaaaaatt ggtagattct
     1621 actgataaag cggatttgcg cttaatctat ttggccttag cgcatatgat taagtttcgt
     1681 ggtcattttt tgattgaggg agatttaaat cctgataata gtgatgtgga caaactattt
     1741 atccagttgg tacaaaccta caatcaatta tttgaagaaa accctattaa cgcaagtgga
     1801 gtagatgcta aagcgattct ttctgcacga ttgagtaaat caagacgatt agaaaatctc
     1861 attgctcagc tccccggtga gaagaaaaat ggcttatttg ggaatctcat tgctttgtca
     1921 ttgggtttga cccctaattt taaatcaaat tttgatttgg cagaagatgc taaattacag
     1981 ctttcaaaag atacttacga tgatgattta gataatttat tggcgcaaat tggagatcaa
     2041 tatgctgatt tgtttttggc agctaagaat ttatcagatg ctattttact ttcagatatc
     2101 ctaagagtaa atactgaaat aactaaggct cccctatcag cttcaatgat taaacgctac
     2161 gatgaacatc atcaagactt gactctttta aaagctttag ttcgacaaca acttccagaa
     2221 aagtataaag aaatcttttt tgatcaatca aaaaacggat atgcaggtta tattgatggg
     2281 ggagctagcc aagaagaatt ttataaattt atcaaaccaa ttttagaaaa aatggatggt
     2341 actgaggaat tattggtgaa actaaatcgt gaagatttgc tgcgcaagca acggaccttt
     2401 gacaacggct ctattcccca tcaaattcac ttgggtgagc tgcatgctat tttgagaaga
     2461 caagaagact tttatccatt tttaaaagac aatcgtgaga agattgaaaa aatcttgact
     2521 tttcgaattc cttattatgt tggtccattg gcgcgtggca atagtcgttt tgcatggatg
     2581 actcggaagt ctgaagaaac aattacccca tggaattttg aagaagttgt cgataaaggt
     2641 gcttcagctc aatcatttat tgaacgcatg acaaactttg ataaaaatct tccaaatgaa
     2701 aaagtactac caaaacatag tttgctttat gagtatttta cggtttataa cgaattgaca
     2761 aaggtcaaat atgttactga aggaatgcga aaaccagcat ttctttcagg tgaacagaag
     2821 aaagccattg ttgatttact cttcaaaaca aatcgaaaag taaccgttaa gcaattaaaa
     2881 gaagattatt tcaaaaaaat agaatgtttt gatagtgttg aaatttcagg agttgaagat
     2941 agatttaatg cttcattagg tacctaccat gatttgctaa aaattattaa agataaagat
     3001 tttttggata atgaagaaaa tgaagatatc ttagaggata ttgttttaac attgacctta
     3061 tttgaagata gggagatgat tgaggaaaga cttaaaacat atgctcacct ctttgatgat
     3121 aaggtgatga aacagcttaa acgtcgccgt tatactggtt ggggacgttt gtctcgaaaa
     3181 ttgattaatg gtattaggga taagcaatct ggcaaaacaa tattagattt tttgaaatca
     3241 gatggttttg ccaatcgcaa ttttatgcag ctgatccatg atgatagttt gacatttaaa
     3301 gaagacattc aaaaagcaca agtgtctgga caaggcgata gtttacatga acatattgca
     3361 aatttagctg gtagccctgc tattaaaaaa ggtattttac agactgtaaa agttgttgat
     3421 gaattggtca aagtaatggg gcggcataag ccagaaaata tcgttattga aatggcacgt
     3481 gaaaatcaga caactcaaaa gggccagaaa aattcgcgag agcgtatgaa acgaatcgaa
     3541 gaaggtatca aagaattagg aagtcagatt cttaaagagc atcctgttga aaatactcaa
     3601 ttgcaaaatg aaaagctcta tctctattat ctccaaaatg gaagagacat gtatgtggac
     3661 caagaattag atattaatcg tttaagtgat tatgatgtcg atgccattgt tccacaaagt
     3721 ttccttaaag acgattcaat agacaataag gtcttaacgc gttctgataa aaatcgtggt
     3781 aaatcggata acgttccaag tgaagaagta gtcaaaaaga tgaaaaacta ttggagacaa
     3841 cttctaaacg ccaagttaat cactcaacgt aagtttgata atttaacgaa agctgaacgt
     3901 ggaggtttga gtgaacttga taaagctggt tttatcaaac gccaattggt tgaaactcgc
     3961 caaatcacta agcatgtggc acaaattttg gatagtcgca tgaatactaa atacgatgaa
     4021 aatgataaac ttattcgaga ggttaaagtg attaccttaa aatctaaatt agtttctgac
     4081 ttccgaaaag atttccaatt ctataaagta cgtgagatta acaattacca tcatgcccat
     4141 gatgcgtatc taaatgccgt cgttggaact gctttgatta agaaatatcc aaaacttgaa
     4201 tcggagtttg tctatggtga ttataaagtt tatgatgttc gtaaaatgat tgctaagtct
     4261 gagcaagaaa taggcaaagc aaccgcaaaa tatttctttt actctaatat catgaacttc
     4321 ttcaaaacag aaattacact tgcaaatgga gagattcgca aacgccctct aatcgaaact
     4381 aatggggaaa ctggagaaat tgtctgggat aaagggcgag attttgccac agtgcgcaaa
     4441 gtattgtcca tgccccaagt caatattgtc aagaaaacag aagtacagac aggcggattc
     4501 tccaaggagt caattttacc aaaaagaaat tcggacaagc ttattgctcg taaaaaagac
     4561 tgggatccaa aaaaatatgg tggttttgat agtccaacgg tagcttattc agtcctagtg
     4621 gttgctaagg tggaaaaagg gaaatcgaag aagttaaaat ccgttaaaga gttactaggg
     4681 atcacaatta tggaaagaag ttcctttgaa aaaaatccga ttgacttttt agaagctaaa
     4741 ggatataagg aagttaaaaa agacttaatc attaaactac ctaaatatag tctttttgag
     4801 ttagaaaacg gtcgtaaacg gatgctggct agtgccggag aattacaaaa aggaaatgag
     4861 ctggctctgc caagcaaata tgtgaatttt ttatatttag ctagtcatta tgaaaagttg
     4921 aagggtagtc cagaagataa cgaacaaaaa caattgtttg tggagcagca taagcattat
     4981 ttagatgaga ttattgagca aatcagtgaa ttttctaagc gtgttatttt agcagatgcc
     5041 aatttagata aagttcttag tgcatataac aaacatagag acaaaccaat acgtgaacaa
     5101 gcagaaaata ttattcattt atttacgttg acgaatcttg gagctcccgc tgcttttaaa
     5161 tattttgata caacaattga tcgtaaacga tatacgtcta caaaagaagt tttagatgcc
     5221 actcttatcc atcaatccat cactggtctt tatgaaacac gcattgattt gagtcagcta
     5281 ggaggtgact ga
//
