LOCUS       sequence-53556-e        4434 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 4434)
  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 4434)
  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: pMJ839
FEATURES             Location/Qualifiers
     source          1..4434
                     /mol_type="genomic DNA"
                     /organism="unspecified"
     CDS             4..21
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             25..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="MKIEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLE
                     EKFPQVAATGDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDAVRYNGKL
                     IAYPIAVEALSLIYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWPLIA
                     ADGGYAFKYENGKYDIKDVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNKGE
                     TAMTINGPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFL
                     ENYLLTDEGLEAVNKDKPLGAVALKSYEEELAKDPRIAATMENAQKGEIMPNIPQMSAF
                     WYAVRTAVINAASGRQTVDEALKDAQT"
     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             1186..4434
                     /codon_start=1
                     /product="Cas9 endonuclease from the Neisseria meningitidis
                     Type II CRISPR/Cas system (Esvelt et al., 2013)"
                     /label=NmCas9
                     /note="generates RNA-guided double strand breaks in DNA"
                     /translation="MAAFKPNPINYILGLDIGIASVGWAMVEIDEDENPICLIDLGVRV
                     FERAEVPKTGDSLAMARRLARSVRRLTRRRAHRLLRARRLLKREGVLQAADFDENGLIK
                     SLPNTPWQLRAAALDRKLTPLEWSAVLLHLIKHRGYLSQRKNEGETADKELGALLKGVA
                     DNAHALQTGDFRTPAELALNKFEKESGHIRNQRGDYSHTFSRKDLQAELILLFEKQKEF
                     GNPHVSGGLKEGIETLLMTQRPALSGDAVQKMLGHCTFEPAEPKAAKNTYTAERFIWLT
                     KLNNLRILEQGSERPLTDTERATLMDEPYRKSKLTYAQARKLLGLEDTAFFKGLRYGKD
                     NAEASTLMEMKAYHAISRALEKEGLKDKKSPLNLSPELQDEIGTAFSLFKTDEDITGRL
                     KDRIQPEILEALLKHISFDKFVQISLKALRRIVPLMEQGKRYDEACAEIYGDHYGKKNT
                     EEKIYLPPIPADEIRNPVVLRALSQARKVINGVVRRYGSPARIHIETAREVGKSFKDRK
                     EIEKRQEENRKDREKAAAKFREYFPNFVGEPKSKDILKLRLYEQQHGKCLYSGKEINLG
                     RLNEKGYVEIDHALPFSRTWDDSFNNKVLVLGSENQNKGNQTPYEYFNGKDNSREWQEF
                     KARVETSRFPRSKKQRILLQKFDEDGFKERNLNDTRYVNRFLCQFVADRMRLTGKGKKR
                     VFASNGQITNLLRGFWGLRKVRAENDRHHALDAVVVACSTVAMQQKITRFVRYKEMNAF
                     DGKTIDKETGEVLHQKTHFPQPWEFFAQEVMIRVFGKPDGKPEFEEADTPEKLRTLLAE
                     KLSSRPEAVHEYVTPLFVSRAPNRKMSGQGHMETVKSAKRLDEGVSVLRVPLTQLKLKD
                     LEKMVNREREPKLYEALKARLEAHKDDPAKAFAEPFYKYDKAGNRTQQVKAVRVEQVQK
                     TGVWVRNHNGIADNATMVRVDVFEKGDKYYLVPIYSWQVAKGILPDRAVVQGKDEEDWQ
                     LIDDSFNFKFSLHPNDLVEVITKKARMFGYFASCHRGTGNINIRIHDLDHKIGKNGILE
                     GIGVKTALSFQKYQIDELGKEIRPCRLKKRPPVR"
     CDS             1186..4431
                     /codon_start=1
                     /product="Cas9 endonuclease from the Neisseria meningitidis
                     Type II CRISPR/Cas system (Esvelt et al., 2013)"
                     /label=NmCas9
                     /note="generates RNA-guided double strand breaks in DNA"
                     /translation="MAAFKPNPINYILGLDIGIASVGWAMVEIDEDENPICLIDLGVRV
                     FERAEVPKTGDSLAMARRLARSVRRLTRRRAHRLLRARRLLKREGVLQAADFDENGLIK
                     SLPNTPWQLRAAALDRKLTPLEWSAVLLHLIKHRGYLSQRKNEGETADKELGALLKGVA
                     DNAHALQTGDFRTPAELALNKFEKESGHIRNQRGDYSHTFSRKDLQAELILLFEKQKEF
                     GNPHVSGGLKEGIETLLMTQRPALSGDAVQKMLGHCTFEPAEPKAAKNTYTAERFIWLT
                     KLNNLRILEQGSERPLTDTERATLMDEPYRKSKLTYAQARKLLGLEDTAFFKGLRYGKD
                     NAEASTLMEMKAYHAISRALEKEGLKDKKSPLNLSPELQDEIGTAFSLFKTDEDITGRL
                     KDRIQPEILEALLKHISFDKFVQISLKALRRIVPLMEQGKRYDEACAEIYGDHYGKKNT
                     EEKIYLPPIPADEIRNPVVLRALSQARKVINGVVRRYGSPARIHIETAREVGKSFKDRK
                     EIEKRQEENRKDREKAAAKFREYFPNFVGEPKSKDILKLRLYEQQHGKCLYSGKEINLG
                     RLNEKGYVEIDHALPFSRTWDDSFNNKVLVLGSENQNKGNQTPYEYFNGKDNSREWQEF
                     KARVETSRFPRSKKQRILLQKFDEDGFKERNLNDTRYVNRFLCQFVADRMRLTGKGKKR
                     VFASNGQITNLLRGFWGLRKVRAENDRHHALDAVVVACSTVAMQQKITRFVRYKEMNAF
                     DGKTIDKETGEVLHQKTHFPQPWEFFAQEVMIRVFGKPDGKPEFEEADTPEKLRTLLAE
                     KLSSRPEAVHEYVTPLFVSRAPNRKMSGQGHMETVKSAKRLDEGVSVLRVPLTQLKLKD
                     LEKMVNREREPKLYEALKARLEAHKDDPAKAFAEPFYKYDKAGNRTQQVKAVRVEQVQK
                     TGVWVRNHNGIADNATMVRVDVFEKGDKYYLVPIYSWQVAKGILPDRAVVQGKDEEDWQ
                     LIDDSFNFKFSLHPNDLVEVITKKARMFGYFASCHRGTGNINIRIHDLDHKIGKNGILE
                     GIGVKTALSFQKYQIDELGKEIRPCRLKKRPPVR"
ORIGIN
        1 atgcatcacc atcaccatca ccccatgaaa 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 cctcgatggc tgccttcaaa
     1201 cctaatccaa tcaactacat cctcggcctc gatatcggca tcgcatccgt cggctgggcg
     1261 atggtagaaa ttgacgaaga tgaaaaccca atctgcctga ttgatttggg cgtgcgcgta
     1321 tttgagcgtg ccgaagtacc gaaaacaggc gactcccttg ccatggcaag gcgtttggcg
     1381 cgcagtgtcc gccgcctgac ccgccgtcgc gcccatcgcc tgcttcgggc ccgccgccta
     1441 ttgaaacgcg aaggcgtatt acaagccgct gattttgacg aaaacggctt gatcaaatcc
     1501 ttaccgaata caccatggca acttcgcgca gccgcattag accgcaaact gacgccttta
     1561 gagtggtcgg cagtcttgtt gcatttaatc aaacaccgcg gttatttgtc gcaaagaaaa
     1621 aacgagggcg aaactgccga taaagagctt ggcgctttgc tcaaaggcgt agccgacaat
     1681 gcccatgcct tacagacagg cgatttccgc acaccggccg aattggcttt aaataaattt
     1741 gagaaagaaa gcggccatat ccgcaatcag cgcggcgatt attcgcatac gttcagccgc
     1801 aaagatttac aggcggagct gattttgctg tttgaaaaac aaaaagaatt tggcaatccg
     1861 catgtttcag gcggccttaa agaaggtatt gaaaccctac tgatgacgca acgccctgcc
     1921 ctgtccggcg atgccgttca aaaaatgttg gggcattgca ccttcgaacc ggcagagccg
     1981 aaagccgcta aaaacaccta cacagccgaa cgtttcatct ggctgaccaa gctgaacaac
     2041 ctgcgtattt tagagcaagg cagcgagcgg ccattgaccg ataccgaacg cgccacgctt
     2101 atggacgagc catacagaaa atccaaactg acttacgcac aagcccgtaa gctgctgggt
     2161 ttagaagata ccgccttttt caaaggcttg cgctatggta aagacaatgc cgaagcctca
     2221 acattgatgg aaatgaaggc ctaccatgcc atcagccgtg cactggaaaa agaaggattg
     2281 aaagacaaaa aatccccatt aaacctttct cccgaattac aagacgaaat cggcacggca
     2341 ttctccctgt tcaaaaccga tgaagacatt acaggccgtc tgaaagaccg tatacagccc
     2401 gaaatcttag aagcgctgtt gaaacacatc agcttcgata agttcgtcca aatttccttg
     2461 aaagcattgc gccgaattgt gcctctaatg gaacaaggca aacgttacga tgaagcctgc
     2521 gccgaaatct acggagacca ttacggcaag aagaatacgg aagaaaagat ttatctgccg
     2581 ccgattcccg ccgacgaaat ccgcaacccc gtcgtcttgc gcgccttatc tcaagcacgt
     2641 aaggtcatta acggcgtggt acgccgttac ggctccccag ctcgtatcca tattgaaact
     2701 gcaagggaag taggtaaatc gtttaaagac cgcaaagaaa ttgagaaacg ccaagaagaa
     2761 aaccgcaaag acagggaaaa agccgccgcc aaattccgag agtatttccc caattttgtc
     2821 ggagaaccca aatccaaaga tattctgaaa ctgcgcctgt acgagcaaca acacggcaaa
     2881 tgcctgtatt cgggcaaaga aatcaactta ggccgtctga acgaaaaagg ctatgtcgaa
     2941 atcgaccatg ccctgccgtt ctcgcgcaca tgggacgaca gtttcaacaa taaagtactg
     3001 gtattgggca gcgaaaacca aaacaaaggc aatcaaaccc cttacgaata cttcaacggc
     3061 aaagacaaca gccgcgaatg gcaggaattt aaagcgcgtg tcgaaaccag ccgtttcccg
     3121 cgcagtaaaa aacaacggat tctgctgcaa aaattcgatg aagacggctt taaagaacgc
     3181 aatctgaacg acacgcgcta cgtcaaccgt ttcctgtgtc aatttgttgc cgaccgtatg
     3241 cggctgacag gtaaaggcaa gaaacgtgtc tttgcatcca acggacaaat taccaatctg
     3301 ttgcgcggct tttggggatt gcgcaaagtg cgtgcggaaa acgaccgcca tcacgccttg
     3361 gacgccgtcg tcgttgcctg ctcgaccgtt gccatgcagc agaaaattac ccgttttgta
     3421 cgctataaag agatgaacgc gtttgacggt aaaaccatag acaaagaaac aggagaagtg
     3481 ctgcatcaaa aaacacactt cccacaacct tgggaatttt tcgcacaaga agtcatgatt
     3541 cgcgtcttcg gcaaaccgga cggcaaaccc gaattcgaag aagccgatac cccagaaaaa
     3601 ctgcgcacgt tgcttgccga aaaattatca tctcgccccg aagccgtaca cgaatacgtt
     3661 acgccactgt ttgtttcacg cgcgcccaat cggaagatga gcgggcaagg gcatatggag
     3721 accgtcaaat ccgccaaacg actggacgaa ggcgtcagcg tgttgcgcgt accgttaaca
     3781 cagttaaaac tgaaagactt ggaaaaaatg gtcaatcggg agcgcgaacc taagctatac
     3841 gaagcactga aagcacggct ggaagcacat aaagacgatc ctgccaaagc ctttgccgag
     3901 ccgttttaca aatacgataa agcaggcaac cgcacccaac aggtaaaagc cgtacgcgta
     3961 gagcaagtac agaaaaccgg cgtatgggtg cgcaaccata acggtattgc cgacaacgca
     4021 accatggtgc gcgtagatgt gtttgagaaa ggcgacaagt attatctggt accgatttac
     4081 agttggcagg tagcgaaagg gattttgccg gatagggctg ttgtacaagg aaaagatgaa
     4141 gaagattggc aacttattga tgatagtttc aactttaaat tctcattaca ccctaatgat
     4201 ttagtcgagg ttataacaaa aaaagctaga atgtttggtt actttgccag ctgccatcga
     4261 ggcaccggta atatcaatat acgcattcat gatcttgatc ataaaattgg caaaaatgga
     4321 atactggaag gtatcggcgt caaaaccgcc ctttcattcc aaaaatacca aattgacgaa
     4381 ctgggcaaag aaatcagacc atgccgtctg aaaaaacgcc cgcctgtccg ttaa
//
