LOCUS       sequence-238731-        4536 bp    DNA     linear   UNA 15-DEC-2025
DEFINITION  obtaining full Cas9 RNP.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      natural DNA sequence
  ORGANISM  unspecified
REFERENCE   1  (bases 1 to 4536)
  AUTHORS   Qiao J, Li W, Lin S, Sun W, Ma L, Liu Y
  TITLE     Co-expression of Cas9 and single-guided RNAs in Escherichia coli 
            streamlines production of Cas9 ribonucleoproteins.
  JOURNAL   Commun Biol. 2019 May 3;2:161. doi: 10.1038/s42003-019-0402-x. 
            eCollection 2019.
  PUBMED    31069270
REFERENCE   2  (bases 1 to 4536)
  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: "Commun 
            Biol."; date: "2019-05-3"; volume: "2:161. doi"; pages: " 
            10.1038/s42003-019-0402-x. eCollection 2019"
COMMENT     Sequence Label: pCold CL7-Cas9
FEATURES             Location/Qualifiers
     source          1..4536
                     /mol_type="genomic DNA"
                     /organism="unspecified"
     CDS             409..432
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     CDS             433..4536
                     /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
                     NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY
                     FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD
                     SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER
                     LKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFM
                     QLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR
                     HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY
                     LYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVP
                     SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH
                     VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL
                     NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT
                     EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK
                     ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI
                     TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE
                     LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD
                     ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL
                     DATLIHQSITGLYETRIDLSQLGGD"
     CDS             433..4536
                     /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
                     NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY
                     FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD
                     SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER
                     LKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFM
                     QLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR
                     HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY
                     LYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVP
                     SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH
                     VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL
                     NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT
                     EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK
                     ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI
                     TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE
                     LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD
                     ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL
                     DATLIHQSITGLYETRIDLSQLGGD"
ORIGIN
        1 atgagcaaaa gcaatgaacc gggtaaagca accggtgaag gtaaaccggt taataacaaa
       61 tggctgaaca atgccggtaa agatctgggt agtccggttc cggatcgtat tgcaaataaa
      121 ctgcgtgata aagaattcga gagcttcgat gattttcgtg aaaccttttg ggaagaagtt
      181 agcaaagatc ctgaactgag caaacagttt agccgcaata acaatgatcg tatgaaagtt
      241 ggtaaagcac cgaaaacacg tacccaggat gttagcggta aacgtacctc atttgaactg
      301 aatcatcaga aaccgattga acagaatggt ggcgtttatg atatggataa cattagcgtt
      361 gttaccccga aacgcaacat tgatattgaa ggcggtggtg gtggttcttt ggaggttttg
      421 ttccagggtc caatggataa gaaatactca ataggcttag atatcggcac aaatagcgtc
      481 ggatgggcgg tgatcactga tgaatataag gttccgtcta aaaagttcaa ggttctggga
      541 aatacagacc gccacagtat caaaaaaaat cttatagggg ctcttttatt tgacagtgga
      601 gagacagcgg aagcgactcg tctcaaacgg acagctcgta gaaggtatac acgtcggaag
      661 aatcgtattt gttatctaca ggagattttt tcaaatgaga tggcgaaagt agatgatagt
      721 ttctttcatc gacttgaaga gtcttttttg gtggaagaag acaagaagca tgaacgtcat
      781 cctatttttg gaaatatagt agatgaagtt gcttatcatg agaaatatcc aactatctat
      841 catctgcgaa aaaaattggt agattctact gataaagcgg atttgcgctt aatctatttg
      901 gccttagcgc atatgattaa gtttcgtggt cattttttga ttgagggaga tttaaatcct
      961 gataatagtg atgtggacaa actatttatc cagttggtac aaacctacaa tcaattattt
     1021 gaagaaaacc ctattaacgc aagtggagta gatgctaaag cgattctttc tgcacgattg
     1081 agtaaatcaa gacgattaga aaatctcatt gctcagctcc ccggtgagaa gaaaaatggc
     1141 ttatttggga atctcattgc tttgtcattg ggtttgaccc ctaattttaa atcaaatttt
     1201 gatttggcag aagatgctaa attacagctt tcaaaagata cttacgatga tgatttagat
     1261 aatttattgg cgcaaattgg agatcaatat gctgatttgt ttttggcagc taagaattta
     1321 tcagatgcta ttttactttc agatatccta agagtaaata ctgaaataac taaggctccc
     1381 ctatcagctt caatgattaa acgctacgat gaacatcatc aagacttgac tcttttaaaa
     1441 gctttagttc gacaacaact tccagaaaag tataaagaaa tcttttttga tcaatcaaaa
     1501 aacggatatg caggttatat tgatggggga gctagccaag aagaatttta taaatttatc
     1561 aaaccaattt tagaaaaaat ggatggtact gaggaattat tggtgaaact aaatcgtgaa
     1621 gatttgctgc gcaagcaacg gacctttgac aacggctcta ttccccatca aattcacttg
     1681 ggtgagctgc atgctatttt gagaagacaa gaagactttt atccattttt aaaagacaat
     1741 cgtgagaaga ttgaaaaaat cttgactttt cgcattcctt attatgttgg tccattggcg
     1801 cgtggcaata gtcgttttgc atggatgact cggaagtctg aagaaacaat taccccatgg
     1861 aattttgaag aagttgtcga taaaggtgct tcagctcaat catttattga acgcatgaca
     1921 aactttgata aaaatcttcc aaatgaaaaa gtactaccaa aacatagttt gctttatgag
     1981 tattttacgg tttataacga attgacaaag gtcaaatatg ttactgaagg aatgcgaaaa
     2041 ccagcatttc tttcaggtga acagaagaaa gccattgttg atttactctt caaaacaaat
     2101 cgaaaagtaa ccgttaagca attaaaagaa gattatttca aaaaaataga atgttttgat
     2161 agtgttgaaa tttcaggagt tgaagataga tttaatgctt cattaggtac ctaccatgat
     2221 ttgctaaaaa ttattaaaga taaagatttt ttggataatg aagaaaatga agatatctta
     2281 gaggatattg ttttaacatt gaccttattt gaagataggg agatgattga ggaaagactt
     2341 aaaacatatg ctcacctctt tgatgataag gtgatgaaac agcttaaacg tcgccgttat
     2401 actggttggg gacgtttgtc tcgaaaattg attaatggta ttagggataa gcaatctggc
     2461 aaaacaatat tagatttttt gaaatcagat ggttttgcca atcgcaattt tatgcagctg
     2521 atccatgatg atagtttgac atttaaagaa gacattcaaa aagcacaagt gtctggacaa
     2581 ggcgatagtt tacatgaaca tattgcaaat ttagctggta gccctgctat taaaaaaggt
     2641 attttacaga ctgtaaaagt tgttgatgaa ttggtcaaag taatggggcg gcataagcca
     2701 gaaaatatcg ttattgaaat ggcacgtgaa aatcagacaa ctcaaaaggg ccagaaaaat
     2761 tcgcgagagc gtatgaaacg aatcgaagaa ggtatcaaag aattaggaag tcagattctt
     2821 aaagagcatc ctgttgaaaa tactcaattg caaaatgaaa agctctatct ctattatctc
     2881 caaaatggaa gagacatgta tgtggaccaa gaattagata ttaatcgttt aagtgattat
     2941 gatgtcgatc acattgttcc acaaagtttc cttaaagacg attcaataga caataaggtc
     3001 ttaacgcgtt ctgataaaaa tcgtggtaaa tcggataacg ttccaagtga agaagtagtc
     3061 aaaaagatga aaaactattg gagacaactt ctaaacgcca agttaatcac tcaacgtaag
     3121 tttgataatt taacgaaagc tgaacgtgga ggtttgagtg aacttgataa agctggtttt
     3181 atcaaacgcc aattggttga aactcgccaa atcactaagc atgtggcaca aattttggat
     3241 agtcgcatga atactaaata cgatgaaaat gataaactta ttcgagaggt taaagtgatt
     3301 accttaaaat ctaaattagt ttctgacttc cgaaaagatt tccaattcta taaagtacgt
     3361 gagattaaca attaccatca tgcccatgat gcgtatctaa atgccgtcgt tggaactgct
     3421 ttgattaaga aatatccaaa acttgaatcg gagtttgtct atggtgatta taaagtttat
     3481 gatgttcgta aaatgattgc taagtctgag caagaaatag gcaaagcaac cgcaaaatat
     3541 ttcttttact ctaatatcat gaacttcttc aaaacagaaa ttacacttgc aaatggagag
     3601 attcgcaaac gccctctaat cgaaactaat ggggaaactg gagaaattgt ctgggataaa
     3661 gggcgagatt ttgccacagt gcgcaaagta ttgtccatgc cccaagtcaa tattgtcaag
     3721 aaaacagaag tacagacagg cggattctcc aaggagtcaa ttttaccaaa aagaaattcg
     3781 gacaagctta ttgctcgtaa aaaagactgg gatccaaaaa aatatggtgg ttttgatagt
     3841 ccaacggtag cttattcagt cctagtggtt gctaaggtgg aaaaagggaa atcgaagaag
     3901 ttaaaatccg ttaaagagtt actagggatc acaattatgg aaagaagttc ctttgaaaaa
     3961 aatccgattg actttttaga agctaaagga tataaggaag ttaaaaaaga cttaatcatt
     4021 aaactaccta aatatagtct ttttgagtta gaaaacggtc gtaaacggat gctggctagt
     4081 gccggagaat tacaaaaagg aaatgagctg gctctgccaa gcaaatatgt gaatttttta
     4141 tatttagcta gtcattatga aaagttgaag ggtagtccag aagataacga acaaaaacaa
     4201 ttgtttgtgg agcagcataa gcattattta gatgagatta ttgagcaaat cagtgaattt
     4261 tctaagcgtg ttattttagc agatgccaat ttagataaag ttcttagtgc atataacaaa
     4321 catagagaca aaccaatacg tgaacaagca gaaaatatta ttcatttatt tacgttgacg
     4381 aatcttggag ctcccgctgc ttttaaatat tttgatacaa caattgatcg taaacgatat
     4441 acgtctacaa aagaagtttt agatgccact cttatccatc aatccatcac tggtctttat
     4501 gaaacacgca ttgatttgag tcagctagga ggtgac
//
