LOCUS       sequence-148392-        6201 bp    DNA     linear   UNA 15-DEC-2025
DEFINITION  Expression of His6-MBP-tagged Cas9-NLS-mCherry protein in E. coli.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      natural DNA sequence
  ORGANISM  unspecified
REFERENCE   1  (bases 1 to 6201)
  AUTHORS   Burger A, Lindsay H, Felker A, Hess C, Anders C, Chiavacci E, Zaugg 
            J, Weber LM, Catena R, Jinek M, Robinson MD, Mosimann C
  TITLE     Maximizing mutagenesis with solubilized CRISPR-Cas9 
            ribonucleoprotein complexes.
  JOURNAL   Development. 2016 Apr 29. pii: dev.134809.
  PUBMED    27130213
REFERENCE   2  (bases 1 to 6201)
  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: 
            "Development. 2016 Apr 29. pii: dev.134809."
COMMENT     Sequence Label: pMJ923
FEATURES             Location/Qualifiers
     source          1..6201
                     /mol_type="genomic DNA"
                     /organism="unspecified"
     CDS             13..30
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             40..1140
                     /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     1114..1137
                     /label=MBP-F
                     /note="Maltose binding protein, forward primer"
     CDS             1195..1215
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQS"
     regulatory      1219..1228
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1225..5328
                     /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             1225..5328
                     /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             5332..5358
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             5338..5364
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /note="This is a variant of the standard HA tag, which has 
                     sequence YPYDVPDYA. Some anti-HA antibodies may not 
                     recognize this variant."
                     /translation="YDVPDYASL"
     CDS             5377..5397
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             5404..5424
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             5464..6168
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="VSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEGT
                     QTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFE
                     DGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALKG
                     EIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERAE
                     GRHSTGGMDELYK"
     primer_bind     complement(5606..5624)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     5874..5893
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     CDS             6175..6195
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
ORIGIN
        1 atgaaatctt ctcaccatca ccatcaccat ggttcttcta tgaaaatcga agaaggtaaa
       61 ctggtaatct ggattaacgg cgataaaggc tataacggtc tcgctgaagt cggtaagaaa
      121 ttcgagaaag ataccggaat taaagtcacc gttgagcatc cggataaact ggaagagaaa
      181 ttcccacagg ttgcggcaac tggcgatggc cctgacatta tcttctgggc acacgaccgc
      241 tttggtggct acgctcaatc tggcctgttg gctgaaatca ccccggacaa agcgttccag
      301 gacaagctgt atccgtttac ctgggatgcc gtacgttaca acggcaagct gattgcttac
      361 ccgatcgctg ttgaagcgtt atcgctgatt tataacaaag atctgctgcc gaacccgcca
      421 aaaacctggg aagagatccc ggcgctggat aaagaactga aagcgaaagg taagagcgcg
      481 ctgatgttca acctgcaaga accgtacttc acctggccgc tgattgctgc tgacgggggt
      541 tatgcgttca agtatgaaaa cggcaagtac gacattaaag acgtgggcgt ggataacgct
      601 ggcgcgaaag cgggtctgac cttcctggtt gacctgatta aaaacaaaca catgaatgca
      661 gacaccgatt actccatcgc agaagctgcc tttaataaag gcgaaacagc gatgaccatc
      721 aacggcccgt gggcatggtc caacatcgac accagcaaag tgaattatgg tgtaacggta
      781 ctgccgacct tcaagggtca accatccaaa ccgttcgttg gcgtgctgag cgcaggtatt
      841 aacgccgcca gtccgaacaa agagctggca aaagagttcc tcgaaaacta tctgctgact
      901 gatgaaggtc tggaagcggt taataaagac aaaccgctgg gtgccgtagc gctgaagtct
      961 tacgaggaag agttggcgaa agatccacgt attgccgcca ctatggaaaa cgcccagaaa
     1021 ggtgaaatca tgccgaacat cccgcagatg tccgctttct ggtatgccgt gcgtactgcg
     1081 gtgatcaacg ccgccagcgg tcgtcagact gtcgatgaag ccctgaaaga cgcgcagact
     1141 aattcgagct cgaacaacaa caacaataac aataacaaca acctcgggat cgaggaaaac
     1201 ctgtacttcc aatccaatgc caccatggac aagaagtaca gcatcggcct ggacatcggc
     1261 accaactctg tgggctgggc cgtgatcacc gacgagtaca aggtgcccag caagaaattc
     1321 aaggtgctgg gcaacaccga ccggcacagc atcaagaaga acctgatcgg cgccctgctg
     1381 ttcgacagcg gagaaacagc cgaggccacc cggctgaaga gaaccgccag aagaagatac
     1441 accagacgga agaaccggat ctgctatctg caagagatct tcagcaacga gatggccaag
     1501 gtggacgaca gcttcttcca cagactggaa gagtccttcc tggtggaaga ggataagaag
     1561 cacgagcggc accccatctt cggcaacatc gtggacgagg tggcctacca cgagaagtac
     1621 cccaccatct accacctgag aaagaaactg gtggacagca ccgacaaggc cgacctgcgg
     1681 ctgatctatc tggccctggc ccacatgatc aagttccggg gccacttcct gatcgagggc
     1741 gacctgaacc ccgacaacag cgacgtggac aagctgttca tccagctggt gcagacctac
     1801 aaccagctgt tcgaggaaaa ccccatcaac gccagcggcg tggacgccaa ggccatcctg
     1861 tctgccagac tgagcaagag cagacggctg gaaaatctga tcgcccagct gcccggcgag
     1921 aagaagaatg gcctgttcgg caacctgatt gccctgagcc tgggcctgac ccccaacttc
     1981 aagagcaact tcgacctggc cgaggatgcc aaactgcagc tgagcaagga cacctacgac
     2041 gacgacctgg acaacctgct ggcccagatc ggcgaccagt acgccgacct gtttctggcc
     2101 gccaagaacc tgtccgacgc catcctgctg agcgacatcc tgagagtgaa caccgagatc
     2161 accaaggccc ccctgagcgc ctctatgatc aagagatacg acgagcacca ccaggacctg
     2221 accctgctga aagctctcgt gcggcagcag ctgcctgaga agtacaaaga gattttcttc
     2281 gaccagagca agaacggcta cgccggctac atcgatggcg gagccagcca ggaagagttc
     2341 tacaagttca tcaagcccat cctggaaaag atggacggca ccgaggaact gctcgtgaag
     2401 ctgaacagag aggacctgct gcggaagcag cggaccttcg acaacggcag catcccccac
     2461 cagatccacc tgggagagct gcacgccatt ctgcggcggc aggaagattt ttacccattc
     2521 ctgaaggaca accgggaaaa gatcgagaag atcctgacct tccgcatccc ctactacgtg
     2581 ggccctctgg ccaggggaaa cagcagattc gcctggatga ccagaaagag cgaggaaacc
     2641 atcaccccct ggaacttcga ggaagtggtg gacaagggcg ccagcgccca gagcttcatc
     2701 gagcggatga ccaacttcga taagaacctg cccaacgaga aggtgctgcc caagcacagc
     2761 ctgctgtacg agtacttcac cgtgtacaac gagctgacca aagtgaaata cgtgaccgag
     2821 ggaatgagaa agcccgcctt cctgagcggc gagcagaaaa aagccatcgt ggacctgctg
     2881 ttcaagacca accggaaagt gaccgtgaag cagctgaaag aggactactt caagaaaatc
     2941 gagtgcttcg actccgtgga aatctccggc gtggaagatc ggttcaacgc ctccctgggc
     3001 acataccacg atctgctgaa aattatcaag gacaaggact tcctggacaa tgaggaaaac
     3061 gaggacattc tggaagatat cgtgctgacc ctgacactgt ttgaggacag agagatgatc
     3121 gaggaacggc tgaaaaccta tgcccacctg ttcgacgaca aagtgatgaa gcagctgaag
     3181 cggcggagat acaccggctg gggcaggctg agccggaagc tgatcaacgg catccgggac
     3241 aagcagtccg gcaagacaat cctggatttc ctgaagtccg acggcttcgc caacagaaac
     3301 ttcatgcagc tgatccacga cgacagcctg acctttaaag aggacatcca gaaagcccag
     3361 gtgtccggcc agggcgatag cctgcacgag cacattgcca atctggccgg cagccccgcc
     3421 attaagaagg gcatcctgca gacagtgaag gtggtggacg agctcgtgaa agtgatgggc
     3481 cggcacaagc ccgagaacat cgtgatcgaa atggccagag agaaccagac cacccagaag
     3541 ggacagaaga acagccgcga gagaatgaag cggatcgaag agggcatcaa agagctgggc
     3601 agccagatcc tgaaagaaca ccccgtggaa aacacccagc tgcagaacga gaagctgtac
     3661 ctgtactacc tgcagaatgg gcgggatatg tacgtggacc aggaactgga catcaaccgg
     3721 ctgtccgact acgatgtgga ccatatcgtg cctcagagct ttctgaagga cgactccatc
     3781 gataacaaag tgctgactcg gagcgacaag aaccggggca agagcgacaa cgtgccctcc
     3841 gaagaggtcg tgaagaagat gaagaactac tggcgccagc tgctgaatgc caagctgatt
     3901 acccagagga agttcgacaa tctgaccaag gccgagagag gcggcctgag cgaactggat
     3961 aaggccggct tcatcaagag acagctggtg gaaacccggc agatcacaaa gcacgtggca
     4021 cagatcctgg actcccggat gaacactaag tacgacgaga acgacaaact gatccgggaa
     4081 gtgaaagtga tcaccctgaa gtccaagctg gtgtccgatt tccggaagga tttccagttt
     4141 tacaaagtgc gcgagatcaa caactaccac cacgcccacg acgcctacct gaacgccgtc
     4201 gtgggaaccg ccctgatcaa aaagtaccct aagctggaaa gcgagttcgt gtacggcgac
     4261 tacaaggtgt acgacgtgcg gaagatgatc gccaagagcg agcaggaaat cggcaaggct
     4321 accgccaagt acttcttcta cagcaacatc atgaactttt tcaagaccga gattaccctg
     4381 gccaacggcg agatccggaa gcggcctctg atcgagacaa acggcgaaac aggcgagatc
     4441 gtgtgggata agggccggga ctttgccacc gtgcggaaag tgctgtctat gccccaagtg
     4501 aatatcgtga aaaagaccga ggtgcagaca ggcggcttca gcaaagagtc tatcctgccc
     4561 aagaggaaca gcgacaagct gatcgccaga aagaaggact gggaccctaa gaagtacggc
     4621 ggcttcgaca gccccaccgt ggcctattct gtgctggtgg tggccaaagt ggaaaagggc
     4681 aagtccaaga aactgaagag tgtgaaagag ctgctgggga tcaccatcat ggaaagaagc
     4741 agcttcgaga agaatcccat cgactttctg gaagccaagg gctacaaaga agtgaaaaag
     4801 gacctgatca tcaagctgcc taagtactcc ctgttcgagc tggaaaacgg ccggaagaga
     4861 atgctggcct ctgccggcga actgcagaag ggaaacgaac tggccctgcc ctccaaatat
     4921 gtgaacttcc tgtacctggc cagccactat gagaagctga agggctcccc cgaggataat
     4981 gagcagaaac agctgtttgt ggaacagcac aaacactacc tggacgagat catcgagcag
     5041 atcagcgagt tctccaagag agtgatcctg gccgacgcta atctggacaa ggtgctgagc
     5101 gcctacaaca agcacagaga caagcctatc agagagcagg ccgagaatat catccacctg
     5161 tttaccctga ccaatctggg agcccctgcc gccttcaagt actttgacac caccatcgac
     5221 cggaagaggt acaccagcac caaagaggtg ctggacgcca ccctgatcca ccagagcatc
     5281 accggcctgt acgagacacg gatcgacctg tctcagctgg gaggcgacgc ctatccctat
     5341 gacgtgcccg attatgccag cctgggcagc ggctccccca agaaaaaacg caaggtggaa
     5401 gatcctaaga aaaagcggaa agtggacggc attggtagtg ggagcaacgg cagcagcgga
     5461 tccgtgagca agggcgagga ggataacatg gccatcatca aggagttcat gcgcttcaag
     5521 gtgcacatgg agggctccgt gaacggccac gagttcgaga tcgagggcga gggcgagggc
     5581 cgcccctacg agggcaccca gaccgccaag ctgaaggtga ccaagggtgg ccccctgccc
     5641 ttcgcctggg acatcctgtc ccctcagttc atgtacggct ccaaggccta cgtgaagcac
     5701 cccgccgaca tccccgacta cttgaagctg tccttccccg agggcttcaa gtgggagcgc
     5761 gtgatgaact tcgaggacgg cggcgtggtg accgtgaccc aggactcctc cctccaggac
     5821 ggcgagttca tctacaaggt gaagctgcgc ggcaccaact tcccctccga cggccccgta
     5881 atgcagaaga agaccatggg ctgggaggcc tcctccgagc ggatgtaccc cgaggacggc
     5941 gccctgaagg gcgagatcaa gcagaggctg aagctgaagg acggcggcca ctacgacgct
     6001 gaggtcaaga ccacctacaa ggccaagaag cccgtgcagc tgcccggcgc ctacaacgtc
     6061 aacatcaagt tggacatcac ctcccacaac gaggactaca ccatcgtgga acagtacgaa
     6121 cgcgccgagg gccgccactc caccggcggc atggacgagc tgtacaaggg gtcacctaag
     6181 aaaaaacgaa aagttgagta a
//
