LOCUS       sequence-141364-        4920 bp    DNA     linear   UNA 15-DEC-2025
DEFINITION  Expresses dCas9-2xNLS-EGFP in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      natural DNA sequence
  ORGANISM  unspecified
REFERENCE   1  (bases 1 to 4920)
  AUTHORS   Nelles DA, Fang MY, O'Connell MR, Xu JL, Markmiller SJ, Doudna JA, 
            Yeo GW
  TITLE     Programmable RNA Tracking in Live Cells with CRISPR/Cas9.
  JOURNAL   Cell. 2016 Mar 16. pii: S0092-8674(16)30204-5. doi: 
            10.1016/j.cell.2016.02.054.
  PUBMED    26997482
REFERENCE   2  (bases 1 to 4920)
  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: "Cell. 2016 
            Mar 16. pii: S0092-8674(16)30204-5. doi: 
            10.1016/j.cell.2016.02.054."
COMMENT     Sequence Label: pCDNA3.1- dCas9-2xNLS-EGFP
FEATURES             Location/Qualifiers
     source          1..4920
                     /mol_type="genomic DNA"
                     /organism="unspecified"
     CDS             join(1..27,28..30,31..2517,2518..2520,2521..4104)
                     /codon_start=1
                     /product="catalytically dead mutant of the Cas9 
                     endonuclease from the Streptococcus pyogenes Type II 
                     CRISPR/Cas system"
                     /label=dCas9
                     /note="RNA-guided DNA-binding protein that lacks 
                     endonuclease activity due to the D10A mutation in the RuvC 
                     catalytic domain and the H840A mutation in the HNH 
                     catalytic domain"
                     /translation="MDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK
                     NLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES
                     FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK
                     FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL
                     ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ
                     IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR
                     QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL
                     RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG
                     NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY
                     FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD
                     SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER
                     LKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFM
                     QLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR
                     HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY
                     LYYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVP
                     SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH
                     VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL
                     NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT
                     EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK
                     ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI
                     TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE
                     LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD
                     ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL
                     DATLIHQSITGLYETRIDLSQLGGD"
     CDS             1..4104
                     /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="MDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK
                     NLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES
                     FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK
                     FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL
                     ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ
                     IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR
                     QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL
                     RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG
                     NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY
                     FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD
                     SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER
                     LKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFM
                     QLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR
                     HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY
                     LYYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVP
                     SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH
                     VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL
                     NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT
                     EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK
                     ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI
                     TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE
                     LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD
                     ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL
                     DATLIHQSITGLYETRIDLSQLGGD"
     CDS             4108..4134
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             4114..4140
                     /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             4153..4173
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             4180..4200
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             join(4204..4206,4207..4920)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     CDS             join(4204..4206,4207..4917)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     primer_bind     complement(4507..4526)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     4854..4875
                     /label=EGFP-C
                     /note="EGFP, forward primer"
ORIGIN
        1 atggacaaga agtacagcat cggcctggcc atcggcacca actctgtggg ctgggccgtg
       61 atcaccgacg agtacaaggt gcccagcaag aaattcaagg tgctgggcaa caccgaccgg
      121 cacagcatca agaagaacct gatcggcgcc ctgctgttcg acagcggaga aacagccgag
      181 gccacccggc tgaagagaac cgccagaaga agatacacca gacggaagaa ccggatctgc
      241 tatctgcaag agatcttcag caacgagatg gccaaggtgg acgacagctt cttccacaga
      301 ctggaagagt ccttcctggt ggaagaggat aagaagcacg agcggcaccc catcttcggc
      361 aacatcgtgg acgaggtggc ctaccacgag aagtacccca ccatctacca cctgagaaag
      421 aaactggtgg acagcaccga caaggccgac ctgcggctga tctatctggc cctggcccac
      481 atgatcaagt tccggggcca cttcctgatc gagggcgacc tgaaccccga caacagcgac
      541 gtggacaagc tgttcatcca gctggtgcag acctacaacc agctgttcga ggaaaacccc
      601 atcaacgcca gcggcgtgga cgccaaggcc atcctgtctg ccagactgag caagagcaga
      661 cggctggaaa atctgatcgc ccagctgccc ggcgagaaga agaatggcct gttcggcaac
      721 ctgattgccc tgagcctggg cctgaccccc aacttcaaga gcaacttcga cctggccgag
      781 gatgccaaac tgcagctgag caaggacacc tacgacgacg acctggacaa cctgctggcc
      841 cagatcggcg accagtacgc cgacctgttt ctggccgcca agaacctgtc cgacgccatc
      901 ctgctgagcg acatcctgag agtgaacacc gagatcacca aggcccccct gagcgcctct
      961 atgatcaaga gatacgacga gcaccaccag gacctgaccc tgctgaaagc tctcgtgcgg
     1021 cagcagctgc ctgagaagta caaagagatt ttcttcgacc agagcaagaa cggctacgcc
     1081 ggctacatcg atggcggagc cagccaggaa gagttctaca agttcatcaa gcccatcctg
     1141 gaaaagatgg acggcaccga ggaactgctc gtgaagctga acagagagga cctgctgcgg
     1201 aagcagcgga ccttcgacaa cggcagcatc ccccaccaga tccacctggg agagctgcac
     1261 gccattctgc ggcggcagga agatttttac ccattcctga aggacaaccg ggaaaagatc
     1321 gagaagatcc tgaccttccg catcccctac tacgtgggcc ctctggccag gggaaacagc
     1381 agattcgcct ggatgaccag aaagagcgag gaaaccatca ccccctggaa cttcgaggaa
     1441 gtggtggaca agggcgccag cgcccagagc ttcatcgagc ggatgaccaa cttcgataag
     1501 aacctgccca acgagaaggt gctgcccaag cacagcctgc tgtacgagta cttcaccgtg
     1561 tacaacgagc tgaccaaagt gaaatacgtg accgagggaa tgagaaagcc cgccttcctg
     1621 agcggcgagc agaaaaaagc catcgtggac ctgctgttca agaccaaccg gaaagtgacc
     1681 gtgaagcagc tgaaagagga ctacttcaag aaaatcgagt gcttcgactc cgtggaaatc
     1741 tccggcgtgg aagatcggtt caacgcctcc ctgggcacat accacgatct gctgaaaatt
     1801 atcaaggaca aggacttcct ggacaatgag gaaaacgagg acattctgga agatatcgtg
     1861 ctgaccctga cactgtttga ggacagagag atgatcgagg aacggctgaa aacctatgcc
     1921 cacctgttcg acgacaaagt gatgaagcag ctgaagcggc ggagatacac cggctggggc
     1981 aggctgagcc ggaagctgat caacggcatc cgggacaagc agtccggcaa gacaatcctg
     2041 gatttcctga agtccgacgg cttcgccaac agaaacttca tgcagctgat ccacgacgac
     2101 agcctgacct ttaaagagga catccagaaa gcccaggtgt ccggccaggg cgatagcctg
     2161 cacgagcaca ttgccaatct ggccggcagc cccgccatta agaagggcat cctgcagaca
     2221 gtgaaggtgg tggacgagct cgtgaaagtg atgggccggc acaagcccga gaacatcgtg
     2281 atcgaaatgg ccagagagaa ccagaccacc cagaagggac agaagaacag ccgcgagaga
     2341 atgaagcgga tcgaagaggg catcaaagag ctgggcagcc agatcctgaa agaacacccc
     2401 gtggaaaaca cccagctgca gaacgagaag ctgtacctgt actacctgca gaatgggcgg
     2461 gatatgtacg tggaccagga actggacatc aaccggctgt ccgactacga tgtggacgct
     2521 atcgtgcctc agagctttct gaaggacgac tccatcgata acaaagtgct gactcggagc
     2581 gacaagaacc ggggcaagag cgacaacgtg ccctccgaag aggtcgtgaa gaagatgaag
     2641 aactactggc gccagctgct gaatgccaag ctgattaccc agaggaagtt cgacaatctg
     2701 accaaggccg agagaggcgg cctgagcgaa ctggataagg ccggcttcat caagagacag
     2761 ctggtggaaa cccggcagat cacaaagcac gtggcacaga tcctggactc ccggatgaac
     2821 actaagtacg acgagaacga caaactgatc cgggaagtga aagtgatcac cctgaagtcc
     2881 aagctggtgt ccgatttccg gaaggatttc cagttttaca aagtgcgcga gatcaacaac
     2941 taccaccacg cccacgacgc ctacctgaac gccgtcgtgg gaaccgccct gatcaaaaag
     3001 taccctaagc tggaaagcga gttcgtgtac ggcgactaca aggtgtacga cgtgcggaag
     3061 atgatcgcca agagcgagca ggaaatcggc aaggctaccg ccaagtactt cttctacagc
     3121 aacatcatga actttttcaa gaccgagatt accctggcca acggcgagat ccggaagcgg
     3181 cctctgatcg agacaaacgg cgaaacaggc gagatcgtgt gggataaggg ccgggacttt
     3241 gccaccgtgc ggaaagtgct gtctatgccc caagtgaata tcgtgaaaaa gaccgaggtg
     3301 cagacaggcg gcttcagcaa agagtctatc ctgcccaaga ggaacagcga caagctgatc
     3361 gccagaaaga aggactggga ccctaagaag tacggcggct tcgacagccc caccgtggcc
     3421 tattctgtgc tggtggtggc caaagtggaa aagggcaagt ccaagaaact gaagagtgtg
     3481 aaagagctgc tggggatcac catcatggaa agaagcagct tcgagaagaa tcccatcgac
     3541 tttctggaag ccaagggcta caaagaagtg aaaaaggacc tgatcatcaa gctgcctaag
     3601 tactccctgt tcgagctgga aaacggccgg aagagaatgc tggcctctgc cggcgaactg
     3661 cagaagggaa acgaactggc cctgccctcc aaatatgtga acttcctgta cctggccagc
     3721 cactatgaga agctgaaggg ctcccccgag gataatgagc agaaacagct gtttgtggaa
     3781 cagcacaaac actacctgga cgagatcatc gagcagatca gcgagttctc caagagagtg
     3841 atcctggccg acgctaatct ggacaaggtg ctgagcgcct acaacaagca cagagacaag
     3901 cctatcagag agcaggccga gaatatcatc cacctgttta ccctgaccaa tctgggagcc
     3961 cctgccgcct tcaagtactt tgacaccacc atcgaccgga agaggtacac cagcaccaaa
     4021 gaggtgctgg acgccaccct gatccaccag agcatcaccg gcctgtacga gacacggatc
     4081 gacctgtctc agctgggagg cgacgcctat ccctatgacg tgcccgatta tgccagcctg
     4141 ggcagcggct cccccaagaa aaaacgcaag gtggaagatc ctaagaaaaa gcggaaagtg
     4201 gacgtgagca agggcgagga gctgttcacc ggggtggtgc ccatcctggt cgagctggac
     4261 ggcgacgtaa acggccacaa gttcagcgtg tccggcgagg gcgagggcga tgccacctac
     4321 ggcaagctga ccctgaagtt catctgcacc accggcaagc tgcccgtgcc ctggcccacc
     4381 ctcgtgacca ccctgaccta cggcgtgcag tgcttcagcc gctaccccga ccacatgaag
     4441 cagcacgact tcttcaagtc cgccatgccc gaaggctacg tccaggagcg caccatcttc
     4501 ttcaaggacg acggcaacta caagacccgc gccgaggtga agttcgaggg cgacaccctg
     4561 gtgaaccgca tcgagctgaa gggcatcgac ttcaaggagg acggcaacat cctggggcac
     4621 aagctggagt acaactacaa cagccacaac gtctatatca tggccgacaa gcagaagaac
     4681 ggcatcaagg tgaacttcaa gatccgccac aacatcgagg acggcagcgt gcagctcgcc
     4741 gaccactacc agcagaacac ccccatcggc gacggccccg tgctgctgcc cgacaaccac
     4801 tacctgagca cccagtccgc cctgagcaaa gaccccaacg agaagcgcga tcacatggtc
     4861 ctgctggagt tcgtgaccgc cgccgggatc actctcggca tggacgagct gtacaagtaa
//
