LOCUS       sequence-127370-        5599 bp    DNA     linear   UNA 15-DEC-2025
DEFINITION  Expresses NLS-dCas9-GFP11x7-NLS in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      natural DNA sequence
  ORGANISM  unspecified
REFERENCE   1  (bases 1 to 5599)
  AUTHORS   Kamiyama D, Sekine S, Barsi-Rhyne B, Hu J, Chen B, Gilbert LA, 
            Ishikawa H, Leonetti MD, Marshall WF, Weissman JS, Huang B
  TITLE     Versatile protein tagging in cells with split fluorescent protein.
  JOURNAL   Nat Commun. 2016 Mar 18;7:11046. doi: 10.1038/ncomms11046.
  PUBMED    26988139
REFERENCE   2  (bases 1 to 5599)
  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: "Nat 
            Commun."; date: "2016-03-18"; volume: "7:11046. doi"; pages: " 
            10.1038/ncomms11046"
COMMENT     Sequence Label: pHRm-NLS-dCas9-GFP11x7-NLS
FEATURES             Location/Qualifiers
     source          1..5599
                     /mol_type="genomic DNA"
                     /organism="unspecified"
     CDS             4..24
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     regulatory      73..82
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(79..105,106..108,109..2595,2596..2598,2599..4182)
                     /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             79..4182
                     /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             4186..4212
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             4192..4218
                     /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             4231..4251
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             4258..4278
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             4321..4746
                     /codon_start=1
                     /product="seven tandem copies of the 11th beta-strand of 
                     superfolder GFP"
                     /label=GFP11x7
                     /note="each GFP11 associates with GFP(1-10), consisting of 
                     the first 10 beta-strands, to reconstitute fluorescent GFP 
                     (Kamiyama et al., 2016)"
                     /translation="RDHMVLHEYVNAAGITGGSGGRDHMVLHEYVNAAGITGGSGGRDH
                     MVLHEYVNAAGITGGSGGRDHMVLHEYVNAAGITGGSGGRDHMVLHEYVNAAGITGGSG
                     GRDHMVLHEYVNAAGITGGSGGRDHMVLHEYVNAAGIT"
     CDS             4769..4789
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             4799..4855
                     /codon_start=1
                     /product="2A peptide from porcine teschovirus-1 
                     polyprotein"
                     /label=P2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="ATNFSLLKQAGDVEENPGP"
     CDS             4868..5566
                     /codon_start=1
                     /product="monomeric blue fluorescent protein"
                     /label=TagBFP
                     /note="mammalian codon-optimized"
                     /translation="MSELIKENMHMKLYMEGTVDNHHFKCTSEGEGKPYEGTQTMRIKV
                     VEGGPLPFAFDILATSFLYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLTA
                     TQDTSLQDGCLIYNVKIRGVNFTSNGPVMQKKTLGWEAFTETLYPADGGLEGRNDMALK
                     LVGGSHLIANIKTTYRSKKPAKNLKMPGVYYVDYRLERIKEANNETYVEQHEVAVARYC
                     DLPSKLGHKLN"
     CDS             5576..5596
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
ORIGIN
        1 atgcccaaga agaagcgcaa ggtgggacgc gtctgcagga tatcaagctt gcggtaccgc
       61 gggcccggga tcgccaccat ggacaagaag tacagcatcg gcctggccat cggcaccaac
      121 tctgtgggct gggccgtgat caccgacgag tacaaggtgc ccagcaagaa attcaaggtg
      181 ctgggcaaca ccgaccggca cagcatcaag aagaacctga tcggcgccct gctgttcgac
      241 agcggagaaa cagccgaggc cacccggctg aagagaaccg ccagaagaag atacaccaga
      301 cggaagaacc ggatctgcta tctgcaagag atcttcagca acgagatggc caaggtggac
      361 gacagcttct tccacagact ggaagagtcc ttcctggtgg aagaggataa gaagcacgag
      421 cggcacccca tcttcggcaa catcgtggac gaggtggcct accacgagaa gtaccccacc
      481 atctaccacc tgagaaagaa actggtggac agcaccgaca aggccgacct gcggctgatc
      541 tatctggccc tggcccacat gatcaagttc cggggccact tcctgatcga gggcgacctg
      601 aaccccgaca acagcgacgt ggacaagctg ttcatccagc tggtgcagac ctacaaccag
      661 ctgttcgagg aaaaccccat caacgccagc ggcgtggacg ccaaggccat cctgtctgcc
      721 agactgagca agagcagacg gctggaaaat ctgatcgccc agctgcccgg cgagaagaag
      781 aatggcctgt tcggcaacct gattgccctg agcctgggcc tgacccccaa cttcaagagc
      841 aacttcgacc tggccgagga tgccaaactg cagctgagca aggacaccta cgacgacgac
      901 ctggacaacc tgctggccca gatcggcgac cagtacgccg acctgtttct ggccgccaag
      961 aacctgtccg acgccatcct gctgagcgac atcctgagag tgaacaccga gatcaccaag
     1021 gcccccctga gcgcctctat gatcaagaga tacgacgagc accaccagga cctgaccctg
     1081 ctgaaagctc tcgtgcggca gcagctgcct gagaagtaca aagagatttt cttcgaccag
     1141 agcaagaacg gctacgccgg ctacatcgat ggcggagcca gccaggaaga gttctacaag
     1201 ttcatcaagc ccatcctgga aaagatggac ggcaccgagg aactgctcgt gaagctgaac
     1261 agagaggacc tgctgcggaa gcagcggacc ttcgacaacg gcagcatccc ccaccagatc
     1321 cacctgggag agctgcacgc cattctgcgg cggcaggaag atttttaccc attcctgaag
     1381 gacaaccggg aaaagatcga gaagatcctg accttccgca tcccctacta cgtgggccct
     1441 ctggccaggg gaaacagcag attcgcctgg atgaccagaa agagcgagga aaccatcacc
     1501 ccctggaact tcgaggaagt ggtggacaag ggcgccagcg cccagagctt catcgagcgg
     1561 atgaccaact tcgataagaa cctgcccaac gagaaggtgc tgcccaagca cagcctgctg
     1621 tacgagtact tcaccgtgta caacgagctg accaaagtga aatacgtgac cgagggaatg
     1681 agaaagcccg ccttcctgag cggcgagcag aaaaaagcca tcgtggacct gctgttcaag
     1741 accaaccgga aagtgaccgt gaagcagctg aaagaggact acttcaagaa aatcgagtgc
     1801 ttcgactccg tggaaatctc cggcgtggaa gatcggttca acgcctccct gggcacatac
     1861 cacgatctgc tgaaaattat caaggacaag gacttcctgg acaatgagga aaacgaggac
     1921 attctggaag atatcgtgct gaccctgaca ctgtttgagg acagagagat gatcgaggaa
     1981 cggctgaaaa cctatgccca cctgttcgac gacaaagtga tgaagcagct gaagcggcgg
     2041 agatacaccg gctggggcag gctgagccgg aagctgatca acggcatccg ggacaagcag
     2101 tccggcaaga caatcctgga tttcctgaag tccgacggct tcgccaacag aaacttcatg
     2161 cagctgatcc acgacgacag cctgaccttt aaagaggaca tccagaaagc ccaggtgtcc
     2221 ggccagggcg atagcctgca cgagcacatt gccaatctgg ccggcagccc cgccattaag
     2281 aagggcatcc tgcagacagt gaaggtggtg gacgagctcg tgaaagtgat gggccggcac
     2341 aagcccgaga acatcgtgat cgaaatggcc agagagaacc agaccaccca gaagggacag
     2401 aagaacagcc gcgagagaat gaagcggatc gaagagggca tcaaagagct gggcagccag
     2461 atcctgaaag aacaccccgt ggaaaacacc cagctgcaga acgagaagct gtacctgtac
     2521 tacctgcaga atgggcggga tatgtacgtg gaccaggaac tggacatcaa ccggctgtcc
     2581 gactacgatg tggacgctat cgtgcctcag agctttctga aggacgactc catcgataac
     2641 aaagtgctga ctcggagcga caagaaccgg ggcaagagcg acaacgtgcc ctccgaagag
     2701 gtcgtgaaga agatgaagaa ctactggcgc cagctgctga atgccaagct gattacccag
     2761 aggaagttcg acaatctgac caaggccgag agaggcggcc tgagcgaact ggataaggcc
     2821 ggcttcatca agagacagct ggtggaaacc cggcagatca caaagcacgt ggcacagatc
     2881 ctggactccc ggatgaacac taagtacgac gagaacgaca aactgatccg ggaagtgaaa
     2941 gtgatcaccc tgaagtccaa gctggtgtcc gatttccgga aggatttcca gttttacaaa
     3001 gtgcgcgaga tcaacaacta ccaccacgcc cacgacgcct acctgaacgc cgtcgtggga
     3061 accgccctga tcaaaaagta ccctaagctg gaaagcgagt tcgtgtacgg cgactacaag
     3121 gtgtacgacg tgcggaagat gatcgccaag agcgagcagg aaatcggcaa ggctaccgcc
     3181 aagtacttct tctacagcaa catcatgaac tttttcaaga ccgagattac cctggccaac
     3241 ggcgagatcc ggaagcggcc tctgatcgag acaaacggcg aaacaggcga gatcgtgtgg
     3301 gataagggcc gggactttgc caccgtgcgg aaagtgctgt ctatgcccca agtgaatatc
     3361 gtgaaaaaga ccgaggtgca gacaggcggc ttcagcaaag agtctatcct gcccaagagg
     3421 aacagcgaca agctgatcgc cagaaagaag gactgggacc ctaagaagta cggcggcttc
     3481 gacagcccca ccgtggccta ttctgtgctg gtggtggcca aagtggaaaa gggcaagtcc
     3541 aagaaactga agagtgtgaa agagctgctg gggatcacca tcatggaaag aagcagcttc
     3601 gagaagaatc ccatcgactt tctggaagcc aagggctaca aagaagtgaa aaaggacctg
     3661 atcatcaagc tgcctaagta ctccctgttc gagctggaaa acggccggaa gagaatgctg
     3721 gcctctgccg gcgaactgca gaagggaaac gaactggccc tgccctccaa atatgtgaac
     3781 ttcctgtacc tggccagcca ctatgagaag ctgaagggct cccccgagga taatgagcag
     3841 aaacagctgt ttgtggaaca gcacaaacac tacctggacg agatcatcga gcagatcagc
     3901 gagttctcca agagagtgat cctggccgac gctaatctgg acaaggtgct gagcgcctac
     3961 aacaagcaca gagacaagcc tatcagagag caggccgaga atatcatcca cctgtttacc
     4021 ctgaccaatc tgggagcccc tgccgccttc aagtactttg acaccaccat cgaccggaag
     4081 aggtacacca gcaccaaaga ggtgctggac gccaccctga tccaccagag catcaccggc
     4141 ctgtacgaga cacggatcga cctgtctcag ctgggaggcg acgcctatcc ctatgacgtg
     4201 cccgattatg ccagcctggg cagcggctcc cccaagaaaa aacgcaaggt ggaagatcct
     4261 aagaaaaagc ggaaagtgga cggcattggt agtgggagca acggcagcag cggatccatg
     4321 cgtgaccaca tggtccttca tgagtatgta aatgctgctg ggattacagg tggctctgga
     4381 ggtagagatc atatggttct ccacgaatac gttaacgccg caggcatcac tggcggtagt
     4441 ggaggacgcg accatatggt actacatgaa tatgtcaatg cagccggaat aaccggaggg
     4501 tccggaggcc gggatcacat ggtgctgcat gagtatgtga acgcggcggg tataactggt
     4561 gggtcgggcg gacgagacca tatggtgctt cacgaatacg taaacgcagc tggcattact
     4621 ggcggatcag gtggcaggga tcacatggta ctccatgagt acgtgaacgc tgctggaatc
     4681 acaggcggta gcggcggtcg ggaccatatg gtcctgcacg aatatgtcaa tgctgccggt
     4741 atcaccgcgg ccgcaaggtg gaggtggacc caagaagaag cgcaaggtgg gaagcggagc
     4801 tactaacttc agcctgctga agcaggctgg agacgtggag gagaaccctg gacctgcacc
     4861 aggaagtatg agcgagctga ttaaggagaa catgcacatg aagctgtaca tggagggcac
     4921 cgtggacaac catcacttca agtgcacatc cgagggcgaa ggcaagccct acgagggcac
     4981 ccagaccatg agaatcaagg tggtcgaggg cggccctctc cccttcgcct tcgacatcct
     5041 ggctactagc ttcctctacg gcagcaagac cttcatcaac cacacccagg gcatccccga
     5101 cttcttcaag cagtccttcc ctgagggctt cacatgggag agagtcacca catacgaaga
     5161 cgggggcgtg ctgaccgcta cccaggacac cagcctccag gacggctgcc tcatctacaa
     5221 cgtcaagatc agaggggtga acttcacatc caacggccct gtgatgcaga agaaaacact
     5281 cggctgggag gccttcaccg agacgctgta ccccgctgac ggcggcctgg aaggcagaaa
     5341 cgacatggcc ctgaagctcg tgggcgggag ccatctgatc gcaaacatca agaccacata
     5401 tagatccaag aaacccgcta agaacctcaa gatgcctggc gtctactatg tggactacag
     5461 actggaaaga atcaaggagg ccaacaacga gacctacgtc gagcagcacg aggtggcagt
     5521 ggccagatac tgcgacctcc ctagcaaact ggggcacaag cttaatggtg gaggtccaaa
     5581 aaagaaaagg aaggtctaa
//
