LOCUS       sequence-173514-        5423 bp    DNA     linear   UNA 21-JUL-2025
DEFINITION  Plasmid expression dCas9 protein in fusion with KRAB domain.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      natural DNA sequence
  ORGANISM  unspecified
REFERENCE   1  (bases 1 to 5423)
  AUTHORS   Xie S, Duan J, Li B, Zhou P, Hon GC
  TITLE     Multiplexed Engineering and Analysis of Combinatorial Enhancer 
            Activity in Single Cells.
  JOURNAL   Mol Cell. 2017 Apr 20;66(2):285-299.e5. doi: 
            10.1016/j.molcel.2017.03.007. Epub 2017 Apr 13.
  PUBMED    28416141
REFERENCE   2  (bases 1 to 5423)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Jul 21, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; doi: 
            "10.1016/j.molcel.2017.03.007"; journalName: "Mol Cell"; date: 
            "2017-04-20- 20"; volume: "66"; issue: "2"; pages: "285-299.e5"
COMMENT     Sequence Label: Lenti-dCas9-KRAB-blast
FEATURES             Location/Qualifiers
     source          1..5423
                     /mol_type="genomic DNA"
                     /organism="unspecified"
     promoter        81..1259
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          312..1250
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     1207..1227
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     CDS             1275..1322
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             join(1323..1346,1347..1349,1350..3905,3906..3908,
                     3909..5423)
                     /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 N863A mutation in the HNH 
                     catalytic domain"
                     /translation="DKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN
                     LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF
                     LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF
                     RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE
                     NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI
                     GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
                     QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
                     KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
                     SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF
                     TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS
                     VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL
                     KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ
                     LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH
                     KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL
                     YYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKARGKSDNVPS
                     EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV
                     AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
                     AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE
                     ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE
                     SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT
                     IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL
                     ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA
                     NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD
                     ATLIHQSITGLYETRIDLSQLGGD"
ORIGIN
        1 tgcaaagatg gataaagttt taaacagaga ggaatctttg cagctaatgg accttctagg
       61 tcttgaaagg agtgggaatt ggctccggtg cccgtcagtg ggcagagcgc acatcgccca
      121 cagtccccga gaagttgggg ggaggggtcg gcaattgaac cggtgcctag agaaggtggc
      181 gcggggtaaa ctgggaaagt gatgtcgtgt actggctccg cctttttccc gagggtgggg
      241 gagaaccgta tataagtgca gtagtcgccg tgaacgttct ttttcgcaac gggtttgccg
      301 ccagaacaca ggtaagtgcc gtgtgtggtt cccgcgggcc tggcctcttt acgggttatg
      361 gcccttgcgt gccttgaatt acttccacct ggctgcagta cgtgattctt gatcccgagc
      421 ttcgggttgg aagtgggtgg gagagttcga ggccttgcgc ttaaggagcc ccttcgcctc
      481 gtgcttgagt tgaggcctgg cctgggcgct ggggccgccg cgtgcgaatc tggtggcacc
      541 ttcgcgcctg tctcgctgct ttcgataagt ctctagccat ttaaaatttt tgatgacctg
      601 ctgcgacgct ttttttctgg caagatagtc ttgtaaatgc gggccaagat ctgcacactg
      661 gtatttcggt ttttggggcc gcgggcggcg acggggcccg tgcgtcccag cgcacatgtt
      721 cggcgaggcg gggcctgcga gcgcggccac cgagaatcgg acgggggtag tctcaagctg
      781 gccggcctgc tctggtgcct ggcctcgcgc cgccgtgtat cgccccgccc tgggcggcaa
      841 ggctggcccg gtcggcacca gttgcgtgag cggaaagatg gccgcttccc ggccctgctg
      901 cagggagctc aaaatggagg acgcggcgct cgggagagcg ggcgggtgag tcacccacac
      961 aaaggaaaag ggcctttccg tcctcagccg tcgcttcatg tgactccacg gagtaccggg
     1021 cgccgtccag gcacctcgat tagttctcga gcttttggag tacgtcgtct ttaggttggg
     1081 gggaggggtt ttatgcgatg gagtttcccc acactgagtg ggtggagact gaagttaggc
     1141 cagcttggca cttgatgtaa ttctccttgg aatttgccct ttttgagttt ggatcttggt
     1201 tcattctcaa gcctcagaca gtggttcaaa gtttttttct tccatttcag gtgtcgtgac
     1261 gtacggccac catgaaaagg ccggcggcca cgaaaaaggc cggccaggca aaaaagaaaa
     1321 aggacaagaa gtacagcatc ggcctggcca tcggcaccaa ctctgtgggc tgggccgtga
     1381 tcaccgacga gtacaaggtg cccagcaaga aattcaaggt gctgggcaac accgaccggc
     1441 acagcatcaa gaagaacctg atcggagccc tgctgttcga cagcggcgaa acagccgagg
     1501 ccacccggct gaagagaacc gccagaagaa gatacaccag acggaagaac cggatctgct
     1561 atctgcaaga gatcttcagc aacgagatgg ccaaggtgga cgacagcttc ttccacagac
     1621 tggaagagtc cttcctggtg gaagaggata agaagcacga gcggcacccc atcttcggca
     1681 acatcgtgga cgaggtggcc taccacgaga agtaccccac catctaccac ctgagaaaga
     1741 aactggtgga cagcaccgac aaggccgacc tgcggctgat ctatctggcc ctggcccaca
     1801 tgatcaagtt ccggggccac ttcctgatcg agggcgacct gaaccccgac aacagcgacg
     1861 tggacaagct gttcatccag ctggtgcaga cctacaacca gctgttcgag gaaaacccca
     1921 tcaacgccag cggcgtggac gccaaggcca tcctgtctgc cagactgagc aagagcagac
     1981 ggctggaaaa tctgatcgcc cagctgcccg gcgagaagaa gaatggcctg ttcggcaacc
     2041 tgattgccct gagcctgggc ctgaccccca acttcaagag caacttcgac ctggccgagg
     2101 atgccaaact gcagctgagc aaggacacct acgacgacga cctggacaac ctgctggccc
     2161 agatcggcga ccagtacgcc gacctgtttc tggccgccaa gaacctgtcc gacgccatcc
     2221 tgctgagcga catcctgaga gtgaacaccg agatcaccaa ggcccccctg agcgcctcta
     2281 tgatcaagag atacgacgag caccaccagg acctgaccct gctgaaagct ctcgtgcggc
     2341 agcagctgcc tgagaagtac aaagagattt tcttcgacca gagcaagaac ggctacgccg
     2401 gctacattga cggcggagcc agccaggaag agttctacaa gttcatcaag cccatcctgg
     2461 aaaagatgga cggcaccgag gaactgctcg tgaagctgaa cagagaggac ctgctgcgga
     2521 agcagcggac cttcgacaac ggcagcatcc cccaccagat ccacctggga gagctgcacg
     2581 ccattctgcg gcggcaggaa gatttttacc cattcctgaa ggacaaccgg gaaaagatcg
     2641 agaagatcct gaccttccgc atcccctact acgtgggccc tctggccagg ggaaacagca
     2701 gattcgcctg gatgaccaga aagagcgagg aaaccatcac cccctggaac ttcgaggaag
     2761 tggtggacaa gggcgcttcc gcccagagct tcatcgagcg gatgaccaac ttcgataaga
     2821 acctgcccaa cgagaaggtg ctgcccaagc acagcctgct gtacgagtac ttcaccgtgt
     2881 ataacgagct gaccaaagtg aaatacgtga ccgagggaat gagaaagccc gccttcctga
     2941 gcggcgagca gaaaaaggcc atcgtggacc tgctgttcaa gaccaaccgg aaagtgaccg
     3001 tgaagcagct gaaagaggac tacttcaaga aaatcgagtg cttcgactcc gtggaaatct
     3061 ccggcgtgga agatcggttc aacgcctccc tgggcacata ccacgatctg ctgaaaatta
     3121 tcaaggacaa ggacttcctg gacaatgagg aaaacgagga cattctggaa gatatcgtgc
     3181 tgaccctgac actgtttgag gacagagaga tgatcgagga acggctgaaa acctatgccc
     3241 acctgttcga cgacaaagtg atgaagcagc tgaagcggcg gagatacacc ggctggggca
     3301 ggctgagccg gaagctgatc aacggcatcc gggacaagca gtccggcaag acaatcctgg
     3361 atttcctgaa gtccgacggc ttcgccaaca gaaacttcat gcagctgatc cacgacgaca
     3421 gcctgacctt taaagaggac atccagaaag cccaggtgtc cggccagggc gatagcctgc
     3481 acgagcacat tgccaatctg gccggcagcc ccgccattaa gaagggcatc ctgcagacag
     3541 tgaaggtggt ggacgagctc gtgaaagtga tgggccggca caagcccgag aacatcgtga
     3601 tcgaaatggc cagagagaac cagaccaccc agaagggaca gaagaacagc cgcgagagaa
     3661 tgaagcggat cgaagagggc atcaaagagc tgggcagcca gatcctgaaa gaacaccccg
     3721 tggaaaacac ccagctgcag aacgagaagc tgtacctgta ctacctgcag aatgggcggg
     3781 atatgtacgt ggaccaggaa ctggacatca accggctgtc cgactacgat gtggaccaca
     3841 tcgtgcctca gagctttctg aaggacgact ccatcgacaa caaggtgctg accagaagcg
     3901 acaaggcccg gggcaagagc gacaacgtgc cctccgaaga ggtcgtgaag aagatgaaga
     3961 actactggcg gcagctgctg aacgccaagc tgattaccca gagaaagttc gacaatctga
     4021 ccaaggccga gagaggcggc ctgagcgaac tggataaggc cggcttcatc aagagacagc
     4081 tggtggaaac ccggcagatc acaaagcacg tggcacagat cctggactcc cggatgaaca
     4141 ctaagtacga cgagaatgac aagctgatcc gggaagtgaa agtgatcacc ctgaagtcca
     4201 agctggtgtc cgatttccgg aaggatttcc agttttacaa agtgcgcgag atcaacaact
     4261 accaccacgc ccacgacgcc tacctgaacg ccgtcgtggg aaccgccctg atcaaaaagt
     4321 accctaagct ggaaagcgag ttcgtgtacg gcgactacaa ggtgtacgac gtgcggaaga
     4381 tgatcgccaa gagcgagcag gaaatcggca aggctaccgc caagtacttc ttctacagca
     4441 acatcatgaa ctttttcaag accgagatta ccctggccaa cggcgagatc cggaagcggc
     4501 ctctgatcga gacaaacggc gaaaccgggg agatcgtgtg ggataagggc cgggattttg
     4561 ccaccgtgcg gaaagtgctg agcatgcccc aagtgaatat cgtgaaaaag accgaggtgc
     4621 agacaggcgg cttcagcaaa gagtctatcc tgcccaagag gaacagcgat aagctgatcg
     4681 ccagaaagaa ggactgggac cctaagaagt acggcggctt cgacagcccc accgtggcct
     4741 attctgtgct ggtggtggcc aaagtggaaa agggcaagtc caagaaactg aagagtgtga
     4801 aagagctgct ggggatcacc atcatggaaa gaagcagctt cgagaagaat cccatcgact
     4861 ttctggaagc caagggctac aaagaagtga aaaaggacct gatcatcaag ctgcctaagt
     4921 actccctgtt cgagctggaa aacggccgga agagaatgct ggcctctgcc ggcgaactgc
     4981 agaagggaaa cgaactggcc ctgccctcca aatatgtgaa cttcctgtac ctggccagcc
     5041 actatgagaa gctgaagggc tcccccgagg ataatgagca gaaacagctg tttgtggaac
     5101 agcacaagca ctacctggac gagatcatcg agcagatcag cgagttctcc aagagagtga
     5161 tcctggccga cgctaatctg gacaaagtgc tgtccgccta caacaagcac cgggataagc
     5221 ccatcagaga gcaggccgag aatatcatcc acctgtttac cctgaccaat ctgggagccc
     5281 ctgccgcctt caagtacttt gacaccacca tcgaccggaa gaggtacacc agcaccaaag
     5341 aggtgctgga cgccaccctg atccaccaga gcatcaccgg cctgtacgag acacggatcg
     5401 acctgtctca gctgggaggc gac
//
