LOCUS       sequence-305784-        4899 bp    DNA     linear   UNA 15-DEC-2025
DEFINITION  Express nuclease dead Cas9 fused to KillerRed chromophore in 
            mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      natural DNA sequence
  ORGANISM  unspecified
REFERENCE   1  (bases 1 to 4899)
  AUTHORS   House NCM, Parasuram R, Layer JV, Price BD
  TITLE     Site-specific targeting of a light activated dCas9-KillerRed fusion 
            protein generates transient, localized regions of oxidative DNA 
            damage.
  JOURNAL   PLoS One. 2020 Dec 17;15(12):e0237759. doi: 
            10.1371/journal.pone.0237759. eCollection 2020.
  PUBMED    33332350
REFERENCE   2  (bases 1 to 4899)
  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: "PLoS One.";
            date: "2020-12-17"; volume: "15(12):e0237759. doi"; pages: " 
            10.1371/journal.pone.0237759. eCollection 2020"
COMMENT     Sequence Label: pJ609-dCas9-KR-puro
FEATURES             Location/Qualifiers
     source          1..4899
                     /mol_type="genomic DNA"
                     /organism="unspecified"
     regulatory      16..25
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(22..48,49..51,52..2535,2536..2541,2542..2607,
                     2608..2610,2611..4125)
                     /codon_start=1
                     /product="catalytically dead mutant of the Cas9 
                     endonuclease from the Streptococcus pyogenes Type II 
                     CRISPR/Cas system"
                     /label=Cas9m4
                     /note="RNA-guided DNA-binding protein that lacks 
                     endonuclease activity due to the mutations D10A, D839A, 
                     H840A, and N863A (Mali et al., 2013)"
                     /translation="MDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK
                     NLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES
                     FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK
                     FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL
                     ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ
                     IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR
                     QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL
                     RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG
                     NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY
                     FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD
                     SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER
                     LKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFM
                     QLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR
                     HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY
                     LYYLQNGRDMYVDQELDINRLSDYDVAAIVPQSFLKDDSIDNKVLTRSDKARGKSDNVP
                     SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH
                     VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL
                     NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT
                     EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK
                     ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI
                     TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE
                     LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD
                     ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL
                     DATLIHQSITGLYETRIDLSQLGGD"
     CDS             4138..4158
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     regulatory      4168..4177
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(4174..4176,4177..4182,4183..4893)
                     /codon_start=1
                     /product="red fluorescent protein KillerRed, a 
                     genetically-encoded photosensitizer (Bulina et al., 2006)"
                     /label=KillerRed
                     /note="mammalian codon-optimized"
                     /translation="MGSEGGPALFQSDMTFKIFIDGEVNGQKFTIVADGSSKFPHGDFN
                     VHAVCETGKLPMSWKPICHLIQYGEPFFARYPDGISHFAQECFPEGLSIDRTVRFENDG
                     TMTSHHTYELDDTCVVSRITVNCDGFQPDGPIMRDQLVDILPNETHMFPHGPNAVRQLA
                     FIGFTTADGGLMMGHFDSKMTFNGSRAIEIPGPHFVTIITKQMRDTSDKRDHVCQREVA
                     YAHSVPRITSAIGSDED"
ORIGIN
        1 aagctggcta gcgccgccac catggacaag aagtactcca ttgggctcgc tatcggcaca
       61 aacagcgtcg gctgggccgt cattacggac gagtacaagg tgccgagcaa aaaattcaaa
      121 gttctgggca ataccgatcg ccacagcata aagaagaacc tcattggcgc cctcctgttc
      181 gactccgggg agacggccga agccacgcgg ctcaaaagaa cagcacggcg cagatatacc
      241 cgcagaaaga atcggatctg ctacctgcag gagatcttta gtaatgagat ggctaaggtg
      301 gatgactctt tcttccatag gctggaggag tcctttttgg tggaggagga taaaaagcac
      361 gagcgccacc caatctttgg caatatcgtg gacgaggtgg cgtaccatga aaagtaccca
      421 accatatatc atctgaggaa gaagcttgta gacagtactg ataaggctga cttgcggttg
      481 atctatctcg cgctggcgca tatgatcaaa tttcggggac acttcctcat cgagggggac
      541 ctgaacccag acaacagcga tgtcgacaaa ctctttatcc aactggttca gacttacaat
      601 cagcttttcg aagagaaccc gatcaacgca tccggagttg acgccaaagc aatcctgagc
      661 gctaggctgt ccaaatcccg gcggctcgaa aacctcatcg cacagctccc tggggagaag
      721 aagaacggcc tgtttggtaa tcttatcgcc ctgtcactcg ggctgacccc caactttaaa
      781 tctaacttcg acctggccga agatgccaag cttcaactga gcaaagacac ctacgatgat
      841 gatctcgaca atctgctggc ccagatcggc gaccagtacg cagacctttt tttggcggca
      901 aagaacctgt cagacgccat tctgctgagt gatattctgc gagtgaacac ggagatcacc
      961 aaagctccgc tgagcgctag tatgatcaag cgctatgatg agcaccacca agacttgact
     1021 ttgctgaagg cccttgtcag acagcaactg cctgagaagt acaaggaaat tttcttcgat
     1081 cagtctaaaa atggctacgc cggatacatt gacggcggag caagccagga ggaattttac
     1141 aaatttatta agcccatctt ggaaaaaatg gacggcaccg aggagctgct ggtaaagctt
     1201 aacagagaag atctgttgcg caaacagcgc actttcgaca atggaagcat cccccaccag
     1261 attcacctgg gcgaactgca cgctatcctc aggcggcaag aggatttcta cccctttttg
     1321 aaagataaca gggaaaagat tgagaaaatc ctcacatttc ggatacccta ctatgtaggc
     1381 cccctcgccc ggggaaattc cagattcgcg tggatgactc gcaaatcaga agagaccatc
     1441 actccctgga acttcgagga agtcgtggat aagggggcct ctgcccagtc cttcatcgaa
     1501 aggatgacta actttgataa aaatctgcct aacgaaaagg tgcttcctaa acactctctg
     1561 ctgtacgagt acttcacagt ttataacgag ctcaccaagg tcaaatacgt cacagaaggg
     1621 atgagaaagc cagcattcct gtctggagag cagaagaaag ctatcgtgga cctcctcttc
     1681 aagacgaacc ggaaagttac cgtgaaacag ctcaaagaag actatttcaa aaagattgaa
     1741 tgtttcgact ctgttgaaat cagcggagtg gaggatcgct tcaacgcatc cctgggaacg
     1801 tatcacgatc tcctgaaaat cattaaagac aaggacttcc tggacaatga ggagaacgag
     1861 gacattcttg aggacattgt cctcaccctt acgttgtttg aagataggga gatgattgaa
     1921 gaacgcttga aaacttacgc tcatctcttc gacgacaaag tcatgaaaca gctcaagagg
     1981 cgccgatata caggatgggg gcggctgtca agaaaactga tcaatgggat ccgagacaag
     2041 cagagtggaa agacaatcct ggattttctt aagtccgatg gatttgccaa ccggaacttc
     2101 atgcagttga tccatgatga ctctctcacc tttaaggagg acatccagaa agcacaagtt
     2161 tctggccagg gggacagtct tcacgagcac atcgctaatc ttgcaggtag cccagctatc
     2221 aaaaagggaa tactgcagac cgttaaggtc gtggatgaac tcgtcaaagt aatgggaagg
     2281 cataagcccg agaatatcgt tatcgagatg gcccgagaga accaaactac ccagaaggga
     2341 cagaagaaca gtagggaaag gatgaagagg attgaagagg gtataaaaga actggggtcc
     2401 caaatcctta aggaacaccc agttgaaaac acccagcttc agaatgagaa gctctacctg
     2461 tactacctgc agaacggcag ggacatgtac gtggatcagg aactggacat caatcggctc
     2521 tccgactacg acgtggctgc tatcgtgccc cagtcttttc tcaaagatga ttctattgat
     2581 aataaagtgt tgacaagatc cgataaagct agagggaaga gtgataacgt cccctcagaa
     2641 gaagttgtca agaaaatgaa aaattattgg cggcagctgc tgaacgccaa actgatcaca
     2701 caacggaagt tcgataatct gactaaggct gaacgaggtg gcctgtctga gttggataaa
     2761 gccggcttca tcaaaaggca gcttgttgag acacgccaga tcaccaagca cgtggcccaa
     2821 attctcgatt cacgcatgaa caccaagtac gatgaaaatg acaaactgat tcgagaggtg
     2881 aaagttatta ctctgaagtc taagctggtc tcagatttca gaaaggactt tcagttttat
     2941 aaggtgagag agatcaacaa ttaccaccat gcgcatgatg cctacctgaa tgcagtggta
     3001 ggcactgcac ttatcaaaaa atatcccaag cttgaatctg aatttgttta cggagactat
     3061 aaagtgtacg atgttaggaa aatgatcgca aagtctgagc aggaaatagg caaggccacc
     3121 gctaagtact tcttttacag caatattatg aattttttca agaccgagat tacactggcc
     3181 aatggagaga ttcggaagcg accacttatc gaaacaaacg gagaaacagg agaaatcgtg
     3241 tgggacaagg gtagggattt cgcgacagtc cggaaggtcc tgtccatgcc gcaggtgaac
     3301 atcgttaaaa agaccgaagt acagaccgga ggcttctcca aggaaagtat cctcccgaaa
     3361 aggaacagcg acaagctgat cgcacgcaaa aaagattggg accccaagaa atacggcgga
     3421 ttcgattctc ctacagtcgc ttacagtgta ctggttgtgg ccaaagtgga gaaagggaag
     3481 tctaaaaaac tcaaaagcgt caaggaactg ctgggcatca caatcatgga gcgatcaagc
     3541 ttcgaaaaaa accccatcga ctttctcgag gcgaaaggat ataaagaggt caaaaaagac
     3601 ctcatcatta agcttcccaa gtactctctc tttgagcttg aaaacggccg gaaacgaatg
     3661 ctcgctagtg cgggcgagct gcagaaaggt aacgagctgg cactgccctc taaatacgtt
     3721 aatttcttgt atctggccag ccactatgaa aagctcaaag ggtctcccga agataatgag
     3781 cagaagcagc tgttcgtgga acaacacaaa cactaccttg atgagatcat cgagcaaata
     3841 agcgaattct ccaaaagagt gatcctcgcc gacgctaacc tcgataaggt gctttctgct
     3901 tacaataagc acagggataa gcccatcagg gagcaggcag aaaacattat ccacttgttt
     3961 actctgacca acttgggcgc gcctgcagcc ttcaagtact tcgacaccac catagacaga
     4021 aagcggtaca cctctacaaa ggaggtcctg gacgccacac tgattcatca gtcaattacg
     4081 gggctctatg aaacaagaat cgacctctct cagctcggtg gagacagcag ggctgacccc
     4141 aagaagaaga ggaaggtgaa accggtcgcc accatgggtt cagagggcgg ccccgccctg
     4201 ttccagagcg acatgacctt caaaatcttc atcgacggcg aggtgaacgg ccagaagttc
     4261 accatcgtgg ccgacggcag cagcaagttc ccccacggcg acttcaacgt gcacgccgtg
     4321 tgcgagaccg gcaagctgcc catgagctgg aagcccatct gccacctgat ccagtacggc
     4381 gagcccttct tcgcccgcta ccccgacggc atcagccatt tcgcccagga gtgcttcccc
     4441 gagggcctga gcatcgaccg caccgtgcgc ttcgagaacg acggcaccat gaccagccac
     4501 cacacctacg agctggacga cacctgcgtg gtgagccgca tcaccgtgaa ctgcgacggc
     4561 ttccagcccg acggccccat catgcgcgac cagctggtgg acatcctgcc caacgagacc
     4621 cacatgttcc cccacggccc caacgccgtg cgccagctgg ccttcatcgg cttcaccacc
     4681 gccgacggcg gcctgatgat gggccacttc gacagcaaga tgaccttcaa cggcagccgc
     4741 gccatcgaga tccccggccc acacttcgtg accatcatca ccaagcagat gagggacacc
     4801 agcgacaagc gcgaccacgt gtgccagcgc gaggtggcct acgcccacag cgtgccccgc
     4861 atcaccagcg ccatcggtag cgacgaggat taagaattc
//
