LOCUS       sequence-270660-        5859 bp    DNA     linear   UNA 15-DEC-2025
DEFINITION  Expresses dRNase H1-EGFP-dCas9 fusion protein in human cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      natural DNA sequence
  ORGANISM  unspecified
REFERENCE   1  (bases 1 to 5859)
  AUTHORS   Abraham KJ, Khosraviani N, Chan JNY, Gorthi A, Samman A, Zhao DY, 
            Wang M, Bokros M, Vidya E, Ostrowski LA, Oshidari R, Pietrobon V, 
            Patel PS, Algouneh A, Singhania R, Liu Y, Yerlici VT, De Carvalho 
            DD, Ohh M, Dickson BC, Hakem R, Greenblatt JF, Lee S, Bishop AJR, 
            Mekhail K
  TITLE     Nucleolar RNA polymerase II drives ribosome biogenesis.
  JOURNAL   Nature. 2020 Sep;585(7824):298-302. doi: 10.1038/s41586-020-2497-0. 
            Epub 2020 Jul 15.
  PUBMED    32669707
REFERENCE   2  (bases 1 to 5859)
  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"; doi: 
            "10.1038/s41586-020-2497-0"; journalName: "Nature"; date: "2020-09";
            volume: "585"; issue: "7824"; pages: "298-302"
COMMENT     Sequence Label: dRED
FEATURES             Location/Qualifiers
     source          1..5859
                     /mol_type="genomic DNA"
                     /organism="unspecified"
     CDS             921..941
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             join(951..953,954..956,957..1667)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(951..953,954..956,957..1667)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(996..1017)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1257..1276)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1604..1625
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             1716..1736
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             join(1746..1772,1773..1775,1776..4262,4263..4265,
                     4266..5852)
                     /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"
ORIGIN
        1 gcggccgcgc caccatgagc tggtttctgt tcctggccca cagagtcgcc ttggccgcct
       61 tgccctgccg ccgcggctct cgcaggttcg ggatgttcta tgccgtgagg aggggccgca
      121 agaccggggt ctttctgacc tggaatgagt gcagagcaca ggtggaccgg tttcctgctg
      181 ccagatttaa gaagtttgcc acagaggatg aggccagggc ctttgtcagg aaatctgcaa
      241 gcccggaagt ttcagaaggg catgaaaatc aacatggaca agaatcggag gcgaaagcca
      301 gcaagcgact ccgtgagcca ctggatggag atggacatga aagcgcagag ccgtatgcaa
      361 agcacatgaa gccgagcgtg gagccggcgc ctccagttag cagagacacg ttttcctaca
      421 tgggagactt cgtcgtcgtc tacactgatg gctgctgctc cagtaatggg cgtagaaggc
      481 cgcgagcagg aatcggcgtt tactgggggc cgggccatcc tttaaatgta ggcattagac
      541 ttcctgggcg gcagacaaac caaagagcgg aaattcatgc agcctgcaaa gccattgaac
      601 aagcaaagac tcaaaacatc aataaactgg ttctgtatac aaacagtatg tttacgataa
      661 atggtataac taactgggtt caaggttgga agaaaaatgg gtggaagaca agtgcaggga
      721 aagaggtgat caacaaagag gactttgtgg cactggagag gcttacccag gggatggaca
      781 ttcagtggat gcatgttcct ggtcattcgg gatttatagg caatgaagaa gctgacagat
      841 tagccagaga aggagctaaa caatcggaag acggaggtgg atctggaggt ggatctggag
      901 gtggatctgg aggtggatct cccaagaaga agaggaaggt gggaggttct atggtgagca
      961 agggcgagga gctgttcacc ggggtggtgc ccatcctggt cgagctggac ggcgacgtaa
     1021 acggccacaa gttcagcgtg tccggcgagg gcgagggcga tgccacctac ggcaagctga
     1081 ccctgaagtt catctgcacc accggcaagc tgcccgtgcc ctggcccacc ctcgtgacca
     1141 ccctgaccta cggcgtgcag tgcttcagcc gctaccccga ccacatgaag cagcacgact
     1201 tcttcaagtc cgccatgccc gaaggctacg tccaggagcg caccatcttc ttcaaggacg
     1261 acggcaacta caagacccgc gccgaggtga agttcgaggg cgacaccctg gtgaaccgca
     1321 tcgagctgaa gggcatcgac ttcaaggagg acggcaacat cctggggcac aagctggagt
     1381 acaactacaa cagccacaac gtctatatca tggccgacaa gcagaagaac ggcatcaagg
     1441 tgaacttcaa gatccgccac aacatcgagg acggcagcgt gcagctcgcc gaccactacc
     1501 agcagaacac ccccatcggc gacggccccg tgctgctgcc cgacaaccac tacctgagca
     1561 cccagtccgc cctgagcaaa gaccccaacg agaagcgcga tcacatggtc ctgctggagt
     1621 tcgtgaccgc cgccgggatc actctcggca tggacgagct gtacaaggga ggtggatctg
     1681 gaggtggatc tggaggtgga tctggaggtg gatctcccaa gaagaagagg aaggtgggag
     1741 gttctatgga taagaaatac tcaataggct tagctatcgg cacaaatagc gtcggatggg
     1801 cggtgatcac tgatgaatat aaggttccgt ctaaaaagtt caaggttctg ggaaatacag
     1861 accgccacag tatcaaaaaa aatcttatag gggctctttt atttgacagt ggagagacag
     1921 cggaagcgac tcgtctcaaa cggacagctc gtagaaggta tacacgtcgg aagaatcgta
     1981 tttgttatct acaggagatt ttttcaaatg agatggcgaa agtagatgat agtttctttc
     2041 atcgacttga agagtctttt ttggtggaag aagacaagaa gcatgaacgt catcctattt
     2101 ttggaaatat agtagatgaa gttgcttatc atgagaaata tccaactatc tatcatctgc
     2161 gaaaaaaatt ggtagattct actgataaag cggatttgcg cttaatctat ttggccttag
     2221 cgcatatgat taagtttcgt ggtcattttt tgattgaggg agatttaaat cctgataata
     2281 gtgatgtgga caaactattt atccagttgg tacaaaccta caatcaatta tttgaagaaa
     2341 accctattaa cgcaagtgga gtagatgcta aagcgattct ttctgcacga ttgagtaaat
     2401 caagacgatt agaaaatctc attgctcagc tccccggtga gaagaaaaat ggcttatttg
     2461 ggaatctcat tgctttgtca ttgggtttga cccctaattt taaatcaaat tttgatttgg
     2521 cagaagatgc taaattacag ctttcaaaag atacttacga tgatgattta gataatttat
     2581 tggcgcaaat tggagatcaa tatgctgatt tgtttttggc agctaagaat ttatcagatg
     2641 ctattttact ttcagatatc ctaagagtaa atactgaaat aactaaggct cccctatcag
     2701 cttcaatgat taaacgctac gatgaacatc atcaagactt gactctttta aaagctttag
     2761 ttcgacaaca acttccagaa aagtataaag aaatcttttt tgatcaatca aaaaacggat
     2821 atgcaggtta tattgatggg ggagctagcc aagaagaatt ttataaattt atcaaaccaa
     2881 ttttagaaaa aatggatggt actgaggaat tattggtgaa actaaatcgt gaagatttgc
     2941 tgcgcaagca acggaccttt gacaacggct ctattcccca tcaaattcac ttgggtgagc
     3001 tgcatgctat tttgagaaga caagaagact tttatccatt tttaaaagac aatcgtgaga
     3061 agattgaaaa aatcttgact tttcgaattc cttattatgt tggtccattg gcgcgtggca
     3121 atagtcgttt tgcatggatg actcggaagt ctgaagaaac aattacccca tggaattttg
     3181 aagaagttgt cgataaaggt gcttcagctc aatcatttat tgaacgcatg acaaactttg
     3241 ataaaaatct tccaaatgaa aaagtactac caaaacatag tttgctttat gagtatttta
     3301 cggtttataa cgaattgaca aaggtcaaat atgttactga aggaatgcga aaaccagcat
     3361 ttctttcagg tgaacagaag aaagccattg ttgatttact cttcaaaaca aatcgaaaag
     3421 taaccgttaa gcaattaaaa gaagattatt tcaaaaaaat agaatgtttt gatagtgttg
     3481 aaatttcagg agttgaagat agatttaatg cttcattagg tacctaccat gatttgctaa
     3541 aaattattaa agataaagat tttttggata atgaagaaaa tgaagatatc ttagaggata
     3601 ttgttttaac attgacctta tttgaagata gggagatgat tgaggaaaga cttaaaacat
     3661 atgctcacct ctttgatgat aaggtgatga aacagcttaa acgtcgccgt tatactggtt
     3721 ggggacgttt gtctcgaaaa ttgattaatg gtattaggga taagcaatct ggcaaaacaa
     3781 tattagattt tttgaaatca gatggttttg ccaatcgcaa ttttatgcag ctgatccatg
     3841 atgatagttt gacatttaaa gaagacattc aaaaagcaca agtgtctgga caaggcgata
     3901 gtttacatga acatattgca aatttagctg gtagccctgc tattaaaaaa ggtattttac
     3961 agactgtaaa agttgttgat gaattggtca aagtaatggg gcggcataag ccagaaaata
     4021 tcgttattga aatggcacgt gaaaatcaga caactcaaaa gggccagaaa aattcgcgag
     4081 agcgtatgaa acgaatcgaa gaaggtatca aagaattagg aagtcagatt cttaaagagc
     4141 atcctgttga aaatactcaa ttgcaaaatg aaaagctcta tctctattat ctccaaaatg
     4201 gaagagacat gtatgtggac caagaattag atattaatcg tttaagtgat tatgatgtcg
     4261 atgccattgt tccacaaagt ttccttaaag acgattcaat agacaataag gtcttaacgc
     4321 gttctgataa aaatcgtggt aaatcggata acgttccaag tgaagaagta gtcaaaaaga
     4381 tgaaaaacta ttggagacaa cttctaaacg ccaagttaat cactcaacgt aagtttgata
     4441 atttaacgaa agctgaacgt ggaggtttga gtgaacttga taaagctggt tttatcaaac
     4501 gccaattggt tgaaactcgc caaatcacta agcatgtggc acaaattttg gatagtcgca
     4561 tgaatactaa atacgatgaa aatgataaac ttattcgaga ggttaaagtg attaccttaa
     4621 aatctaaatt agtttctgac ttccgaaaag atttccaatt ctataaagta cgtgagatta
     4681 acaattacca tcatgcccat gatgcgtatc taaatgccgt cgttggaact gctttgatta
     4741 agaaatatcc aaaacttgaa tcggagtttg tctatggtga ttataaagtt tatgatgttc
     4801 gtaaaatgat tgctaagtct gagcaagaaa taggcaaagc aaccgcaaaa tatttctttt
     4861 actctaatat catgaacttc ttcaaaacag aaattacact tgcaaatgga gagattcgca
     4921 aacgccctct aatcgaaact aatggggaaa ctggagaaat tgtctgggat aaagggcgag
     4981 attttgccac agtgcgcaaa gtattgtcca tgccccaagt caatattgtc aagaaaacag
     5041 aagtacagac aggcggattc tccaaggagt caattttacc aaaaagaaat tcggacaagc
     5101 ttattgctcg taaaaaagac tgggatccaa aaaaatatgg tggttttgat agtccaacgg
     5161 tagcttattc agtcctagtg gttgctaagg tggaaaaagg gaaatcgaag aagttaaaat
     5221 ccgttaaaga gttactaggg atcacaatta tggaaagaag ttcctttgaa aaaaatccga
     5281 ttgacttttt agaagctaaa ggatataagg aagttaaaaa agacttaatc attaaactac
     5341 ctaaatatag tctttttgag ttagaaaacg gtcgtaaacg gatgctggct agtgccggag
     5401 aattacaaaa aggaaatgag ctggctctgc caagcaaata tgtgaatttt ttatatttag
     5461 ctagtcatta tgaaaagttg aagggtagtc cagaagataa cgaacaaaaa caattgtttg
     5521 tggagcagca taagcattat ttagatgaga ttattgagca aatcagtgaa ttttctaagc
     5581 gtgttatttt agcagatgcc aatttagata aagttcttag tgcatataac aaacatagag
     5641 acaaaccaat acgtgaacaa gcagaaaata ttattcattt atttacgttg acgaatcttg
     5701 gagctcccgc tgcttttaaa tattttgata caacaattga tcgtaaacga tatacgtcta
     5761 caaaagaagt tttagatgcc actcttatcc atcaatccat cactggtctt tatgaaacac
     5821 gcattgattt gagtcagcta ggaggtgact gagtctaga
//
