LOCUS       sequence-298302-        5802 bp    DNA     linear   UNA 15-DEC-2025
DEFINITION  Lentiviral expression plasmid encoding dCas9-VPR and a P2A linked 
            mCherry marker.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      natural DNA sequence
  ORGANISM  unspecified
REFERENCE   1  (bases 1 to 5802)
  AUTHORS   Umkehrer C, Holstein F, Formenti L, Jude J, Froussios K, Neumann T, 
            Cronin SM, Haas L, Lipp JJ, Burkard TR, Fellner M, Wiesner T, Zuber 
            J, Obenauf AC
  TITLE     Isolating live cell clones from barcoded populations using 
            CRISPRa-inducible reporters.
  JOURNAL   Nat Biotechnol. 2020 Jul 27. pii: 10.1038/s41587-020-0614-0. doi: 
            10.1038/s41587-020-0614-0.
  PUBMED    32719478
REFERENCE   2  (bases 1 to 5802)
  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 
            Biotechnol. 2020 Jul 27. pii: 10.1038/s41587-020-0614-0. doi: 
            10.1038/s41587-020-0614-0."
COMMENT     Sequence Label: dCas9-VPR_P2A_mCherry
FEATURES             Location/Qualifiers
     source          1..5802
                     /mol_type="genomic DNA"
                     /organism="unspecified"
     CDS             join(1..27,28..30,31..2514,2515..2520,2521..2586,2587..2589,
                     2590..4104)
                     /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             4117..4137
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     protein_bind    4174..4198
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             4228..4377
                     /codon_start=1
                     /product="tetrameric repeat of the minimal activation 
                     domain of herpes simplex virus VP16 (Beerli et al., 1998)"
                     /label=VP64
                     /translation="DALDDFDLDMLGSDALDDFDLDMLGSDALDDFDLDMLGSDALDDF
                     DLDML"
     CDS             4402..4422
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             4852..5208
                     /codon_start=1
                     /product="transcriptional activation domain of human RelA, 
                     also known as p65 (O'Shea and Perkins, 2008)"
                     /label=RelA (p65) AD
                     /translation="PTQAGEGTLSEALLQLQFDDEDLGALLGNSTDPAVFTDLASVDNS
                     EFQQLLNQGIPVAPHTTEPMLMEYPEAITRLVTGAQRPPDPAPAPLGAPGLPNGLLSGD
                     EDFSSIADMDFSALL"
     CDS             5227..5796
                     /codon_start=1
                     /product="transcriptional activation domain from the human 
                     herpesvirus 4 (Epstein-Barr virus) replication and 
                     transcription activator Rta/BRLF1 (Hardwick et al., 1992; 
                     Chavez et al., 2015)"
                     /label=Rta AD
                     /translation="RDSREGMFLPKPEAGSAISDVFEGREVCQPKRIRPFHPPGSPWAN
                     RPLPASLAPTPTGPVHEPVGSLTPAPVPQPLDPAPAVTPEASHLLEDPDEETSQAVKAL
                     REMADTVIPQKEEAAICGQMDLSHPPPRGHLDELTTTLESMTEDLNLDSPLTPELNEIL
                     DTFLNDECLLHAMHISTGLSIFDTSLF"
ORIGIN
        1 atggacaaga agtactccat tgggctcgct atcggcacaa acagcgtcgg ctgggccgtc
       61 attacggacg agtacaaggt gccgagcaaa aaattcaaag ttctgggcaa taccgatcgc
      121 cacagcataa agaagaacct cattggcgcc ctcctgttcg actccgggga gacggccgaa
      181 gccacgcggc tcaaaagaac agcacggcgc agatataccc gcagaaagaa tcggatctgc
      241 tacctgcagg agatctttag taatgagatg gctaaggtgg atgactcttt cttccatagg
      301 ctggaggagt cctttttggt ggaggaggat aaaaagcacg agcgccaccc aatctttggc
      361 aatatcgtgg acgaggtggc gtaccatgaa aagtacccaa ccatatatca tctgaggaag
      421 aagcttgtag acagtactga taaggctgac ttgcggttga tctatctcgc gctggcgcat
      481 atgatcaaat ttcggggaca cttcctcatc gagggggacc tgaacccaga caacagcgat
      541 gtcgacaaac tctttatcca actggttcag acttacaatc agcttttcga agagaacccg
      601 atcaacgcat ccggagttga cgccaaagca atcctgagcg ctaggctgtc caaatcccgg
      661 cggctcgaaa acctcatcgc acagctccct ggggagaaga agaacggcct gtttggtaat
      721 cttatcgccc tgtcactcgg gctgaccccc aactttaaat ctaacttcga cctggccgaa
      781 gatgccaagc ttcaactgag caaagacacc tacgatgatg atctcgacaa tctgctggcc
      841 cagatcggcg accagtacgc agaccttttt ttggcggcaa agaacctgtc agacgccatt
      901 ctgctgagtg atattctgcg agtgaacacg gagatcacca aagctccgct gagcgctagt
      961 atgatcaagc gctatgatga gcaccaccaa gacttgactt tgctgaaggc ccttgtcaga
     1021 cagcaactgc ctgagaagta caaggaaatt ttcttcgatc agtctaaaaa tggctacgcc
     1081 ggatacattg acggcggagc aagccaggag gaattttaca aatttattaa gcccatcttg
     1141 gaaaaaatgg acggcaccga ggagctgctg gtaaagctta acagagaaga tctgttgcgc
     1201 aaacagcgca ctttcgacaa tggaagcatc ccccaccaga ttcacctggg cgaactgcac
     1261 gctatcctca ggcggcaaga ggatttctac ccctttttga aagataacag ggaaaagatt
     1321 gagaaaatcc tcacatttcg gataccctac tatgtaggcc ccctcgcccg gggaaattcc
     1381 agattcgcgt ggatgactcg caaatcagaa gagaccatca ctccctggaa cttcgaggaa
     1441 gtcgtggata agggggcctc tgcccagtcc ttcatcgaaa ggatgactaa ctttgataaa
     1501 aatctgccta acgaaaaggt gcttcctaaa cactctctgc tgtacgagta cttcacagtt
     1561 tataacgagc tcaccaaggt caaatacgtc acagaaggga tgagaaagcc agcattcctg
     1621 tctggagagc agaagaaagc tatcgtggac ctcctcttca agacgaaccg gaaagttacc
     1681 gtgaaacagc tcaaagaaga ctatttcaaa aagattgaat gtttcgactc tgttgaaatc
     1741 agcggagtgg aggatcgctt caacgcatcc ctgggaacgt atcacgatct cctgaaaatc
     1801 attaaagaca aggacttcct ggacaatgag gagaacgagg acattcttga ggacattgtc
     1861 ctcaccctta cgttgtttga agatagggag atgattgaag aacgcttgaa aacttacgct
     1921 catctcttcg acgacaaagt catgaaacag ctcaagaggc gccgatatac aggatggggg
     1981 cggctgtcaa gaaaactgat caatgggatc cgagacaagc agagtggaaa gacaatcctg
     2041 gattttctta agtccgatgg atttgccaac cggaacttca tgcagttgat ccatgatgac
     2101 tctctcacct ttaaggagga catccagaaa gcacaagttt ctggccaggg ggacagtctt
     2161 cacgagcaca tcgctaatct tgcaggtagc ccagctatca aaaagggaat actgcagacc
     2221 gttaaggtcg tggatgaact cgtcaaagta atgggaaggc ataagcccga gaatatcgtt
     2281 atcgagatgg cccgagagaa ccaaactacc cagaagggac agaagaacag tagggaaagg
     2341 atgaagagga ttgaagaggg tataaaagaa ctggggtccc aaatccttaa ggaacaccca
     2401 gttgaaaaca cccagcttca gaatgagaag ctctacctgt actacctgca gaacggcagg
     2461 gacatgtacg tggatcagga actggacatc aatcggctct ccgactacga cgtggctgct
     2521 atcgtgcccc agtcttttct caaagatgat tctattgata ataaagtgtt gacaagatcc
     2581 gataaagcta gagggaagag tgataacgtc ccctcagaag aagttgtcaa gaaaatgaaa
     2641 aattattggc ggcagctgct gaacgccaaa ctgatcacac aacggaagtt cgataatctg
     2701 actaaggctg aacgaggtgg cctgtctgag ttggataaag ccggcttcat caaaaggcag
     2761 cttgttgaga cacgccagat caccaagcac gtggcccaaa ttctcgattc acgcatgaac
     2821 accaagtacg atgaaaatga caaactgatt cgagaggtga aagttattac tctgaagtct
     2881 aagctggtct cagatttcag aaaggacttt cagttttata aggtgagaga gatcaacaat
     2941 taccaccatg cgcatgatgc ctacctgaat gcagtggtag gcactgcact tatcaaaaaa
     3001 tatcccaagc ttgaatctga atttgtttac ggagactata aagtgtacga tgttaggaaa
     3061 atgatcgcaa agtctgagca ggaaataggc aaggccaccg ctaagtactt cttttacagc
     3121 aatattatga attttttcaa gaccgagatt acactggcca atggagagat tcggaagcga
     3181 ccacttatcg aaacaaacgg agaaacagga gaaatcgtgt gggacaaggg tagggatttc
     3241 gcgacagtcc ggaaggtcct gtccatgccg caggtgaaca tcgttaaaaa gaccgaagta
     3301 cagaccggag gcttctccaa ggaaagtatc ctcccgaaaa ggaacagcga caagctgatc
     3361 gcacgcaaaa aagattggga ccccaagaaa tacggcggat tcgattctcc tacagtcgct
     3421 tacagtgtac tggttgtggc caaagtggag aaagggaagt ctaaaaaact caaaagcgtc
     3481 aaggaactgc tgggcatcac aatcatggag cgatcaagct tcgaaaaaaa ccccatcgac
     3541 tttctcgagg cgaaaggata taaagaggtc aaaaaagacc tcatcattaa gcttcccaag
     3601 tactctctct ttgagcttga aaacggccgg aaacgaatgc tcgctagtgc gggcgagctg
     3661 cagaaaggta acgagctggc actgccctct aaatacgtta atttcttgta tctggccagc
     3721 cactatgaaa agctcaaagg gtctcccgaa gataatgagc agaagcagct gttcgtggaa
     3781 caacacaaac actaccttga tgagatcatc gagcaaataa gcgaattctc caaaagagtg
     3841 atcctcgccg acgctaacct cgataaggtg ctttctgctt acaataagca cagggataag
     3901 cccatcaggg agcaggcaga aaacattatc cacttgttta ctctgaccaa cttgggcgcg
     3961 cctgcagcct tcaagtactt cgacaccacc atagacagaa agcggtacac ctctacaaag
     4021 gaggtcctgg acgccacact gattcatcag tcaattacgg ggctctatga aacaagaatc
     4081 gacctctctc agctcggtgg agacagcagg gctgacccca agaagaagag gaaggtgtcg
     4141 ccagggatcc gtcgacttga cgcgttgata tcaacaagtt tgtacaaaaa agcaggctac
     4201 aaagaggcca gcggttccgg acgggctgac gcattggacg attttgatct ggatatgctg
     4261 ggaagtgacg ccctcgatga ttttgacctt gacatgcttg gttcggatgc ccttgatgac
     4321 tttgacctcg acatgctcgg cagtgacgcc cttgatgatt tcgacctgga catgctgatt
     4381 aactctagaa gttccggatc tccgaaaaag aaacgcaaag ttggtagcca gtacctgccc
     4441 gacaccgacg accggcaccg gatcgaggaa aagcggaagc ggacctacga gacattcaag
     4501 agcatcatga agaagtcccc cttcagcggc cccaccgacc ctagacctcc acctagaaga
     4561 atcgccgtgc ccagcagatc cagcgccagc gtgccaaaac ctgcccccca gccttacccc
     4621 ttcaccagca gcctgagcac catcaactac gacgagttcc ctaccatggt gttccccagc
     4681 ggccagatct ctcaggcctc tgctctggct ccagcccctc ctcaggtgct gcctcaggct
     4741 cctgctcctg caccagctcc agccatggtg tctgcactgg ctcaggcacc agcacccgtg
     4801 cctgtgctgg ctcctggacc tccacaggct gtggctccac cagcccctaa acctacacag
     4861 gccggcgagg gcacactgtc tgaagctctg ctgcagctgc agttcgacga cgaggatctg
     4921 ggagccctgc tgggaaacag caccgatcct gccgtgttca ccgacctggc cagcgtggac
     4981 aacagcgagt tccagcagct gctgaaccag ggcatccctg tggcccctca caccaccgag
     5041 cccatgctga tggaataccc cgaggccatc acccggctcg tgacaggcgc tcagaggcct
     5101 cctgatccag ctcctgcccc tctgggagca ccaggcctgc ctaatggact gctgtctggc
     5161 gacgaggact tcagctctat cgccgatatg gatttctcag ccttgctggg ctctggcagc
     5221 ggcagccggg attccaggga agggatgttt ttgccgaagc ctgaggccgg ctccgctatt
     5281 agtgacgtgt ttgagggccg cgaggtgtgc cagccaaaac gaatccggcc atttcatcct
     5341 ccaggaagtc catgggccaa ccgcccactc cccgccagcc tcgcaccaac accaaccggt
     5401 ccagtacatg agccagtcgg gtcactgacc ccggcaccag tccctcagcc actggatcca
     5461 gcgcccgcag tgactcccga ggccagtcac ctgttggagg atcccgatga agagacgagc
     5521 caggctgtca aagcccttcg ggagatggcc gatactgtga ttccccagaa ggaagaggct
     5581 gcaatctgtg gccaaatgga cctttcccat ccgcccccaa ggggccatct ggatgagctg
     5641 acaaccacac ttgagtccat gaccgaggat ctgaacctgg actcacccct gaccccggaa
     5701 ttgaacgaga ttctggatac cttcctgaac gacgagtgcc tcttgcatgc catgcatatc
     5761 agcacaggac tgtccatctt cgacacatct ctgtttggat cc
//
