LOCUS       sequence-103928-        5601 bp    DNA     linear   UNA 15-DEC-2025
DEFINITION  Lenti vector for expression of inducible split Cas9.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      natural DNA sequence
  ORGANISM  unspecified
REFERENCE   1  (bases 1 to 5601)
  AUTHORS   Zetsche B, Volz SE, Zhang F
  TITLE     A split-Cas9 architecture for inducible genome editing and 
            transcription modulation.
  JOURNAL   Nat Biotechnol. 2015 Feb 2. doi: 10.1038/nbt.3149.
  PUBMED    25643054
REFERENCE   2  (bases 1 to 5601)
  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. 2015 Feb 2. doi: 10.1038/nbt.3149."
COMMENT     Sequence Label: pLSC-5
FEATURES             Location/Qualifiers
     source          1..5601
                     /mol_type="genomic DNA"
                     /organism="unspecified"
     misc_RNA        91..166
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        228..439
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     regulatory      447..456
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             459..479
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             504..824
                     /codon_start=1
                     /product="human FK506-binding protein FKBP12"
                     /label=FKBP
                     /translation="GVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKFDSSRDRNKP
                     FKFMLGKQEVIRGWEEGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPHATLVFDVELL
                     KLE"
     CDS             870..3254
                     /codon_start=1
                     /product="C-terminal portion of Streptococcus pyogenes Cas9
                     (Zetsche et al., 2015)"
                     /label=Cas9(C)
                     /translation="CFDSVEISGVEDRFNASLGAYHDLLKIIKDKDFLDNEENEDILED
                     IVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGK
                     TILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKG
                     ILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQI
                     LKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDN
                     KVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDK
                     AGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQF
                     YKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGK
                     ATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMP
                     QVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKV
                     EKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELEN
                     GRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLD
                     EIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKY
                     FDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGD"
     CDS             3255..3302
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             3312..3368
                     /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             3393..5108
                     /codon_start=1
                     /product="N-terminal portion of Streptococcus pyogenes Cas9
                     (Zetsche et al., 2015)"
                     /label=Cas9(N)
                     /translation="DKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN
                     LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF
                     LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF
                     RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE
                     NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI
                     GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
                     QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
                     KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
                     SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF
                     TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIE"
     CDS             5154..5423
                     /codon_start=1
                     /product="FKBP-rapamycin binding domain of human FRAP"
                     /label=FRB
                     /translation="EMWHEGLEEASRLYFGERNVKGMFEVLEPLHAMMERGPQTLKETS
                     FNQAYGRDLMEAQEWCRKYMKSGNVKDLTQAWDLYYHVFRRISKQ"
     CDS             5433..5489
                     /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"
ORIGIN
        1 atcatatgct taccgtaact tgaaagtatt tcgatttctt ggctttatat atcttgtgga
       61 aaggacgaaa caccggagac ggacgtctct gttttagagc tagaaatagc aagttaaaat
      121 aaggctagtc cgttatcaac ttgaaaaagt ggcaccgagt cggtgctttt ttgaattcgc
      181 tagctaggtc ttgaaaggag tgggaattgg ctccggtgcc cgtcagtggg cagagcgcac
      241 atcgcccaca gtccccgaga agttgggggg aggggtcggc aattgatccg gtgcctagag
      301 aaggtggcgc ggggtaaact gggaaagtga tgtcgtgtac tggctccgcc tttttcccga
      361 gggtggggga gaaccgtata taagtgcagt agtcgccgtg aacgttcttt ttcgcaacgg
      421 gtttgccgcc agaacacagg accggtgcca ccatggcccc aaagaagaag cggaaggtcg
      481 gtatccacgg agtcccagca gccggcgtgc aggtggagac catcagcccc ggcgacggca
      541 gaaccttccc caagagaggc cagacctgcg tggtgcacta caccggcatg ctggaggacg
      601 gcaagaagtt cgacagcagc agagacagaa acaagccctt caagttcatg ctgggcaagc
      661 aggaggtgat cagaggctgg gaggagggcg tggcccagat gagcgtgggc cagagagcca
      721 agctgaccat cagccccgac tacgcctacg gcgccaccgg ccaccccggc atcatccctc
      781 ctcacgccac cctggtgttc gacgtggagc tgctgaagct ggagggtggc ggtggctcgg
      841 gtggcggtgg ctcgggtggc ggtggctcgt gcttcgactc cgtggaaatc tccggcgtgg
      901 aagatcggtt caacgcctcc ctgggcgcat accacgatct gctgaaaatt atcaaggaca
      961 aggacttcct ggacaatgag gaaaacgagg acattctgga agatatcgtg ctgaccctga
     1021 cactgtttga ggacagagag atgatcgagg aacggctgaa aacctatgcc cacctgttcg
     1081 acgacaaagt gatgaagcag ctgaagcggc ggagatacac cggctggggc aggctgagcc
     1141 ggaagctgat caacggcatc cgggacaagc agtccggcaa gacaatcctg gatttcctga
     1201 agtccgacgg cttcgccaac agaaacttca tgcagctgat ccacgacgac agcctgacct
     1261 ttaaagagga catccagaaa gcccaggtgt ccggccaggg cgatagcctg cacgagcaca
     1321 ttgccaatct ggccggcagc cccgccatta agaagggcat cctgcagaca gtgaaggtgg
     1381 tggacgagct cgtgaaagtg atgggccggc acaagcccga gaacatcgtg atcgaaatgg
     1441 ccagagagaa ccagaccacc cagaagggac agaagaacag ccgcgagaga atgaagcgga
     1501 tcgaagaggg catcaaagag ctgggcagcc agatcctgaa agaacacccc gtggaaaaca
     1561 cccagctgca gaacgagaag ctgtacctgt actacctgca gaatgggcgg gatatgtacg
     1621 tggaccagga actggacatc aaccggctgt ccgactacga tgtggaccat atcgtgcctc
     1681 agagctttct gaaggacgac tccatcgaca acaaggtgct gaccagaagc gacaagaacc
     1741 ggggcaagag cgacaacgtg ccctccgaag aggtcgtgaa gaagatgaag aactactggc
     1801 ggcagctgct gaacgccaag ctgattaccc agagaaagtt cgacaatctg accaaggccg
     1861 agagaggcgg cctgagcgaa ctggataagg ccggcttcat caagagacag ctggtggaaa
     1921 cccggcagat cacaaagcac gtggcacaga tcctggactc ccggatgaac actaagtacg
     1981 acgagaatga caagctgatc cgggaagtga aagtgatcac cctgaagtcc aagctggtgt
     2041 ccgatttccg gaaggatttc cagttttaca aagtgcgcga gatcaacaac taccaccacg
     2101 cccacgacgc ctacctgaac gccgtcgtgg gaaccgccct gatcaaaaag taccctaagc
     2161 tggaaagcga gttcgtgtac ggcgactaca aggtgtacga cgtgcggaag atgatcgcca
     2221 agagcgagca ggaaatcggc aaggctaccg ccaagtactt cttctacagc aacatcatga
     2281 actttttcaa gaccgagatt accctggcca acggcgagat ccggaagcgg cctctgatcg
     2341 agacaaacgg cgaaaccggg gagatcgtgt gggataaggg ccgggatttt gccaccgtgc
     2401 ggaaagtgct gagcatgccc caagtgaata tcgtgaaaaa gaccgaggtg cagacaggcg
     2461 gcttcagcaa agagtctatc ctgcccaaga ggaacagcga taagctgatc gccagaaaga
     2521 aggactggga ccctaagaag tacggcggct tcgacagccc caccgtggcc tattctgtgc
     2581 tggtggtggc caaagtggaa aagggcaagt ccaagaaact gaagagtgtg aaagagctgc
     2641 tggggatcac catcatggaa agaagcagct tcgagaagaa tcccatcgac tttctggaag
     2701 ccaagggcta caaagaagtg aaaaaggacc tgatcatcaa gctgcctaag tactccctgt
     2761 tcgagctgga aaacggccgg aagagaatgc tggcctctgc cggcgaactg cagaagggaa
     2821 acgaactggc cctgccctcc aaatatgtga acttcctgta cctggccagc cactatgaga
     2881 agctgaaggg ctcccccgag gataatgagc agaaacagct gtttgtggaa cagcacaagc
     2941 actacctgga cgagatcatc gagcagatca gcgagttctc caagagagtg atcctggccg
     3001 acgctaatct ggacaaagtg ctgtccgcct acaacaagca ccgggataag cccatcagag
     3061 agcaggccga gaatatcatc cacctgttta ccctgaccaa tctgggagcc cctgccgcct
     3121 tcaagtactt tgacaccacc atcgaccgga agaggtacac cagcaccaaa gaggtgctgg
     3181 atgccaccct gatccaccag agcatcaccg gcctgtacga gacacggatc gacctgtctc
     3241 agctgggagg cgacaaaagg ccggcggcca cgaaaaaggc cggccaggca aaaaagaaaa
     3301 agggatccgg cgcaacaaac ttctctctgc tgaaacaagc cggagatgtc gaagagaatc
     3361 ctggaccgct agatttagct agcttaatcc tcgacaagaa gtacagcatc ggcctggaca
     3421 tcggcaccaa ctctgtgggc tgggccgtga tcaccgacga gtacaaggtg cccagcaaga
     3481 aattcaaggt gctgggcaac accgaccggc acagcatcaa gaagaacctg atcggagccc
     3541 tgctgttcga cagcggcgaa acagccgagg ccacccggct gaagagaacc gccagaagaa
     3601 gatacaccag acggaagaac cggatctgct atctgcaaga gatcttcagc aacgagatgg
     3661 ccaaggtgga cgacagcttc ttccacagac tggaagagtc cttcctggtg gaagaggata
     3721 agaagcacga gcggcacccc atcttcggca acatcgtgga cgaggtggcc taccacgaga
     3781 agtaccccac catctaccac ctgagaaaga aactggtgga cagcaccgac aaggccgacc
     3841 tgcggctgat ctatctggcc ctggcccaca tgatcaagtt ccggggccac ttcctgatcg
     3901 agggcgacct gaaccccgac aacagcgacg tggacaagct gttcatccag ctggtgcaga
     3961 cctacaacca gctgttcgag gaaaacccca tcaacgccag cggcgtggac gccaaggcca
     4021 tcctgtctgc cagactgagc aagagcagac ggctggaaaa tctgatcgcc cagctgcccg
     4081 gcgagaagaa gaatggcctg ttcggaaacc tgattgccct gagcctgggc ctgaccccca
     4141 acttcaagag caacttcgac ctggccgagg atgccaaact gcagctgagc aaggacacct
     4201 acgacgacga cctggacaac ctgctggccc agatcggcga ccagtacgcc gacctgtttc
     4261 tggccgccaa gaacctgtcc gacgccatcc tgctgagcga catcctgaga gtgaacaccg
     4321 agatcaccaa ggcccccctg agcgcctcta tgatcaagag atacgacgag caccaccagg
     4381 acctgaccct gctgaaagct ctcgtgcggc agcagctgcc tgagaagtac aaagagattt
     4441 tcttcgacca gagcaagaac ggctacgccg gctacattga cggcggagcc agccaggaag
     4501 agttctacaa gttcatcaag cccatcctgg aaaagatgga cggcaccgag gaactgctcg
     4561 tgaagctgaa cagagaggac ctgctacgga agcagcggac cttcgacaac ggcagcatcc
     4621 cccaccagat ccacctggga gagctgcacg ccattctgcg gcggcaggaa gatttttacc
     4681 cattcctgaa ggacaaccgg gaaaagatcg agaagatcct gaccttccgc atcccctact
     4741 acgtgggccc tctggccagg ggaaacagca gattcgcctg gatgaccaga aagagcgagg
     4801 aaaccatcac cccctggaac ttcgaggaag tggtggacaa gggcgcttcc gcccagagct
     4861 tcatcgagcg gatgaccaac ttcgataaga acctgcccaa cgagaaggtg ctgcccaagc
     4921 acagcctgct gtacgagtac ttcaccgtgt ataacgagct gaccaaagtg aaatacgtga
     4981 ccgagggaat gagaaagccc gccttcctga gcggcgagca gaaaaaggcc atcgtggacc
     5041 tgctgttcaa gaccaaccgg aaagtgaccg tgaagcagct gaaagaggac tacttcaaga
     5101 aaatcgaggg tggcggtggc tcgggtggcg gtggctcggg tggcggtggc tcggagatgt
     5161 ggcacgaggg cctggaggag gccagcagac tgtacttcgg cgagagaaac gtgaagggca
     5221 tgttcgaggt gctggagccc ctgcacgcca tgatggagag aggcccccag accctgaagg
     5281 agaccagctt caaccaggcc tacggcagag acctgatgga ggcccaggag tggtgcagaa
     5341 agtacatgaa gagcggcaac gtgaaggacc tgacccaggc ctgggacctg tactaccacg
     5401 tgttcagaag aatcagcaag cagggatccg gcgcaacaaa cttctctctg ctgaaacaag
     5461 ccggagatgt cgaagagaat cctggaccga ccgagtacaa gcccacggtg cgcctcgcca
     5521 cccgcgacga cgtccccagg gccgtacgca ccctcgccgc cgcgttcgcc gactaccccg
     5581 ccacgcgcca caccgtcgat c
//
