LOCUS       sequence-211101-        4779 bp    DNA     linear   UNA 15-DEC-2025
DEFINITION  lenti vector encoding D10Acas9-3xflag with T2A Blastcidin resistance
            marker (EF1a-NLS-D10ACas9-3Xflag-T2A-Blast-WPRE).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      natural DNA sequence
  ORGANISM  unspecified
REFERENCE   1  (bases 1 to 4779)
  AUTHORS   Liu LD, Huang M, Dai P, Liu T, Fan S, Cheng X, Zhao Y, Yeap LS, Meng
            FL
  TITLE     Intrinsic Nucleotide Preference of Diversifying Base Editors Guides 
            Antibody Ex Vivo Affinity Maturation.
  JOURNAL   Cell Rep. 2018 Oct 23;25(4):884-892.e3. doi: 
            10.1016/j.celrep.2018.09.090.
  PUBMED    30355495
REFERENCE   2  (bases 1 to 4779)
  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: 
            "10.1016/j.celrep.2018.09"; journalName: "Cell Rep"; date: 
            "2018-10-23- 23"; volume: "25"; issue: "4"; pages: "884-892.e3"
COMMENT     Sequence Label: pLL8 lenti-D10Acas9-3xflag-blast
FEATURES             Location/Qualifiers
     source          1..4779
                     /mol_type="genomic DNA"
                     /organism="unspecified"
     CDS             4..51
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             join(52..75,76..78,79..2634,2635..2637,2638..4152)
                     /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
                     YYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPS
                     EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV
                     AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
                     AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE
                     ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE
                     SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT
                     IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL
                     ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA
                     NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD
                     ATLIHQSITGLYETRIDLSQLGGD"
     CDS             join(52..75,76..78,79..4152)
                     /codon_start=1
                     /product="nickase mutant of the Cas9 endonuclease from the 
                     Streptococcus pyogenes Type II CRISPR/Cas system"
                     /label=Cas9(D10A)
                     /note="generates RNA-guided single strand nicks in DNA due 
                     to the D10A mutation in the RuvC catalytic domain"
                     /translation="DKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN
                     LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF
                     LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF
                     RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE
                     NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI
                     GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
                     QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
                     KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
                     SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF
                     TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS
                     VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL
                     KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ
                     LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH
                     KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL
                     YYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPS
                     EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV
                     AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
                     AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE
                     ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE
                     SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT
                     IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL
                     ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA
                     NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD
                     ATLIHQSITGLYETRIDLSQLGGD"
     CDS             4207..4227
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             4246..4311
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             4327..4380
                     /codon_start=1
                     /product="2A peptide from Thosea asigna virus capsid 
                     protein"
                     /label=T2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="EGRGSLLTCGDVEENPGP"
     CDS             4381..4779
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
                     VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
                     KAIVKDSDGQPTAVGIRELLPSGYVWEG"
ORIGIN
        1 atgaaaaggc cggcggccac gaaaaaggcc ggccaggcaa aaaagaaaaa ggacaagaag
       61 tacagcatcg gcctggccat cggcaccaac tctgtgggct gggccgtgat caccgacgag
      121 tacaaggtgc ccagcaagaa attcaaggtg ctgggcaaca ccgaccggca cagcatcaag
      181 aagaacctga tcggagccct gctgttcgac agcggcgaaa cagccgaggc cacccggctg
      241 aagagaaccg ccagaagaag atacaccaga cggaagaacc ggatctgcta tctgcaagag
      301 atcttcagca acgagatggc caaggtggac gacagcttct tccacagact ggaagagtcc
      361 ttcctggtgg aagaggataa gaagcacgag cggcacccca tcttcggcaa catcgtggac
      421 gaggtggcct accacgagaa gtaccccacc atctaccacc tgagaaagaa actggtggac
      481 agcaccgaca aggccgacct gcggctgatc tatctggccc tggcccacat gatcaagttc
      541 cggggccact tcctgatcga gggcgacctg aaccccgaca acagcgacgt ggacaagctg
      601 ttcatccagc tggtgcagac ctacaaccag ctgttcgagg aaaaccccat caacgccagc
      661 ggcgtggacg ccaaggccat cctgtctgcc agactgagca agagcagacg gctggaaaat
      721 ctgatcgccc agctgcccgg cgagaagaag aatggcctgt tcggcaacct gattgccctg
      781 agcctgggcc tgacccccaa cttcaagagc aacttcgacc tggccgagga tgccaaactg
      841 cagctgagca aggacaccta cgacgacgac ctggacaacc tgctggccca gatcggcgac
      901 cagtacgccg acctgtttct ggccgccaag aacctgtccg acgccatcct gctgagcgac
      961 atcctgagag tgaacaccga gatcaccaag gcccccctga gcgcctctat gatcaagaga
     1021 tacgacgagc accaccagga cctgaccctg ctgaaagctc tcgtgcggca gcagctgcct
     1081 gagaagtaca aagagatttt cttcgaccag agcaagaacg gctacgccgg ctacattgac
     1141 ggcggagcca gccaggaaga gttctacaag ttcatcaagc ccatcctgga aaagatggac
     1201 ggcaccgagg aactgctcgt gaagctgaac agagaggacc tgctgcggaa gcagcggacc
     1261 ttcgacaacg gcagcatccc ccaccagatc cacctgggag agctgcacgc cattctgcgg
     1321 cggcaggaag atttttaccc attcctgaag gacaaccggg aaaagatcga gaagatcctg
     1381 accttccgca tcccctacta cgtgggccct ctggccaggg gaaacagcag attcgcctgg
     1441 atgaccagaa agagcgagga aaccatcacc ccctggaact tcgaggaagt ggtggacaag
     1501 ggcgcttccg cccagagctt catcgagcgg atgaccaact tcgataagaa cctgcccaac
     1561 gagaaggtgc tgcccaagca cagcctgctg tacgagtact tcaccgtgta taacgagctg
     1621 accaaagtga aatacgtgac cgagggaatg agaaagcccg ccttcctgag cggcgagcag
     1681 aaaaaggcca tcgtggacct gctgttcaag accaaccgga aagtgaccgt gaagcagctg
     1741 aaagaggact acttcaagaa aatcgagtgc ttcgactccg tggaaatctc cggcgtggaa
     1801 gatcggttca acgcctccct gggcacatac cacgatctgc tgaaaattat caaggacaag
     1861 gacttcctgg acaatgagga aaacgaggac attctggaag atatcgtgct gaccctgaca
     1921 ctgtttgagg acagagagat gatcgaggaa cggctgaaaa cctatgccca cctgttcgac
     1981 gacaaagtga tgaagcagct gaagcggcgg agatacaccg gctggggcag gctgagccgg
     2041 aagctgatca acggcatccg ggacaagcag tccggcaaga caatcctgga tttcctgaag
     2101 tccgacggct tcgccaacag aaacttcatg cagctgatcc acgacgacag cctgaccttt
     2161 aaagaggaca tccagaaagc ccaggtgtcc ggccagggcg atagcctgca cgagcacatt
     2221 gccaatctgg ccggcagccc cgccattaag aagggcatcc tgcagacagt gaaggtggtg
     2281 gacgagctcg tgaaagtgat gggccggcac aagcccgaga acatcgtgat cgaaatggcc
     2341 agagagaacc agaccaccca gaagggacag aagaacagcc gcgagagaat gaagcggatc
     2401 gaagagggca tcaaagagct gggcagccag atcctgaaag aacaccccgt ggaaaacacc
     2461 cagctgcaga acgagaagct gtacctgtac tacctgcaga atgggcggga tatgtacgtg
     2521 gaccaggaac tggacatcaa ccggctgtcc gactacgatg tggaccacat cgtgcctcag
     2581 agctttctga aggacgactc catcgacaac aaggtgctga ccagaagcga caagaaccgg
     2641 ggcaagagcg acaacgtgcc ctccgaagag gtcgtgaaga agatgaagaa ctactggcgg
     2701 cagctgctga acgccaagct gattacccag agaaagttcg acaatctgac caaggccgag
     2761 agaggcggcc tgagcgaact ggataaggcc ggcttcatca agagacagct ggtggaaacc
     2821 cggcagatca caaagcacgt ggcacagatc ctggactccc ggatgaacac taagtacgac
     2881 gagaatgaca agctgatccg ggaagtgaaa gtgatcaccc tgaagtccaa gctggtgtcc
     2941 gatttccgga aggatttcca gttttacaaa gtgcgcgaga tcaacaacta ccaccacgcc
     3001 cacgacgcct acctgaacgc cgtcgtggga accgccctga tcaaaaagta ccctaagctg
     3061 gaaagcgagt tcgtgtacgg cgactacaag gtgtacgacg tgcggaagat gatcgccaag
     3121 agcgagcagg aaatcggcaa ggctaccgcc aagtacttct tctacagcaa catcatgaac
     3181 tttttcaaga ccgagattac cctggccaac ggcgagatcc ggaagcggcc tctgatcgag
     3241 acaaacggcg aaaccgggga gatcgtgtgg gataagggcc gggattttgc caccgtgcgg
     3301 aaagtgctga gcatgcccca agtgaatatc gtgaaaaaga ccgaggtgca gacaggcggc
     3361 ttcagcaaag agtctatcct gcccaagagg aacagcgata agctgatcgc cagaaagaag
     3421 gactgggacc ctaagaagta cggcggcttc gacagcccca ccgtggccta ttctgtgctg
     3481 gtggtggcca aagtggaaaa gggcaagtcc aagaaactga agagtgtgaa agagctgctg
     3541 gggatcacca tcatggaaag aagcagcttc gagaagaatc ccatcgactt tctggaagcc
     3601 aagggctaca aagaagtgaa aaaggacctg atcatcaagc tgcctaagta ctccctgttc
     3661 gagctggaaa acggccggaa gagaatgctg gcctctgccg gcgaactgca gaagggaaac
     3721 gaactggccc tgccctccaa atatgtgaac ttcctgtacc tggccagcca ctatgagaag
     3781 ctgaagggct cccccgagga taatgagcag aaacagctgt ttgtggaaca gcacaagcac
     3841 tacctggacg agatcatcga gcagatcagc gagttctcca agagagtgat cctggccgac
     3901 gctaatctgg acaaagtgct gtccgcctac aacaagcacc gggataagcc catcagagag
     3961 caggccgaga atatcatcca cctgtttacc ctgaccaatc tgggagcccc tgccgccttc
     4021 aagtactttg acaccaccat cgaccggaag aggtacacca gcaccaaaga ggtgctggac
     4081 gccaccctga tccaccagag catcaccggc ctgtacgaga cacggatcga cctgtctcag
     4141 ctgggaggcg acagcgctgg aggaggtgga agcggaggag gaggaagcgg aggaggaggt
     4201 agcggaccta agaaaaagag gaaggtggcg gccgctggat ccatggacta taaggaccac
     4261 gacggagact acaaggatca tgatattgat tacaaagacg atgacgataa gtgtacaggc
     4321 agtggagagg gcagaggaag tctgctaaca tgcggtgacg tcgaggagaa tcctggccca
     4381 atggccaagc ctttgtctca agaagaatcc accctcattg aaagagcaac ggctacaatc
     4441 aacagcatcc ccatctctga agactacagc gtcgccagcg cagctctctc tagcgacggc
     4501 cgcatcttca ctggtgtcaa tgtatatcat tttactgggg gaccttgtgc agaactcgtg
     4561 gtgctgggca ctgctgctgc tgcggcagct ggcaacctga cttgtatcgt cgcgatcgga
     4621 aatgagaaca ggggcatctt gagcccctgc ggacggtgcc gacaggtgct tctcgatctg
     4681 catcctggga tcaaagccat agtgaaggac agtgatggac agccgacggc agttgggatt
     4741 cgtgaattgc tgccctctgg ttatgtgtgg gagggctaa
//
