LOCUS       sequence-384711-        5490 bp    DNA     linear   UNA 15-DEC-2025
DEFINITION  Expresses human codon-optimized SpCas9-miniCGBE1 and blasticidin 
            resistance: EFS promoter-SpCas9-miniCGBE1-NLS-FLAG-P2A-BSD.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      natural DNA sequence
  ORGANISM  unspecified
REFERENCE   1  (bases 1 to 5490)
  AUTHORS   Kim N, Choi S, Kim S, Song M, Seo JH, Min S, Park J, Cho SR, Kim HH
  TITLE     Deep learning models to predict the editing efficiencies and 
            outcomes of diverse base editors.
  JOURNAL   Nat Biotechnol. 2024 Mar;42(3):484-497. doi: 
            10.1038/s41587-023-01792-x
  PUBMED    37188916
REFERENCE   2  (bases 1 to 5490)
  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"; journalName: 
            "Nat Biotechnol"; date: "2024-03"; volume: "42"; issue: "3"; pages: 
            "484-497"
COMMENT     Sequence Label: SpCas9-miniCGBE1
FEATURES             Location/Qualifiers
     source          1..5490
                     /mol_type="genomic DNA"
                     /organism="unspecified"
     CDS             37..57
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             join(838..861,862..864,865..4938)
                     /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             838..4938
                     /codon_start=1
                     /product="Cas9 (Csn1) endonuclease from the Streptococcus 
                     pyogenes Type II CRISPR/Cas system"
                     /label=Cas9
                     /note="generates RNA-guided double strand breaks in DNA"
                     /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             4981..5001
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             5002..5025
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             5035..5091
                     /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             5092..5490
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
                     VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
                     KAIVKDSDGQPTAVGIRELLPSGYVWEG"
ORIGIN
        1 atgaaacgga cagccgacgg aagcgagttc gagtcaccaa agaagaagcg gaaagtctcc
       61 tcagagactg ggcctgtcgc cgtcgatcca accctgcgcc gccggattga acctcacgag
      121 tttgaagtgt tctttgaccc ccgggagctg gccaaggaga catgcctgct gtacgagatc
      181 aactggggag gcaggcactc catctggagg cacacctctc agaacacaaa taagcacgtg
      241 gaggtgaact tcatcgagaa gtttaccaca gagcggtact tctgccccaa taccagatgt
      301 agcatcacat ggtttctgag ctggtcccct tgcggagagt gtagcagggc catcaccgag
      361 ttcctgtcca gatatccaca cgtgacactg tttatctaca tcgccaggct gtatcaccac
      421 gcagacccaa ggaataggca gggcctgcgc gatctgatca gctccggcgt gaccatccag
      481 atcatgacag agcaggagtc cggctactgc tggcggaact tcgtgaatta ttctcctagc
      541 aacgaggccc actggcctag gtacccacac ctgtgggtgc gcctgtacgt gctggagctg
      601 tattgcatca tcctgggcct gcccccttgt ctgaatatcc tgcggagaaa gcagccccag
      661 ctgaccttct ttacaatcgc cctgcagtct tgtcactatc agaggctgcc accccacatc
      721 ctgtgggcca caggcctgaa gtctggagga tctagcggag gatcctctgg cagcgagaca
      781 ccaggaacaa gcgagtcagc aacaccagag agcagtggcg gcagcagcgg cggcagcgac
      841 aagaagtaca gcatcggcct ggccatcggc accaactctg tgggctgggc cgtgatcacc
      901 gacgagtaca aggtgcccag caagaaattc aaggtgctgg gcaacaccga ccggcacagc
      961 atcaagaaga acctgatcgg agccctgctg ttcgacagcg gcgaaacagc cgaggccacc
     1021 cggctgaaga gaaccgccag aagaagatac accagacgga agaaccggat ctgctatctg
     1081 caagagatct tcagcaacga gatggccaag gtggacgaca gcttcttcca cagactggaa
     1141 gagtccttcc tggtggaaga ggataagaag cacgagcggc accccatctt cggcaacatc
     1201 gtggacgagg tggcctacca cgagaagtac cccaccatct accacctgag aaagaaactg
     1261 gtggacagca ccgacaaggc cgacctgcgg ctgatctatc tggccctggc ccacatgatc
     1321 aagttccggg gccacttcct gatcgagggc gacctgaacc ccgacaacag cgacgtggac
     1381 aagctgttca tccagctggt gcagacctac aaccagctgt tcgaggaaaa ccccatcaac
     1441 gccagcggcg tggacgccaa ggccatcctg tctgccagac tgagcaagag cagacggctg
     1501 gaaaatctga tcgcccagct gcccggcgag aagaagaatg gcctgttcgg aaacctgatt
     1561 gccctgagcc tgggcctgac ccccaacttc aagagcaact tcgacctggc cgaggatgcc
     1621 aaactgcagc tgagcaagga cacctacgac gacgacctgg acaacctgct ggcccagatc
     1681 ggcgaccagt acgccgacct gtttctggcc gccaagaacc tgtccgacgc catcctgctg
     1741 agcgacatcc tgagagtgaa caccgagatc accaaggccc ccctgagcgc ctctatgatc
     1801 aagagatacg acgagcacca ccaggacctg accctgctga aagctctcgt gcggcagcag
     1861 ctgcctgaga agtacaaaga gattttcttc gaccagagca agaacggcta cgccggctac
     1921 attgacggcg gagccagcca ggaagagttc tacaagttca tcaagcccat cctggaaaag
     1981 atggacggca ccgaggaact gctcgtgaag ctgaacagag aggacctgct gcggaagcag
     2041 cggaccttcg acaacggcag catcccccac cagatccacc tgggagagct gcacgccatt
     2101 ctgcggcggc aggaagattt ttacccattc ctgaaggaca accgggaaaa gatcgagaag
     2161 atcctgacct tccgcatccc ctactacgtg ggccctctgg ccaggggaaa cagcagattc
     2221 gcctggatga ccagaaagag cgaggaaacc atcaccccct ggaacttcga ggaagtggtg
     2281 gacaagggcg cttccgccca gagcttcatc gagcggatga ccaacttcga taagaacctg
     2341 cccaacgaga aggtgctgcc caagcacagc ctgctgtacg agtacttcac cgtgtataac
     2401 gagctgacca aagtgaaata cgtgaccgag ggaatgagaa agcccgcctt cctgagcggc
     2461 gagcagaaaa aggccatcgt ggacctgctg ttcaagacca accggaaagt gaccgtgaag
     2521 cagctgaaag aggactactt caagaaaatc gagtgcttcg actccgtgga aatctccggc
     2581 gtggaagatc ggttcaacgc ctccctgggc acataccacg atctgctgaa aattatcaag
     2641 gacaaggact tcctggacaa tgaggaaaac gaggacattc tggaagatat cgtgctgacc
     2701 ctgacactgt ttgaggacag agagatgatc gaggaacggc tgaaaaccta tgcccacctg
     2761 ttcgacgaca aagtgatgaa gcagctgaag cggcggagat acaccggctg gggcaggctg
     2821 agccggaagc tgatcaacgg catccgggac aagcagtccg gcaagacaat cctggatttc
     2881 ctgaagtccg acggcttcgc caacagaaac ttcatgcagc tgatccacga cgacagcctg
     2941 acctttaaag aggacatcca gaaagcccag gtgtccggcc agggcgatag cctgcacgag
     3001 cacattgcca atctggccgg cagccccgcc attaagaagg gcatcctgca gacagtgaag
     3061 gtggtggacg agctcgtgaa agtgatgggc cggcacaagc ccgagaacat cgtgatcgaa
     3121 atggccagag agaaccagac cacccagaag ggacagaaga acagccgcga gagaatgaag
     3181 cggatcgaag agggcatcaa agagctgggc agccagatcc tgaaagaaca ccccgtggaa
     3241 aacacccagc tgcagaacga gaagctgtac ctgtactacc tgcagaatgg gcgggatatg
     3301 tacgtggacc aggaactgga catcaaccgg ctgtccgact acgatgtgga ccatatcgtg
     3361 cctcagagct ttctgaagga cgactccatc gacaacaagg tgctgaccag aagcgacaag
     3421 aaccggggca agagcgacaa cgtgccctcc gaagaggtcg tgaagaagat gaagaactac
     3481 tggcggcagc tgctgaacgc caagctgatt acccagagaa agttcgacaa tctgaccaag
     3541 gccgagagag gcggcctgag cgaactggat aaggccggct tcatcaagag acagctggtg
     3601 gaaacccggc agatcacaaa gcacgtggca cagatcctgg actcccggat gaacactaag
     3661 tacgacgaga atgacaagct gatccgggaa gtgaaagtga tcaccctgaa gtccaagctg
     3721 gtgtccgatt tccggaagga tttccagttt tacaaagtgc gcgagatcaa caactaccac
     3781 cacgcccacg acgcctacct gaacgccgtc gtgggaaccg ccctgatcaa aaagtaccct
     3841 aagctggaaa gcgagttcgt gtacggcgac tacaaggtgt acgacgtgcg gaagatgatc
     3901 gccaagagcg agcaggaaat cggcaaggct accgccaagt acttcttcta cagcaacatc
     3961 atgaactttt tcaagaccga gattaccctg gccaacggcg agatccggaa gcggcctctg
     4021 atcgagacaa acggcgaaac cggggagatc gtgtgggata agggccggga ttttgccacc
     4081 gtgcggaaag tgctgagcat gccccaagtg aatatcgtga aaaagaccga ggtgcagaca
     4141 ggcggcttca gcaaagagtc tatcctgccc aagaggaaca gcgataagct gatcgccaga
     4201 aagaaggact gggaccctaa gaagtacggc ggcttcgaca gccccaccgt ggcctattct
     4261 gtgctggtgg tggccaaagt ggaaaagggc aagtccaaga aactgaagag tgtgaaagag
     4321 ctactgggga tcaccatcat ggaaagaagc agcttcgaga agaatcccat cgactttctg
     4381 gaagccaagg gctacaaaga agtgaaaaag gacctgatca tcaagctgcc taagtactcc
     4441 ctgttcgagc tggaaaacgg ccggaagaga atgctggcct ctgccggcga actgcagaag
     4501 ggaaacgaac tggccctgcc ctccaaatat gtgaacttcc tgtacctggc cagccactat
     4561 gagaagctga agggctcccc cgaggataat gagcagaaac agctgtttgt ggaacagcac
     4621 aagcactacc tggacgagat catcgagcag atcagcgagt tctccaagag agtgatcctg
     4681 gccgacgcta atctggacaa agtgctgtcc gcctacaaca agcaccggga taagcccatc
     4741 agagagcagg ccgagaatat catccacctg tttaccctga ccaatctggg agcccctgcc
     4801 gccttcaagt actttgacac caccatcgac cggaagaggt acaccagcac caaagaggtg
     4861 ctggacgcca ccctgatcca ccagagcatc accggcctgt acgagacacg gatcgacctg
     4921 tctcagctgg gaggcgactc tggcggctca aaaagaaccg ccgacggcag cgaattcgag
     4981 cccaagaaga agaggaaagt cgattacaaa gacgatgacg ataagggatc cggcgcaaca
     5041 aacttctctc tgctgaaaca agccggagat gtcgaagaga atcctggacc gatggccaag
     5101 cctttgtctc aagaagaatc caccctcatt gaaagagcaa cggctacaat caacagcatc
     5161 cccatctctg aagactacag cgtcgccagc gcagctctct ctagcgacgg ccgcatcttc
     5221 actggtgtca atgtatatca ttttactggg ggaccttgtg cagaactcgt ggtgctgggc
     5281 actgctgctg ctgcggcagc tggcaacctg acttgtatcg tcgcgatcgg aaatgagaac
     5341 aggggcatct tgagcccctg cggacggtgc cgacaggtgc ttctcgatct gcatcctggg
     5401 atcaaagcca tagtgaagga cagtgatgga cagccgacgg cagttgggat tcgtgaattg
     5461 ctgccctctg gttatgtgtg ggagggctaa
//
