LOCUS       sequence-384715-        6114 bp    DNA     linear   UNA 15-DEC-2025
DEFINITION  Expresses human codon-optimized SpCas9-NRCH-APOBEC-nCas9-Ung and 
            blasticidin resistance: EFS 
            promoter-SpCas9-NRCH-APOBEC-nCas9-Ung-NLS-FLAG-P2A-BSD.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      natural DNA sequence
  ORGANISM  unspecified
REFERENCE   1  (bases 1 to 6114)
  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 6114)
  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-NRCH-APOBEC-nCas9-Ung
FEATURES             Location/Qualifiers
     source          1..6114
                     /mol_type="genomic DNA"
                     /organism="unspecified"
     CDS             4..24
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             25..708
                     /codon_start=1
                     /product="cytidine deaminase (C to U editing enzyme) from 
                     rat"
                     /label=APOBEC-1
                     /note="can use ssDNA as a substrate (Komor et al., 2016)"
                     /translation="SSETGPVAVDPTLRRRIEPHEFEVFFDPRELRKETCLLYEINWGG
                     RHSIWRHTSQNTNKHVEVNFIEKFTTERYFCPNTRCSITWFLSWSPCGECSRAITEFLS
                     RYPHVTLFIYIARLYHHADPRNRQGLRDLISSGVTIQIMTEQESGYCWRNFVNYSPSNE
                     AHWPRYPHLWVRLYVLELYCIILGLPPCLNILRRKQPQLTFFTIALQSCHYQRLPPHIL
                     WATGLK"
     CDS             760..4860
                     /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
                     GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMVKRYDEHHQDLTLLKALVRQ
                     QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
                     KQRTFDNGIIPHQIHLGELHAILRRQGDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
                     SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF
                     TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS
                     VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL
                     KTYAHLFDDKVMKQLKRLRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ
                     LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGGH
                     KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL
                     YYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPS
                     EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV
                     AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
                     AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE
                     ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE
                     SILPKGNSDKLIARKKDWDPKKYGGFNSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT
                     IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGVLQKGNEL
                     ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA
                     NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTINRKQYNTTKEVLD
                     ATLIRQSITGLYETRIDLSQLGGD"
     CDS             4861..4908
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             5605..5625
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             5626..5649
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             5659..5715
                     /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             5716..6114
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
                     VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
                     KAIVKDSDGQPTAVGIRELLPSGYVWEG"
ORIGIN
        1 atgccaaaga agaagaggaa ggtttcatcg gagaccggcc ctgttgctgt tgaccccacc
       61 ctgcggcgga gaatcgagcc acacgagttc gaggtgttct tcgacccaag ggagctccgc
      121 aaggagacgt gcctcctgta cgagatcaac tggggcggca ggcactccat ctggaggcac
      181 accagccaaa acaccaacaa gcacgtggag gtcaacttca tcgagaagtt caccaccgag
      241 aggtacttct gcccaaacac ccgctgctcc atcacctggt tcctgtcctg gagcccatgc
      301 ggcgagtgct ccagggccat caccgagttc ctcagccgct acccacacgt caccctgttc
      361 atctacatcg ccaggctcta ccaccacgcc gacccaagga acaggcaggg cctccgcgac
      421 ctgatctcca gcggcgtgac catccaaatc atgaccgagc aggagtccgg ctactgctgg
      481 aggaacttcg tcaactactc cccaagcaac gaggcccact ggccaaggta cccacacctc
      541 tgggtgcgcc tctacgtgct cgagctgtac tgcatcatcc tcggcctgcc accatgcctc
      601 aacatcctga ggcgcaagca accacagctg accttcttca ccatcgccct ccaaagctgc
      661 cactaccaga ggctcccacc acacatcctg tgggctaccg gcctcaagtc cggcagcgag
      721 acgccaggca cctccgagag cgctacgcct gaacttaagg acaagaagta cagcatcggc
      781 ctggccatcg gcaccaactc tgtgggctgg gccgtgatca ccgacgagta caaggtgccc
      841 agcaagaaat tcaaggtgct gggcaacacc gaccggcaca gcatcaagaa gaacctgatc
      901 ggagccctgc tgttcgacag cggcgaaaca gccgaggcca cccggctgaa gagaaccgcc
      961 agaagaagat acaccagacg gaagaaccgg atctgctatc tgcaagagat cttcagcaac
     1021 gagatggcca aggtggacga cagcttcttc cacagactgg aagagtcctt cctggtggaa
     1081 gaggataaga agcacgagcg gcaccccatc ttcggcaaca tcgtggacga ggtggcctac
     1141 cacgagaagt accccaccat ctaccacctg agaaagaaac tggtggacag caccgacaag
     1201 gccgacctgc ggctgatcta tctggccctg gcccacatga tcaagttccg gggccacttc
     1261 ctgatcgagg gcgacctgaa ccccgacaac agcgacgtgg acaagctgtt catccagctg
     1321 gtgcagacct acaaccagct gttcgaggaa aaccccatca acgccagcgg cgtggacgcc
     1381 aaggccatcc tgtctgccag actgagcaag agcagacggc tggaaaatct gatcgcccag
     1441 ctgcccggcg agaagaagaa tggcctgttc ggaaacctga ttgccctgag cctgggcctg
     1501 acccccaact tcaagagcaa cttcgacctg gccgaggatg ccaaactgca gctgagcaag
     1561 gacacctacg acgacgacct ggacaacctg ctggcccaga tcggcgacca gtacgccgac
     1621 ctgtttctgg ccgccaagaa cctgtccgac gccatcctgc tgagcgacat cctgagagtg
     1681 aacaccgaga tcaccaaggc ccccctgagc gcctctatgg tgaagagata cgacgagcac
     1741 caccaggacc tgaccctgct gaaagctctc gtgcggcagc agctgcctga gaagtacaaa
     1801 gagattttct tcgaccagag caagaacggc tacgccggct acattgacgg cggagccagc
     1861 caggaagagt tctacaagtt catcaagccc atcctggaaa agatggacgg caccgaggaa
     1921 ctgctcgtga agctgaacag agaggacctg ctgcggaagc agcggacctt cgacaacggc
     1981 atcatccccc accagatcca cctgggagag ctgcacgcca ttctgcggcg gcagggcgat
     2041 ttttacccat tcctgaagga caaccgggaa aagatcgaga agatcctgac cttccgcatc
     2101 ccctactacg tgggccctct ggccagggga aacagcagat tcgcctggat gaccagaaag
     2161 agcgaggaaa ccatcacccc ctggaacttc gaggaagtgg tggacaaggg cgcttccgcc
     2221 cagagcttca tcgagcggat gaccaacttc gataagaacc tgcccaacga gaaggtgctg
     2281 cccaagcaca gcctgctgta cgagtacttc accgtgtata acgagctgac caaagtgaaa
     2341 tacgtgaccg agggaatgag aaagcccgcc ttcctgagcg gcgagcagaa aaaggccatc
     2401 gtggacctgc tgttcaagac caaccggaaa gtgaccgtga agcagctgaa agaggactac
     2461 ttcaagaaaa tcgagtgctt cgactccgtg gaaatctccg gcgtggaaga tcggttcaac
     2521 gcctccctgg gcacatacca cgatctgctg aaaattatca aggacaagga cttcctggac
     2581 aatgaggaaa acgaggacat tctggaagat atcgtgctga ccctgacact gtttgaggac
     2641 agagagatga tcgaggaacg gctgaaaacc tatgcccacc tgttcgacga caaagtgatg
     2701 aagcagctga agcggctgag atacaccggc tggggcaggc tgagccggaa gctgatcaac
     2761 ggcatccggg acaagcagtc cggcaagaca atcctggatt tcctgaagtc cgacggcttc
     2821 gccaacagaa acttcatgca gctgatccac gacgacagcc tgacctttaa agaggacatc
     2881 cagaaagccc aggtgtccgg ccagggcgat agcctgcacg agcacattgc caatctggcc
     2941 ggcagccccg ccattaagaa gggcatcctg cagacagtga aggtggtgga cgagctcgtg
     3001 aaagtgatgg gcggccacaa gcccgagaac atcgtgatcg aaatggccag agagaaccag
     3061 accacccaga agggacagaa gaacagccgc gagagaatga agcggatcga agagggcatc
     3121 aaagagctgg gcagccagat cctgaaagaa caccccgtgg aaaacaccca gctgcagaac
     3181 gagaagctgt acctgtacta cctgcagaat gggcgggata tgtacgtgga ccaggaactg
     3241 gacatcaacc ggctgtccga ctacgatgtg gaccatatcg tgcctcagag ctttctgaag
     3301 gacgactcca tcgacaacaa ggtgctgacc agaagcgaca agaaccgggg caagagcgac
     3361 aacgtgccct ccgaagaggt cgtgaagaag atgaagaact actggcggca gctgctgaac
     3421 gccaagctga ttacccagag aaagttcgac aatctgacca aggccgagag aggcggcctg
     3481 agcgaactgg ataaggccgg cttcatcaag agacagctgg tggaaacccg gcagatcaca
     3541 aagcacgtgg cacagatcct ggactcccgg atgaacacta agtacgacga gaatgacaag
     3601 ctgatccggg aagtgaaagt gatcaccctg aagtccaagc tggtgtccga tttccggaag
     3661 gatttccagt tttacaaagt gcgcgagatc aacaactacc accacgccca cgacgcctac
     3721 ctgaacgccg tcgtgggaac cgccctgatc aaaaagtacc ctaagctgga aagcgagttc
     3781 gtgtacggcg actacaaggt gtacgacgtg cggaagatga tcgccaagag cgagcaggaa
     3841 atcggcaagg ctaccgccaa gtacttcttc tacagcaaca tcatgaactt tttcaagacc
     3901 gagattaccc tggccaacgg cgagatccgg aagcggcctc tgatcgagac aaacggcgaa
     3961 accggggaga tcgtgtggga taagggccgg gattttgcca ccgtgcggaa agtgctgagc
     4021 atgccccaag tgaatatcgt gaaaaagacc gaggtgcaga caggcggctt cagcaaagag
     4081 tctatcctgc ccaagggcaa cagcgataag ctgatcgcca gaaagaagga ctgggaccct
     4141 aagaagtacg gcggcttcaa cagccccacc gtggcctatt ctgtgctggt ggtggccaaa
     4201 gtggaaaagg gcaagtccaa gaaactgaag agtgtgaaag agctactggg gatcaccatc
     4261 atggaaagaa gcagcttcga gaagaatccc atcgactttc tggaagccaa gggctacaaa
     4321 gaagtgaaaa aggacctgat catcaagctg cctaagtact ccctgttcga gctggaaaac
     4381 ggccggaaga gaatgctggc ctctgccggc gtgctgcaga agggaaacga actggccctg
     4441 ccctccaaat atgtgaactt cctgtacctg gccagccact atgagaagct gaagggctcc
     4501 cccgaggata atgagcagaa acagctgttt gtggaacagc acaagcacta cctggacgag
     4561 atcatcgagc agatcagcga gttctccaag agagtgatcc tggccgacgc taatctggac
     4621 aaagtgctgt ccgcctacaa caagcaccgg gataagccca tcagagagca ggccgagaat
     4681 atcatccacc tgtttaccct gaccaatctg ggagcccctg ccgccttcaa gtactttgac
     4741 accaccatca accggaagca gtacaacacc accaaagagg tgctggacgc caccctgatc
     4801 cggcagagca tcaccggcct gtacgagaca cggatcgacc tgtctcagct gggaggcgac
     4861 aagagacccg cagcaaccaa gaaggcaggg caagcaaaga agaagaagac gcgtgactcc
     4921 ggcggcagca tgtttggaga gagctggaag aagcacctca gcggggagtt cgggaaaccg
     4981 tattttatca agctaatggg atttgttgca gaagaaagaa agcattacac tgtttatcca
     5041 cccccacacc aagtcttcac ctggacccag atgtgtgaca taaaagatgt gaaggttgtc
     5101 atcctgggac aggatccata tcatggacct aatcaagctc acgggctctg ctttagtgtt
     5161 caaaggcctg ttccgcctcc gcccagtttg gagaacattt ataaagagtt gtctacagac
     5221 atagaggatt ttgttcatcc tggccatgga gatttatctg ggtgggccaa gcaaggtgtt
     5281 ctccttctca acgctgtcct cacggttcgt gcccatcaag ccaactctca taaggagcga
     5341 ggctgggagc agttcactga tgcagttgtg tcctggctaa atcagaactc gaatggcctt
     5401 gttttcttgc tctggggctc ttatgctcag aagaagggca gtgccattga taggaagcgg
     5461 caccatgtac tacagacggc tcatccctcc cctttgtcag tgtatagagg gttctttgga
     5521 tgtagacact tttcaaagac caatgagctg ctgcagaagt ctggcaagaa gcccattgac
     5581 tggaaggagc tgtcgggggg gagcccaaag aagaagcgga aggtggatta caaagacgat
     5641 gacgataagg gatccggcgc aacaaacttc tctctgctga aacaagccgg agatgtcgaa
     5701 gagaatcctg gaccgatggc caagcctttg tctcaagaag aatccaccct cattgaaaga
     5761 gcaacggcta caatcaacag catccccatc tctgaagact acagcgtcgc cagcgcagct
     5821 ctctctagcg acggccgcat cttcactggt gtcaatgtat atcattttac tgggggacct
     5881 tgtgcagaac tcgtggtgct gggcactgct gctgctgcgg cagctggcaa cctgacttgt
     5941 atcgtcgcga tcggaaatga gaacaggggc atcttgagcc cctgcggacg gtgccgacag
     6001 gtgcttctcg atctgcatcc tgggatcaaa gccatagtga aggacagtga tggacagccg
     6061 acggcagttg ggattcgtga attgctgccc tctggttatg tgtgggaggg ctaa
//
