LOCUS       sequence-266632-        6663 bp    DNA     linear   UNA 15-DEC-2025
DEFINITION  Targeted simultaneous C-to-T and A-to-G in rice protoplasts.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      natural DNA sequence
  ORGANISM  unspecified
REFERENCE   1  (bases 1 to 6663)
  TITLE     Gao Lab Plasmids
REFERENCE   2  (bases 1 to 6663)
  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"
COMMENT     Sequence Label: STEME-4
FEATURES             Location/Qualifiers
     source          1..6663
                     /mol_type="genomic DNA"
                     /organism="unspecified"
     CDS             join(1834..1857,1858..1860,1861..5934)
                     /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"
     RBS             5847..5855
                     /label=Shine-Dalgarno sequence
                     /note="full consensus sequence for ribosome-binding sites 
                     upstream of start codons in E. coli; complementary to a 
                     region in the 3' end of the 16S rRNA (Chen et al., 1994)"
     CDS             5935..5982
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             5989..6042
                     /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             6043..6291
                     /codon_start=1
                     /product="uracil-DNA glycosylase inhibitor from a Bacillus 
                     subtilis bacteriophage (Mol et al., 1995)"
                     /label=UGI
                     /translation="TNLSDIIEKETGKQLVIQESILMLPEEVEEVIGNKPESDILVHTA
                     YDESTDENVMLLTSDAPEYKPWALVIQDSNGENKIKML"
     CDS             6304..6324
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             6325..6378
                     /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             6379..6627
                     /codon_start=1
                     /product="uracil-DNA glycosylase inhibitor from a Bacillus 
                     subtilis bacteriophage (Mol et al., 1995)"
                     /label=UGI
                     /translation="TNLSDIIEKETGKQLVIQESILMLPEEVEEVIGNKPESDILVHTA
                     YDESTDENVMLLTSDAPEYKPWALVIQDSNGENKIKML"
     CDS             6640..6660
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
ORIGIN
        1 atgtccgagg tggagttctc tcacgagtat tggatgaggc acgctcttac acttgctaag
       61 agagcttggg acgaaagaga agtgccagtt ggcgccgttc ttgtgcataa taatagggtg
      121 atcggcgagg gttggaatag accaattgga aggcatgatc caacagctca cgcagagatt
      181 atggctctca gacaaggcgg cctcgttatg cagaactaca ggctcattga cgctacactc
      241 tacgtgacac tcgaaccttg cgttatgtgc gccggagcta tgattcattc taggattggc
      301 agggtcgtgt ttggagctag ggacgctaaa acaggagccg ccggatctct tatggacgtg
      361 ttgcatcatc caggcatgaa ccatagggtg gagattacag agggcattct tgcagacgag
      421 tgcgctgctc ttctttccga tttcttcagg atgagaaggc aggagattaa ggcccagaag
      481 aaggctcagt cttctacaga tagcggagga tcttccggag gatctagcgg ctccgagaca
      541 ccaggaacat ccgaaagcgc tacaccagaa tctagcggag gctcttccgg aggatcttct
      601 gaagtggagt tctcccacga gtattggatg aggcacgctc ttacacttgc taaaagggct
      661 agggacgaaa gggaagttcc agttggagct gttctcgtgc tcaataacag ggtgattggc
      721 gagggttgga atagagccat tggactccat gatccaacag ctcacgcaga gattatggct
      781 cttagacaag gcggcctcgt tatgcagaat tacagactca tcgacgccac actctacgtt
      841 accttcgaac cttgcgttat gtgcgccgga gctatgatcc attctaggat tggcagggtc
      901 gtgttcggcg ttagaaacgc taagacagga gctgcaggct ctcttatgga cgttcttcat
      961 tacccaggca tgaatcatag agtggagatc acagaaggca ttcttgcaga cgagtgcgca
     1021 gctctccttt gctatttctt caggatgccg aggcaagttt tcaacgctca gaagaaggcc
     1081 cagtcttcta cagattccgg cggatcttct ggaggatcta gcggctccga gacaccagga
     1141 acatccgaat ccgctacacc agagtcttct ggaggatcta gcggaggatc tgaggccagc
     1201 ccggctagcg gcccaaggca tctcatggac ccgcacatct tcaccagcaa cttcaacaac
     1261 ggcatcggca ggcacaagac ctacttgtgc tacgaggtgg agaggctcga caacggaacc
     1321 tccgtgaaga tggaccaaca cagggggttc ctccacaacc aagccaagaa cctcctctgc
     1381 ggcttctacg gcaggcacgc cgagttgagg ttcctcgact tggtgccatc cctccaactc
     1441 gatccagccc aaatctaccg cgtgacctgg ttcatctcct ggtccccatg cttctcctgg
     1501 ggttgcgccg gcgaggttcg ggctttcctc caagaaaaca cccacgtccg cctccgcatt
     1561 ttcgccgcca ggatctatga ttacgaccct ctctacaagg aggccctcca gatgctgcgg
     1621 gacgccggtg ctcaggtgag tatcatgacc tacgacgagt tcaagcactg ctgggacacc
     1681 ttcgttgacc accagggctg cccattccaa ccatgggacg gtctggatga acacagccaa
     1741 gccttgtccg gcaggctccg ggccatcctc caaaaccagg ggaactccgg gagcgagacg
     1801 ccaggcacct ccgagtcggc caccccagaa tctgacaaga agtactcgat cggcctcgcc
     1861 attgggacta actctgttgg ctgggccgtg atcaccgacg agtacaaggt gccctcaaag
     1921 aagttcaagg tcctgggcaa caccgatcgg cattccatca agaagaatct cattggcgct
     1981 ctcctgttcg acagcggcga gacggctgag gctacgcggc tcaagcgcac cgcccgcagg
     2041 cggtacacgc gcaggaagaa tcgcatctgc tacctgcagg agattttctc caacgagatg
     2101 gcgaaggttg acgattcttt cttccacagg ctggaggagt cattcctcgt ggaggaggat
     2161 aagaagcacg agcggcatcc aatcttcggc aacattgtcg acgaggttgc ctaccacgag
     2221 aagtacccta cgatctacca tctgcggaag aagctcgtgg actccacaga taaggcggac
     2281 ctccgcctga tctacctcgc tctggcccac atgattaagt tcaggggcca tttcctgatc
     2341 gagggggatc tcaacccgga caatagcgat gttgacaagc tgttcatcca gctcgtgcag
     2401 acgtacaacc agctcttcga ggagaacccc attaatgcgt caggcgtcga cgcgaaggct
     2461 atcctgtccg ctaggctctc gaagtctcgg cgcctcgaga acctgatcgc ccagctgccg
     2521 ggcgagaaga agaacggcct gttcgggaat ctcattgcgc tcagcctggg gctcacgccc
     2581 aacttcaagt cgaatttcga tctcgctgag gacgccaagc tgcagctctc caaggacaca
     2641 tacgacgatg acctggataa cctcctggcc cagatcggcg atcagtacgc ggacctgttc
     2701 ctcgctgcca agaatctgtc ggacgccatc ctcctgtctg atattctcag ggtgaacacc
     2761 gagattacga aggctccgct ctcagcctcc atgatcaagc gctacgacga gcaccatcag
     2821 gatctgaccc tcctgaaggc gctggtcagg cagcagctcc ccgagaagta caaggagatc
     2881 ttcttcgatc agtcgaagaa cggctacgct gggtacattg acggcggggc ctctcaggag
     2941 gagttctaca agttcatcaa gccgattctg gagaagatgg acggcacgga ggagctgctg
     3001 gtgaagctca atcgcgagga cctcctgagg aagcagcgga cattcgataa cggcagcatc
     3061 ccacaccaga ttcatctcgg ggagctgcac gctatcctga ggaggcagga ggacttctac
     3121 cctttcctca aggataaccg cgagaagatc gagaagattc tgactttcag gatcccgtac
     3181 tacgtcggcc cactcgctag gggcaactcc cgcttcgctt ggatgacccg caagtcagag
     3241 gagacgatca cgccgtggaa cttcgaggag gtggtcgaca agggcgctag cgctcagtcg
     3301 ttcatcgaga ggatgacgaa tttcgacaag aacctgccaa atgagaaggt gctccctaag
     3361 cactcgctcc tgtacgagta cttcacagtc tacaacgagc tgactaaggt gaagtatgtg
     3421 accgagggca tgaggaagcc ggctttcctg tctggggagc agaagaaggc catcgtggac
     3481 ctcctgttca agaccaaccg gaaggtcacg gttaagcagc tcaaggagga ctacttcaag
     3541 aagattgagt gcttcgattc ggtcgagatc tctggcgttg aggaccgctt caacgcctcc
     3601 ctggggacct accacgatct cctgaagatc attaaggata aggacttcct ggacaacgag
     3661 gagaatgagg atatcctcga ggacattgtg ctgacactca ctctgttcga ggaccgggag
     3721 atgatcgagg agcgcctgaa gacttacgcc catctcttcg atgacaaggt catgaagcag
     3781 ctcaagagga ggaggtacac cggctggggg aggctgagca ggaagctcat caacggcatt
     3841 cgggacaagc agtccgggaa gacgatcctc gacttcctga agagcgatgg cttcgcgaac
     3901 cgcaatttca tgcagctgat tcacgatgac agcctcacat tcaaggagga tatccagaag
     3961 gctcaggtga gcggccaggg ggactcgctg cacgagcata tcgcgaacct cgctggctcg
     4021 ccagctatca agaaggggat tctgcagacc gtgaaggttg tggacgagct ggtgaaggtc
     4081 atgggcaggc acaagcctga gaacatcgtc attgagatgg cccgggagaa tcagaccacg
     4141 cagaagggcc agaagaactc acgcgagagg atgaagagga tcgaggaggg cattaaggag
     4201 ctggggtccc agatcctcaa ggagcacccg gtggagaaca cgcagctgca gaatgagaag
     4261 ctctacctgt actacctcca gaatggccgc gatatgtatg tggaccagga gctggatatt
     4321 aacaggctca gcgattacga cgtcgatcat atcgttccac agtcattcct gaaggatgac
     4381 tccattgaca acaaggtcct caccaggtcg gacaagaacc ggggcaagtc tgataatgtt
     4441 ccttcagagg aggtcgttaa gaagatgaag aactactggc gccagctcct gaatgccaag
     4501 ctgatcacgc agcggaagtt cgataacctc acaaaggctg agaggggcgg gctctctgag
     4561 ctggacaagg cgggcttcat caagaggcag ctggtcgaga cacggcagat cactaagcac
     4621 gttgcgcaga ttctcgactc acggatgaac actaagtacg atgagaatga caagctgatc
     4681 cgcgaggtga aggtcatcac cctgaagtca aagctcgtct ccgacttcag gaaggatttc
     4741 cagttctaca aggttcggga gatcaacaat taccaccatg cccatgacgc gtacctgaac
     4801 gcggtggtcg gcacagctct gatcaagaag tacccaaagc tcgagagcga gttcgtgtac
     4861 ggggactaca aggtttacga tgtgaggaag atgatcgcca agtcggagca ggagattggc
     4921 aaggctaccg ccaagtactt cttctactct aacattatga atttcttcaa gacagagatc
     4981 actctggcca atggcgagat ccggaagcgc cccctcatcg agacgaacgg cgagacgggg
     5041 gagatcgtgt gggacaaggg cagggatttc gcgaccgtca ggaaggttct ctccatgcca
     5101 caagtgaata tcgtcaagaa gacagaggtc cagactggcg ggttctctaa ggagtcaatt
     5161 ctgcctaagc ggaacagcga caagctcatc gcccgcaaga aggactggga tccgaagaag
     5221 tacggcgggt tcgacagccc cactgtggcc tactcggtcc tggttgtggc gaaggttgag
     5281 aagggcaagt ccaagaagct caagagcgtg aaggagctgc tggggatcac gattatggag
     5341 cgctccagct tcgagaagaa cccgatcgat ttcctggagg cgaagggcta caaggaggtg
     5401 aagaaggacc tgatcattaa gctccccaag tactcactct tcgagctgga gaacggcagg
     5461 aagcggatgc tggcttccgc tggcgagctg cagaagggga acgagctggc tctgccgtcc
     5521 aagtatgtga acttcctcta cctggcctcc cactacgaga agctcaaggg cagccccgag
     5581 gacaacgagc agaagcagct gttcgtcgag cagcacaagc attacctcga cgagatcatt
     5641 gagcagattt ccgagttctc caagcgcgtg atcctggccg acgcgaatct ggataaggtc
     5701 ctctccgcgt acaacaagca ccgcgacaag ccaatcaggg agcaggctga gaatatcatt
     5761 catctcttca ccctgacgaa cctcggcgcc cctgctgctt tcaagtactt cgacacaact
     5821 atcgatcgca agaggtacac aagcactaag gaggtcctgg acgcgaccct catccaccag
     5881 tcgattaccg gcctctacga gacgcgcatc gacctgtctc agctcggggg cgacaagcgg
     5941 ccagcggcga cgaagaaggc ggggcaggcg aagaagaaga aggaattcga gggcagagga
     6001 agtcttctaa catgcggtga cgtggaggag aatcccggcc ctaccaacct gtccgacatc
     6061 atcgagaagg agacgggcaa gcaactcgtg atccaggaga gcatcctcat gctgccagag
     6121 gaggtggagg aggtcatcgg caacaagcca gagtccgaca tcctggtgca caccgcctac
     6181 gacgagtcca ccgacgagaa cgtcatgctc ctgaccagcg acgccccaga gtacaagcca
     6241 tgggccctcg tcatccagga cagcaacggg gagaacaaga tcaagatgct gtcggggggg
     6301 agcccaaaga agaagcggaa ggtggagggc agaggaagtc ttctaacatg cggtgacgtg
     6361 gaggagaatc ccggccctac caacctgtcc gacatcatcg agaaggagac gggcaagcaa
     6421 ctcgtgatcc aggagagcat cctcatgctg ccagaggagg tggaggaggt catcggcaac
     6481 aagccagagt ccgacatcct ggtgcacacc gcctacgacg agtccaccga cgagaacgtc
     6541 atgctcctga ccagcgacgc cccagagtac aagccatggg ccctcgtcat ccaggacagc
     6601 aacggggaga acaagatcaa gatgctgtcg ggggggagcc caaagaagaa gcggaaggtg
     6661 tag
//
