LOCUS       sequence-266630-        6291 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 6291)
  TITLE     Gao Lab Plasmids
REFERENCE   2  (bases 1 to 6291)
  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-2
FEATURES             Location/Qualifiers
     source          1..6291
                     /mol_type="genomic DNA"
                     /organism="unspecified"
     CDS             join(1840..1863,1864..1866,1867..5940)
                     /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             5853..5861
                     /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             5941..5988
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             6007..6255
                     /codon_start=1
                     /product="uracil-DNA glycosylase inhibitor from a Bacillus 
                     subtilis bacteriophage (Mol et al., 1995)"
                     /label=UGI
                     /translation="TNLSDIIEKETGKQLVIQESILMLPEEVEEVIGNKPESDILVHTA
                     YDESTDENVMLLTSDAPEYKPWALVIQDSNGENKIKML"
     CDS             6268..6288
                     /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 tctcttaagg acaagaagta ctcgatcggc
     1861 ctcgccattg ggactaactc tgttggctgg gccgtgatca ccgacgagta caaggtgccc
     1921 tcaaagaagt tcaaggtcct gggcaacacc gatcggcatt ccatcaagaa gaatctcatt
     1981 ggcgctctcc tgttcgacag cggcgagacg gctgaggcta cgcggctcaa gcgcaccgcc
     2041 cgcaggcggt acacgcgcag gaagaatcgc atctgctacc tgcaggagat tttctccaac
     2101 gagatggcga aggttgacga ttctttcttc cacaggctgg aggagtcatt cctcgtggag
     2161 gaggataaga agcacgagcg gcatccaatc ttcggcaaca ttgtcgacga ggttgcctac
     2221 cacgagaagt accctacgat ctaccatctg cggaagaagc tcgtggactc cacagataag
     2281 gcggacctcc gcctgatcta cctcgctctg gcccacatga ttaagttcag gggccatttc
     2341 ctgatcgagg gggatctcaa cccggacaat agcgatgttg acaagctgtt catccagctc
     2401 gtgcagacgt acaaccagct cttcgaggag aaccccatta atgcgtcagg cgtcgacgcg
     2461 aaggctatcc tgtccgctag gctctcgaag tctcggcgcc tcgagaacct gatcgcccag
     2521 ctgccgggcg agaagaagaa cggcctgttc gggaatctca ttgcgctcag cctggggctc
     2581 acgcccaact tcaagtcgaa tttcgatctc gctgaggacg ccaagctgca gctctccaag
     2641 gacacatacg acgatgacct ggataacctc ctggcccaga tcggcgatca gtacgcggac
     2701 ctgttcctcg ctgccaagaa tctgtcggac gccatcctcc tgtctgatat tctcagggtg
     2761 aacaccgaga ttacgaaggc tccgctctca gcctccatga tcaagcgcta cgacgagcac
     2821 catcaggatc tgaccctcct gaaggcgctg gtcaggcagc agctccccga gaagtacaag
     2881 gagatcttct tcgatcagtc gaagaacggc tacgctgggt acattgacgg cggggcctct
     2941 caggaggagt tctacaagtt catcaagccg attctggaga agatggacgg cacggaggag
     3001 ctgctggtga agctcaatcg cgaggacctc ctgaggaagc agcggacatt cgataacggc
     3061 agcatcccac accagattca tctcggggag ctgcacgcta tcctgaggag gcaggaggac
     3121 ttctaccctt tcctcaagga taaccgcgag aagatcgaga agattctgac tttcaggatc
     3181 ccgtactacg tcggcccact cgctaggggc aactcccgct tcgcttggat gacccgcaag
     3241 tcagaggaga cgatcacgcc gtggaacttc gaggaggtgg tcgacaaggg cgctagcgct
     3301 cagtcgttca tcgagaggat gacgaatttc gacaagaacc tgccaaatga gaaggtgctc
     3361 cctaagcact cgctcctgta cgagtacttc acagtctaca acgagctgac taaggtgaag
     3421 tatgtgaccg agggcatgag gaagccggct ttcctgtctg gggagcagaa gaaggccatc
     3481 gtggacctcc tgttcaagac caaccggaag gtcacggtta agcagctcaa ggaggactac
     3541 ttcaagaaga ttgagtgctt cgattcggtc gagatctctg gcgttgagga ccgcttcaac
     3601 gcctccctgg ggacctacca cgatctcctg aagatcatta aggataagga cttcctggac
     3661 aacgaggaga atgaggatat cctcgaggac attgtgctga cactcactct gttcgaggac
     3721 cgggagatga tcgaggagcg cctgaagact tacgcccatc tcttcgatga caaggtcatg
     3781 aagcagctca agaggaggag gtacaccggc tgggggaggc tgagcaggaa gctcatcaac
     3841 ggcattcggg acaagcagtc cgggaagacg atcctcgact tcctgaagag cgatggcttc
     3901 gcgaaccgca atttcatgca gctgattcac gatgacagcc tcacattcaa ggaggatatc
     3961 cagaaggctc aggtgagcgg ccagggggac tcgctgcacg agcatatcgc gaacctcgct
     4021 ggctcgccag ctatcaagaa ggggattctg cagaccgtga aggttgtgga cgagctggtg
     4081 aaggtcatgg gcaggcacaa gcctgagaac atcgtcattg agatggcccg ggagaatcag
     4141 accacgcaga agggccagaa gaactcacgc gagaggatga agaggatcga ggagggcatt
     4201 aaggagctgg ggtcccagat cctcaaggag cacccggtgg agaacacgca gctgcagaat
     4261 gagaagctct acctgtacta cctccagaat ggccgcgata tgtatgtgga ccaggagctg
     4321 gatattaaca ggctcagcga ttacgacgtc gatcatatcg ttccacagtc attcctgaag
     4381 gatgactcca ttgacaacaa ggtcctcacc aggtcggaca agaaccgggg caagtctgat
     4441 aatgttcctt cagaggaggt cgttaagaag atgaagaact actggcgcca gctcctgaat
     4501 gccaagctga tcacgcagcg gaagttcgat aacctcacaa aggctgagag gggcgggctc
     4561 tctgagctgg acaaggcggg cttcatcaag aggcagctgg tcgagacacg gcagatcact
     4621 aagcacgttg cgcagattct cgactcacgg atgaacacta agtacgatga gaatgacaag
     4681 ctgatccgcg aggtgaaggt catcaccctg aagtcaaagc tcgtctccga cttcaggaag
     4741 gatttccagt tctacaaggt tcgggagatc aacaattacc accatgccca tgacgcgtac
     4801 ctgaacgcgg tggtcggcac agctctgatc aagaagtacc caaagctcga gagcgagttc
     4861 gtgtacgggg actacaaggt ttacgatgtg aggaagatga tcgccaagtc ggagcaggag
     4921 attggcaagg ctaccgccaa gtacttcttc tactctaaca ttatgaattt cttcaagaca
     4981 gagatcactc tggccaatgg cgagatccgg aagcgccccc tcatcgagac gaacggcgag
     5041 acgggggaga tcgtgtggga caagggcagg gatttcgcga ccgtcaggaa ggttctctcc
     5101 atgccacaag tgaatatcgt caagaagaca gaggtccaga ctggcgggtt ctctaaggag
     5161 tcaattctgc ctaagcggaa cagcgacaag ctcatcgccc gcaagaagga ctgggatccg
     5221 aagaagtacg gcgggttcga cagccccact gtggcctact cggtcctggt tgtggcgaag
     5281 gttgagaagg gcaagtccaa gaagctcaag agcgtgaagg agctgctggg gatcacgatt
     5341 atggagcgct ccagcttcga gaagaacccg atcgatttcc tggaggcgaa gggctacaag
     5401 gaggtgaaga aggacctgat cattaagctc cccaagtact cactcttcga gctggagaac
     5461 ggcaggaagc ggatgctggc ttccgctggc gagctgcaga aggggaacga gctggctctg
     5521 ccgtccaagt atgtgaactt cctctacctg gcctcccact acgagaagct caagggcagc
     5581 cccgaggaca acgagcagaa gcagctgttc gtcgagcagc acaagcatta cctcgacgag
     5641 atcattgagc agatttccga gttctccaag cgcgtgatcc tggccgacgc gaatctggat
     5701 aaggtcctct ccgcgtacaa caagcaccgc gacaagccaa tcagggagca ggctgagaat
     5761 atcattcatc tcttcaccct gacgaacctc ggcgcccctg ctgctttcaa gtacttcgac
     5821 acaactatcg atcgcaagag gtacacaagc actaaggagg tcctggacgc gaccctcatc
     5881 caccagtcga ttaccggcct ctacgagacg cgcatcgacc tgtctcagct cgggggcgac
     5941 aagcggccag cggcgacgaa gaaggcgggg caggcgaaga agaagaagga attctccggc
     6001 ggcagcacca acctgtccga catcatcgag aaggagacgg gcaagcaact cgtgatccag
     6061 gagagcatcc tcatgctgcc agaggaggtg gaggaggtca tcggcaacaa gccagagtcc
     6121 gacatcctgg tgcacaccgc ctacgacgag tccaccgacg agaacgtcat gctcctgacc
     6181 agcgacgccc cagagtacaa gccatgggcc ctcgtcatcc aggacagcaa cggggagaac
     6241 aagatcaaga tgctgtcggg ggggagccca aagaagaagc ggaaggtgta g
//
