LOCUS       sequence-266631-        6759 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 6759)
  TITLE     Gao Lab Plasmids
REFERENCE   2  (bases 1 to 6759)
  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-3
FEATURES             Location/Qualifiers
     source          1..6759
                     /mol_type="genomic DNA"
                     /organism="unspecified"
     CDS             join(1930..1953,1954..1956,1957..6030)
                     /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             5943..5951
                     /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             6031..6078
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             6085..6138
                     /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             6139..6387
                     /codon_start=1
                     /product="uracil-DNA glycosylase inhibitor from a Bacillus 
                     subtilis bacteriophage (Mol et al., 1995)"
                     /label=UGI
                     /translation="TNLSDIIEKETGKQLVIQESILMLPEEVEEVIGNKPESDILVHTA
                     YDESTDENVMLLTSDAPEYKPWALVIQDSNGENKIKML"
     CDS             6400..6420
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             6421..6474
                     /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             6475..6723
                     /codon_start=1
                     /product="uracil-DNA glycosylase inhibitor from a Bacillus 
                     subtilis bacteriophage (Mol et al., 1995)"
                     /label=UGI
                     /translation="TNLSDIIEKETGKQLVIQESILMLPEEVEEVIGNKPESDILVHTA
                     YDESTDENVMLLTSDAPEYKPWALVIQDSNGENKIKML"
     CDS             6736..6756
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
ORIGIN
        1 atggaggcca gcccggctag cggcccaagg catctcatgg acccgcacat cttcaccagc
       61 aacttcaaca acggcatcgg caggcacaag acctacttgt gctacgaggt ggagaggctc
      121 gacaacggaa cctccgtgaa gatggaccaa cacagggggt tcctccacaa ccaagccaag
      181 aacctcctct gcggcttcta cggcaggcac gccgagttga ggttcctcga cttggtgcca
      241 tccctccaac tcgatccagc ccaaatctac cgcgtgacct ggttcatctc ctggtcccca
      301 tgcttctcct ggggttgcgc cggcgaggtt cgggctttcc tccaagaaaa cacccacgtc
      361 cgcctccgca ttttcgccgc caggatctat gattacgacc ctctctacaa ggaggccctc
      421 cagatgctgc gggacgccgg tgctcaggtg agtatcatga cctacgacga gttcaagcac
      481 tgctgggaca ccttcgttga ccaccagggc tgcccattcc aaccatggga cggtctggat
      541 gaacacagcc aagccttgtc cggcaggctc cgggccatcc tccaaaacca ggggaactcc
      601 gggagcgaga cgccaggcac ctccgagtcg gccaccccag aatcttccgg cggatcttct
      661 ggaggatcta gcggctccga gacaccagga acatccgaat ccgctacacc agagtcttct
      721 ggaggatcta gcggaggatc ttccgaggtg gagttctctc acgagtattg gatgaggcac
      781 gctcttacac ttgctaagag agcttgggac gaaagagaag tgccagttgg cgccgttctt
      841 gtgcataata atagggtgat cggcgagggt tggaatagac caattggaag gcatgatcca
      901 acagctcacg cagagattat ggctctcaga caaggcggcc tcgttatgca gaactacagg
      961 ctcattgacg ctacactcta cgtgacactc gaaccttgcg ttatgtgcgc cggagctatg
     1021 attcattcta ggattggcag ggtcgtgttt ggagctaggg acgctaaaac aggagccgcc
     1081 ggatctctta tggacgtgtt gcatcatcca ggcatgaacc atagggtgga gattacagag
     1141 ggcattcttg cagacgagtg cgctgctctt ctttccgatt tcttcaggat gagaaggcag
     1201 gagattaagg cccagaagaa ggctcagtct tctacagata gcggaggatc ttccggagga
     1261 tctagcggct ccgagacacc aggaacatcc gaaagcgcta caccagaatc tagcggaggc
     1321 tcttccggag gatcttctga agtggagttc tcccacgagt attggatgag gcacgctctt
     1381 acacttgcta aaagggctag ggacgaaagg gaagttccag ttggagctgt tctcgtgctc
     1441 aataacaggg tgattggcga gggttggaat agagccattg gactccatga tccaacagct
     1501 cacgcagaga ttatggctct tagacaaggc ggcctcgtta tgcagaatta cagactcatc
     1561 gacgccacac tctacgttac cttcgaacct tgcgttatgt gcgccggagc tatgatccat
     1621 tctaggattg gcagggtcgt gttcggcgtt agaaacgcta agacaggagc tgcaggctct
     1681 cttatggacg ttcttcatta cccaggcatg aatcatagag tggagatcac agaaggcatt
     1741 cttgcagacg agtgcgcagc tctcctttgc tatttcttca ggatgccgag gcaagttttc
     1801 aacgctcaga agaaggccca gtcttctaca gattccggcg gatcttctgg aggatctagc
     1861 ggctccgaga caccaggaac atccgaatcc gctacaccag agtcttctgg aggatctagc
     1921 ggaggatctg acaagaagta ctcgatcggc ctcgccattg ggactaactc tgttggctgg
     1981 gccgtgatca ccgacgagta caaggtgccc tcaaagaagt tcaaggtcct gggcaacacc
     2041 gatcggcatt ccatcaagaa gaatctcatt ggcgctctcc tgttcgacag cggcgagacg
     2101 gctgaggcta cgcggctcaa gcgcaccgcc cgcaggcggt acacgcgcag gaagaatcgc
     2161 atctgctacc tgcaggagat tttctccaac gagatggcga aggttgacga ttctttcttc
     2221 cacaggctgg aggagtcatt cctcgtggag gaggataaga agcacgagcg gcatccaatc
     2281 ttcggcaaca ttgtcgacga ggttgcctac cacgagaagt accctacgat ctaccatctg
     2341 cggaagaagc tcgtggactc cacagataag gcggacctcc gcctgatcta cctcgctctg
     2401 gcccacatga ttaagttcag gggccatttc ctgatcgagg gggatctcaa cccggacaat
     2461 agcgatgttg acaagctgtt catccagctc gtgcagacgt acaaccagct cttcgaggag
     2521 aaccccatta atgcgtcagg cgtcgacgcg aaggctatcc tgtccgctag gctctcgaag
     2581 tctcggcgcc tcgagaacct gatcgcccag ctgccgggcg agaagaagaa cggcctgttc
     2641 gggaatctca ttgcgctcag cctggggctc acgcccaact tcaagtcgaa tttcgatctc
     2701 gctgaggacg ccaagctgca gctctccaag gacacatacg acgatgacct ggataacctc
     2761 ctggcccaga tcggcgatca gtacgcggac ctgttcctcg ctgccaagaa tctgtcggac
     2821 gccatcctcc tgtctgatat tctcagggtg aacaccgaga ttacgaaggc tccgctctca
     2881 gcctccatga tcaagcgcta cgacgagcac catcaggatc tgaccctcct gaaggcgctg
     2941 gtcaggcagc agctccccga gaagtacaag gagatcttct tcgatcagtc gaagaacggc
     3001 tacgctgggt acattgacgg cggggcctct caggaggagt tctacaagtt catcaagccg
     3061 attctggaga agatggacgg cacggaggag ctgctggtga agctcaatcg cgaggacctc
     3121 ctgaggaagc agcggacatt cgataacggc agcatcccac accagattca tctcggggag
     3181 ctgcacgcta tcctgaggag gcaggaggac ttctaccctt tcctcaagga taaccgcgag
     3241 aagatcgaga agattctgac tttcaggatc ccgtactacg tcggcccact cgctaggggc
     3301 aactcccgct tcgcttggat gacccgcaag tcagaggaga cgatcacgcc gtggaacttc
     3361 gaggaggtgg tcgacaaggg cgctagcgct cagtcgttca tcgagaggat gacgaatttc
     3421 gacaagaacc tgccaaatga gaaggtgctc cctaagcact cgctcctgta cgagtacttc
     3481 acagtctaca acgagctgac taaggtgaag tatgtgaccg agggcatgag gaagccggct
     3541 ttcctgtctg gggagcagaa gaaggccatc gtggacctcc tgttcaagac caaccggaag
     3601 gtcacggtta agcagctcaa ggaggactac ttcaagaaga ttgagtgctt cgattcggtc
     3661 gagatctctg gcgttgagga ccgcttcaac gcctccctgg ggacctacca cgatctcctg
     3721 aagatcatta aggataagga cttcctggac aacgaggaga atgaggatat cctcgaggac
     3781 attgtgctga cactcactct gttcgaggac cgggagatga tcgaggagcg cctgaagact
     3841 tacgcccatc tcttcgatga caaggtcatg aagcagctca agaggaggag gtacaccggc
     3901 tgggggaggc tgagcaggaa gctcatcaac ggcattcggg acaagcagtc cgggaagacg
     3961 atcctcgact tcctgaagag cgatggcttc gcgaaccgca atttcatgca gctgattcac
     4021 gatgacagcc tcacattcaa ggaggatatc cagaaggctc aggtgagcgg ccagggggac
     4081 tcgctgcacg agcatatcgc gaacctcgct ggctcgccag ctatcaagaa ggggattctg
     4141 cagaccgtga aggttgtgga cgagctggtg aaggtcatgg gcaggcacaa gcctgagaac
     4201 atcgtcattg agatggcccg ggagaatcag accacgcaga agggccagaa gaactcacgc
     4261 gagaggatga agaggatcga ggagggcatt aaggagctgg ggtcccagat cctcaaggag
     4321 cacccggtgg agaacacgca gctgcagaat gagaagctct acctgtacta cctccagaat
     4381 ggccgcgata tgtatgtgga ccaggagctg gatattaaca ggctcagcga ttacgacgtc
     4441 gatcatatcg ttccacagtc attcctgaag gatgactcca ttgacaacaa ggtcctcacc
     4501 aggtcggaca agaaccgggg caagtctgat aatgttcctt cagaggaggt cgttaagaag
     4561 atgaagaact actggcgcca gctcctgaat gccaagctga tcacgcagcg gaagttcgat
     4621 aacctcacaa aggctgagag gggcgggctc tctgagctgg acaaggcggg cttcatcaag
     4681 aggcagctgg tcgagacacg gcagatcact aagcacgttg cgcagattct cgactcacgg
     4741 atgaacacta agtacgatga gaatgacaag ctgatccgcg aggtgaaggt catcaccctg
     4801 aagtcaaagc tcgtctccga cttcaggaag gatttccagt tctacaaggt tcgggagatc
     4861 aacaattacc accatgccca tgacgcgtac ctgaacgcgg tggtcggcac agctctgatc
     4921 aagaagtacc caaagctcga gagcgagttc gtgtacgggg actacaaggt ttacgatgtg
     4981 aggaagatga tcgccaagtc ggagcaggag attggcaagg ctaccgccaa gtacttcttc
     5041 tactctaaca ttatgaattt cttcaagaca gagatcactc tggccaatgg cgagatccgg
     5101 aagcgccccc tcatcgagac gaacggcgag acgggggaga tcgtgtggga caagggcagg
     5161 gatttcgcga ccgtcaggaa ggttctctcc atgccacaag tgaatatcgt caagaagaca
     5221 gaggtccaga ctggcgggtt ctctaaggag tcaattctgc ctaagcggaa cagcgacaag
     5281 ctcatcgccc gcaagaagga ctgggatccg aagaagtacg gcgggttcga cagccccact
     5341 gtggcctact cggtcctggt tgtggcgaag gttgagaagg gcaagtccaa gaagctcaag
     5401 agcgtgaagg agctgctggg gatcacgatt atggagcgct ccagcttcga gaagaacccg
     5461 atcgatttcc tggaggcgaa gggctacaag gaggtgaaga aggacctgat cattaagctc
     5521 cccaagtact cactcttcga gctggagaac ggcaggaagc ggatgctggc ttccgctggc
     5581 gagctgcaga aggggaacga gctggctctg ccgtccaagt atgtgaactt cctctacctg
     5641 gcctcccact acgagaagct caagggcagc cccgaggaca acgagcagaa gcagctgttc
     5701 gtcgagcagc acaagcatta cctcgacgag atcattgagc agatttccga gttctccaag
     5761 cgcgtgatcc tggccgacgc gaatctggat aaggtcctct ccgcgtacaa caagcaccgc
     5821 gacaagccaa tcagggagca ggctgagaat atcattcatc tcttcaccct gacgaacctc
     5881 ggcgcccctg ctgctttcaa gtacttcgac acaactatcg atcgcaagag gtacacaagc
     5941 actaaggagg tcctggacgc gaccctcatc caccagtcga ttaccggcct ctacgagacg
     6001 cgcatcgacc tgtctcagct cgggggcgac aagcggccag cggcgacgaa gaaggcgggg
     6061 caggcgaaga agaagaagga attcgagggc agaggaagtc ttctaacatg cggtgacgtg
     6121 gaggagaatc ccggccctac caacctgtcc gacatcatcg agaaggagac gggcaagcaa
     6181 ctcgtgatcc aggagagcat cctcatgctg ccagaggagg tggaggaggt catcggcaac
     6241 aagccagagt ccgacatcct ggtgcacacc gcctacgacg agtccaccga cgagaacgtc
     6301 atgctcctga ccagcgacgc cccagagtac aagccatggg ccctcgtcat ccaggacagc
     6361 aacggggaga acaagatcaa gatgctgtcg ggggggagcc caaagaagaa gcggaaggtg
     6421 gagggcagag gaagtcttct aacatgcggt gacgtggagg agaatcccgg ccctaccaac
     6481 ctgtccgaca tcatcgagaa ggagacgggc aagcaactcg tgatccagga gagcatcctc
     6541 atgctgccag aggaggtgga ggaggtcatc ggcaacaagc cagagtccga catcctggtg
     6601 cacaccgcct acgacgagtc caccgacgag aacgtcatgc tcctgaccag cgacgcccca
     6661 gagtacaagc catgggccct cgtcatccag gacagcaacg gggagaacaa gatcaagatg
     6721 ctgtcggggg ggagcccaaa gaagaagcgg aaggtgtag
//
