LOCUS       sequence-487634-        7852 bp    DNA     circular SYN 01-OCT-2025
DEFINITION  for DNA-free cytosine base editing in rice and wheat or other 
            plants.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7852)
  AUTHORS   Qiu F, Xue C, Liu J, Li B, Gao Q, Liang R, Chen K, Gao C
  TITLE     An efficient mRNA delivery system for genome editing in plants.
  JOURNAL   Plant Biotechnol J. 2025 Apr;23(4):1348-1358. doi: 
            10.1111/pbi.14591. Epub 2025 Feb 10.
  PUBMED    39928528
REFERENCE   2  (bases 1 to 7852)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Oct 1, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; doi: "10.1111/pbi.14591";
            journalName: "Plant Biotechnol J"; date: "2025-04"; volume: "23"; 
            issue: "4"; pages: "1348-1358"
COMMENT     Sequence Label: pIVT-A3A-DEN2
FEATURES             Location/Qualifiers
     source          1..7852
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     13..32
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        13..31
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             join(780..803,804..806,807..4880)
                     /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             4793..4801
                     /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             4881..4928
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             4950..5198
                     /codon_start=1
                     /product="uracil-DNA glycosylase inhibitor from a Bacillus 
                     subtilis bacteriophage (Mol et al., 1995)"
                     /label=UGI
                     /translation="TNLSDIIEKETGKQLVIQESILMLPEEVEEVIGNKPESDILVHTA
                     YDESTDENVMLLTSDAPEYKPWALVIQDSNGENKIKML"
     CDS             5211..5231
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     primer_bind     complement(5682..5698)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5682..5698)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5695..5717)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5706..5722
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     promoter        complement(join(5730..5736,5737..5754,5755..5760))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5775..5796
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5913..5930)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6084..6672)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6164..6183)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6843..7634,7635..7703))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     7466..7485
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7704..7808)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gagctcggta cctaatacga ctcactatag ggagttgtta gtctacgtgg accgacaaag
       61 acagattctt tgagggagct aagctcaacg tagttctaac agttttttaa ttagagagca
      121 gatctctgat ggaggcaagc cctgcatctg gaccccgcca tctcatggat ccacatattt
      181 tcacaagtaa cttcaacaat ggtattggaa ggcataaaac atatttatgt tatgaagttg
      241 aacgccttga caatggaact tctgtcaaga tggatcagca tagaggattc ttgcacaacc
      301 aggcgaaaaa tctactctgt ggcttctacg ggaggcacgc cgagctgaga tttttggatc
      361 tcgtgccgtc gcttcagctg gatcctgctc agatttatcg tgttacatgg ttcatctcat
      421 ggagcccatg cttcagctgg ggctgtgctg gtgaggtaag ggcatttctt caggagaaca
      481 cacatgtgag acttcgaata tttgctgcgc ggatatatga ctatgatcct ctgtacaagg
      541 aggcgttgca gatgctgcgg gacgccggcg cacaagtttc cattatgacg tacgacgagt
      601 tcaagcactg ctgggacacc tttgtcgacc atcagggctg cccctttcag ccatgggatg
      661 gtttagatga acattctcaa gctctttccg gtcgcctccg cgccatcctc caaaatcaag
      721 gcaactcagg atcagaaaca cctgggacga gtgaatcggc caccccagaa agtaggcctg
      781 acaagaagta ctcgatcggc ctcgccattg ggactaactc tgttggctgg gccgtgatca
      841 ccgacgagta caaggtgccc tcaaagaagt tcaaggtcct gggcaacacc gatcggcatt
      901 ccatcaagaa gaatctcatt ggcgctctcc tgttcgacag cggcgagacg gctgaggcta
      961 cgcggctcaa gcgcaccgcc cgcaggcggt acacgcgcag gaagaatcgc atctgctacc
     1021 tgcaggagat tttctccaac gagatggcga aggttgacga ttctttcttc cacaggctgg
     1081 aggagtcatt cctcgtggag gaggataaga agcacgagcg gcatccaatc ttcggcaaca
     1141 ttgtcgacga ggttgcctac cacgagaagt accctacgat ctaccatctg cggaagaagc
     1201 tcgtggactc cacagataag gcggacctcc gcctgatcta cctcgctctg gcccacatga
     1261 ttaagttcag gggccatttc ctgatcgagg gggatctcaa cccggacaat agcgatgttg
     1321 acaagctgtt catccagctc gtgcagacgt acaaccagct cttcgaggag aaccccatta
     1381 atgcgtcagg cgtcgacgcg aaggctatcc tgtccgctag gctctcgaag tctcggcgcc
     1441 tcgagaacct gatcgcccag ctgccgggcg agaagaagaa cggcctgttc gggaatctca
     1501 ttgcgctcag cctggggctc acgcccaact tcaagtcgaa tttcgatctc gctgaggacg
     1561 ccaagctgca gctctccaag gacacatacg acgatgacct ggataacctc ctggcccaga
     1621 tcggcgatca gtacgcggac ctgttcctcg ctgccaagaa tctgtcggac gccatcctcc
     1681 tgtctgatat tctcagggtg aacaccgaga ttacgaaggc tccgctctca gcctccatga
     1741 tcaagcgcta cgacgagcac catcaggatc tgaccctcct gaaggcgctg gtcaggcagc
     1801 agctccccga gaagtacaag gagatcttct tcgatcagtc gaagaacggc tacgctgggt
     1861 acattgacgg cggggcctct caggaggagt tctacaagtt catcaagccg attctggaga
     1921 agatggacgg cacggaggag ctgctggtga agctcaatcg cgaggacctc ctgaggaagc
     1981 agcggacatt cgataacggc agcatcccac accagattca tctcggggag ctgcacgcta
     2041 tcctgaggag gcaggaggac ttctaccctt tcctcaagga taaccgcgag aagatcgaga
     2101 agattctgac tttcaggatc ccgtactacg tcggcccact cgctaggggc aactcccgct
     2161 tcgcttggat gacccgcaag tcagaggaga cgatcacgcc gtggaacttc gaggaggtgg
     2221 tcgacaaggg cgctagcgct cagtcgttca tcgagaggat gacgaatttc gacaagaacc
     2281 tgccaaatga gaaggtgctc cctaagcact cgctcctgta cgagtacttc acagtctaca
     2341 acgagctgac taaggtgaag tatgtgaccg agggcatgag gaagccggct ttcctgtctg
     2401 gggagcagaa gaaggccatc gtggacctcc tgttcaagac caaccggaag gtcacggtta
     2461 agcagctcaa ggaggactac ttcaagaaga ttgagtgctt cgattcggtc gagatctctg
     2521 gcgttgagga ccgcttcaac gcctccctgg ggacctacca cgatctcctg aagatcatta
     2581 aggataagga cttcctggac aacgaggaga atgaggatat cctcgaggac attgtgctga
     2641 cactcactct gttcgaggac cgggagatga tcgaggagcg cctgaagact tacgcccatc
     2701 tcttcgatga caaggtcatg aagcagctca agaggaggag gtacaccggc tgggggaggc
     2761 tgagcaggaa gctcatcaac ggcattcggg acaagcagtc cgggaagacg atcctcgact
     2821 tcctgaagag cgatggcttc gcgaaccgca atttcatgca gctgattcac gatgacagcc
     2881 tcacattcaa ggaggatatc cagaaggctc aggtgagcgg ccagggggac tcgctgcacg
     2941 agcatatcgc gaacctcgct ggctcgccag ctatcaagaa ggggattctg cagaccgtga
     3001 aggttgtgga cgagctggtg aaggtcatgg gcaggcacaa gcctgagaac atcgtcattg
     3061 agatggcccg ggagaatcag accacgcaga agggccagaa gaactcacgc gagaggatga
     3121 agaggatcga ggagggcatt aaggagctgg ggtcccagat cctcaaggag cacccggtgg
     3181 agaacacgca gctgcagaat gagaagctct acctgtacta cctccagaat ggccgcgata
     3241 tgtatgtgga ccaggagctg gatattaaca ggctcagcga ttacgacgtc gatcatatcg
     3301 ttccacagtc attcctgaag gatgactcca ttgacaacaa ggtcctcacc aggtcggaca
     3361 agaaccgggg caagtctgat aatgttcctt cagaggaggt cgttaagaag atgaagaact
     3421 actggcgcca gctcctgaat gccaagctga tcacgcagcg gaagttcgat aacctcacaa
     3481 aggctgagag gggcgggctc tctgagctgg acaaggcggg cttcatcaag aggcagctgg
     3541 tcgagacacg gcagatcact aagcacgttg cgcagattct cgactcacgg atgaacacta
     3601 agtacgatga gaatgacaag ctgatccgcg aggtgaaggt catcaccctg aagtcaaagc
     3661 tcgtctccga cttcaggaag gatttccagt tctacaaggt tcgggagatc aacaattacc
     3721 accatgccca tgacgcgtac ctgaacgcgg tggtcggcac agctctgatc aagaagtacc
     3781 caaagctcga gagcgagttc gtgtacgggg actacaaggt ttacgatgtg aggaagatga
     3841 tcgccaagtc ggagcaggag attggcaagg ctaccgccaa gtacttcttc tactctaaca
     3901 ttatgaattt cttcaagaca gagatcactc tggccaatgg cgagatccgg aagcgccccc
     3961 tcatcgagac gaacggcgag acgggggaga tcgtgtggga caagggcagg gatttcgcga
     4021 ccgtcaggaa ggttctctcc atgccacaag tgaatatcgt caagaagaca gaggtccaga
     4081 ctggcgggtt ctctaaggag tcaattctgc ctaagcggaa cagcgacaag ctcatcgccc
     4141 gcaagaagga ctgggatccg aagaagtacg gcgggttcga cagccccact gtggcctact
     4201 cggtcctggt tgtggcgaag gttgagaagg gcaagtccaa gaagctcaag agcgtgaagg
     4261 agctgctggg gatcacgatt atggagcgct ccagcttcga gaagaacccg atcgatttcc
     4321 tggaggcgaa gggctacaag gaggtgaaga aggacctgat cattaagctc cccaagtact
     4381 cactcttcga gctggagaac ggcaggaagc ggatgctggc ttccgctggc gagctgcaga
     4441 aggggaacga gctggctctg ccgtccaagt atgtgaactt cctctacctg gcctcccact
     4501 acgagaagct caagggcagc cccgaggaca acgagcagaa gcagctgttc gtcgagcagc
     4561 acaagcatta cctcgacgag atcattgagc agatttccga gttctccaag cgcgtgatcc
     4621 tggccgacgc gaatctggat aaggtcctct ccgcgtacaa caagcaccgc gacaagccaa
     4681 tcagggagca ggctgagaat atcattcatc tcttcaccct gacgaacctc ggcgcccctg
     4741 ctgctttcaa gtacttcgac acaactatcg atcgcaagag gtacacaagc actaaggagg
     4801 tcctggacgc gaccctcatc caccagtcga ttaccggcct ctacgagacg cgcatcgacc
     4861 tgtctcagct cgggggcgac aagcggccag cggcgacgaa gaaggcgggg caggcgaaga
     4921 agaagaagac ccgcgactcc ggcggcagca cgaacctctc cgacatcatc gagaaggaga
     4981 cgggcaagca gctcgtgatc caggagagca tcctcatgct gccggaggag gtggaggagg
     5041 tcatcggcaa caagcccgag tccgacatcc tcgtgcacac cgcctacgac gagtccacgg
     5101 acgagaacgt catgctcctg acgagcgacg ctccagagta caagccatgg gctctcgtga
     5161 tccaggacag caacggcgag aacaagatca agatgctgtc cggcggctcc ccaaagaaga
     5221 agcggaaggt gtagactagt gtcatgggtc gtttaagctg ccgatgtgcc tgcgtcgtct
     5281 ggtgccctct ctccatatgg aggttgtcaa agtatctgct gttcgtgtca tgagtcgtgt
     5341 cagtgttggt ttaataatgg accggttgtg ttgtgtgtgc gtactaccca gaactatgac
     5401 aaatcatgaa taagtttgat gtttgaaatt aaagcctgtg ctcattatgt tctgtctttc
     5461 agttgtctcc taatatttgc ctgcagaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa
     5521 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa
     5581 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaagagacct ccctcacacc ggtgacgggg
     5641 atcgcatgcg atatctcgag atctagcttg gcgtaatcat ggtcatagct gtttcctgtg
     5701 tgaaattgtt atccgctcac aattccacac aacatacgag ccggaagcat aaagtgtaaa
     5761 gcctggggtg cctaatgagt gagctaactc acattaattg cgttgcgctc actgcccgct
     5821 ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa tcggccaacg cgcggggaga
     5881 ggcggtttgc gtattgggcg ctcttccgct tcctcgctca ctgactcgct gcgctcggtc
     5941 gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg taatacggtt atccacagaa
     6001 tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt
     6061 aaaaaggccg cgttgctggc gtttttccat aggctccgcc cccctgacga gcatcacaaa
     6121 aatcgacgct caagtcagag gtggcgaaac ccgacaggac tataaagata ccaggcgttt
     6181 ccccctggaa gctccctcgt gcgctctcct gttccgaccc tgccgcttac cggatacctg
     6241 tccgcctttc tcccttcggg aagcgtggcg ctttctcata gctcacgctg taggtatctc
     6301 agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc
     6361 gaccgctgcg ccttatccgg taactatcgt cttgagtcca acccggtaag acacgactta
     6421 tcgccactgg cagcagccac tggtaacagg attagcagag cgaggtatgt aggcggtgct
     6481 acagagttct tgaagtggtg gcctaactac ggctacacta gaagaacagt atttggtatc
     6541 tgcgctctgc tgaagccagt taccttcgga aaaagagttg gtagctcttg atccggcaaa
     6601 caaaccaccg ctggtagcgg tggttttttt gtttgcaagc agcagattac gcgcagaaaa
     6661 aaaggatctc aagaagatcc tttgatcttt tctacggggt ctgacgctca gtggaacgaa
     6721 aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt
     6781 ttaaattaaa aatgaagttt taaatcaatc taaagtatat atgagtaaac ttggtctgac
     6841 agttaccaat gcttaatcag tgaggcacct atctcagcga tctgtctatt tcgttcatcc
     6901 atagttgcct gactccccgt cgtgtagata actacgatac gggagggctt accatctggc
     6961 cccagtgctg caatgatacc gcgagaccca cgctcaccgg ctccagattt atcagcaata
     7021 aaccagccag ccggaagggc cgagcgcaga agtggtcctg caactttatc cgcctccatc
     7081 cagtctatta attgttgccg ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc
     7141 aacgttgttg ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca
     7201 ttcagctccg gttcccaacg atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa
     7261 gcggttagct ccttcggtcc tccgatcgtt gtcagaagta agttggccgc agtgttatca
     7321 ctcatggtta tggcagcact gcataattct cttactgtca tgccatccgt aagatgcttt
     7381 tctgtgactg gtgagtactc aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt
     7441 tgctcttgcc cggcgtcaat acgggataat accgcgccac atagcagaac tttaaaagtg
     7501 ctcatcattg gaaaacgttc ttcggggcga aaactctcaa ggatcttacc gctgttgaga
     7561 tccagttcga tgtaacccac tcgtgcaccc aactgatctt cagcatcttt tactttcacc
     7621 agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg aataagggcg
     7681 acacggaaat gttgaatact catactcttc ctttttcaat attattgaag catttatcag
     7741 ggttattgtc tcatgagcgg atacatattt gaatgtattt agaaaaataa acaaataggg
     7801 gttccgcgca catttccccg aaaagtgcca cctgccagtg ccaagctaat tc
//
