LOCUS       sequence-487632-        7524 bp    DNA     circular SYN 16-OCT-2025
DEFINITION  for DNA-free adenine 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 7524)
  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 7524)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Oct 16, 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-ABE-TMV
FEATURES             Location/Qualifiers
     source          1..7524
                     /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"
     misc_feature    44..99
                     /label=TMV Omega
                     /note="translational enhancer from the tobacco mosaic virus
                     5'-leader sequence (Gallie et al., 1988)"
     CDS             join(704..727,728..730,731..4804)
                     /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             4717..4725
                     /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             4817..4837
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             4850..4870
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             4883..4903
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     primer_bind     complement(5354..5370)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5354..5370)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5367..5389)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5378..5394
                     /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(5402..5408,5409..5426,5427..5432))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5447..5468
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5585..5602)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5756..6344)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5836..5855)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6515..7306,7307..7375))
                     /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     7138..7157
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7376..7480)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gagctcggta cctaatacga ctcactatag gggtattttt acaacaatta ccaacaacaa
       61 caaacaacaa acaacattac aattactatt tacaattaca atgagcgaag ttgaatttag
      121 ccatgaatat tggatgcgcc atgcacttac acttgcaaag agagcccgcg atgaacgcga
      181 agttcctgtt ggagcagttc ttgttcttaa caaccgcgtt atcggagaag gatggaaccg
      241 cgcaatcgga cttcatgatc ctacagcaca tgcagaaatc atggcacttc gccagggagg
      301 acttgttatg cagaactatc gccttatcga tgcaacactt tatgttacat ttgaaccttg
      361 cgttatgtgc gcaggagcaa tgatccatag ccgcatcgga cgcgttgtgt tcggagtacg
      421 taacagcaaa cgcggagcag caggaagcct tatgaacgtt cttaactatc ctggaatgaa
      481 ccatcgcgtt gaaatcacag aaggaatcct tgcagatgaa tgcgcagcac ttctttgcga
      541 tttctaccgt atgcctcgcc aggtgttcaa tgcgcagaag aaggcccaaa gcagcatcaa
      601 ctcaggtggt agctccggcg gctcgtcagg ttccgaaaca cctggaacaa gcgaaagcgc
      661 aacacctgaa tctagtggag gaagttctgg tgggtccctt aaggacaaga agtactcgat
      721 cggcctcgcc attgggacta actctgttgg ctgggccgtg atcaccgacg agtacaaggt
      781 gccctcaaag aagttcaagg tcctgggcaa caccgatcgg cattccatca agaagaatct
      841 cattggcgct ctcctgttcg acagcggcga gacggctgag gctacgcggc tcaagcgcac
      901 cgcccgcagg cggtacacgc gcaggaagaa tcgcatctgc tacctgcagg agattttctc
      961 caacgagatg gcgaaggttg acgattcttt cttccacagg ctggaggagt cattcctcgt
     1021 ggaggaggat aagaagcacg agcggcatcc aatcttcggc aacattgtcg acgaggttgc
     1081 ctaccacgag aagtacccta cgatctacca tctgcggaag aagctcgtgg actccacaga
     1141 taaggcggac ctccgcctga tctacctcgc tctggcccac atgattaagt tcaggggcca
     1201 tttcctgatc gagggggatc tcaacccgga caatagcgat gttgacaagc tgttcatcca
     1261 gctcgtgcag acgtacaacc agctcttcga ggagaacccc attaatgcgt caggcgtcga
     1321 cgcgaaggct atcctgtccg ctaggctctc gaagtctcgg cgcctcgaga acctgatcgc
     1381 ccagctgccg ggcgagaaga agaacggcct gttcgggaat ctcattgcgc tcagcctggg
     1441 gctcacgccc aacttcaagt cgaatttcga tctcgctgag gacgccaagc tgcagctctc
     1501 caaggacaca tacgacgatg acctggataa cctcctggcc cagatcggcg atcagtacgc
     1561 ggacctgttc ctcgctgcca agaatctgtc ggacgccatc ctcctgtctg atattctcag
     1621 ggtgaacacc gagattacga aggctccgct ctcagcctcc atgatcaagc gctacgacga
     1681 gcaccatcag gatctgaccc tcctgaaggc gctggtcagg cagcagctcc ccgagaagta
     1741 caaggagatc ttcttcgatc agtcgaagaa cggctacgct gggtacattg acggcggggc
     1801 ctctcaggag gagttctaca agttcatcaa gccgattctg gagaagatgg acggcacgga
     1861 ggagctgctg gtgaagctca atcgcgagga cctcctgagg aagcagcgga cattcgataa
     1921 cggcagcatc ccacaccaga ttcatctcgg ggagctgcac gctatcctga ggaggcagga
     1981 ggacttctac cctttcctca aggataaccg cgagaagatc gagaagattc tgactttcag
     2041 gatcccgtac tacgtcggcc cactcgctag gggcaactcc cgcttcgctt ggatgacccg
     2101 caagtcagag gagacgatca cgccgtggaa cttcgaggag gtggtcgaca agggcgctag
     2161 cgctcagtcg ttcatcgaga ggatgacgaa tttcgacaag aacctgccaa atgagaaggt
     2221 gctccctaag cactcgctcc tgtacgagta cttcacagtc tacaacgagc tgactaaggt
     2281 gaagtatgtg accgagggca tgaggaagcc ggctttcctg tctggggagc agaagaaggc
     2341 catcgtggac ctcctgttca agaccaaccg gaaggtcacg gttaagcagc tcaaggagga
     2401 ctacttcaag aagattgagt gcttcgattc ggtcgagatc tctggcgttg aggaccgctt
     2461 caacgcctcc ctggggacct accacgatct cctgaagatc attaaggata aggacttcct
     2521 ggacaacgag gagaatgagg atatcctcga ggacattgtg ctgacactca ctctgttcga
     2581 ggaccgggag atgatcgagg agcgcctgaa gacttacgcc catctcttcg atgacaaggt
     2641 catgaagcag ctcaagagga ggaggtacac cggctggggg aggctgagca ggaagctcat
     2701 caacggcatt cgggacaagc agtccgggaa gacgatcctc gacttcctga agagcgatgg
     2761 cttcgcgaac cgcaatttca tgcagctgat tcacgatgac agcctcacat tcaaggagga
     2821 tatccagaag gctcaggtga gcggccaggg ggactcgctg cacgagcata tcgcgaacct
     2881 cgctggctcg ccagctatca agaaggggat tctgcagacc gtgaaggttg tggacgagct
     2941 ggtgaaggtc atgggcaggc acaagcctga gaacatcgtc attgagatgg cccgggagaa
     3001 tcagaccacg cagaagggcc agaagaactc acgcgagagg atgaagagga tcgaggaggg
     3061 cattaaggag ctggggtccc agatcctcaa ggagcacccg gtggagaaca cgcagctgca
     3121 gaatgagaag ctctacctgt actacctcca gaatggccgc gatatgtatg tggaccagga
     3181 gctggatatt aacaggctca gcgattacga cgtcgatcat atcgttccac agtcattcct
     3241 gaaggatgac tccattgaca acaaggtcct caccaggtcg gacaagaacc ggggcaagtc
     3301 tgataatgtt ccttcagagg aggtcgttaa gaagatgaag aactactggc gccagctcct
     3361 gaatgccaag ctgatcacgc agcggaagtt cgataacctc acaaaggctg agaggggcgg
     3421 gctctctgag ctggacaagg cgggcttcat caagaggcag ctggtcgaga cacggcagat
     3481 cactaagcac gttgcgcaga ttctcgactc acggatgaac actaagtacg atgagaatga
     3541 caagctgatc cgcgaggtga aggtcatcac cctgaagtca aagctcgtct ccgacttcag
     3601 gaaggatttc cagttctaca aggttcggga gatcaacaat taccaccatg cccatgacgc
     3661 gtacctgaac gcggtggtcg gcacagctct gatcaagaag tacccaaagc tcgagagcga
     3721 gttcgtgtac ggggactaca aggtttacga tgtgaggaag atgatcgcca agtcggagca
     3781 ggagattggc aaggctaccg ccaagtactt cttctactct aacattatga atttcttcaa
     3841 gacagagatc actctggcca atggcgagat ccggaagcgc cccctcatcg agacgaacgg
     3901 cgagacgggg gagatcgtgt gggacaaggg cagggatttc gcgaccgtca ggaaggttct
     3961 ctccatgcca caagtgaata tcgtcaagaa gacagaggtc cagactggcg ggttctctaa
     4021 ggagtcaatt ctgcctaagc ggaacagcga caagctcatc gcccgcaaga aggactggga
     4081 tccgaagaag tacggcgggt tcgacagccc cactgtggcc tactcggtcc tggttgtggc
     4141 gaaggttgag aagggcaagt ccaagaagct caagagcgtg aaggagctgc tggggatcac
     4201 gattatggag cgctccagct tcgagaagaa cccgatcgat ttcctggagg cgaagggcta
     4261 caaggaggtg aagaaggacc tgatcattaa gctccccaag tactcactct tcgagctgga
     4321 gaacggcagg aagcggatgc tggcttccgc tggcgagctg cagaagggga acgagctggc
     4381 tctgccgtcc aagtatgtga acttcctcta cctggcctcc cactacgaga agctcaaggg
     4441 cagccccgag gacaacgagc agaagcagct gttcgtcgag cagcacaagc attacctcga
     4501 cgagatcatt gagcagattt ccgagttctc caagcgcgtg atcctggccg acgcgaatct
     4561 ggataaggtc ctctccgcgt acaacaagca ccgcgacaag ccaatcaggg agcaggctga
     4621 gaatatcatt catctcttca ccctgacgaa cctcggcgcc cctgctgctt tcaagtactt
     4681 cgacacaact atcgatcgca agaggtacac aagcactaag gaggtcctgg acgcgaccct
     4741 catccaccag tcgattaccg gcctctacga gacgcgcatc gacctgtctc agctcggggg
     4801 cgactccggc ggcagcccaa agaagaagcg gaaggtgtct ggaggttctc ctaagaaaaa
     4861 gagaaaagtg tccggcggct ccccgaagaa gaagcgcaag gtgtagacta gtgtcatggg
     4921 tcgtttaagc tgccgatgtg cctgcgtcgt ctggtgccct ctctccatat ggaggttgtc
     4981 aaagtatctg ctgttcgtgt catgagtcgt gtcagtgttg gtttaataat ggaccggttg
     5041 tgttgtgtgt gcgtactacc cagaactatg acaaatcatg aataagtttg atgtttgaaa
     5101 ttaaagcctg tgctcattat gttctgtctt tcagttgtct cctaatattt gcctgcagaa
     5161 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa
     5221 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa
     5281 aaaaagagac ctccctcaca ccggtgacgg ggatcgcatg cgatatctcg agatctagct
     5341 tggcgtaatc atggtcatag ctgtttcctg tgtgaaattg ttatccgctc acaattccac
     5401 acaacatacg agccggaagc ataaagtgta aagcctgggg tgcctaatga gtgagctaac
     5461 tcacattaat tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg tcgtgccagc
     5521 tgcattaatg aatcggccaa cgcgcgggga gaggcggttt gcgtattggg cgctcttccg
     5581 cttcctcgct cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc
     5641 actcaaaggc ggtaatacgg ttatccacag aatcagggga taacgcagga aagaacatgt
     5701 gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc
     5761 ataggctccg cccccctgac gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa
     5821 acccgacagg actataaaga taccaggcgt ttccccctgg aagctccctc gtgcgctctc
     5881 ctgttccgac cctgccgctt accggatacc tgtccgcctt tctcccttcg ggaagcgtgg
     5941 cgctttctca tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc
     6001 tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc ggtaactatc
     6061 gtcttgagtc caacccggta agacacgact tatcgccact ggcagcagcc actggtaaca
     6121 ggattagcag agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg tggcctaact
     6181 acggctacac tagaagaaca gtatttggta tctgcgctct gctgaagcca gttaccttcg
     6241 gaaaaagagt tggtagctct tgatccggca aacaaaccac cgctggtagc ggtggttttt
     6301 ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat cctttgatct
     6361 tttctacggg gtctgacgct cagtggaacg aaaactcacg ttaagggatt ttggtcatga
     6421 gattatcaaa aaggatcttc acctagatcc ttttaaatta aaaatgaagt tttaaatcaa
     6481 tctaaagtat atatgagtaa acttggtctg acagttacca atgcttaatc agtgaggcac
     6541 ctatctcagc gatctgtcta tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga
     6601 taactacgat acgggagggc ttaccatctg gccccagtgc tgcaatgata ccgcgagacc
     6661 cacgctcacc ggctccagat ttatcagcaa taaaccagcc agccggaagg gccgagcgca
     6721 gaagtggtcc tgcaacttta tccgcctcca tccagtctat taattgttgc cgggaagcta
     6781 gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt tgccattgct acaggcatcg
     6841 tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc cggttcccaa cgatcaaggc
     6901 gagttacatg atcccccatg ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg
     6961 ttgtcagaag taagttggcc gcagtgttat cactcatggt tatggcagca ctgcataatt
     7021 ctcttactgt catgccatcc gtaagatgct tttctgtgac tggtgagtac tcaaccaagt
     7081 cattctgaga atagtgtatg cggcgaccga gttgctcttg cccggcgtca atacgggata
     7141 ataccgcgcc acatagcaga actttaaaag tgctcatcat tggaaaacgt tcttcggggc
     7201 gaaaactctc aaggatctta ccgctgttga gatccagttc gatgtaaccc actcgtgcac
     7261 ccaactgatc ttcagcatct tttactttca ccagcgtttc tgggtgagca aaaacaggaa
     7321 ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa atgttgaata ctcatactct
     7381 tcctttttca atattattga agcatttatc agggttattg tctcatgagc ggatacatat
     7441 ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg cacatttccc cgaaaagtgc
     7501 cacctgccag tgccaagcta attc
//
