LOCUS       sequence-487631-        7552 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 7552)
  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 7552)
  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-DEN2
FEATURES             Location/Qualifiers
     source          1..7552
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     31..50
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        31..49
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             join(750..773,774..776,777..4850)
                     /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             4763..4771
                     /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             4863..4883
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             4896..4916
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             4929..4949
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     primer_bind     complement(5400..5416)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5400..5416)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5413..5435)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5424..5440
                     /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(5448..5454,5455..5472,5473..5478))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5493..5514
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5631..5648)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5802..6390)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5882..5901)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6561..7352,7353..7421))
                     /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     7184..7203
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7422..7526)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 ccagtgccaa gctaattcga gctcggtacc taatacgact cactataggg agttgttagt
       61 ctacgtggac cgacaaagac agattctttg agggagctaa gctcaacgta gttctaacag
      121 ttttttaatt agagagcaga tctctgatga gcgaagttga atttagccat gaatattgga
      181 tgcgccatgc acttacactt gcaaagagag cccgcgatga acgcgaagtt cctgttggag
      241 cagttcttgt tcttaacaac cgcgttatcg gagaaggatg gaaccgcgca atcggacttc
      301 atgatcctac agcacatgca gaaatcatgg cacttcgcca gggaggactt gttatgcaga
      361 actatcgcct tatcgatgca acactttatg ttacatttga accttgcgtt atgtgcgcag
      421 gagcaatgat ccatagccgc atcggacgcg ttgtgttcgg agtacgtaac agcaaacgcg
      481 gagcagcagg aagccttatg aacgttctta actatcctgg aatgaaccat cgcgttgaaa
      541 tcacagaagg aatccttgca gatgaatgcg cagcacttct ttgcgatttc taccgtatgc
      601 ctcgccaggt gttcaatgcg cagaagaagg cccaaagcag catcaactca ggtggtagct
      661 ccggcggctc gtcaggttcc gaaacacctg gaacaagcga aagcgcaaca cctgaatcta
      721 gtggaggaag ttctggtggg tcccttaagg acaagaagta ctcgatcggc ctcgccattg
      781 ggactaactc tgttggctgg gccgtgatca ccgacgagta caaggtgccc tcaaagaagt
      841 tcaaggtcct gggcaacacc gatcggcatt ccatcaagaa gaatctcatt ggcgctctcc
      901 tgttcgacag cggcgagacg gctgaggcta cgcggctcaa gcgcaccgcc cgcaggcggt
      961 acacgcgcag gaagaatcgc atctgctacc tgcaggagat tttctccaac gagatggcga
     1021 aggttgacga ttctttcttc cacaggctgg aggagtcatt cctcgtggag gaggataaga
     1081 agcacgagcg gcatccaatc ttcggcaaca ttgtcgacga ggttgcctac cacgagaagt
     1141 accctacgat ctaccatctg cggaagaagc tcgtggactc cacagataag gcggacctcc
     1201 gcctgatcta cctcgctctg gcccacatga ttaagttcag gggccatttc ctgatcgagg
     1261 gggatctcaa cccggacaat agcgatgttg acaagctgtt catccagctc gtgcagacgt
     1321 acaaccagct cttcgaggag aaccccatta atgcgtcagg cgtcgacgcg aaggctatcc
     1381 tgtccgctag gctctcgaag tctcggcgcc tcgagaacct gatcgcccag ctgccgggcg
     1441 agaagaagaa cggcctgttc gggaatctca ttgcgctcag cctggggctc acgcccaact
     1501 tcaagtcgaa tttcgatctc gctgaggacg ccaagctgca gctctccaag gacacatacg
     1561 acgatgacct ggataacctc ctggcccaga tcggcgatca gtacgcggac ctgttcctcg
     1621 ctgccaagaa tctgtcggac gccatcctcc tgtctgatat tctcagggtg aacaccgaga
     1681 ttacgaaggc tccgctctca gcctccatga tcaagcgcta cgacgagcac catcaggatc
     1741 tgaccctcct gaaggcgctg gtcaggcagc agctccccga gaagtacaag gagatcttct
     1801 tcgatcagtc gaagaacggc tacgctgggt acattgacgg cggggcctct caggaggagt
     1861 tctacaagtt catcaagccg attctggaga agatggacgg cacggaggag ctgctggtga
     1921 agctcaatcg cgaggacctc ctgaggaagc agcggacatt cgataacggc agcatcccac
     1981 accagattca tctcggggag ctgcacgcta tcctgaggag gcaggaggac ttctaccctt
     2041 tcctcaagga taaccgcgag aagatcgaga agattctgac tttcaggatc ccgtactacg
     2101 tcggcccact cgctaggggc aactcccgct tcgcttggat gacccgcaag tcagaggaga
     2161 cgatcacgcc gtggaacttc gaggaggtgg tcgacaaggg cgctagcgct cagtcgttca
     2221 tcgagaggat gacgaatttc gacaagaacc tgccaaatga gaaggtgctc cctaagcact
     2281 cgctcctgta cgagtacttc acagtctaca acgagctgac taaggtgaag tatgtgaccg
     2341 agggcatgag gaagccggct ttcctgtctg gggagcagaa gaaggccatc gtggacctcc
     2401 tgttcaagac caaccggaag gtcacggtta agcagctcaa ggaggactac ttcaagaaga
     2461 ttgagtgctt cgattcggtc gagatctctg gcgttgagga ccgcttcaac gcctccctgg
     2521 ggacctacca cgatctcctg aagatcatta aggataagga cttcctggac aacgaggaga
     2581 atgaggatat cctcgaggac attgtgctga cactcactct gttcgaggac cgggagatga
     2641 tcgaggagcg cctgaagact tacgcccatc tcttcgatga caaggtcatg aagcagctca
     2701 agaggaggag gtacaccggc tgggggaggc tgagcaggaa gctcatcaac ggcattcggg
     2761 acaagcagtc cgggaagacg atcctcgact tcctgaagag cgatggcttc gcgaaccgca
     2821 atttcatgca gctgattcac gatgacagcc tcacattcaa ggaggatatc cagaaggctc
     2881 aggtgagcgg ccagggggac tcgctgcacg agcatatcgc gaacctcgct ggctcgccag
     2941 ctatcaagaa ggggattctg cagaccgtga aggttgtgga cgagctggtg aaggtcatgg
     3001 gcaggcacaa gcctgagaac atcgtcattg agatggcccg ggagaatcag accacgcaga
     3061 agggccagaa gaactcacgc gagaggatga agaggatcga ggagggcatt aaggagctgg
     3121 ggtcccagat cctcaaggag cacccggtgg agaacacgca gctgcagaat gagaagctct
     3181 acctgtacta cctccagaat ggccgcgata tgtatgtgga ccaggagctg gatattaaca
     3241 ggctcagcga ttacgacgtc gatcatatcg ttccacagtc attcctgaag gatgactcca
     3301 ttgacaacaa ggtcctcacc aggtcggaca agaaccgggg caagtctgat aatgttcctt
     3361 cagaggaggt cgttaagaag atgaagaact actggcgcca gctcctgaat gccaagctga
     3421 tcacgcagcg gaagttcgat aacctcacaa aggctgagag gggcgggctc tctgagctgg
     3481 acaaggcggg cttcatcaag aggcagctgg tcgagacacg gcagatcact aagcacgttg
     3541 cgcagattct cgactcacgg atgaacacta agtacgatga gaatgacaag ctgatccgcg
     3601 aggtgaaggt catcaccctg aagtcaaagc tcgtctccga cttcaggaag gatttccagt
     3661 tctacaaggt tcgggagatc aacaattacc accatgccca tgacgcgtac ctgaacgcgg
     3721 tggtcggcac agctctgatc aagaagtacc caaagctcga gagcgagttc gtgtacgggg
     3781 actacaaggt ttacgatgtg aggaagatga tcgccaagtc ggagcaggag attggcaagg
     3841 ctaccgccaa gtacttcttc tactctaaca ttatgaattt cttcaagaca gagatcactc
     3901 tggccaatgg cgagatccgg aagcgccccc tcatcgagac gaacggcgag acgggggaga
     3961 tcgtgtggga caagggcagg gatttcgcga ccgtcaggaa ggttctctcc atgccacaag
     4021 tgaatatcgt caagaagaca gaggtccaga ctggcgggtt ctctaaggag tcaattctgc
     4081 ctaagcggaa cagcgacaag ctcatcgccc gcaagaagga ctgggatccg aagaagtacg
     4141 gcgggttcga cagccccact gtggcctact cggtcctggt tgtggcgaag gttgagaagg
     4201 gcaagtccaa gaagctcaag agcgtgaagg agctgctggg gatcacgatt atggagcgct
     4261 ccagcttcga gaagaacccg atcgatttcc tggaggcgaa gggctacaag gaggtgaaga
     4321 aggacctgat cattaagctc cccaagtact cactcttcga gctggagaac ggcaggaagc
     4381 ggatgctggc ttccgctggc gagctgcaga aggggaacga gctggctctg ccgtccaagt
     4441 atgtgaactt cctctacctg gcctcccact acgagaagct caagggcagc cccgaggaca
     4501 acgagcagaa gcagctgttc gtcgagcagc acaagcatta cctcgacgag atcattgagc
     4561 agatttccga gttctccaag cgcgtgatcc tggccgacgc gaatctggat aaggtcctct
     4621 ccgcgtacaa caagcaccgc gacaagccaa tcagggagca ggctgagaat atcattcatc
     4681 tcttcaccct gacgaacctc ggcgcccctg ctgctttcaa gtacttcgac acaactatcg
     4741 atcgcaagag gtacacaagc actaaggagg tcctggacgc gaccctcatc caccagtcga
     4801 ttaccggcct ctacgagacg cgcatcgacc tgtctcagct cgggggcgac tccggcggca
     4861 gcccaaagaa gaagcggaag gtgtctggag gttctcctaa gaaaaagaga aaagtgtccg
     4921 gcggctcccc gaagaagaag cgcaaggtgt agactagtgt catgggtcgt ttaagctgcc
     4981 gatgtgcctg cgtcgtctgg tgccctctct ccatatggag gttgtcaaag tatctgctgt
     5041 tcgtgtcatg agtcgtgtca gtgttggttt aataatggac cggttgtgtt gtgtgtgcgt
     5101 actacccaga actatgacaa atcatgaata agtttgatgt ttgaaattaa agcctgtgct
     5161 cattatgttc tgtctttcag ttgtctccta atatttgcct gcagaaaaaa aaaaaaaaaa
     5221 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa
     5281 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa agagacctcc
     5341 ctcacaccgg tgacggggat cgcatgcgat atctcgagat ctagcttggc gtaatcatgg
     5401 tcatagctgt ttcctgtgtg aaattgttat ccgctcacaa ttccacacaa catacgagcc
     5461 ggaagcataa agtgtaaagc ctggggtgcc taatgagtga gctaactcac attaattgcg
     5521 ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt gccagctgca ttaatgaatc
     5581 ggccaacgcg cggggagagg cggtttgcgt attgggcgct cttccgcttc ctcgctcact
     5641 gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat cagctcactc aaaggcggta
     5701 atacggttat ccacagaatc aggggataac gcaggaaaga acatgtgagc aaaaggccag
     5761 caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag gctccgcccc
     5821 cctgacgagc atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc gacaggacta
     5881 taaagatacc aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt tccgaccctg
     5941 ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc
     6001 tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac
     6061 gaaccccccg ttcagcccga ccgctgcgcc ttatccggta actatcgtct tgagtccaac
     6121 ccggtaagac acgacttatc gccactggca gcagccactg gtaacaggat tagcagagcg
     6181 aggtatgtag gcggtgctac agagttcttg aagtggtggc ctaactacgg ctacactaga
     6241 agaacagtat ttggtatctg cgctctgctg aagccagtta ccttcggaaa aagagttggt
     6301 agctcttgat ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag
     6361 cagattacgc gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc tacggggtct
     6421 gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt atcaaaaagg
     6481 atcttcacct agatcctttt aaattaaaaa tgaagtttta aatcaatcta aagtatatat
     6541 gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc
     6601 tgtctatttc gttcatccat agttgcctga ctccccgtcg tgtagataac tacgatacgg
     6661 gagggcttac catctggccc cagtgctgca atgataccgc gagacccacg ctcaccggct
     6721 ccagatttat cagcaataaa ccagccagcc ggaagggccg agcgcagaag tggtcctgca
     6781 actttatccg cctccatcca gtctattaat tgttgccggg aagctagagt aagtagttcg
     6841 ccagttaata gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt gtcacgctcg
     6901 tcgtttggta tggcttcatt cagctccggt tcccaacgat caaggcgagt tacatgatcc
     6961 cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag
     7021 ttggccgcag tgttatcact catggttatg gcagcactgc ataattctct tactgtcatg
     7081 ccatccgtaa gatgcttttc tgtgactggt gagtactcaa ccaagtcatt ctgagaatag
     7141 tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac gggataatac cgcgccacat
     7201 agcagaactt taaaagtgct catcattgga aaacgttctt cggggcgaaa actctcaagg
     7261 atcttaccgc tgttgagatc cagttcgatg taacccactc gtgcacccaa ctgatcttca
     7321 gcatctttta ctttcaccag cgtttctggg tgagcaaaaa caggaaggca aaatgccgca
     7381 aaaaagggaa taagggcgac acggaaatgt tgaatactca tactcttcct ttttcaatat
     7441 tattgaagca tttatcaggg ttattgtctc atgagcggat acatatttga atgtatttag
     7501 aaaaataaac aaataggggt tccgcgcaca tttccccgaa aagtgccacc tg
//
