LOCUS       sequence-486666-        4718 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expressed eGFP(S65T) in agrobacteria for tracing in planta.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4718)
  AUTHORS   Liu MS, Huang TK, Wang YC, Wang SC, Wu CH, Kuo CH, Lai EM
  TITLE     Floral stage optimization and immune evasion enhance 
            Agrobacterium-mediated genome editing in Arabidopsis.
  JOURNAL   New Phytol. 2025 Nov 30. doi: 10.1111/nph.70795.
  PUBMED    41321035
REFERENCE   2  (bases 1 to 4718)
  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"; journalName: "New Phytol.
            2025 Nov 30. doi: 10.1111/nph.70795."
COMMENT     Sequence Label: EML6428_ pRL662-eGFP
FEATURES             Location/Qualifiers
     source          1..4718
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      88..859
                     /label=pBBR1 oriV
                     /note="replication origin of the broad-host-range plasmid 
                     pBBR1 from Bordetella bronchiseptica; requires the pBBR1 
                     Rep protein for replication"
     CDS             860..1522
                     /codon_start=1
                     /product="replication protein for the broad-host-range 
                     plasmid pBBR1 from Bordetella bronchiseptica"
                     /label=pBBR1 Rep
                     /translation="MATQSREIGIQAKNKPGHWVQTERKAHEAWAGLIARKPTAAMLLH
                     HLVAQMGHQNAVVVSQKTLSKLIGRSLRTVQYAVKDLVAERWISVVKLNGPGTVSAYVV
                     NDRVAWGQPRDQLRLSVFSAAVVVDHDDQDESLLGHGDLRRIPTLYPGEQQLPTGPGEE
                     PPSQPGIPGMEPDLPALTETEEWERRGQQRLPMPDEPCFLDDGEPLEPPTRVTLPRR"
     primer_bind     2216..2238
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     2230..2247
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     2231..2247
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     promoter        2257..2275
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     2308..2324
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     2308..2324
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     terminator      complement(2388..2435)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     2431..2449
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     CDS             complement(2500..3216)
                     /codon_start=1
                     /product="S65T variant of Aequorea victoria green 
                     fluorescent protein (Heim et al., 1995)"
                     /label=GFP (S65T)
                     /note="excitable with blue light"
                     /translation="MASKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTFTYGVQCFSRYPDHMKRHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     ANFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITHGMDELYK"
     primer_bind     complement(2540..2560)
                     /label=GFP-F
                     /note="GFP, forward primer. Does NOT anneal to EGFP"
     primer_bind     3155..3183
                     /label=GFP-R
                     /note="GFP, reverse primer. Does NOT anneal to EGFP"
     RBS             3224..3246
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     promoter        complement(3278..3296)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(3348..3368)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(3348..3366)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(3387..3403)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3387..3403)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3400..3422)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3411..3427
                     /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(3435..3441,3442..3459,3460..3465))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3480..3501
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     CDS             complement(3694..4485)
                     /codon_start=1
                     /gene="aadA"
                     /product="aminoglycoside adenylyltransferase (Murphy, 
                     1985)"
                     /label=SmR
                     /note="confers resistance to spectinomycin and 
                     streptomycin"
                     /translation="MGEAVIAEVSTQLSEVVGVIERHLEPTLLAVHLYGSAVDGGLKPH
                     SDIDLLVTVTVRLDETTRRALINDLLETSASPGESEILRAVEVTIVVHDDIIPWRYPAK
                     RELQFGEWQRNDILAGIFEPATIDIDLAILLTKAREHSVALVGPAAEELFDPVPEQDLF
                     EALNETLTLWNSPPDWAGDERNVVLTLSRIWYSAVTGKIAPKDVAADWAMERLPAQYQP
                     VILEARQAYLGQEEDRLASRADQLEEFVHYVKGEITKVVGK"
     promoter        complement(join(4674..4679,4680..4696,4697..4702))
                     /gene="intI1 (promoter lies within the coding sequence)"
                     /label=Pc promoter
                     /note="class 1 integron promoter"
ORIGIN
        1 cgccctacgg gcttgctctc cgggcttcgc cctgcgcggt cgctgcgctc ccttgccagc
       61 ccgtggatat gtggacgatg accgcgagcg gccaccggct ggctcgcttc gctcggcccg
      121 tggacaaccc tgctggacaa gctgatggac aggctgcgcc tgcccacgag cttgaccaca
      181 gggattgccc accggctacc cagccttcga ccacataccc accggctcca actgcgcggc
      241 ctgcggcctt gccccatcaa tttttttaat tttctctggg gaaaagcctc cggcctgcgg
      301 cctgcgcgct tcgcttgccg gttggacacc aagtggaagg cgggtcaagg ctcgcgcagc
      361 gaccgcgcag cggcttggcc ttgacgcgcc tggaacgacc caagcctatg cgagtggggg
      421 cagtcgaagg gcgaagcccg cccgcctgcc ccccgagcct cacggcggcg agtgcggggg
      481 ttccaagggg gcagcgccac cttgggcaag gccgaaggcc gcgcagtcga tcaacaagcc
      541 ccggaggggc cactttttgc cggaggggga gccgcgccga aggcgtgggg gaaccccgca
      601 ggggtgccct tctttgggca ccaaagaact agatataggg cgaaatgcga aagacttaaa
      661 aatcaacaac ttaaaaaagg ggggtacgca acagctcatt gcggcacccc ccgcaatagc
      721 tcattgcgta ggttaaagaa aatctgtaat tgactgccac ttttacgcaa cgcataattg
      781 ttgtcgcgct gccgaaaagt tgcagctgat tgcgcatggt gcctcaaccg tgcggcaccc
      841 ctaccgcatg gagataagca tggccacgca gtccagagaa atcggcattc aagccaagaa
      901 caagcccggt cactgggtgc aaacggaacg caaagcgcat gaggcgtggg ccgggcttat
      961 tgcgaggaaa cccacggcgg caatgctgct gcatcacctc gtggcgcaga tgggccacca
     1021 gaacgccgtg gtggtcagcc agaagacact ttccaagctc atcggacgtt ctttgcggac
     1081 ggtccaatac gcagtcaagg acttggtggc cgagcgctgg atctccgtcg tgaagctcaa
     1141 cggccccggc accgtgtcgg cctacgtggt caatgaccgc gtggcgtggg gccagccccg
     1201 cgaccagttg cgcctgtcgg tgttcagtgc cgccgtggtg gttgatcacg acgaccagga
     1261 cgaatcgctg ttggggcatg gcgacctgcg ccgcatcccg accctgtatc cgggcgagca
     1321 gcaactaccg accggccccg gcgaggagcc gcccagccag cccggcattc cgggcatgga
     1381 accagacctg ccagccttga ccgaaacgga ggaatgggaa cggcgcgggc agcagcgctt
     1441 gccgatgccc gatgagccgt gttttctgga cgatggcgag ccgttggagc cgccgacacg
     1501 ggtcacgctg ccgcgccggt agcacttggg ttgcgcagca acccgtaagt gcgctgttcc
     1561 agactatcgg ctgtagccgc ctcgccgccc tataccttgt ctgcctcccc gcgttgcgtc
     1621 gcggtgcatg gagccgggcc acctcgacct gaatggaagc cggcggcacc tcgctaacgg
     1681 attcaccgtt tttatcaggc tctgggaggc agaataaatg atcatatcgt caattattac
     1741 ctccacgggg agagcctgag caaactggcc tcaggcattt gagaagcaca cggtcacact
     1801 gcttccggta gtcaataaac cggtaaacca gcaatagaca taagcggcta tttaacgacc
     1861 ctgccctgaa ccgacgaccg ggtcgaattt gctttcgaat ttctgccatt catccgctta
     1921 ttatcactta ttcaggcgta gcaaccaggc gtttaagggc accaataact gccttaaaaa
     1981 aattacgccc cgccctgcca ctcatcgcag tacggcctat tggttaaaaa atgagctgat
     2041 ttaacaaaaa tttaacgcga attttaacaa aatattaacg cttacaattt ccattcgcca
     2101 ttcaggctgc gcaactgttg ggaagggcga tcggtgcggg cctcttcgct attacgccag
     2161 ctggcgaaag ggggatgtgc tgcaaggcga ttaagttggg taacgccagg gttttcccag
     2221 tcacgacgtt gtaaaacgac ggccagtgag cgcgcgtaat acgactcact atagggcgaa
     2281 ttggagctcc accgcggtgg cggccgctct agaactagtg gatcccccgg gctgcaggaa
     2341 ttcatcgata tctagatctc gaatccggat atagttcctc ctttcagcaa aaaacccctc
     2401 aagacccgtt tagaggcccc aaggggttat gctagttatt gctcagcggt ggcagcagcc
     2461 aactcagctt cctttcgggc tttgttagca gccggatcct ttgtatagtt catccatgcc
     2521 atgtgtaatc ccagcagctg ttacaaactc aagaaggacc atgtggtctc tcttttcgtt
     2581 gggatctttc gaaagggcag attgtgtgga caggtaatgg ttgtctggta aaaggacagg
     2641 gccatcgcca attggagtat tttgttgata atggtctgct agttgaacgc ttccatcttc
     2701 aatgttgtgt ctaattttga agttagcttt gattccattc ttttgtttgt ctgccatgat
     2761 gtatacattg tgtgagttat agttgtattc caatttgtgc ccaagaatgt ttccatcttc
     2821 tttaaaatca atacctttta actcgattct attaacaagg gtatcacctt caaacttgac
     2881 ttcagcacgt gtcttgtagt tcccgtcatc tttgaaaaat atagttcttt cctgtacata
     2941 accttcgggc atggcactct tgaaaaagtc atgccgtttc atatgatccg ggtatcttga
     3001 aaagcactga acaccataag tgaaagtagt gacaagtgtt ggccatggaa caggtagttt
     3061 tccagtagtg caaataaatt taagggtaag ttttccgtat gttgcatcac cttcaccctc
     3121 tccactgaca gaaaatttgt gcccattaac atcaccatct aattcaacaa gaattgggac
     3181 aactccagtg aagagttctt ctcctttgct agccatatgt atatctcctt cttaaagtta
     3241 aacaaaatta tttctagagg gaaaccgttg tggtctccct atagtgagtc gtattaattt
     3301 cgcgggatcg agatctcgag ggggggcccg gtacccagct tttgttccct ttagtgaggg
     3361 ttaattgcgc gcttggcgta atcatggtca tagctgtttc ctgtgtgaaa ttgttatccg
     3421 ctcacaattc cacacaacat acgagccgga agcataaagt gtaaagcctg gggtgcctaa
     3481 tgagtgagct aactcacatt aattgcgttg cgctcactgc ccgctttcca gtcgggaaac
     3541 ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt
     3601 gggcggggta ccgagctcga attggccgcg gcgttgtgac aatttaccga acaactccgc
     3661 ggccgggaag ccgatctcgg cttgaacgaa ttgttatttg ccgactacct tggtgatctc
     3721 gcctttcacg tagtggacaa attcttccaa ctgatctgcg cgcgaggcca agcgatcttc
     3781 ttcttgtcca agataagcct gtctagcttc aagtatgacg ggctgatact gggccggcag
     3841 gcgctccatt gcccagtcgg cagcgacatc cttcggcgcg attttgccgg ttactgcgct
     3901 gtaccaaatg cgggacaacg taagcactac atttcgctca tcgccagccc agtcgggcgg
     3961 cgagttccat agcgttaagg tttcatttag cgcctcaaat agatcctgtt caggaaccgg
     4021 atcaaagagt tcctccgccg ctggacctac caaggcaacg ctatgttctc ttgcttttgt
     4081 cagcaagata gccagatcaa tgtcgatcgt ggctggctcg aagatacctg caagaatgtc
     4141 attgcgctgc cattctccaa attgcagttc gcgcttagct ggataacgcc acggaatgat
     4201 gtcgtcgtgc acaacaatgg tgacttctac agcgcggaga atctcgctct ctccagggga
     4261 agccgaagtt tccaaaaggt cgttgatcaa agctcgccgc gttgtttcat caagccttac
     4321 ggtcaccgta accagcaaat caatatcact gtgtggcttc aggccgccat ccactgcgga
     4381 gccgtacaaa tgtacggcca gcaacgtcgg ttcgagatgg cgctcgatga cgccaactac
     4441 ctctgatagt tgagtcgata cttcggcgat caccgcttcc cccatcgttg ctgctccata
     4501 acatcaaaca tcgacccacg gcgtaacgcg cttgctgctt ggatgcccga ggcatagact
     4561 gtacaaaaaa acagtcataa caagccatga aaaccgccac tgcgccgtta ccaccgctgc
     4621 gttcggtcaa ggttctggac cagttgcgtg agcgcatacg ctacttgcat tacagtttac
     4681 gaaccgaaca ggcttatgtc aattcgagct cggtaccc
//
