LOCUS       sequence-403628-        7321 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Used to produce and express in planta C-terminal fusion proteins 
            with the VP16 activation domain.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7321)
  AUTHORS   Chen J, Luo M, Hands P, Rolland V, Zhang J, Li Z, Outram M, Dodds P,
            Ayliffe M
  TITLE     A split GAL4 RUBY assay for visual in planta detection of 
            protein-protein interactions.
  JOURNAL   Plant J. 2023 Jun;114(6):1209-1226. doi: 10.1111/tpj.16234. Epub 
            2023 Apr 26.
  PUBMED    37323061
REFERENCE   2  (bases 1 to 7321)
  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"; doi: "10.1111/tpj.16234";
            journalName: "Plant J"; date: "2023-06"; volume: "114"; issue: "6"; 
            pages: "1209-1226"
COMMENT     Sequence Label: 35S-VP16-GWY/pAM
FEATURES             Location/Qualifiers
     source          1..7321
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     terminator      complement(108..360)
                     /label=NOS terminator
                     /note="nopaline synthase terminator and poly(A) signal"
     CDS             complement(380..931)
                     /codon_start=1
                     /gene="Streptomyces hygroscopicus bar"
                     /product="phosphinothricin acetyltransferase"
                     /label=BlpR
                     /note="confers resistance to bialophos or phosphinothricin"
                     /translation="MSPERRPADIRRATEADMPAVCTIVNHYIETSTVNFRTEPQEPQE
                     WTDDLVRLRERYPWLVAEVDGEVAGIAYAGPWKARNAYDWTAESTVYVSPRHQRTGLGS
                     TLYTHLLKSLEAQGFKSVVAVIGLPNDPSVRMHEALGYAPRGMLRAAGFKHGNWHDVGF
                     WQLDFSLPVPPRPVLPVTEI"
     promoter        complement(973..1152)
                     /label=NOS promoter
                     /note="nopaline synthase promoter"
     promoter        1621..1965
                     /label=CaMV 35S promoter
                     /note="strong constitutive promoter from cauliflower mosaic
                     virus"
     primer_bind     1909..1929
                     /label=35S promoter
                     /note="CaMV 35S promoter, forward primer"
     CDS             1998..2231
                     /codon_start=1
                     /gene="UL48"
                     /product="transcriptional activation domain of herpes 
                     simplex virus protein VP16 (Triezenberg et al., 1988; 
                     Cousens et al., 1989)"
                     /label=VP16 AD
                     /translation="APPTDVSLGDELHLDGEDVAMAHADALDDFDLDMLGDGDSPGPGF
                     TPHDSAPYGALDMADFEFEQMFTDALGIDEYGG"
     CDS             2232..2252
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             2259..2279
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     protein_bind    2322..2446
                     /gene="mutant version of attR"
                     /label=attR1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     promoter        join(2471..2476,2477..2494,2495..2501)
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an ""up"" mutation"
     CDS             2555..3214
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     complement(2621..2640)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     CDS             3556..3861
                     /codon_start=1
                     /gene="ccdB"
                     /product="CcdB, a bacterial toxin that poisons DNA gyrase"
                     /label=ccdB
                     /note="Plasmids containing the ccdB gene cannot be 
                     propagated in standard E. coli strains."
                     /translation="MQFKVYTYKRESRYRLFVDVQSDIIDTPGRRMVIPLASARLLSDK
                     VSRELYPVVHIGDESWRMMTTDMASVPVSVIGEEVADLSHRENDIKNAINLMFWGI"
     primer_bind     3740..3759
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     protein_bind    complement(3902..4026)
                     /gene="mutant version of attR"
                     /label=attR2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     misc_feature    4370..4394
                     /label=RB T-DNA repeat
                     /note="right border repeat from nopaline C58 T-DNA"
     rep_origin      4506..5215
                     /label=oriV
                     /note="incP origin of replication"
     promoter        5288..5379
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5380..5448,5449..6240)
                     /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     complement(5598..5617)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6411..6999
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6900..6919
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     misc_feature    join(7311..7321,1..14)
                     /label=LB T-DNA repeat
                     /note="left border repeat from nopaline C58 T-DNA"
ORIGIN
        1 attgtggtgt aaacaaattg acgcttagac aacttaataa cacattgcgg acgtttttaa
       61 tgatcgaata ctaacgtctc taccagatat cagcttgcat gccggtcgat ctagtaacat
      121 agatgacacc gcgcgcgata atttatccta gtttgcgcgc tatattttgt tttctatcgc
      181 gtattaaatg tataattgcg ggactctaat cataaaaacc catctcataa ataacgtcat
      241 gcattacatg ttaattatta catgcttaac gtaattcaac agaaattata tgataatcat
      301 cgcaagaccg gcaacaggat tcaatcttaa gaaactttat tgccaaatgt ttgaacgatc
      361 tgcttgactc taggggtcat cagatttcgg tgacgggcag gaccggacgg ggcggcaccg
      421 gcaggctgaa gtccagctgc cagaaaccca cgtcatgcca gttcccgtgc ttgaagccgg
      481 ccgcccgcag catgccgcgg ggggcatatc cgagcgcctc gtgcatgcgc acgctcgggt
      541 cgttgggcag cccgatgaca gcgaccacgc tcttgaagcc ctgtgcctcc agggacttca
      601 gcaggtgggt gtagagcgtg gagcccagtc ccgtccgctg gtggcggggg gagacgtaca
      661 cggtcgactc ggccgtccag tcgtaggcgt tgcgtgcctt ccagggaccc gcgtaggcga
      721 tgccggcgac ctcgccgtcc acctcggcga cgagccaggg atagcgctcc cgcagacgga
      781 cgaggtcgtc cgtccactcc tgcggttcct gcggctcggt acggaagttg accgtgcttg
      841 tctcgatgta gtggttgacg atggtgcaga ccgccggcat gtccgcctcg gtggcacggc
      901 ggatgtcggc cgggcgtcgt tctgggctca tggtagatcc cctcgatcga gttgagagtg
      961 aatatgagac tctaattgga taccgagggg aatttatgga acgtcagtgg agcatttttg
     1021 acaagaaata tttgctagct gatagtgacc ttaggcgact tttgaacgcg caataatggt
     1081 ttctgacgta tgtgcttagc tcattaaact ccagaaaccc gcggctcagt ggctccttca
     1141 acgttgcggt tctgtcagtt ccaaacgtaa aacggcttgt cccgcgtcat cggcgggggt
     1201 cataacgtga ctcccttaat tctccgctca tgatcagatt gtcgtttccc gccttcggtt
     1261 tgggcgcgcc cggttcagaa gaccagaggg ctattgagac ttttcaacaa agggtaatat
     1321 cgggaaacct cctcggattc cattgcccag ctatctgtca cttcatcgaa aggacagtag
     1381 aaaaggaaga tggcttctac aaatgccatc attgcgataa aggaaaggct atcgttcaag
     1441 atgcctctac cgacagtggt cccaaagatg gacccccacc cacgaggaac atcgtggaaa
     1501 aagaagacgt tccaaccacg tcttcaaagc aagtggattg atgtgataca tggtggagca
     1561 cgacactctc gtctactcca agaatatcaa agatacagtc tcagaagacc agagggctat
     1621 tgagactttt caacaaaggg taatatcggg aaacctcctc ggattccatt gcccagctat
     1681 ctgtcacttc atcgaaagga cagtagaaaa ggaagatggc ttctacaaat gccatcattg
     1741 cgataaagga aaggctatcg ttcaagatgc ctctaccgac agtggtccca aagatggacc
     1801 cccacccacg aggaacatcg tggaaaaaga agacgttcca accacgtctt caaagcaagt
     1861 ggattgatgt gatatctcca ctgacgtaag ggatgacgca caatcccact atccttcgca
     1921 agacccttcc tctatataag gaagttcatt tcatttggag aggacctcga ggtcgacggt
     1981 tccgataagc tatcatggcc cccccgaccg atgtcagcct gggggacgag ctccacttag
     2041 acggcgagga cgtggcgatg gcgcatgccg acgcgctaga cgatttcgat ctggacatgt
     2101 tgggggacgg ggattccccg ggtccgggat ttacccccca cgactccgcc ccctacggcg
     2161 ctctggatat ggccgacttc gagtttgagc agatgtttac cgatgccctt ggaattgacg
     2221 agtacggtgg gcctaagaag aagcggaaag tggaggatcc caagaagaag cgcaaagtcg
     2281 gtggtggtag tggtggtggt ggatcaggtg gtggtggatc tacaagtttg tacaaaaaag
     2341 ctgaacgaga aacgtaaaat gatataaata tcaatatatt aaattagatt ttgcataaaa
     2401 aacagactac ataatactgt aaaacacaac atatccagtc actatggcgg ccgcattagg
     2461 caccccaggc tttacacttt atgcttccgg ctcgtataat gtgtggattt tgagttagga
     2521 tccgtcgaga ttttcaggag ctaaggaagc taaaatggag aaaaaaatca ctggatatac
     2581 caccgttgat atatcccaat ggcatcgtaa agaacatttt gaggcatttc agtcagttgc
     2641 tcaatgtacc tataaccaga ccgttcagct ggatattacg gcctttttaa agaccgtaaa
     2701 gaaaaataag cacaagtttt atccggcctt tattcacatt cttgcccgcc tgatgaatgc
     2761 tcatccggaa ttccgtatgg caatgaaaga cggtgagctg gtgatatggg atagtgttca
     2821 cccttgttac accgttttcc atgagcaaac tgaaacgttt tcatcgctct ggagtgaata
     2881 ccacgacgat ttccggcagt ttctacacat atattcgcaa gatgtggcgt gttacggtga
     2941 aaacctggcc tatttcccta aagggtttat tgagaatatg tttttcgtct cagccaatcc
     3001 ctgggtgagt ttcaccagtt ttgatttaaa cgtggccaat atggacaact tcttcgcccc
     3061 cgttttcacc atgggcaaat attatacgca aggcgacaag gtgctgatgc cgctggcgat
     3121 tcaggttcat catgccgttt gtgatggctt ccatgtcggc agaatgctta atgaattaca
     3181 acagtactgc gatgagtggc agggcggggc gtaaacgcgt ggatccggct tactaaaagc
     3241 cagataacag tatgcgtatt tgcgcgctga tttttgcggt ataagaatat atactgatat
     3301 gtatacccga agtatgtcaa aaagaggtat gctatgaagc agcgtattac agtgacagtt
     3361 gacagcgaca gctatcagtt gctcaaggca tatatgatgt caatatctcc ggtctggtaa
     3421 gcacaaccat gcagaatgaa gcccgtcgtc tgcgtgccga acgctggaaa gcggaaaatc
     3481 aggaagggat ggctgaggtc gcccggttta ttgaaatgaa cggctctttt gctgacgaga
     3541 acaggggctg gtgaaatgca gtttaaggtt tacacctata aaagagagag ccgttatcgt
     3601 ctgtttgtgg atgtacagag tgatattatt gacacgcccg ggcgacggat ggtgatcccc
     3661 ctggccagtg cacgtctgct gtcagataaa gtctcccgtg aactttaccc ggtggtgcat
     3721 atcggggatg aaagctggcg catgatgacc accgatatgg ccagtgtgcc ggtctccgtt
     3781 atcggggaag aagtggctga tctcagccac cgcgaaaatg acatcaaaaa cgccattaac
     3841 ctgatgttct ggggaatata aatgtcaggc tcccttatac acagccagtc tgcaggtcga
     3901 ccatagtgac tggatatgtt gtgttttaca gtattatgta gtctgttttt tatgcaaaat
     3961 ctaatttaat atattgatat ttatatcatt ttacgtttct cgttcagctt tcttgtacaa
     4021 agtggtgtag tgataggaat tcaaggcctc tcgagcctct agaactatag tgagtcgtat
     4081 tactagagtc cgcaaaaatc accagtctct ctctacaaat ctatctctct ctatttttct
     4141 ccagaataat gtgtgagtag ttcccagata agggaattag ggttcttata gggtttcgct
     4201 catgtgttga gcatataaga aacccttagt atgtatttgt atttgtaaaa tacttctatc
     4261 aataaaattt ctaattccta aaaccaaaat ccagtgaccg ggcggccgcc accgcggtgg
     4321 agggggatca gattgtcgtt tcccgccttc agtttaaact atcagtgttt gacaggatat
     4381 attggcgggt aaacctaaga gaaaagagcg tttattagaa taatcggata tttaaaaggg
     4441 cgtgaaaagg tttatccgtt cgtccatttg tatgtgtaca tcaccgacga gcaaggcaag
     4501 accgagcgcc tttccgacgc tcaccgggct ggttgccctc gccgctgggc tggcggccgt
     4561 ctatggccct gcaaacgcgc cagaaacgcc gtcgaagccg tgtgcgagac accggccgcc
     4621 ggcgttgtgg atacctcgcg gaaaacttgg ccctcactga cagatgaggg gcggacgttg
     4681 acacttgagg ggccgactca cccggcgcgg cgttgacaga tgaggggcag gctcgatttc
     4741 ggccggcgac gtggagctgg ccagcctcgc aaatcggcga aaacgcctga ttttacgcga
     4801 gtttcccaca gatgatgtgg acaagcctgg ggataagtgc cctgcggtat tgacacttga
     4861 ggggcgcgac tactgacaga tgaggggcgc gatccttgac acttgagggg cagagtgctg
     4921 acagatgagg ggcgcaccta ttgacatttg aggggctgtc cacaggcaga aaatccagca
     4981 tttgcaaggg tttccgcccg tttttcggcc accgctaacc tgtcttttaa cctgctttta
     5041 aaccaatatt tataaacctt gtttttaacc agggctgcgc cctgtgcgcg tgaccgcgca
     5101 cgccgaaggg gggtgccccc ccttctcgaa ccctcccggc ccgctaacgc gggcctccca
     5161 tccccccagg ggctgcgccc ctcggccgcg aacggcctca ccccaaaaat ggcagcgctg
     5221 gcagtccttg ccattgccgg gatcggggca gtaacgggat gggcgatcag cccgaaagct
     5281 acccctattt gtttattttt ctaaatacat tcaaatatgt atccgctcat gagacaataa
     5341 ccctgataaa tgcttcaata atattgaaaa aggaagagta tgagtattca acatttccgt
     5401 gtcgccctta ttcccttttt tgcggcattt tgccttcctg tttttgctca cccagaaacg
     5461 ctggtgaaag taaaagatgc tgaagatcag ttgggtgcac gagtgggtta catcgaactg
     5521 gatctcaaca gcggtaagat ccttgagagt tttcgccccg aagaacgttt tccaatgatg
     5581 agcactttta aagttctgct atgtggcgcg gtattatccc gtattgacgc cgggcaagag
     5641 caactcggtc gccgcataca ctattctcag aatgacttgg ttgagtactc accagtcaca
     5701 gaaaagcatc ttacggatgg catgacagta agagaattat gcagtgctgc cataaccatg
     5761 agtgataaca ctgcggccaa cttacttctg acaacgatcg gaggaccgaa ggagctaacc
     5821 gcttttttgc acaacatggg ggatcatgta actcgccttg atcgttggga accggagctg
     5881 aatgaagcca taccaaacga cgagcgtgac accacgatgc ctgtagcaat ggcaacaacg
     5941 ttgcgcaaac tattaactgg cgaactactt actctagctt cccggcaaca attaatagac
     6001 tggatggagg cggataaagt tgcaggacca cttctgcgct cggcccttcc ggctggctgg
     6061 tttattgctg ataaatctgg agccggtgag cgtgggtctc gcggtatcat tgcagcactg
     6121 gggccagatg gtaagccctc ccgtatcgta gttatctaca cgacggggag tcaggcaact
     6181 atggatgaac gaaatagaca gatcgctgag ataggtgcct cactgattaa gcattggtaa
     6241 ctgtcagacc aagtttactc atatatactt tagattgatt taaaacttca tttttaattt
     6301 aaaaggatct aggtgaagat cctttttgat aatctcatga ccaaaatccc ttaacgtgag
     6361 ttttcgttcc actgagcgtc agaccccgta gaaaagatca aaggatcttc ttgagatcct
     6421 ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt
     6481 tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt cagcagagcg
     6541 cagataccaa atactgttct tctagtgtag ccgtagttag gccaccactt caagaactct
     6601 gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc tgccagtggc
     6661 gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa ggcgcagcgg
     6721 tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac ctacaccgaa
     6781 ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg
     6841 gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga gcttccaggg
     6901 ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact tgagcgtcga
     6961 tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa cgcggccttt
     7021 ttacggttcc tggccttttg ctggcctttt gctcacatgg actctagcta gaggatcaca
     7081 ggcagcaacg ctctgtcatc gttacaatca acatgctacc ctccgcgaga tcatccgtgt
     7141 ttcaaacccg gcagcttagt tgccgttctt ccgaatagca tcggtaacat gagcaaagtc
     7201 tgccgcctta caacggctct cccgctgacg ccgtcccgga ctgatgggct gcctgtatcg
     7261 agtggtgatt ttgtgccgag ctgccggtcg gggagctgtt ggctggctgg tggcaggata
     7321 t
//
