LOCUS       sequence-401511-        7315 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 7315)
  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 7315)
  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-GWY-VP16/pAM
FEATURES             Location/Qualifiers
     source          1..7315
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     terminator      complement(100..352)
                     /label=NOS terminator
                     /note="nopaline synthase terminator and poly(A) signal"
     CDS             complement(372..923)
                     /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(965..1144)
                     /label=NOS promoter
                     /note="nopaline synthase promoter"
     promoter        1613..1957
                     /label=CaMV 35S promoter
                     /note="strong constitutive promoter from cauliflower mosaic
                     virus"
     primer_bind     1901..1921
                     /label=35S promoter
                     /note="CaMV 35S promoter, forward primer"
     primer_bind     1959..1975
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     primer_bind     1960..1976
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    1987..2111
                     /gene="mutant version of attR"
                     /label=attR1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     promoter        join(2136..2141,2142..2159,2160..2166)
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an ""up"" mutation"
     CDS             2220..2879
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     complement(2286..2305)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     CDS             3221..3526
                     /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     3405..3424
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     protein_bind    complement(3567..3691)
                     /gene="mutant version of attR"
                     /label=attR2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     CDS             3738..3971
                     /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             3972..3992
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             3999..4019
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     misc_feature    4356..4380
                     /label=RB T-DNA repeat
                     /note="right border repeat from nopaline C58 T-DNA"
     rep_origin      4492..5201
                     /label=oriV
                     /note="incP origin of replication"
     promoter        5274..5365
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5366..5434,5435..6226)
                     /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(5584..5603)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6397..6985
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6886..6905
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     misc_feature    join(7297..7315,1..6)
                     /label=LB T-DNA repeat
                     /note="left border repeat from nopaline C58 T-DNA"
ORIGIN
        1 gtaaacaaat tgacgcttag acaacttaat aacacattgc ggacgttttt aatgatcgaa
       61 tactaacgtc tctaccagat atcagcttgc atgccggtcg atctagtaac atagatgaca
      121 ccgcgcgcga taatttatcc tagtttgcgc gctatatttt gttttctatc gcgtattaaa
      181 tgtataattg cgggactcta atcataaaaa cccatctcat aaataacgtc atgcattaca
      241 tgttaattat tacatgctta acgtaattca acagaaatta tatgataatc atcgcaagac
      301 cggcaacagg attcaatctt aagaaacttt attgccaaat gtttgaacga tctgcttgac
      361 tctaggggtc atcagatttc ggtgacgggc aggaccggac ggggcggcac cggcaggctg
      421 aagtccagct gccagaaacc cacgtcatgc cagttcccgt gcttgaagcc ggccgcccgc
      481 agcatgccgc ggggggcata tccgagcgcc tcgtgcatgc gcacgctcgg gtcgttgggc
      541 agcccgatga cagcgaccac gctcttgaag ccctgtgcct ccagggactt cagcaggtgg
      601 gtgtagagcg tggagcccag tcccgtccgc tggtggcggg gggagacgta cacggtcgac
      661 tcggccgtcc agtcgtaggc gttgcgtgcc ttccagggac ccgcgtaggc gatgccggcg
      721 acctcgccgt ccacctcggc gacgagccag ggatagcgct cccgcagacg gacgaggtcg
      781 tccgtccact cctgcggttc ctgcggctcg gtacggaagt tgaccgtgct tgtctcgatg
      841 tagtggttga cgatggtgca gaccgccggc atgtccgcct cggtggcacg gcggatgtcg
      901 gccgggcgtc gttctgggct catggtagat cccctcgatc gagttgagag tgaatatgag
      961 actctaattg gataccgagg ggaatttatg gaacgtcagt ggagcatttt tgacaagaaa
     1021 tatttgctag ctgatagtga ccttaggcga cttttgaacg cgcaataatg gtttctgacg
     1081 tatgtgctta gctcattaaa ctccagaaac ccgcggctca gtggctcctt caacgttgcg
     1141 gttctgtcag ttccaaacgt aaaacggctt gtcccgcgtc atcggcgggg gtcataacgt
     1201 gactccctta attctccgct catgatcaga ttgtcgtttc ccgccttcgg tttgggcgcg
     1261 cccggttcag aagaccagag ggctattgag acttttcaac aaagggtaat atcgggaaac
     1321 ctcctcggat tccattgccc agctatctgt cacttcatcg aaaggacagt agaaaaggaa
     1381 gatggcttct acaaatgcca tcattgcgat aaaggaaagg ctatcgttca agatgcctct
     1441 accgacagtg gtcccaaaga tggaccccca cccacgagga acatcgtgga aaaagaagac
     1501 gttccaacca cgtcttcaaa gcaagtggat tgatgtgata catggtggag cacgacactc
     1561 tcgtctactc caagaatatc aaagatacag tctcagaaga ccagagggct attgagactt
     1621 ttcaacaaag ggtaatatcg ggaaacctcc tcggattcca ttgcccagct atctgtcact
     1681 tcatcgaaag gacagtagaa aaggaagatg gcttctacaa atgccatcat tgcgataaag
     1741 gaaaggctat cgttcaagat gcctctaccg acagtggtcc caaagatgga cccccaccca
     1801 cgaggaacat cgtggaaaaa gaagacgttc caaccacgtc ttcaaagcaa gtggattgat
     1861 gtgatatctc cactgacgta agggatgacg cacaatccca ctatccttcg caagaccctt
     1921 cctctatata aggaagttca tttcatttgg agaggacctc gaggtcgacg gtatcgataa
     1981 gctatcacaa gtttgtacaa aaaagctgaa cgagaaacgt aaaatgatat aaatatcaat
     2041 atattaaatt agattttgca taaaaaacag actacataat actgtaaaac acaacatatc
     2101 cagtcactat ggcggccgca ttaggcaccc caggctttac actttatgct tccggctcgt
     2161 ataatgtgtg gattttgagt taggatccgt cgagattttc aggagctaag gaagctaaaa
     2221 tggagaaaaa aatcactgga tataccaccg ttgatatatc ccaatggcat cgtaaagaac
     2281 attttgaggc atttcagtca gttgctcaat gtacctataa ccagaccgtt cagctggata
     2341 ttacggcctt tttaaagacc gtaaagaaaa ataagcacaa gttttatccg gcctttattc
     2401 acattcttgc ccgcctgatg aatgctcatc cggaattccg tatggcaatg aaagacggtg
     2461 agctggtgat atgggatagt gttcaccctt gttacaccgt tttccatgag caaactgaaa
     2521 cgttttcatc gctctggagt gaataccacg acgatttccg gcagtttcta cacatatatt
     2581 cgcaagatgt ggcgtgttac ggtgaaaacc tggcctattt ccctaaaggg tttattgaga
     2641 atatgttttt cgtctcagcc aatccctggg tgagtttcac cagttttgat ttaaacgtgg
     2701 ccaatatgga caacttcttc gcccccgttt tcaccatggg caaatattat acgcaaggcg
     2761 acaaggtgct gatgccgctg gcgattcagg ttcatcatgc cgtttgtgat ggcttccatg
     2821 tcggcagaat gcttaatgaa ttacaacagt actgcgatga gtggcagggc ggggcgtaaa
     2881 cgcgtggatc cggcttacta aaagccagat aacagtatgc gtatttgcgc gctgattttt
     2941 gcggtataag aatatatact gatatgtata cccgaagtat gtcaaaaaga ggtatgctat
     3001 gaagcagcgt attacagtga cagttgacag cgacagctat cagttgctca aggcatatat
     3061 gatgtcaata tctccggtct ggtaagcaca accatgcaga atgaagcccg tcgtctgcgt
     3121 gccgaacgct ggaaagcgga aaatcaggaa gggatggctg aggtcgcccg gtttattgaa
     3181 atgaacggct cttttgctga cgagaacagg ggctggtgaa atgcagttta aggtttacac
     3241 ctataaaaga gagagccgtt atcgtctgtt tgtggatgta cagagtgata ttattgacac
     3301 gcccgggcga cggatggtga tccccctggc cagtgcacgt ctgctgtcag ataaagtctc
     3361 ccgtgaactt tacccggtgg tgcatatcgg ggatgaaagc tggcgcatga tgaccaccga
     3421 tatggccagt gtgccggtct ccgttatcgg ggaagaagtg gctgatctca gccaccgcga
     3481 aaatgacatc aaaaacgcca ttaacctgat gttctgggga atataaatgt caggctccct
     3541 tatacacagc cagtctgcag gtcgaccata gtgactggat atgttgtgtt ttacagtatt
     3601 atgtagtctg ttttttatgc aaaatctaat ttaatatatt gatatttata tcattttacg
     3661 tttctcgttc agctttcttg tacaaagtgg tgatcggtgg tggtagtggt ggtggtggat
     3721 caggtggtgg tggatctgcc cccccgaccg atgtcagcct gggggacgag ctccacttag
     3781 acggcgagga cgtggcgatg gcgcatgccg acgcgctaga cgatttcgat ctggacatgt
     3841 tgggggacgg ggattccccg ggtccgggat ttacccccca cgactccgcc ccctacggcg
     3901 ctctggatat ggccgacttc gagtttgagc agatgtttac cgatgccctt ggaattgacg
     3961 agtacggtgg gcctaagaag aagcggaaag tggaggatcc caagaagaag cgcaaagtct
     4021 aggaattcaa ggcctctcga gcctctagaa ctatagtgag tcgtattact agagtccgca
     4081 aaaatcacca gtctctctct acaaatctat ctctctctat ttttctccag aataatgtgt
     4141 gagtagttcc cagataaggg aattagggtt cttatagggt ttcgctcatg tgttgagcat
     4201 ataagaaacc cttagtatgt atttgtattt gtaaaatact tctatcaata aaatttctaa
     4261 ttcctaaaac caaaatccag tgaccgggcg gccgccaccg cggtggaggg ggatcagatt
     4321 gtcgtttccc gccttcagtt taaactatca gtgtttgaca ggatatattg gcgggtaaac
     4381 ctaagagaaa agagcgttta ttagaataat cggatattta aaagggcgtg aaaaggttta
     4441 tccgttcgtc catttgtatg tgtacatcac cgacgagcaa ggcaagaccg agcgcctttc
     4501 cgacgctcac cgggctggtt gccctcgccg ctgggctggc ggccgtctat ggccctgcaa
     4561 acgcgccaga aacgccgtcg aagccgtgtg cgagacaccg gccgccggcg ttgtggatac
     4621 ctcgcggaaa acttggccct cactgacaga tgaggggcgg acgttgacac ttgaggggcc
     4681 gactcacccg gcgcggcgtt gacagatgag gggcaggctc gatttcggcc ggcgacgtgg
     4741 agctggccag cctcgcaaat cggcgaaaac gcctgatttt acgcgagttt cccacagatg
     4801 atgtggacaa gcctggggat aagtgccctg cggtattgac acttgagggg cgcgactact
     4861 gacagatgag gggcgcgatc cttgacactt gaggggcaga gtgctgacag atgaggggcg
     4921 cacctattga catttgaggg gctgtccaca ggcagaaaat ccagcatttg caagggtttc
     4981 cgcccgtttt tcggccaccg ctaacctgtc ttttaacctg cttttaaacc aatatttata
     5041 aaccttgttt ttaaccaggg ctgcgccctg tgcgcgtgac cgcgcacgcc gaaggggggt
     5101 gccccccctt ctcgaaccct cccggcccgc taacgcgggc ctcccatccc cccaggggct
     5161 gcgcccctcg gccgcgaacg gcctcacccc aaaaatggca gcgctggcag tccttgccat
     5221 tgccgggatc ggggcagtaa cgggatgggc gatcagcccg aaagctaccc ctatttgttt
     5281 atttttctaa atacattcaa atatgtatcc gctcatgaga caataaccct gataaatgct
     5341 tcaataatat tgaaaaagga agagtatgag tattcaacat ttccgtgtcg cccttattcc
     5401 cttttttgcg gcattttgcc ttcctgtttt tgctcaccca gaaacgctgg tgaaagtaaa
     5461 agatgctgaa gatcagttgg gtgcacgagt gggttacatc gaactggatc tcaacagcgg
     5521 taagatcctt gagagttttc gccccgaaga acgttttcca atgatgagca cttttaaagt
     5581 tctgctatgt ggcgcggtat tatcccgtat tgacgccggg caagagcaac tcggtcgccg
     5641 catacactat tctcagaatg acttggttga gtactcacca gtcacagaaa agcatcttac
     5701 ggatggcatg acagtaagag aattatgcag tgctgccata accatgagtg ataacactgc
     5761 ggccaactta cttctgacaa cgatcggagg accgaaggag ctaaccgctt ttttgcacaa
     5821 catgggggat catgtaactc gccttgatcg ttgggaaccg gagctgaatg aagccatacc
     5881 aaacgacgag cgtgacacca cgatgcctgt agcaatggca acaacgttgc gcaaactatt
     5941 aactggcgaa ctacttactc tagcttcccg gcaacaatta atagactgga tggaggcgga
     6001 taaagttgca ggaccacttc tgcgctcggc ccttccggct ggctggttta ttgctgataa
     6061 atctggagcc ggtgagcgtg ggtctcgcgg tatcattgca gcactggggc cagatggtaa
     6121 gccctcccgt atcgtagtta tctacacgac ggggagtcag gcaactatgg atgaacgaaa
     6181 tagacagatc gctgagatag gtgcctcact gattaagcat tggtaactgt cagaccaagt
     6241 ttactcatat atactttaga ttgatttaaa acttcatttt taatttaaaa ggatctaggt
     6301 gaagatcctt tttgataatc tcatgaccaa aatcccttaa cgtgagtttt cgttccactg
     6361 agcgtcagac cccgtagaaa agatcaaagg atcttcttga gatccttttt ttctgcgcgt
     6421 aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt tgccggatca
     6481 agagctacca actctttttc cgaaggtaac tggcttcagc agagcgcaga taccaaatac
     6541 tgttcttcta gtgtagccgt agttaggcca ccacttcaag aactctgtag caccgcctac
     6601 atacctcgct ctgctaatcc tgttaccagt ggctgctgcc agtggcgata agtcgtgtct
     6661 taccgggttg gactcaagac gatagttacc ggataaggcg cagcggtcgg gctgaacggg
     6721 gggttcgtgc acacagccca gcttggagcg aacgacctac accgaactga gatacctaca
     6781 gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca ggtatccggt
     6841 aagcggcagg gtcggaacag gagagcgcac gagggagctt ccagggggaa acgcctggta
     6901 tctttatagt cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc
     6961 gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc
     7021 cttttgctgg ccttttgctc acatggactc tagctagagg atcacaggca gcaacgctct
     7081 gtcatcgtta caatcaacat gctaccctcc gcgagatcat ccgtgtttca aacccggcag
     7141 cttagttgcc gttcttccga atagcatcgg taacatgagc aaagtctgcc gccttacaac
     7201 ggctctcccg ctgacgccgt cccggactga tgggctgcct gtatcgagtg gtgattttgt
     7261 gccgagctgc cggtcgggga gctgttggct ggctggtggc aggatatatt gtggt
//
