LOCUS       sequence-401530-        7474 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Used to produce and express in planta N-terminal fusion proteins 
            with the GAL4 BD.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7474)
  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 7474)
  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-GAL4BD-GWY/pAM
FEATURES             Location/Qualifiers
     source          1..7474
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     terminator      complement(118..370)
                     /label=NOS terminator
                     /note="nopaline synthase terminator and poly(A) signal"
     CDS             complement(390..941)
                     /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(983..1162)
                     /label=NOS promoter
                     /note="nopaline synthase promoter"
     promoter        1631..1975
                     /label=CaMV 35S promoter
                     /note="strong constitutive promoter from cauliflower mosaic
                     virus"
     primer_bind     1919..1939
                     /label=35S promoter
                     /note="CaMV 35S promoter, forward primer"
     CDS             2005..2445
                     /codon_start=1
                     /gene="Saccharomyces cerevisiae GAL4 (truncated)"
                     /product="DNA binding domain of the GAL4 transcriptional 
                     activator"
                     /label=GAL4 DNA binding domain
                     /translation="MKLLSSIEQACDICRLKKLKCSKEKPKCAKCLKNNWECRYSPKTK
                     RSPLTRAHLTEVESRLERLEQLFLLIFPREDLDMILKMDSLQDIKALLTGLFVQDNVNK
                     DAVTDRLASVETDMPLTLRQHRISATSSSEESSNKGQRQLTVS"
     protein_bind    2488..2612
                     /gene="mutant version of attR"
                     /label=attR1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     promoter        join(2637..2642,2643..2660,2661..2667)
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an ""up"" mutation"
     CDS             2721..3380
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     complement(2787..2806)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     CDS             3722..4027
                     /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     3906..3925
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     protein_bind    complement(4068..4192)
                     /gene="mutant version of attR"
                     /label=attR2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     misc_feature    4533..4557
                     /label=RB T-DNA repeat
                     /note="right border repeat from nopaline C58 T-DNA"
     rep_origin      4669..5378
                     /label=oriV
                     /note="incP origin of replication"
     promoter        5451..5542
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5543..5611,5612..6403)
                     /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(5761..5780)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6574..7162
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7063..7082
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     misc_feature    join(7474,1..24)
                     /label=LB T-DNA repeat
                     /note="left border repeat from nopaline C58 T-DNA"
ORIGIN
        1 ggcaggatat attgtggtgt aaacaaattg acgcttagac aacttaataa cacattgcgg
       61 acgtttttaa tgatcgaata ctaacgtctc taccagatat cagcttgcat gccggtcgat
      121 ctagtaacat agatgacacc gcgcgcgata atttatccta gtttgcgcgc tatattttgt
      181 tttctatcgc gtattaaatg tataattgcg ggactctaat cataaaaacc catctcataa
      241 ataacgtcat gcattacatg ttaattatta catgcttaac gtaattcaac agaaattata
      301 tgataatcat cgcaagaccg gcaacaggat tcaatcttaa gaaactttat tgccaaatgt
      361 ttgaacgatc tgcttgactc taggggtcat cagatttcgg tgacgggcag gaccggacgg
      421 ggcggcaccg gcaggctgaa gtccagctgc cagaaaccca cgtcatgcca gttcccgtgc
      481 ttgaagccgg ccgcccgcag catgccgcgg ggggcatatc cgagcgcctc gtgcatgcgc
      541 acgctcgggt cgttgggcag cccgatgaca gcgaccacgc tcttgaagcc ctgtgcctcc
      601 agggacttca gcaggtgggt gtagagcgtg gagcccagtc ccgtccgctg gtggcggggg
      661 gagacgtaca cggtcgactc ggccgtccag tcgtaggcgt tgcgtgcctt ccagggaccc
      721 gcgtaggcga tgccggcgac ctcgccgtcc acctcggcga cgagccaggg atagcgctcc
      781 cgcagacgga cgaggtcgtc cgtccactcc tgcggttcct gcggctcggt acggaagttg
      841 accgtgcttg tctcgatgta gtggttgacg atggtgcaga ccgccggcat gtccgcctcg
      901 gtggcacggc ggatgtcggc cgggcgtcgt tctgggctca tggtagatcc cctcgatcga
      961 gttgagagtg aatatgagac tctaattgga taccgagggg aatttatgga acgtcagtgg
     1021 agcatttttg acaagaaata tttgctagct gatagtgacc ttaggcgact tttgaacgcg
     1081 caataatggt ttctgacgta tgtgcttagc tcattaaact ccagaaaccc gcggctcagt
     1141 ggctccttca acgttgcggt tctgtcagtt ccaaacgtaa aacggcttgt cccgcgtcat
     1201 cggcgggggt cataacgtga ctcccttaat tctccgctca tgatcagatt gtcgtttccc
     1261 gccttcggtt tgggcgcgcc cggttcagaa gaccagaggg ctattgagac ttttcaacaa
     1321 agggtaatat cgggaaacct cctcggattc cattgcccag ctatctgtca cttcatcgaa
     1381 aggacagtag aaaaggaaga tggcttctac aaatgccatc attgcgataa aggaaaggct
     1441 atcgttcaag atgcctctac cgacagtggt cccaaagatg gacccccacc cacgaggaac
     1501 atcgtggaaa aagaagacgt tccaaccacg tcttcaaagc aagtggattg atgtgataca
     1561 tggtggagca cgacactctc gtctactcca agaatatcaa agatacagtc tcagaagacc
     1621 agagggctat tgagactttt caacaaaggg taatatcggg aaacctcctc ggattccatt
     1681 gcccagctat ctgtcacttc atcgaaagga cagtagaaaa ggaagatggc ttctacaaat
     1741 gccatcattg cgataaagga aaggctatcg ttcaagatgc ctctaccgac agtggtccca
     1801 aagatggacc cccacccacg aggaacatcg tggaaaaaga agacgttcca accacgtctt
     1861 caaagcaagt ggattgatgt gatatctcca ctgacgtaag ggatgacgca caatcccact
     1921 atccttcgca agacccttcc tctatataag gaagttcatt tcatttggag aggacctcga
     1981 ggtcgacggt tccgataagc tatcatgaag ctactgtctt ctatcgaaca agcatgcgat
     2041 atttgccgac ttaaaaagct caagtgctcc aaagaaaaac cgaagtgcgc caagtgtctg
     2101 aagaacaact gggagtgtcg ctactctccc aaaaccaaaa ggtctccgct gactagggca
     2161 catctgacag aagtggaatc aaggctagaa agactggaac agctatttct actgattttt
     2221 cctcgagaag accttgacat gattttgaaa atggattctt tacaggatat aaaagcattg
     2281 ttaacaggat tatttgtaca agataatgtg aataaagatg ccgtcacaga tagattggct
     2341 tcagtggaga ctgatatgcc tctaacattg agacagcata gaataagtgc gacatcatca
     2401 tcggaagaga gtagtaacaa aggtcaaaga cagttgactg tatcgggtgg tggtagtggt
     2461 ggtggtggat caggtggtgg tggatctaca agtttgtaca aaaaagctga acgagaaacg
     2521 taaaatgata taaatatcaa tatattaaat tagattttgc ataaaaaaca gactacataa
     2581 tactgtaaaa cacaacatat ccagtcacta tggcggccgc attaggcacc ccaggcttta
     2641 cactttatgc ttccggctcg tataatgtgt ggattttgag ttaggatccg tcgagatttt
     2701 caggagctaa ggaagctaaa atggagaaaa aaatcactgg atataccacc gttgatatat
     2761 cccaatggca tcgtaaagaa cattttgagg catttcagtc agttgctcaa tgtacctata
     2821 accagaccgt tcagctggat attacggcct ttttaaagac cgtaaagaaa aataagcaca
     2881 agttttatcc ggcctttatt cacattcttg cccgcctgat gaatgctcat ccggaattcc
     2941 gtatggcaat gaaagacggt gagctggtga tatgggatag tgttcaccct tgttacaccg
     3001 ttttccatga gcaaactgaa acgttttcat cgctctggag tgaataccac gacgatttcc
     3061 ggcagtttct acacatatat tcgcaagatg tggcgtgtta cggtgaaaac ctggcctatt
     3121 tccctaaagg gtttattgag aatatgtttt tcgtctcagc caatccctgg gtgagtttca
     3181 ccagttttga tttaaacgtg gccaatatgg acaacttctt cgcccccgtt ttcaccatgg
     3241 gcaaatatta tacgcaaggc gacaaggtgc tgatgccgct ggcgattcag gttcatcatg
     3301 ccgtttgtga tggcttccat gtcggcagaa tgcttaatga attacaacag tactgcgatg
     3361 agtggcaggg cggggcgtaa acgcgtggat ccggcttact aaaagccaga taacagtatg
     3421 cgtatttgcg cgctgatttt tgcggtataa gaatatatac tgatatgtat acccgaagta
     3481 tgtcaaaaag aggtatgcta tgaagcagcg tattacagtg acagttgaca gcgacagcta
     3541 tcagttgctc aaggcatata tgatgtcaat atctccggtc tggtaagcac aaccatgcag
     3601 aatgaagccc gtcgtctgcg tgccgaacgc tggaaagcgg aaaatcagga agggatggct
     3661 gaggtcgccc ggtttattga aatgaacggc tcttttgctg acgagaacag gggctggtga
     3721 aatgcagttt aaggtttaca cctataaaag agagagccgt tatcgtctgt ttgtggatgt
     3781 acagagtgat attattgaca cgcccgggcg acggatggtg atccccctgg ccagtgcacg
     3841 tctgctgtca gataaagtct cccgtgaact ttacccggtg gtgcatatcg gggatgaaag
     3901 ctggcgcatg atgaccaccg atatggccag tgtgccggtc tccgttatcg gggaagaagt
     3961 ggctgatctc agccaccgcg aaaatgacat caaaaacgcc attaacctga tgttctgggg
     4021 aatataaatg tcaggctccc ttatacacag ccagtctgca ggtcgaccat agtgactgga
     4081 tatgttgtgt tttacagtat tatgtagtct gttttttatg caaaatctaa tttaatatat
     4141 tgatatttat atcattttac gtttctcgtt cagctttctt gtacaaagtg gtgtagtaag
     4201 aattcaaggc ctctcgagcc tctagaacta tagtgagtcg tattactaga gtccgcaaaa
     4261 atcaccagtc tctctctaca aatctatctc tctctatttt tctccagaat aatgtgtgag
     4321 tagttcccag ataagggaat tagggttctt atagggtttc gctcatgtgt tgagcatata
     4381 agaaaccctt agtatgtatt tgtatttgta aaatacttct atcaataaaa tttctaattc
     4441 ctaaaaccaa aatccagtga ccgggcggcc gccaccgcgg tggaggggga tcagattgtc
     4501 gtttcccgcc ttcagtttaa actatcagtg tttgacagga tatattggcg ggtaaaccta
     4561 agagaaaaga gcgtttatta gaataatcgg atatttaaaa gggcgtgaaa aggtttatcc
     4621 gttcgtccat ttgtatgtgt acatcaccga cgagcaaggc aagaccgagc gcctttccga
     4681 cgctcaccgg gctggttgcc ctcgccgctg ggctggcggc cgtctatggc cctgcaaacg
     4741 cgccagaaac gccgtcgaag ccgtgtgcga gacaccggcc gccggcgttg tggatacctc
     4801 gcggaaaact tggccctcac tgacagatga ggggcggacg ttgacacttg aggggccgac
     4861 tcacccggcg cggcgttgac agatgagggg caggctcgat ttcggccggc gacgtggagc
     4921 tggccagcct cgcaaatcgg cgaaaacgcc tgattttacg cgagtttccc acagatgatg
     4981 tggacaagcc tggggataag tgccctgcgg tattgacact tgaggggcgc gactactgac
     5041 agatgagggg cgcgatcctt gacacttgag gggcagagtg ctgacagatg aggggcgcac
     5101 ctattgacat ttgaggggct gtccacaggc agaaaatcca gcatttgcaa gggtttccgc
     5161 ccgtttttcg gccaccgcta acctgtcttt taacctgctt ttaaaccaat atttataaac
     5221 cttgttttta accagggctg cgccctgtgc gcgtgaccgc gcacgccgaa ggggggtgcc
     5281 cccccttctc gaaccctccc ggcccgctaa cgcgggcctc ccatcccccc aggggctgcg
     5341 cccctcggcc gcgaacggcc tcaccccaaa aatggcagcg ctggcagtcc ttgccattgc
     5401 cgggatcggg gcagtaacgg gatgggcgat cagcccgaaa gctaccccta tttgtttatt
     5461 tttctaaata cattcaaata tgtatccgct catgagacaa taaccctgat aaatgcttca
     5521 ataatattga aaaaggaaga gtatgagtat tcaacatttc cgtgtcgccc ttattccctt
     5581 ttttgcggca ttttgccttc ctgtttttgc tcacccagaa acgctggtga aagtaaaaga
     5641 tgctgaagat cagttgggtg cacgagtggg ttacatcgaa ctggatctca acagcggtaa
     5701 gatccttgag agttttcgcc ccgaagaacg ttttccaatg atgagcactt ttaaagttct
     5761 gctatgtggc gcggtattat cccgtattga cgccgggcaa gagcaactcg gtcgccgcat
     5821 acactattct cagaatgact tggttgagta ctcaccagtc acagaaaagc atcttacgga
     5881 tggcatgaca gtaagagaat tatgcagtgc tgccataacc atgagtgata acactgcggc
     5941 caacttactt ctgacaacga tcggaggacc gaaggagcta accgcttttt tgcacaacat
     6001 gggggatcat gtaactcgcc ttgatcgttg ggaaccggag ctgaatgaag ccataccaaa
     6061 cgacgagcgt gacaccacga tgcctgtagc aatggcaaca acgttgcgca aactattaac
     6121 tggcgaacta cttactctag cttcccggca acaattaata gactggatgg aggcggataa
     6181 agttgcagga ccacttctgc gctcggccct tccggctggc tggtttattg ctgataaatc
     6241 tggagccggt gagcgtgggt ctcgcggtat cattgcagca ctggggccag atggtaagcc
     6301 ctcccgtatc gtagttatct acacgacggg gagtcaggca actatggatg aacgaaatag
     6361 acagatcgct gagataggtg cctcactgat taagcattgg taactgtcag accaagttta
     6421 ctcatatata ctttagattg atttaaaact tcatttttaa tttaaaagga tctaggtgaa
     6481 gatccttttt gataatctca tgaccaaaat cccttaacgt gagttttcgt tccactgagc
     6541 gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat
     6601 ctgctgcttg caaacaaaaa aaccaccgct accagcggtg gtttgtttgc cggatcaaga
     6661 gctaccaact ctttttccga aggtaactgg cttcagcaga gcgcagatac caaatactgt
     6721 tcttctagtg tagccgtagt taggccacca cttcaagaac tctgtagcac cgcctacata
     6781 cctcgctctg ctaatcctgt taccagtggc tgctgccagt ggcgataagt cgtgtcttac
     6841 cgggttggac tcaagacgat agttaccgga taaggcgcag cggtcgggct gaacgggggg
     6901 ttcgtgcaca cagcccagct tggagcgaac gacctacacc gaactgagat acctacagcg
     6961 tgagctatga gaaagcgcca cgcttcccga agggagaaag gcggacaggt atccggtaag
     7021 cggcagggtc ggaacaggag agcgcacgag ggagcttcca gggggaaacg cctggtatct
     7081 ttatagtcct gtcgggtttc gccacctctg acttgagcgt cgatttttgt gatgctcgtc
     7141 aggggggcgg agcctatgga aaaacgccag caacgcggcc tttttacggt tcctggcctt
     7201 ttgctggcct tttgctcaca tggactctag ctagaggatc acaggcagca acgctctgtc
     7261 atcgttacaa tcaacatgct accctccgcg agatcatccg tgtttcaaac ccggcagctt
     7321 agttgccgtt cttccgaata gcatcggtaa catgagcaaa gtctgccgcc ttacaacggc
     7381 tctcccgctg acgccgtccc ggactgatgg gctgcctgta tcgagtggtg attttgtgcc
     7441 gagctgccgg tcggggagct gttggctggc tggt
//
