LOCUS       sequence-482851-        7187 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression plasmid for TEV-Flag-tagged proteins with FAST-Green 
            transgenic seed selection.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7187)
  AUTHORS   Schwartz S, Engstler C, Muhlbauer S, Windenbach E, Wunder T, Kunz 
            HH, Brandt B
  TITLE     
  JOURNAL   Plant J. 2025 Nov;124(3):e70544. doi: 10.1111/tpj.70544.
  PUBMED    41222448
REFERENCE   2  (bases 1 to 7187)
  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: "Plant J."; 
            date: "2025-11"; volume: "124(3):e70544. doi"; pages: " 
            10.1111/tpj.70544"
COMMENT     Sequence Label: pG20_TEV_Flag_FG
FEATURES             Location/Qualifiers
     source          1..7187
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      63..498
                     /label=pSa ori
                     /note="origin of replication from bacterial plasmid pSa"
     misc_feature    625..647
                     /label=LB T-DNA repeat
                     /note="left border repeat from nopaline C58 T-DNA 
                     (truncated)"
     CDS             complement(join(1097..1810,1811..1813,1814..1816))
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             complement(join(1100..1810,1811..1813,1814..1816))
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(1142..1163)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     1491..1510
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1750..1771
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     protein_bind    3794..3815
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(3830..3835,3836..3853,3854..3860)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3868..3884
                     /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)."
     RBS             3902..3913
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             3920..4597
                     /codon_start=1
                     /product="monomeric derivative of DsRed (Campbell et al., 
                     2002)"
                     /label=mRFP1
                     /translation="MASSEDVIKEFMRFKVRMEGSVNGHEFEIEGEGEGRPYEGTQTAK
                     LKVTKGGPLPFAWDILSPQFQYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGV
                     VTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASTERMYPEDGALKGEIKM
                     RLKLKDGGHYDAEVKTTYMAKKPVQLPGAYKTDIKLDITSHNEDYTIVEQYERAEGRHS
                     TGA"
     terminator      4642..4713
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      4729..4756
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     CDS             4784..4804
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             4823..4846
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     misc_feature    5270..5294
                     /label=RB T-DNA repeat
                     /note="right border repeat from nopaline C58 T-DNA"
     rep_origin      complement(5385..5973)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5465..5484)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(6144..6959)
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     CDS             complement(6144..6959)
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     6867..6886
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     6951..6970
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
ORIGIN
        1 agctctcatc aaccgtggct ccctcacttt ctggctggat gatggggcga ttcaggcgat
       61 ccccatccaa cagcccgccg tcgagcgggc ttttttatcc ccggaagcct gtggatagag
      121 ggtagttatc cacgtgaaac cgctaatgcc ccgcaaagcc ttgattcacg gggctttccg
      181 gcccgctcca aaaactatcc acgtgaaatc gctaatcagg gtacgtgaaa tcgctaatcg
      241 gagtacgtga aatcgctaat aaggtcacgt gaaatcgcta atcaaaaagg cacgtgagaa
      301 cgctaatagc cctttcagat caacagcttg caaacacccc tcgctccggc aagtagttac
      361 agcaagtagt atgttcaatt agcttttcaa ttatgaatat atatatcaat tattggtcgc
      421 ccttggcttg tggacaatgc gctacgcgca ccggctccgc ccgtggacaa ccgcaagcgg
      481 ttgcccaccg tcgagcgcct ttgcccacaa cccggcggcc ggccgcaaca gatcgtttta
      541 taaatttttt tttttgaaaa agaaaaagcc cgaaaggcgg caacctctcg ggcttctgga
      601 tttccgatcc ccggaattag atcttggcag gatatattgt ggtgtaacgt tatcactaaa
      661 tggagcaacc tactgttttt gaagtttggg ttgccatgta atcacttgca ataaatatat
      721 gtactaatga ctgcatcggc ttgcgtgtct aaatttgtag cttattgaaa actttttgtg
      781 gttaaagacg aatccagtta actaacataa gtttaacaaa acaagacctt ttccatatat
      841 agaccaccgt taccaactta caaagaagcc aaataaagta caacacaaca cttgcaagtt
      901 acaaaaagag aggaaaaaaa acatggtacc atttgacaac atatagaaac acacataaac
      961 attacttata acaaaaagat tcaaactcgg cttttattat ctccccctga tcaaattcta
     1021 aacactttat tcctcataaa ctaactcccc gacattggag ttattggact atgacgatga
     1081 catcagtggg gtaagctcac ttgtacagct cgtccatgcc gagagtgatc ccggcggcgg
     1141 tcacgaactc cagcaggacc atgtgatcgc gcttctcgtt ggggtctttg ctcagggcgg
     1201 actgggtgct caggtagtgg ttgtcgggca gcagcacggg gccgtcgccg atgggggtgt
     1261 tctgctggta gtggtcggcg agctgcacgc tgccgtcctc gatgttgtgg cggatcttga
     1321 agttcacctt gatgccgttc ttctgcttgt cggccatgat atagacgttg tggctgttgt
     1381 agttgtactc cagcttgtgc cccaggatgt tgccgtcctc cttgaagtcg atgcccttca
     1441 gctcgatgcg gttcaccagg gtgtcgccct cgaacttcac ctcggcgcgg gtcttgtagt
     1501 tgccgtcgtc cttgaagaag atggtgcgct cctggacgta gccttcgggc atggcggact
     1561 tgaagaagtc gtgctgcttc atgtggtcgg ggtagcggct gaagcactgc acgccgtagg
     1621 tcagggtggt cacgagggtg ggccagggca cgggcagctt gccggtggtg cagatgaact
     1681 tcagggtcag cttgccgtag gtggcatcgc cctcgccctc gccggacacg ctgaacttgt
     1741 ggccgtttac gtcgccgtcc agctcgacca ggatgggcac caccccggtg aacagctcct
     1801 cgcccttgct caccatagta gtgtgctggc caccacgagt acggtcacgg tcatgttccc
     1861 caccagtatg ttgctgtccg tagtactgag ctctgtcttt cagatcctga gctttgcttc
     1921 ccaacttcat ccttgcactg tccaacttgt ctgatccctg tgggtgctct cccgttgcgt
     1981 acctattcac aatcaatagg tatcatacca agcattttac ctatatagat gatatacaca
     2041 gacacattca agctagctag gtcaaatttc gtatttagtt aaccaaatgt tccctagaat
     2101 ttgcaatttc ttattgttac atttgttatt cgaccaaaaa aattgatcca aagctactgg
     2161 ctcaacacat gcatgcacat attgcacaaa attatgaagt aagatgataa atgtgtgctt
     2221 acttgtaaat ccaagagaaa acggttatag cggcaatgcc aaaccctcca gaggaaagaa
     2281 aaccggtgat gaggagtgca actgtgatga gagccgggac aaggattggg ctgaagataa
     2341 cgagcagagg tgttgcaaca gtcaaagcta tgacagttcc aacaagggta aggctggaga
     2401 gaacaaggag ggaaccacca gctgtgacag cagttgcagc tttagcaatc tgcctagact
     2461 tggagtagtc agatcctcgt ccggacatct ggtactggtc acgatctcgg cccatcatcg
     2521 ggtactgatc tctgccgatg atatcgtgat gggttcctct agctgtatcc gccatttttt
     2581 tgttcttgtt tactagagag aatgagattt tgaatgagag attgatcaaa aagatgtgta
     2641 tatattttgg aagaggtagg agtgtttgtt agatatatta agaaaggaga cgagaagtca
     2701 cgtgtcaatg gagacatcat gcaattgtag gccacgtgtt aatggcgtca ccatgcagcc
     2761 gctgcatgtg ttaacgagag gagtctattc aaagacacgt ttgttttttg cctagctcag
     2821 aatgtggcgt tggcatcgaa ccgaaacata aaacgcaaaa cgaacctttg ggctcgtcag
     2881 tatgggtttg ggccttcaat agattccttc tatacggccc acatatccac tattggcaac
     2941 aaaattagaa aaatttagga tttagtttta aaatttgttc cgcgacatta taaagcttcg
     3001 acgagtcagt aataaacggc gtcaaagtgg ttgcagccgg cacacacgag tcgtgtttat
     3061 caactcaaag cacaaatact tttcctcaac ctaaaaataa ggcaattagc caaaaacaac
     3121 tttgcgtgta aacaacgctc aatacacgtg tcattttatt attagctatt gcttcaccgc
     3181 cttagctttc tcgtgaccta gtcgtcctcg tcttttcttc ttcttcttct ataaaacaat
     3241 acccaaagac ctcttcttct tcacaattca gatttcaatt tctcaaaatc ttaaaaactt
     3301 tctctcaatt ctctctaccg tgatcaaggt aaatttctgt gttccttatt ctctcaaaat
     3361 cttcgatttt gttttcgttc gatcccaatt tcgtatatgt tctttggttt agattctgtt
     3421 aatcttagat cgaagtcgat tttctgggtt tgatcgttag atatcatctt aattctcgat
     3481 tagggtttca tagatatcat ccgatttgtt caaataattt gagttttgtc gaataattac
     3541 tcttcgattt gtgatttcta tctagatctg gtgttagttt ctagtttgtg cgatcgaatt
     3601 tgtcgattaa tctgagtttt tctgattaac agactagagt taaccgggct caggcctggc
     3661 gcgccactag tggatccgat ggtgtcttcg attcaatacg caaaccgcct ctccccgcgc
     3721 gttggccgat tcattaatgc agctggcacg acaggtttcc cgactggaaa gcgggcagtg
     3781 agcgcaacgc aattaatgtg agttagctca ctcattaggc accccaggct ttacacttta
     3841 tgcttccggc tcgtatgttg tgtggaattg tgagcggata acaatttcac acatactaga
     3901 gaaagaggag aaatactaga tggcttcctc cgaggacgtt atcaaagagt tcatgcgttt
     3961 caaagttcgt atggaaggtt ccgttaacgg tcacgagttc gaaatcgaag gtgaaggtga
     4021 aggtcgtccg tacgaaggta cccagaccgc taaactgaaa gttaccaaag gtggtccgct
     4081 gccgttcgct tgggacatcc tgtccccgca gttccagtac ggttccaagg cttacgttaa
     4141 acacccggct gacatcccgg actacctgaa actgtccttc ccggaaggtt tcaaatggga
     4201 acgtgttatg aacttcgagg acggtggtgt tgttaccgtt acccaggact cctccctgca
     4261 agacggtgag ttcatctaca aagttaaact gcgtggtacc aacttcccgt ccgacggtcc
     4321 ggttatgcag aaaaaaacga tgggttggga agcatccacc gaacgtatgt acccggagga
     4381 cggtgctctg aaaggtgaaa tcaaaatgcg tctgaaactg aaagacggtg gtcactacga
     4441 cgctgaagtt aaaaccacct acatggctaa aaaaccggtt cagctgccgg gtgcttacaa
     4501 aaccgacatc aaactggaca tcacctccca caacgaggac tacaccatcg ttgaacagta
     4561 cgaacgtgct gaaggtcgtc actccaccgg tgcttaataa cgctgatagt gctagtgtag
     4621 atcgctacta gagccaggca tcaaataaaa cgaaaggctc agtcgaaaga ctgggccttt
     4681 cgttttatct gttgtttgtc ggtgaacgct ctctactaga gtcacactgg ctcaccttcg
     4741 ggtgggcctt tctgcgttta tagaagacac tggacccggg agtgagaacc tctacttcca
     4801 aggctccatg ggcggaggat ctgactacaa ggacgacgat gacaagtagg agctcatatg
     4861 aagatgaaga tgaaatattt ggtgtgtcaa ataaatagct tgtgtgctta agtttgtgtt
     4921 tttttcttgg cttgttgtgt tatgaatttg tggctttttc taatattaaa tgaatgtaag
     4981 atctcattat aatgaataaa caaatgtttc tataatccat tgtgaatgtt ttgttggatc
     5041 tcttctgcag catataacta ctgtatgtgc tatggtatgg actatggaat atgattaaag
     5101 ataaggaatt cagatcggct gagtggctcc ttcaacgttg cggttctgtc agttccaaac
     5161 gtaaaacggc ttgtcccgcg tcatcggcgg gggtcataac gtgactccct taattctccg
     5221 ctcatgatca gattgtcgtt tcccgccttc agtttaaact atcagtgttt gacaggatat
     5281 attggcgggt aaactaagtc gctgtatgtg tttgtttgag atctcatgtg agcaaaaggc
     5341 cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca taggctccgc
     5401 ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga
     5461 ctataaagat accaggcgtt tccccctgga agctccctcg tgcgctctcc tgttccgacc
     5521 ctgccgctta ccggatacct gtccgccttt ctcccttcgg gaagcgtggc gctttctcat
     5581 agctcacgct gtaggtatct cagttcggtg taggtcgttc gctccaagct gggctgtgtg
     5641 cacgaacccc ccgttcagcc cgaccgctgc gccttatccg gtaactatcg tcttgagtcc
     5701 aacccggtaa gacacgactt atcgccactg gcagcagcca ctggtaacag gattagcaga
     5761 gcgaggtatg taggcggtgc tacagagttc ttgaagtggt ggcctaacta cggctacact
     5821 agaagaacag tatttggtat ctgcgctctg ctgaagccag ttaccttcgg aagaagagtt
     5881 ggtagctctt gatccggcaa acaaaccacc gctggtagcg gtggtttttt tgtttgcaag
     5941 cagcagatta cgcgcagaaa aaaaggatct caagaagatc ctttgatctt ttctacgggg
     6001 tctgacgctc agtggaacga aaactcacgt taagggattt tggtcatgag attatcaaaa
     6061 aggatcttca cctagatcct tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata
     6121 tatgtgtaac attggtctag tgattagaaa aactcatcga gcatcaaatg aaactgcaat
     6181 ttattcatat caggattatc aataccatat ttttgaaaaa gccgtttctg taatgaagga
     6241 gaaaactcac cgaggcagtt ccataggatg gcaagatcct ggtatcggtc tgcgattccg
     6301 actcgtccaa catcaataca acctattaat ttcccctcgt caaaaataag gttatcaagt
     6361 gagaaatcac catgagtgac gactgaatcc ggtgagaatg gcaaaagttt atgcatttct
     6421 ttccagactt gttcaacagg ccagccatta cgctcgtcat caaaatcact cgcatcaacc
     6481 aaaccgttat tcattcgtga ttgcgcctga gcgagacgaa atacgcgatc gctgttaaaa
     6541 ggacaattac aaacaggaat cgaatgcaac cggcgcagga acactgccag cgcatcaaca
     6601 atattttcac ctgaatcagg atattcttct aatacctgga atgctgtttt ccctgggatc
     6661 gcagtggtga gtaaccatgc atcatcagga gtacggataa aatgcttgat ggtcggaaga
     6721 ggcataaatt ccgtcagcca gtttagtctg accatctcat ctgtaacatc attggcaacg
     6781 ctacctttgc catgtttcag aaacaactct ggcgcatcgg gcttcccata caatcggtag
     6841 attgtcgcac ctgattgccc gacattatcg cgagcccatt tatacccata taaatcagca
     6901 tccatgttgg aatttaatcg cggccttgag caagacgttt cccgttgaat atggctcata
     6961 acaccccttg tattactgtt tatgtaagca gacagtttta ttgttcatga tgatatattt
     7021 ttatcttgtg caatgtaaca tcagagattt tgagacacaa cgtggctttg ttgaataaat
     7081 cgaacttttg ctgagttgaa ggatcagatc acgcatcttc ccgacaacgc agaccgttcc
     7141 gtggcaaagc aaaagttcaa aatcaccaac tggtccacct acaacaa
//
