LOCUS       sequence-482852-        6714 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression plasmid for TEV-2xStrepII-tagged proteins with Hygromycin
            transgenic seed selection.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6714)
  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 6714)
  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_2xStrepII_Hyg
FEATURES             Location/Qualifiers
     source          1..6714
                     /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)"
     polyA_signal    655..831
                     /label=CaMV poly(A) signal
                     /note="cauliflower mosaic virus polyadenylation signal"
     CDS             complement(1063..2088)
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
                     YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
                     ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
                     TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
                     GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
                     NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
                     E"
     promoter        complement(2108..2453)
                     /label=CaMV 35S promoter
                     /note="strong constitutive promoter from cauliflower mosaic
                     virus"
     primer_bind     complement(2145..2165)
                     /label=35S promoter
                     /note="CaMV 35S promoter, forward primer"
     protein_bind    3258..3279
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(3294..3299,3300..3317,3318..3324)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3332..3348
                     /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             3366..3377
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             3384..4061
                     /codon_start=1
                     /product="monomeric derivative of DsRed (Campbell et al., 
                     2002)"
                     /label=mRFP1
                     /translation="MASSEDVIKEFMRFKVRMEGSVNGHEFEIEGEGEGRPYEGTQTAK
                     LKVTKGGPLPFAWDILSPQFQYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGV
                     VTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASTERMYPEDGALKGEIKM
                     RLKLKDGGHYDAEVKTTYMAKKPVQLPGAYKTDIKLDITSHNEDYTIVEQYERAEGRHS
                     TGA"
     terminator      4106..4177
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      4193..4220
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     CDS             4248..4268
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             join(4284..4289,4290..4313,4314..4349,4350..4373)
                     /codon_start=1
                     /product="two Strep-Tag(R)II moieties connected by a linker
                     for enhanced binding to Strep-Tactin(R), an engineered form
                     of streptavidin (Schmidt et al., 2013)"
                     /label=Twin-Strep-tag
                     /translation="SAWSHPQFEKGGGSGGGSGGSAWSHPQFEK"
     misc_feature    4797..4821
                     /label=RB T-DNA repeat
                     /note="right border repeat from nopaline C58 T-DNA"
     rep_origin      complement(4912..5500)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4992..5011)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(5671..6486)
                     /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(5671..6486)
                     /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     6394..6413
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     6478..6497
                     /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 tatcgatctg
      661 gattttagta ctggattttg gttttaggaa ttagaaattt tattgataga agtattttac
      721 aaatacaaat acatactaag ggtttcttat atgctcaaca catgagcgaa accctataag
      781 aaccctaatt tcccttatcg ggaaactact cacacattat ttatggagaa aatagagaga
      841 gatagatttg tagagagaga ctggtgattt cagcgtaccg aattaattcc tgaatcactg
      901 cgaccggccc tcccgcgacc cagccgagcg agcttagcga actgtggacg agaactgtgc
      961 caccaagcgt aaggccgttc tctcgcattt gccttgctag gctcgcgcga gttgctggct
     1021 gaggcgttct cgaaatcagc tcttgttcgg tcggcatcta ctctattcct ttgccctcgg
     1081 acgagtgctg gggcgtcggt ttccactatc ggcgagtact tctacacagc catcggtcca
     1141 gacggccgcg cttctgcggg cgatttgtgt acgcccgaca gtcccggctc cggatcggac
     1201 gattgcgtcg catcgaccct gcgcccaagc tgcatcatcg aaattgccgt caaccaagct
     1261 ctgatagagt tggtcaagac caatgcggag catatacgcc cggagccttg gcgatcctgc
     1321 aagctccgga tgcctccgct cgaagtagcg cgtctgctgc tccatacaag ccaaccacgg
     1381 cctccagaag aagatgttgg cgacctcgta ttgggaatcc ccgaacatcg cctcgctcca
     1441 gtcaatgacc gctgttatgc ggccattgtc cgtcaggaca ttgttggagc cgaaatccgc
     1501 gtgcacgagg tgccggactt cggggcagtc ctcggcccaa agcatcagct catcgagagc
     1561 ctgcgcgacg gacgcactga cggtgtcgtc catcacagtt tgccagtgat acacatgggg
     1621 atcagcaatc gcgcatatga aatcacgcca tgtagtgtat tgaccgattc cttgcggtcc
     1681 gaatgggccg aacccgctcg tctggctaag atcggccgca gcgatagcat ccatagcctc
     1741 cgcgaccggc tgaagaacag cgggcagttc ggtttcaggc aggtcttgca acgtgacacc
     1801 ctgtgcacgg cgggagatgc aataggtcag gctctcgctg aactccccaa tgtcaagcac
     1861 ttccggaatc gggagcgcgg ccgatgcaaa gtgccgataa acataacgat ctttgtagaa
     1921 accatcggcg cagctattta cccgcaggac atatccacgc cctcctacat cgaagctgaa
     1981 agcacgagat tcttcgccct ccgagagctg catcaggtcg gagacgctgt cgaacttttc
     2041 gatcagaaac ttctcgacag acgtcgcggt gagttcaggc tttttcatag gggggatcag
     2101 cttgggctgt cctctccaaa tgaaatgaac ttccttatat agaggaaggg tcttgcgaag
     2161 gatagtggga ttgtgcgtca tcccttacgt cagtggagat gtcacatcaa tccacttgct
     2221 ttgaagtcgt ggttggaacg acttcttttt ccacgatgct cctcgtgggt gggggtccat
     2281 ctttgggacc actgtcggca gaggcatctt gaatgatagc ctttccttta tcgcaatgat
     2341 ggcatttgta ggagccacct tccttttcta ctgtcctttc gatgaagtga cagatagctg
     2401 ggcaatggaa tccgaggagg tttcccgaaa ttaccctttg ttgaaaagtc tcaataaagc
     2461 ttcgacgagt cagtaataaa cggcgtcaaa gtggttgcag ccggcacaca cgagtcgtgt
     2521 ttatcaactc aaagcacaaa tacttttcct caacctaaaa ataaggcaat tagccaaaaa
     2581 caactttgcg tgtaaacaac gctcaataca cgtgtcattt tattattagc tattgcttca
     2641 ccgccttagc tttctcgtga cctagtcgtc ctcgtctttt cttcttcttc ttctataaaa
     2701 caatacccaa agacctcttc ttcttcacaa ttcagatttc aatttctcaa aatcttaaaa
     2761 actttctctc aattctctct accgtgatca aggtaaattt ctgtgttcct tattctctca
     2821 aaatcttcga ttttgttttc gttcgatccc aatttcgtat atgttctttg gtttagattc
     2881 tgttaatctt agatcgaagt cgattttctg ggtttgatcg ttagatatca tcttaattct
     2941 cgattagggt ttcatagata tcatccgatt tgttcaaata atttgagttt tgtcgaataa
     3001 ttactcttcg atttgtgatt tctatctaga tctggtgtta gtttctagtt tgtgcgatcg
     3061 aatttgtcga ttaatctgag tttttctgat taacagacta gagttaaccg ggctcaggcc
     3121 tggcgcgcca ctagtggatc cgatggtgtc ttcgattcaa tacgcaaacc gcctctcccc
     3181 gcgcgttggc cgattcatta atgcagctgg cacgacaggt ttcccgactg gaaagcgggc
     3241 agtgagcgca acgcaattaa tgtgagttag ctcactcatt aggcacccca ggctttacac
     3301 tttatgcttc cggctcgtat gttgtgtgga attgtgagcg gataacaatt tcacacatac
     3361 tagagaaaga ggagaaatac tagatggctt cctccgagga cgttatcaaa gagttcatgc
     3421 gtttcaaagt tcgtatggaa ggttccgtta acggtcacga gttcgaaatc gaaggtgaag
     3481 gtgaaggtcg tccgtacgaa ggtacccaga ccgctaaact gaaagttacc aaaggtggtc
     3541 cgctgccgtt cgcttgggac atcctgtccc cgcagttcca gtacggttcc aaggcttacg
     3601 ttaaacaccc ggctgacatc ccggactacc tgaaactgtc cttcccggaa ggtttcaaat
     3661 gggaacgtgt tatgaacttc gaggacggtg gtgttgttac cgttacccag gactcctccc
     3721 tgcaagacgg tgagttcatc tacaaagtta aactgcgtgg taccaacttc ccgtccgacg
     3781 gtccggttat gcagaaaaaa acgatgggtt gggaagcatc caccgaacgt atgtacccgg
     3841 aggacggtgc tctgaaaggt gaaatcaaaa tgcgtctgaa actgaaagac ggtggtcact
     3901 acgacgctga agttaaaacc acctacatgg ctaaaaaacc ggttcagctg ccgggtgctt
     3961 acaaaaccga catcaaactg gacatcacct cccacaacga ggactacacc atcgttgaac
     4021 agtacgaacg tgctgaaggt cgtcactcca ccggtgctta ataacgctga tagtgctagt
     4081 gtagatcgct actagagcca ggcatcaaat aaaacgaaag gctcagtcga aagactgggc
     4141 ctttcgtttt atctgttgtt tgtcggtgaa cgctctctac tagagtcaca ctggctcacc
     4201 ttcgggtggg cctttctgcg tttatagaag acactggacc cgggagtgag aacctctact
     4261 tccaaggctc catgggtggg tccagcgcat ggtcacatcc gcagtttgaa aaaggtggtg
     4321 gtagcggtgg tggttcaggt ggtagtgctt ggagccatcc tcagttcgag aaataggagc
     4381 tcatatgaag atgaagatga aatatttggt gtgtcaaata aatagcttgt gtgcttaagt
     4441 ttgtgttttt ttcttggctt gttgtgttat gaatttgtgg ctttttctaa tattaaatga
     4501 atgtaagatc tcattataat gaataaacaa atgtttctat aatccattgt gaatgttttg
     4561 ttggatctct tctgcagcat ataactactg tatgtgctat ggtatggact atggaatatg
     4621 attaaagata aggaattcag atcggctgag tggctccttc aacgttgcgg ttctgtcagt
     4681 tccaaacgta aaacggcttg tcccgcgtca tcggcggggg tcataacgtg actcccttaa
     4741 ttctccgctc atgatcagat tgtcgtttcc cgccttcagt ttaaactatc agtgtttgac
     4801 aggatatatt ggcgggtaaa ctaagtcgct gtatgtgttt gtttgagatc tcatgtgagc
     4861 aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag
     4921 gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc
     4981 gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt
     5041 tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct
     5101 ttctcatagc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg
     5161 ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta actatcgtct
     5221 tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg gtaacaggat
     5281 tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggc ctaactacgg
     5341 ctacactaga agaacagtat ttggtatctg cgctctgctg aagccagtta ccttcggaag
     5401 aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt
     5461 ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc
     5521 tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt
     5581 atcaaaaagg atcttcacct agatcctttt aaattaaaaa tgaagtttta aatcaatcta
     5641 aagtatatat gtgtaacatt ggtctagtga ttagaaaaac tcatcgagca tcaaatgaaa
     5701 ctgcaattta ttcatatcag gattatcaat accatatttt tgaaaaagcc gtttctgtaa
     5761 tgaaggagaa aactcaccga ggcagttcca taggatggca agatcctggt atcggtctgc
     5821 gattccgact cgtccaacat caatacaacc tattaatttc ccctcgtcaa aaataaggtt
     5881 atcaagtgag aaatcaccat gagtgacgac tgaatccggt gagaatggca aaagtttatg
     5941 catttctttc cagacttgtt caacaggcca gccattacgc tcgtcatcaa aatcactcgc
     6001 atcaaccaaa ccgttattca ttcgtgattg cgcctgagcg agacgaaata cgcgatcgct
     6061 gttaaaagga caattacaaa caggaatcga atgcaaccgg cgcaggaaca ctgccagcgc
     6121 atcaacaata ttttcacctg aatcaggata ttcttctaat acctggaatg ctgttttccc
     6181 tgggatcgca gtggtgagta accatgcatc atcaggagta cggataaaat gcttgatggt
     6241 cggaagaggc ataaattccg tcagccagtt tagtctgacc atctcatctg taacatcatt
     6301 ggcaacgcta cctttgccat gtttcagaaa caactctggc gcatcgggct tcccatacaa
     6361 tcggtagatt gtcgcacctg attgcccgac attatcgcga gcccatttat acccatataa
     6421 atcagcatcc atgttggaat ttaatcgcgg ccttgagcaa gacgtttccc gttgaatatg
     6481 gctcataaca ccccttgtat tactgtttat gtaagcagac agttttattg ttcatgatga
     6541 tatattttta tcttgtgcaa tgtaacatca gagattttga gacacaacgt ggctttgttg
     6601 aataaatcga acttttgctg agttgaagga tcagatcacg catcttcccg acaacgcaga
     6661 ccgttccgtg gcaaagcaaa agttcaaaat caccaactgg tccacctaca acaa
//
