LOCUS       sequence-482857-        7163 bp    DNA     circular SYN 25-SEP-2025
DEFINITION  Expression plasmid for TEV-Flag-tagged proteins with FAST-Red 
            transgenic seed selection.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7163)
  TITLE     Kunz Lab Plant Expression Plasmids
REFERENCE   2  (bases 1 to 7163)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Sep 25, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"
COMMENT     Sequence Label: pG20_TEV_Flag_FR
FEATURES             Location/Qualifiers
     source          1..7163
                     /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(1097..1792)
                     /codon_start=1
                     /product="monomeric far-red fluorescent protein (Shcherbo 
                     et al., 2009)"
                     /label=mKate2
                     /note="mammalian codon-optimized"
                     /translation="MSELIKENMHMKLYMEGTVNNHHFKCTSEGEGKPYEGTQTMRIKV
                     VEGGPLPFAFDILATSFMYGSRTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLTA
                     TQDTSLQDGCLIYNVKIRGVNFPSNGPVMQKKTLGWEANTEMLYPADGGLEGRSDMALK
                     LVGGGHLICNFKTTYRSKKPAKNLKMPGVYYVDHRLERIKEADKETYVEQHEVAVARYC
                     DLPSKLGHK"
     protein_bind    3770..3791
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(3806..3811,3812..3829,3830..3836)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3844..3860
                     /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             3878..3889
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             3896..4573
                     /codon_start=1
                     /product="monomeric derivative of DsRed (Campbell et al., 
                     2002)"
                     /label=mRFP1
                     /translation="MASSEDVIKEFMRFKVRMEGSVNGHEFEIEGEGEGRPYEGTQTAK
                     LKVTKGGPLPFAWDILSPQFQYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGV
                     VTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASTERMYPEDGALKGEIKM
                     RLKLKDGGHYDAEVKTTYMAKKPVQLPGAYKTDIKLDITSHNEDYTIVEQYERAEGRHS
                     TGA"
     terminator      4618..4689
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      4705..4732
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     CDS             4760..4780
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             4799..4822
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     misc_feature    5246..5270
                     /label=RB T-DNA repeat
                     /note="right border repeat from nopaline C58 T-DNA"
     rep_origin      complement(5361..5949)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5441..5460)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(6120..6935)
                     /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(6120..6935)
                     /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     6843..6862
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     6927..6946
                     /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 ttgtgcccca gtttgctagg gaggtcgcag tatctggcca
     1141 cagccacctc gtgctgctcg acgtaggttt ctttgtcggc ctccttgatt ctttccagtc
     1201 tgtggtccac atagtagacg ccgggcatct tgaggttctt agcgggtttc ttggatctgt
     1261 atgtggtctt gaagttgcag atcaggtggc ccccgcccac gagcttcagg gccatgtcgc
     1321 ttctgccttc caggccgccg tcagcggggt acagcatctc ggtgttggcc tcccagccga
     1381 gtgttttctt ctgcatcaca gggccgttgg atgggaagtt cacccctctg atcttgacgt
     1441 tgtagatgag gcagccgtcc tggaggctgg tgtcctgggt agcggtcagc acgcccccat
     1501 cttcgtatgt ggtgactctc tcccatgtga agccctcagg gaaggactgc ttaaagaagt
     1561 cggggatgcc ctgggtgtgg ttgatgaagg ttctgctgcc gtacatgaag ctggtagcca
     1621 ggatgtcgaa ggcgaagggg agagggccgc cctcgaccac cttgattctc atggtctggg
     1681 tgccctcgta gggcttgcct tcgccctcgg atgtgcactt gaagtggtgg ttgttcacgg
     1741 tgccctccat gtacagcttc atgtgcatgt tctccttaat cagctcgctc atagtagtgt
     1801 gctggccacc acgagtacgg tcacggtcat gttccccacc agtatgttgc tgtccgtagt
     1861 actgagctct gtctttcaga tcctgagctt tgcttcccaa cttcatcctt gcactgtcca
     1921 acttgtctga tccctgtggg tgctctcccg ttgcgtacct attcacaatc aataggtatc
     1981 ataccaagca ttttacctat atagatgata tacacagaca cattcaagct agctaggtca
     2041 aatttcgtat ttagttaacc aaatgttccc tagaatttgc aatttcttat tgttacattt
     2101 gttattcgac caaaaaaatt gatccaaagc tactggctca acacatgcat gcacatattg
     2161 cacaaaatta tgaagtaaga tgataaatgt gtgcttactt gtaaatccaa gagaaaacgg
     2221 ttatagcggc aatgccaaac cctccagagg aaagaaaacc ggtgatgagg agtgcaactg
     2281 tgatgagagc cgggacaagg attgggctga agataacgag cagaggtgtt gcaacagtca
     2341 aagctatgac agttccaaca agggtaaggc tggagagaac aaggagggaa ccaccagctg
     2401 tgacagcagt tgcagcttta gcaatctgcc tagacttgga gtagtcagat cctcgtccgg
     2461 acatctggta ctggtcacga tctcggccca tcatcgggta ctgatctctg ccgatgatat
     2521 cgtgatgggt tcctctagct gtatccgcca tttttttgtt cttgtttact agagagaatg
     2581 agattttgaa tgagagattg atcaaaaaga tgtgtatata ttttggaaga ggtaggagtg
     2641 tttgttagat atattaagaa aggagacgag aagtcacgtg tcaatggaga catcatgcaa
     2701 ttgtaggcca cgtgttaatg gcgtcaccat gcagccgctg catgtgttaa cgagaggagt
     2761 ctattcaaag acacgtttgt tttttgccta gctcagaatg tggcgttggc atcgaaccga
     2821 aacataaaac gcaaaacgaa cctttgggct cgtcagtatg ggtttgggcc ttcaatagat
     2881 tccttctata cggcccacat atccactatt ggcaacaaaa ttagaaaaat ttaggattta
     2941 gttttaaaat ttgttccgcg acattataaa gcttcgacga gtcagtaata aacggcgtca
     3001 aagtggttgc agccggcaca cacgagtcgt gtttatcaac tcaaagcaca aatacttttc
     3061 ctcaacctaa aaataaggca attagccaaa aacaactttg cgtgtaaaca acgctcaata
     3121 cacgtgtcat tttattatta gctattgctt caccgcctta gctttctcgt gacctagtcg
     3181 tcctcgtctt ttcttcttct tcttctataa aacaataccc aaagacctct tcttcttcac
     3241 aattcagatt tcaatttctc aaaatcttaa aaactttctc tcaattctct ctaccgtgat
     3301 caaggtaaat ttctgtgttc cttattctct caaaatcttc gattttgttt tcgttcgatc
     3361 ccaatttcgt atatgttctt tggtttagat tctgttaatc ttagatcgaa gtcgattttc
     3421 tgggtttgat cgttagatat catcttaatt ctcgattagg gtttcataga tatcatccga
     3481 tttgttcaaa taatttgagt tttgtcgaat aattactctt cgatttgtga tttctatcta
     3541 gatctggtgt tagtttctag tttgtgcgat cgaatttgtc gattaatctg agtttttctg
     3601 attaacagac tagagttaac cgggctcagg cctggcgcgc cactagtgga tccgatggtg
     3661 tcttcgattc aatacgcaaa ccgcctctcc ccgcgcgttg gccgattcat taatgcagct
     3721 ggcacgacag gtttcccgac tggaaagcgg gcagtgagcg caacgcaatt aatgtgagtt
     3781 agctcactca ttaggcaccc caggctttac actttatgct tccggctcgt atgttgtgtg
     3841 gaattgtgag cggataacaa tttcacacat actagagaaa gaggagaaat actagatggc
     3901 ttcctccgag gacgttatca aagagttcat gcgtttcaaa gttcgtatgg aaggttccgt
     3961 taacggtcac gagttcgaaa tcgaaggtga aggtgaaggt cgtccgtacg aaggtaccca
     4021 gaccgctaaa ctgaaagtta ccaaaggtgg tccgctgccg ttcgcttggg acatcctgtc
     4081 cccgcagttc cagtacggtt ccaaggctta cgttaaacac ccggctgaca tcccggacta
     4141 cctgaaactg tccttcccgg aaggtttcaa atgggaacgt gttatgaact tcgaggacgg
     4201 tggtgttgtt accgttaccc aggactcctc cctgcaagac ggtgagttca tctacaaagt
     4261 taaactgcgt ggtaccaact tcccgtccga cggtccggtt atgcagaaaa aaacgatggg
     4321 ttgggaagca tccaccgaac gtatgtaccc ggaggacggt gctctgaaag gtgaaatcaa
     4381 aatgcgtctg aaactgaaag acggtggtca ctacgacgct gaagttaaaa ccacctacat
     4441 ggctaaaaaa ccggttcagc tgccgggtgc ttacaaaacc gacatcaaac tggacatcac
     4501 ctcccacaac gaggactaca ccatcgttga acagtacgaa cgtgctgaag gtcgtcactc
     4561 caccggtgct taataacgct gatagtgcta gtgtagatcg ctactagagc caggcatcaa
     4621 ataaaacgaa aggctcagtc gaaagactgg gcctttcgtt ttatctgttg tttgtcggtg
     4681 aacgctctct actagagtca cactggctca ccttcgggtg ggcctttctg cgtttataga
     4741 agacactgga cccgggagtg agaacctcta cttccaaggc tccatgggcg gaggatctga
     4801 ctacaaggac gacgatgaca agtaggagct catatgaaga tgaagatgaa atatttggtg
     4861 tgtcaaataa atagcttgtg tgcttaagtt tgtgtttttt tcttggcttg ttgtgttatg
     4921 aatttgtggc tttttctaat attaaatgaa tgtaagatct cattataatg aataaacaaa
     4981 tgtttctata atccattgtg aatgttttgt tggatctctt ctgcagcata taactactgt
     5041 atgtgctatg gtatggacta tggaatatga ttaaagataa ggaattcaga tcggctgagt
     5101 ggctccttca acgttgcggt tctgtcagtt ccaaacgtaa aacggcttgt cccgcgtcat
     5161 cggcgggggt cataacgtga ctcccttaat tctccgctca tgatcagatt gtcgtttccc
     5221 gccttcagtt taaactatca gtgtttgaca ggatatattg gcgggtaaac taagtcgctg
     5281 tatgtgtttg tttgagatct catgtgagca aaaggccagc aaaaggccag gaaccgtaaa
     5341 aaggccgcgt tgctggcgtt tttccatagg ctccgccccc ctgacgagca tcacaaaaat
     5401 cgacgctcaa gtcagaggtg gcgaaacccg acaggactat aaagatacca ggcgtttccc
     5461 cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc
     5521 gcctttctcc cttcgggaag cgtggcgctt tctcatagct cacgctgtag gtatctcagt
     5581 tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt tcagcccgac
     5641 cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc cggtaagaca cgacttatcg
     5701 ccactggcag cagccactgg taacaggatt agcagagcga ggtatgtagg cggtgctaca
     5761 gagttcttga agtggtggcc taactacggc tacactagaa gaacagtatt tggtatctgc
     5821 gctctgctga agccagttac cttcggaaga agagttggta gctcttgatc cggcaaacaa
     5881 accaccgctg gtagcggtgg tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa
     5941 ggatctcaag aagatccttt gatcttttct acggggtctg acgctcagtg gaacgaaaac
     6001 tcacgttaag ggattttggt catgagatta tcaaaaagga tcttcaccta gatcctttta
     6061 aattaaaaat gaagttttaa atcaatctaa agtatatatg tgtaacattg gtctagtgat
     6121 tagaaaaact catcgagcat caaatgaaac tgcaatttat tcatatcagg attatcaata
     6181 ccatattttt gaaaaagccg tttctgtaat gaaggagaaa actcaccgag gcagttccat
     6241 aggatggcaa gatcctggta tcggtctgcg attccgactc gtccaacatc aatacaacct
     6301 attaatttcc cctcgtcaaa aataaggtta tcaagtgaga aatcaccatg agtgacgact
     6361 gaatccggtg agaatggcaa aagtttatgc atttctttcc agacttgttc aacaggccag
     6421 ccattacgct cgtcatcaaa atcactcgca tcaaccaaac cgttattcat tcgtgattgc
     6481 gcctgagcga gacgaaatac gcgatcgctg ttaaaaggac aattacaaac aggaatcgaa
     6541 tgcaaccggc gcaggaacac tgccagcgca tcaacaatat tttcacctga atcaggatat
     6601 tcttctaata cctggaatgc tgttttccct gggatcgcag tggtgagtaa ccatgcatca
     6661 tcaggagtac ggataaaatg cttgatggtc ggaagaggca taaattccgt cagccagttt
     6721 agtctgacca tctcatctgt aacatcattg gcaacgctac ctttgccatg tttcagaaac
     6781 aactctggcg catcgggctt cccatacaat cggtagattg tcgcacctga ttgcccgaca
     6841 ttatcgcgag cccatttata cccatataaa tcagcatcca tgttggaatt taatcgcggc
     6901 cttgagcaag acgtttcccg ttgaatatgg ctcataacac cccttgtatt actgtttatg
     6961 taagcagaca gttttattgt tcatgatgat atatttttat cttgtgcaat gtaacatcag
     7021 agattttgag acacaacgtg gctttgttga ataaatcgaa cttttgctga gttgaaggat
     7081 cagatcacgc atcttcccga caacgcagac cgttccgtgg caaagcaaaa gttcaaaatc
     7141 accaactggt ccacctacaa caa
//
