LOCUS       sequence-482853-        8073 bp    DNA     circular SYN 20-AUG-2025
DEFINITION  Expression plasmid for 3C-mCherry-TEV-mVenus-tagged proteins with 
            Hygromycin transgenic seed selection.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8073)
  TITLE     Kunz Lab Plant Expression Plasmids
REFERENCE   2  (bases 1 to 8073)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Aug 20, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"
COMMENT     Sequence Label: pG20_3C_mCherry_TEV_mVenus_Hyg
FEATURES             Location/Qualifiers
     source          1..8073
                     /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..4271
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     CDS             4278..4982
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="VSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEGT
                     QTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFE
                     DGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALKG
                     EIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERAE
                     GRHSTGGMDELYK"
     primer_bind     complement(4420..4438)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     CDS             4992..5012
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             join(5016..5018,5019..5021,5022..5735)
                     /codon_start=1
                     /product="yellow fluorescent protein (YFP) with fast and 
                     efficient maturation (Nagai et al., 2002)"
                     /label=Venus
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KLICTTGKLPVPWPTLVTTLGYGLQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYITADKQKNGIK
                     ANFKIRHNIEDGGVQLADHYQQNTPIGDGPVLLPDNHYLSYQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(5061..5082)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(5322..5341)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     5669..5690
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     misc_feature    6156..6180
                     /label=RB T-DNA repeat
                     /note="right border repeat from nopaline C58 T-DNA"
     rep_origin      complement(6271..6859)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6351..6370)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(7030..7845)
                     /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(7030..7845)
                     /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     7753..7772
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     7837..7856
                     /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 cgggagtctg gaagttctgt
     4261 tccaggggcc ctccggggtg agcaagggcg aggaggataa catggccatc atcaaggagt
     4321 tcatgcgctt caaggtgcac atggagggct ccgtgaacgg ccacgagttc gagatcgagg
     4381 gcgagggcga gggccgcccc tacgagggca cccagaccgc caagctgaag gtgaccaagg
     4441 gtggccccct gcccttcgcc tgggacatcc tgtcccctca gttcatgtac ggctccaagg
     4501 cctacgtgaa gcaccccgcc gacatccccg actacttgaa gctgtccttc cccgagggct
     4561 tcaagtggga gcgcgtgatg aacttcgagg acggcggcgt ggtgaccgtg acccaggact
     4621 cctccctgca ggacggcgag ttcatctaca aggtgaagct gcgcggcacc aacttcccct
     4681 ccgacggccc cgtaatgcag aagaaaacga tgggctggga ggcctcctcc gagcggatgt
     4741 accccgagga cggcgccctg aagggcgaga tcaagcagag gctgaagctg aaggacggcg
     4801 gccactacga cgctgaggtc aagaccacct acaaggccaa gaagcccgtg cagctgcccg
     4861 gcgcctacaa cgtcaacatc aagttggaca tcacctccca caacgaggac tacaccatcg
     4921 tggaacagta cgaacgcgcc gagggccgcc actccaccgg cggcatggac gagctgtaca
     4981 agtccgggag tgagaacctc tacttccaag gctccatggt gagcaagggc gaggagctgt
     5041 tcaccggggt ggtgcccatc ctggtcgagc tggacggcga cgtaaacggc cacaagttca
     5101 gcgtgtccgg cgagggcgag ggcgatgcca cctacggcaa gctgaccctg aagctgatct
     5161 gcaccaccgg caagctgccc gtgccctggc ccaccctcgt gaccaccctg ggctacggcc
     5221 tgcagtgctt cgcccgctac cccgaccaca tgaagcagca cgacttcttc aagtccgcca
     5281 tgcccgaagg ctacgtccag gagcgcacca tcttcttcaa ggacgacggc aactacaaga
     5341 cccgcgccga ggtgaagttc gagggcgaca ccctggtgaa ccgcatcgag ctgaagggca
     5401 tcgacttcaa ggaggacggc aacatcctgg ggcacaagct ggagtacaac tacaacagcc
     5461 acaacgtcta tatcaccgcc gacaagcaga agaacggcat caaggccaac ttcaagatcc
     5521 gccacaacat cgaggacggc ggcgtgcagc tcgccgacca ctaccagcag aacaccccca
     5581 tcggcgacgg ccccgtgctg ctgcccgaca accactacct gagctaccag tccgccctga
     5641 gcaaagaccc caacgagaag cgcgatcaca tggtcctgct ggagttcgtg accgccgccg
     5701 ggatcactct cggcatggac gagctgtaca agtaagagct catatgaaga tgaagatgaa
     5761 atatttggtg tgtcaaataa atagcttgtg tgcttaagtt tgtgtttttt tcttggcttg
     5821 ttgtgttatg aatttgtggc tttttctaat attaaatgaa tgtaagatct cattataatg
     5881 aataaacaaa tgtttctata atccattgtg aatgttttgt tggatctctt ctgcagcata
     5941 taactactgt atgtgctatg gtatggacta tggaatatga ttaaagataa ggaattcaga
     6001 tcggctgagt ggctccttca acgttgcggt tctgtcagtt ccaaacgtaa aacggcttgt
     6061 cccgcgtcat cggcgggggt cataacgtga ctcccttaat tctccgctca tgatcagatt
     6121 gtcgtttccc gccttcagtt taaactatca gtgtttgaca ggatatattg gcgggtaaac
     6181 taagtcgctg tatgtgtttg tttgagatct catgtgagca aaaggccagc aaaaggccag
     6241 gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg ctccgccccc ctgacgagca
     6301 tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg acaggactat aaagatacca
     6361 ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg
     6421 atacctgtcc gcctttctcc cttcgggaag cgtggcgctt tctcatagct cacgctgtag
     6481 gtatctcagt tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt
     6541 tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc cggtaagaca
     6601 cgacttatcg ccactggcag cagccactgg taacaggatt agcagagcga ggtatgtagg
     6661 cggtgctaca gagttcttga agtggtggcc taactacggc tacactagaa gaacagtatt
     6721 tggtatctgc gctctgctga agccagttac cttcggaaga agagttggta gctcttgatc
     6781 cggcaaacaa accaccgctg gtagcggtgg tttttttgtt tgcaagcagc agattacgcg
     6841 cagaaaaaaa ggatctcaag aagatccttt gatcttttct acggggtctg acgctcagtg
     6901 gaacgaaaac tcacgttaag ggattttggt catgagatta tcaaaaagga tcttcaccta
     6961 gatcctttta aattaaaaat gaagttttaa atcaatctaa agtatatatg tgtaacattg
     7021 gtctagtgat tagaaaaact catcgagcat caaatgaaac tgcaatttat tcatatcagg
     7081 attatcaata ccatattttt gaaaaagccg tttctgtaat gaaggagaaa actcaccgag
     7141 gcagttccat aggatggcaa gatcctggta tcggtctgcg attccgactc gtccaacatc
     7201 aatacaacct attaatttcc cctcgtcaaa aataaggtta tcaagtgaga aatcaccatg
     7261 agtgacgact gaatccggtg agaatggcaa aagtttatgc atttctttcc agacttgttc
     7321 aacaggccag ccattacgct cgtcatcaaa atcactcgca tcaaccaaac cgttattcat
     7381 tcgtgattgc gcctgagcga gacgaaatac gcgatcgctg ttaaaaggac aattacaaac
     7441 aggaatcgaa tgcaaccggc gcaggaacac tgccagcgca tcaacaatat tttcacctga
     7501 atcaggatat tcttctaata cctggaatgc tgttttccct gggatcgcag tggtgagtaa
     7561 ccatgcatca tcaggagtac ggataaaatg cttgatggtc ggaagaggca taaattccgt
     7621 cagccagttt agtctgacca tctcatctgt aacatcattg gcaacgctac ctttgccatg
     7681 tttcagaaac aactctggcg catcgggctt cccatacaat cggtagattg tcgcacctga
     7741 ttgcccgaca ttatcgcgag cccatttata cccatataaa tcagcatcca tgttggaatt
     7801 taatcgcggc cttgagcaag acgtttcccg ttgaatatgg ctcataacac cccttgtatt
     7861 actgtttatg taagcagaca gttttattgt tcatgatgat atatttttat cttgtgcaat
     7921 gtaacatcag agattttgag acacaacgtg gctttgttga ataaatcgaa cttttgctga
     7981 gttgaaggat cagatcacgc atcttcccga caacgcagac cgttccgtgg caaagcaaaa
     8041 gttcaaaatc accaactggt ccacctacaa caa
//
