LOCUS       sequence-267200-        8367 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  constitutive expression of 2 mCherry molecules: one tagged with HA 
            and another that binds to actin (control plasmid).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8367)
  AUTHORS   Alcantara A, Seitner D, Navarrete F, Djamei A
  TITLE     A high-throughput screening method to identify proteins involved in 
            unfolded protein response of the endoplasmic reticulum in plants.
  JOURNAL   Plant Methods. 2020 Jan 21;16:4. doi: 10.1186/s13007-020-0552-3. 
            eCollection 2020.
  PUBMED    31988651
REFERENCE   2  (bases 1 to 8367)
  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 
            Methods."; date: "2020-01-21"; volume: "16:4. doi"; pages: " 
            10.1186/s13007-020-0552-3. eCollection 2020"
COMMENT     Sequence Label: p35S::mCh-HA-P2A-lifeact-mCh
FEATURES             Location/Qualifiers
     source          1..8367
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        222..567
                     /label=CaMV 35S promoter
                     /note="strong constitutive promoter from cauliflower mosaic
                     virus"
     primer_bind     510..530
                     /label=35S promoter
                     /note="CaMV 35S promoter, forward primer"
     misc_feature    595..650
                     /label=TMV Omega
                     /note="translational enhancer from the tobacco mosaic virus
                     5'-leader sequence (Gallie et al., 1988)"
     protein_bind    658..682
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             688..1395
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG
                     TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF
                     EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK
                     GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERA
                     EGRHSTGGMDELYK"
     CDS             1396..1422
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     protein_bind    complement(1448..1472)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             1489..1545
                     /codon_start=1
                     /product="2A peptide from porcine teschovirus-1 
                     polyprotein"
                     /label=P2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="ATNFSLLKQAGDVEENPGP"
     CDS             1546..1596
                     /codon_start=1
                     /product="peptide that binds filamentous actin (Riedl et 
                     al., 2008)"
                     /label=Lifeact
                     /note="first 17 amino acids of budding yeast Abp140"
                     /translation="MGVADLIKKFESISKEE"
     CDS             1597..2304
                     /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"
     CDS             3616..4644
                     /codon_start=1
                     /gene="aph(4)-Ia"
                     /product="aminoglycoside phosphotransferase from E. coli"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MGKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGR
                     GYVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGAFSESLTYCISRRAQGVTLQDL
                     PETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHW
                     QTVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAM
                     FGDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVD
                     GNFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRA
                     KE"
     terminator      4652..5359
                     /label=OCS terminator
                     /note="octopine synthase terminator"
     promoter        complement(5392..5410)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(5393..5410)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     misc_feature    5459..5483
                     /label=RB T-DNA repeat
                     /note="right border repeat from nopaline C58 T-DNA"
     rep_origin      complement(5574..6162)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5654..5673)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(6333..7121)
                     /codon_start=1
                     /gene="aadA"
                     /product="aminoglycoside adenylyltransferase (Murphy, 
                     1985)"
                     /label=SmR
                     /note="confers resistance to spectinomycin and 
                     streptomycin"
                     /translation="MREAVIAEVSTQLSEVVGVIERHLEPTLLAVHLYGSAVDGGLKPH
                     SDIDLLVTVTVRLDETTRRALINDLLETSASPGESEILRAVEVTIVVHDDIIPWRYPAK
                     RELQFGEWQRNDILAGIFEPATIDIDLAILLTKAREHSVALVGPAAEELFDPVPEQDLF
                     EALNETLTLWNSPPDWAGDERNVVLTLSRIWYSAVTGKIAPKDVAADWAMERLPAQYQP
                     VILEARQAYLGQEDRLASRADQLEEFVHYVKGEITKVVGK"
     rep_origin      7412..7847
                     /label=pSa ori
                     /note="origin of replication from bacterial plasmid pSa"
     misc_feature    7974..7996
                     /label=LB T-DNA repeat
                     /note="left border repeat from nopaline C58 T-DNA 
                     (truncated)"
ORIGIN
        1 ggagtctcta aaaaggtagt tcccactgaa tcaaaggcca tggagtcaaa gattcaaata
       61 gaggacctaa cagaactcgc cgtaaagact ggcgaacagt tcatacagag tctcttacga
      121 ctcaatgaca agaagaaaat cttcgtcaac atggtggagc acgacacact tgtctactcc
      181 aaaaatatca aagatacagt ctcagaagac caaagggcaa ttgagacttt tcaacaaagg
      241 gtaatatccg gaaacctcct cggattccat tgcccagcta tctgtcactt tattgtgaag
      301 atagtggaaa aggaaggtgg ctcctacaaa tgccatcatt gcgataaagg aaaggccatc
      361 gttgaagatg cctctgccga cagtggtccc aaagatggac ccccacccac gaggagcatc
      421 gtggaaaaag aagacgttcc aaccacgtct tcaaagcaag tggattgatg tgatatctcc
      481 actgacgtaa gggatgacgc acaatcccac tatccttcgc aagacccttc ctctatataa
      541 ggaagttcat ttcatttgga gagaacacgg gggacgagct aacatatttt tacaacaatt
      601 accaacaaca acaaacaaca aacaacatta caattactat ttacaattac aggctctaca
      661 agtttgtaca aaaaagcagg ctccaccatg gtgagcaagg gcgaggagga taacatggcc
      721 atcatcaagg agttcatgcg cttcaaggtg cacatggagg gctccgtgaa cggccacgag
      781 ttcgagatcg agggcgaggg cgagggccgc ccctacgagg gcacccagac cgccaagctg
      841 aaggtgacca agggtggccc cctgcccttc gcctgggaca tcctgtcccc tcagttcatg
      901 tacggctcca aggcctacgt gaagcacccc gccgacatcc ccgactactt gaagctgtcc
      961 ttccccgagg gcttcaagtg ggagcgcgtg atgaacttcg aggacggcgg cgtggtgacc
     1021 gtgacccagg actcctccct gcaggacggc gagttcatct acaaggtgaa gctgcgcggc
     1081 accaacttcc cctccgacgg ccccgtaatg cagaagaaga cgatgggctg ggaggcctcc
     1141 tccgagcgga tgtaccccga ggacggcgcc ctgaagggcg agatcaagca gaggctgaag
     1201 ctgaaggacg gcggccacta cgacgctgag gtcaagacca cctacaaggc caagaagccc
     1261 gtgcagctgc ccggcgccta caacgtcaac atcaagttgg acatcacctc ccacaacgag
     1321 gactacacca tcgtggaaca gtacgaacgc gccgagggcc gccactccac cggcggcatg
     1381 gacgagctgt acaagtaccc atacgatgtt ccagattacg ctggcggccg cacatcgaga
     1441 tatctagacc cagctttctt gtacaaagtg gtatcagctg gttctggagc tactaacttc
     1501 tctctcttga agcaagcagg agatgtggaa gaaaaccctg gtccaatggg tgtcgcagat
     1561 ttgatcaaga aattcgaaag catctcaaag gaagaagtga gcaagggcga ggaggataac
     1621 atggccatca tcaaggagtt catgcgcttc aaggtgcaca tggagggctc cgtgaacggc
     1681 cacgagttcg agatcgaggg cgagggcgag ggccgcccct acgagggcac ccagaccgcc
     1741 aagctgaagg tgaccaaggg tggccccctg cccttcgcct gggacatcct gtcccctcag
     1801 ttcatgtacg gctccaaggc ctacgtgaag caccccgccg acatccccga ctacttgaag
     1861 ctgtccttcc ccgagggctt caagtgggag cgcgtgatga acttcgagga cggcggcgtg
     1921 gtgaccgtga cccaggactc ctccctgcag gacggcgagt tcatctacaa ggtgaagctg
     1981 cgcggcacca acttcccctc cgacggcccc gtaatgcaga agaagaccat gggctgggag
     2041 gcctcctccg agcggatgta ccccgaggac ggcgccctga agggcgagat caagcagagg
     2101 ctgaagctga aggacggcgg ccactacgac gctgaggtca agaccaccta caaggccaag
     2161 aagcccgtgc agctgcccgg cgcctacaac gtcaacatca agttggacat cacctcccac
     2221 aacgaggact acaccatcgt ggaacagtac gaacgcgccg agggccgcca ctccaccggc
     2281 ggcatggacg agctgtacaa gtagctgcat ctcgtctctg ttatgcttaa gaagttcaat
     2341 gtttcgtttc atgtaaaact ttggtggttt gtgttttggg gccttgtata atccctgatg
     2401 aataagtgtt ctactatgtt tccgttcctg ttatctcttt ctttctaatg acaagtcgaa
     2461 cttcttcttt atcatcgctt cgtttttatt atctgtgctt cttttgttta atacgcctgc
     2521 aaagtgactc gactctgttt agtgcagttc tgcgaaactt gtaaatagtc caattgttgg
     2581 cctctagtaa tagatgtagc gaaagtgttg agctgttggg ttctaaggat ggcttgaaca
     2641 tgttaatctt ttaggttctg agtatgatga acattcgttg ttgctaagaa atgcctgtaa
     2701 tgtcccacaa atgtagaaaa tggttcgtac ctttgtccaa gcattgatat gtctgatgag
     2761 aggaaactgc aagatactga gcttggttta acgaaggaga ggcagtttct tccttccaaa
     2821 gcatttcatt tgacaatgcc ttgatcatct taagtagagt ttctgttgtg gaaagtttga
     2881 aactttgaag aaacgactct caagtaaatt gatgatcaca agtgaaagtg tatgttacta
     2941 gtcgacgagt cagtaataaa cggcgtcaaa gtggttgcag ccggcacaca cgagtcgtgt
     3001 ttatcaactc aaagcacaaa tacttttcct caacctaaaa ataaggcaat tagccaaaaa
     3061 caactttgcg tgtaaacaac gctcaataca cgtgtcattt tattattagc tattgcttca
     3121 ccgccttagc tttctcgtga cctagtcgtc ctcgtctttt cttcttcttc ttctataaaa
     3181 caatacccaa agagctcttc ttcttcacaa ttcagatttc aatttctcaa aatcttaaaa
     3241 actttctctc aattctctct accgtgatca aggtaaattt ctgtgttcct tattctctca
     3301 aaatcttcga ttttgttttc gttcgatccc aatttcgtat atgttctttg gtttagattc
     3361 tgttaatctt agatcgaaga cgattttctg ggtttgatcg ttagatatca tcttaattct
     3421 cgattagggt ttcatagata tcatccgatt tgttcaaata atttgagttt tgtcgaataa
     3481 ttactcttcg atttgtgatt tctatctaga tctggtgtta gtttctagtt tgtgcgatcg
     3541 aatttgtaga ttaatctgag tttttctgat taacaccaca ccaatctggt accagaaaag
     3601 gggagaagca aaaccatggg taaaaagcct gaactcaccg cgacgtctgt cgagaagttt
     3661 ctgatcgaaa agttcgacag cgtctccgac ctgatgcagc tctcggaggg cgaagaatct
     3721 cgtgctttca gcttcgatgt aggagggcgt ggatatgtcc tgcgggtaaa tagctgcgcc
     3781 gatggtttct acaaagatcg ttatgtttat cggcactttg catcggccgc gctcccgatt
     3841 ccggaagtgc ttgacattgg ggcattcagc gagagcctga cctattgcat ctcccgccgt
     3901 gcacagggtg tcacgttgca agacctgcct gaaaccgaac tgcccgctgt tctgcaaccg
     3961 gtcgcggagg cgatggatgc catcgctgcg gccgatctta gccagacgag cgggttcggc
     4021 ccattcggac cgcaaggaat cggtcaatac actacatggc gtgatttcat atgcgcgatt
     4081 gctgatcccc atgtgtatca ctggcaaact gtgatggacg acaccgtcag tgcgtccgtc
     4141 gcgcaggctc tcgatgagct gatgctttgg gccgaggact gccccgaagt ccggcacctc
     4201 gtgcacgcgg atttcggctc caacaatgtc ctgacggaca atggccgcat aacagcggtc
     4261 attgactgga gcgaggcgat gttcggggat tcccaatacg aggtcgccaa catcttcttc
     4321 tggaggccgt ggttggcttg tatggagcag cagacgcgct acttcgagcg gaggcatccg
     4381 gagcttgcag gatcgccgcg gctccgggcg tatatgctcc gcattggtct tgaccaactc
     4441 tatcagagct tggttgacgg caatttcgat gatgcagctt gggcgcaggg tcgatgcgac
     4501 gcaatcgtcc gatccggagc cgggactgtc gggcgtacac aaatcgcccg cagaagcgcg
     4561 gccgtctgga ccgatggctg tgtagaagta ctcgccgata gtggaaaccg acgccccagc
     4621 actcgtccga gggcaaagga atagatgcat cctgctttaa tgagatatgc gagacgccta
     4681 tgatcgcatg atatttgctt tcaattctgt tgtgcacgtt gtaaaaaacc tgagcatgtg
     4741 tagctcagat ccttaccgcc ggtttcggtt cattctaatg aatatatcac ccgttactat
     4801 cgtattttta tgaataatat tctccgttca atttactgat tgtaccctac tacttatatg
     4861 tacaatatta aaatgaaaac aatatattgt gctgaatagg tttatagcga catctatgat
     4921 agagcgccac aataacaaac aattgcgttt tattattaca aatccaattt taaaaaaagc
     4981 ggcagaaccg gtcaaaccta aaagactgat tacataaatc ttattcaaat ttcaaaagtg
     5041 ccccaggggc tagtatctac gacacaccga gcggcgaact aataacgctc actgaaggga
     5101 actccggttc cccgccggcg cgcatgggtg agattccttg aagttgagta ttggccgtcc
     5161 gctctaccga aagttacggg caccattcaa cccggtccag cacggcggcc gggtaaccga
     5221 cttgctgccc cgagaattat gcagcatttt tttggtgtat gtgggcccca aatgaagtgc
     5281 aggtcaaacc ttgacagtga cgacaaatcg ttgggcgggt ccagggcgaa ttttgcgaca
     5341 acatgtcgag gctcagcagg accggcatgc aagctagctt actagtgata ttctatagtg
     5401 tcacctaaat gtattctaga ccaagcatgc acagctggcg ttctcagcca gatccccttg
     5461 acaggatata ttggcgggta aactaagtcg ctgtatgtgt ttgtttgaga tctcatgtga
     5521 gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc gtttttccat
     5581 aggctccgcc cccctgacga gcatcacaaa aatcgacgct caagtcagag gtggcgaaac
     5641 ccgacaggac tataaagata ccaggcgttt ccccctggaa gctccctcgt gcgctctcct
     5701 gttccgaccc tgccgcttac cggatacctg tccgcctttc tcccttcggg aagcgtggcg
     5761 ctttctcata gctcacgctg taggtatctc agttcggtgt aggtcgttcg ctccaagctg
     5821 ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg ccttatccgg taactatcgt
     5881 cttgagtcca acccggtaag acacgactta tcgccactgg cagcagccac tggtaacagg
     5941 attagcagag cgaggtatgt aggcggtgct acagagttct tgaagtggtg gcctaactac
     6001 ggctacacta gaagaacagt atttggtatc tgcgctctgc tgaagccagt taccttcgga
     6061 agaagagttg gtagctcttg atccggcaaa caaaccaccg ctggtagcgg tggttttttt
     6121 gtttgcaagc agcagattac gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt
     6181 tctacggggt ctgacgctca gtggaacgaa aactcacgtt aagggatttt ggtcatgaga
     6241 ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa aatgaagttt taaatcaatc
     6301 taaagtatat atgtgtaaca ttggtctagt gattatttgc cgactacctt ggtgatctcg
     6361 cctttcacgt agtgaacaaa ttcttccaac tgatctgcgc gcgaggccaa gcgatcttct
     6421 tgtccaagat aagcctgcct agcttcaagt atgacgggct gatactgggc cggcaggcgc
     6481 tccattgccc agtcggcagc gacatccttc ggcgcgattt tgccggttac tgcgctgtac
     6541 caaatgcggg acaacgtaag cactacattt cgctcatcgc cagcccagtc gggcggcgag
     6601 ttccatagcg ttaaggtttc atttagcgcc tcaaatagat cctgttcagg aaccggatca
     6661 aagagttcct ccgccgctgg acctaccaag gcaacgctat gttctcttgc ttttgtcagc
     6721 aagatagcca gatcaatgtc gatcgtggct ggctcgaaga tacctgcaag aatgtcattg
     6781 cgctgccatt ctccaaattg cagttcgcgc ttagctggat aacgccacgg aatgatgtcg
     6841 tcgtgcacaa caatggtgac ttctacagcg cggagaatct cgctctctcc aggggaagcc
     6901 gaagtttcca aaaggtcgtt gatcaaagct cgccgcgttg tttcatcaag ccttacggtc
     6961 accgtaacca gcaaatcaat atcactgtgt ggcttcaggc cgccatccac tgcggagccg
     7021 tacaaatgta cggccagcaa cgtcggttcg agatggcgct cgatgacgcc aactacctct
     7081 gatagttgag tcgatacttc ggcgatcacc gcttccctca taacacccct tgtattactg
     7141 tttatgtaag cagacagttt tattgttcat gatgatatat ttttatcttg tgcaatgtaa
     7201 catcagagat tttgagacac aacgtggctt tgttgaataa atcgaacttt tgctgagttg
     7261 aaggatcaga tcacgcatct tcccgacaac gcagaccgtt ccgtggcaaa gcaaaagttc
     7321 aaaatcacca actggtccac ctacaacaaa gctctcatca accgtggctc cctcactttc
     7381 tggctggatg atggggcgat tcaggcgatc cccatccaac agcccgccgt cgagcgggct
     7441 tttttatccc cggaagcctg tggatagagg gtagttatcc acgtgaaacc gctaatgccc
     7501 cgcaaagcct tgattcacgg ggctttccgg cccgctccaa aaactatcca cgtgaaatcg
     7561 ctaatcaggg tacgtgaaat cgctaatcgg agtacgtgaa atcgctaata aggtcacgtg
     7621 aaatcgctaa tcaaaaaggc acgtgagaac gctaatagcc ctttcagatc aacagcttgc
     7681 aaacacccct cgctccggca agtagttaca gcaagtagta tgttcaatta gcttttcaat
     7741 tatgaatata tatatcaatt attggtcgcc cttggcttgt ggacaatgcg ctacgcgcac
     7801 cggctccgcc cgtggacaac cgcaagcggt tgcccaccgt cgagcgcctt tgcccacaac
     7861 ccggcggccg gccgcaacag atcgttttat aaattttttt ttttgaaaaa gaaaaagccc
     7921 gaaaggcggc aacctctcgg gcttctggat ttccgatccc cggaattaga tcttggcagg
     7981 atatattgtg gtgtaacgtt ctcgaactag gctcggacga agtaagcggc cgcaacctta
     8041 attaaacacg gcgcgccaac agcgatcgca ccaggtacca cctccgatcc attcggtccc
     8101 cagattagcc ttttcaattt cagaaagaat gctaacccac agatggttag agaggcttac
     8161 gcagcacgtc tcatcaagac gatctacccg agcaataatc tccaggaaat caaatacctt
     8221 cccaagaagg ttaaagatgc agtcaaaaga ttcaggacta actgcatcaa gaacacagag
     8281 aaagatatat ttctcaagat cagaagtact attccagtat ggacgattca aggcttgctt
     8341 cacaaaccaa ggcaagtaat agagatt
//
