LOCUS       sequence-482329-        8551 bp    DNA     circular SYN 07-AUG-2025
DEFINITION  Simultaneous monitoring of transcription and translation of genes in
            plant tissues: N- terminus fusion.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8551)
  TITLE     Dual 2A reporters
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 8551)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Aug 7, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: 
            "Unpublished"
COMMENT     Sequence Label: pMDS2
FEATURES             Location/Qualifiers
     source          1..8551
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_feature    77..101
                     /label=RB T-DNA repeat
                     /note="right border repeat from nopaline C58 T-DNA"
     rep_origin      complement(192..780)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(272..291)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(871..1659)
                     /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      2711..3146
                     /label=pSa ori
                     /note="origin of replication from bacterial plasmid pSa"
     misc_feature    3273..3295
                     /label=LB T-DNA repeat
                     /note="left border repeat from nopaline C58 T-DNA 
                     (truncated)"
     promoter        3355..3700
                     /label=CaMV 35S promoter
                     /note="strong constitutive promoter from cauliflower mosaic
                     virus"
     primer_bind     3643..3663
                     /label=35S promoter
                     /note="CaMV 35S promoter, forward primer"
     CDS             4607..5317
                     /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
                     EGRHSTGGMEELYK"
     primer_bind     complement(4752..4770)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     5020..5039
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     terminator      5336..5588
                     /label=NOS terminator
                     /note="nopaline synthase terminator and poly(A) signal"
     CDS             complement(5686..6009)
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITPSLHACRSTLEDPRVPSSNSLAVVLQRRDWENPGVTQLNR
                     LAAHPPFASWRNSEEARTDRPSQQLRSLNGEWRLMRYFLLTHLCGISHRIWCTLSTICS
                     DAA"
     primer_bind     5691..5710
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5904..5926
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     5918..5935
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     5919..5935
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     misc_feature    5936..5992
                     /label=MCS
                     /note="pUC18/19 multiple cloning site"
     primer_bind     complement(6005..6021)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6005..6021)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(6018..6040)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    6029..6045
                     /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)."
     promoter        complement(join(6053..6059,6060..6077,6078..6083))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6098..6119
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(6236..6253)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     CDS             6316..6405
                     /codon_start=1
                     /product="three tandem HA epitope tags"
                     /label=3xHA
                     /translation="YPYDVPDYAGYPYDVPDYAGSYPYDVPDYA"
     CDS             join(6439..6441,6442..7152)
                     /codon_start=1
                     /product="yellow fluorescent protein (YFP) with fast and 
                     efficient maturation (Nagai et al., 2002)"
                     /label=Venus
                     /note="mammalian codon-optimized"
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     LICTTGKLPVPWPTLVTTLGYGLQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYITADKQKNGIKA
                     NFKIRHNIEDGGVQLADHYQQNTPIGDGPVLLPDNHYLSYQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     CDS             join(7273..7275,7276..7983)
                     /codon_start=1
                     /product="enhanced monomeric variant of CFP (Goedhart et 
                     al., 2012)"
                     /label=mTurquoise2
                     /note="mammalian codon-optimized"
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLSWGVQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYFSDNVYITADKQKNGIKA
                     NFKIRHNIEDGGVQLADHYQQNTPIGDGPVLLPDNHYLSTQSKLSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELY"
     polyA_signal    8291..8467
                     /label=CaMV poly(A) signal
                     /note="cauliflower mosaic virus polyadenylation signal"
     primer_bind     complement(8531..8551)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(8531..8549)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
ORIGIN
        1 gcgctagctc actcaaaggc ggtaatacgg ttatccacag aatcagggga taacgcagga
       61 aagaacatga aggccttgac aggatatatt ggcgggtaaa ctaagtcgct gtatgtgttt
      121 gtttgagatc tcatgtgagc aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg
      181 ttgctggcgt ttttccatag gctccgcccc cctgacgagc atcacaaaaa tcgacgctca
      241 agtcagaggt ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggaagc
      301 tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc
      361 ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta ggtatctcag ttcggtgtag
      421 gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc
      481 ttatccggta actatcgtct tgagtccaac ccggtaagac acgacttatc gccactggca
      541 gcagccactg gtaacaggat tagcagagcg aggtatgtag gcggtgctac agagttcttg
      601 aagtggtggc ctaactacgg ctacactaga agaacagtat ttggtatctg cgctctgctg
      661 aagccagtta ccttcggaag aagagttggt agctcttgat ccggcaaaca aaccaccgct
      721 ggtagcggtg gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa
      781 gaagatcctt tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa
      841 gggattttgg tcatgaattc ttgttagaca ttatttgccg actaccttgg tgatctcgcc
      901 tttcacgtag tgaacaaatt cttccaactg atctgcgcgc gaggccaagc gatcttcttg
      961 tccaagataa gcctgcctag cttcaagtat gacgggctga tactgggccg gcaggcgctc
     1021 cattgcccag tcggcagcga catccttcgg cgcgattttg ccggttactg cgctgtacca
     1081 aatgcgggac aacgtaagca ctacatttcg ctcatcgcca gcccagtcgg gcggcgagtt
     1141 ccatagcgtt aaggtttcat ttagcgcctc aaatagatcc tgttcaggaa ccggatcaaa
     1201 gagttcctcc gccgctggac ctaccaaggc aacgctatgt tctcttgctt ttgtcagcaa
     1261 gatagccaga tcaatgtcga tcgtggctgg ctcgaagata cctgcaagaa tgtcattgcg
     1321 ctgccattct ccaaattgca gttcgcgctt agctggataa cgccacggaa tgatgtcgtc
     1381 gtgcacaaca atggtgactt ctacagcgcg gagaatctcg ctctctccag gggaagccga
     1441 agtttccaaa aggtcgttga tcaaagctcg ccgcgttgtt tcatcaagcc ttacggtcac
     1501 cgtaaccagc aaatcaatat cactgtgtgg cttcaggccg ccatccactg cggagccgta
     1561 caaatgtacg gccagcaacg tcggttcgag atggcgctcg atgacgccaa ctacctctga
     1621 tagttgagtc gatacttcgg cgatcaccgc ttccctcatg atgtttaact cctgaattaa
     1681 gccgcgccgc gaagcggtgt cggcttgaat gaattgttag gcgtcatcct gtgctcccga
     1741 gaaccagtac cagtacatcg ctgtttcgtt cgagacttga ggtctagttt tatacgtgaa
     1801 caggtcaatg ccgccgagag taaagccaca ttttgcgtac aaattgcagg caggtacatt
     1861 gttcgtttgt gtctctaatc gtatgccaag gagctgtctg cttagtgccc actttttcgc
     1921 aaattcgatg agactgtgcg cgactccttt gcctcggtgc gtgtgcgaca caacaatgtg
     1981 ttcgatagag gctagatcgt tccatgttga gttgagttca atcttcccga caagctcttg
     2041 gtcgatgaat gtgccatagc aagcagagtc ttcatcagag tcatcatccg agatgtaatc
     2101 cttccggtag gggctcacac ttctggtaga tagttcaaag ccttggtcgg ataggtgcac
     2161 atcgaacact tcacgaacaa tgaaatggtt ctcagcatca aatgtttccg ccacctgctc
     2221 agggatcacc gaaatcttca tatgacgcct aacgcctggc acagcggatc gcaaacctgg
     2281 cgcggctttt ggcacaaaag gcgtgacagg tttgcgaatc cgttgctgcc acttgtttaa
     2341 tagactggat ggaggcggat aaagttgcag gaccacttct gcgctcggcc cttccggctg
     2401 gctggtttat tgctgataaa tctggagccg gtgagcgtgg atctcgcggt atcattgcag
     2461 cactgccatg atgatatatt tttatcttgt gcaatgtaac atcagagatt ttgagacaca
     2521 acgtggcttt gttgaataaa tcgaactttt gctgagttga aggatcagat cacgcatctt
     2581 cccgacaacg cagaccgttc cgtggcaaag caaaagttca aaatcaccaa ctggtccacc
     2641 tacaacaaag ctctcatcaa ccgtggctcc ctcactttct ggctggatga tggggcgatt
     2701 caggagatcc ccatccaaca gcccgccgtc gagcgggctt ttttatcccc ggaagcctgt
     2761 ggatagaggg tagttatcca cgtgaaaccg ctaatgcccc gcaaagcctt gattcacggg
     2821 gctttccggc ccgctccaaa aactatccac gtgaaatcgc taatcagggt acgtgaaatc
     2881 gctaatcgga gtacgtgaaa tcgctaataa ggtcacgtga aatcgctaat caaaaaggca
     2941 cgtgagaacg ctaatagccc tttcagatca acagcttgca aacacccctc gctccggcaa
     3001 gtagttacag caagtagtat gttcaattag cttttcaatt atgaatatat atatcaatta
     3061 ttggtcgccc ttggcttgtg gacaatgcgc tacgcgcacc ggctccgccc gtggacaacc
     3121 gcaagcggtt gcccaccgtc gagcgccttt gcccacaacc cggcggccgg ccgcaacaga
     3181 tcgttttata aatttttttt tttgaaaaag aaaaagcccg aaaggcggca acctctcggg
     3241 cttctggatt tccgatcccc ggaattagat cttggcagga tatattgtgg tgtaacgttg
     3301 gcgcgcctac tccaaaaatg tcaaagatac agtctcagaa gaccaaaggg ctattgagac
     3361 ttttcaacaa agggtaattt cgggaaacct cctcggattc cattgcccag ctatctgtca
     3421 cttcatcgaa aggacagtag aaaaggaagg tggctcctac aaatgccatc attgcgataa
     3481 aggaaaggct atcattcaag atgcctctgc cgacagtggt cccaaagatg gacccccacc
     3541 cacgaggagc atcgtggaaa aagaagacgt tccaaccacg tcttcaaagc aagtggattg
     3601 atgtgacatc tccactgacg taagggatga cgcacaatcc cactatcctt cgcaagaccc
     3661 ttcctctata taaggaagtt catttcattt ggagaggaca gcccagatca ctagtcttaa
     3721 gtccggagct agctctagaa tggcaaagga tgtggaagcc gttcccggag aaggatttca
     3781 gacaagagac tatcaagatc cgccaccagc tccgtttatt gatggagcgg agctaaagaa
     3841 gtggtctttc tacagagcag ttatcgcaga gttcgtagcc actctcctct tcttatacat
     3901 caccgttttg acagtcatcg gttacaagat tcagtccgat actgatgccg gtggcgtaga
     3961 ttgcggcgga gttggaatcc tcggtatcgc ttgggccttt ggtggtatga tcttcatcct
     4021 cgtctactgc accgccggta tctctggtgg tcacattaac ccagcggtga catttgggct
     4081 attcttggca cgtaaagtgt cgttacctag ggccctattg tacataatcg ctcagtgttt
     4141 gggtgcgatt tgtggagttg gttttgtcaa agccttccaa agctcttact acacccgtta
     4201 cggaggtgga gccaactctc tagccgatgg ctacagcaca gggaccggtc tagccgcaga
     4261 gatcattggt actttcgttc ttgtctacac cgtcttctct gccactgacc ccaaacgtag
     4321 tgccagagac tcccacgttc cggtgttggc gccacttcca atcggatttg ccgtgttcat
     4381 ggtacatttg gctaccattc ccattaccgg aaccggaatt aacccggcaa ggagtttcgg
     4441 agctgccgta atctacaaca agagcaagcc atgggatgac cactggatat tttgggttgg
     4501 accattcatt ggagctgcga tagctgcatt ctaccaccaa ttcgttctaa gagcttcagg
     4561 ttctaagtct cttggatcat tcagaagtgc tgccaacgtc ggatccatgg tgagcaaggg
     4621 cgaggaggat aacatggcca tcatcaagga gttcatgcgc ttcaaggtgc acatggaggg
     4681 ctccgtgaac ggccacgagt tcgagatcga gggcgagggc gagggccgcc cctacgaggg
     4741 cacccagacc gccaagctga aggtgaccaa gggtggcccc ctgcccttcg cctgggacat
     4801 cctgtcccct cagttcatgt acggctccaa ggcctacgtg aagcaccccg ccgacatccc
     4861 cgactacttg aagctgtcct tccccgaggg cttcaagtgg gagcgcgtga tgaacttcga
     4921 ggacggcggc gtggtgaccg tgacccagga ctcctccctg caggacggcg agttcatcta
     4981 caaggtgaag ctgcgcggca ccaacttccc ctccgacggc cccgtaatgc agaagaagac
     5041 catgggctgg gaggcctcct ccgagcggat gtaccccgag gacggcgccc tgaagggcga
     5101 gatcaagcag aggctgaagc tgaaggacgg cggccactac gacgctgagg tcaagaccac
     5161 ctacaaggcc aagaagcccg tgcagctgcc cggcgcctac aacgtcaaca tcaagttgga
     5221 catcacctcc cacaacgagg actacaccat cgtggaacag tacgaacgcg ccgagggccg
     5281 ccactccacc ggcggcatgg aggagctgta caagtaaagc ggccgcccgg ctgcagatcg
     5341 ttcaaacatt tggcaataaa gtttcttaag attgaatcct gttgccggtc ttgcgatgat
     5401 tatcatataa tttctgttga attacgttaa gcatgtaata attaacatgt aatgcatgac
     5461 gttatttatg agatgggttt ttatgattag agtcccgcaa ttatacattt aatacgcgat
     5521 agaaaacaaa atatagcgcg caaactagga taaattatcg cgcgcggtgt catctatgtt
     5581 actagatcgg ggcgcgccct cgagagtaga agagccggat gccgggagca gacaagcccg
     5641 tcagggcgcg tcagcgggtg ttggcgggtg tcggggctgg cttaactatg cggcatcaga
     5701 gcagattgta ctgagagtgc accatatgcg gtgtgaaata ccgcacagat gcgtaaggag
     5761 aaaataccgc atcaggcgcc attcgccatt caggctgcgc aactgttggg aagggcgatc
     5821 ggtgcgggcc tcttcgctat tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt
     5881 aagttgggta acgccagggt tttcccagtc acgacgttgt aaaacgacgg ccagtgaatt
     5941 cgagctcggt acccggggat cctctagagt cgacctgcag gcatgcaggc ttggcgtaat
     6001 catggtcata gctgtttcct gtgtgaaatt gttatccgct cacaattcca cacaacatac
     6061 gagccggaag cataaagtgt aaagcctggg gtgcctaatg agtgagctaa ctcacattaa
     6121 ttgcgttgcg ctcactgccc gctttccagt cgggaaacct gtcgtgccag ctgcattaat
     6181 gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg gcgctcttcc gcttcctcgc
     6241 tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc ggtatcgctc ttcatggatg
     6301 ggcattaaca tcttttaccc atacgatgtt cctgactatg cgggctatcc ctatgacgtc
     6361 ccggactatg caggatccta tccatatgac gttccagatt acgcttccgg aggtggtggt
     6421 tctggtgggg gtggctctgt gagcaagggc gaggagctgt tcaccggggt ggtgcccatc
     6481 ctggtcgagc tggacggcga cgtaaacggc cacaagttca gcgtgtccgg cgagggcgag
     6541 ggcgatgcca cctacggcaa gctgaccctg aagctgatct gcaccaccgg caagctgccc
     6601 gtgccctggc ccaccctcgt gaccaccctg ggctacggcc tgcagtgctt cgcccgctac
     6661 cccgaccaca tgaagcagca cgacttcttc aagtccgcca tgcccgaagg ctacgtccag
     6721 gagcgcacca tcttcttcaa ggacgacggc aactacaaga cccgcgccga ggtgaagttc
     6781 gagggcgaca ccctggtgaa ccgcatcgag ctgaagggca tcgacttcaa ggaggacggc
     6841 aacatcctgg ggcacaagct ggagtacaac tacaacagcc acaacgtcta tatcaccgcc
     6901 gacaagcaga agaacggcat caaggccaac ttcaagatcc gccacaacat cgaggacggc
     6961 ggcgtgcagc tcgccgacca ctaccagcag aacaccccca tcggcgacgg ccccgtgctg
     7021 ctgcccgaca accactacct gagctaccag tccgccctga gcaaagaccc caacgagaag
     7081 cgcgatcaca tggtcctgct ggagttcgtg accgccgccg ggatcactct cggcatggac
     7141 gagctgtaca agcatgattc cggaggtggt ggttctggtg ggggtggctc ctcaggtggc
     7201 tcgggccagc tgttgaattt tgaccttctt aagcttgcgg gagacgtcga gtccaaccct
     7261 gggccccccg gggtgagcaa gggcgaggag ctgttcaccg gggtggtgcc catcctggtc
     7321 gagctggacg gcgacgtaaa cggccacaag ttcagcgtgt ccggcgaggg cgagggcgat
     7381 gccacctacg gcaagctgac cctgaagttc atctgcacca ccggcaagct gcccgtgccc
     7441 tggcccaccc tcgtgaccac cctgtcctgg ggcgtgcagt gcttcgcccg ctaccccgac
     7501 cacatgaagc agcacgactt cttcaagtcc gccatgcccg aaggctacgt ccaggagcgc
     7561 accatcttct tcaaggacga cggcaactac aagacccgcg ccgaggtgaa gttcgagggc
     7621 gacaccctgg tgaaccgcat cgagctgaag ggcatcgact tcaaggagga cggcaacatc
     7681 ctggggcaca agctggagta caactacttt agcgacaacg tctatatcac cgccgacaag
     7741 cagaagaacg gcatcaaggc caacttcaag atccgccaca acatcgagga cggcggcgtg
     7801 cagctcgccg accactacca gcagaacacc cccatcggcg acggccccgt gctgctgccc
     7861 gacaaccact acctgagcac ccagtccaag ctgagcaaag accccaacga gaagcgcgat
     7921 cacatggtcc tgctggagtt cgtgaccgcc gccgggatca ctctcggcat ggacgagctg
     7981 tacattgctg cagcggccga attcaagcgt gaggagcaag caaggaaagc taaggtgaac
     8041 aatgagaaaa agacggaaat agtgaaacca gagagttgta gcaatgaagg agatgtcaag
     8101 gatctgaaaa gaaaggactc tgaggatgga aacgagggtg aggaagaaga agcttcttcg
     8161 aaaccgaaaa agccaaaagt tgctctttct catcttcagg acattgacga cacagaagct
     8221 gatcaagaag aagagtaatg tcggtacgct gaaatcacca gtctctctct acaaatctat
     8281 ctctctctat tttctccata aataatgtgt gagtagtttc ccgataaggg aaattagggt
     8341 tcttataggg tttcgctcat gtgttgagca tataagaaac ccttagtatg tatttgtatt
     8401 tgtaaaatac ttctatcaat aaaatttcta attcctaaaa ccaaaatcca gtactaaaat
     8461 ccagatcgat gggctagagc ggactagttc tagagcggcc gccaccgcgg tggagctcca
     8521 gcttttgttc cctttagtga gggttaattg c
//
