LOCUS       sequence-363159-        5840 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses EGFP and tdTomato in mammalian cells under control of a 
            CMV promoter by using a dual 2A peptide sequence (P2AT2A).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5840)
  AUTHORS   Kozisek T, Samuelson L, Hamann A, Pannier AK
  TITLE     Systematic comparison of nonviral gene delivery strategies for 
            efficient co-expression of two transgenes in human mesenchymal stem 
            cells.
  JOURNAL   J Biol Eng. 2023 Dec 7;17(1):76. doi: 10.1186/s13036-023-00394-0.
  PUBMED    38062439
REFERENCE   2  (bases 1 to 5840)
  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: "J Biol 
            Eng."; date: "2023-12-7"; volume: "17(1):76. doi"; pages: " 
            10.1186/s13036-023-00394-0"
COMMENT     Sequence Label: pEGFP_D2A_tdTomato
FEATURES             Location/Qualifiers
     source          1..5840
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        64..367
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        368..571
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     522..542
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      610..619
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(616..618,619..621,622..1332)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(616..618,619..621,622..1332)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(661..682)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(922..941)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1269..1290
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             1372..1428
                     /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             1438..1491
                     /codon_start=1
                     /product="2A peptide from Thosea asigna virus capsid 
                     protein"
                     /label=T2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="EGRGSLLTCGDVEENPGP"
     CDS             1510..2940
                     /codon_start=1
                     /product="tandem dimeric (pseudo-monomeric) derivative of 
                     DsRed (Shaner et al., 2004)"
                     /label=tdTomato
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEEVIKEFMRFKVRMEGSMNGHEFEIEGEGEGRPYEGTQTA
                     KLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYKKLSFPEGFKWERVMNFEDGG
                     LVTVTQDSSLQDGTLIYKVKMRGTNFPPDGPVMQKKTMGWEASTERLYPRDGVLKGEIH
                     QALKLKDGGHYLVEFKTIYMAKKPVQLPGYYYVDTKLDITSHNEDYTIVEQYERSEGRH
                     HLFLGHGTGSTGSGSSGTASSEDNNMAVIKEFMRFKVRMEGSMNGHEFEIEGEGEGRPY
                     EGTQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYKKLSFPEGFKWERVM
                     NFEDGGLVTVTQDSSLQDGTLIYKVKMRGTNFPPDGPVMQKKTMGWEASTERLYPRDGV
                     LKGEIHQALKLKDGGHYLVEFKTIYMAKKPVQLPGYYYVDTKLDITSHNEDYTIVEQYE
                     RSEGRHHLFLYGMDELYK"
     polyA_signal    3087..3208
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3124..3143)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3178..3197
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     promoter        3241..3345
                     /gene="bla"
                     /label=AmpR promoter
     promoter        3347..3704
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        3347..3538
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(3370..3390)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      3555..3690
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             3739..4533
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     CDS             3739..4533
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(3793..3812)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4403..4422
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(4721..4740)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    4765..4812
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      5141..5729
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5630..5649
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 cattagttat taatagtaat caattacggg gtcattagtt catagcccat atatggagtt
       61 ccgcgttaca taacttacgg taaatggccc gcctggctga ccgcccaacg acccccgccc
      121 attgacgtca ataatgacgt atgttcccat agtaacgcca atagggactt tccattgacg
      181 tcaatgggtg gagtatttac ggtaaactgc ccacttggca gtacatcaag tgtatcatat
      241 gccaagtacg ccccctattg acgtcaatga cggtaaatgg cccgcctggc attatgccca
      301 gtacatgacc ttatgggact ttcctacttg gcagtacatc tacgtattag tcatcgctat
      361 taccatggtg atgcggtttt ggcagtacat caatgggcgt ggatagcggt ttgactcacg
      421 gggatttcca agtctccacc ccattgacgt caatgggagt ttgttttggc accaaaatca
      481 acgggacttt ccaaaatgtc gtaacaactc cgccccattg acgcaaatgg gcggtaggcg
      541 tgtacggtgg gaggtctata taagcagagc tggtttagtg aaccgtcaga tccgctagcg
      601 ctaccggtcg ccaccatggt gagcaagggc gaggagctgt tcaccggggt ggtgcccatc
      661 ctggtcgagc tggacggcga cgtaaacggc cacaagttca gcgtgtccgg cgagggcgag
      721 ggcgatgcca cctacggcaa gctgaccctg aagttcatct gcaccaccgg caagctgccc
      781 gtgccctggc ccaccctcgt gaccaccctg acctacggcg tgcagtgctt cagccgctac
      841 cccgaccaca tgaagcagca cgacttcttc aagtccgcca tgcccgaagg ctacgtccag
      901 gagcgcacca tcttcttcaa ggacgacggc aactacaaga cccgcgccga ggtgaagttc
      961 gagggcgaca ccctggtgaa ccgcatcgag ctgaagggca tcgacttcaa ggaggacggc
     1021 aacatcctgg ggcacaagct ggagtacaac tacaacagcc acaacgtcta tatcatggcc
     1081 gacaagcaga agaacggcat caaggtgaac ttcaagatcc gccacaacat cgaggacggc
     1141 agcgtgcagc tcgccgacca ctaccagcag aacaccccca tcggcgacgg ccccgtgctg
     1201 ctgcccgaca accactacct gagcacccag tccgccctga gcaaagaccc caacgagaag
     1261 cgcgatcaca tggtcctgct ggagttcgtg accgccgccg ggatcactct cggcatggac
     1321 gagctgtaca agtccggagg cagaaagctt ggttccggta ccggaagcgg agctactaac
     1381 ttcagcctgc tgaagcaggc tggagacgtg gaggagaacc ctggacctgg aagcggagag
     1441 ggcagaggaa gtctgctaac atgcggtgac gtcgaggaga atcctggacc taccgagctc
     1501 ggatctacca tggtgagcaa gggcgaggag gtcatcaaag agttcatgcg cttcaaggtg
     1561 cgcatggagg gctccatgaa cggccacgag ttcgagatcg agggcgaggg cgagggccgc
     1621 ccctacgagg gcacccagac cgccaagctg aaggtgacca agggcggccc cctgcccttc
     1681 gcctgggaca tcctgtcccc ccagttcatg tacggctcca aggcgtacgt gaagcacccc
     1741 gccgacatcc ccgattacaa gaagctgtcc ttccccgagg gcttcaagtg ggagcgcgtg
     1801 atgaacttcg aggacggcgg tctggtgacc gtgacccagg actcctccct gcaggacggc
     1861 acgctgatct acaaggtgaa gatgcgcggc accaacttcc cccccgacgg ccccgtaatg
     1921 cagaagaaga ccatgggctg ggaggcctcc accgagcgcc tgtacccccg cgacggcgtg
     1981 ctgaagggcg agatccacca ggccctgaag ctgaaggacg gcggccacta cctggtggag
     2041 ttcaagacca tctacatggc caagaagccc gtgcaactgc ccggctacta ctacgtggac
     2101 accaagctgg acatcacctc ccacaacgag gactacacca tcgtggaaca gtacgagcgc
     2161 tccgagggcc gccaccacct gttcctgggg catggcaccg gcagcaccgg cagcggcagc
     2221 tccggcaccg cctcctccga ggacaacaac atggccgtca tcaaagagtt catgcgcttc
     2281 aaggtgcgca tggagggctc catgaacggc cacgagttcg agatcgaggg cgagggcgag
     2341 ggccgcccct acgagggcac ccagaccgcc aagctgaagg tgaccaaggg cggccccctg
     2401 cccttcgcct gggacatcct gtccccccag ttcatgtacg gctccaaggc gtacgtgaag
     2461 caccccgccg acatccccga ttacaagaag ctgtccttcc ccgagggctt caagtgggag
     2521 cgcgtgatga acttcgagga cggcggtctg gtgaccgtga cccaggactc ctccctgcag
     2581 gacggcacgc tgatctacaa ggtgaagatg cgcggcacca acttcccccc cgacggcccc
     2641 gtaatgcaga agaagaccat gggctgggag gcctccaccg agcgcctgta cccccgcgac
     2701 ggcgtgctga agggcgagat ccaccaggcc ctgaagctga aggacggcgg ccactacctg
     2761 gtggagttca agaccatcta catggccaag aagcccgtgc aactgcccgg ctactactac
     2821 gtggacacca agctggacat cacctcccac aacgaggact acaccatcgt ggaacagtac
     2881 gagcgctccg agggccgcca ccacctgttc ctgtacggca tggacgagct gtacaagtag
     2941 taaagcttaa ttctcgaaga tccaccggat ctagataact gatcataatc agccatacca
     3001 catttgtaga ggttttactt gctttaaaaa acctcccaca cctccccctg aacctgaaac
     3061 ataaaatgaa tgcaattgtt gttgttaact tgtttattgc agcttataat ggttacaaat
     3121 aaagcaatag catcacaaat ttcacaaata aagcattttt ttcactgcat tctagttgtg
     3181 gtttgtccaa actcatcaat gtatcttaac gcgtcaggtg gcacttttcg gggaaatgtg
     3241 cgcggaaccc ctatttgttt atttttctaa atacattcaa atatgtatcc gctcatgaga
     3301 caataaccct gataaatgct tcaataatat tgaaaaagga agagtcctga ggcggaaaga
     3361 accagctgtg gaatgtgtgt cagttagggt gtggaaagtc cccaggctcc ccagcaggca
     3421 gaagtatgca aagcatgcat ctcaattagt cagcaaccag gtgtggaaag tccccaggct
     3481 ccccagcagg cagaagtatg caaagcatgc atctcaatta gtcagcaacc atagtcccgc
     3541 ccctaactcc gcccatcccg cccctaactc cgcccagttc cgcccattct ccgccccatg
     3601 gctgactaat tttttttatt tatgcagagg ccgaggccgc ctcggcctct gagctattcc
     3661 agaagtagtg aggaggcttt tttggaggcc taggcttttg caaagatcga tcaagagaca
     3721 ggatgaggat cgtttcgcat gattgaacaa gatggattgc acgcaggttc tccggccgct
     3781 tgggtggaga ggctattcgg ctatgactgg gcacaacaga caatcggctg ctctgatgcc
     3841 gccgtgttcc ggctgtcagc gcaggggcgc ccggttcttt ttgtcaagac cgacctgtcc
     3901 ggtgccctga atgaactgca agacgaggca gcgcggctat cgtggctggc cacgacgggc
     3961 gttccttgcg cagctgtgct cgacgttgtc actgaagcgg gaagggactg gctgctattg
     4021 ggcgaagtgc cggggcagga tctcctgtca tctcaccttg ctcctgccga gaaagtatcc
     4081 atcatggctg atgcaatgcg gcggctgcat acgcttgatc cggctacctg cccattcgac
     4141 caccaagcga aacatcgcat cgagcgagca cgtactcgga tggaagccgg tcttgtcgat
     4201 caggatgatc tggacgaaga gcatcagggg ctcgcgccag ccgaactgtt cgccaggctc
     4261 aaggcgagca tgcccgacgg cgaggatctc gtcgtgaccc atggcgatgc ctgcttgccg
     4321 aatatcatgg tggaaaatgg ccgcttttct ggattcatcg actgtggccg gctgggtgtg
     4381 gcggaccgct atcaggacat agcgttggct acccgtgata ttgctgaaga gcttggcggc
     4441 gaatgggctg accgcttcct cgtgctttac ggtatcgccg ctcccgattc gcagcgcatc
     4501 gccttctatc gccttcttga cgagttcttc tgagcgggac tctggggttc gaaatgaccg
     4561 accaagcgac gcccaacctg ccatcacgag atttcgattc caccgccgcc ttctatgaaa
     4621 ggttgggctt cggaatcgtt ttccgggacg ccggctggat gatcctccag cgcggggatc
     4681 tcatgctgga gttcttcgcc caccctaggg ggaggctaac tgaaacacgg aaggagacaa
     4741 taccggaagg aacccgcgct atgacggcaa taaaaagaca gaataaaacg cacggtgttg
     4801 ggtcgtttgt tcataaacgc ggggttcggt cccagggctg gcactctgtc gataccccac
     4861 cgagacccca ttggggccaa tacgcccgcg tttcttcctt ttccccaccc caccccccaa
     4921 gttcgggtga aggcccaggg ctcgcagcca acgtcggggc ggcaggccct gccatagcct
     4981 caggttactc atatatactt tagattgatt taaaacttca tttttaattt aaaaggatct
     5041 aggtgaagat cctttttgat aatctcatga ccaaaatccc ttaacgtgag ttttcgttcc
     5101 actgagcgtc agaccccgta gaaaagatca aaggatcttc ttgagatcct ttttttctgc
     5161 gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt tgtttgccgg
     5221 atcaagagct accaactctt tttccgaagg taactggctt cagcagagcg cagataccaa
     5281 atactgttct tctagtgtag ccgtagttag gccaccactt caagaactct gtagcaccgc
     5341 ctacatacct cgctctgcta atcctgttac cagtggctgc tgccagtggc gataagtcgt
     5401 gtcttaccgg gttggactca agacgatagt taccggataa ggcgcagcgg tcgggctgaa
     5461 cggggggttc gtgcacacag cccagcttgg agcgaacgac ctacaccgaa ctgagatacc
     5521 tacagcgtga gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc
     5581 cggtaagcgg cagggtcgga acaggagagc gcacgaggga gcttccaggg ggaaacgcct
     5641 ggtatcttta tagtcctgtc gggtttcgcc acctctgact tgagcgtcga tttttgtgat
     5701 gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa cgcggccttt ttacggttcc
     5761 tggccttttg ctggcctttt gctcacatgt tctttcctgc gttatcccct gattctgtgg
     5821 ataaccgtat taccgccatg
//
