LOCUS       sequence-363158-        6262 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses EGFP and tdTomato in mammalian cells under control of a 
            CMV promoter by using an IRES sequence.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6262)
  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 6262)
  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_IRES_tdTomato
FEATURES             Location/Qualifiers
     source          1..6262
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        61..364
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        365..568
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     519..539
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      607..616
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(613..615,616..618,619..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"
     CDS             join(613..615,616..618,619..1329)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(658..679)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(919..938)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1266..1287
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     regulatory      1926..1935
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1932..3362
                     /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    3506..3627
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3543..3562)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3597..3616
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     promoter        3660..3764
                     /gene="bla"
                     /label=AmpR promoter
     promoter        3766..4123
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        3766..3957
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(3789..3809)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      3974..4109
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             4158..4952
                     /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             4158..4952
                     /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(4212..4231)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4822..4841
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(5140..5159)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    5184..5231
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      5560..6148
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6049..6068
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata tggagttccg
       61 cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc cccgcccatt
      121 gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc attgacgtca
      181 atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt atcatatgcc
      241 aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt atgcccagta
      301 catgacctta tgggactttc ctacttggca gtacatctac gtattagtca tcgctattac
      361 catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg actcacgggg
      421 atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc aaaatcaacg
      481 ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg gtaggcgtgt
      541 acggtgggag gtctatataa gcagagctgg tttagtgaac cgtcagatcc gctagcgcta
      601 ccggtcgcca ccatggtgag caagggcgag gagctgttca ccggggtggt gcccatcctg
      661 gtcgagctgg acggcgacgt aaacggccac aagttcagcg tgtccggcga gggcgagggc
      721 gatgccacct acggcaagct gaccctgaag ttcatctgca ccaccggcaa gctgcccgtg
      781 ccctggccca ccctcgtgac caccctgacc tacggcgtgc agtgcttcag ccgctacccc
      841 gaccacatga agcagcacga cttcttcaag tccgccatgc ccgaaggcta cgtccaggag
      901 cgcaccatct tcttcaagga cgacggcaac tacaagaccc gcgccgaggt gaagttcgag
      961 ggcgacaccc tggtgaaccg catcgagctg aagggcatcg acttcaagga ggacggcaac
     1021 atcctggggc acaagctgga gtacaactac aacagccaca acgtctatat catggccgac
     1081 aagcagaaga acggcatcaa ggtgaacttc aagatccgcc acaacatcga ggacggcagc
     1141 gtgcagctcg ccgaccacta ccagcagaac acccccatcg gcgacggccc cgtgctgctg
     1201 cccgacaacc actacctgag cacccagtcc gccctgagca aagaccccaa cgagaagcgc
     1261 gatcacatgg tcctgctgga gttcgtgacc gccgccggga tcactctcgg catggacgag
     1321 ctgtacaagt aactcgagta ctccggtatt gcggtaccct tgtacgcctg ttttatactc
     1381 ccttcccgta acttagacgc acaaaaccaa gttcaataga agggggtaca aaccagtacc
     1441 accacgaaca agcacttctg tttccccggt gatgtcgtat agactgcttg cgtggttgaa
     1501 agcgacggat ccgttatccg cttatgtact tcgagaagcc cagtaccacc tcggaatctt
     1561 cgatgcgttg cgctcagcac tcaaccccag agtgtagctt aggctgatga gtctggacat
     1621 ccctcaccgg tgacggtggt ccaggctgcg ttggcggcct acctatggct aacgccatgg
     1681 gacgctagtt gtgaacaagg tgtgaagagc ctattgagct acataagaat cctccggccc
     1741 ctgaatgcgg ctaatcccaa cctcggagca ggtggtcaca aaccagtgat tggcctgtcg
     1801 taacgcgcaa gtccgtggcg gaaccgacta ctttgggtgt ccgtgtttcc ttttatttta
     1861 ttgtggctgc ttatggtgac aatcacagat tgttatcata aagcgaattg gataggatcc
     1921 gtacggccac catggtgagc aagggcgagg aggtcatcaa agagttcatg cgcttcaagg
     1981 tgcgcatgga gggctccatg aacggccacg agttcgagat cgagggcgag ggcgagggcc
     2041 gcccctacga gggcacccag accgccaagc tgaaggtgac caagggcggc cccctgccct
     2101 tcgcctggga catcctgtcc ccccagttca tgtacggctc caaggcgtac gtgaagcacc
     2161 ccgccgacat ccccgattac aagaagctgt ccttccccga gggcttcaag tgggagcgcg
     2221 tgatgaactt cgaggacggc ggtctggtga ccgtgaccca ggactcctcc ctgcaggacg
     2281 gcacgctgat ctacaaggtg aagatgcgcg gcaccaactt cccccccgac ggccccgtaa
     2341 tgcagaagaa gaccatgggc tgggaggcct ccaccgagcg cctgtacccc cgcgacggcg
     2401 tgctgaaggg cgagatccac caggccctga agctgaagga cggcggccac tacctggtgg
     2461 agttcaagac catctacatg gccaagaagc ccgtgcaact gcccggctac tactacgtgg
     2521 acaccaagct ggacatcacc tcccacaacg aggactacac catcgtggaa cagtacgagc
     2581 gctccgaggg ccgccaccac ctgttcctgg ggcatggcac cggcagcacc ggcagcggca
     2641 gctccggcac cgcctcctcc gaggacaaca acatggccgt catcaaagag ttcatgcgct
     2701 tcaaggtgcg catggagggc tccatgaacg gccacgagtt cgagatcgag ggcgagggcg
     2761 agggccgccc ctacgagggc acccagaccg ccaagctgaa ggtgaccaag ggcggccccc
     2821 tgcccttcgc ctgggacatc ctgtcccccc agttcatgta cggctccaag gcgtacgtga
     2881 agcaccccgc cgacatcccc gattacaaga agctgtcctt ccccgagggc ttcaagtggg
     2941 agcgcgtgat gaacttcgag gacggcggtc tggtgaccgt gacccaggac tcctccctgc
     3001 aggacggcac gctgatctac aaggtgaaga tgcgcggcac caacttcccc cccgacggcc
     3061 ccgtaatgca gaagaagacc atgggctggg aggcctccac cgagcgcctg tacccccgcg
     3121 acggcgtgct gaagggcgag atccaccagg ccctgaagct gaaggacggc ggccactacc
     3181 tggtggagtt caagaccatc tacatggcca agaagcccgt gcaactgccc ggctactact
     3241 acgtggacac caagctggac atcacctccc acaacgagga ctacaccatc gtggaacagt
     3301 acgagcgctc cgagggccgc caccacctgt tcctgtacgg catggacgag ctgtacaagt
     3361 agagcttaat tctcgaagat ccaccggatc tagataactg atcataatca gccataccac
     3421 atttgtagag gttttacttg ctttaaaaaa cctcccacac ctccccctga acctgaaaca
     3481 taaaatgaat gcaattgttg ttgttaactt gtttattgca gcttataatg gttacaaata
     3541 aagcaatagc atcacaaatt tcacaaataa agcatttttt tcactgcatt ctagttgtgg
     3601 tttgtccaaa ctcatcaatg tatcttaacg cgtcaggtgg cacttttcgg ggaaatgtgc
     3661 gcggaacccc tatttgttta tttttctaaa tacattcaaa tatgtatccg ctcatgagac
     3721 aataaccctg ataaatgctt caataatatt gaaaaaggaa gagtcctgag gcggaaagaa
     3781 ccagctgtgg aatgtgtgtc agttagggtg tggaaagtcc ccaggctccc cagcaggcag
     3841 aagtatgcaa agcatgcatc tcaattagtc agcaaccagg tgtggaaagt ccccaggctc
     3901 cccagcaggc agaagtatgc aaagcatgca tctcaattag tcagcaacca tagtcccgcc
     3961 cctaactccg cccatcccgc ccctaactcc gcccagttcc gcccattctc cgccccatgg
     4021 ctgactaatt ttttttattt atgcagaggc cgaggccgcc tcggcctctg agctattcca
     4081 gaagtagtga ggaggctttt ttggaggcct aggcttttgc aaagatcgat caagagacag
     4141 gatgaggatc gtttcgcatg attgaacaag atggattgca cgcaggttct ccggccgctt
     4201 gggtggagag gctattcggc tatgactggg cacaacagac aatcggctgc tctgatgccg
     4261 ccgtgttccg gctgtcagcg caggggcgcc cggttctttt tgtcaagacc gacctgtccg
     4321 gtgccctgaa tgaactgcaa gacgaggcag cgcggctatc gtggctggcc acgacgggcg
     4381 ttccttgcgc agctgtgctc gacgttgtca ctgaagcggg aagggactgg ctgctattgg
     4441 gcgaagtgcc ggggcaggat ctcctgtcat ctcaccttgc tcctgccgag aaagtatcca
     4501 tcatggctga tgcaatgcgg cggctgcata cgcttgatcc ggctacctgc ccattcgacc
     4561 accaagcgaa acatcgcatc gagcgagcac gtactcggat ggaagccggt cttgtcgatc
     4621 aggatgatct ggacgaagag catcaggggc tcgcgccagc cgaactgttc gccaggctca
     4681 aggcgagcat gcccgacggc gaggatctcg tcgtgaccca tggcgatgcc tgcttgccga
     4741 atatcatggt ggaaaatggc cgcttttctg gattcatcga ctgtggccgg ctgggtgtgg
     4801 cggaccgcta tcaggacata gcgttggcta cccgtgatat tgctgaagag cttggcggcg
     4861 aatgggctga ccgcttcctc gtgctttacg gtatcgccgc tcccgattcg cagcgcatcg
     4921 ccttctatcg ccttcttgac gagttcttct gagcgggact ctggggttcg aaatgaccga
     4981 ccaagcgacg cccaacctgc catcacgaga tttcgattcc accgccgcct tctatgaaag
     5041 gttgggcttc ggaatcgttt tccgggacgc cggctggatg atcctccagc gcggggatct
     5101 catgctggag ttcttcgccc accctagggg gaggctaact gaaacacgga aggagacaat
     5161 accggaagga acccgcgcta tgacggcaat aaaaagacag aataaaacgc acggtgttgg
     5221 gtcgtttgtt cataaacgcg gggttcggtc ccagggctgg cactctgtcg ataccccacc
     5281 gagaccccat tggggccaat acgcccgcgt ttcttccttt tccccacccc accccccaag
     5341 ttcgggtgaa ggcccagggc tcgcagccaa cgtcggggcg gcaggccctg ccatagcctc
     5401 aggttactca tatatacttt agattgattt aaaacttcat ttttaattta aaaggatcta
     5461 ggtgaagatc ctttttgata atctcatgac caaaatccct taacgtgagt tttcgttcca
     5521 ctgagcgtca gaccccgtag aaaagatcaa aggatcttct tgagatcctt tttttctgcg
     5581 cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca gcggtggttt gtttgccgga
     5641 tcaagagcta ccaactcttt ttccgaaggt aactggcttc agcagagcgc agataccaaa
     5701 tactgttctt ctagtgtagc cgtagttagg ccaccacttc aagaactctg tagcaccgcc
     5761 tacatacctc gctctgctaa tcctgttacc agtggctgct gccagtggcg ataagtcgtg
     5821 tcttaccggg ttggactcaa gacgatagtt accggataag gcgcagcggt cgggctgaac
     5881 ggggggttcg tgcacacagc ccagcttgga gcgaacgacc tacaccgaac tgagatacct
     5941 acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc
     6001 ggtaagcggc agggtcggaa caggagagcg cacgagggag cttccagggg gaaacgcctg
     6061 gtatctttat agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg
     6121 ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac gcggcctttt tacggttcct
     6181 ggccttttgc tggccttttg ctcacatgtt ctttcctgcg ttatcccctg attctgtgga
     6241 taaccgtatt accgccatgc at
//
