LOCUS       sequence-340555-        5265 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian expression of a protein of interest fused to the 
            C-terminus of EGFP-eDHFR.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5265)
  AUTHORS   Ishida M, Watanabe H, Takigawa K, Kurishita Y, Oki C, Nakamura A, 
            Hamachi I, Tsukiji S
  TITLE     Synthetic self-localizing ligands that control the spatial location 
            of proteins in living cells.
  JOURNAL   J Am Chem Soc. 2013 Aug 28;135(34):12684-9. doi: 10.1021/ja4046907. 
            Epub 2013 Aug 14.
  PUBMED    23941503
REFERENCE   2  (bases 1 to 5265)
  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"; doi: "10.1021/ja4046907";
            journalName: "J Am Chem Soc"; date: "2013-08-28- 28"; volume: "135";
            issue: "34"; pages: "12684-9"
COMMENT     Sequence Label: pEGFP-eDHFR-MCS
FEATURES             Location/Qualifiers
     source          1..5265
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        96..399
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        400..603
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     554..574
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      642..651
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(648..650,651..653,654..1364)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(648..650,651..653,654..1364)
                     /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(693..714)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(954..973)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1301..1322
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             1377..1850
                     /codon_start=1
                     /gene="folA"
                     /product="E. coli dihydrofolate reductase"
                     /label=ecDHFR
                     /translation="ISLIAALAVDRVIGMENAMPWNLPADLAWFKRNTLNKPVIMGRHT
                     WESIGRPLPGRKNIILSSQPGTDDRVTWVKSVDEAIAACGDVPEIMVIGGGRVYEQFLP
                     KAQKLYLTHIDAEVEGDTHFPDYEPDDWESVFSEFHDADAQNSHSYCFEILERR"
     misc_feature    1912..1964
                     /label=MCS
                     /note="multiple cloning site"
     polyA_signal    2088..2209
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2125..2144)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2179..2198
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(2216..2671)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2353..2374)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2565..2584
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        2698..2802
                     /gene="bla"
                     /label=AmpR promoter
     promoter        2804..3161
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        2804..2995
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(2827..2847)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      3012..3147
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             3196..3990
                     /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             3196..3990
                     /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(3250..3269)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3860..3879
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(4178..4197)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    4222..4269
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      4598..5186
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5087..5106
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 ctgattctgt ggataaccgt attaccgcca tgcattagtt attaatagta atcaattacg
       61 gggtcattag ttcatagccc atatatggag ttccgcgtta cataacttac ggtaaatggc
      121 ccgcctggct gaccgcccaa cgacccccgc ccattgacgt caataatgac gtatgttccc
      181 atagtaacgc caatagggac tttccattga cgtcaatggg tggagtattt acggtaaact
      241 gcccacttgg cagtacatca agtgtatcat atgccaagta cgccccctat tgacgtcaat
      301 gacggtaaat ggcccgcctg gcattatgcc cagtacatga ccttatggga ctttcctact
      361 tggcagtaca tctacgtatt agtcatcgct attaccatgg tgatgcggtt ttggcagtac
      421 atcaatgggc gtggatagcg gtttgactca cggggatttc caagtctcca ccccattgac
      481 gtcaatggga gtttgttttg gcaccaaaat caacgggact ttccaaaatg tcgtaacaac
      541 tccgccccat tgacgcaaat gggcggtagg cgtgtacggt gggaggtcta tataagcaga
      601 gctggtttag tgaaccgtca gatccgctag cgctaccggt cgccaccatg gtgagcaagg
      661 gcgaggagct gttcaccggg gtggtgccca tcctggtcga gctggacggc gacgtaaacg
      721 gccacaagtt cagcgtgtcc ggcgagggcg agggcgatgc cacctacggc aagctgaccc
      781 tgaagttcat ctgcaccacc ggcaagctgc ccgtgccctg gcccaccctc gtgaccaccc
      841 tgacctacgg cgtgcagtgc ttcagccgct accccgacca catgaagcag cacgacttct
      901 tcaagtccgc catgcccgaa ggctacgtcc aggagcgcac catcttcttc aaggacgacg
      961 gcaactacaa gacccgcgcc gaggtgaagt tcgagggcga caccctggtg aaccgcatcg
     1021 agctgaaggg catcgacttc aaggaggacg gcaacatcct ggggcacaag ctggagtaca
     1081 actacaacag ccacaacgtc tatatcatgg ccgacaagca gaagaacggc atcaaggtga
     1141 acttcaagat ccgccacaac atcgaggacg gcagcgtgca gctcgccgac cactaccagc
     1201 agaacacccc catcggcgac ggccccgtgc tgctgcccga caaccactac ctgagcaccc
     1261 agtccgccct gagcaaagac cccaacgaga agcgcgatca catggtcctg ctggagttcg
     1321 tgaccgccgc cgggatcact ctcggcatgg acgagctgta caagagtgct ggtggtatca
     1381 gtctgattgc ggcgttagcg gtagatcgcg ttatcggcat ggaaaacgcc atgccgtgga
     1441 acctgcctgc cgatctcgcc tggtttaaac gcaacacctt aaataaaccc gtgattatgg
     1501 gccgccatac ctgggaatca atcggtcgtc cgttgccagg acgcaaaaat attatcctca
     1561 gcagtcaacc gggtacggac gatcgcgtaa cgtgggtgaa gtcggtggat gaagccatcg
     1621 cggcgtgtgg tgacgtacca gaaatcatgg tgattggcgg cggtcgcgtt tatgaacagt
     1681 tcttgccaaa agcgcaaaaa ctgtatctga cgcatatcga cgcagaagtg gaaggcgaca
     1741 cccatttccc ggattacgag ccggatgact gggaatcggt attcagcgaa ttccacgatg
     1801 ctgatgcgca gaactctcac agctattgct ttgagattct ggagcggcgg agtgctggtg
     1861 gtagtgctgg tggtagtgct ggtggtagtg ctggtggtag tgctggtggt cctcgagctc
     1921 aagcttcgaa ttctgcagtc gacggtaccg cgggcccggg atccaccgga tctagataac
     1981 tgatcataat cagccatacc acatttgtag aggttttact tgctttaaaa aacctcccac
     2041 acctccccct gaacctgaaa cataaaatga atgcaattgt tgttgttaac ttgtttattg
     2101 cagcttataa tggttacaaa taaagcaata gcatcacaaa tttcacaaat aaagcatttt
     2161 tttcactgca ttctagttgt ggtttgtcca aactcatcaa tgtatcttaa ggcgtaaatt
     2221 gtaagcgtta atattttgtt aaaattcgcg ttaaattttt gttaaatcag ctcatttttt
     2281 aaccaatagg ccgaaatcgg caaaatccct tataaatcaa aagaatagac cgagataggg
     2341 ttgagtgttg ttccagtttg gaacaagagt ccactattaa agaacgtgga ctccaacgtc
     2401 aaagggcgaa aaaccgtcta tcagggcgat ggcccactac gtgaaccatc accctaatca
     2461 agttttttgg ggtcgaggtg ccgtaaagca ctaaatcgga accctaaagg gagcccccga
     2521 tttagagctt gacggggaaa gccggcgaac gtggcgagaa aggaagggaa gaaagcgaaa
     2581 ggagcgggcg ctagggcgct ggcaagtgta gcggtcacgc tgcgcgtaac caccacaccc
     2641 gccgcgctta atgcgccgct acagggcgcg tcaggtggca cttttcgggg aaatgtgcgc
     2701 ggaaccccta tttgtttatt tttctaaata cattcaaata tgtatccgct catgagacaa
     2761 taaccctgat aaatgcttca ataatattga aaaaggaaga gtcctgaggc ggaaagaacc
     2821 agctgtggaa tgtgtgtcag ttagggtgtg gaaagtcccc aggctcccca gcaggcagaa
     2881 gtatgcaaag catgcatctc aattagtcag caaccaggtg tggaaagtcc ccaggctccc
     2941 cagcaggcag aagtatgcaa agcatgcatc tcaattagtc agcaaccata gtcccgcccc
     3001 taactccgcc catcccgccc ctaactccgc ccagttccgc ccattctccg ccccatggct
     3061 gactaatttt ttttatttat gcagaggccg aggccgcctc ggcctctgag ctattccaga
     3121 agtagtgagg aggctttttt ggaggcctag gcttttgcaa agatcgatca agagacagga
     3181 tgaggatcgt ttcgcatgat tgaacaagat ggattgcacg caggttctcc ggccgcttgg
     3241 gtggagaggc tattcggcta tgactgggca caacagacaa tcggctgctc tgatgccgcc
     3301 gtgttccggc tgtcagcgca ggggcgcccg gttctttttg tcaagaccga cctgtccggt
     3361 gccctgaatg aactgcaaga cgaggcagcg cggctatcgt ggctggccac gacgggcgtt
     3421 ccttgcgcag ctgtgctcga cgttgtcact gaagcgggaa gggactggct gctattgggc
     3481 gaagtgccgg ggcaggatct cctgtcatct caccttgctc ctgccgagaa agtatccatc
     3541 atggctgatg caatgcggcg gctgcatacg cttgatccgg ctacctgccc attcgaccac
     3601 caagcgaaac atcgcatcga gcgagcacgt actcggatgg aagccggtct tgtcgatcag
     3661 gatgatctgg acgaagagca tcaggggctc gcgccagccg aactgttcgc caggctcaag
     3721 gcgagcatgc ccgacggcga ggatctcgtc gtgacccatg gcgatgcctg cttgccgaat
     3781 atcatggtgg aaaatggccg cttttctgga ttcatcgact gtggccggct gggtgtggcg
     3841 gaccgctatc aggacatagc gttggctacc cgtgatattg ctgaagagct tggcggcgaa
     3901 tgggctgacc gcttcctcgt gctttacggt atcgccgctc ccgattcgca gcgcatcgcc
     3961 ttctatcgcc ttcttgacga gttcttctga gcgggactct ggggttcgaa atgaccgacc
     4021 aagcgacgcc caacctgcca tcacgagatt tcgattccac cgccgccttc tatgaaaggt
     4081 tgggcttcgg aatcgttttc cgggacgccg gctggatgat cctccagcgc ggggatctca
     4141 tgctggagtt cttcgcccac cctaggggga ggctaactga aacacggaag gagacaatac
     4201 cggaaggaac ccgcgctatg acggcaataa aaagacagaa taaaacgcac ggtgttgggt
     4261 cgtttgttca taaacgcggg gttcggtccc agggctggca ctctgtcgat accccaccga
     4321 gaccccattg gggccaatac gcccgcgttt cttccttttc cccaccccac cccccaagtt
     4381 cgggtgaagg cccagggctc gcagccaacg tcggggcggc aggccctgcc atagcctcag
     4441 gttactcata tatactttag attgatttaa aacttcattt ttaatttaaa aggatctagg
     4501 tgaagatcct ttttgataat ctcatgacca aaatccctta acgtgagttt tcgttccact
     4561 gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg agatcctttt tttctgcgcg
     4621 taatctgctg cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc
     4681 aagagctacc aactcttttt ccgaaggtaa ctggcttcag cagagcgcag ataccaaata
     4741 ctgttcttct agtgtagccg tagttaggcc accacttcaa gaactctgta tcaccgccta
     4801 catacctcgc tctgctaatc ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc
     4861 ttaccgggtt ggactcaaga cgatagttac cggataaggc gcagcggtcg ggctgaacgg
     4921 ggggttcgtg cacacagccc agcttggagc gaacgaccta caccgaactg agatacctac
     4981 agcgtgagct atgagaaagc gccacgcttc ccgaagggag aaaggcggac aggtatccgg
     5041 taagcggcag ggtcggaaca ggagagcgca cgagggagct tccaggggga aacgcctggt
     5101 atctttatag tcctgtcggg tttcgccacc tctgacttga gcgtcgattt ttgtgatgct
     5161 cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc ggccttttta cggttcctgg
     5221 ccttttgctg gccttttgct cacatgttct ttcctgcgtt atccc
//
