LOCUS       sequence-340529-        5228 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian expression of a protein of interest fused to the 
            N-terminus of eDHFR(69K6)-EGFP.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5228)
  AUTHORS   Suzuki S, Nakamura A, Hatano Y, Yoshikawa M, Yoshii T, Sawada S, 
            Atsuta-Tsunoda K, Aoki K, Tsukiji S
  TITLE     A chemogenetic platform for controlling plasma membrane signaling 
            and synthetic signal oscillation.
  JOURNAL   Cell Chem Biol. 2022 Sep 15;29(9):1446-1464.e10. doi: 
            10.1016/j.chembiol.2022.06.005. Epub 2022 Jul 13.
  PUBMED    35835118
REFERENCE   2  (bases 1 to 5228)
  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: 
            "10.1016/j.chembiol.2022.06.005"; journalName: "Cell Chem Biol"; 
            date: "2022-09-15- 15"; volume: "29"; issue: "9"; pages: 
            "1446-1464.e10"
COMMENT     Sequence Label: pCMV-MCS-eDHFR(69K6)-EGFP
FEATURES             Location/Qualifiers
     source          1..5228
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        73..376
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        377..580
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     531..551
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      1252..1261
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(1258..1260,1261..1263,1264..1977)
                     /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(1258..1260,1261..1263,1264..1974)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(1303..1324)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1564..1583)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1911..1932
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    2100..2221
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2137..2156)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2191..2210
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(2228..2683)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2365..2386)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2577..2596
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        2710..2814
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(2839..2859)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2883..3093
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     promoter        2905..3101
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      2952..3087
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             3136..3930
                     /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             3136..3930
                     /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(3190..3209)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3800..3819
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(4118..4137)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    4162..4209
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      4538..5126
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5027..5046
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 accgccatgc attagttatt aatagtaatc aattacgggg tcattagttc atagcccata
       61 tatggagttc cgcgttacat aacttacggt aaatggcccg cctggctgac cgcccaacga
      121 cccccgccca ttgacgtcaa taatgacgta tgttcccata gtaacgccaa tagggacttt
      181 ccattgacgt caatgggtgg agtatttacg gtaaactgcc cacttggcag tacatcaagt
      241 gtatcatatg ccaagtacgc cccctattga cgtcaatgac ggtaaatggc ccgcctggca
      301 ttatgcccag tacatgacct tatgggactt tcctacttgg cagtacatct acgtattagt
      361 catcgctatt accatggtga tgcggttttg gcagtacatc aatgggcgtg gatagcggtt
      421 tgactcacgg ggatttccaa gtctccaccc cattgacgtc aatgggagtt tgttttggca
      481 ccaaaatcaa cgggactttc caaaatgtcg taacaactcc gccccattga cgcaaatggg
      541 cggtaggcgt gtacggtggg aggtctatat aagcagagct ggtttagtga accgtcagat
      601 ccgctagcgc taccggactc agatctcgag ctcaagcttg cgatcgctgc agcccgggcc
      661 gcggctggct ctggtgctgg tagtggcgct ggttccggtg ctggctctgg cgcgtcgacg
      721 gtacccgcca ccatgatcag tctgattgcg gcgttagcgg tagatcgcgt tatcggcatg
      781 gaaaacgcca tgccgtggaa cctgcctgcc gatctcgcct ggtttaaacg caacacctta
      841 aataaacccg tgattatggg ccgccatacc tgggaatcaa tcggtcgtcc gttgccagga
      901 cgcaaaaata ttatcctcag cagtcaaccg ggtacggaca aaaaaaagaa aaagaaagat
      961 cgcgtaacgt gggtgaagtc ggtggatgaa gccatcgcgg cgtgtggtga cgtaccagaa
     1021 atcatggtga ttggcggcgg tcgcgtttat gaacagttct tgccaaaagc gcaaaaactg
     1081 tatctgacgc atatcgacgc agaagtggaa ggcgacaccc atttcccgga ttacgagccg
     1141 gatgactggg aatcggtatt cagcgaattc cacgatgctg atgcgcagaa ctctcacagc
     1201 tattgctttg agattctgga gcggcggagc ggctccgggg atccaccggt cgccaccatg
     1261 gtgagcaagg gcgaggagct gttcaccggg gtggtgccca tcctggtcga gctggacggc
     1321 gacgtaaacg gccacaagtt cagcgtgtcc ggcgagggcg agggcgatgc cacctacggc
     1381 aagctgaccc tgaagttcat ctgcaccacc ggcaagctgc ccgtgccctg gcccaccctc
     1441 gtgaccaccc tgacctacgg cgtgcagtgc ttcagccgct accccgacca catgaagcag
     1501 cacgacttct tcaagtccgc catgcccgaa ggctacgtcc aggagcgcac catcttcttc
     1561 aaggacgacg gcaactacaa gacccgcgcc gaggtgaagt tcgagggcga caccctggtg
     1621 aaccgcatcg agctgaaggg catcgacttc aaggaggacg gcaacatcct ggggcacaag
     1681 ctggagtaca actacaacag ccacaacgtc tatatcatgg ccgacaagca gaagaacggc
     1741 atcaaggtga acttcaagat ccgccacaac atcgaggacg gcagcgtgca gctcgccgac
     1801 cactaccagc agaacacccc catcggcgac ggccccgtgc tgctgcccga caaccactac
     1861 ctgagcaccc agtccgccct gagcaaagac cccaacgaga agcgcgatca catggtcctg
     1921 ctggagttcg tgaccgccgc cgggatcact ctcggcatgg acgagctgta caagtaaagc
     1981 ggccgcgact ctagatcata atcagccata ccacatttgt agaggtttta cttgctttaa
     2041 aaaacctccc acacctcccc ctgaacctga aacataaaat gaatgcaatt gttgttgtta
     2101 acttgtttat tgcagcttat aatggttaca aataaagcaa tagcatcaca aatttcacaa
     2161 ataaagcatt tttttcactg cattctagtt gtggtttgtc caaactcatc aatgtatctt
     2221 aaggcgtaaa ttgtaagcgt taatattttg ttaaaattcg cgttaaattt ttgttaaatc
     2281 agctcatttt ttaaccaata ggccgaaatc ggcaaaatcc cttataaatc aaaagaatag
     2341 accgagatag ggttgagtgt tgttccagtt tggaacaaga gtccactatt aaagaacgtg
     2401 gactccaacg tcaaagggcg aaaaaccgtc tatcagggcg atggcccact acgtgaacca
     2461 tcaccctaat caagtttttt ggggtcgagg tgccgtaaag cactaaatcg gaaccctaaa
     2521 gggagccccc gatttagagc ttgacgggga aagccggcga acgtggcgag aaaggaaggg
     2581 aagaaagcga aaggagcggg cgctagggcg ctggcaagtg tagcggtcac gctgcgcgta
     2641 accaccacac ccgccgcgct taatgcgccg ctacagggcg cgtcaggtgg cacttttcgg
     2701 ggaaatgtgc gcggaacccc tatttgttta tttttctaaa tacattcaaa tatgtatccg
     2761 ctcatgagac aataaccctg ataaatgctt caataatatt gaaaaaggaa gagtcctgag
     2821 gcggaaagaa ccagctgtgg aatgtgtgtc agttagggtg tggaaagtcc ccaggctccc
     2881 cagcaggcag aagtatgcaa agcatgcatc tcaattagtc agcaaccata gtcccgcccc
     2941 taactccgcc catcccgccc ctaactccgc ccagttccgc ccattctccg ccccatggct
     3001 gactaatttt ttttatttat gcagaggccg aggccgcctc ggcctctgag ctattccaga
     3061 agtagtgagg aggctttttt ggaggcctag gcttttgcaa agatcgatca agagacagga
     3121 tgaggatcgt ttcgcatgat tgaacaagat ggattgcacg caggttctcc ggccgcttgg
     3181 gtggagaggc tattcggcta tgactgggca caacagacaa tcggctgctc tgatgccgcc
     3241 gtgttccggc tgtcagcgca ggggcgcccg gttctttttg tcaagaccga cctgtccggt
     3301 gccctgaatg aactgcaaga cgaggcagcg cggctatcgt ggctggccac gacgggcgtt
     3361 ccttgcgcag ctgtgctcga cgttgtcact gaagcgggaa gggactggct gctattgggc
     3421 gaagtgccgg ggcaggatct cctgtcatct caccttgctc ctgccgagaa agtatccatc
     3481 atggctgatg caatgcggcg gctgcatacg cttgatccgg ctacctgccc attcgaccac
     3541 caagcgaaac atcgcatcga gcgagcacgt actcggatgg aagccggtct tgtcgatcag
     3601 gatgatctgg acgaagagca tcaggggctc gcgccagccg aactgttcgc caggctcaag
     3661 gcgagcatgc ccgacggcga ggatctcgtc gtgacccatg gcgatgcctg cttgccgaat
     3721 atcatggtgg aaaatggccg cttttctgga ttcatcgact gtggccggct gggtgtggcg
     3781 gaccgctatc aggacatagc gttggctacc cgtgatattg ctgaagagct tggcggcgaa
     3841 tgggctgacc gcttcctcgt gctttacggt atcgccgctc ccgattcgca gcgcatcgcc
     3901 ttctatcgcc ttcttgacga gttcttctga gcgggactct ggggttcgaa atgaccgacc
     3961 aagcgacgcc caacctgcca tcacgagatt tcgattccac cgccgccttc tatgaaaggt
     4021 tgggcttcgg aatcgttttc cgggacgccg gctggatgat cctccagcgc ggggatctca
     4081 tgctggagtt cttcgcccac cctaggggga ggctaactga aacacggaag gagacaatac
     4141 cggaaggaac ccgcgctatg acggcaataa aaagacagaa taaaacgcac ggtgttgggt
     4201 cgtttgttca taaacgcggg gttcggtccc agggctggca ctctgtcgat accccaccga
     4261 gaccccattg gggccaatac gcccgcgttt cttccttttc cccaccccac cccccaagtt
     4321 cgggtgaagg cccagggctc gcagccaacg tcggggcggc aggccctgcc atagcctcag
     4381 gttactcata tatactttag attgatttaa aacttcattt ttaatttaaa aggatctagg
     4441 tgaagatcct ttttgataat ctcatgacca aaatccctta acgtgagttt tcgttccact
     4501 gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg agatcctttt tttctgcgcg
     4561 taatctgctg cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc
     4621 aagagctacc aactcttttt ccgaaggtaa ctggcttcag cagagcgcag ataccaaata
     4681 ctgttcttct agtgtagccg tagttaggcc accacttcaa gaactctgta gcaccgccta
     4741 catacctcgc tctgctaatc ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc
     4801 ttaccgggtt ggactcaaga cgatagttac cggataaggc gcagcggtcg ggctgaacgg
     4861 ggggttcgtg cacacagccc agcttggagc gaacgaccta caccgaactg agatacctac
     4921 agcgtgagct atgagaaagc gccacgcttc ccgaagggag aaaggcggac aggtatccgg
     4981 taagcggcag ggtcggaaca ggagagcgca cgagggagct tccaggggga aacgcctggt
     5041 atctttatag tcctgtcggg tttcgccacc tctgacttga gcgtcgattt ttgtgatgct
     5101 cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc ggccttttta cggttcctgg
     5161 ccttttgctg gccttttgct cacatgttct ttcctgcgtt atcccctgat tctgtggata
     5221 accgtatt
//
