LOCUS       sequence-347460-        5306 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian expression of a protein of interest fused to the 
            C-terminus of EGFP-eDHFR(69K6).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5306)
  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 5306)
  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-EGFP-eDHFR(69K6)-MCS
FEATURES             Location/Qualifiers
     source          1..5306
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(144..163)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    188..235
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      564..1152
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1053..1072
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     enhancer        1327..1630
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        1631..1834
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     1785..1805
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      1875..1884
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(1881..1883,1884..1886,1887..2597)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(1881..1883,1884..1886,1887..2597)
                     /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(1926..1947)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2187..2206)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2534..2555
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     misc_feature    3184..3236
                     /label=MCS
                     /note="multiple cloning site"
     polyA_signal    3360..3481
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3397..3416)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3451..3470
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(3488..3943)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3625..3646)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3837..3856
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        3970..4074
                     /gene="bla"
                     /label=AmpR promoter
     promoter        4076..4433
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        4076..4267
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(4099..4119)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      4284..4419
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             4468..5262
                     /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             4468..5262
                     /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(4522..4541)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     5132..5151
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
ORIGIN
        1 ctgccatcac gagatttcga ttccaccgcc gccttctatg aaaggttggg cttcggaatc
       61 gttttccggg acgccggctg gatgatcctc cagcgcgggg atctcatgct ggagttcttc
      121 gcccacccta gggggaggct aactgaaaca cggaaggaga caataccgga aggaacccgc
      181 gctatgacgg caataaaaag acagaataaa acgcacggtg ttgggtcgtt tgttcataaa
      241 cgcggggttc ggtcccaggg ctggcactct gtcgataccc caccgagacc ccattggggc
      301 caatacgccc gcgtttcttc cttttcccca ccccaccccc caagttcggg tgaaggccca
      361 gggctcgcag ccaacgtcgg ggcggcaggc cctgccatag cctcaggtta ctcatatata
      421 ctttagattg atttaaaact tcatttttaa tttaaaagga tctaggtgaa gatccttttt
      481 gataatctca tgaccaaaat cccttaacgt gagttttcgt tccactgagc gtcagacccc
      541 gtagaaaaga tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg
      601 caaacaaaaa aaccaccgct accagcggtg gtttgtttgc cggatcaaga gctaccaact
      661 ctttttccga aggtaactgg cttcagcaga gcgcagatac caaatactgt ccttctagtg
      721 tagccgtagt taggccacca cttcaagaac tctgtagcac cgcctacata cctcgctctg
      781 ctaatcctgt taccagtggc tgctgccagt ggcgataagt cgtgtcttac cgggttggac
      841 tcaagacgat agttaccgga taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca
      901 cagcccagct tggagcgaac gacctacacc gaactgagat acctacagcg tgagctatga
      961 gaaagcgcca cgcttcccga agggagaaag gcggacaggt atccggtaag cggcagggtc
     1021 ggaacaggag agcgcacgag ggagcttcca gggggaaacg cctggtatct ttatagtcct
     1081 gtcgggtttc gccacctctg acttgagcgt cgatttttgt gatgctcgtc aggggggcgg
     1141 agcctatgga aaaacgccag caacgcggcc tttttacggt tcctggcctt ttgctggcct
     1201 tttgctcaca tgttctttcc tgcgttatcc cctgattctg tggataaccg tattaccgcc
     1261 atgcattagt tattaatagt aatcaattac ggggtcatta gttcatagcc catatatgga
     1321 gttccgcgtt acataactta cggtaaatgg cccgcctggc tgaccgccca acgacccccg
     1381 cccattgacg tcaataatga cgtatgttcc catagtaacg ccaataggga ctttccattg
     1441 acgtcaatgg gtggagtatt tacggtaaac tgcccacttg gcagtacatc aagtgtatca
     1501 tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct ggcattatgc
     1561 ccagtacatg accttatggg actttcctac ttggcagtac atctacgtat tagtcatcgc
     1621 tattaccatg gtgatgcggt tttggcagta catcaatggg cgtggatagc ggtttgactc
     1681 acggggattt ccaagtctcc accccattga cgtcaatggg agtttgtttt ggcaccaaaa
     1741 tcaacgggac tttccaaaat gtcgtaacaa ctccgcccca ttgacgcaaa tgggcggtag
     1801 gcgtgtacgg tgggaggtct atataagcag agctggttta gtgaaccgtc agatccgcta
     1861 gcgatccacc ggtcgccacc atggtgagca agggcgagga gctgttcacc ggggtggtgc
     1921 ccatcctggt cgagctggac ggcgacgtaa acggccacaa gttcagcgtg tccggcgagg
     1981 gcgagggcga tgccacctac ggcaagctga ccctgaagtt catctgcacc accggcaagc
     2041 tgcccgtgcc ctggcccacc ctcgtgacca ccctgaccta cggcgtgcag tgcttcagcc
     2101 gctaccccga ccacatgaag cagcacgact tcttcaagtc cgccatgccc gaaggctacg
     2161 tccaggagcg caccatcttc ttcaaggacg acggcaacta caagacccgc gccgaggtga
     2221 agttcgaggg cgacaccctg gtgaaccgca tcgagctgaa gggcatcgac ttcaaggagg
     2281 acggcaacat cctggggcac aagctggagt acaactacaa cagccacaac gtctatatca
     2341 tggccgacaa gcagaagaac ggcatcaagg tgaacttcaa gatccgccac aacatcgagg
     2401 acggcagcgt gcagctcgcc gaccactacc agcagaacac ccccatcggc gacggccccg
     2461 tgctgctgcc cgacaaccac tacctgagca cccagtccgc cctgagcaaa gaccccaacg
     2521 agaagcgcga tcacatggtc ctgctggagt tcgtgaccgc cgccgggatc actctcggca
     2581 tggacgagct gtacaagtct gggtccgcag ccagtagcgc cggaagtatg atcagtctga
     2641 ttgcggcgtt agcggtagat cgcgttatcg gcatggaaaa cgccatgccg tggaacctgc
     2701 ctgccgatct cgcctggttt aaacgcaaca ccttaaataa acccgtgatt atgggccgcc
     2761 atacctggga atcaatcggt cgtccgttgc caggacgcaa aaatattatc ctcagcagtc
     2821 aaccgggtac ggacaaaaaa aagaaaaaga aagatcgcgt aacgtgggtg aagtcggtgg
     2881 atgaagccat cgcggcgtgt ggtgacgtac cagaaatcat ggtgattggc ggcggtcgcg
     2941 tttatgaaca gttcttgcca aaagcgcaaa aactgtatct gacgcatatc gacgcagaag
     3001 tggaaggcga cacccatttc ccggattacg agccggatga ctgggaatcg gtattcagcg
     3061 aattccacga tgctgatgcg cagaactctc acagctattg ctttgagatt ctggagcggc
     3121 ggagtgctgg tggtagtgct ggtggtagtg ctggtggtag tgctggtggt agtgctggtg
     3181 gtcctcgagc tcaagcttcg aattctgcag tcgacggtac cgcgggcccg ggatccaccg
     3241 gatctagata actgatcata atcagccata ccacatttgt agaggtttta cttgctttaa
     3301 aaaacctccc acacctcccc ctgaacctga aacataaaat gaatgcaatt gttgttgtta
     3361 acttgtttat tgcagcttat aatggttaca aataaagcaa tagcatcaca aatttcacaa
     3421 ataaagcatt tttttcactg cattctagtt gtggtttgtc caaactcatc aatgtatctt
     3481 aacgcgtaaa ttgtaagcgt taatattttg ttaaaattcg cgttaaattt ttgttaaatc
     3541 agctcatttt ttaaccaata ggccgaaatc ggcaaaatcc cttataaatc aaaagaatag
     3601 accgagatag ggttgagtgt tgttccagtt tggaacaaga gtccactatt aaagaacgtg
     3661 gactccaacg tcaaagggcg aaaaaccgtc tatcagggcg atggcccact acgtgaacca
     3721 tcaccctaat caagtttttt ggggtcgagg tgccgtaaag cactaaatcg gaaccctaaa
     3781 gggagccccc gatttagagc ttgacgggga aagccggcga acgtggcgag aaaggaaggg
     3841 aagaaagcga aaggagcggg cgctagggcg ctggcaagtg tagcggtcac gctgcgcgta
     3901 accaccacac ccgccgcgct taatgcgccg ctacagggcg cgtcaggtgg cacttttcgg
     3961 ggaaatgtgc gcggaacccc tatttgttta tttttctaaa tacattcaaa tatgtatccg
     4021 ctcatgagac aataaccctg ataaatgctt caataatatt gaaaaaggaa gagtcctgag
     4081 gcggaaagaa ccagctgtgg aatgtgtgtc agttagggtg tggaaagtcc ccaggctccc
     4141 cagcaggcag aagtatgcaa agcatgcatc tcaattagtc agcaaccagg tgtggaaagt
     4201 ccccaggctc cccagcaggc agaagtatgc aaagcatgca tctcaattag tcagcaacca
     4261 tagtcccgcc cctaactccg cccatcccgc ccctaactcc gcccagttcc gcccattctc
     4321 cgccccatgg ctgactaatt ttttttattt atgcagaggc cgaggccgcc tcggcctctg
     4381 agctattcca gaagtagtga ggaggctttt ttggaggcct aggcttttgc aaagatcgat
     4441 caagagacag gatgaggatc gtttcgcatg attgaacaag atggattgca cgcaggttct
     4501 ccggccgctt gggtggagag gctattcggc tatgactggg cacaacagac aatcggctgc
     4561 tctgatgccg ccgtgttccg gctgtcagcg caggggcgcc cggttctttt tgtcaagacc
     4621 gacctgtccg gtgccctgaa tgaactgcaa gacgaggcag cgcggctatc gtggctggcc
     4681 acgacgggcg ttccttgcgc agctgtgctc gacgttgtca ctgaagcggg aagggactgg
     4741 ctgctattgg gcgaagtgcc ggggcaggat ctcctgtcat ctcaccttgc tcctgccgag
     4801 aaagtatcca tcatggctga tgcaatgcgg cggctgcata cgcttgatcc ggctacctgc
     4861 ccattcgacc accaagcgaa acatcgcatc gagcgagcac gtactcggat ggaagccggt
     4921 cttgtcgatc aggatgatct ggacgaagag catcaggggc tcgcgccagc cgaactgttc
     4981 gccaggctca aggcgagcat gcccgacggc gaggatctcg tcgtgaccca tggcgatgcc
     5041 tgcttgccga atatcatggt ggaaaatggc cgcttttctg gattcatcga ctgtggccgg
     5101 ctgggtgtgg cggaccgcta tcaggacata gcgttggcta cccgtgatat tgctgaagag
     5161 cttggcggcg aatgggctga ccgcttcctc gtgctttacg gtatcgccgc tcccgattcg
     5221 cagcgcatcg ccttctatcg ccttcttgac gagttcttct gagcgggact ctggggttcg
     5281 aaatgaccga ccaagcgacg cccaac
//
