LOCUS       sequence-340527-        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(184..203)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    228..275
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      604..1192
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1093..1112
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     enhancer        1367..1670
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        1671..1874
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     1825..1845
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      1915..1924
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(1921..1923,1924..1926,1927..2637)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(1921..1923,1924..1926,1927..2637)
                     /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(1966..1987)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2227..2246)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2574..2595
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     misc_feature    3224..3276
                     /label=MCS
                     /note="multiple cloning site"
     polyA_signal    3400..3521
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3437..3456)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3491..3510
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(3528..3983)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3665..3686)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3877..3896
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        4010..4114
                     /gene="bla"
                     /label=AmpR promoter
     promoter        4116..4473
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        4116..4307
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(4139..4159)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      4324..4459
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             4508..5302
                     /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             4508..5302
                     /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(4562..4581)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     5172..5191
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
ORIGIN
        1 gactctgggg ttcgaaatga ccgaccaagc gacgcccaac ctgccatcac gagatttcga
       61 ttccaccgcc gccttctatg aaaggttggg cttcggaatc gttttccggg acgccggctg
      121 gatgatcctc cagcgcgggg atctcatgct ggagttcttc gcccacccta gggggaggct
      181 aactgaaaca cggaaggaga caataccgga aggaacccgc gctatgacgg caataaaaag
      241 acagaataaa acgcacggtg ttgggtcgtt tgttcataaa cgcggggttc ggtcccaggg
      301 ctggcactct gtcgataccc caccgagacc ccattggggc caatacgccc gcgtttcttc
      361 cttttcccca ccccaccccc caagttcggg tgaaggccca gggctcgcag ccaacgtcgg
      421 ggcggcaggc cctgccatag cctcaggtta ctcatatata ctttagattg atttaaaact
      481 tcatttttaa tttaaaagga tctaggtgaa gatccttttt gataatctca tgaccaaaat
      541 cccttaacgt gagttttcgt tccactgagc gtcagacccc gtagaaaaga tcaaaggatc
      601 ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct
      661 accagcggtg gtttgtttgc cggatcaaga gctaccaact ctttttccga aggtaactgg
      721 cttcagcaga gcgcagatac caaatactgt tcttctagtg tagccgtagt taggccacca
      781 cttcaagaac tctgtatcac cgcctacata cctcgctctg ctaatcctgt taccagtggc
      841 tgctgccagt ggcgataagt cgtgtcttac cgggttggac tcaagacgat agttaccgga
      901 taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca cagcccagct tggagcgaac
      961 gacctacacc gaactgagat acctacagcg tgagctatga gaaagcgcca cgcttcccga
     1021 agggagaaag gcggacaggt atccggtaag cggcagggtc ggaacaggag agcgcacgag
     1081 ggagcttcca gggggaaacg cctggtatct ttatagtcct gtcgggtttc gccacctctg
     1141 acttgagcgt cgatttttgt gatgctcgtc aggggggcgg agcctatgga aaaacgccag
     1201 caacgcggcc tttttacggt tcctggcctt ttgctggcct tttgctcaca tgttctttcc
     1261 tgcgttatcc cctgattctg tggataaccg tattaccgcc atgcattagt tattaatagt
     1321 aatcaattac ggggtcatta gttcatagcc catatatgga gttccgcgtt acataactta
     1381 cggtaaatgg cccgcctggc tgaccgccca acgacccccg cccattgacg tcaataatga
     1441 cgtatgttcc catagtaacg ccaataggga ctttccattg acgtcaatgg gtggagtatt
     1501 tacggtaaac tgcccacttg gcagtacatc aagtgtatca tatgccaagt acgcccccta
     1561 ttgacgtcaa tgacggtaaa tggcccgcct ggcattatgc ccagtacatg accttatggg
     1621 actttcctac ttggcagtac atctacgtat tagtcatcgc tattaccatg gtgatgcggt
     1681 tttggcagta catcaatggg cgtggatagc ggtttgactc acggggattt ccaagtctcc
     1741 accccattga cgtcaatggg agtttgtttt ggcaccaaaa tcaacgggac tttccaaaat
     1801 gtcgtaacaa ctccgcccca ttgacgcaaa tgggcggtag gcgtgtacgg tgggaggtct
     1861 atataagcag agctggttta gtgaaccgtc agatccgcta gcgatccacc ggtcgccacc
     1921 atggtgagca agggcgagga gctgttcacc ggggtggtgc ccatcctggt cgagctggac
     1981 ggcgacgtaa acggccacaa gttcagcgtg tccggcgagg gcgagggcga tgccacctac
     2041 ggcaagctga ccctgaagtt catctgcacc accggcaagc tgcccgtgcc ctggcccacc
     2101 ctcgtgacca ccctgaccta cggcgtgcag tgcttcagcc gctaccccga ccacatgaag
     2161 cagcacgact tcttcaagtc cgccatgccc gaaggctacg tccaggagcg caccatcttc
     2221 ttcaaggacg acggcaacta caagacccgc gccgaggtga agttcgaggg cgacaccctg
     2281 gtgaaccgca tcgagctgaa gggcatcgac ttcaaggagg acggcaacat cctggggcac
     2341 aagctggagt acaactacaa cagccacaac gtctatatca tggccgacaa gcagaagaac
     2401 ggcatcaagg tgaacttcaa gatccgccac aacatcgagg acggcagcgt gcagctcgcc
     2461 gaccactacc agcagaacac ccccatcggc gacggccccg tgctgctgcc cgacaaccac
     2521 tacctgagca cccagtccgc cctgagcaaa gaccccaacg agaagcgcga tcacatggtc
     2581 ctgctggagt tcgtgaccgc cgccgggatc actctcggca tggacgagct gtacaagtct
     2641 gggtccgcag ccagtagcgc cggaagtatg atcagtctga ttgcggcgtt agcggtagat
     2701 cgcgttatcg gcatggaaaa cgccatgccg tggaacctgc ctgccgatct cgcctggttt
     2761 aaacgcaaca ccttaaataa acccgtgatt atgggccgcc atacctggga atcaatcggt
     2821 cgtccgttgc caggacgcaa aaatattatc ctcagcagtc aaccgggtac ggacaaaaaa
     2881 aagaaaaaga aagatcgcgt aacgtgggtg aagtcggtgg atgaagccat cgcggcgtgt
     2941 ggtgacgtac cagaaatcat ggtgattggc ggcggtcgcg tttatgaaca gttcttgcca
     3001 aaagcgcaaa aactgtatct gacgcatatc gacgcagaag tggaaggcga cacccatttc
     3061 ccggattacg agccggatga ctgggaatcg gtattcagcg aattccacga tgctgatgcg
     3121 cagaactctc acagctattg ctttgagatt ctggagcggc ggagtgctgg tggtagtgct
     3181 ggtggtagtg ctggtggtag tgctggtggt agtgctggtg gtcctcgagc tcaagcttcg
     3241 aattctgcag tcgacggtac cgcgggcccg ggatccaccg gatctagata actgatcata
     3301 atcagccata ccacatttgt agaggtttta cttgctttaa aaaacctccc acacctcccc
     3361 ctgaacctga aacataaaat gaatgcaatt gttgttgtta acttgtttat tgcagcttat
     3421 aatggttaca aataaagcaa tagcatcaca aatttcacaa ataaagcatt tttttcactg
     3481 cattctagtt gtggtttgtc caaactcatc aatgtatctt aaggcgtaaa ttgtaagcgt
     3541 taatattttg ttaaaattcg cgttaaattt ttgttaaatc agctcatttt ttaaccaata
     3601 ggccgaaatc ggcaaaatcc cttataaatc aaaagaatag accgagatag ggttgagtgt
     3661 tgttccagtt tggaacaaga gtccactatt aaagaacgtg gactccaacg tcaaagggcg
     3721 aaaaaccgtc tatcagggcg atggcccact acgtgaacca tcaccctaat caagtttttt
     3781 ggggtcgagg tgccgtaaag cactaaatcg gaaccctaaa gggagccccc gatttagagc
     3841 ttgacgggga aagccggcga acgtggcgag aaaggaaggg aagaaagcga aaggagcggg
     3901 cgctagggcg ctggcaagtg tagcggtcac gctgcgcgta accaccacac ccgccgcgct
     3961 taatgcgccg ctacagggcg cgtcaggtgg cacttttcgg ggaaatgtgc gcggaacccc
     4021 tatttgttta tttttctaaa tacattcaaa tatgtatccg ctcatgagac aataaccctg
     4081 ataaatgctt caataatatt gaaaaaggaa gagtcctgag gcggaaagaa ccagctgtgg
     4141 aatgtgtgtc agttagggtg tggaaagtcc ccaggctccc cagcaggcag aagtatgcaa
     4201 agcatgcatc tcaattagtc agcaaccagg tgtggaaagt ccccaggctc cccagcaggc
     4261 agaagtatgc aaagcatgca tctcaattag tcagcaacca tagtcccgcc cctaactccg
     4321 cccatcccgc ccctaactcc gcccagttcc gcccattctc cgccccatgg ctgactaatt
     4381 ttttttattt atgcagaggc cgaggccgcc tcggcctctg agctattcca gaagtagtga
     4441 ggaggctttt ttggaggcct aggcttttgc aaagatcgat caagagacag gatgaggatc
     4501 gtttcgcatg attgaacaag atggattgca cgcaggttct ccggccgctt gggtggagag
     4561 gctattcggc tatgactggg cacaacagac aatcggctgc tctgatgccg ccgtgttccg
     4621 gctgtcagcg caggggcgcc cggttctttt tgtcaagacc gacctgtccg gtgccctgaa
     4681 tgaactgcaa gacgaggcag cgcggctatc gtggctggcc acgacgggcg ttccttgcgc
     4741 agctgtgctc gacgttgtca ctgaagcggg aagggactgg ctgctattgg gcgaagtgcc
     4801 ggggcaggat ctcctgtcat ctcaccttgc tcctgccgag aaagtatcca tcatggctga
     4861 tgcaatgcgg cggctgcata cgcttgatcc ggctacctgc ccattcgacc accaagcgaa
     4921 acatcgcatc gagcgagcac gtactcggat ggaagccggt cttgtcgatc aggatgatct
     4981 ggacgaagag catcaggggc tcgcgccagc cgaactgttc gccaggctca aggcgagcat
     5041 gcccgacggc gaggatctcg tcgtgaccca tggcgatgcc tgcttgccga atatcatggt
     5101 ggaaaatggc cgcttttctg gattcatcga ctgtggccgg ctgggtgtgg cggaccgcta
     5161 tcaggacata gcgttggcta cccgtgatat tgctgaagag cttggcggcg aatgggctga
     5221 ccgcttcctc gtgctttacg gtatcgccgc tcccgattcg cagcgcatcg ccttctatcg
     5281 ccttcttgac gagttcttct gagcgg
//
