LOCUS       sequence-354838-        5026 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Express EGFP with endoplasmic reticulum-targeting sequence in 
            mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5026)
  AUTHORS   Kashiwagi S, Fujioka Y, Satoh AO, Yoshida A, Fujioka M, Nepal P, 
            Tsuzuki A, Aoki O, Paudel S, Sasajima H, Ohba Y
  TITLE     Folding Latency of Fluorescent Proteins Affects the Mitochondrial 
            Localization of Fusion Proteins.
  JOURNAL   Cell Struct Funct. 2019 Dec 26;44(2):183-194. doi: 
            10.1247/csf.19028. Epub 2019 Nov 15.
  PUBMED    31735740
REFERENCE   2  (bases 1 to 5026)
  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: "10.1247/csf.19028";
            journalName: "Cell Struct Funct"; date: "2019-12-26- 26"; volume: 
            "44"; issue: "2"; pages: "183-194"
COMMENT     Sequence Label: pFX-EGFP-Sec61-beta
FEATURES             Location/Qualifiers
     source          1..5026
                     /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"
     CDS             join(627..629,630..632,633..1343)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(627..629,630..632,633..1343)
                     /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(672..693)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(933..952)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1280..1301
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             1377..1664
                     /codon_start=1
                     /gene="SEC61B"
                     /product="human Sec61-beta subunit of the translocon in the
                     endoplasmic reticulum"
                     /label=Sec61-beta
                     /translation="MPGPTPSGTNVGSSGRSPSKAVAARAAGSTVRQRKNASCGTRSAG
                     RTTSAGTGGMWRFYTEDSPGLKVGPVPVLVMSLLFIASVFMLHIWGKYTRS"
     polyA_signal    1814..1935
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1851..1870)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     1905..1924
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(1942..2397)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2079..2100)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2291..2310
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        2424..2528
                     /gene="bla"
                     /label=AmpR promoter
     promoter        2530..2887
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        2530..2721
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(2553..2573)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      2738..2873
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             2922..3716
                     /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             2922..3716
                     /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(2976..2995)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3586..3605
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(3904..3923)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    3948..3995
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      4324..4912
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4813..4832
                     /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 ccggttctag aagtactgaa ttcaccatgg tgagcaaggg cgaggagctg ttcaccgggg
      661 tggtgcccat cctggtcgag ctggacggcg acgtaaacgg ccacaagttc agcgtgtccg
      721 gcgagggcga gggcgatgcc acctacggca agctgaccct gaagttcatc tgcaccaccg
      781 gcaagctgcc cgtgccctgg cccaccctcg tgaccaccct gacctacggc gtgcagtgct
      841 tcagccgcta ccccgaccac atgaagcagc acgacttctt caagtccgcc atgcccgaag
      901 gctacgtcca ggagcgcacc atcttcttca aggacgacgg caactacaag acccgcgccg
      961 aggtgaagtt cgagggcgac accctggtga accgcatcga gctgaagggc atcgacttca
     1021 aggaggacgg caacatcctg gggcacaagc tggagtacaa ctacaacagc cacaacgtct
     1081 atatcatggc cgacaagcag aagaacggca tcaaggtgaa cttcaagatc cgccacaaca
     1141 tcgaggacgg cagcgtgcag ctcgccgacc actaccagca gaacaccccc atcggcgacg
     1201 gccccgtgct gctgcccgac aaccactacc tgagcaccca gtccgccctg agcaaagacc
     1261 ccaacgagaa gcgcgatcac atggtcctgc tggagttcgt gaccgccgcc gggatcactc
     1321 tcggcatgga cgagctgtac aagagatctg gaggatccgg aggactcgag cccaccatgc
     1381 ctggtccgac ccccagtggc actaacgtgg gatcctcagg gcgctctccc agcaaagcag
     1441 tggccgcccg ggcggcggga tccactgtcc ggcagaggaa aaatgccagc tgtgggacaa
     1501 ggagtgcagg ccgcacaacc tcggcaggca ccggggggat gtggcgattc tacacagaag
     1561 attcacctgg gctcaaagtt ggccctgttc cagtattggt tatgagtctt ctgttcatcg
     1621 cttctgtatt tatgttgcac atttggggca agtacactcg ttcgggcggc cgctgactga
     1681 ctgaagatcc accggatcta gataactgat cataatcagc cataccacat ttgtagaggt
     1741 tttacttgct ttaaaaaacc tcccacacct ccccctgaac ctgaaacata aaatgaatgc
     1801 aattgttgtt gttaacttgt ttattgcagc ttataatggt tacaaataaa gcaatagcat
     1861 cacaaatttc acaaataaag catttttttc actgcattct agttgtggtt tgtccaaact
     1921 catcaatgta tcttaaggcg taaattgtaa gcgttaatat tttgttaaaa ttcgcgttaa
     1981 atttttgtta aatcagctca ttttttaacc aataggccga aatcggcaaa atcccttata
     2041 aatcaaaaga atagaccgag atagggttga gtgttgttcc agtttggaac aagagtccac
     2101 tattaaagaa cgtggactcc aacgtcaaag ggcgaaaaac cgtctatcag ggcgatggcc
     2161 cactacgtga accatcaccc taatcaagtt ttttggggtc gaggtgccgt aaagcactaa
     2221 atcggaaccc taaagggagc ccccgattta gagcttgacg gggaaagccg gcgaacgtgg
     2281 cgagaaagga agggaagaaa gcgaaaggag cgggcgctag ggcgctggca agtgtagcgg
     2341 tcacgctgcg cgtaaccacc acacccgccg cgcttaatgc gccgctacag ggcgcgtcag
     2401 gtggcacttt tcggggaaat gtgcgcggaa cccctatttg tttatttttc taaatacatt
     2461 caaatatgta tccgctcatg agacaataac cctgataaat gcttcaataa tattgaaaaa
     2521 ggaagagtcc tgaggcggaa agaaccagct gtggaatgtg tgtcagttag ggtgtggaaa
     2581 gtccccaggc tccccagcag gcagaagtat gcaaagcatg catctcaatt agtcagcaac
     2641 caggtgtgga aagtccccag gctccccagc aggcagaagt atgcaaagca tgcatctcaa
     2701 ttagtcagca accatagtcc cgcccctaac tccgcccatc ccgcccctaa ctccgcccag
     2761 ttccgcccat tctccgcccc atggctgact aatttttttt atttatgcag aggccgaggc
     2821 cgcctcggcc tctgagctat tccagaagta gtgaggaggc ttttttggag gcctaggctt
     2881 ttgcaaagat cgatcaagag acaggatgag gatcgtttcg catgattgaa caagatggat
     2941 tgcacgcagg ttctccggcc gcttgggtgg agaggctatt cggctatgac tgggcacaac
     3001 agacaatcgg ctgctctgat gccgccgtgt tccggctgtc agcgcagggg cgcccggttc
     3061 tttttgtcaa gaccgacctg tccggtgccc tgaatgaact gcaagacgag gcagcgcggc
     3121 tatcgtggct ggccacgacg ggcgttcctt gcgcagctgt gctcgacgtt gtcactgaag
     3181 cgggaaggga ctggctgcta ttgggcgaag tgccggggca ggatctcctg tcatctcacc
     3241 ttgctcctgc cgagaaagta tccatcatgg ctgatgcaat gcggcggctg catacgcttg
     3301 atccggctac ctgcccattc gaccaccaag cgaaacatcg catcgagcga gcacgtactc
     3361 ggatggaagc cggtcttgtc gatcaggatg atctggacga agagcatcag gggctcgcgc
     3421 cagccgaact gttcgccagg ctcaaggcga gcatgcccga cggcgaggat ctcgtcgtga
     3481 cccatggcga tgcctgcttg ccgaatatca tggtggaaaa tggccgcttt tctggattca
     3541 tcgactgtgg ccggctgggt gtggcggacc gctatcagga catagcgttg gctacccgtg
     3601 atattgctga agagcttggc ggcgaatggg ctgaccgctt cctcgtgctt tacggtatcg
     3661 ccgctcccga ttcgcagcgc atcgccttct atcgccttct tgacgagttc ttctgagcgg
     3721 gactctgggg ttcgaaatga ccgaccaagc gacgcccaac ctgccatcac gagatttcga
     3781 ttccaccgcc gccttctatg aaaggttggg cttcggaatc gttttccggg acgccggctg
     3841 gatgatcctc cagcgcgggg atctcatgct ggagttcttc gcccacccta gggggaggct
     3901 aactgaaaca cggaaggaga caataccgga aggaacccgc gctatgacgg caataaaaag
     3961 acagaataaa acgcacggtg ttgggtcgtt tgttcataaa cgcggggttc ggtcccaggg
     4021 ctggcactct gtcgataccc caccgagacc ccattggggc caatacgccc gcgtttcttc
     4081 cttttcccca ccccaccccc caagttcggg tgaaggccca gggctcgcag ccaacgtcgg
     4141 ggcggcaggc cctgccatag cctcaggtta ctcatatata ctttagattg atttaaaact
     4201 tcatttttaa tttaaaagga tctaggtgaa gatccttttt gataatctca tgaccaaaat
     4261 cccttaacgt gagttttcgt tccactgagc gtcagacccc gtagaaaaga tcaaaggatc
     4321 ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct
     4381 accagcggtg gtttgtttgc cggatcaaga gctaccaact ctttttccga aggtaactgg
     4441 cttcagcaga gcgcagatac caaatactgt tcttctagtg tagccgtagt taggccacca
     4501 cttcaagaac tctgtagcac cgcctacata cctcgctctg ctaatcctgt taccagtggc
     4561 tgctgccagt ggcgataagt cgtgtcttac cgggttggac tcaagacgat agttaccgga
     4621 taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca cagcccagct tggagcgaac
     4681 gacctacacc gaactgagat acctacagcg tgagctatga gaaagcgcca cgcttcccga
     4741 agggagaaag gcggacaggt atccggtaag cggcagggtc ggaacaggag agcgcacgag
     4801 ggagcttcca gggggaaacg cctggtatct ttatagtcct gtcgggtttc gccacctctg
     4861 acttgagcgt cgatttttgt gatgctcgtc aggggggcgg agcctatgga aaaacgccag
     4921 caacgcggcc tttttacggt tcctggcctt ttgctggcct tttgctcaca tgttctttcc
     4981 tgcgttatcc cctgattctg tggataaccg tattaccgcc atgcat
//
