LOCUS       sequence-354852-        5254 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Express EGFP with mitochondria-targeting sequence in mammalian 
            cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5254)
  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 5254)
  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-Tom20-EGFP
FEATURES             Location/Qualifiers
     source          1..5254
                     /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(1098..1100,1101..1103,1104..1814)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(1098..1100,1101..1103,1104..1814)
                     /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(1143..1164)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1404..1423)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1751..1772
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    2042..2163
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2079..2098)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2133..2152
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(2170..2625)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2307..2328)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2519..2538
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        2652..2756
                     /gene="bla"
                     /label=AmpR promoter
     promoter        2758..3115
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        2758..2949
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(2781..2801)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      2966..3101
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             3150..3944
                     /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             3150..3944
                     /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(3204..3223)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3814..3833
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(4132..4151)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    4176..4223
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      4552..5140
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5041..5060
                     /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 aagtactctc gagcccgcca tggtgggtcg gaacagcgcc atcgccgccg
      661 gtgtatgcgg ggcccttttc attgggtact gcatctactt cgaccgcaaa agacgaagtg
      721 accccaactt caagaacagg cttcgagaac gaagaaagaa acagaagctt gccaaggaga
      781 gagctgggct ttccaagtta cctgacctta aagatgctga agctgttcag aagttcttcc
      841 ttgaagaaat acagcttggt gaagagttac tagctcaagg tgaatatgag aagggcgtag
      901 accatctgac aaatgcaatt gctgtgtgtg gacagccaca gcagttactg caggtcttac
      961 agcaaactct tccaccacca gtgttccaga tgcttctgac taagctccca acaattagtc
     1021 agagaattgt aagtgctcag agcttggctg aagatgatgt ggaaggcggc cgcggaggat
     1081 ccggaggaga attcaccatg gtgagcaagg gcgaggagct gttcaccggg gtggtgccca
     1141 tcctggtcga gctggacggc gacgtaaacg gccacaagtt cagcgtgtcc ggcgagggcg
     1201 agggcgatgc cacctacggc aagctgaccc tgaagttcat ctgcaccacc ggcaagctgc
     1261 ccgtgccctg gcccaccctc gtgaccaccc tgacctacgg cgtgcagtgc ttcagccgct
     1321 accccgacca catgaagcag cacgacttct tcaagtccgc catgcccgaa ggctacgtcc
     1381 aggagcgcac catcttcttc aaggacgacg gcaactacaa gacccgcgcc gaggtgaagt
     1441 tcgagggcga caccctggtg aaccgcatcg agctgaaggg catcgacttc aaggaggacg
     1501 gcaacatcct ggggcacaag ctggagtaca actacaacag ccacaacgtc tatatcatgg
     1561 ccgacaagca gaagaacggc atcaaggtga acttcaagat ccgccacaac atcgaggacg
     1621 gcagcgtgca gctcgccgac cactaccagc agaacacccc catcggcgac ggccccgtgc
     1681 tgctgcccga caaccactac ctgagcaccc agtccgccct gagcaaagac cccaacgaga
     1741 agcgcgatca catggtcctg ctggagttcg tgaccgccgc cgggatcact ctcggcatgg
     1801 acgagctgta caagagatct ctgactgact gagatgcgcg gatgtcgaac ggcagttcgg
     1861 ccgaataggc cgacagccgg ccgtcggccg acgacacggc cagggccggc ggggatccac
     1921 cggatctaga taactgatca taatcagcca taccacattt gtagaggttt tacttgcttt
     1981 aaaaaacctc ccacacctcc ccctgaacct gaaacataaa atgaatgcaa ttgttgttgt
     2041 taacttgttt attgcagctt ataatggtta caaataaagc aatagcatca caaatttcac
     2101 aaataaagca tttttttcac tgcattctag ttgtggtttg tccaaactca tcaatgtatc
     2161 ttaaggcgta aattgtaagc gttaatattt tgttaaaatt cgcgttaaat ttttgttaaa
     2221 tcagctcatt ttttaaccaa taggccgaaa tcggcaaaat cccttataaa tcaaaagaat
     2281 agaccgagat agggttgagt gttgttccag tttggaacaa gagtccacta ttaaagaacg
     2341 tggactccaa cgtcaaaggg cgaaaaaccg tctatcaggg cgatggccca ctacgtgaac
     2401 catcacccta atcaagtttt ttggggtcga ggtgccgtaa agcactaaat cggaacccta
     2461 aagggagccc ccgatttaga gcttgacggg gaaagccggc gaacgtggcg agaaaggaag
     2521 ggaagaaagc gaaaggagcg ggcgctaggg cgctggcaag tgtagcggtc acgctgcgcg
     2581 taaccaccac acccgccgcg cttaatgcgc cgctacaggg cgcgtcaggt ggcacttttc
     2641 ggggaaatgt gcgcggaacc cctatttgtt tatttttcta aatacattca aatatgtatc
     2701 cgctcatgag acaataaccc tgataaatgc ttcaataata ttgaaaaagg aagagtcctg
     2761 aggcggaaag aaccagctgt ggaatgtgtg tcagttaggg tgtggaaagt ccccaggctc
     2821 cccagcaggc agaagtatgc aaagcatgca tctcaattag tcagcaacca ggtgtggaaa
     2881 gtccccaggc tccccagcag gcagaagtat gcaaagcatg catctcaatt agtcagcaac
     2941 catagtcccg cccctaactc cgcccatccc gcccctaact ccgcccagtt ccgcccattc
     3001 tccgccccat ggctgactaa ttttttttat ttatgcagag gccgaggccg cctcggcctc
     3061 tgagctattc cagaagtagt gaggaggctt ttttggaggc ctaggctttt gcaaagatcg
     3121 atcaagagac aggatgagga tcgtttcgca tgattgaaca agatggattg cacgcaggtt
     3181 ctccggccgc ttgggtggag aggctattcg gctatgactg ggcacaacag acaatcggct
     3241 gctctgatgc cgccgtgttc cggctgtcag cgcaggggcg cccggttctt tttgtcaaga
     3301 ccgacctgtc cggtgccctg aatgaactgc aagacgaggc agcgcggcta tcgtggctgg
     3361 ccacgacggg cgttccttgc gcagctgtgc tcgacgttgt cactgaagcg ggaagggact
     3421 ggctgctatt gggcgaagtg ccggggcagg atctcctgtc atctcacctt gctcctgccg
     3481 agaaagtatc catcatggct gatgcaatgc ggcggctgca tacgcttgat ccggctacct
     3541 gcccattcga ccaccaagcg aaacatcgca tcgagcgagc acgtactcgg atggaagccg
     3601 gtcttgtcga tcaggatgat ctggacgaag agcatcaggg gctcgcgcca gccgaactgt
     3661 tcgccaggct caaggcgagc atgcccgacg gcgaggatct cgtcgtgacc catggcgatg
     3721 cctgcttgcc gaatatcatg gtggaaaatg gccgcttttc tggattcatc gactgtggcc
     3781 ggctgggtgt ggcggaccgc tatcaggaca tagcgttggc tacccgtgat attgctgaag
     3841 agcttggcgg cgaatgggct gaccgcttcc tcgtgcttta cggtatcgcc gctcccgatt
     3901 cgcagcgcat cgccttctat cgccttcttg acgagttctt ctgagcggga ctctggggtt
     3961 cgaaatgacc gaccaagcga cgcccaacct gccatcacga gatttcgatt ccaccgccgc
     4021 cttctatgaa aggttgggct tcggaatcgt tttccgggac gccggctgga tgatcctcca
     4081 gcgcggggat ctcatgctgg agttcttcgc ccaccctagg gggaggctaa ctgaaacacg
     4141 gaaggagaca ataccggaag gaacccgcgc tatgacggca ataaaaagac agaataaaac
     4201 gcacggtgtt gggtcgtttg ttcataaacg cggggttcgg tcccagggct ggcactctgt
     4261 cgatacccca ccgagacccc attggggcca atacgcccgc gtttcttcct tttccccacc
     4321 ccacccccca agttcgggtg aaggcccagg gctcgcagcc aacgtcgggg cggcaggccc
     4381 tgccatagcc tcaggttact catatatact ttagattgat ttaaaacttc atttttaatt
     4441 taaaaggatc taggtgaaga tcctttttga taatctcatg accaaaatcc cttaacgtga
     4501 gttttcgttc cactgagcgt cagaccccgt agaaaagatc aaaggatctt cttgagatcc
     4561 tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt
     4621 ttgtttgccg gatcaagagc taccaactct ttttccgaag gtaactggct tcagcagagc
     4681 gcagatacca aatactgttc ttctagtgta gccgtagtta ggccaccact tcaagaactc
     4741 tgtagcaccg cctacatacc tcgctctgct aatcctgtta ccagtggctg ctgccagtgg
     4801 cgataagtcg tgtcttaccg ggttggactc aagacgatag ttaccggata aggcgcagcg
     4861 gtcgggctga acggggggtt cgtgcacaca gcccagcttg gagcgaacga cctacaccga
     4921 actgagatac ctacagcgtg agctatgaga aagcgccacg cttcccgaag ggagaaaggc
     4981 ggacaggtat ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg agcttccagg
     5041 gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg
     5101 atttttgtga tgctcgtcag gggggcggag cctatggaaa aacgccagca acgcggcctt
     5161 tttacggttc ctggcctttt gctggccttt tgctcacatg ttctttcctg cgttatcccc
     5221 tgattctgtg gataaccgta ttaccgccat gcat
//
