LOCUS       sequence-354857-        4891 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Express EGFP with actin-targeting sequence in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4891)
  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 4891)
  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-Lifeact-EGFP
FEATURES             Location/Qualifiers
     source          1..4891
                     /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"
     regulatory      624..633
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             630..680
                     /codon_start=1
                     /product="peptide that binds filamentous actin (Riedl et 
                     al., 2008)"
                     /label=Lifeact
                     /note="first 17 amino acids of budding yeast Abp140"
                     /translation="MGVADLIKKFESISKEE"
     CDS             join(735..737,738..740,741..1451)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(735..737,738..740,741..1451)
                     /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(780..801)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1041..1060)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1388..1409
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    1679..1800
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1716..1735)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     1770..1789
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(1807..2262)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1944..1965)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2156..2175
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        2289..2393
                     /gene="bla"
                     /label=AmpR promoter
     promoter        2395..2752
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        2395..2586
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(2418..2438)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      2603..2738
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             2787..3581
                     /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             2787..3581
                     /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(2841..2860)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3451..3470
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(3769..3788)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    3813..3860
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      4189..4777
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4678..4697
                     /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 gaggccacca tgggcgtggc cgacttgatc aagaagttcg
      661 agtccatctc caaggaggag ggggatccac cggtggctac ctgcggccgc ggaggatccg
      721 gaggagaatt caccatggtg agcaagggcg aggagctgtt caccggggtg gtgcccatcc
      781 tggtcgagct ggacggcgac gtaaacggcc acaagttcag cgtgtccggc gagggcgagg
      841 gcgatgccac ctacggcaag ctgaccctga agttcatctg caccaccggc aagctgcccg
      901 tgccctggcc caccctcgtg accaccctga cctacggcgt gcagtgcttc agccgctacc
      961 ccgaccacat gaagcagcac gacttcttca agtccgccat gcccgaaggc tacgtccagg
     1021 agcgcaccat cttcttcaag gacgacggca actacaagac ccgcgccgag gtgaagttcg
     1081 agggcgacac cctggtgaac cgcatcgagc tgaagggcat cgacttcaag gaggacggca
     1141 acatcctggg gcacaagctg gagtacaact acaacagcca caacgtctat atcatggccg
     1201 acaagcagaa gaacggcatc aaggtgaact tcaagatccg ccacaacatc gaggacggca
     1261 gcgtgcagct cgccgaccac taccagcaga acacccccat cggcgacggc cccgtgctgc
     1321 tgcccgacaa ccactacctg agcacccagt ccgccctgag caaagacccc aacgagaagc
     1381 gcgatcacat ggtcctgctg gagttcgtga ccgccgccgg gatcactctc ggcatggacg
     1441 agctgtacaa gagatctctg actgactgag atgcgcggat gtcgaacggc agttcggccg
     1501 aataggccga cagccggccg tcggccgacg acacggccag ggccggcggg gatccaccgg
     1561 atctagataa ctgatcataa tcagccatac cacatttgta gaggttttac ttgctttaaa
     1621 aaacctccca cacctccccc tgaacctgaa acataaaatg aatgcaattg ttgttgttaa
     1681 cttgtttatt gcagcttata atggttacaa ataaagcaat agcatcacaa atttcacaaa
     1741 taaagcattt ttttcactgc attctagttg tggtttgtcc aaactcatca atgtatctta
     1801 aggcgtaaat tgtaagcgtt aatattttgt taaaattcgc gttaaatttt tgttaaatca
     1861 gctcattttt taaccaatag gccgaaatcg gcaaaatccc ttataaatca aaagaataga
     1921 ccgagatagg gttgagtgtt gttccagttt ggaacaagag tccactatta aagaacgtgg
     1981 actccaacgt caaagggcga aaaaccgtct atcagggcga tggcccacta cgtgaaccat
     2041 caccctaatc aagttttttg gggtcgaggt gccgtaaagc actaaatcgg aaccctaaag
     2101 ggagcccccg atttagagct tgacggggaa agccggcgaa cgtggcgaga aaggaaggga
     2161 agaaagcgaa aggagcgggc gctagggcgc tggcaagtgt agcggtcacg ctgcgcgtaa
     2221 ccaccacacc cgccgcgctt aatgcgccgc tacagggcgc gtcaggtggc acttttcggg
     2281 gaaatgtgcg cggaacccct atttgtttat ttttctaaat acattcaaat atgtatccgc
     2341 tcatgagaca ataaccctga taaatgcttc aataatattg aaaaaggaag agtcctgagg
     2401 cggaaagaac cagctgtgga atgtgtgtca gttagggtgt ggaaagtccc caggctcccc
     2461 agcaggcaga agtatgcaaa gcatgcatct caattagtca gcaaccaggt gtggaaagtc
     2521 cccaggctcc ccagcaggca gaagtatgca aagcatgcat ctcaattagt cagcaaccat
     2581 agtcccgccc ctaactccgc ccatcccgcc cctaactccg cccagttccg cccattctcc
     2641 gccccatggc tgactaattt tttttattta tgcagaggcc gaggccgcct cggcctctga
     2701 gctattccag aagtagtgag gaggcttttt tggaggccta ggcttttgca aagatcgatc
     2761 aagagacagg atgaggatcg tttcgcatga ttgaacaaga tggattgcac gcaggttctc
     2821 cggccgcttg ggtggagagg ctattcggct atgactgggc acaacagaca atcggctgct
     2881 ctgatgccgc cgtgttccgg ctgtcagcgc aggggcgccc ggttcttttt gtcaagaccg
     2941 acctgtccgg tgccctgaat gaactgcaag acgaggcagc gcggctatcg tggctggcca
     3001 cgacgggcgt tccttgcgca gctgtgctcg acgttgtcac tgaagcggga agggactggc
     3061 tgctattggg cgaagtgccg gggcaggatc tcctgtcatc tcaccttgct cctgccgaga
     3121 aagtatccat catggctgat gcaatgcggc ggctgcatac gcttgatccg gctacctgcc
     3181 cattcgacca ccaagcgaaa catcgcatcg agcgagcacg tactcggatg gaagccggtc
     3241 ttgtcgatca ggatgatctg gacgaagagc atcaggggct cgcgccagcc gaactgttcg
     3301 ccaggctcaa ggcgagcatg cccgacggcg aggatctcgt cgtgacccat ggcgatgcct
     3361 gcttgccgaa tatcatggtg gaaaatggcc gcttttctgg attcatcgac tgtggccggc
     3421 tgggtgtggc ggaccgctat caggacatag cgttggctac ccgtgatatt gctgaagagc
     3481 ttggcggcga atgggctgac cgcttcctcg tgctttacgg tatcgccgct cccgattcgc
     3541 agcgcatcgc cttctatcgc cttcttgacg agttcttctg agcgggactc tggggttcga
     3601 aatgaccgac caagcgacgc ccaacctgcc atcacgagat ttcgattcca ccgccgcctt
     3661 ctatgaaagg ttgggcttcg gaatcgtttt ccgggacgcc ggctggatga tcctccagcg
     3721 cggggatctc atgctggagt tcttcgccca ccctaggggg aggctaactg aaacacggaa
     3781 ggagacaata ccggaaggaa cccgcgctat gacggcaata aaaagacaga ataaaacgca
     3841 cggtgttggg tcgtttgttc ataaacgcgg ggttcggtcc cagggctggc actctgtcga
     3901 taccccaccg agaccccatt ggggccaata cgcccgcgtt tcttcctttt ccccacccca
     3961 ccccccaagt tcgggtgaag gcccagggct cgcagccaac gtcggggcgg caggccctgc
     4021 catagcctca ggttactcat atatacttta gattgattta aaacttcatt tttaatttaa
     4081 aaggatctag gtgaagatcc tttttgataa tctcatgacc aaaatccctt aacgtgagtt
     4141 ttcgttccac tgagcgtcag accccgtaga aaagatcaaa ggatcttctt gagatccttt
     4201 ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg
     4261 tttgccggat caagagctac caactctttt tccgaaggta actggcttca gcagagcgca
     4321 gataccaaat actgttcttc tagtgtagcc gtagttaggc caccacttca agaactctgt
     4381 agcaccgcct acatacctcg ctctgctaat cctgttacca gtggctgctg ccagtggcga
     4441 taagtcgtgt cttaccgggt tggactcaag acgatagtta ccggataagg cgcagcggtc
     4501 gggctgaacg gggggttcgt gcacacagcc cagcttggag cgaacgacct acaccgaact
     4561 gagataccta cagcgtgagc tatgagaaag cgccacgctt cccgaaggga gaaaggcgga
     4621 caggtatccg gtaagcggca gggtcggaac aggagagcgc acgagggagc ttccaggggg
     4681 aaacgcctgg tatctttata gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt
     4741 tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac gccagcaacg cggccttttt
     4801 acggttcctg gccttttgct ggccttttgc tcacatgttc tttcctgcgt tatcccctga
     4861 ttctgtggat aaccgtatta ccgccatgca t
//
