LOCUS       sequence-354832-        4909 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Express SECFP with actin-targeting sequence in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4909)
  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 4909)
  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-SECFP
FEATURES             Location/Qualifiers
     source          1..4909
                     /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(738..740,741..743,744..1454)
                     /codon_start=1
                     /product="cyan variant of GFP"
                     /label=CFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHRFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTWGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIGFKEDGNILGHKLEYNYISHNVYITADKQKNGIK
                     AHFKIRHNIEDGGVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(783..804)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1044..1063)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1391..1412
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    1697..1818
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1734..1753)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     1788..1807
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(1825..2280)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1962..1983)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2174..2193
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        2307..2411
                     /gene="bla"
                     /label=AmpR promoter
     promoter        2413..2770
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        2413..2604
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(2436..2456)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      2621..2756
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             2805..3599
                     /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             2805..3599
                     /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(2859..2878)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3469..3488
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(3787..3806)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    3831..3878
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      4207..4795
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4696..4715
                     /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 agccatgctc gaggccacca tgggcgtggc cgacttgatc aagaagttcg
      661 agtccatctc caaggaggag ggggatccac cggtggctac ctgcggccgc ggaggatccg
      721 gaggagaatt cggatccatg gtgagcaagg gcgaggagct gttcaccggg gtggtgccca
      781 tcctggtcga gctggacggc gacgtaaacg gccacaggtt cagcgtgtcc ggcgagggcg
      841 agggcgatgc cacctacggc aagctgaccc tgaagttcat ctgcaccacc ggcaagctgc
      901 ccgtgccctg gcccaccctc gtgaccaccc tgacctgggg cgtgcagtgc ttcagccgct
      961 accccgacca catgaagcag cacgacttct tcaagtccgc catgcccgaa ggctacgtcc
     1021 aggagcgtac catcttcttc aaggacgacg gcaactacaa gacccgcgcc gaggtgaagt
     1081 tcgagggcga caccctggtg aaccgcatcg agctgaaggg catcggcttc aaggaggacg
     1141 gcaacatcct ggggcacaag ctagagtaca actacatcag ccacaacgtc tatatcaccg
     1201 ccgacaagca gaagaacggc atcaaggccc acttcaagat ccgccacaac atcgaggacg
     1261 gcggcgtgca gctcgccgac cactaccagc agaacacccc catcggcgac ggccccgtgc
     1321 tgctgcccga caaccactac ctgagcaccc agtccgccct gagcaaagac cccaacgaga
     1381 agcgcgatca catggtcctg ctggagttcg tgaccgccgc cgggatcact ctcggcatgg
     1441 acgagctgta caagggagga tctggaggaa gatctctgac tgactgagat gcgcggatgt
     1501 cgaacggcag ttcggccgaa taggccgaca gccggccgtc ggccgacgac acggccaggg
     1561 ccggcgggga tccaccggat ctagataact gatcataatc agccatacca catttgtaga
     1621 ggttttactt gctttaaaaa acctcccaca cctccccctg aacctgaaac ataaaatgaa
     1681 tgcaattgtt gttgttaact tgtttattgc agcttataat ggttacaaat aaagcaatag
     1741 catcacaaat ttcacaaata aagcattttt ttcactgcat tctagttgtg gtttgtccaa
     1801 actcatcaat gtatcttaag gcgtaaattg taagcgttaa tattttgtta aaattcgcgt
     1861 taaatttttg ttaaatcagc tcatttttta accaataggc cgaaatcggc aaaatccctt
     1921 ataaatcaaa agaatagacc gagatagggt tgagtgttgt tccagtttgg aacaagagtc
     1981 cactattaaa gaacgtggac tccaacgtca aagggcgaaa aaccgtctat cagggcgatg
     2041 gcccactacg tgaaccatca ccctaatcaa gttttttggg gtcgaggtgc cgtaaagcac
     2101 taaatcggaa ccctaaaggg agcccccgat ttagagcttg acggggaaag ccggcgaacg
     2161 tggcgagaaa ggaagggaag aaagcgaaag gagcgggcgc tagggcgctg gcaagtgtag
     2221 cggtcacgct gcgcgtaacc accacacccg ccgcgcttaa tgcgccgcta cagggcgcgt
     2281 caggtggcac ttttcgggga aatgtgcgcg gaacccctat ttgtttattt ttctaaatac
     2341 attcaaatat gtatccgctc atgagacaat aaccctgata aatgcttcaa taatattgaa
     2401 aaaggaagag tcctgaggcg gaaagaacca gctgtggaat gtgtgtcagt tagggtgtgg
     2461 aaagtcccca ggctccccag caggcagaag tatgcaaagc atgcatctca attagtcagc
     2521 aaccaggtgt ggaaagtccc caggctcccc agcaggcaga agtatgcaaa gcatgcatct
     2581 caattagtca gcaaccatag tcccgcccct aactccgccc atcccgcccc taactccgcc
     2641 cagttccgcc cattctccgc cccatggctg actaattttt tttatttatg cagaggccga
     2701 ggccgcctcg gcctctgagc tattccagaa gtagtgagga ggcttttttg gaggcctagg
     2761 cttttgcaaa gatcgatcaa gagacaggat gaggatcgtt tcgcatgatt gaacaagatg
     2821 gattgcacgc aggttctccg gccgcttggg tggagaggct attcggctat gactgggcac
     2881 aacagacaat cggctgctct gatgccgccg tgttccggct gtcagcgcag gggcgcccgg
     2941 ttctttttgt caagaccgac ctgtccggtg ccctgaatga actgcaagac gaggcagcgc
     3001 ggctatcgtg gctggccacg acgggcgttc cttgcgcagc tgtgctcgac gttgtcactg
     3061 aagcgggaag ggactggctg ctattgggcg aagtgccggg gcaggatctc ctgtcatctc
     3121 accttgctcc tgccgagaaa gtatccatca tggctgatgc aatgcggcgg ctgcatacgc
     3181 ttgatccggc tacctgccca ttcgaccacc aagcgaaaca tcgcatcgag cgagcacgta
     3241 ctcggatgga agccggtctt gtcgatcagg atgatctgga cgaagagcat caggggctcg
     3301 cgccagccga actgttcgcc aggctcaagg cgagcatgcc cgacggcgag gatctcgtcg
     3361 tgacccatgg cgatgcctgc ttgccgaata tcatggtgga aaatggccgc ttttctggat
     3421 tcatcgactg tggccggctg ggtgtggcgg accgctatca ggacatagcg ttggctaccc
     3481 gtgatattgc tgaagagctt ggcggcgaat gggctgaccg cttcctcgtg ctttacggta
     3541 tcgccgctcc cgattcgcag cgcatcgcct tctatcgcct tcttgacgag ttcttctgag
     3601 cgggactctg gggttcgaaa tgaccgacca agcgacgccc aacctgccat cacgagattt
     3661 cgattccacc gccgccttct atgaaaggtt gggcttcgga atcgttttcc gggacgccgg
     3721 ctggatgatc ctccagcgcg gggatctcat gctggagttc ttcgcccacc ctagggggag
     3781 gctaactgaa acacggaagg agacaatacc ggaaggaacc cgcgctatga cggcaataaa
     3841 aagacagaat aaaacgcacg gtgttgggtc gtttgttcat aaacgcgggg ttcggtccca
     3901 gggctggcac tctgtcgata ccccaccgag accccattgg ggccaatacg cccgcgtttc
     3961 ttccttttcc ccaccccacc ccccaagttc gggtgaaggc ccagggctcg cagccaacgt
     4021 cggggcggca ggccctgcca tagcctcagg ttactcatat atactttaga ttgatttaaa
     4081 acttcatttt taatttaaaa ggatctaggt gaagatcctt tttgataatc tcatgaccaa
     4141 aatcccttaa cgtgagtttt cgttccactg agcgtcagac cccgtagaaa agatcaaagg
     4201 atcttcttga gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc
     4261 gctaccagcg gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac
     4321 tggcttcagc agagcgcaga taccaaatac tgttcttcta gtgtagccgt agttaggcca
     4381 ccacttcaag aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt
     4441 ggctgctgcc agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttacc
     4501 ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc acacagccca gcttggagcg
     4561 aacgacctac accgaactga gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc
     4621 cgaagggaga aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcac
     4681 gagggagctt ccagggggaa acgcctggta tctttatagt cctgtcgggt ttcgccacct
     4741 ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc
     4801 cagcaacgcg gcctttttac ggttcctggc cttttgctgg ccttttgctc acatgttctt
     4861 tcctgcgtta tcccctgatt ctgtggataa ccgtattacc gccatgcat
//
