LOCUS       sequence-353707-        4437 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses LifeAct-pFAST in mammalian cells (actin labeling).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4437)
  AUTHORS   Benaissa H, Ounoughi K, Aujard I, Fischer E, Goiame R, Nguyen J, 
            Tebo AG, Li C, Le Saux T, Bertolin G, Tramier M, Danglot L, 
            Pietrancosta N, Morin X, Jullien L, Gautier A
  TITLE     Engineering of a fluorescent chemogenetic reporter with tunable 
            color for advanced live-cell imaging.
  JOURNAL   Nat Commun. 2021 Nov 30;12(1):6989. doi: 10.1038/s41467-021-27334-0.
  PUBMED    34848727
REFERENCE   2  (bases 1 to 4437)
  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"; journalName: "Nat 
            Commun."; date: "2021-11-30"; volume: "12(1):6989. doi"; pages: " 
            10.1038/s41467-021-27334-0"
COMMENT     Sequence Label: pAG668 lifeAct-pFAST
FEATURES             Location/Qualifiers
     source          1..4437
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        139..442
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        443..646
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     597..617
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      693..702
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             699..749
                     /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             765..1139
                     /codon_start=1
                     /product="small protein tag, derived from photoactive 
                     yellow protein, that allows in vivo fluorescent labeling by
                     addition of a cell-permeant fluorogen (Plamont et al., 
                     2016)"
                     /label=Y-FAST
                     /translation="MEHVAFGSEDIENTLANMDDEQLDRLAFGVIQLDGDGNILLYNAA
                     EGDITGRDPKQVIGKNFFKDVAPGTDTPEFYGKFKEGAASGNLNTMFEWTIPTSRGPTK
                     VKVHLKKALSGDRYWVFVKRV"
     CDS             1146..1175
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     polyA_signal    1303..1424
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1340..1359)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     1394..1413
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(1431..1886)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1568..1589)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     1780..1799
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        1913..2017
                     /gene="bla"
                     /label=AmpR promoter
     promoter        2019..2376
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        2019..2210
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(2042..2062)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      2227..2362
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             2411..3205
                     /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             2411..3205
                     /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(2465..2484)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3075..3094
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(3393..3412)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    3437..3484
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      3813..4401
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4302..4321
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 ttttgctggc cttttgctca catgttcttt cctgcgttat cccctgattc tgtggataac
       61 cgtattaccg ccatgcatta gttattaata gtaatcaatt acggggtcat tagttcatag
      121 cccatatatg gagttccgcg ttacataact tacggtaaat ggcccgcctg gctgaccgcc
      181 caacgacccc cgcccattga cgtcaataat gacgtatgtt cccatagtaa cgccaatagg
      241 gactttccat tgacgtcaat gggtggagta tttacggtaa actgcccact tggcagtaca
      301 tcaagtgtat catatgccaa gtacgccccc tattgacgtc aatgacggta aatggcccgc
      361 ctggcattat gcccagtaca tgaccttatg ggactttcct acttggcagt acatctacgt
      421 attagtcatc gctattacca tggtgatgcg gttttggcag tacatcaatg ggcgtggata
      481 gcggtttgac tcacggggat ttccaagtct ccaccccatt gacgtcaatg ggagtttgtt
      541 ttggcaccaa aatcaacggg actttccaaa atgtcgtaac aactccgccc cattgacgca
      601 aatgggcggt aggcgtgtac ggtgggaggt ctatataagc agagctggtt tagtgaaccg
      661 tcagatccgc tagcgctacc ggactcagat ctgccaccat gggcgtggcc gacttgatca
      721 agaagttcga gtccatctcc aaggaggagg gggatccacc ggtcatggag catgttgcct
      781 ttggcagtga ggacatcgag aacactctgg ccaatatgga cgacgaacaa ctggataggt
      841 tggcctttgg cgtaattcag ctcgatggtg acgggaatat cctgctgtac aatgctgctg
      901 aaggggacat cactggcaga gatcccaaac aggtgattgg gaagaacttc ttcaaggatg
      961 ttgcacctgg aacggatact cccgagtttt acggcaaatt caaggaaggc gcagcgtcag
     1021 ggaatctgaa caccatgttc gaatggacga taccgacaag caggggacca accaaggtca
     1081 aggtgcactt gaagaaagcc ctttccggtg acagatattg ggtctttgtg aaacgggtgg
     1141 gatccgaaca aaagcttatt tctgaagagg acttgtaata ggcggccgcg actctagatc
     1201 ataatcagcc ataccacatt tgtagaggtt ttacttgctt taaaaaacct cccacacctc
     1261 cccctgaacc tgaaacataa aatgaatgca attgttgttg ttaacttgtt tattgcagct
     1321 tataatggtt acaaataaag caatagcatc acaaatttca caaataaagc atttttttca
     1381 ctgcattcta gttgtggttt gtccaaactc atcaatgtat cttaaggcgt aaattgtaag
     1441 cgttaatatt ttgttaaaat tcgcgttaaa tttttgttaa atcagctcat tttttaacca
     1501 ataggccgaa atcggcaaaa tcccttataa atcaaaagaa tagaccgaga tagggttgag
     1561 tgttgttcca gtttggaaca agagtccact attaaagaac gtggactcca acgtcaaagg
     1621 gcgaaaaacc gtctatcagg gcgatggccc actacgtgaa ccatcaccct aatcaagttt
     1681 tttggggtcg aggtgccgta aagcactaaa tcggaaccct aaagggagcc cccgatttag
     1741 agcttgacgg ggaaagccgg cgaacgtggc gagaaaggaa gggaagaaag cgaaaggagc
     1801 gggcgctagg gcgctggcaa gtgtagcggt cacgctgcgc gtaaccacca cacccgccgc
     1861 gcttaatgcg ccgctacagg gcgcgtcagg tggcactttt cggggaaatg tgcgcggaac
     1921 ccctatttgt ttatttttct aaatacattc aaatatgtat ccgctcatga gacaataacc
     1981 ctgataaatg cttcaataat attgaaaaag gaagagtcct gaggcggaaa gaaccagctg
     2041 tggaatgtgt gtcagttagg gtgtggaaag tccccaggct ccccagcagg cagaagtatg
     2101 caaagcatgc atctcaatta gtcagcaacc aggtgtggaa agtccccagg ctccccagca
     2161 ggcagaagta tgcaaagcat gcatctcaat tagtcagcaa ccatagtccc gcccctaact
     2221 ccgcccatcc cgcccctaac tccgcccagt tccgcccatt ctccgcccca tggctgacta
     2281 atttttttta tttatgcaga ggccgaggcc gcctcggcct ctgagctatt ccagaagtag
     2341 tgaggaggct tttttggagg cctaggcttt tgcaaagatc gatcaagaga caggatgagg
     2401 atcgtttcgc atgattgaac aagatggatt gcacgcaggt tctccggccg cttgggtgga
     2461 gaggctattc ggctatgact gggcacaaca gacaatcggc tgctctgatg ccgccgtgtt
     2521 ccggctgtca gcgcaggggc gcccggttct ttttgtcaag accgacctgt ccggtgccct
     2581 gaatgaactg caagacgagg cagcgcggct atcgtggctg gccacgacgg gcgttccttg
     2641 cgcagctgtg ctcgacgttg tcactgaagc gggaagggac tggctgctat tgggcgaagt
     2701 gccggggcag gatctcctgt catctcacct tgctcctgcc gagaaagtat ccatcatggc
     2761 tgatgcaatg cggcggctgc atacgcttga tccggctacc tgcccattcg accaccaagc
     2821 gaaacatcgc atcgagcgag cacgtactcg gatggaagcc ggtcttgtcg atcaggatga
     2881 tctggacgaa gagcatcagg ggctcgcgcc agccgaactg ttcgccaggc tcaaggcgag
     2941 catgcccgac ggcgaggatc tcgtcgtgac ccatggcgat gcctgcttgc cgaatatcat
     3001 ggtggaaaat ggccgctttt ctggattcat cgactgtggc cggctgggtg tggcggaccg
     3061 ctatcaggac atagcgttgg ctacccgtga tattgctgaa gagcttggcg gcgaatgggc
     3121 tgaccgcttc ctcgtgcttt acggtatcgc cgctcccgat tcgcagcgca tcgccttcta
     3181 tcgccttctt gacgagttct tctgagcggg actctggggt tcgaaatgac cgaccaagcg
     3241 acgcccaacc tgccatcacg agatttcgat tccaccgccg ccttctatga aaggttgggc
     3301 ttcggaatcg ttttccggga cgccggctgg atgatcctcc agcgcgggga tctcatgctg
     3361 gagttcttcg cccacactag ggggaggcta actgaaacac ggaaggagac aataccggaa
     3421 ggaacccgcg ctatgacggc aataaaaaga cagaataaaa cgcacggtgt tgggtcgttt
     3481 gttcataaac gcggggttcg gtcccagggc tggcactctg tcgatacccc accgagaccc
     3541 cattggggcc aatacgcccg cgtttcttcc ttttccccac cccacccccc aagttcgggt
     3601 gaaggcccag ggctcgcagc caacgtcggg gcggcaggcc ctgccatagc ctcaggttac
     3661 tcatatatac tttagattga tttaaaactt catttttaat ttaaaaggat ctaggtgaag
     3721 atcctttttg ataatctcat gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg
     3781 tcagaccccg tagaaaagat caaaggatct tcttgagatc ctttttttct gcgcgtaatc
     3841 tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc ggatcaagag
     3901 ctaccaactc tttttccgaa ggtaactggc ttcagcagag cgcagatacc aaatactgtt
     3961 cttctagtgt agccgtagtt aggccaccac ttcaagaact ctgtagcacc gcctacatac
     4021 ctcgctctgc taatcctgtt accagtggct gctgccagtg gcgataagtc gtgtcttacc
     4081 gggttggact caagacgata gttaccggat aaggcgcagc ggtcgggctg aacggggggt
     4141 tcgtgcacac agcccagctt ggagcgaacg acctacaccg aactgagata cctacagcgt
     4201 gagctatgag aaagcgccac gcttcccgaa gggagaaagg cggacaggta tccggtaagc
     4261 ggcagggtcg gaacaggaga gcgcacgagg gagcttccag ggggaaacgc ctggtatctt
     4321 tatagtcctg tcgggtttcg ccacctctga cttgagcgtc gatttttgtg atgctcgtca
     4381 ggggggcgga gcctatggaa aaacgccagc aacgcggcct ttttacggtt cctggcc
//
