LOCUS       sequence-254959-        4446 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses LifeAct-iFAST in mammalian cells (actin labeling).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4446)
  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 4446)
  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: pAG470 LifeAct-iFAST
FEATURES             Location/Qualifiers
     source          1..4446
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        32..335
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        336..539
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     490..510
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      586..595
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             592..642
                     /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             658..1032
                     /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="MEHVAFGSEDIENTLAKMDDGQLDGLAFGAIQLDGDGNILQYNAA
                     EGDITGRDPKQVIGKNFFKDVAPGTDSPEFYGKFKEGVASGNLNTMFEWMIPTSRGPTK
                     VKIHMKKALSGDSYWVFVKRV"
     CDS             1039..1068
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     polyA_signal    1205..1326
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1242..1261)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     1296..1315
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(1333..1788)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1470..1491)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     1682..1701
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        1815..1919
                     /gene="bla"
                     /label=AmpR promoter
     promoter        1921..2278
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        1921..2112
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(1944..1964)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      2129..2264
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             2313..3107
                     /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             2313..3107
                     /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(2367..2386)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     2977..2996
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(3295..3314)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    3339..3386
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      3715..4303
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4204..4223
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 cattagttca tagcccatat atggagttcc gcgttacata acttacggta aatggcccgc
       61 ctggctgacc gcccaacgac ccccgcccat tgacgtcaat aatgacgtat gttcccatag
      121 taacgccaat agggactttc cattgacgtc aatgggtgga gtatttacgg taaactgccc
      181 acttggcagt acatcaagtg tatcatatgc caagtacgcc ccctattgac gtcaatgacg
      241 gtaaatggcc cgcctggcat tatgcccagt acatgacctt atgggacttt cctacttggc
      301 agtacatcta cgtattagtc atcgctatta ccatggtgat gcggttttgg cagtacatca
      361 atgggcgtgg atagcggttt gactcacggg gatttccaag tctccacccc attgacgtca
      421 atgggagttt gttttggcac caaaatcaac gggactttcc aaaatgtcgt aacaactccg
      481 ccccattgac gcaaatgggc ggtaggcgtg tacggtggga ggtctatata agcagagctg
      541 gtttagtgaa ccgtcagatc cgctagcgct accggactca gatctgccac catgggcgtg
      601 gccgacttga tcaagaagtt cgagtccatc tccaaggagg agggggatcc accggtcatg
      661 gagcatgttg cctttggcag tgaggacatc gagaacactc tggccaaaat ggacgacgga
      721 caactggatg ggttggcctt tggcgcaatt cagctcgatg gtgacgggaa tatcctgcag
      781 tacaatgctg ctgaaggaga catcacaggc agagatccca aacaggtgat tgggaagaac
      841 ttcttcaagg atgttgcacc tggaacggat tctcccgagt tttacggcaa attcaaggaa
      901 ggcgtagcgt cagggaatct gaacaccatg ttcgaatgga tgataccgac aagcagggga
      961 ccaaccaagg tcaagataca catgaagaaa gccctttccg gtgacagcta ttgggtcttt
     1021 gtgaaacggg tgggatccga acaaaagctt atttctgaag aggacttgta atagcccggg
     1081 taagcggccg cgactctaga tcataatcag ccataccaca tttgtagagg ttttacttgc
     1141 tttaaaaaac ctcccacacc tccccctgaa cctgaaacat aaaatgaatg caattgttgt
     1201 tgttaacttg tttattgcag cttataatgg ttacaaataa agcaatagca tcacaaattt
     1261 cacaaataaa gcattttttt cactgcattc tagttgtggt ttgtccaaac tcatcaatgt
     1321 atcttaaggc gtaaattgta agcgttaata ttttgttaaa attcgcgtta aatttttgtt
     1381 aaatcagctc attttttaac caataggccg aaatcggcaa aatcccttat aaatcaaaag
     1441 aatagaccga gatagggttg agtgttgttc cagtttggaa caagagtcca ctattaaaga
     1501 acgtggactc caacgtcaaa gggcgaaaaa ccgtctatca gggcgatggc ccactacgtg
     1561 aaccatcacc ctaatcaagt tttttggggt cgaggtgccg taaagcacta aatcggaacc
     1621 ctaaagggag cccccgattt agagcttgac ggggaaagcc ggcgaacgtg gcgagaaagg
     1681 aagggaagaa agcgaaagga gcgggcgcta gggcgctggc aagtgtagcg gtcacgctgc
     1741 gcgtaaccac cacacccgcc gcgcttaatg cgccgctaca gggcgcgtca ggtggcactt
     1801 ttcggggaaa tgtgcgcgga acccctattt gtttattttt ctaaatacat tcaaatatgt
     1861 atccgctcat gagacaataa ccctgataaa tgcttcaata atattgaaaa aggaagagtc
     1921 ctgaggcgga aagaaccagc tgtggaatgt gtgtcagtta gggtgtggaa agtccccagg
     1981 ctccccagca ggcagaagta tgcaaagcat gcatctcaat tagtcagcaa ccaggtgtgg
     2041 aaagtcccca ggctccccag caggcagaag tatgcaaagc atgcatctca attagtcagc
     2101 aaccatagtc ccgcccctaa ctccgcccat cccgccccta actccgccca gttccgccca
     2161 ttctccgccc catggctgac taattttttt tatttatgca gaggccgagg ccgcctcggc
     2221 ctctgagcta ttccagaagt agtgaggagg cttttttgga ggcctaggct tttgcaaaga
     2281 tcgatcaaga gacaggatga ggatcgtttc gcatgattga acaagatgga ttgcacgcag
     2341 gttctccggc cgcttgggtg gagaggctat tcggctatga ctgggcacaa cagacaatcg
     2401 gctgctctga tgccgccgtg ttccggctgt cagcgcaggg gcgcccggtt ctttttgtca
     2461 agaccgacct gtccggtgcc ctgaatgaac tgcaagacga ggcagcgcgg ctatcgtggc
     2521 tggccacgac gggcgttcct tgcgcagctg tgctcgacgt tgtcactgaa gcgggaaggg
     2581 actggctgct attgggcgaa gtgccggggc aggatctcct gtcatctcac cttgctcctg
     2641 ccgagaaagt atccatcatg gctgatgcaa tgcggcggct gcatacgctt gatccggcta
     2701 cctgcccatt cgaccaccaa gcgaaacatc gcatcgagcg agcacgtact cggatggaag
     2761 ccggtcttgt cgatcaggat gatctggacg aagagcatca ggggctcgcg ccagccgaac
     2821 tgttcgccag gctcaaggcg agcatgcccg acggcgagga tctcgtcgtg acccatggcg
     2881 atgcctgctt gccgaatatc atggtggaaa atggccgctt ttctggattc atcgactgtg
     2941 gccggctggg tgtggcggac cgctatcagg acatagcgtt ggctacccgt gatattgctg
     3001 aagagcttgg cggcgaatgg gctgaccgct tcctcgtgct ttacggtatc gccgctcccg
     3061 attcgcagcg catcgccttc tatcgccttc ttgacgagtt cttctgagcg ggactctggg
     3121 gttcgaaatg accgaccaag cgacgcccaa cctgccatca cgagatttcg attccaccgc
     3181 cgccttctat gaaaggttgg gcttcggaat cgttttccgg gacgccggct ggatgatcct
     3241 ccagcgcggg gatctcatgc tggagttctt cgcccacact agggggaggc taactgaaac
     3301 acggaaggag acaataccgg aaggaacccg cgctatgacg gcaataaaaa gacagaataa
     3361 aacgcacggt gttgggtcgt ttgttcataa acgcggggtt cggtcccagg gctggcactc
     3421 tgtcgatacc ccaccgagac cccattgggg ccaatacgcc cgcgtttctt ccttttcccc
     3481 accccacccc ccaagttcgg gtgaaggccc agggctcgca gccaacgtcg gggcggcagg
     3541 ccctgccata gcctcaggtt actcatatat actttagatt gatttaaaac ttcattttta
     3601 atttaaaagg atctaggtga agatcctttt tgataatctc atgaccaaaa tcccttaacg
     3661 tgagttttcg ttccactgag cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga
     3721 tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt
     3781 ggtttgtttg ccggatcaag agctaccaac tctttttccg aaggtaactg gcttcagcag
     3841 agcgcagata ccaaatactg ttcttctagt gtagccgtag ttaggccacc acttcaagaa
     3901 ctctgtagca ccgcctacat acctcgctct gctaatcctg ttaccagtgg ctgctgccag
     3961 tggcgataag tcgtgtctta ccgggttgga ctcaagacga tagttaccgg ataaggcgca
     4021 gcggtcgggc tgaacggggg gttcgtgcac acagcccagc ttggagcgaa cgacctacac
     4081 cgaactgaga tacctacagc gtgagctatg agaaagcgcc acgcttcccg aagggagaaa
     4141 ggcggacagg tatccggtaa gcggcagggt cggaacagga gagcgcacga gggagcttcc
     4201 agggggaaac gcctggtatc tttatagtcc tgtcgggttt cgccacctct gacttgagcg
     4261 tcgatttttg tgatgctcgt caggggggcg gagcctatgg aaaaacgcca gcaacgcggc
     4321 ctttttacgg ttcctggcct tttgctggcc ttttgctcac atgttctttc ctgcgttatc
     4381 ccctgattct gtggataacc gtattaccgc catgcattag ttattaatag taatcaatta
     4441 cggggt
//
