LOCUS       sequence-358681-        4862 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses LifeAct-GFP targeted to the outer mitochondrial membrane 
            via the Fis1 tail anchor sequence.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4862)
  AUTHORS   Schiavon CR, Zhang T, Zhao B, Moore AS, Wales P, Andrade LR, Wu M, 
            Sung TC, Dayn Y, Feng JW, Quintero OA, Shadel GS, Grosse R, Manor U
  TITLE     Actin chromobody imaging reveals sub-organellar actin dynamics.
  JOURNAL   Nat Methods. 2020 Sep;17(9):917-921. doi: 10.1038/s41592-020-0926-5.
            Epub 2020 Aug 10.
  PUBMED    32778832
REFERENCE   2  (bases 1 to 4862)
  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: 
            "10.1038/s41592-020-0926-5"; journalName: "Nat Methods"; date: 
            "2020-09"; volume: "17"; issue: "9"; pages: "917-921"
COMMENT     Sequence Label: LifeAct-GFP-Fis1
FEATURES             Location/Qualifiers
     source          1..4862
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        16..319
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        320..523
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     474..494
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     CDS             590..640
                     /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"
     regulatory      656..665
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(662..664,665..667,668..1378)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(662..664,665..667,668..1378)
                     /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(707..728)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(968..987)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1315..1336
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    1605..1726
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1642..1661)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     1696..1715
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(1733..2188)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1870..1891)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2082..2101
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        2215..2319
                     /gene="bla"
                     /label=AmpR promoter
     promoter        2321..2678
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        2321..2512
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(2344..2364)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      2529..2664
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             2713..3507
                     /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             2713..3507
                     /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(2767..2786)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3377..3396
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(3695..3714)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    3739..3786
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      4115..4703
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4604..4623
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 atatatggag ttccgcgtta cataacttac ggtaaatggc ccgcctggct gaccgcccaa
       61 cgacccccgc ccattgacgt caataatgac gtatgttccc atagtaacgc caatagggac
      121 tttccattga cgtcaatggg tggagtattt acggtaaact gtccacttgg cagtacatca
      181 agtgtatcat atgccaagta cgccccctat tgacgtcaat gacggtaaat ggcccgcctg
      241 gcattatgcc cagtacatga ccttatggga ctttcctact tggcagtaca tctacgtatt
      301 agtcatcgct attaccatgg tgatgcggtt ttggcagtac atcaatgggc gtggatagcg
      361 gtttgactca cggggatttc caagtctcca ccccattgac gtcaatggga gtttgttttg
      421 gcaccaaaat caacgggact ttccaaaatg tcgtaacaac tccgccccat tgacgcaaat
      481 gggcggtagg cgtgtacggt gggaggtcta tataagcaga gctggtttag tgaaccgtca
      541 gatccgctag cgctaccgga ctcagatctc gagctcaagc ttcgaattca tgggtgtcgc
      601 agatttgatc aagaaattcg aaagcatctc aaaggaagaa ggggatccac cggtcgccac
      661 catggtgagc aagggcgagg agctgttcac cggggtggtg cccatcctgg tcgagctgga
      721 cggcgacgta aacggccaca agttcagcgt gtccggcgag ggcgagggcg atgccaccta
      781 cggcaagctg accctgaagt tcatctgcac caccggcaag ctgcccgtgc cctggcccac
      841 cctcgtgacc accctgacct acggcgtgca gtgcttcagc cgctaccccg accacatgaa
      901 gcagcacgac ttcttcaagt ccgccatgcc cgaaggctac gtccaggagc gcaccatctt
      961 cttcaaggac gacggcaact acaagacccg cgccgaggtg aagttcgagg gcgacaccct
     1021 ggtgaaccgc atcgagctga agggcatcga cttcaaggag gacggcaaca tcctggggca
     1081 caagctggag tacaactaca acagccacaa cgtctatatc atggccgaca agcagaagaa
     1141 cggcatcaag gtgaacttca agatccgcca caacatcgag gacggcagcg tgcagctcgc
     1201 cgaccactac cagcagaaca cccccatcgg cgacggcccc gtgctgctgc ccgacaacca
     1261 ctacctgagc acccagtccg ccctgagcaa agaccccaac gagaagcgcg atcacatggt
     1321 cctgctggag ttcgtgaccg ccgccgggat cactctcggc atggacgagc tgtacaaggg
     1381 gccgattcag aaagaaaccc tgaaaggcgt ggtggtggcg ggcggcgtgc tggcgggcgc
     1441 ggtggcggtg gcgagctttt ttctgcgcaa caaacgccgc taagcggccg cgactctaga
     1501 tcataatcag ccataccaca tttgtagagg ttttacttgc tttaaaaaac ctcccacacc
     1561 tccccctgaa cctgaaacat aaaatgaatg caattgttgt tgttaacttg tttattgcag
     1621 cttataatgg ttacaaataa agcaatagca tcacaaattt cacaaataaa gcattttttt
     1681 cactgcattc tagttgtggt ttgtccaaac tcatcaatgt atcttaaggc gtaaattgta
     1741 agcgttaata ttttgttaaa attcgcgtta aatttttgtt aaatcagctc attttttaac
     1801 caataggccg aaatcggcaa aatcccttat aaatcaaaag aatagaccga gatagggttg
     1861 agtgttgttc cagtttggaa caagagtcca ctattaaaga acgtggactc caacgtcaaa
     1921 gggcgaaaaa ccgtctatca gggcgatggc ccactacgtg aaccatcacc ctaatcaagt
     1981 tttttggggt cgaggtgccg taaagcacta aatcggaacc ctaaagggag cccccgattt
     2041 agagcttgac ggggaaagcc ggcgaacgtg gcgagaaagg aagggaagaa agcgaaagga
     2101 gcgggcgcta gggcgctggc aagtgtagcg gtcacgctgc gcgtaaccac cacacccgcc
     2161 gcgcttaatg cgccgctaca gggcgcgtca ggtggcactt ttcggggaaa tgtgcgcgga
     2221 acccctattt gtttattttt ctaaatacat tcaaatatgt atccgctcat gagacaataa
     2281 ccctgataaa tgcttcaata atattgaaaa aggaagagtc ctgaggcgga aagaaccagc
     2341 tgtggaatgt gtgtcagtta gggtgtggaa agtccccagg ctccccagca ggcagaagta
     2401 tgcaaagcat gcatctcaat tagtcagcaa ccaggtgtgg aaagtcccca ggctccccag
     2461 caggcagaag tatgcaaagc atgcatctca attagtcagc aaccatagtc ccgcccctaa
     2521 ctccgcccat cccgccccta actccgccca gttccgccca ttctccgccc catggctgac
     2581 taattttttt tatttatgca gaggccgagg ccgcctcggc ctctgagcta ttccagaagt
     2641 agtgaggagg cttttttgga ggcctaggct tttgcaaaga tcgatcaaga gacaggatga
     2701 ggatcgtttc gcatgattga acaagatgga ttgcacgcag gttctccggc cgcttgggtg
     2761 gagaggctat tcggctatga ctgggcacaa cagacaatcg gctgctctga tgccgccgtg
     2821 ttccggctgt cagcgcaggg gcgcccggtt ctttttgtca agaccgacct gtccggtgcc
     2881 ctgaatgaac tgcaagacga ggcagcgcgg ctatcgtggc tggccacgac gggcgttcct
     2941 tgcgcagctg tgctcgacgt tgtcactgaa gcgggaaggg actggctgct attgggcgaa
     3001 gtgccggggc aggatctcct gtcatctcac cttgctcctg ccgagaaagt atccatcatg
     3061 gctgatgcaa tgcggcggct gcatacgctt gatccggcta cctgcccatt cgaccaccaa
     3121 gcgaaacatc gcatcgagcg agcacgtact cggatggaag ccggtcttgt cgatcaggat
     3181 gatctggacg aagagcatca ggggctcgcg ccagccgaac tgttcgccag gctcaaggcg
     3241 agcatgcccg acggcgagga tctcgtcgtg acccatggcg atgcctgctt gccgaatatc
     3301 atggtggaaa atggccgctt ttctggattc atcgactgtg gccggctggg tgtggcggac
     3361 cgctatcagg acatagcgtt ggctacccgt gatattgctg aagagcttgg cggcgaatgg
     3421 gctgaccgct tcctcgtgct ttacggtatc gccgctcccg attcgcagcg catcgccttc
     3481 tatcgccttc ttgacgagtt cttctgagcg ggactctggg gttcgaaatg accgaccaag
     3541 cgacgcccaa cctgccatca cgagatttcg attccaccgc cgccttctat gaaaggttgg
     3601 gcttcggaat cgttttccgg gacgccggct ggatgatcct ccagcgcggg gatctcatgc
     3661 tggagttctt cgcccaccct agggggaggc taactgaaac acggaaggag acaataccgg
     3721 aaggaacccg cgctatgacg gcaataaaaa gacagaataa aacgcacggt gttgggtcgt
     3781 ttgttcataa acgcggggtt cggtcccagg gctggcactc tgtcgatacc ccaccgagac
     3841 cccattgggg ccaatacgcc cgcgtttctt ccttttcccc accccacccc ccaagttcgg
     3901 gtgaaggccc agggctcgca gccaacgtcg gggcggcagg ccctgccata gcctcaggtt
     3961 actcatatat actttagatt gatttaaaac ttcattttta atttaaaagg atctaggtga
     4021 agatcctttt tgataatctc atgaccaaaa tcccttaacg tgagttttcg ttccactgag
     4081 cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa
     4141 tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg ccggatcaag
     4201 agctaccaac tctttttccg aaggtaactg gcttcagcag agcgcagata ccaaatactg
     4261 ttcttctagt gtagccgtag ttaggccacc acttcaagaa ctctgtagca ccgcctacat
     4321 acctcgctct gctaatcctg ttaccagtgg ctgctgccag tggcgataag tcgtgtctta
     4381 ccgggttgga ctcaagacga tagttaccgg ataaggcgca gcggtcgggc tgaacggggg
     4441 gttcgtgcac acagcccagc ttggagcgaa cgacctacac cgaactgaga tacctacagc
     4501 gtgagctatg agaaagcgcc acgcttcccg aagggagaaa ggcggacagg tatccggtaa
     4561 gcggcagggt cggaacagga gagcgcacga gggagcttcc agggggaaac gcctggtatc
     4621 tttatagtcc tgtcgggttt cgccacctct gacttgagcg tcgatttttg tgatgctcgt
     4681 caggggggcg gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct
     4741 tttgctggcc ttttgctcac atgttctttc ctgcgttatc ccctgattct gtggataacc
     4801 gtattaccgc catgcattag ttattaatag taatcaatta cggggtcatt agttcatagc
     4861 cc
//
