LOCUS       sequence-322477-        4787 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses a cytoplasmic actin filament reporter, the peptide 
            Lifeact, on a CMV promoter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4787)
  AUTHORS   Belin BJ, Goins LM, Mullins RD
  TITLE     Comparative analysis of tools for live cell imaging of actin network
            architecture.
  JOURNAL   Bioarchitecture. 2015 Aug 28:1-14.
  PUBMED    26317264
REFERENCE   2  (bases 1 to 4787)
  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: 
            "Bioarchitecture. 2015 Aug 28:1-14."
COMMENT     Sequence Label: pEGFP-C1 Lifeact-EGFP
FEATURES             Location/Qualifiers
     source          1..4787
                     /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"
     CDS             597..647
                     /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      663..672
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(669..671,672..674,675..1385)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLK
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(714..735)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(975..994)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     misc_feature    1386..1451
                     /label=MCS
                     /note="multiple cloning site"
     polyA_signal    1575..1696
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1612..1631)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     1666..1685
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(1703..2158)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1840..1861)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2052..2071
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        2185..2289
                     /gene="bla"
                     /label=AmpR promoter
     promoter        2291..2648
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        2291..2482
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(2314..2334)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      2499..2634
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             2683..3477
                     /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             2683..3477
                     /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(2737..2756)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3347..3366
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(3665..3684)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    3709..3756
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      4085..4673
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4574..4593
                     /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 gctagcatgg
      601 gcgtggccga cctgatcaag aagttcgaga gcatcagcaa ggaagagggc gacccaccgg
      661 tcgccaccat ggtgagcaag ggcgaggagc tgttcaccgg ggtggtgccc atcctggtcg
      721 agctggacgg cgacgtaaac ggccacaagt tcagcgtgtc cggcgagggc gagggcgatg
      781 ccacctacgg caagctgacc ctgaagttca tctgcaccac cggcaagctg cccgtgccct
      841 ggcccaccct cgtgaccacc ctgacctacg gcgtgcagtg cttcagccgc taccccgacc
      901 acatgaagca gcacgacttc ttcaagtccg ccatgcccga aggctacgtc caggagcgca
      961 ccatcttctt caaggacgac ggcaactaca agacccgcgc cgaggtgaag ttcgagggcg
     1021 acaccctggt gaaccgcatc gagctgaagg gcatcgactt caaggaggac ggcaacatcc
     1081 tggggcacaa gctggagtac aactacaaca gccacaacgt ctatatcatg gccgacaagc
     1141 agaagaacgg catcaaggtg aacttcaaga tccgccacaa catcgaggac ggcagcgtgc
     1201 agctcgccga ccactaccag cagaacaccc ccatcggcga cggccccgtg ctgctgcccg
     1261 acaaccacta cctgagcacc cagtccgccc tgagcaaaga ccccaacgag aagcgcgatc
     1321 acatggtcct taaggagttc gtgaccgccg ccgggatcac tctcggcatg gacgagctgt
     1381 acaagtccgg actcagatct cgagctcaag cttcgaattc tgcagtcgac ggtaccgcgg
     1441 gcccgggatc caccggatct agataactga tcataatcag ccataccaca tttgtagagg
     1501 ttttacttgc tttaaaaaac ctcccacacc tccccctgaa cctgaaacat aaaatgaatg
     1561 caattgttgt tgttaacttg tttattgcag cttataatgg ttacaaataa agcaatagca
     1621 tcacaaattt cacaaataaa gcattttttt cactgcattc tagttgtggt ttgtccaaac
     1681 tcatcaatgt atcttaacgc gtaaattgta agcgttaata ttttgttaaa attcgcgtta
     1741 aatttttgtt aaatcagctc attttttaac caataggccg aaatcggcaa aatcccttat
     1801 aaatcaaaag aatagaccga gatagggttg agtgttgttc cagtttggaa caagagtcca
     1861 ctattaaaga acgtggactc caacgtcaaa gggcgaaaaa ccgtctatca gggcgatggc
     1921 ccactacgtg aaccatcacc ctaatcaagt tttttggggt cgaggtgccg taaagcacta
     1981 aatcggaacc ctaaagggag cccccgattt agagcttgac ggggaaagcc ggcgaacgtg
     2041 gcgagaaagg aagggaagaa agcgaaagga gcgggcgcta gggcgctggc aagtgtagcg
     2101 gtcacgctgc gcgtaaccac cacacccgcc gcgcttaatg cgccgctaca gggcgcgtca
     2161 ggtggcactt ttcggggaaa tgtgcgcgga acccctattt gtttattttt ctaaatacat
     2221 tcaaatatgt atccgctcat gagacaataa ccctgataaa tgcttcaata atattgaaaa
     2281 aggaagagtc ctgaggcgga aagaaccagc tgtggaatgt gtgtcagtta gggtgtggaa
     2341 agtccccagg ctccccagca ggcagaagta tgcaaagcat gcatctcaat tagtcagcaa
     2401 ccaggtgtgg aaagtcccca ggctccccag caggcagaag tatgcaaagc atgcatctca
     2461 attagtcagc aaccatagtc ccgcccctaa ctccgcccat cccgccccta actccgccca
     2521 gttccgccca ttctccgccc catggctgac taattttttt tatttatgca gaggccgagg
     2581 ccgcctcggc ctctgagcta ttccagaagt agtgaggagg cttttttgga ggcctaggct
     2641 tttgcaaaga tcgatcaaga gacaggatga ggatcgtttc gcatgattga acaagatgga
     2701 ttgcacgcag gttctccggc cgcttgggtg gagaggctat tcggctatga ctgggcacaa
     2761 cagacaatcg gctgctctga tgccgccgtg ttccggctgt cagcgcaggg gcgcccggtt
     2821 ctttttgtca agaccgacct gtccggtgcc ctgaatgaac tgcaagacga ggcagcgcgg
     2881 ctatcgtggc tggccacgac gggcgttcct tgcgcagctg tgctcgacgt tgtcactgaa
     2941 gcgggaaggg actggctgct attgggcgaa gtgccggggc aggatctcct gtcatctcac
     3001 cttgctcctg ccgagaaagt atccatcatg gctgatgcaa tgcggcggct gcatacgctt
     3061 gatccggcta cctgcccatt cgaccaccaa gcgaaacatc gcatcgagcg agcacgtact
     3121 cggatggaag ccggtcttgt cgatcaggat gatctggacg aagagcatca ggggctcgcg
     3181 ccagccgaac tgttcgccag gctcaaggcg agcatgcccg acggcgagga tctcgtcgtg
     3241 acccatggcg atgcctgctt gccgaatatc atggtggaaa atggccgctt ttctggattc
     3301 atcgactgtg gccggctggg tgtggcggac cgctatcagg acatagcgtt ggctacccgt
     3361 gatattgctg aagagcttgg cggcgaatgg gctgaccgct tcctcgtgct ttacggtatc
     3421 gccgctcccg attcgcagcg catcgccttc tatcgccttc ttgacgagtt cttctgagcg
     3481 ggactctggg gttcgaaatg accgaccaag cgacgcccaa cctgccatca cgagatttcg
     3541 attccaccgc cgccttctat gaaaggttgg gcttcggaat cgttttccgg gacgccggct
     3601 ggatgatcct ccagcgcggg gatctcatgc tggagttctt cgcccaccct agggggaggc
     3661 taactgaaac acggaaggag acaataccgg aaggaacccg cgctatgacg gcaataaaaa
     3721 gacagaataa aacgcacggt gttgggtcgt ttgttcataa acgcggggtt cggtcccagg
     3781 gctggcactc tgtcgatacc ccaccgagac cccattgggg ccaatacgcc cgcgtttctt
     3841 ccttttcccc accccacccc ccaagttcgg gtgaaggccc agggctcgca gccaacgtcg
     3901 gggcggcagg ccctgccata gcctcaggtt actcatatat actttagatt gatttaaaac
     3961 ttcattttta atttaaaagg atctaggtga agatcctttt tgataatctc atgaccaaaa
     4021 tcccttaacg tgagttttcg ttccactgag cgtcagaccc cgtagaaaag atcaaaggat
     4081 cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc
     4141 taccagcggt ggtttgtttg ccggatcaag agctaccaac tctttttccg aaggtaactg
     4201 gcttcagcag agcgcagata ccaaatactg ttcttctagt gtagccgtag ttaggccacc
     4261 acttcaagaa ctctgtagca ccgcctacat acctcgctct gctaatcctg ttaccagtgg
     4321 ctgctgccag tggcgataag tcgtgtctta ccgggttgga ctcaagacga tagttaccgg
     4381 ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac acagcccagc ttggagcgaa
     4441 cgacctacac cgaactgaga tacctacagc gtgagctatg agaaagcgcc acgcttcccg
     4501 aagggagaaa ggcggacagg tatccggtaa gcggcagggt cggaacagga gagcgcacga
     4561 gggagcttcc agggggaaac gcctggtatc tttatagtcc tgtcgggttt cgccacctct
     4621 gacttgagcg tcgatttttg tgatgctcgt caggggggcg gagcctatgg aaaaacgcca
     4681 gcaacgcggc ctttttacgg ttcctggcct tttgctggcc ttttgctcac atgttctttc
     4741 ctgcgttatc ccctgattct gtggataacc gtattaccgc catgcat
//
