LOCUS       sequence-90305-e        4856 bp    DNA     circular SYN 14-APR-2025
DEFINITION  Expresses a nuclear-targeted actin filament reporter, consisting of 
            the peptide Lifeact, on a CMV promoter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4856)
  AUTHORS   Belin BJ, Cimini BA, Blackburn EH, Mullins RD
  TITLE     Visualization of actin filaments and monomers in somatic cell 
            nuclei.
  JOURNAL   Mol Biol Cell. 2013 Apr;24(7):982-94. doi: 10.1091/mbc.E12-09-0685. 
            Epub 2013 Feb 27.
  PUBMED    23447706
REFERENCE   2  (bases 1 to 4856)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 14, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; doi: 
            "10.1091/mbc.E12-09-0685"; journalName: "Mol Biol Cell"; date: 
            "2013-04"; volume: "24"; issue: "7"; pages: "982-94"
COMMENT     Sequence Label: pEGFP-C1 Lifeact-EGFP-2XNLS
FEATURES             Location/Qualifiers
     source          1..4856
                     /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      666..675
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(672..674,675..677,678..1388)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(672..674,675..677,678..1388)
                     /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(717..738)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(978..997)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1325..1346
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             1425..1445
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             1449..1469
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     polyA_signal    1644..1765
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1681..1700)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     1735..1754
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(1772..2227)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1909..1930)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2121..2140
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        2254..2358
                     /gene="bla"
                     /label=AmpR promoter
     promoter        2360..2717
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        2360..2551
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(2383..2403)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      2568..2703
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             2752..3546
                     /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             2752..3546
                     /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(2806..2825)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3416..3435
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(3734..3753)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    3778..3825
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      4154..4742
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4643..4662
                     /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 gacccagtac
      661 cggtcgccac catggtgagc aagggcgagg agctgttcac cggggtggtg cccatcctgg
      721 tcgagctgga cggcgacgta aacggccaca agttcagcgt gtccggcgag ggcgagggcg
      781 atgccaccta cggcaagctg accctgaagt tcatctgcac caccggcaag ctgcccgtgc
      841 cctggcccac cctcgtgacc accctgacct acggcgtgca gtgcttcagc cgctaccccg
      901 accacatgaa gcagcacgac ttcttcaagt ccgccatgcc cgaaggctac gtccaggagc
      961 gcaccatctt cttcaaggac gacggcaact acaagacccg cgccgaggtg aagttcgagg
     1021 gcgacaccct ggtgaaccgc atcgagctga agggcatcga cttcaaggag gacggcaaca
     1081 tcctggggca caagctggag tacaactaca acagccacaa cgtctatatc atggccgaca
     1141 agcagaagaa cggcatcaag gtgaacttca agatccgcca caacatcgag gacggcagcg
     1201 tgcagctcgc cgaccactac cagcagaaca cccccatcgg cgacggcccc gtgctgctgc
     1261 ccgacaacca ctacctgagc acccagtccg ccctgagcaa agaccccaac gagaagcgcg
     1321 atcacatggt cctgctggag ttcgtgaccg ccgccgggat cactctcggc atggacgagc
     1381 tgtacaagtc cggactcaga tctcgagctc aagcttccat ggatccaaaa aagaagagaa
     1441 aggtagatcc aaaaaagaag agaaaggtag atccaaaaaa gaagagaaag tgataatagg
     1501 gtaccgcggg cccgggatcc accggatcta gataactgat cataatcagc cataccacat
     1561 ttgtagaggt tttacttgct ttaaaaaacc tcccacacct ccccctgaac ctgaaacata
     1621 aaatgaatgc aattgttgtt gttaacttgt ttattgcagc ttataatggt tacaaataaa
     1681 gcaatagcat cacaaatttc acaaataaag catttttttc actgcattct agttgtggtt
     1741 tgtccaaact catcaatgta tcttaacgcg taaattgtaa gcgttaatat tttgttaaaa
     1801 ttcgcgttaa atttttgtta aatcagctca ttttttaacc aataggccga aatcggcaaa
     1861 atcccttata aatcaaaaga atagaccgag atagggttga gtgttgttcc agtttggaac
     1921 aagagtccac tattaaagaa cgtggactcc aacgtcaaag ggcgaaaaac cgtctatcag
     1981 ggcgatggcc cactacgtga accatcaccc taatcaagtt ttttggggtc gaggtgccgt
     2041 aaagcactaa atcggaaccc taaagggagc ccccgattta gagcttgacg gggaaagccg
     2101 gcgaacgtgg cgagaaagga agggaagaaa gcgaaaggag cgggcgctag ggcgctggca
     2161 agtgtagcgg tcacgctgcg cgtaaccacc acacccgccg cgcttaatgc gccgctacag
     2221 ggcgcgtcag gtggcacttt tcggggaaat gtgcgcggaa cccctatttg tttatttttc
     2281 taaatacatt caaatatgta tccgctcatg agacaataac cctgataaat gcttcaataa
     2341 tattgaaaaa ggaagagtcc tgaggcggaa agaaccagct gtggaatgtg tgtcagttag
     2401 ggtgtggaaa gtccccaggc tccccagcag gcagaagtat gcaaagcatg catctcaatt
     2461 agtcagcaac caggtgtgga aagtccccag gctccccagc aggcagaagt atgcaaagca
     2521 tgcatctcaa ttagtcagca accatagtcc cgcccctaac tccgcccatc ccgcccctaa
     2581 ctccgcccag ttccgcccat tctccgcccc atggctgact aatttttttt atttatgcag
     2641 aggccgaggc cgcctcggcc tctgagctat tccagaagta gtgaggaggc ttttttggag
     2701 gcctaggctt ttgcaaagat cgatcaagag acaggatgag gatcgtttcg catgattgaa
     2761 caagatggat tgcacgcagg ttctccggcc gcttgggtgg agaggctatt cggctatgac
     2821 tgggcacaac agacaatcgg ctgctctgat gccgccgtgt tccggctgtc agcgcagggg
     2881 cgcccggttc tttttgtcaa gaccgacctg tccggtgccc tgaatgaact gcaagacgag
     2941 gcagcgcggc tatcgtggct ggccacgacg ggcgttcctt gcgcagctgt gctcgacgtt
     3001 gtcactgaag cgggaaggga ctggctgcta ttgggcgaag tgccggggca ggatctcctg
     3061 tcatctcacc ttgctcctgc cgagaaagta tccatcatgg ctgatgcaat gcggcggctg
     3121 catacgcttg atccggctac ctgcccattc gaccaccaag cgaaacatcg catcgagcga
     3181 gcacgtactc ggatggaagc cggtcttgtc gatcaggatg atctggacga agagcatcag
     3241 gggctcgcgc cagccgaact gttcgccagg ctcaaggcga gcatgcccga cggcgaggat
     3301 ctcgtcgtga cccatggcga tgcctgcttg ccgaatatca tggtggaaaa tggccgcttt
     3361 tctggattca tcgactgtgg ccggctgggt gtggcggacc gctatcagga catagcgttg
     3421 gctacccgtg atattgctga agagcttggc ggcgaatggg ctgaccgctt cctcgtgctt
     3481 tacggtatcg ccgctcccga ttcgcagcgc atcgccttct atcgccttct tgacgagttc
     3541 ttctgagcgg gactctgggg ttcgaaatga ccgaccaagc gacgcccaac ctgccatcac
     3601 gagatttcga ttccaccgcc gccttctatg aaaggttggg cttcggaatc gttttccggg
     3661 acgccggctg gatgatcctc cagcgcgggg atctcatgct ggagttcttc gcccacccta
     3721 gggggaggct aactgaaaca cggaaggaga caataccgga aggaacccgc gctatgacgg
     3781 caataaaaag acagaataaa acgcacggtg ttgggtcgtt tgttcataaa cgcggggttc
     3841 ggtcccaggg ctggcactct gtcgataccc caccgagacc ccattggggc caatacgccc
     3901 gcgtttcttc cttttcccca ccccaccccc caagttcggg tgaaggccca gggctcgcag
     3961 ccaacgtcgg ggcggcaggc cctgccatag cctcaggtta ctcatatata ctttagattg
     4021 atttaaaact tcatttttaa tttaaaagga tctaggtgaa gatccttttt gataatctca
     4081 tgaccaaaat cccttaacgt gagttttcgt tccactgagc gtcagacccc gtagaaaaga
     4141 tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa
     4201 aaccaccgct accagcggtg gtttgtttgc cggatcaaga gctaccaact ctttttccga
     4261 aggtaactgg cttcagcaga gcgcagatac caaatactgt tcttctagtg tagccgtagt
     4321 taggccacca cttcaagaac tctgtagcac cgcctacata cctcgctctg ctaatcctgt
     4381 taccagtggc tgctgccagt ggcgataagt cgtgtcttac cgggttggac tcaagacgat
     4441 agttaccgga taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca cagcccagct
     4501 tggagcgaac gacctacacc gaactgagat acctacagcg tgagctatga gaaagcgcca
     4561 cgcttcccga agggagaaag gcggacaggt atccggtaag cggcagggtc ggaacaggag
     4621 agcgcacgag ggagcttcca gggggaaacg cctggtatct ttatagtcct gtcgggtttc
     4681 gccacctctg acttgagcgt cgatttttgt gatgctcgtc aggggggcgg agcctatgga
     4741 aaaacgccag caacgcggcc tttttacggt tcctggcctt ttgctggcct tttgctcaca
     4801 tgttctttcc tgcgttatcc cctgattctg tggataaccg tattaccgcc atgcat
//
