LOCUS       sequence-439256-        6027 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  To express PLIN3 with mEGFP tag in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6027)
  AUTHORS   Downie L, Ferrandiz N, Courthold E, Jones M, Royle SJ
  TITLE     Nondisruptive inducible labeling of ER-membrane contact sites using 
            the Lamin B receptor.
  JOURNAL   PLoS Biol. 2025 Jul 10;23(7):e3003249. doi: 
            10.1371/journal.pbio.3003249. eCollection 2025 Jul.
  PUBMED    40638559
REFERENCE   2  (bases 1 to 6027)
  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: "PLoS 
            Biol."; date: "2025-07-10"; volume: "23(7):e3003249. doi"; pages: " 
            10.1371/journal.pbio.3003249. eCollection 2025 Jul"
COMMENT     Sequence Label: mEGFP-PLIN3
FEATURES             Location/Qualifiers
     source          1..6027
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        80..271
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     228..248
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        complement(273..377)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      404..859
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(491..510)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     701..722
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     polyA_signal    866..987
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(877..896)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     931..950
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     CDS             complement(join(2473..3183,3184..3186,3187..3189))
                     /codon_start=1
                     /product="enhanced GFP with monomerizing A206K mutation 
                     (Zacharias et al., 2002)"
                     /label=mEGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSKLSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(2515..2536)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     2864..2883
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     3123..3144
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     regulatory      3186..3195
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     promoter        complement(3234..3437)
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     complement(3263..3283)
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     enhancer        3438..3741
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     rep_origin      complement(3916..4504)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3996..4015)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     polyA_signal    4833..4880
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     primer_bind     4905..4924
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     CDS             complement(5112..5906)
                     /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             complement(5112..5906)
                     /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(5223..5242)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     5833..5852
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     promoter        complement(join(5941..6027,1..271))
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      join(5955..6027,1..63)
                     /label=SV40 ori
                     /note="SV40 origin of replication"
ORIGIN
        1 aaaaaaaatt agtcagccat ggggcggaga atgggcggaa ctgggcggag ttaggggcgg
       61 gatgggcgga gttaggggcg ggactatggt tgctgactaa ttgagatgca tgctttgcat
      121 acttctgcct gctggggagc ctggggactt tccacacctg gttgctgact aattgagatg
      181 catgctttgc atacttctgc ctgctgggga gcctggggac tttccacacc ctaactgaca
      241 cacattccac agctggttct ttccgcctca ggactcttcc tttttcaata ttattgaagc
      301 atttatcagg gttattgtct catgagcgga tacatatttg aatgtattta gaaaaataaa
      361 caaatagggg ttccgcgcac atttccccga aaagtgccac ctgacgcgcc ctgtagcggc
      421 gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga ccgctacact tgccagcgcc
      481 ctagcgcccg ctcctttcgc tttcttccct tcctttctcg ccacgttcgc cggctttccc
      541 cgtcaagctc taaatcgggg gctcccttta gggttccgat ttagtgcttt acggcacctc
      601 gaccccaaaa aacttgatta gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg
      661 gtttttcgcc ctttgacgtt ggagtccacg ttctttaata gtggactctt gttccaaact
      721 ggaacaacac tcaaccctat ctcggtctat tcttttgatt tataagggat tttgccgatt
      781 tcggcctatt ggttaaaaaa tgagctgatt taacaaaaat ttaacgcgaa ttttaacaaa
      841 atattaacgc ttacaattta cgcgttaaga tacattgatg agtttggaca aaccacaact
      901 agaatgcagt gaaaaaaatg ctttatttgt gaaatttgtg atgctattgc tttatttgta
      961 accattataa gctgcaataa acaagttaac aacaacaatt gcattcattt tatgtttcag
     1021 gttcaggggg aggtgtggga ggttttttaa agcaagtaaa acctctacaa atgtggtatg
     1081 gctgattatg atcagttatc tagatccggt ggatcccggg cccgcggtac cgtcgactgc
     1141 agaattcgaa gcttgagcct acttcttctc ctccggggct ttctcagtga ttccaggggc
     1201 aaagggtccc acgagccacg tgacaggtgt gttctgggcc acatattcca ccatgtggtc
     1261 cagggcctcg cgggcgctgg cgacacgctc acggctctgg gccagaatgc tgctggacag
     1321 gtcctggaag gagtggatgc tggaaaacgt ggcctggagg tcctccacct ggcggcgggc
     1381 ctgctgcacc tggtccttca cattggtggg gaggccctga atgctggacc ccagggaggt
     1441 acaggtggcc tgcagttgct gggcaatgtc ccggaacatg gtgagcgccc gggactcgac
     1501 ctgctctggc ttgggcggct ccttctcggg gccctggagc tgcttctggt tccagctgag
     1561 ccacatctgg tgcagcttct cctggccttc caccagcttc tgatcaacgc cttgcttgac
     1621 agtttccatc aggcttagga cctgcgacag ctgcagcaga gcctcctgtg ccctctgctt
     1681 ggtggctcga agcttgccca gcgagtgctc ataggcgtgc tgccgcagcc tctccgacag
     1741 ggagcccaga cgtacgaagt agctctgttc ctgccgctgc tgctgcacgg acgcgacgtc
     1801 aaagccatcc agggatgtgg cgatgcgggc cagttcggca tccgtaaggg gcaggtggtt
     1861 gtccgcccac tcctccgact tccccagcac cgtgtcgacc ccactcaaca ccatctggcc
     1921 caagcgggag cccatgaccg attggacgcc gccggtcact acggactttg tcttgtccac
     1981 gccgctctgc acagcaccgc gggtcgcgtc caccgcctcc gacaattggg tggccaccgt
     2041 gtccttggcg ctagacacca tctcttgggc ccccgacacc ttagacgaca caagctcctt
     2101 ggtgtccgcc aggaccttct ccgtgggctg ctgcaggatg gggaggttct cctccaactt
     2161 gtccagcccc ctgtgggcgt attcgctggc tgatgcaatc tggggctcca gcttggagag
     2221 gatcggctga gccccgctga cagcagccgc cgtgagggtc ctcactccct tctctgctgc
     2281 gtcgcagaca gtcttgatgt gcgggtagct ctccttggtg gaggcatagg ctgcggacac
     2341 catgtcgcag gtggagctga tcagaggcat gctggccaca cggtccacca cactgggctg
     2401 ctgtaccggt tcttccactg tcacctgggt gctgccatca gcctctgccc cgtcggcaga
     2461 catgagtccg gacttgtaca gctcgtccat gccgagagtg atcccggcgg cggtcacgaa
     2521 ctccagcagg accatgtgat cgcgcttctc gttggggtct ttgctcagct tggactgggt
     2581 gctcaggtag tggttgtcgg gcagcagcac ggggccgtcg ccgatggggg tgttctgctg
     2641 gtagtggtcg gcgagctgca cgctgccgtc ctcgatgttg tggcggatct tgaagttcac
     2701 cttgatgccg ttcttctgct tgtcggccat gatatagacg ttgtggctgt tgtagttgta
     2761 ctccagcttg tgccccagga tgttgccgtc ctccttgaag tcgatgccct tcagctcgat
     2821 gcggttcacc agggtgtcgc cctcgaactt cacctcggcg cgggtcttgt agttgccgtc
     2881 gtccttgaag aagatggtgc gctcctggac gtagccttcg ggcatggcgg acttgaagaa
     2941 gtcgtgctgc ttcatgtggt cggggtagcg gctgaagcac tgcacgccgt aggtcagggt
     3001 ggtcacgagg gtgggccagg gcacgggcag cttgccggtg gtgcagatga acttcagggt
     3061 cagcttgccg taggtggcat cgccctcgcc ctcgccggac acgctgaact tgtggccgtt
     3121 tacgtcgccg tccagctcga ccaggatggg caccaccccg gtgaacagct cctcgccctt
     3181 gctcaccatg gtggcgaccg gtagcgctag cggatctgac ggttcactaa accagctctg
     3241 cttatataga cctcccaccg tacacgccta ccgcccattt gcgtcaatgg ggcggagttg
     3301 ttacgacatt ttggaaagtc ccgttgattt tggtgccaaa acaaactccc attgacgtca
     3361 atggggtgga gacttggaaa tccccgtgag tcaaaccgct atccacgccc attgatgtac
     3421 tgccaaaacc gcatcaccat ggtaatagcg atgactaata cgtagatgta ctgccaagta
     3481 ggaaagtccc ataaggtcat gtactgggca taatgccagg cgggccattt accgtcattg
     3541 acgtcaatag ggggcgtact tggcatatga tacacttgat gtactgccaa gtgggcagtt
     3601 taccgtaaat actccaccca ttgacgtcaa tggaaagtcc ctattggcgt tactatggga
     3661 acatacgtca ttattgacgt caatgggcgg gggtcgttgg gcggtcagcc aggcgggcca
     3721 tttaccgtaa gttatgtaac gcggaactcc atatatgggc tatgaactaa tgaccccgta
     3781 attgattact attaataact aatgcatggc ggtaatacgg ttatccacag aatcagggga
     3841 taacgcagga aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc
     3901 cgcgttgctg gcgtttttcc ataggctccg cccccctgac gagcatcaca aaaatcgacg
     3961 ctcaagtcag aggtggcgaa acccgacagg actataaaga taccaggcgt ttccccctgg
     4021 aagctccctc gtgcgctctc ctgttccgac cctgccgctt accggatacc tgtccgcctt
     4081 tctcccttcg ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc tcagttcggt
     4141 gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg
     4201 cgccttatcc ggtaactatc gtcttgagtc caacccggta agacacgact tatcgccact
     4261 ggcagcagcc actggtaaca ggattagcag agcgaggtat gtaggcggtg ctacagagtt
     4321 cttgaagtgg tggcctaact acggctacac tagaagaaca gtatttggta tctgcgctct
     4381 gctgaagcca gttaccttcg gaaaaagagt tggtagctct tgatccggca aacaaaccac
     4441 cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc
     4501 tcaagaagat cctttgatct tttctacggg gtctgacgct cagtggaacg aaaactcacg
     4561 ttaagggatt ttggtcatga gattatcaaa aaggatcttc acctagatcc ttttaaatta
     4621 aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa cctgaggcta tggcagggcc
     4681 tgccgccccg acgttggctg cgagccctgg gccttcaccc gaacttgggg ggtggggtgg
     4741 ggaaaaggaa gaaacgcggg cgtattggcc ccaatggggt ctcggtgggg tatcgacaga
     4801 gtgccagccc tgggaccgaa ccccgcgttt atgaacaaac gacccaacac cgtgcgtttt
     4861 attctgtctt tttattgccg tcatagcgcg ggttccttcc ggtattgtct ccttccgtgt
     4921 ttcagttagc ctccccctag ggtgggcgaa gaactccagc atgagatccc cgcgctggag
     4981 gatcatccag ccggcgtccc ggaaaacgat tccgaagccc aacctttcat agaaggcggc
     5041 ggtggaatcg aaatctcgtg atggcaggtt gggcgtcgct tggtcggtca tttcgaaccc
     5101 cagagtcccg ctcagaagaa ctcgtcaaga aggcgataga aggcgatgcg ctgcgaatcg
     5161 ggagcggcga taccgtaaag cacgaggaag cggtcagccc attcgccgcc aagctcttca
     5221 gcaatatcac gggtagccaa cgctatgtcc tgatagcggt ccgccacacc cagccggcca
     5281 cagtcgatga atccagaaaa gcggccattt tccaccatga tattcggcaa gcaggcatcg
     5341 ccatgggtca cgacgagatc ctcgccgtcg ggcatgctcg ccttgagcct ggcgaacagt
     5401 tcggctggcg cgagcccctg atgctcttcg tccagatcat cctgatcgac aagaccggct
     5461 tccatccgag tacgtgctcg ctcgatgcga tgtttcgctt ggtggtcgaa tgggcaggta
     5521 gccggatcaa gcgtatgcag ccgccgcatt gcatcagcca tgatggatac tttctcggca
     5581 ggagcaaggt gagatgacag gagatcctgc cccggcactt cgcccaatag cagccagtcc
     5641 cttcccgctt cagtgacaac gtcgagcaca gctgcgcaag gaacgcccgt cgtggccagc
     5701 cacgatagcc gcgctgcctc gtcttgcagt tcattcaggg caccggacag gtcggtcttg
     5761 acaaaaagaa ccgggcgccc ctgcgctgac agccggaaca cggcggcatc agagcagccg
     5821 attgtctgtt gtgcccagtc atagccgaat agcctctcca cccaagcggc cggagaacct
     5881 gcgtgcaatc catcttgttc aatcatgcga aacgatcctc atcctgtctc ttgatcgatc
     5941 tttgcaaaag cctaggcctc caaaaaagcc tcctcactac ttctggaata gctcagaggc
     6001 cgaggcggcc tcggcctctg cataaat
//
