LOCUS       sequence-439218-        6905 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  To express a cholesterol synthesis point mutant of LBR (N547D) with 
            GFP and a FKBP tag for rapamycin-induced heterodimerisation in 
            mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6905)
  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 6905)
  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: LBR(N547D)-FKBP-GFP
FEATURES             Location/Qualifiers
     source          1..6905
                     /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             join(2512..2514,2515..2835)
                     /codon_start=1
                     /product="human FK506-binding protein FKBP12"
                     /label=FKBP
                     /translation="MGVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKFDSSRDRNK
                     PFKFMLGKQEVIRGWEEGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPHATLVFDVEL
                     LKLE"
     regulatory      2845..2854
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(2851..2853,2854..2856,2857..3570)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(2851..2853,2854..2856,2857..3567)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(2896..2917)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(3157..3176)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     3504..3525
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    3693..3814
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3730..3749)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3784..3803
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(3821..4276)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3958..3979)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     4170..4189
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        4303..4407
                     /gene="bla"
                     /label=AmpR promoter
     promoter        4409..4766
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        4409..4600
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(4432..4452)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      4617..4752
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             4801..5595
                     /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             4801..5595
                     /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(4855..4874)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     5465..5484
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(5783..5802)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    5827..5874
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      6203..6791
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6692..6711
                     /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 gctagcgcta
      601 ccggactcag atctcgagct caagcttcga attctgcagt cgacggtacc catgccaagt
      661 aggaaatttg ccgatggtga agtggtaaga ggtcgatggc ctgggagttc actttattat
      721 gaagtagaaa ttctgagcca cgacagcacc tcccagcttt acactgtgaa gtataaagat
      781 ggaacagagc ttgaattgaa agagaatgat attaagcctt taacttcctt taggcaaagg
      841 aaaggtggct caacttccag ttccccttcc agacgccgag ggagtcgatc aaggtcacgc
      901 tcccgatccc ctggtcgacc acctaaaagt gcccgccgat ctgcttctgc ttcccaccag
      961 gccgacatta aggaagcaag gagggaagtg gaagttaaat tgactccgct gattctgaag
     1021 ccatttggaa atagcatcag cagatataat ggggagcctg agcatattga gagaaatgac
     1081 gcacctcata aaaatacaca ggaaaaattc agtttgtcac aagaaagcag ttacatagca
     1141 acacagtata gccttcgtcc aagaagagaa gaagtcaaat taaaagaaat agattctaag
     1201 gaagaaaaat acgttgcaaa agaactggca gtgagaacct ttgaagtgac ccccatccgg
     1261 gcaaaggact tggagtttgg aggagtacct ggtgtgtttc tcatcatgtt tggcctgcct
     1321 gtgttcctct tcctgttgct gttgatgtgt aaacagaaag atcccagtct tctgaatttc
     1381 cctcctcctt tgccagcttt gtatgagtta tgggaaacca gagtatttgg ggtctacctc
     1441 ctgtggtttt tgattcaagt cctgttctac ctactgccaa ttggaaaggt tgtagaagga
     1501 acgcctctta ttgatggaag aagactcaag tatagattaa atggattcta tgcttttatc
     1561 ctgacatctg cagtcatcgg aacatctctc ttccagggcg tagagtttca ttacgtgtac
     1621 agtcattttc ttcagtttgc acttgcggcc actgtttttt gtgtggtctt gagtgtgtat
     1681 ctctacatgc gctctttgaa agcgccccgg aatgacctgt cgcctgccag ctctggaaat
     1741 gctgtctatg atttcttcat tggccgtgaa ttaaaccctc gaattggtac ttttgatctc
     1801 aaatactttt gtgaattgcg ccccggattg attggatggg tggttattaa cttggtgatg
     1861 cttttggctg aaatgaaaat acaggaccgc gctgttccat ccttggccat gattttagtt
     1921 aatagtttcc agcttctcta tgtggtggat gctctctgga atgaggaagc gttgttgacg
     1981 accatggaca tcatccacga tggatttgga ttcatgctgg cttttggaga cttggtgtgg
     2041 gttcccttta tttacagctt ccaagccttt tatttagtca gtcatccaaa tgaagtgtct
     2101 tggccaatgg cttctctaat tattgttctg aaactttgtg gttatgtaat cttccgaggt
     2161 gcaaattctc agaaaaatgc attccggaaa aatcccagtg atccaaagct tgcacattta
     2221 aaaaccattc atacttcaac gggaaaaaat cttctagttt ctggatggtg gggctttgtt
     2281 cgccaccccg attacttggg tgatctcatc atggccttgg cgtggtccct cccatgtggt
     2341 tttaaccaca ttctgcctta tttctacata atttatttca ccatgttgct tgtccaccga
     2401 gaagctcgtg acgagtacca ctgtaagaag aaatacggcg tggcttggga aaagtactgt
     2461 cagcgtgtgc cctaccgtat atttccatac atctacacgg atccgccacc aatgggagtg
     2521 caggtggaaa ccatctcccc aggagacggg cgcaccttcc ccaagcgcgg ccagacctgc
     2581 gtggtgcact acaccgggat gcttgaagat ggaaagaaat ttgattcctc ccgggacaga
     2641 aacaagccct ttaagtttat gctaggcaag caggaggtga tccgaggctg ggaagaaggg
     2701 gttgcccaga tgagtgtggg tcagagagcc aaactgacta tatctccaga ttatgcctat
     2761 ggtgccactg ggcacccagg catcatccca ccacatgcca ctctcgtctt cgatgtggag
     2821 cttctaaaac tggaacgacc ggtcgccacc atggtgagca agggcgagga gctgttcacc
     2881 ggggtggtgc ccatcctggt cgagctggac ggcgacgtaa acggccacaa gttcagcgtg
     2941 tccggcgagg gcgagggcga tgccacctac ggcaagctga ccctgaagtt catctgcacc
     3001 accggcaagc tgcccgtgcc ctggcccacc ctcgtgacca ccctgaccta cggcgtgcag
     3061 tgcttcagcc gctaccccga ccacatgaag cagcacgact tcttcaagtc cgccatgccc
     3121 gaaggctacg tccaggagcg caccatcttc ttcaaggacg acggcaacta caagacccgc
     3181 gccgaggtga agttcgaggg cgacaccctg gtgaaccgca tcgagctgaa gggcatcgac
     3241 ttcaaggagg acggcaacat cctggggcac aagctggagt acaactacaa cagccacaac
     3301 gtctatatca tggccgacaa gcagaagaac ggcatcaagg tgaacttcaa gatccgccac
     3361 aacatcgagg acggcagcgt gcagctcgcc gaccactacc agcagaacac ccccatcggc
     3421 gacggccccg tgctgctgcc cgacaaccac tacctgagca cccagtccgc cctgagcaaa
     3481 gaccccaacg agaagcgcga tcacatggtc ctgctggagt tcgtgaccgc cgccgggatc
     3541 actctcggca tggacgagct gtacaagtaa agcggccgcg actctagatc ataatcagcc
     3601 ataccacatt tgtagaggtt ttacttgctt taaaaaacct cccacacctc cccctgaacc
     3661 tgaaacataa aatgaatgca attgttgttg ttaacttgtt tattgcagct tataatggtt
     3721 acaaataaag caatagcatc acaaatttca caaataaagc atttttttca ctgcattcta
     3781 gttgtggttt gtccaaactc atcaatgtat cttaaggcgt aaattgtaag cgttaatatt
     3841 ttgttaaaat tcgcgttaaa tttttgttaa atcagctcat tttttaacca ataggccgaa
     3901 atcggcaaaa tcccttataa atcaaaagaa tagaccgaga tagggttgag tgttgttcca
     3961 gtttggaaca agagtccact attaaagaac gtggactcca acgtcaaagg gcgaaaaacc
     4021 gtctatcagg gcgatggccc actacgtgaa ccatcaccct aatcaagttt tttggggtcg
     4081 aggtgccgta aagcactaaa tcggaaccct aaagggagcc cccgatttag agcttgacgg
     4141 ggaaagccgg cgaacgtggc gagaaaggaa gggaagaaag cgaaaggagc gggcgctagg
     4201 gcgctggcaa gtgtagcggt cacgctgcgc gtaaccacca cacccgccgc gcttaatgcg
     4261 ccgctacagg gcgcgtcagg tggcactttt cggggaaatg tgcgcggaac ccctatttgt
     4321 ttatttttct aaatacattc aaatatgtat ccgctcatga gacaataacc ctgataaatg
     4381 cttcaataat attgaaaaag gaagagtcct gaggcggaaa gaaccagctg tggaatgtgt
     4441 gtcagttagg gtgtggaaag tccccaggct ccccagcagg cagaagtatg caaagcatgc
     4501 atctcaatta gtcagcaacc aggtgtggaa agtccccagg ctccccagca ggcagaagta
     4561 tgcaaagcat gcatctcaat tagtcagcaa ccatagtccc gcccctaact ccgcccatcc
     4621 cgcccctaac tccgcccagt tccgcccatt ctccgcccca tggctgacta atttttttta
     4681 tttatgcaga ggccgaggcc gcctcggcct ctgagctatt ccagaagtag tgaggaggct
     4741 tttttggagg cctaggcttt tgcaaagatc gatcaagaga caggatgagg atcgtttcgc
     4801 atgattgaac aagatggatt gcacgcaggt tctccggccg cttgggtgga gaggctattc
     4861 ggctatgact gggcacaaca gacaatcggc tgctctgatg ccgccgtgtt ccggctgtca
     4921 gcgcaggggc gcccggttct ttttgtcaag accgacctgt ccggtgccct gaatgaactg
     4981 caagacgagg cagcgcggct atcgtggctg gccacgacgg gcgttccttg cgcagctgtg
     5041 ctcgacgttg tcactgaagc gggaagggac tggctgctat tgggcgaagt gccggggcag
     5101 gatctcctgt catctcacct tgctcctgcc gagaaagtat ccatcatggc tgatgcaatg
     5161 cggcggctgc atacgcttga tccggctacc tgcccattcg accaccaagc gaaacatcgc
     5221 atcgagcgag cacgtactcg gatggaagcc ggtcttgtcg atcaggatga tctggacgaa
     5281 gagcatcagg ggctcgcgcc agccgaactg ttcgccaggc tcaaggcgag catgcccgac
     5341 ggcgaggatc tcgtcgtgac ccatggcgat gcctgcttgc cgaatatcat ggtggaaaat
     5401 ggccgctttt ctggattcat cgactgtggc cggctgggtg tggcggaccg ctatcaggac
     5461 atagcgttgg ctacccgtga tattgctgaa gagcttggcg gcgaatgggc tgaccgcttc
     5521 ctcgtgcttt acggtatcgc cgctcccgat tcgcagcgca tcgccttcta tcgccttctt
     5581 gacgagttct tctgagcggg actctggggt tcgaaatgac cgaccaagcg acgcccaacc
     5641 tgccatcacg agatttcgat tccaccgccg ccttctatga aaggttgggc ttcggaatcg
     5701 ttttccggga cgccggctgg atgatcctcc agcgcgggga tctcatgctg gagttcttcg
     5761 cccaccctag ggggaggcta actgaaacac ggaaggagac aataccggaa ggaacccgcg
     5821 ctatgacggc aataaaaaga cagaataaaa cgcacggtgt tgggtcgttt gttcataaac
     5881 gcggggttcg gtcccagggc tggcactctg tcgatacccc accgagaccc cattggggcc
     5941 aatacgcccg cgtttcttcc ttttccccac cccacccccc aagttcgggt gaaggcccag
     6001 ggctcgcagc caacgtcggg gcggcaggcc ctgccatagc ctcaggttac tcatatatac
     6061 tttagattga tttaaaactt catttttaat ttaaaaggat ctaggtgaag atcctttttg
     6121 ataatctcat gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg tcagaccccg
     6181 tagaaaagat caaaggatct tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc
     6241 aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc
     6301 tttttccgaa ggtaactggc ttcagcagag cgcagatacc aaatactgtt cttctagtgt
     6361 agccgtagtt aggccaccac ttcaagaact ctgtagcacc gcctacatac ctcgctctgc
     6421 taatcctgtt accagtggct gctgccagtg gcgataagtc gtgtcttacc gggttggact
     6481 caagacgata gttaccggat aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac
     6541 agcccagctt ggagcgaacg acctacaccg aactgagata cctacagcgt gagctatgag
     6601 aaagcgccac gcttcccgaa gggagaaagg cggacaggta tccggtaagc ggcagggtcg
     6661 gaacaggaga gcgcacgagg gagcttccag ggggaaacgc ctggtatctt tatagtcctg
     6721 tcgggtttcg ccacctctga cttgagcgtc gatttttgtg atgctcgtca ggggggcgga
     6781 gcctatggaa aaacgccagc aacgcggcct ttttacggtt cctggccttt tgctggcctt
     6841 ttgctcacat gttctttcct gcgttatccc ctgattctgt ggataaccgt attaccgcca
     6901 tgcat
//
