LOCUS       sequence-439259-        6927 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  To express LBR with N-terminal 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 6927)
  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 6927)
  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: GFP-FKBP-LBR
FEATURES             Location/Qualifiers
     source          1..6927
                     /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"
     regulatory      607..616
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(613..615,616..618,619..1326)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELY"
     CDS             join(613..615,616..618,619..1326)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELY"
     primer_bind     complement(658..679)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(919..938)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1266..1287
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             join(1375..1377,1378..1698)
                     /codon_start=1
                     /product="human FK506-binding protein FKBP12"
                     /label=FKBP
                     /translation="MGVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKFDSSRDRNK
                     PFKFMLGKQEVIRGWEEGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPHATLVFDVEL
                     LKLE"
     polyA_signal    3715..3836
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3752..3771)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3806..3825
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(3843..4298)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3980..4001)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     4192..4211
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        4325..4429
                     /gene="bla"
                     /label=AmpR promoter
     promoter        4431..4788
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        4431..4622
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(4454..4474)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      4639..4774
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             4823..5617
                     /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             4823..5617
                     /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(4877..4896)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     5487..5506
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(5805..5824)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    5849..5896
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      6225..6813
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6714..6733
                     /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 ccggtcgcca ccatggtgag caagggcgag gagctgttca ccggggtggt gcccatcctg
      661 gtcgagctgg acggcgacgt aaacggccac aagttcagcg tgtccggcga gggcgagggc
      721 gatgccacct acggcaagct gaccctgaag ttcatctgca ccaccggcaa gctgcccgtg
      781 ccctggccca ccctcgtgac caccctgacc tacggcgtgc agtgcttcag ccgctacccc
      841 gaccacatga agcagcacga cttcttcaag tccgccatgc ccgaaggcta cgtccaggag
      901 cgcaccatct tcttcaagga cgacggcaac tacaagaccc gcgccgaggt gaagttcgag
      961 ggcgacaccc tggtgaaccg catcgagctg aagggcatcg acttcaagga ggacggcaac
     1021 atcctggggc acaagctgga gtacaactac aacagccaca acgtctatat catggccgac
     1081 aagcagaaga acggcatcaa ggtgaacttc aagatccgcc acaacatcga ggacggcagc
     1141 gtgcagctcg ccgaccacta ccagcagaac acccccatcg gcgacggccc cgtgctgctg
     1201 cccgacaacc actacctgag cacccagtcc gccctgagca aagaccccaa cgagaagcgc
     1261 gatcacatgg tcctgctgga gttcgtgacc gccgccggga tcactctcgg catggacgag
     1321 ctgtaccgaa tcgatgtgaa tgggggccct gaacccgcca agaacctgta caccatggga
     1381 gtgcaggtgg aaaccatctc cccaggagac gggcgcacct tccccaagcg cggccagacc
     1441 tgcgtggtgc actacaccgg gatgcttgaa gatggaaaga aatttgattc ctcccgggac
     1501 agaaacaagc cctttaagtt tatgctaggc aagcaggagg tgatccgagg ctgggaagaa
     1561 ggggttgccc agatgagtgt gggtcagaga gccaaactga ctatatctcc agattatgcc
     1621 tatggtgcca ctgggcaccc aggcatcatc ccaccacatg ccactctcgt cttcgatgtg
     1681 gagcttctaa aactggaacg gtacaagtcc ggactcagat ctatgccaag taggaaattt
     1741 gccgatggtg aagtggtaag aggtcgatgg cctgggagtt cactttatta tgaagtagaa
     1801 attctgagcc acgacagcac ctcccagctt tacactgtga agtataaaga tggaacagag
     1861 cttgaattga aagagaatga tattaagcct ttaacttcct ttaggcaaag gaaaggtggc
     1921 tcaacttcca gttccccttc cagacgccga gggagtcgat caaggtcacg ctcccgatcc
     1981 cctggtcgac cacctaaaag tgcccgccga tctgcttctg cttcccacca ggccgacatt
     2041 aaggaagcaa ggagggaagt ggaagttaaa ttgactccgc tgattctgaa gccatttgga
     2101 aatagcatca gcagatataa tggggagcct gagcatattg agagaaatga cgcacctcat
     2161 aaaaatacac aggaaaaatt cagtttgtca caagaaagca gttacatagc aacacagtat
     2221 agccttcgtc caagaagaga agaagtcaaa ttaaaagaaa tagattctaa ggaagaaaaa
     2281 tacgttgcaa aagaactggc agtgagaacc tttgaagtga cccccatccg ggcaaaggac
     2341 ttggagtttg gaggagtacc tggtgtgttt ctcatcatgt ttggcctgcc tgtgttcctc
     2401 ttcctgttgc tgttgatgtg taaacagaaa gatcccagtc ttctgaattt ccctcctcct
     2461 ttgccagctt tgtatgagtt atgggaaacc agagtatttg gggtctacct cctgtggttt
     2521 ttgattcaag tcctgttcta cctactgcca attggaaagg ttgtagaagg aacgcctctt
     2581 attgatggaa gaagactcaa gtatagatta aatggattct atgcttttat cctgacatct
     2641 gcagtcatcg gaacatctct cttccagggc gtagagtttc attacgtgta cagtcatttt
     2701 cttcagtttg cacttgcggc cactgttttt tgtgtggtct tgagtgtgta tctctacatg
     2761 cgctctttga aagcgccccg gaatgacctg tcgcctgcca gctctggaaa tgctgtctat
     2821 gatttcttca ttggccgtga attaaaccct cgaattggta cttttgatct caaatacttt
     2881 tgtgaattgc gccccggatt gattggatgg gtggttatta acttggtgat gcttttggct
     2941 gaaatgaaaa tacaggaccg cgctgttcca tccttggcca tgattttagt taatagtttc
     3001 cagcttctct atgtggtgga tgctctctgg aatgaggaag cgttgttgac gaccatggac
     3061 atcatccacg atggatttgg attcatgctg gcttttggag acttggtgtg ggttcccttt
     3121 atttacagct tccaagcctt ttatttagtc agtcatccaa atgaagtgtc ttggccaatg
     3181 gcttctctaa ttattgttct gaaactttgt ggttatgtaa tcttccgagg tgcaaattct
     3241 cagaaaaatg cattccggaa aaatcccagt gatccaaagc ttgcacattt aaaaaccatt
     3301 catacttcaa cgggaaaaaa tcttctagtt tctggatggt ggggctttgt tcgccacccc
     3361 aattacttgg gtgatctcat catggccttg gcgtggtccc tcccatgtgg ttttaaccac
     3421 attctgcctt atttctacat aatttatttc accatgttgc ttgtccaccg agaagctcgt
     3481 gacgagtacc actgtaagaa gaaatacggc gtggcttggg aaaagtactg tcagcgtgtg
     3541 ccctaccgta tatttccata catctactaa ggtaccgcgg gcccgggatc caccggatct
     3601 agataactga tcataatcag ccataccaca tttgtagagg ttttacttgc tttaaaaaac
     3661 ctcccacacc tccccctgaa cctgaaacat aaaatgaatg caattgttgt tgttaacttg
     3721 tttattgcag cttataatgg ttacaaataa agcaatagca tcacaaattt cacaaataaa
     3781 gcattttttt cactgcattc tagttgtggt ttgtccaaac tcatcaatgt atcttaacgc
     3841 gtaaattgta agcgttaata ttttgttaaa attcgcgtta aatttttgtt aaatcagctc
     3901 attttttaac caataggccg aaatcggcaa aatcccttat aaatcaaaag aatagaccga
     3961 gatagggttg agtgttgttc cagtttggaa caagagtcca ctattaaaga acgtggactc
     4021 caacgtcaaa gggcgaaaaa ccgtctatca gggcgatggc ccactacgtg aaccatcacc
     4081 ctaatcaagt tttttggggt cgaggtgccg taaagcacta aatcggaacc ctaaagggag
     4141 cccccgattt agagcttgac ggggaaagcc ggcgaacgtg gcgagaaagg aagggaagaa
     4201 agcgaaagga gcgggcgcta gggcgctggc aagtgtagcg gtcacgctgc gcgtaaccac
     4261 cacacccgcc gcgcttaatg cgccgctaca gggcgcgtca ggtggcactt ttcggggaaa
     4321 tgtgcgcgga acccctattt gtttattttt ctaaatacat tcaaatatgt atccgctcat
     4381 gagacaataa ccctgataaa tgcttcaata atattgaaaa aggaagagtc ctgaggcgga
     4441 aagaaccagc tgtggaatgt gtgtcagtta gggtgtggaa agtccccagg ctccccagca
     4501 ggcagaagta tgcaaagcat gcatctcaat tagtcagcaa ccaggtgtgg aaagtcccca
     4561 ggctccccag caggcagaag tatgcaaagc atgcatctca attagtcagc aaccatagtc
     4621 ccgcccctaa ctccgcccat cccgccccta actccgccca gttccgccca ttctccgccc
     4681 catggctgac taattttttt tatttatgca gaggccgagg ccgcctcggc ctctgagcta
     4741 ttccagaagt agtgaggagg cttttttgga ggcctaggct tttgcaaaga tcgatcaaga
     4801 gacaggatga ggatcgtttc gcatgattga acaagatgga ttgcacgcag gttctccggc
     4861 cgcttgggtg gagaggctat tcggctatga ctgggcacaa cagacaatcg gctgctctga
     4921 tgccgccgtg ttccggctgt cagcgcaggg gcgcccggtt ctttttgtca agaccgacct
     4981 gtccggtgcc ctgaatgaac tgcaagacga ggcagcgcgg ctatcgtggc tggccacgac
     5041 gggcgttcct tgcgcagctg tgctcgacgt tgtcactgaa gcgggaaggg actggctgct
     5101 attgggcgaa gtgccggggc aggatctcct gtcatctcac cttgctcctg ccgagaaagt
     5161 atccatcatg gctgatgcaa tgcggcggct gcatacgctt gatccggcta cctgcccatt
     5221 cgaccaccaa gcgaaacatc gcatcgagcg agcacgtact cggatggaag ccggtcttgt
     5281 cgatcaggat gatctggacg aagagcatca ggggctcgcg ccagccgaac tgttcgccag
     5341 gctcaaggcg agcatgcccg acggcgagga tctcgtcgtg acccatggcg atgcctgctt
     5401 gccgaatatc atggtggaaa atggccgctt ttctggattc atcgactgtg gccggctggg
     5461 tgtggcggac cgctatcagg acatagcgtt ggctacccgt gatattgctg aagagcttgg
     5521 cggcgaatgg gctgaccgct tcctcgtgct ttacggtatc gccgctcccg attcgcagcg
     5581 catcgccttc tatcgccttc ttgacgagtt cttctgagcg ggactctggg gttcgaaatg
     5641 accgaccaag cgacgcccaa cctgccatca cgagatttcg attccaccgc cgccttctat
     5701 gaaaggttgg gcttcggaat cgttttccgg gacgccggct ggatgatcct ccagcgcggg
     5761 gatctcatgc tggagttctt cgcccaccct agggggaggc taactgaaac acggaaggag
     5821 acaataccgg aaggaacccg cgctatgacg gcaataaaaa gacagaataa aacgcacggt
     5881 gttgggtcgt ttgttcataa acgcggggtt cggtcccagg gctggcactc tgtcgatacc
     5941 ccaccgagac cccattgggg ccaatacgcc cgcgtttctt ccttttcccc accccacccc
     6001 ccaagttcgg gtgaaggccc agggctcgca gccaacgtcg gggcggcagg ccctgccata
     6061 gcctcaggtt actcatatat actttagatt gatttaaaac ttcattttta atttaaaagg
     6121 atctaggtga agatcctttt tgataatctc atgaccaaaa tcccttaacg tgagttttcg
     6181 ttccactgag cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga tccttttttt
     6241 ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg
     6301 ccggatcaag agctaccaac tctttttccg aaggtaactg gcttcagcag agcgcagata
     6361 ccaaatactg ttcttctagt gtagccgtag ttaggccacc acttcaagaa ctctgtagca
     6421 ccgcctacat acctcgctct gctaatcctg ttaccagtgg ctgctgccag tggcgataag
     6481 tcgtgtctta ccgggttgga ctcaagacga tagttaccgg ataaggcgca gcggtcgggc
     6541 tgaacggggg gttcgtgcac acagcccagc ttggagcgaa cgacctacac cgaactgaga
     6601 tacctacagc gtgagctatg agaaagcgcc acgcttcccg aagggagaaa ggcggacagg
     6661 tatccggtaa gcggcagggt cggaacagga gagcgcacga gggagcttcc agggggaaac
     6721 gcctggtatc tttatagtcc tgtcgggttt cgccacctct gacttgagcg tcgatttttg
     6781 tgatgctcgt caggggggcg gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg
     6841 ttcctggcct tttgctggcc ttttgctcac atgttctttc ctgcgttatc ccctgattct
     6901 gtggataacc gtattaccgc catgcat
//
