LOCUS       sequence-439136-        5786 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  To express emerin 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 5786)
  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 5786)
  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: FKBP-GFP-emerin
FEATURES             Location/Qualifiers
     source          1..5786
                     /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(604..606,607..927)
                     /codon_start=1
                     /product="human FK506-binding protein FKBP12"
                     /label=FKBP
                     /translation="MGVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKFDSSRDRNK
                     PFKFMLGKQEVIRGWEEGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPHATLVFDVEL
                     LKLE"
     regulatory      937..946
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(943..945,946..948,949..1659)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(943..945,946..948,949..1659)
                     /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(988..1009)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1249..1268)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1596..1617
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    2574..2695
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2611..2630)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2665..2684
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(2702..3157)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2839..2860)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3051..3070
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        3184..3288
                     /gene="bla"
                     /label=AmpR promoter
     promoter        3290..3647
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        3290..3481
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(3313..3333)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      3498..3633
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             3682..4476
                     /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             3682..4476
                     /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(3736..3755)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4346..4365
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(4664..4683)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    4708..4755
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      5084..5672
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5573..5592
                     /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 gctagcccca
      601 ccaatgggag tgcaggtgga aaccatctcc ccaggagacg ggcgcacctt ccccaagcgc
      661 ggccagacct gcgtggtgca ctacaccggg atgcttgaag atggaaagaa atttgattcc
      721 tcccgggaca gaaacaagcc ctttaagttt atgctaggca agcaggaggt gatccgaggc
      781 tgggaagaag gggttgccca gatgagtgtg ggtcagagag ccaaactgac tatatctcca
      841 gattatgcct atggtgccac tgggcaccca ggcatcatcc caccacatgc cactctcgtc
      901 ttcgatgtgg agcttctaaa actggaacga ccggtcgcca ccatggtgag caagggcgag
      961 gagctgttca ccggggtggt gcccatcctg gtcgagctgg acggcgacgt aaacggccac
     1021 aagttcagcg tgtccggcga gggcgagggc gatgccacct acggcaagct gaccctgaag
     1081 ttcatctgca ccaccggcaa gctgcccgtg ccctggccca ccctcgtgac caccctgacc
     1141 tacggcgtgc agtgcttcag ccgctacccc gaccacatga agcagcacga cttcttcaag
     1201 tccgccatgc ccgaaggcta cgtccaggag cgcaccatct tcttcaagga cgacggcaac
     1261 tacaagaccc gcgccgaggt gaagttcgag ggcgacaccc tggtgaaccg catcgagctg
     1321 aagggcatcg acttcaagga ggacggcaac atcctggggc acaagctgga gtacaactac
     1381 aacagccaca acgtctatat catggccgac aagcagaaga acggcatcaa ggtgaacttc
     1441 aagatccgcc acaacatcga ggacggcagc gtgcagctcg ccgaccacta ccagcagaac
     1501 acccccatcg gcgacggccc cgtgctgctg cccgacaacc actacctgag cacccagtcc
     1561 gccctgagca aagaccccaa cgagaagcgc gatcacatgg tcctgctgga gttcgtgacc
     1621 gccgccggga tcactctcgg catggacgag ctgtacaagt ccggactcag atctcgagcc
     1681 atggacaact acgcagatct ttcggatacc gagctgacca ccttgctgcg ccggtacaac
     1741 atcccgcacg ggcctgtagt aggatcaact cgtaggcttt acgagaagaa gatcttcgag
     1801 tacgagaccc agaggcggcg gctctcgccc cccagctcgt ccgccgcctc ctcttatagc
     1861 ttctctgact tgaattcgac tagaggggat gcagatatgt atgatcttcc caagaaagag
     1921 gacgctttac tctaccagag caagggctac aatgacgact actatgaaga gagctacttc
     1981 accaccagga cttatgggga gcccgagtct gccggcccgt ccagggctgt ccgccagtca
     2041 gtgacttcat tcccagatgc tgacgctttc catcaccagg tgcatgatga cgatcttttg
     2101 tcttcttctg aagaggagtg caaggatagg gaacgcccca tgtacggccg ggacagtgcc
     2161 taccagagca tcacgcacta ccgccctgtt tcagcctcca ggagctccct ggacctgtcc
     2221 tattatccta cttcctcctc cacctctttt atgtcctcct catcatcttc ctcttcatgg
     2281 ctcacccgcc gtgccatccg gcctgaaaac cgtgctcctg gggctgggct gggccaggat
     2341 cgccaggtcc cgctctgggg ccagctgctg cttttcctgg tctttgtgat cgtcctcttc
     2401 ttcatttacc acttcatgca ggctgaagaa ggcaacccct tctaggatcc accggatcta
     2461 gataactgat cataatcagc cataccacat ttgtagaggt tttacttgct ttaaaaaacc
     2521 tcccacacct ccccctgaac ctgaaacata aaatgaatgc aattgttgtt gttaacttgt
     2581 ttattgcagc ttataatggt tacaaataaa gcaatagcat cacaaatttc acaaataaag
     2641 catttttttc actgcattct agttgtggtt tgtccaaact catcaatgta tcttaacgcg
     2701 taaattgtaa gcgttaatat tttgttaaaa ttcgcgttaa atttttgtta aatcagctca
     2761 ttttttaacc aataggccga aatcggcaaa atcccttata aatcaaaaga atagaccgag
     2821 atagggttga gtgttgttcc agtttggaac aagagtccac tattaaagaa cgtggactcc
     2881 aacgtcaaag ggcgaaaaac cgtctatcag ggcgatggcc cactacgtga accatcaccc
     2941 taatcaagtt ttttggggtc gaggtgccgt aaagcactaa atcggaaccc taaagggagc
     3001 ccccgattta gagcttgacg gggaaagccg gcgaacgtgg cgagaaagga agggaagaaa
     3061 gcgaaaggag cgggcgctag ggcgctggca agtgtagcgg tcacgctgcg cgtaaccacc
     3121 acacccgccg cgcttaatgc gccgctacag ggcgcgtcag gtggcacttt tcggggaaat
     3181 gtgcgcggaa cccctatttg tttatttttc taaatacatt caaatatgta tccgctcatg
     3241 agacaataac cctgataaat gcttcaataa tattgaaaaa ggaagagtcc tgaggcggaa
     3301 agaaccagct gtggaatgtg tgtcagttag ggtgtggaaa gtccccaggc tccccagcag
     3361 gcagaagtat gcaaagcatg catctcaatt agtcagcaac caggtgtgga aagtccccag
     3421 gctccccagc aggcagaagt atgcaaagca tgcatctcaa ttagtcagca accatagtcc
     3481 cgcccctaac tccgcccatc ccgcccctaa ctccgcccag ttccgcccat tctccgcccc
     3541 atggctgact aatttttttt atttatgcag aggccgaggc cgcctcggcc tctgagctat
     3601 tccagaagta gtgaggaggc ttttttggag gcctaggctt ttgcaaagat cgatcaagag
     3661 acaggatgag gatcgtttcg catgattgaa caagatggat tgcacgcagg ttctccggcc
     3721 gcttgggtgg agaggctatt cggctatgac tgggcacaac agacaatcgg ctgctctgat
     3781 gccgccgtgt tccggctgtc agcgcagggg cgcccggttc tttttgtcaa gaccgacctg
     3841 tccggtgccc tgaatgaact gcaagacgag gcagcgcggc tatcgtggct ggccacgacg
     3901 ggcgttcctt gcgcagctgt gctcgacgtt gtcactgaag cgggaaggga ctggctgcta
     3961 ttgggcgaag tgccggggca ggatctcctg tcatctcacc ttgctcctgc cgagaaagta
     4021 tccatcatgg ctgatgcaat gcggcggctg catacgcttg atccggctac ctgcccattc
     4081 gaccaccaag cgaaacatcg catcgagcga gcacgtactc ggatggaagc cggtcttgtc
     4141 gatcaggatg atctggacga agagcatcag gggctcgcgc cagccgaact gttcgccagg
     4201 ctcaaggcga gcatgcccga cggcgaggat ctcgtcgtga cccatggcga tgcctgcttg
     4261 ccgaatatca tggtggaaaa tggccgcttt tctggattca tcgactgtgg ccggctgggt
     4321 gtggcggacc gctatcagga catagcgttg gctacccgtg atattgctga agagcttggc
     4381 ggcgaatggg ctgaccgctt cctcgtgctt tacggtatcg ccgctcccga ttcgcagcgc
     4441 atcgccttct atcgccttct tgacgagttc ttctgagcgg gactctgggg ttcgaaatga
     4501 ccgaccaagc gacgcccaac ctgccatcac gagatttcga ttccaccgcc gccttctatg
     4561 aaaggttggg cttcggaatc gttttccggg acgccggctg gatgatcctc cagcgcgggg
     4621 atctcatgct ggagttcttc gcccacccta gggggaggct aactgaaaca cggaaggaga
     4681 caataccgga aggaacccgc gctatgacgg caataaaaag acagaataaa acgcacggtg
     4741 ttgggtcgtt tgttcataaa cgcggggttc ggtcccaggg ctggcactct gtcgataccc
     4801 caccgagacc ccattggggc caatacgccc gcgtttcttc cttttcccca ccccaccccc
     4861 caagttcggg tgaaggccca gggctcgcag ccaacgtcgg ggcggcaggc cctgccatag
     4921 cctcaggtta ctcatatata ctttagattg atttaaaact tcatttttaa tttaaaagga
     4981 tctaggtgaa gatccttttt gataatctca tgaccaaaat cccttaacgt gagttttcgt
     5041 tccactgagc gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat cctttttttc
     5101 tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct accagcggtg gtttgtttgc
     5161 cggatcaaga gctaccaact ctttttccga aggtaactgg cttcagcaga gcgcagatac
     5221 caaatactgt tcttctagtg tagccgtagt taggccacca cttcaagaac tctgtagcac
     5281 cgcctacata cctcgctctg ctaatcctgt taccagtggc tgctgccagt ggcgataagt
     5341 cgtgtcttac cgggttggac tcaagacgat agttaccgga taaggcgcag cggtcgggct
     5401 gaacgggggg ttcgtgcaca cagcccagct tggagcgaac gacctacacc gaactgagat
     5461 acctacagcg tgagctatga gaaagcgcca cgcttcccga agggagaaag gcggacaggt
     5521 atccggtaag cggcagggtc ggaacaggag agcgcacgag ggagcttcca gggggaaacg
     5581 cctggtatct ttatagtcct gtcgggtttc gccacctctg acttgagcgt cgatttttgt
     5641 gatgctcgtc aggggggcgg agcctatgga aaaacgccag caacgcggcc tttttacggt
     5701 tcctggcctt ttgctggcct tttgctcaca tgttctttcc tgcgttatcc cctgattctg
     5761 tggataaccg tattaccgcc atgcat
//
