LOCUS       sequence-439137-        6399 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  To express LAP2beta 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 6399)
  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 6399)
  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-LAP2beta
FEATURES             Location/Qualifiers
     source          1..6399
                     /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    3187..3308
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3224..3243)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3278..3297
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(3315..3770)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3452..3473)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3664..3683
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        3797..3901
                     /gene="bla"
                     /label=AmpR promoter
     promoter        3903..4260
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        3903..4094
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(3926..3946)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      4111..4246
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             4295..5089
                     /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             4295..5089
                     /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(4349..4368)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4959..4978
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(5277..5296)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    5321..5368
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      5697..6285
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6186..6205
                     /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 atctatgccg
     1681 gagttcctag aggacccttc ggtcctgacc aaagacaagt tgaagagcga gttggtcgcc
     1741 aacaacgtga cgctcccggc cggcgagcag cgcaaggacg tgtacgtgca gctctacctg
     1801 cagcacctca cggcgcgcaa ccggccgccg ctcgccgcgg gagccaacag caaagggccg
     1861 cccgacttct cgagcgacga ggagcgcgag cccaccccag tgcttggctc cggggcctcc
     1921 gtgggtcgcg gccgcggcgc cgtcggcagg aaagccacaa agaaaactga taagcccagg
     1981 ccagaagata aagatgatct ggatgtgaca gagctctcta acgaagaact tctggaacag
     2041 cttgtgagat atggagtgaa tcctggtccc attgtgggaa ccaccaggaa gctgtatgag
     2101 aagaagctgt tgaagctgag ggaacaggga gcagaatcga gatcctctac tcctctccca
     2161 acagtctctt cctctgcaga aaacacgagg cagaatggaa gtaatgactc tgacagatac
     2221 agtgacaatg acgaagactc taaaatagag ctcaagcttg agaagagaga gccgctaaag
     2281 ggcagagcaa agactccagt aacactcaag caaaggagga ttgagcacaa tcagagctat
     2341 tctgaagctg gagtaactga gactgaatgg acaagtggat cttcaaaagg cggacctctg
     2401 caggcattaa ctagggagtc cacgagaggg tcgagaagaa ctccaaggag aagggtggaa
     2461 ccctcacagc attttcgtgt agatggtgca gtaatttcag agagtactcc catagctgag
     2521 actataaagg cttccagcaa cgactcctta gtggccaata ggttgactgg aaatttcaag
     2581 catgcatctt ctattctgcc aatcactgaa ttctcagaca taaccagaag aacaccaaag
     2641 aaaccattga caagagctga agtgggagaa aaaacagagg aaagaagagt agaaagggat
     2701 attctgaagg aaatgttccc gtatgaagcc tctactccaa ccggaattag tgctagttgc
     2761 cgcagaccaa tcaaaggtgc tgccggccgg ccgctcgagc tcagtgactt caggatggaa
     2821 gaatcgttct catctaagta cgtcccgaag tacgttccct tggcagacgt caagtcggaa
     2881 aagacaaaga agggacgctc cgttcccatg tggataaaaa tgctgctgtt tgctctcgtg
     2941 gcgggttttt tgtttttggt ctatcaagct atggaaacca accaaggaaa ccccttcact
     3001 aattttcttc aagatactaa aatatccaac tgctaggtcg acggtaccgc gggcccggga
     3061 tccaccggat ctagataact gatcataatc agccatacca catttgtaga ggttttactt
     3121 gctttaaaaa acctcccaca cctccccctg aacctgaaac ataaaatgaa tgcaattgtt
     3181 gttgttaact tgtttattgc agcttataat ggttacaaat aaagcaatag catcacaaat
     3241 ttcacaaata aagcattttt ttcactgcat tctagttgtg gtttgtccaa actcatcaat
     3301 gtatcttaac gcgtaaattg taagcgttaa tattttgtta aaattcgcgt taaatttttg
     3361 ttaaatcagc tcatttttta accaataggc cgaaatcggc aaaatccctt ataaatcaaa
     3421 agaatagacc gagatagggt tgagtgttgt tccagtttgg aacaagagtc cactattaaa
     3481 gaacgtggac tccaacgtca aagggcgaaa aaccgtctat cagggcgatg gcccactacg
     3541 tgaaccatca ccctaatcaa gttttttggg gtcgaggtgc cgtaaagcac taaatcggaa
     3601 ccctaaaggg agcccccgat ttagagcttg acggggaaag ccggcgaacg tggcgagaaa
     3661 ggaagggaag aaagcgaaag gagcgggcgc tagggcgctg gcaagtgtag cggtcacgct
     3721 gcgcgtaacc accacacccg ccgcgcttaa tgcgccgcta cagggcgcgt caggtggcac
     3781 ttttcgggga aatgtgcgcg gaacccctat ttgtttattt ttctaaatac attcaaatat
     3841 gtatccgctc atgagacaat aaccctgata aatgcttcaa taatattgaa aaaggaagag
     3901 tcctgaggcg gaaagaacca gctgtggaat gtgtgtcagt tagggtgtgg aaagtcccca
     3961 ggctccccag caggcagaag tatgcaaagc atgcatctca attagtcagc aaccaggtgt
     4021 ggaaagtccc caggctcccc agcaggcaga agtatgcaaa gcatgcatct caattagtca
     4081 gcaaccatag tcccgcccct aactccgccc atcccgcccc taactccgcc cagttccgcc
     4141 cattctccgc cccatggctg actaattttt tttatttatg cagaggccga ggccgcctcg
     4201 gcctctgagc tattccagaa gtagtgagga ggcttttttg gaggcctagg cttttgcaaa
     4261 gatcgatcaa gagacaggat gaggatcgtt tcgcatgatt gaacaagatg gattgcacgc
     4321 aggttctccg gccgcttggg tggagaggct attcggctat gactgggcac aacagacaat
     4381 cggctgctct gatgccgccg tgttccggct gtcagcgcag gggcgcccgg ttctttttgt
     4441 caagaccgac ctgtccggtg ccctgaatga actgcaagac gaggcagcgc ggctatcgtg
     4501 gctggccacg acgggcgttc cttgcgcagc tgtgctcgac gttgtcactg aagcgggaag
     4561 ggactggctg ctattgggcg aagtgccggg gcaggatctc ctgtcatctc accttgctcc
     4621 tgccgagaaa gtatccatca tggctgatgc aatgcggcgg ctgcatacgc ttgatccggc
     4681 tacctgccca ttcgaccacc aagcgaaaca tcgcatcgag cgagcacgta ctcggatgga
     4741 agccggtctt gtcgatcagg atgatctgga cgaagagcat caggggctcg cgccagccga
     4801 actgttcgcc aggctcaagg cgagcatgcc cgacggcgag gatctcgtcg tgacccatgg
     4861 cgatgcctgc ttgccgaata tcatggtgga aaatggccgc ttttctggat tcatcgactg
     4921 tggccggctg ggtgtggcgg accgctatca ggacatagcg ttggctaccc gtgatattgc
     4981 tgaagagctt ggcggcgaat gggctgaccg cttcctcgtg ctttacggta tcgccgctcc
     5041 cgattcgcag cgcatcgcct tctatcgcct tcttgacgag ttcttctgag cgggactctg
     5101 gggttcgaaa tgaccgacca agcgacgccc aacctgccat cacgagattt cgattccacc
     5161 gccgccttct atgaaaggtt gggcttcgga atcgttttcc gggacgccgg ctggatgatc
     5221 ctccagcgcg gggatctcat gctggagttc ttcgcccacc ctagggggag gctaactgaa
     5281 acacggaagg agacaatacc ggaaggaacc cgcgctatga cggcaataaa aagacagaat
     5341 aaaacgcacg gtgttgggtc gtttgttcat aaacgcgggg ttcggtccca gggctggcac
     5401 tctgtcgata ccccaccgag accccattgg ggccaatacg cccgcgtttc ttccttttcc
     5461 ccaccccacc ccccaagttc gggtgaaggc ccagggctcg cagccaacgt cggggcggca
     5521 ggccctgcca tagcctcagg ttactcatat atactttaga ttgatttaaa acttcatttt
     5581 taatttaaaa ggatctaggt gaagatcctt tttgataatc tcatgaccaa aatcccttaa
     5641 cgtgagtttt cgttccactg agcgtcagac cccgtagaaa agatcaaagg atcttcttga
     5701 gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg
     5761 gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac tggcttcagc
     5821 agagcgcaga taccaaatac tgttcttcta gtgtagccgt agttaggcca ccacttcaag
     5881 aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt ggctgctgcc
     5941 agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttacc ggataaggcg
     6001 cagcggtcgg gctgaacggg gggttcgtgc acacagccca gcttggagcg aacgacctac
     6061 accgaactga gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga
     6121 aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcac gagggagctt
     6181 ccagggggaa acgcctggta tctttatagt cctgtcgggt ttcgccacct ctgacttgag
     6241 cgtcgatttt tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg
     6301 gcctttttac ggttcctggc cttttgctgg ccttttgctc acatgttctt tcctgcgtta
     6361 tcccctgatt ctgtggataa ccgtattacc gccatgcat
//
