LOCUS       sequence-439141-        6315 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  To express a lipid droplet ""anchor"" for rapamycin-induced 
            heterodimerisation in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6315)
  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 6315)
  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: FRB-mCherry-PLIN3
FEATURES             Location/Qualifiers
     source          1..6315
                     /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             607..885
                     /codon_start=1
                     /product="FKBP-rapamycin binding domain of human FRAP"
                     /label=FRB
                     /translation="ILWHEMWHEGLEEASRLYFGERNVKGMFEVLEPLHAMMERGPQTL
                     KETSFNQAYGRDLMEAQEWCRKYMKSGNVKDLTQAWDLYYHVFRRISK"
     CDS             619..888
                     /codon_start=1
                     /product="FKBP-rapamycin binding domain of human FRAP"
                     /label=FRB
                     /translation="EMWHEGLEEASRLYFGERNVKGMFEVLEPLHAMMERGPQTLKETS
                     FNQAYGRDLMEAQEWCRKYMKSGNVKDLTQAWDLYYHVFRRISKQ"
     regulatory      904..913
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             910..1617
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG
                     TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF
                     EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK
                     GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERA
                     EGRHSTGGMDELYK"
     primer_bind     complement(1055..1073)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     1323..1342
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     polyA_signal    3103..3224
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3140..3159)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3194..3213
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(3231..3686)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3368..3389)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3580..3599
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        3713..3817
                     /gene="bla"
                     /label=AmpR promoter
     promoter        3819..4176
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        3819..4010
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(3842..3862)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      4027..4162
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             4211..5005
                     /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             4211..5005
                     /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(4265..4284)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4875..4894
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(5193..5212)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    5237..5284
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      5613..6201
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6102..6121
                     /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 gctagcggcc
      601 accatgatcc tctggcatga gatgtggcat gaaggcctgg aagaggcatc tcgtttgtac
      661 tttggggaaa ggaacgtgaa aggcatgttt gaggtgctgg agcccttgca tgctatgatg
      721 gaacggggcc cccagactct gaaggaaaca tcctttaatc aggcctatgg tcgagattta
      781 atggaggccc aagagtggtg caggaagtac atgaaatcag ggaatgtcaa ggacctcacc
      841 caagcctggg acctctatta tcatgtgttc cgacgaatct caaagcaggc cggcccaccg
      901 gtcgccacca tggtgagcaa gggcgaggag gataacatgg ccatcatcaa ggagttcatg
      961 cgcttcaagg tgcacatgga gggctccgtg aacggccacg agttcgagat cgagggcgag
     1021 ggcgagggcc gcccctacga gggcacccag accgccaagc tgaaggtgac caagggtggc
     1081 cccctgccct tcgcctggga catcctgtcc cctcagttca tgtacggctc caaggcctac
     1141 gtgaagcacc ccgccgacat ccccgactac ttgaagctgt ccttccccga gggcttcaag
     1201 tgggagcgcg tgatgaactt cgaggacggc ggcgtggtga ccgtgaccca ggactcctcc
     1261 ctgcaggacg gcgagttcat ctacaaggtg aagctgcgcg gcaccaactt cccctccgac
     1321 ggccccgtaa tgcagaagaa gaccatgggc tgggaggcct cctccgagcg gatgtacccc
     1381 gaggacggcg ccctgaaggg cgagatcaag cagaggctga agctgaagga cggcggccac
     1441 tacgacgctg aggtcaagac cacctacaag gccaagaagc ccgtgcagct gcccggcgcc
     1501 tacaacgtca acatcaagtt ggacatcacc tcccacaacg aggactacac catcgtggaa
     1561 cagtacgaac gcgccgaggg ccgccactcc accggcggca tggacgagct gtacaagtcc
     1621 ggactcatgt ctgccgacgg ggcagaggct gatggcagca cccaggtgac agtggaagaa
     1681 ccggtacagc agcccagtgt ggtggaccgt gtggccagca tgcctctgat cagctccacc
     1741 tgcgacatgg tgtccgcagc ctatgcctcc accaaggaga gctacccgca catcaagact
     1801 gtctgcgacg cagcagagaa gggagtgagg accctcacgg cggctgctgt cagcggggct
     1861 cagccgatcc tctccaagct ggagccccag attgcatcag ccagcgaata cgcccacagg
     1921 gggctggaca agttggagga gaacctcccc atcctgcagc agcccacgga gaaggtcctg
     1981 gcggacacca aggagcttgt gtcgtctaag gtgtcggggg cccaagagat ggtgtctagc
     2041 gccaaggaca cggtggccac ccaattgtcg gaggcggtgg acgcgacccg cggtgctgtg
     2101 cagagcggcg tggacaagac aaagtccgta gtgaccggcg gcgtccaatc ggtcatgggc
     2161 tcccgcttgg gccagatggt gttgagtggg gtcgacacgg tgctggggaa gtcggaggag
     2221 tgggcggaca accacctgcc ccttacggat gccgaactgg cccgcatcgc cacatccctg
     2281 gatggctttg acgtcgcgtc cgtgcagcag cagcggcagg aacagagcta cttcgtacgt
     2341 ctgggctccc tgtcggagag gctgcggcag cacgcctatg agcactcgct gggcaagctt
     2401 cgagccacca agcagagggc acaggaggct ctgctgcagc tgtcgcaggt cctaagcctg
     2461 atggaaactg tcaagcaagg cgttgatcag aagctggtgg aaggccagga gaagctgcac
     2521 cagatgtggc tcagctggaa ccagaagcag ctccagggcc ccgagaagga gccgcccaag
     2581 ccagagcagg tcgagtcccg ggcgctcacc atgttccggg acattgccca gcaactgcag
     2641 gccacctgta cctccctggg gtccagcatt cagggcctcc ccaccaatgt gaaggaccag
     2701 gtgcagcagg cccgccgcca ggtggaggac ctccaggcca cgttttccag catccactcc
     2761 ttccaggacc tgtccagcag cattctggcc cagagccgtg agcgtgtcgc cagcgcccgc
     2821 gaggccctgg accacatggt ggaatatgtg gcccagaaca cacctgtcac gtggctcgtg
     2881 ggaccctttg cccctggaat cactgagaaa gccccggagg agaagaagta ggctcaagct
     2941 tcgaattctg cagtcgacgg taccgcgggc ccgggatcca ccggatctag ataactgatc
     3001 ataatcagcc ataccacatt tgtagaggtt ttacttgctt taaaaaacct cccacacctc
     3061 cccctgaacc tgaaacataa aatgaatgca attgttgttg ttaacttgtt tattgcagct
     3121 tataatggtt acaaataaag caatagcatc acaaatttca caaataaagc atttttttca
     3181 ctgcattcta gttgtggttt gtccaaactc atcaatgtat cttaacgcgt aaattgtaag
     3241 cgttaatatt ttgttaaaat tcgcgttaaa tttttgttaa atcagctcat tttttaacca
     3301 ataggccgaa atcggcaaaa tcccttataa atcaaaagaa tagaccgaga tagggttgag
     3361 tgttgttcca gtttggaaca agagtccact attaaagaac gtggactcca acgtcaaagg
     3421 gcgaaaaacc gtctatcagg gcgatggccc actacgtgaa ccatcaccct aatcaagttt
     3481 tttggggtcg aggtgccgta aagcactaaa tcggaaccct aaagggagcc cccgatttag
     3541 agcttgacgg ggaaagccgg cgaacgtggc gagaaaggaa gggaagaaag cgaaaggagc
     3601 gggcgctagg gcgctggcaa gtgtagcggt cacgctgcgc gtaaccacca cacccgccgc
     3661 gcttaatgcg ccgctacagg gcgcgtcagg tggcactttt cggggaaatg tgcgcggaac
     3721 ccctatttgt ttatttttct aaatacattc aaatatgtat ccgctcatga gacaataacc
     3781 ctgataaatg cttcaataat attgaaaaag gaagagtcct gaggcggaaa gaaccagctg
     3841 tggaatgtgt gtcagttagg gtgtggaaag tccccaggct ccccagcagg cagaagtatg
     3901 caaagcatgc atctcaatta gtcagcaacc aggtgtggaa agtccccagg ctccccagca
     3961 ggcagaagta tgcaaagcat gcatctcaat tagtcagcaa ccatagtccc gcccctaact
     4021 ccgcccatcc cgcccctaac tccgcccagt tccgcccatt ctccgcccca tggctgacta
     4081 atttttttta tttatgcaga ggccgaggcc gcctcggcct ctgagctatt ccagaagtag
     4141 tgaggaggct tttttggagg cctaggcttt tgcaaagatc gatcaagaga caggatgagg
     4201 atcgtttcgc atgattgaac aagatggatt gcacgcaggt tctccggccg cttgggtgga
     4261 gaggctattc ggctatgact gggcacaaca gacaatcggc tgctctgatg ccgccgtgtt
     4321 ccggctgtca gcgcaggggc gcccggttct ttttgtcaag accgacctgt ccggtgccct
     4381 gaatgaactg caagacgagg cagcgcggct atcgtggctg gccacgacgg gcgttccttg
     4441 cgcagctgtg ctcgacgttg tcactgaagc gggaagggac tggctgctat tgggcgaagt
     4501 gccggggcag gatctcctgt catctcacct tgctcctgcc gagaaagtat ccatcatggc
     4561 tgatgcaatg cggcggctgc atacgcttga tccggctacc tgcccattcg accaccaagc
     4621 gaaacatcgc atcgagcgag cacgtactcg gatggaagcc ggtcttgtcg atcaggatga
     4681 tctggacgaa gagcatcagg ggctcgcgcc agccgaactg ttcgccaggc tcaaggcgag
     4741 catgcccgac ggcgaggatc tcgtcgtgac ccatggcgat gcctgcttgc cgaatatcat
     4801 ggtggaaaat ggccgctttt ctggattcat cgactgtggc cggctgggtg tggcggaccg
     4861 ctatcaggac atagcgttgg ctacccgtga tattgctgaa gagcttggcg gcgaatgggc
     4921 tgaccgcttc ctcgtgcttt acggtatcgc cgctcccgat tcgcagcgca tcgccttcta
     4981 tcgccttctt gacgagttct tctgagcggg actctggggt tcgaaatgac cgaccaagcg
     5041 acgcccaacc tgccatcacg agatttcgat tccaccgccg ccttctatga aaggttgggc
     5101 ttcggaatcg ttttccggga cgccggctgg atgatcctcc agcgcgggga tctcatgctg
     5161 gagttcttcg cccaccctag ggggaggcta actgaaacac ggaaggagac aataccggaa
     5221 ggaacccgcg ctatgacggc aataaaaaga cagaataaaa cgcacggtgt tgggtcgttt
     5281 gttcataaac gcggggttcg gtcccagggc tggcactctg tcgatacccc accgagaccc
     5341 cattggggcc aatacgcccg cgtttcttcc ttttccccac cccacccccc aagttcgggt
     5401 gaaggcccag ggctcgcagc caacgtcggg gcggcaggcc ctgccatagc ctcaggttac
     5461 tcatatatac tttagattga tttaaaactt catttttaat ttaaaaggat ctaggtgaag
     5521 atcctttttg ataatctcat gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg
     5581 tcagaccccg tagaaaagat caaaggatct tcttgagatc ctttttttct gcgcgtaatc
     5641 tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc ggatcaagag
     5701 ctaccaactc tttttccgaa ggtaactggc ttcagcagag cgcagatacc aaatactgtt
     5761 cttctagtgt agccgtagtt aggccaccac ttcaagaact ctgtagcacc gcctacatac
     5821 ctcgctctgc taatcctgtt accagtggct gctgccagtg gcgataagtc gtgtcttacc
     5881 gggttggact caagacgata gttaccggat aaggcgcagc ggtcgggctg aacggggggt
     5941 tcgtgcacac agcccagctt ggagcgaacg acctacaccg aactgagata cctacagcgt
     6001 gagctatgag aaagcgccac gcttcccgaa gggagaaagg cggacaggta tccggtaagc
     6061 ggcagggtcg gaacaggaga gcgcacgagg gagcttccag ggggaaacgc ctggtatctt
     6121 tatagtcctg tcgggtttcg ccacctctga cttgagcgtc gatttttgtg atgctcgtca
     6181 ggggggcgga gcctatggaa aaacgccagc aacgcggcct ttttacggtt cctggccttt
     6241 tgctggcctt ttgctcacat gttctttcct gcgttatccc ctgattctgt ggataaccgt
     6301 attaccgcca tgcat
//
