LOCUS       sequence-439139-        5085 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  To express a plasma membrane ""anchor"" for rapamycin-induced 
            heterodimerisation in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5085)
  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 5085)
  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: SH4-FRB-EBFP2
FEATURES             Location/Qualifiers
     source          1..5085
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(300..319)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    344..391
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      720..1308
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1209..1228
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     enhancer        1483..1786
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        1787..1990
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     1941..1961
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     CDS             complement(2098..2115)
                     /codon_start=1
                     /product="HQHQHQ metal affinity tag"
                     /label=HQ tag
                     /note="The HQ tag binds to nickel and other metal ions much
                     like the 6xHis tag, but may allow elution at lower 
                     concentrations of imidazole (Godat et al., 2008)."
                     /translation="HQHQHQ"
     CDS             2130..2408
                     /codon_start=1
                     /product="FKBP-rapamycin binding domain of human FRAP"
                     /label=FRB
                     /translation="ILWHEMWHEGLEEASRLYFGERNVKGMFEVLEPLHAMMERGPQTL
                     KETSFNQAYGRDLMEAQEWCRKYMKSGNVKDLTQAWDLYYHVFRRISK"
     CDS             2415..2441
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     regulatory      2448..2457
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(2454..2456,2457..2459,2460..3173)
                     /codon_start=1
                     /product="enhanced blue variant of GFP (Ai et al., 2007)"
                     /label=EBFP2
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTL
                     KFICTTGKLPVPWPTLVTTLSHGVQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GTYKTRAEVKFEGDTLVNRIELKGVDFKEDGNILGHKLEYNFNSHNIYIMAVKQKNGIK
                     VNFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDSHYLSTQSVLSKDPNEKRDHMVLL
                     EFRTAAGITLGMDELYK"
     primer_bind     complement(2499..2520)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     polyA_signal    3295..3416
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3332..3351)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3386..3405
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(3423..3878)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3560..3581)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3772..3791
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        3905..4009
                     /gene="bla"
                     /label=AmpR promoter
     promoter        4011..4368
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        4011..4202
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(4034..4054)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      4219..4354
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             join(4403..5085,1..112)
                     /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             join(4403..5085,1..112)
                     /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(4457..4476)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     join(5067..5085,1)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
ORIGIN
        1 tgctgaagag cttggcggcg aatgggctga ccgcttcctc gtgctttacg gtatcgccgc
       61 tcccgattcg cagcgcatcg ccttctatcg ccttcttgac gagttcttct gagcgggact
      121 ctggggttcg aaatgaccga ccaagcgacg cccaacctgc catcacgaga tttcgattcc
      181 accgccgcct tctatgaaag gttgggcttc ggaatcgttt tccgggacgc cggctggatg
      241 atcctccagc gcggggatct catgctggag ttcttcgccc accctagggg gaggctaact
      301 gaaacacgga aggagacaat accggaagga acccgcgcta tgacggcaat aaaaagacag
      361 aataaaacgc acggtgttgg gtcgtttgtt cataaacgcg gggttcggtc ccagggctgg
      421 cactctgtcg ataccccacc gagaccccat tggggccaat acgcccgcgt ttcttccttt
      481 tccccacccc accccccaag ttcgggtgaa ggcccagggc tcgcagccaa cgtcggggcg
      541 gcaggccctg ccatagcctc aggttactca tatatacttt agattgattt aaaacttcat
      601 ttttaattta aaaggatcta ggtgaagatc ctttttgata atctcatgac caaaatccct
      661 taacgtgagt tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa aggatcttct
      721 tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca
      781 gcggtggttt gtttgccgga tcaagagcta ccaactcttt ttccgaaggt aactggcttc
      841 agcagagcgc agataccaaa tactgttctt ctagtgtagc cgtagttagg ccaccacttc
      901 aagaactctg tagcaccgcc tacatacctc gctctgctaa tcctgttacc agtggctgct
      961 gccagtggcg ataagtcgtg tcttaccggg ttggactcaa gacgatagtt accggataag
     1021 gcgcagcggt cgggctgaac ggggggttcg tgcacacagc ccagcttgga gcgaacgacc
     1081 tacaccgaac tgagatacct acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg
     1141 agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa caggagagcg cacgagggag
     1201 cttccagggg gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca cctctgactt
     1261 gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac
     1321 gcggcctttt tacggttcct ggccttttgc tggccttttg ctcacatgtt ctttcctgcg
     1381 ttatcccctg attctgtgga taaccgtatt accgccatgc attagttatt aatagtaatc
     1441 aattacgggg tcattagttc atagcccata tatggagttc cgcgttacat aacttacggt
     1501 aaatggcccg cctggctgac cgcccaacga cccccgccca ttgacgtcaa taatgacgta
     1561 tgttcccata gtaacgccaa tagggacttt ccattgacgt caatgggtgg agtatttacg
     1621 gtaaactgcc cacttggcag tacatcaagt gtatcatatg ccaagtacgc cccctattga
     1681 cgtcaatgac ggtaaatggc ccgcctggca ttatgcccag tacatgacct tatgggactt
     1741 tcctacttgg cagtacatct acgtattagt catcgctatt accatggtga tgcggttttg
     1801 gcagtacatc aatgggcgtg gatagcggtt tgactcacgg ggatttccaa gtctccaccc
     1861 cattgacgtc aatgggagtt tgttttggca ccaaaatcaa cgggactttc caaaatgtcg
     1921 taacaactcc gccccattga cgcaaatggg cggtaggcgt gtacggtggg aggtctatat
     1981 aagcagagct ggtttagtga accgtcagat ccgctagcac catgggctgt gtgcaatgta
     2041 aggataaaga agcaacaaaa ctgacggagg atcccaccag atctgcgaat tcaggtgctg
     2101 gtgctggtgc tggtgctatc ctatctagaa tcctctggca tgagatgtgg catgaaggcc
     2161 tggaagaggc atctcgtttg tactttgggg aaaggaacgt gaaaggcatg tttgaggtgc
     2221 tggagccctt gcatgctatg atggaacggg gcccccagac tctgaaggaa acatccttta
     2281 atcaggccta tggtcgagat ttaatggagg cccaagagtg gtgcaggaag tacatgaaat
     2341 cagggaatgt caaggacctc acccaagcct gggacctcta ttatcatgtg ttccgacgaa
     2401 tctcaaagac tagttatccg tacgacgtac cagactacgc accggtcgcc accatggtga
     2461 gcaagggcga ggagctgttc accggggtgg tgcccatcct ggtcgagctg gacggcgacg
     2521 taaacggcca caagttcagc gtgaggggcg agggcgaggg cgatgccacc aacggcaagc
     2581 tgaccctgaa gttcatctgc accaccggca agctgcccgt gccctggccc accctcgtga
     2641 ccaccctgag ccacggcgtg cagtgcttcg cccgctaccc cgaccacatg aagcagcacg
     2701 acttcttcaa gtccgccatg cccgaaggct acgtccagga gcgcaccatc ttcttcaagg
     2761 acgacggcac ctacaagacc cgcgccgagg tgaagttcga gggcgacacc ctggtgaacc
     2821 gcatcgagct gaagggcgtc gacttcaagg aggacggcaa catcctgggg cacaagctgg
     2881 agtacaactt caacagccac aacatctata tcatggccgt caagcagaag aacggcatca
     2941 aggtgaactt caagatccgc cacaacgtgg aggacggcag cgtgcagctc gccgaccact
     3001 accagcagaa cacccccatc ggcgacggcc ccgtgctgct gcccgacagc cactacctga
     3061 gcacccagtc cgtgctgagc aaagacccca acgagaagcg cgatcacatg gtcctgctgg
     3121 agttccgcac cgccgccggg atcactctcg gcatggacga gctgtacaag taagcggccg
     3181 cgactctaga tcataatcag ccataccaca tttgtagagg ttttacttgc tttaaaaaac
     3241 ctcccacacc tccccctgaa cctgaaacat aaaatgaatg caattgttgt tgttaacttg
     3301 tttattgcag cttataatgg ttacaaataa agcaatagca tcacaaattt cacaaataaa
     3361 gcattttttt cactgcattc tagttgtggt ttgtccaaac tcatcaatgt atcttaaggc
     3421 gtaaattgta agcgttaata ttttgttaaa attcgcgtta aatttttgtt aaatcagctc
     3481 attttttaac caataggccg aaatcggcaa aatcccttat aaatcaaaag aatagaccga
     3541 gatagggttg agtgttgttc cagtttggaa caagagtcca ctattaaaga acgtggactc
     3601 caacgtcaaa gggcgaaaaa ccgtctatca gggcgatggc ccactacgtg aaccatcacc
     3661 ctaatcaagt tttttggggt cgaggtgccg taaagcacta aatcggaacc ctaaagggag
     3721 cccccgattt agagcttgac ggggaaagcc ggcgaacgtg gcgagaaagg aagggaagaa
     3781 agcgaaagga gcgggcgcta gggcgctggc aagtgtagcg gtcacgctgc gcgtaaccac
     3841 cacacccgcc gcgcttaatg cgccgctaca gggcgcgtca ggtggcactt ttcggggaaa
     3901 tgtgcgcgga acccctattt gtttattttt ctaaatacat tcaaatatgt atccgctcat
     3961 gagacaataa ccctgataaa tgcttcaata atattgaaaa aggaagagtc ctgaggcgga
     4021 aagaaccagc tgtggaatgt gtgtcagtta gggtgtggaa agtccccagg ctccccagca
     4081 ggcagaagta tgcaaagcat gcatctcaat tagtcagcaa ccaggtgtgg aaagtcccca
     4141 ggctccccag caggcagaag tatgcaaagc atgcatctca attagtcagc aaccatagtc
     4201 ccgcccctaa ctccgcccat cccgccccta actccgccca gttccgccca ttctccgccc
     4261 catggctgac taattttttt tatttatgca gaggccgagg ccgcctcggc ctctgagcta
     4321 ttccagaagt agtgaggagg cttttttgga ggcctaggct tttgcaaaga tcgatcaaga
     4381 gacaggatga ggatcgtttc gcatgattga acaagatgga ttgcacgcag gttctccggc
     4441 cgcttgggtg gagaggctat tcggctatga ctgggcacaa cagacaatcg gctgctctga
     4501 tgccgccgtg ttccggctgt cagcgcaggg gcgcccggtt ctttttgtca agaccgacct
     4561 gtccggtgcc ctgaatgaac tgcaagacga ggcagcgcgg ctatcgtggc tggccacgac
     4621 gggcgttcct tgcgcagctg tgctcgacgt tgtcactgaa gcgggaaggg actggctgct
     4681 attgggcgaa gtgccggggc aggatctcct gtcatctcac cttgctcctg ccgagaaagt
     4741 atccatcatg gctgatgcaa tgcggcggct gcatacgctt gatccggcta cctgcccatt
     4801 cgaccaccaa gcgaaacatc gcatcgagcg agcacgtact cggatggaag ccggtcttgt
     4861 cgatcaggat gatctggacg aagagcatca ggggctcgcg ccagccgaac tgttcgccag
     4921 gctcaaggcg agcatgcccg acggcgagga tctcgtcgtg acccatggcg atgcctgctt
     4981 gccgaatatc atggtggaaa atggccgctt ttctggattc atcgactgtg gccggctggg
     5041 tgtggcggac cgctatcagg acatagcgtt ggctacccgt gatat
//
