LOCUS       sequence-435594-        6056 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 6056)
  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 6056)
  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: Stargazin-EBFP2-FRB
FEATURES             Location/Qualifiers
     source          1..6056
                     /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"
     regulatory      1624..1633
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(1630..1632,1633..1635,1636..2346)
                     /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(1675..1696)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     CDS             2395..2421
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             2440..2718
                     /codon_start=1
                     /product="FKBP-rapamycin binding domain of human FRAP"
                     /label=FRB
                     /translation="ILWHEMWHEGLEEASRLYFGERNVKGMFEVLEPLHAMMERGPQTL
                     KETSFNQAYGRDLMEAQEWCRKYMKSGNVKDLTQAWDLYYHVFRRISK"
     CDS             2452..2721
                     /codon_start=1
                     /product="FKBP-rapamycin binding domain of human FRAP"
                     /label=FRB
                     /translation="EMWHEGLEEASRLYFGERNVKGMFEVLEPLHAMMERGPQTLKETS
                     FNQAYGRDLMEAQEWCRKYMKSGNVKDLTQAWDLYYHVFRRISKQ"
     polyA_signal    2844..2965
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2881..2900)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2935..2954
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(2972..3427)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3109..3130)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3321..3340
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        3454..3558
                     /gene="bla"
                     /label=AmpR promoter
     promoter        3560..3917
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        3560..3751
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(3583..3603)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      3768..3903
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             3952..4746
                     /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             3952..4746
                     /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(4006..4025)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4616..4635
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(4934..4953)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    4978..5025
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      5354..5942
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5843..5862
                     /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 gctagccatg
      601 gggctgtttg atcgaggtgt tcaaatgctt ttaaccaccg ttggtgcttt cgctgccttc
      661 agcttgatga ccatcgctgt gggaaccgac tattggctgt actccagagg ggtttgcaag
      721 accaaaagtg tcagtgagaa tgaaaccagc aagaagaacg aggaagttat gacccattcc
      781 ggattatgga gaacctgctg cctcgaaggg aacttcaaag gtctgtgcaa gcaaatcgac
      841 cactttccgg aagacgcgga ctacgaagct gacaccgcag agtatttcct ccgggccgtg
      901 agggcctcga gtatcttccc gatcctgagt gtgatcctgc ttttcatggg tggcctctgc
      961 atcgcagcca gcgagttcta caaaccggtg aactgcagga agagctcgaa gaattcgaaa
     1021 agcttgacac gccacaacat catcctgagt gctggcatct tcttcgtgtc tgcaggtctt
     1081 agtaatatca tcgggatcat cgtgtatata tcagccaatg ctggagaccc ctccaagagt
     1141 gactccaaaa agaacagcta ctcctacggc tggtccttct acttcggggc cctgtccttc
     1201 atcatcgccg agatggtcgg ggtgctggcc gtgcacatgt ttatcgaccg ccacaaacag
     1261 ctgcgggcca cggcccgcgc caccgactac ctccaggcct ccgccatcac ccgcatcccc
     1321 agctaccgct accgctacca gcgccgcagc cgctccagct cgcgctccac cgagccctct
     1381 cactccagag acgcctcgcc cgtgggcgtg aagggcttca acaccctgcc gtccacggag
     1441 atctccatgt acaccctcag tagggacccc ctgaaggctg ccaccacgcc caccgccacc
     1501 tacaactcgg acagggataa cagctttctc caggtccaca actgtatcca gaaggacagc
     1561 aaggactctt tccacgccaa cacagccaac cgccggacca cgcccgtaaa ggatccaccg
     1621 gtcgccacca tggtgagcaa gggcgaggag ctgttcaccg gggtggtgcc catcctggtc
     1681 gagctggacg gcgacgtaaa cggccacaag ttcagcgtga ggggcgaggg cgagggcgat
     1741 gccaccaacg gcaagctgac cctgaagttc atctgcacca ccggcaagct gcccgtgccc
     1801 tggcccaccc tcgtgaccac cctgagccac ggcgtgcagt gcttcgcccg ctaccccgac
     1861 cacatgaagc agcacgactt cttcaagtcc gccatgcccg aaggctacgt ccaggagcgc
     1921 accatcttct tcaaggacga cggcacctac aagacccgcg ccgaggtgaa gttcgagggc
     1981 gacaccctgg tgaaccgcat cgagctgaag ggcgtcgact tcaaggagga cggcaacatc
     2041 ctggggcaca agctggagta caacttcaac agccacaaca tctatatcat ggccgtcaag
     2101 cagaagaacg gcatcaaggt gaacttcaag atccgccaca acgtggagga cggcagcgtg
     2161 cagctcgccg accactacca gcagaacacc cccatcggcg acggccccgt gctgctgccc
     2221 gacagccact acctgagcac ccagtccgtg ctgagcaaag accccaacga gaagcgcgat
     2281 cacatggtcc tgctggagtt ccgcaccgcc gccgggatca ctctcggcat ggacgagctg
     2341 tacaaggggg taccgcgggc ccgggatcca ccggtgctgc catgtctgta cacttacccc
     2401 tacgacgtgc ccgactacgc aggctccgca cgagtggcca tcctctggca tgagatgtgg
     2461 catgaaggcc tggaagaggc atctcgtttg tactttgggg aaaggaacgt gaaaggcatg
     2521 tttgaggtgc tggagccctt gcatgctatg atggaacggg gcccccagac tctgaaggaa
     2581 acatccttta atcaggccta tggtcgagat ttaatggagg cccaagagtg gtgcaggaag
     2641 tacatgaaat cagggaatgt caaggacctc acccaagcct gggacctcta ttatcatgtg
     2701 ttccgacgaa tctcaaagca ggccggctga taatctagat cataatcagc cataccacat
     2761 ttgtagaggt tttacttgct ttaaaaaacc tcccacacct ccccctgaac ctgaaacata
     2821 aaatgaatgc aattgttgtt gttaacttgt ttattgcagc ttataatggt tacaaataaa
     2881 gcaatagcat cacaaatttc acaaataaag catttttttc actgcattct agttgtggtt
     2941 tgtccaaact catcaatgta tcttaaggcg taaattgtaa gcgttaatat tttgttaaaa
     3001 ttcgcgttaa atttttgtta aatcagctca ttttttaacc aataggccga aatcggcaaa
     3061 atcccttata aatcaaaaga atagaccgag atagggttga gtgttgttcc agtttggaac
     3121 aagagtccac tattaaagaa cgtggactcc aacgtcaaag ggcgaaaaac cgtctatcag
     3181 ggcgatggcc cactacgtga accatcaccc taatcaagtt ttttggggtc gaggtgccgt
     3241 aaagcactaa atcggaaccc taaagggagc ccccgattta gagcttgacg gggaaagccg
     3301 gcgaacgtgg cgagaaagga agggaagaaa gcgaaaggag cgggcgctag ggcgctggca
     3361 agtgtagcgg tcacgctgcg cgtaaccacc acacccgccg cgcttaatgc gccgctacag
     3421 ggcgcgtcag gtggcacttt tcggggaaat gtgcgcggaa cccctatttg tttatttttc
     3481 taaatacatt caaatatgta tccgctcatg agacaataac cctgataaat gcttcaataa
     3541 tattgaaaaa ggaagagtcc tgaggcggaa agaaccagct gtggaatgtg tgtcagttag
     3601 ggtgtggaaa gtccccaggc tccccagcag gcagaagtat gcaaagcatg catctcaatt
     3661 agtcagcaac caggtgtgga aagtccccag gctccccagc aggcagaagt atgcaaagca
     3721 tgcatctcaa ttagtcagca accatagtcc cgcccctaac tccgcccatc ccgcccctaa
     3781 ctccgcccag ttccgcccat tctccgcccc atggctgact aatttttttt atttatgcag
     3841 aggccgaggc cgcctcggcc tctgagctat tccagaagta gtgaggaggc ttttttggag
     3901 gcctaggctt ttgcaaagat cgatcaagag acaggatgag gatcgtttcg catgattgaa
     3961 caagatggat tgcacgcagg ttctccggcc gcttgggtgg agaggctatt cggctatgac
     4021 tgggcacaac agacaatcgg ctgctctgat gccgccgtgt tccggctgtc agcgcagggg
     4081 cgcccggttc tttttgtcaa gaccgacctg tccggtgccc tgaatgaact gcaagacgag
     4141 gcagcgcggc tatcgtggct ggccacgacg ggcgttcctt gcgcagctgt gctcgacgtt
     4201 gtcactgaag cgggaaggga ctggctgcta ttgggcgaag tgccggggca ggatctcctg
     4261 tcatctcacc ttgctcctgc cgagaaagta tccatcatgg ctgatgcaat gcggcggctg
     4321 catacgcttg atccggctac ctgcccattc gaccaccaag cgaaacatcg catcgagcga
     4381 gcacgtactc ggatggaagc cggtcttgtc gatcaggatg atctggacga agagcatcag
     4441 gggctcgcgc cagccgaact gttcgccagg ctcaaggcga gcatgcccga cggcgaggat
     4501 ctcgtcgtga cccatggcga tgcctgcttg ccgaatatca tggtggaaaa tggccgcttt
     4561 tctggattca tcgactgtgg ccggctgggt gtggcggacc gctatcagga catagcgttg
     4621 gctacccgtg atattgctga agagcttggc ggcgaatggg ctgaccgctt cctcgtgctt
     4681 tacggtatcg ccgctcccga ttcgcagcgc atcgccttct atcgccttct tgacgagttc
     4741 ttctgagcgg gactctgggg ttcgaaatga ccgaccaagc gacgcccaac ctgccatcac
     4801 gagatttcga ttccaccgcc gccttctatg aaaggttggg cttcggaatc gttttccggg
     4861 acgccggctg gatgatcctc cagcgcgggg atctcatgct ggagttcttc gcccacccta
     4921 gggggaggct aactgaaaca cggaaggaga caataccgga aggaacccgc gctatgacgg
     4981 caataaaaag acagaataaa acgcacggtg ttgggtcgtt tgttcataaa cgcggggttc
     5041 ggtcccaggg ctggcactct gtcgataccc caccgagacc ccattggggc caatacgccc
     5101 gcgtttcttc cttttcccca ccccaccccc caagttcggg tgaaggccca gggctcgcag
     5161 ccaacgtcgg ggcggcaggc cctgccatag cctcaggtta ctcatatata ctttagattg
     5221 atttaaaact tcatttttaa tttaaaagga tctaggtgaa gatccttttt gataatctca
     5281 tgaccaaaat cccttaacgt gagttttcgt tccactgagc gtcagacccc gtagaaaaga
     5341 tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa
     5401 aaccaccgct accagcggtg gtttgtttgc cggatcaaga gctaccaact ctttttccga
     5461 aggtaactgg cttcagcaga gcgcagatac caaatactgt ccttctagtg tagccgtagt
     5521 taggccacca cttcaagaac tctgtagcac cgcctacata cctcgctctg ctaatcctgt
     5581 taccagtggc tgctgccagt ggcgataagt cgtgtcttac cgggttggac tcaagacgat
     5641 agttaccgga taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca cagcccagct
     5701 tggagcgaac gacctacacc gaactgagat acctacagcg tgagctatga gaaagcgcca
     5761 cgcttcccga agggagaaag gcggacaggt atccggtaag cggcagggtc ggaacaggag
     5821 agcgcacgag ggagcttcca gggggaaacg cctggtatct ttatagtcct gtcgggtttc
     5881 gccacctctg acttgagcgt cgatttttgt gatgctcgtc aggggggcgg agcctatgga
     5941 aaaacgccag caacgcggcc tttttacggt tcctggcctt ttgctggcct tttgctcaca
     6001 tgttctttcc tgcgttatcc cctgattctg tggataaccg tattaccgcc atgcat
//
