LOCUS       sequence-419227-        4728 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of a dimerization-dependent fluorescent protein (ddFP) 
            subunit (RA) fused with plasma membrane (PM) targetting motif of Lck
            in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4728)
  AUTHORS   Koh DHZ, Naito T, Na M, Yeap YJ, Rozario P, Zhong FL, Lim KL, Saheki
            Y
  TITLE     Visualization of accessible cholesterol using a GRAM domain-based 
            biosensor.
  JOURNAL   Nat Commun. 2023 Oct 25;14(1):6773. doi: 10.1038/s41467-023-42498-7.
  PUBMED    37880244
REFERENCE   2  (bases 1 to 4728)
  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: "Nat 
            Commun."; date: "2023-10-25"; volume: "14(1):6773. doi"; pages: " 
            10.1038/s41467-023-42498-7"
COMMENT     Sequence Label: PM-RA
FEATURES             Location/Qualifiers
     source          1..4728
                     /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"
     regulatory      595..604
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     primer_bind     complement(773..791)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     1041..1060
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     misc_feature    1340..1392
                     /label=MCS
                     /note="multiple cloning site"
     polyA_signal    1516..1637
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1553..1572)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     1607..1626
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(1644..2099)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1781..1802)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     1993..2012
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        2126..2230
                     /gene="bla"
                     /label=AmpR promoter
     promoter        2232..2589
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        2232..2423
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(2255..2275)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      2440..2575
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             2624..3418
                     /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             2624..3418
                     /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(2678..2697)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3288..3307
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(3606..3625)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    3650..3697
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      4026..4614
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4515..4534
                     /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 gctagccacc
      601 atgggctgcg tgtgcagcag caaccccgag ctaccggtca tggtgagcaa gagcgaggag
      661 gtcatcaaag agttcatgcg cttcaaggtg cgcttggagg gctccatgaa cggccacgag
      721 ttcgagatcg agggcgaggg cgagggccgc ccctacgagg gcacccagac cgccaagctg
      781 aaggtgacca agggcggccc cctgcccttc gcctgggaca tcctgtcccc ccagatcatg
      841 tacggctcca aggcgtacgt gaagcacccc gccgacgtcc ccgattacat gaagctgtcc
      901 ttccccgagg gcttcaagtg ggagcgcgtg atgcacttcg aggacggcgg tctggtgacc
      961 gtgactcagg acacctccct gcaggacggc acgctgatct acaaggtgaa gatgcgcggc
     1021 accaacttcc cccccgacgg ccccgtaatg cagaagaaga ccttgggctg ggattatgcc
     1081 accgagcgcc tgtaccccga agacggcgtg ctgaagggcg agcttctggg gcgcctgaag
     1141 ctgaaggacg gcggcctcaa cctggtggag ttcaagacca tctacatggc caagaagccc
     1201 gtgcaactgc ccggctacta cttcgtggac accaagctgg gcatcacctc ccacaacgag
     1261 gactacacca tcgtggaaca gtacgagcgc tccgagggcc gccaccacct gggcatggac
     1321 gagctgtaca agggtacctc tcgagctcaa gcttcgaatt ctgcagtcga cggtaccgcg
     1381 ggcccgggat ccaccggatc tagataactg atcataatca gccataccac atttgtagag
     1441 gttttacttg ctttaaaaaa cctcccacac ctccccctga acctgaaaca taaaatgaat
     1501 gcaattgttg ttgttaactt gtttattgca gcttataatg gttacaaata aagcaatagc
     1561 atcacaaatt tcacaaataa agcatttttt tcactgcatt ctagttgtgg tttgtccaaa
     1621 ctcatcaatg tatcttaagg cgtaaattgt aagcgttaat attttgttaa aattcgcgtt
     1681 aaatttttgt taaatcagct cattttttaa ccaataggcc gaaatcggca aaatccctta
     1741 taaatcaaaa gaatagaccg agatagggtt gagtgttgtt ccagtttgga acaagagtcc
     1801 actattaaag aacgtggact ccaacgtcaa agggcgaaaa accgtctatc agggcgatgg
     1861 cccactacgt gaaccatcac cctaatcaag ttttttgggg tcgaggtgcc gtaaagcact
     1921 aaatcggaac cctaaaggga gcccccgatt tagagcttga cggggaaagc cggcgaacgt
     1981 ggcgagaaag gaagggaaga aagcgaaagg agcgggcgct agggcgctgg caagtgtagc
     2041 ggtcacgctg cgcgtaacca ccacacccgc cgcgcttaat gcgccgctac agggcgcgtc
     2101 aggtggcact tttcggggaa atgtgcgcgg aacccctatt tgtttatttt tctaaataca
     2161 ttcaaatatg tatccgctca tgagacaata accctgataa atgcttcaat aatattgaaa
     2221 aaggaagagt cctgaggcgg aaagaaccag ctgtggaatg tgtgtcagtt agggtgtgga
     2281 aagtccccag gctccccagc aggcagaagt atgcaaagca tgcatctcaa ttagtcagca
     2341 accaggtgtg gaaagtcccc aggctcccca gcaggcagaa gtatgcaaag catgcatctc
     2401 aattagtcag caaccatagt cccgccccta actccgccca tcccgcccct aactccgccc
     2461 agttccgccc attctccgcc ccatggctga ctaatttttt ttatttatgc agaggccgag
     2521 gccgcctcgg cctctgagct attccagaag tagtgaggag gcttttttgg aggcctaggc
     2581 ttttgcaaag atcgatcaag agacaggatg aggatcgttt cgcatgattg aacaagatgg
     2641 attgcacgca ggttctccgg ccgcttgggt ggagaggcta ttcggctatg actgggcaca
     2701 acagacaatc ggctgctctg atgccgccgt gttccggctg tcagcgcagg ggcgcccggt
     2761 tctttttgtc aagaccgacc tgtccggtgc cctgaatgaa ctgcaagacg aggcagcgcg
     2821 gctatcgtgg ctggccacga cgggcgttcc ttgcgcagct gtgctcgacg ttgtcactga
     2881 agcgggaagg gactggctgc tattgggcga agtgccgggg caggatctcc tgtcatctca
     2941 ccttgctcct gccgagaaag tatccatcat ggctgatgca atgcggcggc tgcatacgct
     3001 tgatccggct acctgcccat tcgaccacca agcgaaacat cgcatcgagc gagcacgtac
     3061 tcggatggaa gccggtcttg tcgatcagga tgatctggac gaagagcatc aggggctcgc
     3121 gccagccgaa ctgttcgcca ggctcaaggc gagcatgccc gacggcgagg atctcgtcgt
     3181 gacccatggc gatgcctgct tgccgaatat catggtggaa aatggccgct tttctggatt
     3241 catcgactgt ggccggctgg gtgtggcgga ccgctatcag gacatagcgt tggctacccg
     3301 tgatattgct gaagagcttg gcggcgaatg ggctgaccgc ttcctcgtgc tttacggtat
     3361 cgccgctccc gattcgcagc gcatcgcctt ctatcgcctt cttgacgagt tcttctgagc
     3421 gggactctgg ggttcgaaat gaccgaccaa gcgacgccca acctgccatc acgagatttc
     3481 gattccaccg ccgccttcta tgaaaggttg ggcttcggaa tcgttttccg ggacgccggc
     3541 tggatgatcc tccagcgcgg ggatctcatg ctggagttct tcgcccaccc tagggggagg
     3601 ctaactgaaa cacggaagga gacaataccg gaaggaaccc gcgctatgac ggcaataaaa
     3661 agacagaata aaacgcacgg tgttgggtcg tttgttcata aacgcggggt tcggtcccag
     3721 ggctggcact ctgtcgatac cccaccgaga ccccattggg gccaatacgc ccgcgtttct
     3781 tccttttccc caccccaccc cccaagttcg ggtgaaggcc cagggctcgc agccaacgtc
     3841 ggggcggcag gccctgccat agcctcaggt tactcatata tactttagat tgatttaaaa
     3901 cttcattttt aatttaaaag gatctaggtg aagatccttt ttgataatct catgaccaaa
     3961 atcccttaac gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga
     4021 tcttcttgag atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg
     4081 ctaccagcgg tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact
     4141 ggcttcagca gagcgcagat accaaatact gttcttctag tgtagccgta gttaggccac
     4201 cacttcaaga actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg
     4261 gctgctgcca gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg
     4321 gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga
     4381 acgacctaca ccgaactgag atacctacag cgtgagctat gagaaagcgc cacgcttccc
     4441 gaagggagaa aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg
     4501 agggagcttc cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc
     4561 tgacttgagc gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc
     4621 agcaacgcgg cctttttacg gttcctggcc ttttgctggc cttttgctca catgttcttt
     4681 cctgcgttat cccctgattc tgtggataac cgtattaccg ccatgcat
//
