LOCUS       sequence-419219-        5035 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of a dimerization-dependent fluorescent protein (ddFP) 
            subunit (B) fused with the GRAM domain of GRAMD1b carrying a G187W 
            mutation (B-GRAM-W) in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5035)
  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 5035)
  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: B-GRAM-W
FEATURES             Location/Qualifiers
     source          1..5035
                     /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      607..616
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     primer_bind     complement(746..764)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     1014..1033
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     1208..1231
                     /label=DsRed1-C
                     /note="DsRed1, forward primer"
     polyA_signal    1823..1944
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1860..1879)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     1914..1933
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(1951..2406)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2088..2109)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2300..2319
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        2433..2537
                     /gene="bla"
                     /label=AmpR promoter
     promoter        2539..2896
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        2539..2730
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(2562..2582)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      2747..2882
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             2931..3725
                     /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             2931..3725
                     /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(2985..3004)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3595..3614
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(3913..3932)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    3957..4004
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      4333..4921
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4822..4841
                     /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 gctagcgcta
      601 ccggtcgcca ccatggtgag caagggcgag gagaccatca aagagttcat gcgcttcaag
      661 gtgcgcatgg agggctccat gaacggccac gagttcgaga tcgagggcga gggcgagggc
      721 cgcccctacg agggcaccca gaccgccaag ctgaaggtga ccaagggcgg ccccctgccc
      781 ttcgcctggg acatcctgtc cccccagttc atgtacggct ccgaggcgta cgtgaggcac
      841 cccgccgaca tccccgatta caagaagctg cccttccccg agggcttcaa gtgggagcgc
      901 gtgatgaact tcgaagacgg cggtctggtg accgttaccc aggactcctc cctgcaggac
      961 ggcacgctga tctgcaaggt gaagatgcgc ggcaccaact tcccccccga cggccccgta
     1021 atgcagaaga agaccatggg ctgggaggcc tccaccgaga tgctgtaccc cgaagacggc
     1081 gtgctgaagg gccatagcta tcaggccctg aagctgaagg acggcggcca ctacctggtg
     1141 gagttcgaga ccatctacat ggccaagaag cccgtgcaac tgcccggcga ttactgtgtg
     1201 gacaccaagc tggacatcac ctcccacaac gaggactaca ccatcgtgga acagtacggg
     1261 cgctccgagg gccgccaccg tctgggcatg gacgagcggt acaagggtac caagcagaga
     1321 aatgaagact tcagaaagct ctttaagcag cttccagaca cggagcgcct cattgttgat
     1381 tactcatgtg cactccaaag agacattctc cttcagggcc gactctacct ctctgaaaat
     1441 tggatctgct tctacagcaa catcttccgc tgggaaactc tgctgacagt ccgtttgaaa
     1501 gacatctgtt ccatgactaa agaaaaaaca gctcgcctca ttcccaatgc catccaagtt
     1561 tgcactgatt cagaaaagca cttcttcact tcgttttggg cccgggatag gacatatatg
     1621 atgatgttcc ggctctggca gaatgctctc cttgaaaagt gagaattctg cagtcgacgg
     1681 taccgcgggc ccgggatcca ccggatctag ataactgatc ataatcagcc ataccacatt
     1741 tgtagaggtt ttacttgctt taaaaaacct cccacacctc cccctgaacc tgaaacataa
     1801 aatgaatgca attgttgttg ttaacttgtt tattgcagct tataatggtt acaaataaag
     1861 caatagcatc acaaatttca caaataaagc atttttttca ctgcattcta gttgtggttt
     1921 gtccaaactc atcaatgtat cttaacgcgt aaattgtaag cgttaatatt ttgttaaaat
     1981 tcgcgttaaa tttttgttaa atcagctcat tttttaacca ataggccgaa atcggcaaaa
     2041 tcccttataa atcaaaagaa tagaccgaga tagggttgag tgttgttcca gtttggaaca
     2101 agagtccact attaaagaac gtggactcca acgtcaaagg gcgaaaaacc gtctatcagg
     2161 gcgatggccc actacgtgaa ccatcaccct aatcaagttt tttggggtcg aggtgccgta
     2221 aagcactaaa tcggaaccct aaagggagcc cccgatttag agcttgacgg ggaaagccgg
     2281 cgaacgtggc gagaaaggaa gggaagaaag cgaaaggagc gggcgctagg gcgctggcaa
     2341 gtgtagcggt cacgctgcgc gtaaccacca cacccgccgc gcttaatgcg ccgctacagg
     2401 gcgcgtcagg tggcactttt cggggaaatg tgcgcggaac ccctatttgt ttatttttct
     2461 aaatacattc aaatatgtat ccgctcatga gacaataacc ctgataaatg cttcaataat
     2521 attgaaaaag gaagagtcct gaggcggaaa gaaccagctg tggaatgtgt gtcagttagg
     2581 gtgtggaaag tccccaggct ccccagcagg cagaagtatg caaagcatgc atctcaatta
     2641 gtcagcaacc aggtgtggaa agtccccagg ctccccagca ggcagaagta tgcaaagcat
     2701 gcatctcaat tagtcagcaa ccatagtccc gcccctaact ccgcccatcc cgcccctaac
     2761 tccgcccagt tccgcccatt ctccgcccca tggctgacta atttttttta tttatgcaga
     2821 ggccgaggcc gcctcggcct ctgagctatt ccagaagtag tgaggaggct tttttggagg
     2881 cctaggcttt tgcaaagatc gatcaagaga caggatgagg atcgtttcgc atgattgaac
     2941 aagatggatt gcacgcaggt tctccggccg cttgggtgga gaggctattc ggctatgact
     3001 gggcacaaca gacaatcggc tgctctgatg ccgccgtgtt ccggctgtca gcgcaggggc
     3061 gcccggttct ttttgtcaag accgacctgt ccggtgccct gaatgaactg caagacgagg
     3121 cagcgcggct atcgtggctg gccacgacgg gcgttccttg cgcagctgtg ctcgacgttg
     3181 tcactgaagc gggaagggac tggctgctat tgggcgaagt gccggggcag gatctcctgt
     3241 catctcacct tgctcctgcc gagaaagtat ccatcatggc tgatgcaatg cggcggctgc
     3301 atacgcttga tccggctacc tgcccattcg accaccaagc gaaacatcgc atcgagcgag
     3361 cacgtactcg gatggaagcc ggtcttgtcg atcaggatga tctggacgaa gagcatcagg
     3421 ggctcgcgcc agccgaactg ttcgccaggc tcaaggcgag catgcccgac ggcgaggatc
     3481 tcgtcgtgac ccatggcgat gcctgcttgc cgaatatcat ggtggaaaat ggccgctttt
     3541 ctggattcat cgactgtggc cggctgggtg tggcggaccg ctatcaggac atagcgttgg
     3601 ctacccgtga tattgctgaa gagcttggcg gcgaatgggc tgaccgcttc ctcgtgcttt
     3661 acggtatcgc cgctcccgat tcgcagcgca tcgccttcta tcgccttctt gacgagttct
     3721 tctgagcggg actctggggt tcgaaatgac cgaccaagcg acgcccaacc tgccatcacg
     3781 agatttcgat tccaccgccg ccttctatga aaggttgggc ttcggaatcg ttttccggga
     3841 cgccggctgg atgatcctcc agcgcgggga tctcatgctg gagttcttcg cccaccctag
     3901 ggggaggcta actgaaacac ggaaggagac aataccggaa ggaacccgcg ctatgacggc
     3961 aataaaaaga cagaataaaa cgcacggtgt tgggtcgttt gttcataaac gcggggttcg
     4021 gtcccagggc tggcactctg tcgatacccc accgagaccc cattggggcc aatacgcccg
     4081 cgtttcttcc ttttccccac cccacccccc aagttcgggt gaaggcccag ggctcgcagc
     4141 caacgtcggg gcggcaggcc ctgccatagc ctcaggttac tcatatatac tttagattga
     4201 tttaaaactt catttttaat ttaaaaggat ctaggtgaag atcctttttg ataatctcat
     4261 gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg tcagaccccg tagaaaagat
     4321 caaaggatct tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa
     4381 accaccgcta ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc tttttccgaa
     4441 ggtaactggc ttcagcagag cgcagatacc aaatactgtt cttctagtgt agccgtagtt
     4501 aggccaccac ttcaagaact ctgtagcacc gcctacatac ctcgctctgc taatcctgtt
     4561 accagtggct gctgccagtg gcgataagtc gtgtcttacc gggttggact caagacgata
     4621 gttaccggat aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac agcccagctt
     4681 ggagcgaacg acctacaccg aactgagata cctacagcgt gagctatgag aaagcgccac
     4741 gcttcccgaa gggagaaagg cggacaggta tccggtaagc ggcagggtcg gaacaggaga
     4801 gcgcacgagg gagcttccag ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg
     4861 ccacctctga cttgagcgtc gatttttgtg atgctcgtca ggggggcgga gcctatggaa
     4921 aaacgccagc aacgcggcct ttttacggtt cctggccttt tgctggcctt ttgctcacat
     4981 gttctttcct gcgttatccc ctgattctgt ggataaccgt attaccgcca tgcat
//
