LOCUS       sequence-423505-        5080 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian expression of blue fluorescent PI4P reporter 
            mTagBFP2-ORP9-PH.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5080)
  AUTHORS   Ajiki M, Yoshikawa M, Miyazaki T, Kawasaki A, Aoki K, Nakatsu F, 
            Tsukiji S
  TITLE     ORP9-PH domain-based fluorescent reporters for visualizing 
            phosphatidylinositol 4-phosphate dynamics in living cells.
  JOURNAL   RSC Chem Biol. 2024 Apr 24;5(6):544-555. doi: 10.1039/d3cb00232b. 
            eCollection 2024 Jun 5.
  PUBMED    38846081
REFERENCE   2  (bases 1 to 5080)
  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"; doi: 
            "10.1039/d3cb00232b"; journalName: "RSC Chem Biol"; date: 
            "2024-04-24- 24"; volume: "5"; issue: "6"; pages: "544-555"
COMMENT     Sequence Label: pCMV-mTagBFP2-ORP9-PH
FEATURES             Location/Qualifiers
     source          1..5080
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        6..309
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        310..513
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     464..484
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      552..561
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             558..1268
                     /codon_start=1
                     /product="enhanced monomeric blue fluorescent protein 
                     (Subach et al., 2011)"
                     /label=mTagBFP2
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELIKENMHMKLYMEGTVDNHHFKCTSEGEGKPYEGTQTM
                     RIKVVEGGPLPFAFDILATSFLYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGG
                     VLTATQDTSLQDGCLIYNVKIRGVNFTSNGPVMQKKTLGWEAFTETLYPADGGLEGRND
                     MALKLVGGSHLIANAKTTYRSKKPAKNLKMPGVYYVDYRLERIKEANNETYVEQHEVAV
                     ARYCDLPSKLGHKLN"
     polyA_signal    1813..1934
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1850..1869)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     1904..1923
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(1941..2396)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2078..2099)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2290..2309
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        2423..2527
                     /gene="bla"
                     /label=AmpR promoter
     promoter        2529..2886
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        2529..2720
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(2552..2572)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      2737..2872
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             2921..3715
                     /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             2921..3715
                     /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(2975..2994)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3585..3604
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(3903..3922)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    3947..3994
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      4323..4911
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4812..4831
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 ttccgcgtta cataacttac ggtaaatggc ccgcctggct gaccgcccaa cgacccccgc
       61 ccattgacgt caataatgac gtatgttccc atagtaacgc caatagggac tttccattga
      121 cgtcaatggg tggagtattt acggtaaact gcccacttgg cagtacatca agtgtatcat
      181 atgccaagta cgccccctat tgacgtcaat gacggtaaat ggcccgcctg gcattatgcc
      241 cagtacatga ccttatggga ctttcctact tggcagtaca tctacgtatt agtcatcgct
      301 attaccatgg tgatgcggtt ttggcagtac atcaatgggc gtggatagcg gtttgactca
      361 cggggatttc caagtctcca ccccattgac gtcaatggga gtttgttttg gcaccaaaat
      421 caacgggact ttccaaaatg tcgtaacaac tccgccccat tgacgcaaat gggcggtagg
      481 cgtgtacggt gggaggtcta tataagcaga gctggtttag tgaaccgtca gatccgctag
      541 cgctaccggt cgccaccatg gtgtctaagg gcgaagagct gattaaggag aacatgcaca
      601 tgaagctgta catggagggc accgtggaca accatcactt caagtgcaca tccgagggcg
      661 aaggcaagcc ctacgagggc acccagacca tgagaatcaa ggtggtcgag ggcggccctc
      721 tccccttcgc cttcgacatc ctggctacta gcttcctcta cggcagcaag accttcatca
      781 accacaccca gggcatcccc gacttcttca agcagtcctt ccctgagggc ttcacatggg
      841 agagagtcac cacatacgaa gacgggggcg tgctgaccgc tacccaggac accagcctcc
      901 aggacggctg cctcatctac aacgtcaaga tcagaggggt gaacttcaca tccaacggcc
      961 ctgtgatgca gaagaaaaca ctcggctggg aggccttcac cgagacgctg taccccgctg
     1021 acggcggcct ggaaggcaga aacgacatgg ccctgaagct cgtgggcggg agccatctga
     1081 tcgcaaacgc caagaccaca tatagatcca agaaacccgc taagaacctc aagatgcctg
     1141 gcgtctacta tgtggactac agactggaaa gaatcaagga ggccaacaac gagacctacg
     1201 tcgagcagca cgaggtggca gtggccagat actgcgacct ccctagcaaa ctggggcaca
     1261 agcttaattc cggactcaga tctggtggcg gaggctcggg cggaggtggg tcgggtggcg
     1321 gcggatctcg agctcaagct tcgatggcgt ccatcgtgga agggccgctg agcaaatgga
     1381 ctaacgtgat gaagggatgg cagtatcgtt ggttcgtgct ggactacaat gcagggctgc
     1441 tctcctacta cacgtccaag gacaaaatga tgagaggctc tcgaagagga tgcgttagac
     1501 tcagaggagc tgtgattggt atagacgacg aggacgacag caccttcaca atcactgtcg
     1561 atcagaaaac cttccacttc caggctcgag atgcagacga gcgagagaag tggatccatg
     1621 ccttagaaga aactattctt cgccatactc ttcagcttca aggtttggat tcaggatgac
     1681 ccgggatcca ccggatctag ataactgatc ataatcagcc ataccacatt tgtagaggtt
     1741 ttacttgctt taaaaaacct cccacacctc cccctgaacc tgaaacataa aatgaatgca
     1801 attgttgttg ttaacttgtt tattgcagct tataatggtt acaaataaag caatagcatc
     1861 acaaatttca caaataaagc atttttttca ctgcattcta gttgtggttt gtccaaactc
     1921 atcaatgtat cttaacgcgt aaattgtaag cgttaatatt ttgttaaaat tcgcgttaaa
     1981 tttttgttaa atcagctcat tttttaacca ataggccgaa atcggcaaaa tcccttataa
     2041 atcaaaagaa tagaccgaga tagggttgag tgttgttcca gtttggaaca agagtccact
     2101 attaaagaac gtggactcca acgtcaaagg gcgaaaaacc gtctatcagg gcgatggccc
     2161 actacgtgaa ccatcaccct aatcaagttt tttggggtcg aggtgccgta aagcactaaa
     2221 tcggaaccct aaagggagcc cccgatttag agcttgacgg ggaaagccgg cgaacgtggc
     2281 gagaaaggaa gggaagaaag cgaaaggagc gggcgctagg gcgctggcaa gtgtagcggt
     2341 cacgctgcgc gtaaccacca cacccgccgc gcttaatgcg ccgctacagg gcgcgtcagg
     2401 tggcactttt cggggaaatg tgcgcggaac ccctatttgt ttatttttct aaatacattc
     2461 aaatatgtat ccgctcatga gacaataacc ctgataaatg cttcaataat attgaaaaag
     2521 gaagagtcct gaggcggaaa gaaccagctg tggaatgtgt gtcagttagg gtgtggaaag
     2581 tccccaggct ccccagcagg cagaagtatg caaagcatgc atctcaatta gtcagcaacc
     2641 aggtgtggaa agtccccagg ctccccagca ggcagaagta tgcaaagcat gcatctcaat
     2701 tagtcagcaa ccatagtccc gcccctaact ccgcccatcc cgcccctaac tccgcccagt
     2761 tccgcccatt ctccgcccca tggctgacta atttttttta tttatgcaga ggccgaggcc
     2821 gcctcggcct ctgagctatt ccagaagtag tgaggaggct tttttggagg cctaggcttt
     2881 tgcaaagatc gatcaagaga caggatgagg atcgtttcgc atgattgaac aagatggatt
     2941 gcacgcaggt tctccggccg cttgggtgga gaggctattc ggctatgact gggcacaaca
     3001 gacaatcggc tgctctgatg ccgccgtgtt ccggctgtca gcgcaggggc gcccggttct
     3061 ttttgtcaag accgacctgt ccggtgccct gaatgaactg caagacgagg cagcgcggct
     3121 atcgtggctg gccacgacgg gcgttccttg cgcagctgtg ctcgacgttg tcactgaagc
     3181 gggaagggac tggctgctat tgggcgaagt gccggggcag gatctcctgt catctcacct
     3241 tgctcctgcc gagaaagtat ccatcatggc tgatgcaatg cggcggctgc atacgcttga
     3301 tccggctacc tgcccattcg accaccaagc gaaacatcgc atcgagcgag cacgtactcg
     3361 gatggaagcc ggtcttgtcg atcaggatga tctggacgaa gagcatcagg ggctcgcgcc
     3421 agccgaactg ttcgccaggc tcaaggcgag catgcccgac ggcgaggatc tcgtcgtgac
     3481 ccatggcgat gcctgcttgc cgaatatcat ggtggaaaat ggccgctttt ctggattcat
     3541 cgactgtggc cggctgggtg tggcggaccg ctatcaggac atagcgttgg ctacccgtga
     3601 tattgctgaa gagcttggcg gcgaatgggc tgaccgcttc ctcgtgcttt acggtatcgc
     3661 cgctcccgat tcgcagcgca tcgccttcta tcgccttctt gacgagttct tctgagcggg
     3721 actctggggt tcgaaatgac cgaccaagcg acgcccaacc tgccatcacg agatttcgat
     3781 tccaccgccg ccttctatga aaggttgggc ttcggaatcg ttttccggga cgccggctgg
     3841 atgatcctcc agcgcgggga tctcatgctg gagttcttcg cccaccctag ggggaggcta
     3901 actgaaacac ggaaggagac aataccggaa ggaacccgcg ctatgacggc aataaaaaga
     3961 cagaataaaa cgcacggtgt tgggtcgttt gttcataaac gcggggttcg gtcccagggc
     4021 tggcactctg tcgatacccc accgagaccc cattggggcc aatacgcccg cgtttcttcc
     4081 ttttccccac cccacccccc aagttcgggt gaaggcccag ggctcgcagc caacgtcggg
     4141 gcggcaggcc ctgccatagc ctcaggttac tcatatatac tttagattga tttaaaactt
     4201 catttttaat ttaaaaggat ctaggtgaag atcctttttg ataatctcat gaccaaaatc
     4261 ccttaacgtg agttttcgtt ccactgagcg tcagaccccg tagaaaagat caaaggatct
     4321 tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta
     4381 ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc tttttccgaa ggtaactggc
     4441 ttcagcagag cgcagatacc aaatactgtt cttctagtgt agccgtagtt aggccaccac
     4501 ttcaagaact ctgtagcacc gcctacatac ctcgctctgc taatcctgtt accagtggct
     4561 gctgccagtg gcgataagtc gtgtcttacc gggttggact caagacgata gttaccggat
     4621 aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac agcccagctt ggagcgaacg
     4681 acctacaccg aactgagata cctacagcgt gagctatgag aaagcgccac gcttcccgaa
     4741 gggagaaagg cggacaggta tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg
     4801 gagcttccag ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg ccacctctga
     4861 cttgagcgtc gatttttgtg atgctcgtca ggggggcgga gcctatggaa aaacgccagc
     4921 aacgcggcct ttttacggtt cctggccttt tgctggcctt ttgctcacat gttctttcct
     4981 gcgttatccc ctgattctgt ggataaccgt attaccgcca tgcattagtt attaatagta
     5041 atcaattacg gggtcattag ttcatagccc atatatggag
//
