LOCUS       sequence-423503-        5071 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian expression of red fluorescent PI4P reporter 
            dTomato-ORP9-PH.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5071)
  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 5071)
  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-dTomato-ORP9-PH
FEATURES             Location/Qualifiers
     source          1..5071
                     /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..1259
                     /codon_start=1
                     /product="dimeric variant of DsRed fluorescent protein 
                     (Shaner et al., 2004)"
                     /label=dTomato
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEEVIKEFMRFKVRMEGSMNGHEFEIEGEGEGRPYEGTQTA
                     KLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYKKLSFPEGFKWERVMNFEDGG
                     LVTVTQDSSLQDGTLIYKVKMRGTNFPPDGPVMQKKTMGWEASTERLYPRDGVLKGEIH
                     QALKLKDGGHYLVEFKTIYMAKKPVQLPGYYYVDTKLDITSHNEDYTIVEQYERSEGRH
                     HLFLYGMDELYK"
     primer_bind     complement(691..709)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     959..978
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     1153..1176
                     /label=DsRed1-C
                     /note="DsRed1, forward primer"
     polyA_signal    1804..1925
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1841..1860)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     1895..1914
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(1932..2387)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2069..2090)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2281..2300
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        2414..2518
                     /gene="bla"
                     /label=AmpR promoter
     promoter        2520..2877
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        2520..2711
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(2543..2563)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      2728..2863
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             2912..3706
                     /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             2912..3706
                     /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(2966..2985)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3576..3595
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(3894..3913)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    3938..3985
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      4314..4902
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4803..4822
                     /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 gtgagcaagg gcgaggaggt catcaaagag ttcatgcgct
      601 tcaaggtgcg catggagggc tccatgaacg gccacgagtt cgagatcgag ggcgagggcg
      661 agggccgccc ctacgagggc acccagaccg ccaagctgaa ggtgaccaag ggcggccccc
      721 tgcccttcgc ctgggacatc ctgtcccccc agttcatgta cggctccaag gcgtacgtga
      781 agcaccccgc cgacatcccc gattacaaga agctgtcctt ccccgagggc ttcaagtggg
      841 agcgcgtgat gaacttcgag gacggcggtc tggtgaccgt gacccaggac tcctccctgc
      901 aggacggcac gctgatctac aaggtgaaga tgcgcggcac caacttcccc cccgacggcc
      961 ccgtaatgca gaagaagacc atgggctggg aggcctccac cgagcgcctg tacccccgcg
     1021 acggcgtgct gaagggcgag atccaccagg ccctgaagct gaaggacggc ggccactacc
     1081 tggtggagtt caagaccatc tacatggcca agaagcccgt gcaactgccc ggctactact
     1141 acgtggacac caagctggac atcacctccc acaacgagga ctacaccatc gtggaacagt
     1201 acgagcgctc cgagggccgc caccacctgt tcctgtacgg catggacgag ctgtacaagt
     1261 ccggactcag atctggtggc ggaggctcgg gcggaggtgg gtcgggtggc ggcggatctc
     1321 gagctcaagc ttcgatggcg tccatcgtgg aagggccgct gagcaaatgg actaacgtga
     1381 tgaagggatg gcagtatcgt tggttcgtgc tggactacaa tgcagggctg ctctcctact
     1441 acacgtccaa ggacaaaatg atgagaggct ctcgaagagg atgcgttaga ctcagaggag
     1501 ctgtgattgg tatagacgac gaggacgaca gcaccttcac aatcactgtc gatcagaaaa
     1561 ccttccactt ccaggctcga gatgcagacg agcgagagaa gtggatccat gccttagaag
     1621 aaactattct tcgccatact cttcagcttc aaggtttgga ttcaggatga cccgggatcc
     1681 accggatcta gataactgat cataatcagc cataccacat ttgtagaggt tttacttgct
     1741 ttaaaaaacc tcccacacct ccccctgaac ctgaaacata aaatgaatgc aattgttgtt
     1801 gttaacttgt ttattgcagc ttataatggt tacaaataaa gcaatagcat cacaaatttc
     1861 acaaataaag catttttttc actgcattct agttgtggtt tgtccaaact catcaatgta
     1921 tcttaacgcg taaattgtaa gcgttaatat tttgttaaaa ttcgcgttaa atttttgtta
     1981 aatcagctca ttttttaacc aataggccga aatcggcaaa atcccttata aatcaaaaga
     2041 atagaccgag atagggttga gtgttgttcc agtttggaac aagagtccac tattaaagaa
     2101 cgtggactcc aacgtcaaag ggcgaaaaac cgtctatcag ggcgatggcc cactacgtga
     2161 accatcaccc taatcaagtt ttttggggtc gaggtgccgt aaagcactaa atcggaaccc
     2221 taaagggagc ccccgattta gagcttgacg gggaaagccg gcgaacgtgg cgagaaagga
     2281 agggaagaaa gcgaaaggag cgggcgctag ggcgctggca agtgtagcgg tcacgctgcg
     2341 cgtaaccacc acacccgccg cgcttaatgc gccgctacag ggcgcgtcag gtggcacttt
     2401 tcggggaaat gtgcgcggaa cccctatttg tttatttttc taaatacatt caaatatgta
     2461 tccgctcatg agacaataac cctgataaat gcttcaataa tattgaaaaa ggaagagtcc
     2521 tgaggcggaa agaaccagct gtggaatgtg tgtcagttag ggtgtggaaa gtccccaggc
     2581 tccccagcag gcagaagtat gcaaagcatg catctcaatt agtcagcaac caggtgtgga
     2641 aagtccccag gctccccagc aggcagaagt atgcaaagca tgcatctcaa ttagtcagca
     2701 accatagtcc cgcccctaac tccgcccatc ccgcccctaa ctccgcccag ttccgcccat
     2761 tctccgcccc atggctgact aatttttttt atttatgcag aggccgaggc cgcctcggcc
     2821 tctgagctat tccagaagta gtgaggaggc ttttttggag gcctaggctt ttgcaaagat
     2881 cgatcaagag acaggatgag gatcgtttcg catgattgaa caagatggat tgcacgcagg
     2941 ttctccggcc gcttgggtgg agaggctatt cggctatgac tgggcacaac agacaatcgg
     3001 ctgctctgat gccgccgtgt tccggctgtc agcgcagggg cgcccggttc tttttgtcaa
     3061 gaccgacctg tccggtgccc tgaatgaact gcaagacgag gcagcgcggc tatcgtggct
     3121 ggccacgacg ggcgttcctt gcgcagctgt gctcgacgtt gtcactgaag cgggaaggga
     3181 ctggctgcta ttgggcgaag tgccggggca ggatctcctg tcatctcacc ttgctcctgc
     3241 cgagaaagta tccatcatgg ctgatgcaat gcggcggctg catacgcttg atccggctac
     3301 ctgcccattc gaccaccaag cgaaacatcg catcgagcga gcacgtactc ggatggaagc
     3361 cggtcttgtc gatcaggatg atctggacga agagcatcag gggctcgcgc cagccgaact
     3421 gttcgccagg ctcaaggcga gcatgcccga cggcgaggat ctcgtcgtga cccatggcga
     3481 tgcctgcttg ccgaatatca tggtggaaaa tggccgcttt tctggattca tcgactgtgg
     3541 ccggctgggt gtggcggacc gctatcagga catagcgttg gctacccgtg atattgctga
     3601 agagcttggc ggcgaatggg ctgaccgctt cctcgtgctt tacggtatcg ccgctcccga
     3661 ttcgcagcgc atcgccttct atcgccttct tgacgagttc ttctgagcgg gactctgggg
     3721 ttcgaaatga ccgaccaagc gacgcccaac ctgccatcac gagatttcga ttccaccgcc
     3781 gccttctatg aaaggttggg cttcggaatc gttttccggg acgccggctg gatgatcctc
     3841 cagcgcgggg atctcatgct ggagttcttc gcccacccta gggggaggct aactgaaaca
     3901 cggaaggaga caataccgga aggaacccgc gctatgacgg caataaaaag acagaataaa
     3961 acgcacggtg ttgggtcgtt tgttcataaa cgcggggttc ggtcccaggg ctggcactct
     4021 gtcgataccc caccgagacc ccattggggc caatacgccc gcgtttcttc cttttcccca
     4081 ccccaccccc caagttcggg tgaaggccca gggctcgcag ccaacgtcgg ggcggcaggc
     4141 cctgccatag cctcaggtta ctcatatata ctttagattg atttaaaact tcatttttaa
     4201 tttaaaagga tctaggtgaa gatccttttt gataatctca tgaccaaaat cccttaacgt
     4261 gagttttcgt tccactgagc gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat
     4321 cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct accagcggtg
     4381 gtttgtttgc cggatcaaga gctaccaact ctttttccga aggtaactgg cttcagcaga
     4441 gcgcagatac caaatactgt tcttctagtg tagccgtagt taggccacca cttcaagaac
     4501 tctgtagcac cgcctacata cctcgctctg ctaatcctgt taccagtggc tgctgccagt
     4561 ggcgataagt cgtgtcttac cgggttggac tcaagacgat agttaccgga taaggcgcag
     4621 cggtcgggct gaacgggggg ttcgtgcaca cagcccagct tggagcgaac gacctacacc
     4681 gaactgagat acctacagcg tgagctatga gaaagcgcca cgcttcccga agggagaaag
     4741 gcggacaggt atccggtaag cggcagggtc ggaacaggag agcgcacgag ggagcttcca
     4801 gggggaaacg cctggtatct ttatagtcct gtcgggtttc gccacctctg acttgagcgt
     4861 cgatttttgt gatgctcgtc aggggggcgg agcctatgga aaaacgccag caacgcggcc
     4921 tttttacggt tcctggcctt ttgctggcct tttgctcaca tgttctttcc tgcgttatcc
     4981 cctgattctg tggataaccg tattaccgcc atgcattagt tattaatagt aatcaattac
     5041 ggggtcatta gttcatagcc catatatgga g
//
