LOCUS       sequence-423502-        5086 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian expression of green fluorescent PI4P reporter 
            AcGFP1-ORP9-PH.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5086)
  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 5086)
  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-AcGFP1-ORP9-PH
FEATURES             Location/Qualifiers
     source          1..5086
                     /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..1274
                     /codon_start=1
                     /product="Aequorea coerulescens GFP"
                     /label=AcGFP1
                     /note="mammalian codon-optimized"
                     /translation="MVSKGAELFTGIVPILIELNGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLSYGVQCFSRYPDHMKQHDFFKSAMPEGYIQERTIFFEDD
                     GNYKSRAEVKFEGDTLVNRIELTGTDFKEDGNILGNKMEYNYNAHNVYIMTDKAKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMIYF
                     GFVTAAAITHGMDELYK"
     polyA_signal    1819..1940
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1856..1875)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     1910..1929
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(1947..2402)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2084..2105)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2296..2315
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        2429..2533
                     /gene="bla"
                     /label=AmpR promoter
     promoter        2535..2892
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        2535..2726
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(2558..2578)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      2743..2878
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             2927..3721
                     /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             2927..3721
                     /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(2981..3000)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3591..3610
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(3909..3928)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    3953..4000
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      4329..4917
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4818..4837
                     /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 gcgccgagct gttcaccggc atcgtgccca
      601 tcctgatcga gctgaatggc gatgtgaatg gccacaagtt cagcgtgagc ggcgagggcg
      661 agggcgatgc cacctacggc aagctgaccc tgaagttcat ctgcaccacc ggcaagctgc
      721 ctgtgccctg gcccaccctg gtgaccaccc tgagctacgg cgtgcagtgc ttctcacgct
      781 accccgatca catgaagcag cacgacttct tcaagagcgc catgcctgag ggctacatcc
      841 aggagcgcac catcttcttc gaggatgacg gcaactacaa gtcgcgcgcc gaggtgaagt
      901 tcgagggcga taccctggtg aatcgcatcg agctgaccgg caccgatttc aaggaggatg
      961 gcaacatcct gggcaataag atggagtaca actacaacgc ccacaatgtg tacatcatga
     1021 ccgacaaggc caagaatggc atcaaggtga acttcaagat ccgccacaac atcgaggatg
     1081 gcagcgtgca gctggccgac cactaccagc agaatacccc catcggcgat ggccctgtgc
     1141 tgctgcccga taaccactac ctgtccaccc agagcgccct gtccaaggac cccaacgaga
     1201 agcgcgatca catgatctac ttcggcttcg tgaccgccgc cgccatcacc cacggcatgg
     1261 atgagctgta caagtccgga ctcagatctg gtggcggagg ctcgggcgga ggtgggtcgg
     1321 gtggcggcgg atctcgagct caagcttcga tggcgtccat cgtggaaggg ccgctgagca
     1381 aatggactaa cgtgatgaag ggatggcagt atcgttggtt cgtgctggac tacaatgcag
     1441 ggctgctctc ctactacacg tccaaggaca aaatgatgag aggctctcga agaggatgcg
     1501 ttagactcag aggagctgtg attggtatag acgacgagga cgacagcacc ttcacaatca
     1561 ctgtcgatca gaaaaccttc cacttccagg ctcgagatgc agacgagcga gagaagtgga
     1621 tccatgcctt agaagaaact attcttcgcc atactcttca gcttcaaggt ttggattcag
     1681 gatgacccgg gatccaccgg atctagataa ctgatcataa tcagccatac cacatttgta
     1741 gaggttttac ttgctttaaa aaacctccca cacctccccc tgaacctgaa acataaaatg
     1801 aatgcaattg ttgttgttaa cttgtttatt gcagcttata atggttacaa ataaagcaat
     1861 agcatcacaa atttcacaaa taaagcattt ttttcactgc attctagttg tggtttgtcc
     1921 aaactcatca atgtatctta acgcgtaaat tgtaagcgtt aatattttgt taaaattcgc
     1981 gttaaatttt tgttaaatca gctcattttt taaccaatag gccgaaatcg gcaaaatccc
     2041 ttataaatca aaagaataga ccgagatagg gttgagtgtt gttccagttt ggaacaagag
     2101 tccactatta aagaacgtgg actccaacgt caaagggcga aaaaccgtct atcagggcga
     2161 tggcccacta cgtgaaccat caccctaatc aagttttttg gggtcgaggt gccgtaaagc
     2221 actaaatcgg aaccctaaag ggagcccccg atttagagct tgacggggaa agccggcgaa
     2281 cgtggcgaga aaggaaggga agaaagcgaa aggagcgggc gctagggcgc tggcaagtgt
     2341 agcggtcacg ctgcgcgtaa ccaccacacc cgccgcgctt aatgcgccgc tacagggcgc
     2401 gtcaggtggc acttttcggg gaaatgtgcg cggaacccct atttgtttat ttttctaaat
     2461 acattcaaat atgtatccgc tcatgagaca ataaccctga taaatgcttc aataatattg
     2521 aaaaaggaag agtcctgagg cggaaagaac cagctgtgga atgtgtgtca gttagggtgt
     2581 ggaaagtccc caggctcccc agcaggcaga agtatgcaaa gcatgcatct caattagtca
     2641 gcaaccaggt gtggaaagtc cccaggctcc ccagcaggca gaagtatgca aagcatgcat
     2701 ctcaattagt cagcaaccat agtcccgccc ctaactccgc ccatcccgcc cctaactccg
     2761 cccagttccg cccattctcc gccccatggc tgactaattt tttttattta tgcagaggcc
     2821 gaggccgcct cggcctctga gctattccag aagtagtgag gaggcttttt tggaggccta
     2881 ggcttttgca aagatcgatc aagagacagg atgaggatcg tttcgcatga ttgaacaaga
     2941 tggattgcac gcaggttctc cggccgcttg ggtggagagg ctattcggct atgactgggc
     3001 acaacagaca atcggctgct ctgatgccgc cgtgttccgg ctgtcagcgc aggggcgccc
     3061 ggttcttttt gtcaagaccg acctgtccgg tgccctgaat gaactgcaag acgaggcagc
     3121 gcggctatcg tggctggcca cgacgggcgt tccttgcgca gctgtgctcg acgttgtcac
     3181 tgaagcggga agggactggc tgctattggg cgaagtgccg gggcaggatc tcctgtcatc
     3241 tcaccttgct cctgccgaga aagtatccat catggctgat gcaatgcggc ggctgcatac
     3301 gcttgatccg gctacctgcc cattcgacca ccaagcgaaa catcgcatcg agcgagcacg
     3361 tactcggatg gaagccggtc ttgtcgatca ggatgatctg gacgaagagc atcaggggct
     3421 cgcgccagcc gaactgttcg ccaggctcaa ggcgagcatg cccgacggcg aggatctcgt
     3481 cgtgacccat ggcgatgcct gcttgccgaa tatcatggtg gaaaatggcc gcttttctgg
     3541 attcatcgac tgtggccggc tgggtgtggc ggaccgctat caggacatag cgttggctac
     3601 ccgtgatatt gctgaagagc ttggcggcga atgggctgac cgcttcctcg tgctttacgg
     3661 tatcgccgct cccgattcgc agcgcatcgc cttctatcgc cttcttgacg agttcttctg
     3721 agcgggactc tggggttcga aatgaccgac caagcgacgc ccaacctgcc atcacgagat
     3781 ttcgattcca ccgccgcctt ctatgaaagg ttgggcttcg gaatcgtttt ccgggacgcc
     3841 ggctggatga tcctccagcg cggggatctc atgctggagt tcttcgccca ccctaggggg
     3901 aggctaactg aaacacggaa ggagacaata ccggaaggaa cccgcgctat gacggcaata
     3961 aaaagacaga ataaaacgca cggtgttggg tcgtttgttc ataaacgcgg ggttcggtcc
     4021 cagggctggc actctgtcga taccccaccg agaccccatt ggggccaata cgcccgcgtt
     4081 tcttcctttt ccccacccca ccccccaagt tcgggtgaag gcccagggct cgcagccaac
     4141 gtcggggcgg caggccctgc catagcctca ggttactcat atatacttta gattgattta
     4201 aaacttcatt tttaatttaa aaggatctag gtgaagatcc tttttgataa tctcatgacc
     4261 aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag accccgtaga aaagatcaaa
     4321 ggatcttctt gagatccttt ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca
     4381 ccgctaccag cggtggtttg tttgccggat caagagctac caactctttt tccgaaggta
     4441 actggcttca gcagagcgca gataccaaat actgttcttc tagtgtagcc gtagttaggc
     4501 caccacttca agaactctgt agcaccgcct acatacctcg ctctgctaat cctgttacca
     4561 gtggctgctg ccagtggcga taagtcgtgt cttaccgggt tggactcaag acgatagtta
     4621 ccggataagg cgcagcggtc gggctgaacg gggggttcgt gcacacagcc cagcttggag
     4681 cgaacgacct acaccgaact gagataccta cagcgtgagc tatgagaaag cgccacgctt
     4741 cccgaaggga gaaaggcgga caggtatccg gtaagcggca gggtcggaac aggagagcgc
     4801 acgagggagc ttccaggggg aaacgcctgg tatctttata gtcctgtcgg gtttcgccac
     4861 ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac
     4921 gccagcaacg cggccttttt acggttcctg gccttttgct ggccttttgc tcacatgttc
     4981 tttcctgcgt tatcccctga ttctgtggat aaccgtatta ccgccatgca ttagttatta
     5041 atagtaatca attacggggt cattagttca tagcccatat atggag
//
