LOCUS       sequence-423508-        5247 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian expression of blue fluorescent PI(4,5)P2 reporter 
            mTagBFP2-PLC-delta-PH.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5247)
  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 5247)
  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-PLC-delta-PH
FEATURES             Location/Qualifiers
     source          1..5247
                     /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"
     CDS             613..1323
                     /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"
     misc_feature    1324..1389
                     /label=MCS
                     /note="multiple cloning site"
     polyA_signal    2035..2156
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2072..2091)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2126..2145
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(2163..2618)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2300..2321)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2512..2531
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        2645..2749
                     /gene="bla"
                     /label=AmpR promoter
     promoter        2751..3108
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        2751..2942
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(2774..2794)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      2959..3094
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             3143..3937
                     /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             3143..3937
                     /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(3197..3216)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3807..3826
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(4125..4144)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    4169..4216
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      4545..5133
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5034..5053
                     /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 ccatggtgtc taagggcgaa gagctgatta aggagaacat gcacatgaag
      661 ctgtacatgg agggcaccgt ggacaaccat cacttcaagt gcacatccga gggcgaaggc
      721 aagccctacg agggcaccca gaccatgaga atcaaggtgg tcgagggcgg ccctctcccc
      781 ttcgccttcg acatcctggc tactagcttc ctctacggca gcaagacctt catcaaccac
      841 acccagggca tccccgactt cttcaagcag tccttccctg agggcttcac atgggagaga
      901 gtcaccacat acgaagacgg gggcgtgctg accgctaccc aggacaccag cctccaggac
      961 ggctgcctca tctacaacgt caagatcaga ggggtgaact tcacatccaa cggccctgtg
     1021 atgcagaaga aaacactcgg ctgggaggcc ttcaccgaga cgctgtaccc cgctgacggc
     1081 ggcctggaag gcagaaacga catggccctg aagctcgtgg gcgggagcca tctgatcgca
     1141 aacgccaaga ccacatatag atccaagaaa cccgctaaga acctcaagat gcctggcgtc
     1201 tactatgtgg actacagact ggaaagaatc aaggaggcca acaacgagac ctacgtcgag
     1261 cagcacgagg tggcagtggc cagatactgc gacctcccta gcaaactggg gcacaagctt
     1321 aattccggac tcagatctcg agctcaagct tcgaattctg cagtcgacgg taccgcgggc
     1381 ccgggatcca tggactcggg ccgggacttc ctgaccctgc acggcctaca ggatgatgag
     1441 gatctacagg cgctgctgaa gggcagccag ctcctgaagg tgaagtccag ctcatggagg
     1501 agagagcgct tctacaagtt gcaggaggac tgcaagacca tctggcagga gtcccgcaag
     1561 gtcatgcgga ccccggagtc ccagctgttc tccatcgagg acattcagga ggtgcgaatg
     1621 gggcaccgca cggagggtct ggagaagttc gcccgtgatg tgcccgagga ccgctgcttc
     1681 tccattgtct tcaaggacca gcgcaataca ctagacctca tcgccccatc gccagctgat
     1741 gcccagcact gggtgctggg gctgcacaag atcatccacc actcaggctc catggaccag
     1801 cgtcagaagc tacagcactg gattcactcc tgcttgcgaa aagctgacaa aaacaaggac
     1861 aacaagatga gcttcaagga gctgcagaac ttcctgaagg agctcaacat ccagtaatct
     1921 agataactga tcataatcag ccataccaca tttgtagagg ttttacttgc tttaaaaaac
     1981 ctcccacacc tccccctgaa cctgaaacat aaaatgaatg caattgttgt tgttaacttg
     2041 tttattgcag cttataatgg ttacaaataa agcaatagca tcacaaattt cacaaataaa
     2101 gcattttttt cactgcattc tagttgtggt ttgtccaaac tcatcaatgt atcttaacgc
     2161 gtaaattgta agcgttaata ttttgttaaa attcgcgtta aatttttgtt aaatcagctc
     2221 attttttaac caataggccg aaatcggcaa aatcccttat aaatcaaaag aatagaccga
     2281 gatagggttg agtgttgttc cagtttggaa caagagtcca ctattaaaga acgtggactc
     2341 caacgtcaaa gggcgaaaaa ccgtctatca gggcgatggc ccactacgtg aaccatcacc
     2401 ctaatcaagt tttttggggt cgaggtgccg taaagcacta aatcggaacc ctaaagggag
     2461 cccccgattt agagcttgac ggggaaagcc ggcgaacgtg gcgagaaagg aagggaagaa
     2521 agcgaaagga gcgggcgcta gggcgctggc aagtgtagcg gtcacgctgc gcgtaaccac
     2581 cacacccgcc gcgcttaatg cgccgctaca gggcgcgtca ggtggcactt ttcggggaaa
     2641 tgtgcgcgga acccctattt gtttattttt ctaaatacat tcaaatatgt atccgctcat
     2701 gagacaataa ccctgataaa tgcttcaata atattgaaaa aggaagagtc ctgaggcgga
     2761 aagaaccagc tgtggaatgt gtgtcagtta gggtgtggaa agtccccagg ctccccagca
     2821 ggcagaagta tgcaaagcat gcatctcaat tagtcagcaa ccaggtgtgg aaagtcccca
     2881 ggctccccag caggcagaag tatgcaaagc atgcatctca attagtcagc aaccatagtc
     2941 ccgcccctaa ctccgcccat cccgccccta actccgccca gttccgccca ttctccgccc
     3001 catggctgac taattttttt tatttatgca gaggccgagg ccgcctcggc ctctgagcta
     3061 ttccagaagt agtgaggagg cttttttgga ggcctaggct tttgcaaaga tcgatcaaga
     3121 gacaggatga ggatcgtttc gcatgattga acaagatgga ttgcacgcag gttctccggc
     3181 cgcttgggtg gagaggctat tcggctatga ctgggcacaa cagacaatcg gctgctctga
     3241 tgccgccgtg ttccggctgt cagcgcaggg gcgcccggtt ctttttgtca agaccgacct
     3301 gtccggtgcc ctgaatgaac tgcaagacga ggcagcgcgg ctatcgtggc tggccacgac
     3361 gggcgttcct tgcgcagctg tgctcgacgt tgtcactgaa gcgggaaggg actggctgct
     3421 attgggcgaa gtgccggggc aggatctcct gtcatctcac cttgctcctg ccgagaaagt
     3481 atccatcatg gctgatgcaa tgcggcggct gcatacgctt gatccggcta cctgcccatt
     3541 cgaccaccaa gcgaaacatc gcatcgagcg agcacgtact cggatggaag ccggtcttgt
     3601 cgatcaggat gatctggacg aagagcatca ggggctcgcg ccagccgaac tgttcgccag
     3661 gctcaaggcg agcatgcccg acggcgagga tctcgtcgtg acccatggcg atgcctgctt
     3721 gccgaatatc atggtggaaa atggccgctt ttctggattc atcgactgtg gccggctggg
     3781 tgtggcggac cgctatcagg acatagcgtt ggctacccgt gatattgctg aagagcttgg
     3841 cggcgaatgg gctgaccgct tcctcgtgct ttacggtatc gccgctcccg attcgcagcg
     3901 catcgccttc tatcgccttc ttgacgagtt cttctgagcg ggactctggg gttcgaaatg
     3961 accgaccaag cgacgcccaa cctgccatca cgagatttcg attccaccgc cgccttctat
     4021 gaaaggttgg gcttcggaat cgttttccgg gacgccggct ggatgatcct ccagcgcggg
     4081 gatctcatgc tggagttctt cgcccaccct agggggaggc taactgaaac acggaaggag
     4141 acaataccgg aaggaacccg cgctatgacg gcaataaaaa gacagaataa aacgcacggt
     4201 gttgggtcgt ttgttcataa acgcggggtt cggtcccagg gctggcactc tgtcgatacc
     4261 ccaccgagac cccattgggg ccaatacgcc cgcgtttctt ccttttcccc accccacccc
     4321 ccaagttcgg gtgaaggccc agggctcgca gccaacgtcg gggcggcagg ccctgccata
     4381 gcctcaggtt actcatatat actttagatt gatttaaaac ttcattttta atttaaaagg
     4441 atctaggtga agatcctttt tgataatctc atgaccaaaa tcccttaacg tgagttttcg
     4501 ttccactgag cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga tccttttttt
     4561 ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg
     4621 ccggatcaag agctaccaac tctttttccg aaggtaactg gcttcagcag agcgcagata
     4681 ccaaatactg ttcttctagt gtagccgtag ttaggccacc acttcaagaa ctctgtagca
     4741 ccgcctacat acctcgctct gctaatcctg ttaccagtgg ctgctgccag tggcgataag
     4801 tcgtgtctta ccgggttgga ctcaagacga tagttaccgg ataaggcgca gcggtcgggc
     4861 tgaacggggg gttcgtgcac acagcccagc ttggagcgaa cgacctacac cgaactgaga
     4921 tacctacagc gtgagctatg agaaagcgcc acgcttcccg aagggagaaa ggcggacagg
     4981 tatccggtaa gcggcagggt cggaacagga gagcgcacga gggagcttcc agggggaaac
     5041 gcctggtatc tttatagtcc tgtcgggttt cgccacctct gacttgagcg tcgatttttg
     5101 tgatgctcgt caggggggcg gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg
     5161 ttcctggcct tttgctggcc ttttgctcac atgttctttc ctgcgttatc ccctgattct
     5221 gtggataacc gtattaccgc catgcat
//
