LOCUS       sequence-287378-        5765 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  N-terminal half of split PI-PLC targeted to plasma membrane.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5765)
  AUTHORS   Zewe JP, Miller AM, Sangappa S, Wills RC, Goulden BD, Hammond GRV
  TITLE     Probing the subcellular distribution of phosphatidylinositol reveals
            a surprising lack at the plasma membrane.
  JOURNAL   J Cell Biol. 2020 Mar 2;219(3). pii: 133808. doi: 
            10.1083/jcb.201906127.
  PUBMED    32211893
REFERENCE   2  (bases 1 to 5765)
  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: "J Cell 
            Biol."; date: "2020-03-2"; volume: "219(3). pii: 133808. doi"; 
            pages: " 10.1083/jcb.201906127"
COMMENT     Sequence Label: pTagBFP2-C1-lynN11-FKBP-PI-PLCN187
FEATURES             Location/Qualifiers
     source          1..5765
                     /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"
     CDS             complement(652..669)
                     /codon_start=1
                     /product="HQHQHQ metal affinity tag"
                     /label=HQ tag
                     /note="The HQ tag binds to nickel and other metal ions much
                     like the 6xHis tag, but may allow elution at lower 
                     concentrations of imidazole (Godat et al., 2008)."
                     /translation="HQHQHQ"
     CDS             675..1370
                     /codon_start=1
                     /product="monomeric blue fluorescent protein"
                     /label=TagBFP
                     /note="mammalian codon-optimized"
                     /translation="SELIKENMHMKLYMEGTVDNHHFKCTSEGEGKPYEGTQTMRIKVV
                     EGGPLPFAFDILATSFLYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLTAT
                     QDTSLQDGCLIYNVKIRGVNFTSNGPVMQKKTLGWEAFTETLYPADGGLEGRNDMALKL
                     VGGSHLIANAKTTYRSKKPAKNLKMPGVYYVDYRLERIKEANNETYVEQHEVAVARYCD
                     LPSKLGHKLN"
     CDS             1431..1751
                     /codon_start=1
                     /product="human FK506-binding protein FKBP12"
                     /label=FKBP
                     /translation="GVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKFDSSRDRNKP
                     FKFMLGKQEVIRGWEEGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPHATLVFDVELL
                     KLE"
     polyA_signal    2553..2674
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2590..2609)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2644..2663
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(2681..3136)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2818..2839)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3030..3049
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        3163..3267
                     /gene="bla"
                     /label=AmpR promoter
     promoter        3269..3626
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        3269..3460
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(3292..3312)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      3477..3612
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             3661..4455
                     /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             3661..4455
                     /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(3715..3734)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4325..4344
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(4643..4662)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    4687..4734
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      5063..5651
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5552..5571
                     /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 gctagcacca
      601 tgggatgtat aaaatcaaaa gggaaagaca gcagatctgc gaattcaggt gctggtgctg
      661 gtgctggtgc tgtgagcgag ctgattaagg agaacatgca catgaagctg tacatggagg
      721 gcaccgtgga caaccatcac ttcaagtgca catccgaggg cgaaggcaag ccctacgagg
      781 gcacccagac catgagaatc aaggtggtcg agggcggccc tctccccttc gccttcgaca
      841 tcctggctac tagcttcctc tacggcagca agaccttcat caaccacacc cagggcatcc
      901 ccgacttctt caagcagtcc ttccctgagg gcttcacatg ggagagagtc accacatacg
      961 aagacggggg cgtgctgacc gctacccagg acaccagcct ccaggacggc tgcctcatct
     1021 acaacgtcaa gatcagaggg gtgaacttca catccaacgg ccctgtgatg cagaagaaaa
     1081 cactcggctg ggaggccttc accgagacgc tgtaccccgc tgacggcggc ctggaaggca
     1141 gaaacgacat ggccctgaag ctcgtgggcg ggagccatct gatcgcaaac gccaagacca
     1201 catatagatc caagaaaccc gctaagaacc tcaagatgcc tggcgtctac tatgtggact
     1261 acagactgga aagaatcaag gaggccaaca acgagaccta cgtcgagcag cacgaggtgg
     1321 cagtggccag atactgcgac ctccctagca aactggggca caagcttaat tccggactca
     1381 gatctcgaag cgcggccgcg ggagcaggag gagcagctcg agcggcgctg ggagtgcagg
     1441 tggaaaccat ctccccagga gacgggcgca ccttccccaa gcgcggccag acctgcgtgg
     1501 tgcactacac cgggatgctt gaagatggaa agaaatttga ttcctcccgg gacagaaaca
     1561 agccctttaa gtttatgcta ggcaagcagg aggtgatccg aggctgggaa gaaggggttg
     1621 cccagatgag tgtgggtcag agagccaaac tgactatatc tccagattat gcctatggtg
     1681 ccactgggca cccaggcatc atcccaccac atgccactct cgtcttcgat gtggagcttc
     1741 taaaactgga aagtgctggt ggtagtgctg gtggtagtgc tggtggtagt gctggtggta
     1801 gtgctggtgg tccgcgagct caagcttcgt cgcttaataa ctggaataag ccaataaaga
     1861 actctgtaac tacaaaacaa tggatgtccg ctctacctga cacaacaaac ctagcagcgc
     1921 tctctatacc aggtacacat gatacgatga gctataacgg agacataaca tggacattaa
     1981 ccaaaccact agctcaaaca caaacgatgt cattgtacca acaactagaa gcaggaatac
     2041 ggtacatcga tattagagca aaagacaatc tcaacattta ccatgggcca atttttttaa
     2101 atgcatcact ttcaggtgta ttagaaacga ttactcaatt tttaaagaaa aatccaaaag
     2161 aaaccattat tatgcgttta aaggacgagc aaaacagcaa cgatagtttt gattatcgga
     2221 tccaaccact aatcaacatt tataaagatt atttttacac tactcccaga actgacacga
     2281 gcaataaaat ccctacatta aaagatgtcc gcggaaaaat attattactt tcagagaacc
     2341 acacaaaaaa gccattagtc attaactcac gcaaattcgg catgcagttc tagaattctg
     2401 cagtcgacgg taccgcgggc ccgggatcca ccggatctag ataactgatc ataatcagcc
     2461 ataccacatt tgtagaggtt ttacttgctt taaaaaacct cccacacctc cccctgaacc
     2521 tgaaacataa aatgaatgca attgttgttg ttaacttgtt tattgcagct tataatggtt
     2581 acaaataaag caatagcatc acaaatttca caaataaagc atttttttca ctgcattcta
     2641 gttgtggttt gtccaaactc atcaatgtat cttaacgcgt aaattgtaag cgttaatatt
     2701 ttgttaaaat tcgcgttaaa tttttgttaa atcagctcat tttttaacca ataggccgaa
     2761 atcggcaaaa tcccttataa atcaaaagaa tagaccgaga tagggttgag tgttgttcca
     2821 gtttggaaca agagtccact attaaagaac gtggactcca acgtcaaagg gcgaaaaacc
     2881 gtctatcagg gcgatggccc actacgtgaa ccatcaccct aatcaagttt tttggggtcg
     2941 aggtgccgta aagcactaaa tcggaaccct aaagggagcc cccgatttag agcttgacgg
     3001 ggaaagccgg cgaacgtggc gagaaaggaa gggaagaaag cgaaaggagc gggcgctagg
     3061 gcgctggcaa gtgtagcggt cacgctgcgc gtaaccacca cacccgccgc gcttaatgcg
     3121 ccgctacagg gcgcgtcagg tggcactttt cggggaaatg tgcgcggaac ccctatttgt
     3181 ttatttttct aaatacattc aaatatgtat ccgctcatga gacaataacc ctgataaatg
     3241 cttcaataat attgaaaaag gaagagtcct gaggcggaaa gaaccagctg tggaatgtgt
     3301 gtcagttagg gtgtggaaag tccccaggct ccccagcagg cagaagtatg caaagcatgc
     3361 atctcaatta gtcagcaacc aggtgtggaa agtccccagg ctccccagca ggcagaagta
     3421 tgcaaagcat gcatctcaat tagtcagcaa ccatagtccc gcccctaact ccgcccatcc
     3481 cgcccctaac tccgcccagt tccgcccatt ctccgcccca tggctgacta atttttttta
     3541 tttatgcaga ggccgaggcc gcctcggcct ctgagctatt ccagaagtag tgaggaggct
     3601 tttttggagg cctaggcttt tgcaaagatc gatcaagaga caggatgagg atcgtttcgc
     3661 atgattgaac aagatggatt gcacgcaggt tctccggccg cttgggtgga gaggctattc
     3721 ggctatgact gggcacaaca gacaatcggc tgctctgatg ccgccgtgtt ccggctgtca
     3781 gcgcaggggc gcccggttct ttttgtcaag accgacctgt ccggtgccct gaatgaactg
     3841 caagacgagg cagcgcggct atcgtggctg gccacgacgg gcgttccttg cgcagctgtg
     3901 ctcgacgttg tcactgaagc gggaagggac tggctgctat tgggcgaagt gccggggcag
     3961 gatctcctgt catctcacct tgctcctgcc gagaaagtat ccatcatggc tgatgcaatg
     4021 cggcggctgc atacgcttga tccggctacc tgcccattcg accaccaagc gaaacatcgc
     4081 atcgagcgag cacgtactcg gatggaagcc ggtcttgtcg atcaggatga tctggacgaa
     4141 gagcatcagg ggctcgcgcc agccgaactg ttcgccaggc tcaaggcgag catgcccgac
     4201 ggcgaggatc tcgtcgtgac ccatggcgat gcctgcttgc cgaatatcat ggtggaaaat
     4261 ggccgctttt ctggattcat cgactgtggc cggctgggtg tggcggaccg ctatcaggac
     4321 atagcgttgg ctacccgtga tattgctgaa gagcttggcg gcgaatgggc tgaccgcttc
     4381 ctcgtgcttt acggtatcgc cgctcccgat tcgcagcgca tcgccttcta tcgccttctt
     4441 gacgagttct tctgagcggg actctggggt tcgaaatgac cgaccaagcg acgcccaacc
     4501 tgccatcacg agatttcgat tccaccgccg ccttctatga aaggttgggc ttcggaatcg
     4561 ttttccggga cgccggctgg atgatcctcc agcgcgggga tctcatgctg gagttcttcg
     4621 cccaccctag ggggaggcta actgaaacac ggaaggagac aataccggaa ggaacccgcg
     4681 ctatgacggc aataaaaaga cagaataaaa cgcacggtgt tgggtcgttt gttcataaac
     4741 gcggggttcg gtcccagggc tggcactctg tcgatacccc accgagaccc cattggggcc
     4801 aatacgcccg cgtttcttcc ttttccccac cccacccccc aagttcgggt gaaggcccag
     4861 ggctcgcagc caacgtcggg gcggcaggcc ctgccatagc ctcaggttac tcatatatac
     4921 tttagattga tttaaaactt catttttaat ttaaaaggat ctaggtgaag atcctttttg
     4981 ataatctcat gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg tcagaccccg
     5041 tagaaaagat caaaggatct tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc
     5101 aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc
     5161 tttttccgaa ggtaactggc ttcagcagag cgcagatacc aaatactgtt cttctagtgt
     5221 agccgtagtt aggccaccac ttcaagaact ctgtagcacc gcctacatac ctcgctctgc
     5281 taatcctgtt accagtggct gctgccagtg gcgataagtc gtgtcttacc gggttggact
     5341 caagacgata gttaccggat aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac
     5401 agcccagctt ggagcgaacg acctacaccg aactgagata cctacagcgt gagctatgag
     5461 aaagcgccac gcttcccgaa gggagaaagg cggacaggta tccggtaagc ggcagggtcg
     5521 gaacaggaga gcgcacgagg gagcttccag ggggaaacgc ctggtatctt tatagtcctg
     5581 tcgggtttcg ccacctctga cttgagcgtc gatttttgtg atgctcgtca ggggggcgga
     5641 gcctatggaa aaacgccagc aacgcggcct ttttacggtt cctggccttt tgctggcctt
     5701 ttgctcacat gttctttcct gcgttatccc ctgattctgt ggataaccgt attaccgcca
     5761 tgcat
//
