LOCUS       sequence-269439-        5859 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  N-terminal half of split PI-PLC targeted to mitochondria.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5859)
  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 5859)
  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-AKAP1N31-FKBP-PI-PLCN187
FEATURES             Location/Qualifiers
     source          1..5859
                     /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             complement(731..748)
                     /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             763..1464
                     /codon_start=1
                     /product="monomeric blue fluorescent protein"
                     /label=TagBFP
                     /note="mammalian codon-optimized"
                     /translation="MVSELIKENMHMKLYMEGTVDNHHFKCTSEGEGKPYEGTQTMRIK
                     VVEGGPLPFAFDILATSFLYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLT
                     ATQDTSLQDGCLIYNVKIRGVNFTSNGPVMQKKTLGWEAFTETLYPADGGLEGRNDMAL
                     KLVGGSHLIANAKTTYRSKKPAKNLKMPGVYYVDYRLERIKEANNETYVEQHEVAVARY
                     CDLPSKLGHKLN"
     CDS             1525..1845
                     /codon_start=1
                     /product="human FK506-binding protein FKBP12"
                     /label=FKBP
                     /translation="GVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKFDSSRDRNKP
                     FKFMLGKQEVIRGWEEGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPHATLVFDVELL
                     KLE"
     polyA_signal    2647..2768
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2684..2703)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2738..2757
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(2775..3230)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2912..2933)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3124..3143
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        3257..3361
                     /gene="bla"
                     /label=AmpR promoter
     promoter        3363..3720
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        3363..3554
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(3386..3406)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      3571..3706
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             3755..4549
                     /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             3755..4549
                     /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(3809..3828)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4419..4438
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(4737..4756)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    4781..4828
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      5157..5745
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5646..5665
                     /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 ccatggcaat ccagttgcgt tcgctcttcc ccttggcgtt gcccggaatg
      661 ctggccctcc ttggctggtg gtggtttttc tctcgtaaaa aggatcccac cagatctgcg
      721 aattcaggtg ctggtgctgg tgctggtgct atcctatcta gaatggtgag cgagctgatt
      781 aaggagaaca tgcacatgaa gctgtacatg gagggcaccg tggacaacca tcacttcaag
      841 tgcacatccg agggcgaagg caagccctac gagggcaccc agaccatgag aatcaaggtg
      901 gtcgagggcg gccctctccc cttcgccttc gacatcctgg ctactagctt cctctacggc
      961 agcaagacct tcatcaacca cacccagggc atccccgact tcttcaagca gtccttccct
     1021 gagggcttca catgggagag agtcaccaca tacgaagacg ggggcgtgct gaccgctacc
     1081 caggacacca gcctccagga cggctgcctc atctacaacg tcaagatcag aggggtgaac
     1141 ttcacatcca acggccctgt gatgcagaag aaaacactcg gctgggaggc cttcaccgag
     1201 acgctgtacc ccgctgacgg cggcctggaa ggcagaaacg acatggccct gaagctcgtg
     1261 ggcgggagcc atctgatcgc aaacgccaag accacatata gatccaagaa acccgctaag
     1321 aacctcaaga tgcctggcgt ctactatgtg gactacagac tggaaagaat caaggaggcc
     1381 aacaacgaga cctacgtcga gcagcacgag gtggcagtgg ccagatactg cgacctccct
     1441 agcaaactgg ggcacaagct taattccgga ctcagatctc gaagcgcggc cgcgggagca
     1501 ggaggagcag ctcgagcggc gctgggagtg caggtggaaa ccatctcccc aggagacggg
     1561 cgcaccttcc ccaagcgcgg ccagacctgc gtggtgcact acaccgggat gcttgaagat
     1621 ggaaagaaat ttgattcctc ccgggacaga aacaagccct ttaagtttat gctaggcaag
     1681 caggaggtga tccgaggctg ggaagaaggg gttgcccaga tgagtgtggg tcagagagcc
     1741 aaactgacta tatctccaga ttatgcctat ggtgccactg ggcacccagg catcatccca
     1801 ccacatgcca ctctcgtctt cgatgtggag cttctaaaac tggaaagtgc tggtggtagt
     1861 gctggtggta gtgctggtgg tagtgctggt ggtagtgctg gtggtccgcg agctcaagct
     1921 tcgtcgctta ataactggaa taagccaata aagaactctg taactacaaa acaatggatg
     1981 tccgctctac ctgacacaac aaacctagca gcgctctcta taccaggtac acatgatacg
     2041 atgagctata acggagacat aacatggaca ttaaccaaac cactagctca aacacaaacg
     2101 atgtcattgt accaacaact agaagcagga atacggtaca tcgatattag agcaaaagac
     2161 aatctcaaca tttaccatgg gccaattttt ttaaatgcat cactttcagg tgtattagaa
     2221 acgattactc aatttttaaa gaaaaatcca aaagaaacca ttattatgcg tttaaaggac
     2281 gagcaaaaca gcaacgatag ttttgattat cggatccaac cactaatcaa catttataaa
     2341 gattattttt acactactcc cagaactgac acgagcaata aaatccctac attaaaagat
     2401 gtccgcggaa aaatattatt actttcagag aaccacacaa aaaagccatt agtcattaac
     2461 tcacgcaaat tcggcatgca gttctagaat tctgcagtcg acggtaccgc gggcccggga
     2521 tccaccggat ctagataact gatcataatc agccatacca catttgtaga ggttttactt
     2581 gctttaaaaa acctcccaca cctccccctg aacctgaaac ataaaatgaa tgcaattgtt
     2641 gttgttaact tgtttattgc agcttataat ggttacaaat aaagcaatag catcacaaat
     2701 ttcacaaata aagcattttt ttcactgcat tctagttgtg gtttgtccaa actcatcaat
     2761 gtatcttaac gcgtaaattg taagcgttaa tattttgtta aaattcgcgt taaatttttg
     2821 ttaaatcagc tcatttttta accaataggc cgaaatcggc aaaatccctt ataaatcaaa
     2881 agaatagacc gagatagggt tgagtgttgt tccagtttgg aacaagagtc cactattaaa
     2941 gaacgtggac tccaacgtca aagggcgaaa aaccgtctat cagggcgatg gcccactacg
     3001 tgaaccatca ccctaatcaa gttttttggg gtcgaggtgc cgtaaagcac taaatcggaa
     3061 ccctaaaggg agcccccgat ttagagcttg acggggaaag ccggcgaacg tggcgagaaa
     3121 ggaagggaag aaagcgaaag gagcgggcgc tagggcgctg gcaagtgtag cggtcacgct
     3181 gcgcgtaacc accacacccg ccgcgcttaa tgcgccgcta cagggcgcgt caggtggcac
     3241 ttttcgggga aatgtgcgcg gaacccctat ttgtttattt ttctaaatac attcaaatat
     3301 gtatccgctc atgagacaat aaccctgata aatgcttcaa taatattgaa aaaggaagag
     3361 tcctgaggcg gaaagaacca gctgtggaat gtgtgtcagt tagggtgtgg aaagtcccca
     3421 ggctccccag caggcagaag tatgcaaagc atgcatctca attagtcagc aaccaggtgt
     3481 ggaaagtccc caggctcccc agcaggcaga agtatgcaaa gcatgcatct caattagtca
     3541 gcaaccatag tcccgcccct aactccgccc atcccgcccc taactccgcc cagttccgcc
     3601 cattctccgc cccatggctg actaattttt tttatttatg cagaggccga ggccgcctcg
     3661 gcctctgagc tattccagaa gtagtgagga ggcttttttg gaggcctagg cttttgcaaa
     3721 gatcgatcaa gagacaggat gaggatcgtt tcgcatgatt gaacaagatg gattgcacgc
     3781 aggttctccg gccgcttggg tggagaggct attcggctat gactgggcac aacagacaat
     3841 cggctgctct gatgccgccg tgttccggct gtcagcgcag gggcgcccgg ttctttttgt
     3901 caagaccgac ctgtccggtg ccctgaatga actgcaagac gaggcagcgc ggctatcgtg
     3961 gctggccacg acgggcgttc cttgcgcagc tgtgctcgac gttgtcactg aagcgggaag
     4021 ggactggctg ctattgggcg aagtgccggg gcaggatctc ctgtcatctc accttgctcc
     4081 tgccgagaaa gtatccatca tggctgatgc aatgcggcgg ctgcatacgc ttgatccggc
     4141 tacctgccca ttcgaccacc aagcgaaaca tcgcatcgag cgagcacgta ctcggatgga
     4201 agccggtctt gtcgatcagg atgatctgga cgaagagcat caggggctcg cgccagccga
     4261 actgttcgcc aggctcaagg cgagcatgcc cgacggcgag gatctcgtcg tgacccatgg
     4321 cgatgcctgc ttgccgaata tcatggtgga aaatggccgc ttttctggat tcatcgactg
     4381 tggccggctg ggtgtggcgg accgctatca ggacatagcg ttggctaccc gtgatattgc
     4441 tgaagagctt ggcggcgaat gggctgaccg cttcctcgtg ctttacggta tcgccgctcc
     4501 cgattcgcag cgcatcgcct tctatcgcct tcttgacgag ttcttctgag cgggactctg
     4561 gggttcgaaa tgaccgacca agcgacgccc aacctgccat cacgagattt cgattccacc
     4621 gccgccttct atgaaaggtt gggcttcgga atcgttttcc gggacgccgg ctggatgatc
     4681 ctccagcgcg gggatctcat gctggagttc ttcgcccacc ctagggggag gctaactgaa
     4741 acacggaagg agacaatacc ggaaggaacc cgcgctatga cggcaataaa aagacagaat
     4801 aaaacgcacg gtgttgggtc gtttgttcat aaacgcgggg ttcggtccca gggctggcac
     4861 tctgtcgata ccccaccgag accccattgg ggccaatacg cccgcgtttc ttccttttcc
     4921 ccaccccacc ccccaagttc gggtgaaggc ccagggctcg cagccaacgt cggggcggca
     4981 ggccctgcca tagcctcagg ttactcatat atactttaga ttgatttaaa acttcatttt
     5041 taatttaaaa ggatctaggt gaagatcctt tttgataatc tcatgaccaa aatcccttaa
     5101 cgtgagtttt cgttccactg agcgtcagac cccgtagaaa agatcaaagg atcttcttga
     5161 gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg
     5221 gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac tggcttcagc
     5281 agagcgcaga taccaaatac tgtccttcta gtgtagccgt agttaggcca ccacttcaag
     5341 aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt ggctgctgcc
     5401 agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttacc ggataaggcg
     5461 cagcggtcgg gctgaacggg gggttcgtgc acacagccca gcttggagcg aacgacctac
     5521 accgaactga gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga
     5581 aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcac gagggagctt
     5641 ccagggggaa acgcctggta tctttatagt cctgtcgggt ttcgccacct ctgacttgag
     5701 cgtcgatttt tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg
     5761 gcctttttac ggttcctggc cttttgctgg ccttttgctc acatgttctt tcctgcgtta
     5821 tcccctgatt ctgtggataa ccgtattacc gccatgcat
//
