LOCUS       sequence-399472-        6144 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  express plasma membrane targeted fluorescent protein-tagged enzyme.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6144)
  AUTHORS   Wills RC, Doyle CP, Zewe JP, Pacheco J, Hansen SD, Hammond GRV
  TITLE     A novel homeostatic mechanism tunes PI(4,5)P2-dependent signaling at
            the plasma membrane.
  JOURNAL   J Cell Sci. 2023 Aug 3:jcs.261494. doi: 10.1242/jcs.261494.
  PUBMED    37534432
REFERENCE   2  (bases 1 to 6144)
  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 Sci.
            2023 Aug 3:jcs.261494. doi: 10.1242/jcs.261494."
COMMENT     Sequence Label: EGFP-INPP5E-CAAX
FEATURES             Location/Qualifiers
     source          1..6144
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             80..874
                     /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             80..874
                     /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(134..153)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     744..763
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(1062..1081)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    1106..1153
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      1482..2070
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1971..1990
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     enhancer        2245..2548
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        2549..2752
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     2703..2723
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      2791..2800
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(2797..2799,2800..2802,2803..3513)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(2797..2799,2800..2802,2803..3513)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(2842..2863)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(3103..3122)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     3450..3471
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    5116..5237
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5153..5172)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5207..5226
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(5244..5699)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5381..5402)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5593..5612
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        5726..5830
                     /gene="bla"
                     /label=AmpR promoter
     promoter        join(5832..6144,1..45)
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        5832..6023
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(5855..5875)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      join(6040..6144,1..31)
                     /label=SV40 ori
                     /note="SV40 origin of replication"
ORIGIN
        1 cagaagtagt gaggaggctt ttttggaggc ctaggctttt gcaaagatcg atcaagagac
       61 aggatgagga tcgtttcgca tgattgaaca agatggattg cacgcaggtt ctccggccgc
      121 ttgggtggag aggctattcg gctatgactg ggcacaacag acaatcggct gctctgatgc
      181 cgccgtgttc cggctgtcag cgcaggggcg cccggttctt tttgtcaaga ccgacctgtc
      241 cggtgccctg aatgaactgc aagacgaggc agcgcggcta tcgtggctgg ccacgacggg
      301 cgttccttgc gcagctgtgc tcgacgttgt cactgaagcg ggaagggact ggctgctatt
      361 gggcgaagtg ccggggcagg atctcctgtc atctcacctt gctcctgccg agaaagtatc
      421 catcatggct gatgcaatgc ggcggctgca tacgcttgat ccggctacct gcccattcga
      481 ccaccaagcg aaacatcgca tcgagcgagc acgtactcgg atggaagccg gtcttgtcga
      541 tcaggatgat ctggacgaag agcatcaggg gctcgcgcca gccgaactgt tcgccaggct
      601 caaggcgagc atgcccgacg gcgaggatct cgtcgtgacc catggcgatg cctgcttgcc
      661 gaatatcatg gtggaaaatg gccgcttttc tggattcatc gactgtggcc ggctgggtgt
      721 ggcggaccgc tatcaggaca tagcgttggc tacccgtgat attgctgaag agcttggcgg
      781 cgaatgggct gaccgcttcc tcgtgcttta cggtatcgcc gctcccgatt cgcagcgcat
      841 cgccttctat cgccttcttg acgagttctt ctgagcggga ctctggggtt cgaaatgacc
      901 gaccaagcga cgcccaacct gccatcacga gatttcgatt ccaccgccgc cttctatgaa
      961 aggttgggct tcggaatcgt tttccgggac gccggctgga tgatcctcca gcgcggggat
     1021 ctcatgctgg agttcttcgc ccaccctagg gggaggctaa ctgaaacacg gaaggagaca
     1081 ataccggaag gaacccgcgc tatgacggca ataaaaagac agaataaaac gcacggtgtt
     1141 gggtcgtttg ttcataaacg cggggttcgg tcccagggct ggcactctgt cgatacccca
     1201 ccgagacccc attggggcca atacgcccgc gtttcttcct tttccccacc ccacccccca
     1261 agttcgggtg aaggcccagg gctcgcagcc aacgtcgggg cggcaggccc tgccatagcc
     1321 tcaggttact catatatact ttagattgat ttaaaacttc atttttaatt taaaaggatc
     1381 taggtgaaga tcctttttga taatctcatg accaaaatcc cttaacgtga gttttcgttc
     1441 cactgagcgt cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg
     1501 cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg
     1561 gatcaagagc taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca
     1621 aatactgttc ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg
     1681 cctacatacc tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg
     1741 tgtcttaccg ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga
     1801 acggggggtt cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac
     1861 ctacagcgtg agctatgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat
     1921 ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc
     1981 tggtatcttt atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga
     2041 tgctcgtcag gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc
     2101 ctggcctttt gctggccttt tgctcacatg ttctttcctg cgttatcccc tgattctgtg
     2161 gataaccgta ttaccgccat gcattagtta ttaatagtaa tcaattacgg ggtcattagt
     2221 tcatagccca tatatggagt tccgcgttac ataacttacg gtaaatggcc cgcctggctg
     2281 accgcccaac gacccccgcc cattgacgtc aataatgacg tatgttccca tagtaacgcc
     2341 aatagggact ttccattgac gtcaatgggt ggagtattta cggtaaactg cccacttggc
     2401 agtacatcaa gtgtatcata tgccaagtac gccccctatt gacgtcaatg acggtaaatg
     2461 gcccgcctgg cattatgccc agtacatgac cttatgggac tttcctactt ggcagtacat
     2521 ctacgtatta gtcatcgcta ttaccatggt gatgcggttt tggcagtaca tcaatgggcg
     2581 tggatagcgg tttgactcac ggggatttcc aagtctccac cccattgacg tcaatgggag
     2641 tttgttttgg caccaaaatc aacgggactt tccaaaatgt cgtaacaact ccgccccatt
     2701 gacgcaaatg ggcggtaggc gtgtacggtg ggaggtctat ataagcagag ctggtttagt
     2761 gaaccgtcag atccgctagc gctaccggtc gccaccatgg tgagcaaggg cgaggagctg
     2821 ttcaccgggg tggtgcccat cctggtcgag ctggacggcg acgtaaacgg ccacaagttc
     2881 agcgtgtccg gcgagggcga gggcgatgcc acctacggca agctgaccct gaagttcatc
     2941 tgcaccaccg gcaagctgcc cgtgccctgg cccaccctcg tgaccaccct gacctacggc
     3001 gtgcagtgct tcagccgcta ccccgaccac atgaagcagc acgacttctt caagtccgcc
     3061 atgcccgaag gctacgtcca ggagcgcacc atcttcttca aggacgacgg caactacaag
     3121 acccgcgccg aggtgaagtt cgagggcgac accctggtga accgcatcga gctgaagggc
     3181 atcgacttca aggaggacgg caacatcctg gggcacaagc tggagtacaa ctacaacagc
     3241 cacaacgtct atatcatggc cgacaagcag aagaacggca tcaaggtgaa cttcaagatc
     3301 cgccacaaca tcgaggacgg cagcgtgcag ctcgccgacc actaccagca gaacaccccc
     3361 atcggcgacg gccccgtgct gctgcccgac aaccactacc tgagcaccca gtccgccctg
     3421 agcaaagacc ccaacgagaa gcgcgatcac atggtcctgc tggagttcgt gaccgccgcc
     3481 gggatcactc tcggcatgga cgagctgtac aagtccggac tcagatctcg agctcaagct
     3541 tcgaattctt cggatcttgc agactacaag ctccgcgcgc agccgctcct ggtgcgggcc
     3601 cacagcagcc tgggccccgg ccggccgcgg agccccctgg cctgcgacga ctgttccctt
     3661 cgctcggcca aatcctcctt cagcctcctg gcgcccatcc gcagcaagga cgtccgcagc
     3721 aggagttacc tggagggcag cctcctggcc agcggggccc tgttgggggc ggatgagctg
     3781 gcccgctact tcccagaccg gaacgtggca ctcttcgtgg ccacctggaa catgcagggc
     3841 cagaaggagc tcccgcccag cctggacgag ttcctgctcc cagccgaggc cgactatgcc
     3901 caggacctgt atgtcatcgg ggtccaggag ggctgttctg acaggcggga gtgggagact
     3961 cgtctgcagg agacgctggg cccgcactat gtgctgctgt cctcggcggc ccacggcgtg
     4021 ctctacatgt cgctcttcat ccgcagggac ctcatctggt tctgctcaga ggtggagtgc
     4081 tccacggtga ccacacgcat cgtgtctcag atcaagacca agggggcctt gggcatcagc
     4141 ttcacctttt ttggcacttc cttcctcttc atcacgtccc acttcacctc aggtgacggg
     4201 aaggtggcgg agcggctgct ggactacacc aggactgtac aagccctggt cctgcccaga
     4261 aatgtgcccg acaccaaccc ctatcgctcc agcgcagcgg acgtcaccac ccgcttcgat
     4321 gaggtgttct ggtttggaga cttcaacttc cgcctgagtg gcgggcgcac agtcgtggac
     4381 gccctcctgt gccagggcct ggtggtggac gtgccggcgc tgctgcagca cgaccagctc
     4441 atccgggaga tgcggaaagg gtccatcttc aagggcttcc aggagccgga catccacttc
     4501 ctcccatcat acaagtttga catcgggaag gacacgtacg acagcacctc caagcagagg
     4561 acgccctcat acacggaccg cgtcttgtac agaagccgcc acaagggtga catctgtcct
     4621 gtgagctact cttcctgccc cgggatcaag acgtccgacc accgccctgt gtatggcctc
     4681 ttccgggtga aagtgaggcc ggggcgagac aacattccgt tggcagctgg caaatttgat
     4741 agagaactgt acttactagg aattaaaaga cggatttcga aggagattca gaggcagcaa
     4801 gcactacaga gtcagaactc cagcaccatc gcctccgttt ctggtggctc tggaggggaa
     4861 gccgctgcta gagaagctgc cgcaagaggc ggatcagggg gttccggagg cctcgccgcc
     4921 gcagtcgaca accctcctga tgagagtggc cccggctgca tgagctgcaa gtgtgtgctc
     4981 tcctgaggat ccaccggatc tagataactg atcataatca gccataccac atttgtagag
     5041 gttttacttg ctttaaaaaa cctcccacac ctccccctga acctgaaaca taaaatgaat
     5101 gcaattgttg ttgttaactt gtttattgca gcttataatg gttacaaata aagcaatagc
     5161 atcacaaatt tcacaaataa agcatttttt tcactgcatt ctagttgtgg tttgtccaaa
     5221 ctcatcaatg tatcttaacg cgtaaattgt aagcgttaat attttgttaa aattcgcgtt
     5281 aaatttttgt taaatcagct cattttttaa ccaataggcc gaaatcggca aaatccctta
     5341 taaatcaaaa gaatagaccg agatagggtt gagtgttgtt ccagtttgga acaagagtcc
     5401 actattaaag aacgtggact ccaacgtcaa agggcgaaaa accgtctatc agggcgatgg
     5461 cccactacgt gaaccatcac cctaatcaag ttttttgggg tcgaggtgcc gtaaagcact
     5521 aaatcggaac cctaaaggga gcccccgatt tagagcttga cggggaaagc cggcgaacgt
     5581 ggcgagaaag gaagggaaga aagcgaaagg agcgggcgct agggcgctgg caagtgtagc
     5641 ggtcacgctg cgcgtaacca ccacacccgc cgcgcttaat gcgccgctac agggcgcgtc
     5701 aggtggcact tttcggggaa atgtgcgcgg aacccctatt tgtttatttt tctaaataca
     5761 ttcaaatatg tatccgctca tgagacaata accctgataa atgcttcaat aatattgaaa
     5821 aaggaagagt cctgaggcgg aaagaaccag ctgtggaatg tgtgtcagtt agggtgtgga
     5881 aagtccccag gctccccagc aggcagaagt atgcaaagca tgcatctcaa ttagtcagca
     5941 accaggtgtg gaaagtcccc aggctcccca gcaggcagaa gtatgcaaag catgcatctc
     6001 aattagtcag caaccatagt cccgccccta actccgccca tcccgcccct aactccgccc
     6061 agttccgccc attctccgcc ccatggctga ctaatttttt ttatttatgc agaggccgag
     6121 gccgcctcgg cctctgagct attc
//
