LOCUS       sequence-399222-        6053 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  weakly plasma membrane targeted fluorescent protein-tagged enzyme.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6053)
  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 6053)
  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-Lyn11C32S-PIP4K2A
FEATURES             Location/Qualifiers
     source          1..6053
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     44..63
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     promoter        complement(152..509)
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      166..301
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     enhancer        318..509
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     466..486
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        complement(511..615)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      642..1097
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(729..748)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     939..960
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     polyA_signal    1104..1225
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1115..1134)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     1169..1188
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     CDS             complement(join(2621..3331,3332..3334,3335..3337))
                     /codon_start=1
                     /product="enhanced GFP with monomerizing A206K mutation 
                     (Zacharias et al., 2002)"
                     /label=mEGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSKLSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(2663..2684)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     3012..3031
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     3271..3292
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     regulatory      3334..3343
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     promoter        complement(3498..3701)
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     complement(3527..3547)
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     enhancer        3702..4005
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     rep_origin      complement(4180..4768)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4260..4279)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     polyA_signal    5097..5144
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     primer_bind     5169..5188
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     CDS             complement(join(5376..6053,1..117))
                     /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             complement(join(5376..6053,1..117))
                     /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(5487..5506)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
ORIGIN
        1 cagccggaac acggcggcat cagagcagcc gattgtctgt tgtgcccagt catagccgaa
       61 tagcctctcc acccaagcgg ccggagaacc tgcgtgcaat ccatcttgtt caatcatgcg
      121 aaacgatcct catcctgtct cttgatcgat ctttgcaaaa gcctaggcct ccaaaaaagc
      181 ctcctcacta cttctggaat agctcagagg ccgaggcggc ctcggcctct gcataaataa
      241 aaaaaattag tcagccatgg ggcggagaat gggcggaact gggcggagtt aggggcggga
      301 tgggcggagt taggggcggg actatggttg ctgactaatt gagatgcatg ctttgcatac
      361 ttctgcctgc tggggagcct ggggactttc cacacctggt tgctgactaa ttgagatgca
      421 tgctttgcat acttctgcct gctggggagc ctggggactt tccacaccct aactgacaca
      481 cattccacag ctggttcttt ccgcctcagg actcttcctt tttcaatatt attgaagcat
      541 ttatcagggt tattgtctca tgagcggata catatttgaa tgtatttaga aaaataaaca
      601 aataggggtt ccgcgcacat ttccccgaaa agtgccacct gacgcgccct gtagcggcgc
      661 attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg ccagcgccct
      721 agcgcccgct cctttcgctt tcttcccttc ctttctcgcc acgttcgccg gctttccccg
      781 tcaagctcta aatcgggggc tccctttagg gttccgattt agtgctttac ggcacctcga
      841 ccccaaaaaa cttgattagg gtgatggttc acgtagtggg ccatcgccct gatagacggt
      901 ttttcgccct ttgacgttgg agtccacgtt ctttaatagt ggactcttgt tccaaactgg
      961 aacaacactc aaccctatct cggtctattc ttttgattta taagggattt tgccgatttc
     1021 ggcctattgg ttaaaaaatg agctgattta acaaaaattt aacgcgaatt ttaacaaaat
     1081 attaacgctt acaatttacg cgttaagata cattgatgag tttggacaaa ccacaactag
     1141 aatgcagtga aaaaaatgct ttatttgtga aatttgtgat gctattgctt tatttgtaac
     1201 cattataagc tgcaataaac aagttaacaa caacaattgc attcatttta tgtttcaggt
     1261 tcagggggag gtgtgggagg ttttttaaag caagtaaaac ctctacaaat gtggtatggc
     1321 tgattatgat cagttatcta gatccggtgg atccttacgt caagatgtgg ccaataaagt
     1381 ccaaaaagcg ctttgaatac tgttctgggt tcacggtgga gatctccgcg ccagcgccat
     1441 gtttaacagt ttttgcagca tgggcagctt tcttttttgc atcataatga gtaaggatgt
     1501 caataattgc catgaagtac acctccttcc taggcgagtt ttcatggcac ttaattccat
     1561 agacgtcgat gttcggatcg aactccccgg gagccagggg tggtgagctg ttcagtgtat
     1621 tcccggggct atctgggggg gttcccaccg ggtgggtgcc atcgctctcg ccctcctcct
     1681 ccccatcgtt ctcctcacac tccacttcct cctgttcggc tctctccaca tcatgaattc
     1741 ccaccagcag actgtagtcc atgagcttca gctgggccag aaactcaaca tcctttttta
     1801 gtttttccag gaagaccttc ttgctgttgt catcaatata aatcttttgg ccctcattaa
     1861 tgaaatcatt atctttcaga gttggcagtt ctttggcctt ttctttgtca ctagcttctc
     1921 tagccactgt agagcccttt aagtcgtatt tcctatacac agacaaacgg tggctgaata
     1981 catttcttgt aactatcaca tatatttcaa ctccatcaac attaagccgg tacatgccca
     2041 agaactgggg aagaagggtg atcccatgac attccactat gtactggtgg tatttcttca
     2101 ggatgttgtg catttcggcc acgtcttcac tggtaatagt cttgatgatg tatcttttgt
     2161 cgtaggaagt gtgaaaacga gctccactgc gggcctggga gtcgttgggg aggggtgcgc
     2221 tcctggtcag ggaattctgg aaatcttgat catcaattcc aaacctctcc cgcaggttac
     2281 ggaagaccat cgggcagtat tccttaaact tgaaatggct cggcatgttt tctttgttaa
     2341 aaaggtgatt gtccaccttt atttttgaat aggctttgaa gtcatctggc atcaacataa
     2401 cagggatttg aacatggctc agttcattga tcgagtggtt taccccccac atgaggacgc
     2461 tgagcagcgg gtcgctggcc cgaaacagct tcactttctg cgctacgaag tgcttcttct
     2521 tggtcttggt cttgctcgcc aggacggagg accctaggtt gccgggggtc gccatgtcga
     2581 ctgcagaatt cgaagcttga gctcgagatc tgagtccgga cttgtacagc tcgtccatgc
     2641 cgagagtgat cccggcggcg gtcacgaact ccagcaggac catgtgatcg cgcttctcgt
     2701 tggggtcttt gctcagtttg gactgggtgc tcaggtagtg gttgtcgggc agcagcacgg
     2761 ggccgtcgcc gatgggggtg ttctgctggt agtggtcggc gagctgcacg ctgccgtcct
     2821 cgatgttgtg gcggatcttg aagttcacct tgatgccgtt cttctgcttg tcggccatga
     2881 tatagacgtt gtggctgttg tagttgtact ccagcttgtg ccccaggatg ttgccgtcct
     2941 ccttgaagtc gatgcccttc agctcgatgc ggttcaccag ggtgtcgccc tcgaacttca
     3001 cctcggcgcg ggtcttgtag ttgccgtcgt ccttgaagaa gatggtgcgc tcctggacgt
     3061 agccttcggg catggcggac ttgaagaagt cgtgctgctt catgtggtcg gggtagcggc
     3121 tgaagcactg cacgccgtag gtcagggtgg tcacgagggt gggccagggc acgggcagct
     3181 tgccggtggt gcagatgaac ttcagggtca gcttgccgta ggtggcatcg ccctcgccct
     3241 cgccggacac gctgaacttg tggccgttta cgtcgccgtc cagctcgacc aggatgggca
     3301 ccaccccggt gaacagctcc tcgcccttgc tcaccatggc ggcgaggcct ccggaacccc
     3361 ctgatccgcc tcttgcggca gcttctctag cagcggcttc ccctccagag ccaccgctgt
     3421 ctttcccttt tgattttata gatcccatgg tagatctgag tccggtagcg ctagcggatc
     3481 tgacggttca ctaaaccagc tctgcttata tagacctccc accgtacacg cctaccgccc
     3541 atttgcgtca atggggcgga gttgttacga cattttggaa agtcccgttg attttggtgc
     3601 caaaacaaac tcccattgac gtcaatgggg tggagacttg gaaatccccg tgagtcaaac
     3661 cgctatccac gcccattgat gtactgccaa aaccgcatca ccatggtaat agcgatgact
     3721 aatacgtaga tgtactgcca agtaggaaag tcccataagg tcatgtactg ggcataatgc
     3781 caggcgggcc atttaccgtc attgacgtca atagggggcg tacttggcat atgatacact
     3841 tgatgtactg ccaagtgggc agtttaccgt aaatactcca cccattgacg tcaatggaaa
     3901 gtccctattg gcgttactat gggaacatac gtcattattg acgtcaatgg gcgggggtcg
     3961 ttgggcggtc agccaggcgg gccatttacc gtaagttatg taacgcggaa ctccatatat
     4021 gggctatgaa ctaatgaccc cgtaattgat tactattaat aactaatgca tggcggtaat
     4081 acggttatcc acagaatcag gggataacgc aggaaagaac atgtgagcaa aaggccagca
     4141 aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc tccgcccccc
     4201 tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga caggactata
     4261 aagataccag gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc
     4321 gcttaccgga tacctgtccg cctttctccc ttcgggaagc gtggcgcttt ctcatagctc
     4381 acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga
     4441 accccccgtt cagcccgacc gctgcgcctt atccggtaac tatcgtcttg agtccaaccc
     4501 ggtaagacac gacttatcgc cactggcagc agccactggt aacaggatta gcagagcgag
     4561 gtatgtaggc ggtgctacag agttcttgaa gtggtggcct aactacggct acactagaag
     4621 aacagtattt ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa gagttggtag
     4681 ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt gcaagcagca
     4741 gattacgcgc agaaaaaaag gatctcaaga agatcctttg atcttttcta cggggtctga
     4801 cgctcagtgg aacgaaaact cacgttaagg gattttggtc atgagattat caaaaaggat
     4861 cttcacctag atccttttaa attaaaaatg aagttttaaa tcaatctaaa gtatatatga
     4921 gtaacctgag gctatggcag ggcctgccgc cccgacgttg gctgcgagcc ctgggccttc
     4981 acccgaactt ggggggtggg gtggggaaaa ggaagaaacg cgggcgtatt ggccccaatg
     5041 gggtctcggt ggggtatcga cagagtgcca gccctgggac cgaaccccgc gtttatgaac
     5101 aaacgaccca acaccgtgcg ttttattctg tctttttatt gccgtcatag cgcgggttcc
     5161 ttccggtatt gtctccttcc gtgtttcagt tagcctcccc ctagggtggg cgaagaactc
     5221 cagcatgaga tccccgcgct ggaggatcat ccagccggcg tcccggaaaa cgattccgaa
     5281 gcccaacctt tcatagaagg cggcggtgga atcgaaatct cgtgatggca ggttgggcgt
     5341 cgcttggtcg gtcatttcga accccagagt cccgctcaga agaactcgtc aagaaggcga
     5401 tagaaggcga tgcgctgcga atcgggagcg gcgataccgt aaagcacgag gaagcggtca
     5461 gcccattcgc cgccaagctc ttcagcaata tcacgggtag ccaacgctat gtcctgatag
     5521 cggtccgcca cacccagccg gccacagtcg atgaatccag aaaagcggcc attttccacc
     5581 atgatattcg gcaagcaggc atcgccatgg gtcacgacga gatcctcgcc gtcgggcatg
     5641 ctcgccttga gcctggcgaa cagttcggct ggcgcgagcc cctgatgctc ttcgtccaga
     5701 tcatcctgat cgacaagacc ggcttccatc cgagtacgtg ctcgctcgat gcgatgtttc
     5761 gcttggtggt cgaatgggca ggtagccgga tcaagcgtat gcagccgccg cattgcatca
     5821 gccatgatgg atactttctc ggcaggagca aggtgagatg acaggagatc ctgccccggc
     5881 acttcgccca atagcagcca gtcccttccc gcttcagtga caacgtcgag cacagctgcg
     5941 caaggaacgc ccgtcgtggc cagccacgat agccgcgctg cctcgtcttg cagttcattc
     6001 agggcaccgg acaggtcggt cttgacaaaa agaaccgggc gcccctgcgc tga
//
