LOCUS       sequence-360206-        5262 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  PtdIns(3,4,5)P3 biosensor.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5262)
  AUTHORS   Walpole GFW, Pacheco J, Chauhan N, Clark J, Anderson KE, Abbas YM, 
            Brabant-Kirwan D, Montano-Rendon F, Liu Z, Zhu H, Brumell JH, 
            Deiters A, Stephens LR, Hawkins PT, Hammond GRV, Grinstein S, Fairn 
            GD
  TITLE     Kinase-independent synthesis of 3-phosphorylated phosphoinositides 
            by a phosphotransferase.
  JOURNAL   Nat Cell Biol. 2022 Apr 28. pii: 10.1038/s41556-022-00895-y. doi: 
            10.1038/s41556-022-00895-y.
  PUBMED    35484249
REFERENCE   2  (bases 1 to 5262)
  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: "Nat Cell 
            Biol. 2022 Apr 28. pii: 10.1038/s41556-022-00895-y. doi: 
            10.1038/s41556-022-00895-y."
COMMENT     Sequence Label: NES-EGFP-PH-BTKx1
FEATURES             Location/Qualifiers
     source          1..5262
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        77..380
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        381..584
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     535..555
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      611..620
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     regulatory      668..677
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(674..676,677..679,680..1390)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(674..676,677..679,680..1390)
                     /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(719..740)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(980..999)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1327..1348
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    2066..2187
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2103..2122)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2157..2176
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(2194..2649)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2331..2352)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2543..2562
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        2676..2780
                     /gene="bla"
                     /label=AmpR promoter
     promoter        2782..3139
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        2782..2973
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(2805..2825)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      2990..3125
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             3174..3968
                     /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             3174..3968
                     /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(3228..3247)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3838..3857
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(4156..4175)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    4200..4247
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      4576..5164
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5065..5084
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 tattaccgcc atgcattagt tattaatagt aatcaattac ggggtcatta gttcatagcc
       61 catatatgga gttccgcgtt acataactta cggtaaatgg cccgcctggc tgaccgccca
      121 acgacccccg cccattgacg tcaataatga cgtatgttcc catagtaacg ccaataggga
      181 ctttccattg acgtcaatgg gtggagtatt tacggtaaac tgcccacttg gcagtacatc
      241 aagtgtatca tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct
      301 ggcattatgc ccagtacatg accttatggg actttcctac ttggcagtac atctacgtat
      361 tagtcatcgc tattaccatg gtgatgcggt tttggcagta catcaatggg cgtggatagc
      421 ggtttgactc acggggattt ccaagtctcc accccattga cgtcaatggg agtttgtttt
      481 ggcaccaaaa tcaacgggac tttccaaaat gtcgtaacaa ctccgcccca ttgacgcaaa
      541 tgggcggtag gcgtgtacgg tgggaggtct atataagcag agctggttta gtgaaccgtc
      601 agatccgcta gccaccatgg ctctgcagaa aaagttggaa gagcttgagc tggatgaggc
      661 accggtcgcc accatggtga gcaagggcga ggagctgttc accggggtgg tgcccatcct
      721 ggtcgagctg gacggcgacg taaacggcca caagttcagc gtgtccggcg agggcgaggg
      781 cgatgccacc tacggcaagc tgaccctgaa gttcatctgc accaccggca agctgcccgt
      841 gccctggccc accctcgtga ccaccctgac ctacggcgtg cagtgcttca gccgctaccc
      901 cgaccacatg aagcagcacg acttcttcaa gtccgccatg cccgaaggct acgtccagga
      961 gcgcaccatc ttcttcaagg acgacggcaa ctacaagacc cgcgccgagg tgaagttcga
     1021 gggcgacacc ctggtgaacc gcatcgagct gaagggcatc gacttcaagg aggacggcaa
     1081 catcctgggg cacaagctgg agtacaacta caacagccac aacgtctata tcatggccga
     1141 caagcagaag aacggcatca aggtgaactt caagatccgc cacaacatcg aggacggcag
     1201 cgtgcagctc gccgaccact accagcagaa cacccccatc ggcgacggcc ccgtgctgct
     1261 gcccgacaac cactacctga gcacccagtc cgccctgagc aaagacccca acgagaagcg
     1321 cgatcacatg gtcctgctgg agttcgtgac cgccgccggg atcactctcg gcatggacga
     1381 gctgtacaag tccggactca gatctcgagc tcaagcttcg aattctgccg cagtgattct
     1441 ggagagcatc tttctgaagc gatcccaaca gaaaaagaaa acatcacctc taaacttcaa
     1501 gaagcgcctg tttctcttga ccgtgcacaa actctcctac tatgagtatg actttgaacg
     1561 tgggagaaga ggcagtaaga agggttcaat agatgttgag aagatcactt gtgttgaaac
     1621 agtggttcct gaaaaaaatc ctcctccaga aagacagatt ccgagaagag gtgaagagtc
     1681 cagtgaaatg gagcaaattt caatcattga aaggttccct tatcccttcc aggttgtata
     1741 tgatgaaggg cctctctacg tcttctcccc aactgaagaa ctaaggaagc ggtggattca
     1801 ccagctcaaa aacgtaatcc ggtacaacag tgatctggtt cagaaatatc acccttgctt
     1861 ctggatcgat gggcagtatc tctgctgctc tcagacagcc aaaaatgcta tgggctgcca
     1921 aattttggag aactgaggat ccaccggatc tagataactg atcataatca gccataccac
     1981 atttgtagag gttttacttg ctttaaaaaa cctcccacac ctccccctga acctgaaaca
     2041 taaaatgaat gcaattgttg ttgttaactt gtttattgca gcttataatg gttacaaata
     2101 aagcaatagc atcacaaatt tcacaaataa agcatttttt tcactgcatt ctagttgtgg
     2161 tttgtccaaa ctcatcaatg tatcttaacg cgtaaattgt aagcgttaat attttgttaa
     2221 aattcgcgtt aaatttttgt taaatcagct cattttttaa ccaataggcc gaaatcggca
     2281 aaatccctta taaatcaaaa gaatagaccg agatagggtt gagtgttgtt ccagtttgga
     2341 acaagagtcc actattaaag aacgtggact ccaacgtcaa agggcgaaaa accgtctatc
     2401 agggcgatgg cccactacgt gaaccatcac cctaatcaag ttttttgggg tcgaggtgcc
     2461 gtaaagcact aaatcggaac cctaaaggga gcccccgatt tagagcttga cggggaaagc
     2521 cggcgaacgt ggcgagaaag gaagggaaga aagcgaaagg agcgggcgct agggcgctgg
     2581 caagtgtagc ggtcacgctg cgcgtaacca ccacacccgc cgcgcttaat gcgccgctac
     2641 agggcgcgtc aggtggcact tttcggggaa atgtgcgcgg aacccctatt tgtttatttt
     2701 tctaaataca ttcaaatatg tatccgctca tgagacaata accctgataa atgcttcaat
     2761 aatattgaaa aaggaagagt cctgaggcgg aaagaaccag ctgtggaatg tgtgtcagtt
     2821 agggtgtgga aagtccccag gctccccagc aggcagaagt atgcaaagca tgcatctcaa
     2881 ttagtcagca accaggtgtg gaaagtcccc aggctcccca gcaggcagaa gtatgcaaag
     2941 catgcatctc aattagtcag caaccatagt cccgccccta actccgccca tcccgcccct
     3001 aactccgccc agttccgccc attctccgcc ccatggctga ctaatttttt ttatttatgc
     3061 agaggccgag gccgcctcgg cctctgagct attccagaag tagtgaggag gcttttttgg
     3121 aggcctaggc ttttgcaaag atcgatcaag agacaggatg aggatcgttt cgcatgattg
     3181 aacaagatgg attgcacgca ggttctccgg ccgcttgggt ggagaggcta ttcggctatg
     3241 actgggcaca acagacaatc ggctgctctg atgccgccgt gttccggctg tcagcgcagg
     3301 ggcgcccggt tctttttgtc aagaccgacc tgtccggtgc cctgaatgaa ctgcaagacg
     3361 aggcagcgcg gctatcgtgg ctggccacga cgggcgttcc ttgcgcagct gtgctcgacg
     3421 ttgtcactga agcgggaagg gactggctgc tattgggcga agtgccgggg caggatctcc
     3481 tgtcatctca ccttgctcct gccgagaaag tatccatcat ggctgatgca atgcggcggc
     3541 tgcatacgct tgatccggct acctgcccat tcgaccacca agcgaaacat cgcatcgagc
     3601 gagcacgtac tcggatggaa gccggtcttg tcgatcagga tgatctggac gaagagcatc
     3661 aggggctcgc gccagccgaa ctgttcgcca ggctcaaggc gagcatgccc gacggcgagg
     3721 atctcgtcgt gacccatggc gatgcctgct tgccgaatat catggtggaa aatggccgct
     3781 tttctggatt catcgactgt ggccggctgg gtgtggcgga ccgctatcag gacatagcgt
     3841 tggctacccg tgatattgct gaagagcttg gcggcgaatg ggctgaccgc ttcctcgtgc
     3901 tttacggtat cgccgctccc gattcgcagc gcatcgcctt ctatcgcctt cttgacgagt
     3961 tcttctgagc gggactctgg ggttcgaaat gaccgaccaa gcgacgccca acctgccatc
     4021 acgagatttc gattccaccg ccgccttcta tgaaaggttg ggcttcggaa tcgttttccg
     4081 ggacgccggc tggatgatcc tccagcgcgg ggatctcatg ctggagttct tcgcccaccc
     4141 tagggggagg ctaactgaaa cacggaagga gacaataccg gaaggaaccc gcgctatgac
     4201 ggcaataaaa agacagaata aaacgcacgg tgttgggtcg tttgttcata aacgcggggt
     4261 tcggtcccag ggctggcact ctgtcgatac cccaccgaga ccccattggg gccaatacgc
     4321 ccgcgtttct tccttttccc caccccaccc cccaagttcg ggtgaaggcc cagggctcgc
     4381 agccaacgtc ggggcggcag gccctgccat agcctcaggt tactcatata tactttagat
     4441 tgatttaaaa cttcattttt aatttaaaag gatctaggtg aagatccttt ttgataatct
     4501 catgaccaaa atcccttaac gtgagttttc gttccactga gcgtcagacc ccgtagaaaa
     4561 gatcaaagga tcttcttgag atcctttttt tctgcgcgta atctgctgct tgcaaacaaa
     4621 aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa gagctaccaa ctctttttcc
     4681 gaaggtaact ggcttcagca gagcgcagat accaaatact gttcttctag tgtagccgta
     4741 gttaggccac cacttcaaga actctgtagc accgcctaca tacctcgctc tgctaatcct
     4801 gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt accgggttgg actcaagacg
     4861 atagttaccg gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca cacagcccag
     4921 cttggagcga acgacctaca ccgaactgag atacctacag cgtgagctat gagaaagcgc
     4981 cacgcttccc gaagggagaa aggcggacag gtatccggta agcggcaggg tcggaacagg
     5041 agagcgcacg agggagcttc cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt
     5101 tcgccacctc tgacttgagc gtcgattttt gtgatgctcg tcaggggggc ggagcctatg
     5161 gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc ttttgctggc cttttgctca
     5221 catgttcttt cctgcgttat cccctgattc tgtggataac cg
//
