LOCUS       sequence-224727-        5766 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  PI(3,4)P2 biosensor.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5766)
  AUTHORS   Goulden BD, Pacheco J, Dull A, Zewe JP, Deiters A, Hammond GRV
  TITLE     A high-avidity biosensor reveals plasma membrane PI(3,4)P2 is 
            predominantly a class I PI3K signaling product.
  JOURNAL   J Cell Biol. 2019 Mar 4;218(3):1066-1079. doi: 
            10.1083/jcb.201809026. Epub 2018 Dec 27.
  PUBMED    30591513
REFERENCE   2  (bases 1 to 5766)
  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"; doi: 
            "10.1083/jcb.201809026"; journalName: "J Cell Biol"; date: 
            "2019-03-4- 4"; volume: "218"; issue: "3"; pages: "1066-1079"
COMMENT     Sequence Label: NES-EGFP-cPHx2
FEATURES             Location/Qualifiers
     source          1..5766
                     /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      595..604
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     regulatory      652..661
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(658..660,661..663,664..1374)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(658..660,661..663,664..1374)
                     /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(703..724)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(964..983)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1311..1332
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    2554..2675
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2591..2610)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2645..2664
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(2682..3137)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2819..2840)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3031..3050
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        3164..3268
                     /gene="bla"
                     /label=AmpR promoter
     promoter        3270..3627
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        3270..3461
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(3293..3313)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      3478..3613
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             3662..4456
                     /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             3662..4456
                     /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(3716..3735)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4326..4345
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(4644..4663)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    4688..4735
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      5064..5652
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5553..5572
                     /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 gctagccacc
      601 atggctctgc agaaaaagtt ggaagagctt gagctggatg aggcaccggt cgccaccatg
      661 gtgagcaagg gcgaggagct gttcaccggg gtggtgccca tcctggtcga gctggacggc
      721 gacgtaaacg gccacaagtt cagcgtgtcc ggcgagggcg agggcgatgc cacctacggc
      781 aagctgaccc tgaagttcat ctgcaccacc ggcaagctgc ccgtgccctg gcccaccctc
      841 gtgaccaccc tgacctacgg cgtgcagtgc ttcagccgct accccgacca catgaagcag
      901 cacgacttct tcaagtccgc catgcccgaa ggctacgtcc aggagcgcac catcttcttc
      961 aaggacgacg gcaactacaa gacccgcgcc gaggtgaagt tcgagggcga caccctggtg
     1021 aaccgcatcg agctgaaggg catcgacttc aaggaggacg gcaacatcct ggggcacaag
     1081 ctggagtaca actacaacag ccacaacgtc tatatcatgg ccgacaagca gaagaacggc
     1141 atcaaggtga acttcaagat ccgccacaac atcgaggacg gcagcgtgca gctcgccgac
     1201 cactaccagc agaacacccc catcggcgac ggccccgtgc tgctgcccga caaccactac
     1261 ctgagcaccc agtccgccct gagcaaagac cccaacgaga agcgcgatca catggtcctg
     1321 ctggagttcg tgaccgccgc cgggatcact ctcggcatgg acgagctgta caagtccgga
     1381 ctcagatctc gagctcaagc ttcgaattct agaaataatc tgaaacggtc acaaagccat
     1441 cttccttact ttactcctaa accacctcaa gatagtgcgg ttatcaaagc tggatattgt
     1501 gtaaaacaag gagcagtgat gaaaaactgg aagagaagat attttcaatt ggatgaaaac
     1561 acaataggct acttcaaatc tgaactggaa aaggaacctc ttcgtgtaat accacttaaa
     1621 gaggttcata aagtccagga atgtaagcaa agcgacataa tgatgaggga caacctcttt
     1681 gaaattgtaa caacgtctcg aactttctat gtgcaggctg atagccctga agagatgcac
     1741 agttggatta aagcagtctc tggcgccatt gtagcacagc ggggtcccgg cagatctgcg
     1801 tcttctgagc atccccccgg tccttcagaa tccaaacacg ctttccgtcc taccaacgca
     1861 gccaccgcca cctcacattc cacagcctct cgcggaggct cagggggttc cggcggaaga
     1921 aataatctga aacggtcaca aagccatctt ccttacttta ctcctaaacc acctcaagat
     1981 agtgcggtta tcaaagctgg atattgtgta aaacaaggag cagtgatgaa aaactggaag
     2041 agaagatatt ttcaattgga tgaaaacaca ataggctact tcaaatctga actggaaaag
     2101 gaacctcttc gtgtaatacc acttaaagag gttcataaag tccaggaatg taagcaaagc
     2161 gacataatga tgagggacaa cctctttgaa attgtaacaa cgtctcgaac tttctatgtg
     2221 caggctgata gccctgaaga gatgcacagt tggattaaag cagtctctgg cgccattgta
     2281 gcacagcggg gtcccggcag atctgcgtct tctgagcatc cccccggtcc ttcagaatcc
     2341 aaacacgctt tccgtcctac caacgcagcc accgccacct cacattccac agcctctcgc
     2401 tgagtcgacg gtaccgcggg cccgggatcc accggatcta gataactgat cataatcagc
     2461 cataccacat ttgtagaggt tttacttgct ttaaaaaacc tcccacacct ccccctgaac
     2521 ctgaaacata aaatgaatgc aattgttgtt gttaacttgt ttattgcagc ttataatggt
     2581 tacaaataaa gcaatagcat cacaaatttc acaaataaag catttttttc actgcattct
     2641 agttgtggtt tgtccaaact catcaatgta tcttaacgcg taaattgtaa gcgttaatat
     2701 tttgttaaaa ttcgcgttaa atttttgtta aatcagctca ttttttaacc aataggccga
     2761 aatcggcaaa atcccttata aatcaaaaga atagaccgag atagggttga gtgttgttcc
     2821 agtttggaac aagagtccac tattaaagaa cgtggactcc aacgtcaaag ggcgaaaaac
     2881 cgtctatcag ggcgatggcc cactacgtga accatcaccc taatcaagtt ttttggggtc
     2941 gaggtgccgt aaagcactaa atcggaaccc taaagggagc ccccgattta gagcttgacg
     3001 gggaaagccg gcgaacgtgg cgagaaagga agggaagaaa gcgaaaggag cgggcgctag
     3061 ggcgctggca agtgtagcgg tcacgctgcg cgtaaccacc acacccgccg cgcttaatgc
     3121 gccgctacag ggcgcgtcag gtggcacttt tcggggaaat gtgcgcggaa cccctatttg
     3181 tttatttttc taaatacatt caaatatgta tccgctcatg agacaataac cctgataaat
     3241 gcttcaataa tattgaaaaa ggaagagtcc tgaggcggaa agaaccagct gtggaatgtg
     3301 tgtcagttag ggtgtggaaa gtccccaggc tccccagcag gcagaagtat gcaaagcatg
     3361 catctcaatt agtcagcaac caggtgtgga aagtccccag gctccccagc aggcagaagt
     3421 atgcaaagca tgcatctcaa ttagtcagca accatagtcc cgcccctaac tccgcccatc
     3481 ccgcccctaa ctccgcccag ttccgcccat tctccgcccc atggctgact aatttttttt
     3541 atttatgcag aggccgaggc cgcctcggcc tctgagctat tccagaagta gtgaggaggc
     3601 ttttttggag gcctaggctt ttgcaaagat cgatcaagag acaggatgag gatcgtttcg
     3661 catgattgaa caagatggat tgcacgcagg ttctccggcc gcttgggtgg agaggctatt
     3721 cggctatgac tgggcacaac agacaatcgg ctgctctgat gccgccgtgt tccggctgtc
     3781 agcgcagggg cgcccggttc tttttgtcaa gaccgacctg tccggtgccc tgaatgaact
     3841 gcaagacgag gcagcgcggc tatcgtggct ggccacgacg ggcgttcctt gcgcagctgt
     3901 gctcgacgtt gtcactgaag cgggaaggga ctggctgcta ttgggcgaag tgccggggca
     3961 ggatctcctg tcatctcacc ttgctcctgc cgagaaagta tccatcatgg ctgatgcaat
     4021 gcggcggctg catacgcttg atccggctac ctgcccattc gaccaccaag cgaaacatcg
     4081 catcgagcga gcacgtactc ggatggaagc cggtcttgtc gatcaggatg atctggacga
     4141 agagcatcag gggctcgcgc cagccgaact gttcgccagg ctcaaggcga gcatgcccga
     4201 cggcgaggat ctcgtcgtga cccatggcga tgcctgcttg ccgaatatca tggtggaaaa
     4261 tggccgcttt tctggattca tcgactgtgg ccggctgggt gtggcggacc gctatcagga
     4321 catagcgttg gctacccgtg atattgctga agagcttggc ggcgaatggg ctgaccgctt
     4381 cctcgtgctt tacggtatcg ccgctcccga ttcgcagcgc atcgccttct atcgccttct
     4441 tgacgagttc ttctgagcgg gactctgggg ttcgaaatga ccgaccaagc gacgcccaac
     4501 ctgccatcac gagatttcga ttccaccgcc gccttctatg aaaggttggg cttcggaatc
     4561 gttttccggg acgccggctg gatgatcctc cagcgcgggg atctcatgct ggagttcttc
     4621 gcccacccta gggggaggct aactgaaaca cggaaggaga caataccgga aggaacccgc
     4681 gctatgacgg caataaaaag acagaataaa acgcacggtg ttgggtcgtt tgttcataaa
     4741 cgcggggttc ggtcccaggg ctggcactct gtcgataccc caccgagacc ccattggggc
     4801 caatacgccc gcgtttcttc cttttcccca ccccaccccc caagttcggg tgaaggccca
     4861 gggctcgcag ccaacgtcgg ggcggcaggc cctgccatag cctcaggtta ctcatatata
     4921 ctttagattg atttaaaact tcatttttaa tttaaaagga tctaggtgaa gatccttttt
     4981 gataatctca tgaccaaaat cccttaacgt gagttttcgt tccactgagc gtcagacccc
     5041 gtagaaaaga tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg
     5101 caaacaaaaa aaccaccgct accagcggtg gtttgtttgc cggatcaaga gctaccaact
     5161 ctttttccga aggtaactgg cttcagcaga gcgcagatac caaatactgt tcttctagtg
     5221 tagccgtagt taggccacca cttcaagaac tctgtagcac cgcctacata cctcgctctg
     5281 ctaatcctgt taccagtggc tgctgccagt ggcgataagt cgtgtcttac cgggttggac
     5341 tcaagacgat agttaccgga taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca
     5401 cagcccagct tggagcgaac gacctacacc gaactgagat acctacagcg tgagctatga
     5461 gaaagcgcca cgcttcccga agggagaaag gcggacaggt atccggtaag cggcagggtc
     5521 ggaacaggag agcgcacgag ggagcttcca gggggaaacg cctggtatct ttatagtcct
     5581 gtcgggtttc gccacctctg acttgagcgt cgatttttgt gatgctcgtc aggggggcgg
     5641 agcctatgga aaaacgccag caacgcggcc tttttacggt tcctggcctt ttgctggcct
     5701 tttgctcaca tgttctttcc tgcgttatcc cctgattctg tggataaccg tattaccgcc
     5761 atgcat
//
