LOCUS       sequence-224723-        6291 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 6291)
  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 6291)
  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-cPHx3
FEATURES             Location/Qualifiers
     source          1..6291
                     /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    3079..3200
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3116..3135)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3170..3189
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(3207..3662)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3344..3365)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3556..3575
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        3689..3793
                     /gene="bla"
                     /label=AmpR promoter
     promoter        3795..4152
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        3795..3986
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(3818..3838)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      4003..4138
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             4187..4981
                     /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             4187..4981
                     /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(4241..4260)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4851..4870
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(5169..5188)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    5213..5260
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      5589..6177
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6078..6097
                     /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 acctggaaaa tgagctcgag aaataatctg
     1441 aaacggtcac aaagccatct tccttacttt actcctaaac cacctcaaga tagtgcggtt
     1501 atcaaagctg gatattgtgt aaaacaagga gcagtgatga aaaactggaa gagaagatat
     1561 tttcaattgg atgaaaacac aataggctac ttcaaatctg aactggaaaa ggaacctctt
     1621 cgtgtaatac cacttaaaga ggttcataaa gtccaggaat gtaagcaaag cgacataatg
     1681 atgagggaca acctctttga aattgtaaca acgtctcgaa ctttctatgt gcaggctgat
     1741 agccctgaag agatgcacag ttggattaaa gcagtctctg gcgccattgt agcacagcgg
     1801 ggtcccggca gatctgcgtc ttctgagcat ccccccggtc cttcagaatc caaacacgct
     1861 ttccgtccta ccaacgcagc caccgccacc tcacattcca cagcctctcg cggaggctca
     1921 gggggttccg gcggaagaaa taatctgaaa cggtcacaaa gccatcttcc ttactttact
     1981 cctaaaccac ctcaagatag tgcggttatc aaagctggat attgtgtaaa acaaggagca
     2041 gtgatgaaaa actggaagag aagatatttt caattggatg aaaacacaat aggctacttc
     2101 aaatctgaac tggaaaagga acctcttcgt gtaataccac ttaaagaggt tcataaagtc
     2161 caggaatgta agcaaagcga cataatgatg agggacaacc tctttgaaat tgtaacaacg
     2221 tctcgaactt tctatgtgca ggctgatagc cctgaagaga tgcacagttg gattaaagca
     2281 gtctctggcg ccattgtagc acagcggggt cccggcagat ctgcgtcttc tgagcatccc
     2341 cccggtcctt cagaatccaa acacgctttc cgtcctacca acgcagccac cgccacctca
     2401 cattccacag cctctcgcgg aggctcaggg ggttccggcg gaagaaataa tctgaaacgg
     2461 tcacaaagcc atcttcctta ctttactcct aaaccacctc aagatagtgc ggttatcaaa
     2521 gctggatatt gtgtaaaaca aggagcagtg atgaaaaact ggaagagaag atattttcaa
     2581 ttggatgaaa acacaatagg ctacttcaaa tctgaactgg aaaaggaacc tcttcgtgta
     2641 ataccactta aagaggttca taaagtccag gaatgtaagc aaagcgacat aatgatgagg
     2701 gacaacctct ttgaaattgt aacaacgtct cgaactttct atgtgcaggc tgatagccct
     2761 gaagagatgc acagttggat taaagcagtc tctggcgcca ttgtagcaca gcggggtccc
     2821 ggcagatctg cgtcttctga gcatcccccc ggtccttcag aatccaaaca cgctttccgt
     2881 cctaccaacg cagccaccgc cacctcacat tccacagcct ctcgctgagt cgacggtacc
     2941 gcgggcccgg gatccaccgg atctagataa ctgatcataa tcagccatac cacatttgta
     3001 gaggttttac ttgctttaaa aaacctccca cacctccccc tgaacctgaa acataaaatg
     3061 aatgcaattg ttgttgttaa cttgtttatt gcagcttata atggttacaa ataaagcaat
     3121 agcatcacaa atttcacaaa taaagcattt ttttcactgc attctagttg tggtttgtcc
     3181 aaactcatca atgtatctta acgcgtaaat tgtaagcgtt aatattttgt taaaattcgc
     3241 gttaaatttt tgttaaatca gctcattttt taaccaatag gccgaaatcg gcaaaatccc
     3301 ttataaatca aaagaataga ccgagatagg gttgagtgtt gttccagttt ggaacaagag
     3361 tccactatta aagaacgtgg actccaacgt caaagggcga aaaaccgtct atcagggcga
     3421 tggcccacta cgtgaaccat caccctaatc aagttttttg gggtcgaggt gccgtaaagc
     3481 actaaatcgg aaccctaaag ggagcccccg atttagagct tgacggggaa agccggcgaa
     3541 cgtggcgaga aaggaaggga agaaagcgaa aggagcgggc gctagggcgc tggcaagtgt
     3601 agcggtcacg ctgcgcgtaa ccaccacacc cgccgcgctt aatgcgccgc tacagggcgc
     3661 gtcaggtggc acttttcggg gaaatgtgcg cggaacccct atttgtttat ttttctaaat
     3721 acattcaaat atgtatccgc tcatgagaca ataaccctga taaatgcttc aataatattg
     3781 aaaaaggaag agtcctgagg cggaaagaac cagctgtgga atgtgtgtca gttagggtgt
     3841 ggaaagtccc caggctcccc agcaggcaga agtatgcaaa gcatgcatct caattagtca
     3901 gcaaccaggt gtggaaagtc cccaggctcc ccagcaggca gaagtatgca aagcatgcat
     3961 ctcaattagt cagcaaccat agtcccgccc ctaactccgc ccatcccgcc cctaactccg
     4021 cccagttccg cccattctcc gccccatggc tgactaattt tttttattta tgcagaggcc
     4081 gaggccgcct cggcctctga gctattccag aagtagtgag gaggcttttt tggaggccta
     4141 ggcttttgca aagatcgatc aagagacagg atgaggatcg tttcgcatga ttgaacaaga
     4201 tggattgcac gcaggttctc cggccgcttg ggtggagagg ctattcggct atgactgggc
     4261 acaacagaca atcggctgct ctgatgccgc cgtgttccgg ctgtcagcgc aggggcgccc
     4321 ggttcttttt gtcaagaccg acctgtccgg tgccctgaat gaactgcaag acgaggcagc
     4381 gcggctatcg tggctggcca cgacgggcgt tccttgcgca gctgtgctcg acgttgtcac
     4441 tgaagcggga agggactggc tgctattggg cgaagtgccg gggcaggatc tcctgtcatc
     4501 tcaccttgct cctgccgaga aagtatccat catggctgat gcaatgcggc ggctgcatac
     4561 gcttgatccg gctacctgcc cattcgacca ccaagcgaaa catcgcatcg agcgagcacg
     4621 tactcggatg gaagccggtc ttgtcgatca ggatgatctg gacgaagagc atcaggggct
     4681 cgcgccagcc gaactgttcg ccaggctcaa ggcgagcatg cccgacggcg aggatctcgt
     4741 cgtgacccat ggcgatgcct gcttgccgaa tatcatggtg gaaaatggcc gcttttctgg
     4801 attcatcgac tgtggccggc tgggtgtggc ggaccgctat caggacatag cgttggctac
     4861 ccgtgatatt gctgaagagc ttggcggcga atgggctgac cgcttcctcg tgctttacgg
     4921 tatcgccgct cccgattcgc agcgcatcgc cttctatcgc cttcttgacg agttcttctg
     4981 agcgggactc tggggttcga aatgaccgac caagcgacgc ccaacctgcc atcacgagat
     5041 ttcgattcca ccgccgcctt ctatgaaagg ttgggcttcg gaatcgtttt ccgggacgcc
     5101 ggctggatga tcctccagcg cggggatctc atgctggagt tcttcgccca ccctaggggg
     5161 aggctaactg aaacacggaa ggagacaata ccggaaggaa cccgcgctat gacggcaata
     5221 aaaagacaga ataaaacgca cggtgttggg tcgtttgttc ataaacgcgg ggttcggtcc
     5281 cagggctggc actctgtcga taccccaccg agaccccatt ggggccaata cgcccgcgtt
     5341 tcttcctttt ccccacccca ccccccaagt tcgggtgaag gcccagggct cgcagccaac
     5401 gtcggggcgg caggccctgc catagcctca ggttactcat atatacttta gattgattta
     5461 aaacttcatt tttaatttaa aaggatctag gtgaagatcc tttttgataa tctcatgacc
     5521 aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag accccgtaga aaagatcaaa
     5581 ggatcttctt gagatccttt ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca
     5641 ccgctaccag cggtggtttg tttgccggat caagagctac caactctttt tccgaaggta
     5701 actggcttca gcagagcgca gataccaaat actgttcttc tagtgtagcc gtagttaggc
     5761 caccacttca agaactctgt agcaccgcct acatacctcg ctctgctaat cctgttacca
     5821 gtggctgctg ccagtggcga taagtcgtgt cttaccgggt tggactcaag acgatagtta
     5881 ccggataagg cgcagcggtc gggctgaacg gggggttcgt gcacacagcc cagcttggag
     5941 cgaacgacct acaccgaact gagataccta cagcgtgagc tatgagaaag cgccacgctt
     6001 cccgaaggga gaaaggcgga caggtatccg gtaagcggca gggtcggaac aggagagcgc
     6061 acgagggagc ttccaggggg aaacgcctgg tatctttata gtcctgtcgg gtttcgccac
     6121 ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac
     6181 gccagcaacg cggccttttt acggttcctg gccttttgct ggccttttgc tcacatgttc
     6241 tttcctgcgt tatcccctga ttctgtggat aaccgtatta ccgccatgca t
//
