LOCUS       sequence-224780-        5706 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 5706)
  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 5706)
  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-PAmCherry-cPHx2
FEATURES             Location/Qualifiers
     source          1..5706
                     /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      601..610
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             607..1314
                     /codon_start=1
                     /product="photoactivatable monomeric derivative of DsRed 
                     fluorescent protein (Subach et al., 2009)"
                     /label=PAmCherry1
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHVFEIEGEGEGRPYEG
                     TQTAKLKVTKGGPLPFTWDILSPQFMYGSNAYVKHPADIPDYFKLSFPEGFKWERVMKF
                     EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEALSERMYPEDGALK
                     GEVKPRVKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNRKLDITSHNEDYTIVEQYERA
                     EGRHSTGGMDELYK"
     primer_bind     complement(752..770)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     polyA_signal    2494..2615
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2531..2550)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2585..2604
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(2622..3077)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2759..2780)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2971..2990
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        3104..3208
                     /gene="bla"
                     /label=AmpR promoter
     promoter        3210..3567
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        3210..3401
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(3233..3253)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      3418..3553
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             3602..4396
                     /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             3602..4396
                     /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(3656..3675)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4266..4285
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(4584..4603)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    4628..4675
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      5004..5592
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5493..5512
                     /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 gctagcggtc
      601 gccaccatgg tgagcaaggg cgaggaggat aacatggcca tcattaagga gttcatgcgc
      661 ttcaaggtgc acatggaggg gtccgtgaac ggccacgtgt tcgagatcga gggcgagggc
      721 gagggccgcc cctacgaggg cacccagacc gccaagctga aggtgaccaa gggcggcccc
      781 ctgcccttca cctgggacat cctgagccct cagttcatgt acggctccaa tgcctacgtg
      841 aagcaccccg ccgacatccc cgactacttt aagctgtcct tccccgaggg cttcaagtgg
      901 gagcgcgtga tgaaattcga ggacggcggc gtggtgaccg tgacccagga ctcctccctg
      961 caggacggcg agttcatcta caaggtgaag ctgcgcggca ccaacttccc ctccgacggc
     1021 cccgtgatgc agaagaagac catgggctgg gaggccctct ccgagcggat gtaccccgag
     1081 gacggcgccc tgaagggcga ggtcaagccc agagtgaagc tgaaggacgg cggccactac
     1141 gacgctgagg tcaagaccac ctacaaggcc aagaagcccg tgcagctgcc cggcgcctac
     1201 aacgtcaacc gcaagctgga catcaccagc cacaacgagg actacaccat cgtggagcag
     1261 tacgagagag ccgagggccg ccactccacc ggcggcatgg acgagctgta caagtccgga
     1321 ctcagatctc gagctcaagc ttcgaattct agaaataatc tgaaacggtc acaaagccat
     1381 cttccttact ttactcctaa accacctcaa gatagtgcgg ttatcaaagc tggatattgt
     1441 gtaaaacaag gagcagtgat gaaaaactgg aagagaagat attttcaatt ggatgaaaac
     1501 acaataggct acttcaaatc tgaactggaa aaggaacctc ttcgtgtaat accacttaaa
     1561 gaggttcata aagtccagga atgtaagcaa agcgacataa tgatgaggga caacctcttt
     1621 gaaattgtaa caacgtctcg aactttctat gtgcaggctg atagccctga agagatgcac
     1681 agttggatta aagcagtctc tggcgccatt gtagcacagc ggggtcccgg cagatctgcg
     1741 tcttctgagc atccccccgg tccttcagaa tccaaacacg ctttccgtcc taccaacgca
     1801 gccaccgcca cctcacattc cacagcctct cgcggaggct cagggggttc cggcggaaga
     1861 aataatctga aacggtcaca aagccatctt ccttacttta ctcctaaacc acctcaagat
     1921 agtgcggtta tcaaagctgg atattgtgta aaacaaggag cagtgatgaa aaactggaag
     1981 agaagatatt ttcaattgga tgaaaacaca ataggctact tcaaatctga actggaaaag
     2041 gaacctcttc gtgtaatacc acttaaagag gttcataaag tccaggaatg taagcaaagc
     2101 gacataatga tgagggacaa cctctttgaa attgtaacaa cgtctcgaac tttctatgtg
     2161 caggctgata gccctgaaga gatgcacagt tggattaaag cagtctctgg cgccattgta
     2221 gcacagcggg gtcccggcag atctgcgtct tctgagcatc cccccggtcc ttcagaatcc
     2281 aaacacgctt tccgtcctac caacgcagcc accgccacct cacattccac agcctctcgc
     2341 tgagtcgacg gtaccgcggg cccgggatcc accggatcta gataactgat cataatcagc
     2401 cataccacat ttgtagaggt tttacttgct ttaaaaaacc tcccacacct ccccctgaac
     2461 ctgaaacata aaatgaatgc aattgttgtt gttaacttgt ttattgcagc ttataatggt
     2521 tacaaataaa gcaatagcat cacaaatttc acaaataaag catttttttc actgcattct
     2581 agttgtggtt tgtccaaact catcaatgta tcttaacgcg taaattgtaa gcgttaatat
     2641 tttgttaaaa ttcgcgttaa atttttgtta aatcagctca ttttttaacc aataggccga
     2701 aatcggcaaa atcccttata aatcaaaaga atagaccgag atagggttga gtgttgttcc
     2761 agtttggaac aagagtccac tattaaagaa cgtggactcc aacgtcaaag ggcgaaaaac
     2821 cgtctatcag ggcgatggcc cactacgtga accatcaccc taatcaagtt ttttggggtc
     2881 gaggtgccgt aaagcactaa atcggaaccc taaagggagc ccccgattta gagcttgacg
     2941 gggaaagccg gcgaacgtgg cgagaaagga agggaagaaa gcgaaaggag cgggcgctag
     3001 ggcgctggca agtgtagcgg tcacgctgcg cgtaaccacc acacccgccg cgcttaatgc
     3061 gccgctacag ggcgcgtcag gtggcacttt tcggggaaat gtgcgcggaa cccctatttg
     3121 tttatttttc taaatacatt caaatatgta tccgctcatg agacaataac cctgataaat
     3181 gcttcaataa tattgaaaaa ggaagagtcc tgaggcggaa agaaccagct gtggaatgtg
     3241 tgtcagttag ggtgtggaaa gtccccaggc tccccagcag gcagaagtat gcaaagcatg
     3301 catctcaatt agtcagcaac caggtgtgga aagtccccag gctccccagc aggcagaagt
     3361 atgcaaagca tgcatctcaa ttagtcagca accatagtcc cgcccctaac tccgcccatc
     3421 ccgcccctaa ctccgcccag ttccgcccat tctccgcccc atggctgact aatttttttt
     3481 atttatgcag aggccgaggc cgcctcggcc tctgagctat tccagaagta gtgaggaggc
     3541 ttttttggag gcctaggctt ttgcaaagat cgatcaagag acaggatgag gatcgtttcg
     3601 catgattgaa caagatggat tgcacgcagg ttctccggcc gcttgggtgg agaggctatt
     3661 cggctatgac tgggcacaac agacaatcgg ctgctctgat gccgccgtgt tccggctgtc
     3721 agcgcagggg cgcccggttc tttttgtcaa gaccgacctg tccggtgccc tgaatgaact
     3781 gcaagacgag gcagcgcggc tatcgtggct ggccacgacg ggcgttcctt gcgcagctgt
     3841 gctcgacgtt gtcactgaag cgggaaggga ctggctgcta ttgggcgaag tgccggggca
     3901 ggatctcctg tcatctcacc ttgctcctgc cgagaaagta tccatcatgg ctgatgcaat
     3961 gcggcggctg catacgcttg atccggctac ctgcccattc gaccaccaag cgaaacatcg
     4021 catcgagcga gcacgtactc ggatggaagc cggtcttgtc gatcaggatg atctggacga
     4081 agagcatcag gggctcgcgc cagccgaact gttcgccagg ctcaaggcga gcatgcccga
     4141 cggcgaggat ctcgtcgtga cccatggcga tgcctgcttg ccgaatatca tggtggaaaa
     4201 tggccgcttt tctggattca tcgactgtgg ccggctgggt gtggcggacc gctatcagga
     4261 catagcgttg gctacccgtg atattgctga agagcttggc ggcgaatggg ctgaccgctt
     4321 cctcgtgctt tacggtatcg ccgctcccga ttcgcagcgc atcgccttct atcgccttct
     4381 tgacgagttc ttctgagcgg gactctgggg ttcgaaatga ccgaccaagc gacgcccaac
     4441 ctgccatcac gagatttcga ttccaccgcc gccttctatg aaaggttggg cttcggaatc
     4501 gttttccggg acgccggctg gatgatcctc cagcgcgggg atctcatgct ggagttcttc
     4561 gcccacccta gggggaggct aactgaaaca cggaaggaga caataccgga aggaacccgc
     4621 gctatgacgg caataaaaag acagaataaa acgcacggtg ttgggtcgtt tgttcataaa
     4681 cgcggggttc ggtcccaggg ctggcactct gtcgataccc caccgagacc ccattggggc
     4741 caatacgccc gcgtttcttc cttttcccca ccccaccccc caagttcggg tgaaggccca
     4801 gggctcgcag ccaacgtcgg ggcggcaggc cctgccatag cctcaggtta ctcatatata
     4861 ctttagattg atttaaaact tcatttttaa tttaaaagga tctaggtgaa gatccttttt
     4921 gataatctca tgaccaaaat cccttaacgt gagttttcgt tccactgagc gtcagacccc
     4981 gtagaaaaga tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg
     5041 caaacaaaaa aaccaccgct accagcggtg gtttgtttgc cggatcaaga gctaccaact
     5101 ctttttccga aggtaactgg cttcagcaga gcgcagatac caaatactgt tcttctagtg
     5161 tagccgtagt taggccacca cttcaagaac tctgtagcac cgcctacata cctcgctctg
     5221 ctaatcctgt taccagtggc tgctgccagt ggcgataagt cgtgtcttac cgggttggac
     5281 tcaagacgat agttaccgga taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca
     5341 cagcccagct tggagcgaac gacctacacc gaactgagat acctacagcg tgagctatga
     5401 gaaagcgcca cgcttcccga agggagaaag gcggacaggt atccggtaag cggcagggtc
     5461 ggaacaggag agcgcacgag ggagcttcca gggggaaacg cctggtatct ttatagtcct
     5521 gtcgggtttc gccacctctg acttgagcgt cgatttttgt gatgctcgtc aggggggcgg
     5581 agcctatgga aaaacgccag caacgcggcc tttttacggt tcctggcctt ttgctggcct
     5641 tttgctcaca tgttctttcc tgcgttatcc cctgattctg tggataaccg tattaccgcc
     5701 atgcat
//
