LOCUS       sequence-224784-        6231 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 6231)
  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 6231)
  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-cPHx3
FEATURES             Location/Qualifiers
     source          1..6231
                     /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    3019..3140
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3056..3075)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3110..3129
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(3147..3602)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3284..3305)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3496..3515
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        3629..3733
                     /gene="bla"
                     /label=AmpR promoter
     promoter        3735..4092
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        3735..3926
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(3758..3778)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      3943..4078
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             4127..4921
                     /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             4127..4921
                     /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(4181..4200)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4791..4810
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(5109..5128)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    5153..5200
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      5529..6117
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6018..6037
                     /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 acctggaaaa tgagctcgag aaataatctg
     1381 aaacggtcac aaagccatct tccttacttt actcctaaac cacctcaaga tagtgcggtt
     1441 atcaaagctg gatattgtgt aaaacaagga gcagtgatga aaaactggaa gagaagatat
     1501 tttcaattgg atgaaaacac aataggctac ttcaaatctg aactggaaaa ggaacctctt
     1561 cgtgtaatac cacttaaaga ggttcataaa gtccaggaat gtaagcaaag cgacataatg
     1621 atgagggaca acctctttga aattgtaaca acgtctcgaa ctttctatgt gcaggctgat
     1681 agccctgaag agatgcacag ttggattaaa gcagtctctg gcgccattgt agcacagcgg
     1741 ggtcccggca gatctgcgtc ttctgagcat ccccccggtc cttcagaatc caaacacgct
     1801 ttccgtccta ccaacgcagc caccgccacc tcacattcca cagcctctcg cggaggctca
     1861 gggggttccg gcggaagaaa taatctgaaa cggtcacaaa gccatcttcc ttactttact
     1921 cctaaaccac ctcaagatag tgcggttatc aaagctggat attgtgtaaa acaaggagca
     1981 gtgatgaaaa actggaagag aagatatttt caattggatg aaaacacaat aggctacttc
     2041 aaatctgaac tggaaaagga acctcttcgt gtaataccac ttaaagaggt tcataaagtc
     2101 caggaatgta agcaaagcga cataatgatg agggacaacc tctttgaaat tgtaacaacg
     2161 tctcgaactt tctatgtgca ggctgatagc cctgaagaga tgcacagttg gattaaagca
     2221 gtctctggcg ccattgtagc acagcggggt cccggcagat ctgcgtcttc tgagcatccc
     2281 cccggtcctt cagaatccaa acacgctttc cgtcctacca acgcagccac cgccacctca
     2341 cattccacag cctctcgcgg aggctcaggg ggttccggcg gaagaaataa tctgaaacgg
     2401 tcacaaagcc atcttcctta ctttactcct aaaccacctc aagatagtgc ggttatcaaa
     2461 gctggatatt gtgtaaaaca aggagcagtg atgaaaaact ggaagagaag atattttcaa
     2521 ttggatgaaa acacaatagg ctacttcaaa tctgaactgg aaaaggaacc tcttcgtgta
     2581 ataccactta aagaggttca taaagtccag gaatgtaagc aaagcgacat aatgatgagg
     2641 gacaacctct ttgaaattgt aacaacgtct cgaactttct atgtgcaggc tgatagccct
     2701 gaagagatgc acagttggat taaagcagtc tctggcgcca ttgtagcaca gcggggtccc
     2761 ggcagatctg cgtcttctga gcatcccccc ggtccttcag aatccaaaca cgctttccgt
     2821 cctaccaacg cagccaccgc cacctcacat tccacagcct ctcgctgagt cgacggtacc
     2881 gcgggcccgg gatccaccgg atctagataa ctgatcataa tcagccatac cacatttgta
     2941 gaggttttac ttgctttaaa aaacctccca cacctccccc tgaacctgaa acataaaatg
     3001 aatgcaattg ttgttgttaa cttgtttatt gcagcttata atggttacaa ataaagcaat
     3061 agcatcacaa atttcacaaa taaagcattt ttttcactgc attctagttg tggtttgtcc
     3121 aaactcatca atgtatctta acgcgtaaat tgtaagcgtt aatattttgt taaaattcgc
     3181 gttaaatttt tgttaaatca gctcattttt taaccaatag gccgaaatcg gcaaaatccc
     3241 ttataaatca aaagaataga ccgagatagg gttgagtgtt gttccagttt ggaacaagag
     3301 tccactatta aagaacgtgg actccaacgt caaagggcga aaaaccgtct atcagggcga
     3361 tggcccacta cgtgaaccat caccctaatc aagttttttg gggtcgaggt gccgtaaagc
     3421 actaaatcgg aaccctaaag ggagcccccg atttagagct tgacggggaa agccggcgaa
     3481 cgtggcgaga aaggaaggga agaaagcgaa aggagcgggc gctagggcgc tggcaagtgt
     3541 agcggtcacg ctgcgcgtaa ccaccacacc cgccgcgctt aatgcgccgc tacagggcgc
     3601 gtcaggtggc acttttcggg gaaatgtgcg cggaacccct atttgtttat ttttctaaat
     3661 acattcaaat atgtatccgc tcatgagaca ataaccctga taaatgcttc aataatattg
     3721 aaaaaggaag agtcctgagg cggaaagaac cagctgtgga atgtgtgtca gttagggtgt
     3781 ggaaagtccc caggctcccc agcaggcaga agtatgcaaa gcatgcatct caattagtca
     3841 gcaaccaggt gtggaaagtc cccaggctcc ccagcaggca gaagtatgca aagcatgcat
     3901 ctcaattagt cagcaaccat agtcccgccc ctaactccgc ccatcccgcc cctaactccg
     3961 cccagttccg cccattctcc gccccatggc tgactaattt tttttattta tgcagaggcc
     4021 gaggccgcct cggcctctga gctattccag aagtagtgag gaggcttttt tggaggccta
     4081 ggcttttgca aagatcgatc aagagacagg atgaggatcg tttcgcatga ttgaacaaga
     4141 tggattgcac gcaggttctc cggccgcttg ggtggagagg ctattcggct atgactgggc
     4201 acaacagaca atcggctgct ctgatgccgc cgtgttccgg ctgtcagcgc aggggcgccc
     4261 ggttcttttt gtcaagaccg acctgtccgg tgccctgaat gaactgcaag acgaggcagc
     4321 gcggctatcg tggctggcca cgacgggcgt tccttgcgca gctgtgctcg acgttgtcac
     4381 tgaagcggga agggactggc tgctattggg cgaagtgccg gggcaggatc tcctgtcatc
     4441 tcaccttgct cctgccgaga aagtatccat catggctgat gcaatgcggc ggctgcatac
     4501 gcttgatccg gctacctgcc cattcgacca ccaagcgaaa catcgcatcg agcgagcacg
     4561 tactcggatg gaagccggtc ttgtcgatca ggatgatctg gacgaagagc atcaggggct
     4621 cgcgccagcc gaactgttcg ccaggctcaa ggcgagcatg cccgacggcg aggatctcgt
     4681 cgtgacccat ggcgatgcct gcttgccgaa tatcatggtg gaaaatggcc gcttttctgg
     4741 attcatcgac tgtggccggc tgggtgtggc ggaccgctat caggacatag cgttggctac
     4801 ccgtgatatt gctgaagagc ttggcggcga atgggctgac cgcttcctcg tgctttacgg
     4861 tatcgccgct cccgattcgc agcgcatcgc cttctatcgc cttcttgacg agttcttctg
     4921 agcgggactc tggggttcga aatgaccgac caagcgacgc ccaacctgcc atcacgagat
     4981 ttcgattcca ccgccgcctt ctatgaaagg ttgggcttcg gaatcgtttt ccgggacgcc
     5041 ggctggatga tcctccagcg cggggatctc atgctggagt tcttcgccca ccctaggggg
     5101 aggctaactg aaacacggaa ggagacaata ccggaaggaa cccgcgctat gacggcaata
     5161 aaaagacaga ataaaacgca cggtgttggg tcgtttgttc ataaacgcgg ggttcggtcc
     5221 cagggctggc actctgtcga taccccaccg agaccccatt ggggccaata cgcccgcgtt
     5281 tcttcctttt ccccacccca ccccccaagt tcgggtgaag gcccagggct cgcagccaac
     5341 gtcggggcgg caggccctgc catagcctca ggttactcat atatacttta gattgattta
     5401 aaacttcatt tttaatttaa aaggatctag gtgaagatcc tttttgataa tctcatgacc
     5461 aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag accccgtaga aaagatcaaa
     5521 ggatcttctt gagatccttt ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca
     5581 ccgctaccag cggtggtttg tttgccggat caagagctac caactctttt tccgaaggta
     5641 actggcttca gcagagcgca gataccaaat actgttcttc tagtgtagcc gtagttaggc
     5701 caccacttca agaactctgt agcaccgcct acatacctcg ctctgctaat cctgttacca
     5761 gtggctgctg ccagtggcga taagtcgtgt cttaccgggt tggactcaag acgatagtta
     5821 ccggataagg cgcagcggtc gggctgaacg gggggttcgt gcacacagcc cagcttggag
     5881 cgaacgacct acaccgaact gagataccta cagcgtgagc tatgagaaag cgccacgctt
     5941 cccgaaggga gaaaggcgga caggtatccg gtaagcggca gggtcggaac aggagagcgc
     6001 acgagggagc ttccaggggg aaacgcctgg tatctttata gtcctgtcgg gtttcgccac
     6061 ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac
     6121 gccagcaacg cggccttttt acggttcctg gccttttgct ggccttttgc tcacatgttc
     6181 tttcctgcgt tatcccctga ttctgtggat aaccgtatta ccgccatgca t
//
