LOCUS       sequence-473745-        7367 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Genetically encoded FRET-based phosphoinositide sensor for 
            monitoring PI(3,4)P2. Targeted to plasma membrane..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7367)
  AUTHORS   Sahan AZ, Chen M, Su Q, Li Q, Wang D, Zhang J
  TITLE     Lysosomal PIP(3) revealed by genetically encoded lipid biosensors.
  JOURNAL   Proc Natl Acad Sci U S A. 2025 Apr;122(13):e2426929122. doi: 
            10.1073/pnas.2426929122. Epub 2025 Mar 24.
  PUBMED    40127277
REFERENCE   2  (bases 1 to 7367)
  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: "Proc Natl 
            Acad Sci U S A."; date: "2025-04"; volume: "122(13):e2426929122. 
            doi"; pages: " 10.1073/pnas.2426929122. Epub 2025 Mar 24"
COMMENT     Sequence Label: pcDNA3-InPTapp-Kras
FEATURES             Location/Qualifiers
     source          1..7367
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(44..63)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        235..614
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        615..818
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     769..789
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     863..882
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        863..881
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      901..910
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             922..939
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             943..975
                     /codon_start=1
                     /product="leader peptide from bacteriophage T7 gene 10"
                     /label=T7 tag (gene 10 leader)
                     /note="promotes efficient translation in E. coli"
                     /translation="MASMTGGQQMG"
     CDS             join(979..987,988..1002)
                     /codon_start=1
                     /product="Xpress(TM) epitope tag, including an enterokinase
                     recognition and cleavage site"
                     /label=Xpress(TM) tag
                     /translation="DLYDDDDK"
     CDS             join(1009..1011,1012..1014,1015..1527)
                     /codon_start=1
                     /product="N-terminal fragment of mVenus for use in 
                     bimolecular fluorescence complementation (BiFC) (Kodama and
                     Hu, 2010)"
                     /label=VN173
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHRFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTWGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYISHNVYITADKQKNGIK
                     AHFKIRHNIE"
     CDS             join(2140..2142,2143..2145,2146..2856)
                     /codon_start=1
                     /product="enhanced variant of YFP (Heikal et al., 2001)"
                     /label=Citrine
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTSGKLPVPWPTLVTTFGYGLMCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSHQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     promoter        complement(2920..2938)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(2921..2938)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(2958..2975)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2964..3188
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      3234..3662
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3321..3340)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3531..3552
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3671..3691)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3676..4005
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3856..3991
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3918..3937
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             4072..4866
                     /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
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(4126..4145)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4736..4755
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    5040..5161
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5077..5096)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5131..5150
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5210..5226)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5210..5226)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5223..5245)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5234..5250
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     promoter        complement(join(5258..5264,5265..5282,5283..5288))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5303..5324
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5441..5458)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5612..6200)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5692..5711)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6371..7162,7163..7231))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     6994..7013
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7232..7336)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gacggatcgg gagatctccc gatcccctat ggtgcactct cagtacaatc tgctctgatg
       61 ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg
      121 cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc
      181 ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt
      241 gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata
      301 tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc
      361 cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc
      421 attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt
      481 atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt
      541 atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca
      601 tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg
      661 actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc
      721 aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg
      781 gtaggcgtgt acggtgggag gtctatataa gcagagctct ctggctaact agagaaccca
      841 ctgcttactg gcttatcgaa attaatacga ctcactatag ggagacccaa gcttgcggcc
      901 gccaccatgg tgcggggttc tcatcatcat catcatcatg gtatggctag catgactggt
      961 ggacagcaaa tgggtcggga tctgtacgac gatgacgata aggatcccat ggtgagcaag
     1021 ggcgaggagc tgttcaccgg ggtggtgccc atcctggtcg agctggacgg cgacgtaaac
     1081 ggccacaggt tcagcgtgtc cggcgagggc gagggcgatg ccacctacgg caagctgacc
     1141 ctgaagttca tctgcaccac cggcaagctg cccgtgccct ggcccaccct cgtgaccacc
     1201 ctgacctggg gcgtgcagtg cttcagccgc taccccgacc acatgaagca gcacgacttc
     1261 ttcaagtccg ccatgcccga aggctacgtc caggagcgta ccatcttctt caaggacgac
     1321 ggcaactaca agacccgcgc cgaggtgaag ttcgagggcg acaccctggt gaaccgcatc
     1381 gagctgaagg gcatcgactt caaggaggac ggcaacatcc tggggcacaa gctggagtac
     1441 aactacatca gccacaacgt ctatatcacc gccgacaagc agaagaacgg catcaaggcc
     1501 cacttcaaga tccgccacaa catcgaggac ggcagcgtgc agctcgccga ccactaccag
     1561 cagaacaccc ccatcggcga cggccccgtg ctgctgcccg acaaccacta cctgagcacc
     1621 cagtccgccc tgagcaaaga ccccaacgag aagcgcgatc acatggtcct gctggagttc
     1681 gtgaccgccg cccgcatgca ttttactcct aaaccacctc aagatagtgc ggttatcaaa
     1741 gctggatatt gtgtaaaaca aggagcagtg atgaaaaact ggaagagaag atattttcaa
     1801 ttggatgaaa acacaatagg ctacttcaaa tctgaactgg aaaaggaacc tcttcgcgta
     1861 ataccactta aagaggttca taaagtccag gaatgtaagc aaagcgacat aatgatgagg
     1921 gacaacctct ttgaaattgt aacaacgtct cgaactttct atgtgcaggc tgatagccct
     1981 gaagagatgc acagttggat taaagcagtc tctggcgcca ttgtagcaca gcggggtccc
     2041 ggcagatctg cgtcttctga gcatccctct gcaggcggta gcgtggctga ggaagaggat
     2101 gacgaggagg aagacgagga cgatggcggc agcgagctca tggtgagcaa gggcgaggag
     2161 ctgttcaccg gggtggtgcc catcctggtc gagctggacg gcgacgtaaa cggccacaag
     2221 ttcagcgtgt ccggcgaggg cgagggcgat gccacctacg gcaagctgac cctgaagttc
     2281 atctgcacct ccggcaagct gcccgtgccc tggcccaccc tcgtgaccac cttcggctac
     2341 ggcctgatgt gcttcgcccg ctaccccgac cacatgaagc agcacgactt cttcaagtcc
     2401 gccatgcccg aaggctacgt ccaggagcgc accatcttct tcaaggacga cggcaactac
     2461 aagacccgcg ccgaggtgaa gttcgagggc gacaccctgg tgaaccgcat cgagctgaag
     2521 ggcatcgact tcaaggagga cggcaacatc ctggggcaca agctggagta caactacaac
     2581 agccacaatg tctatatcat ggccgacaag cagaagaacg gcatcaaggt gaacttcaag
     2641 atccgccaca acatcgagga cggcagcgtg cagctcgccg accactacca gcagaacacc
     2701 cccatcggcg acggccccgt gctgctgccc gacaaccact acctgagcca ccagtccgcc
     2761 ctgagcaaag accccaacga gaagcgcgat cacatggtcc tgctggagtt cgtgaccgcc
     2821 gccgggatca ctctcggcat ggacgagctg tacaaggaat tcaagaagaa gaagaagagc
     2881 aagaccaagt gcgtgatcat gtaatctaga gggccctatt ctatagtgtc acctaaatgc
     2941 tagagctcgc tgatcagcct cgactgtgcc ttctagttgc cagccatctg ttgtttgccc
     3001 ctcccccgtg ccttccttga ccctggaagg tgccactccc actgtccttt cctaataaaa
     3061 tgaggaaatt gcatcgcatt gtctgagtag gtgtcattct attctggggg gtggggtggg
     3121 gcaggacagc aagggggagg attgggaaga caatagcagg catgctgggg atgcggtggg
     3181 ctctatggct tctgaggcgg aaagaaccag ctggggctct agggggtatc cccacgcgcc
     3241 ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga ccgctacact
     3301 tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct tcctttctcg ccacgttcgc
     3361 cggctttccc cgtcaagctc taaatcgggg gctcccttta gggttccgat ttagtgcttt
     3421 acggcacctc gaccccaaaa aacttgatta gggtgatggt tcacgtagtg ggccatcgcc
     3481 ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg ttctttaata gtggactctt
     3541 gttccaaact ggaacaacac tcaaccctat ctcggtctat tcttttgatt tataagggat
     3601 tttgccgatt tcggcctatt ggttaaaaaa tgagctgatt taacaaaaat ttaacgcgaa
     3661 ttaattctgt ggaatgtgtg tcagttaggg tgtggaaagt ccccaggctc cccagcaggc
     3721 agaagtatgc aaagcatgca tctcaattag tcagcaacca ggtgtggaaa gtccccaggc
     3781 tccccagcag gcagaagtat gcaaagcatg catctcaatt agtcagcaac catagtcccg
     3841 cccctaactc cgcccatccc gcccctaact ccgcccagtt ccgcccattc tccgccccat
     3901 ggctgactaa ttttttttat ttatgcagag gccgaggccg cctctgcctc tgagctattc
     3961 cagaagtagt gaggaggctt ttttggaggc ctaggctttt gcaaaaagct cccgggagct
     4021 tgtatatcca ttttcggatc tgatcaagag acaggatgag gatcgtttcg catgattgaa
     4081 caagatggat tgcacgcagg ttctccggcc gcttgggtgg agaggctatt cggctatgac
     4141 tgggcacaac agacaatcgg ctgctctgat gccgccgtgt tccggctgtc agcgcagggg
     4201 cgcccggttc tttttgtcaa gaccgacctg tccggtgccc tgaatgaact gcaggacgag
     4261 gcagcgcggc tatcgtggct ggccacgacg ggcgttcctt gcgcagctgt gctcgacgtt
     4321 gtcactgaag cgggaaggga ctggctgcta ttgggcgaag tgccggggca ggatctcctg
     4381 tcatctcacc ttgctcctgc cgagaaagta tccatcatgg ctgatgcaat gcggcggctg
     4441 catacgcttg atccggctac ctgcccattc gaccaccaag cgaaacatcg catcgagcga
     4501 gcacgtactc ggatggaagc cggtcttgtc gatcaggatg atctggacga agagcatcag
     4561 gggctcgcgc cagccgaact gttcgccagg ctcaaggcgc gcatgcccga cggcgaggat
     4621 ctcgtcgtga cccatggcga tgcctgcttg ccgaatatca tggtggaaaa tggccgcttt
     4681 tctggattca tcgactgtgg ccggctgggt gtggcggacc gctatcagga catagcgttg
     4741 gctacccgtg atattgctga agagcttggc ggcgaatggg ctgaccgctt cctcgtgctt
     4801 tacggtatcg ccgctcccga ttcgcagcgc atcgccttct atcgccttct tgacgagttc
     4861 ttctgagcgg gactctgggg ttcgaaatga ccgaccaagc gacgcccaac ctgccatcac
     4921 gagatttcga ttccaccgcc gccttctatg aaaggttggg cttcggaatc gttttccggg
     4981 acgccggctg gatgatcctc cagcgcgggg atctcatgct ggagttcttc gcccacccca
     5041 acttgtttat tgcagcttat aatggttaca aataaagcaa tagcatcaca aatttcacaa
     5101 ataaagcatt tttttcactg cattctagtt gtggtttgtc caaactcatc aatgtatctt
     5161 atcatgtctg tataccgtcg acctctagct agagcttggc gtaatcatgg tcatagctgt
     5221 ttcctgtgtg aaattgttat ccgctcacaa ttccacacaa catacgagcc ggaagcataa
     5281 agtgtaaagc ctggggtgcc taatgagtga gctaactcac attaattgcg ttgcgctcac
     5341 tgcccgcttt ccagtcggga aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg
     5401 cggggagagg cggtttgcgt attgggcgct cttccgcttc ctcgctcact gactcgctgc
     5461 gctcggtcgt tcggctgcgg cgagcggtat cagctcactc aaaggcggta atacggttat
     5521 ccacagaatc aggggataac gcaggaaaga acatgtgagc aaaaggccag caaaaggcca
     5581 ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag gctccgcccc cctgacgagc
     5641 atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc gacaggacta taaagatacc
     5701 aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg
     5761 gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta
     5821 ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg
     5881 ttcagcccga ccgctgcgcc ttatccggta actatcgtct tgagtccaac ccggtaagac
     5941 acgacttatc gccactggca gcagccactg gtaacaggat tagcagagcg aggtatgtag
     6001 gcggtgctac agagttcttg aagtggtggc ctaactacgg ctacactaga agaacagtat
     6061 ttggtatctg cgctctgctg aagccagtta ccttcggaaa aagagttggt agctcttgat
     6121 ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag cagattacgc
     6181 gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc tacggggtct gacgctcagt
     6241 ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt atcaaaaagg atcttcacct
     6301 agatcctttt aaattaaaaa tgaagtttta aatcaatcta aagtatatat gagtaaactt
     6361 ggtctgacag ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc tgtctatttc
     6421 gttcatccat agttgcctga ctccccgtcg tgtagataac tacgatacgg gagggcttac
     6481 catctggccc cagtgctgca atgataccgc gagacccacg ctcaccggct ccagatttat
     6541 cagcaataaa ccagccagcc ggaagggccg agcgcagaag tggtcctgca actttatccg
     6601 cctccatcca gtctattaat tgttgccggg aagctagagt aagtagttcg ccagttaata
     6661 gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt gtcacgctcg tcgtttggta
     6721 tggcttcatt cagctccggt tcccaacgat caaggcgagt tacatgatcc cccatgttgt
     6781 gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag
     6841 tgttatcact catggttatg gcagcactgc ataattctct tactgtcatg ccatccgtaa
     6901 gatgcttttc tgtgactggt gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc
     6961 gaccgagttg ctcttgcccg gcgtcaatac gggataatac cgcgccacat agcagaactt
     7021 taaaagtgct catcattgga aaacgttctt cggggcgaaa actctcaagg atcttaccgc
     7081 tgttgagatc cagttcgatg taacccactc gtgcacccaa ctgatcttca gcatctttta
     7141 ctttcaccag cgtttctggg tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa
     7201 taagggcgac acggaaatgt tgaatactca tactcttcct ttttcaatat tattgaagca
     7261 tttatcaggg ttattgtctc atgagcggat acatatttga atgtatttag aaaaataaac
     7321 aaataggggt tccgcgcaca tttccccgaa aagtgccacc tgacgtc
//
