LOCUS       sequence-336133-        6593 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  GFP(ST)-PH fusion used to measure realtime changes in PIP2 levels. .
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6593)
  AUTHORS   van der Wal J, Habets R, Varnai P, Balla T, Jalink K
  TITLE     Monitoring agonist-induced phospholipase C activation in live cells 
            by fluorescence resonance energy transfer.
  JOURNAL   J Biol Chem. 2001 May 4. 276(18):15337-44.
  PUBMED    11152673
REFERENCE   2  (bases 1 to 6593)
  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: "J Biol 
            Chem. 2001 May 4. 276(18):15337-44."
COMMENT     Sequence Label: PIP2-GFP(ST) sensor
FEATURES             Location/Qualifiers
     source          1..6593
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      complement(94..682)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(174..193)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(853..1644,1645..1713))
                     /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     1476..1495
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(1714..1818)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(1893..1912)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        2381..2584
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     2535..2555
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     2629..2648
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        2629..2647
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      2682..2691
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(2688..2690,2691..2693,2694..3404)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(2688..2690,2691..2693,2694..3404)
                     /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(2733..2754)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2994..3013)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     3341..3362
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     promoter        complement(3995..4013)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(3996..4013)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(4033..4050)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    4039..4263
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      4309..4737
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4396..4415)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4606..4627
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4746..4766)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        4751..5080
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      4931..5066
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     4993..5012
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             5147..5941
                     /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(5201..5220)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     5811..5830
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    6115..6236
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(6152..6171)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     6206..6225
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(6285..6301)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6285..6301)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(6298..6320)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    6309..6325
                     /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(6333..6339,6340..6357,6358..6363))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6378..6399
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(6516..6533)
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 atccacagaa tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc
       61 caggaaccgt aaaaaggccg cgttgctggc gtttttccat aggctccgcc cccctgacga
      121 gcatcacaaa aatcgacgct caagtcagag gtggcgaaac ccgacaggac tataaagata
      181 ccaggcgttt ccccctggaa gctccctcgt gcgctctcct gttccgaccc tgccgcttac
      241 cggatacctg tccgcctttc tcccttcggg aagcgtggcg ctttctcata gctcacgctg
      301 taggtatctc agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc
      361 cgttcagccc gaccgctgcg ccttatccgg taactatcgt cttgagtcca acccggtaag
      421 acacgactta tcgccactgg cagcagccac tggtaacagg attagcagag cgaggtatgt
      481 aggcggtgct acagagttct tgaagtggtg gcctaactac ggctacacta gaagaacagt
      541 atttggtatc tgcgctctgc tgaagccagt taccttcgga aaaagagttg gtagctcttg
      601 atccggcaaa caaaccaccg ctggtagcgg tggttttttt gtttgcaagc agcagattac
      661 gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt tctacggggt ctgacgctca
      721 gtggaacgaa aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac
      781 ctagatcctt ttaaattaaa aatgaagttt taaatcaatc taaagtatat atgagtaaac
      841 ttggtctgac agttaccaat gcttaatcag tgaggcacct atctcagcga tctgtctatt
      901 tcgttcatcc atagttgcct gactccccgt cgtgtagata actacgatac gggagggctt
      961 accatctggc cccagtgctg caatgatacc gcgagaccca cgctcaccgg ctccagattt
     1021 atcagcaata aaccagccag ccggaagggc cgagcgcaga agtggtcctg caactttatc
     1081 cgcctccatc cagtctatta attgttgccg ggaagctaga gtaagtagtt cgccagttaa
     1141 tagtttgcgc aacgttgttg ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg
     1201 tatggcttca ttcagctccg gttcccaacg atcaaggcga gttacatgat cccccatgtt
     1261 gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt gtcagaagta agttggccgc
     1321 agtgttatca ctcatggtta tggcagcact gcataattct cttactgtca tgccatccgt
     1381 aagatgcttt tctgtgactg gtgagtactc aaccaagtca ttctgagaat agtgtatgcg
     1441 gcgaccgagt tgctcttgcc cggcgtcaat acgggataat accgcgccac atagcagaac
     1501 tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga aaactctcaa ggatcttacc
     1561 gctgttgaga tccagttcga tgtaacccac tcgtgcaccc aactgatctt cagcatcttt
     1621 tactttcacc agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg
     1681 aataagggcg acacggaaat gttgaatact catactcttc ctttttcaat attattgaag
     1741 catttatcag ggttattgtc tcatgagcgg atacatattt gaatgtattt agaaaaataa
     1801 acaaataggg gttccgcgca catttccccg aaaagtgcca cctgacgtcg acggatcggg
     1861 agatctcccg atcccctatg gtgcactctc agtacaatct gctctgatgc cgcatagtta
     1921 agccagtatc tgctccctgc ttgtgtgttg gaggtcgctg agtagtgcgc gagcaaaatt
     1981 taagctacaa caaggcaagg cttgaccgac aattgcatga agaatctgct tagggttagg
     2041 cgttttgcgc tgcttcgcga tgtacgggcc agatatacgc gttgacattg attattgact
     2101 agttattaat agtaatcaat tacggggtca ttagttcata gcccatatat ggagttccgc
     2161 gttacataac ttacggtaaa tggcccgcct ggctgaccgc ccaacgaccc ccgcccattg
     2221 acgtcaataa tgacgtatgt tcccatagta acgccaatag ggactttcca ttgacgtcaa
     2281 tgacggtaaa tggcccgcct ggcattatgc ccagtacatg accttatggg actttcctac
     2341 ttggcagtac atctacgtat tagtcatcgc tattaccatg gtgatgcggt tttggcagta
     2401 catcaatggg cgtggatagc ggtttgactc acggggattt ccaagtctcc accccattga
     2461 cgtcaatggg agtttgtttt ggcaccaaaa tcaacgggac tttccaaaat gtcgtaacaa
     2521 ctccgcccca ttgacgcaaa tgggcggtag gcgtgtacgg tgggaggtct atataagcag
     2581 agctctctgg ctaactagag aacccactgc ttactggctt atcgaaatta atacgactca
     2641 ctatagggag acccaagctt ggtaccgagc tcggatccat cgccaccatg gtgagcaagg
     2701 gcgaggagct gttcaccggg gtggtgccca tcctggtcga gctggacggc gacgtaaacg
     2761 gccacaagtt cagcgtgtcc ggcgagggcg agggcgatgc cacctacggc aagctgaccc
     2821 tgaagttcat ctgcaccacc ggcaagctgc ccgtgccctg gcccaccctc gtgaccaccc
     2881 tgacctacgg cgtgcagtgc ttcagccgct accccgacca catgaagcag cacgacttct
     2941 tcaagtccgc catgcccgaa ggctacgtcc aggagcgcac catcttcttc aaggacgacg
     3001 gcaactacaa gacccgcgcc gaggtgaagt tcgagggcga caccctggtg aaccgcatcg
     3061 agctgaaggg catcgacttc aaggaggacg gcaacatcct ggggcacaag ctggagtaca
     3121 actacaacag ccacaacgtc tatatcatgg ccgacaagca gaagaacggc atcaaggtga
     3181 acttcaagat ccgccacaac atcgaggacg gcagcgtgca gctcgccgac cactaccagc
     3241 agaacacccc catcggcgac ggccccgtgc tgctgcccga caaccactac ctgagcaccc
     3301 agtccgccct gagcaaagac cccaacgaga agcgcgatca catggtcctg ctggagttcg
     3361 tgaccgccgc cgggatcact ctcggcatgg acgagctgta caagccgaat tcccgggtct
     3421 cagccatgga ctcgggccgg gacttcctga ccctgcacgg cctacaggat gatgaggatc
     3481 tacaggcgct gctgaagggc agccagctcc tgaaggtgaa gtccagctca tggaggagag
     3541 agcgcttcta caagttgcag gaggactgca agaccatctg gcaggagtcc cgcaaggtca
     3601 tgcggacccc ggagtcccag ctgttctcca tcgaggacat tcaggaggtg cgaatggggc
     3661 accgcacgga gggtctggag aagttcgccc gtgatgtgcc cgaggaccgc tgcttctcca
     3721 ttgtcttcaa ggaccagcgc aatacactag acctcatcgc cccatcgcca gctgatgccc
     3781 agcactgggt gctggggctg cacaagatca tccaccactc aggctccatg gaccagcgtc
     3841 agaagctaca gcactggatt cactcctgct tgcgaaaagc tgacaaaaac aaggacaaca
     3901 agatgagctt caaggagctg cagaacttcc tgaaggagct caacatctga tatcggtacc
     3961 gcggccgctc gagcatgcat ctagagggcc ctattctata gtgtcaccta aatgctagag
     4021 ctcgctgatc agcctcgact gtgccttcta gttgccagcc atctgttgtt tgcccctccc
     4081 ccgtgccttc cttgaccctg gaaggtgcca ctcccactgt cctttcctaa taaaatgagg
     4141 aaattgcatc gcattgtctg agtaggtgtc attctattct ggggggtggg gtggggcagg
     4201 acagcaaggg ggaggattgg gaagacaata gcaggcatgc tggggatgcg gtgggctcta
     4261 tggcttctga ggcggaaaga accagctggg gctctagggg gtatccccac gcgccctgta
     4321 gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca
     4381 gcgccctagc gcccgctcct ttcgctttct tcccttcctt tctcgccacg ttcgccggct
     4441 ttccccgtca agctctaaat cgggggctcc ctttagggtt ccgatttagt gctttacggc
     4501 acctcgaccc caaaaaactt gattagggtg atggttcacg tagtgggcca tcgccctgat
     4561 agacggtttt tcgccctttg acgttggagt ccacgttctt taatagtgga ctcttgttcc
     4621 aaactggaac aacaytcaac cctatctcgg tctattcttt tgatttataa gggattttgc
     4681 cgatttcggc ctattggtta aaaaatgagc tgatttaaca aaaatttaac gcgaattaat
     4741 tctgtggaat gtgtgtcagt tagggtgtgg aaagtcccca ggctccccag caggcagaag
     4801 tatgcaaagc atgcatctca attagtcagc aaccaggtgt ggaaagtccc caggctcccc
     4861 agcaggcaga agtatgcaaa gcatgcatct caattagtca gcaaccatag tcccgcccct
     4921 aactccgccc atcccgcccc taactccgcc cagttccgcc cattctccgc cccatggctg
     4981 actaattttt tttatttatg cagaggccga ggccgcctct gcctctgagc tattccagaa
     5041 gtagtgagga ggcttttttg gaggcctagg cttttgcaaa aagctcccgg gagcttgtat
     5101 atccattttc ggatctgatc aagagacagg atgaggatcg tttcgcatga ttgaacaaga
     5161 tggattgcac gcaggttctc cggccgcttg ggtggagagg ctattcggct atgactgggc
     5221 acaacagaca atcggctgct ctgatgccgc cgtgttccgg ctgtcagcgc aggggcgccc
     5281 ggttcttttt gtcaagaccg acctgtccgg tgccctgaat gaactgcagg acgaggcagc
     5341 gcggctatcg tggctggcca cgacgggcgt tccttgcgca gctgtgctcg acgttgtcac
     5401 tgaagcggga agggactggc tgctattggg cgaagtgccg gggcaggatc tcctgtcatc
     5461 tcaccttgct cctgccgaga aagtatccat catggctgat gcaatgcggc ggctgcatac
     5521 gcttgatccg gctacctgcc cattcgacca ccaagcgaaa catcgcatcg agcgagcacg
     5581 tactcggatg gaagccggtc ttgtcgatca ggatgatctg gacgaagagc atcaggggct
     5641 cgcgccagcc gaactgttcg ccaggctcaa ggcgcgcatg cccgacggcg aggatctcgt
     5701 cgtgacccat ggcgatgcct gcttgccgaa tatcatggtg gaaaatggcc gcttttctgg
     5761 attcatcgac tgtggccggc tgggtgtggc ggaccgctat caggacatag cgttggctac
     5821 ccgtgatatt gctgaagagc ttggcggcga atgggctgac cgcttcctcg tgctttacgg
     5881 tatcgccgct cccgattcgc agcgcatcgc cttctatcgc cttcttgacg agttcttctg
     5941 agcgggactc tggggttcga aatgaccgac caagcgacgc ccaacctgcc atcacgagat
     6001 ttcgattcca ccgccgcctt ctatgaaagg ttgggcttcg gaatcgtttt ccgggacgcc
     6061 ggctggatga tcctccagcg cggggatctc atgctggagt tcttcgccca ccccaacttg
     6121 tttattgcag cttataatgg ttacaaataa agcaatagca tcacaaattt cacaaataaa
     6181 gcattttttt cactgcattc tagttgtggt ttgtccaaac tcatcaatgt atcttatcat
     6241 gtctgtatac cgtcgacctc tagctagagc ttggcgtaat catggtcata gctgtttcct
     6301 gtgtgaaatt gttatccgct cacaattcca cacaacatac gagccggaag cataaagtgt
     6361 aaagcctggg gtgcctaatg agtgagctaa ctcacattaa ttgcgttgcg ctcactgccc
     6421 gctttccagt cgggaaacct gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg
     6481 agaggcggtt tgcgtattgg gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg
     6541 gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg cggtaatacg gtt
//
