LOCUS       sequence-399138-        5772 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Photoactivatable and rapamycin recruitable lipid biosensor .
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5772)
  AUTHORS   Pacheco J, Cassidy AC, Zewe JP, Wills RC, Hammond GRV
  TITLE     PI(4,5)P2 diffuses freely in the plasma membrane even within 
            high-density effector protein complexes.
  JOURNAL   J Cell Biol. 2023 Feb 6;222(2):e202204099. doi: 
            10.1083/jcb.202204099. Epub 2022 Nov 23.
  PUBMED    36416724
REFERENCE   2  (bases 1 to 5772)
  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 Cell 
            Biol."; date: "2023-02-6"; volume: "222(2):e202204099. doi"; pages: 
            " 10.1083/jcb.202204099. Epub 2022 Nov 23"
COMMENT     Sequence Label: PAmCherry-N1-FRB-Tubbyc
FEATURES             Location/Qualifiers
     source          1..5772
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        123..426
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        427..630
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     581..601
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     CDS             1505..1783
                     /codon_start=1
                     /product="FKBP-rapamycin binding domain of human FRAP"
                     /label=FRB
                     /translation="ILWHEMWHEGLEEASRLYFGERNVKGMFEVLEPLHAMMERGPQTL
                     KETSFNQAYGRDLMEAQEWCRKYMKSGNVKDLTQAWDLYYHVFRRISK"
     regulatory      1784..1793
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1790..2500
                     /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(1935..1953)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     polyA_signal    2622..2743
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2659..2678)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2713..2732
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(2750..3205)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2887..2908)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3099..3118
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        3232..3336
                     /gene="bla"
                     /label=AmpR promoter
     promoter        3338..3695
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        3338..3529
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(3361..3381)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      3546..3681
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             3730..4524
                     /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             3730..4524
                     /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(3784..3803)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4394..4413
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(4712..4731)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    4756..4803
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      5132..5720
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5621..5640
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 ctcacatgtt ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgccatgc
       61 attagttatt aatagtaatc aattacgggg tcattagttc atagcccata tatggagttc
      121 cgcgttacat aacttacggt aaatggcccg cctggctgac cgcccaacga cccccgccca
      181 ttgacgtcaa taatgacgta tgttcccata gtaacgccaa tagggacttt ccattgacgt
      241 caatgggtgg agtatttacg gtaaactgcc cacttggcag tacatcaagt gtatcatatg
      301 ccaagtacgc cccctattga cgtcaatgac ggtaaatggc ccgcctggca ttatgcccag
      361 tacatgacct tatgggactt tcctacttgg cagtacatct acgtattagt catcgctatt
      421 accatggtga tgcggttttg gcagtacatc aatgggcgtg gatagcggtt tgactcacgg
      481 ggatttccaa gtctccaccc cattgacgtc aatgggagtt tgttttggca ccaaaatcaa
      541 cgggactttc caaaatgtcg taacaactcc gccccattga cgcaaatggg cggtaggcgt
      601 gtacggtggg aggtctatat aagcagagct ggtttagtga accgtcagat ccgctagcgc
      661 taccggactc agatctcgag ctatggtgga tattgaggtc caggatctag aggagtttgc
      721 actgaggcca gccccacaag ggatcaccat caaatgccgc atcactcggg acaagaaggg
      781 gatggaccgc ggcatgtacc ccacctactt tctgcaccta gaccgtgagg atggcaagaa
      841 ggtgttcctc ctggcgggca ggaagagaaa gaagagtaaa acttccaatt acctcatctc
      901 tgtggaccca acagacttgt ctcggggagg cgatagctat atcgggaaat tgcgatccaa
      961 cctgatgggc accaagttca ccgtttatga caatggcgtc aaccctcaga aggcatcctc
     1021 ttccacgctg gaaagcggaa ccttgcgcca ggagctggca gcggtgtgct atgagacaaa
     1081 tgtcctaggc ttcaagggac ctcggaagat gagtgtgatc gtcccaggca tgaacatggt
     1141 tcatgagaga gtctgtatcc gcccccgcaa tgaacatgag accctgttag cacgctggca
     1201 gaacaagaac acggagagca tcattgagct gcagaacaag acgccagtct ggaatgatga
     1261 cacacagtcc tatgtactta acttccacgg ccgtgtcaca caggcttctg tgaagaactt
     1321 ccagatcatc cacggcaatg acccggacta catcgtcatg cagtttggcc gggtagcaga
     1381 agatgtgttc accatggatt acaactaccc actgtgtgca ctgcaggcct ttgccattgc
     1441 tctgtccagc tttgacagca agctggcctg cgagacggta ccgcgggccc gggatccacc
     1501 ggtcatcctc tggcatgaga tgtggcatga aggcctggaa gaggcatctc gtttgtactt
     1561 tggggaaagg aacgtgaaag gcatgtttga ggtgctggag cccttgcatg ctatgatgga
     1621 acggggcccc cagactctga aggaaacatc ctttaatcag gcctatggtc gagatttaat
     1681 ggaggcccaa gagtggtgca ggaagtacat gaaatcaggg aatgtcaagg acctcaccca
     1741 agcctgggac ctctattatc atgtgttccg acgaatctca aaggccacca tggtgagcaa
     1801 gggcgaggag gataacatgg ccatcattaa ggagttcatg cgcttcaagg tgcacatgga
     1861 ggggtccgtg aacggccacg tgttcgagat cgagggcgag ggcgagggcc gcccctacga
     1921 gggcacccag accgccaagc tgaaggtgac caagggcggc cccctgccct tcacctggga
     1981 catcctgagc cctcagttca tgtacggctc caatgcctac gtgaagcacc ccgccgacat
     2041 ccccgactac tttaagctgt ccttccccga gggcttcaag tgggagcgcg tgatgaaatt
     2101 cgaggacggc ggcgtggtga ccgtgaccca ggactcctcc ctgcaggacg gcgagttcat
     2161 ctacaaggtg aagctgcgcg gcaccaactt cccctccgac ggccccgtga tgcagaagaa
     2221 gaccatgggc tgggaggccc tctccgagcg gatgtacccc gaggacggcg ccctgaaggg
     2281 cgaggtcaag cccagagtga agctgaagga cggcggccac tacgacgctg aggtcaagac
     2341 cacctacaag gccaagaagc ccgtgcagct gcccggcgcc tacaacgtca accgcaagct
     2401 ggacatcacc agccacaacg aggactacac catcgtggag cagtacgaga gagccgaggg
     2461 ccgccactcc accggcggca tggacgagct gtacaagtaa gcggccgcga ctctagatca
     2521 taatcagcca taccacattt gtagaggttt tacttgcttt aaaaaacctc ccacacctcc
     2581 ccctgaacct gaaacataaa atgaatgcaa ttgttgttgt taacttgttt attgcagctt
     2641 ataatggtta caaataaagc aatagcatca caaatttcac aaataaagca tttttttcac
     2701 tgcattctag ttgtggtttg tccaaactca tcaatgtatc ttaaggcgta aattgtaagc
     2761 gttaatattt tgttaaaatt cgcgttaaat ttttgttaaa tcagctcatt ttttaaccaa
     2821 taggccgaaa tcggcaaaat cccttataaa tcaaaagaat agaccgagat agggttgagt
     2881 gttgttccag tttggaacaa gagtccacta ttaaagaacg tggactccaa cgtcaaaggg
     2941 cgaaaaaccg tctatcaggg cgatggccca ctacgtgaac catcacccta atcaagtttt
     3001 ttggggtcga ggtgccgtaa agcactaaat cggaacccta aagggagccc ccgatttaga
     3061 gcttgacggg gaaagccggc gaacgtggcg agaaaggaag ggaagaaagc gaaaggagcg
     3121 ggcgctaggg cgctggcaag tgtagcggtc acgctgcgcg taaccaccac acccgccgcg
     3181 cttaatgcgc cgctacaggg cgcgtcaggt ggcacttttc ggggaaatgt gcgcggaacc
     3241 cctatttgtt tatttttcta aatacattca aatatgtatc cgctcatgag acaataaccc
     3301 tgataaatgc ttcaataata ttgaaaaagg aagagtcctg aggcggaaag aaccagctgt
     3361 ggaatgtgtg tcagttaggg tgtggaaagt ccccaggctc cccagcaggc agaagtatgc
     3421 aaagcatgca tctcaattag tcagcaacca ggtgtggaaa gtccccaggc tccccagcag
     3481 gcagaagtat gcaaagcatg catctcaatt agtcagcaac catagtcccg cccctaactc
     3541 cgcccatccc gcccctaact ccgcccagtt ccgcccattc tccgccccat ggctgactaa
     3601 ttttttttat ttatgcagag gccgaggccg cctcggcctc tgagctattc cagaagtagt
     3661 gaggaggctt ttttggaggc ctaggctttt gcaaagatcg atcaagagac aggatgagga
     3721 tcgtttcgca tgattgaaca agatggattg cacgcaggtt ctccggccgc ttgggtggag
     3781 aggctattcg gctatgactg ggcacaacag acaatcggct gctctgatgc cgccgtgttc
     3841 cggctgtcag cgcaggggcg cccggttctt tttgtcaaga ccgacctgtc cggtgccctg
     3901 aatgaactgc aagacgaggc agcgcggcta tcgtggctgg ccacgacggg cgttccttgc
     3961 gcagctgtgc tcgacgttgt cactgaagcg ggaagggact ggctgctatt gggcgaagtg
     4021 ccggggcagg atctcctgtc atctcacctt gctcctgccg agaaagtatc catcatggct
     4081 gatgcaatgc ggcggctgca tacgcttgat ccggctacct gcccattcga ccaccaagcg
     4141 aaacatcgca tcgagcgagc acgtactcgg atggaagccg gtcttgtcga tcaggatgat
     4201 ctggacgaag agcatcaggg gctcgcgcca gccgaactgt tcgccaggct caaggcgagc
     4261 atgcccgacg gcgaggatct cgtcgtgacc catggcgatg cctgcttgcc gaatatcatg
     4321 gtggaaaatg gccgcttttc tggattcatc gactgtggcc ggctgggtgt ggcggaccgc
     4381 tatcaggaca tagcgttggc tacccgtgat attgctgaag agcttggcgg cgaatgggct
     4441 gaccgcttcc tcgtgcttta cggtatcgcc gctcccgatt cgcagcgcat cgccttctat
     4501 cgccttcttg acgagttctt ctgagcggga ctctggggtt cgaaatgacc gaccaagcga
     4561 cgcccaacct gccatcacga gatttcgatt ccaccgccgc cttctatgaa aggttgggct
     4621 tcggaatcgt tttccgggac gccggctgga tgatcctcca gcgcggggat ctcatgctgg
     4681 agttcttcgc ccaccctagg gggaggctaa ctgaaacacg gaaggagaca ataccggaag
     4741 gaacccgcgc tatgacggca ataaaaagac agaataaaac gcacggtgtt gggtcgtttg
     4801 ttcataaacg cggggttcgg tcccagggct ggcactctgt cgatacccca ccgagacccc
     4861 attggggcca atacgcccgc gtttcttcct tttccccacc ccacccccca agttcgggtg
     4921 aaggcccagg gctcgcagcc aacgtcgggg cggcaggccc tgccatagcc tcaggttact
     4981 catatatact ttagattgat ttaaaacttc atttttaatt taaaaggatc taggtgaaga
     5041 tcctttttga taatctcatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt
     5101 cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct
     5161 gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc
     5221 taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgttc
     5281 ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg cctacatacc
     5341 tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg
     5401 ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt
     5461 cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg
     5521 agctatgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg
     5581 gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt
     5641 atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag
     5701 gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt
     5761 gctggccttt tg
//
