LOCUS       sequence-399281-        5379 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Photoactivatable lipid biosensor.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5379)
  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 5379)
  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: PAmCherry1-C1-P4Mx2
FEATURES             Location/Qualifiers
     source          1..5379
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        91..394
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        395..598
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     549..569
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      631..640
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             637..1344
                     /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(782..800)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     CDS             1408..1713
                     /codon_start=1
                     /product="high-affinity phosphatidylinositol 4-phosphate 
                     binding module of SidM (DrrA) from Legionella pneumophila 
                     (Hammond et al., 2014)"
                     /label=P4M
                     /translation="TASTENFKNVKEKYQQMRGDALKTEILADFKDKLAEATDEQSLKQ
                     IVAELKSKDEYRILAKGQGLTTQLLGLKTSSVSSFEKMVEETRESIKSQERQTIKIK"
     CDS             1759..2064
                     /codon_start=1
                     /product="high-affinity phosphatidylinositol 4-phosphate 
                     binding module of SidM (DrrA) from Legionella pneumophila 
                     (Hammond et al., 2014)"
                     /label=P4M
                     /translation="TASTENFKNVKEKYQQMRGDALKTEILADFKDKLAEATDEQSLKQ
                     IVAELKSKDEYRILAKGQGLTTQLLGLKTSSVSSFEKMVEETRESIKSQERQTIKIK"
     polyA_signal    2197..2318
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2234..2253)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2288..2307
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(2325..2780)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2462..2483)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2674..2693
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        2807..2911
                     /gene="bla"
                     /label=AmpR promoter
     promoter        2913..3270
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        2913..3104
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(2936..2956)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      3121..3256
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             3305..4099
                     /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             3305..4099
                     /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(3359..3378)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3969..3988
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(4287..4306)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    4331..4378
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      4707..5295
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5196..5215
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 tctgtggata accgtattac cgccatgcat tagttattaa tagtaatcaa ttacggggtc
       61 attagttcat agcccatata tggagttccg cgttacataa cttacggtaa atggcccgcc
      121 tggctgaccg cccaacgacc cccgcccatt gacgtcaata atgacgtatg ttcccatagt
      181 aacgccaata gggactttcc attgacgtca atgggtggag tatttacggt aaactgccca
      241 cttggcagta catcaagtgt atcatatgcc aagtacgccc cctattgacg tcaatgacgg
      301 taaatggccc gcctggcatt atgcccagta catgacctta tgggactttc ctacttggca
      361 gtacatctac gtattagtca tcgctattac catggtgatg cggttttggc agtacatcaa
      421 tgggcgtgga tagcggtttg actcacgggg atttccaagt ctccacccca ttgacgtcaa
      481 tgggagtttg ttttggcacc aaaatcaacg ggactttcca aaatgtcgta acaactccgc
      541 cccattgacg caaatgggcg gtaggcgtgt acggtgggag gtctatataa gcagagctgg
      601 tttagtgaac cgtcagatcc gctagcggtc gccaccatgg tgagcaaggg cgaggaggat
      661 aacatggcca tcattaagga gttcatgcgc ttcaaggtgc acatggaggg gtccgtgaac
      721 ggccacgtgt tcgagatcga gggcgagggc gagggccgcc cctacgaggg cacccagacc
      781 gccaagctga aggtgaccaa gggcggcccc ctgcccttca cctgggacat cctgagccct
      841 cagttcatgt acggctccaa tgcctacgtg aagcaccccg ccgacatccc cgactacttt
      901 aagctgtcct tccccgaggg cttcaagtgg gagcgcgtga tgaaattcga ggacggcggc
      961 gtggtgaccg tgacccagga ctcctccctg caggacggcg agttcatcta caaggtgaag
     1021 ctgcgcggca ccaacttccc ctccgacggc cccgtgatgc agaagaagac catgggctgg
     1081 gaggccctct ccgagcggat gtaccccgag gacggcgccc tgaagggcga ggtcaagccc
     1141 agagtgaagc tgaaggacgg cggccactac gacgctgagg tcaagaccac ctacaaggcc
     1201 aagaagcccg tgcagctgcc cggcgcctac aacgtcaacc gcaagctgga catcaccagc
     1261 cacaacgagg actacaccat cgtggagcag tacgagagag ccgagggccg ccactccacc
     1321 ggcggcatgg acgagctgta caagtccgga ctcagatctc gagctcaagc ttcgaattct
     1381 ggtggtagtg cttccggact cagatccacg gcaagcacgg aaaactttaa aaatgttaaa
     1441 gaaaaatatc agcaaatgcg aggtgatgct ttaaaaacag aaatcctggc tgatttcaag
     1501 gataaactgg ctgaagctac cgatgaacag agccttaagc aaattgttgc tgaattgaaa
     1561 agtaaagatg aatatagaat tttggctaag gggcaagggt taacaaccca gcttttaggg
     1621 ttaaagacaa gttcagtgtc ttcatttgag aaaatggttg aggaaacaag agaaagtatt
     1681 aagtctcaag aaagacaaac cattaagata aaaggtggta gtgctgtcga cggtggtagt
     1741 gcttccggac tcagatccac ggcaagcacg gaaaacttta aaaatgttaa agaaaaatat
     1801 cagcaaatgc gaggtgatgc tttaaaaaca gaaatcctgg ctgatttcaa ggataaactg
     1861 gctgaagcta ccgatgaaca gagccttaag caaattgttg ctgaattgaa aagtaaagat
     1921 gaatatagaa ttttggctaa ggggcaaggg ttaacaaccc agcttttagg gttaaagaca
     1981 agttcagtgt cttcatttga gaaaatggtt gaggaaacaa gagaaagtat taagtctcaa
     2041 gaaagacaaa ccattaagat aaaataagga tccaccggat ctagataact gatcataatc
     2101 agccatacca catttgtaga ggttttactt gctttaaaaa acctcccaca cctccccctg
     2161 aacctgaaac ataaaatgaa tgcaattgtt gttgttaact tgtttattgc agcttataat
     2221 ggttacaaat aaagcaatag catcacaaat ttcacaaata aagcattttt ttcactgcat
     2281 tctagttgtg gtttgtccaa actcatcaat gtatcttaac gcgtaaattg taagcgttaa
     2341 tattttgtta aaattcgcgt taaatttttg ttaaatcagc tcatttttta accaataggc
     2401 cgaaatcggc aaaatccctt ataaatcaaa agaatagacc gagatagggt tgagtgttgt
     2461 tccagtttgg aacaagagtc cactattaaa gaacgtggac tccaacgtca aagggcgaaa
     2521 aaccgtctat cagggcgatg gcccactacg tgaaccatca ccctaatcaa gttttttggg
     2581 gtcgaggtgc cgtaaagcac taaatcggaa ccctaaaggg agcccccgat ttagagcttg
     2641 acggggaaag ccggcgaacg tggcgagaaa ggaagggaag aaagcgaaag gagcgggcgc
     2701 tagggcgctg gcaagtgtag cggtcacgct gcgcgtaacc accacacccg ccgcgcttaa
     2761 tgcgccgcta cagggcgcgt caggtggcac ttttcgggga aatgtgcgcg gaacccctat
     2821 ttgtttattt ttctaaatac attcaaatat gtatccgctc atgagacaat aaccctgata
     2881 aatgcttcaa taatattgaa aaaggaagag tcctgaggcg gaaagaacca gctgtggaat
     2941 gtgtgtcagt tagggtgtgg aaagtcccca ggctccccag caggcagaag tatgcaaagc
     3001 atgcatctca attagtcagc aaccaggtgt ggaaagtccc caggctcccc agcaggcaga
     3061 agtatgcaaa gcatgcatct caattagtca gcaaccatag tcccgcccct aactccgccc
     3121 atcccgcccc taactccgcc cagttccgcc cattctccgc cccatggctg actaattttt
     3181 tttatttatg cagaggccga ggccgcctcg gcctctgagc tattccagaa gtagtgagga
     3241 ggcttttttg gaggcctagg cttttgcaaa gatcgatcaa gagacaggat gaggatcgtt
     3301 tcgcatgatt gaacaagatg gattgcacgc aggttctccg gccgcttggg tggagaggct
     3361 attcggctat gactgggcac aacagacaat cggctgctct gatgccgccg tgttccggct
     3421 gtcagcgcag gggcgcccgg ttctttttgt caagaccgac ctgtccggtg ccctgaatga
     3481 actgcaagac gaggcagcgc ggctatcgtg gctggccacg acgggcgttc cttgcgcagc
     3541 tgtgctcgac gttgtcactg aagcgggaag ggactggctg ctattgggcg aagtgccggg
     3601 gcaggatctc ctgtcatctc accttgctcc tgccgagaaa gtatccatca tggctgatgc
     3661 aatgcggcgg ctgcatacgc ttgatccggc tacctgccca ttcgaccacc aagcgaaaca
     3721 tcgcatcgag cgagcacgta ctcggatgga agccggtctt gtcgatcagg atgatctgga
     3781 cgaagagcat caggggctcg cgccagccga actgttcgcc aggctcaagg cgagcatgcc
     3841 cgacggcgag gatctcgtcg tgacccatgg cgatgcctgc ttgccgaata tcatggtgga
     3901 aaatggccgc ttttctggat tcatcgactg tggccggctg ggtgtggcgg accgctatca
     3961 ggacatagcg ttggctaccc gtgatattgc tgaagagctt ggcggcgaat gggctgaccg
     4021 cttcctcgtg ctttacggta tcgccgctcc cgattcgcag cgcatcgcct tctatcgcct
     4081 tcttgacgag ttcttctgag cgggactctg gggttcgaaa tgaccgacca agcgacgccc
     4141 aacctgccat cacgagattt cgattccacc gccgccttct atgaaaggtt gggcttcgga
     4201 atcgttttcc gggacgccgg ctggatgatc ctccagcgcg gggatctcat gctggagttc
     4261 ttcgcccacc ctagggggag gctaactgaa acacggaagg agacaatacc ggaaggaacc
     4321 cgcgctatga cggcaataaa aagacagaat aaaacgcacg gtgttgggtc gtttgttcat
     4381 aaacgcgggg ttcggtccca gggctggcac tctgtcgata ccccaccgag accccattgg
     4441 ggccaatacg cccgcgtttc ttccttttcc ccaccccacc ccccaagttc gggtgaaggc
     4501 ccagggctcg cagccaacgt cggggcggca ggccctgcca tagcctcagg ttactcatat
     4561 atactttaga ttgatttaaa acttcatttt taatttaaaa ggatctaggt gaagatcctt
     4621 tttgataatc tcatgaccaa aatcccttaa cgtgagtttt cgttccactg agcgtcagac
     4681 cccgtagaaa agatcaaagg atcttcttga gatccttttt ttctgcgcgt aatctgctgc
     4741 ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt tgccggatca agagctacca
     4801 actctttttc cgaaggtaac tggcttcagc agagcgcaga taccaaatac tgttcttcta
     4861 gtgtagccgt agttaggcca ccacttcaag aactctgtag caccgcctac atacctcgct
     4921 ctgctaatcc tgttaccagt ggctgctgcc agtggcgata agtcgtgtct taccgggttg
     4981 gactcaagac gatagttacc ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc
     5041 acacagccca gcttggagcg aacgacctac accgaactga gatacctaca gcgtgagcta
     5101 tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca ggtatccggt aagcggcagg
     5161 gtcggaacag gagagcgcac gagggagctt ccagggggaa acgcctggta tctttatagt
     5221 cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg
     5281 cggagcctat ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc cttttgctgg
     5341 ccttttgctc acatgttctt tcctgcgtta tcccctgat
//
