LOCUS       sequence-380167-        6039 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses PH domain from PLC delta1 tagged with miRFP703 and two FRB
            mutants (T2098L) in mammalian cells..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6039)
  AUTHORS   Obashi K, Sochacki KA, Strub MP, Taraska JW
  TITLE     A conformational switch in clathrin light chain regulates lattice 
            structure and endocytosis at the plasma membrane of mammalian cells.
  JOURNAL   Nat Commun. 2023 Feb 9;14(1):732. doi: 10.1038/s41467-023-36304-7.
  PUBMED    36759616
REFERENCE   2  (bases 1 to 6039)
  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: "Nat 
            Commun."; date: "2023-02-9"; volume: "14(1):732. doi"; pages: " 
            10.1038/s41467-023-36304-7"
COMMENT     Sequence Label: PH-miRFP-FRBx2
FEATURES             Location/Qualifiers
     source          1..6039
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        66..369
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        370..573
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     524..544
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      1071..1080
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1077..2021
                     /codon_start=1
                     /product="bright monomeric near-infrared fluorescent 
                     protein engineered from a bacterial phytochrome, with an 
                     emission maximum of 703 nm (Shcherbakova et al., 2016)"
                     /label=miRFP703
                     /label=ORF frame 1
                     /translation="MVAGHASGSPAFGTASHSNCEHEEIHLAGSIQPHGALLVVSEHDH
                     RVIQASANAAEFLNLGSVLGVPLAEIDGDLLIKILPHLDPTAEGMPVAVRCRIGNPSTE
                     YCGLMHRPPEGGLIIELERAGPSIDLSGTLAPALERIRTAGSLRALCDDTVLLFQQCTG
                     YDRVMVYRFDEQGHGLVFSECHVPGLESYFGNRYPSSLVPQMARQLYVRQRVRVLVDVT
                     YQPVPLEPRLSPLTGRDLDMSGCFLRSMSPIHLQFLKDMGVRATLAVSLVVGGKLWGLV
                     VCHHYLPRFIRFELRAICKRLAERIATRITALES"
     CDS             2037..2315
                     /codon_start=1
                     /product="T2098L mutant of FKBP-rapamycin binding domain of
                     human FRAP"
                     /label=FRB*
                     /note="binds synthetic ligands such as AP21967"
                     /translation="ILWHEMWHEGLEEASRLYFGERNVKGMFEVLEPLHAMMERGPQTL
                     KETSFNQAYGRDLMEAQEWCRKYMKSGNVKDLLQAWDLYYHVFRRISK"
     CDS             2331..2609
                     /codon_start=1
                     /product="T2098L mutant of FKBP-rapamycin binding domain of
                     human FRAP"
                     /label=FRB*
                     /note="binds synthetic ligands such as AP21967"
                     /translation="ILWHEMWHEGLEEASRLYFGERNVKGMFEVLEPLHAMMERGPQTL
                     KETSFNQAYGRDLMEAQEWCRKYMKSGNVKDLLQAWDLYYHVFRRISK"
     misc_feature    2656..2708
                     /label=MCS
                     /note="multiple cloning site"
     polyA_signal    2832..2953
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2869..2888)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2923..2942
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(2960..3415)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3097..3118)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3309..3328
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        3442..3546
                     /gene="bla"
                     /label=AmpR promoter
     promoter        3548..3905
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        3548..3739
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(3571..3591)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      3756..3891
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             3940..4734
                     /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             3940..4734
                     /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(3994..4013)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4604..4623
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(4922..4941)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    4966..5013
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      5342..5930
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5831..5850
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 tgcattagtt attaatagta atcaattacg gggtcattag ttcatagccc atatatggag
       61 ttccgcgtta cataacttac ggtaaatggc ccgcctggct gaccgcccaa cgacccccgc
      121 ccattgacgt caataatgac gtatgttccc atagtaacgc caatagggac tttccattga
      181 cgtcaatggg tggagtattt acggtaaact gcccacttgg cagtacatca agtgtatcat
      241 atgccaagta cgccccctat tgacgtcaat gacggtaaat ggcccgcctg gcattatgcc
      301 cagtacatga ccttatggga ctttcctact tggcagtaca tctacgtatt agtcatcgct
      361 attaccatgg tgatgcggtt ttggcagtac atcaatgggc gtggatagcg gtttgactca
      421 cggggatttc caagtctcca ccccattgac gtcaatggga gtttgttttg gcaccaaaat
      481 caacgggact ttccaaaatg tcgtaacaac tccgccccat tgacgcaaat gggcggtagg
      541 cgtgtacggt gggaggtcta tataagcaga gctggtttag tgaaccgtca gatccgctag
      601 cgctaccgga ctcagatctc gagctcaagc ttcgaattcc ggccaccatg tccggactca
      661 gatcccacgg gctccaggat gacccggacc ttcaggccct tctgaagggc agccagcttc
      721 tgaaggtgaa gtccagctcg tggcgtaggg aacgcttcta caagctacag gaggactgca
      781 agaccatctg gcaggaatct cgaaaggtca tgaggtcccc ggagtcgcag ctgttctcca
      841 tcgaggacat tcaggaggta cggatgggac accgcacaga aggcctggag aagtttgccc
      901 gagacatccc cgaggatcga tgcttctcca ttgtcttcaa ggaccagcgc aacaccctag
      961 acctcattgc cccatcacca gctgacgctc agcactgggt gcagggcctg cgcaagatca
     1021 tccaccactc cggctccatg gaccagcggc agaaggcgga tccaccggtc gccaccatgg
     1081 tagcaggtca tgcctctggc agccccgcat tcgggaccgc ctctcattcg aattgcgaac
     1141 atgaagagat ccacctcgcc ggctcgatcc agccgcatgg cgcgcttctg gtcgtcagcg
     1201 aacatgatca tcgcgtcatc caggccagcg ccaacgccgc ggaatttctg aatctcggaa
     1261 gcgtactcgg cgttccgctc gccgagatcg acggcgatct gttgatcaag atcctgccgc
     1321 atctcgatcc caccgccgaa ggcatgccgg tcgcggtgcg ctgccggatc ggcaatccct
     1381 ctacggagta ctgcggtctg atgcatcggc ctccggaagg cgggctgatc atcgaactcg
     1441 aacgtgccgg cccgtcgatc gatctgtcag gcacgctggc gccggcgctg gagcggatcc
     1501 gcacggcggg ttcactgcgc gcgctgtgcg atgacaccgt gctgctgttt cagcagtgca
     1561 ccggctacga ccgggtgatg gtgtatcgtt tcgatgagca aggccacggc ctggtattct
     1621 ccgagtgcca tgtgcctggg ctcgaatcct atttcggcaa ccgctatccg tcgtcgctgg
     1681 tcccgcagat ggcgcggcag ctgtacgtgc ggcagcgcgt ccgcgtgctg gtcgacgtca
     1741 cctatcagcc ggtgccgctg gagccgcggc tgtcgccgct gaccgggcgc gatctcgaca
     1801 tgtcgggctg cttcctgcgc tcgatgtcgc cgatccatct gcagttcctg aaggacatgg
     1861 gcgtgcgcgc caccctggcg gtgtcgctgg tggtcggcgg caagctgtgg ggcctggttg
     1921 tctgtcacca ttatctgccg cgcttcatcc gtttcgagct gcgggcgatc tgcaaacggc
     1981 tcgccgaaag gatcgcgacg cggatcaccg cgcttgagag caagggagca agtggaatcc
     2041 tctggcatga gatgtggcat gaaggcctgg aagaggcatc tcgtttgtac tttggggaaa
     2101 ggaacgtgaa aggcatgttt gaggtgctgg agcccttgca tgctatgatg gaacggggcc
     2161 cccagactct gaaggaaaca tcctttaatc aggcctatgg tcgagattta atggaggccc
     2221 aagagtggtg caggaagtac atgaaatcag ggaatgtmaa ggacctcctc caagcctggg
     2281 acctctatta tcatgtgttc cgacgaatct caaagaaggg agcaagtgga atcctctggc
     2341 atgagatgtg gcatgaaggc ctggaagagg catctcgttt gtactttggg gaaaggaacg
     2401 tgaaaggcat gtttgaggtg ctggagccct tgcatgctat gatggaacgg ggcccccaga
     2461 ctctgaagga aacatccttt aatcaggcct atggtcgaga tttaatggag gcccaagagt
     2521 ggtgcaggaa gtacatgaaa tcagggaatg taaaggacct cctccaagcc tgggacctct
     2581 attatcatgt gttccgacga atctcaaaga gtgctggtgg tagtgctggt ggtagtgctg
     2641 gtggtactgg tggtcctcga gctcaagctt cgaattctgc agtcgacggt accgcgggcc
     2701 cgggatccac cggatctaga taactgatca taatcagcca taccacattt gtagaggttt
     2761 tacttgcttt aaaaaacctc ccacacctcc ccctgaacct gaaacataaa atgaatgcaa
     2821 ttgttgttgt taacttgttt attgcagctt ataatggtta caaataaagc aatagcatca
     2881 caaatttcac aaataaagca tttttttcac tgcattctag ttgtggtttg tccaaactca
     2941 tcaatgtatc ttaaggcgta aattgtaagc gttaatattt tgttaaaatt agcgttaaat
     3001 ttttgttaaa tcagctcatt ttttaaccaa taggccgaaa tcggcaaaat cccttataaa
     3061 tcaaaagaat agaccgagat agggttgagt gttgttccag tttggaacaa gagtccacta
     3121 ttaaagaacg tggactccaa cgtcaaaggg cgaaaaaccg tctatcaggg cgatggccca
     3181 ctacgtgaac catcacccta atcaagtttt ttggggtcga ggtgccgtaa agcactaaat
     3241 cggaacccta aagggagccc ccgatttaga gcttgacggg gaaagccggc gaacgtggcg
     3301 agaaaggaag ggaagaaagc gaaaggagcg ggcgctaggg cgctggcaag tgtagcggtc
     3361 acgctgcgcg taaccaccac acccgccgcg cttaatgcgc cgctacaggg cgcgtcaggt
     3421 ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt tatttttcta aatacattca
     3481 aatatgtatc cgctcatgag acaataaccc tgataaatgc ttcaataata ttgaaaaagg
     3541 aagagtcctg aggcggaaag aaccagctgt ggaatgtgtg tcagttaggg tgtggaaagt
     3601 ccccaggctc cccagcaggc agaagtatgc aaagcatgca tctcaattag tcagcaacca
     3661 ggtgtggaaa gtccccaggc tccccagcag gcagaagtat gcaaagcatg catctcaatt
     3721 agtcagcaac catagtcccg cccctaactc cgcccatccc gcccctaact ccgcccagtt
     3781 ccgcccattc tccgccccat ggctgactaa ttttttttat ttatgcagag gccgaggccg
     3841 cctcggcctc tgagctattc cagaagtagt gaggaggctt ttttggaggc ctaggctttt
     3901 gcaaagatcg atcaagagac aggatgagga tcgtttcgca tgattgaaca agatggattg
     3961 cacgcaggtt ctccggccgc ttgggtggag aggctattcg gctatgactg ggcacaacag
     4021 acaatcggct gctctgatgc cgccgtgttc cggctgtcag cgcaggggcg cccggttctt
     4081 tttgtcaaga ccgacctgtc cggtgccctg aatgaactgc aagacgaggc agcgcggcta
     4141 tcgtggctgg ccacgacggg cgttccttgc gcagctgtgc tcgacgttgt cactgaagcg
     4201 ggaagggact ggctgctatt gggcgaagtg ccggggcagg atctcctgtc atctcacctt
     4261 gctcctgccg agaaagtatc catcatggct gatgcaatgc ggcggctgca tacgcttgat
     4321 ccggctacct gcccattcga ccaccaagcg aaacatcgca tcgagcgagc acgtactcgg
     4381 atggaagccg gtcttgtcga tcaggatgat ctggacgaag agcatcaggg gctcgcgcca
     4441 gccgaactgt tcgccaggct caaggcgagc atgcccgacg gcgaggatct cgtcgtgacc
     4501 catggcgatg cctgcttgcc gaatatcatg gtggaaaatg gccgcttttc tggattcatc
     4561 gactgtggcc ggctgggtgt ggcggaccgc tatcaggaca tagcgttggc tacccgtgat
     4621 attgctgaag agcttggcgg cgaatgggct gaccgcttcc tcgtgcttta cggtatcgcc
     4681 gctcccgatt cgcagcgcat cgccttctat cgccttcttg acgagttctt ctgagcggga
     4741 ctctggggtt cgaaatgacc gaccaagcga cgcccaacct gccatcacga gatttcgatt
     4801 ccaccgccgc cttctatgaa aggttgggct tcggaatcgt tttccgggac gccggctgga
     4861 tgatcctcca gcgcggggat ctcatgctgg agttcttcgc ccaccctagg gggaggctaa
     4921 ctgaaacacg gaaggagaca ataccggaag gaacccgcgc tatgacggca ataaaaagac
     4981 agaataaaac gcacggtgtt gggtcgtttg ttcataaacg cggggttcgg tcccagggct
     5041 ggcactctgt cgatacccca ccgagacccc attggggcca atacgcccgc gtttcttcct
     5101 tttccccacc ccacccccca agttcgggtg aaggcccagg gctcgcagcc aacgtcgggg
     5161 cggcaggccc tgccatagcc tcaggttact catatatact ttagattgat ttaaaacttc
     5221 atttttaatt taaaaggatc taggtgaaga tcctttttga taatctcatg accaaaatcc
     5281 cttaacgtga gttttcgttc cactgagcgt cagaccccgt agaaaagatc aaaggatctt
     5341 cttgagatcc tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac
     5401 cagcggtggt ttgtttgccg gatcaagagc taccaactct ttttccgaag gtaactggct
     5461 tcagcagagc gcagatacca aatactgttc ttctagtgta gccgtagtta ggccaccact
     5521 tcaagaactc tgtatcaccg cctacatacc tcgctctgct aatcctgtta ccagtggctg
     5581 ctgccagtgg cgataagtcg tgtcttaccg ggttggactc aagacgatag ttaccggata
     5641 aggcgcagcg gtcgggctga acggggggtt cgtgcacaca gcccagcttg gagcgaacga
     5701 cctacaccga actgagatac ctacagcgtg agctatgaga aagcgccacg cttcccgaag
     5761 ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg
     5821 agcttccagg gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc cacctctgac
     5881 ttgagcgtcg atttttgtga tgctcgtcag gggggcggag cctatggaaa aacgccagca
     5941 acgcggcctt tttacggttc ctggcctttt gctggccttt tgctcacatg ttctttcctg
     6001 cgttatcccc tgattctgtg gataaccgta ttaccgcca
//
