LOCUS       sequence-380166-        5745 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses PH domain from PLC delta1 tagged with miRFP703 and FRB 
            mutant (T2098L) in mammalian cells..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5745)
  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 5745)
  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-FRB
FEATURES             Location/Qualifiers
     source          1..5745
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        81..384
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        385..588
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     539..559
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      1086..1095
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1092..2036
                     /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             2052..2330
                     /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    2377..2429
                     /label=MCS
                     /note="multiple cloning site"
     polyA_signal    2553..2674
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2590..2609)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2644..2663
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(2681..3136)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2818..2839)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3030..3049
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        3163..3267
                     /gene="bla"
                     /label=AmpR promoter
     promoter        3269..3626
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        3269..3460
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(3292..3312)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      3477..3612
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             3661..4455
                     /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             3661..4455
                     /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(3715..3734)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4325..4344
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(4643..4662)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    4687..4734
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      5063..5651
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5552..5571
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 accgtattac cgccatgcat tagttattaa tagtaatcaa ttacggggtc attagttcat
       61 agcccatata tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg
      121 cccaacgacc cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata
      181 gggactttcc attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta
      241 catcaagtgt atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc
      301 gcctggcatt atgcccagta catgacctta tgggactttc ctacttggca gtacatctac
      361 gtattagtca tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga
      421 tagcggtttg actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg
      481 ttttggcacc aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg
      541 caaatgggcg gtaggcgtgt acggtgggag gtctatataa gcagagctgg tttagtgaac
      601 cgtcagatcc gctagcgcta ccggactcag atctcgagct caagcttcga attccggcca
      661 ccatgtccgg actcagatcc cacgggctcc aggatgaccc ggaccttcag gcccttctga
      721 agggcagcca gcttctgaag gtgaagtcca gctcgtggcg tagggaacgc ttctacaagc
      781 tacaggagga ctgcaagacc atctggcagg aatctcgaaa ggtcatgagg tccccggagt
      841 cgcagctgtt ctccatcgag gacattcagg aggtacggat gggacaccgc acagaaggcc
      901 tggagaagtt tgcccgagac atccccgagg atcgatgctt ctccattgtc ttcaaggacc
      961 agcgcaacac cctagacctc attgccccat caccagctga cgctcagcac tgggtgcagg
     1021 gcctgcgcaa gatcatccac cactccggct ccatggacca gcggcagaag gcggatccac
     1081 cggtcgccac catggtagca ggtcatgcct ctggcagccc cgcattcggg accgcctctc
     1141 attcgaattg cgaacatgaa gagatccacc tcgccggctc gatccagccg catggcgcgc
     1201 ttctggtcgt cagcgaacat gatcatcgcg tcatccaggc cagcgccaac gccgcggaat
     1261 ttctgaatct cggaagcgta ctcggcgttc cgctcgccga gatcgacggc gatctgttga
     1321 tcaagatcct gccgcatctc gatcccaccg ccgaaggcat gccggtcgcg gtgcgctgcc
     1381 ggatcggcaa tccctctacg gagtactgcg gtctgatgca tcggcctccg gaaggcgggc
     1441 tgatcatcga actcgaacgt gccggcccgt cgatcgatct gtcaggcacg ctggcgccgg
     1501 cgctggagcg gatccgcacg gcgggttcac tgcgcgcgct gtgcgatgac accgtgctgc
     1561 tgtttcagca gtgcaccggc tacgaccggg tgatggtgta tcgtttcgat gagcaaggcc
     1621 acggcctggt attctccgag tgccatgtgc ctgggctcga atcctatttc ggcaaccgct
     1681 atccgtcgtc gctggtcccg cagatggcgc ggcagctgta cgtgcggcag cgcgtccgcg
     1741 tgctggtcga cgtcacctat cagccggtgc cgctggagcc gcggctgtcg ccgctgaccg
     1801 ggcgcgatct cgacatgtcg ggctgcttcc tgcgctcgat gtcgccgatc catctgcagt
     1861 tcctgaagga catgggcgtg cgcgccaccc tggcggtgtc gctggtggtc ggcggcaagc
     1921 tgtggggcct ggttgtctgt caccattatc tgccgcgctt catccgtttc gagctgcggg
     1981 cgatctgcaa acggctcgcc gaaaggatcg cgacgcggat caccgcgctt gagagcaagg
     2041 gagcaagtgg aatcctctgg catgagatgt ggcatgaagg cctggaagag gcatctcgtt
     2101 tgtactttgg ggaaaggaac gtgaaaggca tgtttgaggt gctggagccc ttgcatgcta
     2161 tgatggaacg gggcccccag actctgaagg aaacatcctt taatcaggcc tatggtcgag
     2221 atttaatgga ggcccaagag tggtgcagga agtacatgaa atcagggaat gtcaaggacc
     2281 tcctccaagc ctgggacctc tattatcatg tgttccgacg aatctcaaag agtgctggtg
     2341 gtagtgctgg tggtagtgct ggtggtactg gtggtcctcg agctcaagct tcgaattctg
     2401 cagtcgacgg taccgcgggc ccgggatcca ccggatctag ataactgatc ataatcagcc
     2461 ataccacatt tgtagaggtt ttacttgctt taaaaaacct cccacacctc cccctgaacc
     2521 tgaaacataa aatgaatgca attgttgttg ttaacttgtt tattgcagct tataatggtt
     2581 acaaataaag caatagcatc acaaatttca caaataaagc atttttttca ctgcattcta
     2641 gttgtggttt gtccaaactc atcaatgtat cttaaggcgt aaattgtaag cgttaatatt
     2701 ttgttaaaat tcgcgttaaa tttttgttaa atcagctcat tttttaacca ataggccgaa
     2761 atcggcaaaa tcccttataa atcaaaagaa tagaccgaga tagggttgag tgttgttcca
     2821 gtttggaaca agagtccact attaaagaac gtggactcca acgtcaaagg gcgaaaaacc
     2881 gtctatcagg gcgatggccc actacgtgaa ccatcaccct aatcaagttt tttggggtcg
     2941 aggtgccgta aagcactaaa tcggaaccct aaagggagcc cccgatttag agcttgacgg
     3001 ggaaagccgg cgaacgtggc gagaaaggaa gggaagaaag cgaaaggagc gggcgctagg
     3061 gcgctggcaa gtgtagcggt cacgctgcgc gtaaccacca cacccgccgc gcttaatgcg
     3121 ccgctacagg gcgcgtcagg tggcactttt cggggaaatg tgcgcggaac ccctatttgt
     3181 ttatttttct aaatacattc aaatatgtat ccgctcatga gacaataacc ctgataaatg
     3241 cttcaataat attgaaaaag gaagagtcct gaggcggaaa gaaccagctg tggaatgtgt
     3301 gtcagttagg gtgtggaaag tccccaggct ccccagcagg cagaagtatg caaagcatgc
     3361 atctcaatta gtcagcaacc aggtgtggaa agtccccagg ctccccagca ggcagaagta
     3421 tgcaaagcat gcatctcaat tagtcagcaa ccatagtccc gcccctaact ccgcccatcc
     3481 cgcccctaac tccgcccagt tccgcccatt ctccgcccca tggctgacta atttttttta
     3541 tttatgcaga ggccgaggcc gcctcggcct ctgagctatt ccagaagtag tgaggaggct
     3601 tttttggagg cctaggcttt tgcaaagatc gatcaagaga caggatgagg atcgtttcgc
     3661 atgattgaac aagatggatt gcacgcaggt tctccggccg cttgggtgga gaggctattc
     3721 ggctatgact gggcacaaca gacaatcggc tgctctgatg ccgccgtgtt ccggctgtca
     3781 gcgcaggggc gaccggttct ttttgtcaag accgacctgt ccggtgccct gaatgaactg
     3841 caagacgagg cagcgcggct atcgtggctg gccacgacgg gcgttccttg cgcagctgtg
     3901 ctcgacgttg tcactgaagc gggaagggac tggctgctat tgggcgaagt gccggggcag
     3961 gatctcctgt catctcacct tgctcctgcc gagaaagtat ccatcatggc tgatgcaatg
     4021 cggcggctgc atacgcttga tccggctacc tgcccattcg accaccaagc gaaacatcgc
     4081 atcgagcgag cacgtactcg gatggaagcc ggtcttgtcg atcaggatga tctggacgaa
     4141 gagcatcagg ggctcgcgcc agccgaactg ttcgccaggc tcaaggcgag catgcccgac
     4201 ggcgaggatc tcgtcgtgac ccatggcgat gcctgcttgc cgaatatcat ggtggaaaat
     4261 ggccgctttt ctggattcat cgactgtggc cggctgggtg tggcggaccg ctatcaggac
     4321 atagcgttgg ctacccgtga tattgctgaa gagcttggcg gcgaatgggc tgaccgcttc
     4381 ctcgtgcttt acggtatcgc cgctcccgat tcgcagcgca tcgccttcta tcgccttctt
     4441 gacgagttct tctgagcggg actctggggt tcgaaatgac cgaccaagcg acgcccaacc
     4501 tgccatcacg agatttcgat tccaccgccg ccttctatga aaggttgggc ttcggaatcg
     4561 ttttccggga cgccggctgg atgatcctcc agcgcgggga tctcatgctg gagttcttcg
     4621 cccaccctag ggggaggcta actgaaacac ggaaggagac aataccggaa ggaacccgcg
     4681 ctatgacggc aataaaaaga cagaataaaa cgcacggtgt tgggtcgttt gttcataaac
     4741 gcggggttcg gtcccagggc tggcactctg tcgatacccc accgagaccc cattggggcc
     4801 aatacgcccg cgtttcttcc ttttccccac cccacccccc aagttcgggt gaaggcccag
     4861 ggctcgcagc caacgtcggg gcggcaggcc ctgccatagc ctcaggttac tcatatatac
     4921 tttagattga tttaaaactt catttttaat ttaaaaggat ctaggtgaag atcctttttg
     4981 ataatctcat gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg tcagaccccg
     5041 tagaaaagat caaaggatct tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc
     5101 aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc
     5161 tttttccgaa ggtaactggc ttcagcagag cgcagatacc aaatactgtt cttctagtgt
     5221 agccgtagtt aggccaccac ttcaagaact ctgtatcacc gcctacatac ctcgctctgc
     5281 taatcctgtt accagtggct gctgccagtg gcgataagtc gtgtcttacc gggttggact
     5341 caagacgata gttaccggat aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac
     5401 agcccagctt ggagcgaacg acctacaccg aactgagata cctacagcgt gagctatgag
     5461 aaagcgccac gcttcccgaa gggagaaagg cggacaggta tccggtaagc ggcagggtcg
     5521 gaacaggaga gcgcacgagg gagcttccag ggggaaacgc ctggtatctt tatagtcctg
     5581 tcgggtttcg ccacctctga cttgagcgtc gatttttgtg atgctcgtca ggggggcgga
     5641 gcctatggaa aaacgccagc aacgcggcct ttttacggtt cctggccttt tgctggcctt
     5701 ttgctcacat gttctttcct gcgttatccc ctgattctgt ggata
//
