LOCUS       sequence-380168-        5799 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses PH domain from PLC delta1 tagged with mCherry and two FRB 
            mutants (T2098L) in mammalian cells..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5799)
  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 5799)
  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-mCherry-FRBx2
FEATURES             Location/Qualifiers
     source          1..5799
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        83..386
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        387..590
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     541..561
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      1088..1097
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1094..1801
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG
                     TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF
                     EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK
                     GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERA
                     EGRHSTGGMDELYK"
     primer_bind     complement(1239..1257)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     1507..1526
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     CDS             1814..2092
                     /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             2108..2386
                     /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    2433..2485
                     /label=MCS
                     /note="multiple cloning site"
     polyA_signal    2609..2730
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2646..2665)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2700..2719
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(2737..3192)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2874..2895)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3086..3105
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        3219..3323
                     /gene="bla"
                     /label=AmpR promoter
     promoter        3325..3682
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        3325..3516
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(3348..3368)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      3533..3668
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             3717..4511
                     /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             3717..4511
                     /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(3771..3790)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4381..4400
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(4699..4718)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    4743..4790
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      5119..5707
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5608..5627
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 taaccgtatt accgccatgc attagttatt aatagtaatc aattacgggg tcattagttc
       61 atagcccata tatggagttc cgcgttacat aacttacggt aaatggcccg cctggctgac
      121 cgcccaacga cccccgccca ttgacgtcaa taatgacgta tgttcccata gtaacgccaa
      181 tagggacttt ccattgacgt caatgggtgg agtatttacg gtaaactgcc cacttggcag
      241 tacatcaagt gtatcatatg ccaagtacgc cccctattga cgtcaatgac ggtaaatggc
      301 ccgcctggca ttatgcccag tacatgacct tatgggactt tcctacttgg cagtacatct
      361 acgtattagt catcgctatt accatggtga tgcggttttg gcagtacatc aatgggcgtg
      421 gatagcggtt tgactcacgg ggatttccaa gtctccaccc cattgacgtc aatgggagtt
      481 tgttttggca ccaaaatcaa cgggactttc caaaatgtcg taacaactcc gccccattga
      541 cgcaaatggg cggtaggcgt gtacggtggg aggtctatat aagcagagct ggtttagtga
      601 accgtcagat ccgctagcgc taccggactc agatctcgag ctcaagcttc gaattccggc
      661 caccatgtcc ggactcagat cccacgggct ccaggatgac ccggaccttc aggcccttct
      721 gaagggcagc cagcttctga aggtgaagtc cagctcgtgg cgtagggaac gcttctacaa
      781 gctacaggag gactgcaaga ccatctggca ggaatctcga aaggtcatga ggtccccgga
      841 gtcgcagctg ttctccatcg aggacattca ggaggtacgg atgggacacc gcacagaagg
      901 cctggagaag tttgcccgag acatccccga ggatcgatgc ttctccattg tcttcaagga
      961 ccagcgcaac accctagacc tcattgcccc atcaccagct gacgctcagc actgggtgca
     1021 gggcctgcgc aagatcatcc accactccgg ctccatggac cagcggcaga aggcggatcc
     1081 accggtcgcc accatggtga gcaagggcga ggaggataac atggccatca tcaaggagtt
     1141 catgcgcttc aaggtgcaca tggagggctc cgtgaacggc cacgagttcg agatcgaggg
     1201 cgagggcgag ggccgcccct acgagggcac ccagaccgcc aagctgaagg tgaccaaggg
     1261 tggccccctg cccttcgcct gggacatcct gtcccctcag ttcatgtacg gctccaaggc
     1321 ctacgtgaag caccccgccg acatccccga ctacttgaag ctgtccttcc ccgagggctt
     1381 caagtgggag cgcgtgatga acttcgagga cggcggcgtg gtgaccgtga cccaggactc
     1441 ctccctgcag gacggcgagt tcatctacaa ggtgaagctg cgcggcacca acttcccctc
     1501 cgacggcccc gtaatgcaga agaagaccat gggctgggag gcctcctccg agcggatgta
     1561 ccccgaggac ggcgccctga agggcgagat caagcagagg ctgaagctga aggacggcgg
     1621 ccactacgac gctgaggtca agaccaccta caaggccaag aagcccgtgc agctgcccgg
     1681 cgcctacaac gtcaacatca agttggacat cacctcccac aacgaggact acaccatcgt
     1741 ggaacagtac gaacgcgccg agggccgcca ctccaccggc ggcatggacg agctgtacaa
     1801 gggagcaagt ggaatcctct ggcatgagat gtggcatgaa ggcctggaag aggcatctcg
     1861 tttgtacttt ggggaaagga acgtgaaagg catgtttgag gtgctggagc ccttgcatgc
     1921 tatgatggaa cggggccccc agactctgaa ggaaacatcc tttaatcagg cctatggtcg
     1981 agatttaatg gaggcccaag agtggtgcag gaagtacatg aaatcaggga atgtcaagga
     2041 cctcctccaa gcctgggacc tctattatca tgtgttccga cgaatctcaa agaagggagc
     2101 aagtggaatc ctctggcatg agatgtggca tgaaggcctg gaagaggcat ctcgtttgta
     2161 ctttggggaa aggaacgtga aaggcatgtt tgaggtgctg gagcccttgc atgctatgat
     2221 ggaacggggc ccccagactc tgaaggaaac atcctttaat caggcctatg gtcgagattt
     2281 aatggaggcc caagagtggt gcaggaagta catgaaatca gggaatgtaa aggacctcct
     2341 ccaagcctgg gacctctatt atcatgtgtt ccgacgaatc tcaaagagtg ctggtggtag
     2401 tgctggtggt agtgctggtg gtactggtgg tcctcgagct caagcttcga attctgcagt
     2461 cgacggtacc gcgggcccgg gatccaccgg atctagataa ctgatcataa tcagccatac
     2521 cacatttgta gaggttttac ttgctttaaa aaacctccca cacctccccc tgaacctgaa
     2581 acataaaatg aatgcaattg ttgttgttaa cttgtttatt gcagcttata atggttacaa
     2641 ataaagcaat agcatcacaa atttcacaaa taaagcattt ttttcactgc attctagttg
     2701 tggtttgtcc aaactcatca atgtatctta aggcgtaaat tgtaagcgtt aatattttgt
     2761 taaaattagc gttaaatttt tgttaaatca gctcattttt taaccaatag gccgaaatcg
     2821 gcaaaatccc ttataaatca aaagaataga ccgagatagg gttgagtgtt gttccagttt
     2881 ggaacaagag tccactatta aagaacgtgg actccaacgt caaagggcga aaaaccgtct
     2941 atcagggcga tggcccacta cgtgaaccat caccctaatc aagttttttg gggtcgaggt
     3001 gccgtaaagc actaaatcgg aaccctaaag ggagcccccg atttagagct tgacggggaa
     3061 agccggcgaa cgtggcgaga aaggaaggga agaaagcgaa aggagcgggc gctagggcgc
     3121 tggcaagtgt agcggtcacg ctgcgcgtaa ccaccacacc cgccgcgctt aatgcgccgc
     3181 tacagggcgc gtcaggtggc acttttcggg gaaatgtgcg cggaacccct atttgtttat
     3241 ttttctaaat acattcaaat atgtatccgc tcatgagaca ataaccctga taaatgcttc
     3301 aataatattg aaaaaggaag agtcctgagg cggaaagaac cagctgtgga atgtgtgtca
     3361 gttagggtgt ggaaagtccc caggctcccc agcaggcaga agtatgcaaa gcatgcatct
     3421 caattagtca gcaaccaggt gtggaaagtc cccaggctcc ccagcaggca gaagtatgca
     3481 aagcatgcat ctcaattagt cagcaaccat agtcccgccc ctaactccgc ccatcccgcc
     3541 cctaactccg cccagttccg cccattctcc gccccatggc tgactaattt tttttattta
     3601 tgcagaggcc gaggccgcct cggcctctga gctattccag aagtagtgag gaggcttttt
     3661 tggaggccta ggcttttgca aagatcgatc aagagacagg atgaggatcg tttcgcatga
     3721 ttgaacaaga tggattgcac gcaggttctc cggccgcttg ggtggagagg ctattcggct
     3781 atgactgggc acaacagaca atcggctgct ctgatgccgc cgtgttccgg ctgtcagcgc
     3841 aggggcgccc ggttcttttt gtcaagaccg acctgtccgg tgccctgaat gaactgcaag
     3901 acgaggcagc gcggctatcg tggctggcca cgacgggcgt tccttgcgca gctgtgctcg
     3961 acgttgtcac tgaagcggga agggactggc tgctattggg cgaagtgccg gggcaggatc
     4021 tcctgtcatc tcaccttgct cctgccgaga aagtatccat catggctgat gcaatgcggc
     4081 ggctgcatac gcttgatccg gctacctgcc cattcgacca ccaagcgaaa catcgcatcg
     4141 agcgagcacg tactcggatg gaagccggtc ttgtcgatca ggatgatctg gacgaagagc
     4201 atcaggggct cgcgccagcc gaactgttcg ccaggctcaa ggcgagcatg cccgacggcg
     4261 aggatctcgt cgtgacccat ggcgatgcct gcttgccgaa tatcatggtg gaaaatggcc
     4321 gcttttctgg attcatcgac tgtggccggc tgggtgtggc ggaccgctat caggacatag
     4381 cgttggctac ccgtgatatt gctgaagagc ttggcggcga atgggctgac cgcttcctcg
     4441 tgctttacgg tatcgccgct cccgattcgc agcgcatcgc cttctatcgc cttcttgacg
     4501 agttcttctg agcgggactc tggggttcga aatgaccgac caagcgacgc ccaacctgcc
     4561 atcacgagat ttcgattcca ccgccgcctt ctatgaaagg ttgggcttcg gaatcgtttt
     4621 ccgggacgcc ggctggatga tcctccagcg cggggatctc atgctggagt tcttcgccca
     4681 ccctaggggg aggctaactg aaacacggaa ggagacaata ccggaaggaa cccgcgctat
     4741 gacggcaata aaaagacaga ataaaacgca cggtgttggg tcgtttgttc ataaacgcgg
     4801 ggttcggtcc cagggctggc actctgtcga taccccaccg agaccccatt ggggccaata
     4861 cgcccgcgtt tcttcctttt ccccacccca ccccccaagt tcgggtgaag gcccagggct
     4921 cgcagccaac gtcggggcgg caggccctgc catagcctca ggttactcat atatacttta
     4981 gattgattta aaacttcatt tttaatttaa aaggatctag gtgaagatcc tttttgataa
     5041 tctcatgacc aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag accccgtaga
     5101 aaagatcaaa ggatcttctt gagatccttt ttttctgcgc gtaatctgct gcttgcaaac
     5161 aaaaaaacca ccgctaccag cggtggtttg tttgccggat caagagctac caactctttt
     5221 tccgaaggta actggcttca gcagagcgca gataccaaat actgttcttc tagtgtagcc
     5281 gtagttaggc caccacttca agaactctgt atcaccgcct acatacctcg ctctgctaat
     5341 cctgttacca gtggctgctg ccagtggcga taagtcgtgt cttaccgggt tggactcaag
     5401 acgatagtta ccggataagg cgcagcggtc gggctgaacg gggggttcgt gcacacagcc
     5461 cagcttggag cgaacgacct acaccgaact gagataccta cagcgtgagc tatgagaaag
     5521 cgccacgctt cccgaaggga gaaaggcgga caggtatccg gtaagcggca gggtcggaac
     5581 aggagagcgc acgagggagc ttccaggggg aaacgcctgg tatctttata gtcctgtcgg
     5641 gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct
     5701 atggaaaaac gccagcaacg cggccttttt acggttcctg gccttttgct ggccttttgc
     5761 tcacatgttc tttcctgcgt tatcccctga ttctgtgga
//
