To construct mammalian expression plasmids, the respective genes of FPs were PCR-amplified as AgeI-NotI fragments and swapped with a gene encoding EGFP in the pEGFP-N1 plasmid (Clontech). IFP2.0-N1 and mIFP-N1 plasmids were acquired from Addgene (#54785 and #54620, respectively).
For protein tagging and labelling of intracellular structures study, miRFPs were amplified, digested with restriction enzymes and then swapped with mTagBFP2 either as C- (for α-tubulin and clathrin) or N-terminal fusions (for keratin, α-actinin, LifeAct, EB3, myosin, vimentin, clathrin, LAMP1, zyxin, H2B and mitochondrial signal) as previously described50 (link). C-terminal fusions (SGGGG)n linker was increased to 30 amino acids. N-terminal fusions linker length was left unchanged.
To create an IκBα reporter plasmid (CMV-IκBα-miRFP703), we used a CMV-IκBα-FLuc plasmid kindly provided by S. Achilefu and D. Piwnica-Worms. A FLuc gene was replaced with one of the miRFP genes. Kozak sequence was deleted in the CMV-IκBα-miRFP703 and CMV-miRFP control plasmids.
miSplit670 and miSplit709 reporter plasmids, which are pC4-RHE-PAS, pC4EN-F1-mGAF670 and pC4EN-F1-mGAF709, were constructed from an iSplit plasmids25 (link) by swapping either PAS or GAF domains. A linker -ggggsggggs- was left unchanged. Where appropriate, an NLS sequence in the pC4EN-F1 plasmid was deleted by site-directed mutagenesis.
For mRNA labelling, a CMV-PAS-MCP plasmid was constructed as follows. PAS-ggggsggggs- without STOP codon was amplified as a single fragment and inserted into the C1 vector backbone using AgeI and KpnI sites, MCP was amplified from an ubc-nls-ha-MCP-VenusN-nls-ha-PCP-VenusC plasmid (Addgene, #52985) and inserted at KpnI and BamHI sites. The cmv-PCP-mGAF670 and cmv-PCP-mGAF709 plasmids were constructed as follows. A PCP without STOP codon was amplified from an ubc-nls-ha-MCP-VenusN-nls-ha-PCP-VenusC plasmid and then inserted into the C1 vector backbone using AgeI and EcoRI restriction sites. A -ggggsggggs-miGAF was amplified as a single fragment and inserted using EcoRI and KpnI sites. A phage-cmv-cfp-12xMBS-PBS was obtained by swapping a 12xMBS-PBS fragment from a Pcr4-12xMBS-PBS (Addgene, #52984) with 24xMS2 in a phage-cmv-cfp-24xms2 plasmid (Addgene, #40651). An ubc-nls-ha-MCP-VenusN-nls-ha-PCP-VenusC, a phage-cmv-cfp-24xMS2 and a Pcr4-12xMBS-PBS plasmids were gifts from B. Wu and R. Singer.
Plasmids encoding several green-red Fucci cell cycle reporters were provided by A. Miyawaki. The mKO2 and mAG genes fused with hCdt1(30–120), hCdt1(1/100), hGem(1/110) and hGem(1/60) sequences in the pCSII-EF-MCS plasmids were swapped with the miRFP709 or miRFP670v1 genes.
For protein tagging and labelling of intracellular structures study, miRFPs were amplified, digested with restriction enzymes and then swapped with mTagBFP2 either as C- (for α-tubulin and clathrin) or N-terminal fusions (for keratin, α-actinin, LifeAct, EB3, myosin, vimentin, clathrin, LAMP1, zyxin, H2B and mitochondrial signal) as previously described50 (link). C-terminal fusions (SGGGG)n linker was increased to 30 amino acids. N-terminal fusions linker length was left unchanged.
To create an IκBα reporter plasmid (CMV-IκBα-miRFP703), we used a CMV-IκBα-FLuc plasmid kindly provided by S. Achilefu and D. Piwnica-Worms. A FLuc gene was replaced with one of the miRFP genes. Kozak sequence was deleted in the CMV-IκBα-miRFP703 and CMV-miRFP control plasmids.
miSplit670 and miSplit709 reporter plasmids, which are pC4-RHE-PAS, pC4EN-F1-mGAF670 and pC4EN-F1-mGAF709, were constructed from an iSplit plasmids25 (link) by swapping either PAS or GAF domains. A linker -ggggsggggs- was left unchanged. Where appropriate, an NLS sequence in the pC4EN-F1 plasmid was deleted by site-directed mutagenesis.
For mRNA labelling, a CMV-PAS-MCP plasmid was constructed as follows. PAS-ggggsggggs- without STOP codon was amplified as a single fragment and inserted into the C1 vector backbone using AgeI and KpnI sites, MCP was amplified from an ubc-nls-ha-MCP-VenusN-nls-ha-PCP-VenusC plasmid (Addgene, #52985) and inserted at KpnI and BamHI sites. The cmv-PCP-mGAF670 and cmv-PCP-mGAF709 plasmids were constructed as follows. A PCP without STOP codon was amplified from an ubc-nls-ha-MCP-VenusN-nls-ha-PCP-VenusC plasmid and then inserted into the C1 vector backbone using AgeI and EcoRI restriction sites. A -ggggsggggs-miGAF was amplified as a single fragment and inserted using EcoRI and KpnI sites. A phage-cmv-cfp-12xMBS-PBS was obtained by swapping a 12xMBS-PBS fragment from a Pcr4-12xMBS-PBS (Addgene, #52984) with 24xMS2 in a phage-cmv-cfp-24xms2 plasmid (Addgene, #40651). An ubc-nls-ha-MCP-VenusN-nls-ha-PCP-VenusC, a phage-cmv-cfp-24xMS2 and a Pcr4-12xMBS-PBS plasmids were gifts from B. Wu and R. Singer.
Plasmids encoding several green-red Fucci cell cycle reporters were provided by A. Miyawaki. The mKO2 and mAG genes fused with hCdt1(30–120), hCdt1(1/100), hGem(1/110) and hGem(1/60) sequences in the pCSII-EF-MCS plasmids were swapped with the miRFP709 or miRFP670v1 genes.
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