The plasmid pcDNA5-FRT-TO-IgM-1.7k-PY-CFPPTS-24MS2SL–I–SceI–24PP7SL was constructed with the restriction site for I–SceI inserted within the exon II of a mouse immunoglobulin μ (IgM) reporter gene, referred to as EX2 reporter. The 24 PP7 stem-loop sequence was excised from pCR4-24xPP7SL [Addgene plasmid no. 31864 (28)] by Bam HI and Bgl II digest and inserted into the Bam HI site of pCMV5-24xMS2SL, generating pCMV5-24xMS2SL-24xPP7SL. To insert the I–SceI restriction site in pCMV5-24xMS2SL-24xPP7SL, two oligo sequences encoding the I–Sce I and two Bam HI sites were hybridized (sequences are shown in table S1) and amplified by polymerase chain reaction (PCR), purified (NZYGelpure kit, NZYTech), and Bam HI digested and ligated into the same site to generate pCMV5-24xMS2SL–I–SceI–24xPP7SL. The Ecl136II– and AleI–generated 24xMS2SL-I–SceI–24xPP7SL fragment was cloned into the blunted Xho I site of the pcDNA5-FRT-TO-IgM-1.7k-PY-CFPPTS reporter (10) using the PaperClip protocol (29). The plasmid pcDNA5-FRT-TO-I–SceI–ExI-24bcMS2-IgM-1.7K.PY-CFPPTS was designed with the I–SceI site in the promoter proximal region of the IgM reporter gene, referred to as the PROP reporter. An array of two tet operator (tetO) sequences followed by an I–SceI site, 24 MS2 stem loops from two nonidentical stem-loop sequences [as described for Addgene plasmid no. 31865 (30)], including five additional nonidentical spacer sequences, to further decrease redundancy was de novo designed (synthesis by GeneArt, Invitrogen). The 2xtetO-(I–SceI)-24 MS2SL fragment was cloned by Ecl 136II and Hind II digest into the same sites in pcDNA5-FRT-TO-IgM-1.7k-PY-CFPPTS. The third reporter gene referred to as EX2-AS was designed on the basis of the human ubiquitin B gene (HGNC:12463; ENST00000302182.7) with an I–SceI site in exon II followed by an open reading frame of two head-to-tail ubiquitin units (synthesis by GeneArt, Invitrogen). The 24 MS2 stem-loop sequence with five nonidentical spacers [lacking the 2xtetO-(I–SceI) part] was ligated in antisense direction into exon I of the reporter gene and the 24 PP7 stem-loop sequence in sense orientation into the exon II 3′ untranslated region. The complete reporter unit was ligated into the pcDNA5-FRT-TO (Invitrogen, Thermo Fisher Scientific) backbone. The plasmids encoding tandem dimers of MS2 and PP7 coat proteins (named MCP and PCP thereafter, respectively) fused to fluorescent proteins were constructed the following way: First, pEGFP-C1 was Vsp I and Bsp 1407I digested, blunted, and religated to remove the CMV promoter and enhanced GFP (EGFP) sequences. The Eco RI–Not I fragment (ubiquitin C promoter) from phage-ubc-nls-ha-tdPCP-gfp [Addgene plasmid no. 40650 (31)] was ligated into the Eco RI–Bsp 120I sites generating a pUBC vector. Upstream restriction sites were removed by religating blunted EcoRI+BglII sites and EGFP including multicloning site were reinserted by ligating a Bsh TI–Xba I EGFP fragment from pEGFP-C1 into Cfr9I–Xba I sites in pUBC to create pUBC-GFP-C1. The pUBC-GFP-nls-tdMCP-GFP plasmid (referred to as MS2-GFP) was generated by ligating a Bsp120I+BglII fragment from phage-ubc-nls-ha-tdMCP-gfp [Addgene plasmid no. 40649 (31)] into Bsp120I+BamHI sites of pUBC-GFP-C1. pUBC-GFP-nls-tdPCP-GFP plasmid (referred to as PP7-GFP) was generated by ligating a Bsp120I+SmaI fragment from phage-ubc-nls-ha-tdPCP-gfp [Addgene plasmid no. 40650 (31)] into Bsp120I+KspAI sites of pUBC-GFP-C1. The pUBC-TagRFPt-nls-tdMCP-TagRFPt (referred to as MS2-RFP) was cloned by PCR amplification of the TagRFPt sequence from phage-UBC-nls-ha-2XMCP-TagRFPt [Addgene plasmid no. 64541 (32)] using primers with Ale I and Bgl II sites (sequences are shown in table S1) to ligate it into the same sites of pUBC-GFP-C1 to generate pUBC-TagRFPt. The Bsp 120I+Bam HI fragment containing nls-tdMCP-TagRFPt from Addgene plasmid no. 64541 was ligated into the same sites in pUBC-TagRFPt. The pI–SceI–GR-near-infrared fluorescent protein (iRFP) construct was made by removing RFP from pI–SceI–GR-RFP [Addgene plasmid no. 17654 (33)] by religating Ecl136II+blunted AarI sites, creating an in-frame deletion. Next, the BamHI+BglII fragment from pIRES-H2BiRFP was ligated into the same sites of the I–SceI–GR containing plasmid to generate pI–Sce I–GR-iRFP.

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