Expression vectors for RelA (pcDNA3-FLAG-RelA and pcDNA3-HA-RelA), NF-κB–responsive reporter (pNF-κB Luc), and Renilla luciferase expression (phRL-TK) vectors were described previously (41). The HA (hemagglutinin)–IKKβ expression vector was provided by H. Ichijo (The University of Tokyo, Japan). The HA-IKKβSS/EE expression vector was constructed by site-directed mutagenesis. The expression vectors for FLAG-tagged Cas variants (Cas full-length, Cas15YF, Cas1-457, Cas1-638, Cas1-671, Cas1-738, Cas458-874, Cas639-874, Cas672-874, and Cas739-874) were constructed using pcDNA3-FLAG as a parent vector. The expression vector for p300 (CMVβ-p300-HA) (42) was a gift from R. H. Goodman (Oregon Health and Science University, OR, USA). The expression vector for NES-Cas was constructed by cloning an NES sequence (MNLVDLQKKLEELELDEQQ) (43) attached Cas into pcDNA3. NF-κB RelA K122/123R, K218R, K310R, and K314/315R were generated by site-directed mutagenesis and cloned into the pcDNA3-FLAG vector. The Myc-tagged RelA WT and K310R cloned in pcDNA3 (provided by J. Anrather) (44) were subcloned into pBabe-hygromycin to construct the retroviral expression vectors for them.

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