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. 2019 Mar 29;2(2):e201800108. doi: 10.26508/lsa.201800108

Figure S1. Conditional expression of BZLF1 in Raji cells.

Figure S1.

(A) The conditional expression plasmid p4816 encodes the unmodified full-length BZLF1 (aa 1–245) protein. The conditional expression plasmids p5693 and p5694 encode FLAG- and tandem Strep-tagged full-length BZLF1 (aa 1–245) and the activation domain (AD) truncated BZLF1 (aa 175–236) protein also termed bZIP, respectively. The bicistronic coding sequences of the tetracycline-controlled repressor rTS and transactivator rtTA2S-M2 are shown. The addition of doxycycline induces bidirectional transcription and concomitant expression of three transgenes: enhanced GFP (eGFP), the human truncated NGF-receptor (tNGF-R), and BZLF1. An internal ribosomal entry site (IRES) separates tNGF-R and eGFP. β-lactamase (amp) and puromycin N-acetyl-transferase (puro) serve as resistance genes in bacteria and Raji cells, respectively. DNA replication in E. coli initiates at the prokaryotic origin of replication (ori). Epstein–Barr nuclear antigen 1 (EBNA1) binds to the origin of plasmid replication (oriP) and supports extrachromosomal DNA replication of the plasmids in Raji cells. (B) The conditional expression plasmids (p4816, p5693, and p5694) were stably introduced into Raji cells, and single-cell clones were selected by limiting dilution, expanded, and analyzed further. GFP expression in Raji cells was a measure of the induced expression of BZLF1. Upon addition of 100 ng/ml doxycycline to the cells for 15 h, GFP expression was analyzed by flow cytometry.