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However, the mechanistic basis of CMV\u2010mediated transcriptional activation in HEK293 cells is unknown and consequently there are no strategies to engineer CMV for controlled expression of recombinant genes. Extensive bioinformatic analyses of transcription factor regulatory elements (TFREs) within the human CMV sequence and transcription factor mRNAs within the HEK293 transcriptome revealed 80 possible regulatory interactions. Through in vitro functional testing using reporter constructs harboring discrete TFREs or CMV deletion variants we identified key TFRE components and clusters of TFREs (cis\u2010regulatory modules) within the CMV sequence. Our data reveal that CMV activity in HEK293 cells is a function of the promoters various constituent TFREs including AhR:ARNT, CREB, E4F, Sp1, ZBED1, JunB, c\u2010Rel, and NF\u2010\u03baB. We also identified critical Sp1\u2010dependent upstream activator elements near the transcriptional start site that were required for efficient transcription and YY1 and RBP\u2010J\u03ba binding sites that mediate transrepression. Our study shows for the first time that novel, compact CMV\u2010derived promoters can be engineered that exhibit up to 50% higher transcriptional efficiency (activity per unit DNA sequence) or 14% increase in total activity compared to the wild\u2010type counterpart.<\/jats:p>","DOI":"10.1002\/biot.202200062","type":"journal-article","created":{"date-parts":[[2022,4,28]],"date-time":"2022-04-28T12:55:05Z","timestamp":1651150505000},"source":"Crossref","is-referenced-by-count":28,"title":["Engineering of the CMV promoter for controlled expression of recombinant genes in HEK293 cells"],"prefix":"10.1002","volume":"17","author":[{"ORCID":"https:\/\/orcid.org\/0000-0001-9933-5764","authenticated-orcid":false,"given":"Yusuf B.","family":"Johari","sequence":"first","affiliation":[{"name":"Department of Chemical and Biological Engineering University of Sheffield  Sheffield UK"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-6046-7687","authenticated-orcid":false,"given":"Joseph M.","family":"Scarrott","sequence":"additional","affiliation":[{"name":"Department of Chemical and Biological Engineering University of Sheffield  Sheffield UK"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-4437-3231","authenticated-orcid":false,"given":"Thilo H.","family":"Pohle","sequence":"additional","affiliation":[{"name":"Department of Chemical and Biological Engineering University of Sheffield  Sheffield UK"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ping","family":"Liu","sequence":"additional","affiliation":[{"name":"Cell Line Development REGENXBIO Inc.  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