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. 2020 Jun 18;8:510. doi: 10.3389/fbioe.2020.00510

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Copyright © 2020 Monteiro, Sanches-Medeiros, Westmann and Silva-Rocha.

This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

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Strategies to construct synthetic complex promoters. (A) DNA sequences harboring the consensus sequence for IHF or Fis binding were selected, along with a control sequence that cannot be recognized by any TF. (B) Double-stranded DNA fragments were produced with cohesive ends specific for each promoter position (numbered from 1 to 4) and assembled together with a weak core promoter harboring the −35/−10 boxes for RNAP recognition (Guazzaroni and Silva-Rocha, 2014). (C) The fragments were cloned into a promoter probe vector (pMR1) harboring resistance to chloramphenicol (CmR), a medium-copy number origin of replication (p15a), and two reporter genes (mCherry and GFPlva). The libraries were introduced into wild-type and mutant strains of E. coli from the KEIO collection (Baba et al., 2006). The resultant strains were analyzed at the population level in a plate reader and the data processed using script in R.