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. Author manuscript; available in PMC: 2019 Apr 10.
Published in final edited form as: ACS Chem Biol. 2016 Jun 21;11(8):2275–2284. doi: 10.1021/acschembio.6b00213

Figure 2.

Figure 2.

Promoter replacements and strategy. (A) A schematic diagram of the replacements made of the native promoters of the inp cluster. (B) and (C) Promoter replacement with marker recycling strategy used to replace the promoters of inpE and inpF. (B) The endogenous promoter is replaced with a fragment carrying AfpyrG, a marker that can be selected for and against, flanked by direct repeats of the alcA promoter [alcA(p)]. (C) Spontaneous recombination between the alcA(p) repeats results in excision of AfpyrG, which is selected for on 5-fluoroorotic acid resulting in a single copy of alcA(p) driving expression of the target gene, inpE. Since AfpyrG is removed, it can be reused as a selectable marker.