Skip to main content
. Author manuscript; available in PMC: 2023 Oct 28.
Published in final edited form as: J Nat Prod. 2022 Sep 29;85(10):2484–2518. doi: 10.1021/acs.jnatprod.2c00487

Figure 24.

Figure 24.

Site-specific chromosomal integration with iterative recombination and maker recycling. (A) Two transforming DNA fragments can be obtained by fusion PCR. The first DNA fragment contains a ~1 kb sequence (blue) homologous to the integration site of the host genome, a selectable marker (maker 1), an inducible PalcA (P), and the 5′ portion of gene A. The second DNA fragment contains the 3′ portion of gene A overlapping by ~1 kb with the 5′ portion and extending ~100 bp downstream of the stop codon (T), marker 2, and another ~1 kb sequence (blue) homologous to the host genome. HR of the two transforming DNA fragments (cotransfomation) results in the integration of the complete expression cassette into the host genome. (B) HR of transforming DNA containing ~900 bp of the 3′ portion of gene A plus ~100 bp of T, a P, a smaller gene (gene B) plus ~100 bp of T, marker 3, and a ~1 kb homologous sequence (blue) results in the integration of the expression cassette of gene B and recycling of marker 2.