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. 2016 Aug 18;6:31601. doi: 10.1038/srep31601

Figure 1. Generation of chromosomal fusions.

Figure 1

Targeting DNA fragments consisting of a kanamycin resistance cassette (aph), a linker of 16 codons (L16) fused 5′ to the coding sequence of either HaloTag or SNAP-tag, were amplified from template vectors p3780 or p3779, respectively. Primers possess 40 bp homology extension for the desired integration site (vertical and diagonal hatching). The targeting constructs were introduced into Salmonella wild type (WT) harbouring plasmid pKD46 for the expression of λ Red recombinase. Due to the design of targeting constructs, Red-mediated recombination results in chromosomal fusions of the gene of interest with either L16-HaloTag aph or L16-SNAP-tag aph replacing the stop codon of the gene of interest. Care should be taken that the RBS of the downstream gene is not affected by the gene fusion. The aph resistance gene may be removed by FLP-mediated recombination.