Recombinases constructs and intrachromosomal recombination (ICR) assay. a The coding sequences of bacterial (RecA, RecG and RuvC) and human (Rad51, Rad52 and DMC1) recombinases (white boxes) were amplified by PCR. Bacterial recombinases were tagged at their N-terminus with SV40 nuclear localisation signal (hatched box). The multigene constructs were made by inserting the 2A sequence from foot and mouth disease virus (black box) between different recombinases in a single open reading frame. These fragments (single and multiple genes) were inserted between the CaMV 35S promoter (35Sp) and nopaline synthase terminator (NosT) of pGSC plasmid containing left (LB) and right (RB) T-DNA borders and the sulphonamide resistant gene (Sul1) for plants selection. Different elements of these constructs schematics are not drawn to scale. b The transgene used as ICR substrate in the tobacco transgenic line N1DC4 is formed of two defective overlapping fragments of β-glucuronidase (GUS) separated by hygromycin resistance gene (hpt). ICR restore a functional GUS gene that can be detected by histochemical staining as blue spots on seedlings (left) and blue pollen (right)
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