Fig. 2: Identification of a loxF8 specific heterodimer clone.

a Overview of the clone selection procedure. Active recombinases for loxF8-L and loxF8-R are cloned to an inducible lentiviral expression plasmid. Human cells are infected with viral particles each coding for a recombinase from the active libraries. After 21 days of constant expression of the recombinases, genomic DNA of the cells is isolated and recombinase genes are retrieved via PCR. Recombinases are combined in a heterodimer and single clones are expressed in E. coli. Activity of the clones is analyzed using a PCR-based assay. The upper band represents the PCR product generated from non-recombined substrate (line with two triangles), whereas the lower band shows the PCR product generated from recombined substrate (line with one triangle). b Plasmid-based activity assay of the loxF8 candidate recombinase heterodimer (D7), Cre and Brec1. Recombinases were expressed in E. coli at four different L-arabinose concentrations (0, 1, 10, and 100 μg/ml). The upper band represents non-recombined substrate (line with two triangles), whereas the lower band shows the recombined plasmid (line with one triangle). The marker lane (M) for 4 kb and 5 kb is shown with arrows. c Mutation analysis of the D7 monomers (one-letter code). The amino acid sequence of Cre recombinase is shown as a reference. Orange and blue letters represent changes found in the left monomer and in the right monomer, respectively. Secondary structure elements are indicated, with alpha-helices displayed as cylinders with letters and beta-sheets represented as numbered arrows. Asterisks denote stop codons. Parts of the figure were created with BioRender.com. Source data are provided as a Source data file.