Map of the targeting plasmid backbone p NosTerFRT-neo and GAPc2ox1/mgfp5-ER targeting cassettes. A: Structure of the targeting plasmid backbone pNosTerFRT-neo, showing the relative positions of the Nopaline synthase polyA signal sequence (NosTer), the FRT-mPGK-Tn5-neo-FRT selection cassette and the restriction sites mapped up and downstream of the NosTer sequence. Upstream sites may be used for cloning any GOI cDNAs to create targeting constructs. Sites shown in green were introduced with the NosTer fragment; all other sites originate from PolyGFP3. Unique sites are underlined. B: Representations of GOI sequences with available upstream and downstream restriction sites as appropriate, together with the cloning strategies employed to clone them into pNosTerFRT-neo to make the targeting cassette constructs.