Existing primer_bind annotation(s) with valid type IIS cut site(s) on the extension Geneious will also assume that you have this “sticky ended” DNA available and so will not design primers for PCR.ģ.
If Geneious detects a pair of appropriately orientated type IIS sites with unique overhangs, then it will assume you wish to use them.If required, Geneious will then design a primer pair for PCR amplification of each Part. Geneious will then choose one, or a combination of these, in order of precedence (see rules 1 to 6 below) to define the insert boundaries to be used for Golden gate recombination.
Geneious will analyse your backbone (if defined), and each sequence passed to it, and will detect existing type IIS restriction sites, overhang annotations, primer_bind annotations and blunt ends. Note that the Geneious Golden gate tool currently does not allow the use of multiple type IIS restriction enzymes for assembly. Usually the “receiving” type IIS sites already present in your backbone will define the site you will use. The following rules apply regardless of the type IIS enzyme selected for assembly. Prior to using the tool you should decide which type IIS restriction enzyme you are going to use. Golden Gate in Geneious Prime: The Basics
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