Figure 2. Level 0 destination vectors and principle for removal of internal sites from level 0 modules.

(A) Level 0 destination vectors. Level 0 modules are made by amplification of selected sequences with primers adding flanking BpiI sites, and cloning of the amplified fragment (shown above the horizontal dotted line) via BpiI into the designated level 0 destination vectors (shown below). In addition to the 5 basic destination vectors, pL0-P, pL0-U, pL0-S, pL0-C and pL0-T, additional destination vectors allow cloning several genetic elements in one module. For example, plasmid pL0-SC can be used to clone sequences encoding cytosolic proteins, which do not contain a signal peptide. (B) Strategy for removing internal type IIS recognition sequences. Removal of a BsaI site in a fragment of interest is done by amplifying two fragments with primers pr1 and 2 and primers pr3 and 4. Primers pr2 and pr3 span the BsaI recognition site and introduce a single nucleotide mismatch (indicated by an arrow). As all primers have BpiI recognition sites in their 5′ extensions, the PCR fragments are cloned with a BpiI-based Golden Gate cloning reaction in the appropriate level 0 destination vector.