Generation of Gateway entry vectors by Golden Gate cloning.

(a) Scheme of entry clone generation in pJOG130. Either PCR products flanked by BsaI restriction sites and suitable 4 bp overhangs or CDS1 Level 0 modules of the Modular Cloning system may be cloned into pJOG130 by BsaI cut/ligation in exchange for a ccdB cassette. (b) as in (a), but when using vector pJOG131 for PCR products with suitable adaptors or CDS1ns modules of the Modular Cloning system. (c) Amino acid sequence encoded by att1 sites / adaptor sequences in pJOG130. The sequence created from using a pJOG130 derivative in a LR recombination reaction is shown. Translation will either initiate at an upstream START codon of an N-terminal epitope tag, or at the ATG codon depicted in bold if no N-terminal tag is fused during LR recombination. (d) as in (c), but when using a pJOG131 derivative during LR recombination. Amino acid sequences encoded at 5’ fusion sites (attB1) are equivalent as in (c) upon fusion of an N-terminal tag. The 3’ fusion site and respective linker sequences are shown. Sequences preceding the TCG (Ser) triplet depicted in bold, which is part of the Golden Gate overhang, will depend on design of PCR product or Level 0 module.