Start-Stop Assembly Toolkit

Start-Stop Assembly is a multi-part, modular DNA assembly method which is functionally scarless, suitable for combinatorial assembly, and ideal for metabolic engineering in diverse organisms. Start-Stop Assembly provides the same key advantages as other Golden Gate-type modular assembly frameworks (efficient, unbiased, multi-part, hierarchical assembly) but also allows designs to be scarless at CDS boundaries, which are highly-sensitive sites where scars affect mRNA structure, activity of the ribosome binding site, and potentially other functional RNA features. The ‘Start-Stop Assembly Toolkit’ contains the 15 core assembly vectors used to assemble constructs of up to 15 coding sequences. It also contains the 25 promoters, RBSs, terminators and carotenoid pathway coding sequences used for assembly of metabolic pathway-encoding constructs in E. coli (described in Taylor et al. 2019). First-time users may find it helpful to assemble carotenoid pathway libraries as a positive control, because the variety of colored E. coli colonies conveniently show that combinatorial assembly is occurring (see Taylor et al. 2019 linked above for more details).