Fig. 1

(a) Vector organization in the different levels of the MoClo system. Level 0 vectors are used to generate a library of parts that can be subsequently assembled in Level 1 transcription units (TU), which in turn serve to generate Level M and P multi-TU constructs. The antibiotic cassettes and the four bases overhangs (fusion sites) of each level are represented by colored boxes. The different genetic parts are represented as pictograms in black boxes. The Type IIs restriction endonucleases used for each cloning step are indicated. (b) Chromosomal integration framework using Conditional-replication, integration, and modular (CRIM) plasmids. The co-transformation of a CRIM-based plasmid and the cognate helper plasmid is followed by a temperature shift that induces the expression of the recombinase, driving the site-specific recombination event at the specific phage attachment site. The proprieties of the system allow for the integration and curing of the helper plasmid in the same incubation step and easy selection for recombinant clones after overnight incubation. (c) Joint use of CRIMoClo plasmids (Level M and P) with other vectors in the MoClo system. Any transcription unit generated in Level 1 can be cloned into high/medium copy number MoClo plasmids or be chromosomally integrated using CRIMoClo plasmids. The design of CRIMoClo plasmids allows for a seamless transition between the two systems