Fig. 2

Schematic overview of the design of Gene Doctoring mutagenesis cassettes in pDOC-GG, using our donor vector for insertion of sfgfp into the glmS site of E. coli as an example. Golden Gate assembly is used to replace the lacZα cassette with the DNA sequence that will form the linear recombination fragment upon digestion with I-SceI in vivo. The top mutagenesis cassette shows the sequence that we assembled, with tetA upstream of sfgfp. The bottom cassette shows an alternative design, using our second tetA genetic part to locate the FRT scar sequence downstream of sfgfp. Mutagenesis cassettes are always flanked by homologous regions (HR1 and HR2) to direct them to the correct location in the chromosome. By designing appropriate 4-bp BsaI overhangs (vertical text), the number and order of the DNA parts can be varied as desired