Nucleic Acids Research, 2010, Vol. 38, No. 8 2635 Figure 6. Simultaneous construction of a fusion protein and insertion into a transcriptional cascade via three-way assembly. (a) I731014 is analogous to the T9002 transcriptional cascade, but uses the mCherry reporter rather than GFP. GFP was inserted downstream of luxR with a long linker component incorporated between the two coding sequences. (b) Detailed schematic showing regions of homology incorporated on 50 tails of primers. The linker component was ampliﬁed from a commercial cloning vector and included several epitope tags. (c) A 1:10:10 vector:insert:insert ratio was optimal for this three-way assembly, resulting in a 9 out of 24 success rate. The success rate was evaluated by colony PCR using VF2/VR primers. Negative control reactions were performed on the original I731014 and T9002-F templates. Background from the pBAD/HisA vector was not a factor because this vector has a diﬀerent selection marker than the ﬁnal construct. The desired construct exhibits a VF2/VR amplicon of 2.9 kb and a noticeable mobility shift compared to the negative controls (correct size indicated by arrow). SUPPLEMENTARY DATA Supplementary Data are available at NAR Online. ACKNOWLEDGEMENTS The authors thank Deepak Chandran for creating the primer design software tool and to Frank Bergmann for putting this tool online. We also would like to thank members of the Sauro and Klavins synthetic biology groups at the University of Washington for materials and useful discussions. FUNDING Sauro Lab start-up budget (University of Washington) and National Science Foundation (NSF) Grant in Theoretical