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. Author manuscript; available in PMC: 2010 Oct 15.
Published in final edited form as: Anal Biochem. 2009 Jul 4;393(2):234–241. doi: 10.1016/j.ab.2009.06.039

Figure 2.

Figure 2

A diagram illustrating the steps involved in constructing the CCH1 expression vector (pCMV-CCH1-GFP). Strategy 1) A fragment consisting of a CMV promoter, a GFP tag and a polyadenylation sequence was PCR-amplified from pcDNA3.1/CT-GFP-topo and cloned into a SacII site of a yeast shuttle vector (pRS425) generating the new vector, pYCMV-GFP. This vector was linearized with restriction enzyme Nhe1 and homologous end sequences of CMV and GFP were placed at the 5’ and 3’ ends of the CCH1 open reading frame, respectively. Strategy 2) Homologous end sequences of CCH1 were located on the pYCMV-GFP vector - at the end of CMV and at the beginning of GFP. To target CCH1 to pYCMV-GFP, primers were designed to amplify pYCMV-GFP with overhangs that corresponded to the 5’ end and the 3’ end of CCH1. Recombination between the ends of CCH1 and the vector was promoted by in vitro and in vivo homologous recombination reactions resulting in pCMV-CCH1-GFP (bottom of Figure 2).