Figure 2.

Construction of Multisite Expression Clones using LR reaction. Three Entry Clones with the DNA fragments of interest flanked by attL4 and attR1 (Promoter), attL1 and attL2 (Reporter gene), and attR2 and attL3 (transcriptional terminator) recombine with a Destination Vector containing attR4 and attR3. During the LR reaction attL sites react only with their cognate attR sites resulting in a Destination Clone containing all three fragments fused together into the desired expression cassette. Expression Clone constructs are selected on the basis of the antibiotic resistance gene in the Destination vector. The by-product of this reaction is non-replicative and its loss from the cell is further ensured by the presence of the ccdB counter-selectable marker gene.