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Fig. 1 Gateway cloning strategy. A: Schematic of the 3-part LR recombination reaction used to generate expression constructs, using three entry clones and the pDestTol2pA/pA2 destination vector. B: Schematic of pDestTol2CG/CG2, destination vectors that include the cmlc2: EGFP-pA expression cassette. C: Strategy for generating a middle entry clone using pDONR221. Generation of 5′ and 3′ entry clones is similar, but uses different att sequences and donor vectors. D: Transformation of TOP10 cells with an LR recombination reaction yields two classes of colonies: clear (arrowheads) and opaque (arrow). Clear colonies yield the correct recombination product >99% of the time, whereas opaque colonies never do.