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. 2013 Feb 26;41(8):e92. doi: 10.1093/nar/gkt115

Figure 1.

Figure 1.

Assembly of dual expression vectors via dual LR Clonase reaction. (a) Canonical Gateway cloning using the BP Clonase reaction to generate entry vectors (Left) and LR Clonase reaction to generate expression vectors (Right). (b) Initial dual DEST architecture that produces ∼50% correctly recombined clones on dual LR Clonase reaction with pENTR1 and pENTR2 plasmids. (c) Improved dual DEST architecture that produces >98% correctly recombined clones on dual LR Clonase reaction with pENTR1 and pENTR2 plasmids by addition of a kanamycin resistance gene between DEST1 and DEST2. (d) Diagram of BP Clonase reaction to generate pENTR 2 vectors from attB PCR products analogous to canonical Gateway cloning (Left). Diagram of the entry transfer system developed to move cDNAs/shRNAs from pENTR1 vectors into pENTR2 vectors via LR Clonase reaction (Right). This system was generated by inserting the DEST1 cassette with the attL sites of pENTR2 and replacing the kanamycin resistance with Tetracycline in pENTR2. (e) In addition to transferring cDNA/shRNAs between entry vectors, we also developed pENTR2-DEST1-IRES-Marker vectors to include useful selectable markers, such as puromycin, neomycin, hygromycin and fluorescent proteins.