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. 2015 May 26;10(5):e0127504. doi: 10.1371/journal.pone.0127504

Fig 1. In-frame TAG codon replacement.

Fig 1

(A) Step 1 involves trinucleotide deletion followed by TAG donation using a combination of the engineered transposon MuDel [25] and the DNA cassette SubSeq [21] essentially as described previously [23] and outlined in B. The TAG substitution library is then cloned in front of the TEV-sfGFP cassette in plasmid pIFtag (S1 Fig). Step 2 outlines the selection for in-frame TAG substitutions. Initially, cells are grown in the absence of a nAA and non-fluorescent colonies selected; fluorescent colonies are removed at this stage as they are deemed not to have an in-frame TAG due to the generation of a full translation product. The second selection involves plating the selected colonies in the presence of nAA. Those cells that regain fluorescence suppress TAG termination due to nAA incorporation and thus produce sfGFP. (B) Alternate versions of the new SubSeq DNA cassette for donating TAG. The two alternatives are shown in the red boxes at the far left. (C) The two nAAs used in this study, p-azido-L-phenylalanine (azF) and p-iodo-L-phenylalanine (iodoF).