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. Author manuscript; available in PMC: 2022 Nov 18.
Published in final edited form as: Cell Chem Biol. 2021 Feb 12;28(11):1542–1553.e5. doi: 10.1016/j.chembiol.2021.01.013

Figure 4. Engineering of a Highly Efficient Plasmid-based ncAAs Incorporation System in Mammalian Cells.

Figure 4.

(A) Schematics of various expression plasmids. Poly-aaRS is the poly-specific aminoacyl tRNA synthetase, eRF1-E55D is the mutant eRF1 gene, T2A is a self-cleaving 2A peptide, tRNA is the orthogonal tRNA, U6 indicates the U6 promoter, H1 indicates the H1 promoter, CMV is the CMV promoter, EGFPTAG is the EGFP reporter gene with a TAG codon at position 39. > or < denotes the direction of the gene cassettes.

(B) Chemical structure of p-Boronophenylalanine (pBoF).

(C) Quantification of pBoF (1 mM) incorporation into the EGFPTAG reporter gene measured in a cell-based fluorescence assay. The indicated constructs were transiently expressed in HEK 293T cells and the cell lysate green fluorescence was quantified in a plate reader at 515 nm emission with excitation at 490 nm. Data represent the mean ± SD of triplicates.