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. 2021 Feb 3;8:619252. doi: 10.3389/fbioe.2020.619252

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Copyright © 2021 McKenna, Balasuriya, Zhong, Li and O'Donoghue.

This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

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Production of differentially phosphorylated AKT1 variants. (A) To product pAKT1^T308, E. coli was transformed with pCDF-Duet1-AKT1-PDK1 containing AKT1 and the AKT1 T308 upstream kinase PDK1. (B) The plasmid pDS-pSer2 contains the phosphoserine orthogonal translation system (OTS) which was used to genetically incorporate phosphoserine at position 473 in response to a UAG nonsense codon in AKT1. The OTS includes the phosphoseryl-tRNA synthetase (SepRS), an elongation factor Tu mutant (EFSep) and five copies of a tRNA^Sep expression cassette. The OTS is co-expressed (C) with a pCDF-duet1 vector containing AKT1 TAG473 and lacking the PDK1 gene. The doubly phosphorylated AKT1 is then generated by (D) co-expressing pDS-pSer2 with the pCDF-duet1 vector containing AKT1 TAG473 and PDK1.