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Considering the low yield of expressed protein with pAcF incorporated, we engineered pCNFRS to improve the incorporation of pAcF by introducing a quadruple mutation H283T/P284S/M285D/D286V (TSDV) located at the anticodon recognition domain that was proved useful in pAzFRS for improving the incorporation of (p-azido-phenylalanine) pAzF 28

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