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We demonstrate a simple, robust, and low-cost method for producing the PURE cell-free transcription translation system. Our OnePot PURE system achieved a protein synthesis yield of 156 peg/mL at a cost of 0.09 USD/ktL, leading to a 14-fold improvement in cost normalized protein synthesis yield over existing PURE systems. The one pot method makes the PURE system easy to generate and allows it to be readily optimized and modified.

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