PEEK: integrated primer design for silent-mutation and restriction-site engineering, validated in silico and by whole-plasmid sequencing

PEEK, a web-based primer-design toolkit, computes primer melting temperatures with NEB Q5 accuracy (R² = 0.9995; MAE 1.32 C) and validates designed silent edits by whole-plasmid sequencing.

• Why it matters: Molecular cloning needs integrated, thermodynamically consistent primer design and explicit ranking of synonymous edits, but existing tools are fragmented and lack standardized validation against external Tm rules.
• What they did: PEEK unifies silent-mutation discovery, restriction-site insertion, and amplification/screening primer design using one primer Tm model, then checks Tm against NEB Q5 and edits via translationally silent designs and plasmid sequencing.
• The result: Across 17 primers, predicted Tm and Ta matched NEB rules without fitted correction, and 15 silent-mutation designs plus a KpnI insertion into a 9.9 kb lentiviral CAR plasmid were confirmed by whole-plasmid sequencing with no off-target differences or protein changes.

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