Golden Gate compatible plasmid for lineage tracing in mammalian systems

EB04119 (10,917 bp) is a modified version of pEGZ2 REF1,2 domesticated for Golden Gate cloning and containing a DNA barcode. The parental vector was domesticated by removing an internal BsmBI site in the hygromycin cassette via a synonymous mutation (GTC→GTT), and an mScarlet dropout cassette with outward-facing BsmBI sites was inserted downstream of WPRE. EMMA fusion sites at position 17 (GCTC-GCCA) were used. Five fragments were PCR-amplified and assembled by Golden Gate using PaqCI, with overhang fidelity verified via NEBridge. The final construct was sequence-verified.

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