A single-vector monoclonal-antibody expression construct was requested for CHO-K1 stable lines, assembled in one BsaI Golden Gate reaction with zero internal BsaI sites as a non-negotiable requirement and using the trastuzumab variable domains as a public template. The delivered design is a 6,871 bp vector (within the ~7 kb target) provided as five one-pot Golden Gate fragments, with an annotated full-construct GenBank, FASTA and an Excel ordering sheet, five junction colony-PCR primer pairs, and a report with the construct schematic and fragment-level QC. The assembled plasmid carries zero internal BsaI / BsmBI / SapI sites; both antibody chains and the puromycin marker are codon-optimized for CHO-K1 (CAI 0.945-0.970, all above the 0.85 target); the five fusion-site overhangs are mutually orthogonal with no detected cross-ligation; and the junction primers are construct-specific (no CHO-K1 genomic amplicons by in-silico PCR). The main consideration to monitor is the expression architecture: the heavy chain is translated cap-dependently and the light chain via the EMCV IRES, so the heavy:light ratio should be checked during expression, with a light-chain-first swap or a dual-promoter format as the fallback if titre is light-chain-limited. All results are computational and should be confirmed at the bench, including sequence-verification of the assembled plasmid across the five fragment junctions.
