CHO mAb expression construct
A single-vector mAb expression construct, assembly-ready in 1 hour 23 minutes.
Deliverable preview
| Fragment | Length | Tm overhangs | QC |
|---|---|---|---|
| CMV promoter | 612 bp | 60.1 °C | PASS |
| IgG1 heavy chain | 1,398 bp | 60.4 °C | PASS |
| IRES | 586 bp | 59.8 °C | PASS |
| Light chain | 714 bp | 60.0 °C | PASS |
The ask
A biotech collaborator developing a candidate therapeutic mAb needed a CHO-K1 expression construct that combined heavy and light chains under a single transfection, plus puromycin selection. Internal experience: prior versions had failed Golden Gate assembly due to internal BsaI sites in codon-optimized variable regions, requiring multiple rounds of redesign. The brief asked for: a 6-fragment Golden Gate assembly scheme, CHO-optimized codon usage, zero internal BsaI sites, palindrome-free 4-nt overhangs, and validation primers across every junction.
Approach
Construct architecture
Defined a 6-fragment scheme: CMV promoter, secretion signal peptide, IgG1 heavy chain, EMCV IRES, light chain, and puromycin resistance cassette — assembled into an 8.2 kb backbone in a single BsaI Golden Gate reaction.
CHO codon optimization with site avoidance
Optimized heavy and light chain codon usage for CHO-K1 (CAI > 0.85), with hard constraints to avoid BsaI/BsmBI/SapI internal sites. Iterated optimization until 0 internal type IIS sites across the construct.
Overhang design
Selected 12 BsaI fusion-site overhangs from the Potapov 2018 high-fidelity set, screened against the construct for off-target ligation events, and confirmed palindrome-free pairwise compatibility.
Junction validation primers
Generated 6 colony-PCR primer pairs anchored across each fragment junction, Tm-matched to ±0.5 °C, BLAST-checked for genome uniqueness against the CHO-K1 reference (PICRH-1.0).
Deliverables
12-page report: construct schematic, fragment-level QC tables, codon optimization summary, overhang scheme, and assembly protocol.
Full GenBank file with every feature annotated — promoter, signal peptide, CH/CL domains, IRES, selection marker — ready for Benchling or SnapGene.
All 6 fragment-flanking oligos + 6 colony-PCR primer pairs in FASTA, with an Excel workbook of Tm, overhang sequences, and order-ready synthesis sheets.
+ the full project folder. Every numbered script, sequence, intermediate alignment, BLAST output, JSON checkpoint, tool version, timestamped database query, methods write-up, and literature reference — re-runnable end-to-end, fully literature-validated, and independently auditable by specialised reviewer agents. The agent flags its own limitations honestly in every report.
Outcome
Delivered in 1 hour 23 minutes vs. ~3 weeks for a typical internal design cycle and a $4–8K quote from a contract design house. The collaborator assembled the construct on the first attempt with no internal-site retries.
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