# Single-Vector mAb Expression Vector - Golden Gate Design
**Trastuzumab IgG1 (anti-HER2) | CHO-K1 stable expression | one-pot BsaI assembly**  
AI Bioinformatician by Labterna | 29 June 2026 | CHO-K1 PICRH-1.0 | de novo design

> **Status: designed and verified - all design targets met, ready to synthesise.** A 6,871 bp single-vector design (~7 kb, within the ~7 kb target) assembles in one BsaI Golden Gate reaction (5 fragments) with zero internal BsaI/BsmBI/SapI, CAI 0.94-0.97 (CHO-K1), orthogonal fusion overhangs (no cross-ligation detected), and CHO-K1-genome-specific colony-PCR primers. One design consideration to monitor: heavy chain cap-dependent, light chain IRES-driven - track the heavy:light ratio during expression (Section 6).

## 1. Summary of the request
A single-vector mAb construct for stable CHO-K1 lines: heavy + light chain in one transfection with puromycin selection, one-pot BsaI Golden Gate. Architecture CMV -> signal peptide -> IgG1 heavy chain -> EMCV IRES -> light chain -> puromycin cassette, ~7 kb, trastuzumab variable domains as template. Zero internal BsaI was non-negotiable (two prior attempts failed there); BsmBI/SapI also removed. Both chains codon-optimised for CHO-K1 (CAI > 0.85); fusion overhangs from the Potapov 2018 high-fidelity set (no palindromes/collisions); a Tm-matched junction colony-PCR pair per fragment, BLAST-checked vs CHO-K1 PICRH-1.0.

## 2. Construct design and architecture
Delivered vector: **6,871 bp circular**. One CMV promoter drives a bicistronic message - heavy chain (cap-dependent) then EMCV IRES -> light chain (cap-independent) - closed by WPRE + bGH poly(A). Both chains carry a murine Ig-kappa secretion leader (the light chain needs its own; the brief named one but both are present). A separate SV40 -> pac -> SV40 poly(A) cassette gives puromycin selection; pUC ori + AmpR support E. coli propagation. A 5' chimeric intron and a WPRE (standard expression enhancers) bring the vector to production-grade without changing the specified element order.

_Figure 1 (output/figures/figure_A_construct_map.png): linear map - elements, fusion overhangs J1-J5, and the five Golden Gate fragments F1-F5._

| Element | Coordinates (bp) | Length | Function |
|---|---|---|---|
| CMV enhancer/promoter | 1-584 | 584 | Strong constitutive promoter (heavy chain, cap-dependent) |
| Chimeric intron | 585-717 | 133 | 5'UTR intron; boosts mature-mRNA export/expression |
| Heavy chain ORF | 728-2,140 | 1,413 | Ig-kappa leader + trastuzumab VH + human IgG1 C (CHO-opt) |
| EMCV IRES | 2,145-2,733 | 589 | Cap-independent re-initiation; 3' ATG = light-chain start |
| Light chain ORF | 2,731-3,435 | 705 | Ig-kappa leader + trastuzumab VL + kappa C (CHO-opt) |
| WPRE | 3,436-4,024 | 589 | Post-transcriptional regulatory element |
| bGH poly(A) | 4,025-4,249 | 225 | Polyadenylation of the antibody transcript |
| SV40 promoter | 4,254-4,583 | 330 | Drives the puromycin-resistance cassette |
| pac (PuroR) ORF | 4,590-5,189 | 600 | Puromycin N-acetyltransferase (CHO-opt); mammalian selection |
| SV40 poly(A) | 5,190-5,311 | 122 | Polyadenylation of the pac cassette |
| pUC origin | 5,316-5,901 | 586 | E. coli origin of replication |
| AmpR (promoter + bla) | 5,902-6,867 | 966 | Ampicillin resistance (E. coli selection) |

_Five fusion-site scars (J1-J5) and two Kozak sequences fill the remaining positions; the light-chain ORF shares its ATG with the 3' end of the EMCV IRES. ORF spans include the stop codon (3 bp more than the CDS lengths in Section 3)._

## 3. Codon optimisation for CHO-K1 and Type IIS site removal
Heavy chain, light chain and pac were codon-optimised for *Cricetulus griseus* CHO-K1 (taxid 10029, Kazusa) with DNA Chisel, maximising CAI under hard constraints (no BsaI/BsmBI/SapI either strand; GC 40-70% windowed; no long homopolymers). All clear CAI > 0.85.

_Figure 2: per-ORF CAI, GC, and fragment sizes._

| Coding region | aa | bp | CAI | GC % | Internal BsaI/BsmBI/SapI |
|---|---|---|---|---|---|
| Heavy chain (leader+VH+IgG1) | 470 | 1,410 | 0.970 | 63.1 | 0 / 0 / 0 |
| Light chain (leader+VL+Ck) | 234 | 702 | 0.964 | 63.0 | 0 / 0 / 0 |
| pac (PuroR) | 199 | 597 | 0.945 | 67.8 | 0 / 0 / 0 |

**3.1 Non-coding elements and domestication.** Seven of nine regulatory/backbone parts were already site-free. Two single BsaI sites were domesticated without changing function: chimeric intron body (one silent substitution, away from splice signals) and bla/AmpR CDS (synonymous GGG->GGT, Gly). The two identical signal-peptide DNA segments were diverged (15 synonymous swaps) to remove the 60-bp direct repeat.

## 4. Golden Gate assembly design and fusion-site fidelity
Five fragments, one-pot BsaI-HFv2 + T4 ligase. Inward BsaI sites excise each fragment with its designed overhangs and are carried away on the cut ends, so the assembled plasmid has **zero BsaI/BsmBI/SapI sites**. In-silico digestion + re-ligation reproduces the 6,871 bp plasmid exactly.

| Frag | Module (5'->3') | Synth bp | In-plasmid bp | Left OH | Right OH |
|---|---|---|---|---|---|
| F1 | CMV enhancer/promoter + chimeric intron | 751 | 717 | GTTA | AGGA |
| F2 | Kozak + Ig-kappa leader - trastuzumab heavy chain (IgG1) + stop | 1,453 | 1,419 | AGGA | TAGA |
| F3 | EMCV IRES + leader - light chain (kappa) + stop + WPRE + bGH poly(A) | 2,139 | 2,105 | TAGA | GCGA |
| F4 | SV40 promoter + Kozak + pac (PuroR) + stop + SV40 poly(A) | 1,092 | 1,058 | GCGA | TGAA |
| F5 | pUC origin + AmpR (bla) | 1,586 | 1,552 | TGAA | GTTA |

**4.1 Fusion-site overhangs (selected from the Potapov/Pryor BsaI-HFv2 matrix).** Overhangs AGGA, TAGA, GCGA, TGAA, GTTA: none palindromic, none a reverse complement of another, minimum pairwise Hamming distance 2. No cross-ligation detected among the chosen overhangs (model fidelity **100%**, a detection-floor result); the estimator gives ~77% for the plant/MoClo set (Pryor ~81%; runs a few points conservative), so its relative ranking is reliable.

_Figure 3: ligation-specificity matrix - diagonal on-target, all off-diagonal cross-talk = 0._

## 5. Junction colony-PCR primers and specificity
One junction-spanning pair per fragment seam (five pairs); a band forms only if the two adjacent fragments are correctly joined, so the five reactions confirm the whole assembly. All Tm-matched (<=0.5 degC), 3' G/C clamp, 263-412 bp.

| Primer | Sequence (5'->3') | nt | Tm | GC% | Amplicon |
|---|---|---|---|---|---|
| J1-F | AACTGGGCTTGTCGAGACAG | 20 | 60.0 | 55 | 340 bp |
| J1-R | GTACCTGGTGTAGCCGTTGG | 20 | 60.4 | 60 | 340 bp |
| J2-F | AAGCTGACCGTGGACAAGAG | 20 | 60.0 | 55 | 302 bp |
| J2-R | TTTGGCGAGAGGGGAAAGAC | 20 | 60.0 | 55 | 302 bp |
| J3-F | CCACTCCCACTGTCCTTTCC | 20 | 60.0 | 60 | 263 bp |
| J3-R | CTGGGGACTTTCCACACCTG | 20 | 60.2 | 60 | 263 bp |
| J4-F | CTTCCTGGAGACATCTGCCC | 20 | 59.8 | 60 | 412 bp |
| J4-R | GACAGGTATCCGGTAAGCGG | 20 | 60.0 | 60 | 412 bp |
| J5-F | GCGGTATCATTGCAGCACTG | 20 | 60.0 | 55 | 290 bp |
| J5-R | TATTGACGTCAATGGGCGGG | 20 | 60.5 | 55 | 290 bp |

**Specificity vs CHO-K1 PICRH-1.0:** PASS - construct-specific (no CHO-K1 host amplicon). isPcr (minPerfect=8, maxSize 4 kb) on all five pairs + ten single-primer self-pairs gave **0 host amplicons**; longest single-primer perfect 3' match 17 nt (chance-level, no convergent partner). Primers are construct-specific.

## 6. Target biology and design rationale
Trastuzumab is a humanised IgG1/kappa anti-HER2 (ERBB2) mAb with public, structurally-defined variable domains (PDB 1N8Z) - the reason the brief uses them as template. VH/VL were taken from and verified against that structure; since 1N8Z is a Fab, the CH2-CH3 was completed from UniProt P01857 (secreted IgG1 heavy constant) and the light chain uses kappa constant UniProt P01834.

**Heavy:light balance.** Heavy chain is cap-dependent; light chain is IRES-driven (lower, less tunable). Because an excess of light chain usually aids folding/secretion and limits heavy-chain aggregation, the brief-specified heavy-first order is the empirically less-favoured arrangement for single-vector IRES mAbs, and a heavy-over-light ratio can cap titre - consider an LC-first swap or a dual-promoter format if titre matters. Main item to watch.

## 7. Caveats and recommendations
- **Heavy:light balance:** if titre is low / heavy chain aggregates, swap chain order (light cap-dependent, heavy via IRES) - a one-step F2/F3 rearrangement - or use two promoters.
- **pac is GC-rich (67.8%):** CAI 0.945, no Type IIS sites; specify a GC-rich synthesis protocol for F4.
- **De novo CDS screened:** optimised HC/LC/pac ORFs carry no AATAAA polyA signal and no strong GT[AG]AGT splice donor; confirm full-length expression at the bench.
- **IRES initiation:** light chain fused to the wild-type EMCV (M81861) 3' initiator ATG (not an engineered pIRES initiator); confirm second-cistron initiation empirically.
- **In-silico only:** splice/IRES activity, expression and secreted-mAb titre need bench confirmation.
- **Order integrity:** vendor-QC each fragment for exactly its two terminal BsaI sites; sequence all five junctions of the assembled plasmid before scale-up.

## 8. Recommended protocol (starting point)
- **Assembly:** equimolar fragments (~75 fmol), NEBridge BsaI-HFv2 + T4 ligase; 30x (37 degC 5 min / 16 degC 5 min), 60 degC 5 min, 80 degC 5 min; transform recA- E. coli; select on ampicillin.
- **Screen:** colony PCR with the five junction pairs (all five bands = correct); Sanger the junctions.
- **Express:** transfect CHO-K1, select with puromycin (5-10 ug/mL, titrate), quantify secreted IgG and check the heavy:light ratio by SDS-PAGE.

## 9. Output files
- **Trastuzumab_IgG1_GoldenGate_CHO_Report.pdf** - this report.
- **Trastuzumab_IgG1_GoldenGate_CHO_construct.gb** - annotated circular GenBank.
- **Trastuzumab_IgG1_GoldenGate_CHO_Data.xlsx** - fragments, primers, overhangs, codon-opt QC.
- **sequences.fasta** - plasmid + 5 synthesis fragments + 10 primers.
- **figures/** - Figures 1-3 (PNG 300 dpi + PDF).
- _scripts/ contains the reproducible numbered pipeline._
- _Provenance: PDB 1N8Z; UniProt P01857/P01834/P13249; GenBank M81861; CHO-K1 PICRH-1.0 GCF_003668045.3; Kazusa taxid 10029. Tools: DNA Chisel 3.2.16, Primer3-py 2.3.0, Biopython 1.87, BLAST+ 2.12.0, isPcr v39._

## 10. References
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11. Rupp O, MacDonald ML, Li S, et al. (2018) A reference genome of the Chinese hamster based on a hybrid assembly strategy. *Biotechnol Bioeng* 115:2087-2100. (CHO-K1 PICRH-1.0, GCF_003668045.3)
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