TCR vector construct design TCRshows

Turn a TCR’s protein ORF into a DNA insert you can send straight for synthesis and clone into your own vector. Start from the α and β variable regions assembled by the sequence assembler: attach the constant regions, join them into a single ORF with a 2A peptide, choose cloning sites, then codon-optimise for the vector context. That context matters: the genomic RNA of a lentiviral or γ-retroviral vector must not be spliced or polyadenylated early, so cryptic splice sites and poly(A) signals are hard constraints here — which is not the same objective as optimising an mRNA drug. Everything is computed locally in your browser; no sequence is uploaded.

1TCR chain sequences

Paste the variable region of the α and β chains separately, as amino acids or DNA — for example the two the sequence assembler builds from V + CDR3 + J. Include the leader peptide if you have it; leave out the constant region.

2Constant regions and the chain linker

Once both chains carry their constant regions, a 2A self-cleaving peptide joins them into a single ORF. The first residue of the constant region is formed by the one base left over from the J segment plus the first two bases of the constant region, so the α chain needs its TRAJ specified (N, D, Y or H are all possible), while β is always E — the tail base of all 16 TRBJ genes is G.

3Cloning sites

The enzymes you pick are added to the “must avoid” list automatically, and the optimiser guarantees they do not occur inside the insert — which is what makes the sites added at each end the only cut sites. After optimisation, every usable enzyme is listed below for the actual sequence.

Restriction enzymes cut poorly too close to the end of a fragment, so a spacer is left at each end. Candidate spacers are tried one by one and one is chosen that does not create a hazardous motif at the junction.

4Vector and elements

These choices determine which sequence features count as hazardous, and how much packaging capacity is left.

5Codon optimisation and avoidance

The objective is to drive hard-constraint violations to zero and push CAI as high as possible, while leaving the amino acid sequence byte-for-byte unchanged. Hard constraints are weighted far above CAI, so they are close to inviolable.

Sequence features to avoid

Additional restriction sites to avoid

The two cloning enzymes chosen in step 3 are already included, so there is no need to tick them again here. Tick the other sites you want to keep usable downstream.

iMethod, evidence and limits

What each check tests

Limits you need to know. ① Splice-site detection is motif matching, not a splicing predictor. What is matched here is the accepted consensus core, which produces false positives (many matches are never actually used) and misses sites that work through splicing enhancers. Its purpose is to clear the obvious mines before synthesis, not to replace an experiment.
② This page does not supply the actual sequences of promoters, WPRE or LTRs. Those elements run from hundreds to thousands of bases, and one mistyped base gives you a silently dead construct — so only length, capacity and suitability are computed from the elements you select. Take the real sequences from the map of the vector you are using. What this page outputs is the insert itself: cloning sites + Kozak + ORF + stop codons.
③ Packaging capacity is an estimate. The fixed backbone overhead uses typical values for common lentiviral and γ-retroviral vectors, and different vectors can differ by hundreds of bases. Titre drops noticeably near the limit, so measure it.
④ Codon optimisation only guarantees that the translated product is unchanged, residue for residue. It does not guarantee higher expression — the relationship between CAI and expression level is statistical.