CODEHOP hybrid primers from a protein alignment

1. 01Hybrid primers from an alignment of PROTEINS, for homologues whose DNA no longer aligns
2. 02A 3-prime degenerate core over three to four conserved residues, under a degeneracy ceiling
3. 03A 5-prime non-degenerate consensus clamp, back-translated through any of 127 codon-usage tables
4. 04Ungapped blocks found and ranked by identity, so you can see which windows are designable
5. 05Both strands, ranked by core degeneracy then clamp score, as the method states
6. 06The whole oligo pool expanded, so you can see every molecule the synthesis makes
7. 07The motif highlighted on the alignment canvas, and TSV plus pool-FASTA export

The method is implemented from the published CODEHOP papers (Rose 1998 and 2003, iCODEHOP 2009), with the clamp melting temperature computed by the same nearest-neighbour engine the rest of the primer tools use. Every number reported is recomputed independently in its tests over a real protein-kinase family alignment and the whole set of published codon-usage tables. The panel runs on @ports/enzymes, @ports/oligo-structure, @ports/primer.

Everything runs client-side. Files are parsed in the browser and projects are saved on this machine, so an unpublished sequence is never uploaded.

When would I use this instead of ordinary degenerate primers?

When the family has diverged so far that its DNA no longer aligns usefully. Silent changes at the third base of each codon accumulate quickly, so a nucleotide-level degenerate primer for distant homologues becomes so degenerate that no single correct molecule is abundant enough to prime. The protein is still conserved, so the design starts there.

Why is part of the primer written in lower case?

That is the 5-prime consensus clamp, and it is not degenerate: one fixed sequence, the most likely nucleotide at each position. Upper case is the 3-prime degenerate core, written in IUPAC letters. The clamp adds the duplex stability that lets a short degenerate core anneal at a useful temperature.

Which codon usage table should I choose?

The organism you are amplifying FROM, not the organisms in your alignment. The alignment is the family across species; the template is the one species on your bench, and the clamp is back-translated for it.

Why did it return nothing for my alignment?

It says which criterion each candidate failed: no invariant nucleotide at the 3-prime end, a core past the degeneracy ceiling, a clamp that could not reach the target temperature, or too long a single-base run. A more conserved block, a higher base cutoff or a wider ceiling is usually the answer.