PCA Gene Assembly — overlapping oligo sets for polymerase cycling assembly

Give it the sequence you want to build and it returns the oligonucleotides that assemble it. The construct is tiled by oligos that alternate between the two strands and overlap their neighbours; in the first cycles those overlaps anneal and the polymerase extends each oligo along its partner, so the fragments grow towards full length. End primers are then added for a second round of cycling, which amplifies the finished construct away from everything incomplete.

What the design actually optimises is the melting temperature of the junction overlaps — one cycling program has to serve every junction at once, so an even Tm across them matters more than even oligo lengths. Method: polymerase cycling assembly; Stemmer et al. (Gene 1995). Long constructs are split into blocks that are assembled separately and joined — for that last step see Gibson Assembly.

One sequence, 5′→3′, with or without a FASTA header — the finished construct as you want it to end up. Anything that is not A/C/G/T is dropped and reported: an ambiguity code cannot be built as a single oligo set.

Oligos and overlaps

Oligo length range (nt)

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Overlap length range (nt)

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Overlap Tm target (°C)

Tm spread allowed (°C)

Junction search window (nt)

Misannealing seed (nt)

Repeats and complex sequence

Mask before placing junctions

Repeated sequence

Low complexity (SSR, telomere-like)

Blocks

Assemble each copy of a long repeat in a separate block

Long repeat from (bp)

Blocks and end primers — a long construct is assembled in pieces and joined

Max block length (bp, 0 = one block)

Block overlap (bp)

End primer Tm (°C)

End primer length (nt)

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Need details? The junction geometry, what the Tm spread means and every parameter are documented in Help: PCA gene assembly. To join finished blocks, or to put the construct into a vector, see Gibson Assembly; to check the end primers against a genome or plasmid, In silico PCR.
Export: use Copy / Download above each report, or click inside a result area → Ctrl+A → Ctrl+C.