Give it an engineered locus and it returns the two assays that genotype it. The knock-in assay puts one primer inside the cassette and its partner in the flanking genome, so the product exists only on the engineered allele. The empty-site assay puts both primers in the flanks, spanning the insertion point, with the reporter across it — short product from wild type, longer by the whole cassette from the engineered allele.
Designed as a pair, because that is what reads out zygosity: wild type gives the empty-site signal only, a homozygote the knock-in signal only, a heterozygote both — and a well with neither is a failed reaction rather than a genotype. The tool knows which bases belong to the cassette and which to the genome, either from the markup or by comparing the two alleles, and every finished assay is checked in silico against both of them. For a substitution or a SNP rather than an insertion use qPCR probes; for the recombinase sites themselves, loxP / FRT assays.
Two ways to give the locus, 5′→3′. One sequence with the cassette in brackets and a slash — …flank[CASSETTE/]flank… — where the bracketed sequence is present on one allele and absent on the other. Or two FASTA records, engineered allele first and wild type second, and the cassette is found by comparing them. Wrap a stretch in /…/ to keep every oligo out of it.
Design
Balance rule for the empty-site reporter — the half carrying the insertion point is made the stronger one
These three numbers are a convention rather than a measurement. The defaults are the conventional ones for this class of assay; they are evaluated here at this site's Tm conditions, and because the halves are short oligos the two conventions agree closely at these temperatures.
Mask before placing oligos
Export: use Copy / Download above each report, or click inside a result area → Ctrl+A → Ctrl+C.