| k | No | potassium concentration. In mM. Advanced. | |
| mg | No | magnesium concentration — the term the Owczarzy corrections are most sensitive to. In mM. Advanced. | |
| na | No | sodium concentration the salt correction is computed for. In mM. Advanced. | |
| cds | Yes | the CDS nucleotide sequence, in frame from the start codon (length must be a multiple of 3). | |
| dntp | No | total dNTP concentration, which chelates magnesium and so lowers the effective Mg²⁺. In mM. Advanced. | |
| tris | No | Tris concentration. In mM. Advanced. | |
| dnac1 | No | the higher-concentration strand (the primer, in a large excess). In nM. Advanced. | |
| dnac2 | No | the lower-concentration strand (the template). In nM. Advanced. | |
| sites | Yes | the saturation sites, comma- or space-separated, as [WT][pos]X — e.g. "V2X, K107X". 1-based residue positions. | |
| _files | No | The project: record references shaped like _subject. Their records become library documents a tool names in sequences/parts/document; a trace or alignment is found by file name. | |
| solver | No | which of Mutation Maker's two solvers designs the pairs. `fast` grows one candidate per site per temperature combination (upstream's own default, milliseconds). `exhaustive` enumerates every primer of every allowed length at every allowed start and scores every geometrically valid pair — hundreds of thousands per site, seconds rather than milliseconds, and it reports what it searched. Values: fast (Fast approximation), exhaustive (Exhaustive search). | fast |
| _subject | No | The record to act on: exactly one of sequence, accession (+source), sample (an id from list_samples) or file ({name, text} or {name, base64}); record_index picks one record of several. | |
| precision | No | decimal places the reported Tm is rounded to. In dp. Advanced. | |
| oligo_prefix | No | prefix for the designed oligo names. Advanced. | ssm |
| five_end_flank | No | explicit 5' flanking sequence — only for a free-standing gene with no plasmid open, and then the 3' flank is required too. Advanced. | |
| forward_primer | No | 5' flanking primer, 5'→3'. Left empty, it is read off the open plasmid 56 bp either side of the CDS (22 nt, and only when it occurs there exactly once). | |
| max_gc_content | No | highest GC accepted. In %. Advanced. | |
| min_gc_content | No | lowest primer GC accepted before a primer is flagged out of range. In %. Advanced. | |
| reverse_primer | No | 3' flanking primer, 5'→3' on its own strand. Left empty, it is read off the open plasmid 56 bp either side of the CDS (22 nt, and only when it occurs there exactly once). | |
| max_primer_size | No | longest whole primer. In nt. Advanced. | |
| min_primer_size | No | shortest whole primer. In nt. Advanced. | |
| salt_correction | No | salt-correction method, in Biopython's `saltcorr` order. Values: No (None), Schildkraut_1965 (Schildkraut & Lifson 1965 (16.6 · log[Na⁺])), Wetmur_1991 (Wetmur 1991 (16.6 · log[Na⁺/(1 + 0.7 · Na⁺)])), SantaLucia_1996 (SantaLucia 1996 (12.5 · log[Na⁺])), SantaLucia_1998 (SantaLucia 1998 (11.7 · log[Na⁺])), SantaLucia_DeltaS_1998 (SantaLucia 1998, entropy (corrects ΔS, not Tm)), Owczarzy_2004 (Owczarzy 2004 (GC-dependent, corrects 1/Tm)), Owczarzy_2008 (Owczarzy 2008 (Mg²⁺ decision tree, dNTP-corrected)). Advanced. | Owczarzy_2004 |
| three_end_flank | No | explicit 3' flanking sequence, the partner of `five_end_flank`. Advanced. | |
| degenerate_codon | No | the degenerate codon installed at every site (NNK is the common 32-codon choice). | NNK |
| max_overlap_size | No | longest overlap. Upstream's scan stops two short of this (a reproduced quirk, see the port header). In nt. Advanced. | |
| min_overlap_size | No | shortest shared overlap. In nt. Advanced. | |
| gc_content_weight | No | score weight on GC overflow. Upstream's default is 0, and its GC term counts G twice and never counts C (a reproduced bug, see the port header) — raising this weight makes that quirk observable. Advanced. | |
| max_five_end_size | No | longest 5' arm before the mutated codon. In nt. Only applies when solver is exhaustive. Advanced. | |
| min_five_end_size | No | shortest 5' arm before the mutated codon. In nt. Advanced. | |
| max_three_end_size | No | longest 3' arm beyond the mutated codon. Bounds the search area and discards candidates whose 3' arm overruns it. In nt. Only applies when solver is exhaustive. Advanced. | |
| min_three_end_size | No | shortest 3' arm beyond the mutated codon. In nt. Advanced. | |
| overlap_temp_range | No | width of the overlap Tm window. In °C. Advanced. | |
| overlap_temp_weight | No | score weight on overlap Tm deviation. Advanced. | |
| three_end_temp_range | No | width of the 3'-end Tm window scanned around each candidate temperature. In °C. Advanced. | |
| three_end_size_weight | No | score weight on 3'-end length deviation. Advanced. | |
| three_end_temp_weight | No | score weight on 3'-end Tm deviation. Advanced. | |
| max_overlap_temperature | No | highest overlap Tm allowed. In °C. | |
| min_overlap_temperature | No | lowest overlap Tm the reaction may settle on. In °C. | |
| overlap_temp_range_step | No | step between overlap temperatures tried. In °C. Advanced. | |
| exclude_flanking_primers | No | take the 3' Tm window from the explicit min/max above instead of from the flanking pair's own Tm. Advanced. | |
| compute_hairpin_homodimer | No | add the hairpin/homodimer/heterodimer penalty to the score (roughly doubles the run; off upstream). Advanced. | |
| max_three_end_temperature | No | highest 3'-end Tm allowed. In °C. | |
| min_three_end_temperature | No | lowest 3'-end Tm (used when `exclude_flanking_primers` is on; otherwise the window comes from the flanking pair). In °C. Only applies when exclude_flanking_primers is true. | |
| three_end_temp_range_step | No | step between 3'-end temperatures tried. In °C. Advanced. | |
| hairpin_temperature_weight | No | score weight on hairpin Tm. Only applies when compute_hairpin_homodimer is true. Advanced. | |
| primer_dimer_temperature_weight | No | score weight on homo/heterodimer Tm. Only applies when compute_hairpin_homodimer is true. Advanced. | |
| separate_forward_reverse_temperatures | No | optimise the forward and reverse 3' temperatures independently. Advanced. | |