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design_multisite_primers

Design QCLM (quick-change lightning multi-site) oligos: one primer per mutated site, all annealing in one reaction, with the codons drawn from an organism's usage table. A design/write.

Input Schema

NameRequiredDescriptionDefault
kNopotassium concentration. In mM. Advanced.
mgNomagnesium concentration — the term the Owczarzy corrections are most sensitive to. In mM. Advanced.
naNosodium concentration the salt correction is computed for. In mM. Only applies when gc_value_set is Schildkraut_1965 or Primer3 or Ahsen_2001. Advanced.
cdsYesthe CDS nucleotide sequence, in frame from the start codon.
dntpNototal dNTP concentration, which chelates magnesium and so lowers the effective Mg²⁺. In mM. Advanced.
seedNoseed for the codon draw. The same seed and the same request give the same oligos; change it to re-roll.
trisNoTris concentration. In mM. Only applies when gc_value_set is Schildkraut_1965 or Primer3 or Ahsen_2001. Advanced.
_filesNoThe 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.
_subjectNoThe 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.
organismNocodon-usage table the synonymous choice is drawn from.e-coli
mutationsYesthe substitutions, comma- or space-separated, as [WT][pos][MUT] — e.g. "E42L, E42W, K107M". Several entries at one position are ONE site delivering several aminos.
precisionNodecimal places the reported Tm is rounded to. In dp. Advanced.
temp_weightNoscore weight on Tm deviation. Advanced.
use_primer3Noapply primer3 hairpin / homodimer / heterodimer penalties. Advanced.
gc_value_setNowhich published GC-formula parameter set the melting temperature is computed under. Values: QuickChange (QuikChange (the kit manufacturer’s own formula)), Chester_1993 (Marmur-Doty (Chester & Marshak 1993)), Schildkraut_1965 (Schildkraut & Lifson 1965 (Marmur-Doty + log[Na⁺])), Primer3 (Primer3Plus product Tm (Biopython’s default set)), Ahsen_2001 (von Ahsen 2001 (accuracy against ease of use)). Advanced.QuickChange
five_end_flankNoexplicit 5' context. Left empty, 60 bp is read off the open record either side of the CDS. Advanced.
max_gc_contentNohighest GC accepted. In %. Advanced.
min_gc_contentNolowest oligo GC accepted. In %. Advanced.
max_primer_sizeNolongest oligo. In nt. Advanced.
max_temperatureNohighest Tm threshold swept. In °C.
min_primer_sizeNoshortest oligo. In nt. Advanced.
min_temperatureNolowest Tm threshold the growth scan sweeps. In °C.
temp_range_sizeNowidth of the acceptable Tm window around the reaction temperature. In °C. Advanced.
three_end_flankNoexplicit 3' context, the partner of `five_end_flank`. Advanced.
gc_content_weightNoscore weight on GC overflow. Advanced.
max_five_end_sizeNolongest 5' lead. In nt. Advanced.
min_five_end_sizeNobases between the 5' terminus and the first mutated codon, minimum. In nt. Advanced.
max_three_end_sizeNolongest 3' tail. In nt. Advanced.
min_three_end_sizeNobases between the last mutated codon and the 3' terminus, minimum. In nt. Advanced.
primer_size_weightNoscore weight on oligo length deviation. Advanced.
temp_threshold_stepNostep between Tm thresholds tried. In °C. Advanced.
five_end_size_weightNoscore weight on 5'-lead length deviation. Advanced.
use_degeneracy_codonNocollapse a site's codons into one IUPAC-degenerate codon (upstream's default). Off, each amino gets its own oligo.
three_end_size_weightNoscore weight on 3'-tail length deviation. Advanced.
non_overlapping_primersNoforbid oligos from overlapping each other. Upstream ranks only the LAST split's solutions with this on (a reproduced bug, see the port header). Advanced.
mutation_coverage_weightNoscore weight on how many of the requested aminos the design actually delivers — upstream's dominant term. Advanced.
hairpin_temperature_weightNoscore weight on hairpin Tm. Advanced.
codon_usage_frequency_thresholdNodiscard synonymous codons rarer than this fraction in the chosen organism.
primer_dimer_temperature_weightNoscore weight on dimer Tm. Advanced.

Output Schema

NameRequiredDescriptionDefault
seedYesthe seed this design was drawn with — change it to re-roll, keep it to reproduce
oligosYesone oligo per site, with its span, Tm, GC and the degenerate codons it installs
failureCountYesconstraint breaches in the winning solution (0 = every oligo inside its limits)
fullSequenceYesthe construct the oligo coordinates are on: flank + CDS + flank
mutationCoverageYesfraction of the requested amino acids the design actually delivers, 0–1
reactionTemperatureYesthe ONE annealing temperature the winning solution is built for

Schema Changelog

Changes observed during successful MCP inspections.

  1. Changed1 schema field changed
    • addedOutput schema / properties / reactionTemperature / x-unit
      Added value: +"°C"
  2. First observed

TDQS

B3.2/5.0
Behavior3/5

Does the description disclose side effects, auth requirements, rate limits, or destructive behavior?

Annotations already declare readOnlyHint=false and destructiveHint=false, so the description's 'A design/write' only confirms what is structured. It adds real value by describing the design behavior (one oligo per site, single-reaction annealing, codon-usage-based synonymous choice), but says nothing about what the design returns being unsaved, or about failure modes when coverage cannot be satisfied.

Agents need to know what a tool does to the world before calling it. Descriptions should go beyond structured annotations to explain consequences.

Conciseness4/5

Is the description appropriately sized, front-loaded, and free of redundancy?

Two tight sentences with the mechanism front-loaded and no filler. The trailing fragment 'A design/write.' is terse to the point of being cryptic but costs little.

Shorter descriptions cost fewer tokens and are easier for agents to parse. Every sentence should earn its place.

Completeness3/5

Given the tool's complexity, does the description cover enough for an agent to succeed on first attempt?

An output schema exists and the schema documents every parameter, so the description does not need to explain returns. However, for a 39-parameter combinatorial design tool it does not warn that default constraints may fail to produce a design, nor point at the two required inputs (CDS, mutation list) that an agent must get right.

Complex tools with many parameters or behaviors need more documentation. Simple tools need less. This dimension scales expectations accordingly.

Parameters3/5

Does the description clarify parameter syntax, constraints, interactions, or defaults beyond what the schema provides?

Schema description coverage is 100%, so all 39 parameters already carry documentation, including defaults, units, and 'Advanced' markers. The description adds no parameter-level meaning beyond echoing the organism codon table concept; baseline 3 is appropriate.

Input schemas describe structure but not intent. Descriptions should explain non-obvious parameter relationships and valid value ranges.

Purpose4/5

Does the description clearly state what the tool does and how it differs from similar tools?

States a specific verb and resource ('Design QCLM ... oligos') and adds the defining mechanism: one primer per mutated site, all annealing in one reaction, codons drawn from an organism's usage table. That mechanism implicitly distinguishes it from sibling primer designers like design_mutagenesis_primers and design_saturation_primers, though no sibling is named.

Agents choose between tools based on descriptions. A clear purpose with a specific verb and resource helps agents select the right tool.

Usage Guidelines2/5

Does the description explain when to use this tool, when not to, or what alternatives exist?

There is no explicit when-to-use guidance, no prerequisites, and no named alternative among the many sibling design_*_primers tools. An agent must infer from 'multi-site' that this is for several mutations at once rather than a single-site mutagenesis design.

Agents often have multiple tools that could apply. Explicit usage guidance like "use X instead of Y when Z" prevents misuse.

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