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Validation

Status

FactorForge is an in-silico only CDS design tool. It optimizes codon usage, GC content, and sequence constraints, but it cannot guarantee protein expression, yield, solubility, folding, secretion, or biological activity in any specific system.

Warning

Wet-lab validation is always required before use in production workflows.

Public validation entries are manually reviewed, non-confidential wet-lab feedback summaries. They are not controlled validation studies, regulatory claims, or guarantees of FactorForge performance.

What FactorForge Checks

Checks are grouped by domain (see rule-engine-roadmap.md for full per-rule status).

Sequence integrity (hard fail in the production design pipeline):

Check Covered Notes
Internal stop codons Yes Hard fail
Amino acid identity Yes Hard fail
Invalid / partial codons Yes Hard fail
Reading-frame / length consistency Yes Hard fail

Configured constraints:

Check Covered Notes
GC% range Yes Host-dependent; default N. benthamiana: 55-65% (legacy engine-output-calibrated band, v1; see Codon Reference Contract below). N. tabacum (BY-2): 55-65% (unchanged). This range is a configured target metric, not a hard gate outside the benchmark scoring contract.

Codon Reference Contract (v1 / v2)

codon_reference_contract_version tracks which codon usage table and GC reference band a result was generated against — distinct from scoring_contract_version (currently v1.1), which defines the unrelated multi_constraint_pass pass/fail formula (biological_pass AND assembly_pass AND gc_in_target_range). A v1→v2 codon-reference change does not change what "pass" means; it changes the codon table and GC band a result is scored against.

Contract version Codon reference asset GC reference band (N. benthamiana) Status
v1 nbenthamiana_legacy_kazusa_sgn_v101 (legacy, circular-derived) 55-65% historical comparator retained for v3.2.x continuity and sensitivity interpretation; not the current default
v2 nbenthamiana_nbev11_hc_v2 (NbeV1.1 LAB-strain, native genome-composition anchor; reference-policy audit) 40-47% current production software default; public overrides remain disabled; not experimental validation or comparative biological-performance evidence

The active default is recorded in data/reference/active_codon_reference.json and synchronized with current_parameter_registry.yaml's codon_reference.active block (see tests/test_registry_production_sync.py for the sync guard). Public CLI and REST API optimize endpoints do not expose a codon-reference override; they always use the active default and reject request fields such as codon_reference, codon_table_id, or codon_table_path. Python/benchmark research paths can still pass an explicit codon_table_path for controlled comparator runs, but those packaged assets are not public production options unless a separate activation gate enables them.

Advisory sequence-risk scans (all 9 run by default; findings are reported, never gating):

Check Covered Notes
PolyA-like motifs Yes Heuristic
AU-rich elements (ARE) Yes Heuristic
AT-rich runs Yes Minimum run length 6 nt default
Homopolymers Yes Synthesis-risk threshold
Tandem repeats Yes >=15 nt, recombination risk
Local GC extremes Yes 50-nt window, 25-75% synthesis guard
Splice-like motifs Yes Heuristic; false positive risk noted
CpG/TpA dinucleotides Yes CAI-budgeted reduction
Rare codon runs Yes Ribosome stalling risk detection

Assembly review:

Check Covered Notes
Forbidden restriction sites Yes BsaI, BsmBI, BpiI (Golden Gate); halts the production design pipeline when unresolvable
MoClo overhang validity / collision Yes (opt-in) Not run by default; reports warnings only, never halts the pipeline

Outside current scope (requires wet-lab validation):

Check Covered Notes
Protein folding No Requires wet-lab validation
Actual expression level No Requires wet-lab validation
Yield / solubility No Requires wet-lab validation
Signal peptide behavior No Requires wet-lab validation
Membrane protein topology No Requires wet-lab validation

Contribute Wet-Lab Results

Public-safe validation summaries are welcome. Positive, negative, equivalent, reduced, failed-expression, and inconclusive outcomes are all useful.

Public GitHub Issues are public. Use them only for public-safe summaries. Do not submit private or sensitive wet-lab feedback through public GitHub Issues.

Default public fields should use protein class rather than protein name. Public credit requires explicit approval and maintainer review.

Do not submit:

  • Raw DNA, RNA, or protein sequences.
  • Proprietary or unpublished construct sequences.
  • Internal batch IDs.
  • Confidential partner/customer information.
  • Patient or clinical subject data.
  • Private contact information.
  • Exact confidential process parameters.
  • Confidential assay protocols.
  • Unpublished construct-identifying labels.

Public-safe summaries may include:

  • FactorForge version.
  • Host organism.
  • Optimization profile.
  • Protein class.
  • Coarse assay category.
  • Replicate category.
  • Comparison result.
  • Expression result.
  • Non-confidential notes only.

Submission channels:

All public entries require manual review before publication.