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Uricase Cassette Ranking, Computational Analysis (comp-022)

1. Question

Across the A. oryzae uricase expression cassette design space, parameterized as 6 promoters × 12 signal peptides × 10 codon variants × 60 secretion scaffolds = 43,200 combinations, which cassettes survive a multi-model concordance gate and warrant promotion to the §1.9 dual-cassette wet-lab feasibility test?

This is the first comp-NNN to instantiate the ClockBase exhaustive-search-then-rank pattern at full cardinality. Prior cassette work in the corpus (comp-010, comp-011) examined two specific candidate designs each; comp-022 ranks the full combinatorial space. The intent is not to replace §1.9, but to ensure that the §1.9 cassette design is the right point in the design space before gene synthesis dollars commit.

2. Verdict

The §1.9 architecture is robust. The composite ranking converges on the architecture comp-010 already recommended (PamyB + amyB signal peptide + direct-secretion, no fusion), and adds three gene-synthesis-time refinements:

  1. Codon variant: prefer "5'-softened" over pure max-CAI. A two-zone codon optimization (low-GC + min-secondary-structure first 30 codons, max-CAI thereafter) ranks at the top of the concordance gate across all four models. Pure max-CAI gives slightly higher CAI (1.000 vs 0.915) but worse 5' mRNA structure (0.594 vs 0.733); the 5'-softened variant wins on N-of-4 concordance.
  2. C-terminal tag: block the PTS1 signal explicitly. Append 3×Ala or His6 to mask the native C-terminal SKL PTS1 motif. This addresses the routing risk flagged in comp-010 §3 at the cassette-design level rather than waiting for the §1.9 anti-uricase ELISA to detect peroxisomal misrouting.
  3. N-glycosylation: ablate N191. The one predicted N-glycosylation sequon in uricase (NSS at position 191; comp-010 §5) is unlikely to be occupied in the native protein but adds residual chaperone-pathway load if it is. A single N191Q point mutation removes the sequon at zero design cost.

Evidence level: Mechanistic Extrapolation (in silico only). Verdict gates a wet-lab confirmation in §1.9; the cassette-design refinements are gene-synthesis-time decisions that can be incorporated in the §1.9 construct order at no marginal cost.

Design-space size: 43,200 candidates enumerated (matches brief's Pass-2 estimate exactly). Concordance pass rate N-of-4 = 4: 195 candidates (0.45%). Concordance pass rate N-of-4 ≥ 3: 2,421 candidates (5.6%), which collapse to 501 unique cassette designs after removing degeneracy across propeptide and N-glyc modifier states.

3. Method

3.1 Design-space parameterization

Axis Cardinality Range
Promoter 6 PamyB (Tada 1991 PMID 1937733), PglaA (Ward 1995 PMID 9634791), PenoA (Toda 2001), PgpdA (Punt 1990 PMID 2113023), PtefI (Kitamoto 1998), PnmtA (Shoji 2005)
Signal peptide 12 6 native koji SPs (amyB, glaA, pepO, alpA, lipase) + foreign cbhI (T. reesei), each ×
Codon variant 10 native_uaZ approx, cai_max, cai_balanced, cai_max_gc54, harmonized (Angov 2008), rare_avoid, low_gc, high_gc, 5p_softened (Kudla 2009), 5p_softened_balanced
Secretion scaffold 60 10 base scaffolds (direct × 3 C-term tag variants; glaA-full KEX2 × 4 KEX2-linker variants; glaA-truncated KEX2 × 2; tandem-KEX2 × 1) × 3 propeptide states × 2 N-glyc states
Product 43,200

Full part-by-part provenance (sequences, citations, fetch dates) is in experiments/comp-022-clockbase-uricase-cassette-ranking/inputs/provenance.md.

3.2 Four scoring models

Model Tier Direction Method Primary source
Codon Adaptation Index (CAI) 1 Higher better Geometric mean of per-codon w-values (w = freq/max-freq-synonym) under A. oryzae table Sharp & Li 1987 PMID 3547335
mRNA 5' structure proxy 1 Higher better GC-content + GC-clamp + palindromic-4mer count over first 120 nt Kudla 2009 PMID 19359587
Architecture-adjusted chaperone load 2 Lower better Σ (disulfide_count × α) + glycosylation + KEX2 + PTS1 penalties chaperone-orthogonal-stacking.md §3.5
Promoter × SP prior 4 Higher better Literature-derived bounded multiplier per promoter and SP (See §3.4)

Tiers reflect the cascading-filter design from the brief. In practice, Tier 1 and Tier 2 are both per-component (per codon variant; per scaffold base) rather than per-candidate, so the full 43,200-candidate evaluation is computationally cheap. The cascading framing matters when Tier 3 fold-quality is included; in this v1 run, Tier 3 is deferred.

3.3 Concordance gate (N-of-M)

Each candidate is assigned a binary "top quintile" flag per model (1 if it falls in the top 20% by that model's score). N-of-4 concordance = sum of flags. The promotion threshold is N-of-4 ≥ 3 (75%), chosen a priori from the ClockBase precedent (30/40 ≈ 75%; autonomous-screening-methodology.md §"Computational-to-wet-lab handoff: N-of-M concordance").

Within the concordance-passing shortlist, candidates are ranked by a continuous composite score (mean of per-model min-max normalized values) to break ties.

3.4 Promoter and signal peptide priors

Promoter Relative strength Citation
PamyB 1.00 (reference) Tada 1991 PMID 1937733; canonical workhorse
PglaA 0.85 Ward 1995 PMID 9634791; Huynh 2020 PMC7257131
PenoA 0.70 Toda 2001
PtefI 0.65 Kitamoto 1998; orthogonal constitutive
PnmtA 0.60 Shoji 2005; tunable repressible
PgpdA 0.55 Punt 1990 PMID 2113023; selection-marker promoter

Signal peptide efficiencies: native koji SPs (amyB, glaA, alpA, lipase, pepO) score 0.82-1.02; foreign cbhI from T. reesei scores 0.65-0.68; pro-region present adds a small bonus (2-5%) for KEX2-mediated N-terminal-homogeneity benefit. All values are bounded literature estimates; the ordinal ranking is robust, the scalar values are priors not measurements. See experiments/comp-022-.../inputs/provenance.md.

4. Key Results

4.1 Per-codon-variant scores (10 variants)

Codon Variant CAI mRNA-5' proxy Rare-codon clusters Headline
native_uaZ (approx) 0.464 0.667 14 Native rate; many rare clusters in A. oryzae context
cai_max 1.000 0.594 0 Pure max-CAI; degraded 5' structure
cai_balanced 0.724 0.589 1 Frequency-weighted; middling on both axes
cai_max_gc54 0.757 0.800 2 GC-constrained; good 5' structure but lower CAI
harmonized (Angov 2008) 0.695 0.617 2 Mid-rank harmonized
rare_avoid 0.781 0.489 0 RSCU≥0.4 filter; worst 5' structure of any cluster
low_gc 0.386 0.667 26 GC-poor; very poor CAI
high_gc 1.000 0.594 0 GC-rich; equivalent to cai_max here
5p_softened 0.915 0.733 2 Top-quintile on both axes; the headline variant
5p_softened_balanced 0.679 0.733 2 5'-softened but mid-rank tail; worse CAI

The "5'-softened" variant (low-GC + max-CAI two-zone optimization) is the only codon variant that lands in the top quintile on both CAI and 5' structure simultaneously. Pure max-CAI lands top on CAI but bottom on 5' structure.

4.2 Per-scaffold chaperone load (10 base scaffolds)

Scaffold Fusion KEX2 site PTS1 blocked Effective load
direct_natag_pts1ok none none False 0.50
direct_3xAla_pts1blk none none True 0.20
direct_his6_pts1ok none none True 0.20
glaA_KR_pts1ok glaA_full KR False 5.10
glaA_KR_3xAla glaA_full KR True 4.80
glaA_KRGGG_pts1ok glaA_full KRGGG False 5.05
glaA_KRGGG_3xAla glaA_full KRGGG True 4.75
glaA_trunc_KR_pts1ok glaA_truncated KR False 2.60
glaA_trunc_KR_3xAla glaA_truncated KR True 2.30
tandem_KEX2_pts1ok glaA_full double_KR False 5.40

Direct-secretion with PTS1-blocking C-terminal tag is the lowest chaperone-load architecture. Glucoamylase-KEX2 fusion variants carry 10-25× the chaperone load because they impose the carrier's disulfides and glycosylation on top of uricase's near-zero intrinsic load. Tandem-KEX2 saturates KEX2 capacity (Spencer 1998 precedent) and adds 5-10% additional load.

Architectural implication: the Ward 1995 fusion architecture is correct for lactoferrin (where it boosts secretion of a complex glycoprotein) but is the wrong architecture for uricase, which has zero intrinsic chaperone load and benefits most from a minimum-overhead direct-secretion design. Comp-010 reached this conclusion via design analysis; comp-022 confirms it via full design-space ranking.

4.3 Top-5 unique cassettes (after collapsing propeptide and N-glyc modifier degeneracy)

Rank Promoter Signal Peptide Codon Variant Scaffold Propeptide N-glyc N-of-4 Composite
1 PamyB SPamyB_pro 5p_softened direct_3xAla_pts1blk none ablated 4 0.912
2 PamyB SPamyB_pro 5p_softened direct_his6_pts1ok none ablated 4 0.912
3 PamyB SPamyB 5p_softened direct_3xAla_pts1blk none ablated 4 0.904
4 PamyB SPamyB 5p_softened direct_his6_pts1ok none ablated 4 0.904
5 PamyB SPglaA_pro 5p_softened direct_3xAla_pts1blk none ablated 4 0.904

195 candidates pass N-of-4 = 4 (all four models top quintile). The top cluster is tightly constrained: PamyB promoter, amyB or glaA signal peptide (with optional pro-region), 5'-softened codon variant, direct-secretion scaffold with PTS1-blocking C-terminal tag, no propeptide between SP and mature N-terminus, N191 glycosylation sequon ablated.

The full top-25 table is in experiments/comp-022-.../outputs/top25.md. The full 501-cassette concordance-passing shortlist is in unique_cassette_shortlist.csv.

4.4 Concordance distribution

N-of-4 Candidates Share
4 195 0.45%
3 2,226 5.15%
2 9,438 21.8%
1 17,946 41.5%
0 13,395 31.0%

The mass of the design space (72.5%) lands at N-of-4 ≤ 1; i.e., most cassette designs are bad on at least three of four axes simultaneously. This is the expected pattern for a high-dimensional design space with orthogonal scoring axes; it is also the failure mode that comp-022's exhaustive enumeration is intended to surface (a priori one-at-a-time selection is path-dependent and tends to lock in a single axis's preferred value while ignoring the others).

5. Limitations

The brief authorized these v1 simplifications; each is documented for downstream readers.

  1. Fold-quality model (Tier 3) deferred to wet-lab. ESMFold and AlphaFold pLDDT are not callable from this subagent's environment (no GPU, no API key, sandbox restricts network). ColabFold and ESM2 perplexity were considered as proxies and also unavailable. Brief authorized this deferral. Implication: the "structural foldability" axis is unscored in this v1. For uricase specifically the consequence is minor because every candidate uses the same AA sequence (Q00511), so fold-quality differences would be driven entirely by cassette-context effects (signal-peptide cleavage, propeptide processing, glycosylation occupancy) rather than by primary-sequence variation. The wet-lab fold quality readout in §1.9 is anti-uricase ELISA + uricase activity assay (UA-disappearance spectrophotometry), which directly tests folded-and-secreted protein. This supersedes any in silico pLDDT proxy at the cassette-design stage.

  2. N-of-M threshold = 3 of 4 (75%), not 4 of 5 (80%). Because the fold-quality model is deferred, M=4 not 5. The 75% concordance fraction is preserved (matches ClockBase 30/40 precedent). An a priori choice; no retrospective calibration was attempted because comp-001 through comp-014 were not cassette-ranking experiments (TCM and complement-modulator literature mining, protease stability, transporter modulation). The retrospective-calibration framing in the brief was based on a misreading of those comps. This is documented in experiments/comp-022-.../README.md.

  3. Promoter strengths and SP efficiencies are bounded literature estimates. They are priors, not measurements in NSlD-ΔP10. They affect the absolute composite scores but not the ordinal ranking robustness of the top cluster: PamyB dominates in the literature consensus by a sufficient margin that any reasonable bounded estimate places it at the top. PglaA's 0.85 vs 0.95 multiplier would swap rank 1 vs rank 3 but not the architectural class of the headline cluster.

  4. mRNA 5' secondary structure is a GC-content + GC-clamp + palindromic-4mer count proxy, not a true minimum-free-energy calculation. ViennaRNA is not installable in this environment (no pre-built binary, network-restricted pip). The proxy is defensible per Kudla 2009 PMID 19359587 (5' GC dominates translation initiation in E. coli; replicated in S. cerevisiae and filamentous fungi), but a true MFE calculation on the surviving shortlist would refine the ranking. Recommended follow-up: retrofit ViennaRNA MFE on the 501-cassette shortlist when an environment with the binary is available.

  5. Native A. flavus codon usage approximated. The "native_uaZ" variant is back-translated using the A. oryzae table with mid-rank-codon biasing rather than from an A. flavus codon usage table directly. This affects only the native_uaZ variant's ranking and does not affect the headline cluster (which uses the 5p_softened variant, optimized for A. oryzae from first principles).

  6. Single payload only. comp-022 ranks uricase cassettes in isolation. The §1.9 wet-lab test is a dual-cassette uricase + lactoferrin design, where pairwise chaperone-pathway competition is governed by chaperone-orthogonal-stacking.md §3.5.3 (effective PDI load 0 + 24-40 = 24-40 total, within demonstrated NSlD-ΔP10 capacity). Comp-022 does not re-evaluate the pairwise interaction; it inherits the comp-010 finding that the uricase + lactoferrin pair is architecturally compatible.

  7. Cascading-filter framing partially redundant for this experiment. Brief described a cascading filter where Tier 1 evaluates all 43,200, Tier 2 evaluates top 5,000, Tier 3 evaluates top 100. In practice, Tier 1 (CAI + mRNA-5') is per-codon-variant only (10 evaluations) and Tier 2 (chaperone-load) is per-scaffold-base only (10 evaluations), so the full 43,200-candidate evaluation is O(1) per candidate after the per-component pre-computations. Cascading matters when Tier 3 fold-quality (per-candidate ESMFold) is in scope; in this v1 it is not.

6. Impact on Experimental Priorities

6.1 §1.9 wet-lab cassette design

The §1.9 architecture stands. The uricase cassette in the existing §1.9 design (validation-experiments.md §1.9) is already [PTEF1 - amyB signal peptide - A. flavus uaZ codon-optimized - TgpdA], which is in the comp-022 top cluster. Three gene-synthesis-time refinements should be incorporated when the construct is ordered:

Refinement Current §1.9 comp-022 recommendation Cost delta
Promoter PTEF1 Substitute PamyB OR keep PTEF1 as orthogonal-promoter design choice $0 (same gene-synthesis cost; PTEF1 is justified by orthogonal-promoter rationale per koji-endgame-strain.md §3.4 even though PamyB is the single-promoter optimum)
Codon variant "codon-optimized for A. oryzae" (unspecified strategy) 5'-softened (low-GC first 30 codons + max-CAI thereafter) $0
C-terminal tag none (native SKL) append 3×Ala or His6 to block PTS1 $0 (single codon trio extension)
N-glycosylation sequon native N191 NSS N191Q ablation $0 (single point mutation)

None of these refinements require new wet-lab infrastructure or change the §1.9 cost or timeline ($3-5K / 8-12 weeks). They are added to the gene-synthesis order at no marginal cost.

6.2 The §1.9 architecture decision: PamyB vs PTEF1 for uricase

Comp-022 single-cassette ranking favors PamyB for both cassettes. The current §1.9 design uses PTEF1 for the uricase cassette specifically to separate transcriptional programs from the PamyB-driven lactoferrin cassette (koji-endgame-strain.md §3.4: "Distinct promoter (TEF1, constitutive) to avoid direct competition with the starch-inducible PamyB of Cassette A"). This is a dual-cassette-specific rationale that comp-022's single-payload ranking does not override.

Decision rule for the §1.9 construct: keep PTEF1 for the uricase cassette per the orthogonal-promoter rationale. If the §1.9 Lf-alone arm titer falls below the 500 mg/L threshold per chaperone-orthogonal-stacking.md §3.5.4 calibration framework, revisit the dual-PamyB design via comp-022's promoter-strength ranking (PamyB-PamyB cassette pair, integrated at paralogous loci per Li 2024 PMID 39830075; brief noted as a "symmetric alternative" in koji-endgame-strain.md §3.4).

6.3 Beyond §1.9: when this framework adds value

Comp-022's value is highest for future cassettes where the right architecture is not yet obvious from manual design:

  • DAF SCR1-4 single-cassette design (validation-experiments.md §1.25): would benefit from a comp-022-style ranking with a fold-quality Tier 3 included (DAF SCR1-4 has 8 disulfides and is sensitive to fold quality, unlike uricase). Recommended follow-up.
  • Engineered C1-INH cassette (parallel CP0 thread surfaced in comp-018): comparable disulfide load + glycosylation + protease-sensitivity profile to lactoferrin. comp-022-style ranking would be load-bearing for first-pass cassette selection.
  • Complestatin NRPS LBP cassette (comp-024 when authored): different chassis (Bacteroides / E. coli Nissle), different codon optimization context, but the same combinatorial structure applies.

For uricase specifically, comp-022 is partly retrospective validation that the cassette-design intuition in the corpus (comp-010 + koji-endgame-strain.md §3.4) was correct. The headline architecture was not surprising; the value is the 501-cassette concordance shortlist as a reproducible artifact plus the codon-variant refinement (5'-softened beats pure max-CAI) which was not in the existing design.

7. Cross-References

8. Status

Complete (v1 + v2 retrofit, 2026-05-14). See §9 for the v2 update.

9. v2 update (2026-05-14)

Both v1-deferred models are now retrofitted on the existing 501-cassette shortlist. The concordance gate tightens from v1's N-of-4 ≥ 3 (75%) to v2's N-of-5 ≥ 4 (80%) because the new models are real, not proxies. Inputs at v2/inputs/; outputs at v2/outputs/.

9.1 Methodology additions

  • ESM2 t33 650M pseudo-likelihood as the Tier 3 fold-quality proxy. The brief authorized ESMFold v1 as first-choice, but ESMFold v1 in fair-esm 2.0.0 requires openfold which the subagent environment could not install cleanly; ESM2 pseudo-likelihood is the documented fallback (Verkuil 2022; Hsu 2022). ESM2 is the language model that ESMFold uses internally, and per-residue masked-LM log-probability is a direct readout of the model's confidence in each residue's local fold context. Rank-preserving across cassettes of similar length (all ~320-400 aa here). Score range presented as a rescaled pseudo-pLDDT [50, 90] for interpretability; the top-quintile flag uses the rank-preserving raw log-prob.
  • ViennaRNA 2.7.2 (Python binding) for real mRNA 5' MFE, replacing v1's GC-clamp proxy. 150-nt window: 61-nt generic A-rich placeholder 5'UTR + signal peptide ORF + first 30 codons of mature uricase. UTR held constant across cassettes.
  • MPS unavailable on the Python 3.13 + torch 2.12 stack in the v2 env. Fell back to CPU; ESM2 inference on all 106 protein-distinct sequences completed in ~70 seconds.

9.2 Headline numbers

Tier v1 v2
Pass concordance 501 cassettes (N-of-4 ≥ 3) 71 cassettes (N-of-5 ≥ 4)
Strictest tier 45 cassettes (N-of-4 = 4) 4 cassettes (N-of-5 = 5)
v1 top-cluster survival in v2 strictest tier n/a 4 of 4 (100%)
v1 shortlisted cassettes dropped by v2 gate n/a 430

v2 concordance distribution across the 501 v1-shortlisted cassettes: 5/5 = 4; ⅘ = 67; ⅗ = 203; ⅖ = 227.

9.3 v1 architecture verdict survives, and the v2 top tier IS the v1 top cluster

The v1 top cluster (PamyB + SPamyB or SPamyB_pro + 5p_softened codon + direct_3xAla_pts1blk or direct_his6_pts1ok scaffold + nglyc_ablated) has 4 cassettes in v1, all 4 surviving into v2's strictest N-of-5 = 5 tier (100% survival). The v2 strictest tier IS the v1 top cluster; the retrofit did not surface new top-tier cassettes outside the v1-promoted set. The v1 verdict that the §1.9 architecture should stand, and that the three gene-synthesis-time refinements (5'-softened codon, PTS1-blocking C-terminal tag, N191Q glycosylation-sequon ablation) are real, are confirmed under v2. (Mechanistic Extrapolation: ESM2 pseudo-likelihood + ViennaRNA MFE; not yet wet-lab validated.)

9.4 v1 GC-clamp proxy was weak; the v2 retrofit materially shifts the mRNA model

Spearman correlation between v1's GC-clamp mrna_5p proxy and v2's real ViennaRNA MFE, computed per (codon, sp) pair (n = 52): rho = 0.241. Weak. The v1 model on this axis was essentially noise. v2's top-quintile MFE cutoff is MFE ≥ -16.4 kcal/mol (less negative = looser 5' structure = faster ribosome initiation per Kudla 2009 PMID 19359587).

9.5 v2 cutoffs on the 501-cassette shortlist cohort

  • ESM2 raw pll_mean top-quintile cutoff: -0.2214 (rescaled pseudo-pLDDT cutoff: 87.53)
  • ViennaRNA MFE top-quintile cutoff: ≥ -16.4 kcal/mol
  • CAI, chaperone load, and promoter-SP prior inherit v1 cutoffs from report.json.

Scoping: v2 quintile cutoffs are computed on the 501-cassette v1 shortlist cohort, not the original 43,200 cohort. v2 is a tighter gate on v1 promotions, not a re-enumeration.

9.6 §1.9 wet-lab impact

The §1.9 design from v1 stands. The three gene-synthesis-time refinements are confirmed. The actionable v2 change is scope reduction: the v1 shortlist of 501 cassettes drops to 71 under N-of-5 ≥ 4, and to 4 under N-of-5 = 5. If gene-synthesis budget allows only a few candidates, the v2 N-of-5 = 5 tier names exactly which ones (the four v1-top-cluster members). The 430-cassette difference between v1 shortlist and v2 shortlist represents cassettes that would have been promoted under v1's deferred-fold-quality methodology but are rejected once real fold-quality and real MFE signals are in the loop.

9.7 v2 limitations honest accounting

  • ESM2 pseudo-likelihood is a fold-quality proxy, not direct pLDDT. ESMFold would have been preferable; openfold install was blocked. Rank-preserving across the similar-length cohort here but not the direct per-residue confidence score ESMFold produces. v2.5 should retrofit real ESMFold once openfold install is unblocked.
  • Pseudo-likelihood from a single masked-LM forward pass per sequence, not from per-position masking. Bias is rank-preserving; absolute values are inflated.
  • 5' MFE window is 150 nt with a 61-nt placeholder UTR. Promoter-specific UTR modeling and longer-window MFE are v2.5 work.
  • CPU inference (MPS unavailable) was acceptable at N=106 sequences (~70 sec); for the full 43,200 design space, MPS or GPU would be required.
  • v2 quintile cutoffs scoped to the 501-cassette shortlist cohort, not the full 43,200 cohort. v2 is the right tool for "is the v1 shortlist over-promoted?" but not for "what new cassettes outside the v1 shortlist should we promote?"; the latter requires running ESM2 + ViennaRNA on the full design space.

9.8 Promoted artifacts

  • experiments/comp-022-clockbase-uricase-cassette-ranking/v2/inputs/protein_shortlist.fasta (106 protein-distinct sequences)
  • experiments/comp-022-clockbase-uricase-cassette-ranking/v2/inputs/protein_shortlist_keymap.csv (sequence_id ↔ cassette-spec keys)
  • experiments/comp-022-clockbase-uricase-cassette-ranking/v2/outputs/esmfold_pLDDT.csv (106 rows, ESM2 pseudo-pLDDT + raw pll_mean)
  • experiments/comp-022-clockbase-uricase-cassette-ranking/v2/outputs/viennarna_mfe.csv (52 rows, real ViennaRNA MFE + v1 GC-clamp proxy)
  • experiments/comp-022-clockbase-uricase-cassette-ranking/v2/outputs/v2_shortlist.csv (71 cassettes passing N-of-5 ≥ 4)
  • experiments/comp-022-clockbase-uricase-cassette-ranking/v2/outputs/v2_top25.md
  • experiments/comp-022-clockbase-uricase-cassette-ranking/v2/outputs/v2_summary.json
  • experiments/comp-022-clockbase-uricase-cassette-ranking/v2/analyze_v2.py (re-ranking pipeline, reproducible)

9.9 v2 status

v2 complete. Tier 3 fold-quality retrofitted (ESM2 pseudo-likelihood fallback; see §9.7). Tier 1' mRNA model retrofitted (real ViennaRNA MFE replacing v1 GC-clamp proxy).

v2.5 deferred (2026-05-14, explicit decision). Real ESMFold, full per-position masking, promoter-specific UTR, MPS/GPU for full-cohort retrofit are deferred until either (a) §1.9 wet-lab data arrives and retrospective validation of in silico scorers becomes possible, or (b) a fresh design problem (different target) requires the same machinery. Reasoning: the v2 output is sufficient to commit gene-synthesis dollars (4 strict winners + 71 looser shortlist); v1 architectural cluster survives v2 at 100%, so the marginal value of a stricter fold-quality model on the same cohort is bounded; wet-lab yield data will dominate any further in silico refinement. The deferral is a real-data-imminent diminishing-returns call, not an infrastructure block — Python 3.12 venv + HuggingFace facebook/esmfold_v1 via transformers is the documented path forward when v2.5 is unblocked.

Verification-agent pass complete per CLAUDE.md Rule 4; all load-bearing numbers in §9 are grep-verified against the v2 output CSVs and JSON in experiments/comp-022-.../v2/outputs/ and the v2 provenance log.