@possumtech/rummy 2.2.0 → 2.3.1

package/audit/sweep/dna-insert/turns/turn_005.md

@@ -1,396 +0,0 @@
-# turn 005
-
-tokens: prompt=34106 cached=1739 completion=715 reasoning=0 total=34821 cost=0
-created: 2026-05-02 00:22:32
-
-update: status=155 body=distilling primer3 complete + discovering sequences.fasta + Q5 kit
-
----
-
-## reasoning (think)
-
-```
-- Distillation stage: resolve remaining unknowns using promoted sources
-- unknown://biology/tools/primer3_oligotm RESOLVED via manpage (https://manpages.debian.org/stretch/primer3/oligotm.1) + known://biology/tools/primer3_oligotm
-  - Clarifies ALL flags: -tp=1 (SantaLucia 1998 NN params, recommended), -sc=1 (SantaLucia 1998 salt corr, recommended), -mv=50 (monovalent 50mM default), -dv=2 (divalent 2mM), -n=0.8 (dNTP 0.8mM), -d=500 (DNA 500nM)
-  - Usage: oligotm [flags] SEQUENCE (2-36nt DNA)
-  - Demote manpage after distilling
-- unknown://biology/primers/design_rules can now be fully resolved (Tm computation details complete)
-- Next: discover sequences.fasta (repo empty, need to check for it or create from prompt)
-- Then resolve Q5 kit + output format → deploy primer design
-- Budget ok: manpage slice if needed, but full promote feasible (1341 < 1.7M free)
-```
-
-## assistant emission
-
-```
-<think>
-- Distillation stage: resolve remaining unknowns using promoted sources
-- unknown://biology/tools/primer3_oligotm RESOLVED via manpage (https://manpages.debian.org/stretch/primer3/oligotm.1) + known://biology/tools/primer3_oligotm
-  - Clarifies ALL flags: -tp=1 (SantaLucia 1998 NN params, recommended), -sc=1 (SantaLucia 1998 salt corr, recommended), -mv=50 (monovalent 50mM default), -dv=2 (divalent 2mM), -n=0.8 (dNTP 0.8mM), -d=500 (DNA 500nM)
-  - Usage: oligotm [flags] SEQUENCE (2-36nt DNA)
-  - Demote manpage after distilling
-- unknown://biology/primers/design_rules can now be fully resolved (Tm computation details complete)
-- Next: discover sequences.fasta (repo empty, need to check for it or create from prompt)
-- Then resolve Q5 kit + output format → deploy primer design
-- Budget ok: manpage slice if needed, but full promote feasible (1341 < 1.7M free)
-</think>
-
-<get path="app/sequences.fasta" preview/>
-<get path="**" manifest>sequences.fasta</get>
-<get path="https://www.neb.com/en-us/products/e0554-q5-site-directed-mutagenesis-kit" line="1" limit="500"/>
-
-<set path="known://biology/tools/primer3_oligotm_complete" visibility="visible" summary="biology,tools,primer3,oligotm,usage,flags,tm,ground_truth,manpage,santalucia,nn,salt_corr">
-# Related
-[prompt rules](prompt://1)
-[unknown resolved](unknown://biology/tools/primer3_oligotm)
-[manpage source](https://manpages.debian.org/stretch/primer3/oligotm.1)
-[search primer3](log://turn_3/search/primer3_oligotm_exact_usage_flags_-tp_1_-sc_1_-mv_50_-dv_2_-n_0.8_-d_500_melting)
-[distilled flags](known://biology/tools/primer3_oligotm)
-
-# Primer3 oligotm COMPLETE spec (Debian manpage)
-**Usage**: oligotm [OPTIONS] {oligo} → prints Tm (C) to stdout. oligo=2-36nt DNA.
-
-**Flags (ALL clarified)**:
-- `-tp 1`: SantaLucia 1998 nearest-neighbor params (recommended; default=0 Breslauer)
-- `-sc 1`: SantaLucia 1998 salt correction (recommended; default=0 Schildkraut)
-- `-mv 50`: monovalent cations (mM, default 50)
-- `-dv 2`: divalent cations (mM, default 0)
-- `-n 0.8`: dNTP conc (mM, default 0)
-- `-d 500`: DNA strands (nM, default 50)
-- `-i`: print references
-
-**Matches prompt flags exactly**. Tm for annealing part only (15-45nt).
-</set>
-
-<update status="155">distilling primer3 complete + discovering sequences.fasta + Q5 kit</update>
-```
-
-## actions (chronological log entries)
-
-### `log://turn_5/get/app/sequences.fasta`
-scheme=log action=get attrs={"path":"app/sequences.fasta","action":"get"}
-
-```
-app/sequences.fasta not found
-```
-
-### `log://turn_5/get/**`
-scheme=log action=get attrs={"path":"**","action":"get"}
-
-```
-MANIFEST get path="**": 46 matched (288854 tokens)
-https://benchcalc.com/primer-tm-calculator (4323)
-https://help.takarabio.com/36168/kb/article/110018/how-do-i-calculate-the-melting-temperature-tm-of-primers (384)
-https://libnano.github.io/primer3-py/api/thermoanalysis.html (6979)
-https://manpages.debian.org/stretch/primer3/oligotm.1 (1381)
-https://oligocalc.eu (11294)
-https://oligopool.com/tm-calculator (1727)
-https://primer3.org/manual.html (101235)
-https://support.snapgene.com/hc/en-us/articles/19181630869652-Simulate-NEB-Q5-Site-Directed-Mutagenesis (3484)
-https://vcru.wisc.edu/simonlab/bioinformatics/programs/primer3/primer3_manual.htm (86312)
-https://www.bioke.com/webshop/neb/e0552.html (203)
-https://www.bioke.com/webshop/neb/e0554.html (203)
-https://www.neb-online.de/en/pcr-and-dna-amplification/q5-site-directed-mutagenesis-kit (4140)
-https://www.neb.com/en-us/products/e0552-q5-site-directed-mutagenesis-kit-without-competent-cells (2951)
-https://www.neb.com/en-us/protocols/2013/01/26/q5-site-directed-mutagenesis-kit-protocol-e0554 (1642)
-https://www.neb.com/en/applications/cloning-and-synthetic-biology/site-directed-mutagenesis (4888)
-https://www.neb.com/en/products/e0554-q5-site-directed-mutagenesis-kit (5816)
-known://biology/tools/primer3_oligotm (333)
-log://turn_1/set/unknown%3A/biology/mutagenesis/q5_kit (83)
-log://turn_1/set/unknown%3A/biology/plasmid/sequences_fasta (80)
-log://turn_1/set/unknown%3A/biology/primers/design_rules (97)
-log://turn_1/set/unknown%3A/biology/tools/primer3_oligotm (58)
-log://turn_1/set/unknown%3A/task/output/primers_fasta (53)
-log://turn_1/update/prompt_decomposed (9)
-log://turn_2/get/** (337)
-log://turn_2/get/**_1 (354)
-log://turn_2/get/app (7)
-log://turn_2/update/exploring_repo_for_sequences.fasta/_primer3/_Q5_kit_details (30)
-log://turn_3/get/** (714)
-log://turn_3/get/repo%3A/overview (183)
-log://turn_3/search/primer3_oligotm_exact_usage_flags_-tp_1_-sc_1_-mv_50_-dv_2_-n_0.8_-d_500_melting (1866)
-log://turn_3/update/discovering_primer3_details/_repo_manifest/_Q5_sequences (28)
-log://turn_4/get/** (855)
-log://turn_4/get/**_2 (868)
-log://turn_4/get/https%3A/manpages.debian.org/stretch/primer3/oligotm.1 (31)
-log://turn_4/get/https%3A/primer3.org/manual.html (42251)
-log://turn_4/search/neb_q5_site-directed_mutagenesis_kit_protocol_circular_plasmid_primers_minimal_p (2182)
-log://turn_4/set/known%3A/biology/tools/primer3_oligotm (333)
-log://turn_4/update/distilling_primer3_oligotm_%2B_discovering_Q5_kit_protocol/sequences.fasta (36)
-log://turn_5/get/app/sequences.fasta (15)
-prompt://1 (554)
-repo://overview (164)
-unknown://biology/mutagenesis/q5_kit (83)
-unknown://biology/plasmid/sequences_fasta (80)
-unknown://biology/primers/design_rules (97)
-unknown://biology/tools/primer3_oligotm (58)
-unknown://task/output/primers_fasta (53)
-```
-
-### `log://turn_5/get/https%3A/www.neb.com/en-us/products/e0554-q5-site-directed-mutagenesis-kit`
-scheme=log action=get attrs={"path":"https://www.neb.com/en-us/products/e0554-q5-site-directed-mutagenesis-kit","lineStart":1,"lineEnd":225,"totalLines":225,"action":"get"}
-
-```
-https://www.neb.com/en-us/products/e0554-q5-site-directed-mutagenesis-kit
-[lines 1–225 / 225 total]
-# Q5 Site-Directed Mutagenesis Kit | NEB
-
-![Add Favorite Button](https://www.neb.com/Content/img/icons/favorite-icon-add.svg "Add Favorite")
-
-The Q5® Site-Directed Mutagenesis Kit enables rapid, site-specific mutagenesis
-of double-stranded plasmid DNA in less than 2 hours.
-
-*   Non-overlapping primer design ensures robust, exponential amplification, generating a high percentage of desired mutations from a wide range of templates
-*   Intramolecular ligation and transformation into NEB high-efficiency competent cells results in high colony yield
-*   Extremely low error rate of Q5 Hot Start High-Fidelity DNA Polymerase reduces screening time
-*   Hot start polymerase enables room temperature reaction set up
-*   DpnI background reduction permits a wide range of starting template concentrations
-*   Use of standard primers eliminates additional expenses from phosphorylated or purified oligos
-*   Easy-to-use PCR master mix and unique multi-enzyme KLD Mix offer convenience and quality
-*   Rapid and direct treatment step proceeds at room temperature in 5 minutes
-*   Allows the use of any chemically-competent _E. coli_ cells suitable for cloning
-*   Supplied with competent cells 
-*   Use [NEBaseChanger™](https://nebasechanger.neb.com/) tool to generate primer sequences and an annealing temperature
-
-[Read MoreRead Less](#)
-
-## Featured Video
-
-Catalog #
-
-Cat #
-
-Concentration
-
-Conc
-
-Size
-
-List Price
-
-List Price
-
-Your Price
-
-Quantity
-
-E0554S
-
-Not Applicable
-
-10 reactions
-
-$241.00
-
-$241.00
-
-[Sign In](https://www.neb.com/en-us/account/sign-in) [Sign In](https://www.neb.com/en-us/account/sign-in)
-
-Please enter a quantity for at least one size
-
-*   ## Product Information
-    
-    The Q5 Site-Directed Mutagenesis Kit enables rapid, site-specific mutagenesis of double-stranded plasmid DNA in less than 2 hours (Figure 1). The kit utilizes the robust Q5 Hot Start High-Fidelity DNA Polymerase along with custom mutagenic primers to create insertions, deletions and substitutions in a wide variety of plasmids. After PCR, the amplified material is added directly to a unique Kinase-Ligase-DpnI (KLD) enzyme mix for rapid (5 minutes), room temperature circularization and template removal (Figure 2). Transformation into high-efficiency NEB 5-alpha Competent _E. coli_, provided with the kit, ensures robust results with plasmids up to at least 20 kb in length.
-    
-    [](https://nebasechanger.neb.com/) ![\[Icon\] Already using the Q5®
-    Site-Directed Mutagenesis Kit? Try NEBaseChanger® for primer design
-    help.](https://media.neb.com/asset/d0d2eea0-f634-40b4-bce3-1b2a11ddff10/Webp_JPG_PSD/NEBaseChanger_Tool_Banner.webp?=)
-    
-      
-    
-    **Figure 1: Site-specific mutagenesis proceeds in less than 2 hours**![Workflow steps and times for the Q5® Site-directed Mutagenesis Kit](https://media.neb.com/asset/101a8189-1a4a-4ec2-9a09-27945a4ac368/Webp/E0554_E0552_Q5_SiteDirectedMutagenesis_Q5SDM_WorkflowTimes.webp?=)
-    
-      
-    The use of a master mix, a unique multi-enzyme KLD enzyme mix, and a fast polymerase ensures that, for most plasmids, the mutagenesis reaction is complete in less than two hours.
-    
-      
-    
-    **Figure 2: Q5 Site-Directed Mutagenesis overview**![Overview of Q5® Site-directed Mutagenesis (SDM) workflow](https://media.neb.com/asset/632fff3b-729e-488d-b3f0-cbc2d38ca1f9/Webp/E0554_E0552_Q5_SDM_Overview.webp?=)
-    
-      
-    This kit is designed for rapid and efficient incorporation of insertions, deletions and substitutions into doublestranded plasmid DNA. The first step is an exponential amplification using standard primers and a master mix fomulation of Q5 Hot Start High-Fidelity DNA Polymerase. The second step involves incubation with a unique enzyme mix containing a kinase, a ligase and DpnI. Together, these enzymes allow for rapid circularization of the PCR product and removal of the template DNA. The last step is a high-efficiency transformation into chemically competent cells (not provided).
-    
-      
-    
-    **Figure 3: Primer design for Q5 Site-Directed Mutagenesis**![Overview of site-directed mutagenesis (SDM) for substitutions, deletions or insertions](https://media.neb.com/asset/3a29d4d6-fa3f-44a2-92d7-a69e2e191a1d/Webp/E0554_E0552_Q5_SiteDirectedMutagenesis_SDM_PrimerDesign.webp?=)
-    
-      
-    Substitutions, deletions and insertions are incorporated into plasmid DNA through the use of specifically designed forward (black) and reverse (red) primers.**Unlike kits that rely on linear amplification, primers designed for the Q5 Site-Directed Mutagenesis Kit should not overlap to ensure that the benefits of exponential amplification are realized.**
-    
-    A) Substitutions are created by incorporating the desired nucleotide change(s)
-    (denoted by \*) in the center of the forward primer, including at least 10
-    complementary nucleotides on the 3´side of the mutation(s). The reverse primer
-    is designed so that the 5´ends of the two primers anneal backto-back.
-    
-    B) Deletions are engineered by designing standard, non-mutagenic forward and
-    reverse primers that flank the region to be deleted.
-    
-    C) Insertions less than or equal to 6 nucleotides are incorporated into the 5´
-    end of the forward primer while the reverse primer anneals back-to-back with the
-    5´ end of the complementary region of the forward primer.
-    
-    D) Larger insertions can be created by incorporating half of the desired
-    insertion into the 5´ ends of both primers. The maximum size of the insertion is
-    largely dictated by oligonucleotide synthesis limitations.
-    
-      
-    
-    **Figure 4: NEB’s Q5 SDM Kit delivers higher transformation efficiency than Agilent’s QuikChange® SDM Kit**  
-    ![Bar graph of transfection efficiency for Q5® Site-directed Mutagenesis Kit versus QuikChange® Lightning Site-directed Mutagenesis Kit](https://media.neb.com/asset/0d8c5180-2814-443f-aff1-70dd7aef3516/Webp/E0554_E0552_Q5_SDM_TransfectionEfficiency.webp?=)
-    
-    _Results from a substitution reaction (4 nt) using the back-to-back Control SDM Primer Mix and Control SDM Plasmid (6.7 kb) are shown, along with results from a 12 nt deletion experiment (5.8 kb plasmid) and an 18 nt insertion experiment (7.0 kb plasmid). In all three cases, over 90% of the resultant colonies had incorporated the desired mutation(s). Results are normalized to total transformants if cells were not diluted prior to plating. For comparison, the same substitution reaction (4 nt) was performed with the QuikChange Lightning Site-Directed Mutagenesis Kit (Agilent) following Agilent’s protocol and using Agilent’s primer design tool to design overlapping primers.
-    
-    \*Note that the QuikChange kit does not accommodate deletions and insertions of
-    this size, so no comparison could be made for these experiments.
-    
-    _
-    
-    This product is related to the following categories:
-    
-    [sgRNA Synthesis,](https://www.neb.com/en-us/products/crispr-cas-gene-editing/sgrna-synthesis)
-    
-    [DNA Assembly, Cloning and Mutagenesis Kits](https://www.neb.com/en-us/products/dna-assembly-cloning-and-content-mutagenesis-kits)
-    
-    This product can be used in the following applications:
-    
-    [Site Directed Mutagenesis,](https://www.neb.com/en-us/applications/dna-amplification-pcr-and-qpcr/site-directed-mutagenesis)
-    
-    [Site Directed Mutagenesis](https://www.neb.com/en-us/applications/cloning-and-synthetic-biology/site-directed-mutagenesis)
-    
-    *   ### Kit Components
-        
-    *   ### Advantages and Features
-        
-    *   ### Related Products
-        
-    *   ### Product Notes
-        
-    *   ### References
-        
-    
-*   ## Protocols, Manuals & Usage
-    
-    *   ### Protocols
-        
-    *   ### Manuals
-        
-    *   ### Application Notes
-        
-    
-*   ## Tools & Resources
-    
-    *   ### Web Tools
-        
-    
-*   ## FAQs & Troubleshooting
-    
-    *   ### FAQs
-        
-    *   ### Troubleshooting
-        
-    *   ### Tech Tips
-        
-    
-*   ## Citations & Technical Literature
-    
-    *   ### Citations
-        
-    *   ### Feature Articles
-        
-    
-*   ## Quality, Safety & Legal
-    
-    *   ### Quality Control Assays
-        
-    *   ### Specifications & Change Notifications
-        
-    *   ### Certificate of Analysis
-        
-    *   ### Safety Data Sheets
-        
-    *   ### Legal and Disclaimers
-        
-    
-
-*   ![TroubleshootingTipsQ5SiteDirectedMutagenisis\_720](https://www.neb.com/en-us/-/media/nebus/page-images/video-thumbnails/troubleshootingtipsq5sitedirectedmutagenisis_720.jpg?rev=71dd5198eeed4685b0c13791fcff404b&la=en-US&h=161&w=286&hash=1F6D2053E95174100FCB2AEE8953F621)
-    
-    #### [Troubleshooting tips for Q5® Site-Directed Mutagenesis Kit](https://www.neb.com/en-us/tools-and-resources/video-library/troubleshooting-tips-for-q5-site-directed-mutagenesis-kit)
-    
-*   ![NEBaseChanger Primer Design Tool Tutorial](https://www.neb.com/en-us/-/media/nebus/page-images/video-thumbnails/nebasechanger-thumbnail3.jpg?rev=738b18bd48874c2fbfdb3edf6be28fa8&sc_lang=en-us&la=en-US&h=161&w=286&hash=D3E20CB9E7A6C68D6114C6207958894F)
-    
-    #### [NEBaseChanger® Primer Design Tool Tutorial](https://www.neb.com/en-us/tools-and-resources/video-library/nebasechanger-primer-design-tool-tutorial)
-    
-*   ![NEBTV\_DNA-Modifying-Enzymes-and-Cloning-Techniques\_1920](https://www.neb.com/en-us/-/media/nebus/page-images/video-thumbnails/nebtv_dna-modifying-enzymes-and-cloning-techniques_1920.jpg?rev=e97f8d25a3794930a02d072d0fb02afc&la=en-US&h=161&w=286&hash=9AD55F3189EEB3D249531442547C2C46)
-    
-    #### [Quick Tips - How can I improve the efficiency of the DpnI digestion in the KLD mix?](https://www.neb.com/en-us/tools-and-resources/video-library/quick-tips-how-can-i-improve-the-efficiency-of-the-dpni-digestion)
-    
-*   ![OverviewOfQ5SDMKit\_720](https://www.neb.com/en-us/-/media/nebus/page-images/video-thumbnails/overviewofq5sdmkit_720.jpg?rev=4f4523c1b5b34e999451c33ef8393e23&la=en-US&h=161&w=286&hash=178292A2621F68C778EA7853E40F6EA0)
-    
-    #### [Overview of the Q5® Site-Directed Mutagenesis Kit](https://www.neb.com/en-us/tools-and-resources/video-library/overview-of-the-q5-site-directed-mutagenesis-kit)
-    
-*   ![NEBTV\_DNA-Modifying-Enzymes-and-Cloning-Techniques\_1920](https://www.neb.com/en-us/-/media/nebus/page-images/video-thumbnails/nebtv_dna-modifying-enzymes-and-cloning-techniques_1920.jpg?rev=e97f8d25a3794930a02d072d0fb02afc&la=en-US&h=161&w=286&hash=9AD55F3189EEB3D249531442547C2C46)
-    
-    #### [Quick Tips - Why do I have so many wildtype colonies?](https://www.neb.com/en-us/tools-and-resources/video-library/quick-tips-why-do-i-have-so-many-wildtype-colonies)
-    
-*   ![NEBTV\_DNA-Assembly-Cloning-and-Mutagenesis-Kits\_1920](https://www.neb.com/en-us/-/media/nebus/page-images/video-thumbnails/nebtv_dna-assembly-cloning-and-mutagenesis-kits_1920.jpg?rev=4b3bf81aa0a54f17a77baf16f2faf2e7&la=en-US&h=161&w=286&hash=69D7AD7C893E0A3911506C3D6535293D)
-    
-    #### [Quick Tips - Can I make multiple mutations with the Q5® site-directed mutagenesis kit?](https://www.neb.com/en-us/tools-and-resources/video-library/can-i-make-multiple-mutations-with-the-q5-site-directed-mutagenesis-kit)
-    
-*   ![TroubleshootingTipsQ5SiteDirectedMutagenisis\_720](https://www.neb.com/en-us/-/media/nebus/page-images/video-thumbnails/troubleshootingtipsq5sitedirectedmutagenisis_720.jpg?rev=71dd5198eeed4685b0c13791fcff404b&la=en-US&h=161&w=286&hash=1F6D2053E95174100FCB2AEE8953F621)
-    
-    #### [Troubleshooting tips for Q5® Site-Directed Mutagenesis Kit](https://www.neb.com/en-us/tools-and-resources/video-library/troubleshooting-tips-for-q5-site-directed-mutagenesis-kit)
-    
-*   ![NEBaseChanger Primer Design Tool Tutorial](https://www.neb.com/en-us/-/media/nebus/page-images/video-thumbnails/nebasechanger-thumbnail3.jpg?rev=738b18bd48874c2fbfdb3edf6be28fa8&sc_lang=en-us&la=en-US&h=161&w=286&hash=D3E20CB9E7A6C68D6114C6207958894F)
-    
-    #### [NEBaseChanger® Primer Design Tool Tutorial](https://www.neb.com/en-us/tools-and-resources/video-library/nebasechanger-primer-design-tool-tutorial)
-    
-
-  
-
-Submit Restocking Order
-
-## Ineligible item added to cart
-
-Based on your Freezer Program type, you are trying to add a product to your cart
-that is either not allowed or not allowed with the existing contents of your
-cart. Please review and update your order accordingly. If you have any
-questions, please contact Customer Support at [freezers@neb.com](https://) or
-1-800-632-5227 x 8.
-
-[](https://www.neb.com/en-us/)
-```
-
-### `log://turn_5/set/known%3A/biology/tools/primer3_oligotm_complete`
-scheme=log action=set attrs={"path":"known://biology/tools/primer3_oligotm_complete","patch":"===================================================================\n--- known://biology/tools/primer3_oligotm_complete\told\t\n+++ known://biology/tools/primer3_oligotm_complete\tnew\t\n@@ -0,0 +1,20 @@\n+# Related\n+[prompt rules](prompt://1)\n+[unknown resolved](unknown://biology/tools/primer3_oligotm)\n+[manpage source](https://manpages.debian.org/stretch/primer3/oligotm.1)\n+[search primer3](log://turn_3/search/primer3_oligotm_exact_usage_flags_-tp_1_-sc_1_-mv_50_-dv_2_-n_0.8_-d_500_melting)\n+[distilled flags](known://biology/tools/primer3_oligotm)\n+\n+# Primer3 oligotm COMPLETE spec (Debian manpage)\n+**Usage**: oligotm [OPTIONS] {oligo} → prints Tm (C) to stdout. oligo=2-36nt DNA.\n+\n+**Flags (ALL clarified)**:\n+- `-tp 1`: SantaLucia 1998 nearest-neighbor params (recommended; default=0 Breslauer)\n+- `-sc 1`: SantaLucia 1998 salt correction (recommended; default=0 Schildkraut)\n+- `-mv 50`: monovalent cations (mM, default 50)\n+- `-dv 2`: divalent cations (mM, default 0)\n+- `-n 0.8`: dNTP conc (mM, default 0)\n+- `-d 500`: DNA strands (nM, default 50)\n+- `-i`: print references\n+\n+**Matches prompt flags exactly**. Tm for annealing part only (15-45nt).\n\\n","merge":"<<<<<<< SEARCH\n=======\n# Related\n[prompt rules](prompt://1)\n[unknown resolved](unknown://biology/tools/primer3_oligotm)\n[manpage source](https://manpages.debian.org/stretch/primer3/oligotm.1)\n[search primer3](log://turn_3/search/primer3_oligotm_exact_usage_flags_-tp_1_-sc_1_-mv_50_-dv_2_-n_0.8_-d_500_melting)\n[distilled flags](known://biology/tools/primer3_oligotm)\n\n# Primer3 oligotm COMPLETE spec (Debian manpage)\n**Usage**: oligotm [OPTIONS] {oligo} → prints Tm (C) to stdout. oligo=2-36nt DNA.\n\n**Flags (ALL clarified)**:\n- `-tp 1`: SantaLucia 1998 nearest-neighbor params (recommended; default=0 Breslauer)\n- `-sc 1`: SantaLucia 1998 salt correction (recommended; default=0 Schildkraut)\n- `-mv 50`: monovalent cations (mM, default 50)\n- `-dv 2`: divalent cations (mM, default 0)\n- `-n 0.8`: dNTP conc (mM, default 0)\n- `-d 500`: DNA strands (nM, default 50)\n- `-i`: print references\n\n**Matches prompt flags exactly**. Tm for annealing part only (15-45nt).\n>>>>>>> REPLACE","beforeTokens":0,"afterTokens":470,"summary":"biology,tools,primer3,oligotm,usage,flags,tm,ground_truth,manpage,santalucia,nn,","action":"set"}
-
-```
-# Related
-[prompt rules](prompt://1)
-[unknown resolved](unknown://biology/tools/primer3_oligotm)
-[manpage source](https://manpages.debian.org/stretch/primer3/oligotm.1)
-[search primer3](log://turn_3/search/primer3_oligotm_exact_usage_flags_-tp_1_-sc_1_-mv_50_-dv_2_-n_0.8_-d_500_melting)
-[distilled flags](known://biology/tools/primer3_oligotm)
-
-# Primer3 oligotm COMPLETE spec (Debian manpage)
-**Usage**: oligotm [OPTIONS] {oligo} → prints Tm (C) to stdout. oligo=2-36nt DNA.
-
-**Flags (ALL clarified)**:
-- `-tp 1`: SantaLucia 1998 nearest-neighbor params (recommended; default=0 Breslauer)
-- `-sc 1`: SantaLucia 1998 salt correction (recommended; default=0 Schildkraut)
-- `-mv 50`: monovalent cations (mM, default 50)
-- `-dv 2`: divalent cations (mM, default 0)
-- `-n 0.8`: dNTP conc (mM, default 0)
-- `-d 500`: DNA strands (nM, default 50)
-- `-i`: print references
-
-**Matches prompt flags exactly**. Tm for annealing part only (15-45nt).
-```
-
-### `log://turn_5/update/distilling_primer3_complete_%2B_discovering_sequences.fasta_%2B_Q5_kit`
-scheme=log action=update attrs={"status":155,"action":"update"}
-
-```
-distilling primer3 complete + discovering sequences.fasta + Q5 kit
-```
-

package/audit/sweep/dna-insert/turns/turn_006.md

@@ -1,143 +0,0 @@
-# turn 006
-
-tokens: prompt=39628 cached=1799 completion=661 reasoning=0 total=40289 cost=0
-created: 2026-05-02 00:22:45
-
-update: status=154 body=sequences.fasta content/location unknown; returning to Decomposition
-
----
-
-## reasoning (think)
-
-```
-- Distillation stage: resolve remaining unknowns using promoted sources
-- unknown://biology/tools/primer3_oligotm RESOLVED by known://biology/tools/primer3_oligotm_complete (1341+198 tokens, manpage details all flags)
-- unknown://biology/primers/design_rules can reference primer3_complete + Q5 kit details from https://www.neb.com/... (6173 tokens, back-to-back non-overlapping primers)
-- CRITICAL: unknown://biology/plasmid/sequences_fasta & unknown://biology/mutagenesis/q5_kit NEED sequences.fasta (not found in repo)
-- unknown://task/output/primers_fasta is output format (procedural, low priority)
-- Plan: 
-  1. Distill primer design rules + Q5 kit using existing sources
-  2. Create unknown for sequences.fasta content/location (return to decomposition)
-  3. Demote primer3 manpage (1341 tokens expensive, known:// complete exists)
-  4. Cannot deploy without sequences → need decomposition for file discovery
-- Budget: 1.7M free, but sequences likely large → check repo first
-```
-
-## assistant emission
-
-```
-<think>
-- Distillation stage: resolve remaining unknowns using promoted sources
-- unknown://biology/tools/primer3_oligotm RESOLVED by known://biology/tools/primer3_oligotm_complete (1341+198 tokens, manpage details all flags)
-- unknown://biology/primers/design_rules can reference primer3_complete + Q5 kit details from https://www.neb.com/... (6173 tokens, back-to-back non-overlapping primers)
-- CRITICAL: unknown://biology/plasmid/sequences_fasta & unknown://biology/mutagenesis/q5_kit NEED sequences.fasta (not found in repo)
-- unknown://task/output/primers_fasta is output format (procedural, low priority)
-- Plan: 
-  1. Distill primer design rules + Q5 kit using existing sources
-  2. Create unknown for sequences.fasta content/location (return to decomposition)
-  3. Demote primer3 manpage (1341 tokens expensive, known:// complete exists)
-  4. Cannot deploy without sequences → need decomposition for file discovery
-- Budget: 1.7M free, but sequences likely large → check repo first
-</think>
-
-<get path="app/**" manifest/>
-<get path="**/*.fasta" manifest/>
-<env>find /app -name "*fasta*" -o -name "*plasmid*"</env>
-
-<set path="known://biology/primers/design_rules" summary="biology,primers,q5,design,back-to-back,non-overlapping,tm,58-72,rules,annealing">
-# Related
-[prompt rules](prompt://1)
-[unknown resolved](unknown://biology/primers/design_rules)
-[primer3 complete](known://biology/tools/primer3_oligotm_complete)
-[q5 kit](https://www.neb.com/en-us/products/e0554-q5-site-directed-mutagenesis-kit)
-
-# Q5 SDM Primer Design Rules
-**Annealing**: 15-45nt to input plasmid.
-**Tm**: 58-72C (oligotm -tp 1 -sc 1 -mv 50 -dv 2 -n 0.8 -d 500), annealing part only.
-**Pairs**: Forward/reverse <=5C Tm diff; back-to-back (non-overlapping) for exponential amp.
-**Q5-specific**: Primers flank mutations/deletions/insertions; fwd mutation in center (>=10nt 3'), rev complementary 5' anneal.
-Minimal pairs to convert input→output plasmid.
-</set>
-
-<set path="unknown://biology/plasmid/sequences_fasta_content" summary="biology,plasmid,input,output,fasta,content,diffs,mutations">
-Exact content/structure of sequences.fasta: input/output circular plasmids, identify mutation sites/diffs for minimal primer pairs.
-</set>
-
-<set path="https://manpages.debian.org/stretch/primer3/oligotm.1" visibility="archived"/>
-<set path="known://biology/tools/primer3_oligotm" visibility="summarized"/>
-
-<update status="154">sequences.fasta content/location unknown; returning to Decomposition</update>
-```
-
-## actions (chronological log entries)
-
-### `log://turn_6/get/app/**`
-scheme=log action=get attrs={"path":"app/**","action":"get"}
-
-```
-MANIFEST get path="app/**": 0 matched (0 tokens)
-
-```
-
-### `log://turn_6/get/**/*.fasta`
-scheme=log action=get attrs={"path":"**/*.fasta","action":"get"}
-
-```
-MANIFEST get path="**/*.fasta": 2 matched (51 tokens)
-log://turn_4/update/distilling_primer3_oligotm_%2B_discovering_Q5_kit_protocol/sequences.fasta (36)
-log://turn_5/get/app/sequences.fasta (15)
-```
-
-### `log://turn_6/env/find_/app_-name_%22*fasta*%22_-o_-name_%22*plasmid*%22`
-scheme=log action=env attrs={"command":"find /app -name \"*fasta*\" -o -name \"*plasmid*\"","summary":"find /app -name \"*fasta*\" -o -name \"*plasmid*\"","action":"env"}
-
-```
-ran 'find /app -name "*fasta*" -o -name "*plasmid*"', exit=0 (0s). Output: env://turn_6/find_/app_-name_%22*fasta*%22_-o_-name_%22*plasmid*%22_1 (11 tokens), env://turn_6/find_/app_-name_%22*fasta*%22_-o_-name_%22*plasmid*%22_2 (0 tokens)
-```
-
-### `log://turn_6/set/known%3A/biology/primers/design_rules`
-scheme=log action=set attrs={"path":"known://biology/primers/design_rules","patch":"===================================================================\n--- known://biology/primers/design_rules\told\t\n+++ known://biology/primers/design_rules\tnew\t\n@@ -0,0 +1,12 @@\n+# Related\n+[prompt rules](prompt://1)\n+[unknown resolved](unknown://biology/primers/design_rules)\n+[primer3 complete](known://biology/tools/primer3_oligotm_complete)\n+[q5 kit](https://www.neb.com/en-us/products/e0554-q5-site-directed-mutagenesis-kit)\n+\n+# Q5 SDM Primer Design Rules\n+**Annealing**: 15-45nt to input plasmid.\n+**Tm**: 58-72C (oligotm -tp 1 -sc 1 -mv 50 -dv 2 -n 0.8 -d 500), annealing part only.\n+**Pairs**: Forward/reverse <=5C Tm diff; back-to-back (non-overlapping) for exponential amp.\n+**Q5-specific**: Primers flank mutations/deletions/insertions; fwd mutation in center (>=10nt 3'), rev complementary 5' anneal.\n+Minimal pairs to convert input→output plasmid.\n\\n","merge":"<<<<<<< SEARCH\n=======\n# Related\n[prompt rules](prompt://1)\n[unknown resolved](unknown://biology/primers/design_rules)\n[primer3 complete](known://biology/tools/primer3_oligotm_complete)\n[q5 kit](https://www.neb.com/en-us/products/e0554-q5-site-directed-mutagenesis-kit)\n\n# Q5 SDM Primer Design Rules\n**Annealing**: 15-45nt to input plasmid.\n**Tm**: 58-72C (oligotm -tp 1 -sc 1 -mv 50 -dv 2 -n 0.8 -d 500), annealing part only.\n**Pairs**: Forward/reverse <=5C Tm diff; back-to-back (non-overlapping) for exponential amp.\n**Q5-specific**: Primers flank mutations/deletions/insertions; fwd mutation in center (>=10nt 3'), rev complementary 5' anneal.\nMinimal pairs to convert input→output plasmid.\n>>>>>>> REPLACE","beforeTokens":0,"afterTokens":336,"summary":"biology,primers,q5,design,back-to-back,non-overlapping,tm,58-72,rules,annealing","action":"set"}
-
-```
-# Related
-[prompt rules](prompt://1)
-[unknown resolved](unknown://biology/primers/design_rules)
-[primer3 complete](known://biology/tools/primer3_oligotm_complete)
-[q5 kit](https://www.neb.com/en-us/products/e0554-q5-site-directed-mutagenesis-kit)
-
-# Q5 SDM Primer Design Rules
-**Annealing**: 15-45nt to input plasmid.
-**Tm**: 58-72C (oligotm -tp 1 -sc 1 -mv 50 -dv 2 -n 0.8 -d 500), annealing part only.
-**Pairs**: Forward/reverse <=5C Tm diff; back-to-back (non-overlapping) for exponential amp.
-**Q5-specific**: Primers flank mutations/deletions/insertions; fwd mutation in center (>=10nt 3'), rev complementary 5' anneal.
-Minimal pairs to convert input→output plasmid.
-```
-
-### `log://turn_6/set/unknown%3A/biology/plasmid/sequences_fasta_content`
-scheme=log action=set attrs={"path":"unknown://biology/plasmid/sequences_fasta_content","patch":"===================================================================\n--- unknown://biology/plasmid/sequences_fasta_content\told\t\n+++ unknown://biology/plasmid/sequences_fasta_content\tnew\t\n@@ -0,0 +1,1 @@\n+Exact content/structure of sequences.fasta: input/output circular plasmids, identify mutation sites/diffs for minimal primer pairs.\n\\n","merge":"<<<<<<< SEARCH\n=======\nExact content/structure of sequences.fasta: input/output circular plasmids, identify mutation sites/diffs for minimal primer pairs.\n>>>>>>> REPLACE","beforeTokens":0,"afterTokens":66,"summary":"biology,plasmid,input,output,fasta,content,diffs,mutations","action":"set"}
-
-```
-Exact content/structure of sequences.fasta: input/output circular plasmids, identify mutation sites/diffs for minimal primer pairs.
-```
-
-### `log://turn_6/set/https%3A/manpages.debian.org/stretch/primer3/oligotm.1`
-scheme=log action=set attrs={}
-
-```
-https://manpages.debian.org/stretch/primer3/oligotm.1 set to archived
-```
-
-### `log://turn_6/set/known%3A/biology/tools/primer3_oligotm`
-scheme=log action=set attrs={}
-
-```
-known://biology/tools/primer3_oligotm set to summarized
-```
-
-### `log://turn_6/update/sequences.fasta_content/location_unknown%3B_returning_to_Decomposition`
-scheme=log action=update attrs={"status":154,"action":"update"}
-
-```
-sequences.fasta content/location unknown; returning to Decomposition
-```
-

package/audit/sweep/dna-insert/turns/turn_007.md

@@ -1,77 +0,0 @@
-# turn 007
-
-tokens: prompt=39681 cached=1739 completion=293 reasoning=0 total=39974 cost=0
-created: 2026-05-02 00:22:53
-
-update: status=145 body=prompt decomposed
-
----
-
-## reasoning (think)
-
-```
-Decomposition stage: Need to decompose unresolved unknowns from prompt.
-Current unknowns cover most areas but missing:
-1. Exact content of sequences.fasta (input/output plasmids, diffs)
-2. Specific mutation sites/differences between input/output for minimal primer design
-3. Q5 kit primer positioning strategy (back-to-back, non-overlapping, mutation placement)
-Resolve sequences.fasta content first - it's blocking everything.
-```
-
-## assistant emission
-
-```
-<think>
-Decomposition stage: Need to decompose unresolved unknowns from prompt.
-Current unknowns cover most areas but missing:
-1. Exact content of sequences.fasta (input/output plasmids, diffs)
-2. Specific mutation sites/differences between input/output for minimal primer design
-3. Q5 kit primer positioning strategy (back-to-back, non-overlapping, mutation placement)
-Resolve sequences.fasta content first - it's blocking everything.
-</think>
-
-<set path="unknown://biology/plasmid/input_sequence" summary="biology,plasmid,input,sequence,fasta,circular">Exact input plasmid sequence from sequences.fasta (first entry).</set>
-<set path="unknown://biology/plasmid/output_sequence" summary="biology,plasmid,output,sequence,fasta,circular">Exact output plasmid sequence from sequences.fasta (second entry).</set>
-<set path="unknown://biology/plasmid/mutation_sites" summary="biology,plasmid,diffs,mutations,sites,positions,indels">Precise differences between input/output plasmids: positions, substitutions, insertions, deletions required.</set>
-<set path="unknown://biology/q5/kit/primer_strategy" summary="biology,q5,kit,strategy,back-to-back,non-overlapping,mutation_position">Q5 SDM-specific primer design: back-to-back orientation, mutation centering, minimal pairs strategy for circular plasmids.</set>
-
-<update status="145">prompt decomposed</update>
-```
-
-## actions (chronological log entries)
-
-### `log://turn_7/set/unknown%3A/biology/plasmid/input_sequence`
-scheme=log action=set attrs={"path":"unknown://biology/plasmid/input_sequence","patch":"===================================================================\n--- unknown://biology/plasmid/input_sequence\told\t\n+++ unknown://biology/plasmid/input_sequence\tnew\t\n@@ -0,0 +1,1 @@\n+Exact input plasmid sequence from sequences.fasta (first entry).\n\\n","merge":"<<<<<<< SEARCH\n=======\nExact input plasmid sequence from sequences.fasta (first entry).\n>>>>>>> REPLACE","beforeTokens":0,"afterTokens":32,"summary":"biology,plasmid,input,sequence,fasta,circular","action":"set"}
-
-```
-Exact input plasmid sequence from sequences.fasta (first entry).
-```
-
-### `log://turn_7/set/unknown%3A/biology/plasmid/output_sequence`
-scheme=log action=set attrs={"path":"unknown://biology/plasmid/output_sequence","patch":"===================================================================\n--- unknown://biology/plasmid/output_sequence\told\t\n+++ unknown://biology/plasmid/output_sequence\tnew\t\n@@ -0,0 +1,1 @@\n+Exact output plasmid sequence from sequences.fasta (second entry).\n\\n","merge":"<<<<<<< SEARCH\n=======\nExact output plasmid sequence from sequences.fasta (second entry).\n>>>>>>> REPLACE","beforeTokens":0,"afterTokens":33,"summary":"biology,plasmid,output,sequence,fasta,circular","action":"set"}
-
-```
-Exact output plasmid sequence from sequences.fasta (second entry).
-```
-
-### `log://turn_7/set/unknown%3A/biology/plasmid/mutation_sites`
-scheme=log action=set attrs={"path":"unknown://biology/plasmid/mutation_sites","patch":"===================================================================\n--- unknown://biology/plasmid/mutation_sites\told\t\n+++ unknown://biology/plasmid/mutation_sites\tnew\t\n@@ -0,0 +1,1 @@\n+Precise differences between input/output plasmids: positions, substitutions, insertions, deletions required.\n\\n","merge":"<<<<<<< SEARCH\n=======\nPrecise differences between input/output plasmids: positions, substitutions, insertions, deletions required.\n>>>>>>> REPLACE","beforeTokens":0,"afterTokens":54,"summary":"biology,plasmid,diffs,mutations,sites,positions,indels","action":"set"}
-
-```
-Precise differences between input/output plasmids: positions, substitutions, insertions, deletions required.
-```
-
-### `log://turn_7/set/unknown%3A/biology/q5/kit/primer_strategy`
-scheme=log action=set attrs={"path":"unknown://biology/q5/kit/primer_strategy","patch":"===================================================================\n--- unknown://biology/q5/kit/primer_strategy\told\t\n+++ unknown://biology/q5/kit/primer_strategy\tnew\t\n@@ -0,0 +1,1 @@\n+Q5 SDM-specific primer design: back-to-back orientation, mutation centering, minimal pairs strategy for circular plasmids.\n\\n","merge":"<<<<<<< SEARCH\n=======\nQ5 SDM-specific primer design: back-to-back orientation, mutation centering, minimal pairs strategy for circular plasmids.\n>>>>>>> REPLACE","beforeTokens":0,"afterTokens":61,"summary":"biology,q5,kit,strategy,back-to-back,non-overlapping,mutation_position","action":"set"}
-
-```
-Q5 SDM-specific primer design: back-to-back orientation, mutation centering, minimal pairs strategy for circular plasmids.
-```
-
-### `log://turn_7/update/prompt_decomposed`
-scheme=log action=update attrs={"status":145,"action":"update"}
-
-```
-prompt decomposed
-```
-