{ "about": { "generatedBy": "pipeline/sequence-toolkit/primer_design.py", "scriptSha256": "5f4daeed831507adfce10b9eb07abd3adeded576b461e94890da43709a389971", "recordedOn": "2026-10-11", "oracle": "Biopython 1.88 MeltingTemp.Tm_NN (DNA_NN3; Owczarzy 2004 as saltcorr 6, Owczarzy 2008 as saltcorr 7; dnac1 = the oligo concentration, dnac2 = 0) for every Tm; the windows, GC, penalties, caps and counts derived in this script from the conventions below; exact sites and products by pipeline/sequence-toolkit/exact_matches.py (reverse complements by Biopython, every window tested directly); one case worked by hand with a write-refusal guard", "margins": "A case is written only when every eligible window's Tm is at least 1e-06 °C from the Tm bounds, every pair's ΔTm at least 1e-06 °C from its limit, and every ranking gap at a cap or between returned pairs at least 1e-06: the engine's Tm agrees with Biopython's within 0.1 °C, so no decision here can turn on that tolerance.", "conventions": [ "Input: one A/C/G/T template of 1 to 50,000 bases with explicit topology; a target region (1-based, inclusive, crossing the origin only on a circular template); up to 100 excluded regions; the flank searched beyond each target edge (0 to 200 bases); annealing length bounds and preferred length (8 to 40); Tm bounds and preferred Tm; the pair's maximum ΔTm (0 to 30 °C); GC bounds (percent); product length bounds including both tails (16 to template length + 400); optional 5′ tails of A/C/G/T up to 200 bases each; and the Tm conditions.", "Refused before any enumeration, with a message naming the field: a template outside the alphabet, length or topology; target or excluded coordinates outside 1..n, or a target crossing the origin of a linear template; a constraint outside its range or inconsistent with another; a tail outside A/C/G/T or over 200 bases. Nothing is relaxed.", "Forward windows: for each gap 0..flank and each length minLength..maxLength, the window whose 3′ end sits gap bases before the target's first base; reverse windows the mirror, whose 3′ end (on the bottom strand) sits gap bases after the target's last base; both read around the circle when circular. Windows are counted in this order of rejection, the first reason counting: boundary (off a linear template, or longer than the template), excluded (any base inside the target or an excluded region), gc (outside the GC bounds), tm (outside the Tm bounds, or refused by the Tm engine).", "A window's annealing sequence is the top strand for a forward window and the reverse complement for a reverse window, 5′ to 3′. Its start and end are the primer's 5′ and 3′ ends on the top strand, 1-based, so a reverse window has start > end unless it crosses the origin. GC is G + C over the annealing length, in percent. Tm is the nearest-neighbour Tm of the annealing part alone (the tail never counts), under the recorded conditions, with the primer in excess.", "A window's penalty is |Tm − preferred Tm| + 0.2 × |length − preferred length|. Each side is sorted by penalty, then start, then length, and cut to the first 24 (perSide).", "Pairs: every forward window with every reverse window of the kept sides. The template span runs from the forward 5′ end to the reverse 5′ end inclusive; a span longer than the template, or a product length (span plus both tails) outside the product bounds, counts productSize; |Tm_f − Tm_r| over the ΔTm limit counts deltaTm. The remaining pairs (eligiblePairs) are ranked by total = (|Tm_f − preferred| + |Tm_r − preferred|) + 0.2 × (|len_f − preferred| + |len_r − preferred|) + |Tm_f − Tm_r|, then forward start, then reverse start.", "The first 24 ranked pairs (evaluatedPairs) are each checked by the exact-pair method on the whole template: exactChecked counts them, exactRefused those the exact method refuses or whose own product is not among its candidates. exactSites are the exact method's site counts per primer on both strands; exactProducts its candidate count. The first 5 pairs that pass are returned (returnedPairs).", "capped is true when either side had more than 24 eligible windows or fewer pairs were checked than were eligible: the search is then not exhaustive, and the returned pairs are the best among those evaluated.", "No Tm of a tailed oligo, no annealing temperature, no genome specificity, no mismatched binding and no amplification outcome is derived: exact binding is checked only within the supplied template, and a returned pair is a sequence design, not evidence that it amplifies." ], "notGated": "Secondary-structure checks (hairpins, self- and cross-dimers at 37 °C) are the structure method's and are gated by its own fixtures (hairpin-dimer.json); they are not recorded here.", "primer3": "Primer3 (primer3-py 2.3.1, GPL-2.0-or-later) was run offline on the real-template cases as a comparison, recorded under docs/science/evidence/primer-design-2026-10-11/; it is not an oracle of this set and nothing from it is shipped.", "templates": { "hand-24": "a 24-base sequence written for the hand-worked case", "J01636.1 1..600": "GenBank J01636.1 (E. coli lac operon), bases 1 to 600, from pipeline/plasmid-map/records (NCBI; no restrictions, depositor terms may apply)", "L09137.2 (pUC19, circular)": "GenBank L09137.2 (cloning vector pUC19c), 2,686 bp, circular, from pipeline/plasmid-map/records" }, "limits": { "template": 50000, "flank": 200, "perSide": 24, "evaluatedPairs": 24, "returnedPairs": 5 } }, "cases": [ { "id": "design-hand-unique", "input": { "templateName": "hand-24", "template": "ATGACCTAGCTATTCGCTAGTCAC", "topology": "linear", "settings": { "target": { "start": 9, "end": 16 }, "excluded": [], "forwardTail": "AA", "reverseTail": "CG", "conditions": { "oligo_nM": 250, "na_mM": 50, "mg_mM": 0, "dntp_mM": 0, "model": "primer-excess", "saltCorrection": "owczarzy-2004" }, "flank": 0, "minLength": 8, "maxLength": 8, "targetLength": 8, "minTm": 0, "maxTm": 100, "targetTm": 40, "maxDeltaTm": 30, "minGc": 0, "maxGc": 100, "minProduct": 24, "maxProduct": 30 } }, "expected": { "method": "bounded-flanks-v1", "pcrMethod": "exact-pair-v1", "templateHash": "2e921837395a2a3194a559cbbef1cea25f272da48e86448e889d2a93ada8e2bc", "topology": "linear", "templateLength": 24, "pairs": [ { "id": "+:1:8|-:24:17", "forward": { "annealing": "ATGACCTA", "tail": "AA", "start": 1, "end": 8, "tm": 8.925696836801308, "gc": 37.5, "penalty": 31.074303163198692 }, "reverse": { "annealing": "GTGACTAG", "tail": "CG", "start": 24, "end": 17, "tm": 12.24154515770067, "gc": 50.0, "penalty": 27.75845484229933 }, "product": { "id": "+:1:8|-:24:17", "sequence": "AAATGACCTAGCTATTCGCTAGTCACCG", "length": 28, "templateLength": 24, "templateSpan": 24, "orientation": "+", "crossesOrigin": false, "forward": { "strand": "+", "start": 1, "end": 8, "length": 8 }, "reverse": { "strand": "-", "start": 24, "end": 17, "length": 8 } }, "ranking": { "tmDeviation": 58.83275800549802, "lengthDeviation": 0.0, "deltaTm": 3.315848320899363, "total": 62.148606326397385 }, "exactSites": { "forward": 1, "reverse": 1 }, "exactProducts": 1 } ], "counts": { "windows": 2, "boundary": 0, "excluded": 0, "gc": 0, "tm": 0, "validForward": 1, "validReverse": 1, "productSize": 0, "deltaTm": 0, "eligiblePairs": 1, "exactChecked": 1, "exactRefused": 0 }, "capped": false }, "method": "bounded-flanks-v1", "oracle": "primer_design.py (Biopython MeltingTemp.Tm_NN for Tm; windows, GC, ranking and caps derived in the script; exact binding by exact_matches.py)", "oracleVersion": "Biopython 1.88", "recordedOn": "2026-10-11", "notes": "Worked by hand in the script's HAND_WORKED block; the script refuses to write unless its derivation agrees with the hand work." }, { "id": "design-lac-linear", "input": { "templateName": "J01636.1 1..600", "template": "GACACCATCGAATGGCGCAAAACCTTTCGCGGTATGGCATGATAGCGCCCGGAAGAGAGTCAATTCAGGGTGGTGAATGTGAAACCAGTAACGTTATACGATGTCGCAGAGTATGCCGGTGTCTCTTATCAGACCGTTTCCCGCGTGGTGAACCAGGCCAGCCACGTTTCTGCGAAAACGCGGGAAAAAGTGGAAGCGGCGATGGCGGAGCTGAATTACATTCCCAACCGCGTGGCACAACAACTGGCGGGCAAACAGTCGTTGCTGATTGGCGTTGCCACCTCCAGTCTGGCCCTGCACGCGCCGTCGCAAATTGTCGCGGCGATTAAATCTCGCGCCGATCAACTGGGTGCCAGCGTGGTGGTGTCGATGGTAGAACGAAGCGGCGTCGAAGCCTGTAAAGCGGCGGTGCACAATCTTCTCGCGCAACGCGTCAGTGGGCTGATCATTAACTATCCGCTGGATGACCAGGATGCCATTGCTGTGGAAGCTGCCTGCACTAATGTTCCGGCGTTATTTCTTGATGTCTCTGACCAGACACCCATCAACAGTATTATTTTCTCCCATGAAGACGGTACGCGACTGGGCGTGGAGCATCTG", "topology": "linear", "settings": { "target": { "start": 201, "end": 400 }, "excluded": [], "forwardTail": "", "reverseTail": "", "conditions": { "oligo_nM": 250, "na_mM": 50, "mg_mM": 0, "dntp_mM": 0, "model": "primer-excess", "saltCorrection": "owczarzy-2004" }, "flank": 50, "minLength": 18, "maxLength": 26, "targetLength": 20, "minTm": 55, "maxTm": 68, "targetTm": 59.95, "maxDeltaTm": 3, "minGc": 30, "maxGc": 70, "minProduct": 180, "maxProduct": 420 } }, "expected": { "method": "bounded-flanks-v1", "pcrMethod": "exact-pair-v1", "templateHash": "fd182829946e68ba90d7c14906883ef31f663537dade5f617f804b8ce46dc316", "topology": "linear", "templateLength": 600, "pairs": [ { "id": "+:158:177|-:428:409", "forward": { "annealing": "CCAGCCACGTTTCTGCGAAA", "tail": "", "start": 158, "end": 177, "tm": 59.99861335790035, "gc": 55.0, "penalty": 0.04861335790035071 }, "reverse": { "annealing": "TGCGCGAGAAGATTGTGCAC", "tail": "", "start": 428, "end": 409, "tm": 59.96600984591282, "gc": 55.0, "penalty": 0.016009845912819287 }, "product": { "id": "+:158:177|-:428:409", "sequence": "CCAGCCACGTTTCTGCGAAAACGCGGGAAAAAGTGGAAGCGGCGATGGCGGAGCTGAATTACATTCCCAACCGCGTGGCACAACAACTGGCGGGCAAACAGTCGTTGCTGATTGGCGTTGCCACCTCCAGTCTGGCCCTGCACGCGCCGTCGCAAATTGTCGCGGCGATTAAATCTCGCGCCGATCAACTGGGTGCCAGCGTGGTGGTGTCGATGGTAGAACGAAGCGGCGTCGAAGCCTGTAAAGCGGCGGTGCACAATCTTCTCGCGCA", "length": 271, "templateLength": 600, "templateSpan": 271, "orientation": "+", "crossesOrigin": false, "forward": { "strand": "+", "start": 158, "end": 177, "length": 20 }, "reverse": { "strand": "-", "start": 428, "end": 409, "length": 20 } }, "ranking": { "tmDeviation": 0.06462320381317, "lengthDeviation": 0.0, "deltaTm": 0.03260351198753142, "total": 0.09722671580070141 }, "exactSites": { "forward": 1, "reverse": 1 }, "exactProducts": 1 }, { "id": "+:158:178|-:428:409", "forward": { "annealing": "CCAGCCACGTTTCTGCGAAAA", "tail": "", "start": 158, "end": 178, "tm": 59.93537222125224, "gc": 52.38095238095238, "penalty": 0.21462777874776579 }, "reverse": { "annealing": "TGCGCGAGAAGATTGTGCAC", "tail": "", "start": 428, "end": 409, "tm": 59.96600984591282, "gc": 55.0, "penalty": 0.016009845912819287 }, "product": { "id": "+:158:178|-:428:409", "sequence": "CCAGCCACGTTTCTGCGAAAACGCGGGAAAAAGTGGAAGCGGCGATGGCGGAGCTGAATTACATTCCCAACCGCGTGGCACAACAACTGGCGGGCAAACAGTCGTTGCTGATTGGCGTTGCCACCTCCAGTCTGGCCCTGCACGCGCCGTCGCAAATTGTCGCGGCGATTAAATCTCGCGCCGATCAACTGGGTGCCAGCGTGGTGGTGTCGATGGTAGAACGAAGCGGCGTCGAAGCCTGTAAAGCGGCGGTGCACAATCTTCTCGCGCA", "length": 271, "templateLength": 600, "templateSpan": 271, "orientation": "+", "crossesOrigin": false, "forward": { "strand": "+", "start": 158, "end": 178, "length": 21 }, "reverse": { "strand": "-", "start": 428, "end": 409, "length": 20 } }, "ranking": { "tmDeviation": 0.03063762466058506, "lengthDeviation": 0.2, "deltaTm": 0.03063762466058506, "total": 0.26127524932117013 }, "exactSites": { "forward": 1, "reverse": 1 }, "exactProducts": 1 }, { "id": "+:178:197|-:428:409", "forward": { "annealing": "ACGCGGGAAAAAGTGGAAGC", "tail": "", "start": 178, "end": 197, "tm": 60.09860053729648, "gc": 55.0, "penalty": 0.14860053729647404 }, "reverse": { "annealing": "TGCGCGAGAAGATTGTGCAC", "tail": "", "start": 428, "end": 409, "tm": 59.96600984591282, "gc": 55.0, "penalty": 0.016009845912819287 }, "product": { "id": "+:178:197|-:428:409", "sequence": "ACGCGGGAAAAAGTGGAAGCGGCGATGGCGGAGCTGAATTACATTCCCAACCGCGTGGCACAACAACTGGCGGGCAAACAGTCGTTGCTGATTGGCGTTGCCACCTCCAGTCTGGCCCTGCACGCGCCGTCGCAAATTGTCGCGGCGATTAAATCTCGCGCCGATCAACTGGGTGCCAGCGTGGTGGTGTCGATGGTAGAACGAAGCGGCGTCGAAGCCTGTAAAGCGGCGGTGCACAATCTTCTCGCGCA", "length": 251, "templateLength": 600, "templateSpan": 251, "orientation": "+", "crossesOrigin": false, "forward": { "strand": "+", "start": 178, "end": 197, "length": 20 }, "reverse": { "strand": "-", "start": 428, "end": 409, "length": 20 } }, "ranking": { "tmDeviation": 0.16461038320929333, "lengthDeviation": 0.0, "deltaTm": 0.13259069138365476, "total": 0.2972010745929481 }, "exactSites": { "forward": 1, "reverse": 1 }, "exactProducts": 1 }, { "id": "+:177:197|-:428:409", "forward": { "annealing": "AACGCGGGAAAAAGTGGAAGC", "tail": "", "start": 177, "end": 197, "tm": 60.03263800822515, "gc": 52.38095238095238, "penalty": 0.2826380082251461 }, "reverse": { "annealing": "TGCGCGAGAAGATTGTGCAC", "tail": "", "start": 428, "end": 409, "tm": 59.96600984591282, "gc": 55.0, "penalty": 0.016009845912819287 }, "product": { "id": "+:177:197|-:428:409", "sequence": "AACGCGGGAAAAAGTGGAAGCGGCGATGGCGGAGCTGAATTACATTCCCAACCGCGTGGCACAACAACTGGCGGGCAAACAGTCGTTGCTGATTGGCGTTGCCACCTCCAGTCTGGCCCTGCACGCGCCGTCGCAAATTGTCGCGGCGATTAAATCTCGCGCCGATCAACTGGGTGCCAGCGTGGTGGTGTCGATGGTAGAACGAAGCGGCGTCGAAGCCTGTAAAGCGGCGGTGCACAATCTTCTCGCGCA", "length": 252, "templateLength": 600, "templateSpan": 252, "orientation": "+", "crossesOrigin": false, "forward": { "strand": "+", "start": 177, "end": 197, "length": 21 }, "reverse": { "strand": "-", "start": 428, "end": 409, "length": 20 } }, "ranking": { "tmDeviation": 0.0986478541379654, "lengthDeviation": 0.2, "deltaTm": 0.06662816231232682, "total": 0.36527601645029223 }, "exactSites": { "forward": 1, "reverse": 1 }, "exactProducts": 1 }, { "id": "+:158:177|-:422:404", "forward": { "annealing": "CCAGCCACGTTTCTGCGAAA", "tail": "", "start": 158, "end": 177, "tm": 59.99861335790035, "gc": 55.0, "penalty": 0.04861335790035071 }, "reverse": { "annealing": "AGAAGATTGTGCACCGCCG", "tail": "", "start": 422, "end": 404, "tm": 59.914723905422534, "gc": 57.89473684210526, "penalty": 0.23527609457746906 }, "product": { "id": "+:158:177|-:422:404", "sequence": "CCAGCCACGTTTCTGCGAAAACGCGGGAAAAAGTGGAAGCGGCGATGGCGGAGCTGAATTACATTCCCAACCGCGTGGCACAACAACTGGCGGGCAAACAGTCGTTGCTGATTGGCGTTGCCACCTCCAGTCTGGCCCTGCACGCGCCGTCGCAAATTGTCGCGGCGATTAAATCTCGCGCCGATCAACTGGGTGCCAGCGTGGTGGTGTCGATGGTAGAACGAAGCGGCGTCGAAGCCTGTAAAGCGGCGGTGCACAATCTTCT", "length": 265, "templateLength": 600, "templateSpan": 265, "orientation": "+", "crossesOrigin": false, "forward": { "strand": "+", "start": 158, "end": 177, "length": 20 }, "reverse": { "strand": "-", "start": 422, "end": 404, "length": 19 } }, "ranking": { "tmDeviation": 0.08388945247781976, "lengthDeviation": 0.2, "deltaTm": 0.08388945247781976, "total": 0.36777890495563953 }, "exactSites": { "forward": 1, "reverse": 1 }, "exactProducts": 1 } ], "counts": { "windows": 918, "boundary": 0, "excluded": 0, "gc": 40, "tm": 234, "validForward": 353, "validReverse": 291, "productSize": 0, "deltaTm": 0, "eligiblePairs": 576, "exactChecked": 5, "exactRefused": 0 }, "capped": true }, "method": "bounded-flanks-v1", "oracle": "primer_design.py (Biopython MeltingTemp.Tm_NN for Tm; windows, GC, ranking and caps derived in the script; exact binding by exact_matches.py)", "oracleVersion": "Biopython 1.88", "recordedOn": "2026-10-11", "notes": "The panel's default constraints on the first 600 bases of the E. coli lac operon, target 201..400." }, { "id": "design-lac-excluded", "input": { "templateName": "J01636.1 1..600", "template": "GACACCATCGAATGGCGCAAAACCTTTCGCGGTATGGCATGATAGCGCCCGGAAGAGAGTCAATTCAGGGTGGTGAATGTGAAACCAGTAACGTTATACGATGTCGCAGAGTATGCCGGTGTCTCTTATCAGACCGTTTCCCGCGTGGTGAACCAGGCCAGCCACGTTTCTGCGAAAACGCGGGAAAAAGTGGAAGCGGCGATGGCGGAGCTGAATTACATTCCCAACCGCGTGGCACAACAACTGGCGGGCAAACAGTCGTTGCTGATTGGCGTTGCCACCTCCAGTCTGGCCCTGCACGCGCCGTCGCAAATTGTCGCGGCGATTAAATCTCGCGCCGATCAACTGGGTGCCAGCGTGGTGGTGTCGATGGTAGAACGAAGCGGCGTCGAAGCCTGTAAAGCGGCGGTGCACAATCTTCTCGCGCAACGCGTCAGTGGGCTGATCATTAACTATCCGCTGGATGACCAGGATGCCATTGCTGTGGAAGCTGCCTGCACTAATGTTCCGGCGTTATTTCTTGATGTCTCTGACCAGACACCCATCAACAGTATTATTTTCTCCCATGAAGACGGTACGCGACTGGGCGTGGAGCATCTG", "topology": "linear", "settings": { "target": { "start": 201, "end": 400 }, "excluded": [ { "start": 120, "end": 170 }, { "start": 420, "end": 470 } ], "forwardTail": "", "reverseTail": "", "conditions": { "oligo_nM": 250, "na_mM": 50, "mg_mM": 0, "dntp_mM": 0, "model": "primer-excess", "saltCorrection": "owczarzy-2004" }, "flank": 50, "minLength": 18, "maxLength": 26, "targetLength": 20, "minTm": 55, "maxTm": 68, "targetTm": 62.0, "maxDeltaTm": 3, "minGc": 30, "maxGc": 70, "minProduct": 180, "maxProduct": 420 } }, "expected": { "method": "bounded-flanks-v1", "pcrMethod": "exact-pair-v1", "templateHash": "fd182829946e68ba90d7c14906883ef31f663537dade5f617f804b8ce46dc316", "topology": "linear", "templateLength": 600, "pairs": [ { "id": "+:172:194|-:419:401", "forward": { "annealing": "GCGAAAACGCGGGAAAAAGTGGA", "tail": "", "start": 172, "end": 194, "tm": 62.14537329359223, "gc": 52.17391304347826, "penalty": 0.745373293592229 }, "reverse": { "annealing": "AGATTGTGCACCGCCGCTT", "tail": "", "start": 419, "end": 401, "tm": 62.113664810140506, "gc": 57.89473684210526, "penalty": 0.3136648101405058 }, "product": { "id": "+:172:194|-:419:401", "sequence": "GCGAAAACGCGGGAAAAAGTGGAAGCGGCGATGGCGGAGCTGAATTACATTCCCAACCGCGTGGCACAACAACTGGCGGGCAAACAGTCGTTGCTGATTGGCGTTGCCACCTCCAGTCTGGCCCTGCACGCGCCGTCGCAAATTGTCGCGGCGATTAAATCTCGCGCCGATCAACTGGGTGCCAGCGTGGTGGTGTCGATGGTAGAACGAAGCGGCGTCGAAGCCTGTAAAGCGGCGGTGCACAATCT", "length": 248, "templateLength": 600, "templateSpan": 248, "orientation": "+", "crossesOrigin": false, "forward": { "strand": "+", "start": 172, "end": 194, "length": 23 }, "reverse": { "strand": "-", "start": 419, "end": 401, "length": 19 } }, "ranking": { "tmDeviation": 0.2590381037327347, "lengthDeviation": 0.8, "deltaTm": 0.03170848345172317, "total": 1.090746587184458 }, "exactSites": { "forward": 1, "reverse": 1 }, "exactProducts": 1 }, { "id": "+:176:198|-:419:401", "forward": { "annealing": "AAACGCGGGAAAAAGTGGAAGCG", "tail": "", "start": 176, "end": 198, "tm": 62.18249482665317, "gc": 52.17391304347826, "penalty": 0.7824948266531693 }, "reverse": { "annealing": "AGATTGTGCACCGCCGCTT", "tail": "", "start": 419, "end": 401, "tm": 62.113664810140506, "gc": 57.89473684210526, "penalty": 0.3136648101405058 }, "product": { "id": "+:176:198|-:419:401", "sequence": "AAACGCGGGAAAAAGTGGAAGCGGCGATGGCGGAGCTGAATTACATTCCCAACCGCGTGGCACAACAACTGGCGGGCAAACAGTCGTTGCTGATTGGCGTTGCCACCTCCAGTCTGGCCCTGCACGCGCCGTCGCAAATTGTCGCGGCGATTAAATCTCGCGCCGATCAACTGGGTGCCAGCGTGGTGGTGTCGATGGTAGAACGAAGCGGCGTCGAAGCCTGTAAAGCGGCGGTGCACAATCT", "length": 244, "templateLength": 600, "templateSpan": 244, "orientation": "+", "crossesOrigin": false, "forward": { "strand": "+", "start": 176, "end": 198, "length": 23 }, "reverse": { "strand": "-", "start": 419, "end": 401, "length": 19 } }, "ranking": { "tmDeviation": 0.29615963679367496, "lengthDeviation": 0.8, "deltaTm": 0.0688300165126634, "total": 1.1649896533063384 }, "exactSites": { "forward": 1, "reverse": 1 }, "exactProducts": 1 }, { "id": "+:175:198|-:419:401", "forward": { "annealing": "AAAACGCGGGAAAAAGTGGAAGCG", "tail": "", "start": 175, "end": 198, "tm": 62.03527481139599, "gc": 50.0, "penalty": 0.8352748113959934 }, "reverse": { "annealing": "AGATTGTGCACCGCCGCTT", "tail": "", "start": 419, "end": 401, "tm": 62.113664810140506, "gc": 57.89473684210526, "penalty": 0.3136648101405058 }, "product": { "id": "+:175:198|-:419:401", "sequence": "AAAACGCGGGAAAAAGTGGAAGCGGCGATGGCGGAGCTGAATTACATTCCCAACCGCGTGGCACAACAACTGGCGGGCAAACAGTCGTTGCTGATTGGCGTTGCCACCTCCAGTCTGGCCCTGCACGCGCCGTCGCAAATTGTCGCGGCGATTAAATCTCGCGCCGATCAACTGGGTGCCAGCGTGGTGGTGTCGATGGTAGAACGAAGCGGCGTCGAAGCCTGTAAAGCGGCGGTGCACAATCT", "length": 245, "templateLength": 600, "templateSpan": 245, "orientation": "+", "crossesOrigin": false, "forward": { "strand": "+", "start": 175, "end": 198, "length": 24 }, "reverse": { "strand": "-", "start": 419, "end": 401, "length": 19 } }, "ranking": { "tmDeviation": 0.1489396215364991, "lengthDeviation": 1.0, "deltaTm": 0.07838999874451247, "total": 1.2273296202810116 }, "exactSites": { "forward": 1, "reverse": 1 }, "exactProducts": 1 }, { "id": "+:172:195|-:419:401", "forward": { "annealing": "GCGAAAACGCGGGAAAAAGTGGAA", "tail": "", "start": 172, "end": 195, "tm": 61.99935292676395, "gc": 50.0, "penalty": 0.8006470732360527 }, "reverse": { "annealing": "AGATTGTGCACCGCCGCTT", "tail": "", "start": 419, "end": 401, "tm": 62.113664810140506, "gc": 57.89473684210526, "penalty": 0.3136648101405058 }, "product": { "id": "+:172:195|-:419:401", "sequence": "GCGAAAACGCGGGAAAAAGTGGAAGCGGCGATGGCGGAGCTGAATTACATTCCCAACCGCGTGGCACAACAACTGGCGGGCAAACAGTCGTTGCTGATTGGCGTTGCCACCTCCAGTCTGGCCCTGCACGCGCCGTCGCAAATTGTCGCGGCGATTAAATCTCGCGCCGATCAACTGGGTGCCAGCGTGGTGGTGTCGATGGTAGAACGAAGCGGCGTCGAAGCCTGTAAAGCGGCGGTGCACAATCT", "length": 248, "templateLength": 600, "templateSpan": 248, "orientation": "+", "crossesOrigin": false, "forward": { "strand": "+", "start": 172, "end": 195, "length": 24 }, "reverse": { "strand": "-", "start": 419, "end": 401, "length": 19 } }, "ranking": { "tmDeviation": 0.1143118833765584, "lengthDeviation": 1.0, "deltaTm": 0.1143118833765584, "total": 1.2286237667531168 }, "exactSites": { "forward": 1, "reverse": 1 }, "exactProducts": 1 }, { "id": "+:177:198|-:419:401", "forward": { "annealing": "AACGCGGGAAAAAGTGGAAGCG", "tail": "", "start": 177, "end": 198, "tm": 62.34357931563284, "gc": 54.54545454545455, "penalty": 0.7435793156328373 }, "reverse": { "annealing": "AGATTGTGCACCGCCGCTT", "tail": "", "start": 419, "end": 401, "tm": 62.113664810140506, "gc": 57.89473684210526, "penalty": 0.3136648101405058 }, "product": { "id": "+:177:198|-:419:401", "sequence": "AACGCGGGAAAAAGTGGAAGCGGCGATGGCGGAGCTGAATTACATTCCCAACCGCGTGGCACAACAACTGGCGGGCAAACAGTCGTTGCTGATTGGCGTTGCCACCTCCAGTCTGGCCCTGCACGCGCCGTCGCAAATTGTCGCGGCGATTAAATCTCGCGCCGATCAACTGGGTGCCAGCGTGGTGGTGTCGATGGTAGAACGAAGCGGCGTCGAAGCCTGTAAAGCGGCGGTGCACAATCT", "length": 243, "templateLength": 600, "templateSpan": 243, "orientation": "+", "crossesOrigin": false, "forward": { "strand": "+", "start": 177, "end": 198, "length": 22 }, "reverse": { "strand": "-", "start": 419, "end": 401, "length": 19 } }, "ranking": { "tmDeviation": 0.45724412577334306, "lengthDeviation": 0.6000000000000001, "deltaTm": 0.2299145054923315, "total": 1.2871586312656746 }, "exactSites": { "forward": 1, "reverse": 1 }, "exactProducts": 1 } ], "counts": { "windows": 918, "boundary": 0, "excluded": 834, "gc": 0, "tm": 6, "validForward": 75, "validReverse": 3, "productSize": 0, "deltaTm": 0, "eligiblePairs": 72, "exactChecked": 5, "exactRefused": 0 }, "capped": true }, "method": "bounded-flanks-v1", "oracle": "primer_design.py (Biopython MeltingTemp.Tm_NN for Tm; windows, GC, ranking and caps derived in the script; exact binding by exact_matches.py)", "oracleVersion": "Biopython 1.88", "recordedOn": "2026-10-11", "notes": "As design-lac-linear with two excluded regions inside the flanks: every window touching one is counted under excluded, never relaxed." }, { "id": "design-lac-tails", "input": { "templateName": "J01636.1 1..600", "template": "GACACCATCGAATGGCGCAAAACCTTTCGCGGTATGGCATGATAGCGCCCGGAAGAGAGTCAATTCAGGGTGGTGAATGTGAAACCAGTAACGTTATACGATGTCGCAGAGTATGCCGGTGTCTCTTATCAGACCGTTTCCCGCGTGGTGAACCAGGCCAGCCACGTTTCTGCGAAAACGCGGGAAAAAGTGGAAGCGGCGATGGCGGAGCTGAATTACATTCCCAACCGCGTGGCACAACAACTGGCGGGCAAACAGTCGTTGCTGATTGGCGTTGCCACCTCCAGTCTGGCCCTGCACGCGCCGTCGCAAATTGTCGCGGCGATTAAATCTCGCGCCGATCAACTGGGTGCCAGCGTGGTGGTGTCGATGGTAGAACGAAGCGGCGTCGAAGCCTGTAAAGCGGCGGTGCACAATCTTCTCGCGCAACGCGTCAGTGGGCTGATCATTAACTATCCGCTGGATGACCAGGATGCCATTGCTGTGGAAGCTGCCTGCACTAATGTTCCGGCGTTATTTCTTGATGTCTCTGACCAGACACCCATCAACAGTATTATTTTCTCCCATGAAGACGGTACGCGACTGGGCGTGGAGCATCTG", "topology": "linear", "settings": { "target": { "start": 201, "end": 400 }, "excluded": [], "forwardTail": "GCGAATTC", "reverseTail": "GCAAGCTT", "conditions": { "oligo_nM": 250, "na_mM": 50, "mg_mM": 0, "dntp_mM": 0, "model": "primer-excess", "saltCorrection": "owczarzy-2004" }, "flank": 50, "minLength": 18, "maxLength": 26, "targetLength": 20, "minTm": 55, "maxTm": 68, "targetTm": 59.95, "maxDeltaTm": 3, "minGc": 30, "maxGc": 70, "minProduct": 190, "maxProduct": 440 } }, "expected": { "method": "bounded-flanks-v1", "pcrMethod": "exact-pair-v1", "templateHash": "fd182829946e68ba90d7c14906883ef31f663537dade5f617f804b8ce46dc316", "topology": "linear", "templateLength": 600, "pairs": [ { "id": "+:158:177|-:428:409", "forward": { "annealing": "CCAGCCACGTTTCTGCGAAA", "tail": "GCGAATTC", "start": 158, "end": 177, "tm": 59.99861335790035, "gc": 55.0, "penalty": 0.04861335790035071 }, "reverse": { "annealing": "TGCGCGAGAAGATTGTGCAC", "tail": "GCAAGCTT", "start": 428, "end": 409, "tm": 59.96600984591282, "gc": 55.0, "penalty": 0.016009845912819287 }, "product": { "id": "+:158:177|-:428:409", "sequence": "GCGAATTCCCAGCCACGTTTCTGCGAAAACGCGGGAAAAAGTGGAAGCGGCGATGGCGGAGCTGAATTACATTCCCAACCGCGTGGCACAACAACTGGCGGGCAAACAGTCGTTGCTGATTGGCGTTGCCACCTCCAGTCTGGCCCTGCACGCGCCGTCGCAAATTGTCGCGGCGATTAAATCTCGCGCCGATCAACTGGGTGCCAGCGTGGTGGTGTCGATGGTAGAACGAAGCGGCGTCGAAGCCTGTAAAGCGGCGGTGCACAATCTTCTCGCGCAAAGCTTGC", "length": 287, "templateLength": 600, "templateSpan": 271, "orientation": "+", "crossesOrigin": false, "forward": { "strand": "+", "start": 158, "end": 177, "length": 20 }, "reverse": { "strand": "-", "start": 428, "end": 409, "length": 20 } }, "ranking": { "tmDeviation": 0.06462320381317, "lengthDeviation": 0.0, "deltaTm": 0.03260351198753142, "total": 0.09722671580070141 }, "exactSites": { "forward": 1, "reverse": 1 }, "exactProducts": 1 }, { "id": "+:158:178|-:428:409", "forward": { "annealing": "CCAGCCACGTTTCTGCGAAAA", "tail": "GCGAATTC", "start": 158, "end": 178, "tm": 59.93537222125224, "gc": 52.38095238095238, "penalty": 0.21462777874776579 }, "reverse": { "annealing": "TGCGCGAGAAGATTGTGCAC", "tail": "GCAAGCTT", "start": 428, "end": 409, "tm": 59.96600984591282, "gc": 55.0, "penalty": 0.016009845912819287 }, "product": { "id": "+:158:178|-:428:409", "sequence": "GCGAATTCCCAGCCACGTTTCTGCGAAAACGCGGGAAAAAGTGGAAGCGGCGATGGCGGAGCTGAATTACATTCCCAACCGCGTGGCACAACAACTGGCGGGCAAACAGTCGTTGCTGATTGGCGTTGCCACCTCCAGTCTGGCCCTGCACGCGCCGTCGCAAATTGTCGCGGCGATTAAATCTCGCGCCGATCAACTGGGTGCCAGCGTGGTGGTGTCGATGGTAGAACGAAGCGGCGTCGAAGCCTGTAAAGCGGCGGTGCACAATCTTCTCGCGCAAAGCTTGC", "length": 287, "templateLength": 600, "templateSpan": 271, "orientation": "+", "crossesOrigin": false, "forward": { "strand": "+", "start": 158, "end": 178, "length": 21 }, "reverse": { "strand": "-", "start": 428, "end": 409, "length": 20 } }, "ranking": { "tmDeviation": 0.03063762466058506, "lengthDeviation": 0.2, "deltaTm": 0.03063762466058506, "total": 0.26127524932117013 }, "exactSites": { "forward": 1, "reverse": 1 }, "exactProducts": 1 }, { "id": "+:178:197|-:428:409", "forward": { "annealing": "ACGCGGGAAAAAGTGGAAGC", "tail": "GCGAATTC", "start": 178, "end": 197, "tm": 60.09860053729648, "gc": 55.0, "penalty": 0.14860053729647404 }, "reverse": { "annealing": "TGCGCGAGAAGATTGTGCAC", "tail": "GCAAGCTT", "start": 428, "end": 409, "tm": 59.96600984591282, "gc": 55.0, "penalty": 0.016009845912819287 }, "product": { "id": "+:178:197|-:428:409", "sequence": "GCGAATTCACGCGGGAAAAAGTGGAAGCGGCGATGGCGGAGCTGAATTACATTCCCAACCGCGTGGCACAACAACTGGCGGGCAAACAGTCGTTGCTGATTGGCGTTGCCACCTCCAGTCTGGCCCTGCACGCGCCGTCGCAAATTGTCGCGGCGATTAAATCTCGCGCCGATCAACTGGGTGCCAGCGTGGTGGTGTCGATGGTAGAACGAAGCGGCGTCGAAGCCTGTAAAGCGGCGGTGCACAATCTTCTCGCGCAAAGCTTGC", "length": 267, "templateLength": 600, "templateSpan": 251, "orientation": "+", "crossesOrigin": false, "forward": { "strand": "+", "start": 178, "end": 197, "length": 20 }, "reverse": { "strand": "-", "start": 428, "end": 409, "length": 20 } }, "ranking": { "tmDeviation": 0.16461038320929333, "lengthDeviation": 0.0, "deltaTm": 0.13259069138365476, "total": 0.2972010745929481 }, "exactSites": { "forward": 1, "reverse": 1 }, "exactProducts": 1 }, { "id": "+:177:197|-:428:409", "forward": { "annealing": "AACGCGGGAAAAAGTGGAAGC", "tail": "GCGAATTC", "start": 177, "end": 197, "tm": 60.03263800822515, "gc": 52.38095238095238, "penalty": 0.2826380082251461 }, "reverse": { "annealing": "TGCGCGAGAAGATTGTGCAC", "tail": "GCAAGCTT", "start": 428, "end": 409, "tm": 59.96600984591282, "gc": 55.0, "penalty": 0.016009845912819287 }, "product": { "id": "+:177:197|-:428:409", "sequence": "GCGAATTCAACGCGGGAAAAAGTGGAAGCGGCGATGGCGGAGCTGAATTACATTCCCAACCGCGTGGCACAACAACTGGCGGGCAAACAGTCGTTGCTGATTGGCGTTGCCACCTCCAGTCTGGCCCTGCACGCGCCGTCGCAAATTGTCGCGGCGATTAAATCTCGCGCCGATCAACTGGGTGCCAGCGTGGTGGTGTCGATGGTAGAACGAAGCGGCGTCGAAGCCTGTAAAGCGGCGGTGCACAATCTTCTCGCGCAAAGCTTGC", "length": 268, "templateLength": 600, "templateSpan": 252, "orientation": "+", "crossesOrigin": false, "forward": { "strand": "+", "start": 177, "end": 197, "length": 21 }, "reverse": { "strand": "-", "start": 428, "end": 409, "length": 20 } }, "ranking": { "tmDeviation": 0.0986478541379654, "lengthDeviation": 0.2, "deltaTm": 0.06662816231232682, "total": 0.36527601645029223 }, "exactSites": { "forward": 1, "reverse": 1 }, "exactProducts": 1 }, { "id": "+:158:177|-:422:404", "forward": { "annealing": "CCAGCCACGTTTCTGCGAAA", "tail": "GCGAATTC", "start": 158, "end": 177, "tm": 59.99861335790035, "gc": 55.0, "penalty": 0.04861335790035071 }, "reverse": { "annealing": "AGAAGATTGTGCACCGCCG", "tail": "GCAAGCTT", "start": 422, "end": 404, "tm": 59.914723905422534, "gc": 57.89473684210526, "penalty": 0.23527609457746906 }, "product": { "id": "+:158:177|-:422:404", "sequence": "GCGAATTCCCAGCCACGTTTCTGCGAAAACGCGGGAAAAAGTGGAAGCGGCGATGGCGGAGCTGAATTACATTCCCAACCGCGTGGCACAACAACTGGCGGGCAAACAGTCGTTGCTGATTGGCGTTGCCACCTCCAGTCTGGCCCTGCACGCGCCGTCGCAAATTGTCGCGGCGATTAAATCTCGCGCCGATCAACTGGGTGCCAGCGTGGTGGTGTCGATGGTAGAACGAAGCGGCGTCGAAGCCTGTAAAGCGGCGGTGCACAATCTTCTAAGCTTGC", "length": 281, "templateLength": 600, "templateSpan": 265, "orientation": "+", "crossesOrigin": false, "forward": { "strand": "+", "start": 158, "end": 177, "length": 20 }, "reverse": { "strand": "-", "start": 422, "end": 404, "length": 19 } }, "ranking": { "tmDeviation": 0.08388945247781976, "lengthDeviation": 0.2, "deltaTm": 0.08388945247781976, "total": 0.36777890495563953 }, "exactSites": { "forward": 1, "reverse": 1 }, "exactProducts": 1 } ], "counts": { "windows": 918, "boundary": 0, "excluded": 0, "gc": 40, "tm": 234, "validForward": 353, "validReverse": 291, "productSize": 0, "deltaTm": 0, "eligiblePairs": 576, "exactChecked": 5, "exactRefused": 0 }, "capped": true }, "method": "bounded-flanks-v1", "oracle": "primer_design.py (Biopython MeltingTemp.Tm_NN for Tm; windows, GC, ranking and caps derived in the script; exact binding by exact_matches.py)", "oracleVersion": "Biopython 1.88", "recordedOn": "2026-10-11", "notes": "As design-lac-linear with 5′ tails (an EcoRI and a HindIII site behind a GC clamp): the tails count towards the product length and never towards Tm or GC; the product carries the forward tail first and the reverse tail reverse complemented last." }, { "id": "design-lac-mg", "input": { "templateName": "J01636.1 1..600", "template": "GACACCATCGAATGGCGCAAAACCTTTCGCGGTATGGCATGATAGCGCCCGGAAGAGAGTCAATTCAGGGTGGTGAATGTGAAACCAGTAACGTTATACGATGTCGCAGAGTATGCCGGTGTCTCTTATCAGACCGTTTCCCGCGTGGTGAACCAGGCCAGCCACGTTTCTGCGAAAACGCGGGAAAAAGTGGAAGCGGCGATGGCGGAGCTGAATTACATTCCCAACCGCGTGGCACAACAACTGGCGGGCAAACAGTCGTTGCTGATTGGCGTTGCCACCTCCAGTCTGGCCCTGCACGCGCCGTCGCAAATTGTCGCGGCGATTAAATCTCGCGCCGATCAACTGGGTGCCAGCGTGGTGGTGTCGATGGTAGAACGAAGCGGCGTCGAAGCCTGTAAAGCGGCGGTGCACAATCTTCTCGCGCAACGCGTCAGTGGGCTGATCATTAACTATCCGCTGGATGACCAGGATGCCATTGCTGTGGAAGCTGCCTGCACTAATGTTCCGGCGTTATTTCTTGATGTCTCTGACCAGACACCCATCAACAGTATTATTTTCTCCCATGAAGACGGTACGCGACTGGGCGTGGAGCATCTG", "topology": "linear", "settings": { "target": { "start": 201, "end": 400 }, "excluded": [], "forwardTail": "", "reverseTail": "", "conditions": { "oligo_nM": 250, "na_mM": 50, "mg_mM": 1.5, "dntp_mM": 0.2, "model": "primer-excess", "saltCorrection": "owczarzy-2008" }, "flank": 50, "minLength": 18, "maxLength": 26, "targetLength": 20, "minTm": 55, "maxTm": 68, "targetTm": 60.95, "maxDeltaTm": 3, "minGc": 30, "maxGc": 70, "minProduct": 180, "maxProduct": 420 } }, "expected": { "method": "bounded-flanks-v1", "pcrMethod": "exact-pair-v1", "templateHash": "fd182829946e68ba90d7c14906883ef31f663537dade5f617f804b8ce46dc316", "topology": "linear", "templateLength": 600, "pairs": [ { "id": "+:176:193|-:469:450", "forward": { "annealing": "AAACGCGGGAAAAAGTGG", "tail": "", "start": 176, "end": 193, "tm": 61.15758568510165, "gc": 50.0, "penalty": 0.6075856851016482 }, "reverse": { "annealing": "GGTCATCCAGCGGATAGTTA", "tail": "", "start": 469, "end": 450, "tm": 61.11705567100347, "gc": 50.0, "penalty": 0.1670556710034674 }, "product": { "id": "+:176:193|-:469:450", "sequence": "AAACGCGGGAAAAAGTGGAAGCGGCGATGGCGGAGCTGAATTACATTCCCAACCGCGTGGCACAACAACTGGCGGGCAAACAGTCGTTGCTGATTGGCGTTGCCACCTCCAGTCTGGCCCTGCACGCGCCGTCGCAAATTGTCGCGGCGATTAAATCTCGCGCCGATCAACTGGGTGCCAGCGTGGTGGTGTCGATGGTAGAACGAAGCGGCGTCGAAGCCTGTAAAGCGGCGGTGCACAATCTTCTCGCGCAACGCGTCAGTGGGCTGATCATTAACTATCCGCTGGATGACC", "length": 294, "templateLength": 600, "templateSpan": 294, "orientation": "+", "crossesOrigin": false, "forward": { "strand": "+", "start": 176, "end": 193, "length": 18 }, "reverse": { "strand": "-", "start": 469, "end": 450, "length": 20 } }, "ranking": { "tmDeviation": 0.3746413561051156, "lengthDeviation": 0.4, "deltaTm": 0.0405300140981808, "total": 0.8151713702032964 }, "exactSites": { "forward": 1, "reverse": 1 }, "exactProducts": 1 }, { "id": "+:176:193|-:454:434", "forward": { "annealing": "AAACGCGGGAAAAAGTGG", "tail": "", "start": 176, "end": 193, "tm": 61.15758568510165, "gc": 50.0, "penalty": 0.6075856851016482 }, "reverse": { "annealing": "AGTTAATGATCAGCCCACTGA", "tail": "", "start": 454, "end": 434, "tm": 60.991115029483694, "gc": 42.857142857142854, "penalty": 0.2411150294836915 }, "product": { "id": "+:176:193|-:454:434", "sequence": "AAACGCGGGAAAAAGTGGAAGCGGCGATGGCGGAGCTGAATTACATTCCCAACCGCGTGGCACAACAACTGGCGGGCAAACAGTCGTTGCTGATTGGCGTTGCCACCTCCAGTCTGGCCCTGCACGCGCCGTCGCAAATTGTCGCGGCGATTAAATCTCGCGCCGATCAACTGGGTGCCAGCGTGGTGGTGTCGATGGTAGAACGAAGCGGCGTCGAAGCCTGTAAAGCGGCGGTGCACAATCTTCTCGCGCAACGCGTCAGTGGGCTGATCATTAACT", "length": 279, "templateLength": 600, "templateSpan": 279, "orientation": "+", "crossesOrigin": false, "forward": { "strand": "+", "start": 176, "end": 193, "length": 18 }, "reverse": { "strand": "-", "start": 454, "end": 434, "length": 21 } }, "ranking": { "tmDeviation": 0.2487007145853397, "lengthDeviation": 0.6000000000000001, "deltaTm": 0.1664706556179567, "total": 1.0151713702032965 }, "exactSites": { "forward": 1, "reverse": 1 }, "exactProducts": 1 }, { "id": "+:182:199|-:469:450", "forward": { "annealing": "GGGAAAAAGTGGAAGCGG", "tail": "", "start": 182, "end": 199, "tm": 61.293900774555254, "gc": 55.55555555555556, "penalty": 0.7439007745552516 }, "reverse": { "annealing": "GGTCATCCAGCGGATAGTTA", "tail": "", "start": 469, "end": 450, "tm": 61.11705567100347, "gc": 50.0, "penalty": 0.1670556710034674 }, "product": { "id": "+:182:199|-:469:450", "sequence": "GGGAAAAAGTGGAAGCGGCGATGGCGGAGCTGAATTACATTCCCAACCGCGTGGCACAACAACTGGCGGGCAAACAGTCGTTGCTGATTGGCGTTGCCACCTCCAGTCTGGCCCTGCACGCGCCGTCGCAAATTGTCGCGGCGATTAAATCTCGCGCCGATCAACTGGGTGCCAGCGTGGTGGTGTCGATGGTAGAACGAAGCGGCGTCGAAGCCTGTAAAGCGGCGGTGCACAATCTTCTCGCGCAACGCGTCAGTGGGCTGATCATTAACTATCCGCTGGATGACC", "length": 288, "templateLength": 600, "templateSpan": 288, "orientation": "+", "crossesOrigin": false, "forward": { "strand": "+", "start": 182, "end": 199, "length": 18 }, "reverse": { "strand": "-", "start": 469, "end": 450, "length": 20 } }, "ranking": { "tmDeviation": 0.510956445558719, "lengthDeviation": 0.4, "deltaTm": 0.17684510355178418, "total": 1.087801549110503 }, "exactSites": { "forward": 1, "reverse": 1 }, "exactProducts": 1 }, { "id": "+:161:178|-:469:450", "forward": { "annealing": "GCCACGTTTCTGCGAAAA", "tail": "", "start": 161, "end": 178, "tm": 61.3660651215726, "gc": 50.0, "penalty": 0.8160651215726006 }, "reverse": { "annealing": "GGTCATCCAGCGGATAGTTA", "tail": "", "start": 469, "end": 450, "tm": 61.11705567100347, "gc": 50.0, "penalty": 0.1670556710034674 }, "product": { "id": "+:161:178|-:469:450", "sequence": "GCCACGTTTCTGCGAAAACGCGGGAAAAAGTGGAAGCGGCGATGGCGGAGCTGAATTACATTCCCAACCGCGTGGCACAACAACTGGCGGGCAAACAGTCGTTGCTGATTGGCGTTGCCACCTCCAGTCTGGCCCTGCACGCGCCGTCGCAAATTGTCGCGGCGATTAAATCTCGCGCCGATCAACTGGGTGCCAGCGTGGTGGTGTCGATGGTAGAACGAAGCGGCGTCGAAGCCTGTAAAGCGGCGGTGCACAATCTTCTCGCGCAACGCGTCAGTGGGCTGATCATTAACTATCCGCTGGATGACC", "length": 309, "templateLength": 600, "templateSpan": 309, "orientation": "+", "crossesOrigin": false, "forward": { "strand": "+", "start": 161, "end": 178, "length": 18 }, "reverse": { "strand": "-", "start": 469, "end": 450, "length": 20 } }, "ranking": { "tmDeviation": 0.583120792576068, "lengthDeviation": 0.4, "deltaTm": 0.24900945056913315, "total": 1.232130243145201 }, "exactSites": { "forward": 1, "reverse": 1 }, "exactProducts": 1 }, { "id": "+:176:193|-:453:433", "forward": { "annealing": "AAACGCGGGAAAAAGTGG", "tail": "", "start": 176, "end": 193, "tm": 61.15758568510165, "gc": 50.0, "penalty": 0.6075856851016482 }, "reverse": { "annealing": "GTTAATGATCAGCCCACTGAC", "tail": "", "start": 453, "end": 433, "tm": 60.81993868631338, "gc": 47.61904761904762, "penalty": 0.33006131368662467 }, "product": { "id": "+:176:193|-:453:433", "sequence": "AAACGCGGGAAAAAGTGGAAGCGGCGATGGCGGAGCTGAATTACATTCCCAACCGCGTGGCACAACAACTGGCGGGCAAACAGTCGTTGCTGATTGGCGTTGCCACCTCCAGTCTGGCCCTGCACGCGCCGTCGCAAATTGTCGCGGCGATTAAATCTCGCGCCGATCAACTGGGTGCCAGCGTGGTGGTGTCGATGGTAGAACGAAGCGGCGTCGAAGCCTGTAAAGCGGCGGTGCACAATCTTCTCGCGCAACGCGTCAGTGGGCTGATCATTAAC", "length": 278, "templateLength": 600, "templateSpan": 278, "orientation": "+", "crossesOrigin": false, "forward": { "strand": "+", "start": 176, "end": 193, "length": 18 }, "reverse": { "strand": "-", "start": 453, "end": 433, "length": 21 } }, "ranking": { "tmDeviation": 0.33764699878827287, "lengthDeviation": 0.6000000000000001, "deltaTm": 0.33764699878827287, "total": 1.2752939975765458 }, "exactSites": { "forward": 1, "reverse": 1 }, "exactProducts": 1 } ], "counts": { "windows": 918, "boundary": 0, "excluded": 0, "gc": 40, "tm": 507, "validForward": 141, "validReverse": 230, "productSize": 0, "deltaTm": 0, "eligiblePairs": 576, "exactChecked": 5, "exactRefused": 0 }, "capped": true }, "method": "bounded-flanks-v1", "oracle": "primer_design.py (Biopython MeltingTemp.Tm_NN for Tm; windows, GC, ranking and caps derived in the script; exact binding by exact_matches.py)", "oracleVersion": "Biopython 1.88", "recordedOn": "2026-10-11", "notes": "As design-lac-linear under 1.5 mM Mg²⁺ and 0.2 mM dNTPs: the Owczarzy 2008 correction (Biopython saltcorr 7), so the Tm bounds select different windows." }, { "id": "design-lac-capped", "input": { "templateName": "J01636.1 1..600", "template": "GACACCATCGAATGGCGCAAAACCTTTCGCGGTATGGCATGATAGCGCCCGGAAGAGAGTCAATTCAGGGTGGTGAATGTGAAACCAGTAACGTTATACGATGTCGCAGAGTATGCCGGTGTCTCTTATCAGACCGTTTCCCGCGTGGTGAACCAGGCCAGCCACGTTTCTGCGAAAACGCGGGAAAAAGTGGAAGCGGCGATGGCGGAGCTGAATTACATTCCCAACCGCGTGGCACAACAACTGGCGGGCAAACAGTCGTTGCTGATTGGCGTTGCCACCTCCAGTCTGGCCCTGCACGCGCCGTCGCAAATTGTCGCGGCGATTAAATCTCGCGCCGATCAACTGGGTGCCAGCGTGGTGGTGTCGATGGTAGAACGAAGCGGCGTCGAAGCCTGTAAAGCGGCGGTGCACAATCTTCTCGCGCAACGCGTCAGTGGGCTGATCATTAACTATCCGCTGGATGACCAGGATGCCATTGCTGTGGAAGCTGCCTGCACTAATGTTCCGGCGTTATTTCTTGATGTCTCTGACCAGACACCCATCAACAGTATTATTTTCTCCCATGAAGACGGTACGCGACTGGGCGTGGAGCATCTG", "topology": "linear", "settings": { "target": { "start": 201, "end": 400 }, "excluded": [], "forwardTail": "", "reverseTail": "", "conditions": { "oligo_nM": 250, "na_mM": 50, "mg_mM": 0, "dntp_mM": 0, "model": "primer-excess", "saltCorrection": "owczarzy-2004" }, "flank": 200, "minLength": 18, "maxLength": 26, "targetLength": 20, "minTm": 55, "maxTm": 68, "targetTm": 61.05, "maxDeltaTm": 3, "minGc": 30, "maxGc": 70, "minProduct": 180, "maxProduct": 800 } }, "expected": { "method": "bounded-flanks-v1", "pcrMethod": "exact-pair-v1", "templateHash": "fd182829946e68ba90d7c14906883ef31f663537dade5f617f804b8ce46dc316", "topology": "linear", "templateLength": 600, "pairs": [ { "id": "+:12:31|-:586:567", "forward": { "annealing": "ATGGCGCAAAACCTTTCGCG", "tail": "", "start": 12, "end": 31, "tm": 61.065643199402075, "gc": 55.0, "penalty": 0.015643199402077812 }, "reverse": { "annealing": "CCAGTCGCGTACCGTCTTCA", "tail": "", "start": 586, "end": 567, "tm": 61.0566792127554, "gc": 60.0, "penalty": 0.006679212755400954 }, "product": { "id": "+:12:31|-:586:567", "sequence": "ATGGCGCAAAACCTTTCGCGGTATGGCATGATAGCGCCCGGAAGAGAGTCAATTCAGGGTGGTGAATGTGAAACCAGTAACGTTATACGATGTCGCAGAGTATGCCGGTGTCTCTTATCAGACCGTTTCCCGCGTGGTGAACCAGGCCAGCCACGTTTCTGCGAAAACGCGGGAAAAAGTGGAAGCGGCGATGGCGGAGCTGAATTACATTCCCAACCGCGTGGCACAACAACTGGCGGGCAAACAGTCGTTGCTGATTGGCGTTGCCACCTCCAGTCTGGCCCTGCACGCGCCGTCGCAAATTGTCGCGGCGATTAAATCTCGCGCCGATCAACTGGGTGCCAGCGTGGTGGTGTCGATGGTAGAACGAAGCGGCGTCGAAGCCTGTAAAGCGGCGGTGCACAATCTTCTCGCGCAACGCGTCAGTGGGCTGATCATTAACTATCCGCTGGATGACCAGGATGCCATTGCTGTGGAAGCTGCCTGCACTAATGTTCCGGCGTTATTTCTTGATGTCTCTGACCAGACACCCATCAACAGTATTATTTTCTCCCATGAAGACGGTACGCGACTGG", "length": 575, "templateLength": 600, "templateSpan": 575, "orientation": "+", "crossesOrigin": false, "forward": { "strand": "+", "start": 12, "end": 31, "length": 20 }, "reverse": { "strand": "-", "start": 586, "end": 567, "length": 20 } }, "ranking": { "tmDeviation": 0.022322412157478766, "lengthDeviation": 0.0, "deltaTm": 0.008963986646676858, "total": 0.031286398804155624 }, "exactSites": { "forward": 1, "reverse": 1 }, "exactProducts": 1 }, { "id": "+:12:31|-:475:456", "forward": { "annealing": "ATGGCGCAAAACCTTTCGCG", "tail": "", "start": 12, "end": 31, "tm": 61.065643199402075, "gc": 55.0, "penalty": 0.015643199402077812 }, "reverse": { "annealing": "CATCCTGGTCATCCAGCGGA", "tail": "", "start": 475, "end": 456, "tm": 61.04745311032701, "gc": 60.0, "penalty": 0.0025468896729847756 }, "product": { "id": "+:12:31|-:475:456", "sequence": "ATGGCGCAAAACCTTTCGCGGTATGGCATGATAGCGCCCGGAAGAGAGTCAATTCAGGGTGGTGAATGTGAAACCAGTAACGTTATACGATGTCGCAGAGTATGCCGGTGTCTCTTATCAGACCGTTTCCCGCGTGGTGAACCAGGCCAGCCACGTTTCTGCGAAAACGCGGGAAAAAGTGGAAGCGGCGATGGCGGAGCTGAATTACATTCCCAACCGCGTGGCACAACAACTGGCGGGCAAACAGTCGTTGCTGATTGGCGTTGCCACCTCCAGTCTGGCCCTGCACGCGCCGTCGCAAATTGTCGCGGCGATTAAATCTCGCGCCGATCAACTGGGTGCCAGCGTGGTGGTGTCGATGGTAGAACGAAGCGGCGTCGAAGCCTGTAAAGCGGCGGTGCACAATCTTCTCGCGCAACGCGTCAGTGGGCTGATCATTAACTATCCGCTGGATGACCAGGATG", "length": 464, "templateLength": 600, "templateSpan": 464, "orientation": "+", "crossesOrigin": false, "forward": { "strand": "+", "start": 12, "end": 31, "length": 20 }, "reverse": { "strand": "-", "start": 475, "end": 456, "length": 20 } }, "ranking": { "tmDeviation": 0.018190089075062588, "lengthDeviation": 0.0, "deltaTm": 0.018190089075062588, "total": 0.036380178150125175 }, "exactSites": { "forward": 1, "reverse": 1 }, "exactProducts": 1 }, { "id": "+:12:31|-:512:493", "forward": { "annealing": "ATGGCGCAAAACCTTTCGCG", "tail": "", "start": 12, "end": 31, "tm": 61.065643199402075, "gc": 55.0, "penalty": 0.015643199402077812 }, "reverse": { "annealing": "GCCGGAACATTAGTGCAGGC", "tail": "", "start": 512, "end": 493, "tm": 60.94050494377336, "gc": 60.0, "penalty": 0.1094950562266348 }, "product": { "id": "+:12:31|-:512:493", "sequence": "ATGGCGCAAAACCTTTCGCGGTATGGCATGATAGCGCCCGGAAGAGAGTCAATTCAGGGTGGTGAATGTGAAACCAGTAACGTTATACGATGTCGCAGAGTATGCCGGTGTCTCTTATCAGACCGTTTCCCGCGTGGTGAACCAGGCCAGCCACGTTTCTGCGAAAACGCGGGAAAAAGTGGAAGCGGCGATGGCGGAGCTGAATTACATTCCCAACCGCGTGGCACAACAACTGGCGGGCAAACAGTCGTTGCTGATTGGCGTTGCCACCTCCAGTCTGGCCCTGCACGCGCCGTCGCAAATTGTCGCGGCGATTAAATCTCGCGCCGATCAACTGGGTGCCAGCGTGGTGGTGTCGATGGTAGAACGAAGCGGCGTCGAAGCCTGTAAAGCGGCGGTGCACAATCTTCTCGCGCAACGCGTCAGTGGGCTGATCATTAACTATCCGCTGGATGACCAGGATGCCATTGCTGTGGAAGCTGCCTGCACTAATGTTCCGGC", "length": 501, "templateLength": 600, "templateSpan": 501, "orientation": "+", "crossesOrigin": false, "forward": { "strand": "+", "start": 12, "end": 31, "length": 20 }, "reverse": { "strand": "-", "start": 512, "end": 493, "length": 20 } }, "ranking": { "tmDeviation": 0.1251382556287126, "lengthDeviation": 0.0, "deltaTm": 0.1251382556287126, "total": 0.2502765112574252 }, "exactSites": { "forward": 1, "reverse": 1 }, "exactProducts": 1 }, { "id": "+:98:118|-:586:567", "forward": { "annealing": "ACGATGTCGCAGAGTATGCCG", "tail": "", "start": 98, "end": 118, "tm": 61.09416535585274, "gc": 57.142857142857146, "penalty": 0.24416535585274063 }, "reverse": { "annealing": "CCAGTCGCGTACCGTCTTCA", "tail": "", "start": 586, "end": 567, "tm": 61.0566792127554, "gc": 60.0, "penalty": 0.006679212755400954 }, "product": { "id": "+:98:118|-:586:567", "sequence": "ACGATGTCGCAGAGTATGCCGGTGTCTCTTATCAGACCGTTTCCCGCGTGGTGAACCAGGCCAGCCACGTTTCTGCGAAAACGCGGGAAAAAGTGGAAGCGGCGATGGCGGAGCTGAATTACATTCCCAACCGCGTGGCACAACAACTGGCGGGCAAACAGTCGTTGCTGATTGGCGTTGCCACCTCCAGTCTGGCCCTGCACGCGCCGTCGCAAATTGTCGCGGCGATTAAATCTCGCGCCGATCAACTGGGTGCCAGCGTGGTGGTGTCGATGGTAGAACGAAGCGGCGTCGAAGCCTGTAAAGCGGCGGTGCACAATCTTCTCGCGCAACGCGTCAGTGGGCTGATCATTAACTATCCGCTGGATGACCAGGATGCCATTGCTGTGGAAGCTGCCTGCACTAATGTTCCGGCGTTATTTCTTGATGTCTCTGACCAGACACCCATCAACAGTATTATTTTCTCCCATGAAGACGGTACGCGACTGG", "length": 489, "templateLength": 600, "templateSpan": 489, "orientation": "+", "crossesOrigin": false, "forward": { "strand": "+", "start": 98, "end": 118, "length": 21 }, "reverse": { "strand": "-", "start": 586, "end": 567, "length": 20 } }, "ranking": { "tmDeviation": 0.05084456860814157, "lengthDeviation": 0.2, "deltaTm": 0.037486143097339664, "total": 0.28833071170548125 }, "exactSites": { "forward": 1, "reverse": 1 }, "exactProducts": 1 }, { "id": "+:98:118|-:475:456", "forward": { "annealing": "ACGATGTCGCAGAGTATGCCG", "tail": "", "start": 98, "end": 118, "tm": 61.09416535585274, "gc": 57.142857142857146, "penalty": 0.24416535585274063 }, "reverse": { "annealing": "CATCCTGGTCATCCAGCGGA", "tail": "", "start": 475, "end": 456, "tm": 61.04745311032701, "gc": 60.0, "penalty": 0.0025468896729847756 }, "product": { "id": "+:98:118|-:475:456", "sequence": "ACGATGTCGCAGAGTATGCCGGTGTCTCTTATCAGACCGTTTCCCGCGTGGTGAACCAGGCCAGCCACGTTTCTGCGAAAACGCGGGAAAAAGTGGAAGCGGCGATGGCGGAGCTGAATTACATTCCCAACCGCGTGGCACAACAACTGGCGGGCAAACAGTCGTTGCTGATTGGCGTTGCCACCTCCAGTCTGGCCCTGCACGCGCCGTCGCAAATTGTCGCGGCGATTAAATCTCGCGCCGATCAACTGGGTGCCAGCGTGGTGGTGTCGATGGTAGAACGAAGCGGCGTCGAAGCCTGTAAAGCGGCGGTGCACAATCTTCTCGCGCAACGCGTCAGTGGGCTGATCATTAACTATCCGCTGGATGACCAGGATG", "length": 378, "templateLength": 600, "templateSpan": 378, "orientation": "+", "crossesOrigin": false, "forward": { "strand": "+", "start": 98, "end": 118, "length": 21 }, "reverse": { "strand": "-", "start": 475, "end": 456, "length": 20 } }, "ranking": { "tmDeviation": 0.04671224552572539, "lengthDeviation": 0.2, "deltaTm": 0.04671224552572539, "total": 0.2934244910514508 }, "exactSites": { "forward": 1, "reverse": 1 }, "exactProducts": 1 } ], "counts": { "windows": 3618, "boundary": 396, "excluded": 0, "gc": 94, "tm": 962, "validForward": 1172, "validReverse": 994, "productSize": 0, "deltaTm": 0, "eligiblePairs": 576, "exactChecked": 5, "exactRefused": 0 }, "capped": true }, "method": "bounded-flanks-v1", "oracle": "primer_design.py (Biopython MeltingTemp.Tm_NN for Tm; windows, GC, ranking and caps derived in the script; exact binding by exact_matches.py)", "oracleVersion": "Biopython 1.88", "recordedOn": "2026-10-11", "notes": "A 200-base flank on each side gives more than 24 eligible windows a side: the sides are cut to 24 by penalty and the search is capped." }, { "id": "design-puc19-origin", "input": { "templateName": "L09137.2 (pUC19, circular)", "template": "TCGCGCGTTTCGGTGATGACGGTGAAAACCTCTGACACATGCAGCTCCCGGAGACGGTCACAGCTTGTCTGTAAGCGGATGCCGGGAGCAGACAAGCCCGTCAGGGCGCGTCAGCGGGTGTTGGCGGGTGTCGGGGCTGGCTTAACTATGCGGCATCAGAGCAGATTGTACTGAGAGTGCACCATATGCGGTGTGAAATACCGCACAGATGCGTAAGGAGAAAATACCGCATCAGGCGCCATTCGCCATTCAGGCTGCGCAACTGTTGGGAAGGGCGATCGGTGCGGGCCTCTTCGCTATTACGCCAGCTGGCGAAAGGGGGATGTGCTGCAAGGCGATTAAGTTGGGTAACGCCAGGGTTTTCCCAGTCACGACGTTGTAAAACGACGGCCAGTGAATTCGAGCTCGGTACCCGGGGATCCTCTAGAGTCGACCTGCAGGCATGCAAGCTTGGCGTAATCATGGTCATAGCTGTTTCCTGTGTGAAATTGTTATCCGCTCACAATTCCACACAACATACGAGCCGGAAGCATAAAGTGTAAAGCCTGGGGTGCCTAATGAGTGAGCTAACTCACATTAATTGCGTTGCGCTCACTGCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCAGCTGCATTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCTCTTCCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGAACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAGGCACCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGTGTAGATAACTACGATACGGGAGGGCTTACCATCTGGCCCCAGTGCTGCAATGATACCGCGAGACCCACGCTCACCGGCTCCAGATTTATCAGCAATAAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCTCCATCCAGTCTATTAATTGTTGCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTTGCCATTGCTACAGGCATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCCCAACGATCAAGGCGAGTTACATGATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCATGGTTATGGCAGCACTGCATAATTCTCTTACTGTCATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAGTACTCAACCAAGTCATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCTCTTGCCCGGCGTCAATACGGGATAATACCGCGCCACATAGCAGAACTTTAAAAGTGCTCATCATTGGAAAACGTTCTTCGGGGCGAAAACTCTCAAGGATCTTACCGCTGTTGAGATCCAGTTCGATGTAACCCACTCGTGCACCCAACTGATCTTCAGCATCTTTTACTTTCACCAGCGTTTCTGGGTGAGCAAAAACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGGCGACACGGAAATGTTGAATACTCATACTCTTCCTTTTTCAATATTATTGAAGCATTTATCAGGGTTATTGTCTCATGAGCGGATACATATTTGAATGTATTTAGAAAAATAAACAAATAGGGGTTCCGCGCACATTTCCCCGAAAAGTGCCACCTGACGTCTAAGAAACCATTATTATCATGACATTAACCTATAAAAATAGGCGTATCACGAGGCCCTTTCGTC", "topology": "circular", "settings": { "target": { "start": 2660, "end": 40 }, "excluded": [], "forwardTail": "", "reverseTail": "", "conditions": { "oligo_nM": 250, "na_mM": 50, "mg_mM": 0, "dntp_mM": 0, "model": "primer-excess", "saltCorrection": "owczarzy-2004" }, "flank": 60, "minLength": 18, "maxLength": 26, "targetLength": 20, "minTm": 55, "maxTm": 68, "targetTm": 60.05, "maxDeltaTm": 3, "minGc": 30, "maxGc": 70, "minProduct": 120, "maxProduct": 400 } }, "expected": { "method": "bounded-flanks-v1", "pcrMethod": "exact-pair-v1", "templateHash": "8a205af08518dae1b0045c7b098986dca18824ef654e75233603602ab89cad04", "topology": "circular", "templateLength": 2686, "pairs": [ { "id": "+:2589:2609|-:84:64", "forward": { "annealing": "GCGCACATTTCCCCGAAAAGT", "tail": "", "start": 2589, "end": 2609, "tm": 60.09908493176272, "gc": 52.38095238095238, "penalty": 0.2490849317627209 }, "reverse": { "annealing": "CGGCATCCGCTTACAGACAAG", "tail": "", "start": 84, "end": 64, "tm": 59.961942060960894, "gc": 57.142857142857146, "penalty": 0.28805793903910343 }, "product": { "id": "+:2589:2609|-:84:64", "sequence": "GCGCACATTTCCCCGAAAAGTGCCACCTGACGTCTAAGAAACCATTATTATCATGACATTAACCTATAAAAATAGGCGTATCACGAGGCCCTTTCGTCTCGCGCGTTTCGGTGATGACGGTGAAAACCTCTGACACATGCAGCTCCCGGAGACGGTCACAGCTTGTCTGTAAGCGGATGCCG", "length": 182, "templateLength": 2686, "templateSpan": 182, "orientation": "+", "crossesOrigin": true, "forward": { "strand": "+", "start": 2589, "end": 2609, "length": 21 }, "reverse": { "strand": "-", "start": 84, "end": 64, "length": 21 } }, "ranking": { "tmDeviation": 0.1371428708018243, "lengthDeviation": 0.4, "deltaTm": 0.1371428708018243, "total": 0.6742857416036486 }, "exactSites": { "forward": 1, "reverse": 1 }, "exactProducts": 1 }, { "id": "+:2596:2615|-:69:50", "forward": { "annealing": "TTTCCCCGAAAAGTGCCACC", "tail": "", "start": 2596, "end": 2615, "tm": 59.731633561719605, "gc": 55.0, "penalty": 0.31836643828039257 }, "reverse": { "annealing": "GACAAGCTGTGACCGTCTCC", "tail": "", "start": 69, "end": 50, "tm": 59.67973345994426, "gc": 60.0, "penalty": 0.3702665400557379 }, "product": { "id": "+:2596:2615|-:69:50", "sequence": "TTTCCCCGAAAAGTGCCACCTGACGTCTAAGAAACCATTATTATCATGACATTAACCTATAAAAATAGGCGTATCACGAGGCCCTTTCGTCTCGCGCGTTTCGGTGATGACGGTGAAAACCTCTGACACATGCAGCTCCCGGAGACGGTCACAGCTTGTC", "length": 160, "templateLength": 2686, "templateSpan": 160, "orientation": "+", "crossesOrigin": true, "forward": { "strand": "+", "start": 2596, "end": 2615, "length": 20 }, "reverse": { "strand": "-", "start": 69, "end": 50, "length": 20 } }, "ranking": { "tmDeviation": 0.6886329783361305, "lengthDeviation": 0.0, "deltaTm": 0.05190010177534532, "total": 0.7405330801114758 }, "exactSites": { "forward": 1, "reverse": 1 }, "exactProducts": 1 }, { "id": "+:2589:2609|-:67:49", "forward": { "annealing": "GCGCACATTTCCCCGAAAAGT", "tail": "", "start": 2589, "end": 2609, "tm": 60.09908493176272, "gc": 52.38095238095238, "penalty": 0.2490849317627209 }, "reverse": { "annealing": "CAAGCTGTGACCGTCTCCG", "tail": "", "start": 67, "end": 49, "tm": 60.22135733315076, "gc": 63.1578947368421, "penalty": 0.3713573331507661 }, "product": { "id": "+:2589:2609|-:67:49", "sequence": "GCGCACATTTCCCCGAAAAGTGCCACCTGACGTCTAAGAAACCATTATTATCATGACATTAACCTATAAAAATAGGCGTATCACGAGGCCCTTTCGTCTCGCGCGTTTCGGTGATGACGGTGAAAACCTCTGACACATGCAGCTCCCGGAGACGGTCACAGCTTG", "length": 165, "templateLength": 2686, "templateSpan": 165, "orientation": "+", "crossesOrigin": true, "forward": { "strand": "+", "start": 2589, "end": 2609, "length": 21 }, "reverse": { "strand": "-", "start": 67, "end": 49, "length": 19 } }, "ranking": { "tmDeviation": 0.22044226491348695, "lengthDeviation": 0.4, "deltaTm": 0.12227240138804518, "total": 0.7427146663015322 }, "exactSites": { "forward": 1, "reverse": 1 }, "exactProducts": 1 }, { "id": "+:2596:2615|-:84:64", "forward": { "annealing": "TTTCCCCGAAAAGTGCCACC", "tail": "", "start": 2596, "end": 2615, "tm": 59.731633561719605, "gc": 55.0, "penalty": 0.31836643828039257 }, "reverse": { "annealing": "CGGCATCCGCTTACAGACAAG", "tail": "", "start": 84, "end": 64, "tm": 59.961942060960894, "gc": 57.142857142857146, "penalty": 0.28805793903910343 }, "product": { "id": "+:2596:2615|-:84:64", "sequence": "TTTCCCCGAAAAGTGCCACCTGACGTCTAAGAAACCATTATTATCATGACATTAACCTATAAAAATAGGCGTATCACGAGGCCCTTTCGTCTCGCGCGTTTCGGTGATGACGGTGAAAACCTCTGACACATGCAGCTCCCGGAGACGGTCACAGCTTGTCTGTAAGCGGATGCCG", "length": 175, "templateLength": 2686, "templateSpan": 175, "orientation": "+", "crossesOrigin": true, "forward": { "strand": "+", "start": 2596, "end": 2615, "length": 20 }, "reverse": { "strand": "-", "start": 84, "end": 64, "length": 21 } }, "ranking": { "tmDeviation": 0.406424377319496, "lengthDeviation": 0.2, "deltaTm": 0.23030849924128916, "total": 0.8367328765607851 }, "exactSites": { "forward": 1, "reverse": 1 }, "exactProducts": 1 }, { "id": "+:2602:2622|-:67:49", "forward": { "annealing": "CGAAAAGTGCCACCTGACGTC", "tail": "", "start": 2602, "end": 2622, "tm": 60.27949512360135, "gc": 57.142857142857146, "penalty": 0.42949512360135317 }, "reverse": { "annealing": "CAAGCTGTGACCGTCTCCG", "tail": "", "start": 67, "end": 49, "tm": 60.22135733315076, "gc": 63.1578947368421, "penalty": 0.3713573331507661 }, "product": { "id": "+:2602:2622|-:67:49", "sequence": "CGAAAAGTGCCACCTGACGTCTAAGAAACCATTATTATCATGACATTAACCTATAAAAATAGGCGTATCACGAGGCCCTTTCGTCTCGCGCGTTTCGGTGATGACGGTGAAAACCTCTGACACATGCAGCTCCCGGAGACGGTCACAGCTTG", "length": 152, "templateLength": 2686, "templateSpan": 152, "orientation": "+", "crossesOrigin": true, "forward": { "strand": "+", "start": 2602, "end": 2622, "length": 21 }, "reverse": { "strand": "-", "start": 67, "end": 49, "length": 19 } }, "ranking": { "tmDeviation": 0.4008524567521192, "lengthDeviation": 0.4, "deltaTm": 0.05813779045058709, "total": 0.8589902472027063 }, "exactSites": { "forward": 1, "reverse": 1 }, "exactProducts": 1 } ], "counts": { "windows": 1098, "boundary": 0, "excluded": 0, "gc": 321, "tm": 193, "validForward": 281, "validReverse": 303, "productSize": 0, "deltaTm": 0, "eligiblePairs": 576, "exactChecked": 5, "exactRefused": 0 }, "capped": true }, "method": "bounded-flanks-v1", "oracle": "primer_design.py (Biopython MeltingTemp.Tm_NN for Tm; windows, GC, ranking and caps derived in the script; exact binding by exact_matches.py)", "oracleVersion": "Biopython 1.88", "recordedOn": "2026-10-11", "notes": "A circular template with the target across the origin (2660..40): the reverse windows wrap, and the product crosses the origin." }, { "id": "design-lac-no-window", "input": { "templateName": "J01636.1 1..600", "template": "GACACCATCGAATGGCGCAAAACCTTTCGCGGTATGGCATGATAGCGCCCGGAAGAGAGTCAATTCAGGGTGGTGAATGTGAAACCAGTAACGTTATACGATGTCGCAGAGTATGCCGGTGTCTCTTATCAGACCGTTTCCCGCGTGGTGAACCAGGCCAGCCACGTTTCTGCGAAAACGCGGGAAAAAGTGGAAGCGGCGATGGCGGAGCTGAATTACATTCCCAACCGCGTGGCACAACAACTGGCGGGCAAACAGTCGTTGCTGATTGGCGTTGCCACCTCCAGTCTGGCCCTGCACGCGCCGTCGCAAATTGTCGCGGCGATTAAATCTCGCGCCGATCAACTGGGTGCCAGCGTGGTGGTGTCGATGGTAGAACGAAGCGGCGTCGAAGCCTGTAAAGCGGCGGTGCACAATCTTCTCGCGCAACGCGTCAGTGGGCTGATCATTAACTATCCGCTGGATGACCAGGATGCCATTGCTGTGGAAGCTGCCTGCACTAATGTTCCGGCGTTATTTCTTGATGTCTCTGACCAGACACCCATCAACAGTATTATTTTCTCCCATGAAGACGGTACGCGACTGGGCGTGGAGCATCTG", "topology": "linear", "settings": { "target": { "start": 201, "end": 400 }, "excluded": [], "forwardTail": "", "reverseTail": "", "conditions": { "oligo_nM": 250, "na_mM": 50, "mg_mM": 0, "dntp_mM": 0, "model": "primer-excess", "saltCorrection": "owczarzy-2004" }, "flank": 50, "minLength": 18, "maxLength": 26, "targetLength": 20, "minTm": 80, "maxTm": 95, "targetTm": 85, "maxDeltaTm": 3, "minGc": 30, "maxGc": 70, "minProduct": 180, "maxProduct": 420 } }, "expected": { "method": "bounded-flanks-v1", "pcrMethod": "exact-pair-v1", "templateHash": "fd182829946e68ba90d7c14906883ef31f663537dade5f617f804b8ce46dc316", "topology": "linear", "templateLength": 600, "pairs": [], "counts": { "windows": 918, "boundary": 0, "excluded": 0, "gc": 40, "tm": 878, "validForward": 0, "validReverse": 0, "productSize": 0, "deltaTm": 0, "eligiblePairs": 0, "exactChecked": 0, "exactRefused": 0 }, "capped": false }, "method": "bounded-flanks-v1", "oracle": "primer_design.py (Biopython MeltingTemp.Tm_NN for Tm; windows, GC, ranking and caps derived in the script; exact binding by exact_matches.py)", "oracleVersion": "Biopython 1.88", "recordedOn": "2026-10-11", "notes": "Tm bounds no window of 18 to 26 bases reaches under these conditions: every window is counted under tm, no pair exists, nothing is relaxed." }, { "id": "design-lac-no-product", "input": { "templateName": "J01636.1 1..600", "template": "GACACCATCGAATGGCGCAAAACCTTTCGCGGTATGGCATGATAGCGCCCGGAAGAGAGTCAATTCAGGGTGGTGAATGTGAAACCAGTAACGTTATACGATGTCGCAGAGTATGCCGGTGTCTCTTATCAGACCGTTTCCCGCGTGGTGAACCAGGCCAGCCACGTTTCTGCGAAAACGCGGGAAAAAGTGGAAGCGGCGATGGCGGAGCTGAATTACATTCCCAACCGCGTGGCACAACAACTGGCGGGCAAACAGTCGTTGCTGATTGGCGTTGCCACCTCCAGTCTGGCCCTGCACGCGCCGTCGCAAATTGTCGCGGCGATTAAATCTCGCGCCGATCAACTGGGTGCCAGCGTGGTGGTGTCGATGGTAGAACGAAGCGGCGTCGAAGCCTGTAAAGCGGCGGTGCACAATCTTCTCGCGCAACGCGTCAGTGGGCTGATCATTAACTATCCGCTGGATGACCAGGATGCCATTGCTGTGGAAGCTGCCTGCACTAATGTTCCGGCGTTATTTCTTGATGTCTCTGACCAGACACCCATCAACAGTATTATTTTCTCCCATGAAGACGGTACGCGACTGGGCGTGGAGCATCTG", "topology": "linear", "settings": { "target": { "start": 201, "end": 400 }, "excluded": [], "forwardTail": "", "reverseTail": "", "conditions": { "oligo_nM": 250, "na_mM": 50, "mg_mM": 0, "dntp_mM": 0, "model": "primer-excess", "saltCorrection": "owczarzy-2004" }, "flank": 50, "minLength": 18, "maxLength": 26, "targetLength": 20, "minTm": 55, "maxTm": 68, "targetTm": 60, "maxDeltaTm": 3, "minGc": 30, "maxGc": 70, "minProduct": 900, "maxProduct": 1000 } }, "expected": { "method": "bounded-flanks-v1", "pcrMethod": "exact-pair-v1", "templateHash": "fd182829946e68ba90d7c14906883ef31f663537dade5f617f804b8ce46dc316", "topology": "linear", "templateLength": 600, "pairs": [], "counts": { "windows": 918, "boundary": 0, "excluded": 0, "gc": 40, "tm": 234, "validForward": 353, "validReverse": 291, "productSize": 576, "deltaTm": 0, "eligiblePairs": 0, "exactChecked": 0, "exactRefused": 0 }, "capped": true }, "method": "bounded-flanks-v1", "oracle": "primer_design.py (Biopython MeltingTemp.Tm_NN for Tm; windows, GC, ranking and caps derived in the script; exact binding by exact_matches.py)", "oracleVersion": "Biopython 1.88", "recordedOn": "2026-10-11", "notes": "Eligible windows on both sides, but no product can reach 900 bases on a 600-base template: every pair counts under productSize and none is returned." }, { "id": "refuse-template-letter", "input": { "template": "ATGACCTAGCTATTCGCTAGTCAN", "topology": "linear", "settings": { "target": { "start": 9, "end": 16 }, "excluded": [], "forwardTail": "", "reverseTail": "", "conditions": { "oligo_nM": 250, "na_mM": 50, "mg_mM": 0, "dntp_mM": 0, "model": "primer-excess", "saltCorrection": "owczarzy-2004" }, "flank": 0, "minLength": 8, "maxLength": 8, "targetLength": 8, "minTm": 0, "maxTm": 100, "targetTm": 40, "maxDeltaTm": 30, "minGc": 0, "maxGc": 100, "minProduct": 24, "maxProduct": 30 } }, "expected": { "refused": true, "field": "template", "messagePattern": "ACGT" }, "notes": "An N in the template refuses before enumeration; nothing is dropped or guessed." }, { "id": "refuse-linear-crossing", "input": { "template": "ATGACCTAGCTATTCGCTAGTCAC", "topology": "linear", "settings": { "target": { "start": 20, "end": 5 }, "excluded": [], "forwardTail": "", "reverseTail": "", "conditions": { "oligo_nM": 250, "na_mM": 50, "mg_mM": 0, "dntp_mM": 0, "model": "primer-excess", "saltCorrection": "owczarzy-2004" }, "flank": 0, "minLength": 8, "maxLength": 8, "targetLength": 8, "minTm": 0, "maxTm": 100, "targetTm": 40, "maxDeltaTm": 30, "minGc": 0, "maxGc": 100, "minProduct": 24, "maxProduct": 30 } }, "expected": { "refused": true, "field": "target", "messagePattern": "coordinates" }, "notes": "A target with start > end crosses the origin, which a linear template has not." }, { "id": "refuse-constraint-range", "input": { "template": "ATGACCTAGCTATTCGCTAGTCAC", "topology": "linear", "settings": { "target": { "start": 9, "end": 16 }, "excluded": [], "forwardTail": "", "reverseTail": "", "conditions": { "oligo_nM": 250, "na_mM": 50, "mg_mM": 0, "dntp_mM": 0, "model": "primer-excess", "saltCorrection": "owczarzy-2004" }, "flank": 0, "minLength": 5, "maxLength": 8, "targetLength": 8, "minTm": 0, "maxTm": 100, "targetTm": 40, "maxDeltaTm": 30, "minGc": 0, "maxGc": 100, "minProduct": 24, "maxProduct": 30 } }, "expected": { "refused": true, "field": "constraints", "messagePattern": "constraints" }, "notes": "A minimum annealing length below 8 is outside the supported range and is refused, not clamped." }, { "id": "refuse-tail-letter", "input": { "template": "ATGACCTAGCTATTCGCTAGTCAC", "topology": "linear", "settings": { "target": { "start": 9, "end": 16 }, "excluded": [], "forwardTail": "ACGN", "reverseTail": "", "conditions": { "oligo_nM": 250, "na_mM": 50, "mg_mM": 0, "dntp_mM": 0, "model": "primer-excess", "saltCorrection": "owczarzy-2004" }, "flank": 0, "minLength": 8, "maxLength": 8, "targetLength": 8, "minTm": 0, "maxTm": 100, "targetTm": 40, "maxDeltaTm": 30, "minGc": 0, "maxGc": 100, "minProduct": 24, "maxProduct": 30 } }, "expected": { "refused": true, "field": "tails", "messagePattern": "Tails" }, "notes": "A tail outside A/C/G/T is refused." } ] }