# Drybench Sequence: methods and checks

How Drybench Sequence reaches its results: every calculator, analysis or view with its methods, the references behind them, how far each has been checked, and the app's limits.

[Open Drybench Sequence](https://www.drybench.com/sequence/app) · [What the levels mean](https://www.drybench.com/methods/levels) · [Every app's methods and checks](https://www.drybench.com/methods)

## Reverse complement

For DNA. Get the matching strand, written in the opposite direction. The output is another DNA sequence.

No method record is kept for this tool, so it carries no level.

### How it is made and tested

- **Reverse complement:** Each base to its Watson-Crick complement and each IUPAC code to the code of its complementary set (R to Y, K to M, B to V, D to H; S, W and N to themselves), the result read 3′ to 5′; U pairs with A for RNA.

## Translate

For DNA or RNA. Convert the sequence into protein letters using the selected reading frame and genetic code.

Its result goes through 1 method. The lowest level among them: Checked against a reference.

### Methods

#### Translation with NCBI's genetic code tables

- **Level:** Checked against a reference
- **Reference:** NCBI. The Genetic Codes, gc.prt version 4.6 (last modified 10 June 2020), fetched 2026-10-06. Public domain.
- **How it was checked:** Checked against Biopython 1.88 exactly on 108 frame translations of three sequences in genetic codes 1, 2 and 11, full length and to the first stop, and on 5 annotated coding sequences (6 October 2026).

## Six frames

For DNA. Compare the six ways to read the sequence as a protein, three on each strand.

Its result goes through 1 method. The lowest level among them: Checked against a reference.

### Methods

#### Translation with NCBI's genetic code tables

- **Level:** Checked against a reference
- **Reference:** NCBI. The Genetic Codes, gc.prt version 4.6 (last modified 10 June 2020), fetched 2026-10-06. Public domain.
- **How it was checked:** Checked against Biopython 1.88 exactly on 108 frame translations of three sequences in genetic codes 1, 2 and 11, full length and to the first stop, and on 5 annotated coding sequences (6 October 2026).

## Open reading frames

For DNA or RNA. Find stretches with start and stop signals that could encode a protein. A match is a candidate, not proof of a gene.

Its result goes through 2 methods. The lowest level among them: Checked against a reference.

### Methods

#### Open reading frames

- **Level:** Checked against a reference
- **Reference:** NCBI ORFfinder conventions as the CSO set them on 6 October 2026: ATG (or the table's initiation codons) to the table's stop, 75 nt counted to the codon before the stop, nested ORFs hidden, ORFs without a stop listed apart.
- **How it was checked:** Checked field by field against an independent scan cross-checked with Biopython 1.88 and NCBI ORFfinder 0.4.3 on 18 cases (6 October 2026).

#### Translation with NCBI's genetic code tables

- **Level:** Checked against a reference
- **Reference:** NCBI. The Genetic Codes, gc.prt version 4.6 (last modified 10 June 2020), fetched 2026-10-06. Public domain.
- **How it was checked:** Checked against Biopython 1.88 exactly on 108 frame translations of three sequences in genetic codes 1, 2 and 11, full length and to the first stop, and on 5 annotated coding sequences (6 October 2026).

## Protein MW and pI

For a protein sequence. Calculate its molecular weight and the pH where its net electrical charge is zero. If you have DNA, use Translate first.

Its result goes through 5 methods. The lowest level among them: Tested, not reference-checked.

Below the top level: Tags and protease sites by motif (Tested, not reference-checked).

### Methods

#### Average molecular weight from the sequence

- **Level:** Checked against a reference
- **Reference:** ExPASy ProtParam (Gasteiger et al. 2005); the fixtures are ProtParam's printed weights, retrieved 5 October 2026.
- **How it was checked:** Checked against Expasy ProtParam within 0.01 Da on 22 sequences with ProtParam's residue masses (6 October 2026). ProtParam is a tool of the SIB Swiss Institute of Bioinformatics; not affiliated.

#### Theoretical isoelectric point

- **Level:** Checked against a reference
- **Reference:** Bjellqvist B et al. Electrophoresis 1993;14:1023-1031; 1994;15:529-539; as ExPASy ProtParam applies them.
- **How it was checked:** Checked against Expasy ProtParam within 0.01 on 22 sequences, never a bound of the pH 0 to 14 search (6 October 2026). ProtParam is a tool of the SIB Swiss Institute of Bioinformatics; not affiliated.

#### Molar extinction coefficient at 280 nm

- **Level:** Checked against a reference
- **Reference:** Pace CN, Vajdos F, Fee L, Grimsley G, Gray T. How to measure and predict the molar absorption coefficient of a protein. Protein Sci 1995;4:2411-2423 (read 5 October 2026). [doi:10.1002/pro.5560041120](https://doi.org/10.1002/pro.5560041120)
- **How it was checked:** Checked against Expasy ProtParam exactly on 22 sequences in both cysteine conventions (6 October 2026). ProtParam is a tool of the SIB Swiss Institute of Bioinformatics; not affiliated.

#### Tags and protease sites by motif

- **Level:** Tested, not reference-checked
- **Reference:** Peptide tag and protease recognition motifs from the cited papers; a cut is proposed at the site nearest an end and confirmed by the person, never inferred from the fold.
- **How it was checked:** Unit-tested on the fixture construct: the His6-TEV construct's cleavage product is the untagged fixture sequence (6 October 2026).

#### Peptide as SMILES

- **Level:** Checked against a reference
- **Reference:** A linear peptide of L-amino acids with free amine and acid ends, neutral, cysteines as free thiols, drawn as RDKit's MolFromSequence draws it (Daylight SMILES; RDKit 2026.03.6 as the oracle).
- **How it was checked:** Checked against RDKit 2026.03.6 MolFromSequence: the same canonical SMILES on 89 peptides and the 12 refusals exact (6 October 2026).

## ε280

For a protein sequence. Estimate how strongly it absorbs UV light at 280 nm, useful for measuring protein concentration.

Its result goes through 2 methods. The lowest level among them: Checked against a reference.

### Methods

#### Molar extinction coefficient at 280 nm

- **Level:** Checked against a reference
- **Reference:** Pace CN, Vajdos F, Fee L, Grimsley G, Gray T. How to measure and predict the molar absorption coefficient of a protein. Protein Sci 1995;4:2411-2423 (read 5 October 2026). [doi:10.1002/pro.5560041120](https://doi.org/10.1002/pro.5560041120)
- **How it was checked:** Checked against Expasy ProtParam exactly on 22 sequences in both cysteine conventions (6 October 2026). ProtParam is a tool of the SIB Swiss Institute of Bioinformatics; not affiliated.

#### Average molecular weight from the sequence

- **Level:** Checked against a reference
- **Reference:** ExPASy ProtParam (Gasteiger et al. 2005); the fixtures are ProtParam's printed weights, retrieved 5 October 2026.
- **How it was checked:** Checked against Expasy ProtParam within 0.01 Da on 22 sequences with ProtParam's residue masses (6 October 2026). ProtParam is a tool of the SIB Swiss Institute of Bioinformatics; not affiliated.

## GC percent

For DNA or RNA. Calculate the percentage of the sequence made up of G and C letters.

No method record is kept for this tool, so it carries no level.

### How it is made and tested

- **GC percent:** (G + C + S) over (A + C + G + T + U + S + W); the other IUPAC codes are left out of both counts, and the result is undefined when nothing can be counted.

## DNA molecular weight

For DNA or RNA. Calculate the molecular weight using the strand and end-group settings.

Its result goes through 1 method. The lowest level among them: Checked against a reference.

### Methods

#### Nucleic acid molecular weight from the sequence

- **Level:** Checked against a reference
- **Reference:** Residue formulas from the nucleoside 5′-monophosphates (PubChem, read 6 October 2026) less water, summed with the IUPAC CIAAW 2021 atomic weights; 5′-OH and 3′-OH ends as a neutral free acid.
- **How it was checked:** Checked against hand arithmetic from the cited residue masses within 0.01 g/mol on 5 oligos (6 October 2026).

## Motif search

For DNA, RNA or protein. Enter the letter pattern in the output panel to find where it occurs in your sequence.

No method record is kept for this tool, so it carries no level.

### How it is made and tested

- **Motif search and binding sites:** Each IUPAC code matches its set of bases, on both strands of DNA when asked; a protein motif matches letter for letter. A primer's binding sites are the places on either strand where its 3′ six bases match exactly and the rest has at most the allowed mismatches; a product runs from a forward site to the next reverse site.

## Oligo Tm

For a short DNA sequence. Estimate the temperature where half the paired strands separate, using the salt and concentration settings.

Its result goes through 4 methods. The lowest level among them: Checked against a reference.

### Methods

#### Oligo melting temperature by nearest neighbour

- **Level:** Checked against a reference
- **Reference:** SantaLucia J Jr. A unified view of polymer, dumbbell, and oligonucleotide DNA nearest-neighbor thermodynamics. Proc Natl Acad Sci USA 1998;95:1460-1465 (Table 2; read 6 October 2026); concentration models after SantaLucia J Jr, Hicks D. Annu Rev Biophys Biomol Struct 2004;33:415-440. [doi:10.1073/pnas.95.4.1460](https://doi.org/10.1073/pnas.95.4.1460)
- **How it was checked:** Checked against Biopython 1.88 MeltingTemp.Tm\_NN within 0.1 °C on 45 oligos under both concentration models, with the Owczarzy 2008 regime exact and the seven refusals exact (6 October 2026).

#### Monovalent salt correction

- **Level:** Checked against a reference
- **Reference:** Owczarzy R, You Y, Moreira BG, et al. Effects of sodium ions on DNA duplex oligomers: improved predictions of melting temperatures. Biochemistry 2004;43:3537-3554 (equation 22). [doi:10.1021/bi034621r](https://doi.org/10.1021/bi034621r)
- **How it was checked:** Checked against Biopython 1.88 MeltingTemp.Tm\_NN within 0.1 °C on 45 oligos under both concentration models, with the Owczarzy 2008 regime exact and the seven refusals exact (6 October 2026).

#### Magnesium and mixed-cation salt correction

- **Level:** Checked against a reference
- **Reference:** Owczarzy R, Moreira BG, You Y, Behlke MA, Walder JA. Predicting stability of DNA duplexes in solutions containing magnesium and monovalent cations. Biochemistry 2008;47:5336-5353 (equations 16 and 18 to 20). [doi:10.1021/bi702363u](https://doi.org/10.1021/bi702363u)
- **How it was checked:** Checked against Biopython 1.88 MeltingTemp.Tm\_NN within 0.1 °C on 45 oligos under both concentration models, with the Owczarzy 2008 regime exact and the seven refusals exact (6 October 2026).

#### Entropy salt correction

- **Level:** Checked against a reference
- **Reference:** SantaLucia J Jr. Proc Natl Acad Sci USA 1998;95:1460-1465 (equation 7): ΔS° + 0.368 (N − 1) ln\[Na⁺\]. [doi:10.1073/pnas.95.4.1460](https://doi.org/10.1073/pnas.95.4.1460)
- **How it was checked:** Checked against Biopython 1.88 MeltingTemp.Tm\_NN within 0.1 °C on 45 oligos under both concentration models, with the Owczarzy 2008 regime exact and the seven refusals exact (6 October 2026).

## Primer check

For DNA primers. Paste the forward primer above, then add the reverse primer in the output panel. Add a template to predict the DNA they could copy.

Its result goes through 5 methods. The lowest level among them: Tested, not reference-checked.

Below the top level: Pair ΔTm and the annealing temperature rule of thumb (Tested, not reference-checked).

### Methods

#### Oligo melting temperature by nearest neighbour

- **Level:** Checked against a reference
- **Reference:** SantaLucia J Jr. A unified view of polymer, dumbbell, and oligonucleotide DNA nearest-neighbor thermodynamics. Proc Natl Acad Sci USA 1998;95:1460-1465 (Table 2; read 6 October 2026); concentration models after SantaLucia J Jr, Hicks D. Annu Rev Biophys Biomol Struct 2004;33:415-440. [doi:10.1073/pnas.95.4.1460](https://doi.org/10.1073/pnas.95.4.1460)
- **How it was checked:** Checked against Biopython 1.88 MeltingTemp.Tm\_NN within 0.1 °C on 45 oligos under both concentration models, with the Owczarzy 2008 regime exact and the seven refusals exact (6 October 2026).

#### Pair ΔTm and the annealing temperature rule of thumb

- **Level:** Tested, not reference-checked
- **Reference:** Arithmetic: ΔTm is the difference of the two Tm values; Ta about 5 °C below the lower Tm is a rule of thumb, not a prediction.

#### Hairpin and dimer ΔG°37 by nearest-neighbour thermodynamics

- **Level:** Checked against a reference
- **Reference:** SantaLucia J Jr, Hicks D. The thermodynamics of DNA structural motifs. Annu Rev Biophys Biomol Struct 2004;33:415-440; stacking from SantaLucia J Jr. Proc Natl Acad Sci USA 1998;95:1460-1465; dangling ends from Bommarito S, Peyret N, SantaLucia J Jr. Nucleic Acids Res 2000;28:1929-1934; salt as a sodium equivalent after von Ahsen N, Wittwer CT, Schütz E. Clin Chem 2001;47:1956-1961. [doi:10.1146/annurev.biophys.32.110601.141800](https://doi.org/10.1146/annurev.biophys.32.110601.141800)
- **How it was checked:** Hairpins = ntthal 2.6.1 within 0.4 kcal/mol on ntthal's own structures, 15 fixtures (6 October 2026); dimers = ntthal 2.6.1 within 0.1 kcal/mol on ntthal's own structures, 16 fixtures (6 October 2026). ntthal reports the structure with the highest melting temperature and this card the lowest ΔG°37.

#### Monovalent salt correction

- **Level:** Checked against a reference
- **Reference:** Owczarzy R, You Y, Moreira BG, et al. Effects of sodium ions on DNA duplex oligomers: improved predictions of melting temperatures. Biochemistry 2004;43:3537-3554 (equation 22). [doi:10.1021/bi034621r](https://doi.org/10.1021/bi034621r)
- **How it was checked:** Checked against Biopython 1.88 MeltingTemp.Tm\_NN within 0.1 °C on 45 oligos under both concentration models, with the Owczarzy 2008 regime exact and the seven refusals exact (6 October 2026).

#### Magnesium and mixed-cation salt correction

- **Level:** Checked against a reference
- **Reference:** Owczarzy R, Moreira BG, You Y, Behlke MA, Walder JA. Predicting stability of DNA duplexes in solutions containing magnesium and monovalent cations. Biochemistry 2008;47:5336-5353 (equations 16 and 18 to 20). [doi:10.1021/bi702363u](https://doi.org/10.1021/bi702363u)
- **How it was checked:** Checked against Biopython 1.88 MeltingTemp.Tm\_NN within 0.1 °C on 45 oligos under both concentration models, with the Owczarzy 2008 regime exact and the seven refusals exact (6 October 2026).

## Primer design

For one DNA template of A, C, G and T. Choose a target region and the constraints; candidate primer pairs are enumerated within the template, ranked by stated terms and checked for one exact product. A pair is a design, not evidence that it amplifies.

Its result goes through 6 methods. The lowest level among them: Checked against a reference.

### Methods

#### Bounded primer design for a target region

- **Level:** Checked against a reference
- **Reference:** A bounded enumeration within the supplied A/C/G/T template (at most 50,000 bases, linear or circular): candidate windows of the chosen lengths whose 3′ ends sit 0 to the chosen flank (at most 200 bases) before the target's first base and after its last, read around the circle when circular; a window is rejected, under the first reason that applies and counted, when it falls off a linear template, touches the target or an excluded region, or has a GC or a nearest-neighbour Tm (annealing part alone, the primer in excess, the recorded conditions) outside the bounds. Each side is ranked by |Tm − preferred Tm| + 0.2 × |length − preferred length| and cut to 24 windows; the pairs are ranked by the two Tm departures, 0.2 times the two length departures and the pair's ΔTm, with the product length (tails included) and ΔTm bounds applied first; the best 24 pairs are checked by the exact-pair method for one exact product at the designed sites and the first 5 that pass are returned. A search cut at any cap is reported as capped. Each retained annealing-part Tm includes its method version, conditions and qualifications; historical v1 receipts remain unchanged. No Tm of a tailed oligo, no annealing temperature, no genome specificity, no mismatched binding and no amplification outcome is derived: a returned pair is a sequence design, not evidence that it amplifies.
- **How it was checked:** Checked against 9 design cases and 4 refusals published and hashed from an independent derivation (every melting temperature from Biopython 1.88 under the recorded conditions; the windows, counts, ranking and caps derived in the script; exact binding by the exact-pair derivation; one case worked by hand): every returned pair, coordinate, product, count and cap exact, Tm and ranking terms within 1e-6 °C (11 October 2026). A separate primer-design program was run offline as a recorded comparison, not as the reference.

#### Oligo melting temperature by nearest neighbour

- **Level:** Checked against a reference
- **Reference:** SantaLucia J Jr. A unified view of polymer, dumbbell, and oligonucleotide DNA nearest-neighbor thermodynamics. Proc Natl Acad Sci USA 1998;95:1460-1465 (Table 2; read 6 October 2026); concentration models after SantaLucia J Jr, Hicks D. Annu Rev Biophys Biomol Struct 2004;33:415-440. [doi:10.1073/pnas.95.4.1460](https://doi.org/10.1073/pnas.95.4.1460)
- **How it was checked:** Checked against Biopython 1.88 MeltingTemp.Tm\_NN within 0.1 °C on 45 oligos under both concentration models, with the Owczarzy 2008 regime exact and the seven refusals exact (6 October 2026).

#### Monovalent salt correction

- **Level:** Checked against a reference
- **Reference:** Owczarzy R, You Y, Moreira BG, et al. Effects of sodium ions on DNA duplex oligomers: improved predictions of melting temperatures. Biochemistry 2004;43:3537-3554 (equation 22). [doi:10.1021/bi034621r](https://doi.org/10.1021/bi034621r)
- **How it was checked:** Checked against Biopython 1.88 MeltingTemp.Tm\_NN within 0.1 °C on 45 oligos under both concentration models, with the Owczarzy 2008 regime exact and the seven refusals exact (6 October 2026).

#### Magnesium and mixed-cation salt correction

- **Level:** Checked against a reference
- **Reference:** Owczarzy R, Moreira BG, You Y, Behlke MA, Walder JA. Predicting stability of DNA duplexes in solutions containing magnesium and monovalent cations. Biochemistry 2008;47:5336-5353 (equations 16 and 18 to 20). [doi:10.1021/bi702363u](https://doi.org/10.1021/bi702363u)
- **How it was checked:** Checked against Biopython 1.88 MeltingTemp.Tm\_NN within 0.1 °C on 45 oligos under both concentration models, with the Owczarzy 2008 regime exact and the seven refusals exact (6 October 2026).

#### Predicted PCR product from a supplied primer pair

- **Level:** Checked against a reference
- **Reference:** Exact matches only: each primer's annealing part, or its reverse complement, matched base for base on an A/C/G/T template of at most 50,000 bases, on both strands, across the origin of a circular template once. A product runs from the forward primer's 5′ end to the reverse primer's 5′ end on the template, with each 5′ tail added once (the reverse tail reverse complemented); pairs whose footprints overlap are counted and build no product; every product is listed, with more than 100 sites per primer or 100 products refused as incomplete. A sequence construction, not a prediction of amplification or of specificity in a genome.
- **How it was checked:** Checked exactly against an independent Python derivation (Biopython 1.88 reverse complements, every window tested directly, one case worked by hand) on 30 cases: every site, product and status, and 13 refusals (10 October 2026).

#### Hairpin and dimer ΔG°37 by nearest-neighbour thermodynamics

- **Level:** Checked against a reference
- **Reference:** SantaLucia J Jr, Hicks D. The thermodynamics of DNA structural motifs. Annu Rev Biophys Biomol Struct 2004;33:415-440; stacking from SantaLucia J Jr. Proc Natl Acad Sci USA 1998;95:1460-1465; dangling ends from Bommarito S, Peyret N, SantaLucia J Jr. Nucleic Acids Res 2000;28:1929-1934; salt as a sodium equivalent after von Ahsen N, Wittwer CT, Schütz E. Clin Chem 2001;47:1956-1961. [doi:10.1146/annurev.biophys.32.110601.141800](https://doi.org/10.1146/annurev.biophys.32.110601.141800)
- **How it was checked:** Hairpins = ntthal 2.6.1 within 0.4 kcal/mol on ntthal's own structures, 15 fixtures (6 October 2026); dimers = ntthal 2.6.1 within 0.1 kcal/mol on ntthal's own structures, 16 fixtures (6 October 2026). ntthal reports the structure with the highest melting temperature and this card the lowest ΔG°37.

## Mutagenesis primers

For one DNA template of A, C, G and T. Give one substitution, insertion or deletion and choose a scheme; primers that carry the change are enumerated within the template under the scheme's stated rules. Tm is given against the mutant and, for the matched 3′ segment, against the template; a primer's Tm on a template it does not fully match is not given. A primer is a design, not evidence that the change will be made.

Its result goes through 4 methods. The lowest level among them: Checked against a reference.

### Methods

#### Site-directed mutagenesis primers

- **Level:** Checked against a reference
- **Reference:** Two schemes, each a sequence design under the rules stated here. Overlapping complementary primers: a window of the mutant top strand containing the change with its exact reverse complement, annealing length 25 to 45 bases measured on the mutant, at least 10 perfectly matched bases on each side of the change (12 to 15 preferred), the change central to within 3 bases, GC 40 to 60 per cent preferred and 30 to 70 per cent required. Back-to-back primers, circular templates only: the changed bases within the 5′-most six bases of the forward primer with at least 15 perfectly matched bases 3′ of the last change, 18 to 30 perfectly matched bases on each primer, each primer's GC fraction 30 to 70 per cent as in the overlapping scheme, the partner primer's 5′ end abutting the forward primer's, and the pair's matched-part melting temperatures within 3 °C; the product is the linear whole-template amplicon and requires circularisation before it is a plasmid. Substitutions of 1 to 3 bases, insertions and deletions of up to 12 bases, templates of 2 to 50,000 bases. Melting temperatures come from the suite's nearest-neighbour model at the conditions shown, and every one of them is of a perfectly matched duplex: for each primer, its melting temperature against the mutant sequence it matches exactly, and the melting temperature of its longest perfectly matched 3′ segment against the template. The melting temperature of a primer against a template it does not fully match is outside this model and is not given. Candidates are ranked by flank balance, the preferred flank band, GC distance and length, each term compared at a published precision: that precision is the agreement between this engine and its oracle, not a physical resolution, and a melting-temperature difference of 1e-6 °C is not a difference anyone can measure. The counts of candidates rejected under each rule are reported. Nothing is relaxed to produce a result.
- **How it was checked:** Checked against 10 design cases over the two schemes and 8 refusals published and hashed from an independent derivation written before the engine (every melting temperature and reverse complement from Biopython 1.88 under the recorded conditions; the mutant, the candidate windows, the matched flanks, GC, the ranking and the counts by reason derived in the script; one case per scheme worked by hand with a write refusal): every sequence, coordinate, flank count, count and refusal exact, each melting temperature within 1e-6 °C (17 October 2026). The cases include a three-base substitution with an unchanged middle base, a twelve-base insertion, a twelve-base deletion crossing a circular origin, a short linear template whose only window carries unequal flanks, and a GC-rich stretch where no window passes: 405 of its 735 candidates fall to the matched-flank minimum, 242 to the central tolerance, 73 to the GC range and 15 to the template's ends.

#### Oligo melting temperature by nearest neighbour

- **Level:** Checked against a reference
- **Reference:** SantaLucia J Jr. A unified view of polymer, dumbbell, and oligonucleotide DNA nearest-neighbor thermodynamics. Proc Natl Acad Sci USA 1998;95:1460-1465 (Table 2; read 6 October 2026); concentration models after SantaLucia J Jr, Hicks D. Annu Rev Biophys Biomol Struct 2004;33:415-440. [doi:10.1073/pnas.95.4.1460](https://doi.org/10.1073/pnas.95.4.1460)
- **How it was checked:** Checked against Biopython 1.88 MeltingTemp.Tm\_NN within 0.1 °C on 45 oligos under both concentration models, with the Owczarzy 2008 regime exact and the seven refusals exact (6 October 2026).

#### Monovalent salt correction

- **Level:** Checked against a reference
- **Reference:** Owczarzy R, You Y, Moreira BG, et al. Effects of sodium ions on DNA duplex oligomers: improved predictions of melting temperatures. Biochemistry 2004;43:3537-3554 (equation 22). [doi:10.1021/bi034621r](https://doi.org/10.1021/bi034621r)
- **How it was checked:** Checked against Biopython 1.88 MeltingTemp.Tm\_NN within 0.1 °C on 45 oligos under both concentration models, with the Owczarzy 2008 regime exact and the seven refusals exact (6 October 2026).

#### Magnesium and mixed-cation salt correction

- **Level:** Checked against a reference
- **Reference:** Owczarzy R, Moreira BG, You Y, Behlke MA, Walder JA. Predicting stability of DNA duplexes in solutions containing magnesium and monovalent cations. Biochemistry 2008;47:5336-5353 (equations 16 and 18 to 20). [doi:10.1021/bi702363u](https://doi.org/10.1021/bi702363u)
- **How it was checked:** Checked against Biopython 1.88 MeltingTemp.Tm\_NN within 0.1 °C on 45 oligos under both concentration models, with the Owczarzy 2008 regime exact and the seven refusals exact (6 October 2026).

## Review sequence revisions

Compare two exact Sequence records without changing either. Residue changes and annotation changes are reviewed separately. This does not verify a physical clone.

Its result goes through 1 method. The lowest level among them: Checked against a reference.

### Methods

#### Design revision review

- **Level:** Checked against a reference
- **Reference:** Two immutable revisions are compared and the correspondence is recorded as data: both SHA-256 sequence hashes, the method id and the kind (identical, origin rotation, reverse complement, edits or unresolved). An exact circular rotation or reverse complement transforms coordinates without inventing mutations; a non-exact circular change is withheld (unresolved, every residue unmapped) rather than resolved at an arbitrary origin; within-region edits are a bounded unit-cost global traceback on the preserved common flanks, diagonal-first, which is descriptive and not a unique biological history. Residue edits and annotation-only changes are separate findings; a note-only change makes no residue edit; an edit overlapping a CDS is qualified and never transferred automatically. A comparison of designs, not evidence about a physical construct.
- **How it was checked:** Checked against 10 revision transforms published and hashed: the coordinate maps worked by hand (origin rotation, reverse complement, a joined CDS, repeated-sequence ambiguity, a note-only change that makes no residue edit, a non-exact circular change withheld as data, a topology change) and an independent unit-cost edit distance for the traceback cases, with each sequence's SHA-256; every kind, map, hash, withheld state and annotation finding exact (10 October 2026).

## Find DNA fragment

Find an exact fragment in the latest saved sequences and constructs in this Workspace. Searches both strands, including across the origin of circular DNA.

Its result goes through 1 method. The lowest level among them: Checked against a reference.

### Methods

#### Exact fragment search in saved sequences

- **Level:** Checked against a reference
- **Reference:** A query of 4 to 10,000 A/C/G/T bases, or its reverse complement, matched base for base on the saved, presented strand of each saved DNA sequence; IUPAC codes in a saved sequence are literal letters that no query base matches, never wildcards; overlapping hits are all kept; a palindromic query's hit is reported once, as both strands; on a circular molecule each start is read once and a hit may cross the origin. 1-based inclusive coordinates on the saved strand.
- **How it was checked:** Checked exactly against an independent Python derivation (Biopython 1.88 reverse complements, every window tested directly) on 26 cases: every hit, strand and origin crossing, and 9 refusals (10 October 2026).

## Data sources

- [NCBI genetic code tables](https://www.ncbi.nlm.nih.gov/Taxonomy/Utils/wprintgc.cgi): Public domain (NCBI)
- [ExPASy ProtParam residue masses and pK values](https://web.expasy.org/protparam/protparam-doc.html): Values cited from the documentation
- [Bjellqvist et al. 1993 and 1994, isoelectric point](https://doi.org/10.1002/elps.11501401163): Method (cited)
- [Pace et al. 1995, molar absorption coefficients](https://doi.org/10.1002/pro.5560041120): Method (cited)
- [SantaLucia 1998, unified nearest-neighbour parameters](https://doi.org/10.1073/pnas.95.4.1460): Values cited from the published table
- [Owczarzy et al. 2004, sodium correction](https://doi.org/10.1021/bi034621r): Method (cited)
- [Owczarzy et al. 2008, magnesium correction](https://doi.org/10.1021/bi702363u): Method (cited)
- [SantaLucia and Hicks 2004, DNA structural motifs](https://doi.org/10.1146/annurev.biophys.32.110601.141800): Values cited from the published tables
- [Bommarito, Peyret and SantaLucia 2000, dangling ends](https://doi.org/10.1093/nar/28.9.1929): Values cited from the published table
- [Nearest Neighbor Database (NNDB), DNA terminal mismatches](https://rna.urmc.rochester.edu/NNDB/dna/dna_hairpin_loops.html): Values cited from the public table; licence to be checked
- [IUPAC CIAAW standard atomic weights 2021](https://www.ciaaw.org/atomic-weights.htm): Values cited from the published table
- [UniProtKB (UniProt REST API)](https://www.uniprot.org/help/license): CC BY 4.0

## Limits

> Not medical advice. Nothing here is advice about any medicine, treatment or condition, and nothing here is an assessment of any person's variant, sample or molecule. Decisions made with it are yours.

- For research and education only, and not medical advice: nothing here is an assessment of any person's variant, sample or molecule, and a difference reported from a reference is a difference, not an interpretation.
- NCBI genetic codes 27, 28 and 31 contain context-dependent stop codons. Translation can show their amino-acid meaning, but stop-terminated translation and ORF detection are refused because the required biological context is not modelled.
- Tm and structure calculations require finite, nonnegative ionic concentrations and a positive model-supported ionic strength. Zero ionic strength is refused rather than silently treated as a salt correction of zero.
- pI, Tm and molecular weight depend on the method; the method and its parameter set are named on every card, and no alternative pK set is offered in this version.
- Buffer conditions you type approximate a reaction buffer, not a vendor's calculator; use the vendor's calculator for its exact value, and treat the annealing temperature as the rule of thumb it is labelled.
- Hairpin and dimer ΔG are two-state estimates at 37 °C from the SantaLucia and Hicks 2004 rules without the special triloop and tetraloop bonuses, so hairpins with GNA or GNRA loops are estimated less stable than they are.
- Sequence operations run locally. Optional UniProt lookup sends the submitted query and selected accession to rest.uniprot.org, never the separate pasted sequence. The Verify at ProtParam and BLAST at NCBI links upload what you send, and say so; an optional Bench backup uploads the Study you choose.
- Tags and protease sites are found by their published motifs and a cut is proposed at the site nearest an end; confirm it, since a motif inside the protein is not a cleavage site.
- Inputs over 1 Mb are refused; six-frame, ORF and motif scans of inputs over 100 kb run in a Web Worker with progress and a Cancel. The primer check's near-match binding-site diagnostics are not run on templates of 100 kb or more.
- Ambiguity codes are refused by name where a number cannot be computed from them (Tm, DNA molecular weight, hairpins and dimers) and kept where it can (reverse complement, translation to X, GC with S counted).
- No offline use: the pages are served by the site, and nothing is installed.
- A link keeps the inputs after the # of the address; it is not sent to a server, but it does stay in browser history and in any chat the link is pasted into.
- Primer design is a bounded enumeration within the pasted template: candidate windows within the chosen flank of each target edge, kept inside the stated length, GC and Tm bounds, 24 windows a side and 24 pairs checked, 5 returned, and the search says when it is capped. A returned pair is a sequence design checked for one exact product in this template; it establishes no genome specificity, no mismatched binding and no amplification outcome, and no annealing temperature is chosen for it.
- A sequence that arrives from another app is read with the alphabet its record states; residues without a stated alphabet are detected from their letters and the detection is shown.
- The genetic code tables are NCBI's version 4.6 (gc.prt, last modified 10 June 2020); a table id travels with every translation.