DNA, RNA, and protein sequences

Composition and transform — length, GC%, molecular weight (ssDNA with a 5′-OH; RNA as a 5′-triphosphate transcript), and the reverse complement, complement, reverse, transcription, and back-transcription.

Translate — all six reading frames with the chosen NCBI genetic code (standard, vertebrate, yeast, invertebrate and other mitochondrial codes, bacterial). Ambiguous codons translate when every possibility gives the same amino acid; otherwise X.

ORFs — start codon to in-frame stop on both strands, longer than a minimum length, with a map. Coordinates are 1-based on the forward strand. Alternative start codons follow the chosen genetic code.

Restriction sites — about 60 common enzymes, including Type IIS enzymes (BsaI, BsmBI, BbsI, SapI) that cut outside their site. Positions are top-strand cut positions; (−) marks a site on the reverse strand. Choose enzymes for a virtual digest; tick Circular for plasmids.

Primer — length, GC%, GC clamp, and three Tm estimates: nearest-neighbour (SantaLucia 1998 unified parameters with the SantaLucia entropy salt correction; Mg²⁺ converted to Na⁺-equivalents as in von Ahsen et al. 2001), salt-adjusted, and the basic Wallace/Marmur rule. Self-dimer and hairpin checks are simple run-length heuristics.

Protein — average molecular weight, pI (choose the pKa set; EMBOSS by default), net charge at pH 7, molar extinction coefficient at 280 nm (Pace et al. 1995: Trp 5500, Tyr 1490, cystine 125), GRAVY, composition, and a Kyte–Doolittle hydropathy plot. A DNA sequence is translated from frame +1 to its first stop.

Codon usage — counts, per-thousand frequencies, and the fraction of each codon among its synonyms, in frame +1.