Work with a DNA sequence

Reverse complement, translation and GC content are the three operations you reach for without thinking, which is exactly why their conventions are worth stating once.

Reverse complement is not the same as complement. Sequences are written 5′→3′ by convention, so the partner strand has to be reversed as well as complemented to be written the same way. Getting this wrong produces a sequence that looks entirely reasonable and orders a primer that anneals nowhere.

Translation needs a reading frame and a genetic code, and neither is safe to assume. Six frames exist for any sequence, three on each strand. The standard code is not universal: vertebrate mitochondria read TGA as tryptophan rather than stop, and several ciliates reassign the stop codons entirely — so translating a mitochondrial gene with the standard table truncates it at the first TGA.

GC content is arithmetic, but decide in advance what ambiguity codes should do. Counting them, ignoring them, or refusing them all give different denominators.