“REV primer” is basically flexible, but there are some points to be considered.
・The “taatga” after the C-terminus of the ORF means two stop codons.
・The length is more important than the sequence. If the length is shorter than 10 bases, the efficiency of protein synthesis will be affected, but if it is longer than 10 bases(like 20 bases or more), it will be fine.
・Avoid sequences that form a rigid secondary structure (ex, high GC content, etc.) just after the stop codon as much as possible.
・For example, a restriction enzyme site can be inserted in this region.