T7 promoter sequence (5'-TAA TAC GAC TCA CTA TAG GG-3').
Required for transcription of the DNA template.
ATG start codon (5'-ATG-3') if not present in the sequence being
amplified. Needed for translation initiation.
Gene-specific sequence. Needed to allow priming of the target
gene.
Highly Desirable:
Kozak consensus sequence (5'-CCACCATGG-3') OR Eukaryotic
translation initiation sequences from sequence being amplified.
Increases efficiency of translation initiation.
6–10 bases upstream of promoter. Improves efficiency of promoter.
3- to 6-base spacer between promoter sequence and Kozak sequence.
Ensures transcription starts a few bases upstream of the Kozak sequence
and allows better ribosome binding to RNA.
Gene-specific sequence. Needed to allow priming of the target gene.
Desirable:
Reverse complement of stop codon (TTA, CTA or TCA) if not presentin
the sequence being amplified. Terminates translation, allowing efficient
release of ribosomes for further rounds of translation.
Addition of a poly(A) tail results in greater RNA stability and
higher levels of translation.
