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Primer Design Considerations for Incorporating a T7 Promoter into a PCR Product TECHNICAL REFERENCE Primer Design Considerations for Incorporating a T7 Promoter into a PCR Product for Subsequent in vitro Transcription/Translation. Forward Primer Required: • 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. Completed Forward Primer Design: 5′- (N6–10)TAATACGACTCACTATAGGG (N3–6) CCACCATGG (N17–22)-3′ Reverse Primer Required: • Gene-specific sequence. Needed to allow priming of the target gene. Desirable: • Reverse complement of stop codon (TTA, CTA or TCA) if not present in 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. ©2010 ALL RIGHTS RESERVED PART #GE636 www.promega.com 2800 WOODS HOLLOW ROAD MADISON, WI 53711-5399 USA TELEPHONE 608-274-4330 PROMEGA CORPORATION Completed Reverse Primer Design: 5′-T30 stop anticodon (N17–22)-3′