Takara Template Vector (BspQ I) for T7 mRNA Synthesis

The prelinearized template vector contains a T7 promoter, transcription start sequence (AGG), 5’-untranslated region (UTR), 3’-UTR, poly(A) sequence, and BspQ I digestion site, which allows the seamless preparation of an in vitro transcription (IVT) template.

The length of additional base pairs (scars) stemming from the poly(A) tail directly correlates with the translation efficiency.Three types of DNA templates encoding Firefly Luciferase (FLuc) mRNA (pFLuc-142A: BspQ I Vector with a 142 bp of poly(A)[Cat. # 6146], pFLuc-102A: BspQ I Vector with a 102 bp of poly(A) [not sold], and pFLuc-105A: Hind III vector with a 105 bp of poly(A) [part of Cat. # 6143]) were digested with specific restriction enzymes as indicated. The linearized templates, with differing numbers of additional base pairs downstream of the poly(A) tail as shown in parenthesis, were then employed in an in vitro transcription process with CleanCap. The resulting in vitro transcribed mRNA was transfected into HEK293T cells to assess the efficiency of mRNA translation, which was quantified by FLuc measurements. The DNA template digested by BspQ I enzyme, lacking any extra sequence at the 3’ end of the poly(A) tail, exhibits the highest capacity for protein translation.

The length of poly(A) tail positively affects the translation efficiency.Two types of DNA templates encoding Firefly Luciferase (FLuc) mRNA, pFLuc-142A: BspQ I Vector with a 142 bp of poly(A) (Cat. # 6146) and pFLuc-102A: BspQ I Vector with a 102 bp of poly(A) (not sold), were digested with BspQ I restriction enzyme. The linearized templates were then employed in an in vitro transcription process with CleanCap. Subsequently, the resulting in vitro transcribed mRNA was transfected into HEK293T cells to assess the efficiency of mRNA translation, quantified by FLuc measurements. The DNA template with a longer poly(A) tail (Cat. # 6146) exhibits the higher capacity for protein translation.