Our in vitro transcription vectors are simple and efficient systems for RNA synthesis, applicable for a variety of research purposes. The mRNA version of the in vitro transcription vector system is ideally suited for producing mRNAs for in vitro translation, biochemical studies, protein expression after injection into cells or embryos, and other applications requiring mRNAs of defined sequences.
This system utilizes a T7 promoter upstream of your sequence of interest, facilitating highly efficient production of mRNA by T7 bacteriophage RNA polymerase (T7 RNAP) under appropriate reaction conditions and in the presence of nucleotide triphosphates. We recommend that you follow established in vitro transcription protocols available in published literature.
T7 RNAP has certain base requirements for efficient transcription initiation which have been already incorporated into the vector design. The first two nucleotides of the transcribed mRNA will be GG, corresponding to the 3’-end of the T7 promoter sequence, followed by your transcript sequence of interest.
Our in vitro transcription vector for mRNA synthesis is engineered for run-off transcription. This means that the T7 RNAP proceeds to the end of the DNA template during transcription, and does not terminate at any specific site within the plasmid. For this reason, the circular plasmid template should be linearized by restriction digestion with SapI, BsiWI, or AscI prior to in vitro transcription for ensuring the length and sequence of the generated transcripts. Any of these enzymes will cut the plasmid at unique sites immediately downstream of the poly(A) sequence. Care should be taken that the sequence to be transcribed does not contain any restriction sites for the linearization enzyme used, as this would result in unstable, truncated mRNA transcripts. Contaminants from the digestion reaction may inhibit the subsequent T7 RNAP transcription reaction, therefore purification via column or phenol:chloroform extraction following digestion is recommended. However, it is generally not necessary to purify the promoter and insert fragment away from other fragments, because only the fragment containing the T7 promoter sequence will serve as a template for transcription.
In vivo, the addition of a 7-methylguanosine cap to the 5' end of mRNA transcripts is essential for RNA stability, efficient translation, nuclear transport, and splicing. Likewise, for majority of research applications of in vitro transcribed mRNA, capped RNA is desired. RNA capping of your transcripts can be carried out either co-transcriptionally, using cap analogs, or post-transcriptionally, using capping enzymes. Both methods produce functional, capped RNA suitable for injection, in vitro translation, and other applications.
For further information about this vector system, please refer to the papers below.
