**T7 RNA Polymerase: Recombinant Protein for In Vitro RNA Synthesis**
**Overview:**
T7 RNA polymerase is an enzyme derived from recombinant protein expression in *Escherichia coli*. It is widely used for in vitro transcription due to its high specificity and efficiency in synthesizing RNA from a DNA template with a T7 promoter sequence. The enzyme catalyzes the 5'→3' synthesis of RNA, utilizing nucleoside triphosphates (NTPs) as substrates.
**Promoter Sequence:**
The T7 promoter sequence required for transcription initiation is:
5'-TAATACGACTCACTATAG*-3'
(*Note: G* indicates the first base of the RNA transcript.*)
**Template Compatibility:**
- DNA templates with double-stranded linear blunt ends or 5' protruding ends.
- Linearized plasmids and PCR products can serve as templates for T7 RNA polymerase.
**GMP Compliance:**
This product is manufactured following Good Manufacturing Practice (GMP) regulations, ensuring high quality and consistency. It is provided in liquid form.
**Features:**
- Capable of synthesizing both long and short RNA transcripts.
- High yield: Produces 100-200 μg of RNA from 1 μg of DNA template.
- Easy to operate with high efficiency.
- Low endotoxin levels.
- Tested to confirm the absence of endonucleases, exonucleases, and RNases.
**Applications:**
- Preparation of radiolabeled RNA probes.
- RNA generation for studies of RNA structure, processing, and catalysis.
- Expression control via antisense RNA.
- Synthesis of mRNA and single-guide RNA (sgRNA).
**Protocol Highlights:**
1. **Template Preparation:**
- Use linearized plasmids or PCR products with a T7 promoter sequence.
2. **Transcription Reaction Setup:**
- Mix the DNA template with T7 RNA polymerase, NTPs, and other necessary transcription components.
3. **Incubation:**
- Allow the reaction to proceed under optimal conditions for RNA synthesis.
4. **RNA Purification:**
- Purify the synthesized RNA to remove any residual enzymes and unincorporated nucleotides.
By following the detailed protocol, users can efficiently produce high-quality RNA suitable for various research applications.