Optimized for in vitro transcription and translation of fusion proteins.
Thermo Scientific T7 Cell-Free Expression Vectors (pT7CFE1) are cloning plasmids optimized to use with the Thermo Scientific 1-Step Human In Vitro Protein Expression System for in vitro translation (IVT) of tagged fusion proteins.
The pT7CFE1 Expression Vectors contain the Encephalomyocarditis virus (EMCV) internal ribosome entry site (IRES) element that is critical for high levels of cap-independent protein expression in the Human In Vitro Translation System. Each vector features a highly-compatible multiple cloning site (MCS) to facilitate easy insertion of protein coding sequences into and between vectors. The pT7CFE1 Vector is available with single or tandem affinity tags at the N- or C- terminus to facilitate protein purification and detection. The pT7CFE Vectors are suitable for insertion of cloned genes, cDNAs, ORFs or PCR products for in vitro transcription and translation. Custom cloning services are also available.
Optimized performance – designed to provide the highest yield in the human in vitro translation system
Many options – multiple tag and tag-location options available
Modular MCS – multiple cloning site is maintained across vector family to facilitate subcloning
Cleavable tags – HRV 3C cleavage site available on select vectors
pT7CFE1 Plasmid Features:
EMCV IRES at the 5' UTR promotes high-level translation of mRNAs
MCS accommodates gene insertion via ten different restriction sites: Msc1, Nde1, BamH1, EcoR1, EcoRV, Pac1, Pst1, Sac1, Sal1, Not1 and Xho1
Poly A sequence in the 3' region promotes mRNA stabilization and protection from nucleases
T7 terminator ensures synthesis of accurate size mRNA transcripts
Plasmid linearization may be accomplished with restriction sites between Poly A sequence and the T7 terminator region
Map of the Thermo Scientific T7 Cell-free Expression Vector elements. Data sheets with features and maps of individual plasmids and complete sequence information are available for download (see table).
Thermo Scientific pT7CFE1 Vectors for cell-free protein expression and available affinity and cleavage tag options.
IRES-mediated protein expression is significantly greater than 5' capped mRNA. tGFP mRNA was transcribed from the pCFE expression plasmids containing an upstream IRES element (+ IRES) or another plasmid without an IRES element. The mRNA generated without an IRES element was either used directly (- IRES, - cap) or chemically modified to have an N-terminal Anti-Reverse Cap Analog (ARCA, + 5' cap). Equal amounts of all the three mRNA's were used in human in vitro translation reactions for 2 hours at 30°C and the relative amount of GFP was determined by fluorescence.
Yadavalli, R., Ledger, C., Sam-Yellowe, TY. (2012). In vitro human cell-free expression system for synthesis of malaria proteins. Parasitol Res. 2012 Jul 11. [Epub ahead of print] DOl I 0.1007 /s00436-0 12-3014-7.