The T7 promoter is a sequence of DNA 18 base pairs long up to transcription start site at +1 (5’ – TAATACGACTCACTATAG – 3’) that is recognized by T7 RNA polymerase1 . The T7 promoter is commonly used to regulate gene expression of recombinant proteins, which can be subsequently used for a variety of downstream research applications2.
Bacterial T7 promoter-based vectors allow for expression, detection and purification of recombinant FLAG® and MAT™ (Metal Affinity Tag) fusions in E. coli. Several vectors containing the T7 promoter offer dual tag options for FLAG® and MAT™-tagged fusion proteins. These vectors confer ampicillin resistance for easy selection of positive transformants. Additionally, the vectors contain the T1T2 transcriptional terminator, the pMB1 (derivative of pBR322) origin of replication, the f1 origin and the lacI gene for repression of the T7 promoter.
pT7-FLAG® and pT7-MAT™ vectors offer the very strong T7/lac promoter. These expression vectors produce even higher yields of recombinant protein than the tac promoter system. However, the T7 promoter is known for background ("leaky") expression, which can be a drawback when recombinant proteins are toxic to the host cell. Therefore, our vectors contain the lac operator (lacO) sequences immediately downstream from the promoter to reduce leaky expression. Unlike the tac promoter system, pT7 vectors must be expressed in hosts containing a source of the T7 polymerase such as (DE3) lysogenic strains.
The MAT™ tag or Metal Affinity Tag (HNHRHKH) has been created for purification of recombinant MAT™ fusion proteins using HIS-Select Nickel and Cobalt Affinity Gels. HIS-Select products allow for highly selective purification of histidine-tagged fusion proteins such as MAT™ fusions. Many of our newest vectors make use of the MAT™ tag, often in combination with the well-known FLAG® tag. MAT™ tag containing vectors are offered in formats for N-terminal or C-terminal tagging.
Figure 1.Kanamycin gene and ampicillin gene for greater selection flexibility in E. coli
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