This plasmid contains the T7 RNA polymerase promoter and E. coli tRNA His and is used as a template for in vitro transcription of E. coli tRNA His. The tRNA gene of interest can be inserted into the multiple cloning site of the expression plasmid, downstream from the T7 RNA promoter. The tRNA gene terminates with a FokI restriction site, such that run-off transcription of the linearized plasmid produces a tRNA transcript with the correct CCA end.
From the laboratory of Christopher S. Francklyn, PhD, University of Vermont.
Part of The Investigator's Annexe program.
|Gene/insert name:||T7 RNA polymerase promoter + E.coli tRNA His|
|Grow in E. coli at 37 C:||Yes|
|Cloning Site 5':||EcoRI|
|Cloning Site 3':||BamHI|
|Insert Size:||115 bp|
|Vector Backbone and Size:||pUC19, 2686 bp|
|High or low copy:||High|
|Shipped:||Ambient temperature (Liquid plasmid DNA in water for domestic orders, spotted on filter paper for international orders)|
Linearize plasmid with FokI prior to in vitro transcription as described in Francklyn et. al, Methods, 2008
Schematic of synthetic template transcription. Overlapping synthetic nucleotides are used as a template for Klenow fragment extension by annealing and extension cycles. The template strand contains 2'-O-methyl modifications on the two terminal 5' residues (inset). The doublestranded fragment then acts as a template for T7 RNA polymerase. The DNA modifications are thought to cause the polymerase to dissociate from the template before adding nontemplated residues, thereby increasing product homogeneity.
Adapted from: Francklyn et. al, Methods, Vol 44, No.2, pp 100-118, 2008.
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