|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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- Yan, W., Augustine, J., and Francklyn, C. A tRNA identity switch mediated by the binding interaction between a tRNA anticodon and the accessory domain of a class II aminoacyl-tRNA synthetase. Biochemistry, 35:21, 6559-6568 (1996).
- Arnez, J.G., Augustine, J.G., Moras, D., and Francklyn, C. The first step of aminoacylation at the atomic level in histidyl-tRNA synthase. PNAS, 94, 7144-7149 (1997).
- Francklyn, C., Musier-Forsyth, K., and Martinis, S.A. Aminoacyl-tRNA synthetases in biology and disease: new evidence for structural and functional diversity in an ancient family of enzymes RNA, 3, 945-960 (1997).
- Francklyn, C., Adams, J., and Augustine, J. Catalytic defects in mutants of class II histidyl-tRNA synthetase from Salmonella typhimurium previously linked to decreased control of histidine biosynthesis regulation. Journal of Molecular Biology, 280, 847-858 (1998).
- Hawko, S.A., and Francklyn, C.S. Covariation of a specificity-determining structural motif in an aminoacyl-tRNA synthetase and a tRNA identity element. Biochemistry, 40:7, 1930-1936 (2001).
- Connoly, S.A., Rosen, A.E., Musier-Forsyth, K., and Francklyn, C.S. G-1:C73 recognition by an arginine cluster in the active site of Escherichia coli histidyl-tRNA synthetase. Biochemistry, 43: 4, 962-969 (2004).
- Guth, E., Connoly, S.H., Bovee, M., and Francklyn, C.S. A substrate-assisted concerted mechanism for aminoacylation by a class II aminoacyl-tRNA synthetase. Biochemistry, 44:10, 3785-3794 (2005)
- Guth, E. and Francklyn, C.S. Kinetic discrimination of tRNA identity by the conserved motif 2 loop of a class II aminoacyl-tRNA synthetase. Molecular Cell, 25, 531-542 (2007).
- Francklyn, C.S., First, E.A., Perona, J.J., and Hou, Y. Methods for kinetic and thermodynamic analysis of aminoacyl-tRNA synthetases. Methods, 44:2, 100-118 (2008).
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