期刊
ACS SYNTHETIC BIOLOGY
卷 4, 期 3, 页码 342-349出版社
AMER CHEMICAL SOC
DOI: 10.1021/sb500194z
关键词
DNA assembly; USER cloning; primer design; site-directed mutagenesis; point mutation; Web server
资金
- Novo Nordisk Foundation
- Novo Nordisk Fonden [NNF10CC1016517] Funding Source: researchfish
USER cloning is a fast and versatile method for engineering of plasmid DNA. We have developed a user friendly Web server tool that automates the design of optimal PCR primers for several distinct USER cloning-based applications. Our Web server, named AMUSER (Automated DNA Modifications with USER cloning), facilitates DNA assembly and introduction of virtually any type of site-directed mutagenesis by designing optimal PCR primers for the desired genetic changes. To demonstrate the utility, we designed primers for a simultaneous two-position site-directed mutagenesis of green fluorescent protein (GFP) to yellow fluorescent protein (YFP), which in a single step reaction resulted in a 94% cloning efficiency. AMUSER also supports degenerate nucleotide primers, single insert combinatorial assembly, and flexible parameters for PCR amplification. AMUSER is freely available online at http://www.cbs.dtu.dk/services/AMUSER/.
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