The NEBridge Golden Gate Assembly Kit (BsmBI-v2) contains an optimized mix of BsmBI-v2 and T4 DNA Ligase. BsmBI-v2 has been engineered by NEB and outperforms BsmBI in Golden Gate Assemblies. Together these enzymes can direct the assembly of multiple inserts/modules and also single insert/library generation cloning with single insert(s) using the Golden Gate approach. The pGGAselect destination plasmid is also provided, which provides a backbone for your assembly. This versatile destination construct has flanking recognition sites in the correct orientation for BsmBI-directed assemblies, and also BsaI- and BbsI-directed assemblies, enabling the destination plasmid to conveniently be used with all three of the most commonly used Type IIS restriction enzymes used for Golden Gate Assembly. It features convenient restriction enzyme sites for subcloning, and has T7/SP6 promoter sequences to enable in vitro transcription.The efficient and seamless assembly of DNA fragments, commonly referred to as Golden Gate Assembly ^(1,2), had its origins in 1996, when for the first time it was shown that multiple inserts could be assembled into a vector backbone using only the sequential ⁽³⁾ or simultaneous ⁽⁴⁾ activities of a single Type IIS restriction enzyme and T4 DNA Ligase.Type IIS restriction enzymes bind to their recognition sites but cut the DNA downstream from that site at a positional, not sequence- specific, cut site. Thus, a single Type IIS restriction enzyme can be used to generate DNA fragments with unique overhangs. As an example, BsmBI has a recognition site of CGTCTC(N1/N5), where the CGTCTC represents the recognition/binding site, and the N1/N5 indicates the cut site is one base downstream on the top strand, and five bases downstream on the bottom strand. Assembly of digested fragments proceeds through annealing of complementary four base overhangs on adjacent fragments. The digested fragments and the final assembly no longer contain Type IIS restriction enzyme recognition sites, so no further cutting is possible. The assembly product accumulates with time.While particularly useful for multi-fragment assemblies such as Transcription Activator Like Effectors (TALEs)⁽⁵⁾ and TALEs fused to a FokI nuclease catalytic domain (TALENs)⁽⁶⁾, the Golden Gate method can also be used for cloning of single inserts and inserts from diverse populations that enable library creation, and multi-site mutagenesis involved in directed evolution ⁽⁷⁾.Please note that while general descriptions regarding Golden Gate Assembly use the BsmBI nomenclature, this kit and protocols feature the specific engineered form optimized for Golden Gate Assembly, BsmBI-v2.To learn more about the Golden Gate Assembly workflow, watch this video tutorial.

Figure 1. Overview: Assembly Protocol of Golden Gate Assembly using BsmBI-v2

Figure 2. Golden Gate Workflow

Figure 3. High Efficiency and High Fidelity Golden Gate Assembly with BsmBI-v2

Figure 4. Complex Assembly by BsmBI-v2 and T4 DNA Ligase

Figure 5.

This product is related to the following categories:

DNA Assembly, Cloning and Mutagenesis Kits Products

This product can be used in the following applications:

DNA Assembly and Cloning,

High-throughput cloning and automation solutions,

NEBridge® Golden Gate Assembly

NEB公司成立于二十世纪七十年代中期，拥有众多经验丰富的科学家，是生产生命科学试剂的领导者。目前，NEB为基因组研究提供最齐全的重组酶和天然酶，并且公司业务范围已延伸至蛋白质组学和药品开发领域。回顾三十余年来的历程，NEB公司作为先驱公司之一，为促进生物科技工业的发展做出了巨大的贡献。NEB美国总部乔 迁新址后拥有最尖端的设备，有一座现代化的发酵中心及设备齐全的实验室，这些实验室主要用于产品生产、质量监控、 产品开发和基础科研之用。作为首批以商业规模生产限制性内切酶的公司之一，NEB一直专注于内切酶的研究，并保持 业内领先水平。NEB公司一贯坚持以科学为本的原则，公司生产的试剂因其高质量、高性价比享誉世界。NEB（北京）有限公司 New England Biolabs (Beijing) LTD. NEB（北京）有限公司New England Biolabs (Beijing) LTD. 为美国New England Biolabs, Inc.在华投资兴建的独资子公司，负责处理其在中国内地及香港地区的全部业务，同时承担部分新产品的研发工作。纽英伦生物技术（北京）有限公司成立于2001年11月，为中国生命科学研究者提供一流的产品和专业的技术服务，促进国内外生命科学学术交流，与中国科研工作者一起将中国推入到二十一世纪生命科学的前沿中去。