Part:BBa_K2908670:Design

- √ Golden Gate assay to construct the plasmids
- First step: Construction of pocket plasmid
- Next: ‘Golden Gate’
- In 2008, Engler etc. reported a kind of cloned strategy through IIS restriction enzymes , called IIS cloning, namely ‘Golden Gate’ cloning. Type IIS restriction enzyme can specifically identify the target sites on double-stranded DNA, and non-specifically cut the DNA double-stranded downstream of the target sites to produce a cohesive end at the 5 'or 3' end of the DNA double-stranded.
- "Golden Gate" cloning method is in the same reaction system, using IIS type restriction endonuclease, cut beyond recognition site of DNA, DNA fragments containing cohesive end, at the same time by ligase connect several DNA fragments according to the established order, and spliced into excluding enzyme recognition site of DNA fragments, is like a linear puzzles are correctly spliced together, make the multiple objective DNA fragments in the order set to realize "seamless connection”.