Author: João Pedro Fonseca; Alain R. Bonny; G. Renuka Kumar; Andrew H. Ng; Jason Town; Qiu Chang Wu; Elham Aslankoohi; Susan Y. Chen; Patrick Harrigan; Lindsey C. Osimiri; Amy L. Kistler; Hana El-Samad
Title: A Toolkit for Rapid Modular Construction of Biological Circuits in Mammalian Cells Document date: 2018_12_26
ID: 1kugu5zk_5
Snippet: The basis for the MTK is a library of parts that are "domesticated" from source DNA using a Golden Gate (GG) reaction 12 into a standard vector. The resulting plasmid is then sequenced once to ensure fidelity to the source sequence, and then can later be reused as a modular part in a variety of genetic constructs. Similar to previously published work 6 , 3 , 10 , 12 , we utilize the type-IIS restriction enzymes BsaI and BsmbI due to their "reach .....
Document: The basis for the MTK is a library of parts that are "domesticated" from source DNA using a Golden Gate (GG) reaction 12 into a standard vector. The resulting plasmid is then sequenced once to ensure fidelity to the source sequence, and then can later be reused as a modular part in a variety of genetic constructs. Similar to previously published work 6 , 3 , 10 , 12 , we utilize the type-IIS restriction enzymes BsaI and BsmbI due to their "reach over" endonuclease activity that leaves arbitrary four base overhangs adjacent to the recognition site. The defining feature for a part vector is a unique four base overhang that categorizes it to similar parts, and ensures the sequential 5' to 3' ligation of parts into a transcriptional unit (TU) of multiple parts.
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