Silver: Assembly

Summary

Unique nucleotide sequence (UNS)-guided DNA assembly is a variation on Gibson/isothermal assembly that is more tolerant to repeated sequence elements, e.g. those common in synthetic metabolic pathways and genetic circuits. In this approach, 40 bp UNSes are designed computationally to be unstructured and biologically inert, then attached to the termini of each DNA part to facilitate accurate recombination-based assembly.

Previous Implementations of UNS-Guided Assembly

The details required for implementation of UNS-guided assembly are described in Torella et al. 2014, "Rapid construction of insulated genetic circuits via synthetic sequence-guided isothermal assembly", as are examples of its use for the construction and optimization of bacterial metabolic pathways.

We have also used this approach to build and genomically integrate mammalian genetic circuits in Lienert et al. 2014, "Two- and three-input TALE-based AND logic computation in embryonic stem cells."

A new manuscript detailing the protocol we use, as well as troubleshooting steps, is forthcoming.

Implementing UNS-Guided Assembly

To add: The ten already-existing UNSes. Directions (use pFL with pRmce, pJT with pDest, and PCR solely on its own. For mix-and-match, consult Nat Protoc).

Obtaining Vectors for UNS-Guided Assembly

A list of available Part and Destination Vectors can be found at our Available Vectors page.

Part and Destination vectors can be obtained from the Silver Lab. E-mail jtorella@fas.harvard.edu.

Software for Designing new UNSes

To add: explanation of UNAFold stuff