Part:BBa_K2333401
Cloning ready protein degradation tag A (strong) with double terminator
This part is designed to easily facilitate appending the pdt#3 tag to the end of an arbitrary protein using Gibson assembly, without requiring multiple steps. This part contains a tail which can be degrade Mesoplasum florum Lon protease (Link mf-Lon here), which is orthogonal to E. Coli proteases. As this part contains both a double stop codon and the B0015 double terminator, this part can be added before the stop codons of an arbitrary protein, preventing a multistep assembly to incorporate double stop codons and a double terminator.
This part is on the William and Mary iGEM standard backbone, which contains 40bp Universal Nucleotide Sequences (UNS) on the inside of the prefix/suffix, which are designed to facilitate PCR and gibson assembly. See Torella, J. P., Boehm, C. R., Lienert, F., Chen, J. H., Way, J. C., & Silver, P. A. (2013).
This part is part of a series comprising 6 parts with pdt tags of different strengths BBa_ K2333401-K2333406. Of this series, the pdt in this part has the highest degradation rate. See characterization from William and Mary 2017, and from Collins et al. 2014 "Tunable Protein Degradation in Bacteria".
This part was designed to facilitate ease of Gibson assembly onto the end of an arbitrary protein, using either William and Mary's UNS backbone system or a standard Biobrick vector. Using a reverse primer with a pdt overhang to the end of a given protein, creates a linear fragment that can be used with any of the parts BBa_ K2333401-K2333406, to append a pdt tag onto a protein in a given circuit, without changing any other underlying architecture.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI site found at 41
Illegal BsaI.rc site found at 263
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