Part:BBa_K2333404

Cloning ready protein degradation tag D (medium) 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 cloning steps. UNS pdt#3 DT contains a tail that can be degrade Mesoplasma florum’s Lon protease Link mf-Lon here, which is orthogonal to E. Coli’s own degradation machinery. As this part contains both a double stop codon and the B0015 double terminator, it can be added before the stop codons of an arbitrary protein, preventing a multistep assembly to incorporate double stop codons and a double terminator.

UNS pdt#3c DT is on the William and Mary iGEM standard backbone, which contains 40bp Universal Nucleotide Sequences (UNS) on the inside of the prefix/suffix as a standardized flanking region to facilitate oligo-based cloning methods like 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 belongs to a series comprising 6 parts with pdt tags of different strengths BBa_ K2333401-K2333406. Of this series, the pdt in this part has a moderate degradation rate similar to pdt #3b. See characterization from William and Mary 2017, and also Collins et al. 2014 "Tunable Protein Degradation in Bacteria" for background informaiton.

This design significantly increases the accessibility of the mf- Lon tags, which can be easily added to the end of any arbitrary protein on 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, any team can easily create and amplify their own linear fragment with parts BBa_ K2333401-K2333406 to append a pdt tag onto a protein in a given circuit without changing other underlying architecture.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI site found at 41
  Illegal BsaI.rc site found at 263