Part:BBa_K2333442:Design
Copper sensor with Protein Degradation Tag F
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 7
Illegal NheI site found at 30 - 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 1229
Illegal AgeI site found at 1341 - 1000COMPATIBLE WITH RFC[1000]
Design Notes
UMaryland 2017 ordered parts of this as a G-block and assembled via Gibson Assembly. RFP with the stop codon removed was attached to protein degradation tag (pdt) F to allow for characterization of pdt F.
Source
The CueR and PcopA sequences were taken from E. coli. CueR was codon optimized.
Pdt F was originally generated by mutagenesis from the endogenous Lon degraded tags from the bacteria Mycoplasma florum by Collins et al. 2014 "Tunable Protein Degradation in Bacteria". Pdt F corresponds to Collins et al.'s tag pdt#3e. To create pdt F, the amino acid sequence was taken from Collins et al. and was codon optimized for E. coli, then synthesized by IDT.
UNS sequences are from Torella, J. P., Boehm, C. R., Lienert, F., Chen, J. H., Way, J. C., & Silver, P. A. (2013). Rapid construction of insulated genetic circuits via synthetic sequence-guided isothermal assembly.
References
[1] Cameron DE, Collins JJ. Tunable protein degradation in bacteria. Nature Biotechnology. 2014;32(12):1276–1281.
[2] Torella JP, Boehm CR, Lienert F, Chen J-H, Way JC, Silver PA. Rapid construction of insulated genetic circuits via synthetic sequence-guided isothermal assembly. Nucleic Acids Research. 2013;42(1):681–689.