Part:BBa_K2333408:Design
UNS J23100 sfGFP pdt B
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 47
Illegal NheI site found at 70 - 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000INCOMPATIBLE WITH RFC[1000]Illegal SapI.rc site found at 106
Design Notes
This part was designed with a constitutive promoter for sfGFP attached to protein degradation tag (pdt) B to allow for characterization of pdt B. The sfGFP reporters have been codon-optimized for E. coli and feature a double stop codon for enhanced efficiency.
Source
Pdt B 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 B corresponds to Collins et al.'s tag pdt#3a. To create pdt B, 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.
The sequence for this sfGFP (BBa_K2333007) was obtained and modified from the sequence in Supplement section V of Lou et al. "2015 Ribozyme-based insulator parts buffer synthetic circuits from genetic context." The sequence was modified to remove any restriction enzymes and then synthesized by IDT.
References
[1] 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.
[2] Cameron DE, Collins JJ. Tunable protein degradation in bacteria. Nature Biotechnology. 2014;32(12):1276–1281.
[3] Lou, C., Stanton, B., Chen, Y.-J., Munsky, B., & Voigt, C. A. (2012). Ribozyme-based insulator parts buffer synthetic circuits from genetic context. Nature Biotechnology, 30(11), 1137–1142. http://doi.org/10.1038/nbt.2401