Part:BBa_K2538009:Design
Synthetic minimal adenovirus major late promoter (pMLPm)
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 8
- 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 178
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
Design Considerations
PAM sequence was changed from NGG to NNGRRT to allow SadCas9 VP64 to target this promoter. Site-directed mutagenesis was done to remove XbaI restriction site (nucleotide #175 was changed from T to G)
Design Notes
Designed using Geneious to meet registry standards. Part was synthesized by Syntezza, Site-directed mutagenesis was done to remove XbaI restriction site (nucleotide #175 was replaced from T to G) and cloned into pSB1C3 using EcoRI and PstI restriction sites.
Source
Synthetic pMLPm sequence: Farzadfard F, Perli SD, Lu TK. Tunable and Multifunctional Eukaryotic Transcription Factors Based on CRISPR/Cas. ACS Synthetic Biology. 2013;2(10):604-613. doi:10.1021/sb400081r. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3805333/?report=classic
References
1. Farzadfard, Fahim, Samuel D. Perli, and Timothy K. Lu. "Tunable and multifunctional eukaryotic transcription factors based on CRISPR/Cas." ACS synthetic biology 2.10 (2013): 604-613.
2. La Russa, Marie F., and Lei S. Qi. "The new state of the art: CRISPR for gene activation and repression." Molecular and cellular biology (2015): MCB-00512.
3. Kim, Jin Hee, et al. "High cleavage efficiency of a 2A peptide derived from porcine teschovirus-1 in human cell lines, zebrafish and mice." PloS one 6.4 (2011): e18556.