Part:BBa_K563000:Design
pHIS4 (Multifunctional Histidine biosynthesis enzyme promoter)
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal XhoI site found at 520
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
Design Notes
In order to determine the appropriate assembly standard which the part can be used with, it's necessary to check the sequence for each of the restriction enzymes. Sequence scanning of pHis4 show that no EcoRI XhoI XbaI SpeI NcoI PstI BamHI exists. As our target gene comes from yeast and contains common restriction sites like EcoRI, NotI and SpeI, the stantard BioBrick prefix and suffix is no longer appropriate. Furthermore, the size of 7800bp makes it pretty expensive and inconvenient for chemical synthesis and codon optimization to avoid certain restriction sites. To match with the operation of downstream gene, the promoter parts also give up standard prefix and suffix. However, based on the modified plasmid backbone pSB1A11,we bring in some other common restriction sites, which also renders functionality and ease-of-use of promoter parts.
Source
We isolate the sequece by PCR on Saccharomyces cerevisiae strain BY4742. For standard operation of BioBricks and easy-use for other teams, we add restriction sites at upstream and downstream of the sequence ( NcoI and XhoI respectively) by designing specific primers (details in sequence and feature annotations). Forward Primer(with restriction site and protective bases): (TCATGCCATGG)ACAAGATTCCTCATCGGAAGAGGTGG Reverse Primer(with restriction site and protective bases): (GGCCGCTCGAG)TATTCAGAAAAAAAATTTTGTAAAC
References
Gcn4p, a Master Regulator of Gene Expression, Is Controlled at Multiple Levels by Diverse Signals of Starvation and Stress. Alan G. Hinnebusch* and Krishnamurthy Natarajan†. EUKARYOTIC CELL, Feb. 2002, p. 22–32