Part:BBa_K2030003:Design

Design Notes

The complete sequence of Saccharomyces cerevisiae S288c chromosome XV (Genbank accession number BK006948) was used to design primers for amplification of this promoter.

FW: gccgcttctagagCGCTTTTATGAACATATTCCG

RV: ttcttcctgcagcggccgctactagtaCGATAGTGCTTTTGTTGTAATCTTAC

The primers were designed to anneal at 64 °C, using Phusion High-Fidelity DNA Polymerase.

The uppercase nucleotides hybridizes with the template and the lowercase nucleotides contains the overhang. The overhang in the FW primer contains the last 13 bp of the prefix, which includes 6 protective bases before the XbaI site to ensure efficient cutting. The overhang in the RV primer contains the suffix with additional 6 protective bases to ensure efficient cutting with PstI.

For assembly of this biobrick, the vector pSB1C3 (BBa_J04450) was cut with XbaI and PstI and the 2044 bp band purified from gel. The PCR product of pPCK1 was cut with XbaI and PstI and spin column purified. The two pieces was ligated and chemically transformed into E. coli DH5α and plated on LB + chloramphenicol. False positive colonies still contain the RFP from BBa_J04450 and are red. White colonies were restreaked on LB + chloramphenicol and their plasmids extracted using miniprep. The plasmids were verified by digestion with XbaI and PstI, resulting in two bands: 2044 (vector) and 462 (pPYK2). The biobrick was then sent for sequencing for further verification.

Source

Genomic DNA from Saccharomyces cerevisiae CEN.PK113-5D.