Part:BBa_K1861040:Design

Fusion protein of SpyCatcher and RPD3 in an expression cassette

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BamHI site found at 260
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 826
  
• 1000
  COMPATIBLE WITH RFC[1000]

Design Notes

In order to clone the gene for the fusion protein of SpyCatcher and rpd3 into this part, we had to add the restriction sites for BamHI to BBa_1159200 and for SacI to BBa_K1861401. We used the following primers to achieve this:

5' BamHI primer - GCC AGG ATC CGT TGA TAC CTT ATC

3' SacI primer - ACA TGC TCA GAG CTC TCC ACT GC
The restriction sites are in italic.
To synthesize the rpd3 gene, we ordered both halves of it from IDT (the first half also included BBa_K1159200) and fused them together by an overlap-PCR. The following sequence (102 nucleotides) was used as overlap:
GCTGTCAACTATGCGGGTGGTTTGCATCATGCAAAAAAATCGGAAGCATCTGGGTTTTGTTATTTAAATGACATAGTACTGGGCATTATTGAGCTACTACGA

Source

The rpd3 gene is located on chromosome XIV of S. cerevisiae. Its sequence was obtained through the Saccharomyces Genome Database.
For the SpyCatcher and the terminator, we used the sequences of BBa_K1159200 and BBa_K392003, respectively.

References

https://eu.idtdna.com/site, visited on 2015-09-19
http://www.yeastgenome.org/locus/S000005274/overview, visited on 2015-09-21

http://web.archive.org/web/20150921180338/http://www.yeastgenome.org/locus/S000005274/overview