Coding

Part:BBa_K1603000:Design

Designed by: John Hellgren   Group: iGEM15_Chalmers-Gothenburg   (2015-09-18)
Revision as of 13:25, 18 September 2015 by Jannie (Talk | contribs) (→‎Design Notes)


Fusion GPCR STE2MAM2


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 408
  • 1000
    COMPATIBLE WITH RFC[1000]


Design Notes

20 base pair overlapping sequence between the signal peptide from STE2 (SPSTE2) and MAM2 without signaling peptide (MAM2-SP) had to be constructed through PCR to allow Gibson assembly of the two gene fragments. Added Prefix and Suffix through PCR.


Primers for isolation of SPSTE2 with Prefix and overlap to MAM2-SP from genomic DNA:

FW:GCTTCTAGATGTCTGATGCGGCTCC

RV:AGTTGGGCAGACAAAGTCATCTGAGTAACAGTACTGTTAACTAAACCTTGC


Primers for isolation of MAM2-SP with Suffix from genomic DNA:

FW:ATGACTTTGTCTGCCCAACT

RV:AGCCTGCAGCGGCCGCTACTAGTATTACGTCCACTTTTTAGTTTCAGATTC

Source

The non-cytoplasmic N-terminal signal peptide from STE2 is a genomic sequence from Saccharomyces cerevisiae CEN.PK2 and the Pheromone P-factor receptor MAM2, without its signaling peptide, is a genomic sequence from Schizosaccharomyces pombe Δ8 h-.

References