Difference between revisions of "Part:BBa K1603000:Design"
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| − | The construction of the final biobrick | + | The construction of the final biobrick was initiated by cutting the vector (BBa_J04450) with XbaI, PstI, KnpI and FastAP and the insert with XbaI and PstI. |
===Source=== | ===Source=== | ||
Revision as of 13:28, 18 September 2015
Fusion GPCR STE2MAM2
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 408
- 1000COMPATIBLE WITH RFC[1000]
Design Notes
Added 20 overlapping base pairs between the two gene fragments through PCR with primers with overhangs. This allowed the two gene fragments to be combined through Gibson assembly. Added Prefix and Suffix through PCR with primers with overhangs.
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
The primers were designed to remove the EcoRI site from the insert when constructing the biobrick to reduce the amount of base pairs in the primers. The primers also add 3 extra protective basepairs to the PstI site in the insert.
The construction of the final biobrick was initiated by cutting the vector (BBa_J04450) with XbaI, PstI, KnpI and FastAP and the insert with XbaI and PstI.
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-.