Part:BBa_K3271011
Gal4::EX-RFP-SP pSB1K3
RFP is flanked by a prefix and a suffix at the 5' and 3' ends, respectively. The prefix and suffix (EXSP) of the pSB1K3 backbone are flanked by the upstream and downstream homology sequences of the Gal4 locus from S. cerevisiae, respectively. To target a DNA part of interest to the Gal4 locus in S. cerevisiae, replace the RFP casstte with the DNA of interest (the insert) within the prefix and the suffix using any method of your choice. Transform the resultant plasmid in E. coli. Successful clones are white colonies (as opposed to red). Screen for the appropriate clone and isolate the desired plasmid via miniprep plasmid extraction. Then, digest the plasmid using PmeI. This creates a DNA fragment comprised of the DNA part of interest flanked by the Gal4 locus upstream homology and the downstream homology at the 5' and 3' ends of the part, respectively. Transform the digest into S. cerevisiae using a lithium acetate protocol of choice. Homologous recombination will facilitate the insertion of the DNA part of interest in the Gal4 locus.
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal prefix found in sequence at 2290
Illegal suffix found in sequence at 3381 - 12INCOMPATIBLE WITH RFC[12]Illegal EcoRI site found at 2290
Illegal SpeI site found at 3382
Illegal PstI site found at 3396
Illegal NotI site found at 2296
Illegal NotI site found at 3389 - 21INCOMPATIBLE WITH RFC[21]Illegal EcoRI site found at 2290
Illegal XhoI site found at 1026
Illegal XhoI site found at 2052 - 23INCOMPATIBLE WITH RFC[23]Illegal prefix found in sequence at 2290
Illegal suffix found in sequence at 3382 - 25INCOMPATIBLE WITH RFC[25]Illegal prefix found in sequence at 2290
Illegal XbaI site found at 2305
Illegal SpeI site found at 3382
Illegal PstI site found at 3396
Illegal AgeI site found at 3092
Illegal AgeI site found at 3204 - 1000COMPATIBLE WITH RFC[1000]
biology | Saccharomyces cerevisiae |
chassis | Saccharomyces cerevisiae and Escherichia coli |
device_type | Plasmid backbone for homologous recombination in Saccharomyces cerevisiae |