Part:BBa_K4190012
mg-Int-leu2-kan_1420 Backbone
Vector for gene insertion and subsequent protein expression at S. cerevisiae Leucine homology site within the genome.
Usage and Biology
For yeast, we used the plasmid mg-Int-leu2-kan-1420, which we obtained from the Kamakaka lab at UCSC. This plasmid has an origin of replication for E. coli. This enables replication of our plasmid in E. coli before we introduced it to S. cerevisiae. Since the plasmid does not have an origin of replication for S. cerevisiae, expression of the plasmid’s features in S. cerevisiae cells is only attainable through genomic integration. The plasmid also contains yeast G418 resistance as a selection marker and leu2 homology sites for integration into the host genome. Our insert was placed in between the two BsaI cut sites [1] already present on the plasmid. Because the promoter and terminator were cut out by our restriction enzyme digest, we added these parts as well during GGA.
We also used mg-int-trp1-hyg-1432-4a plasmid from the Kamakaka Lab. It is nearly identical to the previous plasmid, except that it has hygromycin resistance in place of G418 resistance and trp homology sites in place of leu2. This means that this plasmid integrated at a different spot inside the genome.
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal EcoRI site found at 5007
Illegal XbaI site found at 5016
Illegal SpeI site found at 776
Illegal PstI site found at 785 - 12INCOMPATIBLE WITH RFC[12]Illegal EcoRI site found at 5007
Illegal SpeI site found at 776
Illegal PstI site found at 785
Illegal NotI site found at 2708
Illegal NotI site found at 4471 - 21INCOMPATIBLE WITH RFC[21]Illegal EcoRI site found at 5007
- 23INCOMPATIBLE WITH RFC[23]Illegal EcoRI site found at 5007
Illegal XbaI site found at 5016
Illegal SpeI site found at 776
Illegal PstI site found at 785 - 25INCOMPATIBLE WITH RFC[25]Illegal EcoRI site found at 5007
Illegal XbaI site found at 5016
Illegal SpeI site found at 776
Illegal PstI site found at 785
Illegal NgoMIV site found at 701
Illegal NgoMIV site found at 4801 - 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI site found at 590
Illegal BsaI.rc site found at 5181
Illegal SapI.rc site found at 5365
References
[1] J. H. Lee, H. J. Won, E.-S. Oh, M.-H. Oh, and J. H. Jung, “Golden Gate Cloning- Compatible DNA Replicon/2A-Mediated Polycistronic Vectors for Plants,” Front. Plant Sci., vol. 11, p. 559365, Oct. 2020, doi: 10.3389/fpls.2020.559365.
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