Plasmid

Part:BBa_K4190012

Designed by: N/A   Group: iGEM22_UCSC   (2022-09-21)


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


Assembly Compatibility:
  • 10
    INCOMPATIBLE 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
  • 12
    INCOMPATIBLE 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
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal EcoRI site found at 5007
  • 23
    INCOMPATIBLE 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
  • 25
    INCOMPATIBLE 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
  • 1000
    INCOMPATIBLE 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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