Plasmid

Part:BBa_K165060

Designed by: Aaron Glieberman   Group: iGEM08_BrownTwo   (2008-10-29)
Revision as of 05:49, 30 October 2008 by Jszymans (Talk | contribs)

pRS303 yeast shuttle vector, HIS3

Ampicillin resistance when grown in bacteria. This plasmid is not currently Biobrick compatible, but is useful for inserting a final construct into a yeast cell.

Useful for chromosomal integration of a device into yeast strains. This will insert the vector at the specific locus of the HIS3 gene. To be used in conjunction with histidine drop-out media for positive selection of transformed cells. Transformation using this vector requires linearization of the plasmid by cutting with PstI.

To incorporate a single Biobrick part into this vector for subsequent transformation into yeast, one should cut both vector and part with EcoRI and SpeI.

Alternatively, one can do the final ligation step between two Biobrick parts into this vector by cutting with the following:

Vector: EcoRI, Not I

Prefix: EcoRI, SpeI

Suffix: XbaI, NotI

Original Sikorski vector was described in R. S. Sikorski and P. Hieter, Genetics, 122: 19-27 (1989). http://www.genetics.org/cgi/reprint/122/1/19.pdf


Yeast transformation protocol: R Daniel Gietz & Robert H Schiestl. High-efficiency yeast transformation using the LiAc/SS carrier DNA/PEG method . Nature protocols (2007)


Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal EcoRI site found at 2127
    Illegal XbaI site found at 2097
    Illegal SpeI site found at 2103
    Illegal PstI site found at 1182
    Illegal PstI site found at 2121
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal EcoRI site found at 2127
    Illegal NheI site found at 1007
    Illegal SpeI site found at 2103
    Illegal PstI site found at 1182
    Illegal PstI site found at 2121
    Illegal NotI site found at 2089
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal EcoRI site found at 2127
    Illegal BglII site found at 898
    Illegal BglII site found at 958
    Illegal BamHI site found at 2109
    Illegal XhoI site found at 2160
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal EcoRI site found at 2127
    Illegal XbaI site found at 2097
    Illegal SpeI site found at 2103
    Illegal PstI site found at 1182
    Illegal PstI site found at 2121
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal EcoRI site found at 2127
    Illegal XbaI site found at 2097
    Illegal SpeI site found at 2103
    Illegal PstI site found at 1182
    Illegal PstI site found at 2121
    Illegal NgoMIV site found at 1748
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI.rc site found at 3533
    Illegal SapI site found at 2450


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