Difference between revisions of "Part:BBa K165061"

 
 
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<partinfo>BBa_K165061 short</partinfo>
 
<partinfo>BBa_K165061 short</partinfo>
  
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This is a vector specifically designed for integration into a yeast genome at the LEU2 gene.
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Ampicillin resistance when grown in bacteria.  This plasmid is not compatible with Biobrick construction, but can be used for shuttling Biobrick parts into the yeast genome.
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This integrating vector inserts at the specific locus of the TRP13 gene. To be used in conjunction with tryptophan drop-out media for positive selection of transformed cells.  Transformation using this vector requires linearization of the plasmid by cutting with PstI.
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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.
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Alternatively, one can do the final ligation step between two Biobrick parts into this vector by cutting with the following:
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Vector: EcoRI, Not I
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Prefix: EcoRI, SpeI
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Suffix: XbaI, NotI
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Original Sikorski vector was described in R. S. [http://www.pubmedcentral.nih.gov/articlerender.fcgi?tool=pubmed&pubmedid=2659436 Sikorski and P. Hieter, Genetics, 122: 19-27 (1989)].
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Vector NTI annotated sequence: [[Image:PRS304star.gb]]
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The final construction step of a Biobrick device can be made into this vector but cutting with the following scheme:
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Yeast transformation protocol:
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R Daniel Gietz & Robert H Schiestl. [http://www.natureprotocols.com/2007/01/31/highefficiency_yeast_transform.php High-efficiency yeast transformation using the LiAc/SS carrier DNA/PEG method]. Nature protocols (2007)
  
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The system works in a yeast strain that has a mutation in the LEU2 gene, meaning that it is incapable of producing leucine on its own.  Untransformed yeast cells with this mutation must be grown in media that contains leucine in order to be viable.  However, after a transformation protocol in which competent yeast cells are treated with the pRS304* plasmid
 
  
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Yeast integration
 
  
 
<!-- Add more about the biology of this part here
 
<!-- Add more about the biology of this part here

Latest revision as of 19:39, 31 October 2008

pRS304* yeast shuttle vector, TRP1 selection

Ampicillin resistance when grown in bacteria. This plasmid is not compatible with Biobrick construction, but can be used for shuttling Biobrick parts into the yeast genome.

This integrating vector inserts at the specific locus of the TRP13 gene. To be used in conjunction with tryptophan 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. [http://www.pubmedcentral.nih.gov/articlerender.fcgi?tool=pubmed&pubmedid=2659436 Sikorski and P. Hieter, Genetics, 122: 19-27 (1989)].


Vector NTI annotated sequence: File:PRS304star.gb


Yeast transformation protocol: R Daniel Gietz & Robert H Schiestl. [http://www.natureprotocols.com/2007/01/31/highefficiency_yeast_transform.php 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 1942
    Illegal XbaI site found at 550
    Illegal XbaI site found at 1912
    Illegal SpeI site found at 1918
    Illegal PstI site found at 251
    Illegal PstI site found at 1936
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal EcoRI site found at 1942
    Illegal SpeI site found at 1918
    Illegal PstI site found at 251
    Illegal PstI site found at 1936
    Illegal NotI site found at 1904
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal EcoRI site found at 1942
    Illegal BamHI site found at 1924
    Illegal XhoI site found at 1975
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal EcoRI site found at 1942
    Illegal XbaI site found at 550
    Illegal XbaI site found at 1912
    Illegal SpeI site found at 1918
    Illegal PstI site found at 251
    Illegal PstI site found at 1936
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal EcoRI site found at 1942
    Illegal XbaI site found at 550
    Illegal XbaI site found at 1912
    Illegal SpeI site found at 1918
    Illegal PstI site found at 251
    Illegal PstI site found at 1936
    Illegal NgoMIV site found at 1563
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI.rc site found at 3348
    Illegal SapI site found at 2265