Difference between revisions of "Part:BBa K165046"

 
 
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The GAL1 promoter is induced by the presence of galactose and repressed by glucose.  Raffinose is used as a neutral carbon source, with no effect on pGAL1.  The promoter normally has an all-or-nothing response.  To make it tunable, knock out the Gal2 gene.[http://www.jbc.org/cgi/content/abstract/M512317200v1]
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Use these primers on a Kan resistance gene template: (the portion in bold binds to the template, the rest is homologous to the Gal2 gene on the yeast chromosome.)
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GAL2ko.fwd: AGCAGCAAAA CATTAATTTT GCTTCCAAGA CGACAGTAAT ATGTCTCCTA CAATACCAGT '''GGCAGATCCGCTAGGGATAA'''<br>
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GAL2ko.rev:  AATACTCTGATATATGTACACAAATAATAGGTTTAGGTAAGGAATTTATATAATCGTAAG '''TTCGAGCTCGTTTAAAC'''
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Transform the product into yeast by standard procedure [http://www.natureprotocols.com/2007/01/31/highefficiency_yeast_transform.php], and select transformants with G418 YPD plates
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To check for proper knockout, amplify the section of the chromosome where the insert should be.  Use these primers:
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GAL2kosc.fwd: TGTGCATGTT ATCTATATCC TTCTTTATAT AGATGCTGTT<br>
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GAL2kosc.rev: ATT AAT TGT ATG TTA GCT CAG GAA TTC AAC TGG AAG AAA G
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''Thanks to the lab of Christina Smolke for these primer sequences.''
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<!-- Add more about the biology of this part here
 
<!-- Add more about the biology of this part here

Latest revision as of 03:58, 31 October 2008

pGAL1+ Kozak

The GAL1 promoter is induced by the presence of galactose and repressed by glucose. Raffinose is used as a neutral carbon source, with no effect on pGAL1. The promoter normally has an all-or-nothing response. To make it tunable, knock out the Gal2 gene.[http://www.jbc.org/cgi/content/abstract/M512317200v1]

Use these primers on a Kan resistance gene template: (the portion in bold binds to the template, the rest is homologous to the Gal2 gene on the yeast chromosome.)

GAL2ko.fwd: AGCAGCAAAA CATTAATTTT GCTTCCAAGA CGACAGTAAT ATGTCTCCTA CAATACCAGT GGCAGATCCGCTAGGGATAA
GAL2ko.rev: AATACTCTGATATATGTACACAAATAATAGGTTTAGGTAAGGAATTTATATAATCGTAAG TTCGAGCTCGTTTAAAC


Transform the product into yeast by standard procedure [http://www.natureprotocols.com/2007/01/31/highefficiency_yeast_transform.php], and select transformants with G418 YPD plates


To check for proper knockout, amplify the section of the chromosome where the insert should be. Use these primers:

GAL2kosc.fwd: TGTGCATGTT ATCTATATCC TTCTTTATAT AGATGCTGTT
GAL2kosc.rev: ATT AAT TGT ATG TTA GCT CAG GAA TTC AAC TGG AAG AAA G


Thanks to the lab of Christina Smolke for these primer sequences.



Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 150
  • 1000
    COMPATIBLE WITH RFC[1000]