Primers Design For Yeast Integration
From 2007.igem.org
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2XOREint Rv CAGAAGAGAAGAGATGAGCAAAGGAGACAGAACAAGAGTAGCAGAAAGTCATCGATGAATTCGAGCTCG | 2XOREint Rv CAGAAGAGAAGAGATGAGCAAAGGAGACAGAACAAGAGTAGCAGAAAGTCATCGATGAATTCGAGCTCG | ||
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+ | We tagged Pho8gene with GFP,integrating GFP-KanMX6 cassette in Pho8's ORF | ||
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+ | Fv TTACATATCAGCATACGGGACATTATTTGAACGCGCATTAGCAGCATGAGTAAAGGAGAAGAACTTTTC | ||
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+ | Rv TCAGATCCAGGAACAAGACGTGTCTGTTCGCTTGGTAATGTGTGAGTCATATCGATGAATTCGAGCTCG | ||
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+ | To delete Pho80 endogenous and Pho81, we integrate into their CDS hphMX4 and Kltrip resistence,respective. | ||
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+ | hphMX4-Pho80Fv ATGGAAAGCACATCAGGAGAACGTTCCGAAAATATACATGAGGATCAAGGCGTACGCTGCAGGTCGAC | ||
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+ | hphMX4-Pho80Rv TTAATCTGGCTTTGATCGCTTGTTATAGATATGTGCGTCCTTTGCCTCAGATCGATGAATTCGAGCTCG | ||
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+ | Kltrip-Pho81Fv ATGAAATTCGGCAAGTATTTGGAAGCCAGGCAGTTGGAACTGGCGGAGTAACTAGCTAAAAAGTGGAACGA | ||
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+ | Kltrip-Pho81Rv TTACATATCAATATTATTCTCAAAAAGCAACTCACAAGCGTAGTAGATCCCTCGAGGCCAGAAGACT |
Revision as of 18:49, 18 October 2007
To create our circuite in yeast cell, we have to integrate '2XORE' promoter instead of Pho4 endogenous promoter
First we cloned 2XORE into Hind-Bam E.R. site of p2UG25 plasmid that has natMX4 resistence, so we create a 2XORE-natMX4 cassette,that after we can integrate in the Pho4's ORF.
2XORE Fv Hind CGCGGATCCATCAGTATTACCCTCGACCTC
2XORE Rv Bam CCCAAGCTTTGTTTGCGTGTCTATAGAAG
2XOREint Fv TCTAGATCGTCCACAAAACCGTGTATTCCCTCAGAAGTTGTACGGCCCATTGTTTGCGTGTCTATAGAAG
2XOREint Rv CAGAAGAGAAGAGATGAGCAAAGGAGACAGAACAAGAGTAGCAGAAAGTCATCGATGAATTCGAGCTCG
We tagged Pho8gene with GFP,integrating GFP-KanMX6 cassette in Pho8's ORF
Fv TTACATATCAGCATACGGGACATTATTTGAACGCGCATTAGCAGCATGAGTAAAGGAGAAGAACTTTTC
Rv TCAGATCCAGGAACAAGACGTGTCTGTTCGCTTGGTAATGTGTGAGTCATATCGATGAATTCGAGCTCG
To delete Pho80 endogenous and Pho81, we integrate into their CDS hphMX4 and Kltrip resistence,respective.
hphMX4-Pho80Fv ATGGAAAGCACATCAGGAGAACGTTCCGAAAATATACATGAGGATCAAGGCGTACGCTGCAGGTCGAC
hphMX4-Pho80Rv TTAATCTGGCTTTGATCGCTTGTTATAGATATGTGCGTCCTTTGCCTCAGATCGATGAATTCGAGCTCG
Kltrip-Pho81Fv ATGAAATTCGGCAAGTATTTGGAAGCCAGGCAGTTGGAACTGGCGGAGTAACTAGCTAAAAAGTGGAACGA
Kltrip-Pho81Rv TTACATATCAATATTATTCTCAAAAAGCAACTCACAAGCGTAGTAGATCCCTCGAGGCCAGAAGACT