Difference between revisions of "Team:Czech Republic/Project/Signal transduction/Mata"
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| − | + | ;TetR on ADH1 promoter | |
Tetracycline repressor ORF sequence was taken from [https://www.addgene.org/vector-database/2525/ Addgene databese], the ADH1 promoter sequence was taken from [https://www.addgene.org/15970/ same page]. CYC1 terminator is included after TetR, it’s sequence was also taken from [https://www.addgene.org/15970/ this page]. | Tetracycline repressor ORF sequence was taken from [https://www.addgene.org/vector-database/2525/ Addgene databese], the ADH1 promoter sequence was taken from [https://www.addgene.org/15970/ same page]. CYC1 terminator is included after TetR, it’s sequence was also taken from [https://www.addgene.org/15970/ this page]. | ||
Show sequence | Show sequence | ||
| − | + | ;STE12 on a-specific CYC1 | |
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ORF of ST12 is a genomic sequence that was extracted from chromosome also with the 3’UTR. Sequence of a-specific CYC1 promoter was obtained from Prof. Vershon, who presented series of a-specific promoters{{:Team:Czech_Republic/Template:ReferenceRef|Vershon1999}}. It is a CYC1 promoter with inserted α2-Mcm1 binding sequence from AGA2 gene (α2-Mcm1 complex represses a-sg). | ORF of ST12 is a genomic sequence that was extracted from chromosome also with the 3’UTR. Sequence of a-specific CYC1 promoter was obtained from Prof. Vershon, who presented series of a-specific promoters{{:Team:Czech_Republic/Template:ReferenceRef|Vershon1999}}. It is a CYC1 promoter with inserted α2-Mcm1 binding sequence from AGA2 gene (α2-Mcm1 complex represses a-sg). | ||
Show sequence | Show sequence | ||
| − | + | ;Parts of a1 ORF | |
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Two parts of a1 OFR are included as homologous parts for chromosomal integration. The plasmid integrates in the center of a1 ORF and disrupts the gene. | Two parts of a1 OFR are included as homologous parts for chromosomal integration. The plasmid integrates in the center of a1 ORF and disrupts the gene. | ||
Show sequence | Show sequence | ||
Revision as of 12:33, 7 September 2015
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MATa integration plasmid
MATα integration plasmid carries a synthetic MATa locus that integrates into the wild-type locus inserting TetR and STE12 with their promoters into the center of the a1 ORF and thus disrupting it.
Integrated parts
All part are cloned into pRSII406 integrating vector (from Addgene). The final plasmid shown in the picture includes SnabI restriction site for linearization before chromosomal integration. The whole plasmid is integrated within the a1 ORF.
- TetR on ADH1 promoter
Tetracycline repressor ORF sequence was taken from Addgene databese, the ADH1 promoter sequence was taken from same page. CYC1 terminator is included after TetR, it’s sequence was also taken from this page. Show sequence
- STE12 on a-specific CYC1
ORF of ST12 is a genomic sequence that was extracted from chromosome also with the 3’UTR. Sequence of a-specific CYC1 promoter was obtained from Prof. Vershon, who presented series of a-specific promoters[Vershon1999]. It is a CYC1 promoter with inserted α2-Mcm1 binding sequence from AGA2 gene (α2-Mcm1 complex represses a-sg). Show sequence
- Parts of a1 ORF
Two parts of a1 OFR are included as homologous parts for chromosomal integration. The plasmid integrates in the center of a1 ORF and disrupts the gene. Show sequence
References
- ↑ Hualin Zhong, Ron McCord, and Andrew K. Vershon. Identification of target sites of the alpha2-Mcm1 repressor complex in the yeast genome. doi: 10.1101/gr.9.11.1040 Genome Res. 1999. 9: 1040-1047 Cold Spring Harbor Laboratory Press