Difference between revisions of "Part:BBa K864444"
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Use this system to screen for downregulation of your gene by a random small RNA library. PCR amplify your 5’UTR, adding XbaI and EcoRI sites upstream and a BamHI site downstream, and clone into any backbone carrying this part, replacing the RFP cassette. By cloning with EcoRI and BamHI, the cloning will function as a red/white screening system. You have now created a novel BioBrick, that can be used to screen for translational downregulation of your gene of interest. | Use this system to screen for downregulation of your gene by a random small RNA library. PCR amplify your 5’UTR, adding XbaI and EcoRI sites upstream and a BamHI site downstream, and clone into any backbone carrying this part, replacing the RFP cassette. By cloning with EcoRI and BamHI, the cloning will function as a red/white screening system. You have now created a novel BioBrick, that can be used to screen for translational downregulation of your gene of interest. | ||
| − | SYFP2 <partinfo>BBa_K864100</partinfo> is situated downstream of the RFP cassette<partinfo>BBa_J04450</partinfo> and is connected in frame with a 10 aa glycine-serine repeated linker <partinfo>BBa_J18922</partinfo>, to prevent folding errors in the SYFP2. Before the linker there is a BamHI restriction site (GGATCC), which translates into glycine-serine in E coli. | + | SYFP2 <partinfo>BBa_K864100</partinfo> is situated downstream of the RFP cassette <partinfo>BBa_J04450</partinfo> and is connected in frame with a 10 aa glycine-serine repeated linker <partinfo>BBa_J18922</partinfo>, to prevent folding errors in the SYFP2. Before the linker there is a BamHI restriction site (GGATCC), which translates into glycine-serine in E coli. |
When screening for working small RNAs, it's recommended to include the first 15 codons of your gene of interest. | When screening for working small RNAs, it's recommended to include the first 15 codons of your gene of interest. | ||
Revision as of 16:22, 29 September 2012
RFP-Linker-SYFP2
This part is a component of the Uppsala University iGEM 2012 modular small RNA screening system, together with the spott42 sRNA BBa_K864440. It can also be used for studing up- or downregulation of genes by other mechanisms.
Use this system to screen for downregulation of your gene by a random small RNA library. PCR amplify your 5’UTR, adding XbaI and EcoRI sites upstream and a BamHI site downstream, and clone into any backbone carrying this part, replacing the RFP cassette. By cloning with EcoRI and BamHI, the cloning will function as a red/white screening system. You have now created a novel BioBrick, that can be used to screen for translational downregulation of your gene of interest.
SYFP2 BBa_K864100 is situated downstream of the RFP cassette BBa_J04450 and is connected in frame with a 10 aa glycine-serine repeated linker BBa_J18922, to prevent folding errors in the SYFP2. Before the linker there is a BamHI restriction site (GGATCC), which translates into glycine-serine in E coli.
When screening for working small RNAs, it's recommended to include the first 15 codons of your gene of interest.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 1070
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 781
Illegal AgeI site found at 893 - 1000COMPATIBLE WITH RFC[1000]