Part:BBa_K259004
BioScaffold Linker - Removes Stop Codons & scars & replaces with a Gly-Ser linker
This part is part of a family of Bioscaffold - Linkers.
This part will help users interested in protein-fusion. The usage of the part will allow in frame assembly of 2(+) proteins by:
- Removing the stop codons from the upstream protein.
- Removing the scar created by the Assembly Standard 10 (RFC10) construction of 2(+) protein coding sequences.
- Maintaining the start codon of the downstream protein.
- Adding a flexible (Gly-Ser-Gly) linker between the two protein coding sequences.
- Acting as a translational buffer if ligation is incorrect.
Assembly Instructions
- Create an Assembly Standard 10 fusion on a pSB2k3 plasmid backbone as shown below ;
**Upstream Protein Part - Bioscaffold Linker - Downstream Protein Part**
- Use the BioScaffold Specific Enzymes
(i)Restrict with BpuEI to remove upstream scar (created between Upstream Protein - Bioscaffold) and stop codons from upstream protein part.
(ii)Ligate and you will get the intermediate product;
**Upstream Protein (w/out Stop codons) - Tyr - BioScaffold Linker - Downstream Protein**
- Restrict with CspCI to remove downstream scar.
- Ligate to get the final product;
**Upstream Protein - Tyr-Gly-Ser-Gly- Downstream Protein**
N.B.
CspCI cuts with a 1 b.p discrepancy depending on the sequence. However this should not affect the restriction/ligation process as this has been taken into account and incorporated into the design. Even with the discrepancy involved the restriction/ligation process will still give the same product and in-frame fusion.
The other restriction variants of CspCI have also been taken into account and relevant BioScaffolds are being produced to be tested. (see parts BBa_K259010 BBa_K259009)
Also see part BBa_K259002; the BpuEI - BseRI Bioscaffold.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
//direction/forward
//dna/bioscaffold
//dna/restrictionsite
//dna/spacer
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