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**

The Product of Assembly between 2 proteins and the Bioscaffold Linker.

• 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**

The Reaction and the product.

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

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]