Protein_Domain

Part:BBa_K3114003:Design

Designed by: Cassandra Sillner, Sara Far, Sravya Kakumanu, Nimaya De Silva, Andrew Symes   Group: iGEM19_Calgary   (2019-10-08)


Signal peptide from PhoA (secretion tag)


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
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 1
    Illegal BsaI.rc site found at 79


Design Notes

When designing this part and the rest of our collection, we were interested in creating parts that could be used in Golden Gate assembly right out of the distribution kit without the need to first domesticate them in a Golden Gate entry vector. As such, these parts are not compatible with the iGEM Type IIS RFC[1000] assembly standard because we included the BsaI restriction site and MoClo standard fusion site in the part’s sequence.

As per the MoClo standard, the 5’ signal peptide fusion sequence included in this part is AATG, and the 3’ signal peptide fusion sequence is AGGT (Weber et al., 2011).

Figure 1. Fusion sites used in the MoClo standard for Golden Gate assembly (Weber et al., 2011).

This part includes a start codon. A Gly-Gly spacer sequence was added after to the end of the signal peptide sequence in order to ensure that the fused protein of interest will be in-frame following Golden Gate assembly.

This sequence has been codon optimized for high expression in E. coli.

Source

This part was synthesized.

References

Weber E, Engler C, Gruetzner R, Werner S, Marillonnet S (2011) A modular cloning system for standardized assembly of multigene constructs. PLoS One. doi: 10.1371/journal.pone.0016765