Part:BBa_K215002

pLac+RBS+Secretion Signal and Streptavidin Binding Tags

For example, assume our gene (E0040) starts with: 5'-atgcgtaaaggagaagaacttt...-3'

The design process would be:

1. Start with the XbaI site: 5'-TCTAGA-3'
2. Add ~20bp of the gene immediately after the XbaI site, starting _after_ the start codon (atg), and try to end on a G or C: 5'-TCTAGAcgtaaaggagaagaacttt-3' for E0040 from above. These ~20bp will determine the melting temperature for the primer.
3. Add 6-8 random nucleotides at the start. Try to balance out the primer to ~%50 g&c to a&t: 5'-cgggcTCTAGAcgtaaaggagaagaacttt-3'
4. Tweak the number of nucleotides until the melting point roughly matches that of Vr.

Then to add the gene to the construct, again assuming the gene is a biobrick with standard suffix:

1. PCR with the designed forward primer and Vr
2. Then run the PCR product in a digest with XbaI and PstI, and digest this part (the display construct) with NheI and PstI. The XbaI site has a sticky end that binds with NheI.
3. Standard ligation and transformation.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NheI site found at 196
  
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]