Choose the type of tag you want to add or remove.

Please select the organism in that the tag should be used.

Which tag would you like to use?

Do you want to add or remove the tag?

At which position do you want to addremove the tag?

Does your protein of interest contain methionin as a first amino acid?

Does your protein of interest contain a stop codon?

How do you want to induce the trans-splicing reaction?

Protocol

• Get BBa_K1362105 (RBS + NpuDnaE C-intein RFC [] assembly construct) from the registry of standard biological parts.
• Get BBa_K1362104 (RBS + NpuDnaE N-intein RFC [] assembly construct) from the registry of standard biological parts.
• Get your tag from the registry of standard biological parts.
• Get the DNA of your protein of interest.
• Use Golden Gate Assembly to replace the mRFP selection marker in BBa_K1362105 (RBS + NpuDnaE C-intein RFC [] assembly construct) with the C-terminal tag. Use Golden Gate Assembly to replace the mRFP selection marker in BBa_K1362104 (RBS + NpuDnaE N-intein RFC [] assembly construct) with the N-terminal tag.

• Design and order primers for your tag:

  In 5'-3 direction, the forward primer should have the following sequence:

  NNNNNNGGTCTCCCAACTGCTGGGAA + binding part NNNNNNGGTCTCAGATG + binding part NNNNNNGGTCTCAGATG + binding part

  In 5'-3' direction, the reverse primer should have the following sequence:

  NNNNNNGGTCTCTATTA + binding part NNNNNNGGTCTCGAGCACTTGCCCCT + binding part
  Be careful not to include stop codons of your tag into the binding part.

• Use PCR to create your insert and purify it.

• Use Golden Gate Assembly to replace the mRFP selection marker in BBa_K1362105 (RBS + SspDnaE C-intein RFC [] assembly construct) with your C-terminal tag BBa_K1362104 (RBS + SspDnaE N-intein RFC [] assembly construct) with your N-terminal tag.

• Backtranslate the amino acid sequence of your protein of interest into a nucleotide sequence.

• Design and order primers:

  In 5'-3' direction, the forward primer should have the following sequence:

  NNNNNNGGTCTCAGATG + binding part NNNNNNGGTCTCAG + binding part

  In 5'-3' direction, the reverse primer should have the following sequence:

  NNNNNNGGTCTCGAGCACTTGCCCCT + binding part

  Be careful not to include stop codons of your protein of interest into the binding part.

  NNNNNNGGTCTCCCAACTGCTGGGAA + binding part

  In 5'-3' direction, the reverse primer should have the following sequence:

  NNNNNNGGTCTCTATTA + binding part

• Use PCR to create your insert and purify it.

• Use Golden Gate assembly to to exchange the mRFP selection marker in BBa_K1362104 ( SspDnaE N-intein RFC [] assembly construct) with your insert. BBa_K1362105 ( SspDnaE C-intein RFC [] assembly construct) with your insert.

  Since there is a BsaI site in your protein, you have to religate after the Golden Gate reaction.

• Clone both assembly constructs into compatible expression vectors.

  You can use [verweise zu pSBXen].

  Please use: LOV to induce splicing