How to design a host-specific TargeTron donor plasmid?

You will need:

• A stable replicating E. coli / host-specific shuttle vector
• A strong host-specific inducible promoter sequence
• TargeTron and intron encoded protein (IEP) sequence

Make the host-specific donor plasmid as follows:

1. Amplify by PCR or order the promoter sequence
2. Clone the promoter sequence into your shuttle vector
3. Verify the inserted promoter sequence by sequencing
4. Amplify the TargeTron and the IEP sequence by PCR
   (use e.g. the pACD4-G or pACD3-EcI5G as a template)
5. Clone the TargeTron & IEP sequence downstream of the promoter
6. Validate your new TargeTron donor plasmid by sequencing and restriction digest
7. Test your new donor plasmid (target a non-essential gene; use a TargeTron with the highest score)