Dear Sebastian

several pet vector contain the salI in theMCS (pet20, pet24, pet28) and you can remove the tags at the n-term by double enzime digestion.

However I suggest to you to test PIPE cloning approach (https://www.ncbi.nlm.nih.gov/pubmed/18988020).

You can simply apply this protocol to every plasmid containing  the ccdb gene (e.g all EXPRESSION  vectors suitable for GATEWAY CLONING) and just a smart design of your primers you will be free from the use of restriction enzime at least for the cloning of gene with size up to 6-8KDa. 

If your plasmid do not contain the ccdb, you can also use the PIPE but you have just to minimize the amount of plasmid template in the VectorPCR and digest it with dpnI before to proceed with the PIPE reaction.

Hi there,

What kind of promoter would you like to use for controlling the transcription of your gene of interest?

hi Dominique,

honestly I haven´t thought about the promoter, but usually we use IPTG inducible systems, however I heard that there are also arbainose inducible systems?

Hi, can you use pUC18/19? They have SalI in MCS and are IPTG inducable/no tag, plus blue/white selection for cloning. There are plasmids with arabinose inducable promoters like: http://www.synthesisgene.com/vector/pBAD.pdf; but they have His and or myc- tag included. However, you can always PCR amplify your gene without His tag added, and add whatever restriction site you want through primers. Alternative is to amplify any expression vector you have in inverse PCR, skip whatever tag it has, and add in primers SalI restriction site if you must use it. If vector has C terminal tag, you can just add stop codon. I am also cloning without restriction enzymes with Takara in fusion kit, it works very well, and is easy and very efficient. With this kit you can clone anything in any vector, whatever you can imagine; no restriction enzymes, no scars etc: I can highly recommend.