Do you just want to co-express the two genes? If so, you may want to use the polycistronic vectors from Dr. Song Tan's lab [see Protein Expression and Purification 21, 224-234 (2001)]. I am not quite sure, but the region between BglI and Tth111 doesn't seem to contain important sequences for plasmid propagation.

I agree with Hua Xiao: if possible, try to get your hands on a bicistronic expression vector like pETDuet. If you want to tinker with it, you may engineer it such that it encodes a Tev protease cleavage site to remove the His-tag.

Thanks @ Hua Xiao and Pierre Béguin,

I aim to produce the specific recombinant protein by gene 1 and the RNA molecule by gene 2 in the same cell. Therefore, polycistronic vectors will not work in my experiment.

Hi @ Dung Viet Nguyen,

1. The Rop protein (product of the rop gene) helps to keep the plasmid at relatively low copy number.

2. What do you mean by Tet (in pET28a)?! Perhaps you are referring to the bom (basis of mobility) region... The Rop and bom sequences are important for the efficiency and overall functionality of the plasmid as intended by its design.

3. If your intent is to co-express your two genes of interest using the T7 inducible promoter, you can just get a co-expression vector such as the Duets (e.g. pCDFDuet, pRSFDuet, pACYCDuet, pETDuet; see the attached figure) with multiple cloning sites and you don't have to go through the stress of engineering the pET-28a vector... Otherwise, you can just use splicing by overlap extension PCR or homologous recombination (using a recombinase like Exnase) to put the two genes and the other part of the vector together...