Hi,

I cant really get why you are unable to clone it under T7 promoter (inactive in non-DE3 bacterial strains such as DH5a or JM109) and CMV promoter (also inactive in E. coli). 

I don't know about the  CMV promoter; for T7, there is indeed a problem with host strains expressing T7 RNA polymerase under the control of a lac promoter, even if they carry the pLysS plasmid. You may have better luck with a strain like BL21AI (L-arabinose-inducible) from Invitrogen (now part of life technologies), which affords a more stringent control of expression.

Hi

Wich strain of E coli are you using ? E coli 10G strain is suitable for your first cloning.

But subcloning into T7 promoter, you can use BL21 strain with enginnered version of the lacI availaible at Lucigen too .

 High-Control(tm) BL21(DE3).  or High-Control(tm) 10G (Lucigen) for more stable T7 cloning

take a look here

http://openwetware.org/wiki/E._coli_genotypes#High-Control.28tm.29_10G_.28Lucigen.29

Thanks for your suggestions to change my current bacteria strain (which is Invitrogen Top10). I also did some research which I write down for future reference:

Lucigen tech support scientist Beth was very knowledgeable and recommended:

1) Grow bacteria at RT or 30C

2) in liquid medium never grow to high density.

3) If using synthetic DNA (not my case, my gen is 6 Kb) use codon optimization to decrease secondary/tertiary structure that may lead to rearrangements.

4) She suggested using Endura™ Electrocompetent Cells. I found that this cells are equivalent to Invitrogen Stbl3™ and Agilent SURE strains. NEB also has "Stable" strain but the tech support wasn't able to give advice beyond reading the catalogue.

5) They can offer a pJazz vector with a CMV promoter+polyA although it is not in the catalogue if requested.

Other tips from different source that may help with cloning of unstable inserts such as many ion channels: 

5) For unstable insert glycerol stocks of transformed bacteria are not recommended. It is better to store the purified plasmid

6) Store your purified plasmid at 4C (do not freeze). 

7) Sequence your plasmid before in-vitro transcription to make sure it has not been modified. this is especially important after subcloning, but also after a new prep or after storage.

8) From IDT: grow unstable genes in half the concentration of ampicillin (?)

I know it's kind of a old question, but maybe somebody else will encounter similar problem. We designed method to clone CDS of toxic proteins in bacteria: Article A novel method for cloning of coding sequences of highly tox...