Judging from the delightful hand-drawn map, that LZRS plasmid is older than I am. What exactly are you trying to reproduce? Do you just need to express CNTF by any means or are you actually making lentivirus?

If you don't need to make lentivirus then a lentiviral plasmid isn't ideal, they often don't transfect as efficiently as ordinary plasmids.

One major factor is the promoter, if you want to reproduce a particular study where CNTF was expressed then be sure to use the same cellular promoter.

By the time you're done messing around with cloning it might be cheaper just to buy what you need off the shelf

Hi Brian,

I don't know about other vectors but if it is the cloning that is your problem then I have an alternative method which is restriction free.

It is quite easy and works very well sometimes you may get the results much faster than the restriction based method.

All you need to do is do two rounds of PCR.

The primers for first PCR contains the complementary sequences for your gene of interest, similar to what you would use for PCR amplification of your gene. In the primers you need to keep about 24-30 bp overhangs that are complementary to your site of insertion in the plasmid. So, when you amplify your gene of interest, the PCR product will have 24-30 bp regions flanking the gene segment that are complementary to the site of insertion, and by using this PCR product as primers for second round of PCR, your gene will be inserted into the plasmid.

The original plasmid is then destroyed by DpnI digestion and the remaining plasmid formed by the PCR reaction can be easily transformed and selected.

Let me know if you need any more information on this.

good luck

S