Pcmv6 ac trim3 gfp trueorf clone

FUW-TRIM3 construct was used in all experiments (5 (link)). The TRIM3 cDNA was obtained from pCMV6–AC–TRIM3–GFP TrueORF clone (RG211739; Origene). To clone the TRIM3 cDNA, the gene was amplified from the TrueORF clone and re-cloned in FUW vector using BamH1 and Hpa1 sites. The following primers were used: BamHI–TRIM3 50-GGATCCGCCATGGCAAAGAGGGAGGACAGC-30 and HpaI–TRIM3 50-GTTAACCTACTGGAGGTAGCGATAGGCTTT-30.

Transient expression constructs: pRNAT-Trim3 includes a 3.2 kb full length Trim3 cDNA; pLenti6-Trim3 includes only the coding region (open reading frame) and a V5 tag; and pFUW-Trim3 includes the open reading frame, but no tag.
Lentivirus constructs: FUW-Trim3-GFP and FUW-Trim3 expression constructs were used. The Trim3-GFP fusion cDNA construct was derived from pCMV6-AC-Trim3-GFP TrueORF clone (RG211739, Origene). The vector was partially digested with FseI to avoid the cutting the other FseI site inside the GFP coding region and with BamHI. The resulting 3 kb Trim3-GFP fusion cDNA fragment was ligated into the BamHI and FseI sites of the FUW vector, generating FUW-Trim3-GFP. The FUW-Trim3 construct was generated by amplifying the Trim3 coding region and cloning into the BamHI and HpaI sites of the FUW vector. The following primers were used:

BamHI-TRIM3 5'-GGATCCGCCATGGCAAAGAGGGAGGACAGC-3' and

HpaI-TRIM3 5'-GTTAACCTACTGGAGGTAGCGATAGGCTTT-3'

The FUW-GFP expression vector was previously described [23 (link)].