Constructing sequences for expression plasmids

Full description

Data files contain construct sequences for expression plasmids related to the manuscript by Heider et al .The pRRL.sin.EF1α.eGFP plasmid was derived from an HIV-1 based pRRL.sin backbone plasmid and the enhanced green fluorescent (eGFP) protein was placed under the control of an elongation factor 1 α promoter (EF1α) (44). Then, pRRL.sin.EF1α.PPI.P2A.eGFP was generated from pRRL.sin.EF1α.eGFP plasmid. The encoding sequence for human preproinsulin (hPPI, aa1-110) was inserted upstream of the eGFP sequence and separated from it by a peptide 2A cleavage site sequence. pRLL.sin.HLA-DRα.eGFP.EFS.mTagBFP2 plasmid was then generated from pRRL.sin.EF1α.PPI.P2A.eGFP, by replacing the EF1α promoter with that of a HLA-DRα. The mTagBFP2 sequence (45) from pRRL.sin.EF1α.Hygro.P2A.mTagBFP2 (46) plasmid (AddGene #99374) was placed under the control of an intron-less EF1α promoter (EFS) (47) and then inserted. The hPPI sequence was then added upstream of the eGFP sequence and separated by a P2A cleavage site sequence to generate pRLL.sin.HLA-DRα.PPI.P2A.eGFP.EFS.mTagBFP2. pRRL.sin.HLA-DRα.pp65.P2A.EFS.mTagBFP2 was derived from the pRLL.sin.HLA-DRα.PPI.P2A.eGFP.EFS.mTagBFP2 plasmid, but the sequence encoding NY-ESO-1157-165 CMV pp65495-503 with linkers (48, 49) replaced the hPPI2-24 sequence so that a NY-ESO-1157-165 pp65495-503 PPI24- 110 fusion protein was encoded.