Heider et al construct sequences

file containing construct sequences for expression plasmids related to Heider et al manuscript:
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.

本文件包含与Heider等人研究手稿相关的表达质粒构建序列：
pRRL.sin.EF1α.eGFP质粒源自基于人类免疫缺陷病毒1型（HIV-1）的pRRL.sin骨架质粒，将增强型绿色荧光蛋白（enhanced green fluorescent protein, eGFP）置于延伸因子1α启动子（elongation factor 1 α promoter, EF1α）的调控之下(44)。
随后以pRRL.sin.EF1α.eGFP质粒为基础构建得到pRRL.sin.EF1α.PPI.P2A.eGFP：将人胰岛素原（human preproinsulin, hPPI，氨基酸序列1-110）的编码序列插入至eGFP序列上游，并通过2A肽切割位点序列将二者分隔开。
随后以pRRL.sin.EF1α.PPI.P2A.eGFP为骨架，将EF1α启动子替换为人类白细胞抗原DRα（HLA-DRα）的启动子，构建得到pRLL.sin.HLA-DRα.eGFP.EFS.mTagBFP2质粒。将来自pRRL.sin.EF1α.Hygro.P2A.mTagBFP2（AddGene #99374）质粒(46)的mTagBFP2序列(45)置于无内含子EF1α启动子（intron-less EF1α promoter, EFS）(47)的调控之下，随后将其插入载体。
随后在eGFP序列上游插入hPPI编码序列，并通过P2A切割位点序列将二者分隔，由此得到pRLL.sin.HLA-DRα.PPI.P2A.eGFP.EFS.mTagBFP2质粒。
pRRL.sin.HLA-DRα.pp65.P2A.EFS.mTagBFP2质粒源自pRLL.sin.HLA-DRα.PPI.P2A.eGFP.EFS.mTagBFP2质粒，将其中编码带有连接肽的NY-ESO-1₁₅₇₋₁₆₅与巨细胞病毒（CMV）pp65₄₉₅₋₅₀₃的序列替换了hPPI₂₋₂₄序列，使其编码NY-ESO-1₁₅₇₋₁₆₅-pp65₄₉₅₋₅₀₃-PPI₂₄₋₁₁₀融合蛋白。