Plasmid Construction Using Gibson Assembly

The control vector pEGFP was generated by Gibson assembly [107 (link)] of the following DNA fragments: the EF-1 alpha promoter (amplified from pEF1a-mRor2WT, Addgene #2261, a gift from Roel Nusse [108 (link)]) and the EGFP-ori-AmpR fragment (amplified from pCMV_ABEmax_P2A_GFP, Addgene #112101, a gift from David Liu [109 (link)]).
The pPRKRA plasmid, expressing N-terminal 3XFLAG-tagged PRKRA (NCBI Reference Sequence: NM_011871) mouse protein, was generated by Gibson assembly of the following fragments: the EF-1 alpha promoter (from Addgene #2261), the Kozak-3XFLAG fragment (from c3GIC9, Addgene #62191, a gift from Lukas Dow [110 (link)]), the PRKRA CDS (amplified from a Mael^-/- testis cDNA sample) and the P2A-EGFP-ori-AmpR fragment (from Addgene #112101).
pTN201, pCEPsmL1 and pCEPsmL1mut plasmids have been described elsewhere [20 (link),76 (link)] and were kindly provided by Jeffrey Han (Tulane University); pPAGFP-C1 plasmid (Addgene #11910) was a gift from Svetlana Deryusheva (Carnegie Institution for Science, Embryology).

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De Luca C., Gupta A, & Bortvin A. (2023). Retrotransposon LINE-1 bodies in the cytoplasm of piRNA-deficient mouse spermatocytes: Ribonucleoproteins overcoming the integrated stress response. PLOS Genetics, 19(6), e1010797.

Publication 2023

EF-1 alpha promoter pCMV_ABEmax_P2A_GFP

Cdna Ef 1 alpha Mouse Plasmid Protein Testis

Corresponding Organization : Sidney Kimmel Comprehensive Cancer Center

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Variable analysis

independent variables

Gibson assembly of DNA fragments to generate the control vector pEGFP and the pPRKRA plasmid

dependent variables

Not explicitly mentioned

control variables

The EF-1 alpha promoter (from Addgene #2261)
The Kozak-3XFLAG fragment (from c3GIC9, Addgene #62191)
The PRKRA CDS (amplified from a Mael-/- testis cDNA sample)
The P2A-EGFP-ori-AmpR fragment (from Addgene #112101)
The pTN201, pCEPsmL1 and pCEPsmL1mut plasmids (described elsewhere and provided by Jeffrey Han)
The pPAGFP-C1 plasmid (Addgene #11910, a gift from Svetlana Deryusheva)

positive controls

Not explicitly mentioned

negative controls

Not explicitly mentioned

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