Publication protocol
In converting SP6 to CMV promoter-based plasmids, the CMV promoter of pRL-CMV (Promega) was first inserted into a plasmid with an opposite-orientation Ori (opposite to the coding region sense strand), followed by transfer of each FMR1(nAGG)-FL cassette into this invOri plasmid; both transfers required intermediate linker plasmids. First, the Tet gene of pBR322 (Invitrogen) was removed by HindIII/PvuII (NEB) double digestion, followed by insertion of the linker, 5′-AGCTTAGATCTTGATCAGGATCCCAG-3′, which contains BglII and BamHI sites, to create pBR-Linker. Second, the BamHI and BglII fragment of pRL-CMV, which contains the CMV promoter, was inserted into the corresponding sites in pBR-Linker, resulting in pBR-CMV. pBR-CMV was then digested with SacI and PstI (near the 3′-end of the CMV promoter) followed by insertion of the linker, 5′-CGTTTAGTGAACCGTCAGATCAGTCAGGCGCTCAGCCTGCA-3′, which restores the 3′-end of CMV promoter and the first few bases of the FMR1 5′UTR (to the BlpI site), creating pBR-CMV-Linker. This plasmid was then digested with BlpI and XbaI to remove the entire coding region. Each pSP6-FMR1(nAGG)-FL plasmid was likewise digested with BlpI and XbaI, and the fragment containing FMR1 5′UTR-FMR1exon1-FL was inserted into pBR-CMV-Linker, generating pBR-CMV-FMR1-5′UTR[65–66CGG/(AGG)0–2]-FMR1exon1-FL, designated pCMV-FMR1(nAGG)-FL. The FMR1 5′UTR begins directly after the CMV promoter, eight bases after the transcription start site at base 741 of pRL-CMV (38). All constructs were confirmed by sequencing.
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