Engineered LINE-1 Expression Constructs

The following plasmids are based on the previously described pJM101/L1.3 and pDK101 constructs [4] (link), [16] (link). The amino acid and nucleotide numbers indicate the mutation position based on L1.3 accession number L19088 [63] (link). The constructs were cloned into the pCEP4 expression vector (Invitrogen) and contain the mneoI indicator cassette [32] (link), [47] (link) in the L1 3′UTR unless otherwise indicated. PCR followed by subcloning was used to introduce the respective epitope tag sequences onto the 3′ end of ORF2. As a result of this procedure, we deleted a portion of the L1 3′UTR (nts 5818 to 5953). Oligonucleotides used in our cloning strategies are available upon request.
pADO2Tt contains a Tandem Affinity Purification epitope tag (TAP tag) [43] (link) on ORF2p and was cloned from the pZome-1-C vector (Euroscarf).
pAD2TE1 is derived from pDK101 (L1.3) [16] (link) and contains both the T7 gene 10 epitope tag on the carboxyl-terminus of ORF1p and a TAP tag on the carboxyl-terminus of ORF2p.
pAD2TE1-Δ2 is derived from pAD2TE1, but lacks CMV promoter and SV40 polyadenylation signal present in the original pCEP4 vector.
pAD2TE1-NT is identical to pAD2TE1, but lacks the mneoI indicator cassette.
pES2TE1 is identical to pAD2TE1, but contains a tandem affinity FLAG-HA tag on the carboxyl-terminus ORF2p [44] (link).
pAD500 is derived from L1.3ΔORF1NN [37] (link), and contains a TAP tag on the carboxyl-terminus of ORF2p.
pADL1MT is derived from pJM101/L1.3 and contains 24 repeats of the MS2 stem-loop (MS2 tag) upstream of the mneoI indicator cassette in the L1 3′UTR. The MS2 repeats were subcloned from the pTRIP vector [64] (link).
pAD3TE1 is identical to pAD2TE1, but contains the MS2 tag in the 3′UTR (at the same position as in pADL1MT).
pADO1S is identical to pAD2TE1, but contains three stop codons in ORF1. The first two stop codons (R₇Stop; K₈Stop) were generated by introducing a thymidine at nucleotide position 928 to create a frameshift mutation and by mutating an A to a T at nucleotide position 930. The third stop codon is from the construct pJM108/L1.3 carrying the mutation S₁₁₉Stop [19] (link), [32] (link).
pADLZC is identical to pAD2TE1, but contains four leucine to valine mutations (L_{93,100,107,114}V) in the ORF1p putative leucine zipper domain.
pAD102 is identical to pAD2TE1, but contains the REKG_235–238AAAA mutations in the ORF1p RRM domain [16] (link), [32] (link).
pAD105 is identical to pAD2TE1, but contains the RR_261–262AA mutations in the ORF1p C-terminal domain [16] (link), [19] (link), [32] (link).
pAD106 is identical to pAD2TE1, but contains the RR_261–262KK mutations in the ORF1p C-terminal domain [16] (link).
pAD107 is identical to pAD2TE1, but contains the RR_261–262KR mutation in the ORF1p C-terminal domain [16] (link).
pAD113 is identical to pAD2TE1, but contains the NLR_157–159ALA mutations in the ORF1p RRM domain [31] (link).
pAD116 is identical to pAD2TE1, but contains the YPAKLS_282–287AAAALA substitution in the ORF1p C-terminal domain [16] (link), [32] (link).
pAD135 is identical to pAD2TE1, but contains the D₇₀₂A mutation in the putative ORF2p RT active site [19] (link).
pAD136 is identical to pAD2TE1, but contains the H₂₃₀A mutation in the ORF2p EN domain [19] (link).
pAD162 is identical to pAD2TE1, but contains the CWWDC_1143–1147SWWDS mutations in the ORF2p C-domain [32] (link).
pADL/R is identical to pAD2TE1, but contains a putative leucine zipper domain as well as a C-terminal domain mutant (L_{93,100,107,114}V; RR_261–262AA) in ORF1p.
pADL/C is identical to pAD2TE1, but contains a putative leucine zipper domain mutation (L_{93,100,107,114}V) in ORF1p as well as a C-domain mutation (CWWDC_1143–1147SWWDS) in ORF2p.
LZC is derived from pDK101 and contains four leucine to valine mutations (L_{93,100,107,114}V) in the ORF1p putative leucine zipper domain.
LZ1/2 is derived from pDK101 and contains two leucine to valine mutations (L_93,100V) in the ORF1p putative leucine zipper domain.
LZ2/3 is derived from pDK101 and contains two leucine to valine mutations (L_100,107V) in the ORF1p putative leucine zipper domain.
LZ3/4 is derived from pDK101 and contains two leucine to valine mutations (L_107,114V) in the ORF1p putative leucine zipper domain.
pDK101,pDK102,pDK105,pDK106,pDK107,pDK108,pDK116,pDK135,and pDK500 were described previously [16] (link).
pMS2-GFP-nls, pMS2-CFP, and pTRIP were generous gifts from Edouard Bertrand [64] (link)–[66] (link).
pAgo2-GFP and pDCP1α-GFP were generous gifts from Gregory Hannon [54] (link).
pG3BP-GFP was a generous gift from Jamal Tazi [53] (link).