epitope tag multimer plasmids  (Addgene inc)

Journal: G3: Genes | Genomes | Genetics

Article Title: Multimerized epitope tags for high-sensitivity protein detection

doi: 10.1093/g3journal/jkaf070

Figure Lengend Snippet: Epitope multimerization strategy. ai) Five tandem repeats of the V5 epitope tag were commercially synthesized in the pUC57Kan vector. The gene synthesis plasmids also included unique Asc I and Xho I restriction sites on the upstream side as well as unique Sal I and Not I restriction sites on the downstream side of the tandem repeats. aii) To double the number of V5 repeats, the plasmid was cut with Asc I and Xho I to generate the vector and separately with Asc I and Sal I to create the insert. The vector and insert were ligated together using T4 DNA ligase by virtue of the compatible cohesive ends of Xho I and Sal I . aiii) The resulting plasmid contains 10 in-frame tandem copies of the V5 epitope tag. The hybrid Xho I / Sal I site between the 5XV5 repeats is not recognized by either restriction enzyme, thus regenerating the original configuration of unique restriction sites in ai) and allowing the same cloning strategy to be iteratively repeated with a doubling of the epitope tag copy number with each round of cloning. This cloning strategy was sequentially repeated 4 times to produce plasmids with 10X, 20X, 40X, and 80X tandem epitope tag multimer repeats. This cloning strategy should also be generalizable for multimerizing other types of DNA sequences. b) Reading frames on either side of the epitope tag multimers. To be in frame with the epitope tag coding sequences, codons spanning the Asc I site include an immediately upstream nucleotide and the first 2 base pairs of the Asc I recognition sequence, NGG, followed by CGC and GCC. The reading frame on the Not I side includes the first three codons of the Not I recognition sequence, GCG, followed by GCC, and GCN where N is the next adjacent downstream nucleotide. Note that the open reading frame on the Not I side is open. Carboxy-terminal fusions should thus include a downstream STOP codon. The open reading frame on the downstream Not I side allows the incorporation of epitope tag multimers both at the amino-terminus as well as sites internal to the protein of interest.

Article Snippet: All epitope tag multimer plasmids have been submitted to Addgene for distribution.

Techniques: Synthesized, Plasmid Preparation, Cloning, Sequencing

Journal: G3: Genes | Genomes | Genetics

Article Title: Multimerized epitope tags for high-sensitivity protein detection

doi: 10.1093/g3journal/jkaf070

Figure Lengend Snippet: Genome editing strategy and vGlut epitope multimer-tagged expression in adult Drosophila brain. ai) genomic intron/exon structure of the vGlut gene with exons as rectangles and coding sequences in black. aii) the genome edits to vGlut included a STOP cassette flanked by B2 recombinase target sites (B2RTs) in the intron between exons 6 and 7 and 40 in-frame tandem repeats of either the V5 or MYC epitope tags immediately preceding the TAA stop codon. aiii) conditional expression of vGlut-40XV5 or vGlut-40XMYC results from selective expression of the B2 recombinase in neurons of interest using a GAL4 or split-GAL4 driver in combination with a UAS-B2 recombinase transgene. bi) vGlut-40XV5; bii) Synapsin; biii) overlay; biv) vGlut-40XV5; ci) vGlut-40XMYC; cii) Synapsin; ciii) overlay; civ) vGlut-40XMYC; di) smFLAG-vGlut; dii) Synapsin; diii) overlay; div) smFLAG-vGlut; ei) vGlut; eii) vGlut. Bi, Ci, Di, and Ei images were adjusted post-acquisition to comparable levels of intensity. biv, civ, div, and eii were acquired and processed identically to reveal the relative differences in signal intensity. Scale bar: 100 μm. Complete genotype: b) yw ; B2RT-vGlut-40XV5 GE / + ; c) yw ; B2RT-vGlut-40XMYC GE / + ; d) yw; sm FLAG-vGlut GE / + ; e) yw .

Article Snippet: All epitope tag multimer plasmids have been submitted to Addgene for distribution.

Techniques: Expressing

Journal: G3: Genes | Genomes | Genetics

Article Title: Multimerized epitope tags for high-sensitivity protein detection

doi: 10.1093/g3journal/jkaf070

Figure Lengend Snippet: vGlut epitope multimer expression at the type I and type II neuromuscular junction of third instar larval Drosophila . a) larval type I NMJs at muscle 6/7 boundary. ai) vGlut-40XV5; aii) Synapsin; aiii) overlay; aiv) vGlut-40XV5; av) vGlut-40XMYC; avi) Synapsin; avii) overlay; aviii) vGlut-40XMYC; aix) smFLAG-vGlut; ax) Synapsin; axii) smFLAG-vGlut; axiii) vGlut; axiv) vGlut. Images shown in ai), av), aix), and axiii) were processed post-acquisition to comparable levels of intensity. aiv), aviii), axii), and axiv) were imaged and processed identically to reveal the relative differences in signal intensity. b) larval type II NMJs at muscle 5. bi) vGlut-40XV5; bii) Tdc2; biii) overlay; biv) vGlut-40XV5; bv) vGlut-40XMYC; bvi) Tdc2; bvii) overlay; bviii) vGlut-40XMYC; bix) smFLAG-vGlut; bx) Tdc2; bxi) overlay; bxii) smFLAG-vGlut; bxiii) vGlut; bxiv) Tdc2; bxv) overlay; bxvi) vGlut. Images in bi), bv), bix), an bxiii) were processed post-acquisition to similar levels of signal intensity (to the extent possible). Images shown in biv), bviii), bxii), and bxvi) were acquired and processed identically to reveal the relative differences in signal intensity. Scale bar: 20μm. Complete genotype: ai-iv) yw; B2RT-vGlut-40XV5 GE/+; av-viii) yw; B2RT-vGlut-40XMYC GE/+; aix-xii) yw; smFLAG-vGlut GE/+; axiii, xiv) yw. bi-iv) yw; B2RT-vGlut-40XV5 GE/+; bv-viii) yw; B2RT-vGlut-40XMYC GE/+; bix-xii) yw; smFLAG-vGlut GE/+; bxiii, xiv) yw.

Article Snippet: All epitope tag multimer plasmids have been submitted to Addgene for distribution.

Techniques: Expressing

Journal: G3: Genes | Genomes | Genetics

Article Title: Multimerized epitope tags for high-sensitivity protein detection

doi: 10.1093/g3journal/jkaf070

Figure Lengend Snippet: Epitope tag multimer plasmids. Ethidium bromide-stained agarose gels of Xho I / Not I restriction digests of each epitope tag multimer plasmid. a) ALFA; b) V5; c) OLLAS; d) FLAG; e) MYC; and f) HA. DNA 1 kb ladder plus fragment sizes are indicated on the left side of a) and d). The locations of the sizes of the indicated repeat multimers are indicated on the right side of each gel. The 80X MYC repeat is not entirely stable, e) lane 5. The 80V5, b) lane 5, and 80X OLLAS, c) lane 5, multimer inserts run as a doublet at a similar molecular weight as the vector. Epitope tag multimers were assembled in either the 2.6 kb pUC57Kan plasmid or the 4.6 kb pUC57Kan vGlut donor vector. Some multimer repeats were empirically determined to exhibit greater stability in the latter plasmid. Attempts to generate an 80XHA multimer were not successful.

Article Snippet: All epitope tag multimer plasmids have been submitted to Addgene for distribution.

Techniques: Staining, Plasmid Preparation, Molecular Weight

Journal: G3: Genes | Genomes | Genetics

Article Title: Multimerized epitope tags for high-sensitivity protein detection

doi: 10.1093/g3journal/jkaf070

Figure Lengend Snippet: Epitope multimerization strategy. ai) Five tandem repeats of the V5 epitope tag were commercially synthesized in the pUC57Kan vector. The gene synthesis plasmids also included unique Asc I and Xho I restriction sites on the upstream side as well as unique Sal I and Not I restriction sites on the downstream side of the tandem repeats. aii) To double the number of V5 repeats, the plasmid was cut with Asc I and Xho I to generate the vector and separately with Asc I and Sal I to create the insert. The vector and insert were ligated together using T4 DNA ligase by virtue of the compatible cohesive ends of Xho I and Sal I . aiii) The resulting plasmid contains 10 in-frame tandem copies of the V5 epitope tag. The hybrid Xho I / Sal I site between the 5XV5 repeats is not recognized by either restriction enzyme, thus regenerating the original configuration of unique restriction sites in ai) and allowing the same cloning strategy to be iteratively repeated with a doubling of the epitope tag copy number with each round of cloning. This cloning strategy was sequentially repeated 4 times to produce plasmids with 10X, 20X, 40X, and 80X tandem epitope tag multimer repeats. This cloning strategy should also be generalizable for multimerizing other types of DNA sequences. b) Reading frames on either side of the epitope tag multimers. To be in frame with the epitope tag coding sequences, codons spanning the Asc I site include an immediately upstream nucleotide and the first 2 base pairs of the Asc I recognition sequence, NGG, followed by CGC and GCC. The reading frame on the Not I side includes the first three codons of the Not I recognition sequence, GCG, followed by GCC, and GCN where N is the next adjacent downstream nucleotide. Note that the open reading frame on the Not I side is open. Carboxy-terminal fusions should thus include a downstream STOP codon. The open reading frame on the downstream Not I side allows the incorporation of epitope tag multimers both at the amino-terminus as well as sites internal to the protein of interest.

Article Snippet: A complete list of each epitope tag multimer plasmid and its corresponding Addgene accession number is included in .

Techniques: Synthesized, Plasmid Preparation, Cloning, Sequencing

Journal: G3: Genes | Genomes | Genetics

Article Title: Multimerized epitope tags for high-sensitivity protein detection

doi: 10.1093/g3journal/jkaf070

Figure Lengend Snippet: Genome editing strategy and vGlut epitope multimer-tagged expression in adult Drosophila brain. ai) genomic intron/exon structure of the vGlut gene with exons as rectangles and coding sequences in black. aii) the genome edits to vGlut included a STOP cassette flanked by B2 recombinase target sites (B2RTs) in the intron between exons 6 and 7 and 40 in-frame tandem repeats of either the V5 or MYC epitope tags immediately preceding the TAA stop codon. aiii) conditional expression of vGlut-40XV5 or vGlut-40XMYC results from selective expression of the B2 recombinase in neurons of interest using a GAL4 or split-GAL4 driver in combination with a UAS-B2 recombinase transgene. bi) vGlut-40XV5; bii) Synapsin; biii) overlay; biv) vGlut-40XV5; ci) vGlut-40XMYC; cii) Synapsin; ciii) overlay; civ) vGlut-40XMYC; di) smFLAG-vGlut; dii) Synapsin; diii) overlay; div) smFLAG-vGlut; ei) vGlut; eii) vGlut. Bi, Ci, Di, and Ei images were adjusted post-acquisition to comparable levels of intensity. biv, civ, div, and eii were acquired and processed identically to reveal the relative differences in signal intensity. Scale bar: 100 μm. Complete genotype: b) yw ; B2RT-vGlut-40XV5 GE / + ; c) yw ; B2RT-vGlut-40XMYC GE / + ; d) yw; sm FLAG-vGlut GE / + ; e) yw .

Article Snippet: A complete list of each epitope tag multimer plasmid and its corresponding Addgene accession number is included in .

Techniques: Expressing

Journal: G3: Genes | Genomes | Genetics

Article Title: Multimerized epitope tags for high-sensitivity protein detection

doi: 10.1093/g3journal/jkaf070

Figure Lengend Snippet: vGlut epitope multimer expression at the type I and type II neuromuscular junction of third instar larval Drosophila . a) larval type I NMJs at muscle 6/7 boundary. ai) vGlut-40XV5; aii) Synapsin; aiii) overlay; aiv) vGlut-40XV5; av) vGlut-40XMYC; avi) Synapsin; avii) overlay; aviii) vGlut-40XMYC; aix) smFLAG-vGlut; ax) Synapsin; axii) smFLAG-vGlut; axiii) vGlut; axiv) vGlut. Images shown in ai), av), aix), and axiii) were processed post-acquisition to comparable levels of intensity. aiv), aviii), axii), and axiv) were imaged and processed identically to reveal the relative differences in signal intensity. b) larval type II NMJs at muscle 5. bi) vGlut-40XV5; bii) Tdc2; biii) overlay; biv) vGlut-40XV5; bv) vGlut-40XMYC; bvi) Tdc2; bvii) overlay; bviii) vGlut-40XMYC; bix) smFLAG-vGlut; bx) Tdc2; bxi) overlay; bxii) smFLAG-vGlut; bxiii) vGlut; bxiv) Tdc2; bxv) overlay; bxvi) vGlut. Images in bi), bv), bix), an bxiii) were processed post-acquisition to similar levels of signal intensity (to the extent possible). Images shown in biv), bviii), bxii), and bxvi) were acquired and processed identically to reveal the relative differences in signal intensity. Scale bar: 20μm. Complete genotype: ai-iv) yw; B2RT-vGlut-40XV5 GE/+; av-viii) yw; B2RT-vGlut-40XMYC GE/+; aix-xii) yw; smFLAG-vGlut GE/+; axiii, xiv) yw. bi-iv) yw; B2RT-vGlut-40XV5 GE/+; bv-viii) yw; B2RT-vGlut-40XMYC GE/+; bix-xii) yw; smFLAG-vGlut GE/+; bxiii, xiv) yw.

Article Snippet: A complete list of each epitope tag multimer plasmid and its corresponding Addgene accession number is included in .

Techniques: Expressing

Journal: G3: Genes | Genomes | Genetics

Article Title: Multimerized epitope tags for high-sensitivity protein detection

doi: 10.1093/g3journal/jkaf070

Figure Lengend Snippet: Epitope tag multimer plasmids. Ethidium bromide-stained agarose gels of Xho I / Not I restriction digests of each epitope tag multimer plasmid. a) ALFA; b) V5; c) OLLAS; d) FLAG; e) MYC; and f) HA. DNA 1 kb ladder plus fragment sizes are indicated on the left side of a) and d). The locations of the sizes of the indicated repeat multimers are indicated on the right side of each gel. The 80X MYC repeat is not entirely stable, e) lane 5. The 80V5, b) lane 5, and 80X OLLAS, c) lane 5, multimer inserts run as a doublet at a similar molecular weight as the vector. Epitope tag multimers were assembled in either the 2.6 kb pUC57Kan plasmid or the 4.6 kb pUC57Kan vGlut donor vector. Some multimer repeats were empirically determined to exhibit greater stability in the latter plasmid. Attempts to generate an 80XHA multimer were not successful.

Article Snippet: A complete list of each epitope tag multimer plasmid and its corresponding Addgene accession number is included in .

Techniques: Staining, Plasmid Preparation, Molecular Weight