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24x ms2v6 cassettes  (Addgene inc)


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    Structured Review

    Addgene inc 24x ms2v6 cassettes
    (A) Vectors to append homology regions upstream and downstream of the <t>MS2V6</t> repeats; vc591 <t>(24x</t> MS2V6) and vc592 (12x MS2V6) are slightly modified versions of Addgene plasmids # 104393 and # 104392 (Tutucci et al., Nat Methods 2017). An EcoRI site was added downstream of the MS2V6 repeats. Homology regions can be integrated at the BamHI, EcoRI, and XhoI sites. (B,C,D) Strategies to integrate the 12-24x MS2V6 repeats into the genome: (B) CRISPR/Cas9-facilitated integration without resistance gene; (C) replacement of a counterselectable cassette, e.g. when working with a non-essential gene; (D) integration using the kanMX resistance gene present on the vector.
    24x Ms2v6 Cassettes, supplied by Addgene inc, used in various techniques. Bioz Stars score: 93/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/product/kanr/pET264-pUC+24xMS2V6+Loxp+KANr+Loxp+(Plasmid+%23104393)/bio_rxiv__64898__2026__03__09__710516-100-15-23
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    Images

    1) Product Images from "Establishing MS2-MCP-based single-molecule RNA visualization in Schizosaccharomyces pombe"

    Article Title: Establishing MS2-MCP-based single-molecule RNA visualization in Schizosaccharomyces pombe

    Journal: bioRxiv

    doi: 10.64898/2026.03.09.710516

    (A) Vectors to append homology regions upstream and downstream of the MS2V6 repeats; vc591 (24x MS2V6) and vc592 (12x MS2V6) are slightly modified versions of Addgene plasmids # 104393 and # 104392 (Tutucci et al., Nat Methods 2017). An EcoRI site was added downstream of the MS2V6 repeats. Homology regions can be integrated at the BamHI, EcoRI, and XhoI sites. (B,C,D) Strategies to integrate the 12-24x MS2V6 repeats into the genome: (B) CRISPR/Cas9-facilitated integration without resistance gene; (C) replacement of a counterselectable cassette, e.g. when working with a non-essential gene; (D) integration using the kanMX resistance gene present on the vector.
    Figure Legend Snippet: (A) Vectors to append homology regions upstream and downstream of the MS2V6 repeats; vc591 (24x MS2V6) and vc592 (12x MS2V6) are slightly modified versions of Addgene plasmids # 104393 and # 104392 (Tutucci et al., Nat Methods 2017). An EcoRI site was added downstream of the MS2V6 repeats. Homology regions can be integrated at the BamHI, EcoRI, and XhoI sites. (B,C,D) Strategies to integrate the 12-24x MS2V6 repeats into the genome: (B) CRISPR/Cas9-facilitated integration without resistance gene; (C) replacement of a counterselectable cassette, e.g. when working with a non-essential gene; (D) integration using the kanMX resistance gene present on the vector.

    Techniques Used: Modification, CRISPR, Plasmid Preparation

    (A) The mad2 gene was tagged in the 3’UTR with 24x MS2V6, and MCP-tdSG was expressed from different promoters. (B) Short kymographs from live-cell imaging; longer sequences are shown in Fig. S3. For each MCP-tdSG construct, a strain without integration of MS2 repeats is shown as control. Far left: strain expressing mad2 -24xMS2, but no MCP-tdSG. Images are maximum intensity projections of the Z-stack. Note the different scaling setting for P.cdc2.short-MCP-tdSG. (C) Quantification of the maximum spot intensity over maximum background intensity. Two replicates per strain, 56-162 spots per replicate. Points: individual spots; box plot: summary statistics (center line, median; box boundaries, 1st and 3rd quartiles). (D) The cdc13 gene was internally tagged with circularly permuted superfolder GFP (sfGFPcp) and 12x or 24x MS2V6 cassettes were inserted in the 3’UTR. The construct was expressed under endogenous cdc13 regulatory sequences (promoter, terminator) from the leu1 locus. MCP-tdSG was expressed from the mad3 promoter. (E) Schematic illustrating Cdc13 protein localization. Cdc13 accumulates during interphase and is strongly enriched in the nucleus. Cdc13 localizes to spindle pole bodies and the spindle during early mitosis and becomes degraded at the metaphase-to-anaphase transition. (F,G) Kymographs from live-cell imaging of cells undergoing mitosis; cdc13 -sfGFP tagged with either 24x MS2V6 (F) or 12x MS2V6 (G). A strain without MS2 repeats is shown as control. Images were recorded every 5 sec (F) or 12 sec (G); every second image (every 10 sec, F) or every 10 th image (every 2 min, G) is shown. Images are maximum intensity projections of the Z-stack. (H) Similar to (G), but showing an interphase cell; loss of the nuclear sfGFPcp signal is due to photobleaching, not degradation.
    Figure Legend Snippet: (A) The mad2 gene was tagged in the 3’UTR with 24x MS2V6, and MCP-tdSG was expressed from different promoters. (B) Short kymographs from live-cell imaging; longer sequences are shown in Fig. S3. For each MCP-tdSG construct, a strain without integration of MS2 repeats is shown as control. Far left: strain expressing mad2 -24xMS2, but no MCP-tdSG. Images are maximum intensity projections of the Z-stack. Note the different scaling setting for P.cdc2.short-MCP-tdSG. (C) Quantification of the maximum spot intensity over maximum background intensity. Two replicates per strain, 56-162 spots per replicate. Points: individual spots; box plot: summary statistics (center line, median; box boundaries, 1st and 3rd quartiles). (D) The cdc13 gene was internally tagged with circularly permuted superfolder GFP (sfGFPcp) and 12x or 24x MS2V6 cassettes were inserted in the 3’UTR. The construct was expressed under endogenous cdc13 regulatory sequences (promoter, terminator) from the leu1 locus. MCP-tdSG was expressed from the mad3 promoter. (E) Schematic illustrating Cdc13 protein localization. Cdc13 accumulates during interphase and is strongly enriched in the nucleus. Cdc13 localizes to spindle pole bodies and the spindle during early mitosis and becomes degraded at the metaphase-to-anaphase transition. (F,G) Kymographs from live-cell imaging of cells undergoing mitosis; cdc13 -sfGFP tagged with either 24x MS2V6 (F) or 12x MS2V6 (G). A strain without MS2 repeats is shown as control. Images were recorded every 5 sec (F) or 12 sec (G); every second image (every 10 sec, F) or every 10 th image (every 2 min, G) is shown. Images are maximum intensity projections of the Z-stack. (H) Similar to (G), but showing an interphase cell; loss of the nuclear sfGFPcp signal is due to photobleaching, not degradation.

    Techniques Used: Live Cell Imaging, Construct, Control, Expressing

    Related Articles

    Sequencing:

    Article Title: Orthogonal Versatile Interacting Peptide Tags for Imaging Cellular Proteins.
    Article Snippet: .. E. coli Strains Characteristics Source Top10 F- mcrA Δ(mrr-hsdRMS-mcrBC) Φ80lacZΔM15 Δ lacX74 recA1 araD139 Δ(araleu)7697 galU galK rpsL (StrR) endA1 nupG Thermo Fisher Scientific BL21(DE3) F- ompT hsdSB (rBmB-) gal dcm (DE3) Thermo Fisher Scientific NEB-5alpha fhuA2Δ(argF-lacZ)U169 phoA glnV44 Φ80Δ(lacZ)M15 gyrA96 recA1 relA1 endA1 thi-1 hsdR17 New England Biolabs Plasmids Characteristics Source pET28b (+) T7 promoter, His-tag coding sequence, MCS, lacI coding sequence, (KanR) Novagen pcDNA3.1 CMV promoter, MCS, BGH polyadenylation signal, SV40 origin, (AmpR, KanR) Thermo Fisher Sci mCherry-TOMM20-N-10 CMV promoter, TOMM20, mCherry (C terminal on backbone), (KanR, NeoR) Addgene: 55146 H2B-6-mEmerald CMV promoter, HIST1H2BJ, mEmerald (C terminal on backbone), (KanR,NeoR) Addgene: 54111 mEmerald-Actin-C-18 CMV promoter, actin, mEmerald (N terminal on the backbone), (KanR, NeoR) Addgene: 53978 .. Fluorophore Vendor Excitation Maximum Emission Maximum Quantum Yield Extinction Coefficient (ε) Correction Factor280 Sulfo-Cyanine5-Maleimide (Sulfo-Cy5) Lumiprobe 646 nm 662 nm 0.28 271,000 0.04 Sulfo-Cyanine3-Maleimide (Sulfo-Cy3) Lumiprobe 548 nm 563 nm 0.1 162,000 0.06 AlexaFluor 488 C5 Maleimide (AF488) Thermo Fisher Sci 493 nm 516 nm 0.92 72,000 0.11 Table S4: Summary of probe peptides.

    Polymerase Chain Reaction:

    Article Title: A supernumerary synthetic chromosome in Komagataella phaffii as a repository for extraneous genetic material.
    Article Snippet: .. To achieve this, the 13.5-kb SphI-digested and gel-extracted eDA83 was ligated with a 980-bp PCR-amplified KanR (using oligos 321/322 and vector pUC57-Kan (Addgene) as a template). ..

    CRISPR:

    Article Title: A versatile one-step CRISPR-Cas9 based approach to plasmid-curing
    Article Snippet: .. Plasmids used in this study Oligo ID Description Reference/source pMAZ-SK CRISPR array and tracrRNA expression plasmid, CRISPR array under control of rhamnose- inducible promoter and tracrRNA constitutively expressed, KanR [1] / Addgene #73962 pMA7CR_2.0 Cas9 expression plasmid, Cas9 gene expressed under control of inducible aTc promoter, AmpR [1] / Addgene #73950 pZS4Int-tetR Tetracycline repressor expression plasmid, constitutively expressed, SpecR EXPRESSSYS [1] pFREE Curing plasmid - aTc inducible expression of Cas9 and rhamnose inducible expression of CRISPR array. ..

    Expressing:

    Article Title: A versatile one-step CRISPR-Cas9 based approach to plasmid-curing
    Article Snippet: .. Plasmids used in this study Oligo ID Description Reference/source pMAZ-SK CRISPR array and tracrRNA expression plasmid, CRISPR array under control of rhamnose- inducible promoter and tracrRNA constitutively expressed, KanR [1] / Addgene #73962 pMA7CR_2.0 Cas9 expression plasmid, Cas9 gene expressed under control of inducible aTc promoter, AmpR [1] / Addgene #73950 pZS4Int-tetR Tetracycline repressor expression plasmid, constitutively expressed, SpecR EXPRESSSYS [1] pFREE Curing plasmid - aTc inducible expression of Cas9 and rhamnose inducible expression of CRISPR array. ..

    Plasmid Preparation:

    Article Title: A versatile one-step CRISPR-Cas9 based approach to plasmid-curing
    Article Snippet: .. Plasmids used in this study Oligo ID Description Reference/source pMAZ-SK CRISPR array and tracrRNA expression plasmid, CRISPR array under control of rhamnose- inducible promoter and tracrRNA constitutively expressed, KanR [1] / Addgene #73962 pMA7CR_2.0 Cas9 expression plasmid, Cas9 gene expressed under control of inducible aTc promoter, AmpR [1] / Addgene #73950 pZS4Int-tetR Tetracycline repressor expression plasmid, constitutively expressed, SpecR EXPRESSSYS [1] pFREE Curing plasmid - aTc inducible expression of Cas9 and rhamnose inducible expression of CRISPR array. ..

    Control:

    Article Title: A versatile one-step CRISPR-Cas9 based approach to plasmid-curing
    Article Snippet: .. Plasmids used in this study Oligo ID Description Reference/source pMAZ-SK CRISPR array and tracrRNA expression plasmid, CRISPR array under control of rhamnose- inducible promoter and tracrRNA constitutively expressed, KanR [1] / Addgene #73962 pMA7CR_2.0 Cas9 expression plasmid, Cas9 gene expressed under control of inducible aTc promoter, AmpR [1] / Addgene #73950 pZS4Int-tetR Tetracycline repressor expression plasmid, constitutively expressed, SpecR EXPRESSSYS [1] pFREE Curing plasmid - aTc inducible expression of Cas9 and rhamnose inducible expression of CRISPR array. ..



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    Image Search Results


    (A) Vectors to append homology regions upstream and downstream of the MS2V6 repeats; vc591 (24x MS2V6) and vc592 (12x MS2V6) are slightly modified versions of Addgene plasmids # 104393 and # 104392 (Tutucci et al., Nat Methods 2017). An EcoRI site was added downstream of the MS2V6 repeats. Homology regions can be integrated at the BamHI, EcoRI, and XhoI sites. (B,C,D) Strategies to integrate the 12-24x MS2V6 repeats into the genome: (B) CRISPR/Cas9-facilitated integration without resistance gene; (C) replacement of a counterselectable cassette, e.g. when working with a non-essential gene; (D) integration using the kanMX resistance gene present on the vector.

    Journal: bioRxiv

    Article Title: Establishing MS2-MCP-based single-molecule RNA visualization in Schizosaccharomyces pombe

    doi: 10.64898/2026.03.09.710516

    Figure Lengend Snippet: (A) Vectors to append homology regions upstream and downstream of the MS2V6 repeats; vc591 (24x MS2V6) and vc592 (12x MS2V6) are slightly modified versions of Addgene plasmids # 104393 and # 104392 (Tutucci et al., Nat Methods 2017). An EcoRI site was added downstream of the MS2V6 repeats. Homology regions can be integrated at the BamHI, EcoRI, and XhoI sites. (B,C,D) Strategies to integrate the 12-24x MS2V6 repeats into the genome: (B) CRISPR/Cas9-facilitated integration without resistance gene; (C) replacement of a counterselectable cassette, e.g. when working with a non-essential gene; (D) integration using the kanMX resistance gene present on the vector.

    Article Snippet: The vectors containing 12x MSV6 (pET251-pUC 12xMS2V6 Loxp KANr Loxp, Addgene plasmid # 104392) and 24x MS2V6 cassettes (pET264-pUC 24xMS2V6 Loxp KANr Loxp, Addgene plasmid # 104393) were a gift from Robert Singer and Evelina Tutucci ( ).

    Techniques: Modification, CRISPR, Plasmid Preparation

    (A) The mad2 gene was tagged in the 3’UTR with 24x MS2V6, and MCP-tdSG was expressed from different promoters. (B) Short kymographs from live-cell imaging; longer sequences are shown in Fig. S3. For each MCP-tdSG construct, a strain without integration of MS2 repeats is shown as control. Far left: strain expressing mad2 -24xMS2, but no MCP-tdSG. Images are maximum intensity projections of the Z-stack. Note the different scaling setting for P.cdc2.short-MCP-tdSG. (C) Quantification of the maximum spot intensity over maximum background intensity. Two replicates per strain, 56-162 spots per replicate. Points: individual spots; box plot: summary statistics (center line, median; box boundaries, 1st and 3rd quartiles). (D) The cdc13 gene was internally tagged with circularly permuted superfolder GFP (sfGFPcp) and 12x or 24x MS2V6 cassettes were inserted in the 3’UTR. The construct was expressed under endogenous cdc13 regulatory sequences (promoter, terminator) from the leu1 locus. MCP-tdSG was expressed from the mad3 promoter. (E) Schematic illustrating Cdc13 protein localization. Cdc13 accumulates during interphase and is strongly enriched in the nucleus. Cdc13 localizes to spindle pole bodies and the spindle during early mitosis and becomes degraded at the metaphase-to-anaphase transition. (F,G) Kymographs from live-cell imaging of cells undergoing mitosis; cdc13 -sfGFP tagged with either 24x MS2V6 (F) or 12x MS2V6 (G). A strain without MS2 repeats is shown as control. Images were recorded every 5 sec (F) or 12 sec (G); every second image (every 10 sec, F) or every 10 th image (every 2 min, G) is shown. Images are maximum intensity projections of the Z-stack. (H) Similar to (G), but showing an interphase cell; loss of the nuclear sfGFPcp signal is due to photobleaching, not degradation.

    Journal: bioRxiv

    Article Title: Establishing MS2-MCP-based single-molecule RNA visualization in Schizosaccharomyces pombe

    doi: 10.64898/2026.03.09.710516

    Figure Lengend Snippet: (A) The mad2 gene was tagged in the 3’UTR with 24x MS2V6, and MCP-tdSG was expressed from different promoters. (B) Short kymographs from live-cell imaging; longer sequences are shown in Fig. S3. For each MCP-tdSG construct, a strain without integration of MS2 repeats is shown as control. Far left: strain expressing mad2 -24xMS2, but no MCP-tdSG. Images are maximum intensity projections of the Z-stack. Note the different scaling setting for P.cdc2.short-MCP-tdSG. (C) Quantification of the maximum spot intensity over maximum background intensity. Two replicates per strain, 56-162 spots per replicate. Points: individual spots; box plot: summary statistics (center line, median; box boundaries, 1st and 3rd quartiles). (D) The cdc13 gene was internally tagged with circularly permuted superfolder GFP (sfGFPcp) and 12x or 24x MS2V6 cassettes were inserted in the 3’UTR. The construct was expressed under endogenous cdc13 regulatory sequences (promoter, terminator) from the leu1 locus. MCP-tdSG was expressed from the mad3 promoter. (E) Schematic illustrating Cdc13 protein localization. Cdc13 accumulates during interphase and is strongly enriched in the nucleus. Cdc13 localizes to spindle pole bodies and the spindle during early mitosis and becomes degraded at the metaphase-to-anaphase transition. (F,G) Kymographs from live-cell imaging of cells undergoing mitosis; cdc13 -sfGFP tagged with either 24x MS2V6 (F) or 12x MS2V6 (G). A strain without MS2 repeats is shown as control. Images were recorded every 5 sec (F) or 12 sec (G); every second image (every 10 sec, F) or every 10 th image (every 2 min, G) is shown. Images are maximum intensity projections of the Z-stack. (H) Similar to (G), but showing an interphase cell; loss of the nuclear sfGFPcp signal is due to photobleaching, not degradation.

    Article Snippet: The vectors containing 12x MSV6 (pET251-pUC 12xMS2V6 Loxp KANr Loxp, Addgene plasmid # 104392) and 24x MS2V6 cassettes (pET264-pUC 24xMS2V6 Loxp KANr Loxp, Addgene plasmid # 104393) were a gift from Robert Singer and Evelina Tutucci ( ).

    Techniques: Live Cell Imaging, Construct, Control, Expressing

    (A) Vectors to append homology regions upstream and downstream of the MS2V6 repeats; vc591 (24x MS2V6) and vc592 (12x MS2V6) are slightly modified versions of Addgene plasmids # 104393 and # 104392 (Tutucci et al., Nat Methods 2017). An EcoRI site was added downstream of the MS2V6 repeats. Homology regions can be integrated at the BamHI, EcoRI, and XhoI sites. (B,C,D) Strategies to integrate the 12-24x MS2V6 repeats into the genome: (B) CRISPR/Cas9-facilitated integration without resistance gene; (C) replacement of a counterselectable cassette, e.g. when working with a non-essential gene; (D) integration using the kanMX resistance gene present on the vector.

    Journal: bioRxiv

    Article Title: Establishing MS2-MCP-based single-molecule RNA visualization in Schizosaccharomyces pombe

    doi: 10.64898/2026.03.09.710516

    Figure Lengend Snippet: (A) Vectors to append homology regions upstream and downstream of the MS2V6 repeats; vc591 (24x MS2V6) and vc592 (12x MS2V6) are slightly modified versions of Addgene plasmids # 104393 and # 104392 (Tutucci et al., Nat Methods 2017). An EcoRI site was added downstream of the MS2V6 repeats. Homology regions can be integrated at the BamHI, EcoRI, and XhoI sites. (B,C,D) Strategies to integrate the 12-24x MS2V6 repeats into the genome: (B) CRISPR/Cas9-facilitated integration without resistance gene; (C) replacement of a counterselectable cassette, e.g. when working with a non-essential gene; (D) integration using the kanMX resistance gene present on the vector.

    Article Snippet: The vectors containing 12x MSV6 (pET251-pUC 12xMS2V6 Loxp KANr Loxp, Addgene plasmid # 104392) and 24x MS2V6 cassettes (pET264-pUC 24xMS2V6 Loxp KANr Loxp, Addgene plasmid # 104393) were a gift from Robert Singer and Evelina Tutucci ( ).

    Techniques: Modification, CRISPR, Plasmid Preparation

    (A) The mad2 gene was tagged in the 3’UTR with 24x MS2V6, and MCP-tdSG was expressed from different promoters. (B) Short kymographs from live-cell imaging; longer sequences are shown in Fig. S3. For each MCP-tdSG construct, a strain without integration of MS2 repeats is shown as control. Far left: strain expressing mad2 -24xMS2, but no MCP-tdSG. Images are maximum intensity projections of the Z-stack. Note the different scaling setting for P.cdc2.short-MCP-tdSG. (C) Quantification of the maximum spot intensity over maximum background intensity. Two replicates per strain, 56-162 spots per replicate. Points: individual spots; box plot: summary statistics (center line, median; box boundaries, 1st and 3rd quartiles). (D) The cdc13 gene was internally tagged with circularly permuted superfolder GFP (sfGFPcp) and 12x or 24x MS2V6 cassettes were inserted in the 3’UTR. The construct was expressed under endogenous cdc13 regulatory sequences (promoter, terminator) from the leu1 locus. MCP-tdSG was expressed from the mad3 promoter. (E) Schematic illustrating Cdc13 protein localization. Cdc13 accumulates during interphase and is strongly enriched in the nucleus. Cdc13 localizes to spindle pole bodies and the spindle during early mitosis and becomes degraded at the metaphase-to-anaphase transition. (F,G) Kymographs from live-cell imaging of cells undergoing mitosis; cdc13 -sfGFP tagged with either 24x MS2V6 (F) or 12x MS2V6 (G). A strain without MS2 repeats is shown as control. Images were recorded every 5 sec (F) or 12 sec (G); every second image (every 10 sec, F) or every 10 th image (every 2 min, G) is shown. Images are maximum intensity projections of the Z-stack. (H) Similar to (G), but showing an interphase cell; loss of the nuclear sfGFPcp signal is due to photobleaching, not degradation.

    Journal: bioRxiv

    Article Title: Establishing MS2-MCP-based single-molecule RNA visualization in Schizosaccharomyces pombe

    doi: 10.64898/2026.03.09.710516

    Figure Lengend Snippet: (A) The mad2 gene was tagged in the 3’UTR with 24x MS2V6, and MCP-tdSG was expressed from different promoters. (B) Short kymographs from live-cell imaging; longer sequences are shown in Fig. S3. For each MCP-tdSG construct, a strain without integration of MS2 repeats is shown as control. Far left: strain expressing mad2 -24xMS2, but no MCP-tdSG. Images are maximum intensity projections of the Z-stack. Note the different scaling setting for P.cdc2.short-MCP-tdSG. (C) Quantification of the maximum spot intensity over maximum background intensity. Two replicates per strain, 56-162 spots per replicate. Points: individual spots; box plot: summary statistics (center line, median; box boundaries, 1st and 3rd quartiles). (D) The cdc13 gene was internally tagged with circularly permuted superfolder GFP (sfGFPcp) and 12x or 24x MS2V6 cassettes were inserted in the 3’UTR. The construct was expressed under endogenous cdc13 regulatory sequences (promoter, terminator) from the leu1 locus. MCP-tdSG was expressed from the mad3 promoter. (E) Schematic illustrating Cdc13 protein localization. Cdc13 accumulates during interphase and is strongly enriched in the nucleus. Cdc13 localizes to spindle pole bodies and the spindle during early mitosis and becomes degraded at the metaphase-to-anaphase transition. (F,G) Kymographs from live-cell imaging of cells undergoing mitosis; cdc13 -sfGFP tagged with either 24x MS2V6 (F) or 12x MS2V6 (G). A strain without MS2 repeats is shown as control. Images were recorded every 5 sec (F) or 12 sec (G); every second image (every 10 sec, F) or every 10 th image (every 2 min, G) is shown. Images are maximum intensity projections of the Z-stack. (H) Similar to (G), but showing an interphase cell; loss of the nuclear sfGFPcp signal is due to photobleaching, not degradation.

    Article Snippet: The vectors containing 12x MSV6 (pET251-pUC 12xMS2V6 Loxp KANr Loxp, Addgene plasmid # 104392) and 24x MS2V6 cassettes (pET264-pUC 24xMS2V6 Loxp KANr Loxp, Addgene plasmid # 104393) were a gift from Robert Singer and Evelina Tutucci ( ).

    Techniques: Live Cell Imaging, Construct, Control, Expressing