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Pcr 8 gateway entry vector

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The PCR™8 Gateway entry vector is a plasmid used for cloning DNA sequences into the Gateway entry vector system. It provides a simple and efficient way to transfer DNA fragments between different expression vectors. The vector contains attL1 and attL2 recombination sites that allow for directional cloning of DNA fragments using the Gateway recombination technology.

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Lab products found in correlation

Pcmv sport plasmid, by Thermo Fisher Scientific (3 mentions) Pegfp c1 plasmid, by Takara Bio (3 mentions) Pegfp n1, by Addgene (2 mentions) Pfastbac 6xhis mbp lic expression vector, by Addgene (2 mentions) Pegfp n1, by Takara Bio (1 mentions)

4 protocols using pcr 8 gateway entry vector

1

Rescuing ATAT-2 Expression in C. elegans

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For atat-2 rescues with the native promoter, the atat-2 locus (including promoter, open reading frame, and 3’ UTR) was PCR amplified from N2 genomic DNA. Primer sequences used for cloning are available upon request. For atat-2 rescue with mechanosensory neuron promoters, atat-2 cDNA was amplified from C. elegans RNA and TOPO cloned into pCR8 Gateway entry vector (Invitrogen) to generate pBG-GY896. pBG-GY896 was recombined into a destination vector containing the mec-7 promoter, pBG-GY119, to generate pBG-GY897 (Pmec-7ATAT-2). Site-directed mutagenesis was performed on pBG-GY896 to change two glycine residues into tryptophan (G125W and G127W) resulting in pBG-GY898. Mutation of these conserved glycine residues in the ATAT-2 paralog MEC-17 (G121W and G123W) was previously shown to render MEC-17 catalytically inactive (Topalidou et al., 2012 (link)). After mutagenesis, pBG-GY898 was recombined with the Pmec-7 destination vector to yield pBG-GY899 (Pmec-7ATAT-2 dead). For expression of SYD-2::mScarlet, syd-2 genomic DNA was cloned from C. elegans and TOPO cloned into pCR8 Gateway entry vector to generate pBG-GY699. pBG-GY699 was recombined into a destination vector containing the mec-7 promoter and a C-terminal mScarlet tag, pBG-GY880, to generate pBG-GY936 (Pmec-7SYD-2::mScarlet).
Borgen M.A., Giles A.C., Wang D, & Grill B. (2019). Synapse maintenance is impacted by ATAT-2 tubulin acetyltransferase activity and the RPM-1 signaling hub. eLife, 8, e44040.
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2

Gateway Cloning of Cytoskeletal Proteins

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The full-length human MISP sequence harbored in a pCMV-SPORT plasmid (Harvard PlasmID Database; HsCD00326629) was subcloned by PCR and TOPO-cloned into a pCR™8 Gateway entry vector (Invitrogen; 46–0899). In-frame sequence insertion was confirmed by sequencing. MISP was then shuttled into Gateway-adapted plasmids: pEGFP-C1, pmCherry-C1, and pHALO-C1. Similarly, the human beta-actin and UtrCH sequences were cloned and shuttled into a Gateway adapted HALO-C1 plasmid. To create lentiviral expression vectors, the human MISP sequence was subcloned by PCR and inserted into a puromycin-resistant pLVX1-EGFP backbone by restriction enzyme digestion using XhoI and BamHI. The human fimbrin and villin sequences were cloned into a pEGFP-C1 plasmid (Clontech; 6084–1). The pEGFP-N1 construct harboring the human ezrin sequence was purchased from Addgene, plasmid# 20680. The pEGFP-C1-espin (rat small espin) was a generous gift from Dr. Jim Bartles. To create baculovirus expression vectors, the MISP and EGFP-MISP sequences were subcloned into modified pFastBac-6xHis-MBP LIC expression vector (Addgene; plasmid #30116). All constructs were confirmed by sequencing.
Morales E.A., Arnaiz C., Krystofiak E.S., Zanic M, & Tyska M.J. (2022). Mitotic Spindle Positioning (MISP) is an actin bundler that selectively stabilizes the rootlets of epithelial microvilli. Cell reports, 39(3), 110692.
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3

Gateway Cloning of Cytoskeletal Proteins

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The full-length human MISP sequence harbored in a pCMV-SPORT plasmid (Harvard PlasmID Database; HsCD00326629) was subcloned by PCR and TOPO-cloned into a pCR™8 Gateway entry vector (Invitrogen; 46–0899). In-frame sequence insertion was confirmed by sequencing. MISP was then shuttled into Gateway-adapted plasmids: pEGFP-C1, pmCherry-C1, and pHALO-C1. Similarly, the human beta-actin and UtrCH sequences were cloned and shuttled into a Gateway adapted HALO-C1 plasmid. To create lentiviral expression vectors, the human MISP sequence was subcloned by PCR and inserted into a puromycin-resistant pLVX1-EGFP backbone by restriction enzyme digestion using XhoI and BamHI. The human fimbrin and villin sequences were cloned into a pEGFP-C1 plasmid (Clontech; 6084–1). The pEGFP-N1 construct harboring the human ezrin sequence was purchased from Addgene, plasmid# 20680. The pEGFP-C1-espin (rat small espin) was a generous gift from Dr. Jim Bartles. To create baculovirus expression vectors, the MISP and EGFP-MISP sequences were subcloned into modified pFastBac-6xHis-MBP LIC expression vector (Addgene; plasmid #30116). All constructs were confirmed by sequencing.
Morales E.A., Arnaiz C., Krystofiak E.S., Zanic M, & Tyska M.J. (2022). Mitotic Spindle Positioning (MISP) is an actin bundler that selectively stabilizes the rootlets of epithelial microvilli. Cell reports, 39(3), 110692.
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4

Gateway Cloning of Human Cytoskeletal Proteins

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The human MISP sequence harbored in a pCMV-SPORT plasmid (Harvard PlasmID Database; HsCD00326629) was subcloned by PCR and TOPOcloned into a pCR™8 Gateway entry vector (Invitrogen; 46-0899). In-frame sequence insertion was confirmed by sequencing. MISP was then shuttled into Gateway-adapted plasmids: pEGFP-C1, pmCherry-C1, and pHALO-C1. Similarly, the human beta-actin and UtrCH sequences were cloned and shuttled into a Gateway adapted HALO-C1 plasmid. The human fimbrin sequence was cloned into a pEGFP-C1 plasmid (Clontech; 6084-1). The human ezrin sequence was cloned into a pEGFP-N1 (Clontech; 6085-1). The human MISP and EGFP-MISP sequences were subcloned into modified pFastBac-6xHis-MBP plasmid LIC expression vector (Addgene; plasmid #30116). All constructs were confirmed by sequencing.
Morales E.A., Arnaiz C., Krystofiak E., Žanić M., & Tyska M.J. (2021). Mitotic Spindle Positioning (MISP) is an actin bundler that selectively stabilizes the rootlets of epithelial microvilli.
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