r0560  (New England Biolabs)


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    PmeI
    Description:
    PmeI 2 500 units
    Catalog Number:
    R0560L
    Price:
    302
    Category:
    Restriction Enzymes
    Size:
    2 500 units
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    Structured Review

    New England Biolabs r0560
    PmeI
    PmeI 2 500 units
    https://www.bioz.com/result/r0560/product/New England Biolabs
    Average 99 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    r0560 - by Bioz Stars, 2021-06
    99/100 stars

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    Transformation Assay:

    Article Title: LPS ligand and culture additives improve production of monomeric MD-1 and 2 in Pichia pastoris by decreasing aggregation and intermolecular disulfide bonding
    Article Snippet: .. The empty host vectors pPIC9K and pPICZ〈 were prepared for transformation in parallel, for use as a negative control for MD-1 and MD-2 expression. pPIC9K constructs were linearized by digestion with either SacI , PmeI , or SalI (New England Biolabs). pPICZ〈 constructs were linearized by digestion with SacI or PmeI . ..

    Negative Control:

    Article Title: LPS ligand and culture additives improve production of monomeric MD-1 and 2 in Pichia pastoris by decreasing aggregation and intermolecular disulfide bonding
    Article Snippet: .. The empty host vectors pPIC9K and pPICZ〈 were prepared for transformation in parallel, for use as a negative control for MD-1 and MD-2 expression. pPIC9K constructs were linearized by digestion with either SacI , PmeI , or SalI (New England Biolabs). pPICZ〈 constructs were linearized by digestion with SacI or PmeI . ..

    Expressing:

    Article Title: LPS ligand and culture additives improve production of monomeric MD-1 and 2 in Pichia pastoris by decreasing aggregation and intermolecular disulfide bonding
    Article Snippet: .. The empty host vectors pPIC9K and pPICZ〈 were prepared for transformation in parallel, for use as a negative control for MD-1 and MD-2 expression. pPIC9K constructs were linearized by digestion with either SacI , PmeI , or SalI (New England Biolabs). pPICZ〈 constructs were linearized by digestion with SacI or PmeI . ..

    Construct:

    Article Title: LPS ligand and culture additives improve production of monomeric MD-1 and 2 in Pichia pastoris by decreasing aggregation and intermolecular disulfide bonding
    Article Snippet: .. The empty host vectors pPIC9K and pPICZ〈 were prepared for transformation in parallel, for use as a negative control for MD-1 and MD-2 expression. pPIC9K constructs were linearized by digestion with either SacI , PmeI , or SalI (New England Biolabs). pPICZ〈 constructs were linearized by digestion with SacI or PmeI . ..

    Article Title: Development of a recombinant Newcastle disease virus-vectored vaccine for infectious bronchitis virus variant strains circulating in Egypt
    Article Snippet: Briefly, the codon-optimized IBV S gene—preceded with GE, intergenic sequence (IGS), GS and Kozak sequences, and flanked by PmeI sites—was synthesized and cloned in pUC57 (pUC57-CO.S) by GenScript® (Additional file A). .. To construct rLaSota/wt.S, pUC57-CO.S was digested with PmeI (NEB), and the released insert was gel purified and ligated into full-length rLaSota clone which was predigested by PmeI and dephosphorylated by thermostable shrimp alkaline phosphatase (TSAP) (Promega). .. To construct rLaSota/S(Y1145A) + Fct12 , primer sets (1 and 2) and (3 and 4) were used to induce the “Y1145A-mutation” and add the last 12 aa of NDV F protein using pUC57-CO.S as template (Table ).

    Purification:

    Article Title: Development of a recombinant Newcastle disease virus-vectored vaccine for infectious bronchitis virus variant strains circulating in Egypt
    Article Snippet: Briefly, the codon-optimized IBV S gene—preceded with GE, intergenic sequence (IGS), GS and Kozak sequences, and flanked by PmeI sites—was synthesized and cloned in pUC57 (pUC57-CO.S) by GenScript® (Additional file A). .. To construct rLaSota/wt.S, pUC57-CO.S was digested with PmeI (NEB), and the released insert was gel purified and ligated into full-length rLaSota clone which was predigested by PmeI and dephosphorylated by thermostable shrimp alkaline phosphatase (TSAP) (Promega). .. To construct rLaSota/S(Y1145A) + Fct12 , primer sets (1 and 2) and (3 and 4) were used to induce the “Y1145A-mutation” and add the last 12 aa of NDV F protein using pUC57-CO.S as template (Table ).

    Polymerase Chain Reaction:

    Article Title: Human pluripotent stem cell modeling of tuberous sclerosis complex reveals lineage-specific therapeutic vulnerabilities
    Article Snippet: Agarose gel electrophoresis was utilized to resolve digested amplicons indicating successful integration of ‘stop codon’ donor sequence. .. PCR amplicons were then digested with PmeI restriction enzyme (New England BioLabs #R0560S) in OneTaq buffer for 1h at 37°C. .. Agarose gel electrophoresis was utilized to resolve digested amplicons indicating successful integration of ‘stop codon’ donor sequence.

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    New England Biolabs pme i
    Multigene cloning with Plant X-tender expression vectors. Two expression cassettes were cloned into pCAMBIA_ASX and introduced into N . benthamiana . (A-F) Scheme of cloning procedure. (A) Amplification of expression cassette from template plasmid using primers with appropriate 5’ and 3’ extension homologies in the case of p35S::H2BRFP_tNOS expression cassette. PCR amplification of subunits (pNOS, ECFP, t35S) u sing custom-designed primers with appropriate 5’ extensions to add overlaps between the individual subunits and chosen Level 0 plasmid in the case of pNOS::ECFP_t35S expression cassette. (B) Assembly of subunits into Hin dIII digested Level 0 vectors by NEBuilder HiFi assembly method. Only the restriction of Level 0 vector with A0/A1 homology regions is shown. (C) Assembled cassettes flanked by homology regions were released from the backbone using <t>Pme</t> I. (D) Assembly of expression cassettes into Pac I digested Level 1 vector by TAR or NEBuilder HiFi. (E) Release of the multigene construct from Level 1 vector using I- Sce I homing endonuclease, cutting outside the homology regions A0 and B0. (F) Assembly of two expression cassettes and yeast selection marker ( URA3 ) into Hin dIII digested Plant X-tender expression vectors with SLiCE of NEBuilder HiFi. (G–J) Images of agroinfiltrated N . benthamiana leaves obtained by laser scanning confocal microscopy. Leaves were agroinfiltrated with agrobacteria containing pCAMBIA_ASX_multigene (upper panel) or with empty A . tumefaciens (bottom panel). (G) Nuclear localisation of RFP. Fluorescence is represented as a maximum projection of z-stacks. (H) ECFP is localised in the cytoplasm. Fluorescence is represented as maximum projections of z-stacks. (I) Bright field. (J) Overlay of G, H and I. Scale bars are 100 μm. p35S: cauliflower mosaic virus CaMV 35S promoter, H2BRFP: histon sequence fused to red fluorescence protein (mRFP1), tNOS: nopaline synthase terminator, pNOS: nopaline synthase promoter, ECFP: cyan fluorescent protein, t35S: cauliflower mosaic virus CaMV 35S terminator, A0, A1 AR, B0: homology regions, Rp: selection marker conferring hygromycin resistance in plants, Re: selection marker conferring kanamycin resistance in E . coli and A . tumefaciens , Amp: selection marker conferring ampicillin resistance in E . coli and A . tumefaciens , Kan: selection marker conferring kanamycin resistance in E . coli and A . tumefaciens , LB: left border of T-DNA, RB: right border of T-DNA, Hin dIII, I- Sce I, Pac I, Asc I, Sbf I, Swa I, Fse I, Pme I: restriction enzyme recognition sites, URA3 : yeast selection marker, ccd B: bacterial suicide gene, SLiCE: Seamless ligation cloning extract cloning method, HiFi: NEBuilder HiFi DNA assembly method, Gibson: Gibson DNA assembly method. TAR: cloning based on transformation-associated recombination, PCR: Polymerase chain reaction, ASX: Plant X-tender expression vector.
    Pme I, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Multigene cloning with Plant X-tender expression vectors. Two expression cassettes were cloned into pCAMBIA_ASX and introduced into N . benthamiana . (A-F) Scheme of cloning procedure. (A) Amplification of expression cassette from template plasmid using primers with appropriate 5’ and 3’ extension homologies in the case of p35S::H2BRFP_tNOS expression cassette. PCR amplification of subunits (pNOS, ECFP, t35S) u sing custom-designed primers with appropriate 5’ extensions to add overlaps between the individual subunits and chosen Level 0 plasmid in the case of pNOS::ECFP_t35S expression cassette. (B) Assembly of subunits into Hin dIII digested Level 0 vectors by NEBuilder HiFi assembly method. Only the restriction of Level 0 vector with A0/A1 homology regions is shown. (C) Assembled cassettes flanked by homology regions were released from the backbone using Pme I. (D) Assembly of expression cassettes into Pac I digested Level 1 vector by TAR or NEBuilder HiFi. (E) Release of the multigene construct from Level 1 vector using I- Sce I homing endonuclease, cutting outside the homology regions A0 and B0. (F) Assembly of two expression cassettes and yeast selection marker ( URA3 ) into Hin dIII digested Plant X-tender expression vectors with SLiCE of NEBuilder HiFi. (G–J) Images of agroinfiltrated N . benthamiana leaves obtained by laser scanning confocal microscopy. Leaves were agroinfiltrated with agrobacteria containing pCAMBIA_ASX_multigene (upper panel) or with empty A . tumefaciens (bottom panel). (G) Nuclear localisation of RFP. Fluorescence is represented as a maximum projection of z-stacks. (H) ECFP is localised in the cytoplasm. Fluorescence is represented as maximum projections of z-stacks. (I) Bright field. (J) Overlay of G, H and I. Scale bars are 100 μm. p35S: cauliflower mosaic virus CaMV 35S promoter, H2BRFP: histon sequence fused to red fluorescence protein (mRFP1), tNOS: nopaline synthase terminator, pNOS: nopaline synthase promoter, ECFP: cyan fluorescent protein, t35S: cauliflower mosaic virus CaMV 35S terminator, A0, A1 AR, B0: homology regions, Rp: selection marker conferring hygromycin resistance in plants, Re: selection marker conferring kanamycin resistance in E . coli and A . tumefaciens , Amp: selection marker conferring ampicillin resistance in E . coli and A . tumefaciens , Kan: selection marker conferring kanamycin resistance in E . coli and A . tumefaciens , LB: left border of T-DNA, RB: right border of T-DNA, Hin dIII, I- Sce I, Pac I, Asc I, Sbf I, Swa I, Fse I, Pme I: restriction enzyme recognition sites, URA3 : yeast selection marker, ccd B: bacterial suicide gene, SLiCE: Seamless ligation cloning extract cloning method, HiFi: NEBuilder HiFi DNA assembly method, Gibson: Gibson DNA assembly method. TAR: cloning based on transformation-associated recombination, PCR: Polymerase chain reaction, ASX: Plant X-tender expression vector.

    Journal: PLoS ONE

    Article Title: Plant X-tender: An extension of the AssemblX system for the assembly and expression of multigene constructs in plants

    doi: 10.1371/journal.pone.0190526

    Figure Lengend Snippet: Multigene cloning with Plant X-tender expression vectors. Two expression cassettes were cloned into pCAMBIA_ASX and introduced into N . benthamiana . (A-F) Scheme of cloning procedure. (A) Amplification of expression cassette from template plasmid using primers with appropriate 5’ and 3’ extension homologies in the case of p35S::H2BRFP_tNOS expression cassette. PCR amplification of subunits (pNOS, ECFP, t35S) u sing custom-designed primers with appropriate 5’ extensions to add overlaps between the individual subunits and chosen Level 0 plasmid in the case of pNOS::ECFP_t35S expression cassette. (B) Assembly of subunits into Hin dIII digested Level 0 vectors by NEBuilder HiFi assembly method. Only the restriction of Level 0 vector with A0/A1 homology regions is shown. (C) Assembled cassettes flanked by homology regions were released from the backbone using Pme I. (D) Assembly of expression cassettes into Pac I digested Level 1 vector by TAR or NEBuilder HiFi. (E) Release of the multigene construct from Level 1 vector using I- Sce I homing endonuclease, cutting outside the homology regions A0 and B0. (F) Assembly of two expression cassettes and yeast selection marker ( URA3 ) into Hin dIII digested Plant X-tender expression vectors with SLiCE of NEBuilder HiFi. (G–J) Images of agroinfiltrated N . benthamiana leaves obtained by laser scanning confocal microscopy. Leaves were agroinfiltrated with agrobacteria containing pCAMBIA_ASX_multigene (upper panel) or with empty A . tumefaciens (bottom panel). (G) Nuclear localisation of RFP. Fluorescence is represented as a maximum projection of z-stacks. (H) ECFP is localised in the cytoplasm. Fluorescence is represented as maximum projections of z-stacks. (I) Bright field. (J) Overlay of G, H and I. Scale bars are 100 μm. p35S: cauliflower mosaic virus CaMV 35S promoter, H2BRFP: histon sequence fused to red fluorescence protein (mRFP1), tNOS: nopaline synthase terminator, pNOS: nopaline synthase promoter, ECFP: cyan fluorescent protein, t35S: cauliflower mosaic virus CaMV 35S terminator, A0, A1 AR, B0: homology regions, Rp: selection marker conferring hygromycin resistance in plants, Re: selection marker conferring kanamycin resistance in E . coli and A . tumefaciens , Amp: selection marker conferring ampicillin resistance in E . coli and A . tumefaciens , Kan: selection marker conferring kanamycin resistance in E . coli and A . tumefaciens , LB: left border of T-DNA, RB: right border of T-DNA, Hin dIII, I- Sce I, Pac I, Asc I, Sbf I, Swa I, Fse I, Pme I: restriction enzyme recognition sites, URA3 : yeast selection marker, ccd B: bacterial suicide gene, SLiCE: Seamless ligation cloning extract cloning method, HiFi: NEBuilder HiFi DNA assembly method, Gibson: Gibson DNA assembly method. TAR: cloning based on transformation-associated recombination, PCR: Polymerase chain reaction, ASX: Plant X-tender expression vector.

    Article Snippet: Expression cassettes were released from Level 0 vectors using Pme I (NEB), separated by agarose gel electrophoresis and purified from the gel as described above.

    Techniques: Clone Assay, Expressing, Amplification, Plasmid Preparation, Polymerase Chain Reaction, Construct, Selection, Marker, Confocal Microscopy, Fluorescence, Sequencing, Ligation, Transformation Assay

    Functional evaluation of constructed vectors by cloning expression cassette p35S::H2BRFP_tNOS into Plant X-tender expression vectors. (A-F) Scheme of the cloning procedure. (A) Amplification of expression cassette from template plasmid using primers with appropriate 5’ and 3’ extensions to add A0 and AR homology regions. (B) Expression cassette assembly in Hin dIII restricted pL0A_0-R Level 0 vector by NEBuilder HiFi assembly method. (C) Release of expression cassette with flanking homology regions A0 and AR from Level 0 vector by Pme I digestion. (D) Assembly of expression cassette with flanking homology regions A0 and AR into Pac I digested pL1A-hc / pL1A-lc (A0/AR) Level 1 vector by TAR or NEBuilder HiFi. (E) Release of expression cassette flanked by URA3 yeast selection marker and homology regions A0 and B0 from Level 1 vector by I- Sce I digestion. (F) Assembly of expression cassette flanked by URA3 yeast selection marker and homology regions A0 and B0 into Plant X-tender expression vectors by SLiCE or NEBuilder HiFi. (G-I) Images of agroinfiltrated N . benthamiana leaves obtained by laser scanning confocal microscopy. Leaves were agroinfiltrated with agrobacteria containing pCAMBIA_ASX_cassette, pK7WG_ASX_cassette, pH7WG_ASX_cassette, pB7WG_ASX_cassette or empty agrobacteria (top to bottom). (G) Nuclear localisation of RFP. Fluorescence is represented as maximum projections of z-stacks. (H) Bright field. (I) Overlay of G with H. Scale bars are 100 μm. p35S: cauliflower mosaic virus CaMV 35S promoter, H2BRFP: histon sequence fused to red fluorescence protein (mRFP1), tNOS: nopaline synthase terminator, A0, AR, B0: homology regions, Rp: selection marker conferring resistance in plants (hygromycin in the case of pCAMBIA_ASX and pH7WG_ASX, kanamycin in the case of pK7WG_ASX, glufosinate-ammonium in the case of pB7WG_ASX), Re: selection marker conferring resistance in E . coli and A . tumefaciens (kanamycin in the case of pCAMBIA_ASX, spectinomycinin in the case of pK7WG_ASX, pH7WG_ASX and pB7WG_ASX), Amp: selection marker conferring ampicillin resistance in E . coli and A . tumefaciens , Kan: selection marker conferring kanamycin resistance in E . coli and A . tumefaciens , LB: left border of T-DNA, RB: right border of T-DNA, Hin dIII, I- Sce I, Pac I, Pme I: restriction enzyme recognition sites, URA3 : yeast selection marker, ccd B: bacterial suicide gene, SLiCE: Seamless ligation cloning extract cloning method, HiFi: NEBuilder HiFi DNA assembly method, Gibson: Gibson DNA assembly method, TAR: cloning based on transformation-associated recombination, PCR: Polymerase chain reaction, ASX: Plant X-tender expression vector.

    Journal: PLoS ONE

    Article Title: Plant X-tender: An extension of the AssemblX system for the assembly and expression of multigene constructs in plants

    doi: 10.1371/journal.pone.0190526

    Figure Lengend Snippet: Functional evaluation of constructed vectors by cloning expression cassette p35S::H2BRFP_tNOS into Plant X-tender expression vectors. (A-F) Scheme of the cloning procedure. (A) Amplification of expression cassette from template plasmid using primers with appropriate 5’ and 3’ extensions to add A0 and AR homology regions. (B) Expression cassette assembly in Hin dIII restricted pL0A_0-R Level 0 vector by NEBuilder HiFi assembly method. (C) Release of expression cassette with flanking homology regions A0 and AR from Level 0 vector by Pme I digestion. (D) Assembly of expression cassette with flanking homology regions A0 and AR into Pac I digested pL1A-hc / pL1A-lc (A0/AR) Level 1 vector by TAR or NEBuilder HiFi. (E) Release of expression cassette flanked by URA3 yeast selection marker and homology regions A0 and B0 from Level 1 vector by I- Sce I digestion. (F) Assembly of expression cassette flanked by URA3 yeast selection marker and homology regions A0 and B0 into Plant X-tender expression vectors by SLiCE or NEBuilder HiFi. (G-I) Images of agroinfiltrated N . benthamiana leaves obtained by laser scanning confocal microscopy. Leaves were agroinfiltrated with agrobacteria containing pCAMBIA_ASX_cassette, pK7WG_ASX_cassette, pH7WG_ASX_cassette, pB7WG_ASX_cassette or empty agrobacteria (top to bottom). (G) Nuclear localisation of RFP. Fluorescence is represented as maximum projections of z-stacks. (H) Bright field. (I) Overlay of G with H. Scale bars are 100 μm. p35S: cauliflower mosaic virus CaMV 35S promoter, H2BRFP: histon sequence fused to red fluorescence protein (mRFP1), tNOS: nopaline synthase terminator, A0, AR, B0: homology regions, Rp: selection marker conferring resistance in plants (hygromycin in the case of pCAMBIA_ASX and pH7WG_ASX, kanamycin in the case of pK7WG_ASX, glufosinate-ammonium in the case of pB7WG_ASX), Re: selection marker conferring resistance in E . coli and A . tumefaciens (kanamycin in the case of pCAMBIA_ASX, spectinomycinin in the case of pK7WG_ASX, pH7WG_ASX and pB7WG_ASX), Amp: selection marker conferring ampicillin resistance in E . coli and A . tumefaciens , Kan: selection marker conferring kanamycin resistance in E . coli and A . tumefaciens , LB: left border of T-DNA, RB: right border of T-DNA, Hin dIII, I- Sce I, Pac I, Pme I: restriction enzyme recognition sites, URA3 : yeast selection marker, ccd B: bacterial suicide gene, SLiCE: Seamless ligation cloning extract cloning method, HiFi: NEBuilder HiFi DNA assembly method, Gibson: Gibson DNA assembly method, TAR: cloning based on transformation-associated recombination, PCR: Polymerase chain reaction, ASX: Plant X-tender expression vector.

    Article Snippet: Expression cassettes were released from Level 0 vectors using Pme I (NEB), separated by agarose gel electrophoresis and purified from the gel as described above.

    Techniques: Functional Assay, Construct, Clone Assay, Expressing, Amplification, Plasmid Preparation, Selection, Marker, Confocal Microscopy, Fluorescence, Sequencing, Ligation, Transformation Assay, Polymerase Chain Reaction

    Plant X-tender cloning strategy. Diagram showing example of assembly of two expression cassettes into a plant expression vector using Plant X-tender. Definition of parts and design of Level 0 units is done using GenoCAD. Design of multigene cassettes and computation of primers is performed using the AssemblX webtool. (A-D) Assembly of two expression cassettes into a Level 1 vector. (A) PCR amplification of subunits (e.g. promoter, CDS, terminator) using custom-designed primers with appropriate 5’ extensions to add overlaps between the individual subunits and chosen Level 0 plasmid. (B) Assembly of subunits into Hin dIII digested Level 0 vectors via overlap-based assembly methods. Only the restriction of Level 0 vector with A0/A1 homology regions is shown. (C) Assembled cassettes flanked by homology regions are released from the backbone using one of five rare 8-base cutter recognition sites ( Asc I, Sbf I, Swa I, Fsa I, Pme I) flanking the homology regions. (D) Assembly of expression cassettes into Pac I digested Level 1 vector by of the preferred overlap-based assembly method. (E-G) Multigene assembly into Plant X-tender expression vector. (E) Digestion with I- Sce I allows the release of a multigene construct flanked by homology regions A0 and B0 from the Level 1 AssemblX vector. (F) Hin dIII digestion enables the linearization of Plant X-tender expression vector and the release of ccd B cassette prior the assembly. (G) Assembly of a multigene construct and a yeast selection marker ( URA3 ) flanked by homology regions into Plant X-tender expression vector by overlap-based methods exploiting homologous recombination between the homology regions A0 and B0 of the Plant X-tender expression vector and the homology regions A0 and B0 of the insert. A0, A1, AR, B0: homology regions, Hin dIII, I- Sce I, Pac I, Asc I, Sbf I, Swa I, Fse I, Pme I: restriction enzyme recognition sites, Rp: selection marker conferring resistance in plants, Re: selection marker conferring resistance in E . coli and A . tumefaciens , Amp: selection marker conferring ampicillin resistance in E . coli and A . tumefaciens , Kan: selection marker conferring kanamycin resistance in E . coli and A . tumefaciens , URA3 : yeast selection marker, LB: left border of T-DNA, RB: right border of T-DNA, ccd B: bacterial suicide gene, SLiCE: Seamless ligation cloning extract cloning method, HiFi: HiFi DNA assembly method, Gibson: Gibson DNA assembly method, TAR: cloning based on transformation-associated recombination, PCR: Polymerase chain reaction, CDS: coding sequence, ASX: Plant X-tender expression vector.

    Journal: PLoS ONE

    Article Title: Plant X-tender: An extension of the AssemblX system for the assembly and expression of multigene constructs in plants

    doi: 10.1371/journal.pone.0190526

    Figure Lengend Snippet: Plant X-tender cloning strategy. Diagram showing example of assembly of two expression cassettes into a plant expression vector using Plant X-tender. Definition of parts and design of Level 0 units is done using GenoCAD. Design of multigene cassettes and computation of primers is performed using the AssemblX webtool. (A-D) Assembly of two expression cassettes into a Level 1 vector. (A) PCR amplification of subunits (e.g. promoter, CDS, terminator) using custom-designed primers with appropriate 5’ extensions to add overlaps between the individual subunits and chosen Level 0 plasmid. (B) Assembly of subunits into Hin dIII digested Level 0 vectors via overlap-based assembly methods. Only the restriction of Level 0 vector with A0/A1 homology regions is shown. (C) Assembled cassettes flanked by homology regions are released from the backbone using one of five rare 8-base cutter recognition sites ( Asc I, Sbf I, Swa I, Fsa I, Pme I) flanking the homology regions. (D) Assembly of expression cassettes into Pac I digested Level 1 vector by of the preferred overlap-based assembly method. (E-G) Multigene assembly into Plant X-tender expression vector. (E) Digestion with I- Sce I allows the release of a multigene construct flanked by homology regions A0 and B0 from the Level 1 AssemblX vector. (F) Hin dIII digestion enables the linearization of Plant X-tender expression vector and the release of ccd B cassette prior the assembly. (G) Assembly of a multigene construct and a yeast selection marker ( URA3 ) flanked by homology regions into Plant X-tender expression vector by overlap-based methods exploiting homologous recombination between the homology regions A0 and B0 of the Plant X-tender expression vector and the homology regions A0 and B0 of the insert. A0, A1, AR, B0: homology regions, Hin dIII, I- Sce I, Pac I, Asc I, Sbf I, Swa I, Fse I, Pme I: restriction enzyme recognition sites, Rp: selection marker conferring resistance in plants, Re: selection marker conferring resistance in E . coli and A . tumefaciens , Amp: selection marker conferring ampicillin resistance in E . coli and A . tumefaciens , Kan: selection marker conferring kanamycin resistance in E . coli and A . tumefaciens , URA3 : yeast selection marker, LB: left border of T-DNA, RB: right border of T-DNA, ccd B: bacterial suicide gene, SLiCE: Seamless ligation cloning extract cloning method, HiFi: HiFi DNA assembly method, Gibson: Gibson DNA assembly method, TAR: cloning based on transformation-associated recombination, PCR: Polymerase chain reaction, CDS: coding sequence, ASX: Plant X-tender expression vector.

    Article Snippet: Expression cassettes were released from Level 0 vectors using Pme I (NEB), separated by agarose gel electrophoresis and purified from the gel as described above.

    Techniques: Clone Assay, Expressing, Plasmid Preparation, Polymerase Chain Reaction, Amplification, Construct, Selection, Marker, Homologous Recombination, Ligation, Transformation Assay, Sequencing

    Schematic illustration of the arrangement of the P-SSP7 genome. (A) Sequencing of the ends of the P-SSP7 genome extracted directly from phage particles. Arrows, and numbers under the arrows, indicate the sequences acquired: Blue from the entire genome and green from end fragments produced by digestion of the genome with the BamHI and PmeI restriction enzymes. The positions of the primers used for sequencing are shown in black type at the beginning of the arrows. Genome numbering for the primers and sequences is that for the originally published sequence [5] . The purple line denotes the 728 bp region found to be upstream of ORF1 in this study, but positioned downstream of ORF54 in the originally published sequence. The repeat regions are shown in red at both ends of the genome. (B) Diagram showing the arrangement of the P-SSP7 genome as originally published (GenBank accession numbers: AY939843.1, [5] and GU071093 [16] . (C) Diagram of the revised genome arrangement based on the results from this study (updated GeneBank submission, accession number: AY939843.2).

    Journal: PLoS ONE

    Article Title: The P-SSP7 Cyanophage Has a Linear Genome with Direct Terminal Repeats

    doi: 10.1371/journal.pone.0036710

    Figure Lengend Snippet: Schematic illustration of the arrangement of the P-SSP7 genome. (A) Sequencing of the ends of the P-SSP7 genome extracted directly from phage particles. Arrows, and numbers under the arrows, indicate the sequences acquired: Blue from the entire genome and green from end fragments produced by digestion of the genome with the BamHI and PmeI restriction enzymes. The positions of the primers used for sequencing are shown in black type at the beginning of the arrows. Genome numbering for the primers and sequences is that for the originally published sequence [5] . The purple line denotes the 728 bp region found to be upstream of ORF1 in this study, but positioned downstream of ORF54 in the originally published sequence. The repeat regions are shown in red at both ends of the genome. (B) Diagram showing the arrangement of the P-SSP7 genome as originally published (GenBank accession numbers: AY939843.1, [5] and GU071093 [16] . (C) Diagram of the revised genome arrangement based on the results from this study (updated GeneBank submission, accession number: AY939843.2).

    Article Snippet: The DNA (0.5 µg per reaction) was digested with BamHI and with a combination of BamHI and PmeI (New England Biolabs).

    Techniques: Sequencing, Produced

    Digestion and Southern analyses of the P-SSP7 genome. (A) Schematic genome map showing the positions of the restriction enzyme cleavage sites (red) and the expected fragment sizes after digestion with BamHI alone (top) and both BamHI and PmeI (bottom) based on the revised genome arrangement shown in Fig. 1C. (B) Restriction digestion of the P-SSP7 genome extracted from phage particles (lanes 3 and 4) and the genome cloned into a fosmid (lanes 5 and 6), with BamHI alone (lanes 3 and 5) or with BamHI and PmeI (lanes 4 and 6), separated by pulse field gel electrophoresis. Note that the only difference for digestion of the cloned genome is the presence of an additional fragment corresponding to the size of the fosmid vector. Fragments corresponding to the expected sizes shown in (A) are marked with the appropriate letter designations (a to f). Fragment size markers (M): 1 kb DNA ladder (lane 1) and Lambda DNA cut with HindIII (lane 2), are shown. (C) Southern analyses of the restriction digested DNA in (B) using 4 probes (denoted above the lanes) show that the repeat region appears twice on the genome on the same fragments as the first and last ORFs. The positions of the gene probes on the genome are shown as light blue boxes and the repeat region probe as green boxes in the top panel of (A). Lane numbering and fragment designations are the same as in (B).

    Journal: PLoS ONE

    Article Title: The P-SSP7 Cyanophage Has a Linear Genome with Direct Terminal Repeats

    doi: 10.1371/journal.pone.0036710

    Figure Lengend Snippet: Digestion and Southern analyses of the P-SSP7 genome. (A) Schematic genome map showing the positions of the restriction enzyme cleavage sites (red) and the expected fragment sizes after digestion with BamHI alone (top) and both BamHI and PmeI (bottom) based on the revised genome arrangement shown in Fig. 1C. (B) Restriction digestion of the P-SSP7 genome extracted from phage particles (lanes 3 and 4) and the genome cloned into a fosmid (lanes 5 and 6), with BamHI alone (lanes 3 and 5) or with BamHI and PmeI (lanes 4 and 6), separated by pulse field gel electrophoresis. Note that the only difference for digestion of the cloned genome is the presence of an additional fragment corresponding to the size of the fosmid vector. Fragments corresponding to the expected sizes shown in (A) are marked with the appropriate letter designations (a to f). Fragment size markers (M): 1 kb DNA ladder (lane 1) and Lambda DNA cut with HindIII (lane 2), are shown. (C) Southern analyses of the restriction digested DNA in (B) using 4 probes (denoted above the lanes) show that the repeat region appears twice on the genome on the same fragments as the first and last ORFs. The positions of the gene probes on the genome are shown as light blue boxes and the repeat region probe as green boxes in the top panel of (A). Lane numbering and fragment designations are the same as in (B).

    Article Snippet: The DNA (0.5 µg per reaction) was digested with BamHI and with a combination of BamHI and PmeI (New England Biolabs).

    Techniques: Clone Assay, Nucleic Acid Electrophoresis, Plasmid Preparation, Lambda DNA Preparation

    The replication profile is altered at the endogenous CFS-FRA16D locus in the absence of FANCD2 , providing additional information about fosmids and primers used to identify the regions. (B) Locus map of RR1- PmeI segment containing a portion of the AT-rich fragility core. The segments are aligned according to the positions of the FISH probes (blue) on the map. (C–F) Top; Locus map of PmeI digested RR1 segment. Middle; Aligned photomicrograph images of labeled DNA molecules from (C) Non-affected 1 (GM02184), (D) FANCD2 −/− -L-1 (PD20), (E) FANCD2 −/− -L-2 (2742) and (F) FANCD2 −/− . Bottom; The percentage of molecules incorporating IdU (red) is calculated from the replication program (middle) and is represented as a histogram.

    Journal: Molecular cell

    Article Title: FANCD2 facilitates replication through common fragile sites

    doi: 10.1016/j.molcel.2016.09.017

    Figure Lengend Snippet: The replication profile is altered at the endogenous CFS-FRA16D locus in the absence of FANCD2 , providing additional information about fosmids and primers used to identify the regions. (B) Locus map of RR1- PmeI segment containing a portion of the AT-rich fragility core. The segments are aligned according to the positions of the FISH probes (blue) on the map. (C–F) Top; Locus map of PmeI digested RR1 segment. Middle; Aligned photomicrograph images of labeled DNA molecules from (C) Non-affected 1 (GM02184), (D) FANCD2 −/− -L-1 (PD20), (E) FANCD2 −/− -L-2 (2742) and (F) FANCD2 −/− . Bottom; The percentage of molecules incorporating IdU (red) is calculated from the replication program (middle) and is represented as a histogram.

    Article Snippet: To prepare the cells for restriction enzyme digestion, the plugs were washed with 10 mM MgCl2 and 10 mM Tris-HCl (pH 8.0) and the genomic DNA in the gel plugs was digested with 80 units of PmeI (New England BioLabs Inc.) at 37°C overnight.

    Techniques: Fluorescence In Situ Hybridization, Labeling

    Generation of a TSC2 −/− hPSC library (A) Schematic summary of TSC stem cell modeling strategy. (B) Homologous recombination strategy to knockout TSC2 utilized an ssODN containing a ‘STOP-codon’ donor sequence containing the unique restriction site, PmeI. PmeI digestion of PCR amplicons containing the target cut site reveals homozygous integration of the donor sequence in TSC2 −/− hPSC lines. (C) Imaging flow cytometry image capture following staining with TSC2 antibody revealing clonal populations of TSC2 −/− cells. (D) Quantification of fluorescence intensity of pluripotency markers OCT4, SOX2, and NANOG in each hPSC line under maintenance conditions, normalized to respective parental WT hPSC lines. Values are the mean ± SEM (n = 12; 3x for each cell line). No statistical significance between WT and TSC −/− samples was observed. (E) Haematoxylins and eosin staining of H9 WT and TSC2 −/− teratoma explants. Arrows indicate examples of immature tissues of ectodermal, endodermal, and mesodermal origin. Scale bar, 100µm. (F) Schematic of expected phosphorylation status of mTORC1 effectors under supportive and stress conditions. Western blots probing phosphorylated mTOR (P-mTOR), phosphorylated S6K (P-S6K), and phosphorylated S6 (P-S6) of samples treated for 6h under no treatment, 1% O 2 , and +100nM rapamycin conditions. Densitometry quantification of western blots displaying the ratio of phosphorylated to total protein of mTOR, S6K, and S6 (n = 8; 2x for each cell line). Statistical significance was determined using two-way ANOVA and Tukey’s post hoc analysis. (G) Schematic representation of monolayer-NPC and EB-NCC differentiation protocols. Red asterisks indicate time points harvested for RNA sequencing.

    Journal: bioRxiv

    Article Title: Human pluripotent stem cell modeling of tuberous sclerosis complex reveals lineage-specific therapeutic vulnerabilities

    doi: 10.1101/683359

    Figure Lengend Snippet: Generation of a TSC2 −/− hPSC library (A) Schematic summary of TSC stem cell modeling strategy. (B) Homologous recombination strategy to knockout TSC2 utilized an ssODN containing a ‘STOP-codon’ donor sequence containing the unique restriction site, PmeI. PmeI digestion of PCR amplicons containing the target cut site reveals homozygous integration of the donor sequence in TSC2 −/− hPSC lines. (C) Imaging flow cytometry image capture following staining with TSC2 antibody revealing clonal populations of TSC2 −/− cells. (D) Quantification of fluorescence intensity of pluripotency markers OCT4, SOX2, and NANOG in each hPSC line under maintenance conditions, normalized to respective parental WT hPSC lines. Values are the mean ± SEM (n = 12; 3x for each cell line). No statistical significance between WT and TSC −/− samples was observed. (E) Haematoxylins and eosin staining of H9 WT and TSC2 −/− teratoma explants. Arrows indicate examples of immature tissues of ectodermal, endodermal, and mesodermal origin. Scale bar, 100µm. (F) Schematic of expected phosphorylation status of mTORC1 effectors under supportive and stress conditions. Western blots probing phosphorylated mTOR (P-mTOR), phosphorylated S6K (P-S6K), and phosphorylated S6 (P-S6) of samples treated for 6h under no treatment, 1% O 2 , and +100nM rapamycin conditions. Densitometry quantification of western blots displaying the ratio of phosphorylated to total protein of mTOR, S6K, and S6 (n = 8; 2x for each cell line). Statistical significance was determined using two-way ANOVA and Tukey’s post hoc analysis. (G) Schematic representation of monolayer-NPC and EB-NCC differentiation protocols. Red asterisks indicate time points harvested for RNA sequencing.

    Article Snippet: PCR amplicons were then digested with PmeI restriction enzyme (New England BioLabs #R0560S) in OneTaq buffer for 1h at 37°C.

    Techniques: Homologous Recombination, Knock-Out, Sequencing, Polymerase Chain Reaction, Imaging, Flow Cytometry, Staining, Fluorescence, Western Blot, RNA Sequencing Assay