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  • 99
    New England Biolabs bl21 t7
    Detection of chloroplast-expressed mTurquoise2 in transgenic lines of Marchantia polymorpha. (A) M. polymorpha protein extracts separated by SDS–PAGE. Protein extracts from wild type (WT) and transplastomic (CL0*b) lines of M. polymorpha were separated by 4–12% SDS–PAGE, and visualized by in-gel fluorescence (left) and subsequent Coomassie stain (right). The fluorescence image was generated using a custom imaging device described in the Materials and Methods for visualization of CFP bands (emission 486/10 nm, green) and marker (emission 540/10 nm, red). (B) Untagged and His 6 -tagged mTurquoise2 separated by SDS–PAGE. Untagged mTurquoise2 was expressed from plasmids pCS CL0*b ( psbA promoter) in <t>BL21</t> E. coli and pCRB SREI (T7 promoter) in T7 Express E. coli. His 6 -tagged mTurquoise2 was expressed from plasmid pCRB SREI 6his (T7 promoter) and purified by affinity chromatography. The rightmost lanes contain 50 ng of unboiled and boiled purified protein, respectively. Visualization by in-gel fluorescence and Coomassie stain was conducted as described above. (C) Standard curve for quantification of mTurquoise2 based on in-gel fluorescence. Serial dilutions of 1 µg purified of mTurquoise2 were separated by 4–12% SDS–PAGE and the band intensity quantified by in-gel fluorescence as described in the Materials and Methods. Levels of mTurquoise2 extracted from transplastomic (CL0*b) M. polymorpha (red cross) were estimated by linear regression analysis on densities of fluorescent target bands as shown in (A). Error bars represent the SD of average band density between three different experiments.
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    Thermo Fisher competent e coli
    A; Diagram of the construction of the entry and expression clones. The HepG2 cell line was used as a source of FVII cDNA. Blunt-end PCR product was TOPO-cloned into the pENTR TOPO/D vector. The construct was used to transform competent  E. coli  and positive clones were selected on LB medium containing an appropriate antibiotic. This construct is called the entry clone. Next, LR recombinant reaction was carried out between pENTR TOPO/D-FVII and pDEST26 vector to construct the expression clone. The resulting pDEST26-FVII, which is called the expression clone, was transfected into CHO cells. Stable clones expressing recombinant FVII were established in the presence of geneticin. B; pDEST26 vector. C; N-terminal sequence of the fusion protein.
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    Bio-Rad competent e coli
    A; Diagram of the construction of the entry and expression clones. The HepG2 cell line was used as a source of FVII cDNA. Blunt-end PCR product was TOPO-cloned into the pENTR TOPO/D vector. The construct was used to transform competent  E. coli  and positive clones were selected on LB medium containing an appropriate antibiotic. This construct is called the entry clone. Next, LR recombinant reaction was carried out between pENTR TOPO/D-FVII and pDEST26 vector to construct the expression clone. The resulting pDEST26-FVII, which is called the expression clone, was transfected into CHO cells. Stable clones expressing recombinant FVII were established in the presence of geneticin. B; pDEST26 vector. C; N-terminal sequence of the fusion protein.
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    Meridian Life Science competent e coli
    A; Diagram of the construction of the entry and expression clones. The HepG2 cell line was used as a source of FVII cDNA. Blunt-end PCR product was TOPO-cloned into the pENTR TOPO/D vector. The construct was used to transform competent  E. coli  and positive clones were selected on LB medium containing an appropriate antibiotic. This construct is called the entry clone. Next, LR recombinant reaction was carried out between pENTR TOPO/D-FVII and pDEST26 vector to construct the expression clone. The resulting pDEST26-FVII, which is called the expression clone, was transfected into CHO cells. Stable clones expressing recombinant FVII were established in the presence of geneticin. B; pDEST26 vector. C; N-terminal sequence of the fusion protein.
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    TransGen biotech co competent e coli
    A; Diagram of the construction of the entry and expression clones. The HepG2 cell line was used as a source of FVII cDNA. Blunt-end PCR product was TOPO-cloned into the pENTR TOPO/D vector. The construct was used to transform competent  E. coli  and positive clones were selected on LB medium containing an appropriate antibiotic. This construct is called the entry clone. Next, LR recombinant reaction was carried out between pENTR TOPO/D-FVII and pDEST26 vector to construct the expression clone. The resulting pDEST26-FVII, which is called the expression clone, was transfected into CHO cells. Stable clones expressing recombinant FVII were established in the presence of geneticin. B; pDEST26 vector. C; N-terminal sequence of the fusion protein.
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    Stratagene competent e coli
    A; Diagram of the construction of the entry and expression clones. The HepG2 cell line was used as a source of FVII cDNA. Blunt-end PCR product was TOPO-cloned into the pENTR TOPO/D vector. The construct was used to transform competent  E. coli  and positive clones were selected on LB medium containing an appropriate antibiotic. This construct is called the entry clone. Next, LR recombinant reaction was carried out between pENTR TOPO/D-FVII and pDEST26 vector to construct the expression clone. The resulting pDEST26-FVII, which is called the expression clone, was transfected into CHO cells. Stable clones expressing recombinant FVII were established in the presence of geneticin. B; pDEST26 vector. C; N-terminal sequence of the fusion protein.
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    Avantor competent e coli
    A; Diagram of the construction of the entry and expression clones. The HepG2 cell line was used as a source of FVII cDNA. Blunt-end PCR product was TOPO-cloned into the pENTR TOPO/D vector. The construct was used to transform competent  E. coli  and positive clones were selected on LB medium containing an appropriate antibiotic. This construct is called the entry clone. Next, LR recombinant reaction was carried out between pENTR TOPO/D-FVII and pDEST26 vector to construct the expression clone. The resulting pDEST26-FVII, which is called the expression clone, was transfected into CHO cells. Stable clones expressing recombinant FVII were established in the presence of geneticin. B; pDEST26 vector. C; N-terminal sequence of the fusion protein.
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    Millipore competent e coli rosetta
    A; Diagram of the construction of the entry and expression clones. The HepG2 cell line was used as a source of FVII cDNA. Blunt-end PCR product was TOPO-cloned into the pENTR TOPO/D vector. The construct was used to transform competent  E. coli  and positive clones were selected on LB medium containing an appropriate antibiotic. This construct is called the entry clone. Next, LR recombinant reaction was carried out between pENTR TOPO/D-FVII and pDEST26 vector to construct the expression clone. The resulting pDEST26-FVII, which is called the expression clone, was transfected into CHO cells. Stable clones expressing recombinant FVII were established in the presence of geneticin. B; pDEST26 vector. C; N-terminal sequence of the fusion protein.
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    Thermo Fisher dh10b competent e coli
    A; Diagram of the construction of the entry and expression clones. The HepG2 cell line was used as a source of FVII cDNA. Blunt-end PCR product was TOPO-cloned into the pENTR TOPO/D vector. The construct was used to transform competent  E. coli  and positive clones were selected on LB medium containing an appropriate antibiotic. This construct is called the entry clone. Next, LR recombinant reaction was carried out between pENTR TOPO/D-FVII and pDEST26 vector to construct the expression clone. The resulting pDEST26-FVII, which is called the expression clone, was transfected into CHO cells. Stable clones expressing recombinant FVII were established in the presence of geneticin. B; pDEST26 vector. C; N-terminal sequence of the fusion protein.
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    Thermo Fisher competent e coli dh5α
    A; Diagram of the construction of the entry and expression clones. The HepG2 cell line was used as a source of FVII cDNA. Blunt-end PCR product was TOPO-cloned into the pENTR TOPO/D vector. The construct was used to transform competent  E. coli  and positive clones were selected on LB medium containing an appropriate antibiotic. This construct is called the entry clone. Next, LR recombinant reaction was carried out between pENTR TOPO/D-FVII and pDEST26 vector to construct the expression clone. The resulting pDEST26-FVII, which is called the expression clone, was transfected into CHO cells. Stable clones expressing recombinant FVII were established in the presence of geneticin. B; pDEST26 vector. C; N-terminal sequence of the fusion protein.
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    93
    Promega dh5α competent e coli
    A; Diagram of the construction of the entry and expression clones. The HepG2 cell line was used as a source of FVII cDNA. Blunt-end PCR product was TOPO-cloned into the pENTR TOPO/D vector. The construct was used to transform competent  E. coli  and positive clones were selected on LB medium containing an appropriate antibiotic. This construct is called the entry clone. Next, LR recombinant reaction was carried out between pENTR TOPO/D-FVII and pDEST26 vector to construct the expression clone. The resulting pDEST26-FVII, which is called the expression clone, was transfected into CHO cells. Stable clones expressing recombinant FVII were established in the presence of geneticin. B; pDEST26 vector. C; N-terminal sequence of the fusion protein.
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    90
    TaKaRa competent e coli dh5α
    A; Diagram of the construction of the entry and expression clones. The HepG2 cell line was used as a source of FVII cDNA. Blunt-end PCR product was TOPO-cloned into the pENTR TOPO/D vector. The construct was used to transform competent  E. coli  and positive clones were selected on LB medium containing an appropriate antibiotic. This construct is called the entry clone. Next, LR recombinant reaction was carried out between pENTR TOPO/D-FVII and pDEST26 vector to construct the expression clone. The resulting pDEST26-FVII, which is called the expression clone, was transfected into CHO cells. Stable clones expressing recombinant FVII were established in the presence of geneticin. B; pDEST26 vector. C; N-terminal sequence of the fusion protein.
    Competent E Coli Dh5α, supplied by TaKaRa, used in various techniques. Bioz Stars score: 90/100, based on 85 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    86
    Thermo Fisher dh10bac competent e coli
    A; Diagram of the construction of the entry and expression clones. The HepG2 cell line was used as a source of FVII cDNA. Blunt-end PCR product was TOPO-cloned into the pENTR TOPO/D vector. The construct was used to transform competent  E. coli  and positive clones were selected on LB medium containing an appropriate antibiotic. This construct is called the entry clone. Next, LR recombinant reaction was carried out between pENTR TOPO/D-FVII and pDEST26 vector to construct the expression clone. The resulting pDEST26-FVII, which is called the expression clone, was transfected into CHO cells. Stable clones expressing recombinant FVII were established in the presence of geneticin. B; pDEST26 vector. C; N-terminal sequence of the fusion protein.
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    92
    Genlantis solubl21 competent e coli
    A; Diagram of the construction of the entry and expression clones. The HepG2 cell line was used as a source of FVII cDNA. Blunt-end PCR product was TOPO-cloned into the pENTR TOPO/D vector. The construct was used to transform competent  E. coli  and positive clones were selected on LB medium containing an appropriate antibiotic. This construct is called the entry clone. Next, LR recombinant reaction was carried out between pENTR TOPO/D-FVII and pDEST26 vector to construct the expression clone. The resulting pDEST26-FVII, which is called the expression clone, was transfected into CHO cells. Stable clones expressing recombinant FVII were established in the presence of geneticin. B; pDEST26 vector. C; N-terminal sequence of the fusion protein.
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    Thermo Fisher competent e coli mc1061
    A; Diagram of the construction of the entry and expression clones. The HepG2 cell line was used as a source of FVII cDNA. Blunt-end PCR product was TOPO-cloned into the pENTR TOPO/D vector. The construct was used to transform competent  E. coli  and positive clones were selected on LB medium containing an appropriate antibiotic. This construct is called the entry clone. Next, LR recombinant reaction was carried out between pENTR TOPO/D-FVII and pDEST26 vector to construct the expression clone. The resulting pDEST26-FVII, which is called the expression clone, was transfected into CHO cells. Stable clones expressing recombinant FVII were established in the presence of geneticin. B; pDEST26 vector. C; N-terminal sequence of the fusion protein.
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    Thermo Fisher mc1061 p3 competent e coli
    A; Diagram of the construction of the entry and expression clones. The HepG2 cell line was used as a source of FVII cDNA. Blunt-end PCR product was TOPO-cloned into the pENTR TOPO/D vector. The construct was used to transform competent  E. coli  and positive clones were selected on LB medium containing an appropriate antibiotic. This construct is called the entry clone. Next, LR recombinant reaction was carried out between pENTR TOPO/D-FVII and pDEST26 vector to construct the expression clone. The resulting pDEST26-FVII, which is called the expression clone, was transfected into CHO cells. Stable clones expressing recombinant FVII were established in the presence of geneticin. B; pDEST26 vector. C; N-terminal sequence of the fusion protein.
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    92
    Millipore competent e coli
    A; Diagram of the construction of the entry and expression clones. The HepG2 cell line was used as a source of FVII cDNA. Blunt-end PCR product was TOPO-cloned into the pENTR TOPO/D vector. The construct was used to transform competent  E. coli  and positive clones were selected on LB medium containing an appropriate antibiotic. This construct is called the entry clone. Next, LR recombinant reaction was carried out between pENTR TOPO/D-FVII and pDEST26 vector to construct the expression clone. The resulting pDEST26-FVII, which is called the expression clone, was transfected into CHO cells. Stable clones expressing recombinant FVII were established in the presence of geneticin. B; pDEST26 vector. C; N-terminal sequence of the fusion protein.
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    Thermo Fisher top10 chemically competent e coli
    A; Diagram of the construction of the entry and expression clones. The HepG2 cell line was used as a source of FVII cDNA. Blunt-end PCR product was TOPO-cloned into the pENTR TOPO/D vector. The construct was used to transform competent  E. coli  and positive clones were selected on LB medium containing an appropriate antibiotic. This construct is called the entry clone. Next, LR recombinant reaction was carried out between pENTR TOPO/D-FVII and pDEST26 vector to construct the expression clone. The resulting pDEST26-FVII, which is called the expression clone, was transfected into CHO cells. Stable clones expressing recombinant FVII were established in the presence of geneticin. B; pDEST26 vector. C; N-terminal sequence of the fusion protein.
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    Millipore competent e coli novablue
    A; Diagram of the construction of the entry and expression clones. The HepG2 cell line was used as a source of FVII cDNA. Blunt-end PCR product was TOPO-cloned into the pENTR TOPO/D vector. The construct was used to transform competent  E. coli  and positive clones were selected on LB medium containing an appropriate antibiotic. This construct is called the entry clone. Next, LR recombinant reaction was carried out between pENTR TOPO/D-FVII and pDEST26 vector to construct the expression clone. The resulting pDEST26-FVII, which is called the expression clone, was transfected into CHO cells. Stable clones expressing recombinant FVII were established in the presence of geneticin. B; pDEST26 vector. C; N-terminal sequence of the fusion protein.
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    Thermo Fisher competent e coli bl21
    A; Diagram of the construction of the entry and expression clones. The HepG2 cell line was used as a source of FVII cDNA. Blunt-end PCR product was TOPO-cloned into the pENTR TOPO/D vector. The construct was used to transform competent  E. coli  and positive clones were selected on LB medium containing an appropriate antibiotic. This construct is called the entry clone. Next, LR recombinant reaction was carried out between pENTR TOPO/D-FVII and pDEST26 vector to construct the expression clone. The resulting pDEST26-FVII, which is called the expression clone, was transfected into CHO cells. Stable clones expressing recombinant FVII were established in the presence of geneticin. B; pDEST26 vector. C; N-terminal sequence of the fusion protein.
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    Thermo Fisher competent e coli dh5a
    A; Diagram of the construction of the entry and expression clones. The HepG2 cell line was used as a source of FVII cDNA. Blunt-end PCR product was TOPO-cloned into the pENTR TOPO/D vector. The construct was used to transform competent  E. coli  and positive clones were selected on LB medium containing an appropriate antibiotic. This construct is called the entry clone. Next, LR recombinant reaction was carried out between pENTR TOPO/D-FVII and pDEST26 vector to construct the expression clone. The resulting pDEST26-FVII, which is called the expression clone, was transfected into CHO cells. Stable clones expressing recombinant FVII were established in the presence of geneticin. B; pDEST26 vector. C; N-terminal sequence of the fusion protein.
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    tiangen biotech co competent e coli bl21
    A; Diagram of the construction of the entry and expression clones. The HepG2 cell line was used as a source of FVII cDNA. Blunt-end PCR product was TOPO-cloned into the pENTR TOPO/D vector. The construct was used to transform competent  E. coli  and positive clones were selected on LB medium containing an appropriate antibiotic. This construct is called the entry clone. Next, LR recombinant reaction was carried out between pENTR TOPO/D-FVII and pDEST26 vector to construct the expression clone. The resulting pDEST26-FVII, which is called the expression clone, was transfected into CHO cells. Stable clones expressing recombinant FVII were established in the presence of geneticin. B; pDEST26 vector. C; N-terminal sequence of the fusion protein.
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    Image Search Results


    Detection of chloroplast-expressed mTurquoise2 in transgenic lines of Marchantia polymorpha. (A) M. polymorpha protein extracts separated by SDS–PAGE. Protein extracts from wild type (WT) and transplastomic (CL0*b) lines of M. polymorpha were separated by 4–12% SDS–PAGE, and visualized by in-gel fluorescence (left) and subsequent Coomassie stain (right). The fluorescence image was generated using a custom imaging device described in the Materials and Methods for visualization of CFP bands (emission 486/10 nm, green) and marker (emission 540/10 nm, red). (B) Untagged and His 6 -tagged mTurquoise2 separated by SDS–PAGE. Untagged mTurquoise2 was expressed from plasmids pCS CL0*b ( psbA promoter) in BL21 E. coli and pCRB SREI (T7 promoter) in T7 Express E. coli. His 6 -tagged mTurquoise2 was expressed from plasmid pCRB SREI 6his (T7 promoter) and purified by affinity chromatography. The rightmost lanes contain 50 ng of unboiled and boiled purified protein, respectively. Visualization by in-gel fluorescence and Coomassie stain was conducted as described above. (C) Standard curve for quantification of mTurquoise2 based on in-gel fluorescence. Serial dilutions of 1 µg purified of mTurquoise2 were separated by 4–12% SDS–PAGE and the band intensity quantified by in-gel fluorescence as described in the Materials and Methods. Levels of mTurquoise2 extracted from transplastomic (CL0*b) M. polymorpha (red cross) were estimated by linear regression analysis on densities of fluorescent target bands as shown in (A). Error bars represent the SD of average band density between three different experiments.

    Journal: Plant and Cell Physiology

    Article Title: A Cyan Fluorescent Reporter Expressed from the Chloroplast Genome of Marchantia polymorpha

    doi: 10.1093/pcp/pcv160

    Figure Lengend Snippet: Detection of chloroplast-expressed mTurquoise2 in transgenic lines of Marchantia polymorpha. (A) M. polymorpha protein extracts separated by SDS–PAGE. Protein extracts from wild type (WT) and transplastomic (CL0*b) lines of M. polymorpha were separated by 4–12% SDS–PAGE, and visualized by in-gel fluorescence (left) and subsequent Coomassie stain (right). The fluorescence image was generated using a custom imaging device described in the Materials and Methods for visualization of CFP bands (emission 486/10 nm, green) and marker (emission 540/10 nm, red). (B) Untagged and His 6 -tagged mTurquoise2 separated by SDS–PAGE. Untagged mTurquoise2 was expressed from plasmids pCS CL0*b ( psbA promoter) in BL21 E. coli and pCRB SREI (T7 promoter) in T7 Express E. coli. His 6 -tagged mTurquoise2 was expressed from plasmid pCRB SREI 6his (T7 promoter) and purified by affinity chromatography. The rightmost lanes contain 50 ng of unboiled and boiled purified protein, respectively. Visualization by in-gel fluorescence and Coomassie stain was conducted as described above. (C) Standard curve for quantification of mTurquoise2 based on in-gel fluorescence. Serial dilutions of 1 µg purified of mTurquoise2 were separated by 4–12% SDS–PAGE and the band intensity quantified by in-gel fluorescence as described in the Materials and Methods. Levels of mTurquoise2 extracted from transplastomic (CL0*b) M. polymorpha (red cross) were estimated by linear regression analysis on densities of fluorescent target bands as shown in (A). Error bars represent the SD of average band density between three different experiments.

    Article Snippet: Visualization of proteins by in-gel fluorescence and coomassie staining For total protein extraction from M. polymorpha tissues, 50 mg (fresh weight) of thallus were ground by liquid nitrogen and the resulting powder vortexed in 200 µl of 2 × Tris–glycine for 30 s. For total protein extraction from BL21 or T7 Express competent E. coli (New England Biolabs), an aliquot of overnight culture containing approximately 0.5–1 × 109 cells was pelleted by centrifugation at 14,000 r.p.m. and 4°C for 1 min, resuspended in 50 µl of 2 × Tris–glycine SDS sample buffer and vortexed for 30 s. In both cases, 2-fold dilutions of crude extracts were centrifuged at 14,000 r.p.m. and 4°C for 20 min to remove cell debris, and unboiled (unless indicated otherwise) supernatants were directly loaded onto a NuPAGE Novex 4–12% Bis–Tris protein minigel (Life Technologies).

    Techniques: Transgenic Assay, SDS Page, Fluorescence, Staining, Generated, Imaging, Marker, Plasmid Preparation, Purification, Affinity Chromatography

    A; Diagram of the construction of the entry and expression clones. The HepG2 cell line was used as a source of FVII cDNA. Blunt-end PCR product was TOPO-cloned into the pENTR TOPO/D vector. The construct was used to transform competent  E. coli  and positive clones were selected on LB medium containing an appropriate antibiotic. This construct is called the entry clone. Next, LR recombinant reaction was carried out between pENTR TOPO/D-FVII and pDEST26 vector to construct the expression clone. The resulting pDEST26-FVII, which is called the expression clone, was transfected into CHO cells. Stable clones expressing recombinant FVII were established in the presence of geneticin. B; pDEST26 vector. C; N-terminal sequence of the fusion protein.

    Journal: Blood Transfusion

    Article Title: Expression and purification of recombinant human coagulation factor VII fused to a histidine tag using Gateway technology

    doi: 10.2450/2009.0081-08

    Figure Lengend Snippet: A; Diagram of the construction of the entry and expression clones. The HepG2 cell line was used as a source of FVII cDNA. Blunt-end PCR product was TOPO-cloned into the pENTR TOPO/D vector. The construct was used to transform competent E. coli and positive clones were selected on LB medium containing an appropriate antibiotic. This construct is called the entry clone. Next, LR recombinant reaction was carried out between pENTR TOPO/D-FVII and pDEST26 vector to construct the expression clone. The resulting pDEST26-FVII, which is called the expression clone, was transfected into CHO cells. Stable clones expressing recombinant FVII were established in the presence of geneticin. B; pDEST26 vector. C; N-terminal sequence of the fusion protein.

    Article Snippet: The reaction was then placed on ice and the pENTR TOPO/D-FVII construct was transformed to competent E. coli according to the manufacturer’s protocol (Invitrogen, USA).

    Techniques: Expressing, Clone Assay, Polymerase Chain Reaction, Plasmid Preparation, Construct, Recombinant, Transfection, Sequencing