Peasy uni seamless cloning and assembly kit

Manufactured by Transgene

Sourced in China

The PEASY-Uni Seamless Cloning and Assembly Kit is a laboratory tool designed for the efficient and seamless cloning and assembly of DNA fragments. It provides a streamlined process for constructing recombinant DNA molecules.

Automatically generated - may contain errors

Lab products found in correlation

Dual luciferase reporter assay system, by Promega (2 mentions) Pet32 plasmid, by Merck Group (2 mentions) Lipofectamine 2000, by Thermo Fisher Scientific (2 mentions) Peasy blunt simple cloning vector, by Transgene (1 mentions) Phusion high fidelity pcr master mix, by New England Biolabs (1 mentions) Polybrene, by Merck Group (1 mentions) Spectinomycin, by Takara Bio (1 mentions) One step cloning kit, by Vazyme (1 mentions) Lsm 5 pascal confocal laser scanning microscopy, by Zeiss (1 mentions) Transstarfastpfu fly dna polymerase, by Transgene (1 mentions) Fast mutagenesis system, by Transgene (1 mentions) Fastdigest xhoi, by Thermo Fisher Scientific (1 mentions) L lactic acid, by Merck Group (1 mentions) D lactic acid, by Merck Group (1 mentions) Fastpfu dna polymerase, by Transgene (1 mentions) Synergy 2 multi mode reader, by Agilent Technologies (1 mentions) Ptk rl, by Promega (1 mentions) Mitotracker orange, by Thermo Fisher Scientific (1 mentions) Tcs sp8 confocal fluorescence microscope, by Leica (1 mentions) Primerscript rt reagent kit, by Takara Bio (1 mentions)

52 protocols using peasy uni seamless cloning and assembly kit

Knockout of AOL_s00188g306 in Arthrobotrys oligospora

Check if the same lab product or an alternative is used in the 5 most similar protocols

AOL_s00188g306 of A. oligospora was knocked out using protoplast transformation based on homologous recombination (Si et al., 2022 (link)). The hygromycin-resistant gene (hygR) was selected as the screening marker gene, and knockout plasmids with the hygR screening marker gene were constructed using the pEASY-Uni-seamless Cloning and Assembly kit (Transgen Biotech, Beijing, China).
The genomic DNA of A. oligospora was extracted using a Fungal Genome Extraction Kit (Solarbio, Beijing, China). The upstream and downstream homologous arm sequences of g306 were amplified using primers g306-3F/g306-3R and g306-5F/g306-5R (Supplementary Table S1) with genomic DNA as a template. The hygR fragment was amplified from the pUC57-HygR plasmid using the primers Hph-F/Hph-F (Supplementary Table S1). The pUC19 plasmid was double digested using restriction enzymes XbaI and BamHI (Transgen Biotech, Beijing, China), and the three fragments and linearized pUC19 plasmid were linked using a pEASY-Uni-seamless Cloning and Assembly kit (Transgen Biotech). The ligation product was transformed into E. coli DH5α, and the recombinant plasmid (pUC19-g306KO-hygR) was screened on LB plates containing ampicillin and verified via sequencing.

Gu T., Lu H., Liu H., Zhang G, & Wang Y. (2023). Function discovery of a non-ribosomal peptide synthetase-like encoding gene in the nematode-trapping fungus Arthrobotrys oligospora. Frontiers in Microbiology, 14, 1210288.

+ Open protocol

+ Expand

Construction and Transformation of PAA3 Plasmids

Check if the same lab product or an alternative is used in the 5 most similar protocols

To construct the complementation plasmid, a 10725 bp genomic fragment that contained the PAA3 coding sequence, coupled with the 3251 bp upstream and 1068 bp downstream sequences, was amplified using the primers PAA3-com-F (EcoR1) and PAA3-com-R (HindIII). The fragment was inserted into the binary vector pCAMBIA1301 using the pEASY ® -Uni Seamless Cloning and Assembly Kit (TransGen, Beijing, China) . The recombinant plasmids were transformed into the paa3 mutant using the Agrobacterium tumefaciens-mediated transformation method as described previously (Hui et al., 2020) . For the PAA3P::GUS assays, the promoter of PAA3 gene (3138 bp) was amplified using the primers PAA3P-GUS-F (EcoR1) and PAA3P-GUS-R (Nco1). The fragment was inserted into the binary vector pCAMBIA1301 using the pEASY ® -Uni Seamless Cloning and Assembly Kit (TransGen, Beijing, China) . The recombinant plasmids were transformed into the japonica cultivar ZHONGHUA 11 (ZH11) using the A. tumefaciens-mediated transformation method as described previously (Hui et al., 2020) . The primers used for vector construction are listed in Table S1.

Yang F., Mao X., Huang M., Li Z., Wang Z., Zhu H., Chen R., Lu L., Cheng Q., Wang Y., Tang J., Zhuang H., & Yang F. (2021). PANICLE APICAL ABORTION 3 Controls Panicle Development and Seed Size in Rice.

+ Open protocol

+ Expand

Constructing MoGSK1-GFP and Fgk3-GFP Fusion Proteins

Check if the same lab product or an alternative is used in the 5 most similar protocols

To construct MoGSK1-GFP, a DNA fragment containing the 5 kb promoter region, and 1.6 kb MoGSK1 ORF were PCR-amplified using primers (MoGSK1/NF and MoGSK1-GFP/R) from Ku80. To construct Fgk3-GFP, the 5 kb MoGSK1 promoter region was amplified using primers (MoGSK1/NF and MoGSK1/NR) from Ku80. The 1.2 kb Fgk3 ORF region was amplified using primers (Fgk3/OF and Fgk3-GFP/R) from wild type F. graminearum PH-1 cDNA and ligated at 3′ end of the MoGSK1 promoter region via overlap PCR approach. Both PCR-amplified fragments of MoGSK1 and Fgk3 under the same MoGSK1 promoter were cloned into KpnI and HindIII double-digested GFP-contained pKNTG by an in vitro recombination approach using the pEASY-Uni Seamless Cloning and Assembly Kit (TransGen Biotech, China). The resulting construct pKNTG-MoGSK1-GFP and pKNTG-Fgk3-GFP were transferred into Δmogsk1 protoplasts, respectively.

Zhou T., Dagdas Y.F., Zhu X., Zheng S., Chen L., Cartwright Z., Talbot N.J, & Wang Z. (2017). The glycogen synthase kinase MoGsk1, regulated by Mps1 MAP kinase, is required for fungal development and pathogenicity in Magnaporthe oryzae. Scientific Reports, 7, 945.

+ Open protocol

+ Expand

Cloning, Expression, and Kinetic Analysis of Candidate Gene

Check if the same lab product or an alternative is used in the 5 most similar protocols

The ORF of candidate gene was cloned using Phusion High-Fidelity PCR Master Mix (New England BioLabs, USA) with the specially designed primers (Supplemental Table 1). The PCR products were purified, and then ligated into the pEASY®-Blunt Simple Cloning Vector (TransGen Biotech, China) and transformed into E. coli DH5α cells. Verified the positive colonies by sequencing (Beijing RuiBo Biotechnology Co. Ltd., China) and then subcloned into the pET-32a (+) expression vector (Novagen, USA) according to the protocol of the pEASY®-Uni Seamless Cloning and Assembly Kit (TransGen Biotech, China) (Supplemental Table 1). Recombinant proteins were expressed and purified following the methods described previously [21 (link)], and the protein samples were assessed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE).
In vitro enzyme assays followed the method described previously [21 (link)]. Time-course experiments were carried out to obtain the initial speed of the enzymatic reaction from 3 to 30 min (Fig. S2). Then, 3 min was used in the kinetic assays. The GraphPad Prism version 5 (GraphPad Software, La Jolla California USA, http://www.graphpad.com) was used to obtain kinetic parameters by fitting the obtained data to the Michaelis-Menten equation. All assays were performed in triplicate.

Ma R., Su P., Ma Q., Guo J., Chen S., Jin B., Zhang H., Tang J., Zhou T., Xiao C., Cui G, & Huang L. (2021). Identification of (-)-bornyl diphosphate synthase from Blumea balsamifera and its application for (-)-borneol biosynthesis in Saccharomyces cerevisiae. Synthetic and Systems Biotechnology, 7(1), 490-497.

+ Open protocol

+ Expand

CRISPR Construct Development for Maize

Check if the same lab product or an alternative is used in the 5 most similar protocols

The constructs used in this study were based on the vector pCXUN‐Cas9 in which the codon‐optimized Cas9 was driven by the ubiquitin gene promoter of maize (Zea mays L.) (Sun et al., 2016). The backbone of pCXUN‐Cas9 contains a hygromycin resistant gene (hptII) for callus selection. The gRNAs expression cassette which is driven by a wheat U6 promoter was introduced into pCXUN‐Cas9 with HindIII, by using pEASY‐Uni Seamless Cloning and Assembly Kit (TransGen Biotech, Beijing, China). All primer sets used in this study were as listed in Table S1.

Li J., Jiao G., Sun Y., Chen J., Zhong Y., Yan L., Jiang D., Ma Y, & Xia L. (2021). Modification of starch composition, structure and properties through editing of TaSBEIIa in both winter and spring wheat varieties by CRISPR/Cas9. Plant Biotechnology Journal, 19(5), 937-951.

+ Open protocol

+ Expand

Lentiviral Transduction of eIF3c in HCC

Check if the same lab product or an alternative is used in the 5 most similar protocols

The ORF of eIF3c was amplified and cloned into the pWPXL lentiviral vector using a pEASY‐Uni Seamless Cloning and Assembly kit (TransGen Biotech, Beijing, China). The construct was confirmed by DNA sequencing. Lentiviruses were generated by cotransfecting the lentiviral vector, the packaging plasmid psPAX2, and the VSV‐G envelope plasmid pMD2.G (gifts from Dr Didier Trono, Laboratory of Virology and Genetics, Ecole Polytechnique Federale De Lausanne, Lausanne, Switzerland) into HEK‐293T cells using Lipofectamine 2000 (Invitrogen). Supernatants were collected 48 h after transfection, filtered through a 0.45‐μm membrane filter, and then used to infect HCC cells. Cells were infected with recombinant lentivirus plus 6 μg/mL polybrene (Sigma‐Aldrich, St. Louis, MO, USA).

Li T., Li S., Chen D., Chen B., Yu T., Zhao F., Wang Q., Yao M., Huang S., Chen Z, & He X. (2017). Transcriptomic analyses of RNA‐binding proteins reveal eIF3c promotes cell proliferation in hepatocellular carcinoma. Cancer Science, 108(5), 877-885.

+ Open protocol

+ Expand

Site-Directed Mutagenesis of TwKO

Check if the same lab product or an alternative is used in the 5 most similar protocols

Site-directed mutants of TwKO, including the L387A, L387D, L387S, L387T, L387G, and L387R mutants (L, leucine; A, alanine; D, aspartic acid; S, serine; T, threonine; G, glycine; R, arginine), were constructed using the pEASY-Uni Seamless Cloning and Assembly Kit (TransGen Biotech). The mutagenic primer pairs are listed in Supplementary Table S1. The constructed mutants were verified by complete sequencing and then transformed into yeast strains containing the module pESC-Trp::TwCPS3/TwKSL2 and one of the TwCPR genes (TwCPR3) with higher catalytic activity. The products were extracted and analyzed by GC–MS as described above.

Su P., Guan H., Zhang Y., Wang X., Gao L., Zhao Y., Hu T., Zhou J., Ma B., Tu L., Tong Y., Huang L, & Gao W. (2017). Probing the Single Key Amino Acid Responsible for the Novel Catalytic Function of ent-Kaurene Oxidase Supported by NADPH-Cytochrome P450 Reductases in Tripterygium wilfordii. Frontiers in Plant Science, 8, 1756.

+ Open protocol

+ Expand

Construction of Complementation Plasmid for PAA3 Analysis

Check if the same lab product or an alternative is used in the 5 most similar protocols

To construct the complementation plasmid, a 10,725 bp genomic fragment that contained the PAA3 coding sequence, coupled with the 3251 bp upstream and 1068 bp downstream sequences, was amplified using the primers PAA3-com-F (EcoR1) and PAA3-com-R (HindIII). The fragment was inserted into the binary vector pCAMBIA1301 using the pEASY®-Uni Seamless Cloning and Assembly Kit (TransGen, Beijing, China). The recombinant plasmids were transformed into the paa3 mutant using the Agrobacterium tumefaciens-mediated transformation method as described previously (Zhuang et al., 2020 (link)). For PAA3::GUS assays, the promoter of PAA3 gene (3138 bp) was amplified. The fragment was inserted into the binary vector pCAMBIA1301. Then the recombinant plasmids were transformed into the japonica cultivar ZHONGHUA 11 (ZH11) using the A. tumefaciens-mediated transformation method as described previously (Zhuang et al., 2020 (link)). The primers used for vector construction are listed in Table S2.

Yang F., Xiong M., Huang M., Li Z., Wang Z., Zhu H., Chen R., Lu L., Cheng Q., Wang Y., Tang J., Zhuang H, & Li Y. (2021). Panicle Apical Abortion 3 Controls Panicle Development and Seed Size in Rice. Rice, 14, 68.

+ Open protocol

+ Expand

Recombinant Plasmid Construction and Expression

Check if the same lab product or an alternative is used in the 5 most similar protocols

The purified PCR product was inserted into the pET28a(+), which had been previously digested by EcoR I and Hind III, using the pEASY-Uni Seamless Cloning and Assembly Kit (TransGen Biotech, China) to construct the recombinant plasmid pET28a-lqbg8. DNA linking products were introduced into E. coli DH5α receptor cells by Ca²⁺ chemical transformation method. The positive clones were screened by colony PCR and sequencing, to obtained the recombinant plasmid pET28a-lqbg8. The plasmid pET28a-lqbg8 was transferred to E. coli BL21(DE3) for gene expression.

Huang Y.Y., Lv Z.H., Zheng H.Z., Zhu Q., Liu M.T., Sang P., Wang F., Zhu D., Xian W.D, & Yin Y.R. (2023). Characterization of a thermophilic and glucose-tolerant GH1 β-glucosidase from hot springs and its prospective application in corn stover degradation. Frontiers in Microbiology, 14, 1286682.

+ Open protocol

+ Expand

Genetic Manipulation of E. coli and P. putida

Check if the same lab product or an alternative is used in the 5 most similar protocols

The strains and plasmids used in this study are given in Additional file 1. All kinds of E. coli, and P. putida KT2440 were grown in LB medium (Liquid and Solid). E. coli DH5α was used for all cloning and maintenance, while E. coli S17-1 served as a helper strain for conjugal transfer. E. coli were grown at 37 °C, and P. putida KT2440 was incubated at 30 °C. Antibiotics were added at the following concentrations: kanamycin, 150 μg/mL (50 μg/mL for E. coli); gentamicin, 50 μg/mL; tetracycline, 25 μg/mL (15 μg/mL for E. coli) and spectinomycin, 100 μg/mL.
The DNA polymerase PrimeSTAR^® Max, all restriction endonucleases and T4 DNA ligase were purchased from Takara Bio Inc (Dalian, China). The One Step Cloning Kit (Vazyme Biotech Co., Ltd, Nanjing, China) and pEASY-Uni Seamless Cloning and Assembly Kit (TransGen Biotech, Beijing, China) were applied for seamless cloning.

Sun J., Wang Q., Jiang Y., Wen Z., Yang L., Wu J, & Yang S. (2018). Genome editing and transcriptional repression in Pseudomonas putida KT2440 via the type II CRISPR system. Microbial Cell Factories, 17, 41.

+ Open protocol

+ Expand

About PubCompare

Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.

We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.

However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.

Ready to get started?

Revolutionizing how scientists
search and build protocols!