The antigen module was expressed in the pGAD-T7 plasmid, in which all elements between the ADH1 promoter and ADH1 terminator were deleted and replaced by the target sequences using a one-step clone kit from Vazyme (one-step clone kit, Code No. C112-01, Vazyme, Nanjing, China).
One step clone kit
Manufactured by Vazyme
Sourced in China
The One-step clone kit is a laboratory tool designed for the rapid and efficient cloning of DNA fragments. It provides a straightforward and streamlined process for seamlessly joining DNA sequences together without the need for complex ligation reactions or lengthy cloning protocols.
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8 protocols using one step clone kit
1
Cloning Antigen Module in pGAD-T7
2
Cloning and Characterization of Porcine TFDP2
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The gene encoding TFDP2 (GenBank Accession No. XM_021069556.1) was amplified from 3D4/21 cells cDNA using the primers listed in Supplementary Table S1 and cloned into pGEM®-T Easy Vector (Transgen, Beijing, China). The CDS region of TFDP2 was amplified by PCR using specific primer pairs (Supplementary Table S1) and subcloned into the pFlag-CMV2 vector using a one-step clone kit (Vazyme, Nanjing, China).
Genomic DNA was extracted from PAMs using a DNA extraction kit (TaKaRa, Otsu, Shiga, Japan). The 1701-bp porcine TFDP2 promoter sequence (NC_010455.5) relative to the transcription initiation site (+1) was amplified by specific primers and ligated into luciferase reporter vector pGL3-Basic (named − 1301/400-Luc) using a one-step clone kit (Vazyme, Nanjing, China). The truncated mutants of TFDP2 promoter were then constructed by PCR using the − 1301/400-Luc plasmid as a template (− 792/400-Luc, − 67/400-Luc, − 17/400-Luc). Site-directed mutagenesis of the C/EBP-β, ATF-1, AP-1, SP1 binding sites was performed by PCR using the − 17/400-Luc vector as a template. The 1291-bp porcine cyclin A promoter (NC_010450.4) luciferase reporter plasmid was constructed as above described. The pRL-TK Renilla luciferase reporter plasmid was used as an internal control. All primers are listed in Supplementary Table S1.
Genomic DNA was extracted from PAMs using a DNA extraction kit (TaKaRa, Otsu, Shiga, Japan). The 1701-bp porcine TFDP2 promoter sequence (NC_010455.5) relative to the transcription initiation site (+1) was amplified by specific primers and ligated into luciferase reporter vector pGL3-Basic (named − 1301/400-Luc) using a one-step clone kit (Vazyme, Nanjing, China). The truncated mutants of TFDP2 promoter were then constructed by PCR using the − 1301/400-Luc plasmid as a template (− 792/400-Luc, − 67/400-Luc, − 17/400-Luc). Site-directed mutagenesis of the C/EBP-β, ATF-1, AP-1, SP1 binding sites was performed by PCR using the − 17/400-Luc vector as a template. The 1291-bp porcine cyclin A promoter (NC_010450.4) luciferase reporter plasmid was constructed as above described. The pRL-TK Renilla luciferase reporter plasmid was used as an internal control. All primers are listed in Supplementary Table S1.
3
Seamless Insertion of CXCR5 and CXCR3 Fragments
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Extracellular fragments of CXCR5 and CXCR3 were determined according to the UniProt database. The sequences are shown in Figure S2 . Insert fragments were cloned seamlessly into recipient genes using the one-step clone kit from Vazyme, Co. China (C112-01). All plasmids were confirmed by sequencing.
4
Establishing TRIM22 Protein Constructs
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The Hek293T cells were obtained from the American Type Culture Collection (ATCC), and were cultured in DMEM (Catalog No. C11965500BT, Gibco) supplemented with 10% fetal bovine serum (FBS; Catalog No. 10270106, Gibco) and 1% penicillin/streptomycin (P/S; Catalog No. 15070063, Gibco) with 5% CO2 at 37 °C.
To establish the constructs expressing human tripartite motif-containing 22 (TRIM22) protein, we cloned the coding sequence of human TRIM22 with FLAG or GFP at its N-terminus into pcDNA3.1(+) vector, using one-step clone kit (Vazyme).
For transfection, PEI reagent was applied with the general ratio of 30 reagents as 10 μg plasmids (5 μg FLAG-TRIM22 and 5 μg GFP-TRIM22) into each 10 cm plate at 70% cell confluence. After 48 h transfection, cells were harvested for the following assays.
To establish the constructs expressing human tripartite motif-containing 22 (TRIM22) protein, we cloned the coding sequence of human TRIM22 with FLAG or GFP at its N-terminus into pcDNA3.1(+) vector, using one-step clone kit (Vazyme).
For transfection, PEI reagent was applied with the general ratio of 30 reagents as 10 μg plasmids (5 μg FLAG-TRIM22 and 5 μg GFP-TRIM22) into each 10 cm plate at 70% cell confluence. After 48 h transfection, cells were harvested for the following assays.
5
Overexpression and knockout of OsGRP3
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The coding sequence of OsGRP3 was cloned and inserted into the vector pCAMBIA1301 under the control of the UBI promoter using a one-step clone kit (Vazyme, Nanjing, China) for the overexpression of OsGRP3. The vector for knockout lines was constructed referring to the CRISPR/Cas9 System [68 (link)]. The plasmids were extracted using the TIANprep mini plasmid kit (TIANGEN, Beijing, China) and sent to BIOGLE GeneTech (Changzhou, Jiangsu, China) to generate the transgenic plant.
6
Constructing Bait Proteins for Yeast Two-Hybrid Screening
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The plasmid was synthesized based on previous studies. The pGAD-T7 plasmid was utilized to express the bait, in which all elements between the ADH1 promoter and ADH1 terminator were deleted, with specific target sequences cloned using a one-step clone kit from Vazyme, Co. Nanjing China. The bait protein consisted of a signal peptide, a ligand protein, a transmembrane peptide, the C-terminal region of yeast ubiquitin (Cub) and reporter genes. In different experiments, a signal peptide derived from either SP CXCL14 (residues 1–28), SPWBP1 (WBP1, residues 1–31) was fused at the N-terminus of ligand The WBP1 transmembrane peptide and flanking sequences (residues 350–430) were used as the transmembrane peptide and linkers and fused between CXCL14 and the reporter genes. To examine subcellular localization, EGFP served as the reporter gene. For detecting PPIs (protein-protein interactions), the Cub-GAL4 cassette functioned as the reporter module.
7
Membrane Protein Interaction Assay
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The bait was expressed by the pGAD-T7 plasmid, in which all elements between the ADH1 promoter and ADH1 terminator were deleted and the target sequences were cloned by a one-step clone kit from Vazyme, Co. China. The bait protein consisted of a signal peptide, a ligand protein, a transmembrane peptide, the C-terminus of yeast ubiquitin (Cub), and reporter genes. A signal peptide from one of three genes, SPCXCL12 (signal peptide of CXCL12, residues 1–28), SPWBP1 (WBP1, residues 1–31) or SPMFAL1 (MFAL1, residues 1–89), was fused at the N-terminus of ligand in different experiments. The WBP1 transmembrane peptide and flanking sequences (residues 350–430) were used as the transmembrane peptide and linkers, and fused between CXCL12 and the reporter genes. EGFP was used as a reporter gene to exam the subcellular localization. The Cub-GAL4 cassette was used as a reporter module for the detection of PPIs (Fig. 1A ).
8
Establishing Stable Cell Line with APA-Reporter and dCas13 Constructs
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APA reporter was constructed by inserting a 232 nt region flanking the PAS of the mouse cdc42 gene (Chr4: 137046876–137047107) into the pRiG vector (38 (link)). To establish the stable cell line, the whole APA-reporter region (Figure 1A ) was then cloned into piggyBac cargo plasmid. After co-transfected with piggyBac cargo and transposase for 7 days, HEK293T cells with the medium expression level of dsRED fluorescence were sorted by flow cytometry.
The plasmids for different dCas13 proteins fused with 3xEGFP and their crRNA backbones were kindly provided by Lingling Chen's Lab (37 (link)). To establish the constructs expressing different dCas13 proteins without EGFP tagged, we cloned the coding sequence of each dCas13 with an HA at C-terminal and nucleus localization signal (NLS) at both terminals into the pHAGE-EF1α vector, using one-step clone kit (Vazyme). For gRNA expression, we designed 22 nt oligos to target core regulatory elements or non-targeted controls (NT) and inserted them into the crRNA vector by T4 ligation (37 (link)). The sequences of gRNAs were listed in Supplementary Table.
The plasmids for different dCas13 proteins fused with 3xEGFP and their crRNA backbones were kindly provided by Lingling Chen's Lab (37 (link)). To establish the constructs expressing different dCas13 proteins without EGFP tagged, we cloned the coding sequence of each dCas13 with an HA at C-terminal and nucleus localization signal (NLS) at both terminals into the pHAGE-EF1α vector, using one-step clone kit (Vazyme). For gRNA expression, we designed 22 nt oligos to target core regulatory elements or non-targeted controls (NT) and inserted them into the crRNA vector by T4 ligation (37 (link)). The sequences of gRNAs were listed in Supplementary Table.
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