Codon-optimized sequences of PACT and TRBP were ordered from GeneArt, and regions corresponding to PACT residues 239–313 (PACT-D3) and 208–313 (PACT-Ext-D3), and TRBP residues 258–366 (TRBP-Ext-D3) were cloned into a vector derived from pET-28a (24 (link)) using an In-Fusion cloning strategy (Clontech). This vector is based on the pET vector series, and results in attachment of an N-terminal hexa-histidine tag, maltose binding protein (MBP) and HRV 3C protease cleavage site. Loqs-D3 (residues 392–463) was cloned into the pGEX-4T-1 plasmid as described previously (20 (link)). Mutations were introduced using QuikChange Lightning mutagenesis kits (Agilent).
In fusion cloning strategy
Manufactured by Takara Bio
In-Fusion cloning strategy is a highly efficient and precise method for joining DNA fragments. It enables the seamless assembly of multiple DNA sequences without the need for restriction enzymes or DNA ligase. The In-Fusion cloning process relies on the homologous recombination capabilities of a proprietary Enzyme Mix to fuse DNA fragments with complementary end sequences.
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Lab products found in correlation
Glutathione sepharose 4b, by GE Healthcare (1 mentions)
Q5 site directed mutagenesis kit, by New England Biolabs (1 mentions)
Pet sumo vector, by Thermo Fisher Scientific (1 mentions)
Phusion polymerase, by New England Biolabs (1 mentions)
Cloneamp hifi pcr premix, by Takara Bio (1 mentions)
Phusion high fidelity dna polymerase, by Thermo Fisher Scientific (1 mentions)
Stellar competent cells, by Takara Bio (1 mentions)
In fusion hd cloning kit, by Takara Bio (1 mentions)
9 protocols using in fusion cloning strategy
1
Cloning and Mutagenesis of PACT, TRBP, and Loqs Domains
2
Quantifying Transcription Factor Binding Affinity
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The DNA‐binding assay was performed as previously described (Meng et al., 2019 ). The CDSs of RH1 and RH2 were amplified and inserted into the BamHI and XhoI sites of the pGEX‐4T‐1 vector using the In‐Fusion cloning strategy (Clontech). The recombinant constructs were transformed into Escherichia coli strain BL21 and induced with 0.2 mM isopropyl‐1‐thio‐d ‐galactopyranoside (IPTG). Recombinant GST‐RH1, GST‐RH2 and GST (glutathione S‐transferase, control) were purified using Glutathione Sepharose 4B (#17‐0756‐01; GE Healthcare, Piscataway, NJ, USA) according to the manufacturer’s protocol and quantified with the GE Healthcare protein assay reagent. The MYB‐core or AC‐rich element binding fragment was incubated with the GST‐RH1, GST‐RH2 and GST proteins, respectively, in Glutathione Sepharose and then the DNA‐binding activity (protein‐bound DNA) was determined by qRT‐PCR after washing and elution. The primers used are listed in Table S1 .
3
Cloning and Mutagenesis of PTCHD1-GFP
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The sequence of full‐length coding Ptchd1 mouse cDNA was amplified from the IMAGE cDNA clone 40095445 (GenBank accession number BC116312; Source BioSience) and cloned in pAcGFP1‐N vectors using In‐Fusion cloning strategy (Catalog no. 632501, Clontech) to generate PTCHD1‐GFP with GFP tag at the C‐terminal end (previously described in Ung et al. (2018 (link))). Importantly, the murine PTCHD1 protein shares the same amino acids length (888) with 98% of sequence identity between murine and human PTCHD1 proteins. The mutant PTCHD1‐GFP plasmids were generated using the Q5® Site‐Directed Mutagenesis Kit (New England Biolabs) according to the manufacturer's recommendations. The primer sequences for each mutagenesis are indicated in Table S1 . The efficiency of the site‐directed mutagenesis was verified by Sanger Sequencing.
4
Overexpression and Tagging of OFP1 Protein
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The DNA sequence containing the promoter and coding sequence of OFP1 was cloned into pCAMBIA2300 vector for OFP1 overexpression (OFP1-OE or 1o). A pCAMBIA2300-35S-GFP vector was used for OFP1-GFP construction. A pCAMBIA1300-35S-Flag vector was used for OFP1-Flag overexpression (OFP1-Flag-OE or 1Fo). OFP1 promoter (2-kb) was introduced into pCAMBIA2391Z vector for OFP1p:GUS construction. CRISPR/Cas9 knock-out vectors were constructed following previous reports (Ma and Liu, 2016 (link)). Primer sequences used for vector constructions and additional plasmid information are listed in the Supplementary Table 1 . Sequences were introduced into vectors by either in-fusion cloning strategy (Clontech) or traditional cut-ligation cloning method. These constructs were used to transform rice plant or to infiltrate tobacco leaf epidermis cells by Agrobacterium-mediated method (Sparkes et al., 2006 (link)).
5
FtsY Cloning and Mutagenesis Protocol
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FtsY was cloned from the pET9a vector coding for Cterminally His-tagged FtsY to a pET-SUMO vector (Invitrogen) coding for FtsY with N-terminal His 6 -tag and a SUMO cleavage site between the first Met and the tag, using the In-fusion cloning strategy (ClonTech). The vector was amplified using primers 5 0 -GATCCGGCTGCTAACAAAGCC CGAAAG-3 0 and 3 0 -ACCACCAATCTGTTCTCTGTGAGCCT CAATAATATC-5 0 . FtsY was amplified using primers 5 0 -GAA CAGATTGGTGGTATGGCGAAAGAAAAAAAACG-3 0 and 3 0 -GTTAGCAGCCGGATCTTAATCCTCTCGGGC-5 0 (underlined are the sequences that overlap between vector and insert). The FtsY mutants F196stop, K207stop, A167C, F196C and V342C, G357C, L407C, S422C, G439C, T451C, as well as mutant SecY(S111C) were generated by site-directed mutagenesis using Phusion polymerase (New England Biolabs).
6
Cloning Zscan4c Promoter Constructs
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To generate pZscan4c-pcDNA3.1/CT-GFP-TOPO constructs, a putative Zscan4c promoter (pZscan4) corresponding to −2,400, −480, and −288 bp, respectively, from the Zscan4c Transcription Start Site (TSS) was amplified from BAC RP23-63I1. The pZscan4 was amplified using primers: 5′-TTCTTAATCTGTGGTCGTCCA-3′; 5′-TGTGGTGACAATGGTGTGAA-3′; 5′-GCCAATCTTGGAATTCCTCTTC-3′; 5′-TTGCTTGTATTTGATTCCCC-3′. Dux-HA was a kindle gift of De Iaco. Duxbl1-V5 was obtained cloning Duxbl1 coding sequence into pcDNA3-V5 His (Invitrogen). Duxbl1_CTD-Flag was obtained using the In-Fusion cloning strategy (Takara).
7
Retroviral and Lentiviral Construct Generation
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Retroviral pBabe-puro-based human wt/mutp53 overexpression and lentiviral pLVHMshp53 vectors were described previously81 (link),82 (link). wt/mutp53s were synthesized by polymerase chain reaction (PCR) and cloned into an XbaI-digested Flag-HA-pcDNA3.1 vector using the In-Fusion cloning strategy (Takara Bio) to get Flag and HA double-tagged constructs. Similarly, MCMs were synthesized by reverse transcription PCR and cloned into EcoRI-digested pcDNA3.1/V5-His-A or pLVX-IRES-mCherry vectors using In-Fusion cloning. A TP53 CRISPR/Cas9 knockout plasmid was obtained from Santa Cruz Biotech. Murine R270H mutp53 was generated by site-directed mutagenesis using pMXs-p53 as a template and then cloned into a pLVX-IRES-hygro vector using In-Fusion cloning. The sequences of all the constructs were validated by sequencing analyses. Lentiviral short hairpin RNAs (shRNAs) against MCM5, CGAS, STING1 (TMEM173), and human and murine RelB were purchased from Dharmacon or Sigma-Aldrich. Information on all the vectors, primers, and shRNAs is listed in Supplementary Data 3 .
8
Versatile pS/MAR Vector Modifications
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All of the vector modifications on pS/MAR were performed using the InFusion cloning strategy (Takara Biotech) following the manufacturer’s guidelines.
The vector pS/MAR ubiquitin C (UbiC)-luciferase was generated from the original pEPI vector. The UbiC promoter was introduced into the plasmid at the PcI restriction site (pS/MAR-UbiC), and the luciferase transgene was subsequently cloned into the vector through the BglII site. The luciferase-p2a-SMAD4 expression cassette was generated via PCR and cloned into the pS/MAR-UbiC plasmid at the BglII cloning site. p/MAR GFP was created replacing the GFP expression cassette of the original pEPI plasmid with the GFP-p2a-puromycin cassette generated by PCR.
nS/MAR-GFP and nS/MAR-SMAD4 were generated by swapping the bacterial backbone of the respective canonical plasmids with the R6K-RNA-OUT system developed at Nature Technology Corporation.
The vector pS/MAR ubiquitin C (UbiC)-luciferase was generated from the original pEPI vector. The UbiC promoter was introduced into the plasmid at the PcI restriction site (pS/MAR-UbiC), and the luciferase transgene was subsequently cloned into the vector through the BglII site. The luciferase-p2a-SMAD4 expression cassette was generated via PCR and cloned into the pS/MAR-UbiC plasmid at the BglII cloning site. p/MAR GFP was created replacing the GFP expression cassette of the original pEPI plasmid with the GFP-p2a-puromycin cassette generated by PCR.
nS/MAR-GFP and nS/MAR-SMAD4 were generated by swapping the bacterial backbone of the respective canonical plasmids with the R6K-RNA-OUT system developed at Nature Technology Corporation.
9
Cloning USH2A cDNA into S/MAR Vector
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All vector modifications on pS/MAR were performed using the In-Fusion cloning strategy (Takara Bio) following the manufacturer's instructions. The USH2A cDNA was kindly provided by Prof. Luk Vandenberghe (Harvard Medical School). To clone the USH2A cDNA into the S/MAR vector backbones, it was initially amplified by PCR using Phusion High-Fidelity DNA Polymerase (Thermo Fisher Scientific, F530S). Five fragments of PCR-amplified USH2A cDNA of similar size ( 3,000 bp) were produced using CloneAmp HiFi PCR Premix (Clontech, 639298) for further in-fusion cloning using the In Fusion HD Cloning Kit (Clontech, 639649), fragment by fragment. Each primer set was designed to introduce 15-bp homologous overhangs for efficient recombination plus an enzymatic restriction site. First, 1-3 μg of vector backbone was digested with 1-3 units of XhoI and BmgBI for 1 h at 37°C. For the recombination reaction, 100 ng of vector and 50 ng of the insert were mixed with the In-Fusion mix and incubated at 50°C for 15 min. Stellar competent cells (Takara Bio) were used for transformation. The cloning was repeated five times to get the complete USH2A cDNA insert into the S/MAR vector backbone. Finally, restriction enzyme digestion with SAPI and Sanger sequencing of the full insert were performed for quality control.
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