The amplified genes (IL-15 and IgHg2a) were separately cloned into PCR2.1 TOPO Vector (Thermo Fisher Scientific). K322A mutations into IgG2 Fc and N72D mutation into IL-15 were introduced by site-directed mutagenesis (Agilent). IL-2 signal peptide (SP) was inserted in pcDNA3.1 vector downstream of CMV promoter by using GeneArt Service, Thermo Fisher Scientific, USA. Sequentially IL-15 CDS and IgHg2 genes were inserted in the IL-2-pcDNA3.1 vector to make IL-2SP-N72DIL-15CDS-K322AIgHg2-pcDNA3.1 construct (chimeric IL-15). All cloning, mutagenesis, and fusion constructs were confirmed by Sanger sequencing.
Geneart service
Manufactured by Thermo Fisher Scientific
Sourced in United States
The GeneArt service is a custom gene synthesis offering from Thermo Fisher Scientific. It provides the capability to synthesize and deliver customized DNA sequences to meet research needs.
Automatically generated - may contain errors
Lab products found in correlation
Lipofectamine 2000, by Thermo Fisher Scientific (4 mentions)
Gfp topo vector, by Thermo Fisher Scientific (3 mentions)
Fluostar optima microplate reader, by BMG Labtech (2 mentions)
Pcr2.1 topo vector, by Thermo Fisher Scientific (1 mentions)
Pgem t easy, by Thermo Fisher Scientific (1 mentions)
Pgem t easy, by Promega (1 mentions)
Qiaex 2 gel extraction kit, by Qiagen (1 mentions)
Xhoi, by New England Biolabs (1 mentions)
Mmessage mmachine t7 transcription kit, by Thermo Fisher Scientific (1 mentions)
Pentr directional topo cloning kit, by Thermo Fisher Scientific (1 mentions)
High fidelity dna polymerase, by Thermo Fisher Scientific (1 mentions)
Geneoptimizer software, by Thermo Fisher Scientific (1 mentions)
Cellfectin 2 reagent, by Thermo Fisher Scientific (1 mentions)
17 protocols using geneart service
1
Generation of Chimeric IL-15 Construct
2
Plasmid Constructs for Poliovirus, Coxsackievirus, and Rhinovirus
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Plasmid pXpA-SH coding for the full-length poliovirus type I (strain Mahoney) cDNA with introduced SalI and HpaI restriction sites in the 5’ and 3’ non-translated regions was described in [41 (link)], and its derivative plasmid pXpA-RenR coding for a poliovirus replicon with Renilla luciferase gene substituting the capsid coding region was described in [42 (link)]. To introduce the BFA-resistant mutations in 2C and 3A either PCR fragments synthesized using isolated RNA of BFA-resistant viruses as a template, or gene fragments synthesized by GeneArt service (Thermo Fisher) were introduced into pXpA-SH and pXpA-RenR using standard cloning techniques. All mutations were verified by sequencing. Plasmids p53CB3T7 and pACYC-RV1A coding for the cDNA of Coxsackievirus B3 (strain Nancy) and rhinovirus 1A (strain ATCC VR-1559) under the control of T7 promotor were kindly provided by Professor Frank van Kuppeveld (University of Utrecht, the Netherlands) and Dr. Margaret Scull (University of Maryland), respectively. Expression plasmids pM1-2Cs coding for 2Cs of poliovirus (wt and BFA-resistant), were constructed using pM1-MT vector for a high level of mammalian expression (Roche Biosciences). Cloning details are available upon request. Plasmids coding for human Arf1, 3, 4, 5, and 6 fused to EGFP were a gift from Dr. Catherine Jackson (Université Paris Diderot, France).
3
Constructing IVT Templates with Poly(A) Tails
4
Generation of Spliceosomal snRNA Mutants
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The mutants of U2 (U2weakNSS, U2noNSS and U2stNSS) were created by site-directed mutagenesis using specific primers listed in Supplementary Data 3 and confirmed by sequencing. The U2-MS2 RNA construct, which includes the promoter sequence, was described previously30 (link). The U2weakNSS-MS2 construct was prepared by site-directed mutagenesis using specific primers listed in Supplementary Data 3 . U1-1 (GRCh38/hg38:chr1:16,840,617–16,840,779), U1-26P (GRCh38/hg38:chr14:35,025,383–35,025,595), U4-1 (GRCh38/hg38:chr12:120,730,865–120,731,040), and U5F-1 (GRCh38/hg38:chr1:44,721,744–44,721,901) pre-snRNAs were designed and synthesized by GeneArt service (Thermo Fisher Scientific) including variants containing mutations strengthening (stNSS) and relaxing NSS (noNSS).
5
Producing Wild-type Gsα Protein
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The expression and purification of Gsα, Gβγ, and mini-Gsα have been described previously (Huang et al., 2021 (link)) with the only difference being that a wild-type Gsα was used in the current work. To generate this construct, a double-stranded DNA fragment for the wild-type Gsα short isoform was codon optimized and synthesized using the GeneArt service from Thermofisher. This fragment carried overlapping sequences with the previously described pET15b MBP-Gsα mutant sequence (Huang et al., 2021 (link)). The plasmid was digested with XhoI and SacI (New England BioLabs, Ipswich, MA, USA) to remove the mutant Gsα sequence and purified via electrophoresis and gel extraction kit (Bio Basic, Markham, Canada). The resulted plasmid backbone and DNA fragment were fused using the pEasy assembly kit from TransGen Biotech following manufacturer’s instructions. The plasmid was transformed into Escherichia coli (E. coli) BL21 (DE3) cells and a resulting colony containing the gene for the wild-type Gsα was selected for protein expression.
6
Predicting and Validating miR-181 Targets
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miR‐181 targets were predicted using TargetScan v.6.2 (http://www.targetscan.org/ , release June 2012) and miRWalk (http://zmf.umm.uni-heidelberg.de/apps/zmf/mirwalk2/ ) using mouse and human searches and broadly conserved microRNA target sites settings. 3′UTR regions with wild‐type or mutated miR‐181a target site were synthesized using GeneArt service (Thermo Scientific) and cloned into a GFP TOPO vector (Thermo Scientific). Sequences cloned are listed in Table S3 . C2C12 myoblasts were cultured in 96‐well plates and transfected using Lipofectamine 2000™ (Thermo Scientific) with WT or mutant sensor (200 ng), with 100 nM miR scrambled or miR‐181a mimic (100 nM, GE Healthcare; Soriano‐Arroquia, House, et al., 2016 ). Each experiment was carried out using at least two independent plasmid preparations in triplicates. GFP fluorescence was measured 48 hr following transfections using FLUOstar Optima microplate reader (BMG Labtech).
7
In vitro mRNA Production and Protein Tagging
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mRNA for transfection of exogenous proteins was produced in vitro from the T7 promoter-containing pGEM vector using the mMESSAGE mMACHINE T7 Transcription Kit (Thermo Fisher Scientific) as per the manufacturer's instructions. Genes encoding proteins of interest were directly synthesized as gene strings using the GeneArt service (ThermoFisher) and ligated into pGEM following digestion with AgeI and HindIII. For HaloTag-fused constructs these were followed by a short sequence encoding a GSGSG flexible linker and then the HaloTag gene at the 3′ terminus. For GNAi2-SNAP-tag, the SNAP-tag gene was inserted between nucleotides 342 and 343, corresponding to residues A114 and E115 in the αB-αC loop of Gαi2, following a short GSG linker. This tagging site has been demonstrated previously to retain Gαi2 activity (van Unen et al., 2016 (link)).
8
Constructing Plasmid Vectors for Gene Expression
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gBlocks gene fragments for the respective target sites or miRNAs (table S3 ) with the restriction site(s) at their ends were purchased from IDT. Restriction site cloning was performed using the standard method. Full length ORC4 expression plasmid was generated using gene synthesis using a full length human ORC4 and cloned into CAG-IRES-GFP vector using the GeneArt service (Thermo Fisher).
9
Constructing Plasmid Vectors for Gene Expression
10
Prdx6-202 transcript miR-24-3p regulation
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5ʹUTR of Prdx6-202 transcript regions with either the wild-type or mutated miR-24-3p target sites were synthesized using GeneArt service (Thermo Scientific). The wild type or mutated sequences were subcloned into a GFP TOPO vector (Thermo Scientific). C2C12 myoblasts were cultured in 96-well plates and transfected using Lipofectamine 2000™ (Thermo Scientific) with either 200ng of the wild type or mutant sensor and with either 100nM of the miR scrambled control or 100nM miR-24 mimic.
Each experiment was carried out using at least two independent plasmid preparations in triplicate.
GFP fluorescence was measured 48 hr following transfections using FLUOstar Optima microplate reader (BMG Labtech).
Each experiment was carried out using at least two independent plasmid preparations in triplicate.
GFP fluorescence was measured 48 hr following transfections using FLUOstar Optima microplate reader (BMG Labtech).
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