Gblock gene fragment

Manufactured by Integrated DNA Technologies

Sourced in United States, Belgium

GBlock gene fragments are synthetic double-stranded DNA molecules produced by Integrated DNA Technologies. They are designed to serve as building blocks for the construction of larger DNA sequences, enabling the rapid assembly of genetic elements for a variety of applications.

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105 protocols using gblock gene fragment

Synthetic Promoters for AAV Gene Therapy

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DNA 2.0 (Menlo Park, CA) was used to design codon optimized human NMNAT1 cDNA, which was synthesized into a gBlock gene fragment (Integrated DNA Technologies, Coralville, IA). The hNMNAT1 gBlock was inserted into an scAAV plasmid. Cell type-specific promoter sequences were inserted into this base plasmid. Synthetic cell type-specific promoters were obtained from the following Addgene plasmids: 161_pAAV-ProD5-CatCh-GFP-WPRE (Addgene plasmid # 125981; http://n2t.net/addgene:125981; RRID:Addgene_125981), 124_pAAV-ProC3-CatCh-GFP-WPRE (Addgene plasmid # 125939; http://n2t.net/addgene:125939; RRID:Addgene_125939), and 123_pAAV-ProC1-CatCh-GFP-WPRE (Addgene plasmid # 125937; http://n2t.net/addgene:125937; RRID:Addgene_125937) which were gifts from Botond Roska.¹⁶ (link) The VMD2 and hGRK1 promoters were synthesized into gBlock gene fragments (Integrated DNA Technologies, Coralville, IA) and inserted into the base of an scAAV plasmid. The CASI promoter construct was developed as described previously.¹⁵ (link) All promoter sequences can be found in Table S1.

Brown E.E., Scandura M.J, & Pierce E.A. (2023). Expression of NMNAT1 in the photoreceptors is sufficient to prevent NMNAT1-associated retinal degeneration. Molecular Therapy. Methods & Clinical Development, 29, 319-328.

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CRISPR Competition Assay Protocol

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For in vitro competition assays, lentiviral vectors expressing a target sgRNA together with mNeonGreen (pUSPmNG) or tagRFP-P2A-Puro (pUSEPR) was generated by Gibson assembly as described previously⁷¹. The U6-filler sequence was amplified by PCR using lentiGuide-puro as a template, the PGK promoter sequence was obtained from the GMAP collection⁷¹, and the mNeonGreen sequence was obtained as a gBlock gene fragment (Integrated DNA Technologies). sgRNAs targeting Slc33a1, Tapt1, Suco, Rpa3, and Olfr102 were cloned into the BsmBI-digested pUSPmNG or pUSEPR vectors, using a previously described protocol⁷². Dual sgRNA sequences were ordered as a gBlock gene fragment (Integrated DNA Technologies) contained an sgRNA (#1) H1 sgRNA (#2) sequence flanked by BsmBI-digest sites. Dual sgRNA sequences were BsmBI-digested and cloned into BsmBI-digested pUSEPR vectors. Cas9-expressing cells were infected with pUSPmNG or pUSEPR vectors expressing each sgRNA at an infection efficiency of ~50%. The percentage of mNeonGreen/tagRFP-positive cells was quantified over time using a Guava easyCyte HT flow cytometer (Millipore). For each sgRNA, the percentage of mNeonGreen/tagRFP-positive cells was normalized to respective values at 2 days post infection (defined as day 2).

Romero R., Sánchez-Rivera F.J., Westcott P.M., Mercer K.L., Bhutkar A., Muir A., González-Robles T.J., Lamboy-Rodríguez S.A., Liao L.Z., Ng S.R., Li L., Colón C.I., Naranjo S., Beytagh M.C., Lewis C.A., Hsu P.P., Bronson R.T., Vander Heiden M.G, & Jacks T. (2020). Keap1 mutation renders lung adenocarcinomas dependent on Slc33a1. Nature cancer, 1(6), 589-602.

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Quantitative Detection of Omicron SARS-CoV-2

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The Omicron RT-dPCR assays (Omn143) was performed using the QuantStudio Absolute Q Digital PCR System (Applied Biosystems, Waltham, MA, USA) in 10 μL reaction mixtures (including 10% overage) containing 2.5 μL of 4× Combinati one-step RT-dPCR MasterMix, 0.4 μL of each 10 μM primer (Omn143-Fwd and Omn143-Rev; Table S2), 0.2 μL of 10 μM probe (Omn143 Probe; Table S2), and 6.5 μL of sample RNA. Reaction mixtures were loaded into QuantStudio Absolute Q MAP16 plates and overlaid with 15 μL of isolation buffer. The assay was performed with thermal cycling conditions: reverse transcription at 50°C for 10 min, activation/denaturation at 95°C for 5 min, followed by 45 cycles of denaturation at 95°C for 5 s and annealing/extension at 54°C for 15 s. Assay validation was performed in three independent experiments using synthesized gBlock gene fragments (Integrated DNA Technologies, Coralville, IA, USA): Omicron BA.1 control and Delta control (Table S2). Microreaction chambers that passed QC, as determined by ROX signal, were analyzed. FAM fluorescence intensity threshold to call Omicron positive detection per microchamber was set at 10,000 (more than two standard deviations above water and Delta negative controls). Absolute quantification (copies) was reported.

Smith M.F., Holland S.C., Lee M.B., Hu J.C., Pham N.C., Sullins R.A., Holland L.A., Mu T., Thomas A.W., Fitch R., Driver E.M., Halden R.U., Villegas-Gold M., Sanders S., Krauss J.L., Nordstrom L., Mulrow M., White M., Murugan V, & Lim E.S. (2023). Baseline Sequencing Surveillance of Public Clinical Testing, Hospitals, and Community Wastewater Reveals Rapid Emergence of SARS-CoV-2 Omicron Variant of Concern in Arizona, USA. mBio, 14(1), e03101-22.

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Production and Characterization of Monoclonal Antibodies

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A set of mAbs for binding to MuSK, AChR, and MOG were used as controls. Cell culture supernatant from either an established murine MuSK mAb (4A3; ref. 32 (link)) or a murine MOG mAb (8-18C5; ref. 64 (link)) hybridoma was applied to a Protein G/Sepharose column (GE Healthcare) to isolate the IgG. We also engineered the MuSK mAb (4A3) and the MOG mAb (8-18C5) as chimeric mouse-human recombinant mAbs. They were produced to contain the murine mAb heavy and light chain variable regions fused to the respective human constant regions using an approach we described (65 (link)). These chimeric recombinant mAbs served as positive controls in the human antibody–binding assays and did not require a substitute (murine-specific) secondary antibody because the constant regions were identical to those of human mAbs. The AChR mAb (clone 637) was derived from a human MG thymus (24 (link), 25 (link), 66 (link)). The variable regions were synthesized as gBlock gene fragments (Integrated DNA Technologies), then subcloned into expression vectors, expressed, and purified using approaches we described (67 (link)).

Takata K., Stathopoulos P., Cao M., Mané-Damas M., Fichtner M.L., Benotti E.S., Jacobson L., Waters P., Irani S.R., Martinez-Martinez P., Beeson D., Losen M., Vincent A., Nowak R.J, & O’Connor K.C. (2019). Characterization of pathogenic monoclonal autoantibodies derived from muscle-specific kinase myasthenia gravis patients. JCI Insight, 4(12), e127167.

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Mammalian Cell Expression Plasmid Construction

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All mammalian cell expression plasmids were constructed by USER cloning from gBlock gene fragments (Integrated DNA Technologies) with USER junctions sized between 14 and 20 nucleotides^{58 (link)}. Phusion U Green Multiplex PCR Master Mix (ThermoFisher) was used for amplification of DNA. sgRNA plasmids were constructed by blunt end ligation of a linear PCR product generated by encoding the 20-nt variable protospacer sequence onto the 5′ end of an amplification primer and treating the resulting piece to KLD Enzyme Mix (New England Biolabs) according to the manufacturers’ instruction. Mach1 chemically competent E. coli (ThermoFisher) cells were used.

Rees H.A., Yeh W.H, & Liu D.R. (2019). Development of hRad51–Cas9 nickase fusions that mediate HDR without double-stranded breaks. Nature Communications, 10, 2212.

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Dual-Fluorophore Construct Design

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All constructs were designed using a combination of overlap extension cloning and gBlock gene fragments (Integrated DNA Technologies). All constructs were flanked by BamHI with downstream Kozak sequence and start codons as well as HindIII for final subcloning onto pAAV-hSynapsin1 vectors. Alternatively, constructs were flanked by SacI and AflII sites for subcloning into custom pAAV-hSynapsin1-FLEx vectors.
Concurrent two-color fluorophore expression was achieved by the use of either two plasmids with mRuby2 as the red reference fluorophore or a bicistronic approach in which the GCaMP construct was separated by a P2A sequence¹ from a downstream mRuby3. The two-plasmid approach was used for all in vitro characterization while the bicistronic approach was used for in vivo characterization. Functional assays were performed with no reference fluorophore.

Broussard G.J., Liang Y., Fridman M., Unger E.K., Meng G., Xiao X., Ji N., Petreanu L, & Tian L. (2018). In vivo measurement of afferent activity with axon-specific calcium imaging. Nature neuroscience, 21(9), 1272-1280.

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SARS-CoV-2 Spike Protein Variant Expression

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The S constructs contained the following mutations compared to the WT variant (Wuhan Hu-1; GenBank: MN908947.3): deletion (Δ) of H69, V70 and Y144, N501Y, A570D, D614G, P681H, T716I, S982A, and D1118H in B.1.1.7; L18F, D80A, D215G, L242H, R246I, K417N, E484K, N501Y, D614G, and A701V in B.1.351; and L18F, T20N, P26S, D138Y, R190S, K417T, E484K, N501Y, D614G, H655Y, and T1027I in P.1. They were ordered as gBlock gene fragments (Integrated DNA Technologies) and cloned Pst I/Not I in a pPPI4 expression vector containing a hexahistidine (his) tag with Gibson Assembly (Thermo Fisher Scientific). All S constructs were verified by Sanger sequencing and subsequently produced in human embryonic kidney (HEK) 293F cells (Thermo Fisher Scientific) and purified as previously described (25 (link)). The RBD constructs contained the following mutations: N501Y in B.1.1.7; K417N, E484K, and N501Y in B.1.351; K417T, E484K, and N501Y in P.1; and E484K as a single mutant. They were made by introducing the mutations into the SARS-CoV-2 RBD (331 to 528 amino acids)–StrepII construct provided by D. Lakshamanane (42 (link)) using the QuikChange Site-Directed Mutagenesis Kit (Agilent Technologies). All RBD constructs were produced in ExpiCHO cells and purified as previously described (43 ).

Caniels T.G., Bontjer I., van der Straten K., Poniman M., Burger J.A., Appelman B., Lavell H.A., Oomen M., Godeke G.J., Valle C., Mögling R., van Willigen H.D., Wynberg E., Schinkel M., van Vught L.A., Guerra D., Snitselaar J.L., Chaturbhuj D.N., Cuella Martin I., Moore J.P., de Jong M.D., Reusken C., Sikkens J.J., Bomers M.K., de Bree G.J., van Gils M.J., Eggink D, & Sanders R.W. (2021). Emerging SARS-CoV-2 variants of concern evade humoral immune responses from infection and vaccination. Science Advances, 7(36), eabj5365.

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Gene-specific CRISPR gRNA Design and HDR Templates

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Gene-specific gRNAs sequences were selected using a combination of prediction tools including gRNA Scorer 1.0 and 2.0 (15 (link),16 (link)), GuideScan (17 (link)), or CRISPRz (18 (link)). We selected gRNA sequences with zero predicted off targets with one base pair mismatch. Design principles for HDR templates included a mutated gRNA recognition sequence, incorporation of barcoded nucleotides, homology arms, and incorporation of heterologous DNA sequence encoding EGFP or mScarlet. In some cases, repetitive sequence in non-coding regions of the genome constrained the length of homology arms, as templates could not be synthesized by the manufacturer. Annotated HDR templates have been deposited in Genbank (Accessions MN832870-MN832873). HDR templates were ordered as gBlock Gene Fragments (Integrated DNA Technologies [IDT]). For single stranded DNA templates Ultramer DNA Oligonucleotides (IDT) were used. In HDR templates containing two gRNA sites (b692 assay), the b692 recognition site was appended to the template end. Similarly the ISce site was appended to the end of the template noted to contain this sequence. gBlocks were resuspended in nuclease-free water to a concentration of 50 ηg/μl and stored at −20°C. gRNA sequences are listed in Supplementary Table S1.

DiNapoli S.E., Martinez-McFaline R., Gribbin C.K., Wrighton P.J., Balgobin C.A., Nelson I., Leonard A., Maskin C.R., Shwartz A., Quenzer E.D., Mailhiot D., Kao C., McConnell S.C., de Jong J.L., Goessling W, & Houvras Y. (2020). Synthetic CRISPR/Cas9 reagents facilitate genome editing and homology directed repair. Nucleic Acids Research, 48(7), e38.

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Ribosome-Tethered GCaMP for Calcium Imaging

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To tether GCaMP6 and the GFP nanobody to ribosomes, GCaMP6m (Chen et al., 2013 (link)) or the GFP nanobody (Ekstrand et al., 2014 (link); Rothbauer et al., 2006 (link)) was linked to ribosomal subunit protein RPL10 through a short linker of amino acid sequence SGRTQISSSS-FEF (Heiman et al., 2008 (link)). The resultant construct is GCaMP6-RPL10 and is referred to as ribo-GCaMP for simplicity in the paper. All constructs were designed using a combination of restriction cloning, Gibson Assembly and gBlock gene fragments (Integrated DNA Technologies). For C. elegans constructs, the sequence of rpl-1 was fused to the C-terminus of GCaMP6m using an overlap PCR strategy. All regions that underwent PCR amplification were checked through sanger sequencing (GeneWitz; Elim Biopharm) following RCA-based amplification (GE Templiphi). Constructs were made into custom AAV through Stanford Vector Core:
AAV8-hSyn-GCaMP6m/f/s (Restriction Cloning: AscI & NheI)
AAV5/8-hSyn-riboGCaMP6m/f/s (Gibson Assembly + gBlock)
AAV5/8-hSyn-DIO-riboGCaMP6m/f/s (Restriction Cloning: AscI & NheI)
Linker-RL10 amino acid sequence: SGRTQISSSSFEFSSKVSRDTLYEAVREVLHGNQRKRRKFLETVELQISLKNYDPQKDKRFSGTVRLKSTPRPKFSVCVLGDQQHCD EAKAVDIPHMDIEALKKLNKNKKLVKKLAKKYDAFLASESLIKQIPRILGPGLNKAGKFPSLLTHNENMVAKVDEVKSTIKFQMKKVLC LAVAVGHVKMTDDELVYNIHLAVNFLVSLLKKNWQNVRALYIKSTMGKPQRLY

Chen Y., Jang H., Spratt P.W., Kosar S., Taylor D.E., Essner R.A., Bai L., Leib D.E., Kuo T.W., Lin Y.C., Patel M., Subkhangulova A., Kato S., Feinberg E.H., Bender K.J., Knight Z.A, & Garrison J.L. (2020). Soma-Targeted Imaging of Neural Circuits by Ribosome Tethering. Neuron, 107(3), 454-469.e6.

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SARS-CoV-2 Pseudovirus Production

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The B.1.1.7, B.1.351, and P.1 pseudovirus constructs contained the same mutations as the S constructs. They were ordered as gBlock gene fragments (Integrated DNA Technologies) and cloned Sac I/Apa I in the pCR3 SARS-CoV-2–S_Δ19 expression plasmid (GenBank: MT449663.1) (44 (link)) using Gibson Assembly (Thermo Fisher Scientific). The D614G pseudovirus construct was made using the QuikChange Site-Directed Mutagenesis Kit (Agilent Technologies). All constructs were verified by Sanger sequencing. Pseudovirus was produced by cotransfecting the pCR3 SARS-CoV-2–S_Δ19 expression plasmid with the pHIV-1_NL43 ΔEnv-NanoLuc reporter virus plasmid in HEK293T cells (American Type Culture Collection, CRL-11268) (44 (link), 45 (link)). Cell supernatant containing the pseudovirus was harvested 48 hours after transfection and stored at −80°C until further use.

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