Gene fragments

Manufactured by Integrated DNA Technologies

Gene fragments are short, synthetic DNA sequences used as building blocks for larger DNA constructs. They are designed to specific sequences and can be used in various molecular biology applications.

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Lab products found in correlation

Enzymes for molecular cloning, by New England Biolabs (1 mentions) Hiscribe t7 quick high yield rna synthesis kit, by New England Biolabs (1 mentions) Hor 250, by ProSpec (1 mentions) Hispur ni nta resin, by Thermo Fisher Scientific (1 mentions) Freestyle293 cells, by Thermo Fisher Scientific (1 mentions) Superdex 200 increase 10 300 gl, by GE Healthcare (1 mentions) Codon optimization tool, by Integrated DNA Technologies (1 mentions)

4 protocols using gene fragments

Molecular Cloning Workflow and Reagents

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Enzymes for molecular cloning were purchased from New England Biolabs and Takara Bio. Gene fragments were purchased from Integrated DNA Technologies and Twist Bioscience. W.M. Keck Foundation at Yale University provided oligonucleotide synthesis (Supplementary Table S1) and DNA plasmid (Supplementary Table S2) sequencing. Antibiotics and media additives were used at the following final concentrations: ampicillin (amp), 100 μg/mL; spectinomycin (spec), 50 μg/mL; glucose, 1% (w/v); arabinose, 0.1% (w/v); sodium selenite, 10 μM; N_ε-acetyl-l-lysine (AcK), 10 mM; nicotinamide, 20 mM.

Morosky P., Comyns C., Nunes L.G., Chung C.Z., Hoffmann P.R., Söll D., Vargas-Rodriguez O, & Krahn N. (2023). Dual incorporation of non-canonical amino acids enables production of post-translationally modified selenoproteins. Frontiers in Molecular Biosciences, 10, 1096261.

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Generating Knockout Mice with CRISPR-Cas9

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Two sgRNAs were synthesized using the HiScribe Quick T7 High Yield RNA Synthesis Kit (NEB #E2050) following the manufacturer’s instructions. Template DNA used in the reaction were two gBlocks Gene Fragments of 125bp in length obtained from Integrated DNA Technologies. These sgRNAs were designed to target Cas9 to the 5’ UTR and intron 5 of the murine IDS gene (Figure 1). Cas9 encoding mRNA was purchased from TriLink Biotechnologies. sgRNAs and Cas9 mRNA were mixed in RNase-free water. Ten NSG female mice aged between 8 - 9 weeks were super-ovulated by first injection of 5 IU of Pregnant Mare Serum Gonadotropin (PMSG, C1063, Sigma), intraperitoneal (i. p.), per female, and 48 hours later, followed by injection of 5 IU of human Chorionic Gonadotropin (hCG, HOR-250, PROSPEC Protein Specialists), i. p., per female, and these mice were immediately crossed to male NSG mice. The next morning, zygotes were collected and washed using standard methods¹¹. The sgRNA+Cas9 mRNA mixture was injected into the pronuclei of zygotes using standard procedures. The injected embryos were then transferred into pseudo-pregnant CD-1 (Charles River Laboratory) females as previously described [11 ]. Blood or tissues of offspring generated from this process were subsequently genotyped for IDS gene knockout as described below.

Smith M.C., Belur L.R., Karlen A.D., Podetz-Pedersen K., Erlanson O., Laoharawee K., Furcich J., Lund T.C., You Y., Seelig D., Webber B.R, & McIvor R.S. (2023). Generation and characterization of an immunodeficient mouse model of mucopolysaccharidosis type II. Molecular genetics and metabolism, 138(4), 107539.

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Recombinant CCHFV GP38 Protein Production

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Recombinant CCHFV GP38 proteins were produced from the following isolates: Oman-199809166 (UniProt: A0A0U3C6Q7), Kosova-Hoti (UniProt: B2BSL7), 200406546-Turkey (UniProt: A0A0U2SQZ0), Afg09–2990 (UniProt: E5FEZ4), and 79121M18 (UniProt: D4NYK3). Gene fragments (Integrated DNA Technologies) of each isolate’s MLD-GP38 sequence encoding for residues 1–515, as denoted by CCHFV IbAr10200 strain GPC numbering, were codon-optimized for human cell expression (GenScript Codon Optimization Tool). Gene fragments were each cloned into a pαH eukaryotic expression vector with a C-terminal HRV 3C protease cleavage site, an 8x HisTag, and a Twin-Strep-tag. The plasmid for CCHFV strain IbAr10200 GP38 was previously reported^{28 (link)}. To ensure cleavage of the MLD from GP38, a pCDNA3.1 plasmid encoding for furin was co-transfected with each clinical GP38 plasmid at a mass ratio of 1:9 furin:GP38. The two plasmids were transiently transfected into FreeStyle 293 cells (Invitrogen) using polyethylenimine followed by treatment with 5 μM kifunensine to ensure uniform high-mannose glycosylation. Soluble GP38 was secreted from the harvested medium and purified via Ni-NTA resin (Thermo Scientific HisPur^™ Ni-NTA Resin). GP38 proteins were further purified via SEC using a Superdex 200 Increase 10/300 GL (GE Healthcare Biosciences) in 2 mM Tris pH 8.0, 200 mM NaCl, and 0.02% NaN₃.

Shin O.S., Monticelli S.R., Hjorth C.K., Hornet V., Doyle M., Abelson D., Kuehne A.I., Wang A., Bakken R.R., Mishra A., Middlecamp M., Champney E., Stuart L., Maurer D.P., Li J., Berrigan J., Barajas J., Balinandi S., Lutwama J.J., Lobel L., Zeitlin L., Walker L.M., Dye J.M., Chandran K., Herbert A.S., Pauli N.T, & McLellan J.S. (2024). Crimean-Congo Hemorrhagic Fever Survivors Elicit Protective Non-Neutralizing Antibodies that Target 11 Overlapping Regions on Viral Glycoprotein GP38. bioRxiv.

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Overexpression of Mutant MMP9 with HA Tag

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Gene fragments encoding the full length human MMP9 with a G100L mutation (to achieve functional proteolysis without a requirement for pro-peptide cleavage) (34 (link)) and C-terminal HA affinity tag was purchased from Integrated DNA Technologies. This gene block was inserted into a MIGR1 retroviral transfer plasmid followed by an IRES sequence and the gene encoding for mNeongreen reporter protein.

Tavernier J., Tuypens T., Verhee A., Plaetinck G., Devos R., Van der Heyden J., Guisez Y, & Oefner C. (1995). Identification of receptor-binding domains on human interleukin 5 and design of an interleukin 5-derived receptor antagonist.. Proceedings of the National Academy of Sciences of the United States of America, 92(11), 5194-5198.

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