Pdonr221

Manufactured by Addgene

Sourced in United States

PDONR221 is a vector designed for Gateway cloning. It can be used for the creation of entry clones.

Automatically generated - may contain errors

Lab products found in correlation

7 protocols using pdonr221

Plasmid Cloning and Mutagenesis Protocols

Check if the same lab product or an alternative is used in the 5 most similar protocols

YFP-Parkin plasmid was from Addgene (#23955). The cDNA encoding human Vps34 was obtained from Invitrogen. The Flag tag and attL1 sites (for BP Gateway reaction) were cloned by polymerase chain reaction (PCR) using standard methods. cDNAs were subcloned into pDONR221 with BP clonase (Invitrogen), and site-directed mutagenesis was performed using QuikChange II XL (Stratagene). Kinase-dead ULK1 was achieved by introduction of a K46I mutation. WT and mutant alleles in pDONR221 were sequenced in their entirety to verify no additional mutations were introduced during PCR or mutagenesis steps and then put into either pcDNA3 Myc or pcDNA3 Flag mammalian expression vectors, or pQCXIN retroviral destination vector from Addgene (#17399) by LR Gateway reaction (Invitrogen). pMXs-3XHA-EGFP-OMP25 was a gift from D. Sabatini (Addgene plasmid, # 83356; http://n2t.net/addgene:83356; RRID:Addgene_83356). pLenti-mt-mKeima was subcloned from pCHAC-mt-mKeima (a gift from R. Youle; Addgene plasmid, # 72342; http://n2t.net/addgene:72342; RRID:Addgene_72342).

Hung C.M., Lombardo P.S., Malik N., Brun S.N., Hellberg K., Van Nostrand J.L., Garcia D., Baumgart J., Diffenderfer K., Asara J.M, & Shaw R.J. (2021). AMPK/ULK1-mediated phosphorylation of Parkin ACT domain mediates an early step in mitophagy. Science Advances, 7(15), eabg4544.

+ Open protocol

+ Expand

Cloning HER2 and HER3 using Gateway Technology

Check if the same lab product or an alternative is used in the 5 most similar protocols

Vector cloning was done using Gateway Cloning technology. HER2 and HER3 ORFs were cloned into entry vectors from Thermo-FisherScientific (pDONR221 or pENTR4) or pDONR223-HER3 (ERBB3) which was a gift from William Hahn & David Root (Addgene plasmid # 23874 ; http://n2t.net/addgene:23874 ; RRID: Addgene_23874). Destination vectors used were pcDNA-DEST40 (contains c-terminal V5-His tags), or in-house modified versions of this vector to express c-terminal 2XFlag tags (pDEST40-2XFlag) or 2XHA (pDEST40-2XHA) tags or nSNAP (pDEST40-nSNAP2XHA) or cSNAP (pDEST40-cSNAP2XFL) tags. Lentiviral infections were done using pLEX-ires-GFP, modified from pLEX_307 destination vector (gift from David Root; Addgene plasmid # 41392 ; http://n2t.net/addgene:41392 ; RRID:Addgene_41392) or pLenti-CMV/TO-Hygro DEST (gift from Eric Campeau & Paul Kaufman (Addgene plasmid # 17291 ; http://n2t.net/addgene:17291 ; RRID:Addgene_17291).

Campbell M.R., Ruiz-Saenz A., Zhang Y., Peterson E., Steri V., Oeffinger J., Sampang M., Jura N, & Moasser M.M. (2022). Extensive conformational and physical plasticity protects HER2-HER3 tumorigenic signaling. Cell reports, 38(5), 110285.

+ Open protocol

+ Expand

UBA1 Variant Cloning and Mutagenesis

Check if the same lab product or an alternative is used in the 5 most similar protocols

UBA1 complementary DNA (NM_003334.4) was obtained from GenScript (CloneID OHu24932) in pcDNA3.1, PCR amplified, and subcloned into pDONR221 (ThermoFisher Scientific, 12536017) using Gateway BP Clonase II (Thermo Fisher Scientific, 11789020). pDONR221-UBA1 was modified using NEB Q5 Site-Directed Mutagenesis Kit (New England Biolabs, E0554S) to generate the following pDONR221-UBA1 constructs: Δ1-40 (UBA1b) and Δ1-40/C632A (catalytically inactive UBA1b).²⁰ (link) Lentiviral expression constructs for UBA1 variants and Renilla lucerifase were generated via Gateway LR Clonase II (Thermo Fisher Scientific, 11791020) reaction between each pDONR221 plasmid and lentiviral destination plasmid pLEX307 (Addgene, 41392). All pDONR221 and pLEX307 constructs were confirmed by Sanger sequencing (Genewiz) and alignment using Benchling Biology Software (2021-2023; https://benchling.com).

Chiaramida A., Obwar S.G., Nordstrom A.E., Ericsson M., Saldanha A., Ivanova E.V., Griffin G.K., Khan D.H, & Belizaire R. (2023). Sensitivity to targeted UBA1 inhibition in a myeloid cell line model of VEXAS syndrome. Blood Advances, 7(24), 7445-7456.

+ Open protocol

+ Expand

Cloning HER2 and HER3 using Gateway Technology

Check if the same lab product or an alternative is used in the 5 most similar protocols

+ Open protocol

+ Expand

Lentiviral Construct Generation and Transduction

Check if the same lab product or an alternative is used in the 5 most similar protocols

The constructs used in this study are listed in SI Appendix, Table S1. All restriction enzymes and T4 ligase used for cloning were from New England Biolabs (NEB, Ipswitch, USA) and the Gateway BP and LR Clonase II were from Thermo Fisher Scientific. All Gateway cloning constructs were cloned via the donor plasmid pDONR221 (Addgene, #12536017, Watertown, USA) into the Doxycycline inducible expression plasmid pINDUCER20 (65 (link)). Constructs were heat-shock transformed into One Shot Stbl3 E. coli bacteria (Thermo Fisher Scientific, #C737303). Proper sequence of all constructs has been confirmed by sequencing (Microsynth, Balgach, Switzerland) (SI Appendix, Table S1).
Constructs generated for mammalian expression were separately packed into lentiviral particles using pdelta8.9 (Addgene, #2221) and pCMV-VSV-G (Addgene, #8454) cotransfected using Lipofectamine 3000 (Thermo Fisher Scientific) into Lenti-X 293T cells (TaKaRa, #632180). Crude lentiviral supernatant was used for infection of target mammalian cells. Cells were selected for stable integration of the constructs via respective antibiotic selection.

Boytsov D., Madej G.M., Horn G., Blaha N., Köcher T., Sitte H.H., Siekhaus D., Ziegler C., Sandtner W, & Roblek M. (2024). Orphan lysosomal solute carrier MFSD1 facilitates highly selective dipeptide transport. Proceedings of the National Academy of Sciences of the United States of America, 121(13), e2319686121.

+ Open protocol

+ Expand

Bnip3 Lentiviral Knockdown and Overexpression

Check if the same lab product or an alternative is used in the 5 most similar protocols

Lentiviral stocks were generated as described elsewhere (28 (link)). pLKO.1-puro SHC004 served as the internal control in our studies. pLKO.1-puro or pLKO_TRC005 short hairpin RNAs (shRNAs) targeting Bnip3 were the following: shB3_1 (no. 229457), shB3_2 (no. 9691), shB3_3 (no. 9689), and shB3_4 (no. 9687). Full-length DNA sequences of Bnip3 and Nix were amplified by PCR and cloned into gateway entry plasmid pDONR221 (Invitrogen). Bnip3 ∆TMD or ∆LIR (W13A/L16A) were generated by site-directed mutagenesis (Stratagene). Inducible expression constructs were generated by attL × attR recombination between pDONR221 and pInducer-Bla, genetically modified from the pINDUCER20 lentiviral vector (a gift from Stephen Elledge [29 (link)]; Addgene plasmid no. 44012), by replacing a neomycin cassette with the blasticidin resistance gene.

Tol M.J., Ottenhoff R., van Eijk M., Zelcer N., Aten J., Houten S.M., Geerts D., van Roomen C., Bierlaagh M.C., Scheij S., Hoeksema M.A., Aerts J.M., Bogan J.S., Dorn GW 2.n.d., Argmann C.A, & Verhoeven A.J. (2016). A PPARγ-Bnip3 Axis Couples Adipose Mitochondrial Fusion-Fission Balance to Systemic Insulin Sensitivity. Diabetes, 65(9), 2591-2605.

+ Open protocol

+ Expand

Overexpression of OsSDG729 in Rice

Check if the same lab product or an alternative is used in the 5 most similar protocols

OsSDG729 (Os01g56540) was amplified from DNA of Oryza sativa L. cv. 'Kitaake' using a high fidelity Taq DNA polymerase (Bioline, VELOCITY DNA Polymerase) and cloned into entry vector pDONR221 (Addgene) using Gateway BP clonase (Thermo Scientific). After confirmation by sequencing, the fragment was subcloned into binary vector PMBb7Fm21GW-UBIL (VIB, UGent, Belgium) using Gateway LR clonase (Thermo Scientific) and introduced into Agrobacterium tumefaciens EHA105 cells using tri-parental mating. Rice transformation was performed according to Langdale's lab protocol adapted for Kitaake rice (https://langdalelab.com/protocols/transformation/). Transformed calli were selected on Luria Broth medium supplemented with 35 mg/mL of DL-Phosphinothricin (Duchefa, The Netherlands). T1 generation plants were selected by spraying 30mg/L of DL-Phosphinothricin. For evaluation of the expression level of OsSDG729 in T1 transgenic plants, qRT-PCR was performed (see further). Two independent lines with the highest induction pattern, namely 128-fold (line F3-5) and 16-fold (line B1-6) induction in comparison with wild-type were selected. All primers are listed in Supplementary file 1.

Atighi M.R., Verstraeten B., Meyer T.D., & Kyndt T. (2020). Genome-wide shifts in histone modifications at early stage of rice infection with Meloidogyne graminicola.

+ Open protocol

+ Expand

About PubCompare

Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.

We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.

However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.

Ready to get started?

Revolutionizing how scientists
search and build protocols!