Purposelenti vector encoding the MS2-P65-HSF1 activator helper complex with a 2A Hygro resistance marker (EF1a-MS2-p65-HSF1-2A-Hygro-WPRE)
|Item||Catalog #||Description||Quantity||Price (USD)|
|Plasmid||61426||Plasmid sent as bacteria in agar stab||1||$65|
|Concentrated Lentiviral Prep||61426-LVC||
Virus (100µL at titer ≥ 5×10⁵ TU/mL)
and Plasmid. More Information
This material is available to academics and nonprofits only.
Vector typeMammalian Expression, Lentiviral, CRISPR
Growth in Bacteria
Copy numberHigh Copy
SpeciesH. sapiens (human), Synthetic
MutationN55K in MS2
- Promoter EF1A
- Cloning method Restriction Enzyme
- 5′ cloning site BsiWI (not destroyed)
- 3′ cloning site EcoRI (not destroyed)
- 5′ sequencing primer GTT TGG ATC TTG GTT CAT TCT CAA GCC TCA G
- 3′ sequencing primer cacatagcgtaaaaggagcaacatag (Common Sequencing Primers)
IMPORTANT NOTE: If you intend to order the SAM library, this plasmid will come included with that order. Please do NOT order this plasmid if you are already planning to order the SAM library.
For additional information, protocols and an activator sgRNA design tool, visit our website:
Information for Concentrated Lentiviral Prep (Catalog # 61426-LVC) ( Back to top )
Ready-to-use Concentrated Lentiviral Prep particles produced from lenti MS2-P65-HSF1_Hygro (#61426). In addition to the viral particles, you will also receive purified lenti MS2-P65-HSF1_Hygro plasmid DNA.<p>Concentrated lentiviral particles carrying MS2-P65-HSF1 and hygromycin resistance.</p>
- Volume 100 µL
- Titer ≥5×10⁵ TU/mL
- Pricing $360 USD for preparation of 100 µL virus + $30 USD for plasmid.
- Storage Store at -80℃. Thaw just before use and keep on ice.
- Shipment Viral particles are shipped frozen on dry ice. Plasmid DNA (≥ 200ng) will also be included in the shipment.
Viral Production & Use
- Packaging Plasmids psPAX2 (plasmid #12260)
- Envelope pMD2.G (plasmid #12259)
- Buffer PBS
- Selectable Marker Hygromycin
- Purification Lentivirus was harvested from cell culture medium (DMEM + 10% FBS). Lentiviral particles were then collected by precipitation in polyethylene glycol (PEG) followed by centrifugation. Precipitated pellets containing viral particles were resuspended in PBS.
Requestor is responsible for compliance with their institution's biosafety regulations. Lentivirus is generally considered BSL-2. AAV is generally considered BSL-1, but may require BSL-2 handling depending on the insert. Biosafety Guide
Viral Quality Control
- Proviral Integration Assay: Lenti-X 293T cells were serially transduced with 61426-LVC or a control virus of known titer. 72 hours after transduction cells were harvested, and gDNA extracted and assessed for integrated copies of WPRE.
- PCR confirmation of insert: PCR was carried out with primers targeting the hygromycin-resistance gene and pBluescript. The PCR product was visualized on an agarose gel for size confirmation.
Forward Primer: Hygro-F GACGGCAATTTCGATGATG
Reverse Primer: pBluescript-KS TCGAGGTCGACGGTATC
- Confirmation of protein expression: A549 cells were singly transduced with 61426-LVC or doubly transduced with 61425-LV and 61426-LVC at an MOI of 1, and treated with hygromycin or hygromycin and blasticidin, respectively. Polyclonal pools of hygromycin-resistant or hygromycin and blasticidin-resistance cells were expanded, collected, lysed and tested for p65 expression via immunoblotting. You can view the stable cell line expression data here or in the image section at the top of this page. Read our protocol for generating stable cell lines here.
Visit our viral production page for more information.
While the typical yield for lentiviral vectors ranges from 10⁶-10⁷ TU/mL, titers for large or toxic inserts, such as for MS2-P65-HSF1, can be 10-fold to 100-fold lower. Scientists generating their own lentiviral particles from MS2-P65-HSF1 should expect similarly low titers.
These plasmids were created by your colleagues. Please acknowledge the Principal Investigator, cite the article in which the plasmids were described, and include Addgene in the Materials and Methods of your future publications.
For your Materials & Methods section:lenti MS2-P65-HSF1_Hygro was a gift from Feng Zhang (Addgene plasmid # 61426)
For your References section:Genome-scale transcriptional activation by an engineered CRISPR-Cas9 complex. Konermann S, Brigham MD, Trevino AE, Joung J, Abudayyeh OO, Barcena C, Hsu PD, Habib N, Gootenberg JS, Nishimasu H, Nureki O, Zhang F. Nature. 2014 Dec 10. doi: 10.1038/nature14136. 10.1038/nature14136 PubMed 25494202
Generated by Addgene from full sequence.
Map uploaded by Addgene staff.
Map uploaded by the depositor.