pKLV2-U6gRNA(BbsI)-ccdB-PGKpuro2ABFP-W
(Plasmid
#153033)
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PurposeModified lentiviral pKLV2-U6gRNA(BbsI)-PGKpuro2ABFP-W (#67974) CRISPR gRNA expression vector which includes the ccdB gene flanked by BbsI sites, to improve efficiency of oligonucleotide cloning.
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Depositing Lab
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Sequence Information
Full plasmid sequence is not available for this item.
Ordering
| Item | Catalog # | Description | Quantity | Price (USD) | |
|---|---|---|---|---|---|
| Plasmid | 153033 | Standard format: Plasmid sent in bacteria as agar stab | 1 | $85 | |
This material is available to academics and nonprofits only.
Backbone
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Vector backboneccdb
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Modifications to backbonethe ccdB resistance gene cassette was PCR amplified from pKLV1-fl-U6gRNA(BbsI)-ccdB-PGKpuro2ABFP
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Vector typeLentiviral
Growth in Bacteria
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Bacterial Resistance(s)Ampicillin, 100 μg/mL
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Growth Temperature37°C
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Growth Strain(s)ccdB Survival
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Copy numberHigh Copy
Gene/Insert
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Gene/Insert nameccdb
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Insert Size (bp)306
- Promoter U6 promoter
Cloning Information
- Cloning method Gibson Cloning
- 5′ sequencing primer CACCGGGTCTTCTTA
- 3′ sequencing primer ATAAAGTGTAAAGAAGACctGTTT (Common Sequencing Primers)
Resource Information
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A portion of this plasmid was derived from a plasmid made bythe ccdB resistance gene cassette was PCR amplified from pKLV1-fl-U6gRNA(BbsI)-ccdB-PGKpuro2ABFP (a kindly provided by E. Metzokapian)
Terms and Licenses
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Academic/Nonprofit Terms
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Industry Terms
- Not Available to Industry
Trademarks:
- Zeocin® is an InvivoGen trademark.
Depositor Comments
The ccdB resistance gene cassette was PCR amplified from pKLV1-fl-U6gRNA(BbsI)-ccdB-PGKpuro2ABFP (a kindly provided by E. Metzokapian) with the following oligos, Gibson_pKLV2-ccdb_Fwd and Gibson_pKLV2-ccdb_Rev, which included appended oligonucleotide sequences complementary to the pKLV2-U6gRNA(BbsI)-PGKpuro2ABFP-W backbone and BbsI restriction enzyme sites. The amplified ccdB gene product was cloned into the pKLV2-U6gRNA(BbsI)-PGKpuro2ABFP-W plasmid, linearized by BbsI digestion, by Gibson Assembly Cloning (New England Biolabs) to generate the modified pKLV2-U6gRNA(BbsI)-ccdB-PGKpuro2ABFP-W plasmid vector. This was subsequently transformed into ccdB Survival E.coli (Invitrogen).
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.
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For your Materials & Methods section:
pKLV2-U6gRNA(BbsI)-ccdB-PGKpuro2ABFP-W was a gift from The Gene Editing Team of Wellcome Sanger (Addgene plasmid # 153033) -
For your References section:
Minimal genome-wide human CRISPR-Cas9 library. Goncalves E, Thomas M, Behan FM, Picco G, Pacini C, Allen F, Vinceti A, Sharma M, Jackson DA, Price S, Beaver CM, Dovey O, Parry-Smith D, Iorio F, Parts L, Yusa K, Garnett MJ. Genome Biol. 2021 Jan 21;22(1):40. doi: 10.1186/s13059-021-02268-4. 10.1186/s13059-021-02268-4 PubMed 33478580
Map uploaded by the depositor.
