Price Information
| Cat No. | Plasmid Name | Availability | Add to cart |
|---|---|---|---|
| V012327 | lentiGuide-Puro | In stock, instant shipping |
Buy one, get one free! |
Two tubes of lyophilized plasmid will be delivered, each tube is about 5µg.
Basic Vector Information
The lentiGuide-Puro is a lentiviral vector, also known as pLentiGuide-Puro. It is for expressing a single guide RNA (sgRNA) in Cas9-expressing cells.
- Vector Name:
- lentiGuide-Puro
- Antibiotic Resistance:
- Ampicillin
- Length:
- 10183 bp
- Type:
- CRISPR Plasmids
- Replication origin:
- ori
- Source/Author:
- Sanjana NE, Shalem O, Zhang F.
- Copy Number:
- High copy number
- Promoter:
- EF-1α
- Growth Strain(s):
- Stbl3
- Growth Temperature:
- 37℃
lentiGuide-Puro vector Map
Plasmid Protocol
1. Centrifuge at 5,000×g for 5 min.
2. Carefully open the tube and add 20 μl of sterile water to dissolve the DNA.
3. Close the tube and incubate for 10 minutes at room temperature.
4. Briefly vortex the tube and then do a quick spin to concentrate the liquid at the bottom. Speed is less than 5000×g.
5. Store the plasmid at -20 ℃.
6. The concentration of plasmid re-measurement sometimes differs from the nominal value, which may be due to the position of the lyophilized plasmid in the tube, the efficiency of the re-dissolution, the measurement bias, and adsorption on the wall of the tube, therefore, it is recommended to transform and extract the plasmid before using it
General Plasmid Transform Protocol
1. Take one 100μl of the competent cells and thaw it on ice for 10min, add 2μl of plasmid, then ice bath for 30min, then heat-shock it at 42℃ for 60s, do not stir, and then ice bath for 2min.
2. Add 900μl of LB liquid medium without antibiotics, and incubate at 37℃ for 45min (30℃ for 1-1.5 hours) with 180rpm shaking.
3. Centrifuge at 6000rpm for 5min, leave only 100μl of supernatant to resuspend the bacterial precipitate and spread it onto the target plasmid-resistant LB plate.
4. Invert the plate and incubate at 37℃ for 14h, or at 30℃ for 20h.
5. Pick a single colony into LB liquid medium, add the corresponding antibiotics, incubate at 220rpm for 14h, and extract the plasmid according to the experimental needs and the instructions of the plasmid extraction kit.
References
- Datlinger P, Rendeiro AF, Schmidl C, Krausgruber T, Traxler P, Klughammer J, Schuster LC, Kuchler A, Alpar D, Bock C. Pooled CRISPR screening with single-cell transcriptome readout. Nat Methods. 2017 Mar;14(3):297-301.
lentiGuide-Puro vector Sequence
LOCUS lentiGuide-Puro. 10183 bp DNA circular SYN 01-JAN-1980
DEFINITION Zhang lab lentiviral vector, also known as pLentiGuide-Puro, for
expressing a single guide RNA (sgRNA) in Cas9-expressing cells. Use
together with lentiCas9-Blast.
ACCESSION .
VERSION .
KEYWORDS lentiGuide-Puro
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 10183)
AUTHORS Sanjana NE, Shalem O, Zhang F.
TITLE Improved vectors and genome-wide libraries for CRISPR screening.
JOURNAL Nat. Methods 2014;11:783-4.
PUBMED 25075903
REFERENCE 2 (bases 1 to 10183)
AUTHORS Zhang Lab / Addgene #52963
TITLE Direct Submission
REFERENCE 3 (bases 1 to 10183)
AUTHORS .
TITLE Direct Submission
COMMENT SGRef: number: 1; type: "Journal Article"; journalName: "Nat.
Methods"; date: "2014"; volume: "11"; pages: "783-4"
COMMENT SGRef: number: 2; type: "Journal Article"
FEATURES Location/Qualifiers
source 1..10183
/mol_type="other DNA"
/organism="synthetic DNA construct"
promoter 87..1265
/label=EF-1-alpha promoter
/note="strong constitutive promoter for human elongation
factor EF-1-alpha"
CDS 1278..1874
/label=PuroR
/note="puromycin N-acetyltransferase"
misc_feature 1893..2481
/label=WPRE
/note="woodchuck hepatitis virus posttranscriptional
regulatory element"
LTR 2553..2786
/label=3' LTR (Delta-U3)
/note="self-inactivating 3' long terminal repeat (LTR) from
HIV-1"
polyA_signal 2864..2998
/label=SV40 poly(A) signal
/note="SV40 polyadenylation signal"
rep_origin 3025..3160
/label=SV40 ori
/note="SV40 origin of replication"
promoter complement(3181..3199)
/label=T7 promoter
/note="promoter for bacteriophage T7 RNA polymerase"
rep_origin 3367..3822
/label=f1 ori
/note="f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
promoter 3848..3952
/label=AmpR promoter
CDS 3953..4810
/label=AmpR
/note="beta-lactamase"
rep_origin 4984..5572
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
protein_bind 5860..5881
/label=CAP binding site
/note="CAP binding activates transcription in the presence
of cAMP."
promoter 5896..5926
/label=lac promoter
/note="promoter for the E. coli lac operon"
promoter 5995..6013
/label=T3 promoter
/note="promoter for bacteriophage T3 RNA polymerase"
promoter 6041..6267
/label=RSV promoter
/note="Rous sarcoma virus enhancer/promoter"
LTR 6268..6448
/label=5' LTR (truncated)
/note="truncated 5' long terminal repeat (LTR) from HIV-1"
misc_feature 6495..6620
/label=HIV-1 Psi
/note="packaging signal of human immunodeficiency virus
type 1"
misc_feature 7113..7346
/label=RRE
/note="The Rev response element (RRE) of HIV-1 allows for
Rev-dependent mRNA export from the nucleus to the
cytoplasm."
CDS 7531..7575
/label=gp41 peptide
/note="antigenic peptide corresponding to amino acids 655
to 669 of the HIV envelope protein gp41 (Lutje Hulsik et
al., 2013)"
promoter 7753..7993
/label=U6 promoter
/note="RNA polymerase III promoter for human U6 snRNA"
misc_feature 7998..9882
/label=filler
/note="filler"
/note="digest with BsmBI to replace the filler with an
sgRNA"
misc_RNA 9883..9958
/label=gRNA scaffold
/note="guide RNA scaffold for the Streptococcus pyogenes
CRISPR/Cas9 system"
terminator 9959..9964
/note="polIII terminator"
/note="RNA polymerase III transcription terminator"
misc_feature 10014..10131
/label=cPPT/CTS
/note="central polypurine tract and central termination
sequence of HIV-1"