Price Information
| Cat No. | Plasmid Name | Availability | Add to cart |
|---|---|---|---|
| V012329 | lentiCas9n(D10A)-Blast | In stock, 1 week for quality controls |
Buy one, get one free! |
Two tubes of lyophilized plasmid will be delivered, each tube is about 5µg.
Basic Vector Information
- Vector Name:
- lentiCas9n(D10A)-Blast
- Antibiotic Resistance:
- Ampicillin
- Length:
- 12860 bp
- Type:
- CRISPR Plasmids
- Replication origin:
- ori
- Source/Author:
- Sanjana NE, Shalem O, Zhang F.
- Copy Number:
- High copy number
lentiCas9n(D10A)-Blast 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.
lentiCas9n(D10A)-Blast vector Sequence
LOCUS V012329 12860 bp DNA circular SYN 01-JAN-1980
DEFINITION Exported.
ACCESSION V012329
VERSION V012329
KEYWORDS lentiCas9n(D10A)-Blast
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
.
REFERENCE 1 (bases 1 to 12860)
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 12860)
AUTHORS Zhang Lab / Addgene #63593
TITLE Direct Submission
REFERENCE 3 (bases 1 to 12860)
AUTHORS .
TITLE Direct Submission
COMMENT SGRef: number: 1; type: "Journal Article"; journalName: "Nat.
Methods"; date: "2014"; volume: "11"; pages: "783-4"
SGRef: number: 2; type: "Journal Article"
FEATURES Location/Qualifiers
source 1..12860
/mol_type="other DNA"
/organism="synthetic DNA construct"
enhancer 238..617
/label="CMV enhancer"
/note="human cytomegalovirus immediate early enhancer"
promoter 618..820
/label="CMV promoter"
/note="human cytomegalovirus (CMV) immediate early
promoter"
LTR 835..1015
/label="5' LTR (truncated)"
/note="truncated 5' long terminal repeat (LTR) from HIV-1"
misc_feature 1062..1187
/label="HIV-1 Psi"
/note="packaging signal of human immunodeficiency virus
type 1"
misc_feature 1680..1913
/label="RRE"
/note="The Rev response element (RRE) of HIV-1 allows for
Rev-dependent mRNA export from the nucleus to the
cytoplasm."
CDS 2098..2142
/label="gp41 peptide"
/note="antigenic peptide corresponding to amino acids 655
to 669 of the HIV envelope protein gp41 (Lutje Hulsik et
al., 2013)"
CDS 2291..2332
/note="Protein Tat from Human immunodeficiency virus type 1
group M subtype B (isolate WMJ22). Accession#: P12509"
/label="Protein Tat"
misc_feature 2440..2557
/label="cPPT/CTS"
/note="central polypurine tract and central termination
sequence of HIV-1"
promoter 2651..2862
/label="EF-1-alpha core promoter"
/note="core promoter for human elongation factor
EF-1-alpha"
CDS 2887..6990
/label="Cas9(D10A)"
/note="nickase mutant of the Cas9 endonuclease from the
Streptococcus pyogenes Type II CRISPR/Cas system"
CDS 6991..7038
/codon_start=1
/product="bipartite nuclear localization signal from
nucleoplasmin"
/label="bipartite nuclear localization signal from nucl"
/note="nucleoplasmin NLS"
/translation="KRPAATKKAGQAKKKK"
CDS 7039..7062
/label="FLAG"
/note="FLAG(R) epitope tag, followed by an enterokinase
cleavage site"
CDS 7072..7128
/label="P2A"
/note="2A peptide from porcine teschovirus-1 polyprotein"
CDS 7129..7524
/label="BSD"
/note="blasticidin S deaminase"
misc_feature 7552..8140
/label="WPRE"
/note="woodchuck hepatitis virus posttranscriptional
regulatory element"
LTR 8665..8845
/label="5' LTR (truncated)"
/note="5' LTR (truncated)"
/note="truncated 5' long terminal repeat (LTR) from HIV-1"
polyA_signal 8877..9101
/label="bGH poly(A) signal"
/note="bovine growth hormone polyadenylation signal"
rep_origin 9147..9575
/label="f1 ori"
/note="f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
promoter 9589..9918
/label="SV40 promoter"
/note="SV40 enhancer and early promoter"
promoter 9966..10013
/note="EM7 promoter"
/note="synthetic bacterial promoter "
CDS 10032..10403
/label="BleoR"
/note="antibiotic-binding protein"
polyA_signal 10536..10669
/label="SV40 poly(A) signal"
/note="SV40 polyadenylation signal"
promoter complement(10754..10784)
/label="lac promoter"
/note="promoter for the E. coli lac operon"
protein_bind complement(10799..10820)
/label="CAP binding site"
/note="CAP binding activates transcription in the presence
of cAMP."
rep_origin complement(11108..11696)
/direction=LEFT
/label="ori"
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
CDS complement(11870..12727)
/label="AmpR"
/note="beta-lactamase"
promoter complement(12728..12832)
/label="AmpR promoter"