Price Information
| Cat No. | Plasmid Name | Availability | Add to cart |
|---|---|---|---|
| V011044 | pX330 | 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
- Vector Name:
- pX330
- Antibiotic Resistance:
- Ampicillin
- Length:
- 8484 bp
- Type:
- Mammalian Expression Vectors
- Replication origin:
- ori
- Source/Author:
- Cong L, Ran FA, Cox D, Lin S, Barretto R, Habib N, Hsu PD, Wu X,
- Copy Number:
- High copy number
- Promoter:
- U6
pX330 vector 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.
pX330 vector Sequence
LOCUS pX330. 8484 bp DNA circular SYN 01-JAN-1980
DEFINITION Zhang lab plasmid for expressing a chimeric guide RNA (gRNA)
together with human codon-optimized Cas9. Also known as
pX330-U6-Chimeric_BB-CBh-hSpCas9.
ACCESSION .
VERSION .
KEYWORDS pX330
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 8484)
AUTHORS Cong L, Ran FA, Cox D, Lin S, Barretto R, Habib N, Hsu PD, Wu X,
Jiang W, Marraffini LA, Zhang F.
TITLE Multiplex genome engineering using CRISPR/Cas systems.
JOURNAL Science 2013;339:819-23.
PUBMED 23287718
REFERENCE 2 (bases 1 to 8484)
AUTHORS Zhang Lab / Addgene #42230
TITLE Direct Submission
REFERENCE 3 (bases 1 to 8484)
AUTHORS .
TITLE Direct Submission
COMMENT SGRef: number: 1; type: "Journal Article"; journalName: "Science";
date: "2013"; volume: "339"; pages: "819-23"
COMMENT SGRef: number: 2; type: "Journal Article"
COMMENT Digest with BbsI to insert annealed oligos encoding the guide
sequence.
NGS sequence provided by Addgene.
FEATURES Location/Qualifiers
source 1..8484
/mol_type="other DNA"
/organism="synthetic DNA construct"
intron 187..414
/label=hybrid intron
/note="hybrid between chicken beta-actin (CBA) and minute
virus of mice (MMV) introns (Gray et al., 2011)"
CDS 432..434
/codon_start=1
/product="start codon"
/label=start codon
/note="ATG"
/translation="M"
CDS 435..500
/codon_start=1
/product="three tandem FLAG(R) epitope tags, followed by an
enterokinase cleavage site"
/label=three tandem FLAG
/note="3xFLAG"
/translation="DYKDHDGDYKDHDIDYKDDDDK"
CDS 507..527
/codon_start=1
/product="nuclear localization signal of SV40 large T
antigen"
/note="SV40 NLS"
/translation="PKKKRKV"
CDS 552..4652
/label=Cas9
/note="Cas9 (Csn1) endonuclease from the Streptococcus
pyogenes Type II CRISPR/Cas system"
CDS 4653..4700
/codon_start=1
/product="bipartite nuclear localization signal from
nucleoplasmin"
/label=bipartite nuclear localization signal from
nucl
/note="nucleoplasmin NLS"
/translation="KRPAATKKAGQAKKKK"
polyA_signal 4734..4941
/label=bGH poly(A) signal
/note="bovine growth hormone polyadenylation signal"
repeat_region 4950..5090
/label=AAV2 ITR
/note="inverted terminal repeat of adeno-associated virus
serotype 2"
rep_origin 5165..5620
/label=f1 ori
/note="f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
promoter 5902..6006
/label=AmpR promoter
CDS 6007..6864
/label=AmpR
/note="beta-lactamase"
rep_origin 7038..7626
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
promoter 7688..7928
/label=U6 promoter
/note="RNA polymerase III promoter for human U6 snRNA"
misc_RNA 7955..8030
/label=gRNA scaffold
/note="guide RNA scaffold for the Streptococcus pyogenes
CRISPR/Cas9 system"
terminator 8031..8036
/note="pol III terminator"
/note="RNA polymerase III transcription terminator"
enhancer 8127..8412
/label=CMV enhancer
/note="human cytomegalovirus immediate early enhancer;
contains an 18-bp deletion relative to the standard CMV
enhancer"