Price Information
| Cat No. | Plasmid Name | Availability | Add to cart |
|---|---|---|---|
| V014421 | pT7-SpCas9_sgRNA-site1 (RTW443) | 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:
- pT7-SpCas9_sgRNA-site1 (RTW443)
- Antibiotic Resistance:
- Kanamycin
- Length:
- 2144 bp
- Type:
- Genome editing
- Replication origin:
- ori
- Host:
- E. coli
- Copy Number:
- Low
- Promoter:
- T7
- Growth Temperature:
- 37℃
pT7-SpCas9_sgRNA-site1 (RTW443) 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.
pT7-SpCas9_sgRNA-site1 (RTW443) vector Sequence
LOCUS 62056_20810 2144 bp DNA circular SYN 01-JAN-1980
DEFINITION synthetic circular DNA.
ACCESSION .
VERSION .
KEYWORDS .
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 2144)
AUTHORS .
TITLE Direct Submission
FEATURES Location/Qualifiers
source 1..2144
/mol_type="other DNA"
/organism="synthetic DNA construct"
primer_bind 26..42
/label=M13 rev
/note="common sequencing primer, one of multiple similar
variants"
promoter 171..189
/label=T7 promoter
/note="promoter for bacteriophage T7 RNA polymerase"
misc_RNA 208..283
/label=gRNA scaffold
/note="guide RNA scaffold for the Streptococcus pyogenes
CRISPR/Cas9 system"
primer_bind complement(465..481)
/label=M13 fwd
/note="common sequencing primer, one of multiple similar
variants"
rep_origin 522..1109
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
terminator complement(1121..1148)
/label=T7Te terminator
/note="phage T7 early transcription terminator"
CDS complement(1175..1987)
/codon_start=1
/label=KanR
/note="aminoglycoside phosphotransferase"
/translation="MSHIQRETSCLKPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNGDRVFRLAQAQSRMNNGLVGA
SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
promoter complement(1988..2090)
/label=cat promoter
/note="promoter of the E. coli cat gene encoding
chloramphenicol acetyltransferase"
terminator complement(2091..2122)
/label=tonB terminator
/note="bidirectional E. coli tonB-P14 transcription
terminator"