Price Information
| Cat No. | Plasmid Name | Availability | Buy one, get one free! (?) |
|---|---|---|---|
| V012754 | pX601 | In stock, 1 week for quality controls |
Two tubes of lyophilized plasmid will be delivered, each tube is about 5µg.
Basic Vector Information
Also known as pX601-AAV-CMV::NLS-SaCas9-NLS-3xHA-bGHpA;U6::BsaI-sgRNA
- Vector Name:
- pX601
- Antibiotic Resistance:
- Ampicillin
- Length:
- 7447 bp
- Type:
- CRISPR/Cas
- Replication origin:
- ori
- Copy Number:
- High copy number
- Promoter:
- U6
- Fusion Tag:
- HA
pX601 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.
pX601 vector Sequence
LOCUS pX601. 7447 bp DNA circular SYN 11-SEP-2021
DEFINITION synthetic circular DNA.
ACCESSION .
VERSION .
KEYWORDS pX601
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 7447)
TITLE Direct Submission
REFERENCE 2 (bases 1 to 7447)
AUTHORS .
TITLE Direct Submission
COMMENT SGRef: number: 1; type: "Journal Article"
FEATURES Location/Qualifiers
source 1..7447
/mol_type="other DNA"
/organism="synthetic DNA construct"
enhancer 154..533
/label=CMV enhancer
/note="human cytomegalovirus immediate early enhancer"
promoter 534..737
/label=CMV promoter
/note="human cytomegalovirus (CMV) immediate early
promoter"
CDS 768..788
/codon_start=1
/product="nuclear localization signal of SV40 large T
antigen"
/label=nuclear localization signal of SV40 large T
ant
/note="SV40 NLS"
/translation="PKKKRKV"
CDS 813..3968
/label=SaCas9
/note="Cas9 endonuclease from the Staphylococcus aureus
Type II CRISPR/Cas system"
CDS 3969..4016
/codon_start=1
/product="bipartite nuclear localization signal from
nucleoplasmin"
/label=bipartite nuclear localization signal from
nucl
/note="nucleoplasmin NLS"
/translation="KRPAATKKAGQAKKKK"
CDS 4023..4103
/label=3xHA
/note="three tandem HA epitope tags"
polyA_signal 4137..4344
/label=bGH poly(A) signal
/note="bovine growth hormone polyadenylation signal"
promoter 4352..4592
/label=U6 promoter
/note="RNA polymerase III promoter for human U6 snRNA"
misc_RNA 4621..4696
/label=Sa gRNA scaffold
/note="guide RNA scaffold for the Staphylococcus aureus
CRISPR/Cas9 system"
repeat_region 4710..4850
/label=AAV2 ITR
/note="inverted terminal repeat of adeno-associated virus
serotype 2"
rep_origin 4925..5380
/label=f1 ori
/note="f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
promoter 5662..5766
/label=AmpR promoter
CDS 5767..6624
/label=AmpR
/note="beta-lactamase"
rep_origin 6798..7386
/direction=RIGHT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"