Price Information
| Cat No. | Plasmid Name | Availability | Add to cart |
|---|---|---|---|
| V012360 | pACRISPR | 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:
- pACRISPR
- Antibiotic Resistance:
- Ampicillin
- Length:
- 6935 bp
- Type:
- CRISPR, Synthetic Biology
- Replication origin:
- ori
- Promoter:
- trc
pACRISPR 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.
pACRISPR vector Sequence
LOCUS 40924_3851 6935 bp DNA circular SYN 13-MAY-2021
DEFINITION A sgRNA expression plasmid for genome editing in Pseudomonas
aeruginosa.
ACCESSION .
VERSION .
KEYWORDS .
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 6935)
AUTHORS Chen W, Zhang Y, Zhang Y, Pi Y, Gu T, Song L, Wang Y, Ji Q
TITLE CRISPR/Cas9-based Genome Editing in Pseudomonas aeruginosa and
Cytidine Deaminase-Mediated Base Editing in Pseudomonas Species.
JOURNAL iScience. 2018 Aug 31;6:222-231. doi: 10.1016/j.isci.2018.07.024.
Epub 2018 Aug 1.
PUBMED 30240613
REFERENCE 2 (bases 1 to 6935)
TITLE Direct Submission
REFERENCE 3 (bases 1 to 6935)
AUTHORS .
TITLE Direct Submission
COMMENT SGRef: number: 1; type: "Journal Article"; journalName: "iScience.";
date: "2018-08-31"; pages: "
10.1016/j.isci.2018.07.024. Epub 2018 Aug 1"
COMMENT SGRef: number: 2; type: "Journal Article"
FEATURES Location/Qualifiers
source 1..6935
/mol_type="other DNA"
/organism="synthetic DNA construct"
primer_bind 475..491
/label=M13 fwd
/note="common sequencing primer, one of multiple similar
variants"
promoter 498..516
/label=T7 promoter
/note="promoter for bacteriophage T7 RNA polymerase"
CDS complement(607..2025)
/codon_start=1
/label=SacB
/note="secreted levansucrase that renders bacterial growth
sensitive to sucrose"
/translation="MNIKKFAKQATVLTFTTALLAGGATQAFAKETNQKPYKETYGISH
ITRHDMLQIPEQQKNEKYQVPEFDSSTIKNISSAKGLDVWDSWPLQNADGTVANYHGYH
IVFALAGDPKNADDTSIYMFYQKVGETSIDSWKNAGRVFKDSDKFDANDSILKDQTQEW
SGSATFTSDGKIRLFYTDFSGKHYGKQTLTTAQVNVSASDSSLNINGVEDYKSIFDGDG
KTYQNVQQFIDEGNYSSGDNHTLRDPHYVEDKGHKYLVFEANTGTEDGYQGEESLFNKA
YYGKSTSFFRQESQKLLQSDKKRTAELANGALGMIELNDDYTLKKVMKPLIASNTVTDE
IERANVFKMNGKWYLFTDSRGSKMTIDGITSNDIYMLGYVSNSLTGPYKPLNKTGLVLK
MDLDPNDVTFTYSHFAVPQAKGNNVVITSYMTNRGFYADKQSTFAPSFLLNIKGKKTSV
VKDSILEQGQLTVNK"
promoter 2487..2516
/label=trc promoter
/note="strong E. coli promoter; hybrid between the trp and
lac UV5 promoters"
misc_RNA 2539..2614
/label=gRNA scaffold
/note="guide RNA scaffold for the Streptococcus pyogenes
CRISPR/Cas9 system"
promoter complement(2694..2712)
/label=SP6 promoter
/note="promoter for bacteriophage SP6 RNA polymerase"
primer_bind complement(2730..2746)
/label=M13 rev
/note="common sequencing primer, one of multiple similar
variants"
protein_bind complement(2754..2770)
/label=lac operator
/note="The lac repressor binds to the lac operator to
inhibit transcription in E. coli. This inhibition can be
relieved by adding lactose or
isopropyl-beta-D-thiogalactopyranoside (IPTG)."
promoter complement(2778..2808)
/label=lac promoter
/note="promoter for the E. coli lac operon"
protein_bind complement(2823..2844)
/label=CAP binding site
/note="CAP binding activates transcription in the presence
of cAMP."
promoter 3001..3105
/label=AmpR promoter
CDS 3106..3963
/codon_start=1
/label=AmpR
/note="beta-lactamase"
/translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
LIKHW"
rep_origin complement(4304..4655)
/direction=LEFT
/label=pRO1600 oriV
/note="broad-host-range origin of replication from
Pseudomonas aeruginosa plasmid pRO1600; requires the
pRO1600 Rep protein for replication (West et al., 1994)"
CDS 4669..5490
/codon_start=1
/label=pRO1600 Rep
/note="replication protein for the broad-host-range plasmid
pRO1600 from Pseudomonas aeruginosa"
/translation="VASPPMVYKSNALVEAAYRLSVQEQRIVLACISQVKRSEPVTDEV
MYSVTAEDIATMAGVPIESSYNQLKEAALRLKRREVRLTQEPNGKGKRPSVMITGWVQT
IIYREGEGRVELRFTKDMLPYLTELTKQFTKYALADVAKMDSTHAIRLYELLMQWDSIG
QREIEIDQLRKWFQLEGRYPSIKDFKLRVLDPAVTQINEHSPLQVEWAQRKTGRKVTHL
LFSFGPKKPAKAVGKAPAKRKAGKISDAEIAKQARPGETWEAARARLTQMPL"
rep_origin 5959..6547
/direction=RIGHT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
rep_origin complement(join(6814..6935,1..334))
/direction=LEFT
/label=f1 ori
/note="f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"