Price Information
| Cat No. | Plasmid Name | Availability | Add to cart |
|---|---|---|---|
| V001084 | pCRISPR-cBEST | 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:
- pCRISPR-cBEST
- Antibiotic Resistance:
- Apramycin
- Length:
- 12247 bp
- Type:
- CRISPR, Synthetic Biology
- Replication origin:
- ori
- Copy Number:
- High Copy
- Promoter:
- ermE*/tipA
- Cloning Method:
- Gibson Cloning
pCRISPR-cBEST 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.
pCRISPR-cBEST vector Sequence
LOCUS V001084 12247 bp DNA circular SYN 13-MAY-2021
DEFINITION Exported.
ACCESSION V001084
VERSION V001084
KEYWORDS pCRISPR-cBEST
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
.
REFERENCE 1 (bases 1 to 12247)
AUTHORS Tong Y, Whitford CM, Robertsen HL, Blin K, Jorgensen TS, Klitgaard
AK, Gren T, Jiang X, Weber T, Lee SY
TITLE Highly efficient DSB-free base editing for streptomycetes with
CRISPR-BEST.
JOURNAL Proc Natl Acad Sci U S A. 2019 Oct 8;116(41):20366-20375. doi:
10.1073/pnas.1913493116. Epub 2019 Sep 23.
PUBMED 31548381
REFERENCE 2 (bases 1 to 12247)
TITLE Direct Submission
REFERENCE 3 (bases 1 to 12247)
AUTHORS .
TITLE Direct Submission
COMMENT SGRef: number: 1; type: "Journal Article"; doi:
"10.1073/pnas.1913493116"; journalName: "Proc Natl Acad Sci U S A";
date: "2019-10-8- 8"; volume: "116"; issue: "41"; pages:
"20366-20375"
SGRef: number: 2; type: "Journal Article"
FEATURES Location/Qualifiers
source 1..12247
/mol_type="other DNA"
/organism="synthetic DNA construct"
oriT complement(466..575)
/direction=LEFT
/label="oriT"
/note="incP origin of transfer"
CDS 891..1691
/label="ApmR"
/note="aminoglycoside 3-N-acetyltransferase type IV"
rep_origin 1993..2580
/label="ori"
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
primer_bind 2734..2751
/label="L4440"
/note="L4440 vector, forward primer"
protein_bind 2868..2889
/label="CAP binding site"
/note="CAP binding activates transcription in the presence
of cAMP."
promoter 2904..2934
/label="lac promoter"
/note="promoter for the E. coli lac operon"
protein_bind 2942..2958
/label="lac operator"
/bound_moiety="lac repressor encoded by lacI"
/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)."
primer_bind 2947..2969
/label="M13/pUC Reverse"
/note="In lacZ gene"
primer_bind 2966..2982
/label="M13 rev"
/note="common sequencing primer, one of multiple similar
variants"
primer_bind 2966..2982
/label="M13 Reverse"
/note="In lacZ gene. Also called M13-rev"
misc_RNA 3119..3194
/label="gRNA scaffold"
/note="guide RNA scaffold for the Streptococcus pyogenes
CRISPR/Cas9 system"
terminator complement(3364..3398)
/label="lambda t0 terminator"
/note="minimal transcription terminator from phage lambda
(Scholtissek and Grosse, 1987)"
CDS 3566..4252
/codon_start=1
/product="cytidine deaminase (C to U editing enzyme) from
rat"
/label="APOBEC-1"
/note="can use ssDNA as a substrate (Komor et al., 2016)"
/translation="MSSETGPVAVDPTLRRRIEPHEFEVFFDPRELRKETCLLYEINWG
GRHSIWRHTSQNTNKHVEVNFIEKFTTERYFCPNTRCSITWFLSWSPCGECSRAITEFL
SRYPHVTLFIYIARLYHHADPRNRQGLRDLISSGVTIQIMTEQESGYCWRNFVNYSPSN
EAHWPRYPHLWVRLYVLELYCIILGLPPCLNILRRKQPQLTFFTIALQSCHYQRLPPHI
LWATGLK"
CDS 4301..8401
/label="Cas9(D10A)"
/note="nickase mutant of the Cas9 endonuclease from the
Streptococcus pyogenes Type II CRISPR/Cas system"
CDS 8414..8665
/codon_start=1
/product="uracil-DNA glycosylase inhibitor from a Bacillus
subtilis bacteriophage (Mol et al., 1995)"
/label="UGI"
/translation="TNLSDIIEKETGKQLVIQESILMLPEEVEEVIGNKPESDILVHTA
YDESTDENVMLLTSDAPEYKPWALVIQDSNGENKIKML"
terminator complement(8846..8894)
/label="fd terminator"
/note="central terminator from bacteriophage fd (Otsuka and
Kunisawa, 1982)"
CDS 9237..10043
/gene="tsnR"
/label="23S rRNA (adenosine(1067)-2'-O)-methyltransferase"
/note="23S rRNA (adenosine(1067)-2'-O)-methyltransferase
from Streptomyces azureus. Accession#: P18644"
CDS 10609..12048
/label="pSG5 Rep"
/note="replication initiator protein from the Streptomyces
ghanaensis plasmid pSG5"