Price Information
| Cat No. | Plasmid Name | Availability | Add to cart |
|---|---|---|---|
| V010156 | 6-His-MBP-TEV-FnCpf1 | 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
Gene/Insert name FnCpf1 (humanized) Alt name type V CRISPR-associated protein Cpf1 (humanized) Alt name Cpf1 (humanized) Alt name Francisella tularensis subsp. novicida U112 Cpf1 (humanized) Species Francisella tularensis subsp. novicida U112 Insert Size (bp) 3910 Promoter T7 Tags / Fusion Proteins 6xHis (N terminal on backbone) MBP (N terminal on insert) TEV site (N terminal on insert) Nucleoplasmin NLS (C terminal on insert) 3xHA (C terminal on insert)
- Vector Name:
- 6-His-MBP-TEV-FnCpf1
- Antibiotic Resistance:
- Ampicillin
- Length:
- 10685 bp
- Type:
- CRISPR Plasmids
- Replication origin:
- ori
- Source/Author:
- Feng Zhang
- Copy Number:
- High copy number
- Promoter:
- T7
- Cloning Method:
- Enzyme Cut
- 5' Primer:
- T7 promoter
- 3' Primer:
- T7 terminator
- Fusion Tag:
- MBP
- Expression Method:
- IPTG induced
6-His-MBP-TEV-FnCpf1 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.
6-His-MBP-TEV-FnCpf1 vector Sequence
LOCUS V010156 10685 bp DNA circular SYN 04-JUL-2018
DEFINITION Exported.
ACCESSION V010156
VERSION V010156
KEYWORDS 6-His-MBP-TEV-FnCpf1
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
.
REFERENCE 1 (bases 1 to 10685)
AUTHORS Zetsche B, Gootenberg JS, Abudayyeh OO, Slaymaker IM, Makarova KS,
Essletzbichler P, Volz SE, Joung J, van der Oost J, Regev A, Koonin
EV, Zhang F
TITLE Cpf1 Is a Single RNA-Guided Endonuclease of a Class 2 CRISPR-Cas
System.
JOURNAL Cell. 2015 Sep 23. pii: S0092-8674(15)01200-3. doi:
10.1016/j.cell.2015.09.038.
PUBMED 26422227
REFERENCE 2 (bases 1 to 10685)
TITLE Direct Submission
REFERENCE 3 (bases 1 to 10685)
AUTHORS .
TITLE Direct Submission
COMMENT SGRef: number: 1; type: "Journal Article"; journalName: "Cell. 2015
Sep 23. pii: S0092-8674(15)01200-3. doi:
10.1016/j.cell.2015.09.038."
SGRef: number: 2; type: "Journal Article"
FEATURES Location/Qualifiers
source 1..10685
/mol_type="other DNA"
/organism="synthetic DNA construct"
rep_origin 532..1120
/label="ori"
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
misc_feature complement(1306..1445)
/label="bom"
/note="basis of mobility region from pBR322"
CDS complement(1550..1738)
/label="rop"
/note="Rop protein, which maintains plasmids at low copy
number"
protein_bind complement(2513..2534)
/label="CAP binding site"
/note="CAP binding activates transcription in the presence
of cAMP."
CDS complement(2550..3629)
/label="lacI"
/note="lac repressor"
promoter complement(3630..3707)
/label="lacI promoter"
promoter 4016..4034
/label="T7 promoter"
/note="promoter for bacteriophage T7 RNA polymerase"
protein_bind 4035..4059
/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)."
RBS 4074..4096
/label="RBS"
/note="efficient ribosome binding site from bacteriophage
T7 gene 10 (Olins and Rangwala, 1989)"
CDS 4110..4127
/label="6xHis"
/note="6xHis affinity tag"
CDS 4128..5225
/label="MBP"
/note="maltose binding protein from E. coli"
CDS 5244..5261
/label="thrombin site"
/note="thrombin recognition and cleavage site"
CDS 5268..5288
/label="TEV site"
/note="tobacco etch virus (TEV) protease recognition and
cleavage site"
CDS 5289..9188
/gene="cas12a"
/label="CRISPR-associated endonuclease Cas12a"
/note="CRISPR-associated endonuclease Cas12a from
Francisella tularensis subsp. novicida (strain U112).
Accession#: A0Q7Q2"
CDS 9189..9236
/label="nucleoplasmin NLS"
/note="bipartite nuclear localization signal from
nucleoplasmin"
CDS 9243..9323
/label="3xHA"
/note="three tandem HA epitope tags"
CDS 9348..9371
/label="FLAG"
/note="FLAG(R) epitope tag, followed by an enterokinase
cleavage site"
CDS 9372..9416
/label="AviTag(TM)"
/note="peptide tag that allows for enzymatic biotinylation"
CDS 9432..9449
/label="6xHis"
/note="6xHis affinity tag"
terminator 9516..9563
/label="T7 terminator"
/note="transcription terminator for bacteriophage T7 RNA
polymerase"
rep_origin 9600..9980
/label="M13 ori"
/note="M13 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
promoter 10081..10185
/label="AmpR promoter"
CDS join(10186..10685,1..358)
/label="AmpR"
/note="beta-lactamase"