Price Information
| Cat No. | Plasmid Name | Availability | Add to cart |
|---|---|---|---|
| V005926 | pFS_0453_pET30_pCat-tetR-Term-ptetA-FsRT-Cas1(opt)-Cas2(opt)_T7term_ARRAY2_FaqI | 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:
- pFS_0453_pET30_pCat-tetR-Term-ptetA-FsRT-Cas1(opt)-Cas2(opt)_T7term_ARRAY2_FaqI
- Antibiotic Resistance:
- Kanamycin
- Length:
- 6346 bp
- Type:
- Bacterial Expression
- Replication origin:
- ori
- Copy Number:
- Low Copy
- Promoter:
- T7
- Cloning Method:
- Restriction Enzyme
- 5' Primer:
- T7
- 3' Primer:
- T7-term
pFS_0453_pET30_pCat-tetR-Term-ptetA-FsRT-Cas1(opt)-Cas2(opt)_T7term_ARRAY2_FaqI 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.
pFS_0453_pET30_pCat-tetR-Term-ptetA-FsRT-Cas1(opt)-Cas2(opt)_T7term_ARRAY2_FaqI vector Sequence
LOCUS pFS_0453_pET30_p 6346 bp DNA circular SYN 13-MAY-2021
DEFINITION Expresses E. coli codon-optimized FsRT-Cas1-Cas2 under ptetA
promoter, encodes FsCRISPR array 2, compatible with SENECA
acquisition readout.
ACCESSION .
VERSION .
KEYWORDS pFS_0453_pET30_pCat-tetR-Term-ptetA-FsRT-Cas1(opt)-Cas2(o.
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 6346)
AUTHORS Schmidt F, Cherepkova MY, Platt RJ
TITLE Transcriptional recording by CRISPR spacer acquisition from RNA.
JOURNAL Nature. 2018 Oct;562(7727):380-385. doi: 10.1038/s41586-018-0569-1.
Epub 2018 Oct 3.
PUBMED 30283135
REFERENCE 2 (bases 1 to 6346)
TITLE Direct Submission
REFERENCE 3 (bases 1 to 6346)
AUTHORS .
TITLE Direct Submission
COMMENT SGRef: number: 1; type: "Journal Article"; doi:
"10.1038/s41586-018-0569-1"; journalName: "Nature"; date: "2018-10";
volume: "562"; issue: "7727"; pages: "380-385"
COMMENT SGRef: number: 2; type: "Journal Article"
FEATURES Location/Qualifiers
source 1..6346
/mol_type="other DNA"
/organism="synthetic DNA construct"
CDS complement(213..1025)
/label=KanR
/note="aminoglycoside phosphotransferase"
rep_origin 1147..1735
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
primer_bind 1889..1906
/label=L4440
/note="L4440 vector, forward primer"
misc_feature complement(1921..2060)
/label=bom
/note="basis of mobility region from pBR322"
primer_bind 2146..2168
/label=pGEX 3'
/note="pGEX vectors, reverse primer"
CDS complement(2165..2353)
/label=rop
/note="Rop protein, which maintains plasmids at low copy
number"
RBS 2883..2894
/note="strong bacterial ribosome binding site (Elowitz and
Leibler, 2000)"
CDS 2901..3521
/label=TetR
/note="tetracycline repressor TetR"
terminator 3569..3640
/label=rrnB T1 terminator
/note="transcription terminator T1 from the E. coli rrnB
gene"
terminator 3656..3683
/label=T7Te terminator
/note="phage T7 early transcription terminator"
protein_bind 3698..3716
/label=tet operator
/note="bacterial operator O2 for the tetR and tetA genes"
protein_bind 3723..3741
/gene="tetO"
/label=tet operator
/bound_moiety="tetracycline repressor TetR"
/note="bacterial operator O2 for the tetR and tetA genes"
terminator 6039..6086
/label=T7 terminator
/note="transcription terminator for bacteriophage T7 RNA
polymerase"