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pFS_0453_pET30_pCat-tetR-Term-ptetA-FsRT-Cas1(opt)-Cas2(opt)_T7term_ARRAY2_FaqI vector (V005926)

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

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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

Copy Sequence View Map
pFS_0453_pET30_pCat-tetR-Term-ptetA-FsRT-Cas1(opt)-Cas2(opt)_T7term_ARRAY2_FaqI6346 bp300600900120015001800210024002700300033003600390042004500480051005400570060006300KanRoriL4440bomropRBSTetRrrnB T1 terminatorT7Te terminatortet operatortet operatorT7 terminator

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"