pTargetF vector (V012144)

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Price Information

Cat No.	Plasmid Name	Availability	Add to cart
V012144	pTargetF	In stock, instant shipping	Buy one, get one free!

Two tubes of lyophilized plasmid will be delivered, each tube is about 5µg.

Basic Vector Information

The pTargetF is for constitutive expression of sgRNA without donor editing template DNA.

Vector Name:: pTargetF

Antibiotic Resistance:: Spectinomycin

Length:: 2117 bp

Type:: Bacterial Expression, CRISPR

Replication origin:: ori

Copy Number:: High Copy

Promoter:: J23119(SpeI)

Cloning Method:: Restriction Enzyme

5' Primer:: MoClo-F (agcgaggaagcggaagagcg)

Growth Strain(s):: DH10B

Growth Temperature:: 37℃

pTargetF vector Map

Copy Sequence View 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.

References

Jiang Y, Chen B, Duan C, Sun B, Yang J, Yang S. Multigene editing in the Escherichia coli genome via the CRISPR-Cas9 system. Appl Environ Microbiol. 2015 Apr;81(7):2506-14. doi: 10.1128/AEM.04023-14. Epub 2015 Jan 30. Erratum in: Appl Environ Microbiol. 2016 Jun 15;82(12):3693.

pTargetF vector Sequence

LOCUS       Exported                2117 bp ds-DNA     circular SYN 12-MAY-2021
DEFINITION  Constitutive expression of sgRNA without donor editing template DNA.
ACCESSION   .
VERSION     .
KEYWORDS    pTargetF
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 2117)
  AUTHORS   Jiang Y, Chen B, Duan C, Sun B, Yang J, Yang S
  TITLE     Multigene editing in the Escherichia coli genome using the 
            CRISPR-Cas9 system.
  JOURNAL   Appl Environ Microbiol. 2015 Jan 30. pii: AEM.04023-14.
  PUBMED    25636838
REFERENCE   2  (bases 1 to 2117)
  AUTHORS   .
  TITLE     Direct Submission
FEATURES             Location/Qualifiers
     source          1..2117
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     primer_bind     98..115
                     /label=L4440
                     /note="L4440 vector, forward primer"
     promoter        192..226
                     /label=J23119(SpeI) promoter
                     /note="bacterial promoter (Registry of Standard Biological 
                     Parts BBa_J23119) modified to end with an SpeI site"
     misc_RNA        247..322
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     CDS             508..1299
                     /codon_start=1
                     /gene="aadA"
                     /product="aminoglycoside adenylyltransferase (Murphy, 
                     1985)"
                     /label=SmR
                     /note="confers resistance to spectinomycin and 
                     streptomycin"
                     /translation="MREAVIAEVSTQLSEVVGVIERHLEPTLLAVHLYGSAVDGGLKPH
                     SDIDLLVTVTVRLDETTRRALINDLLETSASPGESEILRAVEVTIVVHDDIIPWRYPAK
                     RELQFGEWQRNDILAGIFEPATIDIDLAILLTKAREHSVALVGPAAEELFDPVPEQDLF
                     EALNETLTLWNSPPDWAGDERNVVLTLSRIWYSAVTGKIAPKDVAADWAMERLPAQYQP
                     VILEARQAYLGQEEDRLASRADQLEEFVHYVKGEITKVVGK"
     rep_origin      1473..2061
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1962..1981
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"