Cart 2

hRad51(K133R)–Cas9(D10A) vector (V016673)

Price Information

Cat No. Plasmid Name Availability Add to cart
V016673 hRad51(K133R)–Cas9(D10A) 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:
hRad51(K133R)–Cas9(D10A)
Antibiotic Resistance:
Ampicillin
Length:
8712 bp
Type:
Gene knockout
Replication origin:
ori
Host:
Mammalian cells
Promoter:
CMV
Growth Strain(s):
Stbl3
Growth Temperature:
37℃

hRad51(K133R)–Cas9(D10A) vector Map

Copy Sequence View Map
hRad51(K133R)–Cas9(D10A)8712 bp4008001200160020002400280032003600400044004800520056006000640068007200760080008400CMV promoterT7 promoterSV40 NLSDNA repair protein RAD51 homolog 1Cas9(D10A)6xHisbGH poly(A) signalM13 revlac operatorlac promoterCAP binding siteoriAmpRAmpR promoterCMV enhancer

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.

hRad51(K133R)–Cas9(D10A) vector Sequence

LOCUS       V016673                 8712 bp    DNA     circular SYN 01-JAN-1980
DEFINITION  Exported.
ACCESSION   V016673
VERSION     V016673
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
            .
REFERENCE   1  (bases 1 to 8712)
  AUTHORS   .
  TITLE     Direct Submission
FEATURES             Location/Qualifiers
     source          1..8712
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        132..335
                     /label="CMV promoter"
                     /note="human cytomegalovirus (CMV) immediate early
                     promoter"
     promoter        377..395
                     /label="T7 promoter"
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             415..435
                     /label="SV40 NLS"
                     /note="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
     CDS             460..1473
                     /gene="RAD51"
                     /label="DNA repair protein RAD51 homolog 1"
                     /note="DNA repair protein RAD51 homolog 1 from Homo
                     sapiens. Accession#: Q06609"
     CDS             1570..5670
                     /label="Cas9(D10A)"
                     /note="nickase mutant of the Cas9 endonuclease from the
                     Streptococcus pyogenes Type II CRISPR/Cas system"
     CDS             5727..5744
                     /label="6xHis"
                     /note="6xHis affinity tag"
     polyA_signal    5773..5997
                     /label="bGH poly(A) signal"
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(6068..6084)
                     /label="M13 rev"
                     /note="common sequencing primer, one of multiple similar
                     variants"
     protein_bind    complement(6092..6108)
                     /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)."
     promoter        complement(6116..6146)
                     /label="lac promoter"
                     /note="promoter for the E. coli lac operon"
     protein_bind    complement(6161..6182)
                     /label="CAP binding site"
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     rep_origin      complement(6470..7058)
                     /direction=LEFT
                     /label="ori"
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
                     replication"
     CDS             complement(7232..8089)
                     /label="AmpR"
                     /note="beta-lactamase"
     promoter        complement(8090..8194)
                     /label="AmpR promoter"
     enhancer        8464..8712
                     /label="CMV enhancer"
                     /note="human cytomegalovirus immediate early enhancer"