JDS246 vector (V012335)

Price Information

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V012335 JDS246 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:
JDS246
Antibiotic Resistance:
Ampicillin
Length:
7614 bp
Type:
CRISPR Plasmids
Replication origin:
ori
Source/Author:
Joung Lab / Addgene #43861
Copy Number:
High copy number

JDS246 vector Map

JDS2467614 bp3006009001200150018002100240027003000330036003900420045004800510054005700600063006600690072007500CMV promoterT7 promoterCas9SV40 NLS3xFLAG6xHisbGH 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.

JDS246 vector Sequence

Copy Sequence

Download GenBank File(.gb)

LOCUS       JDS246.        7614 bp DNA     circular SYN 01-JAN-1980
DEFINITION  Plasmid for expressing FLAG(R)-tagged human codon-optimized Cas9 in 
            mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    JDS246
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7614)
  AUTHORS   Joung Lab / Addgene #43861
  TITLE     Direct Submission
REFERENCE   2  (bases 1 to 7614)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"
FEATURES             Location/Qualifiers
     source          1..7614
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        132..335
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early
                     promoter"
     promoter        377..395
                     /note="T7 promoter"
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             409..4512
                     /label=Cas9
                     /note="Cas9 (Csn1) endonuclease from the Streptococcus
                     pyogenes Type II CRISPR/Cas system"
     CDS             4519..4539
                     /codon_start=1
                     /product="nuclear localization signal of SV40 large T
                     antigen"
                     /note="SV40 NLS"
                     /translation="PKKKRKV"
     CDS             4546..4611
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=three tandem FLAG
                     /note="3xFLAG"
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             4629..4646
                     /label=6xHis
                     /note="6xHis affinity tag"
     polyA_signal    4675..4899
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(4970..4986)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    complement(4994..5010)
                     /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(5018..5048)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    complement(5063..5084)
                     /label=CAP binding site
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     rep_origin      complement(5372..5960)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(6134..6991)
                     /label=AmpR
                     /note="beta-lactamase"
     promoter        complement(6992..7096)
                     /label=AmpR promoter
     enhancer        7366..7614
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"