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pX602 vector (V012753)

Price Information

Cat No. Plasmid Name Availability Add to cart
V012753 pX602 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

Also known as pX602-AAV-TBG::NLS-SaCas9-NLS-HA-OLLAS-bGHpA;U6::BsaI-sgRNA

Vector Name:
pX602
Antibiotic Resistance:
Ampicillin
Length:
7293 bp
Type:
CRISPR/Cas
Replication origin:
ori
Copy Number:
High copy number
Promoter:
TBG

pX602 vector Map

Copy Sequence View Map
pX6027293 bp300600900120015001800210024002700300033003600390042004500480051005400570060006300660069007200TBG promoterSV40 NLSSaCas9nucleoplasmin NLSHAOLLAS tagbGH poly(A) signalSa gRNA scaffoldU6 promoterAAV2 ITRf1 oriAmpR promoterAmpRori

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.

pX602 vector Sequence

LOCUS       pX602.        7293 bp DNA     circular SYN 11-SEP-2021
DEFINITION  synthetic circular DNA.
ACCESSION   .
VERSION     .
KEYWORDS    pX602
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7293)
  TITLE     Direct Submission
REFERENCE   2  (bases 1 to 7293)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"
FEATURES             Location/Qualifiers
     source          1..7293
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        187..596
                     /label=TBG promoter
                     /note="promoter from the human thyroxine-binding globulin
                     gene (Yan et al., 2012)"
     CDS             627..647
                     /codon_start=1
                     /product="nuclear localization signal of SV40 large T
                     antigen"
                     /label=nuclear localization signal of SV40 large T
                     ant
                     /note="SV40 NLS"
                     /translation="PKKKRKV"
     CDS             672..3827
                     /label=SaCas9
                     /note="Cas9 endonuclease from the Staphylococcus aureus
                     Type II CRISPR/Cas system"
     CDS             3828..3875
                     /codon_start=1
                     /product="bipartite nuclear localization signal from
                     nucleoplasmin"
                     /label=bipartite nuclear localization signal from
                     nucl
                     /note="nucleoplasmin NLS"
                     /translation="KRPAATKKAGQAKKKK"
     CDS             3882..3908
                     /label=HA
                     /note="HA (human influenza hemagglutinin) epitope tag"
     CDS             3909..3950
                     /label=OLLAS tag
                     /note="Escherichia coli OmpF Linker and mouse Langerin
                     fusion sequence tag (Park et al., 2008)"
     polyA_signal    3984..4191
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     misc_RNA        complement(4203..4278)
                     /label=Sa gRNA scaffold
                     /note="guide RNA scaffold for the Staphylococcus aureus 
                     CRISPR/Cas9 system"
     promoter        complement(4307..4547)
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA"
     repeat_region   4556..4696
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     rep_origin      4771..5226
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow
                     indicates direction of (+) strand synthesis"
     promoter        5508..5612
                     /label=AmpR promoter
     CDS             5613..6470
                     /label=AmpR
                     /note="beta-lactamase"
     rep_origin      6644..7232
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"