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pT7-gRNA vector (V000385)

Price Information

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V000385 pT7-gRNA In stock, instant shipping

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

Basic Vector Information

The pT7-gRNA vector is specifically engineered for cloning guide RNAs (gRNAs) and facilitates their in vitro transcription. This vector is tailored for efficiently generating target gRNAs.

Vector Name:
pT7-gRNA
Antibiotic Resistance:
Ampicillin
Length:
2541 bp
Type:
CRISPR ; zebrafish expression
Replication origin:
ori
Copy Number:
High Copy
Promoter:
T7
Cloning Method:
Restriction Enzyme
5' Primer:
M13 R
3' Primer:
M13 F
Growth Strain(s):
DH10B
Growth Temperature:
37℃

pT7-gRNA vector Map

Copy Sequence View Map
pT7-gRNA2541 bp600120018002400M13 fwdgRNA scaffoldT7 promoterM13 revlac operatorlac promoterCAP binding siteL4440oriAmpRAmpR promoter

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

  • Jao LE, Wente SR, Chen W. Efficient multiplex biallelic zebrafish genome editing using a CRISPR nuclease system. Proc Natl Acad Sci U S A. 2013;110(34):13904-13909. doi:10.1073/pnas.1308335110

pT7-gRNA vector Sequence

LOCUS       40924_42334        2541 bp DNA     circular SYN 13-MAY-2021
DEFINITION  gRNA cloning vector for in vitro transcription of target gRNA.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 2541)
  AUTHORS   Jao LE, Wente SR, Chen W
  TITLE     Efficient multiplex biallelic zebrafish genome editing using a 
            CRISPR nuclease system.
  JOURNAL   Proc Natl Acad Sci U S A. 2013 Aug 5.
  PUBMED    23918387
REFERENCE   2  (bases 1 to 2541)
  TITLE     Direct Submission
REFERENCE   3  (bases 1 to 2541)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Proc Natl
            Acad Sci U S A. 2013 Aug 5."
COMMENT     SGRef: number: 2; type: "Journal Article"
FEATURES             Location/Qualifiers
     source          1..2541
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     195..211
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     misc_RNA        complement(239..314)
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        complement(348..366)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(390..406)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    complement(414..430)
                     /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(438..468)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    complement(483..504)
                     /label=CAP binding site
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     primer_bind     complement(621..638)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(792..1380)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(1554..2411)
                     /codon_start=1
                     /label=AmpR
                     /note="beta-lactamase"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     promoter        complement(2412..2516)
                     /label=AmpR promoter