Price Information
| Cat No. | Plasmid Name | Availability | Add to cart |
|---|---|---|---|
| V001211 | pmU6-gRNA | 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:
- pmU6-gRNA
- Antibiotic Resistance:
- Kanamycin
- Length:
- 3542 bp
- Type:
- Mammalian Expression, CRISPR
- Replication origin:
- ori
- Promoter:
- SV40
- Cloning Method:
- Restriction Enzyme
- 3' Primer:
- M13 Reverse
pmU6-gRNA vector 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.
pmU6-gRNA vector Sequence
LOCUS 40924_32703 3542 bp DNA circular SYN 13-MAY-2021
DEFINITION Expresses the S. pyogenes sgRNA from the mouse U6 promoter.
ACCESSION .
VERSION .
KEYWORDS .
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 3542)
AUTHORS Kabadi AM, Ousterout DG, Hilton IB, Gersbach CA
TITLE Multiplex CRISPR/Cas9-based genome engineering from a single
lentiviral vector.
JOURNAL Nucleic Acids Res. 2014 Aug 13. pii: gku749.
PUBMED 25122746
REFERENCE 2 (bases 1 to 3542)
TITLE Direct Submission
REFERENCE 3 (bases 1 to 3542)
AUTHORS .
TITLE Direct Submission
COMMENT SGRef: number: 1; type: "Journal Article"; journalName: "Nucleic
Acids Res. 2014 Aug 13. pii: gku749."
COMMENT SGRef: number: 2; type: "Journal Article"
FEATURES Location/Qualifiers
source 1..3542
/mol_type="other DNA"
/organism="synthetic DNA construct"
promoter 1..314
/label=U6 promoter
/note="RNA polymerase III promoter for mouse U6 snRNA (Das
et al., 1988)"
misc_RNA 332..407
/label=gRNA scaffold
/note="guide RNA scaffold for the Streptococcus pyogenes
CRISPR/Cas9 system"
promoter complement(452..470)
/label=T3 promoter
/note="promoter for bacteriophage T3 RNA polymerase"
primer_bind complement(491..507)
/label=M13 rev
/note="common sequencing primer, one of multiple similar
variants"
protein_bind complement(515..531)
/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(539..569)
/label=lac promoter
/note="promoter for the E. coli lac operon"
protein_bind complement(584..605)
/label=CAP binding site
/note="CAP binding activates transcription in the presence
of cAMP."
primer_bind complement(722..739)
/label=L4440
/note="L4440 vector, forward primer"
rep_origin complement(893..1481)
/direction=LEFT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
CDS complement(1561..2352)
/codon_start=1
/label=NeoR/KanR
/note="aminoglycoside phosphotransferase"
/translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
promoter complement(2387..2744)
/label=SV40 promoter
/note="SV40 enhancer and early promoter"
promoter complement(2746..2850)
/label=AmpR promoter
rep_origin complement(2876..3331)
/direction=LEFT
/label=f1 ori
/note="f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
primer_bind 3473..3489
/label=M13 fwd
/note="common sequencing primer, one of multiple similar
variants"
promoter 3499..3517
/label=T7 promoter
/note="promoter for bacteriophage T7 RNA polymerase"