Price Information
| Cat No. | Plasmid Name | Availability | Add to cart |
|---|---|---|---|
| V007412 | pUC119-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:
- pUC119-gRNA
- Antibiotic Resistance:
- Ampicillin
- Length:
- 3771 bp
- Type:
- CRISPR ; Plant expression
- Replication origin:
- ori
- Host:
- Plants
- Promoter:
- AtU6-1
- Cloning Method:
- Restriction Enzyme
- 5' Primer:
- 5’ TGGAATTGTGAGCGGATA 3’
- 3' Primer:
- 5’ ATTAAGTTGGGTAACGCC 3'
pUC119-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.
pUC119-gRNA vector Sequence
LOCUS 40924_44958 3771 bp DNA circular SYN 13-MAY-2021
DEFINITION Can use a PCR template to assemble new desired guide RNA. Contains
an Arabidopsis U6 promoter to drive guide RNA (targeting AtPDS3 gene
target site 1) expression with a TTTTTT as terminator. .
ACCESSION .
VERSION .
KEYWORDS .
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 3771)
AUTHORS Li JF, Norville JE, Aach J, McCormack M, Zhang D, Bush J, Church GM,
Sheen J
TITLE Multiplex and homologous recombination-mediated genome editing in
Arabidopsis and Nicotiana benthamiana using guide RNA and Cas9.
JOURNAL Nat Biotechnol. 2013 Aug;31(8):688-91. doi: 10.1038/nbt.2654.
PUBMED 23929339
REFERENCE 2 (bases 1 to 3771)
TITLE Direct Submission
REFERENCE 3 (bases 1 to 3771)
AUTHORS .
TITLE Direct Submission
COMMENT SGRef: number: 1; type: "Journal Article"; doi: "10.1038/nbt";
journalName: "Nat Biotechnol"; date: "2013-08"; volume: "31"; issue:
"8"; pages: "688-91"
COMMENT SGRef: number: 2; type: "Journal Article"
FEATURES Location/Qualifiers
source 1..3771
/mol_type="other DNA"
/organism="synthetic DNA construct"
protein_bind 107..128
/label=CAP binding site
/note="CAP binding activates transcription in the presence
of cAMP."
promoter 143..173
/label=lac promoter
/note="promoter for the E. coli lac operon"
protein_bind 181..197
/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)."
primer_bind 205..221
/label=M13 rev
/note="common sequencing primer, one of multiple similar
variants"
promoter 407..710
/label=AtU6-1 promoter
/note="promoter for the Arabidopsis thaliana U6-1 snRNA
gene (Waibel and Filipowicz, 1990)"
misc_RNA 731..806
/label=gRNA scaffold
/note="guide RNA scaffold for the Streptococcus pyogenes
CRISPR/Cas9 system"
protein_bind complement(818..842)
/gene="mutant version of attB"
/label=attB2
/bound_moiety="BP Clonase(TM)"
/note="recombination site for the Gateway(R) BP reaction"
primer_bind complement(900..916)
/label=M13 fwd
/note="common sequencing primer, one of multiple similar
variants"
rep_origin 1129..1584
/label=f1 ori
/note="f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
primer_bind complement(1601..1620)
/label=pRS-marker
/note="pRS vectors, use to sequence yeast selectable
marker"
primer_bind 1720..1742
/label=pGEX 3'
/note="pGEX vectors, reverse primer"
primer_bind complement(1780..1798)
/label=pBRforEco
/note="pBR322 vectors, upsteam of EcoRI site, forward
primer"
promoter 1866..1970
/label=AmpR promoter
CDS 1971..2828
/codon_start=1
/label=AmpR
/note="beta-lactamase"
/translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
LIKHW"
rep_origin 3002..3590
/direction=RIGHT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
primer_bind 3744..3761
/label=L4440
/note="L4440 vector, forward primer"