Price Information
| Cat No. | Plasmid Name | Availability | Add to cart |
|---|---|---|---|
| V012734 | pRGEB32 | In stock, instant shipping |
Buy one, get one free! |
Two tubes of lyophilized plasmid will be delivered, each tube is about 5µg.
Basic Vector Information
Express sgRNA/PTG with rice snoRNA U3 promoter and Cas9 with rice ubiquitin promoter for Agrobacterium-mediated transformation.
- Vector Name:
- pRGEB32
- Antibiotic Resistance:
- Kanamycin
- Length:
- 15905 bp
- Type:
- Plant Vectors
- Replication origin:
- ori
- Host:
- Plants
- Source/Author:
- Yinong Yang
- Selection Marker:
- Hygromycin
- Copy Number:
- High copy number
- Promoter:
- CaMV 35S (enhanced)
pRGEB32 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.
pRGEB32 vector Sequence
LOCUS pRGEB32. 15905 bp DNA circular SYN 06-SEP-2021
DEFINITION synthetic circular DNA.
ACCESSION .
VERSION .
KEYWORDS pRGEB32
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 15905)
TITLE Direct Submission
REFERENCE 2 (bases 1 to 15905)
AUTHORS .
TITLE Direct Submission
COMMENT SGRef: number: 1; type: "Journal Article"
FEATURES Location/Qualifiers
source 1..15905
/mol_type="other DNA"
/organism="synthetic DNA construct"
CDS 26..49
/label=FLAG
/note="FLAG(R) epitope tag, followed by an enterokinase
cleavage site"
terminator 71..323
/label=NOS terminator
/note="nopaline synthase terminator and poly(A) signal"
primer_bind complement(332..348)
/label=M13 fwd
/note="M13 fwd"
/note="common sequencing primer, one of multiple similar
variants"
primer_bind complement(349..365)
/label=M13 fwd
/note="common sequencing primer, one of multiple similar
variants"
misc_feature 568..592
/label=RB T-DNA repeat
/note="right border repeat from nopaline C58 T-DNA"
CDS 1892..2518
/label=pVS1 StaA
/note="stability protein from the Pseudomonas plasmid pVS1
(Heeb et al., 2000)"
CDS 2955..4019
/label=pVS1 RepA
/note="replication protein from the Pseudomonas plasmid
pVS1 (Heeb et al., 2000)"
rep_origin 4088..4282
/label=pVS1 oriV
/note="origin of replication for the Pseudomonas plasmid
pVS1 (Heeb et al., 2000)"
misc_feature 4626..4766
/label=bom
/note="basis of mobility region from pBR322"
rep_origin complement(4952..5540)
/direction=LEFT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
CDS complement(5630..6421)
/label=KanR
/note="aminoglycoside phosphotransferase"
misc_feature 6846..6870
/label=LB T-DNA repeat
/note="left border repeat from nopaline C58 T-DNA"
polyA_signal complement(6948..7122)
/label=CaMV poly(A) signal
/note="cauliflower mosaic virus polyadenylation signal"
CDS complement(7165..8187)
/label=HygR
/note="aminoglycoside phosphotransferase from E. coli"
promoter complement(8255..8932)
/label=CaMV 35S promoter (enhanced)
/note="cauliflower mosaic virus 35S promoter with a
duplicated enhancer region"
protein_bind 9123..9144
/label=CAP binding site
/note="CAP binding activates transcription in the presence
of cAMP."
promoter 9159..9189
/label=lac promoter
/note="promoter for the E. coli lac operon"
protein_bind 9197..9213
/label=lac repressor encoded by lacI binding site
/bound_moiety="lac repressor encoded by lacI"
/note="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 9221..9237
/label=M13 rev
/note="M13 rev"
/note="common sequencing primer, one of multiple similar
variants"
promoter 9256..9636
/label=OsU3 promoter
/note="Oryza sativa (rice) snRNA U3 promoter"
misc_RNA 9653..9728
/label=gRNA scaffold
/note="guide RNA scaffold for the Streptococcus pyogenes
CRISPR/Cas9 system"
intron 10557..11518
/label=intron
/note="intron upstream of the coding sequence for the rice
polyubiquitin gene RUBQ2"
protein_bind 11529..11553
/label=attB1
/note="recombination site for the Gateway(R) BP reaction"
CDS 11598..11663
/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 11670..11690
/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 11715..15815
/label=Cas9
/note="Cas9 (Csn1) endonuclease from the Streptococcus
pyogenes Type II CRISPR/Cas system"
CDS 15816..15863
/codon_start=1
/product="bipartite nuclear localization signal from
nucleoplasmin"
/label=bipartite nuclear localization signal from
nucl
/note="nucleoplasmin NLS"
/translation="KRPAATKKAGQAKKKK"