pRGEB32 vector (Cat. No.: V012734)

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Basic Information

Note: Express sgRNA/PTG with rice snoRNA U3 promoter and Cas9 with rice ubiquitin promoter for Agrobacterium-mediated transformation.

Name:: pRGEB32

Antibiotic Resistance:: Kanamycin

Length:: 15888 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)

Growth Strain(s):: JM108

Growth Temperature:: 37℃

$ 248.8

In stock, instant shipping

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

Resources

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

Zhang M, Liu Q, Yang X, Xu J, Liu G, Yao X, Ren R, Xu J, Lou L. CRISPR/Cas9-mediated mutagenesis of Clpsk1 in watermelon to confer resistance to Fusarium oxysporum f.sp. niveum. Plant Cell Rep. 2020 May;39(5):589-595. doi: 10.1007/s00299-020-02516-0. Epub 2020 Mar 9. PMID: 32152696.

pRGEB32 vector (Cat. No.: V012734) Sequence

LOCUS       pRGEB32                15888 bp    DNA     circular SYN 29-DEC-2025
DEFINITION  synthetic circular DNA.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 15888)
  TITLE     Direct Submission
REFERENCE   2  (bases 1 to 15888)
  TITLE     Direct Submission
REFERENCE   3  (bases 1 to 15888)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"
COMMENT     SGRef: number: 2; type: "Journal Article"
FEATURES             Location/Qualifiers
     source          1..15888
                     /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"
     misc_feature    551..575
                     /label=RB T-DNA repeat
                     /note="right border repeat from nopaline C58 T-DNA"
     CDS             1876..2502
                     /label=pVS1 StaA
                     /note="stability protein from the Pseudomonas plasmid pVS1
                     (Heeb et al., 2000)"
     CDS             2940..4004
                     /label=pVS1 RepA
                     /note="replication protein from the Pseudomonas plasmid
                     pVS1 (Heeb et al., 2000)"
     rep_origin      4073..4267
                     /label=pVS1 oriV
                     /note="origin of replication for the Pseudomonas plasmid
                     pVS1 (Heeb et al., 2000)"
     misc_feature    4611..4751
                     /label=bom
                     /note="basis of mobility region from pBR322"
     rep_origin      complement(4937..5525)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(5615..6406)
                     /label=KanR
                     /note="aminoglycoside phosphotransferase"
     misc_feature    6831..6855
                     /label=LB T-DNA repeat
                     /note="left border repeat from nopaline C58 T-DNA"
     polyA_signal    complement(6933..7107)
                     /label=CaMV poly(A) signal
                     /note="cauliflower mosaic virus polyadenylation signal"
     CDS             complement(7151..8173)
                     /label=HygR
                     /note="aminoglycoside phosphotransferase from E. coli"
     promoter        complement(8240..8916)
                     /label=CaMV 35S promoter (enhanced)
                     /note="cauliflower mosaic virus 35S promoter with a
                     duplicated enhancer region"
     protein_bind    9107..9128
                     /label=CAP binding site
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     promoter        9143..9173
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    9181..9197
                     /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     9205..9221
                     /label=M13 rev
                     /note="M13 rev"
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     promoter        9239..9619
                     /label=OsU3 promoter
                     /note="Oryza sativa (rice) snRNA U3 promoter"
     misc_RNA        9636..9711
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     intron          10540..11501
                     /label=intron
                     /note="intron upstream of the coding sequence for the rice 
                     polyubiquitin gene RUBQ2"
     protein_bind    11512..11536
                     /label=attB1
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             11581..11646
                     /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             11653..11673
                     /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             11698..15798
                     /label=Cas9
                     /note="Cas9 (Csn1) endonuclease from the Streptococcus
                     pyogenes Type II CRISPR/Cas system"
     CDS             15799..15846
                     /codon_start=1
                     /product="bipartite nuclear localization signal from
                     nucleoplasmin"
                     /label=bipartite nuclear localization signal from
                     nucl
                     /note="nucleoplasmin NLS"
                     /translation="KRPAATKKAGQAKKKK"