Price Information
| Cat No. | Plasmid Name | Availability | Add to cart |
|---|---|---|---|
| V012419 | pRHA-67 | 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
- Vector Name:
- pRHA-67
- Antibiotic Resistance:
- Ampicillin
- Length:
- 4745 bp
- Type:
- Cloning Vectors
- Replication origin:
- ori
- Source/Author:
- Giacalone MJ, Gentile AM, Lovitt BT, Berkley NL, Gunderson CW,
- Copy Number:
- High copy number
- Promoter:
- rhaB
- 3' Primer:
- M13 rev
pRHA-67 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.
pRHA-67 vector Sequence
LOCUS pRHA-67. 4745 bp DNA circular SYN 01-JAN-1980
DEFINITION pUC18-derived plasmid encoding the rhaB promoter plus rhaR and rhaS
genes, for rhamnose-inducible protein expression in E. coli.
ACCESSION .
VERSION .
KEYWORDS pRHA-67
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 4745)
AUTHORS Giacalone MJ, Gentile AM, Lovitt BT, Berkley NL, Gunderson CW,
Surber MW.
TITLE Toxic protein expression in Escherichia coli using a rhamnose-based
tightly regulated and tunable promoter system.
JOURNAL BioTechniques 2006;40:355-64.
PUBMED 16568824
REFERENCE 2 (bases 1 to 4745)
AUTHORS Xbrane Bioscience
TITLE Direct Submission
REFERENCE 3 (bases 1 to 4745)
AUTHORS .
TITLE Direct Submission
COMMENT SGRef: number: 1; type: "Journal Article"; journalName:
"BioTechniques"; date: "2006"; volume: "40"; pages: "355-64"
COMMENT SGRef: number: 2; type: "Journal Article"
COMMENT This plasmid sequence was reconstructed from the procedure described
in the original paper. The plasmid size is slightly different than
reported.
FEATURES Location/Qualifiers
source 1..4745
/mol_type="other DNA"
/organism="synthetic DNA construct"
primer_bind 379..395
/label=M13 fwd
/note="common sequencing primer, one of multiple similar
variants"
gene 405..410
/gene="rhaT"
/gene_synonym="ECK3900; JW3878"
/locus_tag="b3907"
/label=rhaT
/note="rhaT"
/db_xref="EcoGene:EG11313"
/db_xref="GeneID:948398"
CDS complement(410..1255)
/label=rhaR
/note="transcriptional activator of rhaR and rhaS"
CDS complement(1332..2165)
/label=rhaS
/note="positive regulator of the rhaB promoter"
promoter 2311..2429
/label=rhaB promoter
/note="promoter of the E. coli rhaBAD operon, conferring
tight induction with L-rhamnose and repression with
D-glucose in the presence of RhaR and RhaS (Giacalone et
al., 2006)"
misc_feature 2439..2444
/label=RBS
/note="RBS"
/note="ribosome binding site"
misc_feature 2447..2508
/label=MCS
/note="MCS"
/note="pUC19 multiple cloning site"
primer_bind complement(2524..2540)
/label=M13 rev
/note="common sequencing primer, one of multiple similar
variants"
protein_bind complement(2548..2564)
/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(2572..2602)
/label=lac promoter
/note="promoter for the E. coli lac operon"
protein_bind complement(2617..2638)
/label=CAP binding site
/note="CAP binding activates transcription in the presence
of cAMP."
rep_origin complement(2926..3514)
/direction=LEFT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
CDS complement(3688..4545)
/label=AmpR
/note="beta-lactamase"
promoter complement(4546..4650)
/label=AmpR promoter