Price Information
| Cat No. | Plasmid Name | Availability | Buy one, get one free! (?) |
|---|---|---|---|
| V012544 | pBad/Myc-His A | In stock, instant shipping |
Two tubes of lyophilized plasmid will be delivered, each tube is about 5µg.
Basic Vector Information
The pBAD/His A vector is an arabinose-regulated expression system designed for controlled gene expression in E. coli. In the absence of glucose, arabinose acts as an inducer, activating the araBAD promoter and driving transcription of the target gene. By titrating arabinose concentrations, users can optimize the soluble expression of recombinant proteins. Vectors pBAD/His A, B, and C are identical except for their reading frames, allowing seamless cloning of genes in the correct translational frame for fusion with an N-terminal His-tag.
- Vector Name:
- pBad/Myc-His A
- Antibiotic Resistance:
- Ampicillin
- Length:
- 4094 bp
- Type:
- E. coli Expression Vectors
- Replication origin:
- ori
- Copy Number:
- High copy number
- Promoter:
- araBAD
- Cloning Method:
- Enzyme digestion and ligation
- 5' Primer:
- pBAD-F: ATGCCATAGCATTTTTATCC
- 3' Primer:
- pBAD-R: GATTTAATCTGTATCAGG
- Fusion Tag:
- C-His, C-Myc
- Growth Strain(s):
- TOP10
pBad/Myc-His A 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.
References
- Dhanji H, Doumith M, Hope R, Livermore DM, Woodford N. ISEcp1-mediated transposition of linked blaCTX-M-3 and blaTEM-1b from the IncI1 plasmid pEK204 found in clinical isolates of Escherichia coli from Belfast, UK. J Antimicrob Chemother. 2011 Oct;66(10):2263-5. doi: 10.1093/jac/dkr310. Epub 2011 Jul 26. PMID: 21795257.
pBad/Myc-His A vector Sequence
LOCUS 40924_5769 4094 bp DNA circular SYN 13-JAN-2022
DEFINITION synthetic circular DNA.
ACCESSION .
VERSION .
KEYWORDS .
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 4094)
TITLE Direct Submission
REFERENCE 2 (bases 1 to 4094)
AUTHORS .
TITLE Direct Submission
COMMENT SGRef: number: 1; type: "Journal Article"
FEATURES Location/Qualifiers
source 1..4094
/mol_type="other DNA"
/organism="synthetic DNA construct"
promoter 1..285
/label=araBAD promoter
/note="promoter of the L-arabinose operon of E. coli; the
araC regulatory gene is transcribed in the opposite
direction (Guzman et al., 1995)"
CDS 377..406
/codon_start=1
/label=Myc
/note="Myc (human c-Myc proto-oncogene) epitope tag"
/translation="EQKLISEEDL"
CDS 422..439
/codon_start=1
/label=6xHis
/note="6xHis affinity tag"
/translation="HHHHHH"
terminator 665..751
/label=rrnB T1 terminator
/note="transcription terminator T1 from the E. coli rrnB
gene"
terminator 843..870
/label=rrnB T2 terminator
/note="transcription terminator T2 from the E. coli rrnB
gene"
promoter 889..980
/label=AmpR promoter
CDS 981..1838
/codon_start=1
/label=AmpR
/note="beta-lactamase"
/translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
LIKHW"
rep_origin 2012..2600
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
misc_feature complement(2786..2926)
/label=bom
/note="basis of mobility region from pBR322"
CDS complement(3193..4068)
/codon_start=1
/label=araC
/note="L-arabinose regulatory protein"
/translation="MAEAQNDPLLPGYSFNAHLVAGLTPIEANGYLDFFIDRPLGMKGY
ILNLTIRGQGVVKNQGREFVCRPGDILLFPPGEIHHYGRHPEAREWYHQWVYFRPRAYW
HEWLNWPSIFANTGFFRPDEAHQPHFSDLFGQIINAGQGEGRYSELLAINLLEQLLLRR
MEAINESLHPPMDNRVREACQYISDHLADSNFDIASVAQHVCLSPSRLSHLFRQQLGIS
VLSWREDQRISQAKLLLSTTRMPIATVGRNVGFDDQLYFSRVFKKCTGASPSEFRAGCE
EKVNDVAVKLS"