Price Information
| Cat No. | Plasmid Name | Availability | Buy one, get one free! (?) |
|---|---|---|---|
| V007385 | pMAD | In stock, instant shipping |
Two tubes of lyophilized plasmid will be delivered, each tube is about 5µg.
Basic Vector Information
The pMAD vector is a shuttle vector designed for efficient allelic replacement in naturally nontransformable, low-GC-content Gram-positive bacteria. It features two key components: (i) a temperature-sensitive replication origin (pE194ts) derived from the Staphylococcus aureus plasmid pE194, which enables replication at permissive temperatures (≤32°C) but is fully blocked at nonpermissive temperatures (≥37°C); and (ii) a constitutive clpB promoter (pclpB) from Staphylococcus aureus that drives expression of the thermostable β-galactosidase gene (bgaB) from Bacillus stearothermophilus, facilitating blue-white screening on X-Gal plates without IPTG induction. Below is the step-by-step protocol for gene deletion using this vector.
1. Recombinant vector construction: Tandemly assemble "upstream homology arm of target gene – selection cassette (in the middle) – downstream homology arm", and clone into pMAD’s multiple cloning site (e.g., BamHI/NcoI).
2. Transformation & primary screening: Electrotransform recombinant plasmid into Gram-positive bacteria; spread on "erythromycin + X-Gal" solid medium, incubate at 30°C for 2–3 days. Blue colonies indicate successful vector entry and free replication (pE194ts functions at permissive temperature; bgaB expressed via pclpB promoter).
3. Single crossover (vector integration): Pick 3–5 blue colonies, inoculate into erythromycin-containing liquid medium, shake-culture at 39–42°C for 6–12h (nonpermissive temperature blocks pE194ts replication, forcing single crossover-mediated chromosomal integration). Serial dilute and spread on "erythromycin + X-Gal" medium, incubate at 39–42°C for 18–24h. Select uniform blue colonies (successful integrants: erythromycin-resistant, vector retained).
4. Double crossover (gene deletion + vector excision): Pick 1–2 blue integrant colonies, inoculate into antibiotic-free liquid medium, culture at 30°C for 6h (permissive temperature promotes double crossover and vector excision from chromosome). Transfer to 42°C for 3–6h (nonpermissive temperature eliminates non-excised vectors).
5. Double crossover screening: Serial dilute and spread on "antibiotic-free + X-Gal" medium. White colonies = successful double crossover (vector lost, bgaB removed, target gene deleted); blue colonies = unexcised integrants (discard).
- Vector Name:
- pMAD
- Antibiotic Resistance:
- Ampicillin
- Length:
- 9664 bp
- Type:
- Prokaryotic Expression Vectors
- Replication origin:
- ori
- Growth Strain(s):
- DH10B
- Growth Temperature:
- 37℃
pMAD 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
- Arnaud M, Chastanet A, Débarbouillé M. New vector for efficient allelic replacement in naturally nontransformable, low-GC-content, gram-positive bacteria. Appl Environ Microbiol. 2004;70(11):6887-6891. doi:10.1128/AEM.70.11.6887-6891.2004
pMAD vector Sequence
LOCUS Exported 9664 bp DNA circular SYN 04-DEC-2025
DEFINITION Exported.
ACCESSION V007385
VERSION .
KEYWORDS .
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 9664)
TITLE Direct Submission
REFERENCE 2 (bases 1 to 9664)
TITLE Direct Submission
REFERENCE 3 (bases 1 to 9664)
TITLE Direct Submission
REFERENCE 4 (bases 1 to 9664)
TITLE Direct Submission
REFERENCE 5 (bases 1 to 9664)
AUTHORS .
TITLE Direct Submission
COMMENT SGRef: number: 1; type: "Journal Article"
COMMENT SGRef: number: 2; type: "Journal Article"
COMMENT SGRef: number: 3; type: "Journal Article"
COMMENT SGRef: number: 4; type: "Journal Article"
FEATURES Location/Qualifiers
source 1..9664
/mol_type="other DNA"
/organism="synthetic DNA construct"
CDS complement(98..829)
/gene="ermC"
/label=rRNA adenine N-6-methyltransferase
/note="rRNA adenine N-6-methyltransferase from
Staphylococcus aureus. Accession#: P02979"
rep_origin 1241..3748
/label=pE194ts
/note="temperature-sensitive replication origin (pE194ts)
derived from the Staphylococcus aureus plasmid pE194, which
enables replication at permissive temperatures (32(o)C)
but is fully blocked at nonpermissive temperatures
(37(o)C)"
CDS 1241..2449
/codon_start=1
/gene="pre"
/label=MobV family relaxase
/label=Plasmid recombination enzyme type 1
/note="Plasmid recombination enzyme type 1 from
Staphylococcus aureus. Accession#: P03857"
/translation="MSHSILRVARVKGSSNTNGIQRHNQRENKNYNNKDINHEETYKNY
DLINAQNIKYKDKIDETIDENYSGKRKIRSDAIRHVDGLVTSDKDFFDDLSGEEIERFF
KDSLEFLENEYGKENMLYATVHLDERVPHMHFGFVPLTEDGRLSAKEQLGNKKDFTQLQ
DRFNEYVNEKGYELERGTSKEVTEREHKAMDQYKKDTVFHKQELQEVKDELQKANKQLQ
SGIEHMRSTKPFDYENERTGLFSGREETGRKILTADEFERLQETISSAERIVDDYENIK
STDYYTENQELKKRRESLKEVVNTWKEGYHEKSKEVNKLKRENDSLNEQLNVSEKFQDS
TVTLYRAARANFPGFEKGFNRLKEKFFNDSKFERVGQFMDVVQDNVQKVDRKREKQRTD
DLEM"
CDS complement(3974..5989)
/gene="bgaB"
/label=Beta-galactosidase bgaB
/note="Beta-galactosidase bgaB from Geobacillus
kaustophilus. Accession#: P19668"
promoter complement(6066..6191)
/label=clpB
/note="clpB promoter from Staphylococcus aureus"
CDS 7221..7409
/label=rop
/note="Rop protein, which maintains plasmids at low copy
number"
misc_feature 7514..7654
/label=bom
/note="basis of mobility region from pBR322"
rep_origin complement(7840..8428)
/direction=LEFT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
CDS complement(8602..9459)
/label=AmpR
/note="beta-lactamase"
promoter complement(9460..9564)
/label=AmpR promoter