pAX01 vector (V000803) Gene synthesis in pAX01 backbone

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Price Information

Cat No.	Plasmid Name	Availability	Buy one, get one free! (?)
V000803	pAX01	In stock, instant shipping

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

Basic Vector Information

pAX01 is a versatile integrative expression vector specifically designed for targeted gene insertion and regulated expression in Bacillus subtilis. It features a modular structure that enables flexible replacement of key components, including its integration arms, expression cassettes, and selectable markers—for instance, the erythromycin resistance gene (erm) can be removed via NotI digestion, and the xylose-inducible expression cassette (with the PxylA promoter and xylR repressor) can be excised using SacI. This vector targets the lacA locus of Bacillus subtilis (a weakly expressed gene encoding β-galactosidase) for stable DNA integration through double-crossover homologous recombination, ensuring minimal interference with host physiological functions. To prevent readthrough transcription, its xylose-inducible cassette is fused to the t0 transcription terminator from phage DNA, resulting in tight regulation: no target gene expression is detected without xylose, while full induction is achieved with 0.5% xylose, and sustained high-level expression can be maintained with 2% xylose. Notably, pAX01 is a non-replicating vector in Bacillus subtilis (lacking replication elements for this host) and relies on the ColE1 replicon for propagation in E. coli. It also carries an ampicillin resistance gene (bla) for selection in E. coli and an erythromycin resistance gene (erm) for identifying integrants in Bacillus subtilis, making it a robust tool for controlled gene expression studies in this bacterium.

Vector Name:: pAX01

Antibiotic Resistance:: Ampicillin, Erythromycin

Length:: 7781 bp

Type:: Bacillus subtilis expression vectors

Replication origin:: ori

Selection Marker:: Erythromycin

Growth Strain(s):: DH10B

Growth Temperature:: 37℃

pAX01 vector Map

Copy Sequence View 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

Härtl B, Wehrl W, Wiegert T, Homuth G, Schumann W. 2001. Development of a New Integration Site within the Bacillus subtilis Chromosome and Construction of Compatible Expression Cassettes. J Bacteriol 183:.https://doi.org/10.1128/jb.183.8.2696-2699.2001

pAX01 vector Sequence

LOCUS       Exported                7781 bp DNA     circular SYN 19-NOV-2025
DEFINITION  Exported.
ACCESSION   V000803
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7781)
  TITLE     Direct Submission
REFERENCE   2  (bases 1 to 7781)
  TITLE     Direct Submission
REFERENCE   3  (bases 1 to 7781)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"
COMMENT     SGRef: number: 2; type: "Journal Article"
FEATURES             Location/Qualifiers
     source          1..7781
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             7..522
                     /codon_start=1
                     /product="beta-galactosidase GanA [Bacillus subtilis]"
                     /label=lacA'
                     /note="beta-galactosidase GanA [Bacillus subtilis]"
                     /translation="MSKLEKTHVTKAKFMLHGGDYNPDQWLDRPDILADDIKLMKLSHT
                     NTFSVGIFAWSALEPEEGVYQFEWLDDIFERIHSIGGRVILATPSGARPAWLSQTYPEV
                     LRVNASRVKQLHGGRHNHCLTSKVYREKTRHINRLLAERYGHHPALLMWHISNEYGGDC
                     HCESMRPL"
     terminator      546..580
                     /note="lambda t0 terminator"
                     /note="minimal transcription terminator from phage lambda 
                     (Scholtissek and Grosse, 1987)"
     terminator      complement(660..687)
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB
                     gene"
     terminator      complement(706..792)
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB
                     gene"
     CDS             complement(965..1699)
                     /gene="ermBP"
                     /label=rRNA adenine N-6-methyltransferase
                     /note="rRNA adenine N-6-methyltransferase from Enterococcus
                     faecalis. Accession#: P0A4D5"
     promoter        2078..2182
                     /label=AmpR promoter
     CDS             2518..3642
                     /codon_start=1
                     /product="transcriptional repressor"
                     /label=XylR
                     /translation="MNQKLILDEILKNSPVSRATLSEITGLNKSTVSSQVNTLLEKDFI
                     FEIGAGQSRGGRRPVMLVFNKNAGYSIGIDIGVDYLNGILTDLEGNIILEKTSDLSSSS
                     ASEVKEILFALIHGFVTHMPESPYGLVGIGICVPGLVDRHQQIIFMPNLNWNIKDLQFL
                     IESEFNVPVFVENEANAGAYGEKVFGMTKNYENIVYISINIGIGTGLVINNELYKGVQG
                     FSGEMGHMTIDFNGPKCSCGNRGCWELYASEKALLASLSKEEKNISRKEIVERANKNDV
                     EMLNALQNFGFYIGIGLTNILNTFDIEAVILRNHIIESHPIVLNTIKNEVSSRVHSHLD
                     NKCELLPSSLGKNAPALGAVSIVIDSFLSVTPIS"
     terminator      complement(3674..3708)
                     /label=lambda t0 terminator
                     /note="minimal transcription terminator from phage lambda 
                     (Scholtissek and Grosse, 1987)"
     CDS             3834..4241
                     /codon_start=1
                     /product="beta-galactosidase GanA [Bacillus subtilis]"
                     /label='lacA
                     /translation="MKDYATVIDVKTASVEAVYQEDFYARTPAVTSHEYQQGKAYFIGA
                     RLEDQFQRDFYEGLITDLSLSPVFPVRHGKGVSVQARQDQDNDYIFVMNFTEEKQLVTF
                     DQSVKDIMTGDILSGDLTMEKYEVRIVVNTH"
     promoter        4352..4456
                     /gene="bla"
                     /label=bla promoter
                     /note="AmpR promoter"
     CDS             4457..5314
                     /label=AmpR
                     /note="beta-lactamase"
     rep_origin      5488..6076
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     misc_feature    complement(6262..6402)
                     /label=bom
                     /note="basis of mobility region from pBR322"
     CDS             complement(6507..6695)
                     /label=rop
                     /note="Rop protein, which maintains plasmids at low copy
                     number"