Price Information
| Cat No. | Plasmid Name | Availability | Buy one, get one free! (?) |
|---|---|---|---|
| V013302 | pJB866 | In stock, instant shipping |
Two tubes of lyophilized plasmid will be delivered, each tube is about 5µg.
Basic Vector Information
pJB866 is a broad-host-range expression vector derived from pJB655, containing the Pm promoter, xylS regulatory gene, and a tetracycline resistance marker, with a polylinker from pJB864 inserted. It is useful for regulated gene expression in various Gram-negative bacteria, featuring flexibility in copy number modulation via trfA mutations.
- Vector Name:
- pJB866
- Antibiotic Resistance:
- Tetracycline
- Length:
- 8189 bp
- Type:
- Protein expression
- Replication origin:
- oriV
- Host:
- Broad host
- Promoter:
- Pm
- Growth Strain(s):
- DH10B
- Growth Temperature:
- 30℃
pJB866 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
- Blatny JM, Brautaset T, Winther-Larsen HC, Karunakaran P, Valla S. Improved broad-host-range RK2 vectors useful for high and low regulated gene expression levels in gram-negative bacteria. Plasmid. 1997;38(1):35-51. doi: 10.1006/plas.1997.1294. PMID: 9281494.
pJB866 vector Sequence
LOCUS Exported 8189 bp DNA circular SYN 30-SEP-2025
DEFINITION synthetic circular DNA
ACCESSION .
VERSION .
KEYWORDS pJB866 vector (V013302)
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 8189)
AUTHORS .
TITLE Direct Submission
FEATURES Location/Qualifiers
source 1..8189
/mol_type="other DNA"
/organism="synthetic DNA construct"
promoter complement(159..239)
/label=Pm promoter
/note="The bacterial Pm promoter is activated by XylS in
the presence of benzoate or m-toluate (Marques et al.,
1999)."
CDS 960..1925
/codon_start=1
/gene="xylS"
/product="XylS regulator encoded by the Pseudomonas putida
TOL plasmid pWWO"
/label=XylS
/note="stimulates transcription from the Pm promoter in the
presence of aromatic compounds such as m-toluate or
benzoate"
/translation="MDFCLLNEKSQIFVHAEPYAVSDYVNQYVGTHSIRLPKGGRPAGR
LHHRIFGCLDLCRISYGGSVRVISPGLETCYHLQIILKGHCLWRGHGQEHYFAPGELLL
LNPDDQADLTYSEDCEKFIVKLPSVVLDRACSDNNWHKPREGIRFAARHNLQQLDGFIN
LLGLVCDEAEHTKSMPRVQEHYAGIIASKLLEMLGSNVSREIFSKGNPSFERVVQFIEE
NLKRNISLERLAELAMMSPRSLYNLFEKHAGTTPKNYIRNRKLESIRACLNDPSANVRS
ITEIALDYGFLHLGRFAENYRSAFGELPSDTLRQCKKEVA"
primer_bind 2362..2378
/label=M13 fwd
/note="common sequencing primer, one of multiple similar
variants"
oriT 2466..2574
/note="incP origin of transfer"
CDS complement(3037..3687)
/codon_start=1
/gene="tetR"
/product="tetracycline resistance regulatory protein"
/label=TetR
/translation="MTKLQPNTVIRAALDLLNEVGVDGLTTRKLAERLGVQQPALYWHF
RNKRALLDALAEAMLAENHTHSVPRADDDWRSFLIGNARSFRQALLAYRDGARIHAGTR
PGAPQMETADAQLRFLCEAGFSAGDAVNALMTISYFTVGAVLEEQAGDSDAGERGGTVE
QAPLSPLLRAAIDAFDEAGPDAAFEQGLAVIVDGLAKRRLVVRNVEGPRKGDD"
CDS 3793..4992
/codon_start=1
/gene="tetA"
/product="tetracycline efflux protein"
/label=TcR
/note="confers resistance to tetracycline"
/translation="MKPNIPLIVILSTVALDAVGIGLIMPVLPGLLRDLVHSNDVTAHY
GILLALYALVQFACAPVLGALSDRFGRRPILLVSLAGATVDYAIMATAPFLWVLYIGRI
VAGITGATGAVAGAYIADITDGDERARHFGFMSACFGFGMVAGPVLGGLMGGFSPHAPF
FAAAALNGLNFLTGCFLLPESHKGERRPLRREALNPLASFRWARGMTVVAALMAVFFIM
QLVGQVPAALWVIFGEDRFHWDATTIGISLAAFGILHSLAQAMITGPVAARLGERRALM
LGMIADGTGYILLAFATRGWMAFPIMVLLASGGIGMPALQAMLSRQVDEERQGQLQGSL
AALTSLTSIVGPLLFTAIYAASITTWNGWAWIAGAALYLLCLPALRRGLWSGAGQRADR
"
rep_origin 5577..6108
/label=oriV
/note="incP origin of replication"
CDS 6501..7649
/codon_start=1
/product="trans-acting replication protein that binds to
and activates oriV"
/label=trfA
/translation="MNRTFDRKAYRQELIDAGFSAEDAETIASRTVMRAPRETFQSVGS
IVQQATAKIERDSVQLAPPALPAPSAAVERSRRLEQEAAGLAKSMTIDTRGTMTTKKRK
TAGEDLAKQVSEAKQAALLKHTKQQIKEMQLSLFDIAPWPDTMRAMPNDTARSALFTTR
NKKIPREALQNKVIFHVNKDVKITYTGVELRADDDELVWQQVLEYAKRTPIGEPITFTF
YELCQDLGWSINGRYYTKAEECLSRLQATAMGFTSDRVGHLESVSLLHRFRVLDRGKKT
SRCQVLIDEEIVVLFAGDHYTKFIWEKYRKLSPTARRMFDYFSSHREPYPLKLETFRLM
CGSDSTRVKKWREQVGEACEELRGSGLVEHAWVNDDLVHCKR"