pWB980-ori vector (V017497)

Price Information

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

Basic Vector Information

pWB980 derived from pUB110 is an expression vector in Bacillus for its high copy number and high stability. It has a size of 3787 base pairs, which is considered manageable and does not impose significant metabolic burden on the host cells. One key feature of the pWB980 plasmid is its bacterial resistance attribute, specifically for kanamycin. This feature is extremely useful as it allows for the selection and identification of bacterial cells that have successfully taken up the plasmid. pWB980 harbors the P43 promoter whose transcription direction is consistent with majority of the pUB110-encoded genes. This high-level promoter is popular for driving gene expression in Bacillus subtilis. It is important to note that the pWB980 vector is not a shuttle vector. Users are recommended to treat bacterial cells carrying this plasmid with lysozyme to maximize the plasmid yield during extraction. pWB980-ori is a shuttle plasmid with an additional replication origin compared to pWB980.

pWB980-ori 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.

pWB980-ori vector Sequence

LOCUS       Exported                4276 bp DNA     circular SYN 14-AUG-2024
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    pWB980-ori
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4276)
  AUTHORS   A
  TITLE     Direct Submission
FEATURES             Location/Qualifiers
     source          1..4276
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             309..1313
                     /codon_start=1
                     /gene="repB"
                     /product="RepB replication protein"
                     /label=repB
                     /note="from Enterococcus faecalis plasmid pAM-alpha-1"
                     /db_xref="GI:22652809"
                     /protein_id="AAN03827.1"
                     /translation="MGVSFNIMCPNSSIYSDEKSRVLVDKTKSGKVRPWREKKIANVDY
                     FELLHILEFKKAERVKDCAEILEYKQNRETGERKLYRVWFCKSRLCPMCNWRRAMKHGI
                     QSQKVVAEVIKQKPTVRWLFLTLTVKNVYDGEELNKSLSDMAQGFRRMMQYKKINKNLV
                     GFMRATEVTINNKDNSYNQHMHVLVCVEPTYFKNTENYVNQKQWIQFWKKAMKLDYDPN
                     VKVQMIRPKNKYKSDIQSAIDETAKYPVKDTDFMTDDEEKNLKRLSDLEEGLHRKRLIS
                     YGGLLKEIHKKLNLDDTEEGDLIHTDDDEKADEDGFSIIAMWNWERKNYFIKE"
     CDS             1473..2243
                     /codon_start=1
                     /product="aminoglycoside O-nucleotidyltransferase 
                     ANT(4')-Ia [Staphylococcus aureus]"
                     /label=Kan
                     /translation="MRIVNGPIIMTREERMKIVHEIKERILDKYGDDVKAIGVYGSLGR
                     QTDGPYSDIEMMCVMSTEEAEFSHEWTTGEWKVEVNFDSEEILLDYASQVESDWPLTHG
                     QFFSILPIYDSGGYLEKVYQTAKSVEAQTFHDAICALIVEELFEYAGKWRNIRVQGPTT
                     FLPSLTVQVAMAGAMLIGLHHRICYTTSASVLTEAVKQSDLPSGYDHLCQFVMSGQLSD
                     SEKLLESLENFWNGIQEWTERHGYIVDVSKRIPF"
     rep_origin      complement(2373..2961)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     misc_feature    3727..3748
                     /label=pwb980-p43-F
     promoter        3772..4063
                     /label=P43
     CDS             4105..4191
                     /codon_start=1
                     /label=sacB signal peptide
                     /translation="MNIKKFAKQATVLTFTTALLAGGATQAFA"