Price Information
| Cat No. | Plasmid Name | Availability | Add to cart |
|---|---|---|---|
| V007225 | pL99 | In stock, 1 week for quality controls |
Buy one, get one free! |
Two tubes of lyophilized plasmid will be delivered, each tube is about 5µg.
Basic Vector Information
- Vector Name:
- pL99
- Antibiotic Resistance:
- Apramycin
- Length:
- 7554 bp
- Type:
- Bacterial Expression ; Streptomyces expression
- Replication origin:
- ori
- Copy Number:
- High Copy
- Promoter:
- inducible PnitA-NitR system
- Cloning Method:
- Restriction Enzyme
- 5' Primer:
- 5'-gcatgctaaaccgatacaatt-3'
pL99 vector 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.
pL99 vector Sequence
LOCUS 40924_27524 7554 bp DNA circular SYN 13-MAY-2021
DEFINITION Over-expression shuttle vector for gene cloning and transfer from E.
coli to Streptomyces. Contains inducible PnitA-NitR system .
ACCESSION .
VERSION .
KEYWORDS .
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 7554)
AUTHORS Sun N, Wang Z-B, Wu H-P, Mao X-M, Li Y-Q
TITLE Construction of over-expression shuttle vectors in Streptomyces
JOURNAL Annals of Microbiology Dec 2012, Volume 62, Issue 4, pp 1541-1546
REFERENCE 2 (bases 1 to 7554)
TITLE Direct Submission
REFERENCE 3 (bases 1 to 7554)
AUTHORS .
TITLE Direct Submission
COMMENT SGRef: number: 1; type: "Journal Article"; journalName: "Annals of
Microbiology Dec 2012, Volume 62, Issue 4, pp 1541-1546"
COMMENT SGRef: number: 2; type: "Journal Article"
FEATURES Location/Qualifiers
source 1..7554
/mol_type="other DNA"
/organism="synthetic DNA construct"
terminator 55..94
/label=fd terminator
/note="central terminator from bacteriophage fd (Otsuka and
Kunisawa, 1982)"
terminator 1177..1216
/label=fd terminator
/note="central terminator from bacteriophage fd (Otsuka and
Kunisawa, 1982)"
primer_bind complement(4297..4313)
/label=M13 rev
/note="common sequencing primer, one of multiple similar
variants"
protein_bind complement(4321..4337)
/label=lac operator
/note="The lac repressor binds to the lac operator to
inhibit transcription in E. coli. This inhibition can be
relieved by adding lactose or
isopropyl-beta-D-thiogalactopyranoside (IPTG)."
promoter complement(4345..4375)
/label=lac promoter
/note="promoter for the E. coli lac operon"
protein_bind complement(4390..4411)
/label=CAP binding site
/note="CAP binding activates transcription in the presence
of cAMP."
primer_bind complement(4528..4545)
/label=L4440
/note="L4440 vector, forward primer"
rep_origin complement(4699..5287)
/direction=LEFT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
CDS 5491..6291
/codon_start=1
/label=ApmR
/note="aminoglycoside 3-N-acetyltransferase type IV"
/translation="MSSAVECNVVQYEWRKAELIGQLLNLGVTPGGVLLVHSSFRSVRP
LEDGPLGLIEALRAALGPGGTLVMPSWSGLDDEPFDPATSPVTPDLGVVSDTFWRLPNV
KRSAHPFAFAAAGPQAEQIISDPLPLPPHSPASPVARVHELDGQVLLLGVGHDANTTLH
LAELMAKVPYGVPRHCTILQDGKLVRVDYLENDHCCERFALADRWLKEKSLQKEGPVGH
AFARLIRSRDIVATALGQLGRDPLIFLHPPEAGCEECDAARQSIG"
CDS complement(6728..7096)
/codon_start=1
/label=traJ
/note="oriT-recognizing protein"
/translation="MADETKPTRKGSPPIKVYCLPDERRAIEEKAAAAGMSLSAYLLAV
GQGYKITGVVDYEHVRELARINGDLGRLGGLLKLWLTDDPRTARFGDATILALLAKIEE
KQDELGKVMMGVVRPRAEP"
oriT complement(7129..7238)
/direction=LEFT
/label=oriT
/note="incP origin of transfer"
terminator 7402..7441
/label=fd terminator
/note="central terminator from bacteriophage fd (Otsuka and
Kunisawa, 1982)"