pET-30 Ek/LIC vector (Cat. No.: V010998)
Note: The pET-30 Ek/LIC vector is prepared for rapid, directional cloning of PCR-amplified DNA for high-level expression of polypeptides. Using specifically designed primers for amplification and the pET-30 Ek/LIC Cloning Kit (Cat. No. 69077-3), inserts can be efficiently cloned without the need for restriction digestion or ligation. Fusion proteins contain N-terminal cleavable His•Tag and S•Tag sequences for detection and purification. Unique sites are shown on the circle map. Note that the sequence is numbered by the pBR322 convention, so the T7 expression region is reversed on the circle map. The cloning/expression region of the coding strand transcribed by T7 RNA polymerase is shown below. The f1 origin is oriented so that infection with helper phage will produce virions containing single stranded DNA that corresponds to the coding strand. Therefore, single stranded sequencing should be performed using the T7 terminator primer (Cat. No. 69337-3).
- Name:
- pET-30 Ek/LIC
- Antibiotic Resistance:
- Kanamycin
- Length:
- 5439 bp
- Type:
- pET & Duet Vectors (Novagen)
- Replication origin:
- ori
- Source/Author:
- Novagen (EMD Millipore)
- Copy Number:
- High copy number
- 5' Primer:
- 5'-TAATACGACTCACTATAGGG-3'
- Fusion Tag:
- N-His, N-S, N-EK, C-His
Resources
Plasmid Protocol
1. Centrifuge at 5,000×g for 5 min.
2. Carefully open the tube and add sterile water to dissolve the DNA: add 20 μl for 5 μg plasmid, and 100 μl for 100 μg plasmid.
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.
pET-30 Ek/LIC vector (Cat. No.: V010998) Sequence
LOCUS pET-30_Ek_LIC. 5439 bp DNA circular SYN 01-JAN-1980
DEFINITION Bacterial vector for ligation-independent cloning (LIC) to express
6xHis- and S-tagged proteins with an enterokinase site.
ACCESSION .
VERSION .
KEYWORDS pET-30 Ek_LIC
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 5439)
AUTHORS Novagen (EMD Millipore)
TITLE Direct Submission
REFERENCE 2 (bases 1 to 5439)
AUTHORS .
TITLE Direct Submission
COMMENT SGRef: number: 1; type: "Journal Article"
COMMENT For LIC, linearize with BseRI and treat with T4 DNA polymerase plus
dTTP.
FEATURES Location/Qualifiers
source 1..5439
/mol_type="other DNA"
/organism="synthetic DNA construct"
terminator complement(26..73)
/label=T7 terminator
/note="transcription terminator for bacteriophage T7 RNA
polymerase"
CDS complement(140..157)
/label=6xHis
/note="6xHis affinity tag"
misc_feature 158..216
/label=MCS
/note="MCS"
/note="multiple cloning site"
CDS complement(236..250)
/label=enterokinase site
/note="enterokinase recognition and cleavage site"
CDS complement(266..310)
/label=S-Tag
/note="affinity and epitope tag derived from pancreatic
ribonuclease A"
CDS complement(317..334)
/label=thrombin site
/note="thrombin recognition and cleavage site"
CDS complement(344..361)
/label=6xHis
/note="6xHis affinity tag"
CDS complement(362..364)
/codon_start=1
/product="start codon"
/label=start codon
/note="ATG"
/translation="M"
RBS complement(372..394)
/label=RBS
/note="efficient ribosome binding site from bacteriophage
T7 gene 10 (Olins and Rangwala, 1989)"
protein_bind complement(409..433)
/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(434..452)
/label=T7 promoter
/note="promoter for bacteriophage T7 RNA polymerase"
promoter 765..842
/label=lacI promoter
CDS 843..1922
/label=lacI
/note="lac repressor"
protein_bind 1938..1959
/label=CAP binding site
/note="CAP binding activates transcription in the presence
of cAMP."
CDS 2734..2922
/label=rop
/note="Rop protein, which maintains plasmids at low copy
number"
misc_feature 3027..3169
/label=bom
/note="basis of mobility region from pBR322"
rep_origin complement(3355..3943)
/direction=LEFT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
CDS 4065..4877
/label=KanR
/note="aminoglycoside phosphotransferase"
rep_origin complement(4973..5428)
/direction=LEFT
/label=f1 ori
/note="f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"