Price Information
| Cat No. | Plasmid Name | Availability | Add to cart |
|---|---|---|---|
| V011638 | pIEx/Bac-1 | 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
The dual-purpose pIEx/Bac vectors are designed for cloning and high-level expression of proteins by transiently transfecting Spodoptera-derived insect cells or by generating baculovirus recombinants. Transient transfection and early baculovirus expression is driven by a promoter/enhancer combination that recruits endogenous insect cell transcription machinery, the AcNPV derived hr5 enhancer and ie1 promoter. Late/very late expression in the baculovirus mode is driven by the strong p10 promoter. The pIEx/Bac-1 vector carries an N-terminal Strep•Tag II coding sequence (1) followed by a recognition site for enterokinase.The multiple cloning region is followed by an optional C-terminal His•Tag coding sequence. The presence of two 'gentle elution' tags at both the N- and C-terminus is ideal for dual purification strategies designed to isolate full-length fusion proteins (2). Unique restriction sites are shown on the circle map.
- Vector Name:
- pIEx/Bac-1
- Antibiotic Resistance:
- Ampicillin
- Length:
- 6796 bp
- Type:
- Insect Cell Vectors
- Replication origin:
- ori
- Source/Author:
- Novagen (EMD Millipore)
- Copy Number:
- High copy number
- Promoter:
- IE1
- Cloning Method:
- Enzyme digestion and ligation
- Fusion Tag:
- C-His,N-Strep
pIEx/Bac-1 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
- Hirota Y, Nakagawa K, Sawada N, Okuda N, Suhara Y, Uchino Y, Kimoto T, Funahashi N, Kamao M, Tsugawa N, Okano T. Functional characterization of the vitamin K2 biosynthetic enzyme UBIAD1. PLoS One. 2015 Apr 15;10(4):e0125737.
pIEx/Bac-1 vector Sequence
LOCUS pIEx_Bac-1. 6796 bp DNA circular SYN 01-JAN-1980
DEFINITION Insect cell and baculovirus vector encoding a cleavable N-terminal
Strep-Tag(R) II and a C-terminal 10xHis tag.
ACCESSION .
VERSION .
KEYWORDS pIEx Bac-1
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 6796)
AUTHORS Novagen (EMD Millipore)
TITLE Direct Submission
REFERENCE 2 (bases 1 to 6796)
AUTHORS .
TITLE Direct Submission
COMMENT SGRef: number: 1; type: "Journal Article"
FEATURES Location/Qualifiers
source 1..6796
/mol_type="other DNA"
/organism="synthetic DNA construct"
misc_recomb 329..1156
/label=baculovirus recombination region (lef2/ORF603)
/note="contains ORF603 and part of lef2"
enhancer 1196..1678
/label=hr5 enhancer
/note="baculovirus early transcription enhancer"
promoter 1682..2273
/label=IE1 promoter
/note="promoter of the ie1 gene from the baculovirus
Autographa californica"
promoter 2283..2392
/label=p10 promoter
/note="baculovirus promoter for expression in insect cells"
CDS 2394..2396
/codon_start=1
/product="start codon"
/label=start codon
/note="ATG"
/translation="M"
CDS 2403..2426
/label=Strep-Tag II
/note="peptide that binds Strep-Tactin(R), an engineered
form
of streptavidin"
CDS 2433..2447
/label=enterokinase site
/note="enterokinase recognition and cleavage site"
misc_feature 2447..2506
/label=MCS
/note="MCS"
/note="multiple cloning site"
CDS 2517..2543
/label=9xHis
/note="9xHis affinity tag"
terminator 2565..2871
/label=IE1 terminator
/note="terminator of the ie1 gene from the baculovirus
Autographa californica"
misc_recomb 2900..4257
/label=baculovirus recombination region (ORF1629)
/note="contains part of ORF1629"
rep_origin complement(4520..5108)
/direction=LEFT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
CDS complement(5282..6139)
/label=AmpR
/note="beta-lactamase"
promoter complement(6140..6244)
/label=AmpR promoter
rep_origin 6271..6726
/direction=RIGHT
/label=f1 ori
/note="f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"