Price Information
| Cat No. | Plasmid Name | Availability | Add to cart |
|---|---|---|---|
| V009104 | pCMV-BD | 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 pCMV-BD and pCMV-AD vectors are designed for the construction and expression of gene fusions with the GAL4 DNA binding domain and NF-κB transcriptional activation domain, respectively. The pCMV-BD vector contains DNA encoding amino acids 1–147 of the GAL4 gene (DNA binding domain)2 and unique 3′ cloning sites. It is used for the construction of bait plasmids containing a DNA insert encoding a bait protein. The pCMV-AD vector contains DNA encoding the nuclear localization sequence (NLS) from SV40 large T-antigen (PKKKRKV), amino acids 364–550 of the mouse NF-κB gene (transcriptional activation domain)3 and unique 3′ cloning sites. It is used for constructing a target plasmid containing a DNA insert encoding the interacting proteins. Both pCMV-BD and pCMV-AD contain the pUC origin for replication in E. coli. The pCMV-BD vector contains the kanamycin-resistance gene and the pCMV-AD vector contains the ampicillin-resistance gene. The constitutively-active cytomegalovirus (CMV) promoter governs the expression of the bait and target proteins in the pCMV-BD and pCMV-AD vectors. The SV40 poly (A) provide the signals necessary for transcriptional termination and polyadenylation of the bait and target genes in mammalian cells.
- Vector Name:
- pCMV-BD
- Antibiotic Resistance:
- Kanamycin
- Length:
- 4730 bp
- Type:
- Mammalian Two-Hybrid System
- Replication origin:
- ori
- Selection Marker:
- Neomycin
- Copy Number:
- High copy number
- Promoter:
- CMV
- Fusion Tag:
- Gal4-DBD
pCMV-BD 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.
pCMV-BD vector Sequence
LOCUS 40924_11591 4730 bp DNA circular SYN 13-JAN-2022
DEFINITION synthetic circular DNA.
ACCESSION .
VERSION .
KEYWORDS .
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 4730)
TITLE Direct Submission
REFERENCE 2 (bases 1 to 4730)
AUTHORS .
TITLE Direct Submission
COMMENT SGRef: number: 1; type: "Journal Article"
FEATURES Location/Qualifiers
source 1..4730
/mol_type="other DNA"
/organism="synthetic DNA construct"
enhancer 67..370
/label=CMV enhancer
/note="human cytomegalovirus immediate early enhancer"
promoter 371..574
/label=CMV promoter
/note="human cytomegalovirus (CMV) immediate early
promoter"
promoter 620..638
/label=T3 promoter
/note="promoter for bacteriophage T3 RNA polymerase"
CDS 675..1115
/codon_start=1
/label=GAL4 DNA binding domain
/note="DNA binding domain of the GAL4 transcriptional
activator"
/translation="MKLLSSIEQACDICRLKKLKCSKEKPKCAKCLKNNWECRYSPKTK
RSPLTRAHLTEVESRLERLEQLFLLIFPREDLDMILKMDSLQDIKALLTGLFVQDNVNK
DAVTDRLASVETDMPLTLRQHRISATSSSEESSNKGQRQLTVS"
promoter complement(1232..1250)
/label=T7 promoter
/note="promoter for bacteriophage T7 RNA polymerase"
polyA_signal 1524..1645
/label=SV40 poly(A) signal
/note="SV40 polyadenylation signal"
rep_origin complement(1652..2107)
/direction=LEFT
/label=f1 ori
/note="f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
promoter 2134..2236
/label=AmpR promoter
promoter 2240..2597
/label=SV40 promoter
/note="SV40 enhancer and early promoter"
CDS 2632..3423
/codon_start=1
/label=NeoR/KanR
/note="aminoglycoside phosphotransferase"
/translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
polyA_signal 3658..3705
/label=HSV TK poly(A) signal
/note="herpes simplex virus thymidine kinase
polyadenylation signal (Cole and Stacy, 1985)"
rep_origin 4034..4622
/direction=RIGHT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"