Price Information
| Cat No. | Plasmid Name | Availability | Add to cart |
|---|---|---|---|
| V005971 | pCVD442 | 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
pCVD442 is a suicide vector used for negative selection. Sucrose-resistant colonies indicate the loss of suicide vector sequences.
- Vector Name:
- pCVD442
- Antibiotic Resistance:
- Ampicillin
- Length:
- 6345 bp
- Type:
- Bacterial Expression
- Replication origin:
- R6K γ ori
- Source/Author:
- M S Donnenberg, J B Kaper
- Selection Marker:
- SacB
- Copy Number:
- High Copy
- Promoter:
- sacB
- Cloning Method:
- Restriction Enzyme
- Growth Strain(s):
- S17-1λpir
- Growth Temperature:
- 37℃
pCVD442 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.
References
- Donnenberg MS, Kaper JB. Construction of an eae deletion mutant of enteropathogenic Escherichia coli by using a positive-selection suicide vector. Infect Immun. 1991 Dec;59(12):4310-7. doi: 10.1128/iai.59.12.4310-4317.1991. PMID: 1937792; PMCID: PMC259042.
pCVD442 vector Sequence
LOCUS 40924_13855 6345 bp DNA circular SYN 13-MAY-2021
DEFINITION synthetic circular DNA.
ACCESSION .
VERSION .
KEYWORDS .
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 6345)
AUTHORS Donnenberg MS, Kaper JB
TITLE Construction of an eae deletion mutant of enteropathogenic
Escherichia coli by using a positive-selection suicide vector.
JOURNAL Infect Immun. 1991 Dec . 59(12):4310-7.
PUBMED 1937792
REFERENCE 2 (bases 1 to 6345)
TITLE Direct Submission
REFERENCE 3 (bases 1 to 6345)
AUTHORS .
TITLE Direct Submission
COMMENT SGRef: number: 1; type: "Journal Article"; journalName: "Infect
Immun. 1991 Dec . 59(12):4310-7."
COMMENT SGRef: number: 2; type: "Journal Article"
FEATURES Location/Qualifiers
source 1..6345
/mol_type="other DNA"
/organism="synthetic DNA construct"
primer_bind 143..162
/label=pBRrevBam
/note="pBR322 vectors, tet region, downstream of BamHI,
reverse primer"
oriT 843..952
/label=oriT
/note="incP origin of transfer"
CDS 985..1353
/codon_start=1
/label=traJ
/note="oriT-recognizing protein"
/translation="MADETKPTRKGSPPIKVYCLPDERRAIEEKAAAAGMSLSAYLLAV
GQGYKITGVVDYEHVRELARINGDLGRLGGLLKLWLTDDPRTARFGDATILALLAKIEE
KQDELGKVMMGVVRPRAEP"
rep_origin 1974..2362
/label=R6K gamma ori
/note="gamma replication origin from E. coli plasmid R6K;
requires the R6K initiator protein pi for replication"
protein_bind 2397..2413
/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 2539..2984
/label=sacB promoter
/note="sacB promoter and control region"
CDS 2985..4403
/codon_start=1
/label=SacB
/note="secreted levansucrase that renders bacterial growth
sensitive to sucrose"
/translation="MNIKKFAKQATVLTFTTALLAGGATQAFAKETNQKPYKETYGISH
ITRHDMLQIPEQQKNEKYQVPEFDSSTIKNISSAKGLDVWDSWPLQNADGTVANYHGYH
IVFALAGDPKNADDTSIYMFYQKVGETSIDSWKNAGRVFKDSDKFDANDSILKDQTQEW
SGSATFTSDGKIRLFYTDFSGKHYGKQTLTTAQVNVSASDSSLNINGVEDYKSIFDGDG
KTYQNVQQFIDEGNYSSGDNHTLRDPHYVEDKGHKYLVFEANTGTEDGYQGEESLFNKA
YYGKSTSFFRQESQKLLQSDKKRTAELANGALGMIELNDDYTLKKVMKPLIASNTVTDE
IERANVFKMNGKWYLFTDSRGSKMTIDGITSNDIYMLGYVSNSLTGPYKPLNKTGLVLK
MDLDPNDVTFTYSHFAVPQAKGNNVVITSYMTNRGFYADKQSTFAPSFLLNIKGKKTSV
VKDSILEQGQLTVNK"
promoter 5145..5249
/label=AmpR promoter
CDS 5250..6107
/codon_start=1
/label=AmpR
/note="beta-lactamase"
/translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
LIKHW"
primer_bind complement(6167..6186)
/label=pRS-marker
/note="pRS vectors, use to sequence yeast selectable
marker"