Price Information
| Cat No. | Plasmid Name | Availability | Buy one, get one free! (?) |
|---|---|---|---|
| V003329 | pSEVA321 | In stock, instant shipping |
Two tubes of lyophilized plasmid will be delivered, each tube is about 5µg.
Basic Vector Information
- Vector Name:
- pSEVA321
- Antibiotic Resistance:
- Chloramphenicol
- Length:
- 3668 bp
- Type:
- Cloning vector
- Replication origin:
- oriV
- Source/Author:
- Silva-Rocha R, Martinez-Garcia E, Chavarria M, Calles B, Arce-Rodriguez A, de las Heras A, Paez-Espino D, Durante-Rodriguez G, Kim J, Nikel PI, Platero R, de Lorenzo V.
- Growth Strain(s):
- DH10B
- Growth Temperature:
- 37℃
pSEVA321 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
- Xu T, Chen J, Mitra R, Lin L, Xie Z, Chen GQ, Xiang H, Han J. Deficiency of exopolysaccharides and O-antigen makes Halomonas bluephagenesis self-flocculating and amenable to electrotransformation. Commun Biol. 2022 Jun 24;5(1):623. doi: 10.1038/s42003-022-03570-y. PMID: 35750760; PMCID: PMC9232590.
pSEVA321 vector Sequence
LOCUS 40924_39438 3668 bp DNA circular SYN 18-DEC-2018
DEFINITION Cloning vector pSEVA321, complete sequence.
ACCESSION .
VERSION .
KEYWORDS .
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 3668)
AUTHORS Silva-Rocha R, Martinez-Garcia E, Chavarria M, Calles B,
Arce-Rodriguez A, de las Heras A, Paez-Espino D, Durante-Rodriguez
G, Kim J, Nikel PI, Platero R, de Lorenzo V.
TITLE The Standard European Vector Architecture (SEVA): a coherent
platform for analysis and deployment of complex prokaryotic
phenotypes
JOURNAL Unpublished
REFERENCE 2 (bases 1 to 3668)
AUTHORS Silva-Rocha R, Martinez-Garcia E, Chavarria M, Calles B,
Arce-Rodriguez A, de las Heras A, Paez-Espino D, Durante-Rodriguez
G, Kim J, Nikel PI, Platero R, de Lorenzo V.
TITLE Direct Submission
JOURNAL Submitted (28-AUG-2012) Systems Biology Program, Centro Nacional de
Biotecnologia-CSIC, C/ Darwin 3, Campus de Cantoblanco, Madrid,
Madrid 28049, Spain
REFERENCE 3 (bases 1 to 3668)
TITLE Direct Submission
REFERENCE 4 (bases 1 to 3668)
AUTHORS .
TITLE Direct Submission
COMMENT SGRef: number: 1; type: "Journal Article"; journalName:
"Unpublished"
COMMENT SGRef: number: 2; type: "Journal Article"; journalName: "Submitted
(28-AUG-2012) Systems Biology Program, Centro Nacional de
Biotecnologia-CSIC, C/ Darwin 3, Campus de Cantoblanco, Madrid,
Madrid 28049, Spain"
COMMENT SGRef: number: 3; type: "Journal Article"
COMMENT ##Assembly-Data-START##
Assembly Method :: ApE v. v2.0.40
Sequencing Technology :: Sanger dideoxy sequencing
##Assembly-Data-END##
FEATURES Location/Qualifiers
source 1..3668
/mol_type="other DNA"
/organism="synthetic DNA construct"
primer_bind 9..32
/label=R24
/note="R24"
misc_feature 56..112
/label=MCS
/note="pUC18/19 multiple cloning site"
primer_bind complement(121..144)
/label=F24
/note="F24"
terminator 155..249
/label=lambda t0 terminator
/note="transcription terminator from phage lambda"
promoter 297..399
/label=cat promoter
/note="promoter of the E. coli cat gene encoding
chloramphenicol acetyltransferase"
CDS 400..1056
/codon_start=1
/label=CmR
/note="chloramphenicol acetyltransferase"
/translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
oriT 1208..1316
/label=oriT
/note="incP origin of transfer"
CDS complement(1380..2525)
/codon_start=1
/label=trfA
/note="trans-acting replication protein that binds to and
activates oriV"
/translation="MNRTFDRKAYRQELIDAGFSAEDAETIASRTVMRAPRETFQSVGS
IVQQATAKIERDSVQLAPPALPAPSAAVERSRRLEQEAAGLAKSMTIDTRGTMTTKKRK
TAGEDLAKQVSEAKQAALLKHTKQQIKEMQLSLFDIAPWPDTMRAMPNDTARSALFTTR
NKKIPREALQNKVIFHVNKDVKITYTGVELRADDDELVWQQVLEYAKRTPIGEPITFTF
YELCQDLGWSINGRYYTKAEECLSRLQATAMGFTSDRVGHLESVSLLHRFRVLDRGKKT
SRCQVLIDEEIVVLFAGDHYTKFIWEKYRKLSPTARRMFDYFSSHREPYPLKLETFRLM
CGSDSTRVKKWREQVGEACEELRGSGLVEHAWVNDDLVHCKR"
rep_origin complement(2918..3449)
/direction=LEFT
/label=oriV
/note="incP origin of replication"
terminator complement(3576..3662)
/label=rrnB T1 terminator
/note="transcription terminator T1 from the E. coli rrnB
gene"