Price Information
| Cat No. | Plasmid Name | Availability | Add to cart |
|---|---|---|---|
| V006454 | pETM-14_DnaA_wt | In stock, instant shipping |
Buy one, get one free! |
Two tubes of lyophilized plasmid will be delivered, each tube is about 5µg.
Basic Vector Information
- Vector Name:
- pETM-14_DnaA_wt
- Antibiotic Resistance:
- Kanamycin
- Length:
- 6744 bp
- Type:
- Cloning vector
- Replication origin:
- ori
- Source/Author:
- Paetzold B, Carolis C, Ferrar T, Serrano L, Lluch-Senar M.
pETM-14_DnaA_wt 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.
pETM-14_DnaA_wt vector Sequence
LOCUS V006454 6744 bp DNA circular SYN 18-DEC-2018
DEFINITION Exported.
ACCESSION V006454
VERSION V006454
KEYWORDS .
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
.
REFERENCE 1 (bases 1 to 6744)
AUTHORS Paetzold B, Carolis C, Ferrar T, Serrano L, Lluch-Senar M.
TITLE In situ overlap and sequence synthesis during DNA assembly
JOURNAL ACS Synth Biol 2 (12), 750-755 (2013)
PUBMED 24161008
REFERENCE 2 (bases 1 to 6744)
AUTHORS Paetzold B.
TITLE Direct Submission
JOURNAL Submitted (21-MAR-2013) EMBL-CRG Systems Biology Research Unit,
Centre for Genomic Regulation, Dr. Aiguader 88, Barcelona 08003,
Spain
REFERENCE 3 (bases 1 to 6744)
TITLE Direct Submission
REFERENCE 4 (bases 1 to 6744)
AUTHORS .
TITLE Direct Submission
COMMENT ##Assembly-Data-START##
Sequencing Technology :: Sanger dideoxy sequencing
##Assembly-Data-END##
SGRef: number: 1; type: "Journal Article"; journalName: "ACS Synth
Biol"; date: "2013"; volume: "2"; issue: "12"; pages: "750-755"
SGRef: number: 2; type: "Journal Article"; journalName: "Submitted
(21-MAR-2013) EMBL-CRG Systems Biology Research Unit, Centre for
Genomic Regulation, Dr. Aiguader 88, Barcelona 08003, Spain"
SGRef: number: 3; type: "Journal Article"
FEATURES Location/Qualifiers
source 1..6744
/mol_type="other DNA"
/organism="synthetic DNA construct"
promoter 216..234
/label="T7 promoter"
/note="promoter for bacteriophage T7 RNA polymerase"
protein_bind 235..259
/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)."
RBS 274..296
/label="RBS"
/note="efficient ribosome binding site from bacteriophage
T7 gene 10 (Olins and Rangwala, 1989)"
CDS 309..326
/label="6xHis"
/note="6xHis affinity tag"
CDS 336..359
/label="HRV 3C site"
/note="recognition and cleavage site for human rhinovirus
3C and PreScission proteases"
CDS 360..1760
/gene="dnaA"
/label="Chromosomal replication initiator protein DnaA"
/note="Chromosomal replication initiator protein DnaA from
Escherichia coli (strain K12). Accession#: P03004"
CDS 1820..1837
/label="6xHis"
/note="6xHis affinity tag"
terminator 1904..1951
/label="T7 terminator"
/note="transcription terminator for bacteriophage T7 RNA
polymerase"
rep_origin 1988..2443
/label="f1 ori"
/note="f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
CDS complement(2539..3351)
/label="KanR"
/note="aminoglycoside phosphotransferase"
rep_origin 3473..4061
/label="ori"
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
misc_feature complement(4247..4389)
/label="bom"
/note="basis of mobility region from pBR322"
CDS complement(4494..4682)
/label="rop"
/note="Rop protein, which maintains plasmids at low copy
number"
protein_bind complement(5457..5478)
/label="CAP binding site"
/note="CAP binding activates transcription in the presence
of cAMP."
CDS complement(5494..6573)
/label="lacI"
/note="lac repressor"
promoter complement(6574..6651)
/label="lacI promoter"