Price Information
| Cat No. | Plasmid Name | Availability | Add to cart |
|---|---|---|---|
| V002615 | pTKRED | 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:
- pTKRED
- Antibiotic Resistance:
- Streptomycin
- Length:
- 10726 bp
- Type:
- Helper vector
- Replication origin:
- pSC101 ori
- Source/Author:
- Kuhlman TE, Cox EC.
- Promoter:
- araBAD
- Growth Strain(s):
- Top10
- Growth Temperature:
- 37℃
pTKRED 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
- Kuhlman TE, Cox EC. Site-specific chromosomal integration of large synthetic constructs. Nucleic Acids Res. 2010 Apr;38(6):e92. doi: 10.1093/nar/gkp1193. Epub 2010 Jan 4. PMID: 20047970; PMCID: PMC2847246.
pTKRED vector Sequence
LOCUS V002615 10726 bp DNA circular SYN 18-DEC-2018
DEFINITION Exported.
ACCESSION V002615
VERSION V002615
KEYWORDS .
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
.
REFERENCE 1 (bases 1 to 10726)
AUTHORS Kuhlman TE, Cox EC.
TITLE Site-specific chromosomal integration of large synthetic constructs
JOURNAL Nucleic Acids Res. 38 (6), E92 (2010)
PUBMED 20047970
REFERENCE 2 (bases 1 to 10726)
AUTHORS Kuhlman TE, Cox EC.
TITLE Direct Submission
JOURNAL Submitted (17-DEC-2009) Molecular Biology, Princeton University,
Washington Rd., Princeton, NJ 08544, USA
REFERENCE 3 (bases 1 to 10726)
TITLE Direct Submission
REFERENCE 4 (bases 1 to 10726)
AUTHORS .
TITLE Direct Submission
COMMENT SGRef: number: 1; type: "Journal Article"; journalName: "Nucleic
Acids Res."; date: "2010"; volume: "38"; issue: "6"; pages: "E92"
SGRef: number: 2; type: "Journal Article"; journalName: "Submitted
(17-DEC-2009) Molecular Biology, Princeton University, Washington
Rd., Princeton, NJ 08544, USA"
SGRef: number: 3; type: "Journal Article"
FEATURES Location/Qualifiers
source 1..10726
/mol_type="other DNA"
/organism="synthetic DNA construct"
CDS complement(147..1022)
/label="araC"
/note="L-arabinose regulatory protein"
promoter 1049..1333
/label="araBAD promoter"
/note="promoter of the L-arabinose operon of E. coli; the
araC regulatory gene is transcribed in the opposite
direction (Guzman et al., 1995)"
RBS 1360..1368
/label="Shine-Dalgarno sequence"
/note="full consensus sequence for ribosome-binding sites
upstream of start codons in E. coli; complementary to a
region in the 3' end of the 16S rRNA (Chen et al., 1994)"
CDS 1383..2075
/gene="SCEI"
/label="Intron-encoded endonuclease I-SceI"
/note="Intron-encoded endonuclease I-SceI from
Saccharomyces cerevisiae (strain ATCC 204508 / S288c).
Accession#: P03882"
CDS 2663..3451
/label="SmR"
/note="aminoglycoside adenylyltransferase (Murphy, 1985)"
CDS complement(3781..4458)
/label="Exo"
/note="5' to 3' double-stranded DNA exonuclease in the
lambda Red system"
CDS complement(4458..5240)
/label="Beta"
/note="single-stranded DNA binding recombinase in the
lambda Red system"
CDS complement(5249..5662)
/label="Gam"
/note="inhibitor of the host RecBCD nuclease in the lambda
Red system"
regulatory complement(5687..5714)
/label="Plac"
/note="Plac"
/regulatory_class="promoter"
protein_bind 5696..5712
/label="lac operator"
/bound_moiety="lac repressor encoded by lacI"
/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 complement(5720..5750)
/label="lac UV5 promoter"
/note="E. coli lac promoter with an 'up' mutation"
CDS complement(5879..6937)
/label="RecA"
/note="DNA repair protein from E. coli"
CDS complement(7429..8508)
/label="lacI"
/note="lac repressor"
regulatory complement(8542..8574)
/label="PlacI"
/note="PlacI"
/regulatory_class="promoter"
CDS complement(8697..9644)
/label="Rep101"
/note="RepA protein needed for replication with the pSC101
origin"
rep_origin complement(9692..9914)
/direction=LEFT
/label="pSC101 ori"
/note="low-copy replication origin that requires the Rep101
protein"