Price Information
| Cat No. | Plasmid Name | Availability | Add to cart |
|---|---|---|---|
| V012683 | pTrc99a | 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
Tightly regulated trc promoter vectors useful for the expression of unfused and fused proteins in Escherichia coli
- Vector Name:
- pTrc99a
- Antibiotic Resistance:
- Ampicillin
- Length:
- 4175 bp
- Type:
- Bacterial expression vector
- Replication origin:
- ori
- Copy Number:
- High copy number
- Promoter:
- trc
- Cloning Method:
- Enzyme Cut
- 5' Primer:
- GAGCGGATAACAATTTCACACAGG
- 3' Primer:
- GATTTAATCTGTATCAGG
- Growth Strain(s):
- stbl3
- Growth Temperature:
- 37℃
- Expression Method:
- IPTG induced
pTrc99a 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
- Dai G, Li R, Chen H, Jiang C, You X, Wu Y. A ferritin-like protein with antioxidant activity in Ureaplasma urealyticum. BMC Microbiol. 2015 Jul 26;15:145. doi: 10.1186/s12866-015-0485-6. PMID: 26209240; PMCID: PMC4515015.
- Xu J, Xu X, Xu Q, Zhang Z, Jiang L, Huang H. Efficient production of lycopene by engineered E. coli strains harboring different types of plasmids. Bioprocess Biosyst Eng. 2018 Apr;41(4):489-499. doi: 10.1007/s00449-017-1883-y. Epub 2018 Jan 8. PMID: 29313097.
- Jeon E, Lee S, Won JI, Han SO, Kim J, Lee J. Development of Escherichia coli MG1655 strains to produce long chain fatty acids by engineering fatty acid synthesis (FAS) metabolism. Enzyme Microb Technol. 2011 Jun 10;49(1):44-51. doi: 10.1016/j.enzmictec.2011.04.001. Epub 2011 Apr 8. PMID: 22112270.
pTrc99a vector Sequence
LOCUS Exported 4175 bp DNA circular SYN 26-AUG-2024
DEFINITION synthetic circular DNA.
ACCESSION .
VERSION .
KEYWORDS .
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 4175)
TITLE Direct Submission
REFERENCE 2 (bases 1 to 4175)
TITLE Direct Submission
REFERENCE 3 (bases 1 to 4175)
AUTHORS .
TITLE Direct Submission
COMMENT SGRef: number: 1; type: "Journal Article"
COMMENT SGRef: number: 2; type: "Journal Article"
FEATURES Location/Qualifiers
source 1..4175
/mol_type="other DNA"
/organism="synthetic DNA construct"
promoter 193..222
/label=trc promoter
/note="strong E. coli promoter; hybrid between the trp and
lac UV5 promoters"
protein_bind 230..246
/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)."
misc_feature 270..326
/label=MCS
/note="pUC18/19 multiple cloning site"
terminator 529..615
/label=rrnB T1 terminator
/note="transcription terminator T1 from the E. coli rrnB
gene"
terminator 707..734
/label=rrnB T2 terminator
/note="transcription terminator T2 from the E. coli rrnB
gene"
promoter 754..844
/label=AmpR promoter
CDS 845..1702
/codon_start=1
/label=AmpR
/note="beta-lactamase"
/translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
LIKHW"
rep_origin 1876..2464
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
misc_feature complement(2650..2790)
/label=bom
/note="basis of mobility region from pBR322"
promoter 2976..3053
/label=lacIq promoter
/note="In the lacIq allele, a single base change in the
promoter boosts expression of the lacI gene about 10-fold."
CDS 3054..4133
/codon_start=1
/label=lacI
/note="lac repressor"
/translation="VKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
ALADSLMQLARQVSRLESGQ"