Price Information
| Cat No. | Plasmid Name | Availability | Add to cart |
|---|---|---|---|
| V006626 | pKAR2-Br512 | 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:
- pKAR2-Br512
- Antibiotic Resistance:
- Ampicillin
- Length:
- 6218 bp
- Type:
- Bacterial Expression
- Replication origin:
- ori
- Copy Number:
- High Copy
- Promoter:
- T7, tetPA
- Cloning Method:
- Restriction Enzyme
- 5' Primer:
- GCATCGTCTCATCGGTCTCATATGCACCATCATCACGGTCATC
- 3' Primer:
- ATGCCGTCTCAGGTCTCAGGATCCTTATTACTTGGCGTCATACCAGGTG
pKAR2-Br512 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.
pKAR2-Br512 vector Sequence
LOCUS 40924_26551 6218 bp DNA circular SYN 13-MAY-2021
DEFINITION Expresses Br512 polymerase in E.coli.
ACCESSION .
VERSION .
KEYWORDS .
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 6218)
AUTHORS Maranhao A,Bhadra S, Paik I, Walker D, Ellington AD
TITLE An improved and readily available version of Bst DNA Polymerase for
LAMP, and applications to COVID-19 diagnostics
JOURNAL medRxiv
REFERENCE 2 (bases 1 to 6218)
TITLE Direct Submission
REFERENCE 3 (bases 1 to 6218)
AUTHORS .
TITLE Direct Submission
COMMENT SGRef: number: 1; type: "Journal Article"; journalName: "medRxiv"
COMMENT SGRef: number: 2; type: "Journal Article"
FEATURES Location/Qualifiers
source 1..6218
/mol_type="other DNA"
/organism="synthetic DNA construct"
terminator 15..62
/label=T7 terminator
/note="transcription terminator for bacteriophage T7 RNA
polymerase"
CDS complement(362..985)
/codon_start=1
/label=TetR
/note="tetracycline repressor TetR"
/translation="MMSRLDKSKVINSALELLNEVGIEGLTTRKLAQKLGVEQPTLYWH
VKNKRALLDALAIEMLDRHHTHFCPLEGESWQDFLRNNAKSFRCALLSHRDGAKVHLGT
RPTEKQYETLENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEDQEHQVAKEERETPT
TDSMPPLLRQAIELFDHQGAEPAFLFGLELIICGLEKQLKCESGS"
CDS complement(1017..1874)
/codon_start=1
/label=AmpR
/note="beta-lactamase"
/translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
LIKHW"
promoter complement(1875..1946)
/label=AmpR promoter
terminator 2023..2109
/label=rrnB T1 terminator
/note="transcription terminator T1 from the E. coli rrnB
gene"
primer_bind complement(2253..2270)
/label=L4440
/note="L4440 vector, forward primer"
rep_origin complement(2424..3012)
/direction=LEFT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
primer_bind 3822..3841
/label=Kan-R
/note="Kanamycin resistance gene, reverse primer"
primer_bind 3906..3925
/label=pENTR-R
/note="pENTR vectors, reverse primer"
primer_bind 4069..4087
/label=pBRforEco
/note="pBR322 vectors, upsteam of EcoRI site, forward
primer"
protein_bind 4108..4126
/label=tet operator
/note="bacterial operator O1 for the tetR and tetA genes"
promoter 4127..4145
/label=T7 promoter
/note="promoter for bacteriophage T7 RNA polymerase"
protein_bind 4147..4165
/label=tet operator
/note="bacterial operator O2 for the tetR and tetA genes"
misc_RNA 4184..4234
/label=sTRSV HHRz
/note="hammerhead ribozyme from the tobacco ringspot virus
satellite RNA (Khvorova et al., 2003)"
RBS 4268..4276
/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)"