Price Information
| Cat No. | Plasmid Name | Availability | Add to cart |
|---|---|---|---|
| V011794 | pET161-DEST | In stock, 1 week for quality controls |
Buy one, get one free! |
Two tubes of lyophilized plasmid will be delivered, each tube is about 5µg.
Basic Vector Information
pET161-DEST is for proteins high-level bacterial expression, with a C-terminal tetracysteine tag.
- Vector Name:
- pET161-DEST
- Antibiotic Resistance:
- Ampicillin
- Length:
- 7480 bp
- Type:
- Gateway Cloning Vectors
- Replication origin:
- ori
- Source/Author:
- Invitrogen (Life Technologies)
- Copy Number:
- High copy number
- Promoter:
- tet
pET161-DEST 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
- Ota R, Kotani T, Yamashita M. Biochemical characterization of Pumilio1 and Pumilio2 in Xenopus oocytes. J Biol Chem. 2011 Jan 28;286(4):2853-63.
pET161-DEST vector Sequence
LOCUS 40924_18336 7480 bp DNA circular SYN 01-JAN-1980
DEFINITION Gateway(R) destination vector for high-level bacterial expression of
proteins with a C-terminal tetracysteine (Lumio(TM)) tag.
ACCESSION .
VERSION .
KEYWORDS .
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 7480)
AUTHORS Invitrogen (Life Technologies)
TITLE Direct Submission
REFERENCE 2 (bases 1 to 7480)
AUTHORS .
TITLE Direct Submission
COMMENT SGRef: number: 1; type: "Journal Article"
FEATURES Location/Qualifiers
source 1..7480
/mol_type="other DNA"
/organism="synthetic DNA construct"
promoter 21..39
/label=T7 promoter
/note="promoter for bacteriophage T7 RNA polymerase"
protein_bind 40..64
/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 79..101
/label=RBS
/note="efficient ribosome binding site from bacteriophage
T7 gene 10 (Olins and Rangwala, 1989)"
CDS 109..141
/codon_start=1
/label=T7 tag (gene 10 leader)
/note="leader peptide from bacteriophage T7 gene 10"
/translation="MASMTGGQQMG"
protein_bind 154..278
/label=attR1
/note="recombination site for the Gateway(R) LR reaction"
promoter 303..333
/label=lac UV5 promoter
/note="E. coli lac promoter with an 'up' mutation"
CDS 387..1043
/codon_start=1
/label=CmR
/note="chloramphenicol acetyltransferase"
/translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
CDS 1388..1690
/codon_start=1
/label=ccdB
/note="CcdB, a bacterial toxin that poisons DNA gyrase"
/translation="MQFKVYTYKRESRYRLFVDVQSDIIDTPGRRMVIPLASARLLSDK
VSRELYPVVHIGDESWRMMTTDMASVPVSVIGEEVADLSHRENDIKNAINLMFWGI"
protein_bind complement(1734..1858)
/label=attR2
/note="recombination site for the Gateway(R) LR reaction"
CDS 1887..1904
/codon_start=1
/label=tetracysteine tag
/note="tetracysteine peptide that binds biarsenical
labeling reagents"
/translation="CCPGCC"
CDS 1920..1937
/codon_start=1
/label=6xHis
/note="6xHis affinity tag"
/translation="HHHHHH"
terminator 2008..2055
/label=T7 terminator
/note="transcription terminator for bacteriophage T7 RNA
polymerase"
promoter complement(2230..2258)
/label=tet promoter
/note="E. coli promoter for tetracycline efflux protein
gene"
promoter 2375..2479
/label=AmpR promoter
CDS 2480..3337
/codon_start=1
/label=AmpR
/note="beta-lactamase"
/translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
LIKHW"
rep_origin 3511..4099
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
misc_feature complement(4285..4427)
/label=bom
/note="basis of mobility region from pBR322"
CDS complement(4532..4720)
/codon_start=1
/label=rop
/note="Rop protein, which maintains plasmids at low copy
number"
/translation="VTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
DELYRSCLARFGDDGENL"
protein_bind complement(5998..6019)
/label=CAP binding site
/note="CAP binding activates transcription in the presence
of cAMP."
CDS complement(6035..7114)
/codon_start=1
/label=lacI
/note="lac repressor"
/translation="VKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
ALADSLMQLARQVSRLESGQ"
promoter complement(7115..7192)
/label=lacI promoter