Price Information
| Cat No. | Plasmid Name | Availability | Add to cart |
|---|---|---|---|
| V005214 | pKLD66 | 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
The pKLD66 vector facilitates the production of a recombinant protein featuring an N-terminal His6 tag followed by a maltose-binding protein (MBP) tag in sequence. These tags can be cleaved off using TEV protease.
- Vector Name:
- pKLD66
- Antibiotic Resistance:
- Ampicillin
- Length:
- 6608 bp
- Type:
- Expression vector
- Replication origin:
- ori
- Source/Author:
- Rocco CJ, Dennison KL, Klenchin VA, Rayment I, Escalante-Semerena JC.
- Growth Temperature:
- 37℃
pKLD66 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
- Rocco, C J et al. “Construction and use of new cloning vectors for the rapid isolation of recombinant proteins from Escherichia coli.” Plasmid vol. 59,3 (2008): 231-7. doi:10.1016/j.plasmid.2008.01.001
pKLD66 vector Sequence
LOCUS 40924_26864 6608 bp DNA circular SYN 18-DEC-2018
DEFINITION Expression vector pKLD66, complete sequence.
ACCESSION .
VERSION .
KEYWORDS .
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 6608)
AUTHORS Rocco CJ, Dennison KL, Klenchin VA, Rayment I, Escalante-Semerena
JC.
TITLE Construction and use of new cloning vectors for the rapid isolation
of recombinant proteins from Escherichia coli
JOURNAL Plasmid 59 (3), 231-237 (2008)
PUBMED 18295882
REFERENCE 2 (bases 1 to 6608)
AUTHORS Rocco CJ, Dennison KL, Klenchin VA, Rayment I, Escalante-Semerena
JC.
TITLE Direct Submission
JOURNAL Submitted (10-DEC-2007) Bacteriology, University of Wisconsin -
Madison, 1550 Linden Dr., Madison, WI 53706, USA
REFERENCE 3 (bases 1 to 6608)
TITLE Direct Submission
REFERENCE 4 (bases 1 to 6608)
AUTHORS .
TITLE Direct Submission
COMMENT SGRef: number: 1; type: "Journal Article"; journalName: "Plasmid";
date: "2008"; volume: "59"; issue: "3"; pages: "231-237"
COMMENT SGRef: number: 2; type: "Journal Article"; journalName: "Submitted
(10-DEC-2007) Bacteriology, University of Wisconsin - Madison, 1550
Linden Dr., Madison, WI 53706, USA"
COMMENT SGRef: number: 3; type: "Journal Article"
FEATURES Location/Qualifiers
source 1..6608
/mol_type="other DNA"
/organism="synthetic DNA construct"
CDS 13..30
/codon_start=1
/label=6xHis
/note="6xHis affinity tag"
/translation="HHHHHH"
CDS 37..1134
/codon_start=1
/label=MBP
/note="maltose binding protein from E. coli"
/translation="KIEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLEE
KFPQVAATGDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDAVRYNGKLI
AYPIAVEALSLIYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWPLIAA
DGGYAFKYENGKYDIKDVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNKGET
AMTINGPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFLE
NYLLTDEGLEAVNKDKPLGAVALKSYEEELAKDPRIAATMENAQKGEIMPNIPQMSAFW
YAVRTAVINAASGRQTVDEALKDAQT"
CDS 1180..1200
/codon_start=1
/label=TEV site
/note="tobacco etch virus (TEV) protease recognition and
cleavage site"
/translation="ENLYFQG"
CDS 1251..1268
/codon_start=1
/label=6xHis
/note="6xHis affinity tag"
/translation="HHHHHH"
terminator 1335..1382
/label=T7 terminator
/note="transcription terminator for bacteriophage T7 RNA
polymerase"
rep_origin 1419..1874
/label=f1 ori
/note="f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
promoter 1901..2005
/label=AmpR promoter
CDS 2006..2863
/codon_start=1
/label=AmpR
/note="beta-lactamase"
/translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
LIKHW"
rep_origin 3037..3625
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
misc_feature complement(3811..3953)
/label=bom
/note="basis of mobility region from pBR322"
CDS complement(4058..4246)
/codon_start=1
/label=rop
/note="Rop protein, which maintains plasmids at low copy
number"
/translation="VTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
DELYRSCLARFGDDGENL"
protein_bind complement(5021..5042)
/label=CAP binding site
/note="CAP binding activates transcription in the presence
of cAMP."
CDS complement(5058..6137)
/codon_start=1
/label=lacI
/note="lac repressor"
/translation="VKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
ALADSLMQLARQVSRLESGQ"
promoter complement(6138..6215)
/label=lacI promoter
promoter 6524..6542
/label=T7 promoter
/note="promoter for bacteriophage T7 RNA polymerase"
protein_bind 6543..6567
/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 6582..6604
/label=RBS
/note="efficient ribosome binding site from bacteriophage
T7 gene 10 (Olins and Rangwala, 1989)"