Price Information
| Cat No. | Plasmid Name | Availability | Add to cart |
|---|---|---|---|
| V007659 | pEC-XK99E | 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:
- pEC-XK99E
- Antibiotic Resistance:
- Kanamycin
- Length:
- 7018 bp
- Type:
- Shuttle expression vector
- Replication origin:
- ori
- Source/Author:
- Kirchner O, Tauch A.
pEC-XK99E vector 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.
pEC-XK99E vector Sequence
LOCUS 40924_16690 7018 bp DNA circular SYN 17-DEC-2018
DEFINITION Shuttle expression vector pEC-XK99E, complete sequence.
ACCESSION .
VERSION .
KEYWORDS .
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 7018)
AUTHORS Kirchner O, Tauch A.
TITLE Tools for genetic engineering in the amino acid-producing bacterium
Corynebacterium glutamicum
JOURNAL J. Biotechnol. 104 (1-3), 287-299 (2003)
PUBMED 12948646
REFERENCE 2 (bases 1 to 7018)
AUTHORS Kirchner O, Tauch A.
TITLE Direct Submission
JOURNAL Submitted (15-JAN-2003) Department of Genetics, University of
Bielefeld, Universitaetsstrasse 25, Bielefeld D-33615, Germany
REFERENCE 3 (bases 1 to 7018)
TITLE Direct Submission
REFERENCE 4 (bases 1 to 7018)
AUTHORS .
TITLE Direct Submission
COMMENT SGRef: number: 1; type: "Journal Article"; journalName: "J.
Biotechnol. 104 (1-3), 287-299 (2003)"
COMMENT SGRef: number: 2; type: "Journal Article"; journalName: "Submitted
(15-JAN-2003) Department of Genetics, University of Bielefeld,
Universitaetsstrasse 25, Bielefeld D-33615, Germany"
COMMENT SGRef: number: 3; type: "Journal Article"
FEATURES Location/Qualifiers
source 1..7018
/mol_type="other DNA"
/organism="synthetic DNA construct"
misc_feature 1..57
/label=MCS
/note="pUC18/19 multiple cloning site"
terminator 260..346
/label=rrnB T1 terminator
/note="transcription terminator T1 from the E. coli rrnB
gene"
terminator 438..465
/label=rrnB T2 terminator
/note="transcription terminator T2 from the E. coli rrnB
gene"
CDS complement(705..920)
/codon_start=1
/gene="per"
/product="Per"
/label=per
/note="positive effector of replication"
/protein_id="AAO65195.1"
/translation="MDLSDVALESDALDAAVDLKTVIGFFRALDTTDAPASRDWASAAS
DLETLVADLEELADELRARQRQEDAQ"
gene complement(705..920)
/gene="per"
/label=per
CDS complement(1563..3026)
/codon_start=1
/gene="repA"
/product="RepA"
/label=repA
/note="replication protein"
/protein_id="AAO65196.1"
/translation="MTLADPQDTVTASAWKLSADLFDTHPEAMRCGSRGWTAEDRRELL
AHLGRESFQGSKTRDFASAWIKNPDTGETQPKLYRAGSKALTRCQYVALTHAQHAAVIV
LDIDVPSHQAGGKIEHVNPQVYAILEKWARLEKAPAWIGVNPLSGKCQLIWLIDPVYAA
AGKTSPNMRLLAATTEEMTRVFGADQAFSHRLSRWPLHVSDDPTAYKWHCQHDRVDRLA
DLMEIARTMTGSQKPKKYIEQDFSSGRARIEAAQRATAEAKALAILDASLPSALDASGD
LIDGVRVLWTNPERARDETAFRHALTVGYQLKAAGERLKDAKIIDAYEVAYNVAQAVGA
DGREPDLPAMRDRLTMARRVRGYVAKGQPVVPARRVETQSSRGRKALATMGRRGAATSN
ARRWADPESKYAQETRQRLAEANKRREMTGELLELRVKTAILDARSQSVADPSTRELAG
ELGVSERRIQQVRKALGMEAKRGRPRAEN"
gene complement(1563..3026)
/gene="repA"
/label=repA
CDS 3556..4347
/codon_start=1
/label=NeoR/KanR
/note="aminoglycoside phosphotransferase"
/translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
rep_origin 4450..5038
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
misc_feature complement(5224..5364)
/label=bom
/note="basis of mobility region from pBR322"
promoter 5550..5627
/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 5628..6707
/codon_start=1
/label=lacI
/note="lac repressor"
/translation="VKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
YIPPSTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
ALADSLMQLARQVSRLESGQ"
promoter 6942..6971
/label=trc promoter
/note="strong E. coli promoter; hybrid between the trp and
lac UV5 promoters"
protein_bind 6979..6995
/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)."