Cart 2

pEC-XK99E vector (V007659)

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

E. coli/C. glutamicum shuttle expression vectors based on the medium copy number plasmid pGA1 and on Ptrc.

Vector Name:
pEC-XK99E
Antibiotic Resistance:
Kanamycin
Length:
7017 bp
Type:
Shuttle expression vector
Replication origin:
ori
Source/Author:
Kirchner O, Tauch A.
Growth Strain(s):
DH10b
Growth Temperature:
37℃

pEC-XK99E vector Map

Copy Sequence View Map
pEC-XK99E7017 bp30060090012001500180021002400270030003300360039004200450048005100540057006000630066006900MCSrrnB T1 terminatorrrnB T2 terminatorPerRepANeoR/KanRoribomlacIq promoterlacItrc promoterlac operator

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

  • Kirchner O, Tauch A. Tools for genetic engineering in the amino acid-producing bacterium Corynebacterium glutamicum. J Biotechnol. 2003;104(1-3):287-299. doi:10.1016/s0168-1656(03)00148-2

  • Yang C, Peng Z, Yang L, Du B, Guo C, Sui S, Wang J, Li J, Wang J, Li N. Design and application of artificial rare L-lysine codons in Corynebacterium glutamicum. Front Bioeng Biotechnol. 2023 May 30;11:1194511. doi: 10.3389/fbioe.2023.1194511. PMID: 37324439; PMCID: PMC10268032.

pEC-XK99E vector Sequence

LOCUS       Exported                7017 bp DNA     circular SYN 02-SEP-2024
DEFINITION  Shuttle expression vector pEC-XK99E, complete sequence.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7017)
  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 7017)
  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 7017)
  TITLE     Direct Submission
REFERENCE   4  (bases 1 to 7017)
  TITLE     Direct Submission
REFERENCE   5  (bases 1 to 7017)
  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"
COMMENT     SGRef: number: 4; type: "Journal Article"
FEATURES             Location/Qualifiers
     source          1..7017
                     /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(704..919)
                     /codon_start=1
                     /gene="per"
                     /product="Per"
                     /label=per
                     /note="positive effector of replication"
                     /protein_id="AAO65195.1"
                     /translation="MDLSDVALESDALDAAVDLKTVIGFFRALDTTDAPASRDWASAAS
                     DLETLVADLEELADELRARQRQEDAQ"
     gene            complement(704..919)
                     /gene="per"
                     /label=per
     CDS             complement(1562..3025)
                     /codon_start=1
                     /gene="repA"
                     /product="RepA"
                     /label=repA
                     /note="replication protein"
                     /protein_id="AAO65196.1"
                     /translation="MTLADPQDTVTASAWKFSADLFDTHPELALRSRGWTAEDRRELLA
                     HLGRESFQGSKTRDFASAWIKNPDTGETQPKLYRAGSKALTRCQYVALTHAQHAAVIVL
                     DIDVPSHQAGGKIEHVNPQVYAILEKWARLEKAPAWIGVNPLSGKCQLIWLIDPVYAAA
                     GKTSPNMRLLAATTEEMTRVFGADQAFSHRLSRWPLHVSDDPTAYKWHCQHDRVDRLAD
                     LMEIARTMTGSQKPKKYIEQDFSSGRARIEAAQRATAEAKALAILDASLPSALDASGDL
                     IDGVRVLWTNPERAARDETAFRHALTVGYQLKAAGERLKDAKIIDAYEVAYNVAQAVGA
                     DGREPDLPAMRDRLTMARRVRGYVAKGQPVVPARRVETQSSRGRKALATMGRRGAATSN
                     ARRWADPESKYAQETRQRLAEANKRREMTGELLELRVKTAILDARSQSVADPSTRELAG
                     ELGVSERRIQQVRKALGMEAKRGRPRAEN"
     gene            complement(1562..3025)
                     /gene="repA"
                     /label=repA
     CDS             3555..4346
                     /codon_start=1
                     /label=NeoR/KanR
                     /note="aminoglycoside phosphotransferase"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     rep_origin      4449..5037
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     misc_feature    complement(5223..5363)
                     /label=bom
                     /note="basis of mobility region from pBR322"
     promoter        5549..5626
                     /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             5627..6706
                     /codon_start=1
                     /label=lacI
                     /note="lac repressor"
                     /translation="VKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPSTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     promoter        6941..6970
                     /label=trc promoter
                     /note="strong E. coli promoter; hybrid between the trp and
                     lac UV5 promoters"
     protein_bind    6978..6994
                     /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)."