pME6032 vector (Cat. No.: V004775)
Note: pME6032 is an E. coli-Pseudomonasshuttle vector (9,821 bp) featuring the Tac promoter, pVS1 and p15A replicons, and tetracycline resistance. It supports inducible gene expression in diverse Gram-negative bacteria, commonly used in protein expression and bacterial genetics studies.
- Name:
- pME6032
- Antibiotic Resistance:
- Tetracycline
- Length:
- 9754 bp
- Type:
- Shuttle vector
- Replication origin:
- p15A ori
- Source/Author:
- Heeb S, Itoh Y, Nishijyo T, Schnider U, Keel C, Wade J, Walsh U, O'Gara F, Haas D.
- Growth Strain(s):
- DH10B
- Growth Temperature:
- 37℃
Resources
Plasmid Protocol
1. Centrifuge at 5,000×g for 5 min.
2. Carefully open the tube and add sterile water to dissolve the DNA: add 20 μl for 5 μg plasmid, and 100 μl for 100 μg plasmid.
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
- Lin J, Yang J, Cheng J, Zhang W, Yang X, Ding W, Zhang H, Wang Y, Shen X. Pseudomonas aeruginosa H3-T6SS Combats H2O2 Stress by Diminishing the Amount of Intracellular Unincorporated Iron in a Dps-Dependent Manner and Inhibiting the Synthesis of PQS. Int J Mol Sci. 2023 Jan 13;24(2):1614. doi: 10.3390/ijms24021614. PMID: 36675127; PMCID: PMC9866239.
pME6032 vector (Cat. No.: V004775) Sequence
LOCUS Exported 9754 bp DNA circular SYN 20-DEC-2025
DEFINITION Shuttle vector pME6032, complete sequence.
ACCESSION .
VERSION .
KEYWORDS .
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 9754)
AUTHORS Heeb S, Itoh Y, Nishijyo T, Schnider U, Keel C, Wade J, Walsh U,
O'Gara F, Haas D.
TITLE Small, stable shuttle vectors based on the minimal pVS1 replicon for
use in gram-negative, plant-associated bacteria
JOURNAL Mol. Plant Microbe Interact. 13 (2), 232-237 (2000)
PUBMED 10659714
REFERENCE 2 (bases 1 to 9754)
AUTHORS Heeb S, Blumer C, Haas D.
TITLE Regulatory RNA as mediator in GacA/RsmA-dependent global control of
exoproduct formation in Pseudomonas fluorescens CHA0
JOURNAL J. Bacteriol. 184 (4), 1046-1056 (2002)
PUBMED 11807065
REFERENCE 3 (bases 1 to 9754)
AUTHORS Heeb S.
TITLE Direct Submission
JOURNAL Submitted (19-MAY-2006) Institute of Infection, Immunity and
Inflammation, University of Nottingham, Centre for Biomolecular
Sciences, Nottingham NG7 2RD, United Kingdom
REFERENCE 4 (bases 1 to 9754)
TITLE Direct Submission
REFERENCE 5 (bases 1 to 9754)
TITLE Direct Submission
REFERENCE 6 (bases 1 to 9754)
TITLE Direct Submission
REFERENCE 7 (bases 1 to 9754)
TITLE Direct Submission
REFERENCE 8 (bases 1 to 9754)
AUTHORS .
TITLE Direct Submission
COMMENT SGRef: number: 1; type: "Journal Article"; journalName: "Mol. Plant
Microbe Interact."; date: "2000"; volume: "13"; issue: "2"; pages:
"232-237"
COMMENT SGRef: number: 2; type: "Journal Article"; journalName: "J.
Bacteriol."; date: "2002"; volume: "184"; issue: "4"; pages:
"1046-1056"
COMMENT SGRef: number: 3; type: "Journal Article"; journalName: "Submitted
(19-MAY-2006) Institute of Infection, Immunity and Inflammation,
University of Nottingham, Centre for Biomolecular Sciences,
Nottingham NG7 2RD, United Kingdom"
COMMENT SGRef: number: 4; type: "Journal Article"
COMMENT SGRef: number: 5; type: "Journal Article"
COMMENT SGRef: number: 6; type: "Journal Article"
COMMENT SGRef: number: 7; type: "Journal Article"
FEATURES Location/Qualifiers
source 1..9754
/mol_type="other DNA"
/organism="synthetic DNA construct"
CDS 194..880
/codon_start=1
/product="pVS1 resolvase"
/label=pVS1 resolvase
/note="ORF1; not required for stable maintenance"
/protein_id="ABG33931.1"
/translation="MNKSAAAGLLGYARVSTDDQDLTNQRAELHAAGCTKLFSEKITGT
RRDRPELARMLDHLRPGDVVTVTRLDRLARSTRDLLDIAERIQEAGAGLRSLAEPWADT
TTPAGRMVLTVFAGIAEFERSLIIDRTRSGREAAKARGVKFGPRPTLTPAQIAHARELI
DQEGRTVKEAAALLGVHRSTLYRALERSEEVTPTEARRRGAFREDALTEADALAAAENE
RQEEQA"
CDS 877..1092
/codon_start=1
/product="hypothetical protein"
/label=hypothetical protein
/note="ORF2; not required for stable maintenance"
/protein_id="ABG33932.1"
/translation="MKPHQDGQDEPFFITEEIEAEMIAAGYVFEPPAHVSTVRLHEILA
GLSDAKLAAWPASLAAEETERRRLKR"
CDS 1179..1805
/label=pVS1 StaA
/note="stability protein from the Pseudomonas plasmid pVS1
(Heeb et al., 2000)"
CDS 1829..2044
/codon_start=1
/product="ParG"
/label=ParG
/note="pVS1 partitioning protein"
/protein_id="ABG33934.1"
/translation="MSKSTNTLSAGRPSARSSKAATLASLADTPAMKRVNFQLPAEDHT
KLKMYAVRQGKTITELLSEYIAQLPE"
CDS 2237..3307
/label=pVS1 RepA
/note="replication protein from the Pseudomonas plasmid
pVS1 (Heeb et al., 2000)"
rep_origin 3376..3570
/label=pVS1 oriV
/note="origin of replication for the Pseudomonas plasmid
pVS1 (Heeb et al., 2000)"
rep_origin 4279..4824
/label=p15A ori
/note="Plasmids containing the medium-copy-number p15A
origin of replication can be propagated in E. coli cells
that contain a second plasmid with the ColE1 origin."
oriT 5066..5087
/label=p15A origin of transfer
/note="p15A origin of transfer"
regulatory 5145..5411
/label=T4 transcription terminator
/note="T4 transcription terminator"
/regulatory_class="terminator"
misc_feature 5399..5418
/note="sequencing oligonucleotide P6032:
5'-CCCTCACTGATCCGCTAGTC-3'"
misc_feature 5413..5470
/label=multiple cloning site
/note="multiple cloning site"
regulatory complement(5475..5478)
/regulatory_class="ribosome_binding_site"
protein_bind complement(5487..5503)
/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)."
misc_feature complement(5506)
/label=start of transcription
/note="start of transcription"
promoter complement(5511..5539)
/label=tac promoter
/note="strong E. coli promoter; hybrid between the trp and
lac UV5 promoters"
promoter 5773..5850
/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 5974..6790
/codon_start=1
/product="LacIQ repressor"
/label=LacIQ repressor
/note="tac promoter repressor"
/protein_id="ABG33936.1"
/translation="MAELNYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSR
ADQLGASVVVSMVERSGVEACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPA
LFLDVSDQTPINSIIFSHEDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKY
LTRNQIQPIAEREGDWSAMSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLR
VGADISVVGYDDTEDSSCYIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAV"
CDS complement(7760..8407)
/label=TetR
/note="tetracycline resistance regulatory protein"
CDS 8513..9709
/label=TcR
/note="tetracycline efflux protein"