Price Information
| Cat No. | Plasmid Name | Availability | Add to cart |
|---|---|---|---|
| V004456 | pMSP3535 | 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
- Vector Name:
- pMSP3535
- Length:
- 8353 bp
- Type:
- Cloning vector
- Replication origin:
- ori
- Source/Author:
- Bryan EM, Bae T, Kleerebezem M, Dunny GM.
pMSP3535 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.
pMSP3535 vector Sequence
LOCUS V004456 8353 bp DNA circular SYN 18-DEC-2018
DEFINITION Exported.
ACCESSION V004456
VERSION V004456
KEYWORDS .
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
.
REFERENCE 1 (bases 1 to 8353)
AUTHORS Bryan EM, Bae T, Kleerebezem M, Dunny GM.
TITLE Improved vectors for nisin-controlled expression in gram-positive
bacteria
JOURNAL Plasmid 44 (2), 183-190 (2000)
PUBMED 10964628
REFERENCE 2 (bases 1 to 8353)
AUTHORS Staddon JH, Bryan EM, Manias DA, Dunny GM.
TITLE Conserved target for group II intron insertion in relaxase genes of
conjugative elements of gram-positive bacteria
JOURNAL J. Bacteriol. 186 (8), 2393-2401 (2004)
PUBMED 15060042
REFERENCE 3 (bases 1 to 8353)
AUTHORS Staddon JH, Bryan EM, Manias DA, Bae T, Kleerebezem M, Dunny GM.
TITLE Direct Submission
JOURNAL Submitted (21-MAY-2003) Microbiology, University of Minnesota, MMC
196, 420 Delaware Street S.E., Minneapolis, MN 55455, USA
REFERENCE 4 (bases 1 to 8353)
TITLE Direct Submission
REFERENCE 5 (bases 1 to 8353)
AUTHORS .
TITLE Direct Submission
COMMENT SGRef: number: 1; type: "Journal Article"; journalName: "Plasmid";
date: "2000"; volume: "44"; issue: "2"; pages: "183-190"
SGRef: number: 2; type: "Journal Article"; journalName: "J.
Bacteriol."; date: "2004"; volume: "186"; issue: "8"; pages:
"2393-2401"
SGRef: number: 3; type: "Journal Article"; journalName: "Submitted
(21-MAY-2003) Microbiology, University of Minnesota, MMC 196, 420
Delaware Street S.E., Minneapolis, MN 55455, USA"
SGRef: number: 4; type: "Journal Article"
FEATURES Location/Qualifiers
source 1..8353
/mol_type="other DNA"
/organism="synthetic DNA construct"
CDS 332..1066
/gene="ermBP"
/label="rRNA adenine N-6-methyltransferase"
/note="rRNA adenine N-6-methyltransferase from Enterococcus
faecalis. Accession#: P0A4D5"
CDS 2046..2345
/codon_start=1
/product="RepD"
/label="RepD"
/protein_id="AAP60030.1"
/translation="MNHDECKTYIKNSLLEIRKLANIYTLETFKKELEKRNIYLETKSD
KYFSSEGEDYIYKLIENNKIIYSISGKKLTYKGKKSFSKHAILKQLNEKANQVN"
CDS 2388..3878
/codon_start=1
/product="RepE"
/label="RepE"
/note="plasmid replication; pAMbeta1 plasmid replicon"
/protein_id="AAP60031.1"
/translation="MNIPFVVETVLHDGLLKYKFKNSKIRSITTKPGKSKGAIFAYRSK
SSMIGGRGVVLTSEEAIQENQDTFTHWTPNVYRYGTYADENRSYTKGHSENNLRQINTF
FIDFDIHTAKETISASDILTTAIDLGFMPTMIIKSDKGYQAYFVLETPVYVTSKSEFKS
VKAAKIISQNIREYFGKSLPVDLTCNHFGIARIPRTDNVEFFDPNYRYSFKEWQDWSFK
QTDNKGFTRSSLTVLSGTEGKKQVDEPWFNLLLHETKFSGEKGLIGRNNVMFTLSLAYF
SSGYSIETCEYNMFEFNNRLDQPLEEKEVIKIVRSAYSENYQGANREYITILCKAWVSS
DLTSKDLFVRQGWFKFKKKRSERQRVHLSEWKEDLMAYISEKSDVYKPYLVTTKKEIRE
VLGIPERTLDKLLKVLKANQEIFFKIKPGRNGGIQLASVKSLLLSIIKVKKEEKESYIK
ALTNSFDLEHTFIQETLNKLAERPKTDTQLDLFSYDTG"
CDS 4228..4365
/codon_start=1
/product="RepG"
/label="RepG"
/protein_id="AAP60032.1"
/translation="MEKYLKNPITWIGLVLVVTWFLTKSSEFLIFGVCVLLLVFASQSD
"
CDS 4614..5252
/codon_start=1
/product="NisR"
/label="NisR"
/note="response regulator"
/protein_id="AAP60033.1"
/translation="MKTALEMRNYEVATHQNISLPLDITDFQGFDLILLDIMMSNIEGT
EICKRIRREISTPIIFVSAKDTEEDIINGLGIGGDDYITKPFSLKQLVAKVEANIKREE
RNKHAVHVFSEIRRDLGPITFYLEERRVCVNGQTIPLTCREYDILELLSQRTSKVYTRE
DIYDDVYDEYSNALFRSISEYIYQIRSKFAPYDINPIKTVRGLGYQWHG"
CDS 5245..6585
/gene="nisK"
/label="Nisin biosynthesis sensor protein NisK"
/note="Nisin biosynthesis sensor protein NisK from
Lactococcus lactis subsp. lactis. Accession#: P42707"
rep_origin 7090..7678
/direction=RIGHT
/label="ori"
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
promoter 7936..7954
/label="T7 promoter"
/note="promoter for bacteriophage T7 RNA polymerase"
regulatory 8045..8353
/note="PnisA; nisin inducible promoter"
/regulatory_class="promoter"