pMSP3535 vector (V004456)

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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 Map

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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"