Price Information
| Cat No. | Plasmid Name | Availability | Buy one, get one free! (?) |
|---|---|---|---|
| V004415 | pMTL83353 | In stock, 1 week for quality controls |
Two tubes of lyophilized plasmid will be delivered, each tube is about 5µg.
Basic Vector Information
- Vector Name:
- pMTL83353
- Antibiotic Resistance:
- Streptomycin
- Length:
- 4941 bp
- Type:
- Shuttle vector
- Replication origin:
- ori
- Source/Author:
- Heap JT, Pennington OJ, Cartman ST, Minton NP.
pMTL83353 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.
pMTL83353 vector Sequence
LOCUS 40924_32658 4941 bp DNA circular SYN 18-DEC-2018
DEFINITION Shuttle vector pMTL83353, complete sequence.
ACCESSION .
VERSION .
KEYWORDS .
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 4941)
AUTHORS Heap JT, Pennington OJ, Cartman ST, Minton NP.
TITLE A modular system for Clostridium shuttle plasmids
JOURNAL J. Microbiol. Methods 78 (1), 79-85 (2009)
PUBMED 19445976
REFERENCE 2 (bases 1 to 4941)
AUTHORS Pennington OJ, Heap JT, Cartman ST, Minton NP.
TITLE Direct Submission
JOURNAL Submitted (02-MAR-2009) Centre for Biomolecular Sciences, The
University of Nottingham, University Park, Nottingham NG7 2RD,
United Kingdom
REFERENCE 3 (bases 1 to 4941)
TITLE Direct Submission
REFERENCE 4 (bases 1 to 4941)
AUTHORS .
TITLE Direct Submission
COMMENT SGRef: number: 1; type: "Journal Article"; journalName: "J.
Microbiol. Methods"; date: "2009"; volume: "78"; issue: "1"; pages:
"79-85"
COMMENT SGRef: number: 2; type: "Journal Article"; journalName: "Submitted
(02-MAR-2009) Centre for Biomolecular Sciences, The University of
Nottingham, University Park, Nottingham NG7 2RD, United Kingdom"
COMMENT SGRef: number: 3; type: "Journal Article"
FEATURES Location/Qualifiers
source 1..4941
/mol_type="other DNA"
/organism="synthetic DNA construct"
regulatory 9..62
/note="terminator derived from CD0164 gene of Clostridium
difficile"
/regulatory_class="terminator"
primer_bind 63..79
/label=M13 rev
/note="common sequencing primer, one of multiple similar
variants"
regulatory 89..288
/note="promoter derived from fdx gene of Clostridium
sporogenes"
/regulatory_class="promoter"
RBS 274..282
/label=Shine-Dalgarno sequence
/note="full consensus sequence for ribosome-binding sites
upstream of start codons in E. coli; complementary to a
region in the 3' end of the 16S rRNA (Chen et al., 1994)"
primer_bind complement(393..409)
/label=M13 fwd
/note="common sequencing primer, one of multiple similar
variants"
regulatory 557..598
/note="terminator derived from fdx gene of Clostridium
pasteurianum"
/regulatory_class="terminator"
CDS 1325..1642
/codon_start=1
/gene="repH"
/product="putative plasmid replication protein"
/label=repH
/protein_id="ACR43901.1"
/translation="MKRRAYMKTCKNCKEFIKDTEICKIHSLMIHDKTVATYCSKYNES
RCLHKGKVQCINCSKMNRYGWCAIKMRCFTEEEQKKERTCIKYYARSFKKAHVKKSKKK
K"
gene 1325..1642
/gene="repH"
/label=repH
CDS 2434..3186
/codon_start=1
/gene="aad9"
/product="spectinomycin resistance protein"
/label=aad9
/protein_id="ACR43899.1"
/translation="MNTYEQINKVKKILRKHLKNNLIGTYMFGSGVESGLKPNSDLDFL
VVVSEPLTDQSKEILIQKIRPISKKIGDKSNLRYIELTIIIQQEMVPWNHPPKQEFIYG
EWLQELYEQGYIPQKELNSDLTIMLYQAKRKNKRIYGNYDLEELLPDIPFSDVRRAIMD
SSEELIDNYQDDETNSILTLCRMILTMDTGKIIPKDIAGNAVAESSPLEHRERILLAVR
SYLGENIEWTNENVNLTINYLNNRLKKL"
gene 2434..3186
/gene="aad9"
/label=aad9
rep_origin 3385..3973
/direction=RIGHT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
oriT 4294..4403
/label=oriT
/note="incP origin of transfer"
CDS 4436..4804
/label=traJ
/note="oriT-recognizing protein"