Price Information
| Cat No. | Plasmid Name | Availability | Buy one, get one free! (?) |
|---|---|---|---|
| V004413 | pMTL84422 | 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:
- pMTL84422
- Length:
- 7371 bp
- Type:
- Shuttle vector
- Replication origin:
- p15A ori
- Source/Author:
- Heap JT, Pennington OJ, Cartman ST, Minton NP.
pMTL84422 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.
pMTL84422 vector Sequence
LOCUS 40924_32668 7371 bp DNA circular SYN 18-DEC-2018
DEFINITION Shuttle vector pMTL84422, complete sequence.
ACCESSION .
VERSION .
KEYWORDS .
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 7371)
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 7371)
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 7371)
TITLE Direct Submission
REFERENCE 4 (bases 1 to 7371)
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..7371
/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..238
/note="promoter derived from thl gene of Clostridium
acetobutylicum"
/regulatory_class="promoter"
primer_bind complement(343..359)
/label=M13 fwd
/note="common sequencing primer, one of multiple similar
variants"
regulatory 507..548
/note="terminator derived from fdx gene of Clostridium
pasteurianum"
/regulatory_class="terminator"
CDS 713..1360
/codon_start=1
/product="putative plasmid replication protein"
/label=putative plasmid replication protein
/note="orfB"
/protein_id="ACR43910.1"
/translation="MNKKLEKPFVYKREYDLTGYDVEILQKYELEQAIYVYVGSSCAYN
MRARSSKWRYHIRTNNKSICCNIKNFIHNLELFYKMELKLSDNIINDKLYYSNIAEFEE
FETLEKAREVESTIISQYQFLDSINHMLKQKIILLSNKDSVLNITKNGNTNYLKVKNKY
IEKHKNKPIMRYHINCQFNTDGSVKSITQEFEPILELNKKNTLSRPSRVFLK"
CDS 1635..3272
/codon_start=1
/gene="repA"
/product="plasmid replication protein"
/label=repA
/protein_id="ACR43908.1"
/translation="MEQLDSKYKLKKFLMAVFRDGIGQGNNLIDNEYVRVFQNNKSNSK
QLELGEEFKEYSKTTFFKNIDDIVEFTFAKNIYYENTFFNLCTTDGKAGTNENLINRYA
LGFDFDKKELGQGFNYKDIINLFTKIGLHYHILVDSGNGFHVYVLINKTNNIKLVSEVT
NTLINKLGADKQANLSTQVLRVPYTYNIKNTTKQVKIIHQDKNIYRYDIEKLAKKYCKD
VKTVGNTNTKYILDSKLPNCIVDILKNGSKDGHKNLDLQKIVVTLRLRNKSLSQVISVA
REWNYISQNSLSNSELEYQVKYMYEKLKTVNFGCTGCEFNSDCWNKIESDFIYSDEDTL
FNMPHKHSKDLKYKNRKGVKIMTGNQLFIYNVLLNNKDRELNIDDIMELITYKRKKKVK
NIVMSEKTLRETLKELQHNDYITKTKGVTKLGIKDTYNVKEVRCNIDKQYTISYFVTMA
VIWGIISTEELRLYTHMRYKQDLLVKDDKIKGNILRINQEELAKDLGVTQQRISNMIES
LLDTKILDVWETKINDRGFMYYTYRLNK"
gene 1635..3272
/gene="repA"
/label=repA
rep_origin complement(3478..3717)
/direction=LEFT
/label=oriV
/note="oriV"
CDS 4620..5882
/codon_start=1
/gene="tetA"
/product="tetracycline resistance protein"
/label=tetA
/protein_id="ACR43909.1"
/translation="MVNKLSAYKTYLLFSAITAMCFSLVATVMMVYHIEIVHLNPLQLI
LVGTTLELACFIFEIPTAIVADVYSRKLSIVIGGVLTGVGFILEGSISSFVFVLVAQIV
WGLGSTFISGSLEAWIAEEEKNKDLDEIYIKGAQAGQIGAFIGIVLSTVIANFSVRLPI
IVSGVLFIILALFLWLYMPENNFKPSAPGDLNTFKKMVYTFKSGLKFVKSKSIIMILLA
VTLFYGLSSEGYDRLSNAHFLQDTTLPKLGNLSSVTWFGIFGILGMILSFIVMHFMAKN
LKNEDNRKNGKLLLCINILYISSMLIFALTRNFSLMLIAYLATNTFRIINKPIFSAWLN
GHIDDNSRATVLSINGQMNSLGQILGGPIIGIIATNISVSIGIVCTSLLVTPVLVLYIV
AMIIDKKVDDRVGGIDYEENN"
gene 4620..5882
/gene="tetA"
/label=tetA
rep_origin 5931..6476
/direction=RIGHT
/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 6724..6833
/label=oriT
/note="incP origin of transfer"
CDS 6866..7234
/label=traJ
/note="oriT-recognizing protein"