Price Information
| Cat No. | Plasmid Name | Availability | Buy one, get one free! (?) |
|---|---|---|---|
| V002100 | pWW3535 | In stock, instant shipping |
Two tubes of lyophilized plasmid will be delivered, each tube is about 5µg.
Basic Vector Information
pWW3535 is a broad-host-range vector for stable foreign gene expression in Gram-negative bacteria, used in microbial gene function studies.
- Vector Name:
- pWW3535
- Antibiotic Resistance:
- Ampicillin
- Length:
- 6987 bp
- Type:
- Cloning vector
- Replication origin:
- R6K γ ori
- Source/Author:
- Whitaker WR, Shepherd ES, Sonnenburg JL.
- Growth Strain(s):
- GT115
- Growth Temperature:
- 37℃
pWW3535 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.
References
- Functional analysis of the Rhizobium leguminosarum bv. viciae PssN protein, involved in exopolysaccharide synthesis
pWW3535 vector Sequence
LOCUS 40924_46748 6987 bp DNA circular SYN 18-DEC-2018
DEFINITION Cloning vector pWW3535, complete sequence.
ACCESSION .
VERSION .
KEYWORDS .
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 6987)
AUTHORS Whitaker WR, Shepherd ES, Sonnenburg JL.
TITLE Tunable expression tools enable single-cell strain distinction in
the gut microbiome
JOURNAL Cell (2017) In press
REFERENCE 2 (bases 1 to 6987)
AUTHORS Whitaker WR, Shepherd ES, Sonnenburg JL.
TITLE Direct Submission
JOURNAL
REFERENCE 3 (bases 1 to 6987)
TITLE Direct Submission
REFERENCE 4 (bases 1 to 6987)
AUTHORS .
TITLE Direct Submission
COMMENT SGRef: number: 1; type: "Journal Article"; journalName: "Cell (2017)
In press"
COMMENT SGRef: number: 2; type: "Journal Article"; journalName: "Submitted
(15-MAR-2017) Microbiology "
COMMENT SGRef: number: 3; type: "Journal Article"
FEATURES Location/Qualifiers
source 1..6987
/mol_type="other DNA"
/organism="synthetic DNA construct"
terminator complement(44..87)
/label=rrnB T1 terminator
/note="transcription terminator T1 from the E. coli rrnB
gene"
regulatory 211..295
/regulatory_class="ribosome_binding_site"
CDS 293..1006
/label=superfolder GFP
/note="GFP variant that folds robustly even when fused to
poorly folded proteins (Pedelacq et al., 2006)"
CDS 1013..1036
/label=FLAG
/note="FLAG(R) epitope tag, followed by an enterokinase
cleavage site"
CDS 1037..1060
/label=FLAG
/note="FLAG(R) epitope tag, followed by an enterokinase
cleavage site"
CDS 1061..1084
/label=FLAG
/note="FLAG(R) epitope tag, followed by an enterokinase
cleavage site"
CDS 1088..1105
/label=6xHis
/note="6xHis affinity tag"
terminator 1128..1155
/label=rrnB T2 terminator
/note="transcription terminator T2 from the E. coli rrnB
gene"
regulatory 1175..1267
/regulatory_class="promoter"
regulatory 1272..1356
/regulatory_class="ribosome_binding_site"
CDS 1354..2061
/label=mCherry
/note="monomeric derivative of DsRed fluorescent protein
(Shaner et al., 2004)"
terminator 2084..2111
/label=rrnB T2 terminator
/note="transcription terminator T2 from the E. coli rrnB
gene"
regulatory 2086..2115
/regulatory_class="terminator"
CDS complement(2343..3626)
/codon_start=1
/product="NBU integrase"
/label=NBU integrase
/protein_id="ARI71359.1"
/translation="MNIKRNIIFALESRKKNGVPIVENVPIRMRVIFASQRIEFTTGYR
IDVAKWDADKQRVKSGCTNKLKQSAAEINTDLLKYYAEIQNIFKEFEVQEVMPTTQQLK
EAFNMRMKDTSEEQPEEAPVSFWEVFDEFVKECGNQNNWTASTYEKFAAVRNHLKEFKE
DATFNYFNEFGLNEYVNFLRDTKDMRNSTIGKQMGFLKWFLRWSFKKGHHQNIAYDTFK
PKLKTTSKKVIFLTWDELNKLKDYQIPKDKQYLERVRDVFLFCCFTSLRYSDVRNLKRS
DVKSDHIEITTVKTADSLTIELNKYSKAILDKYKDIHFENYMALPVISNQKMNDYLKEL
GELAEINEPVRETYYKGNERIDEVTPKYALLSTHAGRRTFICNALALGIPAQVVMKWTG
HSDYKAMKPYIDIADDIKANAMNKFNQL"
misc_feature 3786..3797
/label=integration site
/note="integration site"
CDS complement(4069..4926)
/label=AmpR
/note="beta-lactamase"
CDS 5414..6148
/codon_start=1
/product="ErmG"
/label=ErmG
/protein_id="ARI71362.1"
/translation="MNKVNIKDSQNFITSKYHIEKIMNCISLDEKDNIFEIGAGKGHFT
AGLVKRCNFVTAIEIDSKLCEVTRNKLLNYPNYQIVNDDILKFTFPSHNPYKIFGSIPY
NISTNIIRKIVFESSATISYLIVEYGFAKMLLDTNRSLALLLMAEVDISILAKIPRYYF
HPKPKVDSTLIVLKRKPAKMAFKERKKYETFVMKWVNKEYEKLFTKNQFNKALKHARIY
DINNISFEQFVSLFNSYKIFNG"
rep_origin complement(6218..6606)
/direction=LEFT
/label=R6K gamma ori
/note="gamma replication origin from E. coli plasmid R6K;
requires the R6K initiator protein pi for replication"
oriT 6777..6886
/label=oriT
/note="incP origin of transfer"