Price Information
| Cat No. | Plasmid Name | Availability | Buy one, get one free! (?) |
|---|---|---|---|
| V016355 | pUC18T-mini-Tn7T-Apr-sfGFP | 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:
- pUC18T-mini-Tn7T-Apr-sfGFP
- Antibiotic Resistance:
- Apramycin
- Length:
- 5670 bp
- Type:
- Cloning vector
- Replication origin:
- ori
- Source/Author:
- Ducas-Mowchun K, De Silva PM, Crisostomo L, Fernando DM
- Promoter:
- Pc
pUC18T-mini-Tn7T-Apr-sfGFP 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.
pUC18T-mini-Tn7T-Apr-sfGFP vector Sequence
LOCUS 62056_21955 5670 bp DNA circular SYN 02-APR-2019
DEFINITION Cloning vector pUC18T-mini-Tn7T-Apr-sfGFP, complete sequence.
ACCESSION MH976505
VERSION .
KEYWORDS .
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 5670)
AUTHORS Ducas-Mowchun K, De Silva PM, Crisostomo L, Fernando DM, Chao TC,
Pelka P, Schweizer HP, Kumar A.
TITLE Next Generation of Tn7-based single copy insertion elements for use
in multi- and pandrug resistant strains of Acinetobacter baumannii
JOURNAL Appl. Environ. Microbiol. (2019) In press
PUBMED 30902859
REFERENCE 2 (bases 1 to 5670)
AUTHORS Ducas-Mowchun K, De Silva PM, Kumar A.
TITLE Direct Submission
JOURNAL Submitted (24-SEP-2018) Department of Microbiology, University of
Manitoba, 45, Chancellor Circle, Winnipeg, Manitoba R3T2N2, Canada
REFERENCE 3 (bases 1 to 5670)
AUTHORS .
TITLE Direct Submission
COMMENT SGRef: number: 1; type: "Journal Article"; journalName: "Appl.
Environ. Microbiol. (2019) In press"
COMMENT SGRef: number: 2; type: "Journal Article"; journalName: "Submitted
(24-SEP-2018) Department of Microbiology, University of Manitoba,
45, Chancellor Circle, Winnipeg, Manitoba R3T2N2, Canada"
FEATURES Location/Qualifiers
source 1..5670
/mol_type="other DNA"
/organism="synthetic DNA construct"
mobile_element 382..580
/mobile_element_type="transposon:Tn7R"
/label=n7R
/note="right end of Tn7"
primer_bind 585..601
/label=KS primer
/note="common sequencing primer, one of multiple similar
variants"
terminator 629..723
/label=lambda t0 terminator
/note="transcription terminator from phage lambda"
terminator 826..912
/label=rrnB T1 terminator
/note="transcription terminator T1 from the E. coli rrnB
gene"
protein_bind 958..1005
/label=FRT
/note="FLP-mediated recombination occurs in the 8-bp core
sequence TCTAGAAA (Turan and Bode, 2011)."
CDS complement(1123..1923)
/label=ApmR
/note="aminoglycoside 3-N-acetyltransferase type IV"
promoter complement(2164..2192)
/label=Pc promoter
/note="class 1 integron promoter"
misc_feature 2233..2280
/note="FRT; Flp recombinase target site"
misc_feature 2307..2355
/note="MCS; multiple cloning site"
CDS complement(2364..3077)
/label=superfolder GFP
/note="GFP variant that folds robustly even when fused to
poorly folded proteins (Nager et al., 2011)"
protein_bind complement(3108..3124)
/label=lac operator
/note="The lac repressor binds to the lac operator to
inhibit transcription in E. coli. This inhibition can be
relieved by adding lactose or
isopropyl-beta-D-thiogalactopyranoside (IPTG)."
promoter complement(3132..3160)
/label=tac promoter
/note="strong E. coli promoter; hybrid between the trp and
lac UV5 promoters"
misc_feature 3166..3178
/note="MCS; multiple cloning site"
mobile_element complement(3187..3352)
/label=Tn7L
/note="mini-Tn7 element (left end of the Tn7 transposon)"
oriT complement(3511..3619)
/direction=LEFT
/label=oriT
/note="incP origin of transfer"
rep_origin complement(3851..4439)
/direction=LEFT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
CDS complement(4613..5470)
/label=AmpR
/note="beta-lactamase"
promoter complement(5471..5575)
/label=AmpR promoter