Cart 2

pUC18R6KT-mini-Tn7T-Km vector (V010744)

Price Information

Cat No. Plasmid Name Availability Buy one, get one free! (?)
V010744 pUC18R6KT-mini-Tn7T-Km 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:
pUC18R6KT-mini-Tn7T-Km
Antibiotic Resistance:
Ampicillin, Kanamycin
Length:
4911 bp
Type:
Bacterial Expression
Replication origin:
R6K γ ori
Cloning Method:
Restriction Enzyme

pUC18R6KT-mini-Tn7T-Km vector Map

Copy Sequence View Map
pUC18R6KT-mini-Tn7T-Km4911 bp6001200180024003000360042004800oriTTn7LNeoR/KanRFRTrrnB T1 terminatorlambda t0 terminatorKS primerAmpR promoterAmpRR6K gamma ori

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.

pUC18R6KT-mini-Tn7T-Km vector Sequence

LOCUS       40924_45043        4911 bp DNA     circular SYN 13-MAY-2021
DEFINITION  mini-Tn7 base vector with transcriptional terminator and oriT.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4911)
  AUTHORS   Choi KH, Gaynor JB, White KG, Lopez C, Bosio CM, Karkhoff-Schweizer 
            RR, Schweizer HP
  TITLE     A Tn7-based broad-range bacterial cloning and expression system.
  JOURNAL   Nat Methods. 2005 Jun;2(6):443-8.
  PUBMED    15908923
REFERENCE   2  (bases 1 to 4911)
  TITLE     Direct Submission
REFERENCE   3  (bases 1 to 4911)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Nat
            Methods."; date: "2005-06"; volume: "2(6)"; pages: "443-8"
COMMENT     SGRef: number: 2; type: "Journal Article"
FEATURES             Location/Qualifiers
     source          1..4911
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     mobile_element  369..534
                     /label=Tn7L
                     /note="mini-Tn7 element (left end of the Tn7 transposon)"
     CDS             1083..1874
                     /codon_start=1
                     /label=NeoR/KanR
                     /note="aminoglycoside phosphotransferase"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     protein_bind    complement(1985..2032)
                     /label=FRT
                     /note="FLP-mediated recombination occurs in the 8-bp core 
                     sequence TCTAGAAA (Turan and Bode, 2011)."
     terminator      complement(2080..2166)
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB
                     gene"
     terminator      complement(2269..2363)
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     primer_bind     complement(2391..2407)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     promoter        3087..3191
                     /label=AmpR promoter
     CDS             3192..4049
                     /codon_start=1
                     /label=AmpR
                     /note="beta-lactamase"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     rep_origin      complement(4461..4845)
                     /direction=LEFT
                     /label=R6K gamma ori
                     /note="gamma replication origin from E. coli plasmid R6K;
                     requires the R6K initiator protein pi for replication"
     oriT            complement(join(4868..4911,1..65))
                     /direction=LEFT
                     /label=oriT
                     /note="incP origin of transfer"