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pUC18-mini-Tn7T-Gm-lacZ vector (V002427)

Price Information

Cat No. Plasmid Name Availability Add to cart
V002427 pUC18-mini-Tn7T-Gm-lacZ In stock, 1 week for quality controls

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Two tubes of lyophilized plasmid will be delivered, each tube is about 5µg.

Basic Vector Information

Vector Name:
pUC18-mini-Tn7T-Gm-lacZ
Antibiotic Resistance:
Ampicillin
Length:
7656 bp
Type:
Cloning vector
Replication origin:
ori
Source/Author:
Choi KH, Gaynor JB, White KG, Lopez C, Bosio CM, Karkhoff-Schweizer RR, Schweizer HP.
Promoter:
Pc

pUC18-mini-Tn7T-Gm-lacZ vector Map

Copy Sequence View Map
pUC18-mini-Tn7T-Gm-lacZ7656 bp3006009001200150018002100240027003000330036003900420045004800510054005700600063006600690072007500n7RKS primerlambda t0 terminatorrrnB T1 terminatorFRTGmRPc promoterFRT; Flp recombinase target siteMCS; multiple cloning siteRNase III processing sitelacZTn7LoriAmpRAmpR promoter

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.

pUC18-mini-Tn7T-Gm-lacZ vector Sequence

LOCUS       40924_44983        7656 bp DNA     circular SYN 18-DEC-2018
DEFINITION  Cloning vector pUC18-mini-Tn7T-Gm-lacZ, complete sequence.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7656)
  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 2 (6), 443-448 (2005)
  PUBMED    15908923
REFERENCE   2  (bases 1 to 7656)
  AUTHORS   Choi KH, Gaynor J, Karkhoff-Schweizer RR, Schweizer HP.
  TITLE     Direct Submission
  JOURNAL   Submitted (14-APR-2004) Microbiology, Immunology and Pathology, 
            Colorado State University, Fort Collins, CO 80523, USA
REFERENCE   3  (bases 1 to 7656)
  TITLE     Direct Submission
REFERENCE   4  (bases 1 to 7656)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Nat. 
            Methods"; date: "2005"; volume: "2"; issue: "6"; pages: "443-448"
COMMENT     SGRef: number: 2; type: "Journal Article"; journalName: "Submitted 
            (14-APR-2004) Microbiology, Immunology and Pathology, Colorado State
            University, Fort Collins, CO 80523, USA"
COMMENT     SGRef: number: 3; type: "Journal Article"
FEATURES             Location/Qualifiers
     source          1..7656
                     /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(1138..1668)
                     /label=GmR
                     /note="gentamycin acetyltransferase"
     promoter        complement(1857..1885)
                     /label=Pc promoter
                     /note="class 1 integron promoter"
     misc_feature    1926..1973
                     /note="FRT; Flp recombinase target site"
     protein_bind    1926..1973
                     /label=FRT
                     /bound_moiety="FLP recombinase from the Saccharomyces
                     cerevisiae 2u plasmid"
                     /note="FLP-mediated recombination occurs in the 8-bp core 
                     sequence TCTAGAAA (Turan and Bode, 2011)."
     misc_feature    2000..2079
                     /note="MCS; multiple cloning site"
     misc_feature    2103..2179
                     /label=RNase III processing site
                     /note="RNase III processing site"
     CDS             2243..5314
                     /label=lacZ
                     /note="beta-galactosidase"
     mobile_element  complement(5402..5567)
                     /label=Tn7L
                     /note="mini-Tn7 element (left end of the Tn7 transposon)"
     rep_origin      complement(5837..6425)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(6599..7456)
                     /label=AmpR
                     /note="beta-lactamase"
     promoter        complement(7457..7561)
                     /label=AmpR promoter