pUC18T-mini-Tn7T-aadA vector (V002416)

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Cat No.	Plasmid Name	Availability	Add to cart
V002416	pUC18T-mini-Tn7T-aadA	In stock, 1 week for quality controls	Buy one, get one free!

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

Basic Vector Information

Vector Name:: pUC18T-mini-Tn7T-aadA

Antibiotic Resistance:: Ampicillin

Length:: 4871 bp

Type:: Cloning vector

Replication origin:: ori

Source/Author:: Lehman SS, Mladinich K, Boonyakanog A, Mima T, Karkhoff-Schweizer RR, Schweizer HP.

pUC18T-mini-Tn7T-aadA vector Map

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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-aadA vector Sequence

LOCUS       40924_45058        4871 bp DNA     circular SYN 18-DEC-2018
DEFINITION  Cloning vector pUC18T-mini-Tn7T-aadA, complete sequence.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4871)
  AUTHORS   Lehman SS, Mladinich K, Boonyakanog A, Mima T, Karkhoff-Schweizer 
            RR, Schweizer HP.
  TITLE     Versatile nourseothricin and streptomycin/spectinomycin resistance 
            gene cassettes and their use in chromosome integration vectors
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 4871)
  AUTHORS   Lehman SS, Mladinich K, Boonyakanog A, Mima T, Karkhoff-Schweizer 
            RR, Schweizer HP.
  TITLE     Direct Submission
  JOURNAL   Submitted (17-MAR-2016) Molecular Genetics and Microbiology, 
            University of Florida, 2055 Mowry Road, Gainesville, FL 32610, USA
REFERENCE   3  (bases 1 to 4871)
  TITLE     Direct Submission
REFERENCE   4  (bases 1 to 4871)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: 
            "Unpublished"
COMMENT     SGRef: number: 2; type: "Journal Article"; journalName: "Submitted 
            (17-MAR-2016) Molecular Genetics and Microbiology, University of 
            Florida, 2055 Mowry Road, Gainesville, FL 32610, USA"
COMMENT     SGRef: number: 3; type: "Journal Article"
COMMENT     ##Assembly-Data-START##
            Sequencing Technology :: Sanger dideoxy sequencing 
            ##Assembly-Data-END##
FEATURES             Location/Qualifiers
     source          1..4871
                     /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"
     misc_feature    955..988
                     /note="loxP; Cre recombinase site"
     CDS             complement(1162..1950)
                     /label=SmR
                     /note="aminoglycoside adenylyltransferase (Murphy, 1985)"
     regulatory      2052..2101
                     /label=aadA gene promoter
                     /note="aadA gene promoter"
                     /regulatory_class="promoter"
     protein_bind    complement(2254..2287)
                     /label=loxP
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     misc_feature    2302..2376
                     /note="MCS; multiple cloning site"
     mobile_element  complement(2388..2553)
                     /label=Tn7L
                     /note="mini-Tn7 element (left end of the Tn7 transposon)"
     oriT            complement(2712..2820)
                     /direction=LEFT
                     /label=oriT
                     /note="incP origin of transfer"
     rep_origin      complement(3052..3640)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(3814..4671)
                     /label=AmpR
                     /note="beta-lactamase"
     promoter        complement(4672..4776)
                     /label=AmpR promoter