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pCMT-flp vector (V008415)

Price Information

Cat No. Plasmid Name Availability Buy one, get one free! (?)
V008415 pCMT-flp In stock, 1 week for quality controls

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

Basic Vector Information

pSC101 ori is a low-copy replication origin that requires the Rep101 protein, which is a temperature-sensitive protein. When bacteria containing a plasmid with this ori is cultured at 37℃, the plasmid will be lost.

Vector Name:
pCMT-flp
Antibiotic Resistance:
Tetracycline
Length:
5813 bp
Type:
Conjugally transferable flp vector
Replication origin:
pSC101 ori
Source/Author:
Hossain MJ, Thurlow CM, Sun D, Nasrin S, Liles MR.
Copy Number:
Low Copy
Promoter:
tet
Growth Temperature:
30℃

pCMT-flp vector Map

Copy Sequence View Map
pCMT-flp5813 bp60012001800240030003600420048005400tonB terminatororiT; origin of transfer sequencepSC101 oriRep101(Ts)T3Te terminatorT7 terminatorFLPSUMO6xHisRBStet promoterTcR

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.

pCMT-flp vector Sequence

LOCUS       40924_11466        5813 bp DNA     circular SYN 17-DEC-2018
DEFINITION  Conjugally transferable flp vector pCMT-flp, complete sequence.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5813)
  AUTHORS   Hossain MJ, Thurlow CM, Sun D, Nasrin S, Liles MR.
  TITLE     Conjugal Transfer of a Recombineering System to Generate and 
            Complement Markerless Mutants
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 5813)
  AUTHORS   Hossain MJ, Thurlow CM, Sun D, Nasrin S, Liles MR.
  TITLE     Direct Submission
  JOURNAL   Submitted (10-JUN-2015) Biological Sciences, Auburn University, 120 
            W Samford Avenue, Auburn, AL 36849, USA
REFERENCE   3  (bases 1 to 5813)
  TITLE     Direct Submission
REFERENCE   4  (bases 1 to 5813)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: 
            "Unpublished"
COMMENT     SGRef: number: 2; type: "Journal Article"; journalName: "Submitted 
            (10-JUN-2015) Biological Sciences, Auburn University, 120 W Samford 
            Avenue, Auburn, AL 36849, USA"
COMMENT     SGRef: number: 3; type: "Journal Article"
COMMENT     ##Assembly-Data-START##
            Assembly Method       :: CLC Genomics Workbench v. 8.1
            Sequencing Technology :: Illumina; Sanger dideoxy sequencing 
            ##Assembly-Data-END##
FEATURES             Location/Qualifiers
     source          1..5813
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     terminator      48..79
                     /label=tonB terminator
                     /note="bidirectional E. coli tonB-P14 transcription
                     terminator"
     misc_feature    90..144
                     /note="oriT; origin of transfer sequence"
     rep_origin      641..863
                     /label=pSC101 ori
                     /note="low-copy replication origin that requires the Rep101
                     protein"
     CDS             911..1858
                     /label=Rep101(Ts)
                     /note="temperature-sensitive version of the RepA protein
                     needed for replication with the pSC101 origin (Armstrong et
                     al., 1984)"
     terminator      complement(2457..2486)
                     /label=T3Te terminator
                     /note="phage T3 early transcription terminator"
     terminator      complement(2584..2631)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     CDS             complement(2663..3928)
                     /label=FLP
                     /note="site-specific recombinase"
     CDS             complement(3929..4216)
                     /label=SUMO
                     /note="cleavable ubiquitin-like protein tag"
     CDS             complement(4229..4246)
                     /label=6xHis
                     /note="6xHis affinity tag"
     RBS             complement(4257..4279)
                     /label=RBS
                     /note="efficient ribosome binding site from bacteriophage
                     T7 gene 10 (Olins and Rangwala, 1989)"
     promoter        4340..4368
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein
                     gene"
     CDS             4416..5603
                     /label=TcR
                     /note="tetracycline efflux protein"