pSC101_TIMER vector (V001033)

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Price Information

Cat No.	Plasmid Name	Availability	Add to cart
V001033	pSC101_TIMER	In stock, instant shipping	Buy one, get one free!

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:: pSC101_TIMER

Antibiotic Resistance:: Kanamycin

Length:: 4946 bp

Type:: Bacterial Expression

Replication origin:: pSC101 ori

Copy Number:: Low Copy

Promoter:: constitutive ybaJp promoter

Cloning Method:: Restriction Enzyme

pSC101_TIMER vector Map

Copy Sequence View 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.

pSC101_TIMER vector Sequence

LOCUS       40924_38838        4946 bp DNA     circular SYN 13-MAY-2021
DEFINITION  low copy plasmid for expression of a variant of the fluorescent 
            timer protein suitable for bacteria under the control of the 
            constitutive ybaJp promoter (works in E. coli and Salmonella 
            enterica).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4946)
  AUTHORS   Claudi B, Sprote P, Chirkova A, Personnic N, Zankl J, Schurmann N, 
            Schmidt A, Bumann D
  TITLE     Phenotypic variation of Salmonella in host tissues delays 
            eradication by antimicrobial chemotherapy.
  JOURNAL   Cell. 2014 Aug 14;158(4):722-733. doi: 10.1016/j.cell.2014.06.045.
  PUBMED    25126781
REFERENCE   2  (bases 1 to 4946)
  TITLE     Direct Submission
REFERENCE   3  (bases 1 to 4946)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; doi: 
            "10.1016/j.cell.2014.06"; journalName: "Cell"; date: "2014-08-14-
            14"; volume: "158"; issue: "4"; pages: "722-733"
COMMENT     SGRef: number: 2; type: "Journal Article"
FEATURES             Location/Qualifiers
     source          1..4946
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     terminator      64..158
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     rep_origin      650..872
                     /label=pSC101 ori
                     /note="low-copy replication origin that requires the Rep101
                     protein"
     CDS             920..1867
                     /codon_start=1
                     /label=Rep101
                     /note="RepA protein needed for replication with the pSC101 
                     origin"
                     /translation="MSELVVFKANELAISRYDLTEHETKLILCCVALLNPTIENPTRKE
                     RTVSFTYNQYAQMMNISRENAYGVLAKATRELMTRTVEIRNPLVKGFEIFQWTNYAKFS
                     SEKLELVFSEEILPYLFQLKKFIKYNLEHVKSFENKYSMRIYEWLLKELTQKKTHKANI
                     EISLDEFKFMLMLENNYHEFKRLNQWVLKPISKDLNTYSNMKLVVDKRGRPTDTLIFQV
                     ELDRQMDLVTELENNQIKMNGDKIPTTITSDSYLHNGLRKTLHDALTAKIQLTSFEAKF
                     LSDMQSKYDLNGSFSWLTQKQRTTLENILAKYGRI"
     terminator      complement(2408..2494)
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB
                     gene"
     CDS             3286..3960
                     /codon_start=1
                     /label=DsRed-Express
                     /note="rapidly maturing tetrameric variant of DsRed
                     fluorescent protein (Bevis and Glick, 2002)"
                     /translation="MASTEDVIKEFMRFKVRMEGSVNGHEFEIEGEGEGRPYEGTQTAK
                     LKVTKGGPLPFAWDILSPQFQYGSKVYVKHPADIPDYKKLSFPEGFKWERVMNFEDGGV
                     VTVTQDSSLQDGCFIYKVKFIGVNFPSDGPVMQKKTMGWEPSTERLYPRDGVLKGEIHK
                     ALKLKDGGHYLVEFKSIYMAKKPVQLPGYYYVDTKLDITSHNEDYTIVEQYERTEGRYH
                     LFL"
     CDS             join(4185..4946,1..30)
                     /codon_start=1
                     /label=NeoR/KanR
                     /note="aminoglycoside phosphotransferase"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"