pRK415 vector (V003726)

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Price Information

Cat No.	Plasmid Name	Availability	Add to cart
V003726	pRK415	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

Vector Name:: pRK415

Antibiotic Resistance:: Tetracycline

Length:: 10690 bp

Type:: Broad host range expression vector

Replication origin:: oriV

Source/Author:: Hulter N, Wackernagel W.

Promoter:: lac

Growth Strain(s):: DH10B

Growth Temperature:: 37℃

pRK415 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.

pRK415 vector Sequence

LOCUS       40924_37123       10690 bp DNA     circular SYN 18-DEC-2018
DEFINITION  Broad host range expression vector pRK415, complete sequence.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 10690)
  AUTHORS   Hulter N, Wackernagel W.
  TITLE     Double illegitimate recombination events integrate DNA segments 
            through two different mechanisms during natural transformation of 
            Acinetobacter baylyi
  JOURNAL   Mol. Microbiol. 67 (5), 984-995 (2008)
  PUBMED    18194157
REFERENCE   2  (bases 1 to 10690)
  AUTHORS   Huelter N, Wackernagel W.
  TITLE     Direct Submission
  JOURNAL   Submitted (13-FEB-2007) Genetics, Department of Biology and 
            Environmental Sciences, Carl von Ossietzky University Oldenburg, 
            Carl-von-Ossietzkystrasse 9-11, Oldenburg 26111, Germany
REFERENCE   3  (bases 1 to 10690)
  TITLE     Direct Submission
REFERENCE   4  (bases 1 to 10690)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Mol. 
            Microbiol."; date: "2008"; volume: "67"; issue: "5"; pages: 
            "984-995"
COMMENT     SGRef: number: 2; type: "Journal Article"; journalName: "Submitted 
            (13-FEB-2007) Genetics, Department of Biology and Environmental 
            Sciences, Carl von Ossietzky University Oldenburg, 
            Carl-von-Ossietzkystrasse 9-11, Oldenburg 26111, Germany"
COMMENT     SGRef: number: 3; type: "Journal Article"
FEATURES             Location/Qualifiers
     source          1..10690
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      complement(71..602)
                     /direction=LEFT
                     /label=oriV
                     /note="incP origin of replication"
     misc_feature    1239..1295
                     /label=MCS
                     /note="pUC18/19 multiple cloning site"
     promoter        complement(1356..1386)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    complement(1401..1422)
                     /label=CAP binding site
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     CDS             complement(1657..2025)
                     /codon_start=1
                     /label=traJ
                     /note="oriT-recognizing protein"
                     /translation="MADETKPTRKGSPPIKVYCLPDERRAIEEKAAAAGMSLSAYLLAV
                     GQGYKITGVVDYEHVRELARINGDLGRLGGLLKLWLTDDPRTARFGDATILALLAKIEE
                     KQDELGKVMMGVVRPRAEP"
     oriT            complement(2058..2167)
                     /direction=LEFT
                     /label=oriT
                     /note="incP origin of transfer"
     CDS             complement(2851..3498)
                     /codon_start=1
                     /label=TetR
                     /note="tetracycline resistance regulatory protein"
                     /translation="MTKLQPNTVIRAALDLLNEVGVDGLTTRKLAERLGVQQPALYWHF
                     RNKRALLDALAEAMLAENHTHSVPRADDDWRSFLIGNARSFRQALLAYRDGARIHAGTR
                     PGAPQMETADAQLRFLCEAGFSAGDAVNALMTISYFTVGAVLEEQAGDSDAGERGGTVE
                     QAPLSPLLRAAIDAFDEAGPDAAFEQGLAVIVDGLAKRRLVVRNVEGPRKGDD"
     CDS             3604..4800
                     /codon_start=1
                     /label=TcR
                     /note="tetracycline efflux protein"
                     /translation="MKPNIPLIVILSTVALDAVGIGLIMPVLPGLLRDLVHSNDVTAHY
                     GILLALYALVQFACAPVLGALSDRFGRRPILLVSLAGATVDYAIMATAPFLWVLYIGRI
                     VAGITGATGAVAGAYIADITDGDERARHFGFMSACFGFGMVAGPVLGGLMGGFSPHAPF
                     FAAAALNGLNFLTGCFLLPESHKGERRPLRREALNPLASFRWARGMTVVAALMAVFFIM
                     QLVGQVPAALWVIFGEDRFHWDATTIGISLAAFGILHSLAQAMITGPVAARLGERRALM
                     LGMIADGTGYILLAFATRGWMAFPIMVLLASGGIGMPALQAMLSRQVDEERQGQLQGSL
                     AALTSLTSIVGPLLFTAIYAASITTWNGWAWIAGAALYLLCLPALRRGLWSGAGQRADR
                     "
     CDS             complement(6077..7222)
                     /codon_start=1
                     /label=trfA
                     /note="trans-acting replication protein that binds to and 
                     activates oriV"
                     /translation="MNRTFDRKAYRQELIDAGFSAEDAETIASRTVMRAPRETFQSVGS
                     MVQQATAKIERDSVQLAPPALPAPSAAVERSRRLEQEAAGLAKSMTIDTRGTMTTKKRK
                     TAGEDLAKQVSEAKQAALLKHTKQQIKEMQLSLFDIAPWPDTMRAMPNDTARSALFTTR
                     NKKIPREALQNKVIFHVNKDVKITYTGVELRADDDELVWQQVLEYAKRTPIGEPITFTF
                     YELCQDLGWSINGRYYTKAEECLSRLQATAMGFTSDRVGHLESVSLLHRFRVLDRGKKT
                     SRCQVLIDEEIVVLFAGDHYTKFIWEKYRKLSPTARRMFDYFSSHREPYPLKLETFRLM
                     CGSDSTRVKKWREQVGEACEELRGSGLVEHAWVNDDLVHCKR"
     rep_origin      complement(join(10071..10690,1..2))
                     /direction=LEFT
                     /label=oriV
                     /note="incP origin of replication"