-
Depositing Labs
-
Publication
-
Sequence Information
Ordering
Item | Catalog # | Description | Quantity | Price (USD) | |
---|---|---|---|---|---|
Plasmid | 34623 | Standard format: Plasmid sent in bacteria as agar stab | 1 | $85 |
Backbone
-
Vector backbonepKK223-3
-
Backbone manufacturerPharmacia
- Backbone size w/o insert (bp) 4300
-
Modifications to backbonepKD is derived from pKK223-3 (Pharmacia). The ampicillin (Amp) resistance gene was replaced with a kanamycin (Kan) resistance gene. The original multiple cloning site (MCS; NcoI-EcoRI-SacI) was modified by adding an additional ribosome binding site (RBS) and a second MCS (NdeI-BamHI-SalI-HindIII), thus enabling simultaneous protein expression from two genes, both under the control of the same tac promoter.
-
Vector typeBacterial Expression
Growth in Bacteria
-
Bacterial Resistance(s)Kanamycin, 50 μg/mL
-
Growth Temperature30°C
-
Growth Strain(s)DH5alpha
-
Copy numberHigh Copy
Gene/Insert 1
-
Gene/Insert nameSepRS
-
Alt namesepS
-
SpeciesMethanocaldococcuc maripaludis
-
Insert Size (bp)1613
-
Entrez GeneMMP0688 (a.k.a. MMP0688)
- Promoter Ptac
Cloning Information for Gene/Insert 1
- Cloning method Restriction Enzyme
- 5′ cloning site NcoI (not destroyed)
- 3′ cloning site SacI (not destroyed)
- 5′ sequencing primer M13_pUC-Rev (Common Sequencing Primers)
Gene/Insert 2
-
Gene/Insert nameEF-Sep
-
Alt nametufB
-
Alt nameEF-Tu
-
SpeciesE. coli
-
Insert Size (bp)1184
-
MutationHis67 to Arg , Glu216 to Asn, Asp217 to Gly, Phe219 to Tyr, Thr229 to Ser, and Asn274 to Trp
-
Entrez GenetufB (a.k.a. B21_03809)
- Promoter Ptac (from SepRS- operon like)
Cloning Information for Gene/Insert 2
- Cloning method Restriction Enzyme
- 5′ cloning site BamHI (not destroyed)
- 3′ cloning site SalI (not destroyed)
- 5′ sequencing primer pJR_F2
- 3′ sequencing primer pBAD_rev (Common Sequencing Primers)
Resource Information
-
Supplemental Documents
-
Addgene Notes
-
Article Citing this Plasmid
Terms and Licenses
-
Academic/Nonprofit Terms
-
Industry Terms
- Not Available to Industry
Trademarks:
- Zeocin® is an InvivoGen trademark.
Depositor Comments
To prevent possible enzymatic dephosphorylation of O-phospho-L-serine (Sep) in vivo, the gene encoding phosphoserine phosphatase (serB), which is catalyzing the last step in serine biosynthesis, was deleted from Escherichia coli strains Top10 (Top10∆serB - Addgene #34928) and BL21 (BL21∆serB - Addgene #34929). These strains are required hosts when using this plasmid.
Certain elements of this plasmid are covered under US patent 9,090,928 to Yale University
These plasmids were created by your colleagues. Please acknowledge the Principal Investigator, cite the article in which the plasmids were described, and include Addgene in the Materials and Methods of your future publications.
-
For your Materials & Methods section:
pKD-SepRS-EFSep was a gift from Jesse Rinehart & Dieter Söll (Addgene plasmid # 34623 ; http://n2t.net/addgene:34623 ; RRID:Addgene_34623) -
For your References section:
Expanding the genetic code of Escherichia coli with phosphoserine. Park HS, Hohn MJ, Umehara T, Guo LT, Osborne EM, Benner J, Noren CJ, Rinehart J, Soll D. Science. 2011 Aug 26;333(6046):1151-4. 10.1126/science.1207203 PubMed 21868676