Addgene - pKD-SepRS-EFSep Plasmid Data

Plasmid 34623: pKD-SepRS-EFSep

Gene/Insert 1
Gene/insert name:
SepRS
Alt name:
sepS
Insert size:
1613
Species:
Methanocaldococcuc maripaludis
Entrez Gene:
MMP0688 (MMP0688)
Gene/Insert 2
Gene/insert name:
EF-Sep
Alt name:
tufB
Alt name:
EF-Tu
Insert size:
1184
Species:
E. coli
Entrez Gene:
tufB (B21_03809)
Mutation:
His67 to Arg , Glu216 to Asn, Asp217 to Gly, Phe219 to Tyr, Thr229 to Ser, and Asn274 to Trp
Vector backbone:
pKK223-3
(Search Vector Database)
Backbone manufacturer:
Pharmacia
Vector type:
Bacterial Expression
Backbone size w/o insert (bp):
4300
Modifications to Backbone:
pKD 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.
Cloning information for Gene/Insert 1
Promoter:
Ptac
Cloning site 5':
NcoI
Site destroyed during cloning:
No
Cloning site 3':
SacI
Site destroyed during cloning:
No
5' sequencing primer:
M13_pUC-Rev List of Sequencing Primers
Cloning information for Gene/Insert 2
Promoter:
Ptac (from SepRS- operon like)
Cloning site 5':
BamHI
Site destroyed during cloning:
No
Cloning site 3':
SalI
Site destroyed during cloning:
No
5' sequencing primer:
pJR_F2 List of Sequencing Primers
3' sequencing primer:
pBAD_rev
Bacterial resistance(s)
Kanamycin
Growth strain(s)
DH5alpha
Growth temperature (℃):
30
High or low copy:
High Copy
Sequence: View sequences (4)
Map:
View map
Supplemental document:
pKD_SepRS_EFSep (chemical/seq-na-genbank)
Supplemental document:
Notes from Addgene (1)
Principal Investigator:
Jesse Rinehart
Principal Investigator:
Dieter Soll
Terms and Licenses:
MTA

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.

Addgene has sequenced a portion of this plasmid for verification. Click here for the sequencing result.

Article: Expanding the genetic code of Escherichia coli with phosphoserine. Park et al (Science. 2011 Aug 26;333(6046):1151-4. PubMed)

Please acknowledge the principal investigator and cite this article if you use this plasmid in a publication. Also, please include the text "Addgene plasmid 34623" in your Materials and Methods section.