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PurposeUsed in phosphoprotein synthesis. Expresses the Sep-accepting tRNA (tRNASep), the Sep-tRNA synthetase (SepRS) and an engineered EF-Tu (EFSep)
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Depositing Lab
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Sequence Information
Ordering
Item | Catalog # | Description | Quantity | Price (USD) | |
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Plasmid | 52054 | Standard format: Plasmid sent in bacteria as agar stab | 1 | $85 |
Backbone
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Vector backbonepKK223-3
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Backbone manufacturerPharmacia
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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.
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Vector typeBacterial Expression
Growth in Bacteria
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Bacterial Resistance(s)Kanamycin, 50 μg/mL
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Growth Temperature30°C
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Growth Strain(s)Top10
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Growth instructionsStrain grows slowly.
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Copy numberUnknown
Gene/Insert 1
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Gene/Insert nameSepRS
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Alt namesepS
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SpeciesMethanocaldococcuc maripaludis
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Insert Size (bp)1613
- 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 #45 M13pUC-Reverse (Common Sequencing Primers)
Gene/Insert 2
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Gene/Insert nameEF-Sep
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Alt nametufB
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Alt nameEF-Tu
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SpeciesE. coli
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Insert Size (bp)1184
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MutationHis67 to Arg , Glu216 to Asn, Asp217 to Gly, Phe219 to Tyr, Thr229 to Ser, and Asn274 to Trp
- Promoter Ptac
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 #61 pBAD-R (Common Sequencing Primers)
Gene/Insert 3
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Gene/Insert name5x tRNA Sep
Cloning Information for Gene/Insert 3
- Cloning method Unknown
- 5′ sequencing primer #207 KanR2-F
- 3′ sequencing primer #35 LacI-R (Common Sequencing Primers)
Resource Information
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Supplemental Documents
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Articles Citing this Plasmid
Terms and Licenses
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Academic/Nonprofit Terms
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Industry Terms
- Not Available to Industry
Trademarks:
- Zeocin® is an InvivoGen trademark.
Depositor Comments
This plasmid is the same as pKD-SepRS-EFSep, Addgene plasmid #34623, with the addition of the 5xtRNA-Sep insert.
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.
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For your Materials & Methods section:
pKD‐SepRS-EFSep-5x tRNASep (B40 OTS) was a gift from Jesse Rinehart (Addgene plasmid # 52054 ; http://n2t.net/addgene:52054 ; RRID:Addgene_52054)