pCDF-BIRA (2 weeks) vector (Cat. No.: V008639)

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Basic Information

Note: The pCDF-BIRA is a prokaryotic expression plasmid derived from the pCDF vector backbone (containing the CloDF13 replicon and streptomycin/spectinomycin resistance). It is designed for the inducible expression of BirA biotin protein ligase in Escherichia coli. BirA catalyzes the site-specific biotinylation of target proteins fused with an Avi-tag, and is widely used for protein labeling, purification, and interaction studies [1,2].

Name:: pCDF-BIRA (2 weeks)

Antibiotic Resistance:: Streptomycin

Length:: 4547 bp

Type:: Co-expression vector

Replication origin:: CloDF13 ori

Source/Author:: Keates T, Cooper CD, Savitsky P, Allerston CK, Phillips C, Hammarstrom M, Daga N, Berridge G, Mahajan P, Burgess-Brown NA, Muller S, Graslund S, Gileadi O.

Growth Temperature:: 37℃

$ 248.2

In stock, 1 week for quality controls

Two tubes of lyophilized plasmid will be delivered, each tube is about 5µg.

Resources

Plasmid Protocol

1. Centrifuge at 5,000×g for 5 min.

2. Carefully open the tube and add sterile water to dissolve the DNA: add 20 μl for 5 μg plasmid, and 100 μl for 100 μg plasmid.

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.

References

[1]Tsao KL, DeBarbieri B, Michel H, Waugh DS. A versatile plasmid expression vector for the production of biotinylated proteins by site-specific, enzymatic modification in Escherichia coli. Gene. 1996;169(1):59-64 DOI: 10.1016/0378-1119(95)00762-8

[2]Li Y, Sousa R. Expression and purification of E. coli BirA biotin ligase for in vitro biotinylation. Protein Expr Purif. 2012;82(1):162-167 DOI: 10.1016/j.pep.2011.12.008

pCDF-BIRA (2 weeks) vector (Cat. No.: V008639) Sequence

LOCUS       Exported                4547 bp DNA     circular SYN 20-MAY-2026
DEFINITION  Co-expression vector pCDF-BIRA, complete sequence.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4547)
  AUTHORS   Keates T, Cooper CD, Savitsky P, Allerston CK, Phillips C, 
            Hammarstrom M, Daga N, Berridge G, Mahajan P, Burgess-Brown NA, 
            Muller S, Graslund S, Gileadi O.
  TITLE     Expressing the human proteome for affinity proteomics: optimising 
            expression of soluble protein domains and in vivo biotinylation
  JOURNAL   N Biotechnol 29 (5), 515-525 (2012)
  PUBMED    22027370
REFERENCE   2  (bases 1 to 4547)
  AUTHORS   Savitsky P, Gileadi O.
  TITLE     Direct Submission
  JOURNAL   Submitted (04-MAY-2011) Structural Genomics Consortium, University 
            of Oxford, Old Road Campus Research Building, Roosevelt Drive, 
            Oxford, Oxfordshire OX3 7DQ, UK
REFERENCE   3  (bases 1 to 4547)
  TITLE     Direct Submission
REFERENCE   4  (bases 1 to 4547)
  TITLE     Direct Submission
REFERENCE   5  (bases 1 to 4547)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "N
            Biotechnol"; date: "2012"; volume: "29"; issue: "5"; pages: 
            "515-525"
COMMENT     SGRef: number: 2; type: "Journal Article"; journalName: "Submitted
            (04-MAY-2011) Structural Genomics Consortium, University of Oxford,
            Old Road Campus Research Building, Roosevelt Drive, Oxford,
            Oxfordshire OX3 7DQ, UK"
COMMENT     SGRef: number: 3; type: "Journal Article"
COMMENT     SGRef: number: 4; type: "Journal Article"
FEATURES             Location/Qualifiers
     source          1..4547
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     source          3082..3146
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        84..102
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    103..127
                     /label=lac operator
                     /note="The lac repressor binds to the lac operator to
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     RBS             142..164
                     /label=RBS
                     /note="efficient ribosome binding site from bacteriophage
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             174..1136
                     /codon_start=1
                     /label=BirA
                     /note="E. coli biotin protein ligase"
                     /translation="VKDNTVPLKLIALLANGEFHSGEQLGETLGMSRAAINKHIQTLRD
                     WGVDVFTVPGKGYSLPEPIQLLNAKQILGQLDGGSVAVLPVIDSTNQYLLDRIGELKSG
                     DACIAEYQQAGRGRRGRKWFSPFGANLYLSMFWRLEQGPAAAIGLSLVIGIVMAEVLRK
                     LGADKVRVKWPNDLYLQDRKLAGILVELTGKTGDAAQIVIGAGINMAMRRVEESVVNQG
                     WITLQEAGINLDRNTLAAMLIRELRAALELFEQEGLAPYLSRWEKLDNFINRPVKLIIG
                     DKEIFGISRGIDKQGALLLEQDGIIKPWMGGEISLRSAEK"
     CDS             1167..1211
                     /codon_start=1
                     /label=S-Tag
                     /note="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /translation="KETAAAKFERQHMDS"
     terminator      1263..1310
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     CDS             complement(1484..2272)
                     /codon_start=1
                     /label=SmR
                     /note="aminoglycoside adenylyltransferase (Murphy, 1985)"
                     /translation="MREAVIAEVSTQLSEVVGVIERHLEPTLLAVHLYGSAVDGGLKPH
                     SDIDLLVTVTVRLDETTRRALINDLLETSASPGESEILRAVEVTIVVHDDIIPWRYPAK
                     RELQFGEWQRNDILAGIFEPATIDIDLAILLTKAREHSVALVGPAAEELFDPVPEQDLF
                     EALNETLTLWNSPPDWAGDERNVVLTLSRIWYSAVTGKIAPKDVAADWAMERLPAQYQP
                     VILEARQAYLGQEEDRLASRADQLEEFVHYVKGEITKVVGK"
     promoter        complement(2273..2364)
                     /label=AmpR promoter
     rep_origin      complement(2412..3215)
                     /direction=LEFT
                     /label=CloDF13 ori
                     /note="Plasmids containing the CloDF13 (CDF) origin of 
                     replication can be propagated in E. coli cells that contain
                     additional plasmids with compatible origins."
     protein_bind    complement(3391..3412)
                     /label=CAP binding site
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     CDS             complement(3428..4507)
                     /codon_start=1
                     /label=lacI
                     /note="lac repressor"
                     /translation="VKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     promoter        complement(join(4508..4547,1..38))
                     /label=lacI promoter