pBJ-383 (ATCC® 77458)

Applications: YE-type (episomal) shuttle vectorYX-type (expression) shuttle vectorencodes removable tag for protein isolationexpression vectorvector permitting RNA synthesis in vitrovector permitting construction of fusion proteinsvector permitting production of single-stranded DNA  /  Depositors: C Hug

Permits and Restrictions

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Designations pBJ-383
Depositors C Hug
Biosafety Level 1
Host
Distribution host: Escherichia coli HB101 (ATCC 33694)
Vector Information
Size (kb): 7.0999999046325680
DESCRIPTION OF VECTOR:
Intact vector size: 7.100
Type of vector: phagemid
Cloning sites: BamHI SpeI EcoRI SacI
Polylinker sites: SacI BstXI SacII EagI NotI XbaI SpeI BamHI: GST-GAL1/GAL10:
EcoRI XhoI ApaI KpnI
Construction: pGEX-3X, pRS42 6
Host range: Saccharomyces cerevisiaeCandida robusta; Escherichia coli
Features (with orientation and position when available):
marker(s): URA3, ->
replicon: f1, <-
promoter for in vitro transcription: T7 (phi10), ->
MCS: KpnI...EcoRI, ->
promoter for expression: GAL10...GAL1, ->
coding sequence: GST, ->
MCS: BamHI...SacI, ->
promoter for in vitro transcription: T3, <-
replicon: pMB1, ->
marker(s): ampR, <-
replicon: 2 micron
Vector: pBJ-383 (phagemid)
Promoters: Promoter for in vitro transcription T7 (phi10)
Construction: pGEX-3X, pRS42 6
Marker(s):URA3,ampR
Construct size (kb): 7.099999904632568
Features: marker(s): URA3
marker(s): ampR
promoter for expression: GAL10...GAL1
promoter for in vitro transcription: T3
promoter for in vitro transcription: T7 (phi10)
replicon: 2 micron
replicon: f1
replicon: pMB1
MCS: BamHI...SacI
MCS: KpnI...EcoRI
coding sequence: GST
Applications
YE-type (episomal) shuttle vector
YX-type (expression) shuttle vector
encodes removable tag for protein isolation
expression vector
vector permitting RNA synthesis in vitro
vector permitting construction of fusion proteins
vector permitting production of single-stranded DNA
Comments
Restriction digests of the clone give the following sizes (kb): EcoRI--7.1; BamHI--7.1; XhoI--7.1.
The factor Xa recognition site of pGEX-3X is maintained and precedes the BamHI cloning site. The BamHI site is in the 1st reading frame (The A in ggatcc is position 1.)
The GAL10 RNA start site is disrupted, thus providing only one direction of transcription. Unless a start site for transcription is provided, little or no message should be made from inserts cloned into the GAL10 side of the promoter cassette.
Do not use this side of the promoter unless you can insure that mRNA will be properly initiated by providing an RNA start site.
Proteins that associate with the expressed GST fusion protein may co-purify after glutathione affinity chromatography.
The polylinker sites listed may not be unique in this vector.
YE-type, high copy number, galactose-inducible, GST fusion protein expression shuttle vector containing a divergent GAL1/GAL10 promoter cassette. Fusion proteins can be recovered by glutathione affinity chromatography.
Can be used to produce ssDNA. Contains promoters for in vitro RNA synthesis, priming sites useful for sequencing, and yeast REP3 and FRT sequences.
An in-frame stop codon occurs in the SpeI site, so 3' sites are not useful.
Restriction digests of the clone give the following sizes (kb): BamHI--7.2; EcoRI--7.2; SacI--7.2.
Media Medium 1227: LB Medium (ATCC medium 1065) with 50 mcg/ml ampicillin
Growth Conditions
Temperature: 37.0°C
References

Christopher Hug, personal communication

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