pATH3


Vector IG Sequence Link :
General : plasmid ds-DNA 3763 BP
Functions : (expression)(fusion)
Selection : ()
Copy Number :
Hosts : (E.coli RR1)(E.coli)
Suppliers : (ATCC)
Misc.Comments : The PvuII-HindIII fragment from the 5' end of the trp operon through nt 1999 of ECOTGP, which is in trpD CDS, was ligated to the HindIII-PvuII fragment of pBR322 containing the bla gene for Amp-resistance and origin of replication, but not the rop gene, which encodes a negative regulator of ColE1 replication. In addition, the EcoRI site in the pBR322 backbone was eliminated to make plasmid pKRS101. The BglII-HindIII fragment (nt 1392 of trpE to the end of the trpD sequence present in pKRS101) was replaced with a BamHI-EcoRI fragment and an EcoRI-HindIII fragment, both from the MCS of M13mp12 to make plasmid pATH1 (see GenBank M32985). The SmaI-SmaI fragment from the MCS of pATH1 was deleted and the remaining plasmid religated to make plasmid pATH2 (see GenBank M33624). An EcoRI linker was inserted at the remaining SmaI site of pATH2 replacing the SmaI site and changing the reading frames of the other sites in the MCS to make pATH3. The nucleotides remaining in the codon after cutting the vector with the given enzyme are: BamHI-1, ClaI-2, EcoRI-3, HindIII-1, SalI-1, XbaI-1. Restriction digests of the clone give the following sizes (kb): BamHI--3.8; EcoRI--3.8; HindIII--3.8. (ATCC staff) Nucleotide 1 is the first in the trp fragment, at the beginning of a PvuII site 261 nucleotides upstream of the transcription start site. The multiple cloning site follows codon 323 of trpE. To prevent recircularization of the vector, the depositors recommend including 30 - 50 U of calf alkaline phosphatase in the restriction enzyme digestion used to prepare the vector. Escherichia coli containing pATH vectors should be grown on medium supplemented with tryptophan to prevent expression. Storage as DNA at -20C is preferred; long term storage as cells may result in selection of non-expressing promoter mutations. Fusion proteins are produced in Escherichia coli usually in an insoluble form which facilitates purification. Hybrid proteins larger than 90 kDa are not expressed as efficiently as shorter ones. This is one of a series of vectors (ATCC 37695-37703) designed to facilitate expression of an open reading frame as a trpE fusion protein. Medium is -1 1065 plus ampicillin (50 ug/ml) & tryptophan (10 ug/ml).
Parents : (pATH2)
Siblings : ()
Descendents : (pATH10)(pATH11)

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