TY - JOUR
T1 - Construction of versatile low-copy-number vectors for cloning, sequencing and gene expression in Escherichia coli
AU - Rong Fu Wang, Fu Wang
AU - Kushner, Sidney R.
N1 - Funding Information:
The authorsa reg ratefutlo them emberos f our laboratory for manyh elpfuld iscussionsT. his work was supportedin part by an NIH grantt o S.R.K. (GM28760).
Copyright:
Copyright 2014 Elsevier B.V., All rights reserved.
PY - 1991/4
Y1 - 1991/4
N2 - Using the polymerase chain reaction and standard recombinant DNA techniques, a series of new multipurpose low-copy-number (Icn) vectors, pWSK29, pWKS30, pWSK129 and pWKS130, have been constructed. Plasmids pWSK29 and pWKS30 carry the ampicillin-resistance marker (ApR), 20 unique cloning sites flanked by T7 and T3 RNA polymerase promoters, the lacZα. gene and the bacteriophage fl origin of replication (ori) for production of single-stranded (ss) DNA in the presence of a helper phage. Plasmids pWSK129 and pWKS130 carry the kanamycin-resistance marker (KmR) and have 16 unique cloning sites flanked by T7 and T3 RNA polymerase promoters positioned within the lacZα gene. Plasmids pWSK129 and pWKS130 also contain the fl ori for the generation of ss DNA in the presence of a helper phage. All of the plasmids have an lcn of six to eight per cell. Each vector can be used for: (i) complementation analysis, (ii) generating unidirectional deletions with exonuclease III/S1 nuclease, (iii) DNA sequencing, (iv) high-level gene expression using T7 RNA polymerase, and (v) run-off transcription. They are very useful for analyzing genes encoding proteins which are toxic in Escherichia coli in high copy number.
AB - Using the polymerase chain reaction and standard recombinant DNA techniques, a series of new multipurpose low-copy-number (Icn) vectors, pWSK29, pWKS30, pWSK129 and pWKS130, have been constructed. Plasmids pWSK29 and pWKS30 carry the ampicillin-resistance marker (ApR), 20 unique cloning sites flanked by T7 and T3 RNA polymerase promoters, the lacZα. gene and the bacteriophage fl origin of replication (ori) for production of single-stranded (ss) DNA in the presence of a helper phage. Plasmids pWSK129 and pWKS130 carry the kanamycin-resistance marker (KmR) and have 16 unique cloning sites flanked by T7 and T3 RNA polymerase promoters positioned within the lacZα gene. Plasmids pWSK129 and pWKS130 also contain the fl ori for the generation of ss DNA in the presence of a helper phage. All of the plasmids have an lcn of six to eight per cell. Each vector can be used for: (i) complementation analysis, (ii) generating unidirectional deletions with exonuclease III/S1 nuclease, (iii) DNA sequencing, (iv) high-level gene expression using T7 RNA polymerase, and (v) run-off transcription. They are very useful for analyzing genes encoding proteins which are toxic in Escherichia coli in high copy number.
KW - ampicillin resistance
KW - deletion analysis
KW - kanamycin resistance
KW - polymerase chain reaction
KW - Recombinant DNA
KW - single-stranded DNA
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U2 - 10.1016/0378-1119(91)90366-J
DO - 10.1016/0378-1119(91)90366-J
M3 - Article
C2 - 2055470
AN - SCOPUS:0025782890
VL - 100
SP - 195
EP - 199
JO - Gene
JF - Gene
SN - 0378-1119
IS - C
ER -