Promoter and plasmid system for genetic engineering

Abstract

This invention provides a series of low-copy number plasmids comprising restriction endonuclease recognition sites useful for cloning at least three different genes or operons, each flanked by a terminator sequence, the plasmids containing variants of glucose isomerase promoters for varying levels of protein expression. The materials and methods are useful for genetic engineering in microorganisms, especially where multiple genetic insertions are sought.

1 - 5 . (canceled) 
     
     
         6 . A DNA construct comprising at least three transcriptional terminators and at least one cloning site situated between any two transcriptional terminators. 
     
     
         7 . The DNA construct of  claim 6  wherein the transcriptional terminators are tonB, thrA, or aspA, and the cloning sites are selected from the group consisting of AvrII, NheI, BfaI, Cac81, BsaJI, and StyI. 
     
     
         8 . A library of DNA constructs, each DNA construct comprising at least three transcriptional terminators and at least one cloning site situated between any two transcriptional terminators. 
     
     
         9 . A DNA construct selected from the group consisting of
 a) A pSYCO109mcs plasmid consisting of SEQ ID NO:30,   b) A DNA construct for the short 1.5 GI promoter consisting of SEQ ID NO:31,   c) A DNA construct for the short 1.20 GI promoter consisting of SEQ ID NO:32,   d) A DNA construct for the pAH105 plasmid consisting of SEQ ID NO:70,   e) A DNA construct for the pSYCO101 plasmid consisting of SEQ ID NO:71,   f) A DNA construct for the pSYCO103 plasmid consisting of SEQ ID NO:72,   g) A DNA construct for the pSYCO106 plasmid consisting of SEQ ID NO:73,   h) A DNA construct for the pSYCO109 plasmid consisting of SEQ ID NO:74,   i) A DNA construct for the pSCYO106mcs plasmid consisting of SEQ ID NO:78, and   j) A DNA construct for the pRJ50 plasmid consisting of SEQ ID NO:79.   
     
     
         10 . A vector having a multiple cloning site containing restriction recognition site sequences specific for the restriction endonucleases AscI, NheI, PacI, RsrII, NsiI, SacI, MluI, AgeI, SapI, and SnaBI. 
     
     
         11 . The vector of  claim 10  wherein the multiple cloning site has the nucleotide sequence of SEQ ID NO:77. 
     
     
         12 - 13 . (canceled)