Cloning vector with 'lethal' gene?

Duncan Clark Duncan at genesys.demon.co.uk
Fri Jun 9 05:37:13 EST 1995


In article: <v01510100abf87c7d5936@[143.48.11.21]>  lodhi at CSHL.ORG (Muhammad Lodhi) writes:
> 
> Could someone give me the info on the commercial source of a newly
> introduced cloning vector that has a 'lethal' gene on the cloning site.  I
> heard that in this cloning system only the cells with the cloned fragments
> can grow that way all the background is clear.  Any help will be highly
> appreciated

The Invitrogen vector is pUC18 with Zeocin antibiotic selection rather than
Ampicillin, F1 origin and a lacZ alpha E.coli ccdB fusion. This fusion is 
lethal unless interrupted by cloning into the polylinker. A search of Genbank 
for ccbD gave two plasmids, one of which is listed below. 


LOCUS       PK8CCDB      3024 bp ds-DNA             BCT       04-OCT-1994
DEFINITION  Plasmid KIL18 (from E.coli) cytotoxic protein (ccdB) gene, 
complete
            cds.
ACCESSION   L27083
KEYWORDS    ccdB gene; cytotoxic protein.
SOURCE      Plasmid pKIL18 (plasmid Plasmid pKIL18, kingdom Prokaryotae) DNA.
  ORGANISM  Plasmid pKIL18
            Prokaryotae.
REFERENCE   1  (bases 1 to 3024)
  AUTHORS   Bernard,P., Gabant,P., Bahassi,E.M. and Couturier,M.
  TITLE     Positive-selection vectors using the F plasmid ccdB killer gene
  JOURNAL   Gene 148, 71-74 (1994)
  STANDARD  full automatic
COMMENT     NCBI gi: 548209
FEATURES             Location/Qualifiers
     source          1..3024
                     /organism="Plasmid pKIL18"
                     /plasmid="Plasmid pKIL18"
                     /sequenced_mol="DNA"
                     /specific_host="K-12"
     CDS             1..381
                     /gene="ccdB"
                     /note="NCBI gi: 548210"
                     /codon_start=1
                     /product="cytotoxic protein"
                     
/translation="MTMITNSSSVPGDPLESTCRHASLSLQFKVYTYKRESRYRLFVD
                     
VQSDIIDTPGRRMVIPLASARLLSDKVSRELYPVVHIGDESWRMMTTDMASVPVSVIG
                     EEVADLSHRENDIKNAINLMFWGI"

With something so small it should be easy with PCR to make your own
lacZ alpha fusion etc.

Duncan 


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