Part:BBa_K2569034
Constitutively expressed CcdB protein in pSB1C3, is lethal to most of the BioBrick cell strains.
This Biobrick is suitable for fast and seamless promoter part construction. The promoter sequence is added to the pSB1C3 vector by PCR. There are 10 bp homologous at both ends of the PCR product. If the toxin CcdB is expressed in the absence of its antitoxin CcdA or the gyrAArg462Cys mutation, the E. coli host will be killed. Consequently, plasmids including ccdB must be grown in an E. coli host carrying the antitoxin CcdA or the gyrAArg462Cys mutation and will not re-enter the experiment when the recombineering host is used. By Red/ET recombination system, the PCR product is self-ligated to obtain the correct clone, and the enzyme digestion result can reach a correct rate of nearly 100%. Ordinary restriction enzymes or site-specific recombination systems, Cre/IoxP, Flp/FRT, Dre/rox, will always leave a scar at the modification site. However, Red/ET recombination and reverse screening can be used for seamless modification.
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal prefix found at 2049
- 12INCOMPATIBLE WITH RFC[12]Plasmid lacks a prefix.
Plasmid lacks a suffix.
Illegal EcoRI site found at 2049
Illegal SpeI site found at 2
Illegal PstI site found at 16
Illegal NotI site found at 9
Illegal NotI site found at 2055 - 21INCOMPATIBLE WITH RFC[21]Plasmid lacks a prefix.
Plasmid lacks a suffix.
Illegal EcoRI site found at 2049
Illegal BamHI site found at 3028
Illegal XhoI site found at 1033
Illegal XhoI site found at 1925 - 23INCOMPATIBLE WITH RFC[23]Illegal prefix found at 2049
Illegal suffix found at 2 - 25INCOMPATIBLE WITH RFC[25]Illegal prefix found at 2049
Plasmid lacks a suffix.
Illegal XbaI site found at 2064
Illegal SpeI site found at 2
Illegal PstI site found at 16 - 1000INCOMPATIBLE WITH RFC[1000]Plasmid lacks a prefix.
Plasmid lacks a suffix.
Illegal BsaI site found at 2878
Illegal BsaI site found at 3287
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