Difference between revisions of "Part:BBa K864017"
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<partinfo>BBa_K864017 short</partinfo> | <partinfo>BBa_K864017 short</partinfo> | ||
| − | pSB8C15 is a BioBrick standard vector with low copy pSC101ts replication origin <partinfo>BBa_K864050</partinfo> (~5 copies per cell) and | + | pSB8C15 is a BioBrick standard vector with low copy pSC101ts replication origin <partinfo>BBa_K864050</partinfo> (~5 copies per cell) and chloramphenicol antibiotic resistance marker, especially usable for Lambda Red recombineering in E coli. The pSC101ts is a temperature sensitive pSC101 replication origin and propagates at 30’C but not at at 42’C [1]. The backbone sequence is based on pSB3T5, but the E coli His operon terminator BBa_B0053 has been replaced with the late terminator of the Salmonella phage P22. |
===Usage and Biology=== | ===Usage and Biology=== | ||
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A problem when doing chromosomal intergration is that some clones may take up a plasmid instead of recombineering it into the chromosome. When doing recombineering with pSB8x15 backbones, any such clones can be removed by incubation at 42° C. | A problem when doing chromosomal intergration is that some clones may take up a plasmid instead of recombineering it into the chromosome. When doing recombineering with pSB8x15 backbones, any such clones can be removed by incubation at 42° C. | ||
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<span class='h3bb'>Sequence and Features</span> | <span class='h3bb'>Sequence and Features</span> | ||
Revision as of 07:14, 28 September 2012
Low copy BioBrick temperature sensitive standard vector
pSB8C15 is a BioBrick standard vector with low copy pSC101ts replication origin BBa_K864050 (~5 copies per cell) and chloramphenicol antibiotic resistance marker, especially usable for Lambda Red recombineering in E coli. The pSC101ts is a temperature sensitive pSC101 replication origin and propagates at 30’C but not at at 42’C [1]. The backbone sequence is based on pSB3T5, but the E coli His operon terminator BBa_B0053 has been replaced with the late terminator of the Salmonella phage P22.
Usage and Biology
This part is available as pSB8C15-pUCori-redq. This facilitates fast growth at 37° C, high plasmid yields and fast red color expression on transformation plates.
FLP recombinase
The pSB8X15 series are especially suitable for carrying the FLP recombinase, since plasmids can be removed by incubation at 42°C.
Origin switching
Easy ori replacement is possible in the pSB8x15 backbones. The plasmid can have the pSC101ts ori cut out with NheI and MluI and another ori ligated in. To remove any religated pSC101ts, the transformants can be grown at 42° C.
More reliable recombineering
A problem when doing chromosomal intergration is that some clones may take up a plasmid instead of recombineering it into the chromosome. When doing recombineering with pSB8x15 backbones, any such clones can be removed by incubation at 42° C.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Plasmid lacks a prefix.
Plasmid lacks a suffix.
Illegal EcoRI site found at 3445
Illegal NheI site found at 2425
Illegal SpeI site found at 2
Illegal PstI site found at 16
Illegal NotI site found at 9
Illegal NotI site found at 3451 - 21INCOMPATIBLE WITH RFC[21]Plasmid lacks a prefix.
Plasmid lacks a suffix.
Illegal EcoRI site found at 3445 - 23INCOMPATIBLE WITH RFC[23]Illegal prefix found at 3445
Illegal suffix found at 2 - 25INCOMPATIBLE WITH RFC[25]Illegal prefix found at 3445
Plasmid lacks a suffix.
Illegal XbaI site found at 3460
Illegal SpeI site found at 2
Illegal PstI site found at 16
Illegal NgoMIV site found at 2319 - 1000INCOMPATIBLE WITH RFC[1000]Plasmid lacks a prefix.
Plasmid lacks a suffix.