Difference between revisions of "Part:BBa I50042:Design"

 
 
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__NOTOC__
 
__NOTOC__
 
<partinfo>BBa_I50042 short</partinfo>
 
<partinfo>BBa_I50042 short</partinfo>
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===Design Notes===
 
===Design Notes===
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The replication origin boundaries were chosen quite liberally (i.e. they might include more sequence than is strictly necessary) because a different version of this origin (BBa_I50040) was shown to be non-functional.
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The original design for [[Part:BBa_I50042|BBa_I50042]] was [[Part:BBa_I50040|BBa_I50040]]. Synthesis of [[Part:BBa_I50040|BBa_I50040]] (designed pSC101 origin) was straightforward but testing revealed that [[Part:BBa_I50040|BBa_I50040]] was nonfunctional as a replication origin presumably due to introduced mutations.
  
 +
In constructing [[Part:BBa_I50042|BBa_I50042]], the replication origin boundaries were chosen quite liberally (i.e. they might include more sequence than is strictly necessary) in part because [[Part:BBa_I50040|BBa_I50040]] was shown to be non-functional.
  
 +
[[Part:BBa_I50042|BBa_I50042]] is very similar to the replication origin used in Bujard's pZS4Int-1 vector and in Michael Elowitz's repressilator. However, a mutation was introduced to eliminate a SpeI site for compatibility with the BioBrick assembly standard. [[Part:BBa_I50042|BBa_I50042]] is also the origin of replication in pSB4* series plasmids.
 +
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*'''[[User:Rshetty|RS]] 17:55, 21 June 2010 (UTC)''': The sequence of this part was updated based on resequencing of pSB4C5-I52002.  Four point differences were found between the reference sequence in the Registry and the sequence data.  These four point differences were consistent across multiple samples as well as a totally separate vector carrying a pSC101 origin (pSIM5, a recombineering vector not in the Registry).  Hence, I believe that these four differences reflect sequence errors in the canonical pSC101 vector sequence in Genbank.  Features were added to this part to indicate the point differences. 
  
 
===Source===
 
===Source===
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We constructed [[Part:BBa_I50042|BBa_I50042]] by PCR using [[Part:pSB4A3|pSB4A3]]-[[Part:BBa_P1010|P1010]] as a template and amplification primers I50042-f (<code>5'-GTTTCTTCGAATTCGCGGCCGCTTCTAGAGCTGTCAGACCAAGTTTACGAG-3'</code>) and I50042-r (<code>5'-GTTTCTTCCTGCAGCGGCCGCTACTAGTAGTTACATTGTCGATCTGTTC-3'</code>).
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This part was amplified from pSB4A3-P1010.
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===References===
 
===References===
 +
<biblio>
 +
#Cohen-J-Bacteriol-1977 pmid=334752
 +
#Lutz-Nucleic-Acids-Res-1997 pmid=9092630
 +
#Elowitz-Nature-2000 pmid=10659856
 +
</biblio>

Latest revision as of 17:55, 21 June 2010

pSC101 origin of replication


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]


Design Notes

The original design for BBa_I50042 was BBa_I50040. Synthesis of BBa_I50040 (designed pSC101 origin) was straightforward but testing revealed that BBa_I50040 was nonfunctional as a replication origin presumably due to introduced mutations.

In constructing BBa_I50042, the replication origin boundaries were chosen quite liberally (i.e. they might include more sequence than is strictly necessary) in part because BBa_I50040 was shown to be non-functional.

BBa_I50042 is very similar to the replication origin used in Bujard's pZS4Int-1 vector and in Michael Elowitz's repressilator. However, a mutation was introduced to eliminate a SpeI site for compatibility with the BioBrick assembly standard. BBa_I50042 is also the origin of replication in pSB4* series plasmids.

  • RS 17:55, 21 June 2010 (UTC): The sequence of this part was updated based on resequencing of pSB4C5-I52002. Four point differences were found between the reference sequence in the Registry and the sequence data. These four point differences were consistent across multiple samples as well as a totally separate vector carrying a pSC101 origin (pSIM5, a recombineering vector not in the Registry). Hence, I believe that these four differences reflect sequence errors in the canonical pSC101 vector sequence in Genbank. Features were added to this part to indicate the point differences.

Source

We constructed BBa_I50042 by PCR using pSB4A3-P1010 as a template and amplification primers I50042-f (5'-GTTTCTTCGAATTCGCGGCCGCTTCTAGAGCTGTCAGACCAAGTTTACGAG-3') and I50042-r (5'-GTTTCTTCCTGCAGCGGCCGCTACTAGTAGTTACATTGTCGATCTGTTC-3').

References

<biblio>

  1. Cohen-J-Bacteriol-1977 pmid=334752
  2. Lutz-Nucleic-Acids-Res-1997 pmid=9092630
  3. Elowitz-Nature-2000 pmid=10659856

</biblio>