Difference between revisions of "Part:BBa K174006:Design"

 
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<partinfo>BBa_K174006 short</partinfo>
 
<partinfo>BBa_K174006 short</partinfo>
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<partinfo>BBa_K174006 SequenceAndFeatures</partinfo>
 
<partinfo>BBa_K174006 SequenceAndFeatures</partinfo>
  
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===Construction===
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366 bp long sacA sequence is selected from 1440 bp long sacA CDS (from base 392 to base 757). This sacA sequence is amplified by PCR with dangling end primers with EcoRI-NotI-XbaI restriction site at 5’ and SpeI at 3’ and inserted into a Biobrick compatible vector. The sequence is taken from B. subtilis strain 168. Genbank accession number is AL009126 http://www.ncbi.nlm.nih.gov/nuccore/AL009126?ordinalpos=1&itool=EntrezSystem2.PEntrez.Sequence.Sequence_ResultsPanel.Sequence_RVDocSum
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Forward primer used: GATCTG-GAATTCGCGGCCGCTTCTAGAG-TTCTCATTTCTGCGCATGG (Clamp sequence - Standard Biobrick prefix - first 19 base from the Biobrick)
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Reverse primer used: TGTGAC-ACTAGTA-AGGATCGGATGTGCTGATTG (Clamp sequence - SpeI site - last 20 base from the Biobrick)
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Start of biobrick suffix TACTAGT is written as complemantary which is ACTAGTA (speI site is ACTAGT) to have the scar as in Biobrick standards.
  
 
===Design Notes===
 
===Design Notes===

Revision as of 18:53, 10 October 2009

Sac single crossover site for Bacillus subtilis


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]

Construction

366 bp long sacA sequence is selected from 1440 bp long sacA CDS (from base 392 to base 757). This sacA sequence is amplified by PCR with dangling end primers with EcoRI-NotI-XbaI restriction site at 5’ and SpeI at 3’ and inserted into a Biobrick compatible vector. The sequence is taken from B. subtilis strain 168. Genbank accession number is AL009126 http://www.ncbi.nlm.nih.gov/nuccore/AL009126?ordinalpos=1&itool=EntrezSystem2.PEntrez.Sequence.Sequence_ResultsPanel.Sequence_RVDocSum

Forward primer used: GATCTG-GAATTCGCGGCCGCTTCTAGAG-TTCTCATTTCTGCGCATGG (Clamp sequence - Standard Biobrick prefix - first 19 base from the Biobrick)

Reverse primer used: TGTGAC-ACTAGTA-AGGATCGGATGTGCTGATTG (Clamp sequence - SpeI site - last 20 base from the Biobrick) Start of biobrick suffix TACTAGT is written as complemantary which is ACTAGTA (speI site is ACTAGT) to have the scar as in Biobrick standards.

Design Notes

The sequence selected does not contain any restriction site specific to Biobrick standards


Source

The sequence is taken from 'B. subtilis' 168' sacA gene.

References