Difference between revisions of "Part:BBa K2205002:Design"

 
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===Design Notes===
 
===Design Notes===
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This part was designed as a gBlock and Gibson assembled into pSB1C3 using HiFi assembly. Overhangs were designed to esnure that the biobrick prefix and suffix were re-generated.
+
The deGFP sequence was taken from the Addgene database (Plasmid #40019). The sequence was found to have no illegal restriction sites (i.e. no EcoRI, XbaI, SpeI, or PstI sites). A strong, standard Anderson promoter (J23100) and RBS (B0034) was added before the deGFP sequence with biobrick scar sites between each part. A double terminator (B0015) was added after the deGFP sequence. The entire construct was flanked by 30 bp overhangs with the pSB1C3 plasmid, such that the construct could be Gibson assembled into a pSB1C3 plasmid digested with XbaI and Spe. Extra bases were added between the overhangs and the construct so that once the part was assembled into the plasmid, the XbaI and SpeI sites could be regenerated and the biobrick prefix and suffix restored. This construct (J23100-deGFP) with the overhangs was submitted to IDT for synthesis as a gBlock.
  
  

Revision as of 22:08, 1 November 2017


J23100-deGFP


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 7
    Illegal NheI site found at 30
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]


Design Notes

The deGFP sequence was taken from the Addgene database (Plasmid #40019). The sequence was found to have no illegal restriction sites (i.e. no EcoRI, XbaI, SpeI, or PstI sites). A strong, standard Anderson promoter (J23100) and RBS (B0034) was added before the deGFP sequence with biobrick scar sites between each part. A double terminator (B0015) was added after the deGFP sequence. The entire construct was flanked by 30 bp overhangs with the pSB1C3 plasmid, such that the construct could be Gibson assembled into a pSB1C3 plasmid digested with XbaI and Spe. Extra bases were added between the overhangs and the construct so that once the part was assembled into the plasmid, the XbaI and SpeI sites could be regenerated and the biobrick prefix and suffix restored. This construct (J23100-deGFP) with the overhangs was submitted to IDT for synthesis as a gBlock.


Source

Efficient cell-free expression with the endogenous E. Coli RNA polymerase and sigma factor 70. Shin J, Noireaux V. J Biol Eng. 2010 Jun 24;4:8. 10.1186/1754-1611-4-8 PubMed 20576148

References