Difference between revisions of "Part:BBa J119388"

Latest revision as of 23:24, 11 June 2015

rClone Tet Red: Device for GGA Cloning and Testing RBS elements and Riboswitches

rClone TetRed allows users to clone and test new RBS elements and riboswitches without gel purification or other preparation of DNA. It is a destination vector for Golden Gate Assembly (GGA) using BsaI and ligase. A new RBS or riboswitch can be derived from synthetic oligos, PCR, or a plasmid clone. For proper assembly, the new insert must have the appropriate 4 nt sticky ends or be flanked by BsaI sites that produce the sticky ends. With reference to the top strand, the left site must be 5' CGAC 3' and the right site must be 5' GCGG 3'. BsaI always produces a 5' overhang sticky end. Successful GGA assembly replaces the reverse promoter driving GFP expression with the new RBS or riboswitch. Transcription will be initiated in the direction of the TetA-RFP fusion protein coding sequence. The level of expression of TetA-RFP fusion protein will depend on the efficiency of the newly cloned RBS or riboswitch.

Sequence and Features

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
INCOMPATIBLE WITH RFC[12]
Illegal NheI site found at 1008
21
INCOMPATIBLE WITH RFC[21]
Illegal BamHI site found at 1154
23
COMPATIBLE WITH RFC[23]
25
INCOMPATIBLE WITH RFC[25]
Illegal NgoMIV site found at 1180
Illegal NgoMIV site found at 1548
Illegal NgoMIV site found at 1708
Illegal AgeI site found at 2655
Illegal AgeI site found at 2767
1000
INCOMPATIBLE WITH RFC[1000]
Illegal BsaI site found at 849
Illegal BsaI.rc site found at 42

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 __NOTOC__
 <partinfo>BBa_J119388 short</partinfo>
-rClone Tet Red allows users to clone and test new RBS elements and riboswitches without gel purification or other preparation of DNA. It is a destination vector for Golden Gate Assembly (GGA) using BsaI and ligase. A new RBS or riboswitch can be derived from synthetic oligos, PCR, or a plasmid clone. For proper assembly, the new insert must have the appropriate 4 nt sticky ends or be flanked by BsaI sites that produce the sticky ends. With reference to the top strand, the left site must be 5' CGAC 3' and the right site must be 5' GCGG 3'. BsaI always produces a 5' overhang sticky end. Successful GGA assembly replaces the reverse promoter driving GFP expression with the new RBS or riboswitch. Transcription will be initiated in the direction of the TetA-RFP fusion protein coding sequence. The level of expression of TetA-RFP fusion protein will depend on the efficiency of the newly cloned RBS or riboswitch.
+rClone TetRed allows users to clone and test new RBS elements and riboswitches without gel purification or other preparation of DNA. It is a destination vector for Golden Gate Assembly (GGA) using BsaI and ligase. A new RBS or riboswitch can be derived from synthetic oligos, PCR, or a plasmid clone. For proper assembly, the new insert must have the appropriate 4 nt sticky ends or be flanked by BsaI sites that produce the sticky ends. With reference to the top strand, the left site must be 5' CGAC 3' and the right site must be 5' GCGG 3'. BsaI always produces a 5' overhang sticky end. Successful GGA assembly replaces the reverse promoter driving GFP expression with the new RBS or riboswitch. Transcription will be initiated in the direction of the TetA-RFP fusion protein coding sequence. The level of expression of TetA-RFP fusion protein will depend on the efficiency of the newly cloned RBS or riboswitch.
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