Difference between revisions of "Part:BBa J31006:Design"

Revision as of 20:57, 6 December 2006

tetracycline resistance protein TetA(C) (backwards) [cf. BBa_J31007]

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
INCOMPATIBLE WITH RFC[12]
Illegal NheI site found at 1043
21
INCOMPATIBLE WITH RFC[21]
Illegal BamHI site found at 897
23
COMPATIBLE WITH RFC[23]
25
INCOMPATIBLE WITH RFC[25]
Illegal NgoMIV site found at 343
Illegal NgoMIV site found at 503
Illegal NgoMIV site found at 871
1000
COMPATIBLE WITH RFC[1000]

Design Notes

This part was PCR amplified from pSB1AT3 using the following primers. The primers have non-annealing 5'- extensions that introduce a SpeI site to the left and an XbaI site to the right of the coding region (allowing BioBrick cloning in the reverse orientation). Primer annealing sites are shown in bold.
Forward: 5’ ATGCACTAGTATGAAATCTAACAATGCGCTCATC (SpeI site)
Reverse: 5’ GCATTCTAGATTAGGTCGAGGTGGCCCGGC (XbaI site)

The final part was cloned into vector pSB1A2. The Biobricks on this part are not wild type, but the cut sites are still viable.

Standard BioBrick Cloning Sites (Knight)	5'--GAATTC GCGGCCGC T TCTAGA G ----insert---- T ACTAGT A GCGGCCG CTGCAG-- 3'--CTTAAG CGCCGGCG A AGATCT C -------------- A TGATCA T CGCCGGC GACGTC--
BBa_J31001 Cloning Sites	5'--GAATTC GCGGCCGC T TCTAGA * --Tet coding-- * ACTAGT A GCGGCCG CTGCAG-- 3'--CTTAAG CGCCGGCG A AGATCT * -------------- * TGATCA T CGCCGGC GACGTC-- Prefix There is no G spacer () between the XbaI and the Tet coding region. Suffix* There is no T spacer (*) between the Tet coding region and the SpeI site.

Data

Once placed to the right of a reverse RBS, TetB conveys tetrcycline resistance to JM109 cells (see BBa_S03532).

Source

The tetracycline resistance coding region was PCR amplified from pSB1AT3.

References

Knight, Tom. Idempotent Vector Design for Standard Assembly of Biobricks

@@ Line 7: / Line 7: @@
 ===Design Notes===
-BBa J31006 was cloned into vector pSB1A2. The Biobricks on this part are not wild type, but the cut sites are still viable.
+This part was PCR amplified from <partinfo>pSB1AT3</partinfo> using the following primers. The primers have non-annealing 5'- extensions that introduce a SpeI site to the left and an XbaI site to the right of the coding region (allowing BioBrick cloning in the reverse orientation). Primer annealing sites are shown in bold.
+<br>Forward: 5’ ATGC<font color='blue'>ACTAGT</font><b>ATGAAATCTAACAATGCGCTCATC</b> (<font color='blue'>SpeI site</font>)
+<br>Reverse: 5’ GCAT<font color='red'>TCTAGA</font><b>TTAGGTCGAGGTGGCCCGGC</b> (<font color='red'>XbaI site</font>)
+The final part was cloned into vector pSB1A2. The Biobricks on this part are not wild type, but the cut sites are still viable.
 {| width="800px" cellspacing="5"
@@ Line 16: / Line 20: @@
 | style="width:180px" | '''BBa_J31001 Cloning Sites'''
 | style="background:lightgrey" |<font face="courier">5'--GAATTC GCGGCCGC T TCTAGA <font color='blue'>*</font> --Tet coding-- <font color='purple'>*</font> ACTAGT A GCGGCCG CTGCAG--<br>3'--CTTAAG CGCCGGCG A AGATCT <font color='blue'>*</font> -------------- <font color='purple'>*</font> TGATCA T CGCCGGC GACGTC--</font><br><br>
-'''Prefix'''<br>There is <font color='blue'>no G spacer (*)</font> between the XbaI and the Hin coding region.<br>
+'''Prefix'''<br>There is <font color='blue'>no G spacer (*)</font> between the XbaI and the Tet coding region.<br>
-'''Suffix'''<br>There is <font color='purple'>no T spacer (*)</font> between the Hin coding region and the SpeI site.
+'''Suffix'''<br>There is <font color='purple'>no T spacer (*)</font> between the Tet coding region and the SpeI site.
 |}