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

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__NOTOC__
 
__NOTOC__
 
<partinfo>BBa_J31006 short</partinfo>
 
<partinfo>BBa_J31006 short</partinfo>
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===Design Notes===
 
===Design Notes===
  
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It was cloned into PsB1A2 plasmids.
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This part was PCR-amplified from <partinfo>pSB1AT3</partinfo> using the following primers. The primers have non-annealing 5'- extensions that introduce a <font color='blue'>SpeI site</font> to the left and an <font color='red'>XbaI site</font> to the right of the coding region (allowing BioBrick cloning in the reverse orientation). Primer annealing sites are shown in bold.  
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<br>Forward: 5’ ATGC<font color='blue'>ACTAGT</font><b>ATGAAATCTAACAATGCGCTCATC</b>
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===Biobricks===
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<br>Reverse: 5’ GCAT<font color='red'>TCTAGA</font><b>TTAGGTCGAGGTGGCCCGGC</b>
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The Biobricks on this part are not wild type, but the cut sites are still viable.  
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<font size='4'>5'</font size><font color='blue'>GAATTC</font color><font color='pink'> GCGGCCGC</font color><font color='gold'> T</font color> <font color='orange'> TCTAGA</font color><font color='red'> -</font color> <u>aatccagctccaccgggccgaggtacgtggcgctgcgttgcgcccctccgtctgttccatatcccgccgcggatgttaggtacggttgggcaaggtacac
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The final part was cloned into vector pSB1A2. The BioBricks on this part are not wild-type, but the cut sites are still viable.
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gagcggctccgccgtatttagcggcactgctagtcgccaggttactagcttcaatccgaccattctcggcgctcgctaggaacttcgacagggactacca
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gcagtagatggacggacctgtcgtaccggacgttgcgcccgtagggctacggcggccttcgctcttcttagtattaccccttccggtaggtcggagcgca
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gcgcttgcggtcgttctgcatcgggtcgcgcagccggcggtacggccgctattaccggacgaagagcggctttgcaaaccaccgccccggtcactgcttc
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cgaactcgctcccgcacgttctaaggcttatggcgttcgctgtccggctagtagcagcgcgaggtcgctttcgccaggagcggcttttactgggtctcgc
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gacggccgtggacaggatgctcaacgtactatttcttctgtcagtattcacgccgctgctatcagtacggggcgcgggtggccttcctcgactgacccaa
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cttccgagagttcccgtagccagctgcgagagggaatacgctgaggacgtaatccttcgtcgggtcatcatccaactccggcaactcgtggcggcggcgt
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tccttaccacgtacgttcctctaccgcgggttgtcagggggccggtgccccggacggtggtatgggtgcggctttgttcgcgagtactcgggcttcaccg
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ctcgggctagaaggggtagccactacagccgctatatccgcggtcgttggcgtggacaccgcggccactacggccggtgctacgcaggccgcatctccta
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ggtgtcctgcccacaccagcggtactagcgcatcagctatcaccgaggttcatcgcttcgctcgtcctgacccgccgccggtttcgccagcctgtcacga
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ggctcttgcccacgcgtatctttaacgtagttgcgtatatcgcgatcgtcgtgcggtatcactgaccgctacgacagccttacctgctatagggcgttct
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ccgggccgtcatggccgtattggttcggatacggatgtcgtaggtcccactgccacggctcctactgctactcgcgtaacaatctaaagta</u><font color='purple'> -</font color><font color='cyan'> ACTAGT</font color><font color='gold'> A</font color> <font color='magenta'>GCGGCCG</font color><font color='lime green'> CTGCAG</font color>
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<font color='blue'> EcoRI </font color> <font color='pink'> NotI</font color><font color='gold'> Spacer</font color><font color ='orange'> XbaI</font color> <font color='red'> Missing G spacer</font color><u> Coding Region</u> <font color='purple'> Missing T spacer</font color><font color='cyan'> SpeI</font color> <font color='gold'> Spacer</font color> <font color='magenta'> NotI</font color><font color='lime green'> PstI</font color>
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{| width="800px" cellspacing="5"
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|- valign="top"
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| style="width:180px" | '''Standard BioBrick Cloning Sites''' (Knight)
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| style="background:lightgrey"|<font face="courier">5'--GAATTC GCGGCCGC T TCTAGA G ----insert---- T ACTAGT A GCGGCCG CTGCAG--<br>3'--CTTAAG CGCCGGCG A AGATCT C -------------- A TGATCA T CGCCGGC GACGTC--</font>
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|- valign="top"
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| style="width:180px" | '''BBa_J31001 Cloning Sites'''
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| 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>
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'''Prefix'''<br>There is <font color='blue'>no G spacer (*)</font> between the XbaI and the Tet coding region.<br>
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'''Suffix'''<br>There is <font color='purple'>no T spacer (*)</font> between the Tet coding region and the SpeI site.
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|}
  
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We compared our BioBricks with those from Tom Knight's paper,<u> Idempotent Vector Design for Standard Assembly of Biobricks</u>. As seen below
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===Data===
  
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[[Image:BioBricks_from_paper.png]]
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Once placed to the right of a reverse RBS, TetB conveys tetrcycline resistance to JM109 cells (see <partinfo>BBa_S03532</partinfo>).
  
 
===Source===
 
===Source===
  
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PsB1AT3
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The tetracycline resistance coding region was PCR amplified from <partinfo>pSB1AT3</partinfo>.
  
 
===References===
 
===References===
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[https://dspace.mit.edu/handle/1721.1/21168|  Knight, Tom. Idempotent Vector Design for Standard Assembly of Biobricks]
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* [https://dspace.mit.edu/handle/1721.1/21168|  Knight, Tom. Idempotent Vector Design for Standard Assembly of Biobricks]

Latest revision as of 19:57, 11 April 2007

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
Reverse: 5’ GCATTCTAGATTAGGTCGAGGTGGCCCGGC

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