Team:Harvard/fences/parts

From 2010.igem.org

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<h1>parts and primers</h1>
<h1>parts and primers</h1>
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<h2>Vectors</h2>
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<h2>Parts</h2>
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For more information on the vectors visit our "<a href="vectors.html">meet the vectors</a>" page<br /><br />
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<table cellspacing="0">
<table cellspacing="0">
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<tr><td><b>Name</b></td><td><b>Original Name</b></td><td><b>Description</b></td><td><b>Resistance</b></td><td><b>Resistance (Plants)</b></td><td><b>Sequence</b></td></tr>
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<tr><td><b>Name</b></td><td><b>Biobrick Name</b></td><td><b>Description</b></td><td><b>Resistance</b></td><td><b>Sequence</b></td></tr>
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<tr><td>V1</td><td>pORE Open Series 1 (O1)</td><td>Agrobacterium vector open series</td><td>Kanamycin</td><td>pat</td><td></td></tr>
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<tr><td>Barnase</td><td><center>-</center></td><td> Fatal gene for the genetic fence </td><td>chloramphenicol</td><td><a href="https://2010.igem.org/Team:Harvard/f1">link</a></td></tr>
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<tr><td>V2</td><td>pORE Open Series 2 (O2)</td><td>Agrobacterium vector open series</td><td>Kanamycin</td><td>nptII</td><td></td></tr>
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<tr><td>Barstar</td><td><center>-</center></td><td>Protein inhibitor of Barnase</td><td>chloramphenicol</td><td><a href="https://2010.igem.org/Team:Harvard/f2">link</a></td></tr>
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<tr><td>V3</td><td>pORE Expression Series 3 (E3)</td><td>Agrobacterium vector with ENTCUP2 promoter</td><td>Kanamycin</td><td>pat</td><td></td></tr>
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<tr><td>LacIN</td><td>BBa_K382035</td><td>Lac Inhibitor with Nuclear Localization sequence - transcriptional inhibitor of the Act2LacO promoter</td><td>chloramphenicol</td><td><a href="https://2010.igem.org/Team:Harvard/f3">link</a></td></tr>
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<tr><td>V4</td><td>pORE Expression Series 4 (E4)</td><td>Agrobacterium vector with ENTCUP2 promoter</td><td>Kanamycin</td><td>nptII</td><td></td></tr>
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<tr><td>RXRLc</td><td>BBa_K382031</td><td> Part of the Locust Retinoic Acid Receptor and VP16 activation domain fusion protein. RXRLc binds EcR in presence of Methoxyfenozide</td><td>chloramphenicol</td><td><a href="https://2010.igem.org/Team:Harvard/f4">link</a></td></tr>
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<tr><td>V5</td><td>pORE Reporter Series 1 (R1)</td><td>Agrobacterium vector with gusA reporter</td><td>Kanamycin</td><td>nptII</td><td></td></tr>
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<tr><td>RXRHm</td><td>BBa_K382036</td><td>Human Retinoic Acid Receptor performs a function analogous to RXRLc. RXRHm has been shown to be sensitive to lower level of methoxyfenozide, but also to have higher background activation in the absence of methoxyfenozide.</td><td>chloramphenicol</td><td><a href="https://2010.igem.org/Team:Harvard/f5">link</a></td></tr>
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<tr><td>V6</td><td>pORE Reporter Series 3 (R3)</td><td>Agrobacterium vector with smgfp reporter</td><td>Kanamycin</td><td>nptII</td><td></td></tr>
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<tr><td>VP16</td><td><center>-</center></td><td> Part of the Retinoic Acid Receptor and VP16 activation domain fusion protein. VP16 recruits transcription factors, and when colocalized with the Gal4 Upstream Activating Sequence initiates transcription downstream of the 5xGal435s promoter.</td><td>chloramphenicol</td><td><a href="https://2010.igem.org/Team:Harvard/f6">link</a></td></tr>
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<tr><td>V7</td><td><center>-</center></td><td>Agrobacterium vector open series with biobrick cloning site</td><td>Kanamycin</td><td>pat</td><td></td></tr>
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<tr><td>Ecdysone Receptor</td><td>BBa_K382033</td><td> Part of the fusion protein of Ecdysone receptor and Gal4 DNA binding domain. Ecdysone receptor binds RXR-VP16 in presence of Methoxyfenozide </td><td>chloramphenicol</td><td><a href="https://2010.igem.org/Team:Harvard/f7">link</a></td></tr>
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<tr><td>V8</td><td><center>-</center></td><td>Agrobacterium vector open series with biobrick cloning site</td><td>Kanamycin</td><td>nptII</td><td></td></tr>
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<tr><td>Gal4 DNA binding domain</td><td><center>-</center></td><td> Part of the Gal4-EcR fusion protein. Gal4 associates with the Gal4 upstream activating sequence, and when RXR-VP16 is bound, it localizes VP16 to the Gal4UAS35s promoter resulting in transcription downstream. </td><td>chloramphenicol</td><td><a href="https://2010.igem.org/Team:Harvard/f8">link</a></td></tr>
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<tr><td>V9</td><td><center>-</center></td><td>Agrobacterium vector with ENTCUP2 promoter and biobrick cloning site</td><td>Kanamycin</td><td>pat</td><td></td></tr>
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</table>
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<tr><td>V10</td><td><center>-</center></td><td>Agrobacterium vector with ENTCUP2 promoter and biobrick cloning site</td><td>Kanamycin</td><td>nptII</td><td></td></tr>
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<tr><td>V11</td><td><center>-</center></td><td>Agrobacterium vector with gusA reporter and biobrick cloning site</td><td>Kanamycin</td><td>nptII</td><td></td></tr>
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<br>
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<tr><td>V12</td><td><center>-</center></td><td>Agrobacterium vector with smgfp reporter and biobrick cloning site</td><td>Kanamycin</td><td>nptII</td><td></td></tr>
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<h2>Promoters</h2>
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<table cellspacing="0">
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<tr><td><b>Name</b></td><td><b>Description</b></td><td>Active conditions</td><td><b>Resistance</b></td><td><b>Sequence</b></td></tr>
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<tr><td>pENT CUP</td><td> Constitutive Plant Promoter</td><td>Constitutive</td><td>chloramphenicol</td><td><a href="https://2010.igem.org/Team:Harvard/f10">link</a></td></tr>
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<tr><td>5xGal UAS</td><td> 5 time repeat of Gal4 upstream activating sequence</td><td>On in presense of dimerized EcR-Gal and RXRLc-VP16</td><td>chloramphenicol</td><td><a href="https://2010.igem.org/Team:Harvard/f11">link</a></td></tr>
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        <tr><td>Act2lacO</td><td>Actin2 promoter with Lac Inhibitor responsive sites (Lac O sites</td><td>Constitutive, inhibited by LacIN</td><td>chloramphenicol</td><td><a href="https://2010.igem.org/Team:Harvard/f12">link</a></td></tr>
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</table>
</table>
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<table cellspacing="0">
<table cellspacing="0">
<tr><td><b>Name</b></td><td><b>Description</b></td><td><b>Sequence</b></td></tr>
<tr><td><b>Name</b></td><td><b>Description</b></td><td><b>Sequence</b></td></tr>
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<tr><td>VP3</td><td>HindIII-pENTCUP2 forward primer for Expression Series insert</td><td></td></tr>
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<tr><td>Arp2mutForward</td><td>Point mutagenesis to remove restriction enzyme site</td><td>5'-CAA GAA GCT TGA CTT GTC TAC GAA CTC TTC CAT TTC-3'</td></tr>
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<tr><td>VP4</td><td>pENTCUP2-biobrick multiple cloning site-NheI reverse primer for Expression Series insert</td><td></td></tr>
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<tr><td>Arp2mutReverse</td><td>Point mutagenesis to remove restriction enzyme site</td><td>5'-GAA ATG GAA GAA CTC GTA GAC AAG TCA AGC TCC TTG-3'</td></tr>
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<tr><td>VP5</td><td>HindIII-biobrick multiple cloning site-gusA-forward primer for Reporter Series 1 insert</td><td></td></tr>
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        <tr><td>Exp2MutForward</td><td>Point mutagenesis to remove restriction enzyme site</td><td>5'-CCT ACA CAC GGA ACT CCT GGA AAT TAA GAC CG-3'</td></tr>
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<tr><td>VP6</td><td>gusA-NheI reverse primer for Reporter Series 1 insert</td><td></td></tr>
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        <tr><td>Exp2mutReverse</td><td>Point mutagenesis to remove restriction enzyme site</td><td>5'-CGG TCT TAA TTT CCA GGA ACT CCG TGT GTA GG-3'</td></tr>
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<tr><td>VP7</td><td>HindIII-biobrick multiple cloning site-smGFP-forward primer for Reporter Series 1 insert</td><td></td></tr>
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<tr><td>Bam.Gal4.Fwd</td><td>Attaching BamH1 sites to Gal4 in preparation for yeast vector ligation</td><td>5'-AAA AGG ATC CAA AAA AAT GAA GCT ACT GTC TTC TAT C-3'</td></tr>
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<tr><td>VP8</td><td>smGFP-NheI reverse primer for Reporter Series 1 insert</td><td></td></tr>
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<tr><td>HmRXR.Hind.Rev</td><td>Attaching HindIII sites to HmRXR in preparation for yeast vector ligation</td><td>5'-AAT TAA GCT TGG ATC CCT CGA GCT GCA GC-3'</td></tr>
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<tr><td>VP9</td><td>Biobrick multiple cloning site for annealing with VP10 to create Open Series Insert</td><td></td></tr>
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<tr><td>LcRXR.Hind.Rev</td><td>Attaching HindIII sites to LcRXR in preparation for yeast vector ligation</td><td>5'-GAT GAA GCT TGG ATC CCT CGA GCT GC-3'</td></tr>
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<tr><td>VP11</td><td>Biobrick multiple cloning site for annealing with VP10 to create Open Series Insert</td><td></td></tr>
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<tr><td>VP16.Hind.Fwd</td><td>Attaching HindIII sites to VP16 in preparation for yeast vector ligation</td><td>5'-AAA AAA GCT TAA AAA AAT GGC TAG CGC CGC CAC CAT G-3'</td></tr>
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<tr><td>VP12</td><td>1 of 5 for sequencing V12 biobrick cloning site and smgfp reporter</td><td></td></tr>
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<tr><td>VP16.Rev</td><td>Reverse primer for VP16.Hind.Fwd PCR - no modification</td><td>5'-CTG CAG CGG CCG CTA CTA GTG CCC TTG GAA TTG ACG AGT AC-3'</td></tr>
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<tr><td>VP13</td><td>2 of 5 for sequencing V12 biobrick cloning site and smgfp reporter</td><td></td></tr>
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<tr><td>NLS.serine.BB.Fwd</td><td>Oligo to anneal NLS, with one bp difference to avoid self-complementarity </td><td>5'-/5Phos/CTA GAA GTT CTG TTG TTC ATC CTA AGA AGA AGA GAA AGG TTT GAA CTA GTA GCG GCC GCT GCA-3'</td></tr>
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<tr><td>VP14</td><td>3 of 5 for sequencing V12 biobrick cloning site and smgfp reporter</td><td></td></tr>
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<tr><td>NLS.serine.BB.Rev</td><td>Complement to NLS.serine.BB.Fwd. for NLS annealing</td><td>5'-/5Phos/GCG GCC GCT ACT AGT TCA AAC CTT TCT CTT CTT CTT AGG ATG AAC AAC AGA ACT T-3'</td></tr>
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<tr><td>VP15</td><td>4 of 5 for sequencing V12 biobrick cloning site and smgfp reporter</td><td></td></tr>
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<tr><td>Fwd.BB.AtEXP2</td><td>for amplification/extraction of an arabidopsis germination-specific promoter</td><td>5'-CCT TTC TAG ACC TGG TGT GGT TTC TTT G-3'</td></tr>
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<tr><td>VP16</td><td>5 of 5 for sequencing V12 biobrick cloning site and smgfp reporter</td><td></td></tr>
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<tr><td>Rev.BB.AtEXP2</td><td>for amplification/extraction of an arabidopsis germination-specific promoter</td><td>5'-AAG GCT GCA GCG GCC GCT ACT GAT ATT TGC TGA TGG GCT AAA G-3'</td></tr>
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<tr><td>VP17</td><td>1 of 9 for sequencing V11 biobrick cloning site and gusA reporter</td><td></td></tr>
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<tr><td>Fwd.BB.AtARP2</td><td>for amplification/extraction of the arabidopsis ARP2 promoter</td><td>5'-CCT TTC TAG ACC ATC ACA TAT TTG TAG AAT G-3'</td></tr>
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<tr><td>VP18</td><td>2 of 9 for sequencing V11 biobrick cloning site and gusA reporter</td><td></td></tr>
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<tr><td>Rev.BB.AtARP2</td><td>for amplification/extraction of the arabidopsis ARP2 promoter</td><td>5'-AAG GCT GCA GCG GCC GCT ACT AGT CTT CTC CGA TTT CTA TAG AG-3'</td></tr>
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<tr><td>VP19</td><td>3 of 9 for sequencing V11 biobrick cloning site and gusA reporter</td><td></td></tr>
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<tr><td>barnase2.BB.fwd</td><td>addition of biobrick sites/ends to barnase gene</td><td>5'-CCT TTC TAG AAT GGT ACC GGT TAT CAA CAC G-3'</td></tr>
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<tr><td>VP20</td><td>4 of 9 for sequencing V11 biobrick cloning site and gusA reporter</td><td></td></tr>
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<tr><td>BB.Barnase.Rev</td><td>addition of biobrick sites/ends to barnase gene</td><td>5'-AAG GCT GCA GCG GCC GCT ACT AGT TTA TCT GAT TTT TGT AAA GGT CTG-3'</td></tr>
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<tr><td>VP21</td><td>5 of 9 for sequencing V11 biobrick cloning site and gusA reporter</td><td></td></tr>
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<tr><td>NLS.BB.Fwd</td><td>addition of biobrick sites/ends to the arabidopsis NLS</td><td>5'-/5Phos/CTA GAT CTT CTG TTG TTC ATC CAT AGA AGA AGA GAA AGG TTT GAA CTA GTA GCG GCC GCT GCA-3'</td></tr>
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<tr><td>VP22</td><td>6 of 9 for sequencing V11 biobrick cloning site and gusA reporter</td><td></td></tr>
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<tr><td>NLS.BB.Rev</td><td>addition of biobrick sites/ends to the arabidopsis NLS</td><td>5'/5Phos/GCG GCC GCT ACT AGT TCA AAC CTT TCT CTT CTT CTT AGG ATG AAC AAC AGA AGA-3'</td></tr>
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<tr><td>VP23</td><td>7 of 9 for sequencing V11 biobrick cloning site and gusA reporter</td><td></td></tr>
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<tr><td>LacIn.BB.Fwd</td><td>Addition of NLS to the Lac Inhibitor gene to produce LacIN</td><td>5'-CCT TGA ATT CGC GGC CGC ATC TAG AAT GAA AC AGT AAC GTT ATA CGA TGT C-3'</td></tr>
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<tr><td>VP24</td><td>8 of 9 for sequencing V11 biobrick cloning site and gusA reporter</td><td></td></tr>
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<tr><td>LacIN.BB.Rev</td><td>Addition of NLS to the Lac Inhibitor gene to produce LacIN</td><td>5' AAG GTC GCA GCA GCG GCC GCT ACT AGT TCA AAC CTT TCT CTT CTT CTT AGG ATG AAC AAC AGA AGA CTG CCC GCT TTC CAG TCG GGA AA-3'</td></tr>
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<tr><td>VP25</td><td>9 of 9 for sequencing V11 biobrick cloning site and gusA reporter</td><td></td></tr>
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<tr><td>BB.Gal4DBD.Fwd</td><td>Adding biobrick sites to Gal4 DNA Binding Domain</td><td>5'-CTT TTC TAG AAT GAA GCT ACT GTC TTC TAT C-3'</td></tr>
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<tr><td>VP26</td><td>1 of 5 for sequencing V9 and V10 biobrick cloning site and promoter</td><td></td></tr>
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<tr><td>BB.Gal4DBD.Rev</td><td>Adding biobrick sites to Gal4 DNA Binding Domain</td><td>5'-AAG GCT GCA GCG GCC GCT ACT AGT CGA TAC AGT CAA CTG TCT TTG-3'</td></tr>
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<tr><td>VP27</td><td>2 of 5 for sequencing V9 and V10 biobrick cloning site and promoter</td><td></td></tr>
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<tr><td>BB.Barstar.Fwd</td><td>addition of biobrick sites/ends to barstar gene</td><td>5'CCT TTC TAG AAT GAA AAA AGC AGT CAT TAA C-3'</td></tr>
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<tr><td>VP28</td><td>3 of 5 for sequencing V9 and V10 biobrick cloning site and promoter</td><td></td></tr>
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<tr><td>BB.Barstar.Rev</td><td>addition of biobrick sites/ends to barstar gene</td><td>5'-AAG GCT GCA GCG GCC GCT ACT AGT TTA AGA AAG TATA GAT GGT GAT GTC-3'</td></tr>
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<tr><td>VP29</td><td>4 of 5 for sequencing V9 and V10 biobrick cloning site and promoter</td><td></td></tr>
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<tr><td>VP30</td><td>5 of 5 for sequencing V9 and V10 biobrick cloning site and promoter</td><td></td></tr>
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<tr><td>VP33</td><td>1 of 2 for sequencing V7 and V8 biobrick cloning site</td><td></td></tr>
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<tr><td>VP34</td><td>2 of 2 for sequencing V7 and V8 biobrick cloning site</td><td></td></tr>
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Latest revision as of 01:12, 27 October 2010


parts and primers

Parts

NameBiobrick NameDescriptionResistanceSequence
Barnase
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 Fatal gene for the genetic fence chloramphenicollink
Barstar
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Protein inhibitor of Barnasechloramphenicollink
LacINBBa_K382035Lac Inhibitor with Nuclear Localization sequence - transcriptional inhibitor of the Act2LacO promoterchloramphenicollink
RXRLcBBa_K382031 Part of the Locust Retinoic Acid Receptor and VP16 activation domain fusion protein. RXRLc binds EcR in presence of Methoxyfenozidechloramphenicollink
RXRHmBBa_K382036Human Retinoic Acid Receptor performs a function analogous to RXRLc. RXRHm has been shown to be sensitive to lower level of methoxyfenozide, but also to have higher background activation in the absence of methoxyfenozide.chloramphenicollink
VP16
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 Part of the Retinoic Acid Receptor and VP16 activation domain fusion protein. VP16 recruits transcription factors, and when colocalized with the Gal4 Upstream Activating Sequence initiates transcription downstream of the 5xGal435s promoter.chloramphenicollink
Ecdysone ReceptorBBa_K382033 Part of the fusion protein of Ecdysone receptor and Gal4 DNA binding domain. Ecdysone receptor binds RXR-VP16 in presence of Methoxyfenozide chloramphenicollink
Gal4 DNA binding domain
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 Part of the Gal4-EcR fusion protein. Gal4 associates with the Gal4 upstream activating sequence, and when RXR-VP16 is bound, it localizes VP16 to the Gal4UAS35s promoter resulting in transcription downstream. chloramphenicollink

Promoters

NameDescriptionActive conditionsResistanceSequence
pENT CUP Constitutive Plant PromoterConstitutivechloramphenicollink
5xGal UAS 5 time repeat of Gal4 upstream activating sequenceOn in presense of dimerized EcR-Gal and RXRLc-VP16chloramphenicollink
Act2lacOActin2 promoter with Lac Inhibitor responsive sites (Lac O sitesConstitutive, inhibited by LacINchloramphenicollink


Primers

NameDescriptionSequence
Arp2mutForwardPoint mutagenesis to remove restriction enzyme site5'-CAA GAA GCT TGA CTT GTC TAC GAA CTC TTC CAT TTC-3'
Arp2mutReversePoint mutagenesis to remove restriction enzyme site5'-GAA ATG GAA GAA CTC GTA GAC AAG TCA AGC TCC TTG-3'
Exp2MutForwardPoint mutagenesis to remove restriction enzyme site5'-CCT ACA CAC GGA ACT CCT GGA AAT TAA GAC CG-3'
Exp2mutReversePoint mutagenesis to remove restriction enzyme site5'-CGG TCT TAA TTT CCA GGA ACT CCG TGT GTA GG-3'
Bam.Gal4.FwdAttaching BamH1 sites to Gal4 in preparation for yeast vector ligation5'-AAA AGG ATC CAA AAA AAT GAA GCT ACT GTC TTC TAT C-3'
HmRXR.Hind.RevAttaching HindIII sites to HmRXR in preparation for yeast vector ligation5'-AAT TAA GCT TGG ATC CCT CGA GCT GCA GC-3'
LcRXR.Hind.RevAttaching HindIII sites to LcRXR in preparation for yeast vector ligation5'-GAT GAA GCT TGG ATC CCT CGA GCT GC-3'
VP16.Hind.FwdAttaching HindIII sites to VP16 in preparation for yeast vector ligation5'-AAA AAA GCT TAA AAA AAT GGC TAG CGC CGC CAC CAT G-3'
VP16.RevReverse primer for VP16.Hind.Fwd PCR - no modification5'-CTG CAG CGG CCG CTA CTA GTG CCC TTG GAA TTG ACG AGT AC-3'
NLS.serine.BB.FwdOligo to anneal NLS, with one bp difference to avoid self-complementarity 5'-/5Phos/CTA GAA GTT CTG TTG TTC ATC CTA AGA AGA AGA GAA AGG TTT GAA CTA GTA GCG GCC GCT GCA-3'
NLS.serine.BB.RevComplement to NLS.serine.BB.Fwd. for NLS annealing5'-/5Phos/GCG GCC GCT ACT AGT TCA AAC CTT TCT CTT CTT CTT AGG ATG AAC AAC AGA ACT T-3'
Fwd.BB.AtEXP2for amplification/extraction of an arabidopsis germination-specific promoter5'-CCT TTC TAG ACC TGG TGT GGT TTC TTT G-3'
Rev.BB.AtEXP2for amplification/extraction of an arabidopsis germination-specific promoter5'-AAG GCT GCA GCG GCC GCT ACT GAT ATT TGC TGA TGG GCT AAA G-3'
Fwd.BB.AtARP2for amplification/extraction of the arabidopsis ARP2 promoter5'-CCT TTC TAG ACC ATC ACA TAT TTG TAG AAT G-3'
Rev.BB.AtARP2for amplification/extraction of the arabidopsis ARP2 promoter5'-AAG GCT GCA GCG GCC GCT ACT AGT CTT CTC CGA TTT CTA TAG AG-3'
barnase2.BB.fwdaddition of biobrick sites/ends to barnase gene5'-CCT TTC TAG AAT GGT ACC GGT TAT CAA CAC G-3'
BB.Barnase.Revaddition of biobrick sites/ends to barnase gene5'-AAG GCT GCA GCG GCC GCT ACT AGT TTA TCT GAT TTT TGT AAA GGT CTG-3'
NLS.BB.Fwdaddition of biobrick sites/ends to the arabidopsis NLS5'-/5Phos/CTA GAT CTT CTG TTG TTC ATC CAT AGA AGA AGA GAA AGG TTT GAA CTA GTA GCG GCC GCT GCA-3'
NLS.BB.Revaddition of biobrick sites/ends to the arabidopsis NLS5'/5Phos/GCG GCC GCT ACT AGT TCA AAC CTT TCT CTT CTT CTT AGG ATG AAC AAC AGA AGA-3'
LacIn.BB.FwdAddition of NLS to the Lac Inhibitor gene to produce LacIN5'-CCT TGA ATT CGC GGC CGC ATC TAG AAT GAA AC AGT AAC GTT ATA CGA TGT C-3'
LacIN.BB.RevAddition of NLS to the Lac Inhibitor gene to produce LacIN5' AAG GTC GCA GCA GCG GCC GCT ACT AGT TCA AAC CTT TCT CTT CTT CTT AGG ATG AAC AAC AGA AGA CTG CCC GCT TTC CAG TCG GGA AA-3'
BB.Gal4DBD.FwdAdding biobrick sites to Gal4 DNA Binding Domain5'-CTT TTC TAG AAT GAA GCT ACT GTC TTC TAT C-3'
BB.Gal4DBD.RevAdding biobrick sites to Gal4 DNA Binding Domain5'-AAG GCT GCA GCG GCC GCT ACT AGT CGA TAC AGT CAA CTG TCT TTG-3'
BB.Barstar.Fwdaddition of biobrick sites/ends to barstar gene5'CCT TTC TAG AAT GAA AAA AGC AGT CAT TAA C-3'
BB.Barstar.Revaddition of biobrick sites/ends to barstar gene5'-AAG GCT GCA GCG GCC GCT ACT AGT TTA AGA AAG TATA GAT GGT GAT GTC-3'

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