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>
<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> TEXT </td><td> INSERT TEXT HERE </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> TEXT </td><td> INSERT TEXT HERE </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> TEXT </td><td> INSERT TEXT HERE </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> TEXT</td><td> INSERT TEXT HERE </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> TEXT </td><td> INSERT TEXT HERE </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>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>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>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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</table>
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<br>
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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>
<br /><br />
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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>
<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>
<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>
<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><tdExp2MutForward</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>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>
         <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>
         <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>Bam.Gal4.Fwd</td><td>INSERT DESCRIPTION HERE</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>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>HmRXR.Hind.Rev</td><td>INSERT DESCRIPTION HERE</td><td>5'-AAT TAA GCT TGG ATC CCT CGA GCT GCA GC-3'</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>LCRXR</td><td>INSERT DESCRIPTION HERE</td><td>5'-GAT GAA GCT TGG ATC CCT CGA GCT GC-3'</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>VP16.Hind.Fwd</td><td>INSERT DESCRIPTION HERE</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>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>VP16.Rev</td><td>INSERT DESCRIPTION HERE</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>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>NLS.serine.BB.Fwd</td><td>INSERT DESCRIPTION HERE</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>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>NLS.serine.BB.Rev</td><td>INSERT DESCRIPTION HERE</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>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>
<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>
<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>
<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>
<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>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>
<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>
<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>
<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>LacIn.BB.Fwd</td><td>INSERT DESCRIPTION HERE</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>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>LacIN.BB.Rev</td><td>INSERT DESCRIPTION HERE</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>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>BB.Gal3DBD.Fwd</td><td>INSERT DESCRIPTION HERE</td><td>5'-CTT TTC TAG AAT GAA GCT ACT GTC TTC TAT C-3'</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>BB.Gal4DBD.Rev</td><td>INSERT DESCRIPTION HERE</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>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>
<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>
<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>
<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>
<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></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>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>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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