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=== Choosing A Plasmid Backbone ===
When choosing what plasmid backbone to use, you have many elements to consider. Here is a [https://www.addgene.org/empty-backbones/ guide to Addgene's empty vector backbones]. For the most part, we will assume that you want to express a gene; however, we have a section at the end for if you are studying a different genetic element or want to express shRNA.


<html>
<section id="host">
  <h2>Species-Specific Expression</h2>


  <p>If you want to drive expression of your favorite gene, you will need
    a plasmid with a promoter that will be functional in your host
    organism.</p>


  <table class="empty-backbone-table table table-striped table-hover">
    <thead>
      <tr>
        <th>Host</th>


        <th>Relevant Promoters</th>


        <th>Representative Empty Backbones</th>
      </tr>
    </thead>


    <tbody>
      <tr>
        <td>Mammalian</td>


== Choosing A Plasmid Backbone ==
        <td>CMV, SV40, EF1a, CAG</td>


Background
        <td>
          <ul>
            <li><a href="http://www.addgene.org/10791/">pSG5L Flag HA</a> - Transient
              expression</li>


When choosing what plasmid backbone to use, you have many elements to consider. Here is a guide to Addgene's empty vector backbones. For the most part, we will assume that you want to express a gene; however, we have a section at the end for if you are studying a different genetic element or want to express shRNA.
            <li><a href="http://www.addgene.org/1764/">pBABE-puro</a> - Retroviral
              expression</li>
 
            <li><a href="http://www.addgene.org/12257/">pWPXL</a> - Lentiviral
              expression</li>
 
            <li><a href="http://www.addgene.org/16542/">pBI-MCS-EGFP</a> -
              Tet-inducible</li>
 
            <li><a href="http://www.addgene.org/browse/article/4218/">Transient
                expression</a> - Gradia Lab plasmids for mammalian
              expression, Ligation Independent Cloning (LIC)</li>
          </ul>
        </td>
      </tr>
 
      <tr>
        <td>Bacteria</td>
 
        <td>Lac, T7, araBAD</td>
 
        <td>
          <ul>
            <li><a href="http://www.addgene.org/37501/">pBAD LIC cloning vector</a> -
              Bacterial expression (see <a href=
                "/Scott_Gradia/">all expression vectors</a> from
              the Gradia lab)</li>
 
            <li><a href="http://www.addgene.org/26092/">p15TV-L</a> - His-tagged
              bacterial expression vector (see <a href=
                "/sgc/">tagged expression vectors</a> from the
              SGC)</li>
 
            <li><a href="http://www.addgene.org/17806/">pPro18</a> -
              Propionate-inducible expression vector (see entire
              <a href="http://www.addgene.org/browse/article/2026/">pPro
                collection</a>)</li>
 
            <li><a href="http://www.addgene.org/browse/article/3609/">Arrowsmith Lab
                Plasmids</a> - More LIC bacterial cloning
              vectors</li>
 
            <li><a href="http://www.addgene.org/browse/article/6833/">pTD plasmid
                series</a> - Expression with Strep, His or
              optimized YFP tags for purification or localization
              experiments</li>
          </ul>
        </td>
      </tr>
 
      <tr>
        <td>Yeast</td>
 
        <td>GAL4, PGK, ADH1, ADE2, TRP1</td>
 
        <td>
          <ul>
            <li><a href="http://www.addgene.org/yeast-gateway/">Gateway destination
                vectors</a> - Inducible expression, fusion
              proteins, and more</li>
 
            <li><a href="http://www.addgene.org/browse/article/3784/">Sandmeyer Lab
                Plasmids</a> - Set of yeast expression vectors with
              various markers, promoters, etc</li>
 
            <li><a href="http://www.addgene.org/11259/">pRS420</a> - Yeast expression
              vector (see <a href="http://www.addgene.org/browse/article/509/">Cross
                lab collection</a> for more)</li>
 
            <li><a href="http://www.addgene.org/11342/">pLexA</a> and <a href=
                "/11343/">pACT2.2</a> - Yeast 2-hybrid
              plasmids</li>
          </ul>
        </td>
      </tr>
 
      <tr>
        <td>Fly</td>
 
        <td>UAS, MT</td>
 
        <td>
          <ul>
            <li><a href="http://www.addgene.org/browse/article/3631/">Targeted Gene
                Expression</a> - Rubin lab plasmids for drosophila
              expression</li>
 
            <li><a href="http://www.addgene.org/17923/">pMT-puro</a> - Inducible gene
              expression under the metallothionein promoter</li>
 
            <li><a href="http://www.addgene.org/31223/">pACU2</a> - Modified pUAST
              vector containing both P-elements and attB site for
              high expression of UAS-driven transgenes</li>
          </ul>
        </td>
      </tr>
 
      <tr>
        <td>Worm</td>
 
        <td>unc-54, variety of worm gene promoters</td>
 
        <td>
          <ul>
            <li><a href="http://www.addgene.org/firelab/">C. elegans vector kit</a> -
              Collection of plasmids from Dr. Andrew Fire</li>
 
            <li><a href="http://www.addgene.org/1654/">L4440</a> - RNAi in C.
              elegans</li>
 
            <li><a href="http://www.addgene.org/browse/article/3655/">Dupuy Lab</a>
              and <a href="http://www.addgene.org/browse/article/5402/">Lehner Lab</a>
              vectors - Tools for nematode transgenesis</li>
          </ul>
        </td>
      </tr>
 
      <tr>
        <td>Insect / Baculovirus</td>
 
        <td>Polyhedrin</td>
 
        <td>
          <ul>
            <li><a href="http://www.addgene.org/browse/article/4215/">pFastBac LIC</a> and <a href=
                "/browse/article/4217/">pFastBac Dual LIC</a> -
              Vectors from the Gradia lab</li>
 
            <li><a href="http://www.addgene.org/26108/">pFB-LIC-Bse</a> - Insect
              expression vector from Gileadi lab</li>
          </ul>
        </td>
      </tr>
 
      <tr>
        <td>Xenopus</td>
 
        <td>SP6 for transcription and injection</td>
 
        <td>
          <ul>
            <li><a href="http://www.addgene.org/17095/">pCS2P+</a> - Transcription
              from the SP6 promoter</li>
 
            <li><a href="http://www.addgene.org/15681/">pCS2 mt-GFP</a> - Create
              myc-tag, GFP-tag fusions for transcription</li>
 
            <li><a href="http://www.addgene.org/22423/">pCSDest</a> - Easy cloning of
              your gene using Gateway recombination</li>
          </ul>
        </td>
      </tr>
    </tbody>
  </table><a href="#top">Back to Top</a>
</section>
 
<section id="tag">
  <h2>Epitope Tag or Fusion Protein</h2>
 
  <p>Tags and fusion proteins are excellent tools for further
    understanding the function of your favorite gene. For example, fusing
    your protein to an epitope tag, such as Flag or HA, will allow you to
    easily identify your protein using an antibody against that epitope.
    This could allow you to conduct western blots or immunoprecipitations
    of your favorite gene even if you do not have an antibody against it.
    Another common scenario is fusing your protein to another protein, such
    as GFP, which allows you to visualize the cellular localization of your
    protein.</p>
 
  <p>Just remember that when you are designing your plasmid you should
    keep your gene "in frame" with the fusion protein. This means that the
    final product should be translated as a single string of amino acids
    that preserves the sequence of your gene and of the fusion protein.</p>
 
  <table class="empty-backbone-table table table-striped table-hover">
    <thead>
      <tr>
        <th>Tag or Fusion
          Protein</th>
 
        <th>Common uses</th>
 
        <th>Representative Empty
          Backbones</th>
      </tr>
    </thead>
    <tbody>
 
      <tr>
        <td>Flag</td>
 
        <td>Epitope tag</td>
 
        <td>
          <ul>
            <li><a href="http://www.addgene.org/10792/">pcDNA3 Flag HA</a> -
              N-terminal Flag-HA for mammalian expression</li>
 
            <li><a href="http://www.addgene.org/26105/">pNIC-CTHF</a> - C-terminal
              TEV-His6-Flag for bacterial expression</li>
 
            <li><a href="http://www.addgene.org/29715/">pET Flag TEV</a> - N-terminal
              Flag-TEV for bacterial expression</li>
 
            <li><a href="http://www.addgene.org/16331/">pCS2FLAG</a> - N or C-terminal
              2xFLAG tag in pCS for a variety of systems,
              including Xenopus</li>
          </ul>
        </td>
      </tr>
 
      <tr>
        <td>HA</td>
 
        <td>Epitope Tag</td>
 
        <td>
          <ul>
            <li><a href="http://www.addgene.org/12555/">pKH3</a> - N or C-terminal
              3xHA tag for mammalian expression</li>
 
            <li><a href="http://www.addgene.org/12517/">pHAHA</a> - N-terminal double
              HA tag for mammalian expression</li>
 
            <li><a href="http://www.addgene.org/16330/">pCS2HA</a> - N or C-terminal
              2xHA tag in pCS for a variety of systems, including
              Xenopus</li>
 
            <li><a href="http://www.addgene.org/14246/">pAG423GPD-ccdB-HA</a> -
              C-terminal 3xHA tag for yeast expression</li>
          </ul>
        </td>
      </tr>
 
      <tr>
        <td>Myc</td>
 
        <td>Epitope Tag</td>
 
        <td>
            <ul>
                <li><a href="http://www.addgene.org/19400/">pKMyc</a> - N-terminal Myc
              tag for mammalian expression</li>
 
            </ul>
        </td>
      </tr>
 
      <tr>
        <td>6xHis</td>
 
        <td>Epitope Tag</td>
 
        <td>
          <ul>
            <li><a href="http://www.addgene.org/18701/">pEZYmyc-His</a> - C-terminal
              Myc-His tag for mammalian expression (Gateway)</li>
 
            <li><a href="http://www.addgene.org/sgc/">SGC plasmids</a> - A variety of
              His tagged bacterial expression vectors</li>
 
            <li><a href="http://www.addgene.org/29653/">pET His6 TEV</a> - N-terminal
              His6-TEV tag for bacterial expression</li>
 
            <li><a href="http://www.addgene.org/37236/">pET His6</a> - C-terminal
              His6 tag for bacterial expression</li>
 
            <li><a href="http://www.addgene.org/11518/">pDest-527</a>- N-terminal
              His6 tag for bacterial expression (Gateway)</li>
          </ul>
        </td>
      </tr>
 
      <tr>
        <td>GST</td>
 
        <td>Protein Purification</td>
 
        <td>
          <ul>
            <li><a href="http://www.addgene.org/22227/">pEBG</a> - N-terminal GST for
              mammalian expression</li>
 
            <li><a href="http://www.addgene.org/29655/">pET His6 GST TEV</a> -
              N-terminal His6-GST-TEV for bacterial
              expression</li>
 
            <li><a href="http://www.addgene.org/11520/">pDest-565</a> - N-terminal
              His6-GST for bacterial expression (Gateway)</li>
 
            <li><a href="http://www.addgene.org/8908/">pPS892</a> - N-terminal GST
              for yeast expression</li>
 
            <li><a href="http://www.addgene.org/31240/">pDM#834</a> - C-terminal GST
              for C elegans expression</li>
          </ul>
        </td>
      </tr>
 
      <tr>
        <td>GFP</td>
 
        <td>Localization</td>
 
        <td>
          <ul>
            <li><a href="http://www.addgene.org/13031/">pcDNA3-EGFP</a> - C-terminal
              GFP for mammalian expression</li>
 
            <li><a href="http://www.addgene.org/30127/">pcDNA3 GFP LIC cloning
                vector</a> - C-terminal GFP for mammalian
              expression</li>
 
            <li><a href="http://www.addgene.org/8856/">pPS808</a> - N-terminal GFP
              for yeast expression</li>
 
            <li><a href="http://www.addgene.org/29772/">pET GFP</a> - C-terminal GFP
              for bacterial expression</li>
 
            <li><a href="http://www.addgene.org/1494/">pPD95_75</a> - C-terminal GFP
              for C elegans expression</li>
          </ul>
        </td>
      </tr>
 
      <tr>
        <td>Other Fluorescent Proteins</td>
 
        <td>Localization</td>
 
        <td>See full <a href="http://www.addgene.org/fluorescent-proteins/">Fluorescent
            Protein Guide</a></td>
      </tr>
 
      <tr>
        <td>NLS</td>
 
        <td>Nuclear Localization</td>
 
        <td>
          <ul>
            <li><a href="http://www.addgene.org/20026/">pENTR4-myc-nuc</a> - pEntry
              vector that adds a C-terminal nuclear localization
              signal</li>
 
            <li><a href="http://www.addgene.org/45346/">FNpcDNA3</a> - N-terminal
              FLAG and SV40 nuclear localization signal</li>
          </ul>
        </td>
      </tr>
 
      <tr>
        <td>Myr</td>
 
        <td>Membrane Localization</td>
 
        <td>
          <ul>
            <li><a href="http://www.addgene.org/15300/">pWZL-Neo-Myr-Flag-DEST</a> -
              pDest vector that adds a N-terminal myristoylation
              signal</li>
          </ul>
        </td>
      </tr>
    </tbody>
  </table>
 
  <p>For more information about tags, including amino acid sequences, see
    our <a href="http://www.addgene.org/mol_bio_reference/genetic_code/#tags">list of common
      tags</a>.</p><a href="#top">Back to Top</a>
</section>
 
<section id="selection">
  <h2>Selectable Markers</h2>
 
 
  <p>Regardless of your delivery method, it's unlikely that all of your
    cells will take up your plasmid. Thus, many plasmids have markers on
    them so that you can find or select for only the cells that received
    the plasmid.</p>
 
  <table class="empty-backbone-table table table-striped table-hover">
    <thead>
      <tr>
        <th>Selectable
          Marker</th>
 
        <th>Typical Host
          Organism</th>
 
        <th>Representative Empty
          Backbones</th>
      </tr>
    </thead>
    <tbody>
 
      <tr>
        <td>Neomycin (G418)</td>
 
        <td>Mammalian</td>
 
        <td>
          <ul>
            <li><a href="http://www.addgene.org/1767/">pBABE-neo</a> - Retroviral
              gene expression</li>
 
            <li><a href="http://www.addgene.org/21916/">Tet-pLKO-neo</a> -
              Tet-inducible lentiviral shRNA expression</li>
 
            <li><a href="http://www.addgene.org/17392/">pLenti CMV Neo DEST</a> -
              Lentiviral Gateway destination vector for gene
              expression</li>
          </ul>
        </td>
      </tr>
 
      <tr>
        <td>Puromycin</td>
 
        <td>Mammalian</td>
 
        <td>
          <ul>
            <li><a href="http://www.addgene.org/1764/">pBABE-puro</a> - Retroviral
              gene expression</li>
 
            <li><a href="http://www.addgene.org/10878/">pLKO.1 - TRC cloning
                vector</a> - Lentiviral shRNA expression</li>
 
            <li><a href="http://www.addgene.org/21915/">Tet-pLKO-puro</a> -
              Tet-inducible lentiviral shRNA expression</li>
 
            <li><a href="http://www.addgene.org/8452/">pMKO.1 puro</a> - Retroviral
              shRNA expression</li>
 
            <li><a href="http://www.addgene.org/17452/">pLenti CMV Puro DEST</a> -
              Lentiviral Gateway destination vector for gene
              expression</li>
          </ul>
        </td>
      </tr>
 
      <tr>
        <td>Hygromycin</td>
 
        <td>Mammalian</td>
 
        <td>
          <ul>
            <li><a href="http://www.addgene.org/1765/">pBABE-hygro</a> - Retroviral
              gene expression</li>
 
            <li><a href="http://www.addgene.org/24150/">pLKO.1 hygro</a> - Lentiviral
              shRNA expression</li>
 
            <li><a href="http://www.addgene.org/21915/">Tet-pLKO-puro</a> -
              Tet-inducible lentiviral shRNA expression</li>
 
            <li><a href="http://www.addgene.org/17454/">pLenti CMV Hygro DEST</a> -
              Lentiviral Gateway destination vector for gene
              expression</li>
 
            <li><a href="http://www.addgene.org/17484/">pLenti CMV/TO Hygro</a> -
              Lentiviral Gateway destination vector for gene
              expression (tet inducible)</li>
          </ul>
        </td>
      </tr>
 
      <tr>
        <td>Zeocin/Bleo</td>
 
        <td>Mammalian</td>
 
        <td>
          <ul>
            <li><a href="http://www.addgene.org/1766/">pBABE-zeo</a> - Retroviral
              gene expression</li>
 
            <li><a href="http://www.addgene.org/19277/">pLenti X1 GFP-Zeo DEST</a> -
              Lentiviral Gateway destination for shRNA
              expression</li>
 
            <li><a href="http://www.addgene.org/17294/">pLenti CMV/TO Zeo DEST</a> -
              Tet-inducible lentiviral Gateway destination vector
              for gene expression</li>
          </ul>
        </td>
      </tr>
 
      <tr>
        <td>GFP</td>
 
        <td>Varies</td>
 
        <td>
          <ul>
            <li><a href="http://www.addgene.org/10668/">pBABE GFP</a> - Retroviral
              gene expression</li>
 
            <li><a href="http://www.addgene.org/10676/">pMKO.1 GFP</a> - Retroviral
              shRNA expression</li>
 
            <li><a href="http://www.addgene.org/12093/">pSico PGK GFP</a> -
              Conditional shRNA expression through lentiviral
              delivery</li>
 
            <li><a href="http://www.addgene.org/19732/">pLenti CMV GFP DEST</a> -
              Lentiviral Gateway destination vector for gene
              expression</li>
          </ul>
        </td>
      </tr>
 
      <tr>
        <td>URA3</td>
 
        <td>Yeast</td>
 
        <td>
          <ul>
            <li><a href="http://www.addgene.org/26846/">pXP518</a> - Yeast expression
              vector</li>
 
            <li><a href="http://www.addgene.org/8856/">pPS808</a> - Yeast expression
              with N-terminal GFP</li>
 
            <li><a href="http://www.addgene.org/14139/">pAG306GAL-ccdB</a> - Yeast
              Gateway expression vector (see <a href=
                "/yeast-gateway/browse/">more</a> Gateway
              vectors)</li>
          </ul>
        </td>
      </tr>
 
      <tr>
        <td>TRP1</td>
 
        <td>Yeast</td>
 
        <td>
          <ul>
            <li><a href="http://www.addgene.org/26830/">pXP216</a> - Yeast expression
              vector (PGK1 promoter, 2&mu;)</li>
 
            <li><a href="http://www.addgene.org/26836/">pXP316</a> - Yeast expression
              vector (TEF1 promoter, CEN/ARS)</li>
 
            <li><a href="http://www.addgene.org/14135/">pAG304GAL-ccdB</a> - Yeast
              Gateway expression vector (see <a href=
                "/yeast-gateway/browse/">more</a> Gateway
              vectors)</li>
          </ul>
        </td>
      </tr>
 
      <tr>
        <td>LEU2</td>
 
        <td>Yeast</td>
 
        <td>
          <ul>
            <li><a href="http://www.addgene.org/26827/">pXP122</a> - Yeast expression
              vector (PGK1 promoter, CEN/ARS)</li>
 
            <li><a href="http://www.addgene.org/26833/">pXP222</a> - Yeast expression
              vector (PGK1 promoter, 2&mu;)</li>
 
            <li><a href="http://www.addgene.org/14137/">pAG305GAL-ccdB</a> - Yeast
              Gateway expression vector (see <a href=
                "/yeast-gateway/browse/">more</a> Gateway
              vectors)</li>
          </ul>
        </td>
      </tr>
 
      <tr>
        <td>HIS3</td>
 
        <td>Yeast</td>
 
        <td>
          <ul>
            <li><a href="http://www.addgene.org/26832/">pXP220</a> - Yeast expression
              vector (PGK1 promoter, 2&mu;)</li>
 
            <li><a href="http://www.addgene.org/14133/">pAG303GAL-ccdB</a> - Yeast
              Gateway expression vector (see <a href=
                "/yeast-gateway/browse/">more</a> Gateway
              vectors)</li>
          </ul>
        </td>
      </tr>
    </tbody>
  </table><a href="#top">Back to Top</a>
</section>
 
<section id="viral">
  <h2>Viral Expression and Packaging</h2>
 
  <p>Although transient expression is sufficient for some experiments,
    scientists often want to create stable cell lines in which the plasmid
    is incorporated into the host DNA. For mammalian cells, you can do this
    through viral delivery. <a href="http://www.addgene.org/viral-vectors/">Visit our viral vector page for more information.</a> Below are some common delivery methods:</p>
 
  <table class="empty-backbone-table table table-striped table-hover">
    <tbody>
      <thead>
      <tr>
        <th>Delivery
          method</th>
 
        <th>Advantages</th>
 
        <th>Representative Empty
          Backbones</th>
      </tr>
    </thead>
 
    <tbody>
      <tr>
        <td>Lentiviral</td>
 
        <td>Can transduce both dividing and nondividing
          cells</td>
 
        <td>Addgene has an extensive collection of plasmids for
          packaging and expression. See our dedicated <a href=
            "/viral-vectors/lentivirus/">lentiviral plasmid page</a>.</td>
      </tr>
 
      <tr>
        <td>Retroviral</td>
 
        <td>Easy and safe to use</td>
 
        <td>
          <ul>
            <li><a href="http://www.addgene.org/browse/article/102/">pBabe
                plasmids</a> - Mammalian retroviral gene
              expression, multiple selectable markers</li>
 
            <li><a href="http://www.addgene.org/8452/">pMKO.1 puro</a> - Mammalian
              retroviral shRNA expression</li>
 
            <li><a href="http://www.addgene.org/20672/">MSCV-IRES-GFP</a> - Mammalian
              retroviral gene expression with GFP selectable
              marker</li>
 
            <li><a href="http://www.addgene.org/31601/">LZRS-Rfa</a> or <a href=
                "/18656/">pMXs-gw</a> - Mammalian retroviral Gateway
              cloning vectors</li>
          </ul>
        </td>
      </tr>
 
      <tr>
        <td>Adenoviral</td>
 
        <td>High transduction efficiency, but do NOT integrate into
          host genome. <a href="http://www.addgene.org/viral-vectors/aav/">View our AAV page for more information.</a></td>
 
        <td>
          <ul>
            <li><a href="http://www.addgene.org/16405/">pAdTrack-CMV</a> - Shuttle
              vector for transgene expression under a CMV
              promoter</li>
 
            <li><a href="http://www.addgene.org/16404/">pAdTrack</a> - Shuttle vector
              for transgene expression under your chosen
              promoter</li>
 
            <li><a href="http://www.addgene.org/16400/">pAdEasy&reg;-1</a> - Recombine
              plasmids from the shuttle vectors into pAdEasy for
              viral production</li>
 
            <li><a href="http://www.addgene.org/16399/">AdEasier&reg;-1 cells
                (strain)</a> - Bacterial strain that contains
              AdEasy&reg;-1 plasmid</li>
          </ul>
        </td>
      </tr>
    </tbody>
  </table><a href="#top">Back to Top</a>
</section>
 
<section id="special">
  <h2>Reporters, shRNA expression, transgenics and genome
    modification</h2>
 
  <table class="empty-backbone-table table table-striped table-hover">
    <thead>
      <tr>
        <th>Element</th>
 
        <th>Details</th>
 
        <th>Representative Empty
          Backbones</th>
      </tr>
    </thead>
    <tbody>
 
      <tr>
        <td>Promoter</td>
 
        <td>Measure promoter strength</td>
 
        <td>
          <ul>
            <li><a href="http://www.addgene.org/16539/">pBV-Luc</a> - Luciferase
              reporter plasmid with very low basal activity</li>
          </ul>
        </td>
      </tr>
 
      <tr>
        <td>shRNA/RNAi</td>
 
        <td>For gene silencing experiments</td>
 
        <td>
          <ul>
            <li><a href="http://www.addgene.org/10878/">pLKO.1 - TRC cloning
                vector</a> - Lentiviral plasmid for mammalian gene
              silencing</li>
 
            <li><a href="http://www.addgene.org/8452/">pMKO.1 puro</a> - Retroviral
              plasmid for mammalian gene silencing</li>
 
            <li><a href="http://www.addgene.org/12247/">pLVTHM</a> - lentiviral
              plasmid for mammalian gene silencing
              (tet-regulated)</li>
 
            <li><a href="http://www.addgene.org/1654/">L4440</a> - RNAi in C.
              elegans</li>
          </ul>
        </td>
      </tr>
 
      <tr>
        <td>Transgenic</td>
 
        <td>Expression in organisms</td>
 
        <td>
          <ul>
            <li><a href="http://www.addgene.org/21270/">pBigT</a> - For making
              transgenic mice. Contains a loxP-flanked
              cassette</li>
 
            <li><a href="http://www.addgene.org/15037/">pBTG (pBigT-IRES-GFP)</a> -
              pBigT modified to contain IRES-GFP</li>
 
            <li><a href="http://www.addgene.org/browse/article/2323/">Hynes Lab
                Plasmids</a> - A system for Cre-regulated RNAi in
              vivo</li>
 
            <li><a href="http://www.addgene.org/browse/article/3655/">Dupuy Lab</a>
              and <a href="http://www.addgene.org/browse/article/5402/">Lehner Lab</a>
              vectors - Tools for nematode transgenesis</li>
 
            <li><a href="http://www.addgene.org/browse/article/4030/">Rosenberg Lab
                Plasmids</a> - Transgenic single-copy gene
              expression in E. coli</li>
          </ul>
        </td>
      </tr>
 
      <tr>
        <td>TALENs</td>
 
        <td>Gene targeting</td>
 
        <td>
          <ul>
            <li><a href="http://www.addgene.org/TALEN/">TALEN kits</a> - Construct a
              custom TALEN array for genomic engineering</li>
          </ul>
        </td>
      </tr>
    </tbody>
  </table>
</section>
</html>
 
 
[[Category:Neurobiology]] [[Category:Synaptic Plasticity]] [[Category:Neuroinformatics]] [[Category:Genetics]] [[Category:Neuroscience Methods]] [[Category:Methods]]

Latest revision as of 14:19, 5 August 2015

Choosing A Plasmid Backbone

When choosing what plasmid backbone to use, you have many elements to consider. Here is a guide to Addgene's empty vector backbones. For the most part, we will assume that you want to express a gene; however, we have a section at the end for if you are studying a different genetic element or want to express shRNA.

<html> <section id="host">

Species-Specific Expression

If you want to drive expression of your favorite gene, you will need a plasmid with a promoter that will be functional in your host organism.

<thead> </thead> <tbody> </tbody>
Host Relevant Promoters Representative Empty Backbones
Mammalian CMV, SV40, EF1a, CAG
Bacteria Lac, T7, araBAD
Yeast GAL4, PGK, ADH1, ADE2, TRP1
Fly UAS, MT
Worm unc-54, variety of worm gene promoters
Insect / Baculovirus Polyhedrin
Xenopus SP6 for transcription and injection

<a href="#top">Back to Top</a>

</section>

<section id="tag">

Epitope Tag or Fusion Protein

Tags and fusion proteins are excellent tools for further understanding the function of your favorite gene. For example, fusing your protein to an epitope tag, such as Flag or HA, will allow you to easily identify your protein using an antibody against that epitope. This could allow you to conduct western blots or immunoprecipitations of your favorite gene even if you do not have an antibody against it. Another common scenario is fusing your protein to another protein, such as GFP, which allows you to visualize the cellular localization of your protein.

Just remember that when you are designing your plasmid you should keep your gene "in frame" with the fusion protein. This means that the final product should be translated as a single string of amino acids that preserves the sequence of your gene and of the fusion protein.

<thead> </thead> <tbody> </tbody>
Tag or Fusion Protein Common uses Representative Empty Backbones
Flag Epitope tag
HA Epitope Tag
Myc Epitope Tag
6xHis Epitope Tag
GST Protein Purification
GFP Localization
Other Fluorescent Proteins Localization See full <a href="http://www.addgene.org/fluorescent-proteins/">Fluorescent Protein Guide</a>
NLS Nuclear Localization
Myr Membrane Localization

For more information about tags, including amino acid sequences, see our <a href="http://www.addgene.org/mol_bio_reference/genetic_code/#tags">list of common tags</a>.

<a href="#top">Back to Top</a>

</section>

<section id="selection">

Selectable Markers


Regardless of your delivery method, it's unlikely that all of your cells will take up your plasmid. Thus, many plasmids have markers on them so that you can find or select for only the cells that received the plasmid.

<thead> </thead> <tbody> </tbody>
Selectable Marker Typical Host Organism Representative Empty Backbones
Neomycin (G418) Mammalian
Puromycin Mammalian
Hygromycin Mammalian
Zeocin/Bleo Mammalian
GFP Varies
URA3 Yeast
TRP1 Yeast
LEU2 Yeast
HIS3 Yeast

<a href="#top">Back to Top</a>

</section>

<section id="viral">

Viral Expression and Packaging

Although transient expression is sufficient for some experiments, scientists often want to create stable cell lines in which the plasmid is incorporated into the host DNA. For mammalian cells, you can do this through viral delivery. <a href="http://www.addgene.org/viral-vectors/">Visit our viral vector page for more information.</a> Below are some common delivery methods:

<tbody> <thead> </thead> <tbody> </tbody>
Delivery method Advantages Representative Empty Backbones
Lentiviral Can transduce both dividing and nondividing cells Addgene has an extensive collection of plasmids for 
         packaging and expression. See our dedicated <a href=
"/viral-vectors/lentivirus/">lentiviral plasmid page</a>.
Retroviral Easy and safe to use
Adenoviral High transduction efficiency, but do NOT integrate into host genome. <a href="http://www.addgene.org/viral-vectors/aav/">View our AAV page for more information.</a>

<a href="#top">Back to Top</a>

</section>

<section id="special">

Reporters, shRNA expression, transgenics and genome modification

<thead> </thead> <tbody> </tbody>
Element Details Representative Empty Backbones
Promoter Measure promoter strength
shRNA/RNAi For gene silencing experiments
Transgenic Expression in organisms
TALENs Gene targeting

</section> </html>

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