Reference Icon RedbookChoosing Your Perfect Plasmid Backbone


Background

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.

Choose by:

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.

Host Relevant Promoters Representative Empty Backbones
Mammalian CMV, SV40, EF1a, CAG
Bacteria Lac, T7, araBAD
Yeast GAL4, PGK, ADH1, ADE2, TRP1
Fly UAS, MT
  • Targeted Gene Expression - Rubin lab plasmids for drosophila expression
  • pMT-puro - Inducible gene expression under the metallothionein promoter
  • pACU2 - Modified pUAST vector containing both P-elements and attB site for high expression of UAS-driven transgenes
Worm unc-54, variety of worm gene promoters
Insect / Baculovirus Polyhedrin
Xenopus SP6 for transcription and injection
  • pCS2P+ - Transcription from the SP6 promoter
  • pCS2 mt-GFP - Create myc-tag, GFP-tag fusions for transcription
  • pCSDest - Easy cloning of your gene using Gateway recombination
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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.

Tag or Fusion Protein Common uses Representative Empty Backbones
Flag Epitope tag
  • pcDNA3 Flag HA - N-terminal Flag-HA for mammalian expression
  • pNIC-CTHF - C-terminal TEV-His6-Flag for bacterial expression
  • pET Flag TEV - N-terminal Flag-TEV for bacterial expression
  • pCS2FLAG - N or C-terminal 2xFLAG tag in pCS for a variety of systems, including Xenopus
HA Epitope Tag
  • pKH3 - N or C-terminal 3xHA tag for mammalian expression
  • pHAHA - N-terminal double HA tag for mammalian expression
  • pCS2HA - N or C-terminal 2xHA tag in pCS for a variety of systems, including Xenopus
  • pAG423GPD-ccdB-HA - C-terminal 3xHA tag for yeast expression
Myc Epitope Tag
  • pKMyc - N-terminal Myc tag for mammalian expression
6xHis Epitope Tag
  • pEZYmyc-His - C-terminal Myc-His tag for mammalian expression (Gateway)
  • SGC plasmids - A variety of His tagged bacterial expression vectors
  • pET His6 TEV - N-terminal His6-TEV tag for bacterial expression
  • pET His6 - C-terminal His6 tag for bacterial expression
  • pDest-527- N-terminal His6 tag for bacterial expression (Gateway)
GST Protein Purification
  • pEBG - N-terminal GST for mammalian expression
  • pET His6 GST TEV - N-terminal His6-GST-TEV for bacterial expression
  • pDest-565 - N-terminal His6-GST for bacterial expression (Gateway)
  • pPS892 - N-terminal GST for yeast expression
  • pDM#834 - C-terminal GST for C elegans expression
GFP Localization
Other Fluorescent Proteins Localization See full Fluorescent Protein Guide
NLS Nuclear Localization
  • pENTR4-myc-nuc - pEntry vector that adds a C-terminal nuclear localization signal
  • FNpcDNA3 - N-terminal FLAG and SV40 nuclear localization signal
Myr Membrane Localization

For more information about tags, including amino acid sequences, see our list of common tags.

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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.

Selectable Marker Typical Host Organism Representative Empty Backbones
Neomycin (G418) Mammalian
Puromycin Mammalian
Hygromycin Mammalian
Zeocin/Bleo Mammalian
GFP Varies
URA3 Yeast
  • pXP518 - Yeast expression vector
  • pPS808 - Yeast expression with N-terminal GFP
  • pAG306GAL-ccdB - Yeast Gateway expression vector (see more Gateway vectors)
TRP1 Yeast
  • pXP216 - Yeast expression vector (PGK1 promoter, 2μ)
  • pXP316 - Yeast expression vector (TEF1 promoter, CEN/ARS)
  • pAG304GAL-ccdB - Yeast Gateway expression vector (see more Gateway vectors)
LEU2 Yeast
  • pXP122 - Yeast expression vector (PGK1 promoter, CEN/ARS)
  • pXP222 - Yeast expression vector (PGK1 promoter, 2μ)
  • pAG305GAL-ccdB - Yeast Gateway expression vector (see more Gateway vectors)
HIS3 Yeast
  • pXP220 - Yeast expression vector (PGK1 promoter, 2μ)
  • pAG303GAL-ccdB - Yeast Gateway expression vector (see more Gateway vectors)
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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. Below are some common delivery methods.

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 lentiviral resource page
Retroviral Easy and safe to use
  • pBabe plasmids - Mammalian retroviral gene expression, multiple selectable markers
  • pMKO.1 puro - Mammalian retroviral shRNA expression
  • MSCV-IRES-GFP - Mammalian retroviral gene expression with GFP selectable marker
  • LZRS-Rfa or pMXs-gw - Mammalian retroviral Gateway cloning vectors
Adenoviral High transduction efficiency, but do NOT integrate into host genome
  • pAdTrack-CMV - Shuttle vector for transgene expression under a CMV promoter
  • pAdTrack - Shuttle vector for transgene expression under your chosen promoter
  • pAdEasy®-1 - Recombine plasmids from the shuttle vectors into pAdEasy for viral production
  • AdEasier®-1 cells (strain) - Bacterial strain that contains AdEasy®-1 plasmid
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Reporters, shRNA expression, transgenics and genome modification

Element Details Representative Empty Backbones
Promoter Measure promoter strength
  • pBV-Luc - Luciferase reporter plasmid with very low basal activity
shRNA/RNAi For gene silencing experiments
Transgenic Expression in organisms
TALENs Gene targeting
  • TALEN kits - Construct a custom TALEN array for genomic engineering

Do you have suggestions for other plasmids or categories that should be added to this list?

Email help@addgene.org to help us improve this resource!