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Choosing Your Perfect Plasmid Backbone

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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

Epitope tag or fusion protein

Selectable or fluorescent marker

Viral expression and packaging

Reporters, shRNA expression, transgenics and genome modification

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
pSG5L Flag HA - Transient expression

pBABE-puro- Retroviral expression

pWPXL - lentiviral expression

pBI-MCS-EGFP- Tet-inducible

Transient expression - Gradia Lab plasmids for mammalian expression, Ligation Independent Cloning (LIC)

Bacteria
Lac, T7, araBAD
pBAD LIC cloning vector - Bacterial expression (see all expression vectors from the Gradia lab)

pET15-MHL - His-tagged bacterial expression vector (see tagged expression vectors from the SGC)

pPro18 - Propionate-inducible expression vector (see entire pPro collection)

Arrowsmith Lab Plasmids - More LIC bacterial cloning vectors

pTD plasmid series - Expression with Strep, His or optimized YFP tags for purification or localization experiments
Yeast
GAL4, PGK, ADH1, ADE2, TRP1
Gateway destination vectors - Inducible expression, fusion proteins, and more

Sandmeyer Lab Plasmids- Set of yeast expression vectors with various markers, promoters, etc

pRS420 - Yeast expression vector (see Cross lab collection for more)

LexA and pACT2.2 : yeast 2-hybrid plasmids
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
C elegans vector kit - Collection of plasmids from Dr. Andrew Fire

L4440- RNAi in C elegans

Dupuy Lab and Lehner Lab vectors - Tools for nematode transgenesis
Insect / Baculovirus
Polyhedrin
pFastBac LIC and pFastBac Dual LIC- Vectors from the Gradia lab

pFB-LIC-Bse- Insect expression vector from Gileadi lab
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

pEZYmyc - C-terminal myc tag for mammalian expression (Gateway)

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
pcDNA3-EGFP - C-terminal GFP for mammalian expression

pcDNA3 GFP LIC cloning vector - C-terminal GFP for mammalian expression

pPS808 - N-terminal GFP for yeast expression

pET GFP - C-terminal GFP for bacterial expression

pPD95_75 - C-terminal GFP for C elegans expression
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
pWZL-Neo-Myr-Flag-DEST - pDest vector that adds a N-terminal myristoylation signal
For more information about tags, including amino acid sequences, see our list of common tags.

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Selectable or Fluorescent Marker

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
pBABE-neo - retroviral gene expression

Tet-pLKO-neo - Tet-inducible lentiviral shRNA expression

pLenti CMV Neo DEST - Lentiviral Gateway destination vector for gene expression
Puromycin
Mammalian
pBABE-puro - Retroviral gene expression

pLKO.1 - TRC cloning vector - Lentiviral shRNA expression

Tet-pLKO-puro - Tet-inducible lentiviral shRNA expression

pMKO.1 puro - Retroviral shRNA expression

pLenti CMV Puro DEST - Lentiviral Gateway destination vector for gene expression
Hygromycin
Mammalian
pBABE-hygro - Retroviral gene expression

pLKO.1 hygro - Lentiviral shRNA expression

Tet-pLKO-puro - Tet-inducible lentiviral shRNA expression

pLenti CMV Hygro DEST - Lentiviral Gateway destination vector for gene expression

pLenti CMV/TO Hygro - Lentiviral Gateway destination vector for gene expression (tet inducible)
Zeocin/Bleo
Mammalian
pBABE-zeo - Retroviral gene expression

pLenti X1 GFP-Zeo DEST - Lentiviral Gateway destination for shRNA expression

pLenti CMV/TO Zeo DEST - Tet-inducible lentiviral Gateway destination vector for gene expression
GFP
Varies
pBABE GFP - Retroviral gene expression

pMKO.1 GFP - Retroviral shRNA expression

pSico PGK GFP - Conditional shRNA expression through lentiviral delivery

pLenti CMV GFP DEST - Lentiviral Gateway destination vector for gene expression
URA3
Yeast
pXP518 - Yeast expression vector (HX7−391 promoter, CEN/ARS - confirm with depositor?)

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

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
pLKO.1 - TRC cloning vector - Lentiviral plasmid for mammalian gene silencing

pMKO.1 puro - Retroviral plasmid for mammalian gene silencing

pLVTHM - lentiviral plasmid for mammalian gene silencing (tet-regulated)

L4440 - RNAi in C. elegans
Transgenic
Expression in organisms
pBigT - For making transgenic mice. Contains a loxP-flanked cassette

pBTG (pBigT-IRES-GFP) - pBigT modified to contain IRES-GFP

Hynes Lab Plasmids - A system for Cre-regulated RNAi in vivo

Dupuy Lab and Lehner Lab vectors - Tools for nematode transgenesis

Rosenberg Lab Plasmids - Transgenic single-copy gene expression in E. coli
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!

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