Choosing 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
• Epitope tag or fusion protein
• Selectable markers
• 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 pWPXL - Lentiviral expression pBI-MCS-EGFP - Tet-inducible Transient expression - Gradia Lab plasmids for mammalian expression, Ligation Independent Cloning (LIC)
Bacteria	Lac, T7, araBAD	p15TV-L - 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) pLexA 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

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	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.

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	Tet-pLKO-neo - Tet-inducible lentiviral shRNA expression pLenti CMV Neo DEST - Lentiviral Gateway destination vector for gene expression
Puromycin	Mammalian	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	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	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 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. Visit our viral vector page for more information. 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 plasmid 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. View our AAV page for more information.	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