Choosing Your Perfect Plasmid Backbone

Empty backbones are plasmids containing only the elements needed for replication in the host cell and are designed for a user to insert a gene of interest. They may contain tags or fusion proteins, a multiple cloning site (MCS), an inducible promoter, and/or a selectable marker. They are frequently used in molecular biology to isolate, multiply, or express the insert they carry in the target cell. These vectors allow you to test the function of your gene of interest in a controlled environment under various conditions.

When choosing what plasmid backbone to use, you have many elements to consider. Here is a guide to a selection of Addgene's empty vector backbones. For the most part, we will assume that you want to express a gene; however, we have included a function section for if you want to stably express an insert using viral vectors, are studying a different genetic element, or want to express shRNA.

Choose by:

• Species-Specific Expression
• Epitope Tag or Fusion Protein
• Function: Viral Vector Delivery, Genome Modification, mRNA Regulation, and More
• Selectable Markers

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, Ubc	Transient expression - Gradia Lab plasmids for mammalian expression, Ligation Independent Cloning (LIC) pcDNA3.1-mCherry - Expresses mCherry tag in mammalian cells pBI-MCS-EGFP - Tet-inducible Find more Tet-inducible empty backbones on our Tetracycline (Tet) Inducible Expression page
Bacteria	Lac, T7, araBAD, trp	mTagBFP2-pBAD - Protein expression vector with C-terminal mTagBFP2 tag pNIC-GST-TEV-GG - Cloning of target gene with N-terminal GST tag and TEV cleavage Transient expression - Gradia Lab plasmids for bacterial expression, Ligation Independent Cloning (LIC) Also see our Bacterial Expression collection page
Plant	CaMV35S, RPS5A, Ubiquitin	pYPQ203 (pMDC32-Ubi1) - Multisite Gateway T-DNA entry plasmid (attR1 & attR2); Zea mays Ubi1 promoter and Hygromiycin resistance marker Also see more empty backbones on our Plant Plasmids and Resources collection page
Yeast	GAL4, PGK, ADH1, ADE2, TRP1	Gateway destination vectors - Inducible expression, fusion proteins, and more pGBKT7-GW - Gateway compatible Y2H vector - BD pRPR1_gRNA_handle_RPR1t - gRNA expression empty vector pFA6a vectors - Epitope tagging in S. pombe
Insect/Baculovirus	UAS, MT, Polyhedrin	Targeted Gene Expression - Rubin lab plasmids for Drosophila expression pMT-puro - Inducible gene expression under the metallothionein promoter pFastBac LIC and pFastBac Dual LIC - Vectors from the Gradia lab pFB-LIC-Bse - Insect expression vector from Gileadi lab
Worm	unc-54, variety of worm gene promoters	C. elegans vector kit - Collection of plasmids from the Fire Lab L4440 - RNAi in C. elegans Also see our Worm Plasmids and Resources collection page
Zebrafish	CMV, h2afv, XlEef1a1	See our dedicated Zebrafish Plasmids and Resources collection page

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.

Additionally, tags are commonly added to aid in protein purification. Just remember to remove the stop codon for C-terminal tags and omit the start codon for N-terminal tags. And 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.

Read more about epitope tags and protein tags.

Tag or Fusion Protein	Common uses	Representative Empty Backbones
Flag	Epitope tag	c-Flag pcDNA3 or FLAG-HA-pcDNA3.1 - C-terminal Flag or N-terminal FLAG-HA for mammalian expression pNIC-CTHF - C-terminal TEV-His6-Flag for bacterial expression pFA6a vectors - PCR-based C-terminal tagging in S. cerevisiae pCS2FLAG - N- or C-terminal 2xFLAG tag in pCS for a variety of systems, including Xenopus
HA	Epitope tag	pcDNA3.1-HA or c-Flag pcDNA3 - Mammalian expression vector with N- or C-terminal HA tag pInducer20 - Tet-inducible HA-tagged lentiviral vector for ORF expression pDest-565 - Gateway destination vector for bacterial expression; His,GST tag pE2c - Modified pENTR vector for C-terminal 3x HA tag fusion with your gene of interest in plants
Myc	Epitope tag	pKMyc - N-terminal Myc tag for mammalian expression pGEX-4T-1-3xMyc - Bacterial vector for Myc tag pETcon(-) - Yeast surface display vector with a C-terminal Myc tag pPMW-attB - pUASp plasmid with N-terminal Myc tag and attB for Drosophila transgenesis
His	Epitope tag	pEZYmyc-His - C-terminal Myc-His tag for mammalian expression (Gateway) pYIC - Bicistronic fluorescent reporter gene with cap-dependent 3Myc-EYFP-HA-His6 and IRES-dependent ECFP-HA-His6 translation for mammalian expression pDest-527 - N-terminal His6 tag for bacterial expression (Gateway) pYD1 - Expression, secretion, and display of proteins on the extracellular surface of S. cerevisiae cells His-tagged versions of pFastBac LIC vectors and pFastBac Dual LIC vectors for insect expression
Fluorescent proteins (GFP, mCherry, etc)	Localization	pcDNA3-EGFP - C-terminal GFP for mammalian expression pLV-mCherry - 3rd gen lentiviral plasmid expressing mCherry pBad-EBFP2 - Bacterial expression of EBFP2 blue fluorescent protein 35S-eGFP-nosT - Transient expression vector for eGFP in plants Find FP tagging vectors in budding yeast pHT101-mCherry - Expresses mCherry tagged fusion proteins in worms ins:MCS; cryaa:RFP - A zebrafish insulin promoter vector with multiple cloning sites Browse our full Fluorescent Protein (FP) Guide and the Michael Davidson FP Collection
NLS	Nuclear localization	dCas9 plasmid - dCas9 expression plasmid without effector fusions; 3X Flag tag; 2NLS; pCDNA3 vector backbone, mammalian expression pENTR4-myc-nuc - pEntry vector that adds a C-terminal nuclear localization signal pGADCg - Gateway yeast expression vector that adds a SV40 NLS
Myr	Membrane localization	pWZL-Neo-Myr-Flag-DEST - pDest vector that adds an N-terminal myristoylation signal
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) pE4n - Modified pENTR vector for plant expression of an N-terminal GST tag fusion with your gene of interest pFastbac1 Nterminal GST - Baculovirus expression vector with N-terminal GST pGCS-N5(GST) - GST tagged Gateway destination vector for mammalian, avian, xenopus, or zebrafish expression GST plasmids made by Addgene
MBP	Protein purification	pET28-MBP-TEV - Bacterial expression with MBP-TEV N-terminal fusions pDEST Gateway vectors - For the production of His6-MBP-tagged fusion proteins in E. coli
SUMO	Protein purification	pCIOX - Bacterial expression vector for His8-SUMO tagged recombinant protein pDest-Sumo - For prokaryotic expression of target protein as N-terminal fusion form with Sumo tag
Trx (thioredoxin)	Protein purification	pNH-TrxT - LIC cloning in bacteria with N-terminal His-TrxA-TEV tag
NusA	Protein purification	pNIC-HIS6-NUSA-TEV-GG - Cloning of target gene with N-terminal His6 NUSA tag and TEV cleavage pExp-NusA - Expression of protein in E. coli as TEV cleavable N-terminal His and NusA fusions

Function

Besides functions such as protein purification mentioned above, empty backbones can also be used for for viral vector delivery, genome modification, reporter assays, shRNA expression, transgenics, and more. See the next two tables for some other common uses.

Viral Vector Delivery

Although transient expression is sufficient for some experiments, scientists often want to create stable cell lines in which the expression cassette of interest is incorporated into the host DNA. For mammalian cells, you can do this through viral vector delivery. Visit our viral vector page for more information. Below are some common delivery methods.

Delivery method	Advantages	Representative Empty Backbones
AAV	High transduction efficiency, but do NOT integrate into the host genome	Addgene has an extensive collection of plasmids for packaging and expression, see our dedicated Adeno-associated Virus (AAV) Plasmids page
Lentiviral	Can transduce both dividing and nondividing cells, integrate into host genome	pLenti CMV GFP DEST - Lentiviral Gateway destination vector for gene expression pLenti CMV/TO Zeo DEST - Tet-inducible lentiviral Gateway destination vector for gene expression Lentiviral backbones made by Addgene Addgene has an extensive collection of plasmids for packaging and expression, see our dedicated Lentiviral Plasmids page
Retroviral	Easy and safe to use, integrate into host genome	MSCV-IRES-GFP or pMSCV-IRES-mCherry FP - Mammalian retroviral gene expression with GFP or mCherry selectable marker pMSCV-U6sgRNA(BbsI)-PGKpuro2ABFP - For retroviral delivery of one sgRNA pMKO.1 GFP - Retroviral shRNA expression Retroviral backbones made by Addgene
Adenoviral	High transduction efficiency, but do NOT integrate into host genome	pAdTrack-CMV - Shuttle vector for transgene expression under a CMV 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 View our Adenovirus Plasmids page for more information

Genome Modifications, Reporter Assays, mRNA Regulation, and More

Find empty backbones for other uses such as genome modification, reporter assays, shRNA expression, transgenics, and more.

Element	Details	Representative Empty Backbones
CRISPR	Genome modification	lentiCRISPR v2 - 3rd gen lentiviral vector for mammalian genome editing Tet-pLKO-puro and Tet-pLKO-neo - Tet-inducible lentiviral shRNA expression pnCasPA-BEC - A cytidine deaminase-mediated base-editing plasmid in Pseudomonas species Vectors for RNA-guided genome editing in plants Visit our CRISPR Plasmids and Resources collection, and browse our Empty gRNA Expression Vectors page
Cre-lox	Site-specific recombination	See our dedicated Cre-lox Plasmids page pBF3060 - Metabolic engineering in S. cerevisiae
TALENs	Gene targeting	TALEN kits - Construct a custom TALEN array for genomic engineering
Luciferase	Reporter	See our Luciferase collection of plasmid backbones into which you can clone your regulatory element or gene of interest to create a luciferase reporter
Promoter	Measure promoter strength	pBV-Luc - Luciferase reporter plasmid with very low basal activity
shRNA/RNAi	Gene silencing	Browse RNAi empty vectors for cloning in shRNAs, including plasmids for constitutive, conditional (Cre-lox), or inducible (Tet) expression
miRNA and 3' UTR	mRNA regulation	pIS0 - 3'UTR segments of target genes can be inserted into this firefly luciferase reporter to test for their effects on protein production AAV-GfaABC1D-MCS--4x6T-WPRE - For astrocyte-selective expression in AAV
Dual promoter	Separate expression	pRGEB31 - Deliver sgRNA and Cas9 into plants by Agrobacterium-mediated transformation Dual-sgRNA_hU6-mU6 - Lentiviral expression plasmid for two sgRNAs from an hU6 and an mU6 promoter
Protein-protein interaction	Proximity-dependent biotin identification (BioID)	MCS-BioID2-HA or myc-BioID2-MCS - To fuse your protein of interest to the N-terminus or C-terminus of BioID2 3xHA-TurboID-NLS_pCDNA3 or 3xHA-miniTurbo-NLS_pCDNA3 - Expresses 3xHA-tagged TurboID or miniTurbo in the mammalian nucleus pSF3-ultraID - Expresses ultraID in mammalian cells with the Tet-On or Tet-Off system

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, Varies	Tet-pLKO-neo - Tet-inducible lentiviral shRNA expression Find yeast transformation plasmids with G418 selection Find nematode transgenesis plasmids with Neomycin selection
Puromycin	Mammalian	Tet-pLKO-puro - Tet-inducible lentiviral shRNA expression pLKO.1 - TRC cloning vector - Lentiviral shRNA expression pLJM1-EGFP - 3rd gen lentiviral vector for EGFP fusion; PGK driven puromycin
Hygromycin	Mammalian, Varies	pLKO.1 hygro - Lentiviral shRNA expression lentiCRISPRv2 hygro - Variant of lentiCRISPR v2 that confers hygromycin resistance pRGEB32 - Express sgRNA/PTG with rice snoRNA U3 promoter and Cas9 with rice ubiquitin promoter for Agrobacterium-mediated transformation
Blasticidin	Mammalian	pLX304 - 3rd gen lentiviral vector lentiSAMv2 - Lentiviral sgRNA cloning backbone pLKO.1-blast - 3rd gen lentiviral backbone for cloning and expression of new shRNA sequences
Zeocin®/Bleo	Mammalian	lenti sgRNA(MS2)_zeo backbone - 3rd gen lenti sgRNA cloning backbone with MS2 loops at tetraloop and stemloop 2 and EF1a-zeo resistance marker pLenti CMV/TO GFP-Zeo DEST (719-1) - 3rd gen lentiviral Gateway destination vector, expression, CMV/TO promoter, GFP-Zeo pLenti CMV/TO Zeo DEST - Tet-inducible lentiviral Gateway destination vector for gene expression
Thy1.1	Mammalian	pLKO.3 Thy1.1 - Lentiviral shRNA expression
Gentamicin	Varies	pJL-TRBO - An improved agroinfection-compatible Tobacco mosaic virus (TMV)-based transient expression vector
GFP	Varies	pMKO.1 GFP - Retroviral shRNA expression pLenti CMV GFP DEST - Lentiviral Gateway destination vector for gene expression pINDUCER21 (ORF-EG) - Inducible lentiviral gene expression vector
URA3	Yeast	pAG306GAL-ccdB - Yeast Gateway expression vector (see more Gateway vectors) pCAP03-acc(3)IV - Facilitate the capture and expression of bacterial gene clusters S. cerevisiae plasmids for multiple marker selection
TRP1	Yeast	pGBKCg - Gateway compatible Y2H vector
LEU2	Yeast	pGADT7-GW - Gateway compatible Y2H vector - AD pGADCg - Gateway compatible Y2H vector
BASTA	Plant	pGTQL1211YN - Gateway compatible BiFC vector (pEarleyGate201-YN)

Zeocin® is an InvivoGen trademark.

Content last reviewed 28 May 2025.