Cloning & Expression Vectors — Comparison Table
A quick-reference table of 15 widely used cloning and protein expression vectors — from the classic pUC19 and pET-28a(+) to mammalian, insect, yeast and lentiviral backbones. Each row lists the empty-vector size, origin of replication and copy number, selection marker, promoter and any fusion tag, so you can pick the right backbone for your host and purification strategy.
| Vector | Size | Category | Origin | Copy | Selection | Promoter | Fusion tags | Induction |
|---|---|---|---|---|---|---|---|---|
| pUC19 | 2,686 bp | E. coli cloning | pMB1 (pUC-modified, rop-deleted) | high | Ampicillin | lac (lacZα for blue/white screening) | — | IPTG (for lacZα α-complementation / blue-white) |
| pBR322 | 4,361 bp | E. coli cloning | pMB1 (rop-intact) | medium | Ampicillin + Tetracycline | none (no expression cassette) | — | none |
| pET-28a(+) | 5,369 bp | E. coli expression | pBR322 (ColE1-type) | medium | Kanamycin | T7lac | N-terminal 6xHis, thrombin site, T7-tag, optional C-terminal 6xHis | IPTG (requires T7 RNA polymerase host, e.g. BL21(DE3)) |
| pET-22b(+) | 5,493 bp | E. coli expression | pBR322 (ColE1-type) | medium | Ampicillin | T7lac | N-terminal pelB leader (periplasmic export), optional C-terminal 6xHis | IPTG (T7 RNA polymerase host, e.g. BL21(DE3)) |
| pGEX-6P-1 | 4,984 bp* | E. coli expression | pBR322 (ColE1-type) | medium | Ampicillin | tac | N-terminal GST (glutathione S-transferase) | IPTG |
| pMAL-c5X | 5,677 bp | E. coli expression | pMB1 (ColE1-type) | medium | Ampicillin | Ptac | N-terminal MBP (maltose-binding protein, ~42 kDa) | IPTG |
| pACYCDuet-1 | 4,008 bp | E. coli expression | p15A | low | Chloramphenicol | T7lac (two independent MCS/cassettes) | MCS1: N-terminal 6xHis, MCS2: C-terminal S-tag | IPTG (T7 RNA polymerase host) |
| pCDFDuet-1 | 3,781 bp* | E. coli expression | CloDF13 | medium | Streptomycin/Spectinomycin | T7lac (two independent MCS/cassettes) | MCS1: N-terminal 6xHis, MCS2: C-terminal S-tag | IPTG (T7 RNA polymerase host) |
| pBAD/His B | 4,092 bp | E. coli expression | pBR322 (ColE1-type) | medium | Ampicillin | araBAD (PBAD) | N-terminal 6xHis, Xpress epitope, EK (enterokinase) site | L-arabinose (dose-titratable); repressed by glucose via araC |
| pcDNA3.1(+) | 5,428 bp | mammalian | pUC ori (E. coli) + SV40 ori (episomal in SV40-T cells) + f1 | high | Ampicillin + Neomycin/G418 (mammalian, via SV40-driven neo) | CMV (immediate-early enhancer/promoter) | — | constitutive |
| pEGFP-N1 | 4,733 bp | mammalian | pUC ori (E. coli) + f1 | high | Kanamycin (E. coli) + Neomycin/G418 (mammalian) | CMV | C-terminal EGFP (fuses EGFP to C-terminus of insert) | constitutive |
| pFastBac1 | 4,775 bp | insect | pUC ori (ColE1-type, E. coli) | high | Ampicillin (E. coli) + Gentamicin (bacmid transposition in DH10Bac) | polyhedrin (PPH) | — | baculoviral (late/very-late PPH expression post-infection) |
| pPICZ A | 3,329 bp* | yeast | pUC ori (E. coli); no yeast ori — integrates at AOX1 locus | high | Zeocin (both E. coli and Pichia) | AOX1 (methanol-inducible) | C-terminal myc epitope, C-terminal 6xHis | Methanol (induces PAOX1); repressed by glucose/glycerol |
| pGAPZ A | 3,147 bp | yeast | pUC ori (E. coli); no yeast ori — integrates at GAP locus | high | Zeocin (both E. coli and Pichia) | GAP (glyceraldehyde-3-phosphate dehydrogenase; constitutive) | C-terminal myc epitope, C-terminal 6xHis | constitutive (glucose-driven; no methanol needed) |
| pLKO.1 - TRC | 7,032 bp | lentiviral | pUC ori (E. coli); packaged as lentivirus (HIV-1 based) | high | Ampicillin (E. coli) + Puromycin (mammalian) | U6 (shRNA); hPGK (PuroR) | — | constitutive (U6 Pol III drives shRNA) |
Sizes are for the empty parent vector (no insert). * marks entries where the size or a listed feature could not be pinned to a single authoritative value — confirm against the map for your batch.
Origins, copy number and compatibility
The origin of replication sets both copy number and compatibility. High-copy pMB1/pUC origins (pUC19, pcDNA3.1) yield lots of DNA; p15A and CloDF13 origins are lower-copy and, crucially, belong to different compatibility groups. That is why Duet vectors with distinct origins and distinct selection markers can be co-maintained in one cell to express several proteins at once.
Promoters, tags and induction
Expression vectors pair a promoter with an inducer: T7lac and tac promoters respond to IPTG, araBAD to arabinose, and AOX1 to methanol. Fusion tags (His, GST, MBP) drive affinity purification and often solubility, while a downstream protease cleavage site lets you remove the tag afterwards. If you plan to express in a new host, check the codon usage of your insert first.
Frequently asked questions
What is the difference between a cloning vector and an expression vector?
A cloning vector (e.g. pUC19, pBR322) is designed simply to carry and propagate a DNA fragment in E. coli — it maximizes copy number and screening convenience but has no promoter to drive protein production. An expression vector adds a promoter, ribosome binding site and often a fusion tag so the cloned gene is transcribed and translated into protein in the chosen host.
What does the origin of replication tell me?
The origin (ori) determines how the plasmid is maintained and its copy number in E. coli. High-copy pMB1/pUC origins give hundreds of copies per cell (good DNA yield); p15A, CloDF13 and ColE1-derived low/medium-copy origins give fewer copies. Different origins are also compatibility groups — plasmids with different origins (and different selection markers) can coexist in one cell, which is how Duet co-expression systems work.
Which affinity tag should I choose?
A 6xHis tag (pET-28a, pBAD/His) is small and purifies on Ni-NTA under native or denaturing conditions. GST (pGEX) and MBP (pMAL) are larger solubility-enhancing tags purified on glutathione or amylose resin. Reporter/localization work uses EGFP (pEGFP-N1). Many vectors include a protease site (thrombin, PreScission, Factor Xa, enterokinase) so the tag can be removed after purification.
Why do the sizes here sometimes differ from other databases?
Sizes are for the empty parent vector with no insert. A few entries are flagged as uncertain because the manufacturer product page rounds the figure or lists reading-frame variants that differ by a few bp (e.g. the pBAD A/B/C variants, or pLKO.1 with vs. without its stuffer fragment). Always confirm against the exact map for your batch.
Do I need a special host strain?
Often, yes. T7lac vectors (pET, Duet series) require a host carrying T7 RNA polymerase such as BL21(DE3). Bac-to-Bac transfer vectors (pFastBac) are transposed into a bacmid in DH10Bac. Pichia vectors (pPICZ, pGAPZ) integrate into the yeast genome and are expressed in Pichia pastoris. Lentiviral transfer vectors (pLKO.1) need packaging and envelope plasmids to make virus.
What is IPTG induction?
IPTG (isopropyl β-D-1-thiogalactopyranoside) is a non-metabolisable lactose analogue that relieves lac repression, switching on lac-, tac- and T7lac-driven promoters. Adding IPTG to a growing culture triggers expression of the cloned gene. Arabinose (pBAD) and methanol (pPICZ) are alternative inducers for their respective promoter systems.
Learn more
Sources
- 1GenBank: L09137 (pUC19), J01749 (pBR322), U55762 (pEGFP-N1)NCBI NucleotideThe empty-vector sizes and feature content of the three rows that have a real primary sequence record: pUC19 2,686 bp (L09137, 'Cloning vector pUC19c, complete sequence'), pBR322 4,361 bp (J01749), pEGFP-N1 4,733 bp (U55762). All three LOCUS lines match the page exactly. Note for pBR322: Sutcliffe's original determination (Cold Spring Harb Symp Quant Biol 43:77-90, 1979, doi:10.1101/sqb.1979.043.01.013) reported 4,362 bp; the 4,361 bp the page shows is the corrected figure that GenBank carries, so cite the accession rather than the 1979 paper for that number. The originating papers, if the page wants them, are Yanisch-Perron, Vieira & Messing, Gene 33:103-119 (1985), doi:10.1016/0378-1119(85)90120-9 for pUC19, and Sutcliffe 1979 for pBR322.
- 2Addgene Vector DatabaseAddgeneSizes and headline features for the commercial rows with no GenBank record: pET-28a(+) 5,369 bp / Kan / N- and C-terminal His with thrombin site (record 2565); pGEX-6P-1 4,984 bp / tac / Amp / N-terminal GST + PreScission site (record 2887 — this is the exact reference the repo cites, and the only specific bp figure available since Cytiva's own page says only '~4900 bp'); pcDNA3.1(+) 5,428 bp / CMV / Amp + Neo / pUC ori (record 2093); pFastBac1 4,775 bp / polyhedrin / Amp (record 2712); pACYCDuet-1 4,008 bp / P15A replicon / Cm (record 1680); pCDFDuet-1 3,781 bp / CloDF13 replicon / Sm-Spec / 6xHis + S-tag (record 5878); pBAD/His B / Amp / Invitrogen (record 1828). Caveat to state if this is cited publicly: Addgene labels this database 'no longer actively maintained', and it is a compilation of manufacturer maps rather than a primary determination.
- 3Addgene plasmid #10878 — pLKO.1 - TRC cloning vectorAddgene (deposited by the RNAi Consortium / Root & Sabatini labs)The entire pLKO.1 - TRC row: 7,032 bp empty backbone (Addgene states 7,032 bp backbone, ~8,932 bp total with the stuffer, which is the page's '~8.9 kb parent plasmid'), ampicillin at 100 ug/mL for E. coli, puromycin as the mammalian marker, 3rd-generation lentiviral transfer plasmid requiring separate packaging and envelope plasmids. The originating paper is Moffat J et al., Cell 124(6):1283-1298 (2006), doi:10.1016/j.cell.2006.01.040.
- 4Invitrogen vector product pages: pcDNA3.1(+) (V79020) and pPICZ A/B/C (V19020)Thermo Fisher ScientificThe Origin, Selection, Promoter, Fusion tags and Induction columns for the Invitrogen rows: pcDNA3.1(+) — CMV enhancer-promoter, BGH polyA, SV40 ori, pUC ori, Geneticin (G-418) for mammalian plus ampicillin for bacterial selection; pPICZ A (catalog V19020) — inducible AOX1 promoter, Zeocin selection for multicopy integrants, C-terminal 6xHis and c-myc tags, Pichia pastoris host, methanol induction. These pages do not state bp sizes; the sizes come from elsewhere (see unsourced).
- 5Use of bacteriophage T7 RNA polymerase to direct selective high-level expression of cloned genesStudier FW, Moffatt BA · Journal of Molecular Biology 189(1):113-130 · 1986The T7 / T7lac expression claims rather than any single row: why the pET-28a(+), pET-22b(+), pACYCDuet-1 and pCDFDuet-1 rows read 'T7lac' and 'IPTG (requires T7 RNA polymerase host, e.g. BL21(DE3))', and the FAQ answer 'T7lac vectors (pET, Duet series) require a host carrying T7 RNA polymerase such as BL21(DE3)'. This is the original description of the system; the per-vector feature lists themselves are Novagen's (pET System Manual TB055 and the Duet vectors manual TB340, both Merck-published).
Related tools and references
Tools
Render a circular or linear map with restriction sites.
Auto-detect promoters, tags, origins and resistance markers in a plasmid, then run a deep scan against pLannotate's full feature databases.
Paste a GenBank, RefSeq or UniProt accession and get the FASTA or GenBank record.