Sequencing Primers
Though Addgene mainly uses next-generation sequencing (NGS) for plasmid verification, Addgene has used a number of primers for Sanger sequence verification over the years. Below is a list of popular universal primers available from most sequencing services, along with a more complete list of common sequencing primers. These lists are provided for your reference, Addgene does not distribute primers.
To identify primers that may be useful in your sequencing reaction, find your plasmid’s page and see what primers are listed under "5' sequencing primer" and "3' sequencing primer" or explore the sequence map in SnapGene or other software to see additional annotated primers. You can also use Addgene’s protocol to design your own primers. Still have questions about choosing the best primer for your plasmid? Email us at [email protected].
For additional information on molecular biology, plasmids, and recombinant DNA, please consult Addgene's Molecular Biology Reference page.
Universal Sequencing Primers
| Name | Sequence (5' to 3') | Description | Direction |
|---|---|---|---|
| BGH-R | TAGAAGGCACAGTCGAGG | Bovine growth hormone terminator | Reverse |
| CMV Forward | CGCAAATGGGCGGTAGGCGTG | Human CMV immediate early promoter | Forward |
| EGFP-C | CATGGTCCTGCTGGAGTTCGTG | 3' end of EGFP | Forward |
| EGFP-N | CGTCGCCGTCCAGCTCGACCAG | 5' end of EGFP | Reverse |
| hU6-F | GGGAAACGCCTGGTATCTTT | Human U6 promoter | Forward |
| LKO.1 5' (U6) | GACTATCATATGCTTACCGT | Human U6 promoter | Forward |
| M13 Forward (-21) | TGTAAAACGACGGCCAGT | In lacZ gene | Forward |
| M13 Reverse | CAGGAAACAGCTATGAC | In lacZ gene | Reverse |
| M13/pUC Forward | CCCAGTCACGACGTTGTAAAACG | In lacZ gene | Foward |
| M13/pUC Reverse | AGCGGATAACAATTTCACACAGG | In lacZ gene | Reverse |
| MSCV Forward | CCCTTGAACCTCCTCGTTCGACC | Murine stem cell virus | Forward |
| MSCV Reverse | CAGCGGGGCTGCTAAAGCGCATGC | Murine stem cell virus | Reverse |
| pBABE 5' | CTTTATCCAGCCCTCAC | Psi packaging signal, 5' of MCS in pBABE vectors | Forward |
| pBAD Forward | ATGCCATAGCATTTTTATCC | For vectors with E. coli araBAD promoter | Forward |
| pBAD Reverse | GATTTAATCTGTATCAGG | For vectors with E. coli araBAD promoter | Reverse |
| pBluescript KS | TCGAGGTCGACGGTATC | For pBluescript vector | Forward |
| pBluescript SK | TCTAGAACTAGTGGATC | For pBluescript vector | Reverse |
| pGEX 3' | CCGGGAGCTGCATGTGTCAGAGG | 3' end of MCS in pGEX vectors | Reverse |
| pGEX 5' | GGGCTGGCAAGCCACGTTTGGTG | 3' end of glutathione-S-transferase | Forward |
| SP6 | ATTTAGGTGACACTATAG | SP6 promoter | Forward |
| T3 | GCAATTAACCCTCACTAAAGG | T3 promoter | Forward |
| T7 | TAATACGACTCACTATAGGG | T7 promoter | Forward |
| T7 Terminal | GCTAGTTATTGCTCAGCGG | T7 terminator | Reverse |
Other Common Sequencing Primers
| Name | Sequence (5' to 3') | Description | Direction |
|---|---|---|---|
| 3'AOX1 | GCAAATGGCATTCTGACATCC | For Pichia vectors with AOX1 terminator | Reverse |
| 5'AOX1 | GACTGGTTCCAATTGACAAGC | For Pichia vectors with AOX1 promoter | Forward |
| 35S promoter | CTATCCTTCGCAAGACCCTTC | CaMV 35S promoter | Forward |
| AC5 | ACACAAAGCCGCTCCATCAG | Drosophila Actin 5C promoer | Forward |
| Alpha-factor | TACTATTGCCAGCATTGCTGC | Alpha factor signal sequence | Forward |
| Amp-R | ATAATACCGCGCCACATAGC | 5' end of ampicillin resistance gene | Reverse |
| AUG1 Forward | CAATTTACATCTTTATTTATTAACG | For Pichia vectors with AUG1 promoter | Forward |
| AUG1 Reverse | GAAGAGAAAAACATTAGTTGGC | For Pichia vectors with AUG1 promoter | Reverse |
| BGH-R | TAGAAGGCACAGTCGAGG | Bovine growth hormone terminator | Reverse |
| Bglob-intron-F | CTGGTCATCATCCTGCCTTT | Rabbit beta-globin intron | Forward |
| Bglob-intron-R | TTTGCCCCCTCCATATAACA | Rabbit beta-globin intron | Reverse |
| Bglob-pA-R | TTTTGGCAGAGGGAAAAAGA | Rabbit beta-globin polyA region | Reverse |
| CAT-R | GCAACTGACTGAAATGCCTC | 5' end of chloramphenicol resistance gene | Reverse |
| CMV Forward | CGCAAATGGGCGGTAGGCGTG | Human CMV immediate early promoter | Forward |
| CRE-R | GCAAACGGACAGAAGCATTT | 5' end of Cre recombinase | Reverse |
| CYC1 | GCGTGAATGTAAGCGTGAC | CYC1 transcriptioin termination signal/td> | Reverse |
| DsRed1-C | AGCTGGACATCACCTCCCACAACG | 3' end of DsRed1 | Forward |
| DsRed1-N | GTACTGGAACTGGGGGGACAG | 5' end of DsRed1 | Reverse |
| EBV Reverse | GTGGTTTGTCCAAACTCATC | SV40 polyA terminator | Reverse |
| Ecdysone Forward | CTCTGAATACTTTCAACAAGTTAC | Drosophila heat shock promoter | Forward |
| EF-1α Forward | TCAAGCCTCAGACAGTGGTTC | Human elongation factor-1α promoter | Forward |
| EGFP-C | CATGGTCCTGCTGGAGTTCGTG | 3' end of EGFP | Forward |
| EGFP-N | CGTCGCCGTCCAGCTCGACCAG | 5' end of EGFP | Reverse |
| EXFP-R | GTCTTGTAGTTGCCGTCGTC | For distinguishing EGFP vs ECFP vs EYFP | Reverse |
| F1ori-F | GTGGACTCTTGTTCCAAACTGG | F1 origin | Forward |
| F1ori-R | AGGGAAGAAAGCGAAAGGAG | F1 origin | Reverse |
| GAL1 | AATATACCTCTATACTTTAACGTC | S. cerevisiae GAL1 promoter | Forward |
| Gal10pro-F | GGTGGTAATGCCATGTAATATG | S. cerevisiae GAL10 promoter | Forward |
| Gal4 N-term | GAGTAGTAACAAAGGTCAA | 3' end of Gal4 DNA binding domain | Forward |
| Gal4-AD | AATACCACTACAATGGAT | 3' end of Gal4 activation domain | Forward |
| GFP-F | GGTCCTTCTTGAGTTTGTAAC | 3' end of GFP | Forward |
| GFP-R | CCATCTAATTCAACAAGAATTGGGACAAC | 5' end of GFP | Reverse |
| GPDpro-F | CGGTAGGTATTGATTGTAATTCTG | S. cerevisiae GPD promoter | Forward |
| GW-3' | GCATGATGACCACCGATATG | 3' end of Gateway cassette | Forward |
| GW-5' | AATCTCGCCGGATCCTAACT | 5' end of Gateway cassette | Reverse |
| H1 | TCGCTATGTGTTCTGGGAAA | Human H1 promoter | Forward |
| HA-F | TACCCATACGACGTCCCAGA | HA tag | Forward |
| HA-R | TCTGGGACGTCGTATGGGTA | HA tag | Reverse |
| HAT | GAGGAGCACGCTCATGCCCAC | Histidine affinity tag | Forward |
| hGH-PA-R | CCAGCTTGGTTCCCAATAGA | Human growth hormone terminator | Reverse |
| hU6-F | GGGAAACGCCTGGTATCTTT | Human U6 promoter | Forward |
| hUBCpro-F | TGAAGCTCCGGTTTTGAACT | Human Ubiquitin C (UbC) promoter | Forward |
| IRES-F | TGGCTCTCCTCAAGCGTATT | 3' end of IRES | Forward |
| IRES-R | CCTCACATTGCCAAAAGACG | 5' end of IRES | Reverse |
| L4440 | AGCGAGTCAGTGAGCGAG | 5' of MCS in L4440 vector | Forward |
| LacI-R | GGCATACTCTGCGACATCGT | 5' end of LacI | Reverse |
| LacZ-R | GACAGTATCGGCCTCAGGAA | 5' end of LacZ | Reverse |
| LexA | CGTCAGCAGAGCTTCACCATTG | 3' end of LexA DNA binding domain | Forward |
| LKO.1 5' (U6) | GACTATCATATGCTTACCGT | Human U6 promoter | Forward |
| LNCX | AGCTCGTTTAGTGAACCGTCAGATC | Human CMV promoter | Forward |
| Luc-F | AGTCAAGTAACAACCGCGA | 3' end of luciferase | Forward |
| LucNrev | CCTTATGCAGTTGCTCTCC | 5' end of luciferase | Reverse |
| M13 Forward (-21) | TGTAAAACGACGGCCAGT | In lacZ gene | Forward |
| M13 Forward (-40) | GTTTTCCCAGTCACGAC | In lacZ gene | Forward |
| M13 Reverse | CAGGAAACAGCTATGAC | In lacZ gene | Reverse |
| M13/pUC Forward | CCCAGTCACGACGTTGTAAAACG | In lacZ gene | Forward |
| M13/pUC Reverse | AGCGGATAACAATTTCACACAGG | In lacZ gene | Reverse |
| MBP-F | GATGAAGCCCTGAAAGACGCGCAG | 3' end of maltose binding protein | Forward |
| mCherry-F | CCCCGTAATGCAGAAGAAGA | 3' end of mCherry | Forward |
| mCherry-R | TTGGTCACCTTCAGCTTGG | 5' end of mCherry | Reverse |
| MT Forward | CATCTCAGTGCAACTAAA | Drosophila metallothionein promoter | Forward |
| MMLV-F | ATCAGTTCGCTTCTCGCTTC | Moloney murine leukemia virus (MoMuLV) LTR | Forward |
| mPGK-F | CATTCTGCACGCTTCAAAAG | Mouse PGK promoter | Forward |
| MSCV | CCCTTGAACCTCCTCGTTCGACC | Murine stem cell virus | Forward |
| MSCV-rev | CAGCGGGGCTGCTAAAGCGCATGC | Murine stem cell virus | Reverse |
| MT1-F | GCTGTCCTCTAAGCGTCACC | Mouse metallothionein 1 promoter | Forward |
| mU6-F | CAGCACAAAAGGAAACTCACC | Mouse U6 promoter | Forward |
| Myc | GCATCAATGCAGAAGCTGATCTCA | Myc tag | Forward |
| Neo-F | CGTTGGCTACCCGTGATATT | 3' end of neomycin resistance gene | Forward |
| Neo-R | GCCCAGTCATAGCCGAATAG | 5' end of neomycin resistance gene | Reverse |
| NOS-F | GCGTTCAAAAGTCGCCTAAG | Nopaline synthase promoter | Forward |
| Nmt1-F | GCAATGTGCAGCGAAACTAA | S. pombe nmt1 promoter | Forward |
| OpIE2 Forward | CGCAACGATCTGGTAAACAC | OpIE2 promoter | Forward |
| pACYC-F | TGAAGTCAGCCCCATACGAT | p15A origin | Forward |
| pAd-CMV | GCTAGAGATCTGGTACCGTC | For cloning sites after SalI in pAd-CMV vector | Forward |
| pBABE 3' | ACCCTAACTGACACACATTCC | SV40 enhancer, 3' of MCS in pBABE vectors | Reverse |
| pBABE 5' | CTTTATCCAGCCCTCAC | Psi packaging signal, 5' of MCS in pBABE vectors | Forward |
| pBAD Forward | ATGCCATAGCATTTTTATCC | For vectors with E. coli araBAD promoter | Forward |
| pBAD Reverse | GATTTAATCTGTATCAGG | For vectors with E. coli araBAD promoter | Reverse |
| pBluescriptKS | TCGAGGTCGACGGTATC | For pBluescript vector | Forward |
| pBluescriptSK | TCTAGAACTAGTGGATC | For pBluescript vector | Reverse |
| pBMN 5' | GCTTGGATACACGCCGC
MMLV sequence, for inserts in pBMN retroviral vector |
MMLV sequence, for inserts in pBMN retroviral vector | Forward |
| pBR322ori-F | GGGAAACGCCTGGTATCTTT | pBRS322 origin | Forward |
| pBRforBam | CTTGGAGCCACTATCGAC | In pBR322 tet region, upstream of BamHI site | Forward |
| pBRforEco | AATAGGCGTATCACGAGGC | In pBR322, upstream of EcoRI site | Forward |
| pBRrevBam | GGTGATGTCGGCGATATAGG | In pBR322 tet region, downstream of BamHI site | Reverse |
| pCAG-F | GCAACGTGCTGGTTATTGTG | Rabbit beta-globin intron, for pCAG plasmids | Forward |
| pCasper-F | GGGTTTTATTAACTTACAT | 5' end of Drosophila mini-white gene | Reverse |
| pCasper-hs | GCAACTACTGAAATCTGCCAAG | Drosophila Hsp70 promoter | Forward |
| pcDL-F | GTTGCCTTTACTTCTAGGCCT | 5' of EcoRI site in pcDL vector | Forward |
| pENTR-F | CTACAAACTCTTCCTGTTAGTTAG | 5' of attL1 in pENTR vector | Forward |
| pENTR-R | ATGGCTCATAACACCCCTTG | 3' of attL2 in pENTR vector/td> | Reverse |
| pGEX 3' | CCGGGAGCTGCATGTGTCAGAGG | 3' of MCS in pGEX vectors | Reverse |
| pGEX 5' | GGGCTGGCAAGCCACGTTTGGTG | 3' end of glutathione-S-transferase | Forward |
| pGP704-R | AACAAGCCAGGGATGTAACG | R6K gamma origin, 3' of MCS in pGP704 vector | Reverse |
| pHybLex Reverse | GAGTCACTTTAAAATTTGTATACAC | ADH terminator | Reverse |
| pLTet-F | ACTGAGCACATCAGCAGGAC | Lambda phage early leftward (pL) promoter | Forward |
| pLXSN 5' (MSCV) | CCCTTGAACCTCCTCGTTCGACC | Murine stem cell virus, same as MSCV | Forward |
| pMRB101-F | AAGATGCAGGCAGCTGAGTT | HCMV major immediate-early protein (IE) | Forward |
| pMT2-F | TTGCCTTTCTCTCCACAGGT | 3' end of synthetic intron | Forward |
| pMX-S1811 | GACGGCATCGCAGCTTGGATACAC | MMLV sequence, 5' of MCS in pMXs vector | Forward |
| Polyhedrin forward | AAATGATAACCATCTCGC | Polyhedrin promoter | Forward |
| Polyhedrin reverse | GTCCAAGTTTCCCTG | For baculovirus vector with polyhedrin promoter | Reverse |
| pQE promoter | CCCGAAAAGTGCCACCTG | 5' of MCS in pQE vectors | Forward |
| pREP Forward | GCTCGATACAATAAACGCC | Rous sarcoma virus (RSV) promoter | Forward |
| pRS-marker | CGGCATCAGAGCAGATTGTA | To sequence yeast selectable marker in pRS vectors | Forward |
| Pry1 | CTTAGCATGTCCGTGGGGTTTGAAT | PZ P-element | Reverse |
| pTrcHis Forward | GAGGTATATATTAATGTATCG | 5' of MCS in pTrcHis vector | Forward |
| pTrcHis Reverse | GATTTAATCTGTATCAGG | 3' of MCS in pTrcHis vector, same as pBAD-R | Reverse |
| Puro-F | GCAACCTCCCCTTCTACGAGC | 3' end of puromycin resistance gene | Forward |
| Puro-R | GTGGGCTTGTACTCGGTCAT | 5' end of puromycin resistance gene | Reverse |
| pZIP | TCCTTTCCAGCGAGGTTCTA | Murine leukemia virus (MuLV) | Reverse |
| RCAS-F | ACATGGGTGGTGGTATAGCGCTTGCG | 3' of Rous sarcoma virus (RSV) env gene | Forward |
| Rluc-F | CCAGGATTCTTTTCCAATGC | 3' end of Renilla luciferase | Forward |
| RVprimer3 | CTAGCAAAATAGGCTGTCCC | 5' of MCS in pGL3 vector | Forward |
| SFFV-F | ATTGATTGACTGCCCACCTC | Spleen focus forming virus 5' LTR | Forward |
| SP6 | ATTTAGGTGACACTATAG | SP6 promoter | Forward |
| SV40pA-R | GAAATTTGTGATGCTATTGC | SV40 polyA | Reverse |
| SV40pro-F | TATTTATGCAGAGGCCGAGG | SV40 promoter/origin | Forward |
| SV40-spliceR | CACAAAGATCCGGACCAAAG | SV40 splice sequence | Reverse |
| T3 | GCAATTAACCCTCACTAAAGG | T3 promoter | Forward |
| T7 | TAATACGACTCACTATAGGG | T7 promoter | Forward |
| T7 Terminal | GCTAGTTATTGCTCAGCGG | T7 terminator | Reverse |
| Tac promoter | GAGCGGATAACAATTTCACACAGG | Tac promoter | Forward |
| tdTomato-Fwd | CTGTTCCTGTACGGCATGG | 3' end of tdTomato | Forward |
| tdTomato-Rev | TCTTTGATGACGGCCATGT | 5' end of tdTomato | Reverse |
| Tet-R | GGCGAGTTTACGGGTTGTTA | 5' end of tetracycline resistance gene | Reverse |
| TK-pA-R | TTGTCTCCTTCCGTGTTTCA | Thymidine kinase polyA | Reverse |
| Tn7-end | GGGGTGGAAATGGAGTTTTT | Bacteria transposon Tn7 | Reverse? |
| TRC-F | CAAGGCTGTTAGAGAGATAATTGGA | Human U6 promoter | Forward |
| Ubx-F | AACTCGTACTTTGAACAGGC | Drosophila Ultrabithorax gene | Forward |
| V5 Reverse | ACCGAGGAGAGGGTTAGGGAT | V5 epitope | Reverse |
| WPRE-R | CATAGCGTAAAAGGAGCAACA | 5' end of WPRE | Reverse |
| XBG-R | GACTCCATTCGGGTGTTC | Xenopus beta-globin 3'UTR | Reverse |
| XEF1a | TTTCGCCCTAACTTCGTGAT | Xenopus EF1 alpha enhancer/promoter | Forward |
| Xpress Forward | TATGGCTAGCATGACTGGT | Xpress epitope | Forward |
Content last reviewed: 22 October 2025