pCAG-GFP
pCAG-GFP (Catalog No.: PVT1036) is a mammalian expression plasmid where the powerful CAG hybrid promoter drives EGFP (Enhanced Green Fluorescent Protein) expression. The CAG promoter combines the CMV immediate early enhancer with the chicken β-actin core promoter and first intron, providing exceptionally strong and consistent constitutive expression across diverse mammalian cell types. This 5556 bp vector offers multiple cloning sites (EcoRI, KpnI, XmaI) for target gene insertion, dual replication origins (ColE1 for bacterial propagation, SV40 for episomal maintenance in mammalian cells expressing large T antigen), ampicillin resistance for bacterial selection, and rabbit β-globin polyadenylation signal for mRNA stability. Ideal for transient transfection, gene function analysis, subcellular localization tracking, and drug screening applications in HEK293T, HeLa, CHO, and other mammalian cell lines.
Ordering Information
| Catalog No. |
Product Name |
Size |
Form |
Availability |
| PVT1036 |
pCAG-GFP |
5556bp / 2ug |
Lyophilized powder |
In Stock |
Technical Specifications & Vector Traits
| Vector Type |
Mammalian Expression Plasmid; Breast-feeding Plasmids; pCAG Plasmids |
Total Size |
5556 bp |
| Total Size |
5556 bp |
Replicon |
ColE1 ori, SV40 ori |
| Replicon |
ColE1 ori, SV40 ori |
Promoter |
CAG promoter (CMV enhancer + chicken β-actin promoter) |
| Promoter |
CAG promoter (CMV enhancer + chicken β-actin promoter) |
Selection Marker |
Ampicillin Amp (100 μg/ml) |
| Selection Marker |
Ampicillin Amp (100 μg/ml) |
Cloning Strain |
E. coli DH5α |
| Cloning Strain |
E. coli DH5α |
Culture Conditions |
37°C, aerobic, LB |
| Culture Conditions |
37°C, aerobic, LB |
Expression Host |
293T and other mammalian cells |
| Expression Host |
293T and other mammalian cells |
Induction Mode |
No induction required - constitutive expression |
| Induction Mode |
No induction required - constitutive expression |
Terminator |
β-globin poly(A) signal |
| Terminator |
β-globin poly(A) signal |
Marker |
EGFP (Enhanced Green Fluorescent Protein) |
| Marker |
EGFP (Enhanced Green Fluorescent Protein) |
|
Key Features & Benefits
- Strong CAG hybrid promoter (CMV enhancer + chicken β-actin promoter)
- EGFP reporter for real-time fluorescence visualization and tracking
- Dual replication origins: ColE1 (bacterial) + SV40 (mammalian episomal)
- Rabbit β-globin poly(A) signal for robust mRNA stabilization
- Multiple cloning sites: EcoRI, KpnI, XmaI for flexible insertion
- Ampicillin resistance (100 μg/ml) for bacterial selection
- Optimized for high-efficiency transfection in 293T and related lines
- Constitutive expression — no inducer required
Recommended Applications
- Transient transfection and overexpression in mammalian cells
- Gene function analysis and gain-of-function studies
- Subcellular localization tracking via live-cell GFP imaging
- Promoter activity comparison and characterization in mammalian systems
- Drug screening and compound validation assays
- In vivo and in vitro gene delivery optimization studies
- Cell line development and stable line generation (with SV40 ori)
Handling Guidelines & Cautions:
- This product is FOR RESEARCH USE ONLY! Not for diagnostic or therapeutic applications.
- Reconstitution: Centrifuge at 5000rpm/min for 1min, add 20μl ddH2O.
- Shipping temperature: 2-8°C; Lyophilized form requires careful handling.
- Take 2μl plasmid into 100μl competent cell, centrifuge then full coating on plate.
- For mammalian expression: Transfect using standard methods (lipofection, electroporation). Observe GFP signal within 24-48 hours.
▶ Click to expand/collapse
LOCUS Exported 5556 bp ds-DNA circular SYN 19-9-2016
DEFINITION synthetic circular DNA
KEYWORDS pCAG-GFP
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 5556)
TITLE Direct Submission
JOURNAL Exported 2016-09-19
FEATURES Location/Qualifiers
source 1..5556
/organism="synthetic DNA construct"
/mol_type="other DNA"
enhancer 4..383
/note="CMV enhancer"
/note="human cytomegalovirus immediate early enhancer"
promoter 386..661
/note="chicken beta-actin promoter"
CDS 1773..2492
/codon_start=1
/product="enhanced GFP"
/note="EGFP"
/note="mammalian codon-optimized"
/translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
EFVTAAGITLGMDELYK"
polyA_site 2644..2699
/note="beta-globin poly(A) signal"
/note="rabbit beta-globin polyadenylation signal"
primer_bind complement(3059..3075)
/note="M13 rev"
/note="common sequencing primer, one of multiple similar
variants"
protein_bind 3083..3099
/bound_moiety="lac repressor encoded by lacI"
/note="lac operator"
/note="The lac repressor binds to the lac operator to
inhibit transcription in E. coli. This inhibition can be
relieved by adding lactose or
isopropyl-beta-D-thiogalactopyranoside (IPTG)."
promoter complement(3107..3137)
/note="lac promoter"
/note="promoter for the E. coli lac operon"
protein_bind 3152..3173
/bound_moiety="E. coli catabolite activator protein"
/note="CAP binding site"
/note="CAP binding activates transcription in the presence
of cAMP."
promoter 3232..3427
/note="SV40 promoter"
/note="SV40 early promoter"
rep_origin 3278..3413
/note="SV40 ori"
/note="SV40 origin of replication"
polyA_signal 3433..3567
/note="SV40 poly(A) signal"
/note="SV40 polyadenylation signal"
rep_origin complement(3806..4394)
/direction=LEFT
/note="ori"
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
CDS complement(4565..5425)
/codon_start=1
/gene="bla"
/product="beta-lactamase"
/note="AmpR"
/note="confers resistance to ampicillin, carbenicillin, and
related antibiotics"
/translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
LIKHW"
promoter complement(5426..5530)
/gene="bla"
/note="AmpR promoter"
ORIGIN
1 gtcgacattg attattgact agttattaat agtaatcaat tacggggtca ttagttcata
61 gcccatatat ggagttccgc gttacataac ttacggtaaa tggcccgcct ggctgaccgc
121 ccaacgaccc ccgcccattg acgtcaataa tgacgtatgt tcccatagta acgccaatag
181 ggactttcca ttgacgtcaa tgggtggact atttacggta aactgcccac ttggcagtac
241 atcaagtgta tcatatgcca agtacgcccc ctattgacgt caatgacggt aaatggcccg
301 cctggcatta tgcccagtac atgaccttat gggactttcc tacttggcag tacatctacg
361 tattagtcat cgctattacc atgggtcgag gtgagcccca cgttctgctt cactctcccc
421 atctcccccc cctccccacc cccaattttg tatttattta ttttttaatt attttgtgca
481 gcgatggggg cggggggggg gggggcgcgc gccaggcggg gcggggcggg gcgaggggcg
541 gggcggggcg aggcggagag gtgcggcggc agccaatcag agcggcgcgc tccgaaagtt
601 tccttttatg gcgaggcggc ggcggcggcg gccctataaa aagcgaagcg cgcggcgggc
661 gggagtcgct gcgttgcctt cgccccgtgc cccgctccgc gccgcctcgc gccgcccgcc
721 ccggctctga ctgaccgcgt tactcccaca ggtgagcggg cgggacggcc cttctcctcc
781 gggctgtaat tagcgcttgg tttaatgacg gctcgtttct tttctgtggc tgcgtgaaag
841 ccttaaaggg ctccgggagg gccctttgtg cgggggggag cggctcgggg ggtgcgtgcg
901 tgtgtgtgtg cgtggggagc gccgcgtgcg gcccgcgctg cccggcggct gtgagcgctg
961 cgggcgcggc gcggggcttt gtgcgctccg cgtgtgcgcg aggggagcgc ggccgggggc
1021 ggtgccccgc ggtgcggggg ggctgcgagg ggaacaaagg ctgcgtgcgg ggtgtgtgcg
1081 tgggggggtg agcagggggt gtgggcgcgg cggtcgggct gtaacccccc cctgcacccc
1141 cctccccgag ttgctgagca cggcccggct tcgggtgcgg ggctccgtgc ggggcgtggc
1201 gcggggctcg ccgtgccggg cggggggtgg cggcaggtgg gggtgccggg cggggcgggg
1261 ccgcctcggg ccggggaggg ctcgggggag gggcgcggcg gcccccggag cgccggcggc
1321 tgtcgaggcg cggcgagccg cagccattgc cttttatggt aatcgtgcga gagggcgcag
1381 ggacttcctt tgtcccaaat ctgtgcggag ccgaaatctg ggaggcgccg ccgcaccccc
1441 tctagcgggc gcggggcgaa gcggtgcggc gccggcagga aggaaatggg cggggagggc
1501 cttcgtgcgt cgccgcgccg ccgtcccctt ctccctctcc agcctcgggg ctgtccgcgg
1561 ggggacggct gccttcgggg gggacggggc agggcggggt tcggcttctg gcgtgtgacc
1621 ggcggctcta gagcctctgc taaccatgtt catgccttct tctttttcct acagctcctg
1681 ggcaacgtgc tggttattgt gctgtctcat cattttggca aagaattctg cagtcgacgg
1741 taccgcgggc ccgggatcca ccggtcgcca ccatggtgag caagggcgag gagctgttca
1801 ccggggtggt gcccatcctg gtcgagctgg acggcgacgt aaacggccac aagttcagcg
1861 tgtccggcga gggcgagggc gatgccacct acggcaagct gaccctgaag ttcatctgca
1921 ccaccggcaa gctgcccgtg ccctggccca ccctcgtgac caccctgacc tacggcgtgc
1981 agtgcttcag ccgctacccc gaccacatga agcagcacga cttcttcaag tccgccatgc
2041 ccgaaggcta cgtccaggag cgcaccatct tcttcaagga cgacggcaac tacaagaccc
2101 gcgccgaggt gaagttcgag ggcgacaccc tggtgaaccg catcgagctg aagggcatcg
2161 acttcaagga ggacggcaac atcctggggc acaagctgga gtacaactac aacagccaca
2221 acgtctatat catggccgac aagcagaaga acggcatcaa ggtgaacttc aagatccgcc
2281 acaacatcga ggacggcagc gtgcagctcg ccgaccacta ccagcagaac acccccatcg
2341 gcgacggccc cgtgctgctg cccgacaacc actacctgag cacccagtcc gccctgagca
2401 aagaccccaa cgagaagcgc gatcacatgg tcctgctgga gttcgtgacc gccgccggga
2461 tcactctcgg catggacgag ctgtacaagt aaagcggccg cactcctcag gtgcaggctg
2521 cctatcagaa ggtggtggct ggtgtggcca atgccctggc tcacaaatac cactgagatc
2581 tttttccctc tgccaaaaat tatggggaca tcatgaagcc ccttgagcat ctgacttctg
2641 gctaataaag gaaatttatt ttcattgcaa tagtgtgttg gaattttttg tgtctctcac
2701 tcggaaggac atatgggagg gcaaatcatt taaaacatca gaatgagtat ttggtttaga
2761 gtttggcaac atatgcccat atgctggctg ccatgaacaa aggtggctat aaagaggtca
2821 tcagtatatg aaacagcccc ctgctgtcca ttccttattc catagaaaag ccttgacttg
2881 aggttagatt ttttttatat tttgttttgt gttatttttt tctttaacat ccctaaaatt
2941 ttccttacat gttttactag ccagattttt cctcctctcc tgactactcc cagtcatagc
3001 tgtccctctt ctcttatgaa gatccctcga cctgcagccc aagcttggcg taatcatggt
3061 catagctgtt tcctgtgtga aattgttatc cgctcacaat tccacacaac atacgagccg
3121 gaagcataaa gtgtaaagcc tggggtgcct aatgagtgag ctaactcaca ttaattgcgt
3181 tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg ccagcggatc cgcatctcaa
3241 ttagtcagca accatagtcc cgcccctaac tccgcccatc ccgcccctaa ctccgcccag
3301 ttccgcccat tctccgcccc atggctgact aatttttttt atttatgcag aggccgaggc
3361 cgcctcggcc tctgagctat tccagaagta gtgaggaggc ttttttggag gcctaggctt
3421 ttgcaaaaag ctaacttgtt tattgcagct tataatggtt acaaataaag caatagcatc
3481 acaaatttca caaataaagc atttttttca ctgcattcta gttgtggttt gtccaaactc
3541 atcaatgtat cttatcatgt ctggatccgc tgcattaatg aatcggccaa cgcgcgggga
3601 gaggcggttt gcgtattggg cgctcttccg cttcctcgct cactgactcg ctgcgctcgg
3661 tcgttcggct gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg ttatccacag
3721 aatcagggga taacgcagga aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc
3781 gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg cccccctgac gagcatcaca
3841 aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg actataaaga taccaggcgt
3901 ttccccctgg aagctccctc gtgcgctctc ctgttccgac cctgccgctt accggatacc
3961 tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca atgctcacgc tgtaggtatc
4021 tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc
4081 ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc caacccggta agacacgact
4141 tatcgccact ggcagcagcc actggtaaca ggattagcag agcgaggtat gtaggcggtg
4201 ctacagagtt cttgaagtgg tggcctaact acggctacac tagaaggaca gtatttggta
4261 tctgcgctct gctgaagcca gttaccttcg gaaaaagagt tggtagctct tgatccggca
4321 aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa
4381 aaaaaggatc tcaagaagat cctttgatct tttctacggg gtctgacgct cagtggaacg
4441 aaaactcacg ttaagggatt ttggtcatga gattatcaaa aaggatcttc acctagatcc
4501 ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa acttggtctg
4561 acagttacca atgcttaatc agtgaggcac ctatctcagc gatctgtcta tttcgttcat
4621 ccatagttgc ctgactcccc gtcgtgtaga taactacgat acgggagggc ttaccatctg
4681 gccccagtgc tgcaatgata ccgcgagacc cacgctcacc ggctccagat ttatcagcaa
4741 taaaccagcc agccggaagg gccgagcgca gaagtggtcc tgcaacttta tccgcctcca
4801 tccagtctat taattgttgc cgggaagcta gagtaagtag ttcgccagtt aatagtttgc
4861 gcaacgttgt tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt
4921 cattcagctc cggttcccaa cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa
4981 aagcggttag ctccttcggt cctccgatcg ttgtcagaag taagttggcc gcagtgttat
5041 cactcatggt tatggcagca ctgcataatt ctcttactgt catgccatcc gtaagatgct
5101 tttctgtgac tggtgagtac tcaaccaagt cattctgaga atagtgtatg cggcgaccga
5161 gttgctcttg cccggcgtca atacgggata ataccgcgcc acatagcaga actttaaaag
5221 tgctcatcat tggaaaacgt tcttcggggc gaaaactctc aaggatctta ccgctgttga
5281 gatccagttc gatgtaaccc actcgtgcac ccaactgatc ttcagcatct tttactttca
5341 ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg
5401 cgacacggaa atgttgaata ctcatactct tcctttttca atattattga agcatttatc
5461 agggttattg tctcatgagc ggatacatat ttgaatgtat ttagaaaaat aaacaaatag
5521 gggttccgcg cacatttccc cgaaaagtgc cacctg
//
Scientific References
- PMID: 7649440: Niwa H, et al. (1991). Efficient selection for high-expression transfectants with a novel eukaryotic vector. Gene, 108(2), 193-200.
- PMID: 8625695: Okabe M, et al. (1997). Green fluorescent protein as a marker in transgenic mice. Dev Growth Differ, 39(1), 37-45.
- PMID: 15024505: Ikeda H, et al. (2004). The CAG promoter is more efficient than the CMV promoter for gene expression in mouse ES cells. Biochem Biophys Res Commun, 318(3), 882-886.
Frequently Asked Questions (FAQ)
Q: What makes the CAG promoter superior to CMV alone?
The CAG promoter combines the CMV immediate early enhancer with the chicken β-actin core promoter and first intron, providing significantly stronger and more consistent expression across a wider range of mammalian cell types compared to CMV alone, with reduced silencing tendency.
Q: Which mammalian cell lines work best with pCAG-GFP?
HEK293T shows highest transfection efficiency and expression levels. HeLa, CHO-K1, COS-7, NIH-3T3, and most adherent mammalian cell lines also show excellent expression. Optimize transfection reagent and DNA amount for your specific cell type.
Q: Do I need an inducer for expression?
No, the CAG promoter drives constitutive expression without any requirement for chemical inducers. Simply transfect the plasmid and observe GFP fluorescence within 24-48 hours post-transfection.
Q: Can I clone my own gene into this vector?
Yes, utilize the EcoRI, KpnI, or XmaI restriction sites in the multiple cloning region to insert your gene of interest. Note that EGFP is already present in the vector for co-expression or fusion protein strategies.
Q: Can I generate stable cell lines with pCAG-GFP?
Yes, the SV40 origin of replication enables episomal maintenance in mammalian cells expressing SV40 large T antigen (such as COS-7 or engineered 293T lines), facilitating stable expression without genomic integration.
