Regulatory

Part:BBa_K5082008

Designed by: Fangyuan Duan   Group: iGEM24_YiYe-China   (2024-09-07)

pCMV-EGFP plasmid vector

Usage and Biology

Plasmids are double stranded circular DNA molecules, which carry non-essential genes, found in bacterial cells [1]. Plasmids are multifunction, easy to edit, and can be amplified quickly by bacterial cellular machinery [2]. These advantages have made plasmids a popular tool in genetic research.

pCMV-EGFP, also known as pCMV-GFP, is a mammalian expression vector of the GFP reporter gene with a CMV promoter [3]. The pCMV-EGFP plasmid can be amplified by DH5α bacterial cells. Meanwhile, it carries an ampicillin resistance gene for selection. The plasmid map for pCMV-EGFP is shown in Figure 1. Previously, this plasmid has been used in scientific research [4]. 


                      pcmv-egfp-1.png
                      Figure 1. pCMV-EGFP plasmid map.

Design The above characteristics have made pCMV-EGFP an ideal plasmid for our experiment. In our experiment, we fused an HSU structure downstream the Luciferase gene to visualize G3BP1 concentration and thereby diagnosed gastric cancer. Meanwhile, we also transfected empty plasmid backbones and plasmids with MUT regions downstream GFP into stomach cell lines to serve as control groups.

Characterization The pCMV-EGFP plasmid carries an ampicillin resistance gene. Therefore, we cultured bacterial cells containing the plasmid on solid medium with ampicillin. As shown in Figure 2, the bacteria were successfully cultured, proving that the plasmids have been successfully transformed. 

                   pcmv-egfp-2.png
                    Figure 2. pCMV-EGFP bacterial cell culture.

Reference

References

[1] Carattoli, Alessandra. “Plasmids in Gram Negatives: Molecular Typing of Resistance Plasmids.” International Journal of Medical Microbiology, vol. 301, no. 8, Dec. 2011, pp. 654–658

[2] Patron, N.J. Synthetic Biology and Gene Cloning. Elsevier EBooks, Elsevier BV, 1 Jan. 2017, pp. 112–117

[3] “Addgene: PCMV-GFP.” www.addgene.org, www.addgene.org/11153/

[4] Matsuda, Takahiko, and Constance L Cepko. “Electroporation and RNA interference in the rodent retina in vivo and in vitro.” Proceedings of the National Academy of Sciences of the United States of America vol. 101,1 (2004): 16-22. doi:10.1073/pnas.2235688100

Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal EcoRI site found at 1630
    Illegal XbaI site found at 1667
    Illegal SpeI site found at 216
    Illegal PstI site found at 1635
    Illegal PstI site found at 2989
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal EcoRI site found at 1630
    Illegal NheI site found at 862
    Illegal SpeI site found at 216
    Illegal PstI site found at 1635
    Illegal PstI site found at 2989
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal EcoRI site found at 1630
    Illegal BglII site found at 1610
    Illegal BglII site found at 6116
    Illegal BamHI site found at 1661
    Illegal XhoI site found at 1614
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal EcoRI site found at 1630
    Illegal XbaI site found at 1667
    Illegal SpeI site found at 216
    Illegal PstI site found at 1635
    Illegal PstI site found at 2989
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal EcoRI site found at 1630
    Illegal XbaI site found at 1667
    Illegal SpeI site found at 216
    Illegal PstI site found at 1635
    Illegal PstI site found at 2989
    Illegal NgoMIV site found at 2099
    Illegal NgoMIV site found at 3440
    Illegal NgoMIV site found at 3723
    Illegal AgeI site found at 871
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI.rc site found at 849
    Illegal BsaI.rc site found at 5248
    Illegal SapI site found at 4168
    Illegal SapI.rc site found at 3289
    Illegal SapI.rc site found at 3499


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