Plasmid

Part:BBa_K4411020

Designed by: Shi Lifang   Group: iGEM22_SHBS-BANZ   (2022-09-02)


pET28a-C-Target

pET28a-C-Target

Profile

Name: pET28a-C-Target

Base Pairs: 5842 bp

Origin: a common Prokaryotic microorganism plasmid

Properties: a composite part composite by pET28a plasmid backbone and C Target1-TSD-C Target2 DNA sequence

Usage and Biology

BBa_K4411020 is a composite part made up of the pET28a backbone and C Target1-TSD-C Target2 DNA sequence [1-5]. In this part, we insert the C Target1-TSD-C Target2 DNA fragment by designing recombinant primers according to the target sequences and inserted the fragment into the pET28a vector under the regulation of the T7 promoter(Figure 1). What’s more, we also inserted a ccd gene into the plasmid backbone which could be used as a reporter to screen for F plasmid deficit strain in future research.

Figure 1. the plasmid map of the composite part BBa_K4411018..

The pUC19 plasmid backbone is one of the most commonly used expression vectors in E. coli and the Ampicillin resistance could be used to screen the right colonies. It is suitable for DNA fragment cloning, DNA sequencing, and the expression of exogenous genes.


Reference

1.Béguin P, Chekli Y, Sezonov G, Forterre P, Krupovic M. Sequence motifs recognized by the casposon integrase of Aciduliprofundum boonei. Nucleic Acids Res. 2019 Jul 9;47(12):6386-6395.

2.Hickman AB, Dyda F. The casposon-encoded Cas1 protein from Aciduliprofundum boonei is a DNA integrase that generates target site duplications. Nucleic Acids Res. 2015 Dec 15;43(22):10576-87. doi: 10.1093/nar/gkv1180. PMID: 26573596

3.Krupovic M, Shmakov S, Makarova KS, Forterre P, Koonin EV. Recent Mobility of Casposons, Self-Synthesizing Transposons at the Origin of the CRISPR-Cas Immunity. Genome Biol Evol. 2016 Jan 13;8(2):375-86. doi:10.1093/gbe/evw006. PMID: 26764427; PMCID: PMC4779613.

4.Béguin P, Charpin N, Koonin EV, Forterre P, Krupovic M. Casposon integration shows strong target site preference and recapitulates protospacer integration by CRISPR-Cas systems. Nucleic Acids Res. 2016 Dec 1;44(21):10367-10376. doi: 10.1093/nar/gkw821. PMID: 27655632; PMCID: PMC5137440.

5.Krupovic M, Béguin P, Koonin EV. Casposons: mobile genetic elements that gave rise to the CRISPR-Cas adaptation machinery. Curr Opin Microbiol. 2017 Aug; 38:36-43. doi: 10.1016/j.mib.2017.04.004. PMID: 28472712; PMCID: PMC5665730.

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 5293
    Illegal NheI site found at 5769
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 5021
    Illegal BamHI site found at 5047
    Illegal BamHI site found at 5106
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 194
    Illegal NgoMIV site found at 3241
    Illegal NgoMIV site found at 3401
    Illegal NgoMIV site found at 4989
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal SapI.rc site found at 2320


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