Plasmid

Part:BBa_K4411018

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


pUC19-C-Target

pUC19-C-Target

Profile

Name: pUC19-C-Target

Base Pairs: 2690 bp

Origin: modified from pBR322 plasmid

Properties: a composite part composite by pUC19 plasmid backbone and C Target1-TSD-C Target2 DNA sequence

Usage and Biology

BBa_K4411018 is a composite part made up of the pUC19 backbone and C Target1-TSD-C Target2 DNA sequence. In this part, we first extracted the pUC19 plasmid with Ampicillin resistance. Then we did PCR to insert the C Target1-TSD-C Target2 DNA fragment by designing recombinant primers according to the target sequences and inserted the fragment into the HindIII and EcoRI sites of the pUC19 vector (Figure 1).

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.

Profile

Name: C Target1-TSD-C Target2

Base Pairs: 49 bp

Origin: Candidatus Nitrosopumilus koreensis AR1 genome

Properties: target site of the casposons

Usage and Biology

Target site duplication (TSD) is a feature of Target-Primed Reverse Transcription (TPRT) that is necessary to consider when detecting novel insertions [1]. The integration reaction appears to proceed via a two-step mechanism with casposons, whereby the first nucleophilic attack occurs at the TSD segment border by the 3′-OH of the spacer. After the formation of the half-site intermediate, the second nucleophilic attack occurs on the opposite strand at the junction between the TSD segment and the flanking DNA (Figure 2) [2-5].

Figure 2. the working principle of casposons..

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
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]


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Categories
Parameters
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