Difference between revisions of "Part:BBa K3765023"

 
 
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<partinfo>BBa_K3765023 short</partinfo>
 
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Desc.
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OpdA-SpyTag002 (without OpdA signal peptide) fusion protein generator.  
  
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===Usage and Biology===
 
===Usage and Biology===
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This protein generator can be used for expressing the OpdA-SpyTag002 (without OpdA signal peptide) fusion protein under a strong bacteriophage T7 promoter by the T7 RNA Polymerase in suitable bacterial strains like ''E. coli'' BL21(DE3). Transcriptional regulation is by the ''lac'' operator sequence, and expression can be switched on via IPTG induction. Also contains a strong T7 RBS upstream and wild type T7 terminator downstream of the CDS for initiation and termination of translation.
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[[File:T--IISc-Bangalore--images--Map--C2(3.0)--pETMCN-T7.png|thumb|center|800px|Fig 1. Feature Map]]
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To construct this protein generator, we cloned the [https://parts.igem.org/Part:BBa_K3765015 OpdA-SpyTag002 CDS] into the high copy number [https://parts.igem.org/Part:BBa_K3765011 pETMCN] vector. The vector contains SpeI and XbaI restriction sites just downstream of the RBS and upstream of the terminator, respectively. We used Gibson Assembly for cloning, so there were no hindrances with regard to the presence of these restriction sites. In case someone wants to use this part, it is suggested to get it synthesized and use Gibson Assembly (or other such assembly techniques) for cloning instead of restriction digestion-ligation.
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<span class='h3bb'>Sequence and Features</span>
 
<span class='h3bb'>Sequence and Features</span>
 
<partinfo>BBa_K3765023 SequenceAndFeatures</partinfo>
 
<partinfo>BBa_K3765023 SequenceAndFeatures</partinfo>

Latest revision as of 20:16, 20 October 2021


OpdA+SpyTag002 without OpdA signal peptide under T7 promoter

OpdA-SpyTag002 (without OpdA signal peptide) fusion protein generator.

Usage and Biology

This protein generator can be used for expressing the OpdA-SpyTag002 (without OpdA signal peptide) fusion protein under a strong bacteriophage T7 promoter by the T7 RNA Polymerase in suitable bacterial strains like E. coli BL21(DE3). Transcriptional regulation is by the lac operator sequence, and expression can be switched on via IPTG induction. Also contains a strong T7 RBS upstream and wild type T7 terminator downstream of the CDS for initiation and termination of translation.

Fig 1. Feature Map

To construct this protein generator, we cloned the OpdA-SpyTag002 CDS into the high copy number pETMCN vector. The vector contains SpeI and XbaI restriction sites just downstream of the RBS and upstream of the terminator, respectively. We used Gibson Assembly for cloning, so there were no hindrances with regard to the presence of these restriction sites. In case someone wants to use this part, it is suggested to get it synthesized and use Gibson Assembly (or other such assembly techniques) for cloning instead of restriction digestion-ligation.

Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal XbaI site found at 1365
    Illegal SpeI site found at 45
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 1371
    Illegal SpeI site found at 45
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 1353
    Illegal XhoI site found at 1335
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal XbaI site found at 1365
    Illegal SpeI site found at 45
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal XbaI site found at 1365
    Illegal SpeI site found at 45
    Illegal AgeI site found at 1227
    Illegal AgeI site found at 1290
  • 1000
    COMPATIBLE WITH RFC[1000]