Difference between revisions of "Part:BBa K1875005"
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The [http://2016.igem.org/Team:BostonU BostonU 2016] iGEM team synthesized guide operator sequences composed of a 20 base pair target sequence and a PAM sequence.  The target sequence was made using a random DNA generator and entered into the [http://crispr.mit.edu/ CRISPR Design Optimization] tool for the human genome (hg19).  It was chosen among approximately 1000 other randomly generated target sequences due to its high optimization score ( > 98%) and high expression in an initial screen containing iRFP (figure 1).
 
The [http://2016.igem.org/Team:BostonU BostonU 2016] iGEM team synthesized guide operator sequences composed of a 20 base pair target sequence and a PAM sequence.  The target sequence was made using a random DNA generator and entered into the [http://crispr.mit.edu/ CRISPR Design Optimization] tool for the human genome (hg19).  It was chosen among approximately 1000 other randomly generated target sequences due to its high optimization score ( > 98%) and high expression in an initial screen containing iRFP (figure 1).
  
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The target sequence is expressed by a guide RNA (gRNA) expression vector ([https://parts.igem.org/Part:BBa_K1875011 BBa_K1875011]) from the BostonU Gemini Library.  The gRNA binds to dCas9-VPR and targets a paired gRNA operator reporter ([https://parts.igem.org/Part:BBa_K1875014 BBa_K1875014]) to activate a downstream gene of interest (figure 2).
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The target sequence is expressed by a guide RNA (gRNA) expression vector ([https://parts.igem.org/Part:BBa_K1875011 BBa_K1875011]) from the BostonU Gemini Library.  The gRNA binds to dCas9-VPR and targets a paired gRNA operator reporter ([https://parts.igem.org/Part:BBa_K1875014 BBa_K1875013]) to activate a downstream gene of interest (figure 2).
  
 
[[File:T--BostonU--iRFP_screen.png|400px|thumb|left|Figure 1: Initial screen of BostonU's first 18 operator reporters containing GFP]][[File:T--BostonU--ProjectDescription.png|400px|thumb|center|Figure 2: [http://2016.igem.org/Team:BostonU BostonU 2016] Project description]]
 
[[File:T--BostonU--iRFP_screen.png|400px|thumb|left|Figure 1: Initial screen of BostonU's first 18 operator reporters containing GFP]][[File:T--BostonU--ProjectDescription.png|400px|thumb|center|Figure 2: [http://2016.igem.org/Team:BostonU BostonU 2016] Project description]]

Revision as of 05:08, 19 October 2016


A 20 bp target sequence and PAM to make guide operator 1

The [http://2016.igem.org/Team:BostonU BostonU 2016] iGEM team synthesized guide operator sequences composed of a 20 base pair target sequence and a PAM sequence. The target sequence was made using a random DNA generator and entered into the [http://crispr.mit.edu/ CRISPR Design Optimization] tool for the human genome (hg19). It was chosen among approximately 1000 other randomly generated target sequences due to its high optimization score ( > 98%) and high expression in an initial screen containing iRFP (figure 1).

The target sequence is expressed by a guide RNA (gRNA) expression vector (BBa_K1875011) from the BostonU Gemini Library. The gRNA binds to dCas9-VPR and targets a paired gRNA operator reporter (BBa_K1875013) to activate a downstream gene of interest (figure 2).

Figure 1: Initial screen of BostonU's first 18 operator reporters containing GFP
Figure 2: [http://2016.igem.org/Team:BostonU BostonU 2016] Project description

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]