Difference between revisions of "Part:BBa K1875004"

Revision as of 04:50, 19 October 2016

A 20 bp target sequence and PAM to make guide operator 3

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 that targets a paired gRNA operator reporter (BBa_K1875014) to activate a downstream gene of interest.

Figure 1: initial screen of BostonU's first 18 operator reporters containing GFP

[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]

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 <partinfo>BBa_K1875004 short</partinfo>
-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 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).
 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 that targets a paired gRNA operator reporter ([https://parts.igem.org/Part:BBa_K1875014 BBa_K1875014]) to activate a downstream gene of interest.