pwtcas9 vector (V002118)

LOCUS       40924_46658        7692 bp DNA     circular SYN 18-DEC-2018
DEFINITION  Cloning vector pwtcas9, complete sequence.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7692)
  AUTHORS   Chavez A, Pruitt BW, Tuttle M, Shapiro RS, Cecchi RJ, Winston J, 
            Turczyk BM, Tung M, Collins JJ, Church GM.
  TITLE     Precise Cas9 targeting enables genomic mutation prevention
  JOURNAL   Proc. Natl. Acad. Sci. U.S.A. (2018) In press
  PUBMED    29555762
REFERENCE   2  (bases 1 to 7692)
  AUTHORS   Chavez A, Pruitt BW, Tuttle M, Shapiro RS, Cecchi RJ, Winston J, 
            Turczyk BM, Tung M, Collins JJ, Church GM.
  TITLE     Direct Submission
  JOURNAL   Submitted (02-MAR-2018) Synthetic Biology, Wyss Institute, 3 
            Blackfan Circle, Floor 5, Boston, MA 02115, USA
REFERENCE   3  (bases 1 to 7692)
  TITLE     Direct Submission
REFERENCE   4  (bases 1 to 7692)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Proc. Natl.
            Acad. Sci. U.S.A. (2018) In press"
COMMENT     SGRef: number: 2; type: "Journal Article"; journalName: "Submitted 
            (02-MAR-2018) Synthetic Biology, Wyss Institute, 3 Blackfan Circle, 
            Floor 5, Boston, MA 02115, USA"
COMMENT     SGRef: number: 3; type: "Journal Article"
FEATURES             Location/Qualifiers
     source          1..7692
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(157..780)
                     /label=TetR
                     /note="tetracycline repressor TetR"
     promoter        796..851
                     /label=tetR/tetA promoters
                     /note="overlapping promoters for bacterial tetR and tetA"
     RBS             869..880
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             887..4990
                     /label=Cas9
                     /note="Cas9 (Csn1) endonuclease from the Streptococcus
                     pyogenes Type II CRISPR/Cas system"
     misc_feature    4991..5020
                     /label=SGS linker
                     /note="SGS linker"
     CDS             5021..5053
                     /label=ssrA tag
                     /note="C-terminal peptide that mediates degradation in
                     bacteria through the ClpXP and ClpAP proteases (McGinness 
                     et al., 2006)"
     misc_feature    5054..5059
                     /label=double stop
                     /note="double stop"
     terminator      5083..5154
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB
                     gene"
     terminator      5170..5197
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     misc_RNA        5437..5515
                     /label=tracrRNA
                     /note="trans-activating CRISPR RNA for the Streptococcus 
                     pyogenes CRISPR/Cas9 system"
     regulatory      complement(5553..5594)
                     /note="derived from Streptococcus pyogenes; tracrRNA
                     terminator"
                     /regulatory_class="terminator"
     regulatory      5651..5715
                     /label=J23100 promoter
                     /note="J23100 promoter"
                     /regulatory_class="promoter"
     repeat_region   5731..5766
                     /label=DR
                     /note="direct repeat for the Streptococcus pyogenes
                     CRISPR/Cas system"
     rep_origin      5948..6686
                     /label=CloDF13 ori
                     /note="Plasmids containing the CloDF13 (CDF) origin of 
                     replication can be propagated in E. coli cells that contain
                     additional plasmids with compatible origins."
     promoter        6734..6825
                     /label=AmpR promoter
     CDS             6826..7614
                     /label=SmR
                     /note="aminoglycoside adenylyltransferase (Murphy, 1985)"