pCAG-T3-hCasD10A-pA vector (V012293)

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LOCUS       pCAG-T3-hCasD10A        9272 bp DNA     circular SYN 01-JAN-1980
DEFINITION  Plasmid for mammalian cell expression or in vitro transcription of 
            human codon-optimized Cas9 with the D10A nickase mutation.
ACCESSION   .
VERSION     .
KEYWORDS    pCAG-T3-hCasD10A-pA
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 9272)
  AUTHORS   Fujii W, Onuma A, Sugiura K, Naito K.
  TITLE     Efficient generation of genome-modified mice via offset-nicking by 
            CRISPR/Cas system.
  JOURNAL   Biochem. Biophys. Res. Commun. 2014;445:791-4.
  PUBMED    24491566
REFERENCE   2  (bases 1 to 9272)
  AUTHORS   
  TITLE     Direct Submission
REFERENCE   3  (bases 1 to 9272)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Biochem. 
            Biophys. Res. Commun."; date: "2014"; volume: "445"; pages: "791-4"
COMMENT     SGRef: number: 2; type: "Journal Article"
FEATURES             Location/Qualifiers
     source          1..9272
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     polyA_signal    complement(6..140)
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     promoter        complement(146..342)
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     protein_bind    400..421
                     /label=CAP binding site
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     promoter        436..466
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    474..490
                     /label=lac operator
                     /note="The lac repressor binds to the lac operator to
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     primer_bind     498..514
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     polyA_signal    complement(875..930)
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     misc_feature    1103..1197
                     /label=poly(A)
                     /note="poly(A)"
                     /note="enables microinjection of poly(A)-tailed mRNA after
                     in vitro transcription"
     3'UTR           1198..1267
                     /label=portion of the 3' UTR from the human TBPL1 gene
                     /note="portion of the 3' UTR from the human TBPL1 gene"
     CDS             complement(1283..1303)
                     /codon_start=1
                     /product="nuclear localization signal of SV40 large T
                     antigen"
                     /note="SV40 NLS"
                     /translation="PKKKRKV"
     CDS             complement(1316..5416)
                     /label=Cas9(D10A)
                     /note="nickase mutant of the Cas9 endonuclease from the 
                     Streptococcus pyogenes Type II CRISPR/Cas system"
     CDS             complement(5441..5461)
                     /codon_start=1
                     /product="nuclear localization signal of SV40 large T
                     antigen"
                     /note="SV40 NLS"
                     /translation="PKKKRKV"
     CDS             complement(5471..5494)
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase
                     cleavage site"
                     /label=FLAG
                     /note="FLAG"
                     /translation="DYKDDDDK"
     CDS             complement(5495..5497)
                     /codon_start=1
                     /product="start codon"
                     /label=start codon
                     /note="ATG"
                     /translation="M"
     promoter        complement(5555..5573)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     intron          complement(5630..6646)
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and
                     rabbit beta-globin"
     promoter        complement(6647..6903)
                     /note="chicken beta-actin promoter"
                     /note="contains a 21-bp deletion relative to the standard 
                     chicken beta-actin promoter"
     enhancer        6905..7284
                     /note="CMV enhancer"
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        7315..7419
                     /label=AmpR promoter
     CDS             7420..8277
                     /label=AmpR
                     /note="beta-lactamase"
     rep_origin      8451..9039
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"