gRNA_GFP-T1 vector (V012340)

Basic Vector Information

      • Vector Name:
      • gRNA_GFP-T1
      • Antibiotic Resistance:
      • Kanamycin
      • Length:
      • 3973 bp
      • Type:
      • CRISPR Plasmids
      • Source/Author:
      • Mali P, Yang L, Esvelt KM, Aach J, Guell M, DiCarlo JE, Norville JE,
      • Selection Marker:
      • Neomycin/G418(Geneticin)
      • Copy Number:
      • High copy number

gRNA_GFP-T1 vector Vector Map

gRNA_GFP-T13973 bp60012001800240030003600polIII terminatorgRNA scaffoldGFP T1U6 promoterNeoR/KanRBleoRori

Plasmid Resuspension Protocol:

1. Centrifuge at 5,000×g for 5 min.

2. Carefully open the tube and add 20 μl of sterile water to dissolve the DNA.

3. Close the tube and incubate for 10 minutes at room temperature.

4. Briefly vortex the tube and then do a quick spin to concentrate the liquid at the bottom. Speed is less than 5000×g.

5.Store the plasmid at -20 ℃.

gRNA_GFP-T1 vector Sequence

Copy Sequence

Download GeneBank File(.gb)

LOCUS       Exported                3973 bp ds-DNA    circular SYN 06-1-2016
DEFINITION  Plasmid for guide RNA (gRNA) targeting of an integrated GFP gene in 
            human cells using the GFP T1 target sequence.
ACCESSION   .
VERSION     .
KEYWORDS    gRNA_GFP-T1
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3973)
  AUTHORS   Mali P, Yang L, Esvelt KM, Aach J, Guell M, DiCarlo JE, Norville JE,
            Church GM.
  TITLE     RNA-guided human genome engineering via Cas9.
  JOURNAL   Science 2013;339:823-6.
  PUBMED    23287722
REFERENCE   2  (bases 1 to 3973)
  AUTHORS   Church Lab / Addgene #41819
  TITLE     Direct Submission
FEATURES             Location/Qualifiers
     source          1..3973
                     /organism="synthetic DNA construct"
                     /lab_host="Mammalian Cells"
                     /mol_type="other DNA"
     terminator      372..377
                     /note="polIII terminator"
                     /note="RNA polymerase III transcription terminator"
     misc_RNA        complement(378..453)
                     /note="gRNA scaffold"
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     misc_feature    454..473
                     /note="GFP T1"
                     /note="GFP gRNA Target 1 sequence"
     promoter        complement(482..722)
                     /note="U6 promoter"
                     /note="RNA polymerase III promoter for human U6 snRNA"
     CDS             1692..2486
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /note="NeoR/KanR"
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     CDS             2692..3066
                     /codon_start=1
                     /gene="Sh ble from Streptoalloteichus hindustanus"
                     /product="antibiotic-binding protein"
                     /note="BleoR"
                     /note="confers resistance to bleomycin, phleomycin, and 
                     Zeocin(TM)"
                     /translation="MAKLTSAVPVLTARDVAGAVEFWTDRLGFSRDFVEDDFAGVVRDD
                     VTLFISAVQDQVVPDNTLAWVWVRGLDELYAEWSEVVSTNFRDASGPAMTEIGEQPWGR
                     EFALRDPAGNCVHFVAEEQD"
     rep_origin      3204..3792
                     /direction=RIGHT
                     /note="ori"
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"

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