LOCUS       Exported                3973 bp ds-DNA    circular SYN 06-1-2016
DEFINITION  Plasmid for guide RNA (gRNA) targeting of an AAVS1 fragment 
            interrupting an integrated GFP gene in human cells, using the AAVS1 
            T2 target sequence.
ACCESSION   .
VERSION     .
KEYWORDS    gRNA_AAVS1-T2
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 #41818
  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="AAVS1 T2"
                     /note="AAVS1 gRNA Target 2 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"