LOCUS       Exported                3542 bp ds-DNA     circular SYN 13-MAY-2021
DEFINITION  Expresses the S. pyogenes sgRNA from the mouse U6 promoter.
ACCESSION   .
VERSION     .
KEYWORDS    pmU6-gRNA
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3542)
  AUTHORS   Kabadi AM, Ousterout DG, Hilton IB, Gersbach CA
  TITLE     Multiplex CRISPR/Cas9-based genome engineering from a single 
            lentiviral vector.
  JOURNAL   Nucleic Acids Res. 2014 Aug 13. pii: gku749.
  PUBMED    25122746
REFERENCE   2  (bases 1 to 3542)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Nucleic 
            Acids Res. 2014 Aug 13. pii: gku749."
FEATURES             Location/Qualifiers
     source          1..3542
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     promoter        1..314
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for mouse U6 snRNA (Das 
                     et al., 1988)"
     primer_bind     233..253
                     /label=mU6-F
                     /note="Mouse U6 promoter, forward primer"
     misc_RNA        332..407
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     primer_bind     complement(452..472)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(452..470)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(491..507)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(491..507)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(504..526)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    515..531
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /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)."
     promoter        complement(539..569)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    584..605
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(722..739)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(893..1481)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(973..992)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(1558..2352)
                     /codon_start=1
                     /gene="aph(3)-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(1669..1688)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     2279..2298
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     promoter        complement(2387..2744)
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      2401..2536
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(2455..2474)
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     primer_bind     2701..2721
                     /label=pBABE 3
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        complement(2746..2850)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(2876..3331)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3013..3034)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3225..3244
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3458..3480
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     3472..3489
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     3473..3489
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     3499..3518
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        3499..3517
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"