LOCUS       Exported                3515 bp ds-DNA     circular SYN 13-MAY-2021
DEFINITION  Expresses the S. pyogenes sgRNA from the human U6 promoter.
ACCESSION   .
VERSION     .
KEYWORDS    phU6-gRNA
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3515)
  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 3515)
  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..3515
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     rep_origin      complement(3..458)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(140..161)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     352..371
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     585..607
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     599..616
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     600..616
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     626..645
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        626..644
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        696..936
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA"
     primer_bind     696..716
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     867..886
                     /label=LKO.1 5
                     /note="Human U6 promoter, forward primer"
     misc_RNA        963..1038
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     primer_bind     complement(1094..1114)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(1094..1112)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(1133..1149)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1133..1149)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1146..1168)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    1157..1173
                     /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(1181..1211)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1226..1247
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(1364..1381)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1535..2123)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1615..1634)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(2200..2994)
                     /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(2311..2330)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     2921..2940
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     promoter        complement(3029..3386)
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3043..3178
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(3097..3116)
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     primer_bind     3343..3363
                     /label=pBABE 3
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        complement(3388..3492)
                     /gene="bla"
                     /label=AmpR promoter