LOCUS       Exported                2584 bp ds-DNA     circular SYN 13-MAY-2021
DEFINITION  Expression of customizable guide RNA (gRNA) for bacterial gene 
            knockdown.
ACCESSION   .
VERSION     .
KEYWORDS    pgRNA-bacteria
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 2584)
  AUTHORS   Qi LS, Larson MH, Gilbert LA, Doudna JA, Weissman JS, Arkin AP, Lim 
            WA
  TITLE     Repurposing CRISPR as an RNA-Guided Platform for Sequence-Specific 
            Control of Gene Expression.
  JOURNAL   Cell. 2013 Feb 28;152(5):1173-83. doi: 10.1016/j.cell.2013.02.022.
  PUBMED    23452860
REFERENCE   2  (bases 1 to 2584)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; doi: 
            "10.1016/j.cell.2013.02"; journalName: "Cell"; date: "2013-02-28- 
            28"; volume: "152"; issue: "5"; pages: "1173-83"
FEATURES             Location/Qualifiers
     source          1..2584
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     promoter        5..39
                     /label=J23119(SpeI) promoter
                     /note="bacterial promoter (Registry of Standard Biological 
                     Parts BBa_J23119) modified to end with an SpeI site"
     misc_RNA        60..135
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     CDS             156..185
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     CDS             201..218
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(274..291)
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     terminator      444..490
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     complement(543..560)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(714..1302)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(794..813)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(1473..2333)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     2096..2115
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2334..2438)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     2506..2524
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     terminator      2528..2571
                     /label=bacterial terminator
                     /note="putative bacterial transcription terminator"