pKEW212-MBP-TEV-AcrVA5 vector (V012821)

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LOCUS       pKEW212-MBP-TEV-        6337 bp DNA     circular SYN 17-OCT-2023
DEFINITION  Expresses AcrVA5 in bacteria via T7 RNAP, for purification.
ACCESSION   .
VERSION     .
KEYWORDS    pKEW212-MBP-TEV-AcrVA5
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6337)
  AUTHORS   Watters KE, Fellmann C, Bai HB, Ren SM, Doudna JA
  TITLE     Systematic discovery of natural CRISPR-Cas12a inhibitors.
  JOURNAL   Science. 2018 Oct 12;362(6411):236-239. doi: 
            10.1126/science.aau5138. Epub 2018 Sep 6.
  PUBMED    30190307
REFERENCE   2  (bases 1 to 6337)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; doi: 
            "10.1126/science.aau5138"; journalName: "Science"; date: 
            "2018-10-12- 12"; volume: "362"; issue: "6411"; pages: "236-239"
FEATURES             Location/Qualifiers
     source          1..6337
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        1..19
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     RBS             108..130
                     /label=RBS
                     /note="efficient ribosome binding site from bacteriophage
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             150..176
                     /label=9xHis
                     /note="9xHis affinity tag"
     CDS             189..1289
                     /label=MBP
                     /note="maltose binding protein from E. coli"
     CDS             1344..1364
                     /label=TEV site
                     /note="tobacco etch virus (TEV) protease recognition and 
                     cleavage site"
     CDS             1438..1458
                     /label=TEV site
                     /note="tobacco etch virus (TEV) protease recognition and 
                     cleavage site"
     CDS             1465..1740
                     /codon_start=1
                     /label=anti-CRISPR N-acetyltransferase AcrVA5
                     /translation="MKIELSGGYICYSIEEDEVTIDMVEVTTKRQGIGSQLIDMVKDVA
                     REVGLPIGLYAYPQDDSISQEDLIEFYFSNDFEYDPDDVDGRLMRWS"
     terminator      1881..1928
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     promoter        complement(2294..2322)
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein
                     gene"
     primer_bind     complement(2349..2367)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward
                     primer"
     promoter        2435..2539
                     /label=AmpR promoter
     CDS             2540..3397
                     /label=AmpR
                     /note="beta-lactamase"
     rep_origin      3571..4159
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4313..4330
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    complement(4345..4484)
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     4570..4592
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4589..4777)
                     /label=rop
                     /note="Rop protein, which maintains plasmids at low copy
                     number"
     primer_bind     6235..6254
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI,
                     reverse primer"