LOCUS       Exported                3198 bp ds-DNA     circular SYN 31-AUG-2022
DEFINITION  Expresses AcrVA5 in bacteria, under TetR control..
ACCESSION   .
VERSION     .
KEYWORDS    pKEW303-AcrVA5
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3198)
  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 3198)
  AUTHORS   .
  TITLE     Direct Submission
FEATURES             Location/Qualifiers
     source          1..3198
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     promoter        1..56
                     /gene="tetR"
                     /note="tetR/tetA promoters"
                     /note="overlapping promoters for bacterial tetR and tetA"
     protein_bind    37..55
                     /gene="tetO"
                     /bound_moiety="tetracycline repressor TetR"
                     /note="tet operator"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     CDS             83..361
                     /codon_start=1
                     /note="anti-CRISPR N-acetyltransferase AcrVA5"
                     /translation="MKIELSGGYICYSIEEDEVTIDMVEVTTKRQGIGSQLIDMVKDVA
                     REVGLPIGLYAYPQDDSISQEDLIEFYFSNDFEYDPDDVDGRLMRWS"
     terminator      594..621
                     /note="rrnB T2 terminator"
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     rep_origin      complement(747..1335)
                     /direction=LEFT
                     /note="ori"
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(1506..2366)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /note="AmpR"
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     promoter        complement(2367..2471)
                     /gene="bla"
                     /note="AmpR promoter"
     CDS             complement(2557..3180)
                     /codon_start=1
                     /gene="tetR from transposon Tn10"
                     /product="tetracycline repressor TetR"
                     /note="TetR"
                     /note="TetR binds to the tetracycline operator tetO to 
                     inhibit transcription. This inhibition can be relieved by 
                     adding tetracycline or doxycycline."
                     /translation="MSRLDKSKVINSALELLNEVGIEGLTTRKLAQKLGVEQPTLYWHV
                     KNKRALLDALAIEMLDRHHTHFCPLEGESWQDFLRNNAKSFRCALLSHRDGAKVHLGTR
                     PTEKQYETLENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEDQEHQVAKEERETPTT
                     DSMPPLLRQAIELFDHQGAEPAFLFGLELIICGLEKQLKCESGS"