LOCUS       sequence-224436-        3198 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses AcrVA5 in bacteria, under TetR control..
ACCESSION   .
VERSION     .
KEYWORDS    .
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
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
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"
COMMENT     Sequence Label: pKEW303-AcrVA5
FEATURES             Location/Qualifiers
     source          1..3198
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        1..56
                     /gene="tetR"
                     /label=tetR/tetA promoters
                     /note="overlapping promoters for bacterial tetR and tetA"
     protein_bind    37..55
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     terminator      594..621
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     rep_origin      complement(747..1335)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(827..846)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(1506..2297,2298..2366))
                     /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     2129..2148
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2367..2471)
                     /gene="bla"
                     /label=AmpR promoter
     CDS             complement(2557..3183)
                     /codon_start=1
                     /gene="tetR"
                     /product="tetracycline repressor TetR"
                     /label=TetR
                     /note="TetR binds to the tetracycline operator tetO to 
                     inhibit transcription. This inhibition can be relieved by 
                     adding tetracycline or doxycycline."
                     /translation="MMSRLDKSKVINSALELLNEVGIEGLTTRKLAQKLGVEQPTLYWH
                     VKNKRALLDALAIEMLDRHHTHFCPLEGESWQDFLRNNAKSFRCALLSHRDGAKVHLGT
                     RPTEKQYETLENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEDQEHQVAKEERETPT
                     TDSMPPLLRQAIELFDHQGAEPAFLFGLELIICGLEKQLKCESGS"
     primer_bind     3088..3107
                     /label=Tet-R
                     /note="Tetracycline resistance gene, reverse primer"
ORIGIN
        1 gttgacactc tatcgttgat agagttattt taccactccc tatcagtgat agagaaagta
       61 ctaaggtgca ggagttaaga aaatgaaaat cgaactatca ggcggatata tctgctacag
      121 cattgaagaa gatgaagtta ccattgacat ggtagaggtt accaccaaac gtcaaggcat
      181 tggcagtcag ctaattgata tggttaagga tgtagcccgt gaagttggct tgccaattgg
      241 tctatatgcc tacccccaag atgacagcat tagccaagaa gacttgattg agttttattt
      301 ttctaatgac tttgagtatg acccagatga tgtagatggt cgcttgatga gatggagcta
      361 aaactcagaa gtgaaacgcc gccccatgcg agagtaggga actgccaggc atttcagcca
      421 aaaaacttaa gaccgccggt cttgtccact accttgcagt aatgcggtgg acaggatcgg
      481 cggttttctt ttctcttctc aaccgccggg agcggatttg aacgttgcga agcaacggcc
      541 cggagggtgg cgggcaggac gcccgccata aactgccagg catcaaatta agcagaaggc
      601 catcctgacg gatggccttt ttgcgtttct acaaactctg cggtaatacg gttatccaca
      661 gaatcagggg ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac
      721 cgtaaaaagg ccgcgttgct ggcgtttttc cacaggctcc gcccccctga cgagcatcac
      781 aaaaatcgac gctcaagtca gaggtggcga aacccgacag gactataaag ataccaggcg
      841 tttccccctg gaagctccct cgtgcgctct cctgttccga ccctgccgct taccggatac
      901 ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc atagctcacg ctgtaggtat
      961 ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag
     1021 cccgaccgct gcgccttatc cggtaactat cgtcttgagt ccaacccggt aagacacgac
     1081 ttatcgccac tggcagcagc cactggtaac aggattagca gagcgaggta tgtaggcggt
     1141 gctacagagt tcttgaagtg gtggcctaac tacggctaca ctagaagaac agtatttggt
     1201 atctgcgctc tgctgaagcc agttaccttc ggaaaaagag ttggtagctc ttgatccggc
     1261 aaacaaacca ccgctggtag cggtggtttt tttgtttgca agcagcagat tacgcgcaga
     1321 aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc tcagtggaac
     1381 gaaaactcac gttaagggat tttggtcatg agattatcaa aaaggatctt cacctagatc
     1441 cttttaaatt aaaaatgaag ttttaaatca atctaaagta tatatgagta aacttggtct
     1501 gacagttacc aatgcttaat cagtgaggca cctatctcag cgatctgtct atttcgttca
     1561 tccatagttg cctgactccc cgtcgtgtag ataactacga tacgggaggg cttaccatct
     1621 ggccccagtg ctgcaatgat accgcgagac ccacgctcac cggctccaga tttatcagca
     1681 ataaaccagc cagccggaag ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc
     1741 atccagtcta ttaattgttg ccgggaagct agagtaagta gttcgccagt taatagtttg
     1801 cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct
     1861 tcattcagct ccggttccca acgatcaagg cgagttacat gatcccccat gttgtgcaaa
     1921 aaagcggtta gctccttcgg tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta
     1981 tcactcatgg ttatggcagc actgcataat tctcttactg tcatgccatc cgtaagatgc
     2041 ttttctgtga ctggtgagta ctcaaccaag tcattctgag aatagtgtat gcggcgaccg
     2101 agttgctctt gcccggcgtc aatacgggat aataccgcgc cacatagcag aactttaaaa
     2161 gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct caaggatctt accgctgttg
     2221 agatccagtt cgatgtaacc cactcgtgca cccaactgat cttcagcatc ttttactttc
     2281 accagcgttt ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg
     2341 gcgacacgga aatgttgaat actcatactc ttcctttttc aatattattg aagcatttat
     2401 cagggttatt gtctcatgag cggatacata tttgaatgta tttagaaaaa taaacaaata
     2461 ggggttccgc gcacatttcc ccgaaaagtg ccacctgacg tctaagaaac cattattgtg
     2521 gatatacctt actcgagtta gccttgatag gacgtcttaa gacccacttt cacatttaag
     2581 ttgtttttct aatccgcata tgatcaattc aaggccgaat aagaaggctg gctctgcacc
     2641 ttggtgatca aataattcga tagcttgtcg taataatggc ggcatactat cagtagtagg
     2701 tgtttccctt tcttctttag cgacttgatg ctcttgatct tccaatacgc aacctaaagt
     2761 aaaatgcccc acagcgctga gtgcatataa tgcattctct agtgaaaaac cttgttggca
     2821 taaaaaggct aattgatttt cgagagtttc atactgtttt tctgtaggcc gtgtacctaa
     2881 atgtactttt gctccatcgc gatgacttag taaagcacat ctaaaacttt tagcgttatt
     2941 acgtaaaaaa tcttgccagc tttccccttc taaagggcaa aagtgagtat ggtgcctatc
     3001 taacatctca atggctaagg cgtcgagcaa agcccgctta ttttttacat gccaatacaa
     3061 tgtaggctgc tctacaccta gcttctgggc gagtttacgg gttgttaaac cttcgattcc
     3121 gacctcatta agcagctcta atgcgctgtt aatcacttta cttttatcta atctagacat
     3181 cattaattcc taattttt
//
