LOCUS       sequence-226343-        3680 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses AcrVA4 in bacteria, under TetR control..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3680)
  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 3680)
  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: pKEW302-AcrVA4
FEATURES             Location/Qualifiers
     source          1..3680
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(118..744)
                     /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     649..668
                     /label=Tet-R
                     /note="Tetracycline resistance gene, reverse primer"
     promoter        760..815
                     /gene="tetR"
                     /label=tetR/tetA promoters
                     /note="overlapping promoters for bacterial tetR and tetA"
     protein_bind    796..814
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     terminator      1784..1811
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     rep_origin      complement(1937..2525)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2017..2036)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2696..3487,3488..3556))
                     /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     3319..3338
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3557..3661)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 ccacctgacg tctaagaaac cattattgtg gatatacctt actcgagtta gccacctgac
       61 gtctaagaaa ccattattgt ggatatacct tactcgagtt agccttgata ggacgtctta
      121 agacccactt tcacatttaa gttgtttttc taatccgcat atgatcaatt caaggccgaa
      181 taagaaggct ggctctgcac cttggtgatc aaataattcg atagcttgtc gtaataatgg
      241 cggcatacta tcagtagtag gtgtttccct ttcttcttta gcgacttgat gctcttgatc
      301 ttccaatacg caacctaaag taaaatgccc cacagcgctg agtgcatata atgcattctc
      361 tagtgaaaaa ccttgttggc ataaaaaggc taattgattt tcgagagttt catactgttt
      421 ttctgtaggc cgtgtaccta aatgtacttt tgctccatcg cgatgactta gtaaagcaca
      481 tctaaaactt ttagcgttat tacgtaaaaa atcttgccag ctttcccctt ctaaagggca
      541 aaagtgagta tggtgcctat ctaacatctc aatggctaag gcgtcgagca aagcccgctt
      601 attttttaca tgccaataca atgtaggctg ctctacacct agcttctggg cgagtttacg
      661 ggttgttaaa ccttcgattc cgacctcatt aagcagctct aatgcgctgt taatcacttt
      721 acttttatct aatctagaca tcattaattc ctaatttttg ttgacactct atcgttgata
      781 gagttatttt accactccct atcagtgata gagaaaatgg tcgcttgatg agatggagct
      841 aaaaaaatgt atgaaatcaa actaaatgac acccttatac atcagacaga tgatagagtg
      901 aacgcgtttg tggcttatcg ctacctatta agacgtggcg atctaccaaa atgcgagaac
      961 atcgcaagaa tgtattatga tggcaaggtt attaagactg acgttattga ccacgacagt
     1021 gttcacagtg acgaacaagc caaggttagt aacaacgaca tcattaagat ggcaatttcc
     1081 gaacttggcg tgaataactt caagtcgcta atcaagaagc aaggctatcc gttcagcaat
     1141 gggcatatta atagttggtt cacagatgac ccagtcaaat ccaagacaat gcacaatgac
     1201 gaaatgtatt tggttgtgca ggcactgatt agagcatgca taattaaaga gattgattta
     1261 tatacagagc aattatataa catcattaag agcctgccgt acgacaaacg cccaaatgtt
     1321 gtatatagtg accaaccatt agacccgaat aatcttgatt tatccgaacc tgaattatgg
     1381 gcggagcagg tgggcgaatg tatgagatat gcacacaacg accaaccttg cttctacatt
     1441 ggttcgacta agcgtgagtt gcgtgttaat tatattgtgc ctgtaatcgg tgtgagagat
     1501 gagatagaac gtgtaatgac actggaagaa gtgcgaaatt tacacaaata aaactcagaa
     1561 gtgaaacgcc gccccatgcg agagtaggga actgccaggc atttcagcca aaaaacttaa
     1621 gaccgccggt cttgtccact accttgcagt aatgcggtgg acaggatcgg cggytttctt
     1681 ttctcttctc aaccgccggg agcggatttg aacgttgcga agcaacggcc cggagggtgg
     1741 cgggcaggac gcccgccata aactgccagg catcaaatta agcagaaggc catcctgacg
     1801 gatggccttt ttgcgtttct acaaactctg cggtaatacg gttatccaca gaatcagggg
     1861 ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg
     1921 ccgcgttgct ggcgtttttc cacaggctcc gcccccctga cgagcatcac aaaaatcgac
     1981 gctcaagtca gaggtggcga aacccgacag gactataaag ataccaggcg tttccccctg
     2041 gaagctccct cgtgcgctct cctgttccga ccctgccgct taccggatac ctgtccgcct
     2101 ttctcccttc gggaagcgtg gcgctttctc atagctcacg ctgtaggtat ctcagttcgg
     2161 tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct
     2221 gcgccttatc cggtaactat cgtcttgagt ccaacccggt aagacacgac ttatcgccac
     2281 tggcagcagc cactggtaac aggattagca gagcgaggta tgtaggcggt gctacagagt
     2341 tcttgaagtg gtggcctaac tacggctaca ctagaagaac agtatttggt atctgcgctc
     2401 tgctgaagcc agttaccttc ggaaaaagag ttggtagctc ttgatccggc aaacaaacca
     2461 ccgctggtag cggtggtttt tttgtttgca agcagcagat tacgcgcaga aaaaaaggat
     2521 ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc tcagtggaac gaaaactcac
     2581 gttaagggat tttggtcatg agattatcaa aaaggatctt cacctagatc cttttaaatt
     2641 aaaaatgaag ttttaaatca atctaaagta tatatgagta aacttggtct gacagttacc
     2701 aatgcttaat cagtgaggca cctatctcag cgatctgtct atttcgttca tccatagttg
     2761 cctgactccc cgtcgtgtag ataactacga tacgggaggg cttaccatct ggccccagtg
     2821 ctgcaatgat accgcgagac ccacgctcac cggctccaga tttatcagca ataaaccagc
     2881 cagccggaag ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc atccagtcta
     2941 ttaattgttg ccgggaagct agagtaagta gttcgccagt taatagtttg cgcaacgttg
     3001 ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct tcattcagct
     3061 ccggttccca acgatcaagg cgagttacat gatcccccat gttgtgcaaa aaagcggtta
     3121 gctccttcgg tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg
     3181 ttatggcagc actgcataat tctcttactg tcatgccatc cgtaagatgc ttttctgtga
     3241 ctggtgagta ctcaaccaag tcattctgag aatagtgtat gcggcgaccg agttgctctt
     3301 gcccggcgtc aatacgggat aataccgcgc cacatagcag aactttaaaa gtgctcatca
     3361 ttggaaaacg ttcttcgggg cgaaaactct caaggatctt accgctgttg agatccagtt
     3421 cgatgtaacc cactcgtgca cccaactgat cttcagcatc ttttactttc accagcgttt
     3481 ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga
     3541 aatgttgaat actcatactc ttcctttttc aatattattg aagcatttat cagggttatt
     3601 gtctcatgag cggatacata tttgaatgta tttagaaaaa taaacaaata ggggttccgc
     3661 gcacatttcc ccgaaaagtg
//
