LOCUS       sequence-224374-        6483 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses AcrVA1 in bacteria via T7 RNAP, for purification.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6483)
  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 6483)
  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: pKEW189-MBP-TEV-AcrVA1
FEATURES             Location/Qualifiers
     source          1..6483
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     50..69
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        50..68
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     RBS             157..179
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             199..228
                     /codon_start=1
                     /product="10xHis affinity tag"
                     /label=10xHis
                     /translation="HHHHHHHHHH"
     CDS             238..1338
                     /codon_start=1
                     /gene="malE (mutated)"
                     /product="maltose binding protein from E. coli"
                     /label=MBP
                     /note="This version of the gene does not encode a signal 
                     sequence, so MBP will remain in the cytosol."
                     /translation="MKIEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLE
                     EKFPQVAATGDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDAVRYNGKL
                     IAYPIAVEALSLIYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWPLIA
                     ADGGYAFKYENGKYDIKDVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNKGE
                     TAMTINGPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFL
                     ENYLLTDEGLEAVNKDKPLGAVALKSYEEELAKDPRIAATMENAQKGEIMPNIPQMSAF
                     WYAVRTAVINAASGRQTVDEALKDAQT"
     primer_bind     1312..1335
                     /label=MBP-F
                     /note="Maltose binding protein, forward primer"
     CDS             1393..1413
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQS"
     primer_bind     complement(2062..2080)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2076..2123
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     promoter        complement(join(2489..2494,2495..2511,2512..2517))
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
     primer_bind     complement(2544..2562)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        2630..2734
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2735..2803,2804..3595)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(2953..2972)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3766..4354
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4255..4274
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4508..4525
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4540..4679
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4605..4624)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4765..4787
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4781..4972)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     6430..6449
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
ORIGIN
        1 ggccacgatg cgtccggcgt agaggatcga gatctcgatc ccgcgaaatt aatacgactc
       61 actataggga gaccacaacg gtttccctct agtgccggct ccggagagct ctttaattaa
      121 gcggccgccc tgcaggactc gagttctaga aataattttg tttaacttta agaaggagat
      181 atacatatga aatcttctca ccatcaccat caccatcacc atcaccatgg ttcttctatg
      241 aaaatcgaag aaggtaaact ggtaatctgg attaacggcg ataaaggcta taacggtctc
      301 gctgaagtcg gtaagaaatt cgagaaagat accggaatta aagtcaccgt tgagcatccg
      361 gataaactgg aagagaaatt cccacaggtt gcggcaactg gcgatggccc tgacattatc
      421 ttctgggcac acgaccgctt tggtggctac gctcaatctg gcctgttggc tgaaatcacc
      481 ccggacaaag cgttccagga caagctgtat ccgtttacct gggatgccgt acgttacaac
      541 ggcaagctga ttgcttaccc gatcgctgtt gaagcgttat cgctgattta taacaaagat
      601 ctgctgccga acccgccaaa aacctgggaa gagatcccgg cgctggataa agaactgaaa
      661 gcgaaaggta agagcgcgct gatgttcaac ctgcaagaac cgtacttcac ctggccgctg
      721 attgctgctg acgggggtta tgcgttcaag tatgaaaacg gcaagtacga cattaaagac
      781 gtgggcgtgg ataacgctgg cgcgaaagcg ggtctgacct tcctggttga cctgattaaa
      841 aacaaacaca tgaatgcaga caccgattac tccatcgcag aagctgcctt taataaaggc
      901 gaaacagcga tgaccatcaa cggcccgtgg gcatggtcca acatcgacac cagcaaagtg
      961 aattatggtg taacggtact gccgaccttc aagggtcaac catccaaacc gttcgttggc
     1021 gtgctgagcg caggtattaa cgccgccagt ccgaacaaag agctggcaaa agagttcctc
     1081 gaaaactatc tgctgactga tgaaggtctg gaagcggtta ataaagacaa accgctgggt
     1141 gccgtagcgc tgaagtctta cgaggaagag ttggcgaaag atccacgtat tgccgccact
     1201 atggaaaacg cccagaaagg tgaaatcatg ccgaacatcc cgcagatgtc cgctttctgg
     1261 tatgccgtgc gtactgcggt gatcaacgcc gccagcggtc gtcagactgt cgatgaagcc
     1321 ctgaaagacg cgcagactaa ttcgagctcg aacaacaaca acaataacaa taacaacaac
     1381 ctcgggatcg aggaaaacct gtacttccaa tccatgagca aagcaatgta tgaagcgaaa
     1441 gagcgttacg ccaagaaaaa aatgcaagaa aatacaaaaa ttgatacgct aacagatgaa
     1501 cagcatgatg cactagcgca gctttgtgct tttcgccata aattccactc taacaaagat
     1561 tctttatttt tgagtgaaag tgcattttct ggtgaatttt cttttgagat gcagtctgat
     1621 gaaaatagca agcttagaga agttggctta ccaacaatcg aatggtcatt ttatgataat
     1681 tcccacatcc cggatgattc atttagagaa tggtttaatt ttgctaatta ttcagaattg
     1741 tcggaaacca tccaggaaca aggcttagag ctagacctag atgatgatga aacctatgaa
     1801 cttgtgtatg atgaactata tacagaggct atgggcgaat atgaagagct aaatcaagat
     1861 attgaaaaat acctaagacg aattgatgaa gagcatggta cacaatactg tccaacaggg
     1921 tttgcaagat taagataaat attggaagtg gataacggat ccgcgatcgc ggcgcgccac
     1981 ctggtggccg gccggtacca cgcgtgcgcg ctgatccggc tgctaacaaa gcccgaaagg
     2041 aagctgagtt ggctgctgcc accgctgagc aataactagc ataacccctt ggggcctcta
     2101 aacgggtctt gaggggtttt ttgctgaaag gaggaactat atccggatat ccacaggacg
     2161 ggtgtggtcg ccatgatcgc gtagtcgata gtggctccaa gtagcgaagc gagcaggact
     2221 gggcggcggc caaagcggtc ggacagtgct ccgagaacgg gtgcgcatag aaattgcatc
     2281 aacgcatata gcgctagcag cacgccatag tgactggcga tgctgtcgga atggacgata
     2341 tcccgcaaga ggcccggcag taccggcata accaagccta tgcctacagc atccagggtg
     2401 acggtgccga ggatgacgat gagcgcattg ttagatttca tacacggtgc ctgactgcgt
     2461 tagcaattta actgtgataa actaccgcat taaagcttat cgatgataag ctgtcaaaca
     2521 tgagaattct tgaagacgaa agggcctcgt gatacgccta tttttatagg ttaatgtcat
     2581 gataataatg gtttcttaga cgtcaggtgg cacttttcgg ggaaatgtgc gcggaacccc
     2641 tatttgttta tttttctaaa tacattcaaa tatgtatccg ctcatgagac aataaccctg
     2701 ataaatgctt caataacatt gaaaaaggaa gagtatgagt attcaacatt tccgtgtcgc
     2761 ccttattccc ttttttgcgg cattttgcct tcctgttttt gctcacccag aaacgctggt
     2821 gaaagtaaaa gatgctgaag atcagttggg tgcacgagtg ggttacatcg aactggatct
     2881 caacagcggt aagatccttg agagttttcg ccccgaagaa cgttttccaa tgatgagcac
     2941 ttttaaagtt ctgctatgtg gcgcggtatt atcccgtgtt gacgccgggc aagagcaact
     3001 cggtcgccgc atacactatt ctcagaatga cttggttgag tactcaccag tcacagaaaa
     3061 gcatcttacg gatggcatga cagtaagaga attatgcagt gctgccataa ccatgagtga
     3121 taacactgcg gccaacttac ttctgacaac gatcggagga ccgaaggagc taaccgcttt
     3181 tttgcacaac atgggggatc atgtaactcg ccttgatcgt tgggaaccgg agctgaatga
     3241 agccatacca aacgacgagc gtgacaccac gatgcctgca gcaatggcaa caacgttgcg
     3301 caaactatta actggcgaac tacttactct agcttcccgg caacaattaa tagactggat
     3361 ggaggcggat aaagttgcag gaccacttct gcgctcggcc cttccggctg gctggtttat
     3421 tgctgataaa tctggagccg gtgagcgtgg gtctcgcggt atcattgcag cactggggcc
     3481 agatggtaag ccctcccgta tcgtagttat ctacacgacg gggagtcagg caactatgga
     3541 tgaacgaaat agacagatcg ctgagatagg tgcctcactg attaagcatt ggtaactgtc
     3601 agaccaagtt tactcatata tactttagat tgatttaaaa cttcattttt aatttaaaag
     3661 gatctaggtg aagatccttt ttgataatct catgaccaaa atcccttaac gtgagttttc
     3721 gttccactga gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag atcctttttt
     3781 tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt
     3841 gccggatcaa gagctaccaa ctctttttcc gaaggtaact ggcttcagca gagcgcagat
     3901 accaaatact gtccttctag tgtagccgta gttaggccac cacttcaaga actctgtagc
     3961 accgcctaca tacctcgctc tgctaatcct gttaccagtg gctgctgcca gtggcgataa
     4021 gtcgtgtctt accgggttgg actcaagacg atagttaccg gataaggcgc agcggtcggg
     4081 ctgaacgggg ggttcgtgca cacagcccag cttggagcga acgacctaca ccgaactgag
     4141 atacctacag cgtgagctat gagaaagcgc cacgcttccc gaagggagaa aggcggacag
     4201 gtatccggta agcggcaggg tcggaacagg agagcgcacg agggagcttc cagggggaaa
     4261 cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt
     4321 gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg
     4381 gttcctggcc ttttgctggc cttttgctca catgttcttt cctgcgttat cccctgattc
     4441 tgtggataac cgtattaccg cctttgagtg agctgatacc gctcgccgca gccgaacgac
     4501 cgagcgcagc gagtcagtga gcgaggaagc ggaagagcgc ctgatgcggt attttctcct
     4561 tacgcatctg tgcggtattt cacaccgcaa tggtgcactc tcagtacaat ctgctctgat
     4621 gccgcatagt taagccagta tacactccgc tatcgctacg tgactgggtc atggctgcgc
     4681 cccgacaccc gccaacaccc gctgacgcgc cctgacgggc ttgtctgctc ccggcatccg
     4741 cttacagaca agctgtgacc gtctccggga gctgcatgtg tcagaggttt tcaccgtcat
     4801 caccgaaacg cgcgaggcag ctgcggtaaa gctcatcagc gtggtcgtga agcgattcac
     4861 agatgtctgc ctgttcatcc gcgtccagct cgttgagttt ctccagaagc gttaatgtct
     4921 ggcttctgat aaagcgggcc atgttaaggg cggttttttc ctgtttggtc actgatgcct
     4981 ccgtgtaagg gggatttctg ttcatggggg taatgatacc gatgaaacga gagaggatgc
     5041 tcacgatacg ggttactgat gatgaacatg cccggttact ggaacgttgt gagggtaaac
     5101 aactggcggt atggatgcgg cgggaccaga gaaaaatcac tcagggtcaa tgccagcgct
     5161 tcgttaatac agatgtaggt gttccacagg gtagccagca gcatcctgcg atgcagatcc
     5221 ggaacataat ggtgcagggc gctgacttcc gagtttccag actttacgaa acacggaaac
     5281 cgaagaccat tcatgttgtt gctcaggtcg cagacgtttt gcagcagcag tcgcttcacg
     5341 ttcgctcgcg tatcggtgat tcattctgct aaccagtaag gcaaccccgc cagcctagcc
     5401 gggtcctcaa cgacaggagc acgatcatgc gcacccgtgg ccaggaccca acgctgcccg
     5461 agatgcgccg cgtgcggctg ctggagatgg cggacgcgat ggatatgttc tgccaagggt
     5521 tggtttgcgc attcacagtt ctccgcaaga attgattggc tccaattctt ggagtggtga
     5581 atccgttagc gaggtgccgc cggcttccat tcaggtcgag gtggcccggc tccatgcacc
     5641 gcgacgcaac gcggggaggc agacaaggta tagggcggcg cctacaatcc atgccaaccc
     5701 gttccatgtg ctcgccgagg cggcataaat cgccgtgacg atcagcggtc caatgatcga
     5761 agttaggctg gtaagagccg cgagcgatcc ttgaagctgt ccctgatggt cgtcatctac
     5821 ctgcctggac agcatggcct gcaacgcggg catcccgatg ccgccggaag cgagaagaat
     5881 cataatgggg aaggccatcc agcctcgcgt cgcgaacgcc agcaagacgt agcccagcgc
     5941 gtcggccgcc atgccggcga taatggcctg cttctcgccg aaacgtttgg tggcgggacc
     6001 agtgacgaag gcttgagcga gggcgtgcaa gattccgaat accgcaagcg acaggccgat
     6061 catcgtcgcg ctccagcgaa agcggtcctc gccgaaaatg acccagagcg ctgccggcac
     6121 ctgtcctacg agttgcatga taaagaagac agtcataagt gcggcgacga tagtcatgcc
     6181 ccgcgcccac cggaaggagc tgactgggtt gaaggctctc aagggcatcg gtcgacgctc
     6241 tcccttatgc gactcctgca ttaggaagca gcccagtagt aggttgaggc cgttgagcac
     6301 cgccgccgca aggaatggtg catgcaagga gatggcgccc aacagtcccc cggccacggg
     6361 gcctgccacc atacccacgc cgaaacaagc gctcatgagc ccgaagtggc gagcccgatc
     6421 ttccccatcg gtgatgtcgg cgatataggc gccagcaacc gcacctgtgg cgccggtgat
     6481 gcc
//
