LOCUS       sequence-218331-        6672 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses AcrVA4 in bacteria via T7 RNAP, for purification.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6672)
  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 6672)
  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: pKEW212-MBP-TEV-AcrVA4
FEATURES             Location/Qualifiers
     source          1..6672
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        complement(join(139..144,145..161,162..167))
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
     primer_bind     complement(194..212)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        280..384
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(385..453,454..1245)
                     /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(603..622)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      1416..2004
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1905..1924
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2158..2175
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    2190..2332
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(2258..2277)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2418..2440
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(2434..2625)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     4083..4102
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     4186..4205
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        4186..4204
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     RBS             4293..4315
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             4335..4364
                     /codon_start=1
                     /product="10xHis affinity tag"
                     /label=10xHis
                     /translation="HHHHHHHHHH"
     CDS             4374..5474
                     /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     5448..5471
                     /label=MBP-F
                     /note="Maltose binding protein, forward primer"
     CDS             5529..5549
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQS"
     primer_bind     complement(6384..6402)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      6398..6445
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
ORIGIN
        1 ggcccggcag taccggcata accaagccta tgcctacagc atccagggtg acggtgccga
       61 ggatgacgat gagcgcattg ttagatttca tacacggtgc ctgactgcgt tagcaattta
      121 actgtgataa actaccgcat taaagcttat cgatgataag ctgtcaaaca tgagaattct
      181 tgaagacgaa agggcctcgt gatacgccta tttttatagg ttaatgtcat gataataatg
      241 gtttcttaga cgtcaggtgg cacttttcgg ggaaatgtgc gcggaacccc tatttgttta
      301 tttttctaaa tacattcaaa tatgtatccg ctcatgagac aataaccctg ataaatgctt
      361 caataacatt gaaaaaggaa gagtatgagt attcaacatt tccgtgtcgc ccttattccc
      421 ttttttgcgg cattttgcct tcctgttttt gctcacccag aaacgctggt gaaagtaaaa
      481 gatgctgaag atcagttggg tgcacgagtg ggttacatcg aactggatct caacagcggt
      541 aagatccttg agagttttcg ccccgaagaa cgttttccaa tgatgagcac ttttaaagtt
      601 ctgctatgtg gcgcggtatt atcccgtgtt gacgccgggc aagagcaact cggtcgccgc
      661 atacactatt ctcagaatga cttggttgag tactcaccag tcacagaaaa gcatcttacg
      721 gatggcatga cagtaagaga attatgcagt gctgccataa ccatgagtga taacactgcg
      781 gccaacttac ttctgacaac gatcggagga ccgaaggagc taaccgcttt tttgcacaac
      841 atgggggatc atgtaactcg ccttgatcgt tgggaaccgg agctgaatga agccatacca
      901 aacgacgagc gtgacaccac gatgcctgca gcaatggcaa caacgttgcg caaactatta
      961 actggcgaac tacttactct agcttcccgg caacaattaa tagactggat ggaggcggat
     1021 aaagttgcag gaccacttct gcgctcggcc cttccggctg gctggtttat tgctgataaa
     1081 tctggagccg gtgagcgtgg gtctcgcggt atcattgcag cactggggcc agatggtaag
     1141 ccctcccgta tcgtagttat ctacacgacg gggagtcagg caactatgga tgaacgaaat
     1201 agacagatcg ctgagatagg tgcctcactg attaagcatt ggtaactgtc agaccaagtt
     1261 tactcatata tactttagat tgatttaaaa cttcattttt aatttaaaag gatctaggtg
     1321 aagatccttt ttgataatct catgaccaaa atcccttaac gtgagttttc gttccactga
     1381 gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag atcctttttt tctgcgcgta
     1441 atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa
     1501 gagctaccaa ctctttttcc gaaggtaact ggcttcagca gagcgcagat accaaatact
     1561 gtccttctag tgtagccgta gttaggccac cacttcaaga actctgtagc accgcctaca
     1621 tacctcgctc tgctaatcct gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt
     1681 accgggttgg actcaagacg atagttaccg gataaggcgc agcggtcggg ctgaacgggg
     1741 ggttcgtgca cacagcccag cttggagcga acgacctaca ccgaactgag atacctacag
     1801 cgtgagctat gagaaagcgc cacgcttccc gaagggagaa aggcggacag gtatccggta
     1861 agcggcaggg tcggaacagg agagcgcacg agggagcttc cagggggaaa cgcctggtat
     1921 ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt gtgatgctcg
     1981 tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc
     2041 ttttgctggc cttttgctca catgttcttt cctgcgttat cccctgattc tgtggataac
     2101 cgtattaccg cctttgagtg agctgatacc gctcgccgca gccgaacgac cgagcgcagc
     2161 gagtcagtga gcgaggaagc ggaagagcgc ctgatgcggt attttctcct tacgcatctg
     2221 tgcggtattt cacaccgcat atatggtgca ctctcagtac aatctgctct gatgccgcat
     2281 agttaagcca gtatacactc cgctatcgct acgtgactgg gtcatggctg cgccccgaca
     2341 cccgccaaca cccgctgacg cgccctgacg ggcttgtctg ctcccggcat ccgcttacag
     2401 acaagctgtg accgtctccg ggagctgcat gtgtcagagg ttttcaccgt catcaccgaa
     2461 acgcgcgagg cagctgcggt aaagctcatc agcgtggtcg tgaagcgatt cacagatgtc
     2521 tgcctgttca tccgcgtcca gctcgttgag tttctccaga agcgttaatg tctggcttct
     2581 gataaagcgg gccatgttaa gggcggtttt ttcctgtttg gtcactgatg cctccgtgta
     2641 agggggattt ctgttcatgg gggtaatgat accgatgaaa cgagagagga tgctcacgat
     2701 acgggttact gatgatgaac atgcccggtt actggaacgt tgtgagggta aacaactggc
     2761 ggtatggatg cggcgggacc agagaaaaat cactcagggt caatgccagc gcttcgttaa
     2821 tacagatgta ggtgttccac agggtagcca gcagcatcct gcgatgcaga tccggaacat
     2881 aatggtgcag ggcgctgact tccgcgtttc cagactttac gaaacacgga aaccgaagac
     2941 cattcatgtt gttgctcagg tcgcagacgt tttgcagcag cagtcgcttc acgttcgctc
     3001 gcgtatcggt gattcattct gctaaccagt aaggcaaccc cgccagccta gccgggtcct
     3061 caacgacagg agcacgatca tgcgcacccg tggccaggac ccaacgctgc ccgagatgcg
     3121 ccgcgtgcgg ctgctggaga tggcggacgc gatggatatg ttctgccaag ggttggtttg
     3181 cgcattcaca gttctccgca agaattgatt ggctccaatt cttggagtgg tgaatccgtt
     3241 agcgaggtgc cgccggcttc cattcaggtc gaggtggccc ggctccatgc accgcgacgc
     3301 aacgcgggga ggcagacaag gtatagggcg gcgcctacaa tccatgccaa cccgttccat
     3361 gtgctcgccg aggcggcata aatcgccgtg acgatcagcg gtccagtgat cgaagttagg
     3421 ctggtaagag ccgcgagcga tccttgaagc tgtccctgat ggtcgtcatc tacctgcctg
     3481 gacagcatgg cctgcaacgc gggcatcccg atgccgccgg aagcgagaag aatcataatg
     3541 gggaaggcca tccagcctcg cgtcgcgaac gccagcaaga cgtagcccag cgcgtcggcc
     3601 gccatgccgg cgataatggc ctgcttctcg ccgaaacgtt tggtggcggg accagtgacg
     3661 aaggcttgag cgagggcgtg caagattccg aataccgcaa gcgacaggcc gatcatcgtc
     3721 gcgctccagc gaaagcggtc ctcgccgaaa atgacccaga gcgctgccgg cacctgtcct
     3781 acgagttgca tgataaagaa gacagtcata agtgcggcga cgatagtcat gccccgcgcc
     3841 caccggaagg agctgactgg gttgaaggct ctcaagggca tcggtcgacg ctctccctta
     3901 tgcgactcct gcattaggaa gcagcccagt agtaggttga ggccgttgag caccgccgcc
     3961 gcaaggaatg gtgcatgcaa ggagatggcg cccaacagtc ccccggccac ggggcctgcc
     4021 accataccca cgccgaaaca agcgctcatg agcccgaagt ggcgagcccg atcttcccca
     4081 tcggtgatgt cggcgatata ggcgccagca accgcacctg tggcgccggt gatgccggcc
     4141 acgatgcgtc cggcgtagag gatcgagatc tcgatcccgc gaaattaata cgactcacta
     4201 tagggagacc acaacggttt ccctctagtg ccggctccgg agagctcttt aattaagcgg
     4261 ccgccctgca ggactcgagt tctagaaata attttgttta actttaagaa ggagatatac
     4321 atatgaaatc ttctcaccat caccatcacc atcaccatca ccatggttct tctatgaaaa
     4381 tcgaagaagg taaactggta atctggatta acggcgataa aggctataac ggtctcgctg
     4441 aagtcggtaa gaaattcgag aaagataccg gaattaaagt caccgttgag catccggata
     4501 aactggaaga gaaattccca caggttgcgg caactggcga tggccctgac attatcttct
     4561 gggcacacga ccgctttggt ggctacgctc aatctggcct gttggctgaa atcaccccgg
     4621 acaaagcgtt ccaggacaag ctgtatccgt ttacctggga tgccgtacgt tacaacggca
     4681 agctgattgc ttacccgatc gctgttgaag cgttatcgct gatttataac aaagatctgc
     4741 tgccgaaccc gccaaaaacc tgggaagaga tcccggcgct ggataaagaa ctgaaagcga
     4801 aaggtaagag cgcgctgatg ttcaacctgc aagaaccgta cttcacctgg ccgctgattg
     4861 ctgctgacgg gggttatgcg ttcaagtatg aaaacggcaa gtacgacatt aaagacgtgg
     4921 gcgtggataa cgctggcgcg aaagcgggtc tgaccttcct ggttgacctg attaaaaaca
     4981 aacacatgaa tgcagacacc gattactcca tcgcagaagc tgcctttaat aaaggcgaaa
     5041 cagcgatgac catcaacggc ccgtgggcat ggtccaacat cgacaccagc aaagtgaatt
     5101 atggtgtaac ggtactgccg accttcaagg gtcaaccatc caaaccgttc gttggcgtgc
     5161 tgagcgcagg tattaacgcc gccagtccga acaaagagct ggcaaaagag ttcctcgaaa
     5221 actatctgct gactgatgaa ggtctggaag cggttaataa agacaaaccg ctgggtgccg
     5281 tagcgctgaa gtcttacgag gaagagttgg cgaaagatcc acgtattgcc gccactatgg
     5341 aaaacgccca gaaaggtgaa atcatgccga acatcccgca gatgtccgct ttctggtatg
     5401 ccgtgcgtac tgcggtgatc aacgccgcca gcggtcgtca gactgtcgat gaagccctga
     5461 aagacgcgca gactaattcg agctcgaaca acaacaacaa taacaataac aacaacctcg
     5521 ggatcgagga aaacctgtac ttccaatcca atgcaatgta tgaaatcaaa ctaaatgaca
     5581 cccttataca tcagacagat gatagagtga acgcgtttgt ggcttatcgc tacctattaa
     5641 gacgtggcga tctaccaaaa tgcgagaaca tcgcaagaat gtattatgat ggcaaggtta
     5701 ttaagactga cgttattgac cacgacagtg ttcacagtga cgaacaagcc aaggttagta
     5761 acaacgacat cattaagatg gcaatttccg aacttggcgt gaataacttc aagtcgctaa
     5821 tcaagaagca aggctatccg ttcagcaatg ggcatattaa tagttggttc acagatgacc
     5881 cagtcaaatc caagacaatg cacaatgacg aaatgtattt ggttgtgcag gcactgatta
     5941 gagcatgcat aattaaagag attgatttat atacagagca attatataac atcattaaga
     6001 gcctgccgta cgacaaacgc ccaaatgttg tatatagtga ccaaccatta gacccgaata
     6061 atcttgattt atccgaacct gaattatggg cggagcaggt gggcgaatgt atgagatatg
     6121 cacacaacga ccaaccttgc ttctacattg gttcgactaa gcgtgagttg cgtgttaatt
     6181 atattgtgcc tgtaatcggt gtgagagatg agatagaacg tgtaatgaca ctggaagaag
     6241 tgcgaaattt acacaaataa atattggaag tggataacgg atccgcgatc gcggcgcgcc
     6301 acctggtggc cggccggtac cacgcgtgcg cgctgatccg gctgctaaca aagcccgaaa
     6361 ggaagctgag ttggctgctg ccaccgctga gcaataacta gcataacccc ttggggcctc
     6421 taaacgggtc ttgaggggtt ttttgctgaa aggaggaact atatccggat atccacagga
     6481 cgggtgtggt cgccatgatc gcgtagtcga tagtggctcc aagtagcgaa gcgagcagga
     6541 ctgggcggcg gccaaagcgg tcggacagtg ctccgagaac gggtgcgcat agaaattgca
     6601 tcaacgcata tagcgctagc agcacgccat agtgactggc gatgctgtcg gaatggacga
     6661 tatcccgcaa ga
//
