LOCUS       sequence-218329-        6486 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 6486)
  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 6486)
  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..6486
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        complement(join(241..246,247..263,264..269))
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
     primer_bind     complement(296..314)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        382..486
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(487..555,556..1347)
                     /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(705..724)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      1518..2106
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2007..2026
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2260..2277
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    2292..2434
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(2360..2379)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2520..2542
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(2536..2727)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     4185..4204
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     4288..4307
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        4288..4306
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     RBS             4395..4417
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             4437..4466
                     /codon_start=1
                     /product="10xHis affinity tag"
                     /label=10xHis
                     /translation="HHHHHHHHHH"
     CDS             4476..5576
                     /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     5550..5573
                     /label=MBP-F
                     /note="Maltose binding protein, forward primer"
     CDS             5631..5651
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQS"
     primer_bind     complement(6300..6318)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      6314..6361
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
ORIGIN
        1 ctccgagaac gggtgcgcat agaaattgca tcaacgcata tagcgctagc agcacgccat
       61 agtgactggc gatgctgtcg gaatggacga tatcccgcaa gaggcccggc agtaccggca
      121 taaccaagcc tatgcctaca gcatccaggg tgacggtgcc gaggatgacg atgagcgcat
      181 tgttagattt catacacggt gcctgactgc gttagcaatt taactgtgat aaactaccgc
      241 attaaagctt atcgatgata agctgtcaaa catgagaatt cttgaagacg aaagggcctc
      301 gtgatacgcc tatttttata ggttaatgtc atgataataa tggtttctta gacgtcaggt
      361 ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt tatttttcta aatacattca
      421 aatatgtatc cgctcatgag acaataaccc tgataaatgc ttcaataaca ttgaaaaagg
      481 aagagtatga gtattcaaca tttccgtgtc gcccttattc ccttttttgc ggcattttgc
      541 cttcctgttt ttgctcaccc agaaacgctg gtgaaagtaa aagatgctga agatcagttg
      601 ggtgcacgag tgggttacat cgaactggat ctcaacagcg gtaagatcct tgagagtttt
      661 cgccccgaag aacgttttcc aatgatgagc acttttaaag ttctgctatg tggcgcggta
      721 ttatcccgtg ttgacgccgg gcaagagcaa ctcggtcgcc gcatacacta ttctcagaat
      781 gacttggttg agtactcacc agtcacagaa aagcatctta cggatggcat gacagtaaga
      841 gaattatgca gtgctgccat aaccatgagt gataacactg cggccaactt acttctgaca
      901 acgatcggag gaccgaagga gctaaccgct tttttgcaca acatggggga tcatgtaact
      961 cgccttgatc gttgggaacc ggagctgaat gaagccatac caaacgacga gcgtgacacc
     1021 acgatgcctg cagcaatggc aacaacgttg cgcaaactat taactggcga actacttact
     1081 ctagcttccc ggcaacaatt aatagactgg atggaggcgg ataaagttgc aggaccactt
     1141 ctgcgctcgg cccttccggc tggctggttt attgctgata aatctggagc cggtgagcgt
     1201 gggtctcgcg gtatcattgc agcactgggg ccagatggta agccctcccg tatcgtagtt
     1261 atctacacga cggggagtca ggcaactatg gatgaacgaa atagacagat cgctgagata
     1321 ggtgcctcac tgattaagca ttggtaactg tcagaccaag tttactcata tatactttag
     1381 attgatttaa aacttcattt ttaatttaaa aggatctagg tgaagatcct ttttgataat
     1441 ctcatgacca aaatccctta acgtgagttt tcgttccact gagcgtcaga ccccgtagaa
     1501 aagatcaaag gatcttcttg agatcctttt tttctgcgcg taatctgctg cttgcaaaca
     1561 aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc aagagctacc aactcttttt
     1621 ccgaaggtaa ctggcttcag cagagcgcag ataccaaata ctgtccttct agtgtagccg
     1681 tagttaggcc accacttcaa gaactctgta gcaccgccta catacctcgc tctgctaatc
     1741 ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga
     1801 cgatagttac cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc
     1861 agcttggagc gaacgaccta caccgaactg agatacctac agcgtgagct atgagaaagc
     1921 gccacgcttc ccgaagggag aaaggcggac aggtatccgg taagcggcag ggtcggaaca
     1981 ggagagcgca cgagggagct tccaggggga aacgcctggt atctttatag tcctgtcggg
     2041 tttcgccacc tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta
     2101 tggaaaaacg ccagcaacgc ggccttttta cggttcctgg ccttttgctg gccttttgct
     2161 cacatgttct ttcctgcgtt atcccctgat tctgtggata accgtattac cgcctttgag
     2221 tgagctgata ccgctcgccg cagccgaacg accgagcgca gcgagtcagt gagcgaggaa
     2281 gcggaagagc gcctgatgcg gtattttctc cttacgcatc tgtgcggtat ttcacaccgc
     2341 atatatggtg cactctcagt acaatctgct ctgatgccgc atagttaagc cagtatacac
     2401 tccgctatcg ctacgtgact gggtcatggc tgcgccccga cacccgccaa cacccgctga
     2461 cgcgccctga cgggcttgtc tgctcccggc atccgcttac agacaagctg tgaccgtctc
     2521 cgggagctgc atgtgtcaga ggttttcacc gtcatcaccg aaacgcgcga ggcagctgcg
     2581 gtaaagctca tcagcgtggt cgtgaagcga ttcacagatg tctgcctgtt catccgcgtc
     2641 cagctcgttg agtttctcca gaagcgttaa tgtctggctt ctgataaagc gggccatgtt
     2701 aagggcggtt ttttcctgtt tggtcactga tgcctccgtg taagggggat ttctgttcat
     2761 gggggtaatg ataccgatga aacgagagag gatgctcacg atacgggtta ctgatgatga
     2821 acatgcccgg ttactggaac gttgtgaggg taaacaactg gcggtatgga tgcggcggga
     2881 ccagagaaaa atcactcagg gtcaatgcca gcgcttcgtt aatacagatg taggtgttcc
     2941 acagggtagc cagcagcatc ctgcgatgca gatccggaac ataatggtgc agggcgctga
     3001 cttccgcgtt tccagacttt acgaaacacg gaaaccgaag accattcatg ttgttgctca
     3061 ggtcgcagac gttttgcagc agcagtcgct tcacgttcgc tcgcgtatcg gtgattcatt
     3121 ctgctaacca gtaaggcaac cccgccagcc tagccgggtc ctcaacgaca ggagcacgat
     3181 catgcgcacc cgtggccagg acccaacgct gcccgagatg cgccgcgtgc ggctgctgga
     3241 gatggcggac gcgatggata tgttctgcca agggttggtt tgcgcattca cagttctccg
     3301 caagaattga ttggctccaa ttcttggagt ggtgaatccg ttagcgaggt gccgccggct
     3361 tccattcagg tcgaggtggc ccggctccat gcaccgcgac gcaacgcggg gaggcagaca
     3421 aggtataggg cggcgcctac aatccatgcc aacccgttcc atgtgctcgc cgaggcggca
     3481 taaatcgccg tgacgatcag cggtccagtg atcgaagtta ggctggtaag agccgcgagc
     3541 gatccttgaa gctgtccctg atggtcgtca tctacctgcc tggacagcat ggcctgcaac
     3601 gcgggcatcc cgatgccgcc ggaagcgaga agaatcataa tggggaaggc catccagcct
     3661 cgcgtcgcga acgccagcaa gacgtagccc agcgcgtcgg ccgccatgcc ggcgataatg
     3721 gcctgcttct cgccgaaacg tttggtggcg ggaccagtga cgaaggcttg agcgagggcg
     3781 tgcaagattc cgaataccgc aagcgacagg ccgatcatcg tcgcgctcca gcgaaagcgg
     3841 tcctcgccga aaatgaccca gagcgctgcc ggcacctgtc ctacgagttg catgataaag
     3901 aagacagtca taagtgcggc gacgatagtc atgccccgcg cccaccggaa ggagctgact
     3961 gggttgaagg ctctcaaggg catcggtcga cgctctccct tatgcgactc ctgcattagg
     4021 aagcagccca gtagtaggtt gaggccgttg agcaccgccg ccgcaaggaa tggtgcatgc
     4081 aaggagatgg cgcccaacag tcccccggcc acggggcctg ccaccatacc cacgccgaaa
     4141 caagcgctca tgagcccgaa gtggcgagcc cgatcttccc catcggtgat gtcggcgata
     4201 taggcgccag caaccgcacc tgtggcgccg gtgatgccgg ccacgatgcg tccggcgtag
     4261 aggatcgaga tctcgatccc gcgaaattaa tacgactcac tatagggaga ccacaacggt
     4321 ttccctctag tgccggctcc ggagagctct ttaattaagc ggccgccctg caggactcga
     4381 gttctagaaa taattttgtt taactttaag aaggagatat acatatgaaa tcttctcacc
     4441 atcaccatca ccatcaccat caccatggtt cttctatgaa aatcgaagaa ggtaaactgg
     4501 taatctggat taacggcgat aaaggctata acggtctcgc tgaagtcggt aagaaattcg
     4561 agaaagatac cggaattaaa gtcaccgttg agcatccgga taaactggaa gagaaattcc
     4621 cacaggttgc ggcaactggc gatggccctg acattatctt ctgggcacac gaccgctttg
     4681 gtggctacgc tcaatctggc ctgttggctg aaatcacccc ggacaaagcg ttccaggaca
     4741 agctgtatcc gtttacctgg gatgccgtac gttacaacgg caagctgatt gcttacccga
     4801 tcgctgttga agcgttatcg ctgatttata acaaagatct gctgccgaac ccgccaaaaa
     4861 cctgggaaga gatcccggcg ctggataaag aactgaaagc gaaaggtaag agcgcgctga
     4921 tgttcaacct gcaagaaccg tacttcacct ggccgctgat tgctgctgac gggggttatg
     4981 cgttcaagta tgaaaacggc aagtacgaca ttaaagacgt gggcgtggat aacgctggcg
     5041 cgaaagcggg tctgaccttc ctggttgacc tgattaaaaa caaacacatg aatgcagaca
     5101 ccgattactc catcgcagaa gctgccttta ataaaggcga aacagcgatg accatcaacg
     5161 gcccgtgggc atggtccaac atcgacacca gcaaagtgaa ttatggtgta acggtactgc
     5221 cgaccttcaa gggtcaacca tccaaaccgt tcgttggcgt gctgagcgca ggtattaacg
     5281 ccgccagtcc gaacaaagag ctggcaaaag agttcctcga aaactatctg ctgactgatg
     5341 aaggtctgga agcggttaat aaagacaaac cgctgggtgc cgtagcgctg aagtcttacg
     5401 aggaagagtt ggcgaaagat ccacgtattg ccgccactat ggaaaacgcc cagaaaggtg
     5461 aaatcatgcc gaacatcccg cagatgtccg ctttctggta tgccgtgcgt actgcggtga
     5521 tcaacgccgc cagcggtcgt cagactgtcg atgaagccct gaaagacgcg cagactaatt
     5581 cgagctcgaa caacaacaac aataacaata acaacaacct cgggatcgag gaaaacctgt
     5641 acttccaatc catgagcaaa gcaatgtatg aagcgaaaga gcgttacgcc aagaaaaaaa
     5701 tgcaagaaaa tacaaaaatt gatacgctaa cagatgaaca gcatgatgca ctagcgcagc
     5761 tttgtgcttt tcgccataaa ttccactcta acaaagattc tttatttttg agtgaaagtg
     5821 cattttctgg tgaattttct tttgagatgc agtctgatga aaatagcaag cttagagaag
     5881 ttggcttacc aacaatcgaa tggtcatttt atgataattc ccacatcccg gatgattcat
     5941 ttagagaatg gtttaatttt gctaattatt cagaattgtc ggaaaccatc caggaacaag
     6001 gcttagagct agacctagat gatgatgaaa cctatgaact tgtgtatgat gaactatata
     6061 cagaggctat gggcgaatat gaagagctaa atcaagatat tgaaaaatac ctaagacgaa
     6121 ttgatgaaga gcatggtaca caatactgtc caacagggtt tgcaagatta agataaatat
     6181 tggaagtgga taacggatcc gcgatcgcgg cgcgccacct ggtggccggc cggtaccacg
     6241 cgtgcgcgct gatccggctg ctaacaaagc ccgaaaggaa gctgagttgg ctgctgccac
     6301 cgctgagcaa taactagcat aaccccttgg ggcctctaaa cgggtcttga ggggtttttt
     6361 gctgaaagga ggaactatat ccggatatcc acaggacggg tgtggtcgcc atgatcgcgt
     6421 agtcgatagt ggctccaagt agcgaagcga gcaggactgg gcggcggcca aagcggtcgg
     6481 acagtg
//
