LOCUS       sequence-218330-        6246 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses AcrVA5 in bacteria via T7 RNAP, for purification.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6246)
  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 6246)
  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-AcrVA5
FEATURES             Location/Qualifiers
     source          1..6246
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        complement(join(142..147,148..164,165..170))
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
     primer_bind     complement(197..215)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        283..387
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(388..456,457..1248)
                     /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(606..625)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      1419..2007
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1908..1927
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2161..2178
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    2193..2335
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(2261..2280)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2421..2443
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(2437..2628)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     4086..4105
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     4189..4208
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        4189..4207
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     RBS             4296..4318
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             4338..4367
                     /codon_start=1
                     /product="10xHis affinity tag"
                     /label=10xHis
                     /translation="HHHHHHHHHH"
     CDS             4377..5477
                     /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     5451..5474
                     /label=MBP-F
                     /note="Maltose binding protein, forward primer"
     CDS             5532..5552
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQS"
     primer_bind     complement(5961..5979)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      5975..6022
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
ORIGIN
        1 agaggcccgg cagtaccggc ataaccaagc ctatgcctac agcatccagg gtgacggtgc
       61 cgaggatgac gatgagcgca ttgttagatt tcatacacgg tgcctgactg cgttagcaat
      121 ttaactgtga taaactaccg cattaaagct tatcgatgat aagctgtcaa acatgagaat
      181 tcttgaagac gaaagggcct cgtgatacgc ctatttttat aggttaatgt catgataata
      241 atggtttctt agacgtcagg tggcactttt cggggaaatg tgcgcggaac ccctatttgt
      301 ttatttttct aaatacattc aaatatgtat ccgctcatga gacaataacc ctgataaatg
      361 cttcaataac attgaaaaag gaagagtatg agtattcaac atttccgtgt cgcccttatt
      421 cccttttttg cggcattttg ccttcctgtt tttgctcacc cagaaacgct ggtgaaagta
      481 aaagatgctg aagatcagtt gggtgcacga gtgggttaca tcgaactgga tctcaacagc
      541 ggtaagatcc ttgagagttt tcgccccgaa gaacgttttc caatgatgag cacttttaaa
      601 gttctgctat gtggcgcggt attatcccgt gttgacgccg ggcaagagca actcggtcgc
      661 cgcatacact attctcagaa tgacttggtt gagtactcac cagtcacaga aaagcatctt
      721 acggatggca tgacagtaag agaattatgc agtgctgcca taaccatgag tgataacact
      781 gcggccaact tacttctgac aacgatcgga ggaccgaagg agctaaccgc ttttttgcac
      841 aacatggggg atcatgtaac tcgccttgat cgttgggaac cggagctgaa tgaagccata
      901 ccaaacgacg agcgtgacac cacgatgcct gcagcaatgg caacaacgtt gcgcaaacta
      961 ttaactggcg aactacttac tctagcttcc cggcaacaat taatagactg gatggaggcg
     1021 gataaagttg caggaccact tctgcgctcg gcccttccgg ctggctggtt tattgctgat
     1081 aaatctggag ccggtgagcg tgggtctcgc ggtatcattg cagcactggg gccagatggt
     1141 aagccctccc gtatcgtagt tatctacacg acggggagtc aggcaactat ggatgaacga
     1201 aatagacaga tcgctgagat aggtgcctca ctgattaagc attggtaact gtcagaccaa
     1261 gtttactcat atatacttta gattgattta aaacttcatt tttaatttaa aaggatctag
     1321 gtgaagatcc tttttgataa tctcatgacc aaaatccctt aacgtgagtt ttcgttccac
     1381 tgagcgtcag accccgtaga aaagatcaaa ggatcttctt gagatccttt ttttctgcgc
     1441 gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg tttgccggat
     1501 caagagctac caactctttt tccgaaggta actggcttca gcagagcgca gataccaaat
     1561 actgtccttc tagtgtagcc gtagttaggc caccacttca agaactctgt agcaccgcct
     1621 acatacctcg ctctgctaat cctgttacca gtggctgctg ccagtggcga taagtcgtgt
     1681 cttaccgggt tggactcaag acgatagtta ccggataagg cgcagcggtc gggctgaacg
     1741 gggggttcgt gcacacagcc cagcttggag cgaacgacct acaccgaact gagataccta
     1801 cagcgtgagc tatgagaaag cgccacgctt cccgaaggga gaaaggcgga caggtatccg
     1861 gtaagcggca gggtcggaac aggagagcgc acgagggagc ttccaggggg aaacgcctgg
     1921 tatctttata gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc
     1981 tcgtcagggg ggcggagcct atggaaaaac gccagcaacg cggccttttt acggttcctg
     2041 gccttttgct ggccttttgc tcacatgttc tttcctgcgt tatcccctga ttctgtggat
     2101 aaccgtatta ccgcctttga gtgagctgat accgctcgcc gcagccgaac gaccgagcgc
     2161 agcgagtcag tgagcgagga agcggaagag cgcctgatgc ggtattttct ccttacgcat
     2221 ctgtgcggta tttcacaccg catatatggt gcactctcag tacaatctgc tctgatgccg
     2281 catagttaag ccagtataca ctccgctatc gctacgtgac tgggtcatgg ctgcgccccg
     2341 acacccgcca acacccgctg acgcgccctg acgggcttgt ctgctcccgg catccgctta
     2401 cagacaagct gtgaccgtct ccgggagctg catgtgtcag aggttttcac cgtcatcacc
     2461 gaaacgcgcg aggcagctgc ggtaaagctc atcagcgtgg tcgtgaagcg attcacagat
     2521 gtctgcctgt tcatccgcgt ccagctcgtt gagtttctcc agaagcgtta atgtctggct
     2581 tctgataaag cgggccatgt taagggcggt tttttcctgt ttggtcactg atgcctccgt
     2641 gtaaggggga tttctgttca tgggggtaat gataccgatg aaacgagaga ggatgctcac
     2701 gatacgggtt actgatgatg aacatgcccg gttactggaa cgttgtgagg gtaaacaact
     2761 ggcggtatgg atgcggcggg accagagaaa aatcactcag ggtcaatgcc agcgcttcgt
     2821 taatacagat gtaggtgttc cacagggtag ccagcagcat cctgcgatgc agatccggaa
     2881 cataatggtg cagggcgctg acttccgcgt ttccagactt tacgaaacac ggaaaccgaa
     2941 gaccattcat gttgttgctc aggtcgcaga cgttttgcag cagcagtcgc ttcacgttcg
     3001 ctcgcgtatc ggtgattcat tctgctaacc agtaaggcaa ccccgccagc ctagccgggt
     3061 cctcaacgac aggagcacga tcatgcgcac ccgtggccag gacccaacgc tgcccgagat
     3121 gcgccgcgtg cggctgctgg agatggcgga cgcgatggat atgttctgcc aagggttggt
     3181 ttgcgcattc acagttctcc gcaagaattg attggctcca attcttggag tggtgaatcc
     3241 gttagcgagg tgccgccggc ttccattcag gtcgaggtgg cccggctcca tgcaccgcga
     3301 cgcaacgcgg ggaggcagac aaggtatagg gcggcgccta caatccatgc caacccgttc
     3361 catgtgctcg ccgaggcggc ataaatcgcc gtgacgatca gcggtccagt gatcgaagtt
     3421 aggctggtaa gagccgcgag cgatccttga agctgtccct gatggtcgtc atctacctgc
     3481 ctggacagca tggcctgcaa cgcgggcatc ccgatgccgc cggaagcgag aagaatcata
     3541 atggggaagg ccatccagcc tcgcgtcgcg aacgccagca agacgtagcc cagcgcgtcg
     3601 gccgccatgc cggcgataat ggcctgcttc tcgccgaaac gtttggtggc gggaccagtg
     3661 acgaaggctt gagcgagggc gtgcaagatt ccgaataccg caagcgacag gccgatcatc
     3721 gtcgcgctcc agcgaaagcg gtcctcgccg aaaatgaccc agagcgctgc cggcacctgt
     3781 cctacgagtt gcatgataaa gaagacagtc ataagtgcgg cgacgatagt catgccccgc
     3841 gcccaccgga aggagctgac tgggttgaag gctctcaagg gcatcggtcg acgctctccc
     3901 ttatgcgact cctgcattag gaagcagccc agtagtaggt tgaggccgtt gagcaccgcc
     3961 gccgcaagga atggtgcatg caaggagatg gcgcccaaca gtcccccggc cacggggcct
     4021 gccaccatac ccacgccgaa acaagcgctc atgagcccga agtggcgagc ccgatcttcc
     4081 ccatcggtga tgtcggcgat ataggcgcca gcaaccgcac ctgtggcgcc ggtgatgccg
     4141 gccacgatgc gtccggcgta gaggatcgag atctcgatcc cgcgaaatta atacgactca
     4201 ctatagggag accacaacgg tttccctcta gtgccggctc cggagagctc tttaattaag
     4261 cggccgccct gcaggactcg agttctagaa ataattttgt ttaactttaa gaaggagata
     4321 tacatatgaa atcttctcac catcaccatc accatcacca tcaccatggt tcttctatga
     4381 aaatcgaaga aggtaaactg gtaatctgga ttaacggcga taaaggctat aacggtctcg
     4441 ctgaagtcgg taagaaattc gagaaagata ccggaattaa agtcaccgtt gagcatccgg
     4501 ataaactgga agagaaattc ccacaggttg cggcaactgg cgatggccct gacattatct
     4561 tctgggcaca cgaccgcttt ggtggctacg ctcaatctgg cctgttggct gaaatcaccc
     4621 cggacaaagc gttccaggac aagctgtatc cgtttacctg ggatgccgta cgttacaacg
     4681 gcaagctgat tgcttacccg atcgctgttg aagcgttatc gctgatttat aacaaagatc
     4741 tgctgccgaa cccgccaaaa acctgggaag agatcccggc gctggataaa gaactgaaag
     4801 cgaaaggtaa gagcgcgctg atgttcaacc tgcaagaacc gtacttcacc tggccgctga
     4861 ttgctgctga cgggggttat gcgttcaagt atgaaaacgg caagtacgac attaaagacg
     4921 tgggcgtgga taacgctggc gcgaaagcgg gtctgacctt cctggttgac ctgattaaaa
     4981 acaaacacat gaatgcagac accgattact ccatcgcaga agctgccttt aataaaggcg
     5041 aaacagcgat gaccatcaac ggcccgtggg catggtccaa catcgacacc agcaaagtga
     5101 attatggtgt aacggtactg ccgaccttca agggtcaacc atccaaaccg ttcgttggcg
     5161 tgctgagcgc aggtattaac gccgccagtc cgaacaaaga gctggcaaaa gagttcctcg
     5221 aaaactatct gctgactgat gaaggtctgg aagcggttaa taaagacaaa ccgctgggtg
     5281 ccgtagcgct gaagtcttac gaggaagagt tggcgaaaga tccacgtatt gccgccacta
     5341 tggaaaacgc ccagaaaggt gaaatcatgc cgaacatccc gcagatgtcc gctttctggt
     5401 atgccgtgcg tactgcggtg atcaacgccg ccagcggtcg tcagactgtc gatgaagccc
     5461 tgaaagacgc gcagactaat tcgagctcga acaacaacaa caataacaat aacaacaacc
     5521 tcgggatcga ggaaaacctg tacttccaat ccaatgcaat gaaaatcgaa ctatcaggcg
     5581 gatatatctg ctacagcatt gaagaagatg aagttaccat tgacatggta gaggttacca
     5641 ccaaacgtca aggcattggc agtcagctaa ttgatatggt taaggatgta gcccgtgaag
     5701 ttggcttgcc aattggtcta tatgcctacc cccaagatga cagcattagc caagaagact
     5761 tgattgagtt ttatttttct aatgactttg agtatgaccc agatgatgta gatggtcgct
     5821 tgatgagatg gagctaaata ttggaagtgg ataacggatc cgcgatcgcg gcgcgccacc
     5881 tggtggccgg ccggtaccac gcgtgcgcgc tgatccggct gctaacaaag cccgaaagga
     5941 agctgagttg gctgctgcca ccgctgagca ataactagca taaccccttg gggcctctaa
     6001 acgggtcttg aggggttttt tgctgaaagg aggaactata tccggatatc cacaggacgg
     6061 gtgtggtcgc catgatcgcg tagtcgatag tggctccaag tagcgaagcg agcaggactg
     6121 ggcggcggcc aaagcggtcg gacagtgctc cgagaacggg tgcgcataga aattgcatca
     6181 acgcatatag cgctagcagc acgccatagt gactggcgat gctgtcggaa tggacgatat
     6241 cccgca
//
