LOCUS       sequence-326607-        6337 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 6337)
  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 6337)
  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..6337
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     1..20
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1..19
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     RBS             108..130
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             150..179
                     /codon_start=1
                     /product="10xHis affinity tag"
                     /label=10xHis
                     /translation="HHHHHHHHHH"
     CDS             189..1289
                     /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     1263..1286
                     /label=MBP-F
                     /note="Maltose binding protein, forward primer"
     CDS             1344..1364
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQS"
     CDS             1438..1458
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQS"
     primer_bind     complement(1867..1885)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1881..1928
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     promoter        complement(join(2294..2299,2300..2316,2317..2322))
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
     primer_bind     complement(2349..2367)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        2435..2539
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2540..2608,2609..3400)
                     /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(2758..2777)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3571..4159
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4060..4079
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4313..4330
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4345..4484
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4410..4429)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4570..4592
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4586..4777)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     6235..6254
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
ORIGIN
        1 taatacgact cactataggg agaccacaac ggtttccctc tagtgccggc tccggagagc
       61 tctttaatta agcggccgcc ctgcaggact cgagttctag aaataatttt gtttaacttt
      121 aagaaggaga tatacatatg aaatcttctc accatcacca tcaccatcac catcaccatg
      181 gttcttctat gaaaatcgaa gaaggtaaac tggtaatctg gattaacggc gataaaggct
      241 ataacggtct cgctgaagtc ggtaagaaat tcgagaaaga taccggaatt aaagtcaccg
      301 ttgagcatcc ggataaactg gaagagaaat tcccacaggt tgcggcaact ggcgatggcc
      361 ctgacattat cttctgggca cacgaccgct ttggtggcta cgctcaatct ggcctgttgg
      421 ctgaaatcac cccggacaaa gcgttccagg acaagctgta tccgtttacc tgggatgccg
      481 tacgttacaa cggcaagctg attgcttacc cgatcgctgt tgaagcgtta tcgctgattt
      541 ataacaaaga tctgctgccg aacccgccaa aaacctggga agagatcccg gcgctggata
      601 aagaactgaa agcgaaaggt aagagcgcgc tgatgttcaa cctgcaagaa ccgtacttca
      661 cctggccgct gattgctgct gacgggggtt atgcgttcaa gtatgaaaac ggcaagtacg
      721 acattaaaga cgtgggcgtg gataacgctg gcgcgaaagc gggtctgacc ttcctggttg
      781 acctgattaa aaacaaacac atgaatgcag acaccgatta ctccatcgca gaagctgcct
      841 ttaataaagg cgaaacagcg atgaccatca acggcccgtg ggcatggtcc aacatcgaca
      901 ccagcaaagt gaattatggt gtaacggtac tgccgacctt caagggtcaa ccatccaaac
      961 cgttcgttgg cgtgctgagc gcaggtatta acgccgccag tccgaacaaa gagctggcaa
     1021 aagagttcct cgaaaactat ctgctgactg atgaaggtct ggaagcggtt aataaagaca
     1081 aaccgctggg tgccgtagcg ctgaagtctt acgaggaaga gttggcgaaa gatccacgta
     1141 ttgccgccac tatggaaaac gcccagaaag gtgaaatcat gccgaacatc ccgcagatgt
     1201 ccgctttctg gtatgccgtg cgtactgcgg tgatcaacgc cgccagcggt cgtcagactg
     1261 tcgatgaagc cctgaaagac gcgcagacta attcgagctc gaacaacaac aacaataaca
     1321 ataacaacaa cctcgggatc gaggaaaacc tgtacttcca atccaatgca atgaaaatcg
     1381 aactatcagg cggatatatc tgctacagca ttgaagaaga acaacctcgg gatcgaggaa
     1441 aacctgtact tccaatccaa tgcaatgaaa atcgaactat caggcggata tatctgctac
     1501 agcattgaag aagatgaagt taccattgac atggtagagg ttaccaccaa acgtcaaggc
     1561 attggcagtc agctaattga tatggttaag gatgtagccc gtgaagttgg cttgccaatt
     1621 ggtctatatg cctaccccca agatgacagc attagccaag aagacttgat tgagttttat
     1681 ttttctaatg actttgagta tgacccagat gatgtagatg gtcgcttgat gagatggagc
     1741 taaatattgg aagtggataa cggatccgcg atcgcggcgc gccacctggt ggccggccgg
     1801 taccacgcgt gcgcgctgat ccggctgcta acaaagcccg aaaggaagct gagttggctg
     1861 ctgccaccgc tgagcaataa ctagcataac cccttggggc ctctaaacgg gtcttgaggg
     1921 gttttttgct gaaaggagga actatatccg gatatccaca ggacgggtgt ggtcgccatg
     1981 atcgcgtagt cgatagtggc tccaagtagc gaagcgagca ggactgggcg gcggccaaag
     2041 cggtcggaca gtgctccgag aacgggtgcg catagaaatt gcatcaacgc atatagcgct
     2101 agcagcacgc catagtgact ggcgatgctg tcggaatgga cgatatcccg caagaggccc
     2161 ggcagtaccg gcataaccaa gcctatgcct acagcatcca gggtgacggt gccgaggatg
     2221 acgatgagcg cattgttaga tttcatacac ggtgcctgac tgcgttagca atttaactgt
     2281 gataaactac cgcattaaag cttatcgatg ataagctgtc aaacatgaga attcttgaag
     2341 acgaaagggc ctcgtgatac gcctattttt ataggttaat gtcatgataa taatggtttc
     2401 ttagacgtca ggtggcactt ttcggggaaa tgtgcgcgga acccctattt gtttattttt
     2461 ctaaatacat tcaaatatgt atccgctcat gagacaataa ccctgataaa tgcttcaata
     2521 acattgaaaa aggaagagta tgagtattca acatttccgt gtcgccctta ttcccttttt
     2581 tgcggcattt tgccttcctg tttttgctca cccagaaacg ctggtgaaag taaaagatgc
     2641 tgaagatcag ttgggtgcac gagtgggtta catcgaactg gatctcaaca gcggtaagat
     2701 ccttgagagt tttcgccccg aagaacgttt tccaatgatg agcactttta aagttctgct
     2761 atgtggcgcg gtattatccc gtgttgacgc cgggcaagag caactcggtc gccgcataca
     2821 ctattctcag aatgacttgg ttgagtactc accagtcaca gaaaagcatc ttacggatgg
     2881 catgacagta agagaattat gcagtgctgc cataaccatg agtgataaca ctgcggccaa
     2941 cttacttctg acaacgatcg gaggaccgaa ggagctaacc gcttttttgc acaacatggg
     3001 ggatcatgta actcgccttg atcgttggga accggagctg aatgaagcca taccaaacga
     3061 cgagcgtgac accacgatgc ctgcagcaat ggcaacaacg ttgcgcaaac tattaactgg
     3121 cgaactactt actctagctt cccggcaaca attaatagac tggatggagg cggataaagt
     3181 tgcaggacca cttctgcgct cggcccttcc ggctggctgg tttattgctg ataaatctgg
     3241 agccggtgag cgtgggtctc gcggtatcat tgcagcactg gggccagatg gtaagccctc
     3301 ccgtatcgta gttatctaca cgacggggag tcaggcaact atggatgaac gaaatagaca
     3361 gatcgctgag ataggtgcct cactgattaa gcattggtaa ctgtcagacc aagtttactc
     3421 atatatactt tagattgatt taaaacttca tttttaattt aaaaggatct aggtgaagat
     3481 cctttttgat aatctcatga ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc
     3541 agaccccgta gaaaagatca aaggatcttc ttgagatcct ttttttctgc gcgtaatctg
     3601 ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt tgtttgccgg atcaagagct
     3661 accaactctt tttccgaagg taactggctt cagcagagcg cagataccaa atactgtcct
     3721 tctagtgtag ccgtagttag gccaccactt caagaactct gtagcaccgc ctacatacct
     3781 cgctctgcta atcctgttac cagtggctgc tgccagtggc gataagtcgt gtcttaccgg
     3841 gttggactca agacgatagt taccggataa ggcgcagcgg tcgggctgaa cggggggttc
     3901 gtgcacacag cccagcttgg agcgaacgac ctacaccgaa ctgagatacc tacagcgtga
     3961 gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg
     4021 cagggtcgga acaggagagc gcacgaggga gcttccaggg ggaaacgcct ggtatcttta
     4081 tagtcctgtc gggtttcgcc acctctgact tgagcgtcga tttttgtgat gctcgtcagg
     4141 ggggcggagc ctatggaaaa acgccagcaa cgcggccttt ttacggttcc tggccttttg
     4201 ctggcctttt gctcacatgt tctttcctgc gttatcccct gattctgtgg ataaccgtat
     4261 taccgccttt gagtgagctg ataccgctcg ccgcagccga acgaccgagc gcagcgagtc
     4321 agtgagcgag gaagcggaag agcgcctgat gcggtatttt ctccttacgc atctgtgcgg
     4381 tatttcacac cgcaatggtg cactctcagt acaatctgct ctgatgccgc atagttaagc
     4441 cagtatacac tccgctatcg ctacgtgact gggtcatggc tgcgccccga cacccgccaa
     4501 cacccgctga cgcgccctga cgggcttgtc tgctcccggc atccgcttac agacaagctg
     4561 tgaccgtctc cgggagctgc atgtgtcaga ggttttcacc gtcatcaccg aaacgcgcga
     4621 ggcagctgcg gtaaagctca tcagcgtggt cgtgaagcga ttcacagatg tctgcctgtt
     4681 catccgcgtc cagctcgttg agtttctcca gaagcgttaa tgtctggctt ctgataaagc
     4741 gggccatgtt aagggcggtt ttttcctgtt tggtcactga tgcctccgtg taagggggat
     4801 ttctgttcat gggggtaatg ataccgatga aacgagagag gatgctcacg atacgggtta
     4861 ctgatgatga acatgcccgg ttactggaac gttgtgaggg taaacaactg gcggtatgga
     4921 tgcggcggga ccagagaaaa atcactcagg gtcaatgcca gcgcttcgtt aatacagatg
     4981 taggtgttcc acagggtagc cagcagcatc ctgcgatgca gatccggaac ataatggtgc
     5041 agggcgctga cttccgagtt tccagacttt acgaaacacg gaaaccgaag accattcatg
     5101 ttgttgctca ggtcgcagac gttttgcagc agcagtcgct tcacgttcgc tcgcgtatcg
     5161 gtgattcatt ctgctaacca gtaaggcaac cccgccagcc tagccgggtc ctcaacgaca
     5221 ggagcacgat catgcgcacc cgtggccagg acccaacgct gcccgagatg cgccgcgtgc
     5281 ggctgctgga gatggcggac gcgatggata tgttctgcca agggttggtt tgcgcattca
     5341 cagttctccg caagaattga ttggctccaa ttcttggagt ggtgaatccg ttagcgaggt
     5401 gccgccggct tccattcagg tcgaggtggc ccggctccat gcaccgcgac gcaacgcggg
     5461 gaggcagaca aggtataggg cggcgcctac aatccatgcc aacccgttcc atgtgctcgc
     5521 cgaggcggca taaatcgccg tgacgatcag cggtccaatg atcgaagtta ggctggtaag
     5581 agccgcgagc gatccttgaa gctgtccctg atggtcgtca tctacctgcc tggacagcat
     5641 ggcctgcaac gcgggcatcc cgatgccgcc ggaagcgaga agaatcataa tggggaaggc
     5701 catccagcct cgcgtcgcga acgccagcaa gacgtagccc agcgcgtcgg ccgccatgcc
     5761 ggcgataatg gcctgcttct cgccgaaacg tttggtggcg ggaccagtga cgaaggcttg
     5821 agcgagggcg tgcaagattc cgaataccgc aagcgacagg ccgatcatcg tcgcgctcca
     5881 gcgaaagcgg tcctcgccga aaatgaccca gagcgctgcc ggcacctgtc ctacgagttg
     5941 catgataaag aagacagtca taagtgcggc gacgatagtc atgccccgcg cccaccggaa
     6001 ggagctgact gggttgaagg ctctcaaggg catcggtcga cgctctccct tatgcgactc
     6061 ctgcattagg aagcagccca gtagtaggtt gaggccgttg agcaccgccg ccgcaaggaa
     6121 tggtgcatgc aaggagatgg cgcccaacag tcccccggcc acggggcctg ccaccatacc
     6181 cacgccgaaa caagcgctca tgagcccgaa gtggcgagcc cgatcttccc catcggtgat
     6241 gtcggcgata taggcgccag caaccgcacc tgtggcgccg gtgatgccgg ccacgatgcg
     6301 tccggcgtag aggatcgaga tctcgatccc gcgaaat
//
