LOCUS       sequence-224386-        6669 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 6669)
  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 6669)
  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..6669
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     96..115
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        96..114
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     RBS             203..225
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             245..274
                     /codon_start=1
                     /product="10xHis affinity tag"
                     /label=10xHis
                     /translation="HHHHHHHHHH"
     CDS             284..1384
                     /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     1358..1381
                     /label=MBP-F
                     /note="Maltose binding protein, forward primer"
     CDS             1439..1459
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQS"
     primer_bind     complement(2294..2312)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2308..2355
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     promoter        complement(join(2721..2726,2727..2743,2744..2749))
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
     primer_bind     complement(2776..2794)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        2862..2966
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2967..3035,3036..3827)
                     /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(3185..3204)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3998..4586
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4487..4506
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4740..4757
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4772..4911
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4837..4856)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4997..5019
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(5013..5204)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     join(6662..6669,1..12)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
ORIGIN
        1 cggcgatata ggcgccagca accgcacctg tggcgccggt gatgccggcc acgatgcgtc
       61 cggcgtagag gatcgagatc tcgatcccgc gaaattaata cgactcacta tagggagacc
      121 acaacggttt ccctctagtg ccggctccgg agagctcttt aattaagcgg ccgccctgca
      181 ggactcgagt tctagaaata attttgttta actttaagaa ggagatatac atatgaaatc
      241 ttctcaccat caccatcacc atcaccatca ccatggttct tctatgaaaa tcgaagaagg
      301 taaactggta atctggatta acggcgataa aggctataac ggtctcgctg aagtcggtaa
      361 gaaattcgag aaagataccg gaattaaagt caccgttgag catccggata aactggaaga
      421 gaaattccca caggttgcgg caactggcga tggccctgac attatcttct gggcacacga
      481 ccgctttggt ggctacgctc aatctggcct gttggctgaa atcaccccgg acaaagcgtt
      541 ccaggacaag ctgtatccgt ttacctggga tgccgtacgt tacaacggca agctgattgc
      601 ttacccgatc gctgttgaag cgttatcgct gatttataac aaagatctgc tgccgaaccc
      661 gccaaaaacc tgggaagaga tcccggcgct ggataaagaa ctgaaagcga aaggtaagag
      721 cgcgctgatg ttcaacctgc aagaaccgta cttcacctgg ccgctgattg ctgctgacgg
      781 gggttatgcg ttcaagtatg aaaacggcaa gtacgacatt aaagacgtgg gcgtggataa
      841 cgctggcgcg aaagcgggtc tgaccttcct ggttgacctg attaaaaaca aacacatgaa
      901 tgcagacacc gattactcca tcgcagaagc tgcctttaat aaaggcgaaa cagcgatgac
      961 catcaacggc ccgtgggcat ggtccaacat cgacaccagc aaagtgaatt atggtgtaac
     1021 ggtactgccg accttcaagg gtcaaccatc caaaccgttc gttggcgtgc tgagcgcagg
     1081 tattaacgcc gccagtccga acaaagagct ggcaaaagag ttcctcgaaa actatctgct
     1141 gactgatgaa ggtctggaag cggttaataa agacaaaccg ctgggtgccg tagcgctgaa
     1201 gtcttacgag gaagagttgg cgaaagatcc acgtattgcc gccactatgg aaaacgccca
     1261 gaaaggtgaa atcatgccga acatcccgca gatgtccgct ttctggtatg ccgtgcgtac
     1321 tgcggtgatc aacgccgcca gcggtcgtca gactgtcgat gaagccctga aagacgcgca
     1381 gactaattcg agctcgaaca acaacaacaa taacaataac aacaacctcg ggatcgagga
     1441 aaacctgtac ttccaatcca atgcaatgta tgaaatcaaa ctaaatgaca cccttataca
     1501 tcagacagat gatagagtga acgcgtttgt ggcttatcgc tacctattaa gacgtggcga
     1561 tctaccaaaa tgcgagaaca tcgcaagaat gtattatgat ggcaaggtta ttaagactga
     1621 cgttattgac cacgacagtg ttcacagtga cgaacaagcc aaggttagta acaacgacat
     1681 cattaagatg gcaatttccg aacttggcgt gaataacttc aagtcgctaa tcaagaagca
     1741 aggctatccg ttcagcaatg ggcatattaa tagttggttc acagatgacc cagtcaaatc
     1801 caagacaatg cacaatgacg aaatgtattt ggttgtgcag gcactgatta gagcatgcat
     1861 aattaaagag attgatttat atacagagca attatataac atcattaaga gcctgccgta
     1921 cgacaaacgc ccaaatgttg tatatagtga ccaaccatta gacccgaata atcttgattt
     1981 atccgaacct gaattatggg cggagcaggt gggcgaatgt atgagatatg cacacaacga
     2041 ccaaccttgc ttctacattg gttcgactaa gcgtgagttg cgtgttaatt atattgtgcc
     2101 tgtaatcggt gtgagagatg agatagaacg tgtaatgaca ctggaagaag tgcgaaattt
     2161 acacaaataa atattggaag tggataacgg atccgcgatc gcggcgcgcc acctggtggc
     2221 cggccggtac cacgcgtgcg cgctgatccg gctgctaaca aagcccgaaa ggaagctgag
     2281 ttggctgctg ccaccgctga gcaataacta gcataacccc ttggggcctc taaacgggtc
     2341 ttgaggggtt ttttgctgaa aggaggaact atatccggat atccacagga cgggtgtggt
     2401 cgccatgatc gcgtagtcga tagtggctcc aagtagcgaa gcgagcagga ctgggcggcg
     2461 gccaaagcgg tcggacagtg ctccgagaac gggtgcgcat agaaattgca tcaacgcata
     2521 tagcgctagc agcacgccat agtgactggc gatgctgtcg gaatggacga tatcccgcaa
     2581 gaggcccggc agtaccggca taaccaagcc tatgcctaca gcatccaggg tgacggtgcc
     2641 gaggatgacg atgagcgcat tgttagattt catacacggt gcctgactgc gttagcaatt
     2701 taactgtgat aaactaccgc attaaagctt atcgatgata agctgtcaaa catgagaatt
     2761 cttgaagacg aaagggcctc gtgatacgcc tatttttata ggttaatgtc atgataataa
     2821 tggtttctta gacgtcaggt ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt
     2881 tatttttcta aatacattca aatatgtatc cgctcatgag acaataaccc tgataaatgc
     2941 ttcaataaca ttgaaaaagg aagagtatga gtattcaaca tttccgtgtc gcccttattc
     3001 ccttttttgc ggcattttgc cttcctgttt ttgctcaccc agaaacgctg gtgaaagtaa
     3061 aagatgctga agatcagttg ggtgcacgag tgggttacat cgaactggat ctcaacagcg
     3121 gtaagatcct tgagagtttt cgccccgaag aacgttttcc aatgatgagc acttttaaag
     3181 ttctgctatg tggcgcggta ttatcccgtg ttgacgccgg gcaagagcaa ctcggtcgcc
     3241 gcatacacta ttctcagaat gacttggttg agtactcacc agtcacagaa aagcatctta
     3301 cggatggcat gacagtaaga gaattatgca gtgctgccat aaccatgagt gataacactg
     3361 cggccaactt acttctgaca acgatcggag gaccgaagga gctaaccgct tttttgcaca
     3421 acatggggga tcatgtaact cgccttgatc gttgggaacc ggagctgaat gaagccatac
     3481 caaacgacga gcgtgacacc acgatgcctg cagcaatggc aacaacgttg cgcaaactat
     3541 taactggcga actacttact ctagcttccc ggcaacaatt aatagactgg atggaggcgg
     3601 ataaagttgc aggaccactt ctgcgctcgg cccttccggc tggctggttt attgctgata
     3661 aatctggagc cggtgagcgt gggtctcgcg gtatcattgc agcactgggg ccagatggta
     3721 agccctcccg tatcgtagtt atctacacga cggggagtca ggcaactatg gatgaacgaa
     3781 atagacagat cgctgagata ggtgcctcac tgattaagca ttggtaactg tcagaccaag
     3841 tttactcata tatactttag attgatttaa aacttcattt ttaatttaaa aggatctagg
     3901 tgaagatcct ttttgataat ctcatgacca aaatccctta acgtgagttt tcgttccact
     3961 gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg agatcctttt tttctgcgcg
     4021 taatctgctg cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc
     4081 aagagctacc aactcttttt ccgaaggtaa ctggcttcag cagagcgcag ataccaaata
     4141 ctgtccttct agtgtagccg tagttaggcc accacttcaa gaactctgta gcaccgccta
     4201 catacctcgc tctgctaatc ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc
     4261 ttaccgggtt ggactcaaga cgatagttac cggataaggc gcagcggtcg ggctgaacgg
     4321 ggggttcgtg cacacagccc agcttggagc gaacgaccta caccgaactg agatacctac
     4381 agcgtgagct atgagaaagc gccacgcttc ccgaagggag aaaggcggac aggtatccgg
     4441 taagcggcag ggtcggaaca ggagagcgca cgagggagct tccaggggga aacgcctggt
     4501 atctttatag tcctgtcggg tttcgccacc tctgacttga gcgtcgattt ttgtgatgct
     4561 cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc ggccttttta cggttcctgg
     4621 ccttttgctg gccttttgct cacatgttct ttcctgcgtt atcccctgat tctgtggata
     4681 accgtattac cgcctttgag tgagctgata ccgctcgccg cagccgaacg accgagcgca
     4741 gcgagtcagt gagcgaggaa gcggaagagc gcctgatgcg gtattttctc cttacgcatc
     4801 tgtgcggtat ttcacaccgc aatggtgcac tctcagtaca atctgctctg atgccgcata
     4861 gttaagccag tatacactcc gctatcgcta cgtgactggg tcatggctgc gccccgacac
     4921 ccgccaacac ccgctgacgc gccctgacgg gcttgtctgc tcccggcatc cgcttacaga
     4981 caagctgtga ccgtctccgg gagctgcatg tgtcagaggt tttcaccgtc atcaccgaaa
     5041 cgcgcgaggc agctgcggta aagctcatca gcgtggtcgt gaagcgattc acagatgtct
     5101 gcctgttcat ccgcgtccag ctcgttgagt ttctccagaa gcgttaatgt ctggcttctg
     5161 ataaagcggg ccatgttaag ggcggttttt tcctgtttgg tcactgatgc ctccgtgtaa
     5221 gggggatttc tgttcatggg ggtaatgata ccgatgaaac gagagaggat gctcacgata
     5281 cgggttactg atgatgaaca tgcccggtta ctggaacgtt gtgagggtaa acaactggcg
     5341 gtatggatgc ggcgggacca gagaaaaatc actcagggtc aatgccagcg cttcgttaat
     5401 acagatgtag gtgttccaca gggtagccag cagcatcctg cgatgcagat ccggaacata
     5461 atggtgcagg gcgctgactt ccgagtttcc agactttacg aaacacggaa accgaagacc
     5521 attcatgttg ttgctcaggt cgcagacgtt ttgcagcagc agtcgcttca cgttcgctcg
     5581 cgtatcggtg attcattctg ctaaccagta aggcaacccc gccagcctag ccgggtcctc
     5641 aacgacagga gcacgatcat gcgcacccgt ggccaggacc caacgctgcc cgagatgcgc
     5701 cgcgtgcggc tgctggagat ggcggacgcg atggatatgt tctgccaagg gttggtttgc
     5761 gcattcacag ttctccgcaa gaattgattg gctccaattc ttggagtggt gaatccgtta
     5821 gcgaggtgcc gccggcttcc attcaggtcg aggtggcccg gctccatgca ccgcgacgca
     5881 acgcggggag gcagacaagg tatagggcgg cgcctacaat ccatgccaac ccgttccatg
     5941 tgctcgccga ggcggcataa atcgccgtga cgatcagcgg tccaatgatc gaagttaggc
     6001 tggtaagagc cgcgagcgat ccttgaagct gtccctgatg gtcgtcatct acctgcctgg
     6061 acagcatggc ctgcaacgcg ggcatcccga tgccgccgga agcgagaaga atcataatgg
     6121 ggaaggccat ccagcctcgc gtcgcgaacg ccagcaagac gtagcccagc gcgtcggccg
     6181 ccatgccggc gataatggcc tgcttctcgc cgaaacgttt ggtggcggga ccagtgacga
     6241 aggcttgagc gagggcgtgc aagattccga ataccgcaag cgacaggccg atcatcgtcg
     6301 cgctccagcg aaagcggtcc tcgccgaaaa tgacccagag cgctgccggc acctgtccta
     6361 cgagttgcat gataaagaag acagtcataa gtgcggcgac gatagtcatg ccccgcgccc
     6421 accggaagga gctgactggg ttgaaggctc tcaagggcat cggtcgacgc tctcccttat
     6481 gcgactcctg cattaggaag cagcccagta gtaggttgag gccgttgagc accgccgccg
     6541 caaggaatgg tgcatgcaag gagatggcgc ccaacagtcc cccggccacg gggcctgcca
     6601 ccatacccac gccgaaacaa gcgctcatga gcccgaagtg gcgagcccga tcttccccat
     6661 cggtgatgt
//
