LOCUS       sequence-274378-        6528 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of AcrIIA13 protein.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6528)
  AUTHORS   Watters KE, Shivram H, Fellmann C, Lew RJ, McMahon B, Doudna JA
  TITLE     Potent CRISPR-Cas9 inhibitors from Staphylococcus genomes.
  JOURNAL   Proc Natl Acad Sci U S A. 2020 Mar 10. pii: 1917668117. doi: 
            10.1073/pnas.1917668117.
  PUBMED    32156733
REFERENCE   2  (bases 1 to 6528)
  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"; journalName: "Proc Natl 
            Acad Sci U S A. 2020 Mar 10. pii: 1917668117. doi: 
            10.1073/pnas.1917668117."
COMMENT     Sequence Label: pKEW215_AcrIIA13
FEATURES             Location/Qualifiers
     source          1..6528
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        complement(join(157..162,163..179,180..185))
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
     primer_bind     complement(212..230)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        298..402
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(403..471,472..1263)
                     /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(621..640)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      1434..2022
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1923..1942
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2176..2193
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    2208..2347
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(2273..2292)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2433..2455
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(2449..2640)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     4098..4117
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     4201..4220
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        4201..4219
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     RBS             4308..4330
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             4350..4379
                     /codon_start=1
                     /product="10xHis affinity tag"
                     /label=10xHis
                     /translation="HHHHHHHHHH"
     CDS             4389..5489
                     /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     5463..5486
                     /label=MBP-F
                     /note="Maltose binding protein, forward primer"
     CDS             5544..5564
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQS"
     primer_bind     complement(6258..6276)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      6272..6319
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
ORIGIN
        1 ggacgatatc ccgcaagagg cccggcagta ccggcataac caagcctatg cctacagcat
       61 ccagggtgac ggtgccgagg atgacgatga gcgcattgtt agatttcata cacggtgcct
      121 gactgcgtta gcaatttaac tgtgataaac taccgcatta aagcttatcg atgataagct
      181 gtcaaacatg agaattcttg aagacgaaag ggcctcgtga tacgcctatt tttataggtt
      241 aatgtcatga taataatggt ttcttagacg tcaggtggca cttttcgggg aaatgtgcgc
      301 ggaaccccta tttgtttatt tttctaaata cattcaaata tgtatccgct catgagacaa
      361 taaccctgat aaatgcttca ataacattga aaaaggaaga gtatgagtat tcaacatttc
      421 cgtgtcgccc ttattccctt ttttgcggca ttttgccttc ctgtttttgc tcacccagaa
      481 acgctggtga aagtaaaaga tgctgaagat cagttgggtg cacgagtggg ttacatcgaa
      541 ctggatctca acagcggtaa gatccttgag agttttcgcc ccgaagaacg ttttccaatg
      601 atgagcactt ttaaagttct gctatgtggc gcggtattat cccgtgttga cgccgggcaa
      661 gagcaactcg gtcgccgcat acactattct cagaatgact tggttgagta ctcaccagtc
      721 acagaaaagc atcttacgga tggcatgaca gtaagagaat tatgcagtgc tgccataacc
      781 atgagtgata acactgcggc caacttactt ctgacaacga tcggaggacc gaaggagcta
      841 accgcttttt tgcacaacat gggggatcat gtaactcgcc ttgatcgttg ggaaccggag
      901 ctgaatgaag ccataccaaa cgacgagcgt gacaccacga tgcctgcagc aatggcaaca
      961 acgttgcgca aactattaac tggcgaacta cttactctag cttcccggca acaattaata
     1021 gactggatgg aggcggataa agttgcagga ccacttctgc gctcggccct tccggctggc
     1081 tggtttattg ctgataaatc tggagccggt gagcgtgggt ctcgcggtat cattgcagca
     1141 ctggggccag atggtaagcc ctcccgtatc gtagttatct acacgacggg gagtcaggca
     1201 actatggatg aacgaaatag acagatcgct gagataggtg cctcactgat taagcattgg
     1261 taactgtcag accaagttta ctcatatata ctttagattg atttaaaact tcatttttaa
     1321 tttaaaagga tctaggtgaa gatccttttt gataatctca tgaccaaaat cccttaacgt
     1381 gagttttcgt tccactgagc gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat
     1441 cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct accagcggtg
     1501 gtttgtttgc cggatcaaga gctaccaact ctttttccga aggtaactgg cttcagcaga
     1561 gcgcagatac caaatactgt ccttctagtg tagccgtagt taggccacca cttcaagaac
     1621 tctgtagcac cgcctacata cctcgctctg ctaatcctgt taccagtggc tgctgccagt
     1681 ggcgataagt cgtgtcttac cgggttggac tcaagacgat agttaccgga taaggcgcag
     1741 cggtcgggct gaacgggggg ttcgtgcaca cagcccagct tggagcgaac gacctacacc
     1801 gaactgagat acctacagcg tgagctatga gaaagcgcca cgcttcccga agggagaaag
     1861 gcggacaggt atccggtaag cggcagggtc ggaacaggag agcgcacgag ggagcttcca
     1921 gggggaaacg cctggtatct ttatagtcct gtcgggtttc gccacctctg acttgagcgt
     1981 cgatttttgt gatgctcgtc aggggggcgg agcctatgga aaaacgccag caacgcggcc
     2041 tttttacggt tcctggcctt ttgctggcct tttgctcaca tgttctttcc tgcgttatcc
     2101 cctgattctg tggataaccg tattaccgcc tttgagtgag ctgataccgc tcgccgcagc
     2161 cgaacgaccg agcgcagcga gtcagtgagc gaggaagcgg aagagcgcct gatgcggtat
     2221 tttctcctta cgcatctgtg cggtatttca caccgcaatg gtgcactctc agtacaatct
     2281 gctctgatgc cgcatagtta agccagtata cactccgcta tcgctacgtg actgggtcat
     2341 ggctgcgccc cgacacccgc caacacccgc tgacgcgccc tgacgggctt gtctgctccc
     2401 ggcatccgct tacagacaag ctgtgaccgt ctccgggagc tgcatgtgtc agaggttttc
     2461 accgtcatca ccgaaacgcg cgaggcagct gcggtaaagc tcatcagcgt ggtcgtgaag
     2521 cgattcacag atgtctgcct gttcatccgc gtccagctcg ttgagtttct ccagaagcgt
     2581 taatgtctgg cttctgataa agcgggccat gttaagggcg gttttttcct gtttggtcac
     2641 tgatgcctcc gtgtaagggg gatttctgtt catgggggta atgataccga tgaaacgaga
     2701 gaggatgctc acgatacggg ttactgatga tgaacatgcc cggttactgg aacgttgtga
     2761 gggtaaacaa ctggcggtat ggatgcggcg ggaccagaga aaaatcactc agggtcaatg
     2821 ccagcgcttc gttaatacag atgtaggtgt tccacagggt agccagcagc atcctgcgat
     2881 gcagatccgg aacataatgg tgcagggcgc tgacttccga gtttccagac tttacgaaac
     2941 acggaaaccg aagaccattc atgttgttgc tcaggtcgca gacgttttgc agcagcagtc
     3001 gcttcacgtt cgctcgcgta tcggtgattc attctgctaa ccagtaaggc aaccccgcca
     3061 gcctagccgg gtcctcaacg acaggagcac gatcatgcgc acccgtggcc aggacccaac
     3121 gctgcccgag atgcgccgcg tgcggctgct ggagatggcg gacgcgatgg atatgttctg
     3181 ccaagggttg gtttgcgcat tcacagttct ccgcaagaat tgattggctc caattcttgg
     3241 agtggtgaat ccgttagcga ggtgccgccg gcttccattc aggtcgaggt ggcccggctc
     3301 catgcaccgc gacgcaacgc ggggaggcag acaaggtata gggcggcgcc tacaatccat
     3361 gccaacccgt tccatgtgct cgccgaggcg gcataaatcg ccgtgacgat cagcggtcca
     3421 atgatcgaag ttaggctggt aagagccgcg agcgatcctt gaagctgtcc ctgatggtcg
     3481 tcatctacct gcctggacag catggcctgc aacgcgggca tcccgatgcc gccggaagcg
     3541 agaagaatca taatggggaa ggccatccag cctcgcgtcg cgaacgccag caagacgtag
     3601 cccagcgcgt cggccgccat gccggcgata atggcctgct tctcgccgaa acgtttggtg
     3661 gcgggaccag tgacgaaggc ttgagcgagg gcgtgcaaga ttccgaatac cgcaagcgac
     3721 aggccgatca tcgtcgcgct ccagcgaaag cggtcctcgc cgaaaatgac ccagagcgct
     3781 gccggcacct gtcctacgag ttgcatgata aagaagacag tcataagtgc ggcgacgata
     3841 gtcatgcccc gcgcccaccg gaaggagctg actgggttga aggctctcaa gggcatcggt
     3901 cgacgctctc ccttatgcga ctcctgcatt aggaagcagc ccagtagtag gttgaggccg
     3961 ttgagcaccg ccgccgcaag gaatggtgca tgcaaggaga tggcgcccaa cagtcccccg
     4021 gccacggggc ctgccaccat acccacgccg aaacaagcgc tcatgagccc gaagtggcga
     4081 gcccgatctt ccccatcggt gatgtcggcg atataggcgc cagcaaccgc acctgtggcg
     4141 ccggtgatgc cggccacgat gcgtccggcg tagaggatcg agatctcgat cccgcgaaat
     4201 taatacgact cactataggg agaccacaac ggtttccctc tagtgccggc tccggagagc
     4261 tctttaatta agcggccgcc ctgcaggact cgagttctag aaataatttt gtttaacttt
     4321 aagaaggaga tatacatatg aaatcttctc accatcacca tcaccatcac catcaccatg
     4381 gttcttctat gaaaatcgaa gaaggtaaac tggtaatctg gattaacggc gataaaggct
     4441 ataacggtct cgctgaagtc ggtaagaaat tcgagaaaga taccggaatt aaagtcaccg
     4501 ttgagcatcc ggataaactg gaagagaaat tcccacaggt tgcggcaact ggcgatggcc
     4561 ctgacattat cttctgggca cacgaccgct ttggtggcta cgctcaatct ggcctgttgg
     4621 ctgaaatcac cccggacaaa gcgttccagg acaagctgta tccgtttacc tgggatgccg
     4681 tacgttacaa cggcaagctg attgcttacc cgatcgctgt tgaagcgtta tcgctgattt
     4741 ataacaaaga tctgctgccg aacccgccaa aaacctggga agagatcccg gcgctggata
     4801 aagaactgaa agcgaaaggt aagagcgcgc tgatgttcaa cctgcaagaa ccgtacttca
     4861 cctggccgct gattgctgct gacgggggtt atgcgttcaa gtatgaaaac ggcaagtacg
     4921 acattaaaga cgtgggcgtg gataacgctg gcgcgaaagc gggtctgacc ttcctggttg
     4981 acctgattaa aaacaaacac atgaatgcag acaccgatta ctccatcgca gaagctgcct
     5041 ttaataaagg cgaaacagcg atgaccatca acggcccgtg ggcatggtcc aacatcgaca
     5101 ccagcaaagt gaattatggt gtaacggtac tgccgacctt caagggtcaa ccatccaaac
     5161 cgttcgttgg cgtgctgagc gcaggtatta acgccgccag tccgaacaaa gagctggcaa
     5221 aagagttcct cgaaaactat ctgctgactg atgaaggtct ggaagcggtt aataaagaca
     5281 aaccgctggg tgccgtagcg ctgaagtctt acgaggaaga gttggcgaaa gatccacgta
     5341 ttgccgccac tatggaaaac gcccagaaag gtgaaatcat gccgaacatc ccgcagatgt
     5401 ccgctttctg gtatgccgtg cgtactgcgg tgatcaacgc cgccagcggt cgtcagactg
     5461 tcgatgaagc cctgaaagac gcgcagacta attcgagctc gaacaacaac aacaataaca
     5521 ataacaacaa cctcgggatc gaggaaaacc tgtacttcca atccaatgca atgagagaga
     5581 caatacaacg attgttagca agtaacttat ccagcaacag tattgcatta gcgactggag
     5641 tatcgcaagc agtgattagt aagttgcgta atgccaagaa agaaatagga aatctaagtt
     5701 tgaacagcgc tgaaaaatta tataattatc aaaaggggtt ggaagtaatg aataaatcaa
     5761 tcgaaattaa agaccaaaat aacatagtat taattgacag tttaggacaa ttctttactg
     5821 acattgagaa cgacaataac ggtcgttaca acattgacta tgttttgttg aatgaagtgg
     5881 aacatgacaa tggcaacacg tattatgaag tgggaatgta cagaaccgaa gaggttcctt
     5941 tcagtgataa agttactcaa gataatgttg aattgctaga agataaatgg cttcaaatag
     6001 atcaacaagg agaaagttat gttgaaagta tctttttcga aaacgaagaa gatgcgagag
     6061 agtatatcaa attagttctg aaaggtcacg aaacttttga agaaactgca aaagcaatcg
     6121 gcgtgattaa ataaatattg gaagtggata acggatccgc gatcgcggcg cgccacctgg
     6181 tggccggccg gtaccacgcg tgcgcgctga tccggctgct aacaaagccc gaaaggaagc
     6241 tgagttggct gctgccaccg ctgagcaata actagcataa ccccttgggg cctctaaacg
     6301 ggtcttgagg ggttttttgc tgaaaggagg aactatatcc ggatatccac aggacgggtg
     6361 tggtcgccat gatcgcgtag tcgatagtgg ctccaagtag cgaagcgagc aggactgggc
     6421 ggcggccaaa gcggtcggac agtgctccga gaacgggtgc gcatagaaat tgcatcaacg
     6481 catatagcgc tagcagcacg ccatagtgac tggcgatgct gtcggaat
//
