LOCUS       sequence-274367-        6267 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of truncated AcrIIA14 protein.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6267)
  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 6267)
  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: pKEW395_AcrIIA14_truncated
FEATURES             Location/Qualifiers
     source          1..6267
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     201..218
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    233..372
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(298..317)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     458..480
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(474..665)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     2123..2142
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     2226..2245
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        2226..2244
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     RBS             2333..2355
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             2375..2404
                     /codon_start=1
                     /product="10xHis affinity tag"
                     /label=10xHis
                     /translation="HHHHHHHHHH"
     CDS             2414..3514
                     /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     3488..3511
                     /label=MBP-F
                     /note="Maltose binding protein, forward primer"
     CDS             3569..3589
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQS"
     primer_bind     complement(4022..4040)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      4036..4083
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     promoter        complement(join(4449..4454,4455..4471,4472..4477))
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
     primer_bind     complement(4504..4522)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4590..4694
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4695..4763,4764..5555)
                     /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(4913..4932)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      join(5726..6267,1..47)
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6215..6234
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac gccagcaacg
       61 cggccttttt acggttcctg gccttttgct ggccttttgc tcacatgttc tttcctgcgt
      121 tatcccctga ttctgtggat aaccgtatta ccgcctttga gtgagctgat accgctcgcc
      181 gcagccgaac gaccgagcgc agcgagtcag tgagcgagga agcggaagag cgcctgatgc
      241 ggtattttct ccttacgcat ctgtgcggta tttcacaccg caatggtgca ctctcagtac
      301 aatctgctct gatgccgcat agttaagcca gtatacactc cgctatcgct acgtgactgg
      361 gtcatggctg cgccccgaca cccgccaaca cccgctgacg cgccctgacg ggcttgtctg
      421 ctcccggcat ccgcttacag acaagctgtg accgtctccg ggagctgcat gtgtcagagg
      481 ttttcaccgt catcaccgaa acgcgcgagg cagctgcggt aaagctcatc agcgtggtcg
      541 tgaagcgatt cacagatgtc tgcctgttca tccgcgtcca gctcgttgag tttctccaga
      601 agcgttaatg tctggcttct gataaagcgg gccatgttaa gggcggtttt ttcctgtttg
      661 gtcactgatg cctccgtgta agggggattt ctgttcatgg gggtaatgat accgatgaaa
      721 cgagagagga tgctcacgat acgggttact gatgatgaac atgcccggtt actggaacgt
      781 tgtgagggta aacaactggc ggtatggatg cggcgggacc agagaaaaat cactcagggt
      841 caatgccagc gcttcgttaa tacagatgta ggtgttccac agggtagcca gcagcatcct
      901 gcgatgcaga tccggaacat aatggtgcag ggcgctgact tccgagtttc cagactttac
      961 gaaacacgga aaccgaagac cattcatgtt gttgctcagg tcgcagacgt tttgcagcag
     1021 cagtcgcttc acgttcgctc gcgtatcggt gattcattct gctaaccagt aaggcaaccc
     1081 cgccagccta gccgggtcct caacgacagg agcacgatca tgcgcacccg tggccaggac
     1141 ccaacgctgc ccgagatgcg ccgcgtgcgg ctgctggaga tggcggacgc gatggatatg
     1201 ttctgccaag ggttggtttg cgcattcaca gttctccgca agaattgatt ggctccaatt
     1261 cttggagtgg tgaatccgtt agcgaggtgc cgccggcttc cattcaggtc gaggtggccc
     1321 ggctccatgc accgcgacgc aacgcgggga ggcagacaag gtatagggcg gcgcctacaa
     1381 tccatgccaa cccgttccat gtgctcgccg aggcggcata aatcgccgtg acgatcagcg
     1441 gtccaatgat cgaagttagg ctggtaagag ccgcgagcga tccttgaagc tgtccctgat
     1501 ggtcgtcatc tacctgcctg gacagcatgg cctgcaacgc gggcatcccg atgccgccgg
     1561 aagcgagaag aatcataatg gggaaggcca tccagcctcg cgtcgcgaac gccagcaaga
     1621 cgtagcccag cgcgtcggcc gccatgccgg cgataatggc ctgcttctcg ccgaaacgtt
     1681 tggtggcggg accagtgacg aaggcttgag cgagggcgtg caagattccg aataccgcaa
     1741 gcgacaggcc gatcatcgtc gcgctccagc gaaagcggtc ctcgccgaaa atgacccaga
     1801 gcgctgccgg cacctgtcct acgagttgca tgataaagaa gacagtcata agtgcggcga
     1861 cgatagtcat gccccgcgcc caccggaagg agctgactgg gttgaaggct ctcaagggca
     1921 tcggtcgacg ctctccctta tgcgactcct gcattaggaa gcagcccagt agtaggttga
     1981 ggccgttgag caccgccgcc gcaaggaatg gtgcatgcaa ggagatggcg cccaacagtc
     2041 ccccggccac ggggcctgcc accataccca cgccgaaaca agcgctcatg agcccgaagt
     2101 ggcgagcccg atcttcccca tcggtgatgt cggcgatata ggcgccagca accgcacctg
     2161 tggcgccggt gatgccggcc acgatgcgtc cggcgtagag gatcgagatc tcgatcccgc
     2221 gaaattaata cgactcacta tagggagacc acaacggttt ccctctagtg ccggctccgg
     2281 agagctcttt aattaagcgg ccgccctgca ggactcgagt tctagaaata attttgttta
     2341 actttaagaa ggagatatac atatgaaatc ttctcaccat caccatcacc atcaccatca
     2401 ccatggttct tctatgaaaa tcgaagaagg taaactggta atctggatta acggcgataa
     2461 aggctataac ggtctcgctg aagtcggtaa gaaattcgag aaagataccg gaattaaagt
     2521 caccgttgag catccggata aactggaaga gaaattccca caggttgcgg caactggcga
     2581 tggccctgac attatcttct gggcacacga ccgctttggt ggctacgctc aatctggcct
     2641 gttggctgaa atcaccccgg acaaagcgtt ccaggacaag ctgtatccgt ttacctggga
     2701 tgccgtacgt tacaacggca agctgattgc ttacccgatc gctgttgaag cgttatcgct
     2761 gatttataac aaagatctgc tgccgaaccc gccaaaaacc tgggaagaga tcccggcgct
     2821 ggataaagaa ctgaaagcga aaggtaagag cgcgctgatg ttcaacctgc aagaaccgta
     2881 cttcacctgg ccgctgattg ctgctgacgg gggttatgcg ttcaagtatg aaaacggcaa
     2941 gtacgacatt aaagacgtgg gcgtggataa cgctggcgcg aaagcgggtc tgaccttcct
     3001 ggttgacctg attaaaaaca aacacatgaa tgcagacacc gattactcca tcgcagaagc
     3061 tgcctttaat aaaggcgaaa cagcgatgac catcaacggc ccgtgggcat ggtccaacat
     3121 cgacaccagc aaagtgaatt atggtgtaac ggtactgccg accttcaagg gtcaaccatc
     3181 caaaccgttc gttggcgtgc tgagcgcagg tattaacgcc gccagtccga acaaagagct
     3241 ggcaaaagag ttcctcgaaa actatctgct gactgatgaa ggtctggaag cggttaataa
     3301 agacaaaccg ctgggtgccg tagcgctgaa gtcttacgag gaagagttgg cgaaagatcc
     3361 acgtattgcc gccactatgg aaaacgccca gaaaggtgaa atcatgccga acatcccgca
     3421 gatgtccgct ttctggtatg ccgtgcgtac tgcggtgatc aacgccgcca gcggtcgtca
     3481 gactgtcgat gaagccctga aagacgcgca gactaattcg agctcgaaca acaacaacaa
     3541 taacaataac aacaacctcg ggatcgagga aaacctgtac ttccaatcca atgctatgaa
     3601 gagtgtaaaa tatataagta atatgagtaa acaagaaaaa ggttatagag tttatgtgaa
     3661 tgtcgtgaac gaagatactg acaaagggtt tttgttccct agcgtcccta aagaagttat
     3721 cgagaatgac aaaattgacg aattatttaa ctttgaacat cataaaccat atgttcaaaa
     3781 agctaaatca agatacgata aaaatggcat tggatataaa attgttcaat tagatgaagg
     3841 ttttcaaaaa ttcatcgaat tgaataaaga aaaaatgaaa gagaatttgg attattaaat
     3901 attggaagtg gataacggat ccgcgatcgc ggcgcgccac ctggtggccg gccggtacca
     3961 cgcgtgcgcg ctgatccggc tgctaacaaa gcccgaaagg aagctgagtt ggctgctgcc
     4021 accgctgagc aataactagc ataacccctt ggggcctcta aacgggtctt gaggggtttt
     4081 ttgctgaaag gaggaactat atccggatat ccacaggacg ggtgtggtcg ccatgatcgc
     4141 gtagtcgata gtggctccaa gtagcgaagc gagcaggact gggcggcggc caaagcggtc
     4201 ggacagtgct ccgagaacgg gtgcgcatag aaattgcatc aacgcatata gcgctagcag
     4261 cacgccatag tgactggcga tgctgtcgga atggacgata tcccgcaaga ggcccggcag
     4321 taccggcata accaagccta tgcctacagc atccagggtg acggtgccga ggatgacgat
     4381 gagcgcattg ttagatttca tacacggtgc ctgactgcgt tagcaattta actgtgataa
     4441 actaccgcat taaagcttat cgatgataag ctgtcaaaca tgagaattct tgaagacgaa
     4501 agggcctcgt gatacgccta tttttatagg ttaatgtcat gataataatg gtttcttaga
     4561 cgtcaggtgg cacttttcgg ggaaatgtgc gcggaacccc tatttgttta tttttctaaa
     4621 tacattcaaa tatgtatccg ctcatgagac aataaccctg ataaatgctt caataacatt
     4681 gaaaaaggaa gagtatgagt attcaacatt tccgtgtcgc ccttattccc ttttttgcgg
     4741 cattttgcct tcctgttttt gctcacccag aaacgctggt gaaagtaaaa gatgctgaag
     4801 atcagttggg tgcacgagtg ggttacatcg aactggatct caacagcggt aagatccttg
     4861 agagttttcg ccccgaagaa cgttttccaa tgatgagcac ttttaaagtt ctgctatgtg
     4921 gcgcggtatt atcccgtgtt gacgccgggc aagagcaact cggtcgccgc atacactatt
     4981 ctcagaatga cttggttgag tactcaccag tcacagaaaa gcatcttacg gatggcatga
     5041 cagtaagaga attatgcagt gctgccataa ccatgagtga taacactgcg gccaacttac
     5101 ttctgacaac gatcggagga ccgaaggagc taaccgcttt tttgcacaac atgggggatc
     5161 atgtaactcg ccttgatcgt tgggaaccgg agctgaatga agccatacca aacgacgagc
     5221 gtgacaccac gatgcctgca gcaatggcaa caacgttgcg caaactatta actggcgaac
     5281 tacttactct agcttcccgg caacaattaa tagactggat ggaggcggat aaagttgcag
     5341 gaccacttct gcgctcggcc cttccggctg gctggtttat tgctgataaa tctggagccg
     5401 gtgagcgtgg gtctcgcggt atcattgcag cactggggcc agatggtaag ccctcccgta
     5461 tcgtagttat ctacacgacg gggagtcagg caactatgga tgaacgaaat agacagatcg
     5521 ctgagatagg tgcctcactg attaagcatt ggtaactgtc agaccaagtt tactcatata
     5581 tactttagat tgatttaaaa cttcattttt aatttaaaag gatctaggtg aagatccttt
     5641 ttgataatct catgaccaaa atcccttaac gtgagttttc gttccactga gcgtcagacc
     5701 ccgtagaaaa gatcaaagga tcttcttgag atcctttttt tctgcgcgta atctgctgct
     5761 tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa gagctaccaa
     5821 ctctttttcc gaaggtaact ggcttcagca gagcgcagat accaaatact gtccttctag
     5881 tgtagccgta gttaggccac cacttcaaga actctgtagc accgcctaca tacctcgctc
     5941 tgctaatcct gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt accgggttgg
     6001 actcaagacg atagttaccg gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca
     6061 cacagcccag cttggagcga acgacctaca ccgaactgag atacctacag cgtgagctat
     6121 gagaaagcgc cacgcttccc gaagggagaa aggcggacag gtatccggta agcggcaggg
     6181 tcggaacagg agagcgcacg agggagcttc cagggggaaa cgcctggtat ctttatagtc
     6241 ctgtcgggtt tcgccacctc tgacttg
//
