LOCUS       sequence-274370-        6062 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of AcrIIA14 protein.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6062)
  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 6062)
  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: pKEW394_AcrIIA14
FEATURES             Location/Qualifiers
     source          1..6062
                     /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             join(2414..2416,2417..3127)
                     /codon_start=1
                     /product="GFP variant that folds robustly even when fused 
                     to poorly folded proteins (Pedelacq et al., 2006)"
                     /label=superfolder GFP
                     /note="mammalian codon-optimized"
                     /translation="MSKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKRHDFFKSAMPEGYVQERTISFKDDG
                     TYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHNVYITADKQKNGIKA
                     NFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSVLSKDPNEKRDHMVLLE
                     FVTAAGITHGMDELYK"
     CDS             2414..3127
                     /codon_start=1
                     /product="GFP variant that folds robustly even when fused 
                     to poorly folded proteins (Nager et al., 2011)"
                     /label=superfolder GFP
                     /translation="MSKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKRHDFFKSAMPEGYVQERTISFKDDG
                     TYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHNVYITADKQKNGIKA
                     NFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSVLSKDPNEKRDHMVLLE
                     FVTAAGITHGMDELYK"
     primer_bind     complement(2444..2472)
                     /label=GFP-R
                     /note="GFP, reverse primer. Does NOT anneal to EGFP"
     primer_bind     3067..3087
                     /label=GFP-F
                     /note="GFP, forward primer. Does NOT anneal to EGFP"
     CDS             3182..3202
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQS"
     primer_bind     complement(3817..3835)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      3831..3878
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     promoter        complement(join(4244..4249,4250..4266,4267..4272))
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
     primer_bind     complement(4299..4317)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4385..4489
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4490..4558,4559..5350)
                     /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(4708..4727)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      join(5521..6062,1..47)
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6010..6029
                     /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 tccgcgtttc 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 tctatgagca aaggagaaga acttttcact ggagttgtcc caattcttgt
     2461 tgaattagat ggtgatgtta atgggcacaa attttctgtc cgtggagagg gtgaaggtga
     2521 tgctacaaac ggaaaactca cccttaaatt tatttgcact actggaaaac tacctgttcc
     2581 gtggccaaca cttgtcacta ctctgaccta tggtgttcaa tgcttttccc gttatccgga
     2641 tcacatgaaa cggcatgact ttttcaagag tgccatgccc gaaggttatg tacaggaacg
     2701 cactatatct ttcaaagatg acgggaccta caagacgcgt gctgaagtca agtttgaagg
     2761 tgataccctt gttaatcgta tcgagttaaa gggtattgat tttaaagaag atggaaacat
     2821 tcttggacac aaactcgagt acaactttaa ctcacacaat gtatacatca cggcagacaa
     2881 acaaaagaat ggaatcaaag ctaacttcaa aattcgccac aacgttgaag atggttccgt
     2941 tcaactagca gaccattatc aacaaaatac tccaattggc gatggccctg tccttttacc
     3001 agacaaccat tacctgtcga cacaatctgt cctttcgaaa gatcccaacg aaaagcgtga
     3061 ccacatggtc cttcttgagt ttgtaactgc tgctgggatt acacatggca tggatgagct
     3121 ctacaaaaat tcgagctcga acaacaacaa caataacaat aacaacaacc tcgggatcga
     3181 ggaaaacctg tacttccaat ccaatgcatt gaaaaaaaca atcgaaaaac tattaaacag
     3241 tgacttgaac agtaactaca tcgcaaaaaa aacaggtgtt gaacaaagta caatctatcg
     3301 tttgagaacg ggcgaacgcc agttaggcaa actaggattg gatagtgcag aaagattata
     3361 taattatcaa aaggagatag aaaatatgaa gagtgtaaaa tatataagta atatgagtaa
     3421 acaagaaaaa ggttatagag tttatgtgaa tgtcgtgaac gaagatactg acaaagggtt
     3481 tttgttccct agcgtcccta aagaagttat cgagaatgac aaaattgacg aattatttaa
     3541 ctttgaacat cataaaccat atgttcaaaa agctaaatca agatacgata aaaatggcat
     3601 tggatataaa attgttcaat tagatgaagg ttttcaaaaa ttcatcgaat tgaataaaga
     3661 aaaaatgaaa gagaatttgg attattaata ataacattgg aagtggataa cggatccgcg
     3721 atcgcggcgc gccacctggt ggccggccgg taccacgcgt gcgcgctgat ccggctgcta
     3781 acaaagcccg aaaggaagct gagttggctg ctgccaccgc tgagcaataa ctagcataac
     3841 cccttggggc ctctaaacgg gtcttgaggg gttttttgct gaaaggagga actatatccg
     3901 gatatccaca ggacgggtgt ggtcgccatg atcgcgtagt cgatagtggc tccaagtagc
     3961 gaagcgagca ggactgggcg gcggccaaag cggtcggaca gtgctccgag aacgggtgcg
     4021 catagaaatt gcatcaacgc atatagcgct agcagcacgc catagtgact ggcgatgctg
     4081 tcggaatgga cgatatcccg caagaggccc ggcagtaccg gcataaccaa gcctatgcct
     4141 acagcatcca gggtgacggt gccgaggatg acgatgagcg cattgttaga tttcatacac
     4201 ggtgcctgac tgcgttagca atttaactgt gataaactac cgcattaaag cttatcgatg
     4261 ataagctgtc aaacatgaga attcttgaag acgaaagggc ctcgtgatac gcctattttt
     4321 ataggttaat gtcatgataa taatggtttc ttagacgtca ggtggcactt ttcggggaaa
     4381 tgtgcgcgga acccctattt gtttattttt ctaaatacat tcaaatatgt atccgctcat
     4441 gagacaataa ccctgataaa tgcttcaata acattgaaaa aggaagagta tgagtattca
     4501 acatttccgt gtcgccctta ttcccttttt tgcggcattt tgccttcctg tttttgctca
     4561 cccagaaacg ctggtgaaag taaaagatgc tgaagatcag ttgggtgcac gagtgggtta
     4621 catcgaactg gatctcaaca gcggtaagat ccttgagagt tttcgccccg aagaacgttt
     4681 tccaatgatg agcactttta aagttctgct atgtggcgcg gtattatccc gtgttgacgc
     4741 cgggcaagag caactcggtc gccgcataca ctattctcag aatgacttgg ttgagtactc
     4801 accagtcaca gaaaagcatc ttacggatgg catgacagta agagaattat gcagtgctgc
     4861 cataaccatg agtgataaca ctgcggccaa cttacttctg acaacgatcg gaggaccgaa
     4921 ggagctaacc gcttttttgc acaacatggg ggatcatgta actcgccttg atcgttggga
     4981 accggagctg aatgaagcca taccaaacga cgagcgtgac accacgatgc ctgcagcaat
     5041 ggcaacaacg ttgcgcaaac tattaactgg cgaactactt actctagctt cccggcaaca
     5101 attaatagac tggatggagg cggataaagt tgcaggacca cttctgcgct cggcccttcc
     5161 ggctggctgg tttattgctg ataaatctgg agccggtgag cgtgggtctc gcggtatcat
     5221 tgcagcactg gggccagatg gtaagccctc ccgtatcgta gttatctaca cgacggggag
     5281 tcaggcaact atggatgaac gaaatagaca gatcgctgag ataggtgcct cactgattaa
     5341 gcattggtaa ctgtcagacc aagtttactc atatatactt tagattgatt taaaacttca
     5401 tttttaattt aaaaggatct aggtgaagat cctttttgat aatctcatga ccaaaatccc
     5461 ttaacgtgag ttttcgttcc actgagcgtc agaccccgta gaaaagatca aaggatcttc
     5521 ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc
     5581 agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt
     5641 cagcagagcg cagataccaa atactgtcct tctagtgtag ccgtagttag gccaccactt
     5701 caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc
     5761 tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa
     5821 ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac
     5881 ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg
     5941 gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga
     6001 gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact
     6061 tg
//
