LOCUS       sequence-430359-        7689 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  S. aureus USA300 GyrB mutant T522 residue codon-optimized for E. 
            coli.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7689)
  AUTHORS   French S, Guo ABY, Ellis MJ, Deisinger JP, Johnson JW, Rachwalski K,
            Piquette ZA, Lluka T, Zary M, Gamage S, Magolan J, Brown ED
  TITLE     A platform for predicting mechanism of action based on bacterial 
            transcriptional responses identifies an unusual DNA gyrase 
            inhibitor.
  JOURNAL   Cell Rep. 2024 Apr 4;43(4):114053. doi: 
            10.1016/j.celrep.2024.114053.
  PUBMED    38578824
REFERENCE   2  (bases 1 to 7689)
  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: "Cell Rep.";
            date: "2024-04-4"; volume: "43(4):114053. doi"; pages: " 
            10.1016/j.celrep.2024.114053"
COMMENT     Sequence Label: pET151-GyrB_T522
FEATURES             Location/Qualifiers
     source          1..7689
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(336..356)
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             complement(357..398)
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     CDS             complement(399..416)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     RBS             427..449
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    464..488
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     primer_bind     complement(488..507)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(489..507)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(591..610)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        816..893
                     /gene="lacI"
                     /label=lacI promoter
     CDS             894..1976
                     /codon_start=1
                     /gene="lacI"
                     /product="lac repressor"
                     /label=lacI
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
                     /translation="MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     primer_bind     complement(913..932)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     CDS             3288..3479
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(3473..3495)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    3581..3720
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     3636..3655
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(3735..3752)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3906..4494)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3986..4005)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4665..5456,5457..5525))
                     /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     5288..5307
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5526..5630)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     5698..5716
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     terminator      complement(5954..6001)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     5997..6015
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
ORIGIN
        1 tgcagaacgg tcagaataac ttcaactgcc ggacgaccca ttttctcttg aatatcaacc
       61 ggaataccac gaccattatc ggtaacttta atccagttgt ctttctcgat caccacttca
      121 atctgatttg cataacctgc cagtgcttca tcaatgctgt tatcaacaat ttcccaaacc
      181 agatgatgca gaccacgttc gctggtgcta ccaatataca tacccggacg tttacgaact
      241 gcttccagac cttccagaac ctgaatctga cctgcgccat aattatcggt gttattcaca
      301 tcactcagtg cggtaaccat ggtgaaggga tcaattccct gaaaatacag gttttccgta
      361 gaatcgagac cgaggagagg gttagggata ggcttaccat ggtgatggtg atgatgcata
      421 tgtatatctc cttcttaaag ttaaacaaaa ttatttctag aggggaattg ttatccgctc
      481 acaattcccc tatagtgagt cgtattaatt tcgcgggatc gagatctcga tcctctacgc
      541 cggacgcatc gtggccggca tcaccggcgc cacaggtgcg gttgctggcg cctatatcgc
      601 cgacatcacc gatggggaag atcgggctcg ccacttcggg ctcatgagcg cttgtttcgg
      661 cgtgggtatg gtggcaggcc ccgtggccgg gggactgttg ggcgccatct ccttgcatgc
      721 accattcctt gcggcggcgg tgctcaacgg cctcaaccta ctactgggct gcttcctaat
      781 gcaggagtcg cataagggag agcgtcgaga tcccggacac catcgaatgg cgcaaaacct
      841 ttcgcggtat ggcatgatag cgcccggaag agagtcaatt cagggtggtg aatgtgaaac
      901 cagtaacgtt atacgatgtc gcagagtatg ccggtgtctc ttatcagacc gtttcccgcg
      961 tggtgaacca ggccagccac gtttctgcga aaacgcggga aaaagtggaa gcggcgatgg
     1021 cggagctgaa ttacattccc aaccgcgtgg cacaacaact ggcgggcaaa cagtcgttgc
     1081 tgattggcgt tgccacctcc agtctggccc tgcacgcgcc gtcgcaaatt gtcgcggcga
     1141 ttaaatctcg cgccgatcaa ctgggtgcca gcgtggtggt gtcgatggta gaacgaagcg
     1201 gcgtcgaagc ctgtaaagcg gcggtgcaca atcttctcgc gcaacgcgtc agtgggctga
     1261 tcattaacta tccgctggat gaccaggatg ccattgctgt ggaagctgcc tgcactaatg
     1321 ttccggcgtt atttcttgat gtctctgacc agacacccat caacagtatt attttctccc
     1381 atgaagacgg tacgcgactg ggcgtggagc atctggtcgc attgggtcac cagcaaatcg
     1441 cgctgttagc gggcccatta agttctgtct cggcgcgtct gcgtctggct ggctggcata
     1501 aatatctcac tcgcaatcaa attcagccga tagcggaacg ggaaggcgac tggagtgcca
     1561 tgtccggttt tcaacaaacc atgcaaatgc tgaatgaggg catcgttccc actgcgatgc
     1621 tggttgccaa cgatcagatg gcgctgggcg caatgcgcgc cattaccgag tccgggctgc
     1681 gcgttggtgc ggatatctcg gtagtgggat acgacgatac cgaagacagc tcatgttata
     1741 tcccgccgtt aaccaccatc aaacaggatt ttcgcctgct ggggcaaacc agcgtggacc
     1801 gcttgctgca actctctcag ggccaggcgg tgaagggcaa tcagctgttg cccgtctcac
     1861 tggtgaaaag aaaaaccacc ctggcgccca atacgcaaac cgcctctccc cgcgcgttgg
     1921 ccgattcatt aatgcagctg gcacgacagg tttcccgact ggaaagcggg cagtgagcgc
     1981 aacgcaatta atgtaagtta gctcactcat taggcaccgg gatctcgacc gatgcccttg
     2041 agagccttca acccagtcag ctccttccgg tgggcgcggg gcatgactat cgtcgccgca
     2101 cttatgactg tcttctttat catgcaactc gtaggacagg tgccggcagc gctctgggtc
     2161 attttcggcg aggaccgctt tcgctggagc gcgacgatga tcggcctgtc gcttgcggta
     2221 ttcggaatct tgcacgccct cgctcaagcc ttcgtcactg gtcccgccac caaacgtttc
     2281 ggcgagaagc aggccattat cgccggcatg gcggccgacg cgctgggcta cgtcttgctg
     2341 gcgttcgcga cgcgaggctg gatggccttc cccattatga ttcttctcgc ttccggcggc
     2401 atcgggatgc ccgcgttgca ggccatgctg tccaggcagg tagatgacga ccatcaggga
     2461 cagcttcaag gatcgctcgc ggctcttacc agcctaactt cgatcattgg accgctgatc
     2521 gtcacggcga tttatgccgc ctcggcgagc acatggaacg ggttggcatg gattgtaggc
     2581 gccgccctat accttgtctg cctccccgcg ttgcgtcgcg gtgcatggag ccgggccacc
     2641 tcgacctgaa tggaagccgg cggcacctcg ctaacggatt caccactcca agaattggag
     2701 ccaatcaatt cttgcggaga actgtgaatg cgcaaaccaa cccttggcag aacatatcca
     2761 tcgcgtccgc catctccagc agccgcacgc ggcgcatctc gggcagcgtt gggtcctggc
     2821 cacgggtgcg catgatcgtg ctcctgtcgt tgaggacccg gctaggctgg cggggttgcc
     2881 ttactggtta gcagaatgaa tcaccgatac gcgagcgaac gtgaagcgac tgctgctgca
     2941 aaacgtctgc gacctgagca acaacatgaa tggtcttcgg tttccgtgtt tcgtaaagtc
     3001 tggaaacgcg gaagtcagcg ccctgcacca ttatgttccg gatctgcatc gcaggatgct
     3061 gctggctacc ctgtggaaca cctacatctg tattaacgaa gcgctggcat tgaccctgag
     3121 tgatttttct ctggtcccgc cgcatccata ccgccagttg tttaccctca caacgttcca
     3181 gtaaccgggc atgttcatca tcagtaaccc gtatcgtgag catcctctct cgtttcatcg
     3241 gtatcattac ccccatgaac agaaatcccc cttacacgga ggcatcagtg accaaacagg
     3301 aaaaaaccgc ccttaacatg gcccgcttta tcagaagcca gacattaacg cttctggaga
     3361 aactcaacga gctggacgcg gatgaacagg cagacatctg tgaatcgctt cacgaccacg
     3421 ctgatgagct ttaccgcagc tgcctcgcgc gtttcggtga tgacggtgaa aacctctgac
     3481 acatgcagct cccggagacg gtcacagctt gtctgtaagc ggatgccggg agcagacaag
     3541 cccgtcaggg cgcgtcagcg ggtgttggcg ggtgtcgggg cgcagccatg acccagtcac
     3601 gtagcgatag cggagtgtat actggcttaa ctatgcggca tcagagcaga ttgtactgag
     3661 agtgcaccat tgcggtgtga aataccgcac agatgcgtaa ggagaaaata ccgcatcagg
     3721 cgctcttccg cttcctcgct cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg
     3781 gtatcagctc actcaaaggc ggtaatacgg ttatccacag aatcagggga taacgcagga
     3841 aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg
     3901 gcgtttttcc ataggctccg cccccctgac gagcatcaca aaaatcgacg ctcaagtcag
     3961 aggtggcgaa acccgacagg actataaaga taccaggcgt ttccccctgg aagctccctc
     4021 gtgcgctctc ctgttccgac cctgccgctt accggatacc tgtccgcctt tctcccttcg
     4081 ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt
     4141 cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc
     4201 ggtaactatc gtcttgagtc caacccggta agacacgact tatcgccact ggcagcagcc
     4261 actggtaaca ggattagcag agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg
     4321 tggcctaact acggctacac tagaaggaca gtatttggta tctgcgctct gctgaagcca
     4381 gttaccttcg gaaaaagagt tggtagctct tgatccggca aacaaaccac cgctggtagc
     4441 ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat
     4501 cctttgatct tttctacggg gtctgacgct cagtggaacg aaaactcacg ttaagggatt
     4561 ttggtcatga gattatcaaa aaggatcttc acctagatcc ttttaaatta aaaatgaagt
     4621 tttaaatcaa tctaaagtat atatgagtaa acttggtctg acagttacca atgcttaatc
     4681 agtgaggcac ctatctcagc gatctgtcta tttcgttcat ccatagttgc ctgactcccc
     4741 gtcgtgtaga taactacgat acgggagggc ttaccatctg gccccagtgc tgcaatgata
     4801 ccgcgagacc cacgctcacc ggctccagat ttatcagcaa taaaccagcc agccggaagg
     4861 gccgagcgca gaagtggtcc tgcaacttta tccgcctcca tccagtctat taattgttgc
     4921 cgggaagcta gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt tgccattgct
     4981 gcaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc cggttcccaa
     5041 cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa aagcggttag ctccttcggt
     5101 cctccgatcg ttgtcagaag taagttggcc gcagtgttat cactcatggt tatggcagca
     5161 ctgcataatt ctcttactgt catgccatcc gtaagatgct tttctgtgac tggtgagtac
     5221 tcaaccaagt cattctgaga atagtgtatg cggcgaccga gttgctcttg cccggcgtca
     5281 acacgggata ataccgcgcc acatagcaga actttaaaag tgctcatcat tggaaaacgt
     5341 tcttcggggc gaaaactctc aaggatctta ccgctgttga gatccagttc gatgtaaccc
     5401 actcgtgcac ccaactgatc ttcagcatct tttactttca ccagcgtttc tgggtgagca
     5461 aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa atgttgaata
     5521 ctcatactct tcctttttca atattattga agcatttatc agggttattg tctcatgagc
     5581 ggatacatat ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg cacatttccc
     5641 cgaaaagtgc cacctgacgt ctaagaaacc attattatca tgacattaac ctataaaaat
     5701 aggcgtatca cgaggccctt tcgtcttcaa gaattaattc tcatgtttga cagcttatca
     5761 tcgataagct agctttaatg cggtagttta tcacagttaa attgctaacg cagtcaggca
     5821 ccgtgtatga aatctaacaa tgcgctcatc gtcatcctcg gcaccgtcac cctggatgct
     5881 gtaggcatag gcttggttat gccggtactg ccgggcctct tgcgggatat ccggatatag
     5941 ttcctccttt cagcaaaaaa cccctcaaga cccgtttaga ggccccaagg ggttatgcta
     6001 gttattgctc agcggtggca gcagccaact cagcttcctt tcgggctttg ttagcagccg
     6061 gatctgagct cgcccttaaa gtccaggttg gcataaacgg cattatcttc aataaactgg
     6121 cgacgatttt ccacaacatc acccatcagc atttcaaagg tctgatcggc ttcaattgca
     6181 tcttccagtt taacttgcag cagtgcacga tgttccggat tcatggtggt ttcccacagc
     6241 tgatctgcat tcatttcacc cagaccttta taacgtgcaa ttgaccattt cggtgtcgga
     6301 ttcagttcgc ttttcagttt gtccagctcg cgatcattat acacgtagta tttctgttta
     6361 ccctgggtca gtttatacag cggaggctgt gcaatataaa cataaccggc ttcgatcagc
     6421 ggacgcataa aacgataaaa gaaaatcagc agcagggtac gaatatgtgc accatcaaca
     6481 tcggcatcgg tcataatcac aatcttatga taacgggctt ttgccagatc aaaatcacca
     6541 ccaataccgg tgccaaatgc ggtaatcatc tggcgaattt cattattgtt cagaatgcga
     6601 tccagacgtg ctttttccac attcagaatt ttaccacgca gcggcagaat tgcctgggta
     6661 cgactatcac gaccgctttt ggtactacca ccggcactat caccttccac cagaaaaatt
     6721 tcacattctt ccggtgattt gctgctacaa tctgccagtt taccaggcag gcttgcaaca
     6781 tccagtgcgc ttttacgacg ggtaacttca cgtgctttct ttgctgcaac acgggcacgt
     6841 gctgccataa tacctttttc aacaacggta cgtgcaacct gcggattttc atacagaaag
     6901 cgctcaaagt gttcgctaaa cagtttatcc acaacctgac gaacttcgct attacccagt
     6961 ttggttttgg tctgaccttc aaactgagga tcgccatgtt taatgctaat aattgcggtc
     7021 ataccttcac gggtatcctc accgctcaga cgatcttttt cttctttcat gattttgctg
     7081 ctcagaccat agctattcag aacacgggtc agtgcacgtt taaaaccatc ttcgtgtgta
     7141 ccgccttcat aggtatgaat gttatttgca taggtcagca gattggtggc ataaccgcta
     7201 ttatactgga tggcaatttc cacttcgata tcatctttgc tctgatggat atagatcggc
     7261 tcatcatgaa tcggttcttt gttttcgttc agcagttcca catagctttt aatgccacct
     7321 tcataatggt agctatcttc acgaacattt tcttcgtcac gttcatcacg cagggtaatc
     7381 tgaatgcctt tattcagaaa tgccagttca cgaatacgct gctgcagggt ttcatagtta
     7441 taaacggtgg tttcggtgaa gatttcacca tctgctttaa aacgaataac ggtgccggtt
     7501 ttatcggtgg tgccaacttc tttcagatca aactgcggaa cacccttttt ataggcctgg
     7561 tgataaatgg tttcgttacg atgaacataa acttccagat cctggctcag tgcattaaca
     7621 acgctgctac caacaccatg caggccaccg ctaactttat agccaccacc accaaatttg
     7681 ccaccggca
//
