LOCUS       sequence-430357-        8418 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  S. aureus USA300 GyrA mutant F164 R238 residues codon-optimized for 
            E. coli.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8418)
  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 8418)
  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-GyrA_F164R238
FEATURES             Location/Qualifiers
     source          1..8418
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(1913..1931)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1927..1974
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     complement(2212..2230)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        2298..2402
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2403..2471,2472..3263)
                     /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(2621..2640)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3434..4022
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3923..3942
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4176..4193
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4208..4347
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4273..4292)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4433..4455
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4449..4640)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(5952..7034)
                     /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     6996..7015
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(7035..7112)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     7318..7337
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     7421..7440
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        7421..7439
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    7440..7464
                     /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)."
     RBS             7479..7501
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             7512..7529
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             7530..7571
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     CDS             7572..7592
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
ORIGIN
        1 cccgtatgat tgagaaaatt gccgaactgg ttcgcgataa aaagattgat ggtattaccg
       61 atctgcgtga tgaaaccagc ttaggcaccg gtgttcgtgt tgttattgat gttcgtaaag
      121 atgcaaatgc cagcgtgatt ctgaacaacc tgtataaaca gacaccgctg cagaccagct
      181 ttggtgttaa tatgattgca ctggttaatg gtcgtccgaa actgatcaat ctgaaagaag
      241 cactggttca ctatctggaa catcagaaaa ccgttgttcg tcgtcgtacc cagtataatc
      301 tgcgtaaagc aaaagatcgt gcccatattc tggaaggtct gcgtattgcc ctggatcata
      361 ttgatgaaat tatcagcacc attcgcgaaa gcgataccga taaagttgca atggaaagcc
      421 tgcagcagcg ttttaaactg agcgaaaaac aggcacaggc aattctggat atgcgtctgc
      481 gtcgtctgac cggtctggaa cgcgacaaaa ttgaagcaga atataatgag ctgctgaact
      541 atatcagcga actggaagcc attctggcag atgaagaagt tctgctgcag ctggtacgtg
      601 atgaactgac agaaattcgt gatcgttttg gtgatgatcg tcgtacggaa attcagttag
      661 gtggttttga aaacctggaa gatgaggatc tgattccgga agaacaaatt gttattacgc
      721 tgagccacaa caactacatt aaacgcctgc cggttagcac ctatcgtgca cagaatcgcg
      781 gtggtcgtgg tgttcagggc atgaataccc tggaagaaga ttttgttagc cagctggtta
      841 ccctgagcac ccatgatcac gttctgtttt tcaccaataa aggtcgcgtg tataaactga
      901 aaggttatga agtgccggaa ctgagccgtc agagcaaagg tattccggtt gttaatgcaa
      961 ttgagctgga aaacgatgaa gtgattagca ccatgattgc cgttaaagat ctggaaagcg
     1021 aagataactt tctggtgttt gcaaccaaac gtggtgttgt taaacgtagc gcactgagca
     1081 atttttctcg cattaatcgc aatggcaaaa tcgccattag ctttcgtgaa gatgatgagc
     1141 tgatcgcagt gcgtctgacc agcggtcaag aagatattct gattggcacc agccatgcaa
     1201 gcctgattcg ttttcctgaa agcaccctgc gtccgctggg tcgtaccgca accggtgtta
     1261 aaggtattac cctgcgcgaa ggtgatgaag ttgttggtct ggatgttgca catgcaaata
     1321 gcgttgatga agtgctggtt gtgaccgaaa atggttatgg taaacgtaca ccggtgaatg
     1381 attatcgtct gagcaatcgt ggtggtaaag gcattaaaac cgcaaccatt accgaacgta
     1441 atggtaatgt tgtttgcatt accaccgtta ccggtgaaga ggacctgatg attgttacca
     1501 atgccggtgt tattattcgc ctggatgtgg cagatattag tcagaatggt cgtgcagcac
     1561 agggtgttcg tctgattcgt ctgggtgatg accagtttgt tagcaccgtt gcaaaagtta
     1621 aagaagatgc agaggacgaa accaacgaag atgaacagag caccagcacc gttagcgaag
     1681 atggcaccga acagcagcgt gaagcagttg tgaatgatga aacaccgggt aatgccattc
     1741 ataccgaagt tattgatagc gaagaaaatg atgaagatgg tcgtattgaa gtgcgtcagg
     1801 atttcatgga tcgtgttgaa gaggatattc agcagagcag tgatgaagaa aagggcgagc
     1861 tcagatccgg ctgctaacaa agcccgaaag gaagctgagt tggctgctgc caccgctgag
     1921 caataactag cataacccct tggggcctct aaacgggtct tgaggggttt tttgctgaaa
     1981 ggaggaacta tatccggata tcccgcaaga ggcccggcag taccggcata accaagccta
     2041 tgcctacagc atccagggtg acggtgccga ggatgacgat gagcgcattg ttagatttca
     2101 tacacggtgc ctgactgcgt tagcaattta actgtgataa actaccgcat taaagctagc
     2161 ttatcgatga taagctgtca aacatgagaa ttaattcttg aagacgaaag ggcctcgtga
     2221 tacgcctatt tttataggtt aatgtcatga taataatggt ttcttagacg tcaggtggca
     2281 cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt tttctaaata cattcaaata
     2341 tgtatccgct catgagacaa taaccctgat aaatgcttca ataatattga aaaaggaaga
     2401 gtatgagtat tcaacatttc cgtgtcgccc ttattccctt ttttgcggca ttttgccttc
     2461 ctgtttttgc tcacccagaa acgctggtga aagtaaaaga tgctgaagat cagttgggtg
     2521 cacgagtggg ttacatcgaa ctggatctca acagcggtaa gatccttgag agttttcgcc
     2581 ccgaagaacg ttttccaatg atgagcactt ttaaagttct gctatgtggc gcggtattat
     2641 cccgtgttga cgccgggcaa gagcaactcg gtcgccgcat acactattct cagaatgact
     2701 tggttgagta ctcaccagtc acagaaaagc atcttacgga tggcatgaca gtaagagaat
     2761 tatgcagtgc tgccataacc atgagtgata acactgcggc caacttactt ctgacaacga
     2821 tcggaggacc gaaggagcta accgcttttt tgcacaacat gggggatcat gtaactcgcc
     2881 ttgatcgttg ggaaccggag ctgaatgaag ccataccaaa cgacgagcgt gacaccacga
     2941 tgcctgcagc aatggcaaca acgttgcgca aactattaac tggcgaacta cttactctag
     3001 cttcccggca acaattaata gactggatgg aggcggataa agttgcagga ccacttctgc
     3061 gctcggccct tccggctggc tggtttattg ctgataaatc tggagccggt gagcgtgggt
     3121 ctcgcggtat cattgcagca ctggggccag atggtaagcc ctcccgtatc gtagttatct
     3181 acacgacggg gagtcaggca actatggatg aacgaaatag acagatcgct gagataggtg
     3241 cctcactgat taagcattgg taactgtcag accaagttta ctcatatata ctttagattg
     3301 atttaaaact tcatttttaa tttaaaagga tctaggtgaa gatccttttt gataatctca
     3361 tgaccaaaat cccttaacgt gagttttcgt tccactgagc gtcagacccc gtagaaaaga
     3421 tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa
     3481 aaccaccgct accagcggtg gtttgtttgc cggatcaaga gctaccaact ctttttccga
     3541 aggtaactgg cttcagcaga gcgcagatac caaatactgt ccttctagtg tagccgtagt
     3601 taggccacca cttcaagaac tctgtagcac cgcctacata cctcgctctg ctaatcctgt
     3661 taccagtggc tgctgccagt ggcgataagt cgtgtcttac cgggttggac tcaagacgat
     3721 agttaccgga taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca cagcccagct
     3781 tggagcgaac gacctacacc gaactgagat acctacagcg tgagctatga gaaagcgcca
     3841 cgcttcccga agggagaaag gcggacaggt atccggtaag cggcagggtc ggaacaggag
     3901 agcgcacgag ggagcttcca gggggaaacg cctggtatct ttatagtcct gtcgggtttc
     3961 gccacctctg acttgagcgt cgatttttgt gatgctcgtc aggggggcgg agcctatgga
     4021 aaaacgccag caacgcggcc tttttacggt tcctggcctt ttgctggcct tttgctcaca
     4081 tgttctttcc tgcgttatcc cctgattctg tggataaccg tattaccgcc tttgagtgag
     4141 ctgataccgc tcgccgcagc cgaacgaccg agcgcagcga gtcagtgagc gaggaagcgg
     4201 aagagcgcct gatgcggtat tttctcctta cgcatctgtg cggtatttca caccgcaatg
     4261 gtgcactctc agtacaatct gctctgatgc cgcatagtta agccagtata cactccgcta
     4321 tcgctacgtg actgggtcat ggctgcgccc cgacacccgc caacacccgc tgacgcgccc
     4381 tgacgggctt gtctgctccc ggcatccgct tacagacaag ctgtgaccgt ctccgggagc
     4441 tgcatgtgtc agaggttttc accgtcatca ccgaaacgcg cgaggcagct gcggtaaagc
     4501 tcatcagcgt ggtcgtgaag cgattcacag atgtctgcct gttcatccgc gtccagctcg
     4561 ttgagtttct ccagaagcgt taatgtctgg cttctgataa agcgggccat gttaagggcg
     4621 gttttttcct gtttggtcac tgatgcctcc gtgtaagggg gatttctgtt catgggggta
     4681 atgataccga tgaaacgaga gaggatgctc acgatacggg ttactgatga tgaacatgcc
     4741 cggttactgg aacgttgtga gggtaaacaa ctggcggtat ggatgcggcg ggaccagaga
     4801 aaaatcactc agggtcaatg ccagcgcttc gttaatacag atgtaggtgt tccacagggt
     4861 agccagcagc atcctgcgat gcagatccgg aacataatgg tgcagggcgc tgacttccgc
     4921 gtttccagac tttacgaaac acggaaaccg aagaccattc atgttgttgc tcaggtcgca
     4981 gacgttttgc agcagcagtc gcttcacgtt cgctcgcgta tcggtgattc attctgctaa
     5041 ccagtaaggc aaccccgcca gcctagccgg gtcctcaacg acaggagcac gatcatgcgc
     5101 acccgtggcc aggacccaac gctgcccgag atgcgccgcg tgcggctgct ggagatggcg
     5161 gacgcgatgg atatgttctg ccaagggttg gtttgcgcat tcacagttct ccgcaagaat
     5221 tgattggctc caattcttgg agtggtgaat ccgttagcga ggtgccgccg gcttccattc
     5281 aggtcgaggt ggcccggctc catgcaccgc gacgcaacgc ggggaggcag acaaggtata
     5341 gggcggcgcc tacaatccat gccaacccgt tccatgtgct cgccgaggcg gcataaatcg
     5401 ccgtgacgat cagcggtcca atgatcgaag ttaggctggt aagagccgcg agcgatcctt
     5461 gaagctgtcc ctgatggtcg tcatctacct gcctggacag catggcctgc aacgcgggca
     5521 tcccgatgcc gccggaagcg agaagaatca taatggggaa ggccatccag cctcgcgtcg
     5581 cgaacgccag caagacgtag cccagcgcgt cggccgccat gccggcgata atggcctgct
     5641 tctcgccgaa acgtttggtg gcgggaccag tgacgaaggc ttgagcgagg gcgtgcaaga
     5701 ttccgaatac cgcaagcgac aggccgatca tcgtcgcgct ccagcgaaag cggtcctcgc
     5761 cgaaaatgac ccagagcgct gccggcacct gtcctacgag ttgcatgata aagaagacag
     5821 tcataagtgc ggcgacgata gtcatgcccc gcgcccaccg gaaggagctg actgggttga
     5881 aggctctcaa gggcatcggt cgagatcccg gtgcctaatg agtgagctaa cttacattaa
     5941 ttgcgttgcg ctcactgccc gctttccagt cgggaaacct gtcgtgccag ctgcattaat
     6001 gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg gcgccagggt ggtttttctt
     6061 ttcaccagtg agacgggcaa cagctgattg cccttcaccg cctggccctg agagagttgc
     6121 agcaagcggt ccacgctggt ttgccccagc aggcgaaaat cctgtttgat ggtggttaac
     6181 ggcgggatat aacatgagct gtcttcggta tcgtcgtatc ccactaccga gatatccgca
     6241 ccaacgcgca gcccggactc ggtaatggcg cgcattgcgc ccagcgccat ctgatcgttg
     6301 gcaaccagca tcgcagtggg aacgatgccc tcattcagca tttgcatggt ttgttgaaaa
     6361 ccggacatgg cactccagtc gccttcccgt tccgctatcg gctgaatttg attgcgagtg
     6421 agatatttat gccagccagc cagacgcaga cgcgccgaga cagaacttaa tgggcccgct
     6481 aacagcgcga tttgctggtg acccaatgcg accagatgct ccacgcccag tcgcgtaccg
     6541 tcttcatggg agaaaataat actgttgatg ggtgtctggt cagagacatc aagaaataac
     6601 gccggaacat tagtgcaggc agcttccaca gcaatggcat cctggtcatc cagcggatag
     6661 ttaatgatca gcccactgac gcgttgcgcg agaagattgt gcaccgccgc tttacaggct
     6721 tcgacgccgc ttcgttctac catcgacacc accacgctgg cacccagttg atcggcgcga
     6781 gatttaatcg ccgcgacaat ttgcgacggc gcgtgcaggg ccagactgga ggtggcaacg
     6841 ccaatcagca acgactgttt gcccgccagt tgttgtgcca cgcggttggg aatgtaattc
     6901 agctccgcca tcgccgcttc cactttttcc cgcgttttcg cagaaacgtg gctggcctgg
     6961 ttcaccacgc gggaaacggt ctgataagag acaccggcat actctgcgac atcgtataac
     7021 gttactggtt tcacattcac caccctgaat tgactctctt ccgggcgcta tcatgccata
     7081 ccgcgaaagg ttttgcgcca ttcgatggtg tccgggatct cgacgctctc ccttatgcga
     7141 ctcctgcatt aggaagcagc ccagtagtag gttgaggccg ttgagcaccg ccgccgcaag
     7201 gaatggtgca tgcaaggaga tggcgcccaa cagtcccccg gccacggggc ctgccaccat
     7261 acccacgccg aaacaagcgc tcatgagccc gaagtggcga gcccgatctt ccccatcggt
     7321 gatgtcggcg atataggcgc cagcaaccgc acctgtggcg ccggtgatgc cggccacgat
     7381 gcgtccggcg tagaggatcg agatctcgat cccgcgaaat taatacgact cactataggg
     7441 gaattgtgag cggataacaa ttcccctcta gaaataattt tgtttaactt taagaaggag
     7501 atatacatat gcatcatcac catcaccatg gtaagcctat ccctaaccct ctcctcggtc
     7561 tcgattctac ggaaaacctg tattttcagg gaattgatcc cttcaccatg gcagaactgc
     7621 cgcagagccg tattaatgaa cgcaatatta ccagcgaaat gcgtgaatcc tttctggatt
     7681 atgcaatgag cgttattgtt gcacgtgccc tgccggatgt tcgtgatggt ctgaaaccgg
     7741 ttcatcgtcg tattctgtat ggcctgaatg aacagggtat gacaccggat aaaagctata
     7801 aaaagagcgc acgtattgtc ggtgatgtga tgggtaaata tcatccgcat ggtgatctga
     7861 gcatttatga agcaatggtt cgtatggcac aggattttag ctatcgttat ccgctggttg
     7921 atggtcaggg taattttggt agcatggatg gtgatggtgc agcagcaatg cgttataccg
     7981 aagcacgtat gacaaaaatc accctggaac tgctgcgtga tattaacaaa gataccatcg
     8041 atttcatcga caactatgat ggcaatgaac gtgaaccgag cgttctgcct gcacgtctgc
     8101 cgaatctgct ggcaaatggt gcaagcggta ttgcagttgg tatggcaacc aatattccgc
     8161 ctcataatct gaccgaactg attaatggtg tgctgagcct gagcaaaaat ccggatatta
     8221 gcattgcaga actgatggaa gatattgaag gtcctgattt tccgaccgca ggtctgattc
     8281 tgggtaaaag tggtattcgt cgtgcatatg aaacaggtcg tggtagcatt cagatgcgta
     8341 gccgtgcagt tattgaagaa cgtggtggtg gtcgtcagcg tattgttgtt accgaaattc
     8401 cgtttcaggt taacaaag
//
