LOCUS       sequence-430358-        7689 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  S. aureus USA300 GyrB mutant A467 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_A467
FEATURES             Location/Qualifiers
     source          1..7689
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(1383..1403)
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             complement(1404..1445)
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     CDS             complement(1446..1463)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     RBS             1474..1496
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    1511..1535
                     /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(1535..1554)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1536..1554)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1638..1657)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        1863..1940
                     /gene="lacI"
                     /label=lacI promoter
     CDS             1941..3023
                     /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(1960..1979)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     CDS             4335..4526
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(4520..4542)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    4628..4767
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     4683..4702
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(4782..4799)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4953..5541)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5033..5052)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5712..6503,6504..6572))
                     /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     6335..6354
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6573..6677)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     6745..6763
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     terminator      complement(7001..7048)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     7044..7062
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
ORIGIN
        1 ggcagaattg cctgggtacg actatcacga ccgcttttgg tactaccacc ggcactatca
       61 ccttccacca gaaaaatttc acattcttcc ggtgatttgc tgctacaatc tgccagttta
      121 ccaggcaggc ttgcaacatc cagtgcgctt ttacgacggg taacttcacg tgctttcttt
      181 gctgcaacac gggcacgtgc tgccataata cctttttcaa caacggtacg tgcaacctgc
      241 ggattttcat acagaaagcg ctcaaagtgt tcgctaaaca gtttatccac aacctgacga
      301 acttcgctat tacccagttt ggttttggtc tgaccttcaa actgaggatc gccatgttta
      361 atgctaataa ttgcggtcat accttcacgg gtatcctcac cgctcagacg atctttttct
      421 tctttcatga ttttgctgct cagaccatag ctattcagaa cacgggtcag tgcacgttta
      481 aaaccatctt cgtgtgtacc gccttcatag gtatgaatgt tatttgcata ggtcagcaga
      541 ttggtggcat aaccgctatt atactggatg gcaatttcca cttcgatatc atctttgctc
      601 tgatggatat agatcggctc atcatgaatc ggttctttgt tttcgttcag cagttccaca
      661 tagcttttaa tgccaccttc ataatggtag ctatcttcac gaacattttc ttcgtcacgt
      721 tcatcacgca gggtaatctg aatgccttta ttcagaaatg ccagttcacg aatacgctgc
      781 tgcagggttt catagttata aacggtggtt tcggtgaaga tttcaccatc tgctttaaaa
      841 cgaataacgg tgccggtttt atcggtggtg ccaacttctt tcagatcaaa ctgcggaaca
      901 ccctttttat aggcctggtg ataaatggtt tcgttacgat gaacataaac ttccagatcc
      961 tggctcagtg cattaacaac gctgctacca acaccatgca ggccaccgct aactttatag
     1021 ccaccaccac caaatttgcc accggcatgc agaacggtca gaataacttc aactgccgga
     1081 cgacccattt tctcttgaat atcaaccgga ataccacgac cattatcggt aactttaatc
     1141 cagttgtctt tctcgatcac cacttcaatc tgatttgcat aacctgccag tgcttcatca
     1201 atgctgttat caacaatttc ccaaaccaga tgatgcagac cacgttcgct ggtgctacca
     1261 atatacatac ccggacgttt acgaactgct tccagacctt ccagaacctg aatctgacct
     1321 gcgccataat tatcggtgtt attcacatca ctcagtgcgg taaccatggt gaagggatca
     1381 attccctgaa aatacaggtt ttccgtagaa tcgagaccga ggagagggtt agggataggc
     1441 ttaccatggt gatggtgatg atgcatatgt atatctcctt cttaaagtta aacaaaatta
     1501 tttctagagg ggaattgtta tccgctcaca attcccctat agtgagtcgt attaatttcg
     1561 cgggatcgag atctcgatcc tctacgccgg acgcatcgtg gccggcatca ccggcgccac
     1621 aggtgcggtt gctggcgcct atatcgccga catcaccgat ggggaagatc gggctcgcca
     1681 cttcgggctc atgagcgctt gtttcggcgt gggtatggtg gcaggccccg tggccggggg
     1741 actgttgggc gccatctcct tgcatgcacc attccttgcg gcggcggtgc tcaacggcct
     1801 caacctacta ctgggctgct tcctaatgca ggagtcgcat aagggagagc gtcgagatcc
     1861 cggacaccat cgaatggcgc aaaacctttc gcggtatggc atgatagcgc ccggaagaga
     1921 gtcaattcag ggtggtgaat gtgaaaccag taacgttata cgatgtcgca gagtatgccg
     1981 gtgtctctta tcagaccgtt tcccgcgtgg tgaaccaggc cagccacgtt tctgcgaaaa
     2041 cgcgggaaaa agtggaagcg gcgatggcgg agctgaatta cattcccaac cgcgtggcac
     2101 aacaactggc gggcaaacag tcgttgctga ttggcgttgc cacctccagt ctggccctgc
     2161 acgcgccgtc gcaaattgtc gcggcgatta aatctcgcgc cgatcaactg ggtgccagcg
     2221 tggtggtgtc gatggtagaa cgaagcggcg tcgaagcctg taaagcggcg gtgcacaatc
     2281 ttctcgcgca acgcgtcagt gggctgatca ttaactatcc gctggatgac caggatgcca
     2341 ttgctgtgga agctgcctgc actaatgttc cggcgttatt tcttgatgtc tctgaccaga
     2401 cacccatcaa cagtattatt ttctcccatg aagacggtac gcgactgggc gtggagcatc
     2461 tggtcgcatt gggtcaccag caaatcgcgc tgttagcggg cccattaagt tctgtctcgg
     2521 cgcgtctgcg tctggctggc tggcataaat atctcactcg caatcaaatt cagccgatag
     2581 cggaacggga aggcgactgg agtgccatgt ccggttttca acaaaccatg caaatgctga
     2641 atgagggcat cgttcccact gcgatgctgg ttgccaacga tcagatggcg ctgggcgcaa
     2701 tgcgcgccat taccgagtcc gggctgcgcg ttggtgcgga tatctcggta gtgggatacg
     2761 acgataccga agacagctca tgttatatcc cgccgttaac caccatcaaa caggattttc
     2821 gcctgctggg gcaaaccagc gtggaccgct tgctgcaact ctctcagggc caggcggtga
     2881 agggcaatca gctgttgccc gtctcactgg tgaaaagaaa aaccaccctg gcgcccaata
     2941 cgcaaaccgc ctctccccgc gcgttggccg attcattaat gcagctggca cgacaggttt
     3001 cccgactgga aagcgggcag tgagcgcaac gcaattaatg taagttagct cactcattag
     3061 gcaccgggat ctcgaccgat gcccttgaga gccttcaacc cagtcagctc cttccggtgg
     3121 gcgcggggca tgactatcgt cgccgcactt atgactgtct tctttatcat gcaactcgta
     3181 ggacaggtgc cggcagcgct ctgggtcatt ttcggcgagg accgctttcg ctggagcgcg
     3241 acgatgatcg gcctgtcgct tgcggtattc ggaatcttgc acgccctcgc tcaagccttc
     3301 gtcactggtc ccgccaccaa acgtttcggc gagaagcagg ccattatcgc cggcatggcg
     3361 gccgacgcgc tgggctacgt cttgctggcg ttcgcgacgc gaggctggat ggccttcccc
     3421 attatgattc ttctcgcttc cggcggcatc gggatgcccg cgttgcaggc catgctgtcc
     3481 aggcaggtag atgacgacca tcagggacag cttcaaggat cgctcgcggc tcttaccagc
     3541 ctaacttcga tcattggacc gctgatcgtc acggcgattt atgccgcctc ggcgagcaca
     3601 tggaacgggt tggcatggat tgtaggcgcc gccctatacc ttgtctgcct ccccgcgttg
     3661 cgtcgcggtg catggagccg ggccacctcg acctgaatgg aagccggcgg cacctcgcta
     3721 acggattcac cactccaaga attggagcca atcaattctt gcggagaact gtgaatgcgc
     3781 aaaccaaccc ttggcagaac atatccatcg cgtccgccat ctccagcagc cgcacgcggc
     3841 gcatctcggg cagcgttggg tcctggccac gggtgcgcat gatcgtgctc ctgtcgttga
     3901 ggacccggct aggctggcgg ggttgcctta ctggttagca gaatgaatca ccgatacgcg
     3961 agcgaacgtg aagcgactgc tgctgcaaaa cgtctgcgac ctgagcaaca acatgaatgg
     4021 tcttcggttt ccgtgtttcg taaagtctgg aaacgcggaa gtcagcgccc tgcaccatta
     4081 tgttccggat ctgcatcgca ggatgctgct ggctaccctg tggaacacct acatctgtat
     4141 taacgaagcg ctggcattga ccctgagtga tttttctctg gtcccgccgc atccataccg
     4201 ccagttgttt accctcacaa cgttccagta accgggcatg ttcatcatca gtaacccgta
     4261 tcgtgagcat cctctctcgt ttcatcggta tcattacccc catgaacaga aatccccctt
     4321 acacggaggc atcagtgacc aaacaggaaa aaaccgccct taacatggcc cgctttatca
     4381 gaagccagac attaacgctt ctggagaaac tcaacgagct ggacgcggat gaacaggcag
     4441 acatctgtga atcgcttcac gaccacgctg atgagcttta ccgcagctgc ctcgcgcgtt
     4501 tcggtgatga cggtgaaaac ctctgacaca tgcagctccc ggagacggtc acagcttgtc
     4561 tgtaagcgga tgccgggagc agacaagccc gtcagggcgc gtcagcgggt gttggcgggt
     4621 gtcggggcgc agccatgacc cagtcacgta gcgatagcgg agtgtatact ggcttaacta
     4681 tgcggcatca gagcagattg tactgagagt gcaccattgc ggtgtgaaat accgcacaga
     4741 tgcgtaagga gaaaataccg catcaggcgc tcttccgctt cctcgctcac tgactcgctg
     4801 cgctcggtcg ttcggctgcg gcgagcggta tcagctcact caaaggcggt aatacggtta
     4861 tccacagaat caggggataa cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc
     4921 aggaaccgta aaaaggccgc gttgctggcg tttttccata ggctccgccc ccctgacgag
     4981 catcacaaaa atcgacgctc aagtcagagg tggcgaaacc cgacaggact ataaagatac
     5041 caggcgtttc cccctggaag ctccctcgtg cgctctcctg ttccgaccct gccgcttacc
     5101 ggatacctgt ccgcctttct cccttcggga agcgtggcgc tttctcatag ctcacgctgt
     5161 aggtatctca gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc
     5221 gttcagcccg accgctgcgc cttatccggt aactatcgtc ttgagtccaa cccggtaaga
     5281 cacgacttat cgccactggc agcagccact ggtaacagga ttagcagagc gaggtatgta
     5341 ggcggtgcta cagagttctt gaagtggtgg cctaactacg gctacactag aaggacagta
     5401 tttggtatct gcgctctgct gaagccagtt accttcggaa aaagagttgg tagctcttga
     5461 tccggcaaac aaaccaccgc tggtagcggt ggtttttttg tttgcaagca gcagattacg
     5521 cgcagaaaaa aaggatctca agaagatcct ttgatctttt ctacggggtc tgacgctcag
     5581 tggaacgaaa actcacgtta agggattttg gtcatgagat tatcaaaaag gatcttcacc
     5641 tagatccttt taaattaaaa atgaagtttt aaatcaatct aaagtatata tgagtaaact
     5701 tggtctgaca gttaccaatg cttaatcagt gaggcaccta tctcagcgat ctgtctattt
     5761 cgttcatcca tagttgcctg actccccgtc gtgtagataa ctacgatacg ggagggctta
     5821 ccatctggcc ccagtgctgc aatgataccg cgagacccac gctcaccggc tccagattta
     5881 tcagcaataa accagccagc cggaagggcc gagcgcagaa gtggtcctgc aactttatcc
     5941 gcctccatcc agtctattaa ttgttgccgg gaagctagag taagtagttc gccagttaat
     6001 agtttgcgca acgttgttgc cattgctgca ggcatcgtgg tgtcacgctc gtcgtttggt
     6061 atggcttcat tcagctccgg ttcccaacga tcaaggcgag ttacatgatc ccccatgttg
     6121 tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg tcagaagtaa gttggccgca
     6181 gtgttatcac tcatggttat ggcagcactg cataattctc ttactgtcat gccatccgta
     6241 agatgctttt ctgtgactgg tgagtactca accaagtcat tctgagaata gtgtatgcgg
     6301 cgaccgagtt gctcttgccc ggcgtcaaca cgggataata ccgcgccaca tagcagaact
     6361 ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa aactctcaag gatcttaccg
     6421 ctgttgagat ccagttcgat gtaacccact cgtgcaccca actgatcttc agcatctttt
     6481 actttcacca gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga
     6541 ataagggcga cacggaaatg ttgaatactc atactcttcc tttttcaata ttattgaagc
     6601 atttatcagg gttattgtct catgagcgga tacatatttg aatgtattta gaaaaataaa
     6661 caaatagggg ttccgcgcac atttccccga aaagtgccac ctgacgtcta agaaaccatt
     6721 attatcatga cattaaccta taaaaatagg cgtatcacga ggccctttcg tcttcaagaa
     6781 ttaattctca tgtttgacag cttatcatcg ataagctagc tttaatgcgg tagtttatca
     6841 cagttaaatt gctaacgcag tcaggcaccg tgtatgaaat ctaacaatgc gctcatcgtc
     6901 atcctcggca ccgtcaccct ggatgctgta ggcataggct tggttatgcc ggtactgccg
     6961 ggcctcttgc gggatatccg gatatagttc ctcctttcag caaaaaaccc ctcaagaccc
     7021 gtttagaggc cccaaggggt tatgctagtt attgctcagc ggtggcagca gccaactcag
     7081 cttcctttcg ggctttgtta gcagccggat ctgagctcgc ccttaaagtc caggttggca
     7141 taaacggcat tatcttcaat aaactggcga cgattttcca caacatcacc catcagcatt
     7201 tcaaaggtct gatccgcttc aattgcatct tccagtttaa cttgcagcag tgcacgatgt
     7261 tccggattca tggtggtttc ccacagctga tctgcattca tttcacccag acctttataa
     7321 cgtgcaattg accatttcgg tgtcggattc agttcgcttt tcagtttgtc cagctcgcga
     7381 tcattataca cgtagtattt ctgtttaccc tgggtcagtt tatacagcgg aggctgtgca
     7441 atataaacat aaccggcttc aatcagcgga cgcataaaac gatagaaaaa tgtcagcagc
     7501 agggtacgaa tatgtgcacc atcaacatcg gcatcggtca taatcacaat cttatgataa
     7561 cgggcttttg ccagatcaaa atcaccacca ataccggtgc caaatgcggt aatcatctgg
     7621 cgaatttcat tattgttcag aatgcgatcc agacgcactt tttccacatt cagaatttta
     7681 ccacgcagc
//
