LOCUS       sequence-430356-        7689 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  S. aureus USA300 GyrB 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
FEATURES             Location/Qualifiers
     source          1..7689
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             2153..2344
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(2338..2360)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    2446..2585
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     2501..2520
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(2600..2617)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2771..3359)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2851..2870)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3530..4321,4322..4390))
                     /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     4153..4172
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4391..4495)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     4563..4581
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     terminator      complement(4819..4866)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     4862..4880
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     CDS             complement(6890..6910)
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             complement(6911..6952)
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     CDS             complement(6953..6970)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     RBS             6981..7003
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    7018..7042
                     /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(7042..7061)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(7043..7061)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(7145..7164)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        7370..7447
                     /gene="lacI"
                     /label=lacI promoter
     CDS             join(7448..7689,1..841)
                     /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(7467..7486)
                     /label=LacI-R
                     /note="LacI, reverse primer"
ORIGIN
        1 ggcgattaaa tctcgcgccg atcaactggg tgccagcgtg gtggtgtcga tggtagaacg
       61 aagcggcgtc gaagcctgta aagcggcggt gcacaatctt ctcgcgcaac gcgtcagtgg
      121 gctgatcatt aactatccgc tggatgacca ggatgccatt gctgtggaag ctgcctgcac
      181 taatgttccg gcgttatttc ttgatgtctc tgaccagaca cccatcaaca gtattatttt
      241 ctcccatgaa gacggtacgc gactgggcgt ggagcatctg gtcgcattgg gtcaccagca
      301 aatcgcgctg ttagcgggcc cattaagttc tgtctcggcg cgtctgcgtc tggctggctg
      361 gcataaatat ctcactcgca atcaaattca gccgatagcg gaacgggaag gcgactggag
      421 tgccatgtcc ggttttcaac aaaccatgca aatgctgaat gagggcatcg ttcccactgc
      481 gatgctggtt gccaacgatc agatggcgct gggcgcaatg cgcgccatta ccgagtccgg
      541 gctgcgcgtt ggtgcggata tctcggtagt gggatacgac gataccgaag acagctcatg
      601 ttatatcccg ccgttaacca ccatcaaaca ggattttcgc ctgctggggc aaaccagcgt
      661 ggaccgcttg ctgcaactct ctcagggcca ggcggtgaag ggcaatcagc tgttgcccgt
      721 ctcactggtg aaaagaaaaa ccaccctggc gcccaatacg caaaccgcct ctccccgcgc
      781 gttggccgat tcattaatgc agctggcacg acaggtttcc cgactggaaa gcgggcagtg
      841 agcgcaacgc aattaatgta agttagctca ctcattaggc accgggatct cgaccgatgc
      901 ccttgagagc cttcaaccca gtcagctcct tccggtgggc gcggggcatg actatcgtcg
      961 ccgcacttat gactgtcttc tttatcatgc aactcgtagg acaggtgccg gcagcgctct
     1021 gggtcatttt cggcgaggac cgctttcgct ggagcgcgac gatgatcggc ctgtcgcttg
     1081 cggtattcgg aatcttgcac gccctcgctc aagccttcgt cactggtccc gccaccaaac
     1141 gtttcggcga gaagcaggcc attatcgccg gcatggcggc cgacgcgctg ggctacgtct
     1201 tgctggcgtt cgcgacgcga ggctggatgg ccttccccat tatgattctt ctcgcttccg
     1261 gcggcatcgg gatgcccgcg ttgcaggcca tgctgtccag gcaggtagat gacgaccatc
     1321 agggacagct tcaaggatcg ctcgcggctc ttaccagcct aacttcgatc attggaccgc
     1381 tgatcgtcac ggcgatttat gccgcctcgg cgagcacatg gaacgggttg gcatggattg
     1441 taggcgccgc cctatacctt gtctgcctcc ccgcgttgcg tcgcggtgca tggagccggg
     1501 ccacctcgac ctgaatggaa gccggcggca cctcgctaac ggattcacca ctccaagaat
     1561 tggagccaat caattcttgc ggagaactgt gaatgcgcaa accaaccctt ggcagaacat
     1621 atccatcgcg tccgccatct ccagcagccg cacgcggcgc atctcgggca gcgttgggtc
     1681 ctggccacgg gtgcgcatga tcgtgctcct gtcgttgagg acccggctag gctggcgggg
     1741 ttgccttact ggttagcaga atgaatcacc gatacgcgag cgaacgtgaa gcgactgctg
     1801 ctgcaaaacg tctgcgacct gagcaacaac atgaatggtc ttcggtttcc gtgtttcgta
     1861 aagtctggaa acgcggaagt cagcgccctg caccattatg ttccggatct gcatcgcagg
     1921 atgctgctgg ctaccctgtg gaacacctac atctgtatta acgaagcgct ggcattgacc
     1981 ctgagtgatt tttctctggt cccgccgcat ccataccgcc agttgtttac cctcacaacg
     2041 ttccagtaac cgggcatgtt catcatcagt aacccgtatc gtgagcatcc tctctcgttt
     2101 catcggtatc attaccccca tgaacagaaa tcccccttac acggaggcat cagtgaccaa
     2161 acaggaaaaa accgccctta acatggcccg ctttatcaga agccagacat taacgcttct
     2221 ggagaaactc aacgagctgg acgcggatga acaggcagac atctgtgaat cgcttcacga
     2281 ccacgctgat gagctttacc gcagctgcct cgcgcgtttc ggtgatgacg gtgaaaacct
     2341 ctgacacatg cagctcccgg agacggtcac agcttgtctg taagcggatg ccgggagcag
     2401 acaagcccgt cagggcgcgt cagcgggtgt tggcgggtgt cggggcgcag ccatgaccca
     2461 gtcacgtagc gatagcggag tgtatactgg cttaactatg cggcatcaga gcagattgta
     2521 ctgagagtgc accattgcgg tgtgaaatac cgcacagatg cgtaaggaga aaataccgca
     2581 tcaggcgctc ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc
     2641 gagcggtatc agctcactca aaggcggtaa tacggttatc cacagaatca ggggataacg
     2701 caggaaagaa catgtgagca aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt
     2761 tgctggcgtt tttccatagg ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa
     2821 gtcagaggtg gcgaaacccg acaggactat aaagatacca ggcgtttccc cctggaagct
     2881 ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc
     2941 cttcgggaag cgtggcgctt tctcatagct cacgctgtag gtatctcagt tcggtgtagg
     3001 tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct
     3061 tatccggtaa ctatcgtctt gagtccaacc cggtaagaca cgacttatcg ccactggcag
     3121 cagccactgg taacaggatt agcagagcga ggtatgtagg cggtgctaca gagttcttga
     3181 agtggtggcc taactacggc tacactagaa ggacagtatt tggtatctgc gctctgctga
     3241 agccagttac cttcggaaaa agagttggta gctcttgatc cggcaaacaa accaccgctg
     3301 gtagcggtgg tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag
     3361 aagatccttt gatcttttct acggggtctg acgctcagtg gaacgaaaac tcacgttaag
     3421 ggattttggt catgagatta tcaaaaagga tcttcaccta gatcctttta aattaaaaat
     3481 gaagttttaa atcaatctaa agtatatatg agtaaacttg gtctgacagt taccaatgct
     3541 taatcagtga ggcacctatc tcagcgatct gtctatttcg ttcatccata gttgcctgac
     3601 tccccgtcgt gtagataact acgatacggg agggcttacc atctggcccc agtgctgcaa
     3661 tgataccgcg agacccacgc tcaccggctc cagatttatc agcaataaac cagccagccg
     3721 gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag tctattaatt
     3781 gttgccggga agctagagta agtagttcgc cagttaatag tttgcgcaac gttgttgcca
     3841 ttgctgcagg catcgtggtg tcacgctcgt cgtttggtat ggcttcattc agctccggtt
     3901 cccaacgatc aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg gttagctcct
     3961 tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt gttatcactc atggttatgg
     4021 cagcactgca taattctctt actgtcatgc catccgtaag atgcttttct gtgactggtg
     4081 agtactcaac caagtcattc tgagaatagt gtatgcggcg accgagttgc tcttgcccgg
     4141 cgtcaacacg ggataatacc gcgccacata gcagaacttt aaaagtgctc atcattggaa
     4201 aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc agttcgatgt
     4261 aacccactcg tgcacccaac tgatcttcag catcttttac tttcaccagc gtttctgggt
     4321 gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt
     4381 gaatactcat actcttcctt tttcaatatt attgaagcat ttatcagggt tattgtctca
     4441 tgagcggata catatttgaa tgtatttaga aaaataaaca aataggggtt ccgcgcacat
     4501 ttccccgaaa agtgccacct gacgtctaag aaaccattat tatcatgaca ttaacctata
     4561 aaaataggcg tatcacgagg ccctttcgtc ttcaagaatt aattctcatg tttgacagct
     4621 tatcatcgat aagctagctt taatgcggta gtttatcaca gttaaattgc taacgcagtc
     4681 aggcaccgtg tatgaaatct aacaatgcgc tcatcgtcat cctcggcacc gtcaccctgg
     4741 atgctgtagg cataggcttg gttatgccgg tactgccggg cctcttgcgg gatatccgga
     4801 tatagttcct cctttcagca aaaaacccct caagacccgt ttagaggccc caaggggtta
     4861 tgctagttat tgctcagcgg tggcagcagc caactcagct tcctttcggg ctttgttagc
     4921 agccggatct gagctcgccc ttaaagtcca ggttggcata aacggcatta tcttcaataa
     4981 actggcgacg attttccaca acatcaccca tcagcatttc aaaggtctga tccgcttcaa
     5041 ttgcatcttc cagtttaact tgcagcagtg cacgatgttc cggattcatg gtggtttccc
     5101 acagctgatc tgcattcatt tcacccagac ctttataacg tgcaattgac catttcggtg
     5161 tcggattcag ttcgcttttc agtttgtcca gctcgcgatc attatacacg tagtatttct
     5221 gtttaccctg ggtcagttta tacagcggag gctgtgcaat ataaacataa ccggcttcaa
     5281 tcagcggacg cataaaacga tagaaaaatg tcagcagcag ggtacgaata tgtgcaccat
     5341 caacatcggc atcggtcata atcacaatct tatgataacg ggcttttgcc agatcaaaat
     5401 caccaccaat accggtgcca aatgcggtaa tcatctggcg aatttcatta ttgttcagaa
     5461 tgcgatccag acgtgctttt tccacattca gaattttacc acgcagcggc agaattgcct
     5521 gggtacgact atcacgaccg cttttggtac taccaccggc actatcacct tccaccagaa
     5581 aaatttcaca ttcttccggt gatttgctgc tacaatctgc cagtttacca ggcaggcttg
     5641 caacatccag tgcgctttta cgacgggtaa cttcacgtgc tttctttgct gcaacacggg
     5701 cacgtgctgc cataatacct ttttcaacaa cggtacgtgc aacctgcgga ttttcataca
     5761 gaaagcgctc aaagtgttcg ctaaacagtt tatccacaac ctgacgaact tcgctattac
     5821 ccagtttggt tttggtctga ccttcaaact gaggatcgcc atgtttaatg ctaataattg
     5881 cggtcatacc ttcacgggta tcctcaccgc tcagacgatc tttttcttct ttcatgattt
     5941 tgctgctcag accatagcta ttcagaacac gggtcagtgc acgtttaaaa ccatcttcgt
     6001 gtgtaccgcc ttcataggta tgaatgttat ttgcataggt cagcagattg gtggcataac
     6061 cgctattata ctggatggca atttccactt cgatatcatc tttgctctga tggatataga
     6121 tcggctcatc atgaatcggt tctttgtttt cgttcagcag ttccacatag cttttaatgc
     6181 caccttcata atggtagcta tcttcacgaa cattttcttc gtcacgttca tcacgcaggg
     6241 taatctgaat gcctttattc agaaatgcca gttcacgaat acgctgctgc agggtttcat
     6301 agttataaac ggtggtttcg gtgaagattt caccatctgc tttaaaacga ataacggtgc
     6361 cggttttatc ggtggtgcca acttctttca gatcaaactg cggaacaccc tttttatagg
     6421 cctggtgata aatggtttcg ttacgatgaa cataaacttc cagatcctgg ctcagtgcat
     6481 taacaacgct gctaccaaca ccatgcaggc caccgctaac tttatagcca ccaccaccaa
     6541 atttgccacc ggcatgcaga acggtcagaa taacttcaac tgccggacga cccattttct
     6601 cttgaatatc aaccggaata ccacgaccat tatcggtaac tttaatccag ttgtctttct
     6661 cgatcaccac ttcaatctga tttgcataac ctgccagtgc ttcatcaatg ctgttatcaa
     6721 caatttccca aaccagatga tgcagaccac gttcgctggt gctaccaata tacatacccg
     6781 gacgtttacg aactgcttcc agaccttcca gaacctgaat ctgacctgcg ccataattat
     6841 cggtgttatt cacatcactc agtgcggtaa ccatggtgaa gggatcaatt ccctgaaaat
     6901 acaggttttc cgtagaatcg agaccgagga gagggttagg gataggctta ccatggtgat
     6961 ggtgatgatg catatgtata tctccttctt aaagttaaac aaaattattt ctagagggga
     7021 attgttatcc gctcacaatt cccctatagt gagtcgtatt aatttcgcgg gatcgagatc
     7081 tcgatcctct acgccggacg catcgtggcc ggcatcaccg gcgccacagg tgcggttgct
     7141 ggcgcctata tcgccgacat caccgatggg gaagatcggg ctcgccactt cgggctcatg
     7201 agcgcttgtt tcggcgtggg tatggtggca ggccccgtgg ccgggggact gttgggcgcc
     7261 atctccttgc atgcaccatt ccttgcggcg gcggtgctca acggcctcaa cctactactg
     7321 ggctgcttcc taatgcagga gtcgcataag ggagagcgtc gagatcccgg acaccatcga
     7381 atggcgcaaa acctttcgcg gtatggcatg atagcgcccg gaagagagtc aattcagggt
     7441 ggtgaatgtg aaaccagtaa cgttatacga tgtcgcagag tatgccggtg tctcttatca
     7501 gaccgtttcc cgcgtggtga accaggccag ccacgtttct gcgaaaacgc gggaaaaagt
     7561 ggaagcggcg atggcggagc tgaattacat tcccaaccgc gtggcacaac aactggcggg
     7621 caaacagtcg ttgctgattg gcgttgccac ctccagtctg gccctgcacg cgccgtcgca
     7681 aattgtcgc
//
