LOCUS       sequence-421300-        8418 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  S. aureus USA300 GyrA 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
FEATURES             Location/Qualifiers
     source          1..8418
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     106..125
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     209..228
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        209..227
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    228..252
                     /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             267..289
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             300..317
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             318..359
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     CDS             360..380
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     primer_bind     complement(3119..3137)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      3133..3180
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     complement(3418..3436)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3504..3608
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3609..3677,3678..4469)
                     /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(3827..3846)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4640..5228
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5129..5148
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5382..5399
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    5414..5553
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(5479..5498)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5639..5661
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(5655..5846)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(7158..8240)
                     /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     8202..8221
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(8241..8318)
                     /gene="lacI"
                     /label=lacI promoter
ORIGIN
        1 caaggagatg gcgcccaaca gtcccccggc cacggggcct gccaccatac ccacgccgaa
       61 acaagcgctc atgagcccga agtggcgagc ccgatcttcc ccatcggtga tgtcggcgat
      121 ataggcgcca gcaaccgcac ctgtggcgcc ggtgatgccg gccacgatgc gtccggcgta
      181 gaggatcgag atctcgatcc cgcgaaatta atacgactca ctatagggga attgtgagcg
      241 gataacaatt cccctctaga aataattttg tttaacttta agaaggagat atacatatgc
      301 atcatcacca tcaccatggt aagcctatcc ctaaccctct cctcggtctc gattctacgg
      361 aaaacctgta ttttcaggga attgatccct tcaccatggc agaactgccg cagagccgta
      421 ttaatgaacg caatattacc agcgaaatgc gtgaatcctt tctggattat gcaatgagcg
      481 ttattgttgc acgtgccctg ccggatgttc gtgatggtct gaaaccggtt catcgtcgta
      541 ttctgtatgg cctgaatgaa cagggtatga caccggataa aagctataaa aagagcgcac
      601 gtattgtcgg tgatgtgatg ggtaaatatc atccgcatgg tgatctgagc atttatgaag
      661 caatggttcg tatggcacag gattttagct atcgttatcc gctggttgat ggtcagggta
      721 attttggtag catggatggt gatggtgcag cagcaatgcg ttataccgaa gcacgtatga
      781 caaaaatcac cctggaactg ctgcgtgata ttaacaaaga taccatcgat ttcatcgaca
      841 actatgatgg caatgaacgt gaaccgagcg ttctgcctgc acgttttccg aatctgctgg
      901 caaatggtgc aagcggtatt gcagttggta tggcaaccaa tattccgcct cataatctga
      961 ccgaactgat taatggtgtg ctgagcctga gcaaaaatcc ggatattagc attgcagaac
     1021 tgatggaaga tattgaaggt cctgattttc cgaccgcagg tctgattctg ggtaaaagtg
     1081 gtattcgtcg tgcatatgaa acaggtcgtg gtagcattca gatgcgtagc cgtgcagtta
     1141 ttgaagaacg tggtggtggt cgtcagcgta ttgttgttac cgaaattccg tttcaggtta
     1201 acaaagcccg tatgattgag aaaattgccg aactggttcg cgataaaaag attgatggta
     1261 ttaccgatct gcgtgatgaa accagcctgc gtaccggtgt tcgtgttgtt attgatgttc
     1321 gtaaagatgc aaatgccagc gtgattctga acaacctgta taaacagaca ccgctgcaga
     1381 ccagctttgg tgttaatatg attgcactgg ttaatggtcg tccgaaactg atcaatctga
     1441 aagaagcact ggttcactat ctggaacatc agaaaaccgt tgttcgtcgt cgtacccagt
     1501 ataatctgcg taaagcaaaa gatcgtgccc atattctgga aggtctgcgt attgccctgg
     1561 atcatattga tgaaattatc agcaccattc gcgaaagcga taccgataaa gttgcaatgg
     1621 aaagcctgca gcagcgtttt aaactgagcg aaaaacaggc acaggcaatt ctggatatgc
     1681 gtctgcgtcg tctgaccggt ctggaacgcg acaaaattga agcagaatat aatgagctgc
     1741 tgaactatat cagcgaactg gaagccattc tggcagatga agaagttctg ctgcagctgg
     1801 tacgtgatga actgacagaa attcgtgatc gttttggtga tgatcgtcgt acggaaattc
     1861 agttaggtgg ttttgaaaac ctggaagatg aggatctgat tccggaagaa caaattgtta
     1921 ttacgctgag ccacaacaac tacattaaac gtctgccggt tagcacctat cgtgcacaga
     1981 atcgcggtgg tcgtggtgtt cagggcatga ataccctgga agaagatttt gttagccagc
     2041 tggttaccct gagcacccat gatcacgttc tgtttttcac caataaaggt cgcgtgtata
     2101 aactgaaagg ttatgaagtg ccggaactga gccgtcagag caaaggtatt ccggttgtta
     2161 atgcaattga gctggaaaac gatgaagtga ttagcaccat gattgccgtt aaagatctgg
     2221 aaagcgaaga taactttctg gtgtttgcaa ccaaacgtgg tgttgttaaa cgtagcgcac
     2281 tgagcaattt ttctcgcatt aatcgcaatg gcaaaatcgc cattagcttt cgtgaagatg
     2341 atgagctgat cgcagtgcgt ctgaccagcg gtcaagaaga tattctgatt ggcaccagcc
     2401 atgcaagcct gattcgtttt cctgaaagca ccctgcgtcc gctgggtcgt accgcaaccg
     2461 gtgttaaagg tattaccctg cgcgaaggtg atgaagttgt tggtctggat gttgcacatg
     2521 caaatagcgt tgatgaagtg ctggttgtga ccgaaaatgg ttatggtaaa cgtacaccgg
     2581 tgaatgatta tcgtctgagc aatcgtggtg gtaaaggcat taaaaccgca accattaccg
     2641 aacgtaatgg taatgttgtt tgcattacca ccgttaccgg tgaagaggac ctgatgattg
     2701 ttaccaatgc cggtgttatt attcgcctgg atgtggcaga tattagtcag aatggtcgtg
     2761 cagcacaggg tgttcgtctg attcgtctgg gtgatgacca gtttgttagc accgttgcaa
     2821 aagttaaaga agatgcagag gacgaaacca acgaagatga acagagcacc agcaccgtta
     2881 gcgaagatgg caccgaacag cagcgtgaag cagttgtgaa tgatgaaaca ccgggtaatg
     2941 ccattcatac cgaagttatt gatagcgaag aaaatgatga agatggtcgt attgaagtgc
     3001 gtcaggattt catggatcgt gttgaagagg atattcagca gagcagtgat gaagaaaagg
     3061 gcgagctcag atccggctgc taacaaagcc cgaaaggaag ctgagttggc tgctgccacc
     3121 gctgagcaat aactagcata accccttggg gcctctaaac gggtcttgag gggttttttg
     3181 ctgaaaggag gaactatatc cggatatccc gcaagaggcc cggcagtacc ggcataacca
     3241 agcctatgcc tacagcatcc agggtgacgg tgccgaggat gacgatgagc gcattgttag
     3301 atttcataca cggtgcctga ctgcgttagc aatttaactg tgataaacta ccgcattaaa
     3361 gctagcttat cgatgataag ctgtcaaaca tgagaattaa ttcttgaaga cgaaagggcc
     3421 tcgtgatacg cctattttta taggttaatg tcatgataat aatggtttct tagacgtcag
     3481 gtggcacttt tcggggaaat gtgcgcggaa cccctatttg tttatttttc taaatacatt
     3541 caaatatgta tccgctcatg agacaataac cctgataaat gcttcaataa tattgaaaaa
     3601 ggaagagtat gagtattcaa catttccgtg tcgcccttat tccctttttt gcggcatttt
     3661 gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt
     3721 tgggtgcacg agtgggttac atcgaactgg atctcaacag cggtaagatc cttgagagtt
     3781 ttcgccccga agaacgtttt ccaatgatga gcacttttaa agttctgcta tgtggcgcgg
     3841 tattatcccg tgttgacgcc gggcaagagc aactcggtcg ccgcatacac tattctcaga
     3901 atgacttggt tgagtactca ccagtcacag aaaagcatct tacggatggc atgacagtaa
     3961 gagaattatg cagtgctgcc ataaccatga gtgataacac tgcggccaac ttacttctga
     4021 caacgatcgg aggaccgaag gagctaaccg cttttttgca caacatgggg gatcatgtaa
     4081 ctcgccttga tcgttgggaa ccggagctga atgaagccat accaaacgac gagcgtgaca
     4141 ccacgatgcc tgcagcaatg gcaacaacgt tgcgcaaact attaactggc gaactactta
     4201 ctctagcttc ccggcaacaa ttaatagact ggatggaggc ggataaagtt gcaggaccac
     4261 ttctgcgctc ggcccttccg gctggctggt ttattgctga taaatctgga gccggtgagc
     4321 gtgggtctcg cggtatcatt gcagcactgg ggccagatgg taagccctcc cgtatcgtag
     4381 ttatctacac gacggggagt caggcaacta tggatgaacg aaatagacag atcgctgaga
     4441 taggtgcctc actgattaag cattggtaac tgtcagacca agtttactca tatatacttt
     4501 agattgattt aaaacttcat ttttaattta aaaggatcta ggtgaagatc ctttttgata
     4561 atctcatgac caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag
     4621 aaaagatcaa aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa
     4681 caaaaaaacc accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt
     4741 ttccgaaggt aactggcttc agcagagcgc agataccaaa tactgtcctt ctagtgtagc
     4801 cgtagttagg ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa
     4861 tcctgttacc agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa
     4921 gacgatagtt accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc
     4981 ccagcttgga gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa
     5041 gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa
     5101 caggagagcg cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg
     5161 ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc
     5221 tatggaaaaa cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg
     5281 ctcacatgtt ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg
     5341 agtgagctga taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg
     5401 aagcggaaga gcgcctgatg cggtattttc tccttacgca tctgtgcggt atttcacacc
     5461 gcaatggtgc actctcagta caatctgctc tgatgccgca tagttaagcc agtatacact
     5521 ccgctatcgc tacgtgactg ggtcatggct gcgccccgac acccgccaac acccgctgac
     5581 gcgccctgac gggcttgtct gctcccggca tccgcttaca gacaagctgt gaccgtctcc
     5641 gggagctgca tgtgtcagag gttttcaccg tcatcaccga aacgcgcgag gcagctgcgg
     5701 taaagctcat cagcgtggtc gtgaagcgat tcacagatgt ctgcctgttc atccgcgtcc
     5761 agctcgttga gtttctccag aagcgttaat gtctggcttc tgataaagcg ggccatgtta
     5821 agggcggttt tttcctgttt ggtcactgat gcctccgtgt aagggggatt tctgttcatg
     5881 ggggtaatga taccgatgaa acgagagagg atgctcacga tacgggttac tgatgatgaa
     5941 catgcccggt tactggaacg ttgtgagggt aaacaactgg cggtatggat gcggcgggac
     6001 cagagaaaaa tcactcaggg tcaatgccag cgcttcgtta atacagatgt aggtgttcca
     6061 cagggtagcc agcagcatcc tgcgatgcag atccggaaca taatggtgca gggcgctgac
     6121 ttccgcgttt ccagacttta cgaaacacgg aaaccgaaga ccattcatgt tgttgctcag
     6181 gtcgcagacg ttttgcagca gcagtcgctt cacgttcgct cgcgtatcgg tgattcattc
     6241 tgctaaccag taaggcaacc ccgccagcct agccgggtcc tcaacgacag gagcacgatc
     6301 atgcgcaccc gtggccagga cccaacgctg cccgagatgc gccgcgtgcg gctgctggag
     6361 atggcggacg cgatggatat gttctgccaa gggttggttt gcgcattcac agttctccgc
     6421 aagaattgat tggctccaat tcttggagtg gtgaatccgt tagcgaggtg ccgccggctt
     6481 ccattcaggt cgaggtggcc cggctccatg caccgcgacg caacgcgggg aggcagacaa
     6541 ggtatagggc ggcgcctaca atccatgcca acccgttcca tgtgctcgcc gaggcggcat
     6601 aaatcgccgt gacgatcagc ggtccaatga tcgaagttag gctggtaaga gccgcgagcg
     6661 atccttgaag ctgtccctga tggtcgtcat ctacctgcct ggacagcatg gcctgcaacg
     6721 cgggcatccc gatgccgccg gaagcgagaa gaatcataat ggggaaggcc atccagcctc
     6781 gcgtcgcgaa cgccagcaag acgtagccca gcgcgtcggc cgccatgccg gcgataatgg
     6841 cctgcttctc gccgaaacgt ttggtggcgg gaccagtgac gaaggcttga gcgagggcgt
     6901 gcaagattcc gaataccgca agcgacaggc cgatcatcgt cgcgctccag cgaaagcggt
     6961 cctcgccgaa aatgacccag agcgctgccg gcacctgtcc tacgagttgc atgataaaga
     7021 agacagtcat aagtgcggcg acgatagtca tgccccgcgc ccaccggaag gagctgactg
     7081 ggttgaaggc tctcaagggc atcggtcgag atcccggtgc ctaatgagtg agctaactta
     7141 cattaattgc gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc
     7201 attaatgaat cggccaacgc gcggggagag gcggtttgcg tattgggcgc cagggtggtt
     7261 tttcttttca ccagtgagac gggcaacagc tgattgccct tcaccgcctg gccctgagag
     7321 agttgcagca agcggtccac gctggtttgc cccagcaggc gaaaatcctg tttgatggtg
     7381 gttaacggcg ggatataaca tgagctgtct tcggtatcgt cgtatcccac taccgagata
     7441 tccgcaccaa cgcgcagccc ggactcggta atggcgcgca ttgcgcccag cgccatctga
     7501 tcgttggcaa ccagcatcgc agtgggaacg atgccctcat tcagcatttg catggtttgt
     7561 tgaaaaccgg acatggcact ccagtcgcct tcccgttccg ctatcggctg aatttgattg
     7621 cgagtgagat atttatgcca gccagccaga cgcagacgcg ccgagacaga acttaatggg
     7681 cccgctaaca gcgcgatttg ctggtgaccc aatgcgacca gatgctccac gcccagtcgc
     7741 gtaccgtctt catgggagaa aataatactg ttgatgggtg tctggtcaga gacatcaaga
     7801 aataacgccg gaacattagt gcaggcagct tccacagcaa tggcatcctg gtcatccagc
     7861 ggatagttaa tgatcagccc actgacgcgt tgcgcgagaa gattgtgcac cgccgcttta
     7921 caggcttcga cgccgcttcg ttctaccatc gacaccacca cgctggcacc cagttgatcg
     7981 gcgcgagatt taatcgccgc gacaatttgc gacggcgcgt gcagggccag actggaggtg
     8041 gcaacgccaa tcagcaacga ctgtttgccc gccagttgtt gtgccacgcg gttgggaatg
     8101 taattcagct ccgccatcgc cgcttccact ttttcccgcg ttttcgcaga aacgtggctg
     8161 gcctggttca ccacgcggga aacggtctga taagagacac cggcatactc tgcgacatcg
     8221 tataacgtta ctggtttcac attcaccacc ctgaattgac tctcttccgg gcgctatcat
     8281 gccataccgc gaaaggtttt gcgccattcg atggtgtccg ggatctcgac gctctccctt
     8341 atgcgactcc tgcattagga agcagcccag tagtaggttg aggccgttga gcaccgccgc
     8401 cgcaaggaat ggtgcatg
//
