LOCUS       sequence-211369-        4944 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  one-step modification of chromosomal locous using targocil/tarO gene
            in Staphylococcus aureus.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4944)
  AUTHORS   Lee W, Do T, Zhang G, Kahne D, Meredith TC, Walker S
  TITLE     Antibiotic Combinations That Enable One-Step, Targeted Mutagenesis 
            of Chromosomal Genes.
  JOURNAL   ACS Infect Dis. 2018 Mar 23. doi: 10.1021/acsinfecdis.8b00017.
  PUBMED    29534563
REFERENCE   2  (bases 1 to 4944)
  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: "ACS Infect 
            Dis. 2018 Mar 23. doi: 10.1021/acsinfecdis.8b00017."
COMMENT     Sequence Label: pTarKO
FEATURES             Location/Qualifiers
     source          1..4944
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_feature    1272..1328
                     /label=MCS
                     /note="pUC18/19 multiple cloning site"
     primer_bind     complement(1341..1357)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1341..1357)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1354..1376)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    1365..1381
                     /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)."
     promoter        complement(join(1389..1395,1396..1413,1414..1419))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1434..1455
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(1572..1589)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1743..2331)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1823..1842)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2502..3293,3294..3362))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     3125..3144
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3363..3467)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     3535..3553
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(3591..3613)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     3713..3732
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 taaaatgttg ccttaaatga tatgatgaaa aaatgaataa taggcgatat ataagaaatg
       61 aatcgtatag ttgtaaatat gatatcattg attgaacgaa ttaatttata ataaagctat
      121 aagatatacg tagaaaatag atatatcatt ctataaagac aatattaaat aaatataacg
      181 ttaaaaacaa ttaatatcga tgaaggtgaa taaatggtta cattattact agttgcagta
      241 acaatgattg tcagtttgac gataacacca attgttattg caatatcgaa aagattaaat
      301 ttagttgata aaccaaattt tagaaaagta cacactaaac ctatttcagt tatgggtggt
      361 acagtgattc tcttttcatt tttaataggt atttggattg gtcatcctat tgaaacagaa
      421 atcaaaccac ttattattgg tgcgattatt atgtacgtac ttgggcttgt agatgatatc
      481 tacgatttga aaccgtatat aaaattggct ggtcaaattg ccgctgcctt agtagttgct
      541 ttttatggtg tgactattga ttttatttcg ttgccaatgg gtacaacgat tcattttgga
      601 tttcttagta ttccaattac tgtgatttgg attgttgcta ttacaaatgc aattaactta
      661 attgatggac tcgatggttt ggcgtcgggt gtttctgcaa tcggactcat tacaataggg
      721 ttcattgcaa ttttacaagc taatattttc ataacgatga tttgttgtgt tttattaggc
      781 tctttaattg ggtttttatt ttacaatttc catcctgcca aaatattttt aggtgatagt
      841 ggggctttaa tgattggatt tatcatcgga ttcctttctt tactcggatt caaaaatatt
      901 acaattattg cattgttctt cccaattgtt atcttagcag ttccattcat tgatactttg
      961 ttcgcaatga ttcgacgtgt gaaaaaaggg cagcatataa tgcaagctga taaatcgcat
     1021 ttgcatcata aactattagc tttaggctac acacatagac aaacagtatt attaatctat
     1081 tcaatctcta ttttatttag tctttcgagc attattttgt atgtatcgcc accattaggt
     1141 gttgtattaa tgtttgtatt aatcatattt agtattgaat taattgttga atttacagga
     1201 ttaatagata acaactaccg accaatatta aatttaatta gtcgtaagtc atctcataaa
     1261 gaggaatagg ggaattcgag ctcggtaccc ggggatcctc tagagtcgac ctgcaggcat
     1321 gcaagcttgg cgtaatcatg gtcatagctg tttcctgtgt gaaattgtta tccgctcaca
     1381 attccacaca acatacgagc cggaagcata aagtgtaaag cctggggtgc ctaatgagtg
     1441 agctaactca cattaattgc gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg
     1501 tgccagctgc attaatgaat cggccaacgc gcggggagag gcggtttgcg tattgggcgc
     1561 tcttccgctt cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta
     1621 tcagctcact caaaggcggt aatacggtta tccacagaat caggggataa cgcaggaaag
     1681 aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg
     1741 tttttccata ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg
     1801 tggcgaaacc cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg
     1861 cgctctcctg ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga
     1921 agcgtggcgc tttctcatag ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc
     1981 tccaagctgg gctgtgtgca cgaacccccc gttcagcccg accgctgcgc cttatccggt
     2041 aactatcgtc ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact
     2101 ggtaacagga ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg
     2161 cctaactacg gctacactag aagaacagta tttggtatct gcgctctgct gaagccagtt
     2221 accttcggaa aaagagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt
     2281 ggtttttttg tttgcaagca gcagattacg cgcagaaaaa aaggatctca agaagatcct
     2341 ttgatctttt ctacggggtc tgacgctcag tggaacgaaa actcacgtta agggattttg
     2401 gtcatgagat tatcaaaaag gatcttcacc tagatccttt taaattaaaa atgaagtttt
     2461 aaatcaatct aaagtatata tgagtaaact tggtctgaca gttaccaatg cttaatcagt
     2521 gaggcaccta tctcagcgat ctgtctattt cgttcatcca tagttgcctg actccccgtc
     2581 gtgtagataa ctacgatacg ggagggctta ccatctggcc ccagtgctgc aatgataccg
     2641 cgagacccac gctcaccggc tccagattta tcagcaataa accagccagc cggaagggcc
     2701 gagcgcagaa gtggtcctgc aactttatcc gcctccatcc agtctattaa ttgttgccgg
     2761 gaagctagag taagtagttc gccagttaat agtttgcgca acgttgttgc cattgctaca
     2821 ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat tcagctccgg ttcccaacga
     2881 tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag cggttagctc cttcggtcct
     2941 ccgatcgttg tcagaagtaa gttggccgca gtgttatcac tcatggttat ggcagcactg
     3001 cataattctc ttactgtcat gccatccgta agatgctttt ctgtgactgg tgagtactca
     3061 accaagtcat tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata
     3121 cgggataata ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg aaaacgttct
     3181 tcggggcgaa aactctcaag gatcttaccg ctgttgagat ccagttcgat gtaacccact
     3241 cgtgcaccca actgatcttc agcatctttt actttcacca gcgtttctgg gtgagcaaaa
     3301 acaggaaggc aaaatgccgc aaaaaaggga ataagggcga cacggaaatg ttgaatactc
     3361 atactcttcc tttttcaata ttattgaagc atttatcagg gttattgtct catgagcgga
     3421 tacatatttg aatgtattta gaaaaataaa caaatagggg ttccgcgcac atttccccga
     3481 aaagtgccac ctgacgtcta agaaaccatt attatcatga cattaaccta taaaaatagg
     3541 cgtatcacga ggccctttcg tctcgcgcgt ttcggtgatg acggtgaaaa cctctgacac
     3601 atgcagctcc cggagacggt cacagcttgt ctgtaagcgg atgccgggag cagacaagcc
     3661 cgtcagggcg cgtcagcggg tgttggcggg tgtcggggct ggcttaacta tgcggcatca
     3721 gagcagattg tactgagagt gcaccatatg ccggcaatag ttacccttat tatcaagata
     3781 agaaagaaaa ggatttttcg ctacgctcaa atcctttaaa aaaacacaaa agaccacatt
     3841 ttttaatgtg gtcttttatt cttcaactaa agcacccatt agttcaacaa acgaaaattg
     3901 gataaagtgg gatattttta aaatatatat ttatgttaca gtaatattga cttttaaaaa
     3961 aggattgatt ctaatgaaga aagcagacaa gtaagcctcc taaattcact ttagataaaa
     4021 atttaggagg catatcaaat gaactttaat aaaattgatt tagacaattg gaagagaaaa
     4081 gagatattta atcattattt gaaccaacaa acgactttta gtataaccac agaaattgat
     4141 attagtgttt tataccgaaa cataaaacaa gaaggatata aattttaccc tgcatttatt
     4201 ttcttagtga caagggtgat aaactcaaat acagctttta gaactggtta caatagcgac
     4261 ggagagttag gttattggga taagttagag ccactttata caatttttga tggtgtatct
     4321 aaaacattct ctggtatttg gactcctgta aagaatgact tcaaagagtt ttatgattta
     4381 tacctttctg atgtagagaa atataatggt tcggggaaat tgtttcccaa aacacctata
     4441 cctgaaaatg ctttttctct ttctattatt ccatggactt catttactgg gtttaactta
     4501 aatatcaata ataatagtaa ttaccttcta cccattatta cagcaggaaa attcattaat
     4561 aaaggtaatt caatatattt accgctatct ttacaggtac atcattctgt ttgtgatggt
     4621 tatcatgcag gattgtttat gaactctatt caggaattgt cagataggcc taatgactgg
     4681 cttttataat atgagataat gccgactgta ctttttacag tcggcattat ctcatattat
     4741 aaaagccagt cataccaata acttaagggt aactattgcc ggcatatgcg gtgtgaaata
     4801 ccgcacagat gcgtaaggag aaaataccgc atcaggcgcc gtctgaatcg actcctttaa
     4861 ttgaccacta gctattgtaa gtgaaaactt acaatttgtc attagtttac atataaaatt
     4921 aatgtatgat atagactttg atgt
//
