LOCUS       sequence-438701-        4440 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Enables scarless gene deletions in Streptococcus pyogenes using the 
            counterselection marker pheS*.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4440)
  AUTHORS   Lautenschlager N, Schmidt K, Schiffer C, Wulff TF, Hahnke K, 
            Finstermeier K, Mansour M, Elsholz AKW, Charpentier E
  TITLE     Expanding the genetic toolbox for the obligate human pathogen 
            Streptococcus pyogenes.
  JOURNAL   Front Bioeng Biotechnol. 2024 Jun 7;12:1395659. doi: 
            10.3389/fbioe.2024.1395659. eCollection 2024.
  PUBMED    38911550
REFERENCE   2  (bases 1 to 4440)
  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: "Front 
            Bioeng Biotechnol."; date: "2024-06-7"; volume: "12:1395659. doi"; 
            pages: " 10.3389/fbioe.2024.1395659. eCollection 2024"
COMMENT     Sequence Label: pEC3135 (pERASE)
FEATURES             Location/Qualifiers
     source          1..4440
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      152..740
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     641..660
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     894..911
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     3273..3289
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    3399..3420
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(3435..3440,3441..3458,3459..3465)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3473..3489
                     /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             3507..3518
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             3525..4202
                     /codon_start=1
                     /product="monomeric derivative of DsRed (Campbell et al., 
                     2002)"
                     /label=mRFP1
                     /translation="MASSEDVIKEFMRFKVRMEGSVNGHEFEIEGEGEGRPYEGTQTAK
                     LKVTKGGPLPFAWDILSPQFQYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGV
                     VTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASTERMYPEDGALKGEIKM
                     RLKLKDGGHYDAEVKTTYMAKKPVQLPGAYKTDIKLDITSHNEDYTIVEQYERAEGRHS
                     TGA"
     terminator      4247..4318
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      4334..4361
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     primer_bind     complement(4405..4421)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4405..4421)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
ORIGIN
        1 catatatact ttagattgat ttaaaacttc atttttaatt taaaaggatc taggtgaaga
       61 tcctttttga taatctcatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt
      121 cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct
      181 gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc
      241 taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgttc
      301 ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg cctacatacc
      361 tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg
      421 ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt
      481 cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg
      541 agctatgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg
      601 gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt
      661 atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag
      721 gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt
      781 gctggccttt tgctcacatg ttctttcctg cgttatcccc tgattctgtg gataaccgta
      841 ttaccgcctt tgagtgagct gataccgctc gccgcagccg aacgaccgag cgcagcgagt
      901 cagtgagcga ggaagcggaa gagcgcccaa tacgcaaacc gcctctcttt tttattttac
      961 ataatgatac ataatttacc gaaacttgcg gaacataatt gaggaatcat agaattttgt
     1021 caaaataatt ttattgacaa cgtcttatta acgttgatat aatttaaatt ttatttgaca
     1081 aaaatgggct cgtgttgtac aataaatgta gttaaaggag gataatatgg accttcaagc
     1141 tcaattggaa gaacttaaaa ctaaaacctt ggaaactctt caaagtctta ctggtaacca
     1201 tacaaaagaa ttgcaagatt tgcgtgttgc agttcttggt aagaagggtt cacttactga
     1261 tttgcttaaa ggtcttaagg atttgtcaaa cgatttgcgt cctgtggtag gcaaacaagt
     1321 aaacgaagtt cgtgacttat tgacaaaagc tttcgaagag caagctaaga tcgttgaagc
     1381 cgctaaaatt caagctcaat tggatgctga atcaatcgac gttactcttc ctggacgtca
     1441 aatgactctg gggcatcgtc acgttcttac tcaaacatca gaagaaatcg aagacatctt
     1501 ccttggtatg ggtttccaaa ttgtagatgg tttcgaagtt gaaaaagatt attataactt
     1561 cgaacgtatg aaccttccaa aagaccaccc tgctcgtgac atgcaagaca ctttctacat
     1621 tactgaagag atcttgttac gtactcacac ttcaccagta caagctcgta ctcttgatca
     1681 acacgacttc tctaagggcc ctttaaaaat ggtttcacca ggccgtgtat tccgccgcga
     1741 cactgacgac gctactcact cacaccaatt ccaccaaatc gaaggtcttg tagttggcaa
     1801 aaatatttca atgggtgact tgaaaggcac tttggaaatg atcattaaaa aaatgttcgg
     1861 tgacgaacgt agcatccgtc ttcgcccttc ttatttccct ttctcagaac catcagtaga
     1921 agttgacgta tcatgtttca aatgtggagg taaaggctgt aacgtttgta aaaaaactgg
     1981 ttggatcgaa atcttgggtg ctggtatggt tcacccaagt gttcttgaaa tgtcaggtgt
     2041 tgatgcaaaa gagtactcag gtttcggttt cggcttggga caagagcgta tcgctatgtt
     2101 gcgctacgga attaacgata ttcgtggctt ctaccaaggc gaccaacgtt tctcagaaca
     2161 atttaactaa aaaaaaggcc cacaaaagtg ggcctttttt aaacgaaatg atacaccaat
     2221 cagtgcaaaa aaagatataa tgggagataa gacggttcgt gttcgtgctg acttgcacca
     2281 tatcataaaa atcgaaacag caaagaatgg cggaaacgta aaagaagtta tggaaataag
     2341 acttagaagc aaacttaaga gtgtgttgat agtgcagtat cttaaaattt tgtataatag
     2401 gaattgaagt taaattagat gctaaaaatt tgtaattaag aaggagtgat tacatgaaca
     2461 aaaatataaa atattctcaa aactttttaa cgagtgaaaa agtactcaac caaataataa
     2521 aacaattgaa tttaaaagaa accgataccg tttacgaaat tggaacaggt aaagggcatt
     2581 taacgacgaa actggctaaa ataagtaaac aggtaacgtc tattgaatta gacagtcatc
     2641 tattcaactt atcgtcagaa aaattaaaac tgaatactcg tgtcacttta attcaccaag
     2701 atattctaca gtttcaattc cctaacaaac agaggtataa aattgttggg agtattcctt
     2761 accatttaag cacacaaatt attaaaaaag tggtttttga aagccatgcg tctgacatct
     2821 atctgattgt tgaagaagga ttctacaagc gtaccttgga tattcaccga acactagggt
     2881 tgctcttgca cactcaagtc tcgattcagc aattgcttaa gctgccagcg gaatgctttc
     2941 atcctaaacc aaaagtaaac agtgtcttaa taaaacttac ccgccatacc acagatgttc
     3001 cagataaata ttggaagcta tatacgtact ttgtttcaaa atgggtcaat cgagaatatc
     3061 gtcaactgtt tactaaaaat cagtttcatc aagcaatgaa acacgccaaa gtaaacaatt
     3121 taagtaccgt tacttatgag caagtattgt ctatttttaa tagttatcta ttatttaacg
     3181 ggaggaaata attctatgag tcgcttttgt aaatttggaa agttacacgt tactaaaggg
     3241 aatgtagata aattattagg taaatctaaa acgtaaaacg acggccagtc agcgagacca
     3301 gctgaattca aaaaaaaatc tagatggccg gcgattcatt aatgcagctg gcacgacagg
     3361 tttcccgact ggaaagcggg cagtgagcgc aacgcaatta atgtgagtta gctcactcat
     3421 taggcacccc aggctttaca ctttatgctt ccggctcgta tgttgtgtgg aattgtgagc
     3481 ggataacaat ttcacacata ctagagaaag aggagaaata ctagatggct tcctccgaag
     3541 acgttatcaa agagttcatg cgtttcaaag ttcgtatgga aggttccgtt aacggtcacg
     3601 agttcgaaat cgaaggtgaa ggtgaaggtc gtccgtacga aggtacccag accgctaaac
     3661 tgaaagttac caaaggtggt ccgctgccgt tcgcttggga catcctgtcc ccgcagttcc
     3721 agtacggttc caaagcttac gttaaacacc cggctgacat cccggactac ctgaaactgt
     3781 ccttcccgga aggtttcaaa tgggaacgtg ttatgaactt cgaagacggt ggtgttgtta
     3841 ccgttaccca ggactcctcc ctgcaagacg gtgagttcat ctacaaagtt aaactgcgtg
     3901 gtaccaactt cccgtccgat ggtccggtta tgcagaaaaa aaccatgggt tgggaagctt
     3961 ccaccgaacg tatgtacccg gaagacggtg ctctgaaagg tgaaatcaaa atgcgtctga
     4021 aactgaaaga cggtggtcac tacgacgctg aagttaaaac cacctacatg gctaaaaaac
     4081 cggttcagct gccgggtgct tacaaaaccg acatcaaact ggacatcacc tcccacaacg
     4141 aagactacac catcgttgaa cagtacgaac gtgctgaagg tcgtcactcc accggtgctt
     4201 aataacgctg atagtgctag tgtagatcgc tactagagcc aggcatcaaa taaaacgaaa
     4261 ggctcagtcg aaagactggg cctttcgttt tatctgttgt ttgtcggtga acgctctcta
     4321 ctagagtcac actggctcac cttcgggtgg gcctttctgc gtttataacc ggttaatact
     4381 agtactgcag tgaaggtctc atgtgtcata gctgtttcct gctgtcagac caagtttact
//
