LOCUS       sequence-365361-        2438 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Str resistant,CoE1* ori. CRISPR/Cas9 gRNA under constitutive J23119 
            promoter. gRNA targeting E. coli ptsI codon 2~9. HRT has synonymous 
            mutations in ptsI. (for 1st round editing).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 2438)
  AUTHORS   Yang DD, Rusch LM, Widney KA, Morgenthaler AB, Copley SD
  TITLE     Synonymous edits in the Escherichia coli genome have substantial and
            condition-dependent effects on fitness.
  JOURNAL   Proc Natl Acad Sci U S A. 2024 Jan 30;121(5):e2316834121. doi: 
            10.1073/pnas.2316834121. Epub 2024 Jan 22.
  PUBMED    38252823
REFERENCE   2  (bases 1 to 2438)
  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: "Proc Natl 
            Acad Sci U S A."; date: "2024-01-30"; volume: "121(5):e2316834121. 
            doi"; pages: " 10.1073/pnas.2316834121. Epub 2024 Jan 22"
COMMENT     Sequence Label: pDY279
FEATURES             Location/Qualifiers
     source          1..2438
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     98..115
                     /label=L4440
                     /note="L4440 vector, forward primer"
     promoter        513..547
                     /label=J23119(SpeI) promoter
                     /note="bacterial promoter (Registry of Standard Biological 
                     Parts BBa_J23119) modified to end with an SpeI site"
     misc_RNA        568..643
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     CDS             829..1620
                     /codon_start=1
                     /gene="aadA"
                     /product="aminoglycoside adenylyltransferase (Murphy, 
                     1985)"
                     /label=SmR
                     /note="confers resistance to spectinomycin and 
                     streptomycin"
                     /translation="MREAVIAEVSTQLSEVVGVIERHLEPTLLAVHLYGSAVDGGLKPH
                     SDIDLLVTVTVRLDETTRRALINDLLETSASPGESEILRAVEVTIVVHDDIIPWRYPAK
                     RELQFGEWQRNDILAGIFEPATIDIDLAILLTKAREHSVALVGPAAEELFDPVPEQDLF
                     EALNETLTLWNSPPDWAGDERNVVLTLSRIWYSAVTGKIAPKDVAADWAMERLPAQYQP
                     VILEARQAYLGQEEDRLASRADQLEEFVHYVKGEITKVVGK"
     rep_origin      1794..2382
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2283..2302
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 catgttcttt cctgcgttat cccctgattc tgtggataac cgtattaccg cctttgagtg
       61 agctgatacc gctcgccgca gccgaacgac cgagcgcagc gagtcagtga gcgaggaagc
      121 ggaagagcgc ctgatgcggt attttctcct tacgcatctg tgcggtattt cacaccgcat
      181 atgctggatt gggcctgact caaggtaccg ttgtgactat ctccgcagaa ggcgaagacg
      241 agcagaaagc ggttgaacat ctggttaaac tgatggcgga actcgagtaa tttcccgggt
      301 tcttttaaaa atcagtcaca agtaaggtag ggttatgata tccggaatat tggcttcacc
      361 aggtatcgct ttcggtaaag ctctgcttct gaaagaagac gaaattgtca ttgaccggaa
      421 aaaaatttct gccgaccagg ttgatcagga agttgaacgt tttctgagcg gtcgtgccaa
      481 ggcatcagcc cagctggaaa cgatcaaaac gattgacagc tagctcagtc ctaggtataa
      541 tactagtttc aggcatttta gcatcccgtt ttagagctag aaatagcaag ttaaaataag
      601 gctagtccgt tatcaacttg aaaaagtggc accgagtcgg tgcttttttt gaattctcta
      661 gagtcgacct gcagaagctt agatctatta ccctgttatc cctactcgag ttcatgtgca
      721 gctccataag caaaagggga tgataagttt atcaccaccg actatttgca acagtgccgt
      781 tgatcgtgct atgatcgact gatgtcatca gcggtggagt gcaatgtcat gagggaagcg
      841 gtgatcgccg aagtatcgac tcaactatca gaggtagttg gcgtcatcga gcgccatctc
      901 gaaccgacgt tgctggccgt acatttgtac ggctccgcag tggatggcgg cctgaagcca
      961 cacagtgata ttgatttgct ggttacggtg accgtaaggc ttgatgaaac aacgcggcga
     1021 gctttgatca acgacctttt ggaaacttcg gcttcccctg gagagagcga gattctccgc
     1081 gctgtagaag tcaccattgt tgtgcacgac gacatcattc cgtggcgtta tccagctaag
     1141 cgcgaactgc aatttggaga atggcagcgc aatgacattc ttgcaggtat cttcgagcca
     1201 gccacgatcg acattgatct ggctatcttg ctgacaaaag caagagaaca tagcgttgcc
     1261 ttggtaggtc cagcggcgga ggaactcttt gatccggttc ctgaacagga tctatttgag
     1321 gcgctaaatg aaaccttaac gctatggaac tcgccgcccg actgggctgg cgatgagcga
     1381 aatgtagtgc ttacgttgtc ccgcatttgg tacagcgcag taaccggcaa aatcgcgccg
     1441 aaggatgtcg ctgccgactg ggcaatggag cgcctgccgg cccagtatca gcccgtcata
     1501 cttgaagcta gacaggctta tcttggacaa gaagaagatc gcttggcctc gcgcgcagat
     1561 cagttggaag aatttgtcca ctacgtgaaa ggcgagatca ccaaggtagt cggcaaataa
     1621 gatgccgctc gccagtcgat tggctgagct cataagttcc tattccgaag ttccgcgaac
     1681 gcgtaaagga tctaggtgaa gatccttttt gataatctca tgaccaaaat cccttaacgt
     1741 gagttttcgt tccactgagc gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat
     1801 cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct accagcggtg
     1861 gtttgtttgc cggatcaaga gctaccaact ctttttccga aggtaactgg cttcagcaga
     1921 gcgcagatac caaatactgt ccttctagtg tagccgtagt taggccacca cttcaagaac
     1981 tctgtagcac cgcctacata cctcgctctg ctaatcctgt taccagtggc tgctgccagt
     2041 ggcgataagt cgtgtcttac cgggttggac tcaagacgat agttaccgga taaggcgcag
     2101 cggtcgggct gaacgggggg ttcgtgcaca cagcccagct tggagcgaac gacctacacc
     2161 gaactgagat acctacagcg tgagctatga gaaagcgcca cgcttcccga agggagaaag
     2221 gcggacaggt atccggtaag cggcagggtc ggaacaggag agcgcacgag ggagcttcca
     2281 gggggaaacg cctggtatct ttatagtcct gtcgggtttc gccacctctg acttgagcgt
     2341 cgatttttgt gatgctcgtc aggggggcgg agcctatgga aaaacgccag caacgcggcc
     2401 tttttacggt tcctggcctt ttgctggcct tttgctca
//
