LOCUS       sequence-329519-        3134 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  CRISPR-Cas9-assisted genome editing in Escherichia coli.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3134)
  AUTHORS   Li Q, Sun B, Chen J, Zhang Y, Jiang Y, Yang S
  TITLE     A modified pCas/pTargetF system for CRISPR-Cas9-assisted genome 
            editing in Escherichia coli.
  JOURNAL   Acta Biochim Biophys Sin (Shanghai). 2021 Apr 15;53(5):620-627. doi:
            10.1093/abbs/gmab036.
  PUBMED    33764372
REFERENCE   2  (bases 1 to 3134)
  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"; doi: "10"; journalName: 
            "Acta Biochim Biophys Sin (Shanghai)"; date: "2021-04-15- 15"; 
            volume: "53"; issue: "5"; pages: "620-627"
COMMENT     Sequence Label: pEcgRNA
FEATURES             Location/Qualifiers
     source          1..3134
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     45..62
                     /label=L4440
                     /note="L4440 vector, forward primer"
     promoter        139..173
                     /label=J23119(SpeI) promoter
                     /note="bacterial promoter (Registry of Standard Biological 
                     Parts BBa_J23119) modified to end with an SpeI site"
     CDS             complement(560..865)
                     /codon_start=1
                     /gene="ccdB"
                     /product="CcdB, a bacterial toxin that poisons DNA gyrase"
                     /label=ccdB
                     /note="Plasmids containing the ccdB gene cannot be 
                     propagated in standard E. coli strains."
                     /translation="MQFKVYTYKRESRYRLFVDVQSDIIDTPGRRMVIPLASARLLSDK
                     VSRELYPVVHIGDESWRMMTTDMASVPVSVIGEEVADLSHRENDIKNAINLMFWGI"
     primer_bind     complement(662..681)
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     misc_RNA        1211..1286
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     CDS             1472..2263
                     /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      2437..3025
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2926..2945
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 ttgagtgagc tgataccgct cgccgcagcc gaacgaccga gcgcagcgag tcagtgagcg
       61 aggaagcgga agagcgcctg atgcggtatt ttctccttac gcatctgtgc ggtatttcac
      121 accgcatatg ctggatcctt gacagctagc tcagtcctag gtataatact agttgagacc
      181 cagcagtgaa agataaatga tctgagaccc aggtcgaccg acagccttcc aaatgttctt
      241 ctcaaacgga atcgtcgtat ccagcctact cgctattgtc ctcaatgccg tattaaatca
      301 taaaaagaaa taagaaaaag aggtgcgagc ctcttttttg tgtgacaaaa taaaaacatc
      361 tacctattca tatacgctag tgtcatagtc ctgaaaatca tctgcatcaa gaacaatttc
      421 acaactctta tacttttctc ttacaagtcg ttcggcttca tctggatttt cagcctctat
      481 acttactaaa cgtgataaag tttctgtaat ttctactgta tcgacctgca gactggctgt
      541 gtataaggga gcctgacatt tatattcccc agaacatcag gttaatggcg tttttgatgt
      601 cattttcgcg gtggctgaga tcagccactt cttccccgat aacggagacc ggcacactgg
      661 ccatatcggt ggtcatcatg cgccagcttt catccccgat atgcaccacc gggtaaagtt
      721 cacgggagac tttatctgac agcagacgtg cactggccag ggggatcacc atccgtcgcc
      781 cgggcgtgtc aataatatca ctctgtacat ccacaaacag acgataacgg ctctctcttt
      841 tataggtgta aaccttaaac tgcatttcac cagcccctgt tctcgtcagc aaaagagccg
      901 ttcatttcaa taaaccgggc gacctcagcc atcccttcct gattttccgc tttccagcgt
      961 tcggcacgca gacgacgggc ttcattctgc atggttgtgc ttaccagacc ggagatattg
     1021 acatcatata tgccttgagc aactgatagc tgtcgctgtc aactgtcact gtaatacgct
     1081 gcttcatagc atacctcttt ttgacatact tcgggtatac atatcagtat atattcttat
     1141 accgcaaaaa tcagcgcgca aatacgcata ctgttatctg gcttttagta agccggatcc
     1201 ctgggtctcg gttttagagc tagaaatagc aagttaaaat aaggctagtc cgttatcaac
     1261 ttgaaaaagt ggcaccgagt cggtgctttt tttgaattct ctagagtcga cctgcagaag
     1321 cttagatcta ttaccctgtt atccctactc gagttcatgt gcagctccat aagcaaaagg
     1381 ggatgataag tttatcacca ccgactattt gcaacagtgc cgttgatcgt gctatgatcg
     1441 actgatgtca tcagcggtgg agtgcaatgt catgagggaa gcggtgatcg ccgaagtatc
     1501 gactcaacta tcagaggtag ttggcgtcat cgagcgccat ctcgaaccga cgttgctggc
     1561 cgtacatttg tacggctccg cagtggatgg cggcctgaag ccacacagtg atattgattt
     1621 gctggttacg gtgaccgtaa ggcttgatga aacaacgcgg cgagctttga tcaacgacct
     1681 tttggaaact tcggcttccc ctggagagag cgagattctc cgcgctgtag aagtcaccat
     1741 tgttgtgcac gacgacatca ttccgtggcg ttatccagct aagcgcgaac tgcaatttgg
     1801 agaatggcag cgcaatgaca ttcttgcagg tatcttcgag ccagccacga tcgacattga
     1861 tctggctatc ttgctgacaa aagcaagaga acatagcgtt gccttggtag gtccagcggc
     1921 ggaggaactc tttgatccgg ttcctgaaca ggatctattt gaggcgctaa atgaaacctt
     1981 aacgctatgg aactcgccgc ccgactgggc tggcgatgag cgaaatgtag tgcttacgtt
     2041 gtcccgcatt tggtacagcg cagtaaccgg caaaatcgcg ccgaaggatg tcgctgccga
     2101 ctgggcaatg gagcgcctgc cggcccagta tcagcccgtc atacttgaag ctagacaggc
     2161 ttatcttgga caagaagaag atcgcttggc ctcgcgcgca gatcagttgg aagaatttgt
     2221 ccactacgtg aaaggcgaga tcaccaaggt agtcggcaaa taagatgccg ctcgccagtc
     2281 gattggctga gctcataagt tcctattccg aagttccgcg aacgcgtaaa ggatctaggt
     2341 gaagatcctt tttgataatc tcatgaccaa aatcccttaa cgtgagtttt cgttccactg
     2401 agcgtcagac cccgtagaaa agatcaaagg atcttcttga gatccttttt ttctgcgcgt
     2461 aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt tgccggatca
     2521 agagctacca actctttttc cgaaggtaac tggcttcagc agagcgcaga taccaaatac
     2581 tgtccttcta gtgtagccgt agttaggcca ccacttcaag aactctgtag caccgcctac
     2641 atacctcgct ctgctaatcc tgttaccagt ggctgctgcc agtggcgata agtcgtgtct
     2701 taccgggttg gactcaagac gatagttacc ggataaggcg cagcggtcgg gctgaacggg
     2761 gggttcgtgc acacagccca gcttggagcg aacgacctac accgaactga gatacctaca
     2821 gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca ggtatccggt
     2881 aagcggcagg gtcggaacag gagagcgcac gagggagctt ccagggggaa acgcctggta
     2941 tctttatagt cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc
     3001 gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc
     3061 cttttgctgg ccttttgctc acatgttctt tcctgcgtta tcccctgatt ctgtggataa
     3121 ccgtattacc gcct
//
