LOCUS       Exported                3134 bp ds-DNA     circular SYN 13-MAY-2021
DEFINITION  CRISPR-Cas9-assisted genome editing in Escherichia coli.
ACCESSION   .
VERSION     .
KEYWORDS    pEcgRNA
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3134)
  AUTHORS   Li , Q i, Sun, Bingbing, Chen, Jun, Zhang, Yiwen, Jiang, Y u Jiang, 
            Yang, Sheng
  TITLE     A modified pCas/pTargetF system for CRISPR-Cas9-assisted genome 
            editing in Escherichia coli
  JOURNAL   Acta Biochimica et Biophysica Sinica, 2021
REFERENCE   2  (bases 1 to 3134)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Acta 
            Biochimica et Biophysica Sinica, 2021"
FEATURES             Location/Qualifiers
     source          1..3134
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     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"