LOCUS       sequence-227924-        2117 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Constitutive expression of sgRNA without donor editing template DNA.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 2117)
  AUTHORS   Jiang Y, Chen B, Duan C, Sun B, Yang J, Yang S
  TITLE     Multigene editing in the Escherichia coli genome using the 
            CRISPR-Cas9 system.
  JOURNAL   Appl Environ Microbiol. 2015 Jan 30. pii: AEM.04023-14.
  PUBMED    25636838
REFERENCE   2  (bases 1 to 2117)
  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: "Appl 
            Environ Microbiol. 2015 Jan 30. pii: AEM.04023-14."
COMMENT     Sequence Label: pTargetF
FEATURES             Location/Qualifiers
     source          1..2117
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     98..115
                     /label=L4440
                     /note="L4440 vector, forward primer"
     promoter        192..226
                     /label=J23119(SpeI) promoter
                     /note="bacterial promoter (Registry of Standard Biological 
                     Parts BBa_J23119) modified to end with an SpeI site"
     misc_RNA        247..322
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     CDS             508..1299
                     /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      1473..2061
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1962..1981
                     /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 atgctggatc cttgacagct agctcagtcc taggtataat actagtcatc gccgcagcgg
      241 tttcaggttt tagagctaga aatagcaagt taaaataagg ctagtccgtt atcaacttga
      301 aaaagtggca ccgagtcggt gctttttttg aattctctag agtcgacctg cagaagctta
      361 gatctattac cctgttatcc ctactcgagt tcatgtgcag ctccataagc aaaaggggat
      421 gataagttta tcaccaccga ctatttgcaa cagtgccgtt gatcgtgcta tgatcgactg
      481 atgtcatcag cggtggagtg caatgtcatg agggaagcgg tgatcgccga agtatcgact
      541 caactatcag aggtagttgg cgtcatcgag cgccatctcg aaccgacgtt gctggccgta
      601 catttgtacg gctccgcagt ggatggcggc ctgaagccac acagtgatat tgatttgctg
      661 gttacggtga ccgtaaggct tgatgaaaca acgcggcgag ctttgatcaa cgaccttttg
      721 gaaacttcgg cttcccctgg agagagcgag attctccgcg ctgtagaagt caccattgtt
      781 gtgcacgacg acatcattcc gtggcgttat ccagctaagc gcgaactgca atttggagaa
      841 tggcagcgca atgacattct tgcaggtatc ttcgagccag ccacgatcga cattgatctg
      901 gctatcttgc tgacaaaagc aagagaacat agcgttgcct tggtaggtcc agcggcggag
      961 gaactctttg atccggttcc tgaacaggat ctatttgagg cgctaaatga aaccttaacg
     1021 ctatggaact cgccgcccga ctgggctggc gatgagcgaa atgtagtgct tacgttgtcc
     1081 cgcatttggt acagcgcagt aaccggcaaa atcgcgccga aggatgtcgc tgccgactgg
     1141 gcaatggagc gcctgccggc ccagtatcag cccgtcatac ttgaagctag acaggcttat
     1201 cttggacaag aagaagatcg cttggcctcg cgcgcagatc agttggaaga atttgtccac
     1261 tacgtgaaag gcgagatcac caaggtagtc ggcaaataag atgccgctcg ccagtcgatt
     1321 ggctgagctc ataagttcct attccgaagt tccgcgaacg cgtaaaggat ctaggtgaag
     1381 atcctttttg ataatctcat gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg
     1441 tcagaccccg tagaaaagat caaaggatct tcttgagatc ctttttttct gcgcgtaatc
     1501 tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc ggatcaagag
     1561 ctaccaactc tttttccgaa ggtaactggc ttcagcagag cgcagatacc aaatactgtc
     1621 cttctagtgt agccgtagtt aggccaccac ttcaagaact ctgtagcacc gcctacatac
     1681 ctcgctctgc taatcctgtt accagtggct gctgccagtg gcgataagtc gtgtcttacc
     1741 gggttggact caagacgata gttaccggat aaggcgcagc ggtcgggctg aacggggggt
     1801 tcgtgcacac agcccagctt ggagcgaacg acctacaccg aactgagata cctacagcgt
     1861 gagctatgag aaagcgccac gcttcccgaa gggagaaagg cggacaggta tccggtaagc
     1921 ggcagggtcg gaacaggaga gcgcacgagg gagcttccag ggggaaacgc ctggtatctt
     1981 tatagtcctg tcgggtttcg ccacctctga cttgagcgtc gatttttgtg atgctcgtca
     2041 ggggggcgga gcctatggaa aaacgccagc aacgcggcct ttttacggtt cctggccttt
     2101 tgctggcctt ttgctca
//
