LOCUS       sequence-297225-        3182 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  sgRNA for ebpA gene (NT) under nisin-inducible nisA promoter, 
            barcoded.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3182)
  AUTHORS   Afonina I, Ong J, Chua J, Lu T, Kline KA
  TITLE     Multiplex CRISPRi System Enables the Study of Stage-Specific Biofilm
            Genetic Requirements in Enterococcus faecalis.
  JOURNAL   mBio. 2020 Oct 20;11(5):e01101-20. doi: 10.1128/mBio.01101-20.
  PUBMED    33082254
REFERENCE   2  (bases 1 to 3182)
  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: "10.1128/mBio"; 
            journalName: "mBio"; date: "2020-10-20- 20"; volume: "11"; issue: 
            "5"; pages: "e01101-20"
COMMENT     Sequence Label: pGCP123-EbpA_g1
FEATURES             Location/Qualifiers
     source          1..3182
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_RNA        210..285
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     primer_bind     complement(371..391)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(371..389)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(410..426)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(410..426)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             complement(623..634)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     CDS             complement(2275..3069)
                     /codon_start=1
                     /gene="aphA-3"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin"
                     /translation="MAKMRISPELKKLIEKYRCVKDTEGMSPAKVYKLVGENENLYLKM
                     TDSRYKGTTYDVEREKDMMLWLEGKLPVPKVLHFERHDGWSNLLMSEADGVLCSEEYED
                     EQSPEKIIELYAECIRLFHSIDISDCPYTNSLDSRLAELDYLLNNDLADVDCENWEEDT
                     PFKDPRELYDFLKTEKPEEELVFSHGDLGDSNIFVKDGKVSGFIDLGRSGRADKWYDIA
                     FCVRSIREDIGEEQYVELFFDLLGIKPDWEKIKYYILLDELF"
ORIGIN
        1 aacagctttt tcaaagttgt tttcaaagtt ggcgtataac atagtatcga cggaagatcc
       61 tgcagagatc tatctaaaac agtcttaatt ctatcttgag aaagtattgg taataatatt
      121 attgtcgata acgcgagcat aataaacggc tctgattaaa ttctgaagtt tgttagatac
      181 aatgatttca cgccaggtgc ttttcccgag ttttagagct agaaatagca agttaaaata
      241 aggctagtcc gttatcaact tgaaaaagtg gcaccgagtc ggtgcttttt ttggatccac
      301 tagtggtacc gaattcacat tccgcaattg gcatgcggcc gccaccgcgg tggagctcca
      361 gcttttgttc cctttagtga gggttaattg cgcgcttggc gtaatcatgg tcatagctgt
      421 ttcctggcat gctcgatttt ttattaaaac gtctcaaaat cgtttctgag acgttttagc
      481 gtttatttcg tttagttatc ggcataatcg ttaaaacagg cgttatcgta gcgtaaaagc
      541 ccttgagcgt agcgtggctt tgcagcgaag atgttgtctg ttagattatg aaagccgatg
      601 actgaatgaa ataataagcg cagcgtcctt ctatttcggt tggaggaggc tcaagggagt
      661 ttgagggaat gaaattccct catgggtttg attttaaaaa ttgcttgcaa ttttgccgag
      721 cggtagcgct ggaaaatttt tgaaaaaaat ttggaatttg gaaaaaaatg gggggaaagg
      781 aagcgaattt tgcttccgta ctacgacccc ccattaagtg ccgagtgcca atttttgtgc
      841 caaaaacgct ctatcccaac tggctcaagg gtttgagggg tttttcaatc gccaacgaat
      901 cgccaacgtt ttcgccaacg ttttttataa atctatattt aagtagcttt attgttgttt
      961 ttatgattac aaagtgatac actaatttta taaaattatt tgattggagt tttttaaatg
     1021 gtgatttcag aatcgaaaaa aagagttatg atttctctga caaaagagca agataaaaaa
     1081 ttaacagata tggcgaaaca aaaaggtttt tcaaaatctg cggttgcggc gttagctata
     1141 gaagaatatg caagaaagga atcagaacaa aaaaaataag cgaaagctcg cgtttttaga
     1201 aggatacgag ttttcgctac ttgtttttga taaggtaatt atatcatggc tattaaaaat
     1261 actaaagcta gaaattttgg atttttatta tatcctgact caattcctaa tgattggaaa
     1321 gaaaaattag agagtttggg cgtatctatg gctgtcagtc ctttacacga tatggacgaa
     1381 aaaaaagata aagatacatg gaatagtagt gatgttatac gaaatggaaa gcactataaa
     1441 aaaccacact atcacgttat atatattgca cgaaatcctg taacaataga aagcgttagg
     1501 aacaagatta agcgaaaatt ggggaatagt tcagttgctc atgttgagat acttgattat
     1561 atcaaaggtt catatgaata tttgactcat gaatcaaagg acgctattgc taagaataaa
     1621 catatatacg acaaaaaaga tattttgaac attaatgatt ttgatattga ccgctatata
     1681 acacttgatg aaagccaaaa aagaaaattg aagaatttac ttttagatat agtggatgac
     1741 tataatttgg taaatacaaa agatttaatg gcttttattc gccttagggg agcggagttt
     1801 ggaattttaa atacgaatga tgtaaaagat attgtttcaa caaactctag cgcctttaga
     1861 ttatggtttg agggcaatta tcagtgtgga tatagagcaa gttatgcaaa ggttcttgat
     1921 gctgaaacgg gggaaataaa atgacaaaca aagaaaaaga gttatttgct gaaaatgagg
     1981 aattaaaaaa agaaattaag gacttaaaag agcgtattga aagatacaga gaaatggaag
     2041 ttgaattaag tacaacaata gatttattga gaggagggat tattgaataa ataaaagccc
     2101 ccctgacgaa agtcgacgat acaaattcct cgtaggcgct cgggacccct atctagcgaa
     2161 cttttagaaa agatataaaa catcagagta tggacagttg cggatgtact tcagaaaaga
     2221 ttagatgtct aaaaagcttg tagttaaagc tttttagaca tctaaatcta ggtactaaaa
     2281 caattcatcc agtaaaatat aatattttat tttctcccaa tcaggcttga tccccagtaa
     2341 gtcaaaaaat agctcgacat actgttcttc cccgatatcc tccctgatcg accggacgca
     2401 gaaggcaatg tcataccact tgtccgccct gccgcttctc ccaagatcaa taaagccact
     2461 tactttgcca tctttcacaa agatgttgct gtctcccagg tcgccgtggg aaaagacaag
     2521 ttcctcttcg ggcttttccg tctttaaaaa atcatacagc tcgcgcggat ctttaaatgg
     2581 agtgtcttct tcccagtttt cgcaatccac atcggccaga tcgttattca gtaagtaatc
     2641 caattcggct aagcggctgt ctaagctatt cgtataggga caatccgata tgtcgatgga
     2701 gtgaaagagc ctgatgcact ccgcatacag ctcgataatc ttttcagggc tttgttcatc
     2761 ttcatactct tccgagcaaa ggacgccatc ggcctcactc atgagcagat tgctccagcc
     2821 atcatgccgt tcaaagtgca ggacctttgg aacaggcagc tttccttcca gccatagcat
     2881 catgtccttt tcccgttcca catcataggt ggtcccttta taccggctgt ccgtcatttt
     2941 taaatatagg ttttcatttt ctcccaccag cttatatacc ttagcaggag acattccttc
     3001 cgtatctttt acgcagcggt atttttcgat cagttttttc aattccggtg atattctcat
     3061 tttagccatt tattatttcc ttcctctttt ctacagtatt taaagatacc ccaagaagct
     3121 aattataaca agacgaactc caattcactg ttccttgcat tctaaaacct taaataccag
     3181 aa
//
