LOCUS       sequence-452204-        2890 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Backbone plasmid for cloning sgRNA.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 2890)
  AUTHORS   Sharma A, Omer Aden R, Puhar A, Cisneros DA
  TITLE     CRISPR-Cas-Guided Mutagenesis of Chromosome and Virulence Plasmid in
            Shigella flexneri by Cytosine Base Editing.
  JOURNAL   mSystems. 2022 Dec 21:e0104522. doi: 10.1128/msystems.01045-22.
  PUBMED    36541764
REFERENCE   2  (bases 1 to 2890)
  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: "mSystems. 
            2022 Dec 21:e0104522. doi: 10.1128/msystems.01045-22."
COMMENT     Sequence Label: pgRNA_AT
FEATURES             Location/Qualifiers
     source          1..2890
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        13..47
                     /label=J23119(SpeI) promoter
                     /note="bacterial promoter (Registry of Standard Biological 
                     Parts BBa_J23119) modified to end with an SpeI site"
     CDS             395..700
                     /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     579..598
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     misc_RNA        708..783
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     terminator      799..885
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      977..1004
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     rep_origin      complement(1166..1754)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1246..1265)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(1925..2716,2717..2785))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     2548..2567
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2786..2890)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 cacattgaat tcttgacagc tagctcagtc ctaggtataa tactagtgag accacgcgtg
       61 gatccggctt actaaaagcc agataacagt atgcgtattt gcgcgctgat ttttgcggta
      121 taagaatata tactgatatg tatacccgaa gtatgtcaaa aagaggtatg ctatgaagca
      181 gcgtattaca gtgacagttg acagcgacag ctatcagttg ctcaaggcat atatgatgtc
      241 aatatctccg gtctggtaag cacaaccatg cagaatgaag cccgtcgtct gcgtgccgaa
      301 cgctggaaag cggaaaatca ggaagggatg gctgaggtcg cccggtttat tgaaatgaac
      361 ggctcttttg ctgacgagaa caggggctgg tgaaatgcag tttaaggttt acacctataa
      421 aagagagagc cgttatcgtc tgtttgtgga tgtacagagt gatattattg acacgcccgg
      481 gcgacggatg gtgatccccc tggccagtgc acgtctgctg tcagataaag tctcccgtga
      541 actttacccg gtggtgcata tcggggatga aagctggcgc atgatgacca ccgatatggc
      601 cagtgtgccg gtatccgtta tcggggaaga agtggctgat ctcagccacc gcgaaaatga
      661 catcaaaaac gccattaacc tgatgttctg gggaatataa ggtctccgtt ttagagctag
      721 aaatagcaag ttaaaataag gctagtccgt tatcaacttg aaaaagtggc accgagtcgg
      781 tgcttttttt ccaggcatca aataaaacga aaggctcagt cgaaagactg ggcctttcgt
      841 tttatctgtt gtttgtcggt gaacgctctc ctgagtagga caaatccgcc gggagcggat
      901 ttgaacgttg cgaagcaacg gcccggaggg tggcgggcag gacgcccgcc ataaactgcc
      961 aggcatcaaa ttaagcagaa ggccatcctg acggatggcc tttttgcgtt tctacaaact
     1021 cttttgttta tttttctaaa tacattcaaa tatgtatccg ctcatgaatt aattccgcta
     1081 gatgacgtgc ggcttcgacc tccaagcttt gagcaaaagg ccagcaaaag gccaggaacc
     1141 gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg cccccctgac gagcatcaca
     1201 aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg actataaaga taccaggcgt
     1261 ttccccctgg aagctccctc gtgcgctctc ctgttccgac cctgccgctt accggatacc
     1321 tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc
     1381 tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc
     1441 ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc caacccggta agacacgact
     1501 tatcgccact ggcagcagcc actggtaaca ggattagcag agcgaggtat gtaggcggtg
     1561 ctacagagtt cttgaagtgg tggcctaact acggctacac tagaagaaca gtatttggta
     1621 tctgcgctct gctgaagcca gttaccttcg gaaaaagagt tggtagctct tgatccggca
     1681 aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa
     1741 aaaaaggatc tcaagaagat cctttgatct tttctacggg gtctgacgct cagtggaacg
     1801 aaaactcacg ttaagggatt ttggtcatga gattatcaaa aaggatcttc acctagatcc
     1861 ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa acttggtctg
     1921 acagttacca atgcttaatc agtgaggcac ctatctcagc gatctgtcta tttcgttcat
     1981 ccatagttgc ctgactcccc gtcgtgtaga taactacgat acgggagggc ttaccatctg
     2041 gccccagtgc tgcaatgata ccgcgcgacc cacgctcacc ggctccagat ttatcagcaa
     2101 taaaccagcc agccggaagg gccgagcgca gaagtggtcc tgcaacttta tccgcctcca
     2161 tccagtctat taattgttgc cgggaagcta gagtaagtag ttcgccagtt aatagtttgc
     2221 gcaacgttgt tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt
     2281 cattcagctc cggttcccaa cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa
     2341 aagcggttag ctccttcggt cctccgatcg ttgtcagaag taagttggcc gcagtgttat
     2401 cactcatggt tatggcagca ctgcataatt ctcttactgt catgccatcc gtaagatgct
     2461 tttctgtgac tggtgagtac tcaaccaagt cattctgaga atagtgtatg cggcgaccga
     2521 gttgctcttg cccggcgtca atacgggata ataccgcgcc acatagcaga actttaaaag
     2581 tgctcatcat tggaaaacgt tcttcggggc gaaaactctc aaggatctta ccgctgttga
     2641 gatccagttc gatgtaaccc actcgtgcac ccaactgatc ttcagcatct tttactttca
     2701 ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg
     2761 cgacacggaa atgttgaata ctcatactct tcctttttca atattattga agcatttatc
     2821 agggttattg tctcatgagc ggatacatat ttgaatgtat ttagaaaaat aaacaaatag
     2881 gggttccgcg
//
