LOCUS       sequence-60718-e        2433 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  A crRNA expression plasmid specific to the rpsL allele..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 2433)
  AUTHORS   Jiang W, Bikard D, Cox D, Zhang F, Marraffini LA
  TITLE     RNA-guided editing of bacterial genomes using CRISPR-Cas systems.
  JOURNAL   Nat Biotechnol. 2013 Jan 29. doi: 10.1038/nbt.2508.
  PUBMED    23360965
REFERENCE   2  (bases 1 to 2433)
  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: "Nat 
            Biotechnol. 2013 Jan 29. doi: 10.1038/nbt.2508."
COMMENT     Sequence Label: pCRISPR::rpsL
FEATURES             Location/Qualifiers
     source          1..2433
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(15..33)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     CDS             212..1006
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(266..285)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     876..895
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     terminator      1037..1131
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     rep_origin      1219..1807
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1708..1727
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     terminator      complement(1972..2058)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     misc_feature    2088..2219
                     /label=crRNA leader
                     /note="crRNA leader sequence for the Streptococcus pyogenes
                     CRISPR/Cas system"
     repeat_region   2220..2255
                     /label=DR
                     /note="direct repeat for the Streptococcus pyogenes 
                     CRISPR/Cas system"
     repeat_region   2286..2321
                     /label=DR
                     /note="direct repeat for the Streptococcus pyogenes 
                     CRISPR/Cas system"
     promoter        complement(join(2354..2379,2380..2385,2386..2402,2403..2408,
                     2409..2427))
                     /label=PLtetO-1 promoter
                     /note="modified phage lambda PL promoter with tet operator 
                     sites (Lutz and Bujard, 1997)"
     protein_bind    2384..2402
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    2409..2427
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
ORIGIN
        1 gtgaagacga aagggcctcg tgatacgcct atttttatag gttaatgtca tgataataat
       61 ggtttcttag acgtcggaat tgccagctgg ggcgccctct ggtaaggttg ggaagccctg
      121 caaagtaaac tggatggctt tcttgccgcc aaggatctga tggcgcaggg gatcaagatc
      181 tgatcaagag acaggatgag gatcgtttcg catgattgaa caagatggat tgcacgcagg
      241 ttctccggcc gcttgggtgg agaggctatt cggctatgac tgggcacaac agacaatcgg
      301 ctgctctgat gccgccgtgt tccggctgtc agcgcagggg cgcccggttc tttttgtcaa
      361 gaccgacctg tccggtgccc tgaatgaact gcaggacgag gcagcgcggc tatcgtggct
      421 ggccacgacg ggcgttcctt gcgcagctgt gctcgacgtt gtcactgaag cgggaaggga
      481 ctggctgcta ttgggcgaag tgccggggca ggatctcctg tcatctcacc ttgctcctgc
      541 cgagaaagta tccatcatgg ctgatgcaat gcggcggctg catacgcttg atccggctac
      601 ctgcccattc gaccaccaag cgaaacatcg catcgagcga gcacgtactc ggatggaagc
      661 cggtcttgtc gatcaggatg atctggacga agagcatcag gggctcgcgc cagccgaact
      721 gttcgccagg ctcaaggcgc gcatgcccga cggcgaggat ctcgtcgtga cccatggcga
      781 tgcctgcttg ccgaatatca tggtggaaaa tggccgcttt tctggattca tcgactgtgg
      841 ccggctgggt gtggcggacc gctatcagga catagcgttg gctacccgtg atattgctga
      901 agagcttggc ggcgaatggg ctgaccgctt cctcgtgctt tacggtatcg ccgctcccga
      961 ttcgcagcgc atcgccttct atcgccttct tgacgagttc ttctgagcgg gactctgggg
     1021 ttcgagagct cgcttggact cctgttgata gatccagtaa tgacctcaga actccatctg
     1081 gatttgttca gaacgctcgg ttgccgccgg gcgtttttta ttggtgagaa tccaagcact
     1141 agtcatgacc aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag accccgtaga
     1201 aaagatcaaa ggatcttctt gagatccttt ttttctgcgc gtaatctgct gcttgcaaac
     1261 aaaaaaacca ccgctaccag cggtggtttg tttgccggat caagagctac caactctttt
     1321 tccgaaggta actggcttca gcagagcgca gataccaaat actgtccttc tagtgtagcc
     1381 gtagttaggc caccacttca agaactctgt agcaccgcct acatacctcg ctctgctaat
     1441 cctgttacca gtggctgctg ccagtggcga taagtcgtgt cttaccgggt tggactcaag
     1501 acgatagtta ccggataagg cgcagcggtc gggctgaacg gggggttcgt gcacacagcc
     1561 cagcttggag cgaacgacct acaccgaact gagataccta cagcgtgagc attgagaaag
     1621 cgccacgctt cccgaaggga gaaaggcgga caggtatccg gtaagcggca gggtcggaac
     1681 aggagagcgc acgagggagc ttccaggggg aaacgcctgg tatctttata gtcctgtcgg
     1741 gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct
     1801 atggaaaaac gccagcaacg cggccttttt acggttcctg gccttttgct ggccttttgc
     1861 tcacatgttc tttcctgcgt tatcccctga ttctgtggat aaccgtatta ccgcctttga
     1921 gtgagctgat accgctcgcc gcagccgaac gccctaggtc tagggcggcg gatttgtcct
     1981 actcaggaga gcgttcaccg acaaacaaca gataaaacga aaggcccagt ctttcgactg
     2041 agcctttcgt tttatttgat gcctctagca cgcgtaccat gggatcctat ttcttaataa
     2101 ctaaaaatat ggtataatac tcttaataaa tgcagtaata caggggcttt tcaagactga
     2161 agtctagctg agacaaatag tgcgattacg aaatttttta gacaaaaata gtctacgagg
     2221 ttttagagct atgctgtttt gaatggtccc aaaactactt tacgcagcgc ggagttcggt
     2281 tttttgtttt agagctatgc tgttttgaat ggtcccaaaa cttcagcaca ctgagacttg
     2341 ttgagttgaa ttcggtcagt gcgtcctgct gatgtgctca gtatctctat cactgatagg
     2401 gatgtcaatc tctatcactg atagggactc gag
//
