LOCUS       sequence-197176-        2936 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Repress mCherry and GFP independently, with different strengths.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 2936)
  AUTHORS   Vigouroux A, Oldewurtel E, Cui L, Bikard D, van Teeffelen S
  TITLE     Tuning dCas9's ability to block transcription enables robust, 
            noiseless knockdown of bacterial genes.
  JOURNAL   Mol Syst Biol. 2018 Mar 8;14(3):e7899.
  PUBMED    29519933
REFERENCE   2  (bases 1 to 2936)
  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: "Mol Syst 
            Biol."; date: "2018-03-8"; volume: "14(3)"; pages: "e7899"
COMMENT     Sequence Label: pCRRNA-G14-R11
FEATURES             Location/Qualifiers
     source          1..2936
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     terminator      complement(260..346)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     misc_feature    376..507
                     /label=crRNA leader
                     /note="crRNA leader sequence for the Streptococcus pyogenes
                     CRISPR/Cas system"
     repeat_region   508..543
                     /label=DR
                     /note="direct repeat for the Streptococcus pyogenes 
                     CRISPR/Cas system"
     repeat_region   574..609
                     /label=DR
                     /note="direct repeat for the Streptococcus pyogenes 
                     CRISPR/Cas system"
     repeat_region   640..675
                     /label=DR
                     /note="direct repeat for the Streptococcus pyogenes 
                     CRISPR/Cas system"
     misc_RNA        796..874
                     /label=tracrRNA
                     /note="trans-activating CRISPR RNA for the Streptococcus 
                     pyogenes CRISPR/Cas9 system"
     primer_bind     complement(1019..1037)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     CDS             1216..2010
                     /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(1270..1289)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     1880..1899
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     terminator      2041..2135
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     rep_origin      2223..2811
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2712..2731
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 cgctcgccgc agccgaacgc cccaaaaagc ctcgctttca gcacctgtcg tttcctttct
       61 tttcagaggg tattttaaat aaaaacatta agttatgacg aagaagaacg gaaacgcctt
      121 aaaccggaaa attttcataa atagcgaaaa cccgcgaggt cgccgccccg taacctgtcg
      181 gatcaccgga aaggacccgt aaagtgataa tgattatcat ctacatatca caacgtgcgt
      241 ggagggtcta gggcggcgga tttgtcctac tcaggagagc gttcaccgac aaacaacaga
      301 taaaacgaaa ggcccagtct ttcgactgag cctttcgttt tatttgatgc ctctagcacg
      361 cgtaccatgg gatcctattt cttaataact aaaaatatgg tataatactc ttaataaatg
      421 cagtaataca ggggcttttc aagactgaag tctagctgag acaaatagtg cgattacgaa
      481 attttttaga caaaaatagt ctacgaggtt ttagagctat gctgttttga atggtcccaa
      541 aacccttcac cttgtggtgg aacagagaat ttggttttag agctatgctg ttttgaatgg
      601 tcccaaaaca gtttggcgga gacccacgct tcatacggag ttttagagct atgctgtttt
      661 gaatggtccc aaaacttcag cacactgaga cttgttgagt tgaattcatg agtattaagt
      721 attgttttat ggctgataaa tttctttgaa tttctccttg attatttgtt ataaaagtta
      781 taaaataatc ttgttggaac cattcaaaac agcatagcaa gttaaaataa ggctagtccg
      841 ttatcaactt gaaaaagtgg caccgagtcg gtgctttttt tgatacttct attctactct
      901 gactgcaaac caaaaaaaca agcgctttca aaacgcttgt tttatcattt ttagggaaat
      961 taatctctta atccttttat cattctacat ttaggcgctg ccatcttaag acgaaagggc
     1021 ctcgtgatac gcctattttt ataggttaat gtcatgataa taatggtttc ttagacgtcg
     1081 gaattgccag ctggggcgcc ctctggtaag gttgggaagc cctgcaaagt aaactggatg
     1141 gctttcttgc cgccaaggat ctgatggcgc aggggatcaa gatctgatca agagacagga
     1201 tgaggatcgt ttcgcatgat tgaacaagat ggattgcacg caggttctcc ggccgcttgg
     1261 gtggagaggc tattcggcta tgactgggca caacagacaa tcggctgctc tgatgccgcc
     1321 gtgttccggc tgtcagcgca ggggcgcccg gttctttttg tcaagaccga cctgtccggt
     1381 gccctgaatg aactgcagga cgaggcagcg cggctatcgt ggctggccac gacgggcgtt
     1441 ccttgcgcag ctgtgctcga cgttgtcact gaagcgggaa gggactggct gctattgggc
     1501 gaagtgccgg ggcaggatct cctgtcatct caccttgctc ctgccgagaa agtatccatc
     1561 atggctgatg caatgcggcg gctgcatacg cttgatccgg ctacctgccc attcgaccac
     1621 caagcgaaac atcgcatcga gcgagcacgt actcggatgg aagccggtct tgtcgatcag
     1681 gatgatctgg acgaagagca tcaggggctc gcgccagccg aactgttcgc caggctcaag
     1741 gcgcgcatgc ccgacggcga ggatctcgtc gtgacccatg gcgatgcctg cttgccgaat
     1801 atcatggtgg aaaatggccg cttttctgga ttcatcgact gtggccggct gggtgtggcg
     1861 gaccgctatc aggacatagc gttggctacc cgtgatattg ctgaagagct tggcggcgaa
     1921 tgggctgacc gcttcctcgt gctttacggt atcgccgctc ccgattcgca gcgcatcgcc
     1981 ttctatcgcc ttcttgacga gttcttctga gcgggactct ggggttcgag agctcgcttg
     2041 gactcctgtt gatagatcca gtaatgacct cagaactcca tctggatttg ttcagaacgc
     2101 tcggttgccg ccgggcgttt tttattggtg agaatccaag cactagtcat gaccaaaatc
     2161 ccttaacgtg agttttcgtt ccactgagcg tcagaccccg tagaaaagat caaaggatct
     2221 tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta
     2281 ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc tttttccgaa ggtaactggc
     2341 ttcagcagag cgcagatacc aaatactgtc cttctagtgt agccgtagtt aggccaccac
     2401 ttcaagaact ctgtagcacc gcctacatac ctcgctctgc taatcctgtt accagtggct
     2461 gctgccagtg gcgataagtc gtgtcttacc gggttggact caagacgata gttaccggat
     2521 aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac agcccagctt ggagcgaacg
     2581 acctacaccg aactgagata cctacagcgt gagctatgag aaagcgccac gcttcccgaa
     2641 gggagaaagg cggacaggta tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg
     2701 gagcttccag ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg ccacctctga
     2761 cttgagcgtc gatttttgtg atgctcgtca ggggggcgga gcctatggaa aaacgccagc
     2821 aacgcggcct ttttacggtt cctggccttt tgctggcctt ttgctcacat gttctttcct
     2881 gcgttatccc ctgattctgt ggataaccgt attaccgcct ttgagtgagc tgatac
//
