LOCUS       sequence-192581-        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-G0-R20
FEATURES             Location/Qualifiers
     source          1..2936
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_RNA        complement(134..212)
                     /label=tracrRNA
                     /note="trans-activating CRISPR RNA for the Streptococcus 
                     pyogenes CRISPR/Cas9 system"
     repeat_region   333..368
                     /label=DR
                     /note="direct repeat for the Streptococcus pyogenes 
                     CRISPR/Cas system"
     repeat_region   399..434
                     /label=DR
                     /note="direct repeat for the Streptococcus pyogenes 
                     CRISPR/Cas system"
     repeat_region   465..500
                     /label=DR
                     /note="direct repeat for the Streptococcus pyogenes 
                     CRISPR/Cas system"
     misc_feature    complement(501..632)
                     /label=crRNA leader
                     /note="crRNA leader sequence for the Streptococcus pyogenes
                     CRISPR/Cas system"
     terminator      662..748
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     rep_origin      complement(1133..1721)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1213..1232)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     terminator      complement(1809..1903)
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     CDS             complement(1934..2728)
                     /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(2045..2064)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     2655..2674
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     2907..2925
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
ORIGIN
        1 aagatggcag cgcctaaatg tagaatgata aaaggattaa gagattaatt tccctaaaaa
       61 tgataaaaca agcgttttga aagcgcttgt ttttttggtt tgcagtcaga gtagaataga
      121 agtatcaaaa aaagcaccga ctcggtgcca ctttttcaag ttgataacgg actagcctta
      181 ttttaacttg ctatgctgtt ttgaatggtt ccaacaagat tattttataa cttttataac
      241 aaataatcaa ggagaaattc aaagaaattt atcagccata aaacaatact taatactcat
      301 gaattcaact caacaagtct cagtgtgctg aagttttggg accattcaaa acagcatagc
      361 tctaaaactc cgtatgaagg cacccagacc gccaaactgt tttgggacca ttcaaaacag
      421 catagctcta aaacactctt aatgtcgcga ccaggatgtg cgatgttttg ggaccattca
      481 aaacagcata gctctaaaac ctcgtagact atttttgtct aaaaaatttc gtaatcgcac
      541 tatttgtctc agctagactt cagtcttgaa aagcccctgt attactgcat ttattaagag
      601 tattatacca tatttttagt tattaagaaa taggatccca tggtacgcgt gctagaggca
      661 tcaaataaaa cgaaaggctc agtcgaaaga ctgggccttt cgttttatct gttgtttgtc
      721 ggtgaacgct ctcctgagta ggacaaatcc gccgccctag accctccacg cacgttgtga
      781 tatgtagatg ataatcatta tcactttacg ggtcctttcc ggtgatccga caggttacgg
      841 ggcggcgacc tcgcgggttt tcgctattta tgaaaatttt ccggtttaag gcgtttccgt
      901 tcttcttcgt cataacttaa tgtttttatt taaaataccc tctgaaaaga aaggaaacga
      961 caggtgctga aagcgaggct ttttggggcg ttcggctgcg gcgagcggta tcagctcact
     1021 caaaggcggt aatacggtta tccacagaat caggggataa cgcaggaaag aacatgtgag
     1081 caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg tttttccata
     1141 ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg tggcgaaacc
     1201 cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg cgctctcctg
     1261 ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga agcgtggcgc
     1321 tttctcaatg ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc tccaagctgg
     1381 gctgtgtgca cgaacccccc gttcagcccg accgctgcgc cttatccggt aactatcgtc
     1441 ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact ggtaacagga
     1501 ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg cctaactacg
     1561 gctacactag aaggacagta tttggtatct gcgctctgct gaagccagtt accttcggaa
     1621 aaagagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt ggtttttttg
     1681 tttgcaagca gcagattacg cgcagaaaaa aaggatctca agaagatcct ttgatctttt
     1741 ctacggggtc tgacgctcag tggaacgaaa actcacgtta agggattttg gtcatgacta
     1801 gtgcttggat tctcaccaat aaaaaacgcc cggcggcaac cgagcgttct gaacaaatcc
     1861 agatggagtt ctgaggtcat tactggatct atcaacagga gtccaagcga gctctcgaac
     1921 cccagagtcc cgctcagaag aactcgtcaa gaaggcgata gaaggcgatg cgctgcgaat
     1981 cgggagcggc gataccgtaa agcacgagga agcggtcagc ccattcgccg ccaagctctt
     2041 cagcaatatc acgggtagcc aacgctatgt cctgatagcg gtccgccaca cccagccggc
     2101 cacagtcgat gaatccagaa aagcggccat tttccaccat gatattcggc aagcaggcat
     2161 cgccatgggt cacgacgaga tcctcgccgt cgggcatgcg cgccttgagc ctggcgaaca
     2221 gttcggctgg cgcgagcccc tgatgctctt cgtccagatc atcctgatcg acaagaccgg
     2281 cttccatccg agtacgtgct cgctcgatgc gatgtttcgc ttggtggtcg aatgggcagg
     2341 tagccggatc aagcgtatgc agccgccgca ttgcatcagc catgatggat actttctcgg
     2401 caggagcaag gtgagatgac aggagatcct gccccggcac ttcgcccaat agcagccagt
     2461 cccttcccgc ttcagtgaca acgtcgagca cagctgcgca aggaacgccc gtcgtggcca
     2521 gccacgatag ccgcgctgcc tcgtcctgca gttcattcag ggcaccggac aggtcggtct
     2581 tgacaaaaag aaccgggcgc ccctgcgctg acagccggaa cacggcggca tcagagcagc
     2641 cgattgtctg ttgtgcccag tcatagccga atagcctctc cacccaagcg gccggagaac
     2701 ctgcgtgcaa tccatcttgt tcaatcatgc gaaacgatcc tcatcctgtc tcttgatcag
     2761 atcttgatcc cctgcgccat cagatccttg gcggcaagaa agccatccag tttactttgc
     2821 agggcttccc aaccttacca gagggcgccc cagctggcaa ttccgacgtc taagaaacca
     2881 ttattatcat gacattaacc tataaaaata ggcgtatcac gaggcccttt cgtctt
//
