LOCUS       sequence-197184-        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-G11-R20
FEATURES             Location/Qualifiers
     source          1..2936
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     217..236
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     terminator      complement(701..787)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     misc_feature    817..948
                     /label=crRNA leader
                     /note="crRNA leader sequence for the Streptococcus pyogenes
                     CRISPR/Cas system"
     repeat_region   949..984
                     /label=DR
                     /note="direct repeat for the Streptococcus pyogenes 
                     CRISPR/Cas system"
     repeat_region   1015..1050
                     /label=DR
                     /note="direct repeat for the Streptococcus pyogenes 
                     CRISPR/Cas system"
     repeat_region   1081..1116
                     /label=DR
                     /note="direct repeat for the Streptococcus pyogenes 
                     CRISPR/Cas system"
     misc_RNA        1237..1315
                     /label=tracrRNA
                     /note="trans-activating CRISPR RNA for the Streptococcus 
                     pyogenes CRISPR/Cas9 system"
     primer_bind     complement(1460..1478)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     CDS             1657..2451
                     /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(1711..1730)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     2321..2340
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     terminator      2482..2576
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     rep_origin      join(2664..2936,1..316)
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
ORIGIN
        1 ggactcaaga cgatagttac cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg
       61 cacacagccc agcttggagc gaacgaccta caccgaactg agatacctac agcgtgagct
      121 atgagaaagc gccacgcttc ccgaagggag aaaggcggac aggtatccgg taagcggcag
      181 ggtcggaaca ggagagcgca cgagggagct tccaggggga aacgcctggt atctttatag
      241 tcctgtcggg tttcgccacc tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg
      301 gcggagccta tggaaaaacg ccagcaacgc ggccttttta cggttcctgg ccttttgctg
      361 gccttttgct cacatgttct ttcctgcgtt atcccctgat tctgtggata accgtattac
      421 cgcctttgag tgagctgata ccgctcgccg cagccgaacg ccccaaaaag cctcgctttc
      481 agcacctgtc gtttcctttc ttttcagagg gtattttaaa taaaaacatt aagttatgac
      541 gaagaagaac ggaaacgcct taaaccggaa aattttcata aatagcgaaa acccgcgagg
      601 tcgccgcccc gtaacctgtc ggatcaccgg aagggacccg taaagtgata atgattatca
      661 tctacatatc acaacgtgcg tggagggtct agggcggcgg atttgtccta ctcaggagag
      721 cgttcaccga caaacaacag ataaaacgaa aggcccagtc tttcgactga gcctttcgtt
      781 ttatttgatg cctctagcac gcgtaccatg ggatcctatt tcttaataac taaaaatatg
      841 gtataatact cttaataaat gcagtaatac aggggctttt caagactgaa gtctagctga
      901 gacaaatagt gcgattacga aattttttag acaaaaatag tctacgaggt tttagagcta
      961 tgctgttttg aatggtccca aaacccttca ccttgtggtg cttcagagaa tttggtttta
     1021 gagctatgct gttttgaatg gtcccaaaac agtttggcgg tctgggtgcc ttcatacgga
     1081 gttttagagc tatgctgttt tgaatggtcc caaaacttca gcacactgag acttgttgag
     1141 ttgaattcat gagtattaag tattgtttta tggctgataa atttctttga atttctcctt
     1201 gattatttgt tataaaagtt ataaaataat cttgttggaa ccattcaaaa cagcatagca
     1261 agttaaaata aggctagtcc gttatcaact tgaaaaagtg gcaccgagtc ggtgcttttt
     1321 ttgatacttc tattctactc tgactgcaaa ccaaaaaaac aagcgctttc aaaacgcttg
     1381 ttttatcatt tttagggaaa ttaatctctt aatcctttta tcattctaca tttaggcgct
     1441 gccatcttaa gacgaaaggg cctcgtgata cgcctatttt tataggttaa tgtcatgata
     1501 ataatggttt cttagacgtc ggaattgcca gctggggcgc cctctggtaa ggttgggaag
     1561 ccctgcaaag taaactggat ggctttcttg ccgccaagga tctgatggcg caggggatca
     1621 agatctgatc aagagacagg atgaggatcg tttcgcatga ttgaacaaga tggattgcac
     1681 gcaggttctc cggccgcttg ggtggagagg ctattcggct atgactgggc acaacagaca
     1741 atcggctgct ctgatgccgc cgtgttccgg ctgtcagcgc aggggcgccc ggttcttttt
     1801 gtcaagaccg acctgtccgg tgccctgaat gaactgcagg acgaggcagc gcggctatcg
     1861 tggctggcca cgacgggcgt tccttgcgca gctgtgctcg acgttgtcac tgaagcggga
     1921 agggactggc tgctattggg cgaagtgccg gggcaggatc tcctgtcatc tcaccttgct
     1981 cctgccgaga aagtatccat catggctgat gcaatgcggc ggctgcatac gcttgatccg
     2041 gctacctgcc cattcgacca ccaagcgaaa catcgcatcg agcgagcacg tactcggatg
     2101 gaagccggtc ttgtcgatca ggatgatctg gacgaagagc atcaggggct cgcgccagcc
     2161 gaactgttcg ccaggctcaa ggcgcgcatg cccgacggcg aggatctcgt cgtgacccat
     2221 ggcgatgcct gcttgccgaa tatcatggtg gaaaatggcc gcttttctgg attcatcgac
     2281 tgtggccggc tgggtgtggc ggaccgctat caggacatag cgttggctac ccgtgatatt
     2341 gctgaagagc ttggcggcga atgggctgac cgcttcctcg tgctttacgg tatcgccgct
     2401 cccgattcgc agcgcatcgc cttctatcgc cttcttgacg agttcttctg agcgggactc
     2461 tggggttcga gagctcgctt ggactcctgt tgatagatcc agtaatgacc tcagaactcc
     2521 atctggattt gttcagaacg ctcggttgcc gccgggcgtt ttttattggt gagaatccaa
     2581 gcactagtca tgaccaaaat cccttaacgt gagttttcgt tccactgagc gtcagacccc
     2641 gtagaaaaga tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg
     2701 caaacaaaaa aaccaccgct accagcggtg gtttgtttgc cggatcaaga gctaccaact
     2761 ctttttccga aggtaactgg cttcagcaga gcgcagatac caaatactgt ccttctagtg
     2821 tagccgtagt taggccacca cttcaagaac tctgtagcac cgcctacata cctcgctctg
     2881 ctaatcctgt taccagtggc tgctgccagt ggcgataagt cgtgtcttac cgggtt
//
