LOCUS       sequence-197180-        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-R18
FEATURES             Location/Qualifiers
     source          1..2936
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     terminator      complement(16..102)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     misc_feature    132..263
                     /label=crRNA leader
                     /note="crRNA leader sequence for the Streptococcus pyogenes
                     CRISPR/Cas system"
     repeat_region   264..299
                     /label=DR
                     /note="direct repeat for the Streptococcus pyogenes 
                     CRISPR/Cas system"
     repeat_region   330..365
                     /label=DR
                     /note="direct repeat for the Streptococcus pyogenes 
                     CRISPR/Cas system"
     repeat_region   396..431
                     /label=DR
                     /note="direct repeat for the Streptococcus pyogenes 
                     CRISPR/Cas system"
     misc_RNA        552..630
                     /label=tracrRNA
                     /note="trans-activating CRISPR RNA for the Streptococcus 
                     pyogenes CRISPR/Cas9 system"
     primer_bind     complement(775..793)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     CDS             972..1766
                     /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(1026..1045)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     1636..1655
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     terminator      1797..1891
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     rep_origin      1979..2567
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2468..2487
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 ggtctagggc ggcggatttg tcctactcag gagagcgttc accgacaaac aacagataaa
       61 acgaaaggcc cagtctttcg actgagcctt tcgttttatt tgatgcctct agcacgcgta
      121 ccatgggatc ctatttctta ataactaaaa atatggtata atactcttaa taaatgcagt
      181 aatacagggg cttttcaaga ctgaagtcta gctgagacaa atagtgcgat tacgaaattt
      241 tttagacaaa aatagtctac gaggttttag agctatgctg ttttgaatgg tcccaaaacc
      301 cttcaccttg tggtggaaca gagaatttgg ttttagagct atgctgtttt gaatggtccc
      361 aaaacagttt ggcggagtgg gtgccttcat acggagtttt agagctatgc tgttttgaat
      421 ggtcccaaaa cttcagcaca ctgagacttg ttgagttgaa ttcatgagta ttaagtattg
      481 ttttatggct gataaatttc tttgaatttc tccttgatta tttgttataa aagttataaa
      541 ataatcttgt tggaaccatt caaaacagca tagcaagtta aaataaggct agtccgttat
      601 caacttgaaa aagtggcacc gagtcggtgc tttttttgat acttctattc tactctgact
      661 gcaaaccaaa aaaacaagcg ctttcaaaac gcttgtttta tcatttttag ggaaattaat
      721 ctcttaatcc ttttatcatt ctacatttag gcgctgccat cttaagacga aagggcctcg
      781 tgatacgcct atttttatag gttaatgtca tgataataat ggtttcttag acgtcggaat
      841 tgccagctgg ggcgccctct ggtaaggttg ggaagccctg caaagtaaac tggatggctt
      901 tcttgccgcc aaggatctga tggcgcaggg gatcaagatc tgatcaagag acaggatgag
      961 gatcgtttcg catgattgaa caagatggat tgcacgcagg ttctccggcc gcttgggtgg
     1021 agaggctatt cggctatgac tgggcacaac agacaatcgg ctgctctgat gccgccgtgt
     1081 tccggctgtc agcgcagggg cgcccggttc tttttgtcaa gaccgacctg tccggtgccc
     1141 tgaatgaact gcaggacgag gcagcgcggc tatcgtggct ggccacgacg ggcgttcctt
     1201 gcgcagctgt gctcgacgtt gtcactgaag cgggaaggga ctggctgcta ttgggcgaag
     1261 tgccggggca ggatctcctg tcatctcacc ttgctcctgc cgagaaagta tccatcatgg
     1321 ctgatgcaat gcggcggctg catacgcttg atccggctac ctgcccattc gaccaccaag
     1381 cgaaacatcg catcgagcga gcacgtactc ggatggaagc cggtcttgtc gatcaggatg
     1441 atctggacga agagcatcag gggctcgcgc cagccgaact gttcgccagg ctcaaggcgc
     1501 gcatgcccga cggcgaggat ctcgtcgtga cccatggcga tgcctgcttg ccgaatatca
     1561 tggtggaaaa tggccgcttt tctggattca tcgactgtgg ccggctgggt gtggcggacc
     1621 gctatcagga catagcgttg gctacccgtg atattgctga agagcttggc ggcgaatggg
     1681 ctgaccgctt cctcgtgctt tacggtatcg ccgctcccga ttcgcagcgc atcgccttct
     1741 atcgccttct tgacgagttc ttctgagcgg gactctgggg ttcgagagct cgcttggact
     1801 cctgttgata gatccagtaa tgacctcaga actccatctg gatttgttca gaacgctcgg
     1861 ttgccgccgg gcgtttttta ttggtgagaa tccaagcact agtcatgacc aaaatccctt
     1921 aacgtgagtt ttcgttccac tgagcgtcag accccgtaga aaagatcaaa ggatcttctt
     1981 gagatccttt ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag
     2041 cggtggtttg tttgccggat caagagctac caactctttt tccgaaggta actggcttca
     2101 gcagagcgca gataccaaat actgtccttc tagtgtagcc gtagttaggc caccacttca
     2161 agaactctgt agcaccgcct acatacctcg ctctgctaat cctgttacca gtggctgctg
     2221 ccagtggcga taagtcgtgt cttaccgggt tggactcaag acgatagtta ccggataagg
     2281 cgcagcggtc gggctgaacg gggggttcgt gcacacagcc cagcttggag cgaacgacct
     2341 acaccgaact gagataccta cagcgtgagc tatgagaaag cgccacgctt cccgaaggga
     2401 gaaaggcgga caggtatccg gtaagcggca gggtcggaac aggagagcgc acgagggagc
     2461 ttccaggggg aaacgcctgg tatctttata gtcctgtcgg gtttcgccac ctctgacttg
     2521 agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac gccagcaacg
     2581 cggccttttt acggttcctg gccttttgct ggccttttgc tcacatgttc tttcctgcgt
     2641 tatcccctga ttctgtggat aaccgtatta ccgcctttga gtgagctgat accgctcgcc
     2701 gcagccgaac gccccaaaaa gcctcgcttt cagcacctgt cgtttccttt cttttcagag
     2761 ggtattttaa ataaaaacat taagttatga cgaagaagaa cgggaacgcc ttaaaccgga
     2821 aaattttcat aaatagcgaa aacccgcgag gtcgccgccc cgtaacctgt cggatcaccg
     2881 gaaaggaccc gtaaagtgat aatgattatc atctacatat cacaacgtgc gtggag
//
