LOCUS       sequence-197177-        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-G20-R11
FEATURES             Location/Qualifiers
     source          1..2936
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_feature    8..139
                     /label=crRNA leader
                     /note="crRNA leader sequence for the Streptococcus pyogenes
                     CRISPR/Cas system"
     repeat_region   140..175
                     /label=DR
                     /note="direct repeat for the Streptococcus pyogenes 
                     CRISPR/Cas system"
     repeat_region   206..241
                     /label=DR
                     /note="direct repeat for the Streptococcus pyogenes 
                     CRISPR/Cas system"
     repeat_region   272..307
                     /label=DR
                     /note="direct repeat for the Streptococcus pyogenes 
                     CRISPR/Cas system"
     misc_RNA        428..506
                     /label=tracrRNA
                     /note="trans-activating CRISPR RNA for the Streptococcus 
                     pyogenes CRISPR/Cas9 system"
     primer_bind     complement(651..669)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     CDS             848..1642
                     /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(902..921)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     1512..1531
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     terminator      1673..1767
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     rep_origin      1855..2443
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2344..2363
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     terminator      complement(2828..2914)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
ORIGIN
        1 gggatcctat ttcttaataa ctaaaaatat ggtataatac tcttaataaa tgcagtaata
       61 caggggcttt tcaagactga agtctagctg agacaaatag tgcgattacg aaatttttta
      121 gacaaaaata gtctacgagg ttttagagct atgctgtttt gaatggtccc aaaacccttc
      181 accttcacca cgaacagaga atttggtttt agagctatgc tgttttgaat ggtcccaaaa
      241 cagtttggcg gagacccacg cttcatacgg agttttagag ctatgctgtt ttgaatggtc
      301 ccaaaacttc agcacactga gacttgttga gttgaattca tgagtattaa gtattgtttt
      361 atggctgata aatttctttg aatttctcct tgattatttg ttataaaagt tataaaataa
      421 tcttgttgga accattcaaa acagcatagc aagttaaaat aaggctagtc cgttatcaac
      481 ttgaaaaagt ggcaccgagt cggtgctttt tttgatactt ctattctact ctgactgcaa
      541 accaaaaaaa caagcgcttt caaaacgctt gttttatcat ttttagggaa attaatctct
      601 taatcctttt atcattctac atttaggcgc tgccatctta agacgaaagg gcctcgtgat
      661 acgcctattt ttataggtta atgtcatgat aataatggtt tcttagacgt cggaattgcc
      721 agctggggcg ccctctggta aggttgggaa gccctgcaaa gtaaactgga tggctttctt
      781 gccgccaagg atctgatggc gcaggggatc aagatctgat caagagacag gatgaggatc
      841 gtttcgcatg attgaacaag atggattgca cgcaggttct ccggccgctt gggtggagag
      901 gctattcggc tatgactggg cacaacagac aatcggctgc tctgatgccg ccgtgttccg
      961 gctgtcagcg caggggcgcc cggttctttt tgtcaagacc gacctgtccg gtgccctgaa
     1021 tgaactgcag gacgaggcag cgcggctatc gtggctggcc acgacgggcg ttccttgcgc
     1081 agctgtgctc gacgttgtca ctgaagcggg aagggactgg ctgctattgg gcgaagtgcc
     1141 ggggcaggat ctcctgtcat ctcaccttgc tcctgccgag aaagtatcca tcatggctga
     1201 tgcaatgcgg cggctgcata cgcttgatcc ggctacctgc ccattcgacc accaagcgaa
     1261 acatcgcatc gagcgagcac gtactcggat ggaagccggt cttgtcgatc aggatgatct
     1321 ggacgaagag catcaggggc tcgcgccagc cgaactgttc gccaggctca aggcgcgcat
     1381 gcccgacggc gaggatctcg tcgtgaccca tggcgatgcc tgcttgccga atatcatggt
     1441 ggaaaatggc cgcttttctg gattcatcga ctgtggccgg ctgggtgtgg cggaccgcta
     1501 tcaggacata gcgttggcta cccgtgatat tgctgaagag cttggcggcg aatgggctga
     1561 ccgcttcctc gtgctttacg gtatcgccgc tcccgattcg cagcgcatcg ccttctatcg
     1621 ccttcttgac gagttcttct gagcgggact ctggggttcg agagctcgct tggactcctg
     1681 ttgatagatc cagtaatgac ctcagaactc catctggatt tgttcagaac gctcggttgc
     1741 cgccgggcgt tttttattgg tgagaatcca agcactagtc atgaccaaaa tcccttaacg
     1801 tgagttttcg ttccactgag cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga
     1861 tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt
     1921 ggtttgtttg ccggatcaag agctaccaac tctttttccg aaggtaactg gcttcagcag
     1981 agcgcagata ccaaatactg tccttctagt gtagccgtag ttaggccacc acttcaagaa
     2041 ctctgtagca ccgcctacat acctcgctct gctaatcctg ttaccagtgg ctgctgccag
     2101 tggcgataag tcgtgtctta ccgggttgga ctcaagacga tagttaccgg ataaggcgca
     2161 gcggtcgggc tgaacggggg gttcgtgcac acagcccagc ttggagcgaa cgacctacac
     2221 cgaactgaga tacctacagc gtgagctatg agaaagcgcc acgcttcccg aagggagaaa
     2281 ggcggacagg tatccggtaa gcggcagggt cggaacagga gagcgcacga gggagcttcc
     2341 agggggaaac gcctggtatc tttatagtcc tgtcgggttt cgccacctct gacttgagcg
     2401 tcgatttttg tgatgctcgt caggggggcg gagcctatgg aaaaacgcca gcaacgcggc
     2461 ctttttacgg ttcctggcct tttgctggcc ttttgctcac atgttctttc ctgcgttatc
     2521 ccctgattct gtggataacc gtattaccgc ctttgagtga gctgataccg ctcgccgcag
     2581 ccgaacgccc caaaaagcct cgctttcagc acctgtcgtt tcctttcttt tcagagggta
     2641 ttttaaataa aaacattaag ttatgacgaa gaagaacgga aacgccttaa accggaaaat
     2701 tttcataaat agcgaaaacc cgcgaggtcg ccgccccgta acctgtcgga tcaccggaag
     2761 ggacccgtaa agtgataatg attatcatct acatatcaca acgtgcgtgg agggtctagg
     2821 gcggcggatt tgtcctactc aggagagcgt tcaccgacaa acaacagata aaacgaaagg
     2881 cccagtcttt cgactgagcc tttcgtttta tttgatgcct ctagcacgcg taccat
//
