LOCUS       sequence-197181-        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-R18
FEATURES             Location/Qualifiers
     source          1..2936
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     terminator      complement(94..180)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     misc_feature    210..341
                     /label=crRNA leader
                     /note="crRNA leader sequence for the Streptococcus pyogenes
                     CRISPR/Cas system"
     repeat_region   342..377
                     /label=DR
                     /note="direct repeat for the Streptococcus pyogenes 
                     CRISPR/Cas system"
     repeat_region   408..443
                     /label=DR
                     /note="direct repeat for the Streptococcus pyogenes 
                     CRISPR/Cas system"
     repeat_region   474..509
                     /label=DR
                     /note="direct repeat for the Streptococcus pyogenes 
                     CRISPR/Cas system"
     misc_RNA        630..708
                     /label=tracrRNA
                     /note="trans-activating CRISPR RNA for the Streptococcus 
                     pyogenes CRISPR/Cas9 system"
     primer_bind     complement(853..871)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     CDS             1050..1844
                     /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(1104..1123)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     1714..1733
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     terminator      1875..1969
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     rep_origin      2057..2645
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2546..2565
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 cccgtaacct gtcggatcac cggaaaggac ccgtaaagtg ataatgatta tcatctacat
       61 atcacaacgt gcgtggaggg tctagggcgg cggatttgtc ctactcagga gagcgttcac
      121 cgacaaacaa cagataaaac gaaaggccca gtctttcgac tgagcctttc gttttatttg
      181 atgcctctag cacgcgtacc atgggatcct atttcttaat aactaaaaat atggtataat
      241 actcttaata aatgcagtaa tacaggggct tttcaagact gaagtctagc tgagacaaat
      301 agtgcgatta cgaaattttt tagacaaaaa tagtctacga ggttttagag ctatgctgtt
      361 ttgaatggtc ccaaaaccct tcaccttcac cacgaacaga gaatttggtt ttagagctat
      421 gctgttttga atggtcccaa aacagtttgg cggagtgggt gccttcatac ggagttttag
      481 agctatgctg ttttgaatgg tcccaaaact tcagcacact gagacttgtt gagttgaatt
      541 catgagtatt aagtattgtt ttatggctga taaatttctt tgaatttctc cttgattatt
      601 tgttataaaa gttataaaat aatcttgttg gaaccattca aaacagcata gcaagttaaa
      661 ataaggctag tccgttatca acttgaaaaa gtggcaccga gtcggtgctt tttttgatac
      721 ttctattcta ctctgactgc aaaccaaaaa aacaagcgct ttcaaaacgc ttgttttatc
      781 atttttaggg aaattaatct cttaatcctt ttatcattct acatttaggc gctgccatct
      841 taagacgaaa gggcctcgtg atacgcctat ttttataggt taatgtcatg ataataatgg
      901 tttcttagac gtcggaattg ccagctgggg cgccctctgg taaggttggg aagccctgca
      961 aagtaaactg gatggctttc ttgccgccaa ggatctgatg gcgcagggga tcaagatctg
     1021 atcaagagac aggatgagga tcgtttcgca tgattgaaca agatggattg cacgcaggtt
     1081 ctccggccgc ttgggtggag aggctattcg gctatgactg ggcacaacag acaatcggct
     1141 gctctgatgc cgccgtgttc cggctgtcag cgcaggggcg cccggttctt tttgtcaaga
     1201 ccgacctgtc cggtgccctg aatgaactgc aggacgaggc agcgcggcta tcgtggctgg
     1261 ccacgacggg cgttccttgc gcagctgtgc tcgacgttgt cactgaagcg ggaagggact
     1321 ggctgctatt gggcgaagtg ccggggcagg atctcctgtc atctcacctt gctcctgccg
     1381 agaaagtatc catcatggct gatgcaatgc ggcggctgca tacgcttgat ccggctacct
     1441 gcccattcga ccaccaagcg aaacatcgca tcgagcgagc acgtactcgg atggaagccg
     1501 gtcttgtcga tcaggatgat ctggacgaag agcatcaggg gctcgcgcca gccgaactgt
     1561 tcgccaggct caaggcgcgc atgcccgacg gcgaggatct cgtcgtgacc catggcgatg
     1621 cctgcttgcc gaatatcatg gtggaaaatg gccgcttttc tggattcatc gactgtggcc
     1681 ggctgggtgt ggcggaccgc tatcaggaca tagcgttggc tacccgtgat attgctgaag
     1741 agcttggcgg cgaatgggct gaccgcttcc tcgtgcttta cggtatcgcc gctcccgatt
     1801 cgcagcgcat cgccttctat cgccttcttg acgagttctt ctgagcggga ctctggggtt
     1861 cgagagctcg cttggactcc tgttgataga tccagtaatg acctcagaac tccatctgga
     1921 tttgttcaga acgctcggtt gccgccgggc gttttttatt ggtgagaatc caagcactag
     1981 tcatgaccaa aatcccttaa cgtgagtttt cgttccactg agcgtcagac cccgtagaaa
     2041 agatcaaagg atcttcttga gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa
     2101 aaaaaccacc gctaccagcg gtggtttgtt tgccggatca agagctacca actctttttc
     2161 cgaaggtaac tggcttcagc agagcgcaga taccaaatac tgtccttcta gtgtagccgt
     2221 agttaggcca ccacttcaag aactctgtag caccgcctac atacctcgct ctgctaatcc
     2281 tgttaccagt ggctgctgcc agtggcgata agtcgtgtct taccgggttg gactcaagac
     2341 gatagttacc ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc acacagccca
     2401 gcttggagcg aacgacctac accgaactga gatacctaca gcgtgagcta tgagaaagcg
     2461 ccacgcttcc cgaagggaga aaggcggaca ggtatccggt aagcggcagg gtcggaacag
     2521 gagagcgcac gagggagctt ccagggggaa acgcctggta tctttatagt cctgtcgggt
     2581 ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg cggagcctat
     2641 ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc cttttgctgg ccttttgctc
     2701 acatgttctt tcctgcgtta tcccctgatt ctgtggataa ccgtattacc gcctttgagt
     2761 gagctgatac cgctcgccgc agccgaacgc cccaaaaagc ctcgctttca gcacctgtcg
     2821 tttcctttct tttcagaggg tattttaaat aaaaacatta agttatgacg aagaagaacg
     2881 gaaacgcctt aaaccggaaa attttcataa atagcgaaaa cccgcgaggt cgccgc
//
