LOCUS       sequence-197182-        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"
     primer_bind     complement(186..204)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     CDS             383..1177
                     /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(437..456)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     1047..1066
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     terminator      1208..1302
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     rep_origin      1390..1978
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1879..1898
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     terminator      complement(2363..2449)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     misc_feature    2479..2610
                     /label=crRNA leader
                     /note="crRNA leader sequence for the Streptococcus pyogenes
                     CRISPR/Cas system"
     repeat_region   2611..2646
                     /label=DR
                     /note="direct repeat for the Streptococcus pyogenes 
                     CRISPR/Cas system"
     repeat_region   2677..2712
                     /label=DR
                     /note="direct repeat for the Streptococcus pyogenes 
                     CRISPR/Cas system"
     repeat_region   2743..2778
                     /label=DR
                     /note="direct repeat for the Streptococcus pyogenes 
                     CRISPR/Cas system"
     misc_RNA        join(2899..2936,1..41)
                     /label=tracrRNA
                     /note="trans-activating CRISPR RNA for the Streptococcus 
                     pyogenes CRISPR/Cas9 system"
ORIGIN
        1 tagtccgtta tcaacttgaa aaagtggcac cgagtcggtg ctttttttga tacttctatt
       61 ctactctgac tgcaaaccaa aaaaacaagc gctttcaaaa cgcttgtttt atcattttta
      121 gggaaattaa tctcttaatc cttttatcat tctacattta ggcgctgcca tcttaagacg
      181 aaagggcctc gtgatacgcc tatttttata ggttaatgtc atgataataa tggtttctta
      241 gacgtcggaa ttgccagctg gggcgccctc tggtaaggtt gggaagccct gcaaagtaaa
      301 ctggatggct ttcttgccgc caaggatctg atggcgcagg ggatcaagat ctgatcaaga
      361 gacaggatga ggatcgtttc gcatgattga acaagatgga ttgcacgcag gttctccggc
      421 cgcttgggtg gagaggctat tcggctatga ctgggcacaa cagacaatcg gctgctctga
      481 tgccgccgtg ttccggctgt cagcgcaggg gcgcccggtt ctttttgtca agaccgacct
      541 gtccggtgcc ctgaatgaac tgcaggacga ggcagcgcgg ctatcgtggc tggccacgac
      601 gggcgttcct tgcgcagctg tgctcgacgt tgtcactgaa gcgggaaggg actggctgct
      661 attgggcgaa gtgccggggc aggatctcct gtcatctcac cttgctcctg ccgagaaagt
      721 atccatcatg gctgatgcaa tgcggcggct gcatacgctt gatccggcta cctgcccatt
      781 cgaccaccaa gcgaaacatc gcatcgagcg agcacgtact cggatggaag ccggtcttgt
      841 cgatcaggat gatctggacg aagagcatca ggggctcgcg ccagccgaac tgttcgccag
      901 gctcaaggcg cgcatgcccg acggcgagga tctcgtcgtg acccatggcg atgcctgctt
      961 gccgaatatc atggtggaaa atggccgctt ttctggattc atcgactgtg gccggctggg
     1021 tgtggcggac cgctatcagg acatagcgtt ggctacccgt gatattgctg aagagcttgg
     1081 cggcgaatgg gctgaccgct tcctcgtgct ttacggtatc gccgctcccg attcgcagcg
     1141 catcgccttc tatcgccttc ttgacgagtt cttctgagcg ggactctggg gttcgagagc
     1201 tcgcttggac tcctgttgat agatccagta atgacctcag aactccatct ggatttgttc
     1261 agaacgctcg gttgccgccg ggcgtttttt attggtgaga atccaagcac tagtcatgac
     1321 caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa
     1381 aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc
     1441 accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt ttccgaaggt
     1501 aactggcttc agcagagcgc agataccaaa tactgtcctt ctagtgtagc cgtagttagg
     1561 ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa tcctgttacc
     1621 agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa gacgatagtt
     1681 accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc ccagcttgga
     1741 gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa gcgccacgct
     1801 tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa caggagagcg
     1861 cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca
     1921 cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa
     1981 cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg ctcacatgtt
     2041 ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg agtgagctga
     2101 taccgctcgc cgcagccgaa cgccccaaaa agcctcgctt tcagcacctg tcgtttcctt
     2161 tcttttcaga gggtatttta aataaaaaca ttaagttatg acgaagaaga acgggaacgc
     2221 cttaaaccgg aaaattttca taaatagcga aaacccgcga ggtcgccgcc ccgtaacctg
     2281 tcggatcacc ggaaaggacc cgtaaagtga taatgattat catctacata tcacaacgtg
     2341 cgtggagggt ctagggcggc ggatttgtcc tactcaggag agcgttcacc gacaaacaac
     2401 agataaaacg aaaggcccag tctttcgact gagcctttcg ttttatttga tgcctctagc
     2461 acgcgtacca tgggatccta tttcttaata actaaaaata tggtataata ctcttaataa
     2521 atgcagtaat acaggggctt ttcaagactg aagtctagct gagacaaata gtgcgattac
     2581 gaaatttttt agacaaaaat agtctacgag gttttagagc tatgctgttt tgaatggtcc
     2641 caaaacatcg cacatcctgg tsgykamrwt aakwkkgttt tagagctatg ctgttttgaa
     2701 tggtcccaaa acagtttggc ggwgagggtg ccttcatacg gagttttaga gctatgctgt
     2761 tttgaatggt cccaaaactt cagcacactg agacttgttg agttgaattc atgagtatta
     2821 agtattgttt tatggctgat aaatttcttt gaatttctcc ttgattattt gttataaaag
     2881 ttataaaata atcttgttgg aaccattcaa aacagcatag caagttaaaa taaggc
//
