LOCUS       sequence-197185-        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-R20
FEATURES             Location/Qualifiers
     source          1..2936
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     165..184
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     terminator      326..420
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     rep_origin      508..1096
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     997..1016
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     terminator      complement(1481..1567)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     misc_feature    1597..1728
                     /label=crRNA leader
                     /note="crRNA leader sequence for the Streptococcus pyogenes
                     CRISPR/Cas system"
     repeat_region   1729..1764
                     /label=DR
                     /note="direct repeat for the Streptococcus pyogenes 
                     CRISPR/Cas system"
     repeat_region   1795..1830
                     /label=DR
                     /note="direct repeat for the Streptococcus pyogenes 
                     CRISPR/Cas system"
     repeat_region   1861..1896
                     /label=DR
                     /note="direct repeat for the Streptococcus pyogenes 
                     CRISPR/Cas system"
     misc_RNA        2017..2095
                     /label=tracrRNA
                     /note="trans-activating CRISPR RNA for the Streptococcus 
                     pyogenes CRISPR/Cas9 system"
     primer_bind     complement(2240..2258)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     CDS             join(2437..2936,1..295)
                     /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(2491..2510)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
ORIGIN
        1 agccgaactg ttcgccaggc tcaaggcgcg catgcccgac ggcgaggatc tcgtcgtgac
       61 ccatggcgat gcctgcttgc cgaatatcat ggtggaaaat ggccgctttt ctggattcat
      121 cgactgtggc cggctgggtg tggcggaccg ctatcaggac atagcgttgg ctacccgtga
      181 tattgctgaa gagcttggcg gcgaatgggc tgaccgcttc ctcgtgcttt acggtatcgc
      241 cgctcccgat tcgcagcgca tcgccttcta tcgccttctt gacgagttct tctgagcggg
      301 actctggggt tcgagagctc gcttggactc ctgttgatag atccagtaat gacctcagaa
      361 ctccatctgg atttgttcag aacgctcggt tgccgccggg cgttttttat tggtgagaat
      421 ccaagcacta gtcatgacca aaatccctta acgtgagttt tcgttccact gagcgtcaga
      481 ccccgtagaa aagatcaaag gatcttcttg agatcctttt tttctgcgcg taatctgctg
      541 cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc aagagctacc
      601 aactcttttt ccgaaggtaa ctggcttcag cagagcgcag ataccaaata ctgtccttct
      661 agtgtagccg tagttaggcc accacttcaa gaactctgta gcaccgccta catacctcgc
      721 tctgctaatc ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt
      781 ggactcaaga cgatagttac cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg
      841 cacacagccc agcttggagc gaacgaccta caccgaactg agatacctac agcgtgagct
      901 atgagaaagc gccacgcttc ccgaagggag aaaggcggac aggtatccgg taagcggcag
      961 ggtcggaaca ggagagcgca cgagggagct tccaggggga aacgcctggt atctttatag
     1021 tcctgtcggg tttcgccacc tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg
     1081 gcggagccta tggaaaaacg ccagcaacgc ggccttttta cggttcctgg ccttttgctg
     1141 gccttttgct cacatgttct ttcctgcgtt atcccctgat tctgtggata accgtattac
     1201 cgcctttgag tgagctgata ccgctcgccg cagccgaacg ccccaaaaag cctcgctttc
     1261 agcacctgtc gtttcctttc ttttcagagg gtattttaaa taaaaacatt aagttatgac
     1321 gaagaagaac ggaaacgcct taaaccggaa aattttcata aatagcgaaa acccgcgagg
     1381 tcgccgcccc gtaacctgtc ggatcaccgg aaaggacccg taaagtgata atgattatca
     1441 tctacatatc acaacgtgcg tggagggtct agggcggcgg atttgtccta ctcaggagag
     1501 cgttcaccga caaacaacag ataaaacgaa aggcccagtc tttcgactga gcctttcgtt
     1561 ttatttgatg cctctagcac gcgtaccatg ggatcctatt tcttaataac taaaaatatg
     1621 gtataatact cttaataaat gcagtaatac aggggctttt caagactgaa gtctagctga
     1681 gacaaatagt gcgattacga aattttttag acaaaaatag tctacgaggt tttagagcta
     1741 tgctgttttg aatggtccca aaacccttca ccttgtggtg gaacagtgaa tttggtttta
     1801 gagctatgct gttttgaatg gtcccaaaac agtttggcgg tctgggtgcc ttcatacgga
     1861 gttttagagc tatgctgttt tgaatggtcc caaaacttca gcacactgag acttgttgag
     1921 ttgaattcat gagtattaag tattgtttta tggctgataa atttctttga atttctcctt
     1981 gattatttgt tataaaagtt ataaaataat cttgttggaa ccattcaaaa cagcatagca
     2041 agttaaaata aggctagtcc gttatcaact tgaaaaagtg gcaccgagtc ggtgcttttt
     2101 ttgatacttc tattctactc tgactgcaaa ccaaaaaaac aagcgctttc aaaacgcttg
     2161 ttttatcatt tttagggaaa ttaatctctt aatcctttta tcattctaca tttaggcgct
     2221 gccatcttaa gacgaaaggg cctcgtgata cgcctatttt tataggttaa tgtcatgata
     2281 ataatggttt cttagacgtc ggaattgcca gctggggcgc cctctggtaa ggttgggaag
     2341 ccctgcaaag taaactggat ggctttcttg ccgccaagga tctgatggcg caggggatca
     2401 agatctgatc aagagacagg atgaggatcg tttcgcatga ttgaacaaga tggattgcac
     2461 gcaggttctc cggccgcttg ggtggagagg ctattcggct atgactgggc acaacagaca
     2521 atcggctgct ctgatgccgc cgtgttccgg ctgtcagcgc aggggcgccc ggttcttttt
     2581 gtcaagaccg acctgtccgg tgccctgaat gaactgcagg acgaggcagc gcggctatcg
     2641 tggctggcca cgacgggcgt tccttgcgca gctgtgctcg acgttgtcac tgaagcggga
     2701 agggactggc tgctattggg cgaagtgccg gggcaggatc tcctgtcatc tcaccttgct
     2761 cctgccgaga aagtatccat catggctgat gcaatgcggc ggctgcatac gcttgatccg
     2821 gctacctgcc cattcgacca ccaagcgaaa catcgcatcg agcgagcacg tactcggatg
     2881 gaagccggtc ttgtcgatca ggatgatctg gacgaagagc atcaggggct cgcgcc
//
