LOCUS       sequence-197187-        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-R20
FEATURES             Location/Qualifiers
     source          1..2936
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(120..138)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     CDS             317..1111
                     /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(371..390)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     981..1000
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     terminator      1142..1236
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     rep_origin      1324..1912
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1813..1832
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     terminator      complement(2297..2383)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     misc_feature    2413..2544
                     /label=crRNA leader
                     /note="crRNA leader sequence for the Streptococcus pyogenes
                     CRISPR/Cas system"
     repeat_region   2545..2580
                     /label=DR
                     /note="direct repeat 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"
     misc_RNA        2833..2911
                     /label=tracrRNA
                     /note="trans-activating CRISPR RNA for the Streptococcus 
                     pyogenes CRISPR/Cas9 system"
ORIGIN
        1 tgactgcaaa ccaaaaaaac aagcgctttc aaaacgcttg ttttatcatt tttagggaaa
       61 ttaatctctt aatcctttta tcattctaca tttaggcgct gccatcttaa gacgaaaggg
      121 cctcgtgata cgcctatttt tataggttaa tgtcatgata ataatggttt cttagacgtc
      181 ggaattgcca gctggggcgc cctctggtaa ggttgggaag ccctgcaaag taaactggat
      241 ggctttcttg ccgccaagga tctgatggcg caggggatca agatctgatc aagagacagg
      301 atgaggatcg tttcgcatga ttgaacaaga tggattgcac gcaggttctc cggccgcttg
      361 ggtggagagg ctattcggct atgactgggc acaacagaca atcggctgct ctgatgccgc
      421 cgtgttccgg ctgtcagcgc aggggcgccc ggttcttttt gtcaagaccg acctgtccgg
      481 tgccctgaat gaactgcagg acgaggcagc gcggctatcg tggctggcca cgacgggcgt
      541 tccttgcgca gctgtgctcg acgttgtcac tgaagcggga agggactggc tgctattggg
      601 cgaagtgccg gggcaggatc tcctgtcatc tcaccttgct cctgccgaga aagtatccat
      661 catggctgat gcaatgcggc ggctgcatac gcttgatccg gctacctgcc cattcgacca
      721 ccaagcgaaa catcgcatcg agcgagcacg tactcggatg gaagccggtc ttgtcgatca
      781 ggatgatctg gacgaagagc atcaggggct cgcgccagcc gaactgttcg ccaggctcaa
      841 ggcgcgcatg cccgacggcg aggatctcgt cgtgacccat ggcgatgcct gcttgccgaa
      901 tatcatggtg gaaaatggcc gcttttctgg attcatcgac tgtggccggc tgggtgtggc
      961 ggaccgctat caggacatag cgttggctac ccgtgatatt gctgaagagc ttggcggcga
     1021 atgggctgac cgcttcctcg tgctttacgg tatcgccgct cccgattcgc agcgcatcgc
     1081 cttctatcgc cttcttgacg agttcttctg agcgggactc tggggttcga gagctcgctt
     1141 ggactcctgt tgatagatcc agtaatgacc tcagaactcc atctggattt gttcagaacg
     1201 ctcggttgcc gccgggcgtt ttttattggt gagaatccaa gcactagtca tgaccaaaat
     1261 cccttaacgt gagttttcgt tccactgagc gtcagacccc gtagaaaaga tcaaaggatc
     1321 ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct
     1381 accagcggtg gtttgtttgc cggatcaaga gctaccaact ctttttccga aggtaactgg
     1441 cttcagcaga gcgcagatac caaatactgt ccttctagtg tagccgtagt taggccacca
     1501 cttcaagaac tctgtagcac cgcctacata cctcgctctg ctaatcctgt taccagtggc
     1561 tgctgccagt ggcgataagt cgtgtcttac cgggttggac tcaagacgat agttaccgga
     1621 taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca cagcccagct tggagcgaac
     1681 gacctacacc gaactgagat acctacagcg tgagctatga gaaagcgcca cgcttcccga
     1741 agggagaaag gcggacaggt atccggtaag cggcagggtc ggaacaggag agcgcacgag
     1801 ggagcttcca gggggaaacg cctggtatct ttatagtcct gtcgggtttc gccacctctg
     1861 acttgagcgt cgatttttgt gatgctcgtc aggggggcgg agcctatgga aaaacgccag
     1921 caacgcggcc tttttacggt tcctggcctt ttgctggcct tttgctcaca tgttctttcc
     1981 tgcgttatcc cctgattctg tggataaccg tattaccgcc tttgagtgag ctgataccgc
     2041 tcgccgcagc cgaacgcccc aaaaagcctc gctttcagca cctgtcgttt cctttctttt
     2101 cagagggtat tttaaataaa aacattaagt tatgacgaag aagaacggaa acgccttaaa
     2161 ccggaaaatt ttcataaata gcgaaaaccc gcgaggtcgc cgccccgtaa cctgtcggat
     2221 caccggaagg gacccgtaaa gtgataatga ttatcatcta catatcacaa cgtgcgtgga
     2281 gggtctaggg cggcggattt gtcctactca ggagagcgtt caccgacaaa caacagataa
     2341 aacgaaaggc ccagtctttc gactgagcct ttcgttttat ttgatgcctc tagcacgcgt
     2401 accatgggat cctatttctt aataactaaa aatatggtat aatactctta ataaatgcag
     2461 taatacaggg gcttttcaag actgaagtct agctgagaca aatagtgcga ttacgaaatt
     2521 ttttagacaa aaatagtcta cgaggtttta gagctatgct gttttgaatg gtcccaaaac
     2581 ccttcacctt caccacgaac agagaatttg gttttagagc tatgctgttt tgaatggtcc
     2641 caaaacagtt tggcggtctg ggtgccttca tacggagttt tagagctatg ctgttttgaa
     2701 tggtcccaaa acttcagcac actgagactt gttgagttga attcatgagt attaagtatt
     2761 gttttatggc tgataaattt ctttgaattt ctccttgatt atttgttata aaagttataa
     2821 aataatcttg ttggaaccat tcaaaacagc atagcaagtt aaaataaggc tagtccgtta
     2881 tcaacttgaa aaagtggcac cgagtcggtg ctttttttga tacttctatt ctactc
//
