LOCUS       sequence-197179-        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-G11-R18
FEATURES             Location/Qualifiers
     source          1..2936
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     47..66
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     terminator      208..302
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     rep_origin      390..978
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     879..898
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     terminator      complement(1363..1449)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     misc_feature    1479..1610
                     /label=crRNA leader
                     /note="crRNA leader sequence for the Streptococcus pyogenes
                     CRISPR/Cas system"
     repeat_region   1611..1646
                     /label=DR
                     /note="direct repeat for the Streptococcus pyogenes 
                     CRISPR/Cas system"
     repeat_region   1677..1712
                     /label=DR
                     /note="direct repeat for the Streptococcus pyogenes 
                     CRISPR/Cas system"
     repeat_region   1743..1778
                     /label=DR
                     /note="direct repeat for the Streptococcus pyogenes 
                     CRISPR/Cas system"
     misc_RNA        1899..1977
                     /label=tracrRNA
                     /note="trans-activating CRISPR RNA for the Streptococcus 
                     pyogenes CRISPR/Cas9 system"
     primer_bind     complement(2122..2140)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     CDS             join(2319..2936,1..177)
                     /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(2373..2392)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
ORIGIN
        1 atcgactgtg gccggctggg tgtggcggac cgctatcagg acatagcgtt ggctacccgt
       61 gatattgctg aagagcttgg cggcgaatgg gctgaccgct tcctcgtgct ttacggtatc
      121 gccgctcccg attcgcagcg catcgccttc tatcgccttc ttgacgagtt cttctgagcg
      181 ggactctggg gttcgagagc tcgcttggac tcctgttgat agatccagta atgacctcag
      241 aactccatct ggatttgttc agaacgctcg gttgccgccg ggcgtttttt attggtgaga
      301 atccaagcac tagtcatgac caaaatccct taacgtgagt tttcgttcca ctgagcgtca
      361 gaccccgtag aaaagatcaa aggatcttct tgagatcctt tttttctgcg cgtaatctgc
      421 tgcttgcaaa caaaaaaacc accgctacca gcggtggttt gtttgccgga tcaagagcta
      481 ccaactcttt ttccgaaggt aactggcttc agcagagcgc agataccaaa tactgtcctt
      541 ctagtgtagc cgtagttagg ccaccacttc aagaactctg tagcaccgcc tacatacctc
      601 gctctgctaa tcctgttacc agtggctgct gccagtggcg ataagtcgtg tcttaccggg
      661 ttggactcaa gacgatagtt accggataag gcgcagcggt cgggctgaac ggggggttcg
      721 tgcacacagc ccagcttgga gcgaacgacc tacaccgaac tgagatacct acagcgtgag
      781 ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc
      841 agggtcggaa caggagagcg cacgagggag cttccagggg gaaacgcctg gtatctttat
      901 agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg
      961 gggcggagcc tatggaaaaa cgccagcaac gcggcctttt tacggttcct ggccttttgc
     1021 tggccttttg ctcacatgtt ctttcctgcg ttatcccctg attctgtgga taaccgtatt
     1081 accgcctttg agtgagctga taccgctcgc cgcagccgaa cgccccaaaa agcctcgctt
     1141 tcagcacctg tcgtttcctt tcttttcaga gggtatttta aataaaaaca ttaagttatg
     1201 acgaagaaga acggaaacgc cttaaaccgg aaaattttca taaatagcga aaacccgcga
     1261 ggtcgccgcc ccgtaacctg tcggatcacc ggaaaggacc cgtaaagtga taatgattat
     1321 catctacata tcacaacgtg cgtggagggt ctagggcggc ggatttgtcc tactcaggag
     1381 agcgttcacc gacaaacaac agataaaacg aaaggcccag tctttcgact gagcctttcg
     1441 ttttatttga tgcctctagc acgcgtacca tgggatccta tttcttaata actaaaaata
     1501 tggtataata ctcttaataa atgcagtaat acaggggctt ttcaagactg aagtctagct
     1561 gagacaaata gtgcgattac gaaatttttt agacaaaaat agtctacgag gttttagagc
     1621 tatgctgttt tgaatggtcc caaaaccctt caccttgtgg tgcttcagag aatttggttt
     1681 tagagctatg ctgttttgaa tggtcccaaa acagtttggc ggagtgggtg ccttcatacg
     1741 gagttttaga gctatgctgt tttgaatggt cccaaaactt cagcacactg agacttgttg
     1801 agttgaattc atgagtatta agtattgttt tatggctgat aaatttcttt gaatttctcc
     1861 ttgattattt gttataaaag ttataaaata atcttgttgg aaccattcaa aacagcatag
     1921 caagttaaaa taaggctagt ccgttatcaa cttgaaaaag tggcaccgag tcggtgcttt
     1981 ttttgatact tctattctac tctgactgca aaccaaaaaa acaagcgctt tcaaaacgct
     2041 tgttttatca tttttaggga aattaatctc ttaatccttt tatcattcta catttaggcg
     2101 ctgccatctt aagacgaaag ggcctcgtga tacgcctatt tttataggtt aatgtcatga
     2161 taataatggt ttcttagacg tcggaattgc cagctggggc gccctctggt aaggttggga
     2221 agccctgcaa agtaaactgg atggctttct tgccgccaag gatctgatgg cgcaggggat
     2281 caagatctga tcaagagaca ggatgaggat cgtttcgcat gattgaacaa gatggattgc
     2341 acgcaggttc tccggccgct tgggtggaga ggctattcgg ctatgactgg gcacaacaga
     2401 caatcggctg ctctgatgcc gccgtgttcc ggctgtcagc gcaggggcgc ccggttcttt
     2461 ttgtcaagac cgacctgtcc ggtgccctga atgaactgca ggacgaggca gcgcggctat
     2521 cgtggctggc cacgacgggc gttccttgcg cagctgtgct cgacgttgtc actgaagcgg
     2581 gaagggactg gctgctattg ggcgaagtgc cggggcagga tctcctgtca tctcaccttg
     2641 ctcctgccga gaaagtatcc atcatggctg atgcaatgcg gcggctgcat acgcttgatc
     2701 cggctacctg cccattcgac caccaagcga aacatcgcat cgagcgagca cgtactcgga
     2761 tggaagccgg tcttgtcgat caggatgatc tggacgaaga gcatcagggg ctcgcgccag
     2821 ccgaactgtt cgccaggctc aaggcgcgca tgcccgacgg cgaggatctc gtcgtgaccc
     2881 atggcgatgc ctgcttgccg aatatcatgg tggaaaatgg ccgcttttct ggattc
//
