LOCUS       sequence-197168-        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-R0
FEATURES             Location/Qualifiers
     source          1..2936
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     279..298
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     terminator      complement(763..849)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     misc_feature    879..1010
                     /label=crRNA leader
                     /note="crRNA leader sequence for the Streptococcus pyogenes
                     CRISPR/Cas system"
     repeat_region   1011..1046
                     /label=DR
                     /note="direct repeat for the Streptococcus pyogenes 
                     CRISPR/Cas system"
     repeat_region   1077..1112
                     /label=DR
                     /note="direct repeat for the Streptococcus pyogenes 
                     CRISPR/Cas system"
     repeat_region   1143..1178
                     /label=DR
                     /note="direct repeat for the Streptococcus pyogenes 
                     CRISPR/Cas system"
     misc_RNA        1299..1377
                     /label=tracrRNA
                     /note="trans-activating CRISPR RNA for the Streptococcus 
                     pyogenes CRISPR/Cas9 system"
     primer_bind     complement(1522..1540)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     CDS             1719..2513
                     /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(1773..1792)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     2383..2402
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     terminator      2544..2638
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     rep_origin      join(2726..2936,1..378)
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
ORIGIN
        1 gctctgctaa tcctgttacc agtggctgct gccagtggcg ataagtcgtg tcttaccggg
       61 ttggactcaa gacgatagtt accggataag gcgcagcggt cgggctgaac ggggggttcg
      121 tgcacacagc ccagcttgga gcgaacgacc tacaccgaac tgagatacct acagcgtgag
      181 ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc
      241 agggtcggaa caggagagcg cacgagggag cttccagggg gaaacgcctg gtatctttat
      301 agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg
      361 gggcggagcc tatggaaaaa cgccagcaac gcggcctttt tacggttcct ggccttttgc
      421 tggccttttg ctcacatgtt ctttcctgcg ttatcccctg attctgtgga taaccgtatt
      481 accgcctttg agtgagctga taccgctcgc cgcagccgaa cgccccaaaa agcctcgctt
      541 tcagcacctg tcgtttcctt tcttttcaga gggtatttta aataaaaaca ttaagttatg
      601 acgaagaaga acgggaacgc cttaaaccgg aaaattttca taaatagcga aaacccgcga
      661 ggtcgccgcc ccgtaacctg tcggatcacc ggaaaggacc cgtaaagtga taatgattat
      721 catctacata tcacaacgtg cgtggagggt ctagggcggc ggatttgtcc tactcaggag
      781 agcgttcacc gacaaacaac agataaaacg aaaggcccag tctttcgact gagcctttcg
      841 ttttatttga tgcctctagc acgcgtacca tgggatccta tttcttaata actaaaaata
      901 tggtataata ctcttaataa atgcagtaat acaggggctt ttcaagactg aagtctagct
      961 gagacaaata gtgcgattac gaaatttttt agacaaaaat agtctacgag gttttagagc
     1021 tatgctgttt tgaatggtcc caaaaccctt caccttgtgg tggaacagag aatttggttt
     1081 tagagctatg ctgttttgaa tggtcccaaa acatcgcaca tcctggtcgc gacattaaga
     1141 gtgttttaga gctatgctgt tttgaatggt cccaaaactt cagcacactg agacttgttg
     1201 agttgaattc atgagtatta agtattgttt tatggctgat aaatttcttt gaatttctcc
     1261 ttgattattt gttataaaag ttataaaata atcttgttgg aaccattcaa aacagcatag
     1321 caagttaaaa taaggctagt ccgttatcaa cttgaaaaag tggcaccgag tcggtgcttt
     1381 ttttgatact tctattctac tctgactgca aaccaaaaaa acaagcgctt tcaaaacgct
     1441 tgttttatca tttttaggga aattaatctc ttaatccttt tatcattcta catttaggcg
     1501 ctgccatctt aagacgaaag ggcctcgtga tacgcctatt tttataggtt aatgtcatga
     1561 taataatggt ttcttagacg tcggaattgc cagctggggc gccctctggt aaggttggga
     1621 agccctgcaa agtaaactgg atggctttct tgccgccaag gatctgatgg cgcaggggat
     1681 caagatctga tcaagagaca ggatgaggat cgtttcgcat gattgaacaa gatggattgc
     1741 acgcaggttc tccggccgct tgggtggaga ggctattcgg ctatgactgg gcacaacaga
     1801 caatcggctg ctctgatgcc gccgtgttcc ggctgtcagc gcaggggcgc ccggttcttt
     1861 ttgtcaagac cgacctgtcc ggtgccctga atgaactgca ggacgaggca gcgcggctat
     1921 cgtggctggc cacgacgggc gttccttgcg cagctgtgct cgacgttgtc actgaagcgg
     1981 gaagggactg gctgctattg ggcgaagtgc cggggcagga tctcctgtca tctcaccttg
     2041 ctcctgccga gaaagtatcc atcatggctg atgcaatgcg gcggctgcat acgcttgatc
     2101 cggctacctg cccattcgac caccaagcga aacatcgcat cgagcgagca cgtactcgga
     2161 tggaagccgg tcttgtcgat caggatgatc tggacgaaga gcatcagggg ctcgcgccag
     2221 ccgaactgtt cgccaggctc aaggcgcgca tgcccgacgg cgaggatctc gtcgtgaccc
     2281 atggcgatgc ctgcttgccg aatatcatgg tggaaaatgg ccgcttttct ggattcatcg
     2341 actgtggccg gctgggtgtg gcggaccgct atcaggacat agcgttggct acccgtgata
     2401 ttgctgaaga gcttggcggc gaatgggctg accgcttcct cgtgctttac ggtatcgccg
     2461 ctcccgattc gcagcgcatc gccttctatc gccttcttga cgagttcttc tgagcgggac
     2521 tctggggttc gagagctcgc ttggactcct gttgatagat ccagtaatga cctcagaact
     2581 ccatctggat ttgttcagaa cgctcggttg ccgccgggcg ttttttattg gtgagaatcc
     2641 aagcactagt catgaccaaa atcccttaac gtgagttttc gttccactga gcgtcagacc
     2701 ccgtagaaaa gatcaaagga tcttcttgag atcctttttt tctgcgcgta atctgctgct
     2761 tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa gagctaccaa
     2821 ctctttttcc gaaggtaact ggcttcagca gagcgcagat accaaatact gtccttctag
     2881 tgtagccgta gttaggccac cacttcaaga actctgtagc accgcctaca tacctc
//
