LOCUS       sequence-250942-        3006 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Engineered sgRNA-LEA2-WTmis2 generator circuit including promoter 
            Plux2, 2 BoxB aptamers and 2 mis-matches in sgRNA scaffold.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3006)
  AUTHORS   Liu Y, Wan X, Wang B
  TITLE     Engineered CRISPRa enables programmable eukaryote-like gene 
            activation in bacteria.
  JOURNAL   Nat Commun. 2019 Aug 26;10(1):3693. doi: 10.1038/s41467-019-11479-0.
  PUBMED    31451697
REFERENCE   2  (bases 1 to 3006)
  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: "Nat 
            Commun."; date: "2019-08-26"; volume: "10(1):3693. doi"; pages: " 
            10.1038/s41467-019-11479-0"
COMMENT     Sequence Label: pLY86
FEATURES             Location/Qualifiers
     source          1..3006
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(27..44)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(161..706)
                     /direction=LEFT
                     /label=p15A ori
                     /note="Plasmids containing the medium-copy-number p15A 
                     origin of replication can be propagated in E. coli cells 
                     that contain a second plasmid with the ColE1 origin.
                     "
     primer_bind     complement(241..260)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     terminator      complement(820..914)
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     CDS             complement(935..1594)
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     1509..1528
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     promoter        complement(1595..1697)
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     RBS             2636..2647
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     promoter        complement(2659..2693)
                     /label=BBa_J23117
                     /note="Anderson constitutive promoter 
                     (https://parts.igem.org/Promoters/Catalog/Anderson). 
                     Measured strength relative to BBa_J223100: 0.06"
     terminator      2701..2728
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     protein_bind    2836..2854
                     /label=boxB
                     /bound_moiety="lambda-N peptide"
                     /note="lambda N peptide binding site consisting of an RNA 
                     hairpin within the N-utliization (nut) site of the lambda 
                     bacteriophage transcript (Tan and Frankel, 1995)"
     protein_bind    2891..2909
                     /label=boxB
                     /bound_moiety="lambda-N peptide"
                     /note="lambda N peptide binding site consisting of an RNA 
                     hairpin within the N-utliization (nut) site of the lambda 
                     bacteriophage transcript (Tan and Frankel, 1995)"
ORIGIN
        1 ctaggcctag ggatatattc cgcttcctcg ctcactgact cgctacgctc ggtcgttcga
       61 ctgcggcgag cggaaatggc ttacgaacgg ggcggagatt tcctggaaga tgccaggaag
      121 atacttaaca gggaagtgag agggccgcgg caaagccgtt tttccatagg ctccgccccc
      181 ctgacaagca tcacgaaatc tgacgctcaa atcagtggtg gcgaaacccg acaggactat
      241 aaagatacca ggcgtttccc cctggcggct ccctcgtgcg ctctcctgtt cctgcctttc
      301 ggtttaccgg tgtcattccg ctgttatggc cgcgtttgtc tcattccacg cctgacactc
      361 agttccgggt aggcagttcg ctccaagctg gactgtatgc acgaaccccc cgttcagtcc
      421 gaccgctgcg ccttatccgg taactatcgt cttgagtcca acccggaaag acatgcaaaa
      481 gcaccactgg cagcagccac tggtaattga tttagaggag ttagtcttga agtcatgcgc
      541 cggttaaggc taaactgaaa ggacaagttt tggtgactgc gctcctccaa gccagttacc
      601 tcggttcaaa gagttggtag ctcagagaac cttcgaaaaa ccgccctgca aggcggtttt
      661 ttcgttttca gagcaagaga ttacgcgcag accaaaacga tctcaagaag atcatcttat
      721 taatcagata aaatatttct agatttcagt gcaatttatc tcttcaaatg tagcacctga
      781 agtcagcccc atacgatata agttgttact agtgcttgga ttctcaccaa taaaaaacgc
      841 ccggcggcaa ccgagcgttc tgaacaaatc cagatggagt tctgaggtca ttactggatc
      901 tatcaacagg agtccaagcg agctcgatat caaattacgc cccgccctgc cactcatcgc
      961 agtactgttg taattcatta agcattctgc cgacatggaa gccatcacaa acggcatgat
     1021 gaacctgaat cgccagcggc atcagcacct tgtcgccttg cgtataatat ttgcccatgg
     1081 tgaaaacggg ggcgaagaag ttgtccatat tggccacgtt taaatcaaaa ctggtgaaac
     1141 tcacccaggg attggctgag acgaaaaaca tattctcaat aaacccttta gggaaatagg
     1201 ccaggttttc accgtaacac gccacatctt gcgaatatat gtgtagaaac tgccggaaat
     1261 cgtcgtggta ttcactccag agcgatgaaa acgtttcagt ttgctcatgg aaaacggtgt
     1321 aacaagggtg aacactatcc catatcacca gctcaccgtc tttcattgcc atacgaaatt
     1381 ccggatgagc attcatcagg cgggcaagaa tgtgaataaa ggccggataa aacttgtgct
     1441 tatttttctt tacggtcttt aaaaaggccg taatatccag ctgaacggtc tggttatagg
     1501 tacattgagc aactgactga aatgcctcaa aatgttcttt acgatgccat tgggatatat
     1561 caacggtggt atatccagtg atttttttct ccattttagc ttccttagct cctgaaaatc
     1621 tcgataactc aaaaaatacg cccggtagtg atcttatttc attatggtga aagttggaac
     1681 ctcttacgtg ccgatcaacg tctcattttc gccagatatc taattcgcgg ccgcttctag
     1741 agcctagttg tcttcatgca tgaagacaaa attaatacta gagggaccaa aacgaaaaaa
     1801 gacgcttttc agcgtcttat tgttcgtctt tggtaccgag ctctagtagt gatctacact
     1861 agcactatca gtgttattaa tttttaaagt atgggcaatc aattgctcct gttaaaattg
     1921 ctttagaaat actttggcag cggtttgttg tattgagttt catttgcgca ttggttaaat
     1981 ggaaagtgac agtacgctca ctgcaaccta atatttttga aatatcccaa gagctttttc
     2041 cttcgcatgc ccacgctaaa cattcttttt ctcttttggt taaatcgttg tttgatttat
     2101 tatttgctat atttattttt cgataattat caactagaga aggaacaatt aatggtatgt
     2161 tcatacacgc atgtaaaaat aaactatcta tatagttgtc tttttctgaa tgtgcaaaac
     2221 taagcattcc gaagccattg ttagccgtat gaatagggaa actaaaccca gtgataagac
     2281 ctgatgtttt cgcttcttta attacatttg gagatttttt atttacagca ttgttttcaa
     2341 atatattcca attaattggt gaatgattgg agttagaata atctactata ggatcatatt
     2401 ttattaaatt agcgtcatca taatattgcc tccatttttt agggtaatta tctaggattg
     2461 aaatatcaga tttaaccata gaatgaggat aaatgatcgc gagtaaataa tattcacaat
     2521 gtaccatttt agtcatatca gataagcatt gattaatatc attattgctt ctacaagctt
     2581 taattttatt aattattctg tatgtgtcgt cggcatttat gtttttcatc tagtatttct
     2641 cctctttaat ctctagtagc tagcacaatc cctaggactg agctagctgt caatcacact
     2701 ggctcacctt cgggtgggcc tttctgcgtt tatatactag agacctgtag gatcgtacag
     2761 gtttacgcaa gaaaatggtt tgttactttc gaataaaact agaagttatt atatagttcg
     2821 gtcgttaaag agctagggcc ctgaagaagg gccctagcaa gttaaaataa ggctagtccg
     2881 ttatcaactt gggccctgaa gaagggccca agtggcaccg agtcggtgct ttttttgaag
     2941 cttctcggta ccaaattcca gaaaagaggc ctcccgaaag gggggccttt tttcgttttg
     3001 gtccta
//
