LOCUS       sequence-258930-        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"
     protein_bind    35..53
                     /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)"
     terminator      complement(161..188)
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     promoter        196..230
                     /label=BBa_J23117
                     /note="Anderson constitutive promoter 
                     (https://parts.igem.org/Promoters/Catalog/Anderson). 
                     Measured strength relative to BBa_J223100: 0.06"
     RBS             242..253
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     promoter        1192..1294
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     CDS             1295..1954
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     complement(1361..1380)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     terminator      1975..2069
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     rep_origin      2183..2728
                     /direction=RIGHT
                     /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     2629..2648
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2845..2862
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    2986..3004
                     /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 gttgataacg gactagcctt attttaactt gctagggccc ttcttcaggg ccctagctct
       61 ttaacgaccg aactatataa taacttctag ttttattcga aagtaacaaa ccattttctt
      121 gcgtaaacct gtacgatcct acaggtctct agtatataaa cgcagaaagg cccacccgaa
      181 ggtgagccag tgtgattgac agctagctca gtcctaggga ttgtgctagc tactagagat
      241 taaagaggag aaatactaga tgaaaaacat aaatgccgac gacacataca gaataattaa
      301 taaaattaaa gcttgtagaa gcaataatga tattaatcaa tgcttatctg atatgactaa
      361 aatggtacat tgtgaatatt atttactcgc gatcatttat cctcattcta tggttaaatc
      421 tgatatttca atcctagata attaccctaa aaaatggagg caatattatg atgacgctaa
      481 tttaataaaa tatgatccta tagtagatta ttctaactcc aatcattcac caattaattg
      541 gaatatattt gaaaacaatg ctgtaaataa aaaatctcca aatgtaatta aagaagcgaa
      601 aacatcaggt cttatcactg ggtttagttt ccctattcat acggctaaca atggcttcgg
      661 aatgcttagt tttgcacatt cagaaaaaga caactatata gatagtttat ttttacatgc
      721 gtgtatgaac ataccattaa ttgttccttc tctagttgat aattatcgaa aaataaatat
      781 agcaaataat aaatcaaaca acgatttaac caaaagagaa aaagaatgtt tagcgtgggc
      841 atgcgaagga aaaagctctt gggatatttc aaaaatatta ggttgcagtg agcgtactgt
      901 cactttccat ttaaccaatg cgcaaatgaa actcaataca acaaaccgct gccaaagtat
      961 ttctaaagca attttaacag gagcaattga ttgcccatac tttaaaaatt aataacactg
     1021 atagtgctag tgtagatcac tactagagct cggtaccaaa gacgaacaat aagacgctga
     1081 aaagcgtctt ttttcgtttt ggtccctcta gtattaattt tgtcttcatg catgaagaca
     1141 actaggctct agaagcggcc gcgaattaga tatctggcga aaatgagacg ttgatcggca
     1201 cgtaagaggt tccaactttc accataatga aataagatca ctaccgggcg tattttttga
     1261 gttatcgaga ttttcaggag ctaaggaagc taaaatggag aaaaaaatca ctggatatac
     1321 caccgttgat atatcccaat ggcatcgtaa agaacatttt gaggcatttc agtcagttgc
     1381 tcaatgtacc tataaccaga ccgttcagct ggatattacg gcctttttaa agaccgtaaa
     1441 gaaaaataag cacaagtttt atccggcctt tattcacatt cttgcccgcc tgatgaatgc
     1501 tcatccggaa tttcgtatgg caatgaaaga cggtgagctg gtgatatggg atagtgttca
     1561 cccttgttac accgttttcc atgagcaaac tgaaacgttt tcatcgctct ggagtgaata
     1621 ccacgacgat ttccggcagt ttctacacat atattcgcaa gatgtggcgt gttacggtga
     1681 aaacctggcc tatttcccta aagggtttat tgagaatatg tttttcgtct cagccaatcc
     1741 ctgggtgagt ttcaccagtt ttgatttaaa cgtggccaat atggacaact tcttcgcccc
     1801 cgttttcacc atgggcaaat attatacgca aggcgacaag gtgctgatgc cgctggcgat
     1861 tcaggttcat catgccgttt gtgatggctt ccatgtcggc agaatgctta atgaattaca
     1921 acagtactgc gatgagtggc agggcggggc gtaatttgat atcgagctcg cttggactcc
     1981 tgttgataga tccagtaatg acctcagaac tccatctgga tttgttcaga acgctcggtt
     2041 gccgccgggc gttttttatt ggtgagaatc caagcactag taacaactta tatcgtatgg
     2101 ggctgacttc aggtgctaca tttgaagaga taaattgcac tgaaatctag aaatatttta
     2161 tctgattaat aagatgatct tcttgagatc gttttggtct gcgcgtaatc tcttgctctg
     2221 aaaacgaaaa aaccgccttg cagggcggtt tttcgaaggt tctctgagct accaactctt
     2281 tgaaccgagg taactggctt ggaggagcgc agtcaccaaa acttgtcctt tcagtttagc
     2341 cttaaccggc gcatgacttc aagactaact cctctaaatc aattaccagt ggctgctgcc
     2401 agtggtgctt ttgcatgtct ttccgggttg gactcaagac gatagttacc ggataaggcg
     2461 cagcggtcgg actgaacggg gggttcgtgc atacagtcca gcttggagcg aactgcctac
     2521 ccggaactga gtgtcaggcg tggaatgaga caaacgcggc cataacagcg gaatgacacc
     2581 ggtaaaccga aaggcaggaa caggagagcg cacgagggag ccgccagggg gaaacgcctg
     2641 gtatctttat agtcctgtcg ggtttcgcca ccactgattt gagcgtcaga tttcgtgatg
     2701 cttgtcaggg gggcggagcc tatggaaaaa cggctttgcc gcggccctct cacttccctg
     2761 ttaagtatct tcctggcatc ttccaggaaa tctccgcccc gttcgtaagc catttccgct
     2821 cgccgcagtc gaacgaccga gcgtagcgag tcagtgagcg aggaagcgga atatatccct
     2881 aggcctagta ggaccaaaac gaaaaaaggc ccccctttcg ggaggcctct tttctggaat
     2941 ttggtaccga gaagcttcaa aaaaagcacc gactcggtgc cacttgggcc cttcttcagg
     3001 gcccaa
//
