LOCUS       sequence-258324-        3006 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Engineered sgRNA-LEB2 (2 BoxB aptamers) generator circuit including 
            promoter Plux2.
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: pLY77
FEATURES             Location/Qualifiers
     source          1..3006
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    48..66
                     /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(174..201)
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     promoter        209..243
                     /label=BBa_J23117
                     /note="Anderson constitutive promoter 
                     (https://parts.igem.org/Promoters/Catalog/Anderson). 
                     Measured strength relative to BBa_J223100: 0.06"
     RBS             255..266
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     promoter        1205..1307
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     CDS             1308..1967
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     complement(1374..1393)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     terminator      1988..2082
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     rep_origin      2196..2741
                     /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     2642..2661
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2858..2875
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    join(2999..3006,1..11)
                     /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 cttcagggcc caagttgata acggactagc cttatttgaa cttgctaggg cccttcttca
       61 gggccctagc tctcaaacca aaacaacgtc tcaaaaattc tagttttatt cgaaagtaac
      121 aaaccatttt cttgcgtaaa cctgtacgat cctacaggtc tctagtatat aaacgcagaa
      181 aggcccaccc gaaggtgagc cagtgtgatt gacagctagc tcagtcctag ggattgtgct
      241 agctactaga gattaaagag gagaaatact agatgaaaaa cataaatgcc gacgacacat
      301 acagaataat taataaaatt aaagcttgta gaagcaataa tgatattaat caatgcttat
      361 ctgatatgac taaaatggta cattgtgaat attatttact cgcgatcatt tatcctcatt
      421 ctatggttaa atctgatatt tcaatcctag ataattaccc taaaaaatgg aggcaatatt
      481 atgatgacgc taatttaata aaatatgatc ctatagtaga ttattctaac tccaatcatt
      541 caccaattaa ttggaatata tttgaaaaca atgctgtaaa taaaaaatct ccaaatgtaa
      601 ttaaagaagc gaaaacatca ggtcttatca ctgggtttag tttccctatt catacggcta
      661 acaatggctt cggaatgctt agttttgcac attcagaaaa agacaactat atagatagtt
      721 tatttttaca tgcgtgtatg aacataccat taattgttcc ttctctagtt gataattatc
      781 gaaaaataaa tatagcaaat aataaatcaa acaacgattt aaccaaaaga gaaaaagaat
      841 gtttagcgtg ggcatgcgaa ggaaaaagct cttgggatat ttcaaaaata ttaggttgca
      901 gtgagcgtac tgtcactttc catttaacca atgcgcaaat gaaactcaat acaacaaacc
      961 gctgccaaag tatttctaaa gcaattttaa caggagcaat tgattgccca tactttaaaa
     1021 attaataaca ctgatagtgc tagtgtagat cactactaga gctcggtacc aaagacgaac
     1081 aataagacgc tgaaaagcgt cttttttcgt tttggtccct ctagtattaa ttttgtcttc
     1141 atgcatgaag acaactaggc tctagaagcg gccgcgaatt agatatctgg cgaaaatgag
     1201 acgttgatcg gcacgtaaga ggttccaact ttcaccataa tgaaataaga tcactaccgg
     1261 gcgtattttt tgagttatcg agattttcag gagctaagga agctaaaatg gagaaaaaaa
     1321 tcactggata taccaccgtt gatatatccc aatggcatcg taaagaacat tttgaggcat
     1381 ttcagtcagt tgctcaatgt acctataacc agaccgttca gctggatatt acggcctttt
     1441 taaagaccgt aaagaaaaat aagcacaagt tttatccggc ctttattcac attcttgccc
     1501 gcctgatgaa tgctcatccg gaatttcgta tggcaatgaa agacggtgag ctggtgatat
     1561 gggatagtgt tcacccttgt tacaccgttt tccatgagca aactgaaacg ttttcatcgc
     1621 tctggagtga ataccacgac gatttccggc agtttctaca catatattcg caagatgtgg
     1681 cgtgttacgg tgaaaacctg gcctatttcc ctaaagggtt tattgagaat atgtttttcg
     1741 tctcagccaa tccctgggtg agtttcacca gttttgattt aaacgtggcc aatatggaca
     1801 acttcttcgc ccccgttttc accatgggca aatattatac gcaaggcgac aaggtgctga
     1861 tgccgctggc gattcaggtt catcatgccg tttgtgatgg cttccatgtc ggcagaatgc
     1921 ttaatgaatt acaacagtac tgcgatgagt ggcagggcgg ggcgtaattt gatatcgagc
     1981 tcgcttggac tcctgttgat agatccagta atgacctcag aactccatct ggatttgttc
     2041 agaacgctcg gttgccgccg ggcgtttttt attggtgaga atccaagcac tagtaacaac
     2101 ttatatcgta tggggctgac ttcaggtgct acatttgaag agataaattg cactgaaatc
     2161 tagaaatatt ttatctgatt aataagatga tcttcttgag atcgttttgg tctgcgcgta
     2221 atctcttgct ctgaaaacga aaaaaccgcc ttgcagggcg gtttttcgaa ggttctctga
     2281 gctaccaact ctttgaaccg aggtaactgg cttggaggag cgcagtcacc aaaacttgtc
     2341 ctttcagttt agccttaacc ggcgcatgac ttcaagacta actcctctaa atcaattacc
     2401 agtggctgct gccagtggtg cttttgcatg tctttccggg ttggactcaa gacgatagtt
     2461 accggataag gcgcagcggt cggactgaac ggggggttcg tgcatacagt ccagcttgga
     2521 gcgaactgcc tacccggaac tgagtgtcag gcgtggaatg agacaaacgc ggccataaca
     2581 gcggaatgac accggtaaac cgaaaggcag gaacaggaga gcgcacgagg gagccgccag
     2641 ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg ccaccactga tttgagcgtc
     2701 agatttcgtg atgcttgtca ggggggcgga gcctatggaa aaacggcttt gccgcggccc
     2761 tctcacttcc ctgttaagta tcttcctggc atcttccagg aaatctccgc cccgttcgta
     2821 agccatttcc gctcgccgca gtcgaacgac cgagcgtagc gagtcagtga gcgaggaagc
     2881 ggaatatatc cctaggccta gtaggaccaa aacgaaaaaa ggcccccctt tcgggaggcc
     2941 tcttttctgg aatttggtac cgagaagctt caaaaaaagc accgactcgg tgccacttgg
     3001 gccctt
//
