LOCUS       sequence-258323-        3006 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Engineered sgRNA-LEA2 (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: pLY76
FEATURES             Location/Qualifiers
     source          1..3006
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(3..20)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(137..682)
                     /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(217..236)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     terminator      complement(796..890)
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     CDS             complement(911..1570)
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     1485..1504
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     promoter        complement(1571..1673)
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     RBS             2612..2623
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     promoter        complement(2635..2669)
                     /label=BBa_J23117
                     /note="Anderson constitutive promoter 
                     (https://parts.igem.org/Promoters/Catalog/Anderson). 
                     Measured strength relative to BBa_J223100: 0.06"
     terminator      2677..2704
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     protein_bind    2812..2830
                     /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    2867..2885
                     /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 tcctcgctca ctgactcgct acgctcggtc gttcgactgc ggcgagcgga aatggcttac
       61 gaacggggcg gagatttcct ggaagatgcc aggaagatac ttaacaggga agtgagaggg
      121 ccgcggcaaa gccgtttttc cataggctcc gcccccctga caagcatcac gaaatctgac
      181 gctcaaatca gtggtggcga aacccgacag gactataaag ataccaggcg tttccccctg
      241 gcggctccct cgtgcgctct cctgttcctg cctttcggtt taccggtgtc attccgctgt
      301 tatggccgcg tttgtctcat tccacgcctg acactcagtt ccgggtaggc agttcgctcc
      361 aagctggact gtatgcacga accccccgtt cagtccgacc gctgcgcctt atccggtaac
      421 tatcgtcttg agtccaaccc ggaaagacat gcaaaagcac cactggcagc agccactggt
      481 aattgattta gaggagttag tcttgaagtc atgcgccggt taaggctaaa ctgaaaggac
      541 aagttttggt gactgcgctc ctccaagcca gttacctcgg ttcaaagagt tggtagctca
      601 gagaaccttc gaaaaaccgc cctgcaaggc ggttttttcg ttttcagagc aagagattac
      661 gcgcagacca aaacgatctc aagaagatca tcttattaat cagataaaat atttctagat
      721 ttcagtgcaa tttatctctt caaatgtagc acctgaagtc agccccatac gatataagtt
      781 gttactagtg cttggattct caccaataaa aaacgcccgg cggcaaccga gcgttctgaa
      841 caaatccaga tggagttctg aggtcattac tggatctatc aacaggagtc caagcgagct
      901 cgatatcaaa ttacgccccg ccctgccact catcgcagta ctgttgtaat tcattaagca
      961 ttctgccgac atggaagcca tcacaaacgg catgatgaac ctgaatcgcc agcggcatca
     1021 gcaccttgtc gccttgcgta taatatttgc ccatggtgaa aacgggggcg aagaagttgt
     1081 ccatattggc cacgtttaaa tcaaaactgg tgaaactcac ccagggattg gctgagacga
     1141 aaaacatatt ctcaataaac cctttaggga aataggccag gttttcaccg taacacgcca
     1201 catcttgcga atatatgtgt agaaactgcc ggaaatcgtc gtggtattca ctccagagcg
     1261 atgaaaacgt ttcagtttgc tcatggaaaa cggtgtaaca agggtgaaca ctatcccata
     1321 tcaccagctc accgtctttc attgccatac gaaattccgg atgagcattc atcaggcggg
     1381 caagaatgtg aataaaggcc ggataaaact tgtgcttatt tttctttacg gtctttaaaa
     1441 aggccgtaat atccagctga acggtctggt tataggtaca ttgagcaact gactgaaatg
     1501 cctcaaaatg ttctttacga tgccattggg atatatcaac ggtggtatat ccagtgattt
     1561 ttttctccat tttagcttcc ttagctcctg aaaatctcga taactcaaaa aatacgcccg
     1621 gtagtgatct tatttcatta tggtgaaagt tggaacctct tacgtgccga tcaacgtctc
     1681 attttcgcca gatatctaat tcgcggccgc ttctagagcc tagttgtctt catgcatgaa
     1741 gacaaaatta atactagagg gaccaaaacg aaaaaagacg cttttcagcg tcttattgtt
     1801 cgtctttggt accgagctct agtagtgatc tacactagca ctatcagtgt tattaatttt
     1861 taaagtatgg gcaatcaatt gctcctgtta aaattgcttt agaaatactt tggcagcggt
     1921 ttgttgtatt gagtttcatt tgcgcattgg ttaaatggaa agtgacagta cgctcactgc
     1981 aacctaatat ttttgaaata tcccaagagc tttttccttc gcatgcccac gctaaacatt
     2041 ctttttctct tttggttaaa tcgttgtttg atttattatt tgctatattt atttttcgat
     2101 aattatcaac tagagaagga acaattaatg gtatgttcat acacgcatgt aaaaataaac
     2161 tatctatata gttgtctttt tctgaatgtg caaaactaag cattccgaag ccattgttag
     2221 ccgtatgaat agggaaacta aacccagtga taagacctga tgttttcgct tctttaatta
     2281 catttggaga ttttttattt acagcattgt tttcaaatat attccaatta attggtgaat
     2341 gattggagtt agaataatct actataggat catattttat taaattagcg tcatcataat
     2401 attgcctcca ttttttaggg taattatcta ggattgaaat atcagattta accatagaat
     2461 gaggataaat gatcgcgagt aaataatatt cacaatgtac cattttagtc atatcagata
     2521 agcattgatt aatatcatta ttgcttctac aagctttaat tttattaatt attctgtatg
     2581 tgtcgtcggc atttatgttt ttcatctagt atttctcctc tttaatctct agtagctagc
     2641 acaatcccta ggactgagct agctgtcaat cacactggct caccttcggg tgggcctttc
     2701 tgcgtttata tactagagac ctgtaggatc gtacaggttt acgcaagaaa atggtttgtt
     2761 actttcgaat aaaactagaa gttattatat agttcggtcg tttgagagct agggccctga
     2821 agaagggccc tagcaagttc aaataaggct agtccgttat caacttgggc cctgaagaag
     2881 ggcccaagtg gcaccgagtc ggtgcttttt ttgaagcttc tcggtaccaa attccagaaa
     2941 agaggcctcc cgaaaggggg gccttttttc gttttggtcc tactaggcct agggatatat
     3001 tccgct
//
