LOCUS       sequence-250976-        2470 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Engineered sgRNA-LEB4 generator including Anderson promoter J23100 
            for golden gate assembly.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 2262 to 2296)
  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 2470)
  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: pLY133
FEATURES             Location/Qualifiers
     source          1..2470
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     terminator      2..59
                     /label=his operon terminator
                     /note="This putative transcriptin terminator from the E. 
                     coli his operon has a 2-bp deletion introduced during 
                     synthesis. Its efficiency has not been determined."
     primer_bind     complement(83..100)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(254..842)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(334..353)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(1075..1890)
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     CDS             complement(1075..1890)
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     1798..1817
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     1882..1901
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     primer_bind     2095..2113
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     terminator      complement(2117..2160)
                     /label=bacterial terminator
                     /note="putative bacterial transcription terminator"
     misc_feature    2163..2184
                     /label=BioBrick prefix
                     /note="BioBrick prefix for parts that do not start with 
                     ""ATG"""
     promoter        2262..2296
                     /label=﻿BBa_J23100
                     /note="Anderson constitutive promoter 
                     (https://parts.igem.org/Promoters/Catalog/Anderson). 
                     Measured strength relative to BBa_J223100: 1.0
                     "
                     /citation=[1]
     protein_bind    2334..2352
                     /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    2389..2407
                     /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)"
     misc_feature    join(2451..2470,1)
                     /label=BioBrick suffix
                     /note="universal suffix for all parts"
ORIGIN
        1 gtccggcaaa aaagggcaag gtgtcaccac cctgcccttt ttctttaaaa ccgaaaagat
       61 tacttcgcgt tatgcaggct tcctcgctca ctgactcgct gcgctcggtc gttcggctgc
      121 ggcgagcggt atcagctcac tcaaaggcgg taatacggtt atccacagaa tcaggggata
      181 acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg
      241 cgttgctggc gtttttccac aggctccgcc cccctgacga gcatcacaaa aatcgacgct
      301 caagtcagag gtggcgaaac ccgacaggac tataaagata ccaggcgttt ccccctggaa
      361 gctccctcgt gcgctctcct gttccgaccc tgccgcttac cggatacctg tccgcctttc
      421 tcccttcggg aagcgtggcg ctttctcata gctcacgctg taggtatctc agttcggtgt
      481 aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg
      541 ccttatccgg taactatcgt cttgagtcca acccggtaag acacgactta tcgccactgg
      601 cagcagccac tggtaacagg attagcagag cgaggtatgt aggcggtgct acagagttct
      661 tgaagtggtg gcctaactac ggctacacta gaagaacagt atttggtatc tgcgctctgc
      721 tgaagccagt taccttcgga aaaagagttg gtagctcttg atccggcaaa caaaccaccg
      781 ctggtagcgg tggttttttt gtttgcaagc agcagattac gcgcagaaaa aaaggatctc
      841 aagaagatcc tttgatcttt tctacggggt ctgacgctca gtggaacgaa aactcacgtt
      901 aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa
      961 aatgaagttt taaatcaatc taaagtatat atgagtaaac ttggtctgac agctcgagtc
     1021 ccgtcaagtc agcgtaatgc tctgccagtg ttacaaccaa ttaaccaatt ctgattagaa
     1081 aaactcatcg agcatcaaat gaaactgcaa tttattcata tcaggattat caataccata
     1141 tttttgaaaa agccgtttct gtaatgaagg agaaaactca ccgaggcagt tccataggat
     1201 ggcaagatcc tggtatcggt ctgcgattcc gactcgtcca acatcaatac aacctattaa
     1261 tttcccctcg tcaaaaataa ggttatcaag tgagaaatca ccatgagtga cgactgaatc
     1321 cggtgagaat ggcaaaagct tatgcatttc tttccagact tgttcaacag gccagccatt
     1381 acgctcgtca tcaaaatcac tcgcatcaac caaaccgtta ttcattcgtg attgcgcctg
     1441 agcgagacga aatacgcgat cgctgttaaa aggacaatta caaacaggaa tcgaatgcaa
     1501 ccggcgcagg aacactgcca gcgcatcaac aatattttca cctgaatcag gatattcttc
     1561 taatacctgg aatgctgttt tcccggggat cgcagtggtg agtaaccatg catcatcagg
     1621 agtacggata aaatgcttga tggtcggaag aggcataaat tccgtcagcc agtttagtct
     1681 gaccatctca tctgtaacat cattggcaac gctacctttg ccatgtttca gaaacaactc
     1741 tggcgcatcg ggcttcccat acaatcgata gattgtcgca cctgattgcc cgacattatc
     1801 gcgagcccat ttatacccat ataaatcagc atccatgttg gaatttaatc gcggcctgga
     1861 gcaagacgtt tcccgttgaa tatggctcat aacacccctt gtattactgt ttatgtaagc
     1921 agacagtttt attgttcatg atgatatatt tttatcttgt gcaatgtaac atcagagatt
     1981 ttgagacaca acgtggcttt gttgaataaa tcgaactttt gctgagttga aggatcagct
     2041 cgagtgccac ttgacgtcta agaaaccatt attatcatga cattaaccta taaaaatagg
     2101 cgtatcacga ggcagaattt cagataaaaa aaatccttag ctttcgctaa ggatgatttc
     2161 tggaattcgc ggccgcttct agaggaagac aaacgtggaa acacagaaaa aagcccgcac
     2221 ctgacagtgc gggctttttt tttcgaccaa aggtactaga gttgacggct agctcagtcc
     2281 taggtacagt gctagctact agagtgatcg ttcggaaata agtttgagag ctagggccct
     2341 gaagaagggc cctagcaagt tcaaataagg ctagtccgtt atcaacttgg gccctgaaga
     2401 agggcccaag tggcaccgag tcggtgcttt ttttgaagga cattgtcttc tactagtagc
     2461 ggccgctgca
//
