LOCUS       sequence-266878-        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 956 to 990)
  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"
     primer_bind     429..448
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     682..699
                     /label=L4440
                     /note="L4440 vector, forward primer"
     terminator      complement(723..780)
                     /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."
     misc_feature    781..801
                     /label=BioBrick suffix
                     /note="universal suffix for all parts"
     protein_bind    845..863
                     /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    900..918
                     /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)"
     promoter        complement(956..990)
                     /label=﻿BBa_J23100
                     /note="Anderson constitutive promoter 
                     (https://parts.igem.org/Promoters/Catalog/Anderson). 
                     Measured strength relative to BBa_J223100: 1.0
                     "
                     /citation=[1]
     misc_feature    1068..1089
                     /label=BioBrick prefix
                     /note="BioBrick prefix for parts that do not start with 
                     ""ATG"""
     terminator      1092..1135
                     /label=bacterial terminator
                     /note="putative bacterial transcription terminator"
     primer_bind     complement(1139..1157)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(1351..1370)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             1362..2177
                     /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             1362..2177
                     /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     complement(1435..1454)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      join(2410..2470,1..528)
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
ORIGIN
        1 gcggtggttt gtttgccgga tcaagagcta ccaactcttt ttccgaaggt aactggcttc
       61 agcagagcgc agataccaaa tactgttctt ctagtgtagc cgtagttagg ccaccacttc
      121 aagaactctg tagcaccgcc tacatacctc gctctgctaa tcctgttacc agtggctgct
      181 gccagtggcg ataagtcgtg tcttaccggg ttggactcaa gacgatagtt accggataag
      241 gcgcagcggt cgggctgaac ggggggttcg tgcacacagc ccagcttgga gcgaacgacc
      301 tacaccgaac tgagatacct acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg
      361 agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa caggagagcg cacgagggag
      421 cttccagggg gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca cctctgactt
      481 gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc tgtggaaaaa cgccagcaac
      541 gcggcctttt tacggttcct ggccttttgc tggccttttg ctcacatgtt ctttcctgcg
      601 ttatcccctg attctgtgga taaccgtatt accgcctttg agtgagctga taccgctcgc
      661 cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg aagcctgcat aacgcgaagt
      721 aatcttttcg gttttaaaga aaaagggcag ggtggtgaca ccttgccctt ttttgccgga
      781 ctgcagcggc cgctactagt agaagacaat gtccttcaaa aaaagcaccg actcggtgcc
      841 acttgggccc ttcttcaggg cccaagttga taacggacta gccttatttg aacttgctag
      901 ggcccttctt cagggcccta gctctcaaac ttatttccga acgatcactc tagtagctag
      961 cactgtacct aggactgagc tagccgtcaa ctctagtacc tttggtcgaa aaaaaaagcc
     1021 cgcactgtca ggtgcgggct tttttctgtg tttccacgtt tgtcttcctc tagaagcggc
     1081 cgcgaattcc agaaatcatc cttagcgaaa gctaaggatt ttttttatct gaaattctgc
     1141 ctcgtgatac gcctattttt ataggttaat gtcatgataa taatggtttc ttagacgtca
     1201 agtggcactc gagctgatcc ttcaactcag caaaagttcg atttattcaa caaagccacg
     1261 ttgtgtctca aaatctctga tgttacattg cacaagataa aaatatatca tcatgaacaa
     1321 taaaactgtc tgcttacata aacagtaata caaggggtgt tatgagccat attcaacggg
     1381 aaacgtcttg ctccaggccg cgattaaatt ccaacatgga tgctgattta tatgggtata
     1441 aatgggctcg cgataatgtc gggcaatcag gtgcgacaat ctatcgattg tatgggaagc
     1501 ccgatgcgcc agagttgttt ctgaaacatg gcaaaggtag cgttgccaat gatgttacag
     1561 atgagatggt cagactaaac tggctgacgg aatttatgcc tcttccgacc atcaagcatt
     1621 ttatccgtac tcctgatgat gcatggttac tcaccactgc gatccccggg aaaacagcat
     1681 tccaggtatt agaagaatat cctgattcag gtgaaaatat tgttgatgcg ctggcagtgt
     1741 tcctgcgccg gttgcattcg attcctgttt gtaattgtcc ttttaacagc gatcgcgtat
     1801 ttcgtctcgc tcaggcgcaa tcacgaatga ataacggttt ggttgatgcg agtgattttg
     1861 atgacgagcg taatggctgg cctgttgaac aagtctggaa agaaatgcat aagcttttgc
     1921 cattctcacc ggattcagtc gtcactcatg gtgatttctc acttgataac cttatttttg
     1981 acgaggggaa attaataggt tgtattgatg ttggacgagt cggaatcgca gaccgatacc
     2041 aggatcttgc catcctatgg aactgcctcg gtgagttttc tccttcatta cagaaacggc
     2101 tttttcaaaa atatggtatt gataatcctg atatgaataa attgcagttt catttgatgc
     2161 tcgatgagtt tttctaatca gaattggtta attggttgta acactggcag agcattacgc
     2221 tgacttgacg ggactcgagc tgtcagacca agtttactca tatatacttt agattgattt
     2281 aaaacttcat ttttaattta aaaggatcta ggtgaagatc ctttttgata atctcatgac
     2341 caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa
     2401 aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc
     2461 accgctacca
//
