LOCUS       sequence-337565-        4295 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Toehold switch multi-level gene expression controller (MLC).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4295)
  AUTHORS   Greco FV, Pandi A, Erb TJ, Grierson CS, Gorochowski TE
  TITLE     Harnessing the central dogma for stringent multi-level control of 
            gene expression.
  JOURNAL   Nat Commun. 2021 Mar 19;12(1):1738. doi: 10.1038/s41467-021-21995-7.
  PUBMED    33741937
REFERENCE   2  (bases 1 to 4295)
  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: "2021-03-19"; volume: "12(1):1738. doi"; pages: " 
            10.1038/s41467-021-21995-7"
COMMENT     Sequence Label: pTHS
FEATURES             Location/Qualifiers
     source          1..4295
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     969..988
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        join(1146..1151,1152..1167,1168..1174)
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    1180..1199
                     /label=lac operator (symmetric)
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG). The 
                     symmetric lac operator was optimized for tight binding of 
                     lac repressor."
     terminator      1270..1356
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      1448..1475
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     protein_bind    2396..2415
                     /label=lac operator (symmetric)
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG). The 
                     symmetric lac operator was optimized for tight binding of 
                     lac repressor."
     promoter        complement(join(2421..2427,2428..2443,2444..2449))
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     rep_origin      complement(2568..3156)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2648..2667)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     complement(3267..3286)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             3278..4093
                     /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             3278..4093
                     /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(3351..3370)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     CDS             complement(join(4220..4295,1..1007))
                     /codon_start=1
                     /gene="lacI"
                     /product="lac repressor"
                     /label=lacI
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
                     /translation="MKPVTLYDVAEYAGVSYMTVSRVVNQASHVSAKTREKVEAAMVEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIYSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
ORIGIN
        1 gtttgcgtat tgggcgccag ggtggttttt cttttcacca gtgagactgg caacagctga
       61 ttgcccttca ccgcctggcc ctgagagagt tgcagcaagc ggtccacgct ggtttgcccc
      121 agcaggcgaa aatcctgttt gatggtggtt aacggcggga tataacatga gctatcttcg
      181 gtatcgtcgt atcccactac cgagatatcc gcaccaacgc gcagcccgga ctcggtaatg
      241 gcgcgcattg cgcccagcgc catctgatcg ttggcaacca gcatcgcagt gggaacgatg
      301 ccctcattca gcatttgcat ggtttgttga aaaccggaca tggcactcca gtcgccttcc
      361 cgttccgcta tcggctgaat ttgattgcga gtgagatatt tatgccagcc agccagacgc
      421 agacgcgccg agacagaact taatgggccc gctaacagcg cgatttgctg gtgacccaat
      481 gcgaccagat gctccacgcc cagtcgcgta ccgtcctcat gggagtaaat aatactgttg
      541 atgggtgtct ggtcagagac atcaagaaat aacgccggaa cattagtgca ggcagcttcc
      601 acagcaatgg catcctggtc atccagcgga tagttaatga tcagcccact gacgcgttgc
      661 gcgagaagat tgtgcaccgc cgctttacag gcttcgacgc cgcttcgttc taccatcgac
      721 accaccacgc tggcacccag ttgatcggcg cgagatttaa tcgccgcgac aatttgcgac
      781 ggcgcgtgca gggccagact ggaggtggca acgccaatca gcaacgactg tttgcccgcc
      841 agttgttgtg ccacgcggtt gggaatgtaa ttcagctcca ccatcgccgc ttccactttt
      901 tcccgcgttt tcgcagaaac gtggctggcc tggttcacca cgcgggaaac ggtcatataa
      961 gagacaccgg catactctgc gacatcgtat aacgttactg gtttcatatt cacctccatg
     1021 aattgactct cttccgggcg ctatcatgcc ataccgcgaa aggtttttca ccattcgatg
     1081 gcgcgccgcg gctgggaatc tgagacatga gtcaagatat ttgctcggta acgtatgctg
     1141 gagtgttgac aattaatcat cggctcgtat aatgtgtgga attgtgagcg ctcacaatta
     1201 ggtgggacct attggacccg tttccaatag gtgaacaaga cgatagaaca agcatttgca
     1261 cttatagagc ttataaaacg aaaggctcag tcgaaagact gggcctttcg ttttatctgt
     1321 tgtttgtcgg tgaacgctct cctgagtagg acaaatccgc cgggagcgga tttgaacgtt
     1381 gcgaagcaac ggcccggagg gtggcgggca ggacgcccgc cataaactgc caggcatcaa
     1441 attaagcaga aggccatcct gacggatggc cttttggccc ccgaccctta gactctgtac
     1501 tcagggacca aaacgaaaaa aggcccccct ttcgggaggc ctcttttctg gaatttggta
     1561 ccgagcgcta gttgtatcgt acgtcggtct aggaatcgtt tagccgcctc acttatacaa
     1621 ctcatccatg cctaaagtaa tccccgccgc ggttacgaac tccaaaagga ccatgtgatc
     1681 acgtttctca tttgggtcct tcgataattt ggattgtgta ctcaggtagt gattgtcagg
     1741 aagtaaaacc gggccgtcgc caatgggtgt gttttgttga taatggtcag ccaattgcac
     1801 gctgccatcc tcaatgttgt gacgaatctt gaaatttacc ttaatcccgt tcttctgctt
     1861 gtcagccatg atatacacgt tgtgcgaatt atagttatac tccagtttat gtcccaaaat
     1921 gttgccatct tctttaaagt caatgccttt caattcaatg cgattcacta acgtatcccc
     1981 ctcaaacttg acctcggcgc gtgtcttata gttcccgtca tccttgaaga agattgtgcg
     2041 ctcttgcaca tagccttctg gcattgcaga tttaaagaag tcgtgctgct tcatgtgatc
     2101 tgggtaacgc gagaaacatt ggaccccgta tgtaagtgtt gtaaccagag ttggccaggg
     2161 gactggaagt tttcctgtag tacaaatgaa cttaagcgtc aatttgccgt aagtagcgtc
     2221 gccttcgccc tcgccgctaa ccgaaaactt atgaccatta acgtccccat ccaactcaac
     2281 caagatgggt acaacgcctg taaacagctc ctctccttta ctcaccatca cgcttgttct
     2341 catgtcttgt ctcctctgtt tcaagacgat agaacaagca tttgcactta ttactaattg
     2401 tgagcgctca caattccaca cattatacga gccgatgatt aattgtcaac atgcccaaag
     2461 gcggtaatac ggttatccac agaatcaggg gataacgcag gaaagaacat gtgagcaaaa
     2521 ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc
     2581 cgcccccctg acgagcatca caaaaatcga cgctcaagtc agaggtggcg aaacccgaca
     2641 ggactataaa gataccaggc gtttccccct ggaagctccc tcgtgcgctc tcctgttccg
     2701 accctgccgc ttaccggata cctgtccgcc tttctccctt cgggaagcgt ggcgctttct
     2761 catagctcac gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt
     2821 gtgcacgaac cccccgttca gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag
     2881 tccaacccgg taagacacga cttatcgcca ctggcagcag ccactggtaa caggattagc
     2941 agagcgaggt atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa ctacggctac
     3001 actagaagga cagtatttgg tatctgcgct ctgctgaagc cagttacctt cggaaaaaga
     3061 gttggtagct cttgatccgg caaacaaacc accgctggta gcggtggttt ttttgtttgc
     3121 aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag atcctttgat cttttctacg
     3181 gggtctgacg ctcagtggaa cgaaaactca cgttaaggga ttttggtcat gaacaataaa
     3241 actgtctgct tacataaaca gtaatacaag gggtgttatg agccatattc aacgggaaac
     3301 gtcttgctct aggccgcgat taaattccaa catggatgct gatttatatg ggtataaatg
     3361 ggctcgcgat aatgtcgggc aatcaggtgc gacaatctat cgattgtatg ggaagcccga
     3421 tgcgccagag ttgtttctga aacatggcaa aggtagcgtt gccaatgatg ttacagatga
     3481 gatggtcaga ctaaactggc tgacggaatt tatgcctctt ccgaccatca agcattttat
     3541 ccgtactcct gatgatgcat ggttactcac cactgcgatc cccgggaaaa cagcattcca
     3601 ggtattagaa gaatatcctg attcaggtga aaatattgtt gatgcgctgg cagtgttcct
     3661 gcgccggttg cattcgattc ctgtttgtaa ttgtcctttt aacagcgatc gcgtatttcg
     3721 tctcgctcag gcgcaatcac gaatgaataa cggtttggtt gatgcgagtg attttgatga
     3781 cgagcgtaat ggctggcctg ttgaacaagt ctggaaagaa atgcataaac ttttgccatt
     3841 ctcaccggat tcagtcgtca ctcatggtga tttctcactt gataacctta tttttgacga
     3901 ggggaaatta ataggttgta ttgatgttgg acgagtcgga atcgcagacc gataccagga
     3961 tcttgccatc ctatggaact gcctcggtga gttttctcct tcattacaga aacggctttt
     4021 tcaaaaatat ggtattgata atcctgatat gaataaattg cagtttcatt tgatgctcga
     4081 tgagtttttc taagaattaa ttcatgagcg gatacatatt tgaatgtatt tagaaaaata
     4141 aacaaatagg ggttccgcgg tgaagtgaaa agaaaaaagg ccgcagagcg gcctttttag
     4201 ttagatcgga ccccacatct cactgcccgc tttccagtcg ggaaacctgt cgtgccagct
     4261 gcattaatga atcggccaac gcgcggggag aggcg
//
