LOCUS       sequence-375359-        5766 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Clostridium expression vector with TetR-repressed NanoLuc 
            expression.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5766)
  AUTHORS   Rostain W, Zaplana T, Boutard M, Baum C, Tabuteau S, Sanitha M, 
            Ramya M, Guss A, Ettwiller L, Tolonen AC
  TITLE     Tuning of Gene Expression in Clostridium phytofermentans Using 
            Synthetic Promoters and CRISPRi.
  JOURNAL   ACS Synth Biol. 2022 Dec 16;11(12):4077-4088. doi: 
            10.1021/acssynbio.2c00385. Epub 2022 Nov 25.
  PUBMED    36427328
REFERENCE   2  (bases 1 to 5766)
  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"; doi: 
            "10.1021/acssynbio.2c00385"; journalName: "ACS Synth Biol"; date: 
            "2022-12-16- 16"; volume: "11"; issue: "12"; pages: "4077-4088"
COMMENT     Sequence Label: pQnl_tet
FEATURES             Location/Qualifiers
     source          1..5766
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      2887..3475
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3376..3395
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3629..3646
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     3740..3756
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     3740..3756
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     protein_bind    3826..3844
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O1 for the tetR and tetA genes"
     CDS             3890..4405
                     /codon_start=1
                     /product="NanoLuc(R) luciferase"
                     /label=Nluc
                     /note="human codon-optimized"
                     /translation="MVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQR
                     IVLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGV
                     TPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGW
                     RLCERILA"
     CDS             4569..5189
                     /codon_start=1
                     /gene="tetR from transposon Tn10"
                     /product="tetracycline repressor TetR"
                     /label=TetR
                     /note="TetR binds to the tetracycline operator tetO to 
                     inhibit transcription. This inhibition can be relieved by 
                     adding tetracycline or doxycycline."
                     /translation="SRLDKSKVINSALELLNEVGIEGLTTRKLAQKLGVEQPTLYWHVK
                     NKRALLDALAIEMLDRHHTHFCPLEGESWQDFLRNNAKSFRCALLSHRDGAKVHLGTRP
                     TEKQYETLENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEDQEHQVAKEERETPTTD
                     SMPPLLRQAIELFDHQGAEPAFLFGLELIICGLEKQLKCESG*"
     primer_bind     complement(4639..4658)
                     /label=Tet-R
                     /note="Tetracycline resistance gene, reverse primer"
ORIGIN
        1 atgactaatt ataatcaaaa aaatgaaaat aaacaagagg taaaaactgc tttagagaaa
       61 tgtactgata aaaaaagaaa aaatcctaga tttacgtcat acatagcacc tttaactact
      121 aagaaaaata ttgaaaggac ttccacttgt ggagattatt tgtttatgtt gagtgatgca
      181 gacttagaac attttaaatt acataaaggt aatttttgcg gtaatagatt ttgtccaatg
      241 tgtagttggc gacttgcttg taaggatagt ttagaaatat ctattcttat ggagcattta
      301 agaaaagaag aaaataaaga gtttatattt ttaactctta caactccaaa tgtaaaaagt
      361 tatgatctta attattctat taaacaatat aataaatctt ttaaaaaatt aatggagcgt
      421 aaggaagtta aggatataac taaaggttat ataagaaaat tagaagtaac ttaccaaaag
      481 gaaaaataca taacaaagga tttatggaaa ataaaaaaag attattatca aaaaaaagga
      541 cttgaaattg gtgatttaga acctaatttt gatacttata atcctcattt tcatgtagtt
      601 attgcagtta ataaaagtta ttttacagat aaaaattatt atataaatcg agaaagatgg
      661 ttggaattat ggaagtttgc tactaaggat gattctataa ctcaagttga tgttagaaaa
      721 gcaaaaatta atgattataa agaggtttac gaacttgcga aatattcagc taaagacact
      781 gattatttaa tatcgaggcc agtatttgaa attttttata aagcattaaa aggcaagcag
      841 gtattagttt ttagtggatt ttttaaagat gcacacaaat tgtacaagca aggaaaactt
      901 gatgtttata aaaagaaaga tgaaattaaa tatgtctata tagtttatta taattggtgc
      961 aaaaaacaat atgaaaaaac tagaataagg gaacttacgg aagatgaaaa agaagaatta
     1021 aatcaagatt taatagatgg aatagaaata gattaaagtg taactatact ttatatatat
     1081 atgattaaaa aaataaaaaa caacagccta ttaggttgtt gttttttatt ttctttatta
     1141 atttttttaa tttttagttt ttagttcttt tttaaaataa gtttcagcct ctttttcaat
     1201 attttttaaa gaaggagtat ttgcatgaat tgcctttttt ctaacagact taggaaatat
     1261 tttaacagta tcttcttgcg ccggtgattt tggaacttca taacttacta atttataatt
     1321 attattttct tttttaattg taacagttgc aaaagaagct gaacctgttc cttcaactag
     1381 tttatcatct tcaatataat attcttgacc tatatagtat aaatatattt ttattatatt
     1441 tttacttttt tctgaatcta ttattttata atcataaaaa gttttaccac caaaagaagg
     1501 ttgtactcct tctggtccaa catatttttt tactatatta tctaaataat ttttgggaac
     1561 tggtgttgta atttgattaa tcgaacaacc agttatactt aaaggaatta taactataaa
     1621 aatatatagg attatctttt taaatttcat tattggcctc ctttttatta aatttatgtt
     1681 accataaaaa ggacataacg ggaatatgta gaatattttt aatgtagaca aaattttaca
     1741 taaatataaa gaaaggaagt gtttgtttaa attttatagc aaactatcaa aaattagggg
     1801 gataaaaatt tatgaaaaaa aggttttcga tgttattttt atgtttaact ttaatagttt
     1861 gtggtttatt tacaaattcg gccggccgaa gcaaacttaa gagtgtgttg atagtgcagt
     1921 atcttaaaat tttgtataat aggaattgaa gttaaattag atgctaaaaa tttgtaatta
     1981 agaaggagtg attacatgaa caaaaatata aaatattctc aaaacttttt aacgagtgaa
     2041 aaagtactca accaaataat aaaacaattg aatttaaaag aaaccgatac cgtttacgaa
     2101 attggaacag gtaaagggca tttaacgacg aaactggcta aaataagtaa acaggtaacg
     2161 tctattgaat tagacagtca tctattcaac ttatcgtcag aaaaattaaa actgaatact
     2221 cgtgtcactt taattcacca agatattcta cagtttcaat tccctaacaa acagaggtat
     2281 aaaattgttg ggagtattcc ttaccattta agcacacaaa ttattaaaaa agtggttttt
     2341 gaaagccatg cgtctgacat ctatctgatt gttgaagaag gattctacaa gcgtaccttg
     2401 gatattcacc gaacactagg gttgctcttg cacactcaag tctcgattca gcaattgctt
     2461 aagctgccag cggaatgctt tcatcctaaa ccaaaagtaa acagtgtctt aataaaactt
     2521 acccgccata ccacagatgt tccagataaa tattggaagc tatatacgta ctttgtttca
     2581 aaatgggtca atcgagaata tcgtcaactg tttactaaaa atcagtttca tcaagcaatg
     2641 aaacacgcca aagtaaacaa tttaagtacc gttacttatg agcaagtatt gtctattttt
     2701 aatagttatc tattatttaa cgggaggaaa taattctatg agtcgctttt gtaaatttgg
     2761 aaagttacac gttactaaag ggaatgtgtt taaactcctt tttgataatc tcatgaccaa
     2821 aatcccttaa cgtgagtttt cgttccactg agcgtcagac cccgtagaaa agatcaaagg
     2881 atcttcttga gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc
     2941 gctaccagcg gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac
     3001 tggcttcagc agagcgcaga taccaaatac tgttcttcta gtgtagccgt agttaggcca
     3061 ccacttcaag aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt
     3121 ggctgctgcc agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttacc
     3181 ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc acacagccca gcttggagcg
     3241 aacgacctac accgaactga gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc
     3301 cgaagggaga aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcac
     3361 gagggagctt ccagggggaa acgcctggta tctttatagt cctgtcgggt ttcgccacct
     3421 ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc
     3481 cagcaacgcg gcctttttac ggttcctggc cttttgctgg ccttttgctc acatgttctt
     3541 tcctgcgtta tcccctgatt ctgtggataa ccgtattacc gcctttgagt gagctgatac
     3601 cgctcgccgc agccgaacga ccgagcgcag cgagtcagtg agcgaggaag cggaagagcg
     3661 cccaatacgc agggccccct gcaggataaa aaaattgtag ataaatttta taaaatagtt
     3721 ttatctacaa tttttttatc aggaaacagc tatgaccgcg gccgctgtat ccatatgacc
     3781 atgattacga attcgaagac gcttccctat cagtgataga ttgaaactct atcattgata
     3841 gagtataata tcttcccgaa cacagtatat aaggcaaagg aggtttttta tggtcttcac
     3901 actcgaagat ttcgttgggg actggcgaca gacagccggc tacaacctgg accaagtcct
     3961 tgaacaggga ggtgtgtcca gtttgtttca gaatctcggg gtgtccgtaa ctccgatcca
     4021 aaggattgtc ctgagcggtg aaaatgggct gaagatcgac atccatgtca tcatcccgta
     4081 tgaaggtctg agcggcgacc aaatgggcca gatcgaaaaa atttttaagg tggtgtaccc
     4141 tgtggatgat catcacttta aggtgatcct gcactatggc acactggtaa tcgacggggt
     4201 tacgccgaac atgatcgact atttcggacg gccgtatgaa ggcatcgccg tgttcgacgg
     4261 caaaaagatc actgtaacag ggaccctgtg gaacggcaac aaaattatcg acgagcgcct
     4321 gatcaacccc gacggctccc tgctgttccg agtaaccatc aacggagtga ccggctggcg
     4381 gctgtgcgaa cgcattctgg cgtaatattt atttctattg agtaaaataa aaaagatggt
     4441 gagataatct caccatcttt ttttctttta aatccgtcga ccttagaagc aaacttagga
     4501 tccaaaaata tattgataaa aataataata gtgggtataa ttaagttgtt aggaggttag
     4561 ttagaatatc tagattagat aaaagtaaag tgattaacag cgcattagag ctgcttaatg
     4621 aggtcggaat cgaaggttta acaacccgta aactcgccca gaagctaggt gtagagcagc
     4681 ctacattgta ttggcatgta aaaaataagc gggctttgct cgacgcctta gccattgaga
     4741 tgttagatag gcaccatact cacttttgcc ctttagaagg ggaaagctgg caagattttt
     4801 tacgtaataa cgctaaaagt tttagatgtg ctttactaag tcatcgcgat ggagcaaaag
     4861 tacatttagg tacacggcct acagaaaaac agtatgaaac tctcgaaaat caattagcct
     4921 ttttatgcca acaaggtttt tcactagaga atgcattata tgcactcagc gctgtggggc
     4981 attttacttt aggttgcgta ttggaagatc aagagcatca agtcgctaaa gaagaaaggg
     5041 aaacacctac tactgatagt atgccgccat tattacgaca agctatcgaa ttatttgatc
     5101 accaaggtgc agagccagcc ttcttattcg gccttgaatt gatcatatgc ggattagaaa
     5161 aacaacttaa atgtgaaagt gggtaataaa gcctcttccg ctagcataaa aataagaagc
     5221 ctgcatttgc aggcttctta tttttatggc gcgccgttct gaatccttag ctaatggttc
     5281 aacaggtaac tatgacgaag atagcaccct ggataagtct gtaatggatt ctaaggcatt
     5341 taatgaagac gtgtatataa aatgtgctaa tgaaaaagaa aatgcgttaa aagagcctaa
     5401 aatgagttca aatggttttg aaattgattg gtagtttaat ttaatatatt ttttctattg
     5461 gctatctcga tacctataga atcttctgtt cacttttgtt tttgaaatat aaaaaggggc
     5521 tttttagccc ctttttttta aaactccgga ggagtttctt cattcttgat actatacgta
     5581 actatttcga tttgacttca ttgtcaatta agctagtaaa atcaatggtt aaaaaacaaa
     5641 aaacttgcat ttttctacct agtaatttat aattttaagt gtcgagttta aaagtataat
     5701 ttaccaggaa aggagcaagt tttttaataa ggaaaaattt ttccttttaa aattctattt
     5761 cgttat
//
