LOCUS       sequence-378049-        8290 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Clostridium CRISPRi plasmid with TetR-repressible dCas12a (LbCpf1) 
            expression.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8290)
  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 8290)
  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: pQdC12a
FEATURES             Location/Qualifiers
     source          1..8290
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             1359..1982
                     /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="MSRLDKSKVINSALELLNEVGIEGLTTRKLAQKLGVEQPTLYWHV
                     KNKRALLDALAIEMLDRHHTHFCPLEGESWQDFLRNNAKSFRCALLSHRDGAKVHLGTR
                     PTEKQYETLENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEDQEHQVAKEERETPTT
                     DSMPPLLRQAIELFDHQGAEPAFLFGLELIICGLEKQLKCESG*"
     primer_bind     complement(1432..1451)
                     /label=Tet-R
                     /note="Tetracycline resistance gene, reverse primer"
     rep_origin      4780..5368
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5269..5288
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5522..5539
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     5633..5649
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     5633..5649
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     protein_bind    5720..5738
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O1 for the tetR and tetA genes"
     CDS             join(5798..8290,1..1191)
                     /codon_start=1
                     /product="CRISPR-associated protein from Lachnospiraceae 
                     bacterium ND2006 (Zetsche et al., 2015)"
                     /label=LbCpf1
                     /translation="MSKLEKFTNCYSLSKTLRFKAIPVGKTQENIDNKRLLVEDEKRAE
                     DYKGVKKLLDRYYLSFINDVLHSIKLKNLNNYISLFRKKTRTEKENKELENLEINLRKE
                     IAKAFKGNEGYKSLFKKDIIETILPEFLDDKDEIALVNSFNGFTTAFTGFFDNRENMFS
                     EEAKSTSIAFRCINENLTRYISNMDIFEKVDAIFDKHEVQEIKEKILNSDYDVEDFFEG
                     EFFNFVLTQEGIDVYNAIIGGFVTESGEKIKGLNEYINLYNQKTKQKLPKFKPLYKQVL
                     SDRESLSFYGEGYTSDEEVLEVFRNTLNKNSEIFSSIKKLEKLFKNFDEYSSAGIFVKN
                     GPAISTISKDIFGEWNVIRDKWNAEYDDIHLKKKAVVTEKYEDDRRKSFKKIGSFSLEQ
                     LQEYADADLSVVEKLKEIIIQKVDEIYKVYGSSEKLFDADFVLEKSLKKNDAVVAIMKD
                     LLDSVKSFENYIKAFFGEGKETNRDESFYGDFVLAYDILLKVDHIYDAIRNYVTQKPYS
                     KDKFKLYFQNPQFMGGWDKDKETDYRATILRYGSKYYLAIMDKKYAKCLQKIDKDDVNG
                     NYEKINYKLLPGPNKMLPKVFFSKKWMAYYNPSEDIQKIYKNGTFKKGDMFNLNDCHKL
                     IDFFKDSISRYPKWSNAYDFNFSETEKYKDIAGFYREVEEQGYKVSFESASKKEVDKLV
                     EEGKLYMFQIYNKDFSDKSHGTPNLHTMYFKLLFDENNHGQIRLSGGAELFMRRASLKK
                     EELVVHPANSPIANKNPDNPKKTTTLSYDVYKDKRFSEDQYELHIPIAINKCPKNIFKI
                     NTEVRVLLKHDDNPYVIGIARGERNLLYIVVVDGKGNIVEQYSLNEIINNFNGIRIKTD
                     YHSLLDKKEKERFEARQNWTSIENIKELKAGYISQVVHKICELVEKYDAVIALEDLNSG
                     FKNSRVKVEKQVYQKFEKMLIDKLNYMVDKKSNPCATGGALKGYQITNKFESFKSMSTQ
                     NGFIFYIPAWLTSKIDPSTGFVNLLKTKYTSIADSKKFISSFDRIMYVPEEDLFEFALD
                     YKNFSRTDADYIKKWKLYSYGNRIRIFRNPKKNNVFDWEEVCLTSAYKELFNKYGINYQ
                     QGDIRALLCEQSDKAFYSSFMALMSLMLQMRNSITGRTDVDFLISPVKNSDGIFYDSRN
                     YEAQENAILPKNADANGAYNIARKVLWAIGQFKKAEDEKLDKVKIAISNKEWLEYAQTS
                     VKH"
ORIGIN
        1 gctaggggcg agcgcaatct gctgtatatc gtggtggtgg acggcaaggg caacatcgtg
       61 gagcagtatt ccctgaacga gatcatcaac aacttcaacg gcatcaggat caagacagat
      121 taccactctc tgctggacaa gaaggagaag gagaggttcg aggcccgcca gaactggacc
      181 tccatcgaga atatcaagga gctgaaggcc ggctatatct ctcaggtggt gcacaagatc
      241 tgcgagctgg tggagaagta cgatgccgtg atcgccctgg aggacctgaa ctctggcttt
      301 aagaatagcc gcgtgaaggt ggagaagcag gtgtatcaga agttcgagaa gatgctgatc
      361 gataagctga actacatggt ggacaagaag tctaatcctt gtgcaacagg cggcgccctg
      421 aagggctatc agatcaccaa taagttcgag agctttaagt ccatgtctac ccagaacggc
      481 ttcatctttt acatccctgc ctggctgaca tccaagatcg atccatctac cggctttgtg
      541 aacctgctga aaaccaagta taccagcatc gccgattcca agaagttcat cagctccttt
      601 gacaggatca tgtacgtgcc cgaggaggat ctgttcgagt ttgccctgga ctataagaac
      661 ttctctcgca cagacgccga ttacatcaag aagtggaagc tgtactccta cggcaaccgg
      721 atcagaatct tccggaatcc taagaagaac aacgtgttcg actgggagga ggtgtgcctg
      781 accagcgcct ataaggagct gttcaacaag tacggcatca attatcagca gggcgatatc
      841 agagccctgc tgtgcgagca gtccgacaag gccttctact ctagctttat ggccctgatg
      901 agcctgatgc tgcagatgcg gaacagcatc acaggccgca ccgacgtgga ttttctgatc
      961 agccctgtga agaactccga cggcatcttc tacgatagcc ggaactatga ggcccaggag
     1021 aatgccatcc tgccaaagaa cgccgacgcc aatggcgcct ataacatcgc cagaaaggtg
     1081 ctgtgggcca tcggccagtt caagaaggcc gaggacgaga agctggataa ggtgaagatc
     1141 gccatctcta acaaggagtg gctggagtac gcccagacca gcgtgaagca ctgaatagta
     1201 tttatttcta ttgagtaaaa taaaaaagat ggtgagataa tctcaccatc tttttttctt
     1261 ttaaatccgt cgaccttaga agcaaactta ggatccaaaa atatattgat aaaaataata
     1321 atagtgggta taattaagtt gttaggaggt tagttagaat gtctagatta gataaaagta
     1381 aagtgattaa cagcgcatta gagctgctta atgaggtcgg aatcgaaggt ttaacaaccc
     1441 gtaaactcgc ccagaagcta ggtgtagagc agcctacatt gtattggcat gtaaaaaata
     1501 agcgggcttt gctcgacgcc ttagccattg agatgttaga taggcaccat actcactttt
     1561 gccctttaga aggggaaagc tggcaagatt ttttacgtaa taacgctaaa agttttagat
     1621 gtgctttact aagtcatcgc gatggagcaa aagtacattt aggtacacgg cctacagaaa
     1681 aacagtatga aactctcgaa aatcaattag cctttttatg ccaacaaggt ttttcactag
     1741 agaatgcatt atatgcactc agcgctgtgg ggcattttac tttaggttgc gtattggaag
     1801 atcaagagca tcaagtcgct aaagaagaaa gggaaacacc tactactgat agtatgccgc
     1861 cattattacg acaagctatc gaattatttg atcaccaagg tgcagagcca gccttcttat
     1921 tcggccttga attgatcata tgcggattag aaaaacaact taaatgtgaa agtgggtaat
     1981 aaagcctctt ccgctagcag gctgttttga gggattcaaa acagcctttt tccaaaattc
     2041 gcccttggaa ttctacataa tatattgaca aatttatttt ttaaagttaa aattaagttg
     2101 gtttcaaaga ttaaataatt tctactaagt gtagattagt cttccctact acgatcaacg
     2161 aagacatgtt tcaaagattg aagaatttct acgctagcat agcgatcgaa tcttccgcta
     2221 gcataaaaat aagaagcctg catttgcagg cttcttattt ttatggcgcg ccgccattat
     2281 ttttttgaac aattgacaat tcatttctta ttttttatta agtgatagtc aaaaggcata
     2341 acagtgctga atagaaagaa atttacagaa aagaaaatta tagaatttag tatgattaat
     2401 tatactcatt tatgaatgtt taattgaata caaaaaaaaa tacttgttat gtattcaatt
     2461 acgggttaaa atatagacaa gttgaaaaat ttaataaaaa aataagtcct cagctcttat
     2521 atattaagct accaacttag tatataagcc aaaacttaaa tgtgctacca acacatcaag
     2581 ccgttagaga actctatcta tagcaatatt tcaaatgtac cgacatacaa gagaaacatt
     2641 aactatatat attcaattta tgagattatc ttaacagata taaatgtaaa ttgcaataag
     2701 taagatttag aagtttatag cctttgtgta ttggaagcag tacgcaaagg cttttttatt
     2761 tgataaaaat tagaagtata tttatttttt cataattaat ttatgaaaat gaaagggggt
     2821 gagcaaagtg acagaggaaa gcagtatctt atcaaataac aaggtattag caatatcatt
     2881 attgacttta gcagtaaaca ttatgacttt tatagtgctt gtagctaagt agtacgaaag
     2941 ggggagcttt aaaaagctcc ttggaataca tagaattcat aaattaattt atgaaaagaa
     3001 gggcgtatat gaaaacttgt aaaaattgca aagagtttat taaagatact gaaatatgca
     3061 aaatacattc gttgatgatt catgataaaa cagtagcaac ctattgcagt aaatacaatg
     3121 agtcaagatg tttacataaa gggaaagtcc aatgtattaa ttgttcaaag atgaaccgat
     3181 atggatggtg tgccataaaa atgagatgtt ttacagagga agaacagaaa aaagaacgta
     3241 catgcattaa atattatgca aggagcttta aaaaagctca tgtaaagaag agtaaaaaga
     3301 aaaaataatt tatttattaa tttaatattg agagtgccga cacagtatgc actaaaaaat
     3361 atatctgtgg tgtagtgagc cgatacaaaa ggatagtcac tcgcattttc ataatacatc
     3421 ttatgttatg attatgtgtc ggtgggactt cacgacgaaa acccacaata aaaaaagagt
     3481 tcggggtagg gttaagcata gttgaggcaa ctaaacaatc aagctaggat atgcagtagc
     3541 agaccgtaag gtcgttgttt aggtgtgttg taatacatac gctattaaga tgtaaaaata
     3601 cggataccaa tgaagggaaa agtataattt ttggatgtag tttgtttgtt catctatggg
     3661 caaactacgt ccaaagccgt ttccaaatct gctaaaaagt atatcctttc taaaatcaaa
     3721 gtcaagtatg aaatcataaa taaagtttaa ttttgaagtt attatgatat tatgtttttc
     3781 tattaaaata aattaagtat atagaatagt ttaataatag tatatactta atgtgataag
     3841 tgtctgacag tgtcacagaa aggatgattg ttatggatta taagcggccg gccagtgggc
     3901 aagttgaaaa attcacaaaa atgtggtata atatctttgt tcattagagc gataaacttg
     3961 aatttgagag ggaacttaga tggtatttga aaaaattgat aaaaatagtt ggaacagaaa
     4021 agagtatttt gaccactact ttgcaagtgt accttgtacc tacagcatga ccgttaaagt
     4081 ggatatcaca caaataaagg aaaagggaat gaaactatat cctgcaatgc tttattatat
     4141 tgcaatgatt gtaaaccgcc attcagagtt taggacggca atcaatcaag atggtgaatt
     4201 ggggatatat gatgagatga taccaagcta tacaatattt cacaatgata ctgaaacatt
     4261 ttccagcctt tggactgagt gtaagtctga ctttaaatca tttttagcag attatgaaag
     4321 tgatacgcaa cggtatggaa acaatcatag aatggaagga aagccaaatg ctccggaaaa
     4381 catttttaat gtatctatga taccgtggtc aaccttcgat ggctttaatc tgaatttgca
     4441 gaaaggatat gattatttga ttcctatttt tactatgggg aaatattata aagaagataa
     4501 caaaattata cttcctttgg caattcaagt tcatcacgca gtatgtgacg gatttcacat
     4561 ttgccgtttt gtaaacgaat tgcaggaatt gataaatagt taacttcagg tttgtctgta
     4621 actaaaaaca agtatttaag caaaaacatc gtagaaatac ggtgtttttt gttaccctaa
     4681 gtttaaactc ctttttgata atctcatgac caaaatccct taacgtgagt tttcgttcca
     4741 ctgagcgtca gaccccgtag aaaagatcaa aggatcttct tgagatcctt tttttctgcg
     4801 cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca gcggtggttt gtttgccgga
     4861 tcaagagcta ccaactcttt ttccgaaggt aactggcttc agcagagcgc agataccaaa
     4921 tactgttctt ctagtgtagc cgtagttagg ccaccacttc aagaactctg tagcaccgcc
     4981 tacatacctc gctctgctaa tcctgttacc agtggctgct gccagtggcg ataagtcgtg
     5041 tcttaccggg ttggactcaa gacgatagtt accggataag gcgcagcggt cgggctgaac
     5101 ggggggttcg tgcacacagc ccagcttgga gcgaacgacc tacaccgaac tgagatacct
     5161 acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc
     5221 ggtaagcggc agggtcggaa caggagagcg cacgagggag cttccagggg gaaacgcctg
     5281 gtatctttat agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg
     5341 ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac gcggcctttt tacggttcct
     5401 ggccttttgc tggccttttg ctcacatgtt ctttcctgcg ttatcccctg attctgtgga
     5461 taaccgtatt accgcctttg agtgagctga taccgctcgc cgcagccgaa cgaccgagcg
     5521 cagcgagtca gtgagcgagg aagcggaaga gcgcccaata cgcagggccc cctgcaggat
     5581 aaaaaaattg tagataaatt ttataaaata gttttatcta caattttttt atcaggaaac
     5641 agctatgacc gcggccgctg tatccatatg accatgatta cgaattcgag ctcaggatcc
     5701 ctatcagtga tagattgaaa ctctatcatt gatagagtat aatatcttgg taccttttac
     5761 atttacctgg ggacaggcca gacaaaggag gttttttatg agcaagctgg agaagtttac
     5821 aaactgctac tccctgtcta agaccctgag gttcaaggcc atccctgtgg gcaagaccca
     5881 ggagaacatc gacaataagc ggctgctggt ggaggacgag aagagagccg aggattataa
     5941 gggcgtgaag aagctgctgg atcgctacta tctgtctttt atcaacgacg tgctgcacag
     6001 catcaagctg aagaatctga acaattacat cagcctgttc cggaagaaaa ccagaaccga
     6061 gaaggagaat aaggagctgg agaacctgga gatcaatctg cggaaggaga tcgccaaggc
     6121 cttcaagggc aacgagggct acaagtccct gtttaagaag gatatcatcg agacaatcct
     6181 gccagagttc ctggacgata aggacgagat cgccctggtg aacagcttca atggctttac
     6241 cacagccttc accggcttct ttgataacag agagaatatg ttttccgagg aggccaagag
     6301 cacatccatc gccttcaggt gtatcaacga gaatctgacc cgctacatct ctaatatgga
     6361 catcttcgag aaggtggacg ccatctttga taagcacgag gtgcaggaga tcaaggagaa
     6421 gatcctgaac agcgactatg atgtggagga tttctttgag ggcgagttct ttaactttgt
     6481 gctgacacag gagggcatcg acgtgtataa cgccatcatc ggcggcttcg tgaccgagag
     6541 cggcgagaag atcaagggcc tgaacgagta catcaacctg tataatcaga aaaccaagca
     6601 gaagctgcct aagtttaagc cactgtataa gcaggtgctg agcgatcggg agtctctgag
     6661 cttctacggc gagggctata catccgatga ggaggtgctg gaggtgttta gaaacaccct
     6721 gaacaagaac agcgagatct tcagctccat caagaagctg gagaagctgt tcaagaattt
     6781 tgacgagtac tctagcgccg gcatctttgt gaagaacggc cccgccatca gcacaatctc
     6841 caaggatatc ttcggcgagt ggaacgtgat ccgggacaag tggaatgccg agtatgacga
     6901 tatccacctg aagaagaagg ccgtggtgac cgagaagtac gaggacgatc ggagaaagtc
     6961 cttcaagaag atcggctcct tttctctgga gcagctgcag gagtacgccg acgccgatct
     7021 gtctgtggtg gagaagctga aggagatcat catccagaag gtggatgaga tctacaaggt
     7081 gtatggctcc tctgagaagc tgttcgacgc cgattttgtg ctggagaaga gcctgaagaa
     7141 gaacgacgcc gtggtggcca tcatgaagga cctgctggat tctgtgaaga gcttcgagaa
     7201 ttacatcaag gccttctttg gcgagggcaa ggagacaaac agggacgagt ccttctatgg
     7261 cgattttgtg ctggcctacg acatcctgct gaaggtggac cacatctacg atgccatccg
     7321 caattatgtg acccagaagc cctactctaa ggataagttc aagctgtatt ttcagaaccc
     7381 tcagttcatg ggcggctggg acaaggataa ggagacagac tatcgggcca ccatcctgag
     7441 atacggctcc aagtactatc tggccatcat ggataagaag tacgccaagt gcctgcagaa
     7501 gatcgacaag gacgatgtga acggcaatta cgagaagatc aactataagc tgctgcccgg
     7561 ccctaataag atgctgccaa aggtgttctt ttctaagaag tggatggcct actataaccc
     7621 cagcgaggac atccagaaga tctacaagaa tggcacattc aagaagggcg atatgtttaa
     7681 cctgaatgac tgtcacaagc tgatcgactt ctttaaggat agcatctccc ggtatccaaa
     7741 gtggtccaat gcctacgatt tcaacttttc tgagacagag aagtataagg acatcgccgg
     7801 cttttacaga gaggtggagg agcagggcta taaggtgagc ttcgagtctg ccagcaagaa
     7861 ggaggtggat aagctggtgg aggagggcaa gctgtatatg ttccagatct ataacaagga
     7921 cttttccgat aagtctcacg gcacacccaa tctgcacacc atgtacttca agctgctgtt
     7981 tgacgagaac aatcacggac agatcaggct gagcggagga gcagagctgt tcatgaggcg
     8041 cgcctccctg aagaaggagg agctggtggt gcacccagcc aactccccta tcgccaacaa
     8101 gaatccagat aatcccaaga aaaccacaac cctgtcctac gacgtgtata aggataagag
     8161 gttttctgag gaccagtacg agctgcacat cccaatcgcc atcaataagt gccccaagaa
     8221 catcttcaag atcaatacag aggtgcgcgt gctgctgaag cacgacgata acccctatgt
     8281 gatcggcatc
//
