LOCUS       sequence-375973-        8425 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  E. coli - Eggerthella lenta shuttle plasmid (KanR), cumate-inducible
            promoter Pct5.1-crRNA(hcdR-targeting spacer) hcdR-deleting repair 
            template, used for hcdR deletion.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8425)
  AUTHORS   Dong X, Guthrie BGH, Alexander M, Noecker C, Ramirez L, Glasser NR, 
            Turnbaugh PJ, Balskus EP
  TITLE     Genetic manipulation of the human gut bacterium Eggerthella lenta 
            reveals a widespread family of transcriptional regulators.
  JOURNAL   Nat Commun. 2022 Dec 9;13(1):7624. doi: 10.1038/s41467-022-33576-3.
  PUBMED    36494336
REFERENCE   2  (bases 1 to 8425)
  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: "2022-12-9"; volume: "13(1):7624. doi"; pages: " 
            10.1038/s41467-022-33576-3"
COMMENT     Sequence Label: pXD71Cas10.6RT4-Pct5.1-crRNA(hcdR)-RT(Delta-hcdR)
FEATURES             Location/Qualifiers
     source          1..8425
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             3010..3804
                     /codon_start=1
                     /gene="aphA-3"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin"
                     /translation="MAKMRISPELKKLIEKYRCVKDTEGMSPAKVYKLVGENENLYLKM
                     TDSRYKGTTYDVEREKDMMLWLEGKLPVPKVLHFERHDGWSNLLMSEADGVLCSEEYED
                     EQSPEKIIELYAECIRLFHSIDISDCPYTNSLDSRLAELDYLLNNDLADVDCENWEEDT
                     PFKDPRELYDFLKTEKPEEELVFSHGDLGDSNIFVKDGKVSGFIDLGRSGRADKWYDIA
                     FCVRSIREDIGEEQYVELFFDLLGIKPDWEKIKYYILLDELF"
     terminator      complement(3927..3954)
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     protein_bind    4087..4114
                     /label=CuO
                     /bound_moiety="CymR"
                     /note="CymR-binding P2 operator sequence from the p-cmt 
                     operon of Pseudomonas putida (Mullick et al., 2006)"
     protein_bind    4129..4156
                     /label=CuO
                     /bound_moiety="CymR"
                     /note="CymR-binding P2 operator sequence from the p-cmt 
                     operon of Pseudomonas putida (Mullick et al., 2006)"
     CDS             4494..5105
                     /codon_start=1
                     /gene="cymR from Pseudomonas putida"
                     /product="cumate repressor (Mullick et al., 2006)"
                     /label=CymR
                     /translation="MSPKRRTQAERAMETQGKLIAAALGVLREKGYAGFRIADVPGAAG
                     VSRGAQSHHFPTKLELLLATFEWLYEQITERSRARLAKLKPEDDVIQQMLDDAAEFFLD
                     DDFSISLDLIVAADRDPALREGIQRTVERNRFVVEDMWLGVLVSRGLSRDDAEDILWLI
                     FNSVRGLAVRSLWQKDKERFERVRNSTLEIARERYAKFKR"
     misc_feature    5108..5127
                     /label=BioBrick prefix
                     /note="BioBrick prefix for parts that start with ""ATG"""
     primer_bind     complement(5284..5302)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     rep_origin      7542..8130
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     8031..8050
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     terminator      complement(8311..8382)
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
ORIGIN
        1 gagacaagga gattatagca tggcggcaaa cgctggcgag acgaaggtga agctctggga
       61 gggcatctgc tacccagaga acatgcgcga tgactggcag gacgagatag acgacattct
      121 tcaagtgcct ttcgcttatg cggttcacga catagaccat gaccagaaga gcaagcagcg
      181 caagacgcat gcgcacgtca ttgttgtctg gggcggcaac acgacacgca aggcaatcat
      241 caacgtcttg aaccgattga gcgcggacgg caagaagtgc tgttcgtcag ccgagccaat
      301 caacaacatc aggcatgcgt atgactatct gattcacgac acggcttcct gtcgcaagaa
      361 gggcaaggag ctgtatccgg tcgaagcacg catcgaggga aacaacttcg acattggtca
      421 gctcgaacag ctctcgacta aggacaagca ggacatgctc ttcgagttgg tcggcttcat
      481 catgtgcgag aggttcgaga ccatcaacga tttcacggct gcggcgctga gggagtttcc
      541 cgagcagtac cgcgaaatca tcgtggggta caactcgatt ctcgagcgct attgtcgcgg
      601 gaactatctg aacgccgaac gtaagcgcaa gtgcgtggaa cagtcaggtg ccaaagagta
      661 gtcgggtcaa tgcagcaatg cgtgactacg gggcggcgtg gtctgcgctc gcgcagatct
      721 aagccgactc gttcacggat tgcggaattg acgcgacggc caaccgaata gaccagactt
      781 gattaagggt cgatggacgc tctgctccat cgaccctttc cttgcccgtg tctcgactgg
      841 atgagacctg cagaatcggg cggctcatta tttttcgcta actacgtccg aaaaataatg
      901 tgtccgaaaa ataatgtgac aaatgacaaa tgacaaatga ccacaggggg gccactacga
      961 cggcccccct aatagtgccg tgtgccgtgt gccgaaccgc aggtagattg aggggtggtt
     1021 cggaggggcg aagtccctcc gttcggcgct cgcgccgatg gcccgagggc cgtgagcgaa
     1081 gccgcgaacg ggcaagggct ggggggtctc ggggggaacg cccgagcggg tgcagggctg
     1141 gccctgccac gtccttagcg tagctaaggt ctaacgtgct agactttagt cgattaagtc
     1201 tgtacggttc ggaggtttcc atggctcaca tcgcgaaata caaggctcca tcggtcgggc
     1261 acatgcttgc ccactaccgc cgcgaccgca gcagcctgga gcgcgacaac atcgaccccg
     1321 agcgcatcaa gaacgacttg gtggtcggcc actacacgaa caaggacggc aagctcgttg
     1381 tggggcgcgt ggagccgcgc gatggcgagc cgaactgggg cacggtggag agccgcatcg
     1441 agcgcgtgaa cgaggcccag aaggccgcag ggaagcgtgc cacgcgcaag gatgcggtcg
     1501 ttatggctga cgttgttgtg acgcttcccg acaatgtgcg caagggcgat gaggacaggt
     1561 tcttccgtct cacgtactgg tacctgtcga agaagttcgg catcgagaac atgatgggcg
     1621 ggtacgtcca caaggacgag gtgttgaagg atggcacgcc cgcccgcgac cacatgcacg
     1681 tgccgttcac gcccatccta gatggacggt tcaactacaa gcagatgtgc ccccgcagct
     1741 tctaccggag catgcacaag gagcttgggg attacctaga ggggcgcatg ggctaccgcc
     1801 ctgcaatcga gcttgacgag gagacgcggg cgcagcgtgt atacacggac aagtccgtgg
     1861 atatcgacaa ggtacgctgt gcggtcgata aggccgtggt tgaacctgcg aaggacgaag
     1921 cggcgcgaat cgtcgcggcg gctagggaag aagccgccgc tttactgaat gatgccgaaa
     1981 cgcgtaaggc agagcttgtt accgagattg ccgacaagga gggtgacctt gccgagcttg
     2041 acagccagct cgaggacgtg aagctcgata tcgaagatga gcaagaccga ttggagtgtc
     2101 ttcggcaacg agcggacgga gttgcgcgag acattggaga gcttcaaccc atcgctaccg
     2161 aggttcgggg ctgggaagct gcgggaaaag ctgagcgcgg cgcaatcctc gataacatcg
     2221 ttgttaagtg cgatggactc gcgagccgca tcagagcagg agttgcggga atgcgaatca
     2281 aagtggaaga gcttagaagt cgaatatcgc gcccgctcga atatctgatg cgcagggaac
     2341 agccaaggca acagagcctt aatgacgtgc tgcgcgatgc gcagcgggcg gcggatgcgt
     2401 ggaaccgcga ccacgctgca ccgcagagga cgtatcgcgg gcgggctaga tgatgtgtta
     2461 tagtagtacc tgtaatacac gacaaggagg tgcgacgcga aaaaaccccg ctgaagcggg
     2521 gttttttgcg gagcttcgct tccacgatgc gcatgattcg gaggccccgg cgatcgccgg
     2581 ggcctccgtg cgttgtgtgt cgatttggga tgcagccaga tgggcttcct cggcgcgaac
     2641 cggccgagat ttccgaatgc agacggtctc gcttggacga tgcgctttcg aataatcccg
     2701 gcggagcggg cattccagtc ttcttatcaa tatatctata gaatgggcaa agcataaaaa
     2761 cttgcatgga ctaatgcttg aaacccagga caataacctt atagcttgta aattctatca
     2821 taattgtggt ttcaaaatcg gctccgtcga tactatgtta tacgccaact ttgaaaacaa
     2881 ctttgaaaaa gctgttttct ggtatttaag gttttagaat gcaaggaaca gtgaattgga
     2941 gttcgtcttg ttataattag cttcttgggg tatctttaaa tactgtagaa aagaggaagg
     3001 aaataataaa tggctaaaat gagaatatca ccggaattga aaaaactgat cgaaaaatac
     3061 cgctgcgtaa aagatacgga aggaatgtct cctgctaagg tatataagct ggtgggagaa
     3121 aatgaaaacc tatatttaaa aatgacggac agccggtata aagggaccac ctatgatgtg
     3181 gaacgggaaa aggacatgat gctatggctg gaaggaaagc tgcctgttcc aaaggtcctg
     3241 cactttgaac ggcatgatgg ctggagcaat ctgctcatga gtgaggccga tggcgtcctt
     3301 tgctcggaag agtatgaaga tgaacaaagc cctgaaaaga ttatcgagct gtatgcggag
     3361 tgcatcaggc tctttcactc catcgacata tcggattgtc cctatacgaa tagcttagac
     3421 agccgcttag ccgaattgga ttacttactg aataacgatc tggccgatgt ggattgcgaa
     3481 aactgggaag aagacactcc atttaaagat ccgcgcgagc tgtatgattt tttaaagacg
     3541 gaaaagcccg aagaggaact tgtcttttcc cacggcgacc tgggagacag caacatcttt
     3601 gtgaaagatg gcaaagtaag tggctttatt gatcttggga gaagcggcag ggcggacaag
     3661 tggtatgaca ttgccttctg cgtccggtcg atcagggagg atatcgggga agaacagtat
     3721 gtcgagctat tttttgactt actggggatc aagcctgatt gggagaaaat aaaatattat
     3781 attttactgg atgaattgtt ttagatcgtt aagggatcaa ctttgggaga gagttcaaaa
     3841 ttgatccttt ttttataaca ggaattaaat aataaaaaag ccggattaat aatctggctt
     3901 tttatattct ctctctagta tataaacgca gaaaggccca cccgaaggtg agccagtgtg
     3961 agtcgactag tatttcaacc cgcactcccc atgcggggag tgacgggcca cgatggtcac
     4021 gaggatgact acggaggcat ttcaacccgc actccccatg cggggagtga cattagttaa
     4081 tcataataca aacagaccag attgtctgtt tgttggccgc aacactcgta caaacagacc
     4141 agattgtctg tttgttacta catttattgt acaacacgag cccatttttg tcaaataaaa
     4201 tttaaattat atcaacgtta ataagacgtt gtcaataaaa ttattttgac aaaatagaat
     4261 agtaatacag gatccgaatc gtttcagttg cctaaataat aaaaaagccg gattaataat
     4321 caaataaccg ttccttcaat gtttttttta gaaaatttag ttataatatt agatatgata
     4381 cttttaaata tctaattcaa gcttcaaaaa acaccaactt agttcggtgg ataaacaaag
     4441 gagtggttat tattcaaatt gcagatcaag ctttagtaaa aaaaatggtg atcatgagtc
     4501 caaagagaag aacacaggca gagcgcgcaa tggagaccca gggcaagttg attgcagcgg
     4561 ccctgggggt tttacgggaa aaaggttacg cgggattccg gatcgcagat gtgcccggtg
     4621 ctgctggtgt ctccagagga gcgcagagcc atcatttccc gacaaagctt gagcttctgc
     4681 ttgccacttt tgaatggctt tacgaacaga tcaccgaacg cagtcgggct cgattagcga
     4741 aattgaagcc agaggatgac gtcatccagc aaatgctgga cgacgccgcc gaatttttcc
     4801 tcgacgatga cttctctatc agccttgatt tgattgtggc tgccgaccgg gacccagcgt
     4861 tacgcgaggg tattcagcgc acggtagaga ggaatcggtt tgtcgtcgag gatatgtggc
     4921 ttggtgttct ggtgagccgt ggtctttcgc gtgatgatgc agaagatatc ctttggttga
     4981 tattcaattc ggtgcgtggg cttgctgttc gtagcctatg gcagaaggac aaagaacgct
     5041 ttgagcgtgt caggaactcg acactcgaaa ttgcgcgaga gcggtacgcg aaattcaagc
     5101 gctagatcta gaagcggccg cgaattcaga atagtaatac aggatccgaa tcgtttcagt
     5161 tgccgccgga ttaataatca aataaccgtt cctaaaaaat gccctcttgg gttatcaaga
     5221 gggtcattat atttcgcaaa aaaccccgct tcggcggggt tttttcgcct tgaagacgaa
     5281 agggcctcgt gatacgccta tttttatagg ttaatgtcat gataataatg gtttcttaga
     5341 cgtcaggtgg cacttttcgg ggaaatgtgg agatgggggc gtcgaacatc ttgtcggcag
     5401 cgccctcgaa cgtgtcttcc atcctgcctt cgaatctcga aagaaagccc attgcatctc
     5461 ctttaatggt gcgtggctcg aacctcggcg gatgccgggt tcgcgcacgg gaggcaacgc
     5521 cgcgctggcg tgttccgcgc gatcgcgtgc ccaaacgcaa ccaggttact tcgtgtgtcc
     5581 ctgcatatga ctatgcaatg ttccatcttg ccataaaccc cctgcccgcg ccccatcctt
     5641 tacgcaatcg tcattattgt gaagcggggc ttgcgcgagg gcgtggacga tagggaagcg
     5701 ggatctgcga gggtgcggac gattgggaag caggggttac gccggagtgc gggtggctga
     5761 gaggcggggt ttgcgcgaag gcgtggacga ttgggaagca gggctacgcg aggttgctga
     5821 cgaatttgcg cgctcgttgc gccgtggtgc gagcttgcgt ttctccctcg tagtcgcgcg
     5881 tcgacggtcg gttgcgtacc caaagcggaa atacgacccg tatttgtaag tttttcgact
     5941 tcgtggcacg cgcggcaatc gttttccctg gtcgaggtgt tcgccgattt gtcacggtag
     6001 tgcgtgcgct tttgagatgc ggagaaaagc ctggtcagaa catctcatga ataagaggcg
     6061 ttgccacaag gtcgaaaaat ttgcagaagc gccctcgaaa accgtttctt gctcgcgttg
     6121 acggcgtagc tgcaagggat gtcgtgcggt tgtgcgagcg cgaaggcgcc tggcgtttca
     6181 gcgggagctg gggtgaggca aaccttgcgt cgcgaactgc acgacggcga aggggaacgc
     6241 cgtgcaaccg cgcgagcgtg tcgagatcgt gcgaacgcgc ctgcggatcg acgaccaggt
     6301 ttgtgcacgg aacgcgggat ttgcgagcgg tcgcggaggc cgcgcgctgc aatcggcgag
     6361 gggcaagctc gtcataccgt gcagaggttg ttttttctag cgaggtgagc gagtcgatca
     6421 gggttttgcg ctcgatgcgc aaacgtttga tctggggtaa tagaaaaata atcctctgag
     6481 gatgatgttc ggaagggcgc ccgtctgctt tcgagggcgt gctgaggcgt cgcggtgctg
     6541 catcgggccg gcttgtgaag aataacgggg cgcttgccga gccggaacgg gaggtgtgcg
     6601 ggggcccggc tggtcgtccg aacccccgcg cggcggatta cgccaccgtg aacaccacgc
     6661 tgtcgaagcg ctcgagcggc gtgctctcgc cctcggtgcc gcttccgtcg aacgcggtgt
     6721 gcgtgcccgg cttctggctg tcggccttgt gcaccgtgca cgtgtactgc ttgccggaca
     6781 gctcgtccgc caagctggag acgctgtagt cgtgaatggc cgcaaggccg ttctgactgt
     6841 tgctggaaac cacgctctcg tccaggcaag cagcggcggt gctgccttcg ggaacgatga
     6901 tgagcgaggt cttcacgcct ccgccctgag cggtttcgtc caccgtgcac gtgacctcga
     6961 tgacgccctt gctggttgca gcaacgttct cttcgccgtg gacgtagtag aacgcatagg
     7021 caggaaccgc gatcgaggcg cacaggcagc tcagaaccgc aacggcaaga atcttcttgc
     7081 cccaagctac gttggccatt ggagcttcct ttcctaagaa gccccgcttc tcgcaacgcg
     7141 ccccgattat agggagccgg cgaagcgcgc gtcaaggtga tttcgcggcc ccggcgtatg
     7201 atgcgcgcga atccctgcgt aattacgcct tgtaggatga tgagccgtcg cgcgcagccc
     7261 ctatactcga acgcagcgaa gagagaagaa ggaggaatga tgcctaccgt atcgtttgac
     7321 gacgtgctgg gcttagacga cgagttcgat cctctgatgg ccgacgatct gctgtcagac
     7381 caagtttact catatatact ttagattgat ttaaaacttc atttttaatt taaaaggatc
     7441 taggtgaaga tcctttttga taatctcatg accaaaatcc cttaacgtga gttttcgttc
     7501 cactgagcgt cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg
     7561 cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg
     7621 gatcaagagc taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca
     7681 aatactgtcc ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg
     7741 cctacatacc tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg
     7801 tgtcttaccg ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga
     7861 acggggggtt cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac
     7921 ctacagcgtg agctatgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat
     7981 ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc
     8041 tggtatcttt atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga
     8101 tgctcgtcag gggggcggag cctatggaaa aacgccagca acgcggcccg agatgcgccg
     8161 cgtgcggctg ctggagatgg cggacgcgat ggatatgttc tgccaagggt tggtttgcgc
     8221 attcacagtt ctccgcaaga attgattggc tccaattctt ggagtggtga atccgttagc
     8281 gaggtgccgc cggcatgagc tcctctagta gagagcgttc accgacaaac aacagataaa
     8341 acgaaaggcc cagtctttcg actgagcctt tcgttttatt tgaaaaagaa aagccccagc
     8401 aagcttggcg gcagcagggg cattc
//
