LOCUS       sequence-375976-        8438 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  E. coli - Eggerthella lenta shuttle plasmid (KanR), cumate-inducible
            promoter Pct5.1-crRNA(cgr2-targeting spacer) cgr1/cgr2-deleting 
            repair template, used for cgr1/cgr2 deletion.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8438)
  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 8438)
  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: pXD71Cas10.4RT4-Pct5.1-crRNA(cgr2)-RT(Delta-cgr12)
FEATURES             Location/Qualifiers
     source          1..8438
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             3004..3798
                     /codon_start=1
                     /gene="aphA-3"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin"
                     /translation="MAKMRISPELKKLIEKYRCVKDTEGMSPAKVYKLVGENENLYLKM
                     TDSRYKGTTYDVEREKDMMLWLEGKLPVPKVLHFERHDGWSNLLMSEADGVLCSEEYED
                     EQSPEKIIELYAECIRLFHSIDISDCPYTNSLDSRLAELDYLLNNDLADVDCENWEEDT
                     PFKDPRELYDFLKTEKPEEELVFSHGDLGDSNIFVKDGKVSGFIDLGRSGRADKWYDIA
                     FCVRSIREDIGEEQYVELFFDLLGIKPDWEKIKYYILLDELF"
     terminator      complement(3921..3948)
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     protein_bind    4081..4108
                     /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    4123..4150
                     /label=CuO
                     /bound_moiety="CymR"
                     /note="CymR-binding P2 operator sequence from the p-cmt 
                     operon of Pseudomonas putida (Mullick et al., 2006)"
     CDS             4488..5099
                     /codon_start=1
                     /gene="cymR from Pseudomonas putida"
                     /product="cumate repressor (Mullick et al., 2006)"
                     /label=CymR
                     /translation="MSPKRRTQAERAMETQGKLIAAALGVLREKGYAGFRIADVPGAAG
                     VSRGAQSHHFPTKLELLLATFEWLYEQITERSRARLAKLKPEDDVIQQMLDDAAEFFLD
                     DDFSISLDLIVAADRDPALREGIQRTVERNRFVVEDMWLGVLVSRGLSRDDAEDILWLI
                     FNSVRGLAVRSLWQKDKERFERVRNSTLEIARERYAKFKR"
     misc_feature    5102..5121
                     /label=BioBrick prefix
                     /note="BioBrick prefix for parts that start with ""ATG"""
     primer_bind     complement(5278..5296)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     rep_origin      7549..8137
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     8038..8057
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     terminator      complement(8318..8389)
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
ORIGIN
        1 aggagattat agcatggcgg caaacgctgg cgagacgaag gtgaagctct gggagggcat
       61 ctgctaccca gagaacatgc gcgatgactg gcaggacgag atagacgaca ttcttcaagt
      121 gcctttcgct tatgcggttc acgacataga ccatgaccag aagagcaagc agcgcaagac
      181 gcatgcgcac gtcattgttg tctggggcgg caacacgaca cgcaaggcaa tcatcaacgt
      241 cttgaaccga ttgagcgcgg acggcaagaa gtgctgttcg tcagccgagc caatcaacaa
      301 catcaggcat gcgtatgact atctgattca cgacacggct tcctgtcgca agaagggcaa
      361 ggagctgtat ccggtcgaag cacgcatcga gggaaacaac ttcgacattg gtcagctcga
      421 acagctctcg actaaggaca agcaggacat gctcttcgag ttggtcggct tcatcatgtg
      481 cgagaggttc gagaccatca acgatttcac ggctgcggcg ctgagggagt ttcccgagca
      541 gtaccgcgaa atcatcgtgg ggtacaactc gattctcgag cgctattgtc gcgggaacta
      601 tctgaacgcc gaacgtaagc gcaagtgcgt ggaacagtca ggtgccaaag agtagtcggg
      661 tcaatgcagc aatgcgtgac tacggggcgg cgtggtctgc gctcgcgcag atctaagccg
      721 actcgttcac ggattgcgga attgacgcga cggccaaccg aatagaccag acttgattaa
      781 gggtcgatgg acgctctgct ccatcgaccc tttccttgcc cgtgtctcga ctggatgaga
      841 cctgcagaat cgggcggctc attatttttc gctaactacg tccgaaaaat aatgtgtccg
      901 aaaaataatg tgacaaatga caaatgacaa atgaccacag gggggccact acgacggccc
      961 ccctaatagt gccgtgtgcc gtgtgccgaa ccgcaggtag attgaggggt ggttcggagg
     1021 ggcgaagtcc ctccgttcgg cgctcgcgcc gatggcccga gggccgtgag cgaagccgcg
     1081 aacgggcaag ggctgggggg tctcgggggg aacgcccgag cgggtgcagg gctggccctg
     1141 ccacgtcctt agcgtagcta aggtctaacg tgctagactt tagtcgatta agtctgtacg
     1201 gttcggaggt ttccatggct cacatcgcga aatacaaggc tccatcggtc gggcacatgc
     1261 ttgcccacta ccgccgcgac cgcagcagcc tggagcgcga caacatcgac cccgagcgca
     1321 tcaagaacga cttggtggtc ggccactaca cgaacaagga cggcaagctc gttgtggggc
     1381 gcgtggagcc gcgcgatggc gagccgaact ggggcacggt ggagagccgc atcgagcgcg
     1441 tgaacgaggc ccagaaggcc gcagggaagc gtgccacgcg caaggatgcg gtcgttatgg
     1501 ctgacgttgt tgtgacgctt cccgacaatg tgcgcaaggg cgatgaggac aggttcttcc
     1561 gtctcacgta ctggtacctg tcgaagaagt tcggcatcga gaacatgatg ggcgggtacg
     1621 tccacaagga cgaggtgttg aaggatggca cgcccgcccg cgaccacatg cacgtgccgt
     1681 tcacgcccat cctagatgga cggttcaact acaagcagat gtgcccccgc agcttctacc
     1741 ggagcatgca caaggagctt ggggattacc tagaggggcg catgggctac cgccctgcaa
     1801 tcgagcttga cgaggagacg cgggcgcagc gtgtatacac ggacaagtcc gtggatatcg
     1861 acaaggtacg ctgtgcggtc gataaggccg tggttgaacc tgcgaaggac gaagcggcgc
     1921 gaatcgtcgc ggcggctagg gaagaagccg ccgctttact gaatgatgcc gaaacgcgta
     1981 aggcagagct tgttaccgag attgccgaca aggagggtga ccttgccgag cttgacagcc
     2041 agctcgagga cgtgaagctc gatatcgaag atgagcaaga ccgattggag tgtcttcggc
     2101 aacgagcgga cggagttgcg cgagacattg gagagcttca acccatcgct accgaggttc
     2161 ggggctggga agctgcggga aaagctgagc gcggcgcaat cctcgataac atcgttgtta
     2221 agtgcgatgg actcgcgagc cgcatcagag caggagttgc gggaatgcga atcaaagtgg
     2281 aagagcttag aagtcgaata tcgcgcccgc tcgaatatct gatgcgcagg gaacagccaa
     2341 ggcaacagag ccttaatgac gtgctgcgcg atgcgcagcg ggcggcggat gcgtggaacc
     2401 gcgaccacgc tgcaccgcag aggacgtatc gcgggcgggc tagatgatgt gttatagtag
     2461 tacctgtaat acacgacaag gaggtgcgac gcgaaaaaac cccgctgaag cggggttttt
     2521 tgcggagctt cgcttccacg atgcgcatga ttcggaggcc ccggcgatcg ccggggcctc
     2581 cgtgcgttgt gtgtcgattt gggatgcagc cagatgggct tcctcggcgc gaaccggccg
     2641 agatttccga atgcagacgg tctcgcttgg acgatgcgct ttcgaataat cccggcggag
     2701 cgggcattcc agtcttctta tcaatatatc tatagaatgg gcaaagcata aaaacttgca
     2761 tggactaatg cttgaaaccc aggacaataa ccttatagct tgtaaattct atcataattg
     2821 tggtttcaaa atcggctccg tcgatactat gttatacgcc aactttgaaa acaactttga
     2881 aaaagctgtt ttctggtatt taaggtttta gaatgcaagg aacagtgaat tggagttcgt
     2941 cttgttataa ttagcttctt ggggtatctt taaatactgt agaaaagagg aaggaaataa
     3001 taaatggcta aaatgagaat atcaccggaa ttgaaaaaac tgatcgaaaa ataccgctgc
     3061 gtaaaagata cggaaggaat gtctcctgct aaggtatata agctggtggg agaaaatgaa
     3121 aacctatatt taaaaatgac ggacagccgg tataaaggga ccacctatga tgtggaacgg
     3181 gaaaaggaca tgatgctatg gctggaagga aagctgcctg ttccaaaggt cctgcacttt
     3241 gaacggcatg atggctggag caatctgctc atgagtgagg ccgatggcgt cctttgctcg
     3301 gaagagtatg aagatgaaca aagccctgaa aagattatcg agctgtatgc ggagtgcatc
     3361 aggctctttc actccatcga catatcggat tgtccctata cgaatagctt agacagccgc
     3421 ttagccgaat tggattactt actgaataac gatctggccg atgtggattg cgaaaactgg
     3481 gaagaagaca ctccatttaa agatccgcgc gagctgtatg attttttaaa gacggaaaag
     3541 cccgaagagg aacttgtctt ttcccacggc gacctgggag acagcaacat ctttgtgaaa
     3601 gatggcaaag taagtggctt tattgatctt gggagaagcg gcagggcgga caagtggtat
     3661 gacattgcct tctgcgtccg gtcgatcagg gaggatatcg gggaagaaca gtatgtcgag
     3721 ctattttttg acttactggg gatcaagcct gattgggaga aaataaaata ttatatttta
     3781 ctggatgaat tgttttagat cgttaaggga tcaactttgg gagagagttc aaaattgatc
     3841 ctttttttat aacaggaatt aaataataaa aaagccggat taataatctg gctttttata
     3901 ttctctctct agtatataaa cgcagaaagg cccacccgaa ggtgagccag tgtgagtcga
     3961 ctagtatttc aacccgcact ccccatgcgg ggagtgaccc gagttgccgc cgtacatcgc
     4021 cttgtccttc tcatttcaac ccgcactccc catgcgggga gtgacattag ttaatcataa
     4081 tacaaacaga ccagattgtc tgtttgttgg ccgcaacact cgtacaaaca gaccagattg
     4141 tctgtttgtt actacattta ttgtacaaca cgagcccatt tttgtcaaat aaaatttaaa
     4201 ttatatcaac gttaataaga cgttgtcaat aaaattattt tgacaaaata gaatagtaat
     4261 acaggatccg aatcgtttca gttgcctaaa taataaaaaa gccggattaa taatcaaata
     4321 accgttcctt caatgttttt tttagaaaat ttagttataa tattagatat gatactttta
     4381 aatatctaat tcaagcttca aaaaacacca acttagttcg gtggataaac aaaggagtgg
     4441 ttattattca aattgcagat caagctttag taaaaaaaat ggtgatcatg agtccaaaga
     4501 gaagaacaca ggcagagcgc gcaatggaga cccagggcaa gttgattgca gcggccctgg
     4561 gggttttacg ggaaaaaggt tacgcgggat tccggatcgc agatgtgccc ggtgctgctg
     4621 gtgtctccag aggagcgcag agccatcatt tcccgacaaa gcttgagctt ctgcttgcca
     4681 cttttgaatg gctttacgaa cagatcaccg aacgcagtcg ggctcgatta gcgaaattga
     4741 agccagagga tgacgtcatc cagcaaatgc tggacgacgc cgccgaattt ttcctcgacg
     4801 atgacttctc tatcagcctt gatttgattg tggctgccga ccgggaccca gcgttacgcg
     4861 agggtattca gcgcacggta gagaggaatc ggtttgtcgt cgaggatatg tggcttggtg
     4921 ttctggtgag ccgtggtctt tcgcgtgatg atgcagaaga tatcctttgg ttgatattca
     4981 attcggtgcg tgggcttgct gttcgtagcc tatggcagaa ggacaaagaa cgctttgagc
     5041 gtgtcaggaa ctcgacactc gaaattgcgc gagagcggta cgcgaaattc aagcgctaga
     5101 tctagaagcg gccgcgaatt cagaatagta atacaggatc cgaatcgttt cagttgccgc
     5161 cggattaata atcaaataac cgttcctaaa aaatgccctc ttgggttatc aagagggtca
     5221 ttatatttcg caaaaaaccc cgcttcggcg gggttttttc gccttgaaga cgaaagggcc
     5281 tcgtgatacg cctattttta taggttaatg tcatgataat aatggtttct tagacgtcag
     5341 gtggcacttt tcggggaaat gtggatcgaa gcccccgaag aacggtatcg agccgtcgcc
     5401 accgtacgcg aacgaccagc cgcacaccgt ccacgtgatg ccgacgatgc cgagcaccgc
     5461 aaacgacatg atcatcgtgc tcacgacgtt cttacggcgg ctcaagcccc cgtagaagaa
     5521 cgccagaccc ggcgtcatca gcaggacgag catcgcgcaa acgatcatga agcccgtcga
     5581 ccctgtgtca aacatatgca tctacctcct tggaaatcgc gcgtcgcatc ggttacgctt
     5641 cgatgatccc acgctttgac ctggtgttga gtcacagctg ccactgttaa cacgccggga
     5701 aacgtcttgt aacaaacacg aagcggccgc gttacaataa ggaaacacgc accaccgccc
     5761 cacccgcgaa aggttgcctt ccatgagcat cgcattcgac gccgttcgaa ccgccgacga
     5821 tagacagcgc ctcgccgcgc tggccgacga gatctggcac gagtactggc ccgccctcat
     5881 cggcgacgcg caaaccgact acatggtgga gaacttccaa agcctcggcg ccatcgagcg
     5941 cgacctgcgc gagcacgcct acgagtactg gttcctgcgc gccgaggacg acgggcgcat
     6001 cgtggggtac acggggggcc gcgcggagcc cgagacgaac cgctacttca tctcgaaggt
     6061 gtacctgcag gccgaggaac gcggcaaggg cttcgcctcg caggccatcg ccttctacga
     6121 gcggctgtgc cacgagcgcg gcctcgaggc gatgtacctc acggtgaaca aacacaacga
     6181 ccttggtatt cgcgcctatc tcggcaaggg attcgaaacc atcgacgctg tggaaaccga
     6241 catcggcaac ggcttcgtca tggacgactt catcatggag aaacgggtcg agtagcccga
     6301 gaggccaagt cgccgacagt ccttgcggaa ccgtcggcga caagggtact tatgattcca
     6361 tcgtgcgctt cccgaacact cggaaagtcc cttcgcaatc ccagtctaat cacagcgcaa
     6421 cgtcaggtaa aacacggtat tgaccatttg ttttcgcagg tcaaaatact agttttccgc
     6481 aagtgacgat tttcgacagc agacgcgcgg ccgagaggag cgcggcgtag tatactgcga
     6541 gcgcaacgaa gaccgaaggg acggagatgc cgatgaaggg gttcgccgac gctgcagcgc
     6601 tcgagagcgc gctcgcgctt ctgcatgatg cagagctcat ggaattcgtc caggaacatc
     6661 agcacaacta cctctaccgc gcggatttca tgaaagagcc tatgcccggg gatgcagacc
     6721 cggcgcttat gtgggagctc gtgtctttcg tccgacgcat ggcgggaagg cctaccgtgc
     6781 gggagctgaa caagtcgcag ccaatcggcc ggcaatgctt ctggacggcg acgcccgaga
     6841 tgatcgccgg actgaacgat atcgtgagca ggagcaatcc ctccgcccag ctaacgcagt
     6901 cgctcgcgcg gctcagcacg cgacccgcca tccagcaact tgcgcttgaa gagctggaag
     6961 cggcagggta tcgcgacggc gtcgacgtcg agcatgaaga gctgcgggag atcgtttgcg
     7021 acaagcgagc gccggaaacg cccgaagaac ggctcttcgc caacgcgaga gagctcatcg
     7081 aagaaatcga ggagctcgcg tccgaaccgt tcaacgtcga gctcatcgcc tacatgcacg
     7141 agcgcctcgc gctcgacacg aaggggcttc gcgtcgagcc tcgcccttcc cccgcaccga
     7201 agacctccgt gccgtcacca ccttccgatc agctgctcga atcgatcgtc gcaggatgcg
     7261 agaatccatg cctgtggggt ccccatcctt tattcggcgc gctgataaac gccgacacga
     7321 tttgggtaac gccggcattc gagcgcttca acggactcat ggagttgctc attcgctgct
     7381 gtcagaccaa gtttactcat atatacttta gattgattta aaacttcatt tttaatttaa
     7441 aaggatctag gtgaagatcc tttttgataa tctcatgacc aaaatccctt aacgtgagtt
     7501 ttcgttccac tgagcgtcag accccgtaga aaagatcaaa ggatcttctt gagatccttt
     7561 ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg
     7621 tttgccggat caagagctac caactctttt tccgaaggta actggcttca gcagagcgca
     7681 gataccaaat actgtccttc tagtgtagcc gtagttaggc caccacttca agaactctgt
     7741 agcaccgcct acatacctcg ctctgctaat cctgttacca gtggctgctg ccagtggcga
     7801 taagtcgtgt cttaccgggt tggactcaag acgatagtta ccggataagg cgcagcggtc
     7861 gggctgaacg gggggttcgt gcacacagcc cagcttggag cgaacgacct acaccgaact
     7921 gagataccta cagcgtgagc tatgagaaag cgccacgctt cccgaaggga gaaaggcgga
     7981 caggtatccg gtaagcggca gggtcggaac aggagagcgc acgagggagc ttccaggggg
     8041 aaacgcctgg tatctttata gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt
     8101 tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac gccagcaacg cggcccgaga
     8161 tgcgccgcgt gcggctgctg gagatggcgg acgcgatgga tatgttctgc caagggttgg
     8221 tttgcgcatt cacagttctc cgcaagaatt gattggctcc aattcttgga gtggtgaatc
     8281 cgttagcgag gtgccgccgg catgagctcc tctagtagag agcgttcacc gacaaacaac
     8341 agataaaacg aaaggcccag tctttcgact gagcctttcg ttttatttga aaaagaaaag
     8401 ccccagcaag cttggcggca gcaggggcat tcgagaca
//
