LOCUS       sequence-375980-        8438 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  E. coli - Eggerthella lenta shuttle plasmid (KanR), cumate-inducible
            promoter Pct5.1-crRNA(nontargeting spacer) cgr1/cgr2-deleting repair
            template, used as negative control for pXD71Cas10.4RT4.
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: pXD71Cas10.0RTcgr-Pct5.1-crRNA(AarI)-RT(Delta-cgr12)
FEATURES             Location/Qualifiers
     source          1..8438
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             3008..3802
                     /codon_start=1
                     /gene="aphA-3"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin"
                     /translation="MAKMRISPELKKLIEKYRCVKDTEGMSPAKVYKLVGENENLYLKM
                     TDSRYKGTTYDVEREKDMMLWLEGKLPVPKVLHFERHDGWSNLLMSEADGVLCSEEYED
                     EQSPEKIIELYAECIRLFHSIDISDCPYTNSLDSRLAELDYLLNNDLADVDCENWEEDT
                     PFKDPRELYDFLKTEKPEEELVFSHGDLGDSNIFVKDGKVSGFIDLGRSGRADKWYDIA
                     FCVRSIREDIGEEQYVELFFDLLGIKPDWEKIKYYILLDELF"
     terminator      complement(3925..3952)
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     protein_bind    4085..4112
                     /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    4127..4154
                     /label=CuO
                     /bound_moiety="CymR"
                     /note="CymR-binding P2 operator sequence from the p-cmt 
                     operon of Pseudomonas putida (Mullick et al., 2006)"
     CDS             4492..5103
                     /codon_start=1
                     /gene="cymR from Pseudomonas putida"
                     /product="cumate repressor (Mullick et al., 2006)"
                     /label=CymR
                     /translation="MSPKRRTQAERAMETQGKLIAAALGVLREKGYAGFRIADVPGAAG
                     VSRGAQSHHFPTKLELLLATFEWLYEQITERSRARLAKLKPEDDVIQQMLDDAAEFFLD
                     DDFSISLDLIVAADRDPALREGIQRTVERNRFVVEDMWLGVLVSRGLSRDDAEDILWLI
                     FNSVRGLAVRSLWQKDKERFERVRNSTLEIARERYAKFKR"
     misc_feature    5106..5125
                     /label=BioBrick prefix
                     /note="BioBrick prefix for parts that start with ""ATG"""
     primer_bind     complement(5282..5300)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     rep_origin      7553..8141
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     8042..8061
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     terminator      complement(8322..8393)
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
ORIGIN
        1 gacaaggaga ttatagcatg gcggcaaacg ctggcgagac gaaggtgaag ctctgggagg
       61 gcatctgcta cccagagaac atgcgcgatg actggcagga cgagatagac gacattcttc
      121 aagtgccttt cgcttatgcg gttcacgaca tagaccatga ccagaagagc aagcagcgca
      181 agacgcatgc gcacgtcatt gttgtctggg gcggcaacac gacacgcaag gcaatcatca
      241 acgtcttgaa ccgattgagc gcggacggca agaagtgctg ttcgtcagcc gagccaatca
      301 acaacatcag gcatgcgtat gactatctga ttcacgacac ggcttcctgt cgcaagaagg
      361 gcaaggagct gtatccggtc gaagcacgca tcgagggaaa caacttcgac attggtcagc
      421 tcgaacagct ctcgactaag gacaagcagg acatgctctt cgagttggtc ggcttcatca
      481 tgtgcgagag gttcgagacc atcaacgatt tcacggctgc ggcgctgagg gagtttcccg
      541 agcagtaccg cgaaatcatc gtggggtaca actcgattct cgagcgctat tgtcgcggga
      601 actatctgaa cgccgaacgt aagcgcaagt gcgtggaaca gtcaggtgcc aaagagtagt
      661 cgggtcaatg cagcaatgcg tgactacggg gcggcgtggt ctgcgctcgc gcagatctaa
      721 gccgactcgt tcacggattg cggaattgac gcgacggcca accgaataga ccagacttga
      781 ttaagggtcg atggacgctc tgctccatcg accctttcct tgcccgtgtc tcgactggat
      841 gagacctgca gaatcgggcg gctcattatt tttcgctaac tacgtccgaa aaataatgtg
      901 tccgaaaaat aatgtgacaa atgacaaatg acaaatgacc acaggggggc cactacgacg
      961 gcccccctaa tagtgccgtg tgccgtgtgc cgaaccgcag gtagattgag gggtggttcg
     1021 gaggggcgaa gtccctccgt tcggcgctcg cgccgatggc ccgagggccg tgagcgaagc
     1081 cgcgaacggg caagggctgg ggggtctcgg ggggaacgcc cgagcgggtg cagggctggc
     1141 cctgccacgt ccttagcgta gctaaggtct aacgtgctag actttagtcg attaagtctg
     1201 tacggttcgg aggtttccat ggctcacatc gcgaaataca aggctccatc ggtcgggcac
     1261 atgcttgccc actaccgccg cgaccgcagc agcctggagc gcgacaacat cgaccccgag
     1321 cgcatcaaga acgacttggt ggtcggccac tacacgaaca aggacggcaa gctcgttgtg
     1381 gggcgcgtgg agccgcgcga tggcgagccg aactggggca cggtggagag ccgcatcgag
     1441 cgcgtgaacg aggcccagaa ggccgcaggg aagcgtgcca cgcgcaagga tgcggtcgtt
     1501 atggctgacg ttgttgtgac gcttcccgac aatgtgcgca agggcgatga ggacaggttc
     1561 ttccgtctca cgtactggta cctgtcgaag aagttcggca tcgagaacat gatgggcggg
     1621 tacgtccaca aggacgaggt gttgaaggat ggcacgcccg cccgcgacca catgcacgtg
     1681 ccgttcacgc ccatcctaga tggacggttc aactacaagc agatgtgccc ccgcagcttc
     1741 taccggagca tgcacaagga gcttggggat tacctagagg ggcgcatggg ctaccgccct
     1801 gcaatcgagc ttgacgagga gacgcgggcg cagcgtgtat acacggacaa gtccgtggat
     1861 atcgacaagg tacgctgtgc ggtcgataag gccgtggttg aacctgcgaa ggacgaagcg
     1921 gcgcgaatcg tcgcggcggc tagggaagaa gccgccgctt tactgaatga tgccgaaacg
     1981 cgtaaggcag agcttgttac cgagattgcc gacaaggagg gtgaccttgc cgagcttgac
     2041 agccagctcg aggacgtgaa gctcgatatc gaagatgagc aagaccgatt ggagtgtctt
     2101 cggcaacgag cggacggagt tgcgcgagac attggagagc ttcaacccat cgctaccgag
     2161 gttcggggct gggaagctgc gggaaaagct gagcgcggcg caatcctcga taacatcgtt
     2221 gttaagtgcg atggactcgc gagccgcatc agagcaggag ttgcgggaat gcgaatcaaa
     2281 gtggaagagc ttagaagtcg aatatcgcgc ccgctcgaat atctgatgcg cagggaacag
     2341 ccaaggcaac agagccttaa tgacgtgctg cgcgatgcgc agcgggcggc ggatgcgtgg
     2401 aaccgcgacc acgctgcacc gcagaggacg tatcgcgggc gggctagatg atgtgttata
     2461 gtagtacctg taatacacga caaggaggtg cgacgcgaaa aaaccccgct gaagcggggt
     2521 tttttgcgga gcttcgcttc cacgatgcgc atgattcgga ggccccggcg atcgccgggg
     2581 cctccgtgcg ttgtgtgtcg atttgggatg cagccagatg ggcttcctcg gcgcgaaccg
     2641 gccgagattt ccgaatgcag acggtctcgc ttggacgatg cgctttcgaa taatcccggc
     2701 ggagcgggca ttccagtctt cttatcaata tatctataga atgggcaaag cataaaaact
     2761 tgcatggact aatgcttgaa acccaggaca ataaccttat agcttgtaaa ttctatcata
     2821 attgtggttt caaaatcggc tccgtcgata ctatgttata cgccaacttt gaaaacaact
     2881 ttgaaaaagc tgttttctgg tatttaaggt tttagaatgc aaggaacagt gaattggagt
     2941 tcgtcttgtt ataattagct tcttggggta tctttaaata ctgtagaaaa gaggaaggaa
     3001 ataataaatg gctaaaatga gaatatcacc ggaattgaaa aaactgatcg aaaaataccg
     3061 ctgcgtaaaa gatacggaag gaatgtctcc tgctaaggta tataagctgg tgggagaaaa
     3121 tgaaaaccta tatttaaaaa tgacggacag ccggtataaa gggaccacct atgatgtgga
     3181 acgggaaaag gacatgatgc tatggctgga aggaaagctg cctgttccaa aggtcctgca
     3241 ctttgaacgg catgatggct ggagcaatct gctcatgagt gaggccgatg gcgtcctttg
     3301 ctcggaagag tatgaagatg aacaaagccc tgaaaagatt atcgagctgt atgcggagtg
     3361 catcaggctc tttcactcca tcgacatatc ggattgtccc tatacgaata gcttagacag
     3421 ccgcttagcc gaattggatt acttactgaa taacgatctg gccgatgtgg attgcgaaaa
     3481 ctgggaagaa gacactccat ttaaagatcc gcgcgagctg tatgattttt taaagacgga
     3541 aaagcccgaa gaggaacttg tcttttccca cggcgacctg ggagacagca acatctttgt
     3601 gaaagatggc aaagtaagtg gctttattga tcttgggaga agcggcaggg cggacaagtg
     3661 gtatgacatt gccttctgcg tccggtcgat cagggaggat atcggggaag aacagtatgt
     3721 cgagctattt tttgacttac tggggatcaa gcctgattgg gagaaaataa aatattatat
     3781 tttactggat gaattgtttt agatcgttaa gggatcaact ttgggagaga gttcaaaatt
     3841 gatccttttt ttataacagg aattaaataa taaaaaagcc ggattaataa tctggctttt
     3901 tatattctct ctctagtata taaacgcaga aaggcccacc cgaaggtgag ccagtgtgag
     3961 tcgactagta tttcaacccg cactccccat gcggggagtg acagtcgcag gtgagttcag
     4021 atctgcacct gcgactattt caacccgcac tccccatgcg gggagtgaca ttagttaatc
     4081 ataatacaaa cagaccagat tgtctgtttg ttggccgcaa cactcgtaca aacagaccag
     4141 attgtctgtt tgttactaca tttattgtac aacacgagcc catttttgtc aaataaaatt
     4201 taaattatat caacgttaat aagacgttgt caataaaatt attttgacaa aatagaatag
     4261 taatacagga tccgaatcgt ttcagttgcc taaataataa aaaagccgga ttaataatca
     4321 aataaccgtt ccttcaatgt tttttttaga aaatttagtt ataatattag atatgatact
     4381 tttaaatatc taattcaagc ttcaaaaaac accaacttag ttcggtggat aaacaaagga
     4441 gtggttatta ttcaaattgc agatcaagct ttagtaaaaa aaatggtgat catgagtcca
     4501 aagagaagaa cacaggcaga gcgcgcaatg gagacccagg gcaagttgat tgcagcggcc
     4561 ctgggggttt tacgggaaaa aggttacgcg ggattccgga tcgcagatgt gcccggtgct
     4621 gctggtgtct ccagaggagc gcagagccat catttcccga caaagcttga gcttctgctt
     4681 gccacttttg aatggcttta cgaacagatc accgaacgca gtcgggctcg attagcgaaa
     4741 ttgaagccag aggatgacgt catccagcaa atgctggacg acgccgccga atttttcctc
     4801 gacgatgact tctctatcag ccttgatttg attgtggctg ccgaccggga cccagcgtta
     4861 cgcgagggta ttcagcgcac ggtagagagg aatcggtttg tcgtcgagga tatgtggctt
     4921 ggtgttctgg tgagccgtgg tctttcgcgt gatgatgcag aagatatcct ttggttgata
     4981 ttcaattcgg tgcgtgggct tgctgttcgt agcctatggc agaaggacaa agaacgcttt
     5041 gagcgtgtca ggaactcgac actcgaaatt gcgcgagagc ggtacgcgaa attcaagcgc
     5101 tagatctaga agcggccgcg aattcagaat agtaatacag gatccgaatc gtttcagttg
     5161 ccgccggatt aataatcaaa taaccgttcc taaaaaatgc cctcttgggt tatcaagagg
     5221 gtcattatat ttcgcaaaaa accccgcttc ggcggggttt tttcgccttg aagacgaaag
     5281 ggcctcgtga tacgcctatt tttataggtt aatgtcatga taataatggt ttcttagacg
     5341 tcaggtggca cttttcgggg aaatgtggat cgaagccccc gaagaacggt atcgagccgt
     5401 cgccaccgta cgcgaacgac cagccgcaca ccgtccacgt gatgccgacg atgccgagca
     5461 ccgcaaacga catgatcatc gtgctcacga cgttcttacg gcggctcaag cccccgtaga
     5521 agaacgccag acccggcgtc atcagcagga cgagcatcgc gcaaacgatc atgaagcccg
     5581 tcgaccctgt gtcaaacata tgcatctacc tccttggaaa tcgcgcgtcg catcggttac
     5641 gcttcgatga tcccacgctt tgacctggtg ttgagtcaca gctgccactg ttaacacgcc
     5701 gggaaacgtc ttgtaacaaa cacgaagcgg ccgcgttaca ataaggaaac acgcaccacc
     5761 gccccacccg cgaaaggttg ccttccatga gcatcgcatt cgacgccgtt cgaaccgccg
     5821 acgatagaca gcgcctcgcc gcgctggccg acgagatctg gcacgagtac tggcccgccc
     5881 tcatcggcga cgcgcaaacc gactacatgg tggagaactt ccaaagcctc ggcgccatcg
     5941 agcgcgacct gcgcgagcac gcctacgagt actggttcct gcgcgccgag gacgacgggc
     6001 gcatcgtggg gtacacgggg ggccgcgcgg agcccgagac gaaccgctac ttcatctcga
     6061 aggtgtacct gcaggccgag gaacgcggca agggcttcgc ctcgcaggcc atcgccttct
     6121 acgagcggct gtgccacgag cgcggcctcg aggcgatgta cctcacggtg aacaaacaca
     6181 acgaccttgg tattcgcgcc tatctcggca agggattcga aaccatcgac gctgtggaaa
     6241 ccgacatcgg caacggcttc gtcatggacg acttcatcat ggagaaacgg gtcgagtagc
     6301 ccgagaggcc aagtcgccga cagtccttgc ggaaccgtcg gcgacaaggg tacttatgat
     6361 tccatcgtgc gcttcccgaa cactcggaaa gtcccttcgc aatcccagtc taatcacagc
     6421 gcaacgtcag gtaaaacacg gtattgacca tttgttttcg caggtcaaaa tactagtttt
     6481 ccgcaagtga cgattttcga cagcagacgc gcggccgaga ggagcgcggc gtagtatact
     6541 gcgagcgcaa cgaagaccga agggacggag atgccgatga aggggttcgc cgacgctgca
     6601 gcgctcgaga gcgcgctcgc gcttctgcat gatgcagagc tcatggaatt cgtccaggaa
     6661 catcagcaca actacctcta ccgcgcggat ttcatgaaag agcctatgcc cggggatgca
     6721 gacccggcgc ttatgtggga gctcgtgtct ttcgtccgac gcatggcggg aaggcctacc
     6781 gtgcgggagc tgaacaagtc gcagccaatc ggccggcaat gcttctggac ggcgacgccc
     6841 gagatgatcg ccggactgaa cgatatcgtg agcaggagca atccctccgc ccagctaacg
     6901 cagtcgctcg cgcggctcag cacgcgaccc gccatccagc aacttgcgct tgaagagctg
     6961 gaagcggcag ggtatcgcga cggcgtcgac gtcgagcatg aagagctgcg ggagatcgtt
     7021 tgcgacaagc gagcgccgga aacgcccgaa gaacggctct tcgccaacgc gagagagctc
     7081 atcgaagaaa tcgaggagct cgcgtccgaa ccgttcaacg tcgagctcat cgcctacatg
     7141 cacgagcgcc tcgcgctcga cacgaagggg cttcgcgtcg agcctcgccc ttcccccgca
     7201 ccgaagacct ccgtgccgtc accaccttcc gatcagctgc tcgaatcgat cgtcgcagga
     7261 tgcgagaatc catgcctgtg gggtccccat cctttattcg gcgcgctgat aaacgccgac
     7321 acgatttggg taacgccggc attcgagcgc ttcaacggac tcatggagtt gctcattcgc
     7381 tgctgtcaga ccaagtttac tcatatatac tttagattga tttaaaactt catttttaat
     7441 ttaaaaggat ctaggtgaag atcctttttg ataatctcat gaccaaaatc ccttaacgtg
     7501 agttttcgtt ccactgagcg tcagaccccg tagaaaagat caaaggatct tcttgagatc
     7561 ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg
     7621 tttgtttgcc ggatcaagag ctaccaactc tttttccgaa ggtaactggc ttcagcagag
     7681 cgcagatacc aaatactgtc cttctagtgt agccgtagtt aggccaccac ttcaagaact
     7741 ctgtagcacc gcctacatac ctcgctctgc taatcctgtt accagtggct gctgccagtg
     7801 gcgataagtc gtgtcttacc gggttggact caagacgata gttaccggat aaggcgcagc
     7861 ggtcgggctg aacggggggt tcgtgcacac agcccagctt ggagcgaacg acctacaccg
     7921 aactgagata cctacagcgt gagctatgag aaagcgccac gcttcccgaa gggagaaagg
     7981 cggacaggta tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg gagcttccag
     8041 ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg ccacctctga cttgagcgtc
     8101 gatttttgtg atgctcgtca ggggggcgga gcctatggaa aaacgccagc aacgcggccc
     8161 gagatgcgcc gcgtgcggct gctggagatg gcggacgcga tggatatgtt ctgccaaggg
     8221 ttggtttgcg cattcacagt tctccgcaag aattgattgg ctccaattct tggagtggtg
     8281 aatccgttag cgaggtgccg ccggcatgag ctcctctagt agagagcgtt caccgacaaa
     8341 caacagataa aacgaaaggc ccagtctttc gactgagcct ttcgttttat ttgaaaaaga
     8401 aaagccccag caagcttggc ggcagcaggg gcattcga
//
