LOCUS       sequence-376012-        8279 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  E. coli - Eggerthella lenta shuttle plasmid (KanR), cumate-inducible
            promoter Pct5.1-crRNA(AarI)-RFP, entry plasmid for crRNA cloning.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8279)
  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 8279)
  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: pXD71Cas10RFP-Pct5.1-crRNA(AarI)-RFP
FEATURES             Location/Qualifiers
     source          1..8279
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     terminator      24..95
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     rep_origin      complement(276..864)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(356..375)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(1035..1826,1827..1895))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     1658..1677
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(1896..2000)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     2068..2086
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     misc_feature    2243..2262
                     /label=BioBrick prefix
                     /note="BioBrick prefix for parts that start with ""ATG"""
     CDS             complement(2265..2876)
                     /codon_start=1
                     /gene="cymR from Pseudomonas putida"
                     /product="cumate repressor (Mullick et al., 2006)"
                     /label=CymR
                     /translation="MSPKRRTQAERAMETQGKLIAAALGVLREKGYAGFRIADVPGAAG
                     VSRGAQSHHFPTKLELLLATFEWLYEQITERSRARLAKLKPEDDVIQQMLDDAAEFFLD
                     DDFSISLDLIVAADRDPALREGIQRTVERNRFVVEDMWLGVLVSRGLSRDDAEDILWLI
                     FNSVRGLAVRSLWQKDKERFERVRNSTLEIARERYAKFKR"
     protein_bind    3214..3241
                     /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    3256..3283
                     /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    3441..3462
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(3477..3482,3483..3500,3501..3507)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3515..3531
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     RBS             3549..3560
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             3567..4244
                     /codon_start=1
                     /product="monomeric derivative of DsRed (Campbell et al., 
                     2002)"
                     /label=mRFP1
                     /translation="MASSEDVIKEFMRFKVRMEGSVNGHEFEIEGEGEGRPYEGTQTAK
                     LKVTKGGPLPFAWDILSPQFQYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGV
                     VTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASTERMYPEDGALKGEIKM
                     RLKLKDGGHYDAEVKTTYMAKKPVQLPGAYKTDIKLDITSHNEDYTIVEQYERAEGRHS
                     TGA"
     terminator      4306..4333
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     CDS             complement(4456..5250)
                     /codon_start=1
                     /gene="aphA-3"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin"
                     /translation="MAKMRISPELKKLIEKYRCVKDTEGMSPAKVYKLVGENENLYLKM
                     TDSRYKGTTYDVEREKDMMLWLEGKLPVPKVLHFERHDGWSNLLMSEADGVLCSEEYED
                     EQSPEKIIELYAECIRLFHSIDISDCPYTNSLDSRLAELDYLLNNDLADVDCENWEEDT
                     PFKDPRELYDFLKTEKPEEELVFSHGDLGDSNIFVKDGKVSGFIDLGRSGRADKWYDIA
                     FCVRSIREDIGEEQYVELFFDLLGIKPDWEKIKYYILLDELF"
ORIGIN
        1 agcttgctgg ggcttttctt tttcaaataa aacgaaaggc tcagtcgaaa gactgggcct
       61 ttcgttttat ctgttgtttg tcggtgaacg ctctctacta gaggagctca tgccggcggc
      121 acctcgctaa cggattcacc actccaagaa ttggagccaa tcaattcttg cggagaactg
      181 tgaatgcgca aaccaaccct tggcagaaca tatccatcgc gtccgccatc tccagcagcc
      241 gcacgcggcg catctcgggc cgcgttgctg gcgtttttcc ataggctccg cccccctgac
      301 gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg actataaaga
      361 taccaggcgt ttccccctgg aagctccctc gtgcgctctc ctgttccgac cctgccgctt
      421 accggatacc tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca tagctcacgc
      481 tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc
      541 cccgttcagc ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc caacccggta
      601 agacacgact tatcgccact ggcagcagcc actggtaaca ggattagcag agcgaggtat
      661 gtaggcggtg ctacagagtt cttgaagtgg tggcctaact acggctacac tagaaggaca
      721 gtatttggta tctgcgctct gctgaagcca gttaccttcg gaaaaagagt tggtagctct
      781 tgatccggca aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt
      841 acgcgcagaa aaaaaggatc tcaagaagat cctttgatct tttctacggg gtctgacgct
      901 cagtggaacg aaaactcacg ttaagggatt ttggtcatga gattatcaaa aaggatcttc
      961 acctagatcc ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa
     1021 acttggtctg acagttacca atgcttaatc agtgaggcac ctatctcagc gatctgtcta
     1081 tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga taactacgat acgggagggc
     1141 ttaccatctg gccccagtgc tgcaatgata ccgcgagacc cacgctcacc ggctccagat
     1201 ttatcagcaa taaaccagcc agccggaagg gccgagcgca gaagtggtcc tgcaacttta
     1261 tccgcctcca tccagtctat taattgttgc cgggaagcta gagtaagtag ttcgccagtt
     1321 aatagtttgc gcaacgttgt tgccattgct gcaggcatcg tggtgtcacg ctcgtcgttt
     1381 ggtatggctt cattcagctc cggttcccaa cgatcaaggc gagttacatg atcccccatg
     1441 ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg ttgtcagaag taagttggcc
     1501 gcagtgttat cactcatggt tatggcagca ctgcataatt ctcttactgt catgccatcc
     1561 gtaagatgct tttctgtgac tggtgagtac tcaaccaagt cattctgaga atagtgtatg
     1621 cggcgaccga gttgctcttg cccggcgtca acacgggata ataccgcgcc acatagcaga
     1681 actttaaaag tgctcatcat tggaaaacgt tcttcggggc gaaaactctc aaggatctta
     1741 ccgctgttga gatccagttc gatgtaaccc actcgtgcac ccaactgatc ttcagcatct
     1801 tttactttca ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag
     1861 ggaataaggg cgacacggaa atgttgaata ctcatactct tcctttttca atattattga
     1921 agcatttatc agggttattg tctcatgagc ggatacatat ttgaatgtat ttagaaaaat
     1981 aaacaaatag gggttccgcg cacatttccc cgaaaagtgc cacctgacgt ctaagaaacc
     2041 attattatca tgacattaac ctataaaaat aggcgtatca cgaggccctt tcgtcttcaa
     2101 ggcgaaaaaa ccccgccgaa gcggggtttt ttgcgaaata taatgaccct cttgataacc
     2161 caagagggca ttttttagga acggttattt gattattaat ccggcggcaa ctgaaacgat
     2221 tcggatcctg tattactatt ctgaattcgc ggccgcttct agatctagcg cttgaatttc
     2281 gcgtaccgct ctcgcgcaat ttcgagtgtc gagttcctga cacgctcaaa gcgttctttg
     2341 tccttctgcc ataggctacg aacagcaagc ccacgcaccg aattgaatat caaccaaagg
     2401 atatcttctg catcatcacg cgaaagacca cggctcacca gaacaccaag ccacatatcc
     2461 tcgacgacaa accgattcct ctctaccgtg cgctgaatac cctcgcgtaa cgctgggtcc
     2521 cggtcggcag ccacaatcaa atcaaggctg atagagaagt catcgtcgag gaaaaattcg
     2581 gcggcgtcgt ccagcatttg ctggatgacg tcatcctctg gcttcaattt cgctaatcga
     2641 gcccgactgc gttcggtgat ctgttcgtaa agccattcaa aagtggcaag cagaagctca
     2701 agctttgtcg ggaaatgatg gctctgcgct cctctggaga caccagcagc accgggcaca
     2761 tctgcgatcc ggaatcccgc gtaacctttt tcccgtaaaa cccccagggc cgctgcaatc
     2821 aacttgccct gggtctccat tgcgcgctct gcctgtgttc ttctctttgg actcatgatc
     2881 accatttttt ttactaaagc ttgatctgca atttgaataa taaccactcc tttgtttatc
     2941 caccgaacta agttggtgtt ttttgaagct tgaattagat atttaaaagt atcatatcta
     3001 atattataac taaattttct aaaaaaaaca ttgaaggaac ggttatttga ttattaatcc
     3061 ggctttttta ttatttaggc aactgaaacg attcggatcc tgtattacta ttctattttg
     3121 tcaaaataat tttattgaca acgtcttatt aacgttgata taatttaaat tttatttgac
     3181 aaaaatgggc tcgtgttgta caataaatgt agtaacaaac agacaatctg gtctgtttgt
     3241 acgagtgttg cggccaacaa acagacaatc tggtctgttt gtattatgat taactaatgt
     3301 cactccccgc atggggagtg cgggttgaaa tagtcgcagg tgctacgcaa accgcctctc
     3361 cccgcgcgtt ggccgattca ttaatgcagc tggcacgaca ggtttcccga ctggaaagcg
     3421 ggcagtgagc gcaacgcaat taatgtgagt tagctcactc attaggcacc ccaggcttta
     3481 cactttatgc ttccggctcg tatgttgtgt ggaattgtga gcggataaca atttcacaca
     3541 tactagagaa agaggagaaa tactagatgg cttcctccga agacgttatc aaagagttca
     3601 tgcgtttcaa agttcgtatg gaaggttccg ttaacggtca cgagttcgaa atcgaaggtg
     3661 aaggtgaagg tcgtccgtac gaaggtaccc agaccgctaa actgaaagtt accaaaggtg
     3721 gtccgctgcc gttcgcttgg gacatcctgt ccccgcagtt ccagtacggt tccaaagctt
     3781 acgttaaaca cccggctgac atcccggact acctgaaact gtccttcccg gaaggtttca
     3841 aatgggaacg tgttatgaac ttcgaagacg gtggtgttgt taccgttacc caggactcct
     3901 ccctgcaaga cggtgagttc atctacaaag ttaaactgcg tggtaccaac ttcccgtccg
     3961 atggtccggt tatgcagaaa aaaaccatgg gttgggaagc ttccaccgaa cgtatgtacc
     4021 cggaagacgg tgctctgaaa ggtgaaatca aaatgcgtct gaaactgaaa gacggtggtc
     4081 actacgacgc tgaagttaaa accacctaca tggctaaaaa accggttcag ctgccgggtg
     4141 cttacaaaac cgacatcaaa ctggacatca cctcccacaa cgaagactac accatcgttg
     4201 aacagtacga acgtgctgaa ggtcgtcact ccaccggtgc ttaacacctg cgactgtcac
     4261 tccccgcatg gggagtgcgg gttgaaatac tagtcgactc acactggctc accttcgggt
     4321 gggcctttct gcgtttatat actagagaga gaatataaaa agccagatta ttaatccggc
     4381 ttttttatta tttaattcct gttataaaaa aaggatcaat tttgaactct ctcccaaagt
     4441 tgatccctta acgatctaaa acaattcatc cagtaaaata taatatttta ttttctccca
     4501 atcaggcttg atccccagta agtcaaaaaa tagctcgaca tactgttctt ccccgatatc
     4561 ctccctgatc gaccggacgc agaaggcaat gtcataccac ttgtccgccc tgccgcttct
     4621 cccaagatca ataaagccac ttactttgcc atctttcaca aagatgttgc tgtctcccag
     4681 gtcgccgtgg gaaaagacaa gttcctcttc gggcttttcc gtctttaaaa aatcatacag
     4741 ctcgcgcgga tctttaaatg gagtgtcttc ttcccagttt tcgcaatcca catcggccag
     4801 atcgttattc agtaagtaat ccaattcggc taagcggctg tctaagctat tcgtataggg
     4861 acaatccgat atgtcgatgg agtgaaagag cctgatgcac tccgcataca gctcgataat
     4921 cttttcaggg ctttgttcat cttcatactc ttccgagcaa aggacgccat cggcctcact
     4981 catgagcaga ttgctccagc catcatgccg ttcaaagtgc aggacctttg gaacaggcag
     5041 ctttccttcc agccatagca tcatgtcctt ttcccgttcc acatcatagg tggtcccttt
     5101 ataccggctg tccgtcattt ttaaatatag gttttcattt tctcccacca gcttatatac
     5161 cttagcagga gacattcctt ccgtatcttt tacgcagcgg tatttttcga tcagtttttt
     5221 caattccggt gatattctca ttttagccat ttattatttc cttcctcttt tctacagtat
     5281 ttaaagatac cccaagaagc taattataac aagacgaact ccaattcact gttccttgca
     5341 ttctaaaacc ttaaatacca gaaaacagct ttttcaaagt tgttttcaaa gttggcgtat
     5401 aacatagtat cgacggagcc gattttgaaa ccacaattat gatagaattt acaagctata
     5461 aggttattgt cctgggtttc aagcattagt ccatgcaagt ttttatgctt tgcccattct
     5521 atagatatat tgataagaag actggaatgc ccgctccgcc gggattattc gaaagcgcat
     5581 cgtccaagcg agaccgtctg cattcggaaa tctcggccgg ttcgcgccga ggaagcccat
     5641 ctggctgcat cccaaatcga cacacaacgc acggaggccc cggcgatcgc cggggcctcc
     5701 gaatcatgcg catcgtggaa gcgaagctcc gcaaaaaacc ccgcttcagc ggggtttttt
     5761 cgcgtcgcac ctccttgtcg tgtattacag gtactactat aacacatcat ctagcccgcc
     5821 cgcgatacgt cctctgcggt gcagcgtggt cgcggttcca cgcatccgcc gcccgctgcg
     5881 catcgcgcag cacgtcatta aggctctgtt gccttggctg ttccctgcgc atcagatatt
     5941 cgagcgggcg cgatattcga cttctaagct cttccacttt gattcgcatt cccgcaactc
     6001 ctgctctgat gcggctcgcg agtccatcgc acttaacaac gatgttatcg aggattgcgc
     6061 cgcgctcagc ttttcccgca gcttcccagc cccgaacctc ggtagcgatg ggttgaagct
     6121 ctccaatgtc tcgcgcaact ccgtccgctc gttgccgaag acactccaat cggtcttgct
     6181 catcttcgat atcgagcttc acgtcctcga gctggctgtc aagctcggca aggtcaccct
     6241 ccttgtcggc aatctcggta acaagctctg ccttacgcgt ttcggcatca ttcagtaaag
     6301 cggcggcttc ttccctagcc gccgcgacga ttcgcgccgc ttcgtccttc gcaggttcaa
     6361 ccacggcctt atcgaccgca cagcgtacct tgtcgatatc cacggacttg tccgtgtata
     6421 cacgctgcgc ccgcgtctcc tcgtcaagct cgattgcagg gcggtagccc atgcgcccct
     6481 ctaggtaatc cccaagctcc ttgtgcatgc tccggtagaa gctgcggggg cacatctgct
     6541 tgtagttgaa ccgtccatct aggatgggcg tgaacggcac gtgcatgtgg tcgcgggcgg
     6601 gcgtgccatc cttcaacacc tcgtccttgt ggacgtaccc gcccatcatg ttctcgatgc
     6661 cgaacttctt cgacaggtac cagtacgtga gacggaagaa cctgtcctca tcgcccttgc
     6721 gcacattgtc gggaagcgtc acaacaacgt cagccataac gaccgcatcc ttgcgcgtgg
     6781 cacgcttccc tgcggccttc tgggcctcgt tcacgcgctc gatgcggctc tccaccgtgc
     6841 cccagttcgg ctcgccatcg cgcggctcca cgcgccccac aacgagcttg ccgtccttgt
     6901 tcgtgtagtg gccgaccacc aagtcgttct tgatgcgctc ggggtcgatg ttgtcgcgct
     6961 ccaggctgct gcggtcgcgg cggtagtggg caagcatgtg cccgaccgat ggagccttgt
     7021 atttcgcgat gtgagccatg gaaacctccg aaccgtacag acttaatcga ctaaagtcta
     7081 gcacgttaga ccttagctac gctaaggacg tggcagggcc agccctgcac ccgctcgggc
     7141 gttccccccg agacccccca gcccttgccc gttcgcggct tcgctcacgg ccctcgggcc
     7201 atcggcgcga gcgccgaacg gagggacttc gcccctccga accacccctc aatctacctg
     7261 cggttcggca cacggcacac ggcactatta ggggggccgt cgtagtggcc cccctgtggt
     7321 catttgtcat ttgtcatttg tcacattatt tttcggacac attatttttc ggacgtagtt
     7381 agcgaaaaat aatgagccgc ccgattctgc aggtctcatc cagtcgagac acgggcaagg
     7441 aaagggtcga tggagcagag cgtccatcga cccttaatca agtctggtct attcggttgg
     7501 ccgtcgcgtc aattccgcaa tccgtgaacg agtcggctta gatctgcgcg agcgcagacc
     7561 acgccgcccc gtagtcacgc attgctgcat tgacccgact actctttggc acctgactgt
     7621 tccacgcact tgcgcttacg ttcggcgttc agatagttcc cgcgacaata gcgctcgaga
     7681 atcgagttgt accccacgat gatttcgcgg tactgctcgg gaaactccct cagcgccgca
     7741 gccgtgaaat cgttgatggt ctcgaacctc tcgcacatga tgaagccgac caactcgaag
     7801 agcatgtcct gcttgtcctt agtcgagagc tgttcgagct gaccaatgtc gaagttgttt
     7861 ccctcgatgc gtgcttcgac cggatacagc tccttgccct tcttgcgaca ggaagccgtg
     7921 tcgtgaatca gatagtcata cgcatgcctg atgttgttga ttggctcggc tgacgaacag
     7981 cacttcttgc cgtccgcgct caatcggttc aagacgttga tgattgcctt gcgtgtcgtg
     8041 ttgccgcccc agacaacaat gacgtgcgca tgcgtcttgc gctgcttgct cttctggtca
     8101 tggtctatgt cgtgaaccgc ataagcgaaa ggcacttgaa gaatgtcgtc tatctcgtcc
     8161 tgccagtcat cgcgcatgtt ctctgggtag cagatgccct cccagagctt caccttcgtc
     8221 tcgccagcgt ttgccgccat gctataatct ccttgtctcg aatgcccctg ctgccgcca
//
