LOCUS       sequence-368431-        8990 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses eMutaT7[PmCDA1] and UGI for mutagenizing genes downstream 
            from a T7 promoter in e.coli.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8990)
  AUTHORS   Seo D, Koh B, Eom GE, Kim HW, Kim S
  TITLE     A dual gene-specific mutator system installs all transition 
            mutations at similar frequencies in vivo.
  JOURNAL   Nucleic Acids Res. 2023 Apr 18:gkad266. doi: 10.1093/nar/gkad266.
  PUBMED    37070179
REFERENCE   2  (bases 1 to 8990)
  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: "Nucleic 
            Acids Res. 2023 Apr 18:gkad266. doi: 10.1093/nar/gkad266."
COMMENT     Sequence Label: pDae069 (eMutaT7[PmCDA1] with optimized RBS)
FEATURES             Location/Qualifiers
     source          1..8990
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(96..974)
                     /codon_start=1
                     /gene="araC"
                     /product="L-arabinose regulatory protein"
                     /label=araC
                     /translation="MAEAQNDPLLPGYSFNAHLVAGLTPIEANGYLDFFIDRPLGMKGY
                     ILNLTIRGQGVVKNQGREFVCRPGDILLFPPGEIHHYGRHPEAREWYHQWVYFRPRAYW
                     HEWLNWPSIFANTGFFRPDEAHQPHFSDLFGQIINAGQGEGRYSELLAINLLEQLLLRR
                     MEAINESLHPPMDNRVREACQYISDHLADSNFDIASVAQHVCLSPSRLSHLFRQQLGIS
                     VLSWREDQRISQAKLLLSTTRMPIATVGRNVGFDDQLYFSRVFKKCTGASPSEFRAGCE
                     EKVNDVAVKLS"
     promoter        1001..1285
                     /gene="araBAD"
                     /label=araBAD promoter
                     /note="promoter of the L-arabinose operon of E. coli; the 
                     araC regulatory gene is transcribed in the opposite 
                     direction (Guzman et al., 1995)"
     primer_bind     1208..1227
                     /label=pBAD Forward
                     /note="For vectors with E. coli araBAD promoter, forward 
                     primer"
     CDS             4731..4985
                     /codon_start=1
                     /product="uracil-DNA glycosylase inhibitor from a Bacillus 
                     subtilis bacteriophage (Mol et al., 1995)"
                     /label=UGI
                     /translation="MTNLSDIIEKETGKQLVIQESILMLPEEVEEVIGNKPESDILVHT
                     AYDESTDENVMLLTSDAPEYKPWALVIQDSNGENKIKML"
     primer_bind     complement(5024..5041)
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     terminator      5194..5280
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      5372..5399
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     promoter        5418..5509
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      5967..6422
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6054..6073)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     6264..6285
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(6978..7637)
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     7552..7571
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     promoter        complement(7638..7740)
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     primer_bind     complement(8132..8149)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(8266..8811)
                     /direction=LEFT
                     /label=p15A ori
                     /note="Plasmids containing the medium-copy-number p15A 
                     origin of replication can be propagated in E. coli cells 
                     that contain a second plasmid with the ColE1 origin.
                     "
     primer_bind     complement(8346..8365)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     8887..8906
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
ORIGIN
        1 atcgatgcat aatgtgcctg tcaaatggac gaagcaggga ttctgcaaac cctatgctac
       61 tccgtcaagc cgtcaattgt ctgattcgtt accaattatg acaacttgac ggctacatca
      121 ttcacttttt cttcacaacc ggcacggaac tcgctcgggc tggccccggt gcatttttta
      181 aatacccgcg agaaatagag ttgatcgtca aaaccaacat tgcgaccgac ggtggcgata
      241 ggcatccggg tggtgctcaa aagcagcttc gcctggctga tacgttggtc ctcgcgccag
      301 cttaagacgc taatccctaa ctgctggcgg aaaagatgtg acagacgcga cggcgacaag
      361 caaacatgct gtgcgacgct ggcgatatca aaattgctgt ctgccaggtg atcgctgatg
      421 tactgacaag cctcgcgtac ccgattatcc atcggtggat ggagcgactc gttaatcgct
      481 tccatgcgcc gcagtaacaa ttgctcaagc agatttatcg ccagcagctc cgaatagcgc
      541 ccttcccctt gcccggcgtt aatgatttgc ccaaacaggt cgctgaaatg cggctggtgc
      601 gcttcatccg ggcgaaagaa ccccgtattg gcaaatattg acggccagtt aagccattca
      661 tgccagtagg cgcgcggacg aaagtaaacc cactggtgat accattcgcg agcctccgga
      721 tgacgaccgt agtgatgaat ctctcctggc gggaacagca aaatatcacc cggtcggcaa
      781 acaaattctc gtccctgatt tttcaccacc ccctgaccgc gaatggtgag attgagaata
      841 taacctttca ttcccagcgg tcggtcgata aaaaaatcga gataaccgtt ggcctcaatc
      901 ggcgttaaac ccgccaccag atgggcatta aacgagtatc ccggcagcag gggatcattt
      961 tgcgcttcag ccatactttt catactcccg ccattcagag aagaaaccaa ttgtccatat
     1021 tgcatcagac attgccgtca ctgcgtcttt tactggctct tctcgctaac caaaccggta
     1081 accccgctta ttaaaagcat tctgtaacaa agcgggacca aagccatgac aaaaacgcgt
     1141 aacaaaagtg tctataatca cggcagaaaa gtccacattg attatttgca cggcgtcaca
     1201 ctttgctatg ccatagcatt tttatccata agattagcgg atcctacctg acgcttttta
     1261 tcgcaactct ctactgtttc tccatacccg tttttttggg ctagcgaatt cgagctcagg
     1321 aggaattcac catggcatta tcatatgatg acagacgccg agtacgtgcg cattcatgag
     1381 aaactggata tttacacctt caagaagcag ttcttcaaca acaagaaatc tgtgtcacac
     1441 cgctgctacg tgctgtttga gttgaagcga aggggcgaaa gaagggcttg cttttggggc
     1501 tatgccgtca acaagcccca aagtggcacc gagagaggaa tacacgctga gatattcagt
     1561 atccgaaagg tggaagagta tcttcgggat aatcctgggc agtttacgat caactggtat
     1621 tccagctgga gtccttgcgc tgattgtgcc gagaaaattc tggaatggta taatcaggaa
     1681 cttcggggaa acgggcacac attgaaaatc tgggcctgca agctgtacta cgagaagaat
     1741 gcccggaacc agataggact ctggaatctg agggacaatg gtgtaggcct gaacgtgatg
     1801 gtttccgagc actatcagtg ttgtcggaag attttcatcc aaagctctca taaccagctc
     1861 aatgaaaacc gctggttgga gaaaacactg aaacgtgcgg agaagtggag atccgagctg
     1921 agcatcatga tccaggtcaa gattctgcat accactaagt ctccagccgt tagcgagact
     1981 cccgggacct cagagtccgc cacacccgaa agtaacacga ttaacatcgc taagaacgac
     2041 ttctctgaca tcgaactggc tgctatcccg ttcaacactc tggctgacca ttacggtgag
     2101 cgtttagctc gcgaacagtt ggcccttgag catgagtctt acgagatggg tgaagcacgc
     2161 ttccgcaaga tgtttgagcg tcaacttaaa gctggtgagg ttgcggataa cgctgccgcc
     2221 aagcctctca tcactaccct actccctaag atgattgcac gcatcaacga ctggtttgag
     2281 gaagtgaaag ctaagcgcgg caagcgcccg acagccttcc agttcctgca agaaatcaag
     2341 ccggaagccg tagcgtacat caccattaag accactctgg cttgcctaac cagtgctgac
     2401 aatacaaccg ttcaggctgt agcaagcgca atcggtcggg ccattgagga cgaggctcgc
     2461 ttcggtcgta tccgtgacct tgaagctaag cacttcaaga aaaacgttga ggaacaactc
     2521 aacaagcgcg tagggcacgt ctacaagaaa gcatttatgc aagttgtcga ggctgacatg
     2581 ctctctaagg gtctactcgg tggcgaggcg tggtcttcgt ggcataagga agactctatt
     2641 catgtaggag tacgctgcat cgagatgctc attgagtcaa ccggaatggt tagcttacac
     2701 cgccaaaatg ctggcgtagt aggtcaagac tctgagacta tcgaactcgc acctgaatac
     2761 gctgaggcta tcgcaacccg tgcaggtgcg ctggctggca tctctccgat gttccaacct
     2821 tgcgtagttc ctcctaagcc gtggactggc attactggtg gtggctattg ggctaacggt
     2881 cgtcgtcctc tggcgctggt gcgtactcac agtaagaaag cactgatgcg ctacgaagac
     2941 gtttacatgc ctgaggtgta caaagcgatt aacattgcgc aaaacaccgc atggaaaatc
     3001 aacaagaaag tcctagcggt cgccaacgta atcaccaagt ggaagcattg tccggtcgag
     3061 gacatccctg cgattgagcg tgaagaactc ccgatgaaac cggaagacat cgacatgaat
     3121 cctgaggctc tcaccgcgtg gaaacgtgct gccgctgctg tgtaccgcaa ggacaaggct
     3181 cgcaagtctc gccgtatcag ccttgagttc atgcttgagc aagccaataa gtttgctaac
     3241 cataaggcca tctggttccc ttacaacatg gactggcgcg gtcgtgttta cgctgtgtca
     3301 atgttcaacc cgcaaggtaa cgatatgacc aaaggactgc ttacgctggc gaaaggtaaa
     3361 ccaatcggta aggaaggtta ctactggctg aaaatccacg gtgcaaactg tgcgggtgtc
     3421 gataaggttc cgttccctga gcgcatcaag ttcattgagg aaaaccacga gaacatcatg
     3481 gcttgcgcta agtctccact ggagaacact tggtgggctg agcaagattc tccgttctgc
     3541 ttccttgcgt tctgctttga gtacgctggg gtacagcacc acggcctgag ctataactgc
     3601 tcccttccgc tggcgtttga cgggtcttgc tctggcatcc agcacttctc cgcgatgctc
     3661 cgagatgagg taggtggtcg cgcggttaac ttgcttccta gtgaaaccgt tcaggacatc
     3721 tacgggattg ttgctaagaa agtcaacgag attctacaag cagacgcaat caatgggacc
     3781 gataacgaag tagttaccgt gaccgatgag aacactggtg aaatctctga gaaagtcaag
     3841 ctgggcacta aggcactggc tggtcaatgg ctggcttacg gtgttactcg cagtgtgact
     3901 aagcgttcag tcatgacgct ggcttacggg tccaaagagt tcggcttccg tcaacaagtg
     3961 ctggaagata ccattcagcc agctattgat tccggcaagg gtctgatgtt cactcagccg
     4021 aatcaggctg ctggatacat ggctaagctg atttgggaat ctgtgagcgt gacggtggta
     4081 gctgcggttg aagcaatgaa ctggcttaag tctgctgcta agctgctggc tgctgaggtc
     4141 aaagataaga agactggaga gattcttcgc aagcgttgcg ctgtgcattg ggtaactcct
     4201 gatggtttcc ctgtgtggca ggaatacaag aagcctattc agacgcgctt gaacctgatg
     4261 ttcctcggtc agttccgctt acagcctacc attaacacca acaaagatag cgagattgat
     4321 gcacacaaac aggagtctgg tatcgctcct aactttgtac acagccaaga cggtagccac
     4381 cttcgtaaga ctgtagtgtg ggcacacgag aagtacggaa tcgaatcttt tgcactgatt
     4441 cacgactcct tcggtaccat tccggctgac gctgcgaacc tgttcaaagc agtgcgcgaa
     4501 actatggttg acacctatga gtcttgtgat gtactggctg atttctacga ccagttcgct
     4561 gaccagttgc acgagtctca attggacaaa atgccagcac ttccggctaa aggtaacttg
     4621 aacctccgtg acatcttaga gtcggacttc gcgttcgcgt aactgcagcc cggggatcct
     4681 aacagagcgc gctctgtttg agtactagca ataaataagg aggatttttt atgaccaacc
     4741 tttccgacat catagagaag gaaacaggca aacagttggt catccaagag tcgatactca
     4801 tgcttcctga agaagttgag gaggtcattg ggaataagcc ggaaagtgac attctcgtac
     4861 acactgcgta tgatgagagc accgatgaga acgtgatgct gctcacgtca gatgccccag
     4921 agtacaaacc ctgggctctg gtgattcagg actctaatgg agagaacaag atcaagatgc
     4981 tgtaaaagct tggctgtttt ggcggatgag agaagatttt cagcctgata cagattaaat
     5041 cagaacgcag aagcggtctg ataaaacaga atttgcctgg cggcagtagc gcggtggtcc
     5101 cacctgaccc catgccgaac tcagaagtga aacgccgtag cgccgatggt agtgtggggt
     5161 ctccccatgc gagagtaggg aactgccagg catcaaataa aacgaaaggc tcagtcgaaa
     5221 gactgggcct ttcgttttat ctgttgtttg tcggtgaacg ctctcctgag taggacaaat
     5281 ccgccgggag cggatttgaa cgttgcgaag caacggcccg gagggtggcg ggcaggacgc
     5341 ccgccataaa ctgccaggca tcaaattaag cagaaggcca tcctgacgga tggccttttt
     5401 gcgtttctac aaactctttt gtttattttt ctaaatacat tcaaatatgt atccgctcat
     5461 gagacaataa ccctgataaa tgcttcaata atattgaaaa aggaagagta tgagtattca
     5521 acatttccgt gtcgccctta ttcccttttt tgcggcattt tgccttcctg tttttgctca
     5581 cccagaaacg ctggtgaaag taaaagatgc tgaagatcag ttgggtgcag caaactatta
     5641 actggcgaac tacttactct agcttcccgg caacaattaa tagactggat ggaggcggat
     5701 aaagttgcag gaccacttct gcgctcggcc cttccggctg gctggtttat tgctgataaa
     5761 tctggagccg gtgagcgtgg gtctcgcggt atcattgcag cactggggcc agatggtaag
     5821 ccctcccgta tcgtagttat ctacacgacg gggagtcagg caactatgga tgaacgaaat
     5881 agacagatcg ctgagatagg tgcctcactg attaagcatt ggtaactgtc agaccaagtt
     5941 tactcatata tactttagat tgatttacgc gccctgtagc ggcgcattaa gcgcggcggg
     6001 tgtggtggtt acgcgcagcg tgaccgctac acttgccagc gccctagcgc ccgctccttt
     6061 cgctttcttc ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg
     6121 ggggctccct ttagggttcc gatttagtgc tttacggcac ctcgacccca aaaaacttga
     6181 tttgggtgat ggttcacgta gtgggccatc gccctgatag acggtttttc gccctttgac
     6241 gttggagtcc acgttcttta atagtggact cttgttccaa actggaacaa cactcaaccc
     6301 tatctcgggc tattcttttg atttataagg gattttgccg atttcggcct attggttaaa
     6361 aaatgagctg atttaacaaa aatttaacgc gaattttaac aaaatattaa cgtttacaat
     6421 ttaaaaggat ctaggtgaag atcctttttg ataatctcat gaccaaaatc ccttaacgtg
     6481 agttttcgtt ccactgagcg tcagaccccg tagaaaagat caaaggatct tcttgagatc
     6541 ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg
     6601 tttgtttgcc ggatcaagag ctaccaactc tttttccgaa ggtaactggc ttcagcagag
     6661 cgcagatacc aaatactgtc cttctagtgt agccgtagtt aggccaccac ttcaagaact
     6721 ctgtagcacc gcctacatac ctcgctctgc taatcctgtt accagtcagg catttgagaa
     6781 gcacacggtc acactgcttc cggtagtcaa taaaccggta aaccagcaat agacataagc
     6841 ggctatttaa cgaccctgcc ctgaaccgac gaccgggtcg aatttgcttt cgaatttctg
     6901 ccattcatcc gcttattatc acttattcag gcgtagcaac caggcgttta agggcaccaa
     6961 taactgcctt aaaaaaatta cgccccgccc tgccactcat cgcagtactg ttgtaattca
     7021 ttaagcattc tgccgacatg gaagccatca caaacggcat gatgaacctg aatcgccagc
     7081 ggcatcagca ccttgtcgcc ttgcgtataa tatttgccca tggtgaaaac gggggcgaag
     7141 aagttgtcca tattggccac gtttaaatca aaactggtga aactcaccca gggattggct
     7201 gagacgaaaa acatattctc aataaaccct ttagggaaat aggccaggtt ttcaccgtaa
     7261 cacgccacat cttgcgaata tatgtgtaga aactgccgga aatcgtcgtg gtattcactc
     7321 cagagcgatg aaaacgtttc agtttgctca tggaaaacgg tgtaacaagg gtgaacacta
     7381 tcccatatca ccagctcacc gtctttcatt gccatacgga attccggatg agcattcatc
     7441 aggcgggcaa gaatgtgaat aaaggccgga taaaacttgt gcttattttt ctttacggtc
     7501 tttaaaaagg ccgtaatatc cagctgaacg gtctggttat aggtacattg agcaactgac
     7561 tgaaatgcct caaaatgttc tttacgatgc cattgggata tatcaacggt ggtatatcca
     7621 gtgatttttt tctccatttt agcttcctta gctcctgaaa atctcgataa ctcaaaaaat
     7681 acgcccggta gtgatcttat ttcattatgg tgaaagttgg aacctcttac gtgccgatca
     7741 acgtctcatt ttcgccaaaa gttggcccag ggcttcccgg tatcaacagg gacaccagga
     7801 tttatttatt ctgcgaagtg atcttccgtc acaggtattt attcggcgca aagtgcgtcg
     7861 ggtgatgctg ccaacttact gatttagtgt atgatggtgt ttttgaggtg ctccagtggc
     7921 ttctgtttct atcagctgtc cctcctgttc agctactgac ggggtggtgc gtaacggcaa
     7981 aagcaccgcc ggacatcagc gctagcggag tgtatactgg cttactatgt tggcactgat
     8041 gagggtgtca gtgaagtgct tcatgtggca ggagaaaaaa ggctgcaccg gtgcgtcagc
     8101 agaatatgtg atacaggata tattccgctt cctcgctcac tgactcgcta cgctcggtcg
     8161 ttcgactgcg gcgagcggaa atggcttacg aacggggcgg agatttcctg gaagatgcca
     8221 ggaagatact taacagggaa gtgagagggc cgcggcaaag ccgtttttcc ataggctccg
     8281 cccccctgac aagcatcacg aaatctgacg ctcaaatcag tggtggcgaa acccgacagg
     8341 actataaaga taccaggcgt ttccccctgg cggctccctc gtgcgctctc ctgttcctgc
     8401 ctttcggttt accggtgtca ttccgctgtt atggccgcgt ttgtctcatt ccacgcctga
     8461 cactcagttc cgggtaggca gttcgctcca agctggactg tatgcacgaa ccccccgttc
     8521 agtccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg gaaagacatg
     8581 caaaagcacc actggcagca gccactggta attgatttag aggagttagt cttgaagtca
     8641 tgcgccggtt aaggctaaac tgaaaggaca agttttggtg actgcgctcc tccaagccag
     8701 ttacctcggt tcaaagagtt ggtagctcag agaaccttcg aaaaaccgcc ctgcaaggcg
     8761 gttttttcgt tttcagagca agagattacg cgcagaccaa aacgatctca agaagatcat
     8821 cttattaatc agataaaata tttctaggct catgagcccg aagtggcgag cccgatcttc
     8881 cccatcggtg atgtcggcga tataggcgcc agcaaccgca cctgtggcgc cggtgatgcc
     8941 ggccacgatg cgtccggcgt agaggatctg ctcatgtttg acagcttatc
//
