LOCUS       sequence-342410-        8956 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses eMutaT7 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 8956)
  AUTHORS   Park H, Kim S
  TITLE     Gene-specific mutagenesis enables rapid continuous evolution of 
            enzymes in vivo.
  JOURNAL   Nucleic Acids Res. 2021 Apr 6;49(6):e32. doi: 10.1093/nar/gkaa1231.
  PUBMED    33406230
REFERENCE   2  (bases 1 to 8956)
  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."; date: "2021-04-6"; volume: "49(6):e32. doi"; pages: " 
            10.1093/nar/gkaa1231"
COMMENT     Sequence Label: pHyo094 (eMutaT7)
FEATURES             Location/Qualifiers
     source          1..8956
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     terminator      43..70
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     promoter        89..180
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      638..1093
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(725..744)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     935..956
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(1649..2308)
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     2223..2242
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     promoter        complement(2309..2411)
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     primer_bind     complement(2803..2820)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2937..3482)
                     /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(3017..3036)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3558..3577
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     CDS             complement(3757..4635)
                     /codon_start=1
                     /gene="araC"
                     /product="L-arabinose regulatory protein"
                     /label=araC
                     /translation="MAEAQNDPLLPGYSFNAHLVAGLTPIEANGYLDFFIDRPLGMKGY
                     ILNLTIRGQGVVKNQGREFVCRPGDILLFPPGEIHHYGRHPEAREWYHQWVYFRPRAYW
                     HEWLNWPSIFANTGFFRPDEAHQPHFSDLFGQIINAGQGEGRYSELLAINLLEQLLLRR
                     MEAINESLHPPMDNRVREACQYISDHLADSNFDIASVAQHVCLSPSRLSHLFRQQLGIS
                     VLSWREDQRISQAKLLLSTTRMPIATVGRNVGFDDQLYFSRVFKKCTGASPSEFRAGCE
                     EKVNDVAVKLS"
     promoter        4662..4946
                     /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     4869..4888
                     /label=pBAD Forward
                     /note="For vectors with E. coli araBAD promoter, forward 
                     primer"
     CDS             8358..8612
                     /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(8651..8668)
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     terminator      8821..8907
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
ORIGIN
        1 gggcaggacg cccgccataa actgccaggc atcaaattaa gcagaaggcc atcctgacgg
       61 atggcctttt tgcgtttcta caaactcttt tgtttatttt tctaaataca ttcaaatatg
      121 tatccgctca tgagacaata accctgataa atgcttcaat aatattgaaa aaggaagagt
      181 atgagtattc aacatttccg tgtcgccctt attccctttt ttgcggcatt ttgccttcct
      241 gtttttgctc acccagaaac gctggtgaaa gtaaaagatg ctgaagatca gttgggtgca
      301 gcaaactatt aactggcgaa ctacttactc tagcttcccg gcaacaatta atagactgga
      361 tggaggcgga taaagttgca ggaccacttc tgcgctcggc ccttccggct ggctggttta
      421 ttgctgataa atctggagcc ggtgagcgtg ggtctcgcgg tatcattgca gcactggggc
      481 cagatggtaa gccctcccgt atcgtagtta tctacacgac ggggagtcag gcaactatgg
      541 atgaacgaaa tagacagatc gctgagatag gtgcctcact gattaagcat tggtaactgt
      601 cagaccaagt ttactcatat atactttaga ttgatttacg cgccctgtag cggcgcatta
      661 agcgcggcgg gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag cgccctagcg
      721 cccgctcctt tcgctttctt cccttccttt ctcgccacgt tcgccggctt tccccgtcaa
      781 gctctaaatc gggggctccc tttagggttc cgatttagtg ctttacggca cctcgacccc
      841 aaaaaacttg atttgggtga tggttcacgt agtgggccat cgccctgata gacggttttt
      901 cgccctttga cgttggagtc cacgttcttt aatagtggac tcttgttcca aactggaaca
      961 acactcaacc ctatctcggg ctattctttt gatttataag ggattttgcc gatttcggcc
     1021 tattggttaa aaaatgagct gatttaacaa aaatttaacg cgaattttaa caaaatatta
     1081 acgtttacaa tttaaaagga tctaggtgaa gatccttttt gataatctca tgaccaaaat
     1141 cccttaacgt gagttttcgt tccactgagc gtcagacccc gtagaaaaga tcaaaggatc
     1201 ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct
     1261 accagcggtg gtttgtttgc cggatcaaga gctaccaact ctttttccga aggtaactgg
     1321 cttcagcaga gcgcagatac caaatactgt ccttctagtg tagccgtagt taggccacca
     1381 cttcaagaac tctgtagcac cgcctacata cctcgctctg ctaatcctgt taccagtcag
     1441 gcatttgaga agcacacggt cacactgctt ccggtagtca ataaaccggt aaaccagcaa
     1501 tagacataag cggctattta acgaccctgc cctgaaccga cgaccgggtc gaatttgctt
     1561 tcgaatttct gccattcatc cgcttattat cacttattca ggcgtagcaa ccaggcgttt
     1621 aagggcacca ataactgcct taaaaaaatt acgccccgcc ctgccactca tcgcagtact
     1681 gttgtaattc attaagcatt ctgccgacat ggaagccatc acaaacggca tgatgaacct
     1741 gaatcgccag cggcatcagc accttgtcgc cttgcgtata atatttgccc atggtgaaaa
     1801 cgggggcgaa gaagttgtcc atattggcca cgtttaaatc aaaactggtg aaactcaccc
     1861 agggattggc tgagacgaaa aacatattct caataaaccc tttagggaaa taggccaggt
     1921 tttcaccgta acacgccaca tcttgcgaat atatgtgtag aaactgccgg aaatcgtcgt
     1981 ggtattcact ccagagcgat gaaaacgttt cagtttgctc atggaaaacg gtgtaacaag
     2041 ggtgaacact atcccatatc accagctcac cgtctttcat tgccatacgg aattccggat
     2101 gagcattcat caggcgggca agaatgtgaa taaaggccgg ataaaacttg tgcttatttt
     2161 tctttacggt ctttaaaaag gccgtaatat ccagctgaac ggtctggtta taggtacatt
     2221 gagcaactga ctgaaatgcc tcaaaatgtt ctttacgatg ccattgggat atatcaacgg
     2281 tggtatatcc agtgattttt ttctccattt tagcttcctt agctcctgaa aatctcgata
     2341 actcaaaaaa tacgcccggt agtgatctta tttcattatg gtgaaagttg gaacctctta
     2401 cgtgccgatc aacgtctcat tttcgccaaa agttggccca gggcttcccg gtatcaacag
     2461 ggacaccagg atttatttat tctgcgaagt gatcttccgt cacaggtatt tattcggcgc
     2521 aaagtgcgtc gggtgatgct gccaacttac tgatttagtg tatgatggtg tttttgaggt
     2581 gctccagtgg cttctgtttc tatcagctgt ccctcctgtt cagctactga cggggtggtg
     2641 cgtaacggca aaagcaccgc cggacatcag cgctagcgga gtgtatactg gcttactatg
     2701 ttggcactga tgagggtgtc agtgaagtgc ttcatgtggc aggagaaaaa aggctgcacc
     2761 ggtgcgtcag cagaatatgt gatacaggat atattccgct tcctcgctca ctgactcgct
     2821 acgctcggtc gttcgactgc ggcgagcgga aatggcttac gaacggggcg gagatttcct
     2881 ggaagatgcc aggaagatac ttaacaggga agtgagaggg ccgcggcaaa gccgtttttc
     2941 cataggctcc gcccccctga caagcatcac gaaatctgac gctcaaatca gtggtggcga
     3001 aacccgacag gactataaag ataccaggcg tttccccctg gcggctccct cgtgcgctct
     3061 cctgttcctg cctttcggtt taccggtgtc attccgctgt tatggccgcg tttgtctcat
     3121 tccacgcctg acactcagtt ccgggtaggc agttcgctcc aagctggact gtatgcacga
     3181 accccccgtt cagtccgacc gctgcgcctt atccggtaac tatcgtcttg agtccaaccc
     3241 ggaaagacat gcaaaagcac cactggcagc agccactggt aattgattta gaggagttag
     3301 tcttgaagtc atgcgccggt taaggctaaa ctgaaaggac aagttttggt gactgcgctc
     3361 ctccaagcca gttacctcgg ttcaaagagt tggtagctca gagaaccttc gaaaaaccgc
     3421 cctgcaaggc ggttttttcg ttttcagagc aagagattac gcgcagacca aaacgatctc
     3481 aagaagatca tcttattaat cagataaaat atttctaggc tcatgagccc gaagtggcga
     3541 gcccgatctt ccccatcggt gatgtcggcg atataggcgc cagcaaccgc acctgtggcg
     3601 ccggtgatgc cggccacgat gcgtccggcg tagaggatct gctcatgttt gacagcttat
     3661 catcgatgca taatgtgcct gtcaaatgga cgaagcaggg attctgcaaa ccctatgcta
     3721 ctccgtcaag ccgtcaattg tctgattcgt taccaattat gacaacttga cggctacatc
     3781 attcactttt tcttcacaac cggcacggaa ctcgctcggg ctggccccgg tgcatttttt
     3841 aaatacccgc gagaaataga gttgatcgtc aaaaccaaca ttgcgaccga cggtggcgat
     3901 aggcatccgg gtggtgctca aaagcagctt cgcctggctg atacgttggt cctcgcgcca
     3961 gcttaagacg ctaatcccta actgctggcg gaaaagatgt gacagacgcg acggcgacaa
     4021 gcaaacatgc tgtgcgacgc tggcgatatc aaaattgctg tctgccaggt gatcgctgat
     4081 gtactgacaa gcctcgcgta cccgattatc catcggtgga tggagcgact cgttaatcgc
     4141 ttccatgcgc cgcagtaaca attgctcaag cagatttatc gccagcagct ccgaatagcg
     4201 cccttcccct tgcccggcgt taatgatttg cccaaacagg tcgctgaaat gcggctggtg
     4261 cgcttcatcc gggcgaaaga accccgtatt ggcaaatatt gacggccagt taagccattc
     4321 atgccagtag gcgcgcggac gaaagtaaac ccactggtga taccattcgc gagcctccgg
     4381 atgacgaccg tagtgatgaa tctctcctgg cgggaacagc aaaatatcac ccggtcggca
     4441 aacaaattct cgtccctgat ttttcaccac cccctgaccg cgaatggtga gattgagaat
     4501 ataacctttc attcccagcg gtcggtcgat aaaaaaatcg agataaccgt tggcctcaat
     4561 cggcgttaaa cccgccacca gatgggcatt aaacgagtat cccggcagca ggggatcatt
     4621 ttgcgcttca gccatacttt tcatactccc gccattcaga gaagaaacca attgtccata
     4681 ttgcatcaga cattgccgtc actgcgtctt ttactggctc ttctcgctaa ccaaaccggt
     4741 aaccccgctt attaaaagca ttctgtaaca aagcgggacc aaagccatga caaaaacgcg
     4801 taacaaaagt gtctataatc acggcagaaa agtccacatt gattatttgc acggcgtcac
     4861 actttgctat gccatagcat ttttatccat aagattagcg gatcctacct gacgcttttt
     4921 atcgcaactc tctactgttt ctccataccc gtttttttgg gctagcgaat tcgagctcag
     4981 gaggaattca ccatggcatt atcatatgat gacagacgcc gagtacgtgc gcattcatga
     5041 gaaactggat atttacacct tcaagaagca gttcttcaac aacaagaaat ctgtgtcaca
     5101 ccgctgctac gtgctgtttg agttgaagcg aaggggcgaa agaagggctt gcttttgggg
     5161 ctatgccgtc aacaagcccc aaagtggcac cgagagagga atacacgctg agatattcag
     5221 tatccgaaag gtggaagagt atcttcggga taatcctggg cagtttacga tcaactggta
     5281 ttccagctgg agtccttgcg ctgattgtgc cgagaaaatt ctggaatggt ataatcagga
     5341 acttcgggga aacgggcaca cattgaaaat ctgggcctgc aagctgtact acgagaagaa
     5401 tgcccggaac cagataggac tctggaatct gagggacaat ggtgtaggcc tgaacgtgat
     5461 ggtttccgag cactatcagt gttgtcggaa gattttcatc caaagctctc ataaccagct
     5521 caatgaaaac cgctggttgg agaaaacact gaaacgtgcg gagaagtgga gatccgagct
     5581 gagcatcatg atccaggtca agattctgca taccactaag tctccagccg ttagcgagac
     5641 tcccgggacc tcagagtccg ccacacccga aagtaacacg attaacatcg ctaagaacga
     5701 cttctctgac atcgaactgg ctgctatccc gttcaacact ctggctgacc attacggtga
     5761 gcgtttagct cgcgaacagt tggcccttga gcatgagtct tacgagatgg gtgaagcacg
     5821 cttccgcaag atgtttgagc gtcaacttaa agctggtgag gttgcggata acgctgccgc
     5881 caagcctctc atcactaccc tactccctaa gatgattgca cgcatcaacg actggtttga
     5941 ggaagtgaaa gctaagcgcg gcaagcgccc gacagccttc cagttcctgc aagaaatcaa
     6001 gccggaagcc gtagcgtaca tcaccattaa gaccactctg gcttgcctaa ccagtgctga
     6061 caatacaacc gttcaggctg tagcaagcgc aatcggtcgg gccattgagg acgaggctcg
     6121 cttcggtcgt atccgtgacc ttgaagctaa gcacttcaag aaaaacgttg aggaacaact
     6181 caacaagcgc gtagggcacg tctacaagaa agcatttatg caagttgtcg aggctgacat
     6241 gctctctaag ggtctactcg gtggcgaggc gtggtcttcg tggcataagg aagactctat
     6301 tcatgtagga gtacgctgca tcgagatgct cattgagtca accggaatgg ttagcttaca
     6361 ccgccaaaat gctggcgtag taggtcaaga ctctgagact atcgaactcg cacctgaata
     6421 cgctgaggct atcgcaaccc gtgcaggtgc gctggctggc atctctccga tgttccaacc
     6481 ttgcgtagtt cctcctaagc cgtggactgg cattactggt ggtggctatt gggctaacgg
     6541 tcgtcgtcct ctggcgctgg tgcgtactca cagtaagaaa gcactgatgc gctacgaaga
     6601 cgtttacatg cctgaggtgt acaaagcgat taacattgcg caaaacaccg catggaaaat
     6661 caacaagaaa gtcctagcgg tcgccaacgt aatcaccaag tggaagcatt gtccggtcga
     6721 ggacatccct gcgattgagc gtgaagaact cccgatgaaa ccggaagaca tcgacatgaa
     6781 tcctgaggct ctcaccgcgt ggaaacgtgc tgccgctgct gtgtaccgca aggacaaggc
     6841 tcgcaagtct cgccgtatca gccttgagtt catgcttgag caagccaata agtttgctaa
     6901 ccataaggcc atctggttcc cttacaacat ggactggcgc ggtcgtgttt acgctgtgtc
     6961 aatgttcaac ccgcaaggta acgatatgac caaaggactg cttacgctgg cgaaaggtaa
     7021 accaatcggt aaggaaggtt actactggct gaaaatccac ggtgcaaact gtgcgggtgt
     7081 cgataaggtt ccgttccctg agcgcatcaa gttcattgag gaaaaccacg agaacatcat
     7141 ggcttgcgct aagtctccac tggagaacac ttggtgggct gagcaagatt ctccgttctg
     7201 cttccttgcg ttctgctttg agtacgctgg ggtacagcac cacggcctga gctataactg
     7261 ctcccttccg ctggcgtttg acgggtcttg ctctggcatc cagcacttct ccgcgatgct
     7321 ccgagatgag gtaggtggtc gcgcggttaa cttgcttcct agtgaaaccg ttcaggacat
     7381 ctacgggatt gttgctaaga aagtcaacga gattctacaa gcagacgcaa tcaatgggac
     7441 cgataacgaa gtagttaccg tgaccgatga gaacactggt gaaatctctg agaaagtcaa
     7501 gctgggcact aaggcactgg ctggtcaatg gctggcttac ggtgttactc gcagtgtgac
     7561 taagcgttca gtcatgacgc tggcttacgg gtccaaagag ttcggcttcc gtcaacaagt
     7621 gctggaagat accattcagc cagctattga ttccggcaag ggtctgatgt tcactcagcc
     7681 gaatcaggct gctggataca tggctaagct gatttgggaa tctgtgagcg tgacggtggt
     7741 agctgcggtt gaagcaatga actggcttaa gtctgctgct aagctgctgg ctgctgaggt
     7801 caaagataag aagactggag agattcttcg caagcgttgc gctgtgcatt gggtaactcc
     7861 tgatggtttc cctgtgtggc aggaatacaa gaagcctatt cagacgcgct tgaacctgat
     7921 gttcctcggt cagttccgct tacagcctac cattaacacc aacaaagata gcgagattga
     7981 tgcacacaaa caggagtctg gtatcgctcc taactttgta cacagccaag acggtagcca
     8041 ccttcgtaag actgtagtgt gggcacacga gaagtacgga atcgaatctt ttgcactgat
     8101 tcacgactcc ttcggtacca ttccggctga cgctgcgaac ctgttcaaag cagtgcgcga
     8161 aactatggtt gacacctatg agtcttgtga tgtactggct gatttctacg accagttcgc
     8221 tgaccagttg cacgagtctc aattggacaa aatgccagca cttccggcta aaggtaactt
     8281 gaacctccgt gacatcttag agtcggactt cgcgttcgcg taactgcagc ccggggatcc
     8341 tctagagtcg acctgcgatg accaaccttt ccgacatcat agagaaggaa acaggcaaac
     8401 agttggtcat ccaagagtcg atactcatgc ttcctgaaga agttgaggag gtcattggga
     8461 ataagccgga aagtgacatt ctcgtacaca ctgcgtatga tgagagcacc gatgagaacg
     8521 tgatgctgct cacgtcagat gccccagagt acaaaccctg ggctctggtg attcaggact
     8581 ctaatggaga gaacaagatc aagatgctgt aaaagcttgg ctgttttggc ggatgagaga
     8641 agattttcag cctgatacag attaaatcag aacgcagaag cggtctgata aaacagaatt
     8701 tgcctggcgg cagtagcgcg gtggtcccac ctgaccccat gccgaactca gaagtgaaac
     8761 gccgtagcgc cgatggtagt gtggggtctc cccatgcgag agtagggaac tgccaggcat
     8821 caaataaaac gaaaggctca gtcgaaagac tgggcctttc gttttatctg ttgtttgtcg
     8881 gtgaacgctc tcctgagtag gacaaatccg ccgggagcgg atttgaacgt tgcgaagcaa
     8941 cggcccggag ggtggc
//
