LOCUS       sequence-491604-        6780 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  L-arabinose inducible TadDE-XTEN16-CasB(Cas11)-GSSG-UGI.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6780)
  AUTHORS   Lee C, Kim S
  TITLE     Continuous targeted hypermutation with a tunable mutation window.
  JOURNAL   Nat Commun. 2025 Nov 25. doi: 10.1038/s41467-025-66736-2.
  PUBMED    41290661
REFERENCE   2  (bases 1 to 6780)
  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.
            2025 Nov 25. doi: 10.1038/s41467-025-66736-2."
COMMENT     Sequence Label: pMut19_tadDE16B
FEATURES             Location/Qualifiers
     source          1..6780
                     /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
                     HEWLNWPSIFANTGFFRPDEAHQPHFSDLFG*IINAGQGEGRYSELLAINLLEQLLLRR
                     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             1332..1349
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             2421..2669
                     /codon_start=1
                     /product="uracil-DNA glycosylase inhibitor from a Bacillus 
                     subtilis bacteriophage (Mol et al., 1995)"
                     /label=UGI
                     /translation="TNLSDIIEKETGKQLVIQESILMLPEEVEEVIGNKPESDILVHTA
                     YDESTDENVMLLTSDAPEYKPWALVIQDSNGENKIKML"
     CDS             2682..2702
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     misc_feature    2725..2781
                     /label=MCS
                     /note="pUC18/19 multiple cloning site"
     primer_bind     complement(2814..2831)
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     terminator      2984..3070
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      3162..3189
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     promoter        3208..3299
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      3757..4212
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3844..3863)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4054..4075
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(4768..5427)
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     5342..5361
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     promoter        complement(5428..5530)
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     primer_bind     complement(5922..5939)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6056..6601)
                     /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(6136..6155)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6677..6696
                     /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 aatgatttac 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 tccatacaaa acatccagcg tttcagggag gttagagcca
     1321 tgggcagcag ccatcatcat catcatcacg gaagtcccaa gaaacgtaag gttgggtcaa
     1381 tgagtgaagt tgaattcagc catgaatatt ggatgcgtca tgccctgacc ctggcaaaac
     1441 gtgcccgcga tgaaggtgaa gctccggttg gtgcggtgct ggttttaaat aatcgtgtga
     1501 ttggcgaagg ctggaatcgt cgtattggtc tgcatgatcc taccgcacat gcagaaatca
     1561 tggccctgcg tcagggtggt ctggttatgc aaaattctcg cctgattgat gccaccctgt
     1621 atgtgacctt tgaaccttgt gtgatgtgtg cgggtgcaat gattaatagt cgtattggcc
     1681 gtgttgtgtt tggcgttcgt aatagcaagc gcggtgccgc cggcagcctg atgaatgttc
     1741 tgaattatcc tggcatgaat catcgcgtgg aaattaccga gggtattctg gccgatgaat
     1801 gtgccgcact gctgtgcgat ttttatcgca tgccgcgtca agtgtttaat gcacaaaaga
     1861 aagcacagag cagcattaat agtggtagcg aaacaccggg gacttcagaa tcggccaccc
     1921 cggagtctat ggctgatgaa attgatgcaa tggctttata tcgagcctgg caacaactgg
     1981 ataatggatc atgtgcgcaa attagacgtg tttcagaacc tgatgaatta cgcgatatcc
     2041 ctgcgtttta taggctggtg caaccttttg gttgggaaaa cccacgtcac cagcaggctc
     2101 ttttgcgcat ggtgttttgc ctgagcgcag gaaagaatgt catccgacat caggacaaaa
     2161 aatcggagca aacaacaggt atctcgttgg gaagagcttt agccaatagt ggaagaatta
     2221 acgagcgccg tatctttcaa ttaattcggg ctgacagaac agccgatatg gtccagttac
     2281 gtcgattact tactcacgcc gaacccgtac ttgactggcc attaatggcc aggatgttga
     2341 cctggtgggg aaagcgcgaa cgccagcaac ttctggaaga ttttgtattg accacaaaca
     2401 aaaatgcgtc tggtggttct actaatctgt cagatattat tgaaaaggag accggtaagc
     2461 aactggttat ccaggaatcc atcctcatgc tcccagagga ggtggaagaa gtcattggga
     2521 acaagccgga aagcgatata ctcgtgcaca ccgcctacga cgagagcacc gacgagaatg
     2581 tcatgcttct gactagcgac gcccctgaat acaagccttg ggctctggtc atacaggata
     2641 gcaacggtga gaacaagatt aagatgctct ctggtggttc tcccaagaag aagaggaaag
     2701 tctaatccgt ttttttgggc tagcgaattc gagctcggta cccggggatc ctctagagtc
     2761 gacctgcagg catgcaagct tggctgtttt ggcggatgag agaagatttt cagcctgata
     2821 cagattaaat cagaacgcag aagcggtctg ataaaacaga atttgcctgg cggcagtagc
     2881 gcggtggtcc cacctgaccc catgccgaac tcagaagtga aacgccgtag cgccgatggt
     2941 agtgtggggt ctccccatgc gagagtaggg aactgccagg catcaaataa aacgaaaggc
     3001 tcagtcgaaa gactgggcct ttcgttttat ctgttgtttg tcggtgaacg ctctcctgag
     3061 taggacaaat ccgccgggag cggatttgaa cgttgcgaag caacggcccg gagggtggcg
     3121 ggcaggacgc ccgccataaa ctgccaggca tcaaattaag cagaaggcca tcctgacgga
     3181 tggccttttt gcgtttctac aaactctttt gtttattttt ctaaatacat tcaaatatgt
     3241 atccgctcat gagacaataa ccctgataaa tgcttcaata atattgaaaa aggaagagta
     3301 tgagtattca acatttccgt gtcgccctta ttcccttttt tgcggcattt tgccttcctg
     3361 tttttgctca cccagaaacg ctggtgaaag taaaagatgc tgaagatcag ttgggtgcag
     3421 caaactatta actggcgaac tacttactct agcttcccgg caacaattaa tagactggat
     3481 ggaggcggat aaagttgcag gaccacttct gcgctcggcc cttccggctg gctggtttat
     3541 tgctgataaa tctggagccg gtgagcgtgg gtctcgcggt atcattgcag cactggggcc
     3601 agatggtaag ccctcccgta tcgtagttat ctacacgacg gggagtcagg caactatgga
     3661 tgaacgaaat agacagatcg ctgagatagg tgcctcactg attaagcatt ggtaactgtc
     3721 agaccaagtt tactcatata tactttagat tgatttacgc gccctgtagc ggcgcattaa
     3781 gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac acttgccagc gccctagcgc
     3841 ccgctccttt cgctttcttc ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag
     3901 ctctaaatcg ggggctccct ttagggttcc gatttagtgc tttacggcac ctcgacccca
     3961 aaaaacttga tttgggtgat ggttcacgta gtgggccatc gccctgatag acggtttttc
     4021 gccctttgac gttggagtcc acgttcttta atagtggact cttgttccaa actggaacaa
     4081 cactcaaccc tatctcgggc tattcttttg atttataagg gattttgccg atttcggcct
     4141 attggttaaa aaatgagctg atttaacaaa aatttaacgc gaattttaac aaaatattaa
     4201 cgtttacaat ttaaaaggat ctaggtgaag atcctttttg ataatctcat gaccaaaatc
     4261 ccttaacgtg agttttcgtt ccactgagcg tcagaccccg tagaaaagat caaaggatct
     4321 tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta
     4381 ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc tttttccgaa ggtaactggc
     4441 ttcagcagag cgcagatacc aaatactgtc cttctagtgt agccgtagtt aggccaccac
     4501 ttcaagaact ctgtagcacc gcctacatac ctcgctctgc taatcctgtt accagtcagg
     4561 catttgagaa gcacacggtc acactgcttc cggtagtcaa taaaccggta aaccagcaat
     4621 agacataagc ggctatttaa cgaccctgcc ctgaaccgac gaccgggtcg aatttgcttt
     4681 cgaatttctg ccattcatcc gcttattatc acttattcag gcgtagcaac caggcgttta
     4741 agggcaccaa taactgcctt aaaaaaatta cgccccgccc tgccactcat cgcagtactg
     4801 ttgtaattca ttaagcattc tgccgacatg gaagccatca caaacggcat gatgaacctg
     4861 aatcgccagc ggcatcagca ccttgtcgcc ttgcgtataa tatttgccca tggtgaaaac
     4921 gggggcgaag aagttgtcca tattggccac gtttaaatca aaactggtga aactcaccca
     4981 gggattggct gagacgaaaa acatattctc aataaaccct ttagggaaat aggccaggtt
     5041 ttcaccgtaa cacgccacat cttgcgaata tatgtgtaga aactgccgga aatcgtcgtg
     5101 gtattcactc cagagcgatg aaaacgtttc agtttgctca tggaaaacgg tgtaacaagg
     5161 gtgaacacta tcccatatca ccagctcacc gtctttcatt gccatacgga attccggatg
     5221 agcattcatc aggcgggcaa gaatgtgaat aaaggccgga taaaacttgt gcttattttt
     5281 ctttacggtc tttaaaaagg ccgtaatatc cagctgaacg gtctggttat aggtacattg
     5341 agcaactgac tgaaatgcct caaaatgttc tttacgatgc cattgggata tatcaacggt
     5401 ggtatatcca gtgatttttt tctccatttt agcttcctta gctcctgaaa atctcgataa
     5461 ctcaaaaaat acgcccggta gtgatcttat ttcattatgg tgaaagttgg aacctcttac
     5521 gtgccgatca acgtctcatt ttcgccaaaa gttggcccag ggcttcccgg tatcaacagg
     5581 gacaccagga tttatttatt ctgcgaagtg atcttccgtc acaggtattt attcggcgca
     5641 aagtgcgtcg ggtgatgctg ccaacttact gatttagtgt atgatggtgt ttttgaggtg
     5701 ctccagtggc ttctgtttct atcagctgtc cctcctgttc agctactgac ggggtggtgc
     5761 gtaacggcaa aagcaccgcc ggacatcagc gctagcggag tgtatactgg cttactatgt
     5821 tggcactgat gagggtgtca gtgaagtgct tcatgtggca ggagaaaaaa ggctgcaccg
     5881 gtgcgtcagc agaatatgtg atacaggata tattccgctt cctcgctcac tgactcgcta
     5941 cgctcggtcg ttcgactgcg gcgagcggaa atggcttacg aacggggcgg agatttcctg
     6001 gaagatgcca ggaagatact taacagggaa gtgagagggc cgcggcaaag ccgtttttcc
     6061 ataggctccg cccccctgac aagcatcacg aaatctgacg ctcaaatcag tggtggcgaa
     6121 acccgacagg actataaaga taccaggcgt ttccccctgg cggctccctc gtgcgctctc
     6181 ctgttcctgc ctttcggttt accggtgtca ttccgctgtt atggccgcgt ttgtctcatt
     6241 ccacgcctga cactcagttc cgggtaggca gttcgctcca agctggactg tatgcacgaa
     6301 ccccccgttc agtccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg
     6361 gaaagacatg caaaagcacc actggcagca gccactggta attgatttag aggagttagt
     6421 cttgaagtca tgcgccggtt aaggctaaac tgaaaggaca agttttggtg actgcgctcc
     6481 tccaagccag ttacctcggt tcaaagagtt ggtagctcag agaaccttcg aaaaaccgcc
     6541 ctgcaaggcg gttttttcgt tttcagagca agagattacg cgcagaccaa aacgatctca
     6601 agaagatcat cttattaatc agataaaata tttctaggct catgagcccg aagtggcgag
     6661 cccgatcttc cccatcggtg atgtcggcga tataggcgcc agcaaccgca cctgtggcgc
     6721 cggtgatgcc ggccacgatg cgtccggcgt agaggatctg ctcatgtttg acagcttatc
//
