LOCUS       sequence-491662-        6828 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  L-arabinose inducible TadDE-XTEN32-CasB(Cas11)-GSSG-UGI.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6828)
  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 6828)
  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: pMut20_tadDE32B
FEATURES             Location/Qualifiers
     source          1..6828
                     /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             1332..1349
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             2469..2717
                     /codon_start=1
                     /product="uracil-DNA glycosylase inhibitor from a Bacillus 
                     subtilis bacteriophage (Mol et al., 1995)"
                     /label=UGI
                     /translation="TNLSDIIEKETGKQLVIQESILMLPEEVEEVIGNKPESDILVHTA
                     YDESTDENVMLLTSDAPEYKPWALVIQDSNGENKIKML"
     CDS             2730..2750
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     misc_feature    2773..2829
                     /label=MCS
                     /note="pUC18/19 multiple cloning site"
     primer_bind     complement(2862..2879)
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     terminator      3032..3118
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      3210..3237
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     promoter        3256..3347
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      3805..4260
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3892..3911)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4102..4123
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(4816..5475)
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     5390..5409
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     promoter        complement(5476..5578)
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     primer_bind     complement(5970..5987)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6104..6649)
                     /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(6184..6203)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6725..6744
                     /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 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 cagagtctgg gcctggtacg agtacggagc catcagaggg atctgcacct gggagtatgg
     1981 ctgatgaaat tgatgcaatg gctttatatc gagcctggca acaactggat aatggatcat
     2041 gtgcgcaaat tagacgtgtt tcagaacctg atgaattacg cgatatccct gcgttttata
     2101 ggctggtgca accttttggt tgggaaaacc cacgtcacca gcaggctctt ttgcgcatgg
     2161 tgttttgcct gagcgcagga aagaatgtca tccgacatca ggacaaaaaa tcggagcaaa
     2221 caacaggtat ctcgttggga agagctttag ccaatagtgg aagaattaac gagcgccgta
     2281 tctttcaatt aattcgggct gacagaacag ccgatatggt ccagttacgt cgattactta
     2341 ctcacgccga acccgtactt gactggccat taatggccag gatgttgacc tggtggggaa
     2401 agcgcgaacg ccagcaactt ctggaagatt ttgtattgac cacaaacaaa aatgcgtctg
     2461 gtggttctac taatctgtca gatattattg aaaaggagac cggtaagcaa ctggttatcc
     2521 aggaatccat cctcatgctc ccagaggagg tggaagaagt cattgggaac aagccggaaa
     2581 gcgatatact cgtgcacacc gcctacgacg agagcaccga cgagaatgtc atgcttctga
     2641 ctagcgacgc ccctgaatac aagccttggg ctctggtcat acaggatagc aacggtgaga
     2701 acaagattaa gatgctctct ggtggttctc ccaagaagaa gaggaaagtc taatccgttt
     2761 ttttgggcta gcgaattcga gctcggtacc cggggatcct ctagagtcga cctgcaggca
     2821 tgcaagcttg gctgttttgg cggatgagag aagattttca gcctgataca gattaaatca
     2881 gaacgcagaa gcggtctgat aaaacagaat ttgcctggcg gcagtagcgc ggtggtccca
     2941 cctgacccca tgccgaactc agaagtgaaa cgccgtagcg ccgatggtag tgtggggtct
     3001 ccccatgcga gagtagggaa ctgccaggca tcaaataaaa cgaaaggctc agtcgaaaga
     3061 ctgggccttt cgttttatct gttgtttgtc ggtgaacgct ctcctgagta ggacaaatcc
     3121 gccgggagcg gatttgaacg ttgcgaagca acggcccgga gggtggcggg caggacgccc
     3181 gccataaact gccaggcatc aaattaagca gaaggccatc ctgacggatg gcctttttgc
     3241 gtttctacaa actcttttgt ttatttttct aaatacattc aaatatgtat ccgctcatga
     3301 gacaataacc ctgataaatg cttcaataat attgaaaaag gaagagtatg agtattcaac
     3361 atttccgtgt cgcccttatt cccttttttg cggcattttg ccttcctgtt tttgctcacc
     3421 cagaaacgct ggtgaaagta aaagatgctg aagatcagtt gggtgcagca aactattaac
     3481 tggcgaacta cttactctag cttcccggca acaattaata gactggatgg aggcggataa
     3541 agttgcagga ccacttctgc gctcggccct tccggctggc tggtttattg ctgataaatc
     3601 tggagccggt gagcgtgggt ctcgcggtat cattgcagca ctggggccag atggtaagcc
     3661 ctcccgtatc gtagttatct acacgacggg gagtcaggca actatggatg aacgaaatag
     3721 acagatcgct gagataggtg cctcactgat taagcattgg taactgtcag accaagttta
     3781 ctcatatata ctttagattg atttacgcgc cctgtagcgg cgcattaagc gcggcgggtg
     3841 tggtggttac gcgcagcgtg accgctacac ttgccagcgc cctagcgccc gctcctttcg
     3901 ctttcttccc ttcctttctc gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg
     3961 ggctcccttt agggttccga tttagtgctt tacggcacct cgaccccaaa aaacttgatt
     4021 tgggtgatgg ttcacgtagt gggccatcgc cctgatagac ggtttttcgc cctttgacgt
     4081 tggagtccac gttctttaat agtggactct tgttccaaac tggaacaaca ctcaacccta
     4141 tctcgggcta ttcttttgat ttataaggga ttttgccgat ttcggcctat tggttaaaaa
     4201 atgagctgat ttaacaaaaa tttaacgcga attttaacaa aatattaacg tttacaattt
     4261 aaaaggatct aggtgaagat cctttttgat aatctcatga ccaaaatccc ttaacgtgag
     4321 ttttcgttcc actgagcgtc agaccccgta gaaaagatca aaggatcttc ttgagatcct
     4381 ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt
     4441 tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt cagcagagcg
     4501 cagataccaa atactgtcct tctagtgtag ccgtagttag gccaccactt caagaactct
     4561 gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtcaggca tttgagaagc
     4621 acacggtcac actgcttccg gtagtcaata aaccggtaaa ccagcaatag acataagcgg
     4681 ctatttaacg accctgccct gaaccgacga ccgggtcgaa tttgctttcg aatttctgcc
     4741 attcatccgc ttattatcac ttattcaggc gtagcaacca ggcgtttaag ggcaccaata
     4801 actgccttaa aaaaattatg ccccgccctg ccactcatcg cagtactgtt gtaattcatt
     4861 aagcattctg ccgacatgga agccatcaca aacggcatga tgaacctgaa tcgccagcgg
     4921 catcagcacc ttgtcgcctt gcgtataata tttgcccatg gtgaaaacgg gggcgaagaa
     4981 gttgtccata ttggccacgt ttaaatcaaa actggtgaaa ctcacccagg gattggctga
     5041 gacgaaaaac atattctcaa taaacccttt agggaaatag gccaggtttt caccgtaaca
     5101 cgccacatct tgcgaatata tgtgtagaaa ctgccggaaa tcgtcgtggt attcactcca
     5161 gagcgatgaa aacgtttcag tttgctcatg gaaaacggtg taacaagggt gaacactatc
     5221 ccatatcacc agctcaccgt ctttcattgc catacggaat tccggatgag cattcatcag
     5281 gcgggcaaga atgtgaataa aggccggata aaacttgtgc ttatttttct ttacggtctt
     5341 taaaaaggcc gtaatatcca gctgaacggt ctggttatag gtacattgag caactgactg
     5401 aaatgcctca aaatgttctt tacgatgcca ttgggatata tcaacggtgg tatatccagt
     5461 gatttttttc tccattttag cttccttagc tcctgaaaat ctcgataact caaaaaatac
     5521 gcccggtagt gatcttattt cattatggtg aaagttggaa cctcttacgt gccgatcaac
     5581 gtctcatttt cgccaaaagt tggcccaggg cttcccggta tcaacaggga caccaggatt
     5641 tatttattct gcgaagtgat cttccgtcac aggtatttat tcggcgcaaa gtgcgtcggg
     5701 tgatgctgcc aacttactga tttagtgtat gatggtgttt ttgaggtgct ccagtggctt
     5761 ctgtttctat cagctgtccc tcctgttcag ctactgacgg ggtggtgcgt aacggcaaaa
     5821 gcaccgccgg acatcagcgc tagcggagtg tatactggct tactatgttg gcactgatga
     5881 gggtgtcagt gaagtgcttc atgtggcagg agaaaaaagg ctgcaccggt gcgtcagcag
     5941 aatatgtgat acaggatata ttccgcttcc tcgctcactg actcgctacg ctcggtcgtt
     6001 cgactgcggc gagcggaaat ggcttacgaa cggggcggag atttcctgga agatgccagg
     6061 aagatactta acagggaagt gagagggccg cggcaaagcc gtttttccat aggctccgcc
     6121 cccctgacaa gcatcacgaa atctgacgct caaatcagtg gtggcgaaac ccgacaggac
     6181 tataaagata ccaggcgttt ccccctggcg gctccctcgt gcgctctcct gttcctgcct
     6241 ttcggtttac cggtgtcatt ccgctgttat ggccgcgttt gtctcattcc acgcctgaca
     6301 ctcagttccg ggtaggcagt tcgctccaag ctggactgta tgcacgaacc ccccgttcag
     6361 tccgaccgct gcgccttatc cggtaactat cgtcttgagt ccaacccgga aagacatgca
     6421 aaagcaccac tggcagcagc cactggtaat tgatttagag gagttagtct tgaagtcatg
     6481 cgccggttaa ggctaaactg aaaggacaag ttttggtgac tgcgctcctc caagccagtt
     6541 acctcggttc aaagagttgg tagctcagag aaccttcgaa aaaccgccct gcaaggcggt
     6601 tttttcgttt tcagagcaag agattacgcg cagaccaaaa cgatctcaag aagatcatct
     6661 tattaatcag ataaaatatt tctaggctca tgagcccgaa gtggcgagcc cgatcttccc
     6721 catcggtgat gtcggcgata taggcgccag caaccgcacc tgtggcgccg gtgatgccgg
     6781 ccacgatgcg tccggcgtag aggatctgct catgtttgac agcttatc
//
