LOCUS       sequence-491676-        6954 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  L-arabinose inducible pmCDA-XTEN32-CasB(Cas11)-GSSG-UGI.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6954)
  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 6954)
  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: pMut3_pm32B
FEATURES             Location/Qualifiers
     source          1..6954
                     /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             2595..2843
                     /codon_start=1
                     /product="uracil-DNA glycosylase inhibitor from a Bacillus 
                     subtilis bacteriophage (Mol et al., 1995)"
                     /label=UGI
                     /translation="TNLSDIIEKETGKQLVIQESILMLPEEVEEVIGNKPESDILVHTA
                     YDESTDENVMLLTSDAPEYKPWALVIQDSNGENKIKML"
     CDS             2856..2876
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     misc_feature    2899..2955
                     /label=MCS
                     /note="pUC18/19 multiple cloning site"
     primer_bind     complement(2988..3005)
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     terminator      3158..3244
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      3336..3363
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     promoter        3382..3473
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      3931..4386
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4018..4037)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4228..4249
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(4942..5601)
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     5516..5535
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     promoter        complement(5602..5704)
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     primer_bind     complement(6096..6113)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6230..6775)
                     /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(6310..6329)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6851..6870
                     /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 tgatgacaga cgccgagtac gtgcgcattc atgagaaact ggatatttac accttcaaga
     1441 agcagttctt caacaacaag aaatctgtgt cacaccgctg ctacgtgctg tttgagttga
     1501 agcgaagggg cgaaagaagg gcttgctttt ggggctatgc cgtcaacaag ccccaaagtg
     1561 gcaccgagag aggaatacac gctgagatat tcagtatccg aaaggtggaa gagtatcttc
     1621 gggataatcc tgggcagttt acgatcaact ggtattccag ctggagtcct tgcgctgatt
     1681 gtgccgagaa aattctggaa tggtataatc aggaacttcg gggaaacggg cacacattga
     1741 aaatctgggc ctgcaagctg tactacgaga agaatgcccg gaaccagata ggactctgga
     1801 atctgaggga caatggtgta ggcctgaacg tgatggtttc cgagcactat cagtgttgtc
     1861 ggaagatttt catccaaagc tctcataacc agctcaatga aaaccgctgg ttggagaaaa
     1921 cactgaaacg tgcggagaag tggagatccg agctgagcat catgatccag gtcaagattc
     1981 tgcataccac taagtctcca gccgttagtg gtagcgaaac accggggact tcagaatcgg
     2041 ccaccccaga gtctgggcct ggtacgagta cggagccatc agagggatct gcacctggga
     2101 gtatggctga tgaaattgat gcaatggctt tatatcgagc ctggcaacaa ctggataatg
     2161 gatcatgtgc gcaaattaga cgtgtttcag aacctgatga attacgcgat atccctgcgt
     2221 tttataggct ggtgcaacct tttggttggg aaaacccacg tcaccagcag gctcttttgc
     2281 gcatggtgtt ttgcctgagc gcaggaaaga atgtcatccg acatcaggac aaaaaatcgg
     2341 agcaaacaac aggtatctcg ttgggaagag ctttagccaa tagtggaaga attaacgagc
     2401 gccgtatctt tcaattaatt cgggctgaca gaacagccga tatggtccag ttacgtcgat
     2461 tacttactca cgccgaaccc gtacttgact ggccattaat ggccaggatg ttgacctggt
     2521 ggggaaagcg cgaacgccag caacttctgg aagattttgt attgaccaca aacaaaaatg
     2581 cgtctggtgg ttctactaat ctgtcagata ttattgaaaa ggagaccggt aagcaactgg
     2641 ttatccagga atccatcctc atgctcccag aggaggtgga agaagtcatt gggaacaagc
     2701 cggaaagcga tatactcgtg cacaccgcct acgacgagag caccgacgag aatgtcatgc
     2761 ttctgactag cgacgcccct gaatacaagc cttgggctct ggtcatacag gatagcaacg
     2821 gtgagaacaa gattaagatg ctctctggtg gttctcccaa gaagaagagg aaagtctaat
     2881 ccgttttttt gggctagcga attcgagctc ggtacccggg gatcctctag agtcgacctg
     2941 caggcatgca agcttggctg ttttggcgga tgagagaaga ttttcagcct gatacagatt
     3001 aaatcagaac gcagaagcgg tctgataaaa cagaatttgc ctggcggcag tagcgcggtg
     3061 gtcccacctg accccatgcc gaactcagaa gtgaaacgcc gtagcgccga tggtagtgtg
     3121 gggtctcccc atgcgagagt agggaactgc caggcatcaa ataaaacgaa aggctcagtc
     3181 gaaagactgg gcctttcgtt ttatctgttg tttgtcggtg aacgctctcc tgagtaggac
     3241 aaatccgccg ggagcggatt tgaacgttgc gaagcaacgg cccggagggt ggcgggcagg
     3301 acgcccgcca taaactgcca ggcatcaaat taagcagaag gccatcctga cggatggcct
     3361 ttttgcgttt ctacaaactc ttttgtttat ttttctaaat acattcaaat atgtatccgc
     3421 tcatgagaca ataaccctga taaatgcttc aataatattg aaaaaggaag agtatgagta
     3481 ttcaacattt ccgtgtcgcc cttattccct tttttgcggc attttgcctt cctgtttttg
     3541 ctcacccaga aacgctggtg aaagtaaaag atgctgaaga tcagttgggt gcagcaaact
     3601 attaactggc gaactactta ctctagcttc ccggcaacaa ttaatagact ggatggaggc
     3661 ggataaagtt gcaggaccac ttctgcgctc ggcccttccg gctggctggt ttattgctga
     3721 taaatctgga gccggtgagc gtgggtctcg cggtatcatt gcagcactgg ggccagatgg
     3781 taagccctcc cgtatcgtag ttatctacac gacggggagt caggcaacta tggatgaacg
     3841 aaatagacag atcgctgaga taggtgcctc actgattaag cattggtaac tgtcagacca
     3901 agtttactca tatatacttt agattgattt acgcgccctg tagcggcgca ttaagcgcgg
     3961 cgggtgtggt ggttacgcgc agcgtgaccg ctacacttgc cagcgcccta gcgcccgctc
     4021 ctttcgcttt cttcccttcc tttctcgcca cgttcgccgg ctttccccgt caagctctaa
     4081 atcgggggct ccctttaggg ttccgattta gtgctttacg gcacctcgac cccaaaaaac
     4141 ttgatttggg tgatggttca cgtagtgggc catcgccctg atagacggtt tttcgccctt
     4201 tgacgttgga gtccacgttc tttaatagtg gactcttgtt ccaaactgga acaacactca
     4261 accctatctc gggctattct tttgatttat aagggatttt gccgatttcg gcctattggt
     4321 taaaaaatga gctgatttaa caaaaattta acgcgaattt taacaaaata ttaacgttta
     4381 caatttaaaa ggatctaggt gaagatcctt tttgataatc tcatgaccaa aatcccttaa
     4441 cgtgagtttt cgttccactg agcgtcagac cccgtagaaa agatcaaagg atcttcttga
     4501 gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg
     4561 gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac tggcttcagc
     4621 agagcgcaga taccaaatac tgtccttcta gtgtagccgt agttaggcca ccacttcaag
     4681 aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt caggcatttg
     4741 agaagcacac ggtcacactg cttccggtag tcaataaacc ggtaaaccag caatagacat
     4801 aagcggctat ttaacgaccc tgccctgaac cgacgaccgg gtcgaatttg ctttcgaatt
     4861 tctgccattc atccgcttat tatcacttat tcaggcgtag caaccaggcg tttaagggca
     4921 ccaataactg ccttaaaaaa attatgcccc gccctgccac tcatcgcagt actgttgtaa
     4981 ttcattaagc attctgccga catggaagcc atcacaaacg gcatgatgaa cctgaatcgc
     5041 cagcggcatc agcaccttgt cgccttgcgt ataatatttg cccatggtga aaacgggggc
     5101 gaagaagttg tccatattgg ccacgtttaa atcaaaactg gtgaaactca cccagggatt
     5161 ggctgagacg aaaaacatat tctcaataaa ccctttaggg aaataggcca ggttttcacc
     5221 gtaacacgcc acatcttgcg aatatatgtg tagaaactgc cggaaatcgt cgtggtattc
     5281 actccagagc gatgaaaacg tttcagtttg ctcatggaaa acggtgtaac aagggtgaac
     5341 actatcccat atcaccagct caccgtcttt cattgccata cggaattccg gatgagcatt
     5401 catcaggcgg gcaagaatgt gaataaaggc cggataaaac ttgtgcttat ttttctttac
     5461 ggtctttaaa aaggccgtaa tatccagctg aacggtctgg ttataggtac attgagcaac
     5521 tgactgaaat gcctcaaaat gttctttacg atgccattgg gatatatcaa cggtggtata
     5581 tccagtgatt tttttctcca ttttagcttc cttagctcct gaaaatctcg ataactcaaa
     5641 aaatacgccc ggtagtgatc ttatttcatt atggtgaaag ttggaacctc ttacgtgccg
     5701 atcaacgtct cattttcgcc aaaagttggc ccagggcttc ccggtatcaa cagggacacc
     5761 aggatttatt tattctgcga agtgatcttc cgtcacaggt atttattcgg cgcaaagtgc
     5821 gtcgggtgat gctgccaact tactgattta gtgtatgatg gtgtttttga ggtgctccag
     5881 tggcttctgt ttctatcagc tgtccctcct gttcagctac tgacggggtg gtgcgtaacg
     5941 gcaaaagcac cgccggacat cagcgctagc ggagtgtata ctggcttact atgttggcac
     6001 tgatgagggt gtcagtgaag tgcttcatgt ggcaggagaa aaaaggctgc accggtgcgt
     6061 cagcagaata tgtgatacag gatatattcc gcttcctcgc tcactgactc gctacgctcg
     6121 gtcgttcgac tgcggcgagc ggaaatggct tacgaacggg gcggagattt cctggaagat
     6181 gccaggaaga tacttaacag ggaagtgaga gggccgcggc aaagccgttt ttccataggc
     6241 tccgcccccc tgacaagcat cacgaaatct gacgctcaaa tcagtggtgg cgaaacccga
     6301 caggactata aagataccag gcgtttcccc ctggcggctc cctcgtgcgc tctcctgttc
     6361 ctgcctttcg gtttaccggt gtcattccgc tgttatggcc gcgtttgtct cattccacgc
     6421 ctgacactca gttccgggta ggcagttcgc tccaagctgg actgtatgca cgaacccccc
     6481 gttcagtccg accgctgcgc cttatccggt aactatcgtc ttgagtccaa cccggaaaga
     6541 catgcaaaag caccactggc agcagccact ggtaattgat ttagaggagt tagtcttgaa
     6601 gtcatgcgcc ggttaaggct aaactgaaag gacaagtttt ggtgactgcg ctcctccaag
     6661 ccagttacct cggttcaaag agttggtagc tcagagaacc ttcgaaaaac cgccctgcaa
     6721 ggcggttttt tcgttttcag agcaagagat tacgcgcaga ccaaaacgat ctcaagaaga
     6781 tcatcttatt aatcagataa aatatttcta ggctcatgag cccgaagtgg cgagcccgat
     6841 cttccccatc ggtgatgtcg gcgatatagg cgccagcaac cgcacctgtg gcgccggtga
     6901 tgccggccac gatgcgtccg gcgtagagga tctgctcatg tttgacagct tatc
//
