LOCUS       sequence-491675-        7719 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  L-arabinose inducible rAPOBEC-XTEN64-CasC(Cas7)-GSSG-UGI.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7719)
  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 7719)
  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: pMut12_apo64C
FEATURES             Location/Qualifiers
     source          1..7719
                     /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             1380..2066
                     /codon_start=1
                     /product="cytidine deaminase (C to U editing enzyme) from 
                     rat"
                     /label=APOBEC-1
                     /note="can use ssDNA as a substrate (Komor et al., 2016)"
                     /translation="MSSETGPVAVDPTLRRRIEPHEFEVFFDPRELRKETCLLYEINWG
                     GRHSIWRHTSQNTNKHVEVNFIEKFTTERYFCPNTRCSITWFLSWSPCGECSRAITEFL
                     SRYPHVTLFIYIARLYHHADPRNRQGLRDLISSGVTIQIMTEQESGYCWRNFVNYSPSN
                     EAHWPRYPHLWVRLYVLELYCIILGLPPCLNILRRKQPQLTFFTIALQSCHYQRLPPHI
                     LWATGLK"
     CDS             3360..3608
                     /codon_start=1
                     /product="uracil-DNA glycosylase inhibitor from a Bacillus 
                     subtilis bacteriophage (Mol et al., 1995)"
                     /label=UGI
                     /translation="TNLSDIIEKETGKQLVIQESILMLPEEVEEVIGNKPESDILVHTA
                     YDESTDENVMLLTSDAPEYKPWALVIQDSNGENKIKML"
     CDS             3621..3641
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     misc_feature    3664..3720
                     /label=MCS
                     /note="pUC18/19 multiple cloning site"
     primer_bind     complement(3753..3770)
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     terminator      3923..4009
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      4101..4128
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     promoter        4147..4238
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      4696..5151
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4783..4802)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4993..5014
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(5707..6366)
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     6281..6300
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     promoter        complement(6367..6469)
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     primer_bind     complement(6861..6878)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6995..7540)
                     /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(7075..7094)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     7616..7635
                     /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 tgtcttctga aaccggtccg gttgcggttg acccgaccct gcgtcgtcgt atcgaaccgc
     1441 acgaattcga agttttcttc gacccgcgtg aactgcgtaa agaaacctgc ctgctgtacg
     1501 aaatcaactg gggtggtcgt cactctatct ggcgtcacac ctctcagaac accaacaaac
     1561 acgttgaagt taacttcatc gaaaaattca ccaccgaacg ttacttctgc ccgaacaccc
     1621 gttgctctat cacctggttc ctgtcttggt ctccgtgcgg tgaatgctct cgtgcgatca
     1681 ccgaattcct gtctcgttac ccgcacgtta ccctgttcat ctacatcgcg cgtctgtacc
     1741 accacgcgga cccgcgtaac cgtcagggtc tgcgtgacct gatctcttct ggtgttacca
     1801 tccagatcat gaccgaacag gaatctggtt actgctggcg taacttcgtt aactactctc
     1861 cgtctaacga agcgcactgg ccgcgttacc cgcacctgtg ggttcgtctg tacgttctgg
     1921 aactgtactg catcatcctg ggtctgccgc cgtgcctgaa catcctgcgt cgtaaacagc
     1981 cgcagctgac cttcttcacc atcgcgctgc agtcttgcca ctaccagcgt ctgccgccgc
     2041 acatcctgtg ggcgaccggt ctgaaatccg gtagcgaaac accggggact tcagaatcgg
     2101 ccaccccaga gtctgggcct ggtacgagta cggagccatc agagggatct gcacctggga
     2161 gtccggctgg ttctcctact tcaactgagg agggtacttc ggaaagtgct acccctgagt
     2221 cgggaccagg aagcgaacca gcaacgagtg gcagcgaaat gtctaacttt atcaatattc
     2281 atgttctgat ctctcacagc ccttcatgtc tgaaccgcga cgatatgaac atgcagaaag
     2341 acgctatttt cggcggcaaa agacgagtaa gaatttcaag tcaaagcctt aaacgtgcga
     2401 tgcgtaaaag tggttattac gcacaaaata ttggtgaatc cagtctcaga accattcatc
     2461 ttgcacaatt acgtgatgtt cttcggcaaa aacttggtga acgttttgac caaaaaatca
     2521 tcgataagac attagcgctg ctctccggta aatcagttga tgaagccgaa aagatttctg
     2581 ccgatgcggt tactccctgg gttgtgggag aaatagcctg gttctgtgag caggttgcaa
     2641 aagcagaggc tgataatctg gatgataaaa agctgctcaa agttcttaag gaagatattg
     2701 ccgccatacg tgtgaattta cagcagggtg ttgatattgc gcttagtgga agaatggcaa
     2761 ccagcggcat gatgactgag ttgggaaaag ttgatggtgc aatgtccatt gcgcatgcga
     2821 tcactactca tcaggttgat tctgatattg actggttcac cgctgtagat gatttacagg
     2881 aacaaggttc tgcacatctg ggaactcagg aattttcatc gggtgttttt tatcgttatg
     2941 ccaacattaa cctcgctcaa cttcaggaaa atttaggtgg tgcctccagg gagcaggctc
     3001 tggaaattgc aacccatgtt gttcatatgc tggcaacaga ggtccctgga gcaaaacagc
     3061 gtacttatgc cgcttttaac cctgcggata tggtaatggt taatttctcc gatatgccac
     3121 tttctatggc aaatgctttt gaaaaagcgg ttaaagcgaa agatggcttt ttgcaaccgt
     3181 ctatacaggc gtttaatcaa tattgggatc gcgttgccaa tggatatggt ctgaacggag
     3241 ctgctgcgca attcagctta tctgatgtag acccaattac tgctcaagtt aaacaaatgc
     3301 ctactttaga acagttaaaa tcctgggttc gtaataatgg cgaggcgtct ggtggttcta
     3361 ctaatctgtc agatattatt gaaaaggaga ccggtaagca actggttatc caggaatcca
     3421 tcctcatgct cccagaggag gtggaagaag tcattgggaa caagccggaa agcgatatac
     3481 tcgtgcacac cgcctacgac gagagcaccg acgagaatgt catgcttctg actagcgacg
     3541 cccctgaata caagccttgg gctctggtca tacaggatag caacggtgag aacaagatta
     3601 agatgctctc tggtggttct cccaagaaga agaggaaagt ctaatccgtt tttttgggct
     3661 agcgaattcg agctcggtac ccggggatcc tctagagtcg acctgcaggc atgcaagctt
     3721 ggctgttttg gcggatgaga gaagattttc agcctgatac agattaaatc agaacgcaga
     3781 agcggtctga taaaacagaa tttgcctggc ggcagtagcg cggtggtccc acctgacccc
     3841 atgccgaact cagaagtgaa acgccgtagc gccgatggta gtgtggggtc tccccatgcg
     3901 agagtaggga actgccaggc atcaaataaa acgaaaggct cagtcgaaag actgggcctt
     3961 tcgttttatc tgttgtttgt cggtgaacgc tctcctgagt aggacaaatc cgccgggagc
     4021 ggatttgaac gttgcgaagc aacggcccgg agggtggcgg gcaggacgcc cgccataaac
     4081 tgccaggcat caaattaagc agaaggccat cctgacggat ggcctttttg cgtttctaca
     4141 aactcttttg tttatttttc taaatacatt caaatatgta tccgctcatg agacaataac
     4201 cctgataaat gcttcaataa tattgaaaaa ggaagagtat gagtattcaa catttccgtg
     4261 tcgcccttat tccctttttt gcggcatttt gccttcctgt ttttgctcac ccagaaacgc
     4321 tggtgaaagt aaaagatgct gaagatcagt tgggtgcagc aaactattaa ctggcgaact
     4381 acttactcta gcttcccggc aacaattaat agactggatg gaggcggata aagttgcagg
     4441 accacttctg cgctcggccc ttccggctgg ctggtttatt gctgataaat ctggagccgg
     4501 tgagcgtggg tctcgcggta tcattgcagc actggggcca gatggtaagc cctcccgtat
     4561 cgtagttatc tacacgacgg ggagtcaggc aactatggat gaacgaaata gacagatcgc
     4621 tgagataggt gcctcactga ttaagcattg gtaactgtca gaccaagttt actcatatat
     4681 actttagatt gatttacgcg ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta
     4741 cgcgcagcgt gaccgctaca cttgccagcg ccctagcgcc cgctcctttc gctttcttcc
     4801 cttcctttct cgccacgttc gccggctttc cccgtcaagc tctaaatcgg gggctccctt
     4861 tagggttccg atttagtgct ttacggcacc tcgaccccaa aaaacttgat ttgggtgatg
     4921 gttcacgtag tgggccatcg ccctgataga cggtttttcg ccctttgacg ttggagtcca
     4981 cgttctttaa tagtggactc ttgttccaaa ctggaacaac actcaaccct atctcgggct
     5041 attcttttga tttataaggg attttgccga tttcggccta ttggttaaaa aatgagctga
     5101 tttaacaaaa atttaacgcg aattttaaca aaatattaac gtttacaatt taaaaggatc
     5161 taggtgaaga tcctttttga taatctcatg accaaaatcc cttaacgtga gttttcgttc
     5221 cactgagcgt cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg
     5281 cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg
     5341 gatcaagagc taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca
     5401 aatactgtcc ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg
     5461 cctacatacc tcgctctgct aatcctgtta ccagtcaggc atttgagaag cacacggtca
     5521 cactgcttcc ggtagtcaat aaaccggtaa accagcaata gacataagcg gctatttaac
     5581 gaccctgccc tgaaccgacg accgggtcga atttgctttc gaatttctgc cattcatccg
     5641 cttattatca cttattcagg cgtagcaacc aggcgtttaa gggcaccaat aactgcctta
     5701 aaaaaattac gccccgccct gccactcatc gcagtactgt tgtaattcat taagcattct
     5761 gccgacatgg aagccatcac aaacggcatg atgaacctga atcgccagcg gcatcagcac
     5821 cttgtcgcct tgcgtataat atttgcccat ggtgaaaacg ggggcgaaga agttgtccat
     5881 attggccacg tttaaatcaa aactggtgaa actcacccag ggattggctg agacgaaaaa
     5941 catattctca ataaaccctt tagggaaata ggccaggttt tcaccgtaac acgccacatc
     6001 ttgcgaatat atgtgtagaa actgccggaa atcgtcgtgg tattcactcc agagcgatga
     6061 aaacgtttca gtttgctcat ggaaaacggt gtaacaaggg tgaacactat cccatatcac
     6121 cagctcaccg tctttcattg ccatacggaa ttccggatga gcattcatca ggcgggcaag
     6181 aatgtgaata aaggccggat aaaacttgtg cttatttttc tttacggtct ttaaaaaggc
     6241 cgtaatatcc agctgaacgg tctggttata ggtacattga gcaactgact gaaatgcctc
     6301 aaaatgttct ttacgatgcc attgggatat atcaacggtg gtatatccag tgattttttt
     6361 ctccatttta gcttccttag ctcctgaaaa tctcgataac tcaaaaaata cgcccggtag
     6421 tgatcttatt tcattatggt gaaagttgga acctcttacg tgccgatcaa cgtctcattt
     6481 tcgccaaaag ttggcccagg gcttcccggt atcaacaggg acaccaggat ttatttattc
     6541 tgcgaagtga tcttccgtca caggtattta ttcggcgcaa agtgcgtcgg gtgatgctgc
     6601 caacttactg atttagtgta tgatggtgtt tttgaggtgc tccagtggct tctgtttcta
     6661 tcagctgtcc ctcctgttca gctactgacg gggtggtgcg taacggcaaa agcaccgccg
     6721 gacatcagcg ctagcggagt gtatactggc ttactatgtt ggcactgatg agggtgtcag
     6781 tgaagtgctt catgtggcag gagaaaaaag gctgcaccgg tgcgtcagca gaatatgtga
     6841 tacaggatat attccgcttc ctcgctcact gactcgctac gctcggtcgt tcgactgcgg
     6901 cgagcggaaa tggcttacga acggggcgga gatttcctgg aagatgccag gaagatactt
     6961 aacagggaag tgagagggcc gcggcaaagc cgtttttcca taggctccgc ccccctgaca
     7021 agcatcacga aatctgacgc tcaaatcagt ggtggcgaaa cccgacagga ctataaagat
     7081 accaggcgtt tccccctggc ggctccctcg tgcgctctcc tgttcctgcc tttcggttta
     7141 tcggtgtcat tccgctgtta tggccgcgtt tgtctcattc cacgcctgac actcagttcc
     7201 gggtaggcag ttcgctccaa gctggactgt atgcacgaac cccccgttca gtccgaccgc
     7261 tgcgccttat ccggtaacta tcgtcttgag tccaacccgg aaagacatgc aaaagcacca
     7321 ctggcagcag ccactggtaa ttgatttaga ggagttagtc ttgaagtcat gcgccggtta
     7381 aggctaaact gaaaggacaa gttttggtga ctgcgctcct ccaagccagt tacctcggtt
     7441 caaagagttg gtagctcaga gaaccttcga aaaaccgccc tgcaaggcgg ttttttcgtt
     7501 ttcagagcaa gagattacgc gcagaccaaa acgatctcaa gaagatcatc ttattaatca
     7561 gataaaatat ttctaggctc atgagcccga agtggcgagc ccgatcttcc ccatcggtga
     7621 tgtcggcgat ataggcgcca gcaaccgcac ctgtggcgcc ggtgatgccg gccacgatgc
     7681 gtccggcgta gaggatctgc tcatgtttga cagcttatc
//
