LOCUS       sequence-335282-        8899 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses fusion protein MBPc-AID-T7RNAP in E. coli.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8899)
  AUTHORS   Alvarez B, Mencia M, de Lorenzo V, Fernandez LA
  TITLE     In vivo diversification of target genomic sites using processive 
            base deaminase fusions blocked by dCas9.
  JOURNAL   Nat Commun. 2020 Dec 22;11(1):6436. doi: 10.1038/s41467-020-20230-z.
  PUBMED    33353963
REFERENCE   2  (bases 1 to 8899)
  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."; date: "2020-12-22"; volume: "11(1):6436. doi"; pages: " 
            10.1038/s41467-020-20230-z"
COMMENT     Sequence Label: pSEVA221MBPc-AID-T7RNAP
FEATURES             Location/Qualifiers
     source          1..8899
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     terminator      2291..2385
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     primer_bind     complement(2506..2525)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             2517..3332
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     oriT            3499..3607
                     /note="incP origin of transfer"
     CDS             complement(3668..4816)
                     /codon_start=1
                     /product="trans-acting replication protein that binds to 
                     and activates oriV"
                     /label=trfA
                     /translation="MNRTFDRKAYRQELIDAGFSAEDAETIASRTVMRAPRETFQSVGS
                     IVQQATAKIERDSVQLAPPALPAPSAAVERSRRLEQEAAGLAKSMTIDTRGTMTTKKRK
                     TAGEDLAKQVSEAKQAALLKHTKQQIKEMQLSLFDIAPWPDTMRAMPNDTARSALFTTR
                     NKKIPREALQNKVIFHVNKDVKITYTGVELRADDDELVWQQVLEYAKRTPIGEPITFTF
                     YELCQDLGWSINGRYYTKAEECLSRLQATAMGFTSDRVGHLESVSLLHRFRVLDRGKKT
                     SRCQVLIDEEIVVLFAGDHYTKFIWEKYRKLSPTARRMFDYFSSHREPYPLKLETFRLM
                     CGSDSTRVKKWREQVGEACEELRGSGLVEHAWVNDDLVHCKR"
     rep_origin      5209..5740
                     /label=oriV
                     /note="incP origin of replication"
     terminator      complement(5871..5957)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     CDS             complement(6000..6626)
                     /codon_start=1
                     /gene="tetR"
                     /product="tetracycline repressor TetR"
                     /label=TetR
                     /note="TetR binds to the tetracycline operator tetO to 
                     inhibit transcription. This inhibition can be relieved by 
                     adding tetracycline or doxycycline."
                     /translation="MMSRLDKSKVINSALELLNEVGIEGLTTRKLAQKLGVEQPTLYWH
                     VKNKRALLDALAIEMLDRHHTHFCPLEGESWQDFLRNNAKSLRCALLSHRDGAKVHLGT
                     RPTEKQYETLENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEDQEHQVAKEERETPT
                     TDSMPPLLRQAIELFDHQGAEPAFLFGLELIICGLEKQLKCESGS"
     primer_bind     6531..6550
                     /label=Tet-R
                     /note="Tetracycline resistance gene, reverse primer"
     promoter        6642..6697
                     /gene="tetR"
                     /label=tetR/tetA promoters
                     /note="overlapping promoters for bacterial tetR and tetA"
     protein_bind    6648..6666
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O1 for the tetR and tetA genes"
     protein_bind    6678..6696
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     CDS             6734..7834
                     /codon_start=1
                     /gene="malE (mutated)"
                     /product="maltose binding protein from E. coli"
                     /label=MBP
                     /note="This version of the gene does not encode a signal 
                     sequence, so MBP will remain in the cytosol."
                     /translation="MKTEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLE
                     EKFPQVAATGDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDAVRYNGKL
                     IAYPIAVEALSLIYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWPLIA
                     ADGGYAFKYENGKYDIKDVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNKGE
                     TAMTINGPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFL
                     ENYLLTDEGLEAVNKDKPLGAVALKSYEEELAKDPRIAATMENAQKGEIMPNIPQMSAF
                     WYAVRTAVINAASGRQTVDEALKDAQT"
     primer_bind     7808..7831
                     /label=MBP-F
                     /note="Maltose binding protein, forward primer"
     CDS             7841..8431
                     /codon_start=1
                     /gene="AICDA"
                     /product="human activation-induced cytidine deaminase 
                     (Dickerson et al., 2003)"
                     /label=AID
                     /translation="DSLLMNRRKFLYQFKNVRWAKGRRETYLCYVVKRRDSATSFSLDF
                     GYLRNKNGCHVELLFLRYISDWDLDPGRCYRVTWFTSWSPCYDCARHVADFLRGNPNLS
                     LRIFTARLYFCEDRKAEPEGLRRLHRAGVQIAIMTFKDYFYCWNTFVENHERTFKAWEG
                     LHENSVRLSRQLRRILLPLYEVDDLRDAFRTLGL"
ORIGIN
        1 tgcctaacca gtgctgacaa tacaaccgtt caggctgtag caagcgcaat cggtcgggcc
       61 attgaggacg aggctcgctt cggtcgtatc cgtgaccttg aagctaagca cttcaagaaa
      121 aacgttgagg aacaactcaa caagcgcgta gggcacgtct acaagaaagc atttatgcaa
      181 gttgtcgagg ctgacatgct ctctaagggt ctactcggtg gcgaggcgtg gtcttcgtgg
      241 cataaggaag actctattca tgtaggagta cgctgcatcg agatgctcat tgagtcaacc
      301 ggaatggtta gcttacaccg ccaaaatgct ggcgtagtag gtcaagactc tgagactatc
      361 gaactcgcac ctgaatacgc tgaggctatc gcaacccgtg caggtgcgct ggctggcatc
      421 tctccgatgt tccaaccttg cgtagttcct cctaagccgt ggactggcat tactggtggt
      481 ggctattggg ctaacggtcg tcgtcctctg gcgctggtgc gtactcacag taagaaagca
      541 ctgatgcgct acgaagacgt ttacatgcct gaggtgtaca aagcgattaa cattgcgcaa
      601 aacaccgcat ggaaaatcaa caagaaagtc ctagcggtcg ccaacgtaat caccaagtgg
      661 aagcattgtc cggtcgagga catccctgcg attgagcgtg aagaactccc gatgaaaccg
      721 gaagacatcg acatgaatcc tgaggctctc accgcgtgga aacgtgctgc cgctgctgtg
      781 taccgcaagg acaaggctcg caagtctcgc cgtatcagcc ttgagttcat gcttgagcaa
      841 gccaataagt ttgctaacca taaggccatc tggttccctt acaacatgga ctggcgcggt
      901 cgtgtttacg ctgtgtcaat gttcaacccg caaggtaacg atatgaccaa aggactgctt
      961 acgctggcga aaggtaaacc aatcggtaag gaaggttact actggctgaa aatccacggt
     1021 gcaaactgtg cgggtgtcga taaggttccg ttccctgagc gcatcaagtt cattgaggaa
     1081 aaccacgaga acatcatggc ttgcgctaag tctccactgg agaacacttg gtgggctgag
     1141 caagattctc cgttctgctt ccttgcgttc tgctttgagt acgctggggt acagcaccac
     1201 ggcctgagct ataactgctc ccttccgctg gcgtttgacg ggtcttgctc tggcatccag
     1261 cacttctccg cgatgctccg agatgaggta ggtggtcgcg cggttaactt gcttcctagt
     1321 gaaaccgttc aggacatcta cgggattgtt gctaagaaag tcaacgagat tctacaagca
     1381 gacgcaatca atgggaccga taacgaagta gttaccgtga ccgatgagaa cactggtgaa
     1441 atctctgaga aagtcaagct gggcactaag gcactggctg gtcaatggct ggcttacggt
     1501 gttactcgca gtgtgactaa gcgttcagtc atgacgctgg cttacgggtc caaagagttc
     1561 ggcttccgtc aacaagtgct ggaagatacc attcagccag ctattgattc cggcaagggt
     1621 ctgatgttca ctcagccgaa tcaggctgct ggatacatgg ctaagctgat ttgggaatct
     1681 gtgagcgtga cggtggtagc tgcggttgaa gcaatgaact ggcttaagtc tgctgctaag
     1741 ctgctggctg ctgaggtcaa agataagaag actggagaga ttcttcgcaa gcgttgcgct
     1801 gtgcattggg taactcctga tggtttccct gtgtggcagg aatacaagaa gcctattcag
     1861 acgcgcttga acctgatgtt cctcggtcag ttccgcttac agcctaccat taacaccaac
     1921 aaagatagcg agattgatgc acacaaacag gagtctggta tcgctcctaa ctttgtacac
     1981 agccaagacg gtagccacct tcgtaagact gtagtgtggg cacacgagaa gtacggaatc
     2041 gaatcttttg cactgattca cgactccttc ggtaccattc cggctgacgc tgcgaacctg
     2101 ttcaaagcag tgcgcgaaac tatggttgac acatatgagt cttgtgatgt actggctgat
     2161 ttctacgacc agttcgctga ccagttgcac gagtctcaat tggacaaaat gccagcactt
     2221 ccggctaaag gtaacttgaa cctccgtgac atcttagagt cggacttcgc gttcgcgtaa
     2281 actagtcttg gactcctgtt gatagatcca gtaatgacct cagaactcca tctggatttg
     2341 ttcagaacgc tcggttgccg ccgggcgttt tttattggtg agaatccagg ggtccccaat
     2401 aattacgatt taaatttgtg tctcaaaatc tctgatgtta cattgcacaa gataaaaata
     2461 tatcatcatg aacaataaaa ctgtctgctt acataaacag taatacaagg ggtgttatga
     2521 gccatattca gcgtgaaacg agctgtagcc gtccgcgtct gaacagcaac atggatgcgg
     2581 atctgtatgg ctataaatgg gcgcgtgata acgtgggtca gagcggcgcg accatttatc
     2641 gtctgtatgg caaaccggat gcgccggaac tgtttctgaa acatggcaaa ggcagcgtgg
     2701 cgaacgatgt gaccgatgaa atggtgcgtc tgaactggct gaccgaattt atgccgctgc
     2761 cgaccattaa acattttatt cgcaccccgg atgatgcgtg gctgctgacc accgcgattc
     2821 cgggcaaaac cgcgtttcag gtgctggaag aatatccgga tagcggcgaa aacattgtgg
     2881 atgcgctggc cgtgtttctg cgtcgtctgc atagcattcc ggtgtgcaac tgcccgttta
     2941 acagcgatcg tgtgtttcgt ctggcccagg cgcagagccg tatgaacaac ggcctggtgg
     3001 atgcgagcga ttttgatgat gaacgtaacg gctggccggt ggaacaggtg tggaaagaaa
     3061 tgcataaact gctgccgttt agcccggata gcgtggtgac ccacggcgat tttagcctgg
     3121 ataacctgat tttcgatgaa ggcaaactga ttggctgcat tgatgtgggc cgtgtgggca
     3181 ttgcggatcg ttatcaggat ctggccattc tgtggaactg cctgggcgaa tttagcccga
     3241 gcctgcaaaa acgtctgttt cagaaatatg gcattgataa tccggatatg aacaaactgc
     3301 aatttcatct gatgctggat gaatttttct aataattaat tggaccgcgg tccgcgcgtt
     3361 gtccttttcc gctgcataac cctgcttcgg ggtcattata gcgatttttt cggtatatcc
     3421 atcctttttc gcacgatata caggattttg ccaaagggtt cgtgtagact ttccttggtg
     3481 tatccaacgg cgtcagccgg gcaggatagg tgaagtaggc ccacccgcga gcgggtgttc
     3541 cttcttcact gtcccttatt cgcacctggc ggtgctcaac gggaatcctg ctctgcgagg
     3601 ctggccgtag gccggccgcg atgcaggtgg ctgctgaacc cccagccgga actgacccca
     3661 caaggcccta gcgtttgcaa tgcaccaggt catcattgac ccaggcgtgt tccaccaggc
     3721 cgctgcctcg caactcttcg caggcttcgc cgacctgctc gcgccacttc ttcacgcggg
     3781 tggaatccga tccgcacatg aggcggaagg tttccagctt gagcgggtac ggctcccggt
     3841 gcgagctgaa atagtcgaac atccgtcggg ccgtcggcga cagcttgcgg tacttctccc
     3901 atatgaattt cgtgtagtgg tcgccagcaa acagcacgac gatttcctcg tcgatcagga
     3961 cctggcaacg ggacgttttc ttgccacggt ccaggacgcg gaagcggtgc agcagcgaca
     4021 ccgattccag gtgcccaacg cggtcggacg tgaagcccat cgccgtcgcc tgtaggcgcg
     4081 acaggcattc ctcggccttc gtgtaatacc ggccattgat cgaccagccc aggtcctggc
     4141 aaagctcgta gaacgtgaag gtgatcggct cgccgatagg ggtgcgcttc gcgtactcca
     4201 acacctgctg ccacaccagt tcgtcatcgt cggcccgcag ctcgacgccg gtgtaggtga
     4261 tcttcacgtc cttgttgacg tggaaaatga ccttgttttg cagcgcctcg cgcgggattt
     4321 tcttgttgcg cgtggtgaac agggcagagc gggccgtgtc gtttggcatc gctcgcatcg
     4381 tgtccggcca cggcgcaata tcgaacaagg aaagctgcat ttccttgatc tgctgcttcg
     4441 tgtgtttcag caacgcggcc tgcttggctt cgctgacctg ttttgccagg tcctcgccgg
     4501 cggtttttcg cttcttggtc gtcatagttc ctcgcgtgtc gatggtcatc gacttcgcca
     4561 aacctgccgc ctcctgttcg agacgacgcg aacgctccac ggcggccgat ggcgcgggca
     4621 gggcaggggg agccagttgc acgctgtcgc gctcgatctt ggccgtagct tgctggacta
     4681 tcgagccgac ggactggaag gtttcgcggg gcgcacgcat gacggtgcgg cttgcgatgg
     4741 tttcggcatc ctcggcggaa aaccccgcgt cgatcagttc ttgcctgtat gccttccggt
     4801 caaacgtccg attcattcac cctccttgcg ggattgcccc ggaattaatt ccccggatcg
     4861 atccgtcgat cttgatcccc tgcgccatca gatccttggc ggcaagaaag ccatccagtt
     4921 tactttgcag ggcttcccaa ccttaccaga gggcgcccca gctggcaatt ccggttcgct
     4981 tgctgtccat aaaaccgccc agtctagcta tcgccatgta agcccactgc aagctacctg
     5041 ctttctcttt gcgcttgcgt tttcccttgt ccagatagcc cagtagctga cattcatccg
     5101 gggtcagcac cgtttctgcg gactggcttt ctacgtggct gccatttttg gggtgaggcc
     5161 gttcgcggcc gaggggcgca gcccctgggg ggatgggagg cccgcgttag cgggccggga
     5221 gggttcgaga agggggggca ccccccttcg gcgtgcgcgg tcacgcgcac agggcgcagc
     5281 cctggttaaa aacaaggttt ataaatattg gtttaaaagc aggttaaaag acaggttagc
     5341 ggtggccgaa aaacgggcgg aaacccttgc aaatgctgga ttttctgcct gtggacagcc
     5401 cctcaaatgt caataggtgc gcccctcatc tgtcagcact ctgcccctca agtgtcaagg
     5461 atcgcgcccc tcatctgtca gtagtcgcgc ccctcaagtg tcaataccgc agggcactta
     5521 tccccaggct tgtccacatc atctgtggga aactcgcgta aaatcaggcg ttttcgccga
     5581 tttgcgaggc tggccagctc cacgtcgccg gccgaaatcg agcctgcccc tcatctgtca
     5641 acgccgcgcc gggtgagtcg gcccctcaag tgtcaacgtc cgcccctcat ctgtcagtga
     5701 gggccaagtt ttccgcgagg tatccacaac gccggcggcc ctacatggct ctgctgtagt
     5761 gagtgggttg cgctccggca gcggtcctga tcccccgcag aaaaaaagga tctcaagaag
     5821 atcctttgat cttttctacg gcgcgccgag ctcgaattct agggcggcgg atttgtccta
     5881 ctcaggagag cgttcaccga caaacaacag ataaaacgaa aggcccagtc tttcgactga
     5941 gcctttcgtt ttatttgatg cctctagcac gcctgccagg aattggggat cggagatctt
     6001 taagacccac tttcacattt aagttgtttt tctaatccgc atataatcaa ttcaaggccg
     6061 aataagaagg ctggctctgc accttggtga tcaaataatt cgatagcttg tcgtaataat
     6121 ggcggcatac tatcagtagt aggtgtttcc ctttcttctt tagcgacttg atgctcttga
     6181 tcttccaata cgcaacctaa agtaaaatgc cccacagcgc tgagtgcata taatgcattc
     6241 tctagtgaaa aaccttgttg gcataaaaag gctaattgat tttcgagagt ttcatactgt
     6301 ttttctgtag gccgtgtacc taaatgtact tttgctccat cgcgatgact tagtaaagca
     6361 catctaagac ttttagcgtt attacgtaaa aaatcttgcc agctttcccc ttctaaaggg
     6421 caaaagtgag tatggtgcct atctaacatc tcaatggcta aggcgtcgag caaagcccgc
     6481 ttatttttta catgccaata caatgtaggc tgctctacac ctagcttctg ggcgagttta
     6541 cgggttgtta aaccttcgat tccgacctca ttaagcagct ctaatgcgct gttaatcact
     6601 ttacttttat ctaatctgga catcattaat tcctaatttt tgttgacact ctatcattga
     6661 tagagttatt ttaccactcc ctatcagtga tagagaaaag tgaatctaga gtcgacaaga
     6721 aggagatata catatgaaaa ctgaagaagg taaactggta atctggatta acggcgataa
     6781 aggctataac ggtctcgctg aagtcggtaa gaaattcgag aaagataccg gaattaaagt
     6841 caccgttgag catccggata aactggaaga gaaattccca caggttgcgg caactggcga
     6901 tggccctgac attatcttct gggcacacga ccgctttggt ggctacgctc aatctggcct
     6961 gttggctgaa atcaccccgg acaaagcgtt ccaggacaag ctgtatccgt ttacctggga
     7021 tgccgtacgt tacaacggca agctgattgc ttacccgatc gctgttgaag cgttatcgct
     7081 gatttataac aaagatctgc tgccgaaccc gccaaaaacc tgggaagaga tcccggcgct
     7141 ggataaagaa ctgaaagcga aaggtaagag cgcgctgatg ttcaacctgc aagaaccgta
     7201 cttcacctgg ccgctgattg ctgctgacgg gggttatgcg ttcaagtatg aaaacggcaa
     7261 gtacgacatt aaagacgtgg gcgtggataa cgctggcgcg aaagcgggtc tgaccttcct
     7321 ggttgacctg attaaaaaca aacacatgaa tgcagacacc gattactcca tcgcagaagc
     7381 tgcctttaat aaaggcgaaa cagcgatgac catcaacggc ccgtgggcat ggtccaacat
     7441 cgacaccagc aaagtgaatt atggtgtaac ggtactgccg accttcaagg gtcaaccatc
     7501 caaaccgttc gttggcgtgc tgagcgcagg tattaacgcc gccagtccga acaaagagct
     7561 ggcaaaagag ttcctcgaaa actatctgct gactgatgaa ggtctggaag cggttaataa
     7621 agacaaaccg ctgggtgccg tagcgctgaa gtcttacgag gaagagttgg cgaaagatcc
     7681 acgtattgcc gccactatgg aaaacgccca gaaaggtgaa atcatgccga acatcccgca
     7741 gatgtccgct ttctggtatg ccgtgcgtac tgcggtgatc aacgccgcca gcggtcgtca
     7801 gactgtcgat gaagccctga aagacgcgca gactggatcc gatagcctgc tgatgaatcg
     7861 tcgtaaattt ctgtaccagt ttaaaaacgt tcgttgggca aaaggtcgtc gtgaaaccta
     7921 tctgtgttat gttgttaaac gtcgtgatag cgcaaccagc tttagcctgg attttggtta
     7981 tctgcgtaac aaaaatggtt gccatgttga actgctgttt ctgcgttata ttagcgattg
     8041 ggatttagat ccgggtcgtt gttatcgtgt tacctggttt accagttgga gcccgtgtta
     8101 tgactgtgcc cgtcatgttg cagattttct gcgtggtaat ccgaatctga gcctgcgtat
     8161 ttttaccgca cgtctgtatt tttgcgaaga tcgtaaagca gaaccggaag gtctgcgtcg
     8221 tctgcatcgt gccggtgttc agattgcaat tatgaccttc aaagactact tctactgctg
     8281 gaataccttt gtggaaaatc atgaacgtac ctttaaagca tgggaaggcc tgcatgaaaa
     8341 ttcagttcgt ctgagccgtc agctgcgtcg tattctgctg ccgctgtatg aagttgatga
     8401 tctgcgtgat gcatttcgta ccctgggtct gctgcagggt ggtggtagcg gtggtggttc
     8461 aggtggtggc agtggcggtg gaagtggtgg cggttctggc ggaggatcag gtggcggtag
     8521 caagcttatg aacacgatta acatcgctaa gaacgacttc tctgacatcg aactggctgc
     8581 tatcccgttc aacactctgg ctgaccatta cggtgagcgt ttagctcgcg aacagttggc
     8641 ccttgagcat gagtcttacg agatgggtga agcacgcttc cgcaagatgt ttgagcgtca
     8701 acttaaagct ggtgaggttg cggataacgc tgccgccaag cctctcatca ctaccctact
     8761 ccctaagatg attgcacgca tcaacgactg gtttgaggaa gtgaaagcta agcgcggcaa
     8821 gcgcccgaca gccttccagt tcctgcaaga aatcaagccg gaagccgtag cgtacatcac
     8881 cattaagacc actctggct
//
