LOCUS       sequence-335281-        7918 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses fusion protein rAPOBEC1-T7RNAP in E. coli.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7918)
  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 7918)
  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: pSEVA221rAPOBEC1-T7RNAP
FEATURES             Location/Qualifiers
     source          1..7918
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     terminator      2718..2812
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     primer_bind     complement(2933..2952)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             2944..3759
                     /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            3926..4034
                     /note="incP origin of transfer"
     CDS             complement(4095..5243)
                     /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      5636..6167
                     /label=oriV
                     /note="incP origin of replication"
     terminator      complement(6298..6384)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     CDS             complement(6427..7053)
                     /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     6958..6977
                     /label=Tet-R
                     /note="Tetracycline resistance gene, reverse primer"
     promoter        7069..7124
                     /gene="tetR"
                     /label=tetR/tetA promoters
                     /note="overlapping promoters for bacterial tetR and tetA"
     protein_bind    7075..7093
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O1 for the tetR and tetA genes"
     protein_bind    7105..7123
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     CDS             7188..7877
                     /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="MGSSETGPVAVDPTLRRRIEPHEFEVFFDPRELRKETCLLYEINW
                     GGRHSIWRHTSQNTNKHVEVNFIEKFTTERYFCPNTRCSITWFLSWSPCGECSRAITEF
                     LSRYPHVTLFIYIARLYHHADPRNRQGLRDLISSGVTIQIMTEQESGYCWRNFVNYSPS
                     NEAHWPRYPHLWVRLYVLELYCIILGLPPCLNILRRKQPQLTFFTIALQSCHYQRLPPH
                     ILWATGLK"
ORIGIN
        1 tggcggtgga agtggtggcg gttctggcgg aggatcaggt ggcggtagca agcttatgaa
       61 cacgattaac atcgctaaga acgacttctc tgacatcgaa ctggctgcta tcccgttcaa
      121 cactctggct gaccattacg gtgagcgttt agctcgcgaa cagttggccc ttgagcatga
      181 gtcttacgag atgggtgaag cacgcttccg caagatgttt gagcgtcaac ttaaagctgg
      241 tgaggttgcg gataacgctg ccgccaagcc tctcatcact accctactcc ctaagatgat
      301 tgcacgcatc aacgactggt ttgaggaagt gaaagctaag cgcggcaagc gcccgacagc
      361 cttccagttc ctgcaagaaa tcaagccgga agccgtagcg tacatcacca ttaagaccac
      421 tctggcttgc ctaaccagtg ctgacaatac aaccgttcag gctgtagcaa gcgcaatcgg
      481 tcgggccatt gaggacgagg ctcgcttcgg tcgtatccgt gaccttgaag ctaagcactt
      541 caagaaaaac gttgaggaac aactcaacaa gcgcgtaggg cacgtctaca agaaagcatt
      601 tatgcaagtt gtcgaggctg acatgctctc taagggtcta ctcggtggcg aggcgtggtc
      661 ttcgtggcat aaggaagact ctattcatgt aggagtacgc tgcatcgaga tgctcattga
      721 gtcaaccgga atggttagct tacaccgcca aaatgctggc gtagtaggtc aagactctga
      781 gactatcgaa ctcgcacctg aatacgctga ggctatcgca acccgtgcag gtgcgctggc
      841 tggcatctct ccgatgttcc aaccttgcgt agttcctcct aagccgtgga ctggcattac
      901 tggtggtggc tattgggcta acggtcgtcg tcctctggcg ctggtgcgta ctcacagtaa
      961 gaaagcactg atgcgctacg aagacgttta catgcctgag gtgtacaaag cgattaacat
     1021 tgcgcaaaac accgcatgga aaatcaacaa gaaagtccta gcggtcgcca acgtaatcac
     1081 caagtggaag cattgtccgg tcgaggacat ccctgcgatt gagcgtgaag aactcccgat
     1141 gaaaccggaa gacatcgaca tgaatcctga ggctctcacc gcgtggaaac gtgctgccgc
     1201 tgctgtgtac cgcaaggaca aggctcgcaa gtctcgccgt atcagccttg agttcatgct
     1261 tgagcaagcc aataagtttg ctaaccataa ggccatctgg ttcccttaca acatggactg
     1321 gcgcggtcgt gtttacgctg tgtcaatgtt caacccgcaa ggtaacgata tgaccaaagg
     1381 actgcttacg ctggcgaaag gtaaaccaat cggtaaggaa ggttactact ggctgaaaat
     1441 ccacggtgca aactgtgcgg gtgtcgataa ggttccgttc cctgagcgca tcaagttcat
     1501 tgaggaaaac cacgagaaca tcatggcttg cgctaagtct ccactggaga acacttggtg
     1561 ggctgagcaa gattctccgt tctgcttcct tgcgttctgc tttgagtacg ctggggtaca
     1621 gcaccacggc ctgagctata actgctccct tccgctggcg tttgacgggt cttgctctgg
     1681 catccagcac ttctccgcga tgctccgaga tgaggtaggt ggtcgcgcgg ttaacttgct
     1741 tcctagtgaa accgttcagg acatctacgg gattgttgct aagaaagtca acgagattct
     1801 acaagcagac gcaatcaatg ggaccgataa cgaagtagtt accgtgaccg atgagaacac
     1861 tggtgaaatc tctgagaaag tcaagctggg cactaaggca ctggctggtc aatggctggc
     1921 ttacggtgtt actcgcagtg tgactaagcg ttcagtcatg acgctggctt acgggtccaa
     1981 agagttcggc ttccgtcaac aagtgctgga agataccatt cagccagcta ttgattccgg
     2041 caagggtctg atgttcactc agccgaatca ggctgctgga tacatggcta agctgatttg
     2101 ggaatctgtg agcgtgacgg tggtagctgc ggttgaagca atgaactggc ttaagtctgc
     2161 tgctaagctg ctggctgctg aggtcaaaga taagaagact ggagagattc ttcgcaagcg
     2221 ttgcgctgtg cattgggtaa ctcctgatgg tttccctgtg tggcaggaat acaagaagcc
     2281 tattcagacg cgcttgaacc tgatgttcct cggtcagttc cgcttacagc ctaccattaa
     2341 caccaacaaa gatagcgaga ttgatgcaca caaacaggag tctggtatcg ctcctaactt
     2401 tgtacacagc caagacggta gccaccttcg taagactgta gtgtgggcac acgagaagta
     2461 cggaatcgaa tcttttgcac tgattcacga ctccttcggt accattccgg ctgacgctgc
     2521 gaacctgttc aaagcagtgc gcgaaactat ggttgacaca tatgagtctt gtgatgtact
     2581 ggctgatttc tacgaccagt tcgctgacca gttgcacgag tctcaattgg acaaaatgcc
     2641 agcacttccg gctaaaggta acttgaacct ccgtgacatc ttagagtcgg acttcgcgtt
     2701 cgcgtaaact agtcttggac tcctgttgat agatccagta atgacctcag aactccatct
     2761 ggatttgttc agaacgctcg gttgccgccg ggcgtttttt attggtgaga atccaggggt
     2821 ccccaataat tacgatttaa atttgtgtct caaaatctct gatgttacat tgcacaagat
     2881 aaaaatatat catcatgaac aataaaactg tctgcttaca taaacagtaa tacaaggggt
     2941 gttatgagcc atattcagcg tgaaacgagc tgtagccgtc cgcgtctgaa cagcaacatg
     3001 gatgcggatc tgtatggcta taaatgggcg cgtgataacg tgggtcagag cggcgcgacc
     3061 atttatcgtc tgtatggcaa accggatgcg ccggaactgt ttctgaaaca tggcaaaggc
     3121 agcgtggcga acgatgtgac cgatgaaatg gtgcgtctga actggctgac cgaatttatg
     3181 ccgctgccga ccattaaaca ttttattcgc accccggatg atgcgtggct gctgaccacc
     3241 gcgattccgg gcaaaaccgc gtttcaggtg ctggaagaat atccggatag cggcgaaaac
     3301 attgtggatg cgctggccgt gtttctgcgt cgtctgcata gcattccggt gtgcaactgc
     3361 ccgtttaaca gcgatcgtgt gtttcgtctg gcccaggcgc agagccgtat gaacaacggc
     3421 ctggtggatg cgagcgattt tgatgatgaa cgtaacggct ggccggtgga acaggtgtgg
     3481 aaagaaatgc ataaactgct gccgtttagc ccggatagcg tggtgaccca cggcgatttt
     3541 agcctggata acctgatttt cgatgaaggc aaactgattg gctgcattga tgtgggccgt
     3601 gtgggcattg cggatcgtta tcaggatctg gccattctgt ggaactgcct gggcgaattt
     3661 agcccgagcc tgcaaaaacg tctgtttcag aaatatggca ttgataatcc ggatatgaac
     3721 aaactgcaat ttcatctgat gctggatgaa tttttctaat aattaattgg accgcggtcc
     3781 gcgcgttgtc cttttccgct gcataaccct gcttcggggt cattatagcg attttttcgg
     3841 tatatccatc ctttttcgca cgatatacag gattttgcca aagggttcgt gtagactttc
     3901 cttggtgtat ccaacggcgt cagccgggca ggataggtga agtaggccca cccgcgagcg
     3961 ggtgttcctt cttcactgtc ccttattcgc acctggcggt gctcaacggg aatcctgctc
     4021 tgcgaggctg gccgtaggcc ggccgcgatg caggtggctg ctgaaccccc agccggaact
     4081 gaccccacaa ggccctagcg tttgcaatgc accaggtcat cattgaccca ggcgtgttcc
     4141 accaggccgc tgcctcgcaa ctcttcgcag gcttcgccga cctgctcgcg ccacttcttc
     4201 acgcgggtgg aatccgatcc gcacatgagg cggaaggttt ccagcttgag cgggtacggc
     4261 tcccggtgcg agctgaaata gtcgaacatc cgtcgggccg tcggcgacag cttgcggtac
     4321 ttctcccata tgaatttcgt gtagtggtcg ccagcaaaca gcacgacgat ttcctcgtcg
     4381 atcaggacct ggcaacggga cgttttcttg ccacggtcca ggacgcggaa gcggtgcagc
     4441 agcgacaccg attccaggtg cccaacgcgg tcggacgtga agcccatcgc cgtcgcctgt
     4501 aggcgcgaca ggcattcctc ggccttcgtg taataccggc cattgatcga ccagcccagg
     4561 tcctggcaaa gctcgtagaa cgtgaaggtg atcggctcgc cgataggggt gcgcttcgcg
     4621 tactccaaca cctgctgcca caccagttcg tcatcgtcgg cccgcagctc gacgccggtg
     4681 taggtgatct tcacgtcctt gttgacgtgg aaaatgacct tgttttgcag cgcctcgcgc
     4741 gggattttct tgttgcgcgt ggtgaacagg gcagagcggg ccgtgtcgtt tggcatcgct
     4801 cgcatcgtgt ccggccacgg cgcaatatcg aacaaggaaa gctgcatttc cttgatctgc
     4861 tgcttcgtgt gtttcagcaa cgcggcctgc ttggcttcgc tgacctgttt tgccaggtcc
     4921 tcgccggcgg tttttcgctt cttggtcgtc atagttcctc gcgtgtcgat ggtcatcgac
     4981 ttcgccaaac ctgccgcctc ctgttcgaga cgacgcgaac gctccacggc ggccgatggc
     5041 gcgggcaggg cagggggagc cagttgcacg ctgtcgcgct cgatcttggc cgtagcttgc
     5101 tggactatcg agccgacgga ctggaaggtt tcgcggggcg cacgcatgac ggtgcggctt
     5161 gcgatggttt cggcatcctc ggcggaaaac cccgcgtcga tcagttcttg cctgtatgcc
     5221 ttccggtcaa acgtccgatt cattcaccct ccttgcggga ttgccccgga attaattccc
     5281 cggatcgatc cgtcgatctt gatcccctgc gccatcagat ccttggcggc aagaaagcca
     5341 tccagtttac tttgcagggc ttcccaacct taccagaggg cgccccagct ggcaattccg
     5401 gttcgcttgc tgtccataaa accgcccagt ctagctatcg ccatgtaagc ccactgcaag
     5461 ctacctgctt tctctttgcg cttgcgtttt cccttgtcca gatagcccag tagctgacat
     5521 tcatccgggg tcagcaccgt ttctgcggac tggctttcta cgtggctgcc atttttgggg
     5581 tgaggccgtt cgcggccgag gggcgcagcc cctgggggga tgggaggccc gcgttagcgg
     5641 gccgggaggg ttcgagaagg gggggcaccc cccttcggcg tgcgcggtca cgcgcacagg
     5701 gcgcagccct ggttaaaaac aaggtttata aatattggtt taaaagcagg ttaaaagaca
     5761 ggttagcggt ggccgaaaaa cgggcggaaa cccttgcaaa tgctggattt tctgcctgtg
     5821 gacagcccct caaatgtcaa taggtgcgcc cctcatctgt cagcactctg cccctcaagt
     5881 gtcaaggatc gcgcccctca tctgtcagta gtcgcgcccc tcaagtgtca ataccgcagg
     5941 gcacttatcc ccaggcttgt ccacatcatc tgtgggaaac tcgcgtaaaa tcaggcgttt
     6001 tcgccgattt gcgaggctgg ccagctccac gtcgccggcc gaaatcgagc ctgcccctca
     6061 tctgtcaacg ccgcgccggg tgagtcggcc cctcaagtgt caacgtccgc ccctcatctg
     6121 tcagtgaggg ccaagttttc cgcgaggtat ccacaacgcc ggcggcccta catggctctg
     6181 ctgtagtgag tgggttgcgc tccggcagcg gtcctgatcc cccgcagaaa aaaaggatct
     6241 caagaagatc ctttgatctt ttctacggcg cgccgagctc gaattctagg gcggcggatt
     6301 tgtcctactc aggagagcgt tcaccgacaa acaacagata aaacgaaagg cccagtcttt
     6361 cgactgagcc tttcgtttta tttgatgcct ctagcacgcc tgccaggaat tggggatcgg
     6421 agatctttaa gacccacttt cacatttaag ttgtttttct aatccgcata taatcaattc
     6481 aaggccgaat aagaaggctg gctctgcacc ttggtgatca aataattcga tagcttgtcg
     6541 taataatggc ggcatactat cagtagtagg tgtttccctt tcttctttag cgacttgatg
     6601 ctcttgatct tccaatacgc aacctaaagt aaaatgcccc acagcgctga gtgcatataa
     6661 tgcattctct agtgaaaaac cttgttggca taaaaaggct aattgatttt cgagagtttc
     6721 atactgtttt tctgtaggcc gtgtacctaa atgtactttt gctccatcgc gatgacttag
     6781 taaagcacat ctaagacttt tagcgttatt acgtaaaaaa tcttgccagc tttccccttc
     6841 taaagggcaa aagtgagtat ggtgcctatc taacatctca atggctaagg cgtcgagcaa
     6901 agcccgctta ttttttacat gccaatacaa tgtaggctgc tctacaccta gcttctgggc
     6961 gagtttacgg gttgttaaac cttcgattcc gacctcatta agcagctcta atgcgctgtt
     7021 aatcacttta cttttatcta atctggacat cattaattcc taatttttgt tgacactcta
     7081 tcattgatag agttatttta ccactcccta tcagtgatag agaaaagtga atctagagtc
     7141 gacaagaagg agatatacat atggcaagca tgaccggtgg tcagcagatg ggtagcagcg
     7201 aaaccggtcc ggttgcagtt gatccgacac tgcgtcgtcg tattgaaccg catgaatttg
     7261 aagttttttt tgatccgcgt gagctgcgta aagaaacctg tctgctgtat gaaattaact
     7321 ggggtggtcg tcatagcatt tggcgtcata ccagccagaa taccaataaa catgtggaag
     7381 tgaacttcat cgagaaattt accaccgaac gttatttttg cccgaataca cgttgtagca
     7441 ttacctggtt tctgagctgg tcaccgtgtg gtgaatgtag ccgtgcaatt accgaatttc
     7501 tgagccgtta tccgcatgtt accctgttta tctatattgc ccgtctgtat catcatgcag
     7561 atccgcgtaa tcgtcagggt ctgcgtgatc tgattagcag cggtgttacc attcagatta
     7621 tgaccgaaca agaaagcggt tattgctggc gtaattttgt gaattatagc ccgagcaatg
     7681 aagcacattg gcctcgctat ccgcatctgt gggttcgtct gtatgttctg gaactgtatt
     7741 gtattattct gggtctgcct ccgtgtctga atattctgcg tcgtaaacag ccgcagctga
     7801 ccttttttac cattgcactg caaagctgtc attatcagcg tctgccaccg catattctgt
     7861 gggcaaccgg tctgaaactg cagggtggtg gtagcggtgg tggttcaggt ggtggcag
//
