LOCUS       sequence-335284-        7825 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses fusion protein AID-T7RNAP in E. coli.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7825)
  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 7825)
  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: pSEVA221AID-T7RNAP
FEATURES             Location/Qualifiers
     source          1..7825
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     terminator      2698..2792
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     primer_bind     complement(2913..2932)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             2924..3739
                     /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            3906..4014
                     /note="incP origin of transfer"
     CDS             complement(4075..5223)
                     /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      5616..6147
                     /label=oriV
                     /note="incP origin of replication"
     terminator      complement(6278..6364)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     CDS             complement(6407..7033)
                     /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     6938..6957
                     /label=Tet-R
                     /note="Tetracycline resistance gene, reverse primer"
     promoter        7049..7104
                     /gene="tetR"
                     /label=tetR/tetA promoters
                     /note="overlapping promoters for bacterial tetR and tetA"
     protein_bind    7055..7073
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O1 for the tetR and tetA genes"
     protein_bind    7085..7103
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     CDS             7168..7764
                     /codon_start=1
                     /gene="AICDA"
                     /product="human activation-induced cytidine deaminase 
                     (Dickerson et al., 2003)"
                     /label=AID
                     /translation="MGDSLLMNRRKFLYQFKNVRWAKGRRETYLCYVVKRRDSATSFSL
                     DFGYLRNKNGCHVELLFLRYISDWDLDPGRCYRVTWFTSWSPCYDCARHVADFLRGNPN
                     LSLRIFTARLYFCEDRKAEPEGLRRLHRAGVQIAIMTFKDYFYCWNTFVENHERTFKAW
                     EGLHENSVRLSRQLRRILLPLYEVDDLRDAFRTLGL"
ORIGIN
        1 gttctggcgg aggatcaggt ggcggtagca agcttatgaa cacgattaac atcgctaaga
       61 acgacttctc tgacatcgaa ctggctgcta tcccgttcaa cactctggct gaccattacg
      121 gtgagcgttt agctcgcgaa cagttggccc ttgagcatga gtcttacgag atgggtgaag
      181 cacgcttccg caagatgttt gagcgtcaac ttaaagctgg tgaggttgcg gataacgctg
      241 ccgccaagcc tctcatcact accctactcc ctaagatgat tgcacgcatc aacgactggt
      301 ttgaggaagt gaaagctaag cgcggcaagc gcccgacagc cttccagttc ctgcaagaaa
      361 tcaagccgga agccgtagcg tacatcacca ttaagaccac tctggcttgc ctaaccagtg
      421 ctgacaatac aaccgttcag gctgtagcaa gcgcaatcgg tcgggccatt gaggacgagg
      481 ctcgcttcgg tcgtatccgt gaccttgaag ctaagcactt caagaaaaac gttgaggaac
      541 aactcaacaa gcgcgtaggg cacgtctaca agaaagcatt tatgcaagtt gtcgaggctg
      601 acatgctctc taagggtcta ctcggtggcg aggcgtggtc ttcgtggcat aaggaagact
      661 ctattcatgt aggagtacgc tgcatcgaga tgctcattga gtcaaccgga atggttagct
      721 tacaccgcca aaatgctggc gtagtaggtc aagactctga gactatcgaa ctcgcacctg
      781 aatacgctga ggctatcgca acccgtgcag gtgcgctggc tggcatctct ccgatgttcc
      841 aaccttgcgt agttcctcct aagccgtgga ctggcattac tggtggtggc tattgggcta
      901 acggtcgtcg tcctctggcg ctggtgcgta ctcacagtaa gaaagcactg atgcgctacg
      961 aagacgttta catgcctgag gtgtacaaag cgattaacat tgcgcaaaac accgcatgga
     1021 aaatcaacaa gaaagtccta gcggtcgcca acgtaatcac caagtggaag cattgtccgg
     1081 tcgaggacat ccctgcgatt gagcgtgaag aactcccgat gaaaccggaa gacatcgaca
     1141 tgaatcctga ggctctcacc gcgtggaaac gtgctgccgc tgctgtgtac cgcaaggaca
     1201 aggctcgcaa gtctcgccgt atcagccttg agttcatgct tgagcaagcc aataagtttg
     1261 ctaaccataa ggccatctgg ttcccttaca acatggactg gcgcggtcgt gtttacgctg
     1321 tgtcaatgtt caacccgcaa ggtaacgata tgaccaaagg actgcttacg ctggcgaaag
     1381 gtaaaccaat cggtaaggaa ggttactact ggctgaaaat ccacggtgca aactgtgcgg
     1441 gtgtcgataa ggttccgttc cctgagcgca tcaagttcat tgaggaaaac cacgagaaca
     1501 tcatggcttg cgctaagtct ccactggaga acacttggtg ggctgagcaa gattctccgt
     1561 tctgcttcct tgcgttctgc tttgagtacg ctggggtaca gcaccacggc ctgagctata
     1621 actgctccct tccgctggcg tttgacgggt cttgctctgg catccagcac ttctccgcga
     1681 tgctccgaga tgaggtaggt ggtcgcgcgg ttaacttgct tcctagtgaa accgttcagg
     1741 acatctacgg gattgttgct aagaaagtca acgagattct acaagcagac gcaatcaatg
     1801 ggaccgataa cgaagtagtt accgtgaccg atgagaacac tggtgaaatc tctgagaaag
     1861 tcaagctggg cactaaggca ctggctggtc aatggctggc ttacggtgtt actcgcagtg
     1921 tgactaagcg ttcagtcatg acgctggctt acgggtccaa agagttcggc ttccgtcaac
     1981 aagtgctgga agataccatt cagccagcta ttgattccgg caagggtctg atgttcactc
     2041 agccgaatca ggctgctgga tacatggcta agctgatttg ggaatctgtg agcgtgacgg
     2101 tggtagctgc ggttgaagca atgaactggc ttaagtctgc tgctaagctg ctggctgctg
     2161 aggtcaaaga taagaagact ggagagattc ttcgcaagcg ttgcgctgtg cattgggtaa
     2221 ctcctgatgg tttccctgtg tggcaggaat acaagaagcc tattcagacg cgcttgaacc
     2281 tgatgttcct cggtcagttc cgcttacagc ctaccattaa caccaacaaa gatagcgaga
     2341 ttgatgcaca caaacaggag tctggtatcg ctcctaactt tgtacacagc caagacggta
     2401 gccaccttcg taagactgta gtgtgggcac acgagaagta cggaatcgaa tcttttgcac
     2461 tgattcacga ctccttcggt accattccgg ctgacgctgc gaacctgttc aaagcagtgc
     2521 gcgaaactat ggttgacaca tatgagtctt gtgatgtact ggctgatttc tacgaccagt
     2581 tcgctgacca gttgcacgag tctcaattgg acaaaatgcc agcacttccg gctaaaggta
     2641 acttgaacct ccgtgacatc ttagagtcgg acttcgcgtt cgcgtaaact agtcttggac
     2701 tcctgttgat agatccagta atgacctcag aactccatct ggatttgttc agaacgctcg
     2761 gttgccgccg ggcgtttttt attggtgaga atccaggggt ccccaataat tacgatttaa
     2821 atttgtgtct caaaatctct gatgttacat tgcacaagat aaaaatatat catcatgaac
     2881 aataaaactg tctgcttaca taaacagtaa tacaaggggt gttatgagcc atattcagcg
     2941 tgaaacgagc tgtagccgtc cgcgtctgaa cagcaacatg gatgcggatc tgtatggcta
     3001 taaatgggcg cgtgataacg tgggtcagag cggcgcgacc atttatcgtc tgtatggcaa
     3061 accggatgcg ccggaactgt ttctgaaaca tggcaaaggc agcgtggcga acgatgtgac
     3121 cgatgaaatg gtgcgtctga actggctgac cgaatttatg ccgctgccga ccattaaaca
     3181 ttttattcgc accccggatg atgcgtggct gctgaccacc gcgattccgg gcaaaaccgc
     3241 gtttcaggtg ctggaagaat atccggatag cggcgaaaac attgtggatg cgctggccgt
     3301 gtttctgcgt cgtctgcata gcattccggt gtgcaactgc ccgtttaaca gcgatcgtgt
     3361 gtttcgtctg gcccaggcgc agagccgtat gaacaacggc ctggtggatg cgagcgattt
     3421 tgatgatgaa cgtaacggct ggccggtgga acaggtgtgg aaagaaatgc ataaactgct
     3481 gccgtttagc ccggatagcg tggtgaccca cggcgatttt agcctggata acctgatttt
     3541 cgatgaaggc aaactgattg gctgcattga tgtgggccgt gtgggcattg cggatcgtta
     3601 tcaggatctg gccattctgt ggaactgcct gggcgaattt agcccgagcc tgcaaaaacg
     3661 tctgtttcag aaatatggca ttgataatcc ggatatgaac aaactgcaat ttcatctgat
     3721 gctggatgaa tttttctaat aattaattgg accgcggtcc gcgcgttgtc cttttccgct
     3781 gcataaccct gcttcggggt cattatagcg attttttcgg tatatccatc ctttttcgca
     3841 cgatatacag gattttgcca aagggttcgt gtagactttc cttggtgtat ccaacggcgt
     3901 cagccgggca ggataggtga agtaggccca cccgcgagcg ggtgttcctt cttcactgtc
     3961 ccttattcgc acctggcggt gctcaacggg aatcctgctc tgcgaggctg gccgtaggcc
     4021 ggccgcgatg caggtggctg ctgaaccccc agccggaact gaccccacaa ggccctagcg
     4081 tttgcaatgc accaggtcat cattgaccca ggcgtgttcc accaggccgc tgcctcgcaa
     4141 ctcttcgcag gcttcgccga cctgctcgcg ccacttcttc acgcgggtgg aatccgatcc
     4201 gcacatgagg cggaaggttt ccagcttgag cgggtacggc tcccggtgcg agctgaaata
     4261 gtcgaacatc cgtcgggccg tcggcgacag cttgcggtac ttctcccata tgaatttcgt
     4321 gtagtggtcg ccagcaaaca gcacgacgat ttcctcgtcg atcaggacct ggcaacggga
     4381 cgttttcttg ccacggtcca ggacgcggaa gcggtgcagc agcgacaccg attccaggtg
     4441 cccaacgcgg tcggacgtga agcccatcgc cgtcgcctgt aggcgcgaca ggcattcctc
     4501 ggccttcgtg taataccggc cattgatcga ccagcccagg tcctggcaaa gctcgtagaa
     4561 cgtgaaggtg atcggctcgc cgataggggt gcgcttcgcg tactccaaca cctgctgcca
     4621 caccagttcg tcatcgtcgg cccgcagctc gacgccggtg taggtgatct tcacgtcctt
     4681 gttgacgtgg aaaatgacct tgttttgcag cgcctcgcgc gggattttct tgttgcgcgt
     4741 ggtgaacagg gcagagcggg ccgtgtcgtt tggcatcgct cgcatcgtgt ccggccacgg
     4801 cgcaatatcg aacaaggaaa gctgcatttc cttgatctgc tgcttcgtgt gtttcagcaa
     4861 cgcggcctgc ttggcttcgc tgacctgttt tgccaggtcc tcgccggcgg tttttcgctt
     4921 cttggtcgtc atagttcctc gcgtgtcgat ggtcatcgac ttcgccaaac ctgccgcctc
     4981 ctgttcgaga cgacgcgaac gctccacggc ggccgatggc gcgggcaggg cagggggagc
     5041 cagttgcacg ctgtcgcgct cgatcttggc cgtagcttgc tggactatcg agccgacgga
     5101 ctggaaggtt tcgcggggcg cacgcatgac ggtgcggctt gcgatggttt cggcatcctc
     5161 ggcggaaaac cccgcgtcga tcagttcttg cctgtatgcc ttccggtcaa acgtccgatt
     5221 cattcaccct ccttgcggga ttgccccgga attaattccc cggatcgatc cgtcgatctt
     5281 gatcccctgc gccatcagat ccttggcggc aagaaagcca tccagtttac tttgcagggc
     5341 ttcccaacct taccagaggg cgccccagct ggcaattccg gttcgcttgc tgtccataaa
     5401 accgcccagt ctagctatcg ccatgtaagc ccactgcaag ctacctgctt tctctttgcg
     5461 cttgcgtttt cccttgtcca gatagcccag tagctgacat tcatccgggg tcagcaccgt
     5521 ttctgcggac tggctttcta cgtggctgcc atttttgggg tgaggccgtt cgcggccgag
     5581 gggcgcagcc cctgggggga tgggaggccc gcgttagcgg gccgggaggg ttcgagaagg
     5641 gggggcaccc cccttcggcg tgcgcggtca cgcgcacagg gcgcagccct ggttaaaaac
     5701 aaggtttata aatattggtt taaaagcagg ttaaaagaca ggttagcggt ggccgaaaaa
     5761 cgggcggaaa cccttgcaaa tgctggattt tctgcctgtg gacagcccct caaatgtcaa
     5821 taggtgcgcc cctcatctgt cagcactctg cccctcaagt gtcaaggatc gcgcccctca
     5881 tctgtcagta gtcgcgcccc tcaagtgtca ataccgcagg gcacttatcc ccaggcttgt
     5941 ccacatcatc tgtgggaaac tcgcgtaaaa tcaggcgttt tcgccgattt gcgaggctgg
     6001 ccagctccac gtcgccggcc gaaatcgagc ctgcccctca tctgtcaacg ccgcgccggg
     6061 tgagtcggcc cctcaagtgt caacgtccgc ccctcatctg tcagtgaggg ccaagttttc
     6121 cgcgaggtat ccacaacgcc ggcggcccta catggctctg ctgtagtgag tgggttgcgc
     6181 tccggcagcg gtcctgatcc cccgcagaaa aaaaggatct caagaagatc ctttgatctt
     6241 ttctacggcg cgccgagctc gaattctagg gcggcggatt tgtcctactc aggagagcgt
     6301 tcaccgacaa acaacagata aaacgaaagg cccagtcttt cgactgagcc tttcgtttta
     6361 tttgatgcct ctagcacgcc tgccaggaat tggggatcgg agatctttaa gacccacttt
     6421 cacatttaag ttgtttttct aatccgcata taatcaattc aaggccgaat aagaaggctg
     6481 gctctgcacc ttggtgatca aataattcga tagcttgtcg taataatggc ggcatactat
     6541 cagtagtagg tgtttccctt tcttctttag cgacttgatg ctcttgatct tccaatacgc
     6601 aacctaaagt aaaatgcccc acagcgctga gtgcatataa tgcattctct agtgaaaaac
     6661 cttgttggca taaaaaggct aattgatttt cgagagtttc atactgtttt tctgtaggcc
     6721 gtgtacctaa atgtactttt gctccatcgc gatgacttag taaagcacat ctaagacttt
     6781 tagcgttatt acgtaaaaaa tcttgccagc tttccccttc taaagggcaa aagtgagtat
     6841 ggtgcctatc taacatctca atggctaagg cgtcgagcaa agcccgctta ttttttacat
     6901 gccaatacaa tgtaggctgc tctacaccta gcttctgggc gagtttacgg gttgttaaac
     6961 cttcgattcc gacctcatta agcagctcta atgcgctgtt aatcacttta cttttatcta
     7021 atctggacat cattaattcc taatttttgt tgacactcta tcattgatag agttatttta
     7081 ccactcccta tcagtgatag agaaaagtga atctagagtc gacaagaagg agatatacat
     7141 atggcaagca tgaccggtgg tcagcagatg ggtgatagcc tgctgatgaa tcgtcgtaaa
     7201 tttctgtacc agtttaaaaa cgttcgttgg gcaaaaggtc gtcgtgaaac ctatctgtgt
     7261 tatgttgtta aacgtcgtga tagcgcaacc agctttagcc tggattttgg ttatctgcgt
     7321 aacaaaaatg gttgccatgt tgaactgctg tttctgcgtt atattagcga ttgggattta
     7381 gatccgggtc gttgttatcg tgttacctgg tttaccagtt ggagcccgtg ttatgactgt
     7441 gcccgtcatg ttgcagattt tctgcgtggt aatccgaatc tgagcctgcg tatttttacc
     7501 gcacgtctgt atttttgcga agatcgtaaa gcagaaccgg aaggtctgcg tcgtctgcat
     7561 cgtgccggtg ttcagattgc aattatgacc ttcaaagact acttctactg ctggaatacc
     7621 tttgtggaaa atcatgaacg tacctttaaa gcatgggaag gcctgcatga aaattcagtt
     7681 cgtctgagcc gtcagctgcg tcgtattctg ctgccgctgt atgaagttga tgatctgcgt
     7741 gatgcatttc gtaccctggg tctgctgcag ggtggtggta gcggtggtgg ttcaggtggt
     7801 ggcagtggcg gtggaagtgg tggcg
//
