LOCUS       sequence-335283-        8149 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses fusion protein TrxA-AID-T7RNAP in E. coli.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8149)
  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 8149)
  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: pSEVA221TrxA-AID-T7RNAP
FEATURES             Location/Qualifiers
     source          1..8149
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     terminator      2715..2809
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     primer_bind     complement(2930..2949)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             2941..3756
                     /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            3923..4031
                     /note="incP origin of transfer"
     CDS             complement(4092..5240)
                     /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      5633..6164
                     /label=oriV
                     /note="incP origin of replication"
     terminator      complement(6295..6381)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     CDS             complement(6424..7050)
                     /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     6955..6974
                     /label=Tet-R
                     /note="Tetracycline resistance gene, reverse primer"
     promoter        7066..7121
                     /gene="tetR"
                     /label=tetR/tetA promoters
                     /note="overlapping promoters for bacterial tetR and tetA"
     protein_bind    7072..7090
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O1 for the tetR and tetA genes"
     protein_bind    7102..7120
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     CDS             7158..7484
                     /codon_start=1
                     /product="His-Patch thioredoxin"
                     /label=HP-thioredoxin
                     /note="The histidines at positions 8, 32, and 64 coordinate
                     a divalent cation, allowing HP-thioredoxin to be purified 
                     on metal-chelating resins."
                     /translation="MSDKIIHLTDDSFDTDVLKADGAILVDFWAHWCGPCKMIAPILDE
                     IADEYQGKLTVAKLNIDHNPGTAPKYGIRGIPTLLLFKNGEVAATKVGALSKGQLKEFL
                     DANLA"
     CDS             7500..7514
                     /codon_start=1
                     /product="enterokinase recognition and cleavage site"
                     /label=enterokinase site
                     /translation="DDDDK"
     CDS             7515..8105
                     /codon_start=1
                     /gene="AICDA"
                     /product="human activation-induced cytidine deaminase 
                     (Dickerson et al., 2003)"
                     /label=AID
                     /translation="DSLLMNRRKFLYQFKNVRWAKGRRETYLCYVVKRRDSATSFSLDF
                     GYLRNKNGCHVELLFLRYISDWDLDPGRCYRVTWFTSWSPCYDCARHVADFLRGNPNLS
                     LRIFTARLYFCEDRKAEPEGLRRLHRAGVQIAIMTFKDYFYCWNTFVENHERTFKAWEG
                     LHENSVRLSRQLRRILLPLYEVDDLRDAFRTLGL"
ORIGIN
        1 cggtggaagt ggtggcggtt ctggcggagg atcaggtggc ggtagcaagc ttatgaacac
       61 gattaacatc gctaagaacg acttctctga catcgaactg gctgctatcc cgttcaacac
      121 tctggctgac cattacggtg agcgtttagc tcgcgaacag ttggcccttg agcatgagtc
      181 ttacgagatg ggtgaagcac gcttccgcaa gatgtttgag cgtcaactta aagctggtga
      241 ggttgcggat aacgctgccg ccaagcctct catcactacc ctactcccta agatgattgc
      301 acgcatcaac gactggtttg aggaagtgaa agctaagcgc ggcaagcgcc cgacagcctt
      361 ccagttcctg caagaaatca agccggaagc cgtagcgtac atcaccatta agaccactct
      421 ggcttgccta accagtgctg acaatacaac cgttcaggct gtagcaagcg caatcggtcg
      481 ggccattgag gacgaggctc gcttcggtcg tatccgtgac cttgaagcta agcacttcaa
      541 gaaaaacgtt gaggaacaac tcaacaagcg cgtagggcac gtctacaaga aagcatttat
      601 gcaagttgtc gaggctgaca tgctctctaa gggtctactc ggtggcgagg cgtggtcttc
      661 gtggcataag gaagactcta ttcatgtagg agtacgctgc atcgagatgc tcattgagtc
      721 aaccggaatg gttagcttac accgccaaaa tgctggcgta gtaggtcaag actctgagac
      781 tatcgaactc gcacctgaat acgctgaggc tatcgcaacc cgtgcaggtg cgctggctgg
      841 catctctccg atgttccaac cttgcgtagt tcctcctaag ccgtggactg gcattactgg
      901 tggtggctat tgggctaacg gtcgtcgtcc tctggcgctg gtgcgtactc acagtaagaa
      961 agcactgatg cgctacgaag acgtttacat gcctgaggtg tacaaagcga ttaacattgc
     1021 gcaaaacacc gcatggaaaa tcaacaagaa agtcctagcg gtcgccaacg taatcaccaa
     1081 gtggaagcat tgtccggtcg aggacatccc tgcgattgag cgtgaagaac tcccgatgaa
     1141 accggaagac atcgacatga atcctgaggc tctcaccgcg tggaaacgtg ctgccgctgc
     1201 tgtgtaccgc aaggacaagg ctcgcaagtc tcgccgtatc agccttgagt tcatgcttga
     1261 gcaagccaat aagtttgcta accataaggc catctggttc ccttacaaca tggactggcg
     1321 cggtcgtgtt tacgctgtgt caatgttcaa cccgcaaggt aacgatatga ccaaaggact
     1381 gcttacgctg gcgaaaggta aaccaatcgg taaggaaggt tactactggc tgaaaatcca
     1441 cggtgcaaac tgtgcgggtg tcgataaggt tccgttccct gagcgcatca agttcattga
     1501 ggaaaaccac gagaacatca tggcttgcgc taagtctcca ctggagaaca cttggtgggc
     1561 tgagcaagat tctccgttct gcttccttgc gttctgcttt gagtacgctg gggtacagca
     1621 ccacggcctg agctataact gctcccttcc gctggcgttt gacgggtctt gctctggcat
     1681 ccagcacttc tccgcgatgc tccgagatga ggtaggtggt cgcgcggtta acttgcttcc
     1741 tagtgaaacc gttcaggaca tctacgggat tgttgctaag aaagtcaacg agattctaca
     1801 agcagacgca atcaatggga ccgataacga agtagttacc gtgaccgatg agaacactgg
     1861 tgaaatctct gagaaagtca agctgggcac taaggcactg gctggtcaat ggctggctta
     1921 cggtgttact cgcagtgtga ctaagcgttc agtcatgacg ctggcttacg ggtccaaaga
     1981 gttcggcttc cgtcaacaag tgctggaaga taccattcag ccagctattg attccggcaa
     2041 gggtctgatg ttcactcagc cgaatcaggc tgctggatac atggctaagc tgatttggga
     2101 atctgtgagc gtgacggtgg tagctgcggt tgaagcaatg aactggctta agtctgctgc
     2161 taagctgctg gctgctgagg tcaaagataa gaagactgga gagattcttc gcaagcgttg
     2221 cgctgtgcat tgggtaactc ctgatggttt ccctgtgtgg caggaataca agaagcctat
     2281 tcagacgcgc ttgaacctga tgttcctcgg tcagttccgc ttacagccta ccattaacac
     2341 caacaaagat agcgagattg atgcacacaa acaggagtct ggtatcgctc ctaactttgt
     2401 acacagccaa gacggtagcc accttcgtaa gactgtagtg tgggcacacg agaagtacgg
     2461 aatcgaatct tttgcactga ttcacgactc cttcggtacc attccggctg acgctgcgaa
     2521 cctgttcaaa gcagtgcgcg aaactatggt tgacacatat gagtcttgtg atgtactggc
     2581 tgatttctac gaccagttcg ctgaccagtt gcacgagtct caattggaca aaatgccagc
     2641 acttccggct aaaggtaact tgaacctccg tgacatctta gagtcggact tcgcgttcgc
     2701 gtaaactagt cttggactcc tgttgataga tccagtaatg acctcagaac tccatctgga
     2761 tttgttcaga acgctcggtt gccgccgggc gttttttatt ggtgagaatc caggggtccc
     2821 caataattac gatttaaatt tgtgtctcaa aatctctgat gttacattgc acaagataaa
     2881 aatatatcat catgaacaat aaaactgtct gcttacataa acagtaatac aaggggtgtt
     2941 atgagccata ttcagcgtga aacgagctgt agccgtccgc gtctgaacag caacatggat
     3001 gcggatctgt atggctataa atgggcgcgt gataacgtgg gtcagagcgg cgcgaccatt
     3061 tatcgtctgt atggcaaacc ggatgcgccg gaactgtttc tgaaacatgg caaaggcagc
     3121 gtggcgaacg atgtgaccga tgaaatggtg cgtctgaact ggctgaccga atttatgccg
     3181 ctgccgacca ttaaacattt tattcgcacc ccggatgatg cgtggctgct gaccaccgcg
     3241 attccgggca aaaccgcgtt tcaggtgctg gaagaatatc cggatagcgg cgaaaacatt
     3301 gtggatgcgc tggccgtgtt tctgcgtcgt ctgcatagca ttccggtgtg caactgcccg
     3361 tttaacagcg atcgtgtgtt tcgtctggcc caggcgcaga gccgtatgaa caacggcctg
     3421 gtggatgcga gcgattttga tgatgaacgt aacggctggc cggtggaaca ggtgtggaaa
     3481 gaaatgcata aactgctgcc gtttagcccg gatagcgtgg tgacccacgg cgattttagc
     3541 ctggataacc tgattttcga tgaaggcaaa ctgattggct gcattgatgt gggccgtgtg
     3601 ggcattgcgg atcgttatca ggatctggcc attctgtgga actgcctggg cgaatttagc
     3661 ccgagcctgc aaaaacgtct gtttcagaaa tatggcattg ataatccgga tatgaacaaa
     3721 ctgcaatttc atctgatgct ggatgaattt ttctaataat taattggacc gcggtccgcg
     3781 cgttgtcctt ttccgctgca taaccctgct tcggggtcat tatagcgatt ttttcggtat
     3841 atccatcctt tttcgcacga tatacaggat tttgccaaag ggttcgtgta gactttcctt
     3901 ggtgtatcca acggcgtcag ccgggcagga taggtgaagt aggcccaccc gcgagcgggt
     3961 gttccttctt cactgtccct tattcgcacc tggcggtgct caacgggaat cctgctctgc
     4021 gaggctggcc gtaggccggc cgcgatgcag gtggctgctg aacccccagc cggaactgac
     4081 cccacaaggc cctagcgttt gcaatgcacc aggtcatcat tgacccaggc gtgttccacc
     4141 aggccgctgc ctcgcaactc ttcgcaggct tcgccgacct gctcgcgcca cttcttcacg
     4201 cgggtggaat ccgatccgca catgaggcgg aaggtttcca gcttgagcgg gtacggctcc
     4261 cggtgcgagc tgaaatagtc gaacatccgt cgggccgtcg gcgacagctt gcggtacttc
     4321 tcccatatga atttcgtgta gtggtcgcca gcaaacagca cgacgatttc ctcgtcgatc
     4381 aggacctggc aacgggacgt tttcttgcca cggtccagga cgcggaagcg gtgcagcagc
     4441 gacaccgatt ccaggtgccc aacgcggtcg gacgtgaagc ccatcgccgt cgcctgtagg
     4501 cgcgacaggc attcctcggc cttcgtgtaa taccggccat tgatcgacca gcccaggtcc
     4561 tggcaaagct cgtagaacgt gaaggtgatc ggctcgccga taggggtgcg cttcgcgtac
     4621 tccaacacct gctgccacac cagttcgtca tcgtcggccc gcagctcgac gccggtgtag
     4681 gtgatcttca cgtccttgtt gacgtggaaa atgaccttgt tttgcagcgc ctcgcgcggg
     4741 attttcttgt tgcgcgtggt gaacagggca gagcgggccg tgtcgtttgg catcgctcgc
     4801 atcgtgtccg gccacggcgc aatatcgaac aaggaaagct gcatttcctt gatctgctgc
     4861 ttcgtgtgtt tcagcaacgc ggcctgcttg gcttcgctga cctgttttgc caggtcctcg
     4921 ccggcggttt ttcgcttctt ggtcgtcata gttcctcgcg tgtcgatggt catcgacttc
     4981 gccaaacctg ccgcctcctg ttcgagacga cgcgaacgct ccacggcggc cgatggcgcg
     5041 ggcagggcag ggggagccag ttgcacgctg tcgcgctcga tcttggccgt agcttgctgg
     5101 actatcgagc cgacggactg gaaggtttcg cggggcgcac gcatgacggt gcggcttgcg
     5161 atggtttcgg catcctcggc ggaaaacccc gcgtcgatca gttcttgcct gtatgccttc
     5221 cggtcaaacg tccgattcat tcaccctcct tgcgggattg ccccggaatt aattccccgg
     5281 atcgatccgt cgatcttgat cccctgcgcc atcagatcct tggcggcaag aaagccatcc
     5341 agtttacttt gcagggcttc ccaaccttac cagagggcgc cccagctggc aattccggtt
     5401 cgcttgctgt ccataaaacc gcccagtcta gctatcgcca tgtaagccca ctgcaagcta
     5461 cctgctttct ctttgcgctt gcgttttccc ttgtccagat agcccagtag ctgacattca
     5521 tccggggtca gcaccgtttc tgcggactgg ctttctacgt ggctgccatt tttggggtga
     5581 ggccgttcgc ggccgagggg cgcagcccct ggggggatgg gaggcccgcg ttagcgggcc
     5641 gggagggttc gagaaggggg ggcacccccc ttcggcgtgc gcggtcacgc gcacagggcg
     5701 cagccctggt taaaaacaag gtttataaat attggtttaa aagcaggtta aaagacaggt
     5761 tagcggtggc cgaaaaacgg gcggaaaccc ttgcaaatgc tggattttct gcctgtggac
     5821 agcccctcaa atgtcaatag gtgcgcccct catctgtcag cactctgccc ctcaagtgtc
     5881 aaggatcgcg cccctcatct gtcagtagtc gcgcccctca agtgtcaata ccgcagggca
     5941 cttatcccca ggcttgtcca catcatctgt gggaaactcg cgtaaaatca ggcgttttcg
     6001 ccgatttgcg aggctggcca gctccacgtc gccggccgaa atcgagcctg cccctcatct
     6061 gtcaacgccg cgccgggtga gtcggcccct caagtgtcaa cgtccgcccc tcatctgtca
     6121 gtgagggcca agttttccgc gaggtatcca caacgccggc ggccctacat ggctctgctg
     6181 tagtgagtgg gttgcgctcc ggcagcggtc ctgatccccc gcagaaaaaa aggatctcaa
     6241 gaagatcctt tgatcttttc tacggcgcgc cgagctcgaa ttctagggcg gcggatttgt
     6301 cctactcagg agagcgttca ccgacaaaca acagataaaa cgaaaggccc agtctttcga
     6361 ctgagccttt cgttttattt gatgcctcta gcacgcctgc caggaattgg ggatcggaga
     6421 tctttaagac ccactttcac atttaagttg tttttctaat ccgcatataa tcaattcaag
     6481 gccgaataag aaggctggct ctgcaccttg gtgatcaaat aattcgatag cttgtcgtaa
     6541 taatggcggc atactatcag tagtaggtgt ttccctttct tctttagcga cttgatgctc
     6601 ttgatcttcc aatacgcaac ctaaagtaaa atgccccaca gcgctgagtg catataatgc
     6661 attctctagt gaaaaacctt gttggcataa aaaggctaat tgattttcga gagtttcata
     6721 ctgtttttct gtaggccgtg tacctaaatg tacttttgct ccatcgcgat gacttagtaa
     6781 agcacatcta agacttttag cgttattacg taaaaaatct tgccagcttt ccccttctaa
     6841 agggcaaaag tgagtatggt gcctatctaa catctcaatg gctaaggcgt cgagcaaagc
     6901 ccgcttattt tttacatgcc aatacaatgt aggctgctct acacctagct tctgggcgag
     6961 tttacgggtt gttaaacctt cgattccgac ctcattaagc agctctaatg cgctgttaat
     7021 cactttactt ttatctaatc tggacatcat taattcctaa tttttgttga cactctatca
     7081 ttgatagagt tattttacca ctccctatca gtgatagaga aaagtgaatc tagagtcgac
     7141 aagaaggaga tatacatatg tctgataaaa ttattcatct gactgatgat tcttttgata
     7201 ctgatgtact taaggcagat ggtgcaatcc tggttgattt ctgggcacac tggtgcggtc
     7261 cgtgcaaaat gatcgctccg attctggatg aaatcgctga cgaatatcag ggcaaactga
     7321 ccgttgcaaa actgaacatc gatcacaacc cgggcactgc gccgaaatat ggcatccgtg
     7381 gtatcccgac tctgctgctg ttcaaaaacg gtgaagtggc ggcaaccaaa gtgggtgcac
     7441 tgtctaaagg tcagttgaaa gagttcctcg acgctaacct ggccggctct ggatccggtg
     7501 atgacgatga caaggatagc ctgctgatga atcgtcgtaa atttctgtac cagtttaaaa
     7561 acgttcgttg ggcaaaaggt cgtcgtgaaa cctatctgtg ttatgttgtt aaacgtcgtg
     7621 atagcgcaac cagctttagc ctggattttg gttatctgcg taacaaaaat ggttgccatg
     7681 ttgaactgct gtttctgcgt tatattagcg attgggattt agatccgggt cgttgttatc
     7741 gtgttacctg gtttaccagt tggagcccgt gttatgactg tgcccgtcat gttgcagatt
     7801 ttctgcgtgg taatccgaat ctgagcctgc gtatttttac cgcacgtctg tatttttgcg
     7861 aagatcgtaa agcagaaccg gaaggtctgc gtcgtctgca tcgtgccggt gttcagattg
     7921 caattatgac cttcaaagac tacttctact gctggaatac ctttgtggaa aatcatgaac
     7981 gtacctttaa agcatgggaa ggcctgcatg aaaattcagt tcgtctgagc cgtcagctgc
     8041 gtcgtattct gctgccgctg tatgaagttg atgatctgcg tgatgcattt cgtaccctgg
     8101 gtctgctgca gggtggtggt agcggtggtg gttcaggtgg tggcagtgg
//
