LOCUS       sequence-335279-        7729 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses fusion protein TadA*-T7RNAP in E. coli.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7729)
  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 7729)
  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: pSEVA221TadA*-T7RNAP
FEATURES             Location/Qualifiers
     source          1..7729
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     terminator      2714..2808
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     primer_bind     complement(2929..2948)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             2940..3755
                     /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            3922..4030
                     /note="incP origin of transfer"
     CDS             complement(4091..5239)
                     /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      5632..6163
                     /label=oriV
                     /note="incP origin of replication"
     terminator      complement(6294..6380)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     CDS             complement(6423..7049)
                     /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     6954..6973
                     /label=Tet-R
                     /note="Tetracycline resistance gene, reverse primer"
     promoter        7065..7120
                     /gene="tetR"
                     /label=tetR/tetA promoters
                     /note="overlapping promoters for bacterial tetR and tetA"
     protein_bind    7071..7089
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O1 for the tetR and tetA genes"
     protein_bind    7101..7119
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
ORIGIN
        1 ggtggaagtg gtggcggttc tggcggagga tcaggtggcg gtagcaagct tatgaacacg
       61 attaacatcg ctaagaacga cttctctgac atcgaactgg ctgctatccc gttcaacact
      121 ctggctgacc attacggtga gcgtttagct cgcgaacagt tggcccttga gcatgagtct
      181 tacgagatgg gtgaagcacg cttccgcaag atgtttgagc gtcaacttaa agctggtgag
      241 gttgcggata acgctgccgc caagcctctc atcactaccc tactccctaa gatgattgca
      301 cgcatcaacg actggtttga ggaagtgaaa gctaagcgcg gcaagcgccc gacagccttc
      361 cagttcctgc aagaaatcaa gccggaagcc gtagcgtaca tcaccattaa gaccactctg
      421 gcttgcctaa ccagtgctga caatacaacc gttcaggctg tagcaagcgc aatcggtcgg
      481 gccattgagg acgaggctcg cttcggtcgt atccgtgacc ttgaagctaa gcacttcaag
      541 aaaaacgttg aggaacaact caacaagcgc gtagggcacg tctacaagaa agcatttatg
      601 caagttgtcg aggctgacat gctctctaag ggtctactcg gtggcgaggc gtggtcttcg
      661 tggcataagg aagactctat tcatgtagga gtacgctgca tcgagatgct cattgagtca
      721 accggaatgg ttagcttaca ccgccaaaat gctggcgtag taggtcaaga ctctgagact
      781 atcgaactcg cacctgaata cgctgaggct atcgcaaccc gtgcaggtgc gctggctggc
      841 atctctccga tgttccaacc ttgcgtagtt cctcctaagc cgtggactgg cattactggt
      901 ggtggctatt gggctaacgg tcgtcgtcct ctggcgctgg tgcgtactca cagtaagaaa
      961 gcactgatgc gctacgaaga cgtttacatg cctgaggtgt acaaagcgat taacattgcg
     1021 caaaacaccg catggaaaat caacaagaaa gtcctagcgg tcgccaacgt aatcaccaag
     1081 tggaagcatt gtccggtcga ggacatccct gcgattgagc gtgaagaact cccgatgaaa
     1141 ccggaagaca tcgacatgaa tcctgaggct ctcaccgcgt ggaaacgtgc tgccgctgct
     1201 gtgtaccgca aggacaaggc tcgcaagtct cgccgtatca gccttgagtt catgcttgag
     1261 caagccaata agtttgctaa ccataaggcc atctggttcc cttacaacat ggactggcgc
     1321 ggtcgtgttt acgctgtgtc aatgttcaac ccgcaaggta acgatatgac caaaggactg
     1381 cttacgctgg cgaaaggtaa accaatcggt aaggaaggtt actactggct gaaaatccac
     1441 ggtgcaaact gtgcgggtgt cgataaggtt ccgttccctg agcgcatcaa gttcattgag
     1501 gaaaaccacg agaacatcat ggcttgcgct aagtctccac tggagaacac ttggtgggct
     1561 gagcaagatt ctccgttctg cttccttgcg ttctgctttg agtacgctgg ggtacagcac
     1621 cacggcctga gctataactg ctcccttccg ctggcgtttg acgggtcttg ctctggcatc
     1681 cagcacttct ccgcgatgct ccgagatgag gtaggtggtc gcgcggttaa cttgcttcct
     1741 agtgaaaccg ttcaggacat ctacgggatt gttgctaaga aagtcaacga gattctacaa
     1801 gcagacgcaa tcaatgggac cgataacgaa gtagttaccg tgaccgatga gaacactggt
     1861 gaaatctctg agaaagtcaa gctgggcact aaggcactgg ctggtcaatg gctggcttac
     1921 ggtgttactc gcagtgtgac taagcgttca gtcatgacgc tggcttacgg gtccaaagag
     1981 ttcggcttcc gtcaacaagt gctggaagat accattcagc cagctattga ttccggcaag
     2041 ggtctgatgt tcactcagcc gaatcaggct gctggataca tggctaagct gatttgggaa
     2101 tctgtgagcg tgacggtggt agctgcggtt gaagcaatga actggcttaa gtctgctgct
     2161 aagctgctgg ctgctgaggt caaagataag aagactggag agattcttcg caagcgttgc
     2221 gctgtgcatt gggtaactcc tgatggtttc cctgtgtggc aggaatacaa gaagcctatt
     2281 cagacgcgct tgaacctgat gttcctcggt cagttccgct tacagcctac cattaacacc
     2341 aacaaagata gcgagattga tgcacacaaa caggagtctg gtatcgctcc taactttgta
     2401 cacagccaag acggtagcca ccttcgtaag actgtagtgt gggcacacga gaagtacgga
     2461 atcgaatctt ttgcactgat tcacgactcc ttcggtacca ttccggctga cgctgcgaac
     2521 ctgttcaaag cagtgcgcga aactatggtt gacacatatg agtcttgtga tgtactggct
     2581 gatttctacg accagttcgc tgaccagttg cacgagtctc aattggacaa aatgccagca
     2641 cttccggcta aaggtaactt gaacctccgt gacatcttag agtcggactt cgcgttcgcg
     2701 taaactagtc ttggactcct gttgatagat ccagtaatga cctcagaact ccatctggat
     2761 ttgttcagaa cgctcggttg ccgccgggcg ttttttattg gtgagaatcc aggggtcccc
     2821 aataattacg atttaaattt gtgtctcaaa atctctgatg ttacattgca caagataaaa
     2881 atatatcatc atgaacaata aaactgtctg cttacataaa cagtaataca aggggtgtta
     2941 tgagccatat tcagcgtgaa acgagctgta gccgtccgcg tctgaacagc aacatggatg
     3001 cggatctgta tggctataaa tgggcgcgtg ataacgtggg tcagagcggc gcgaccattt
     3061 atcgtctgta tggcaaaccg gatgcgccgg aactgtttct gaaacatggc aaaggcagcg
     3121 tggcgaacga tgtgaccgat gaaatggtgc gtctgaactg gctgaccgaa tttatgccgc
     3181 tgccgaccat taaacatttt attcgcaccc cggatgatgc gtggctgctg accaccgcga
     3241 ttccgggcaa aaccgcgttt caggtgctgg aagaatatcc ggatagcggc gaaaacattg
     3301 tggatgcgct ggccgtgttt ctgcgtcgtc tgcatagcat tccggtgtgc aactgcccgt
     3361 ttaacagcga tcgtgtgttt cgtctggccc aggcgcagag ccgtatgaac aacggcctgg
     3421 tggatgcgag cgattttgat gatgaacgta acggctggcc ggtggaacag gtgtggaaag
     3481 aaatgcataa actgctgccg tttagcccgg atagcgtggt gacccacggc gattttagcc
     3541 tggataacct gattttcgat gaaggcaaac tgattggctg cattgatgtg ggccgtgtgg
     3601 gcattgcgga tcgttatcag gatctggcca ttctgtggaa ctgcctgggc gaatttagcc
     3661 cgagcctgca aaaacgtctg tttcagaaat atggcattga taatccggat atgaacaaac
     3721 tgcaatttca tctgatgctg gatgaatttt tctaataatt aattggaccg cggtccgcgc
     3781 gttgtccttt tccgctgcat aaccctgctt cggggtcatt atagcgattt tttcggtata
     3841 tccatccttt ttcgcacgat atacaggatt ttgccaaagg gttcgtgtag actttccttg
     3901 gtgtatccaa cggcgtcagc cgggcaggat aggtgaagta ggcccacccg cgagcgggtg
     3961 ttccttcttc actgtccctt attcgcacct ggcggtgctc aacgggaatc ctgctctgcg
     4021 aggctggccg taggccggcc gcgatgcagg tggctgctga acccccagcc ggaactgacc
     4081 ccacaaggcc ctagcgtttg caatgcacca ggtcatcatt gacccaggcg tgttccacca
     4141 ggccgctgcc tcgcaactct tcgcaggctt cgccgacctg ctcgcgccac ttcttcacgc
     4201 gggtggaatc cgatccgcac atgaggcgga aggtttccag cttgagcggg tacggctccc
     4261 ggtgcgagct gaaatagtcg aacatccgtc gggccgtcgg cgacagcttg cggtacttct
     4321 cccatatgaa tttcgtgtag tggtcgccag caaacagcac gacgatttcc tcgtcgatca
     4381 ggacctggca acgggacgtt ttcttgccac ggtccaggac gcggaagcgg tgcagcagcg
     4441 acaccgattc caggtgccca acgcggtcgg acgtgaagcc catcgccgtc gcctgtaggc
     4501 gcgacaggca ttcctcggcc ttcgtgtaat accggccatt gatcgaccag cccaggtcct
     4561 ggcaaagctc gtagaacgtg aaggtgatcg gctcgccgat aggggtgcgc ttcgcgtact
     4621 ccaacacctg ctgccacacc agttcgtcat cgtcggcccg cagctcgacg ccggtgtagg
     4681 tgatcttcac gtccttgttg acgtggaaaa tgaccttgtt ttgcagcgcc tcgcgcggga
     4741 ttttcttgtt gcgcgtggtg aacagggcag agcgggccgt gtcgtttggc atcgctcgca
     4801 tcgtgtccgg ccacggcgca atatcgaaca aggaaagctg catttccttg atctgctgct
     4861 tcgtgtgttt cagcaacgcg gcctgcttgg cttcgctgac ctgttttgcc aggtcctcgc
     4921 cggcggtttt tcgcttcttg gtcgtcatag ttcctcgcgt gtcgatggtc atcgacttcg
     4981 ccaaacctgc cgcctcctgt tcgagacgac gcgaacgctc cacggcggcc gatggcgcgg
     5041 gcagggcagg gggagccagt tgcacgctgt cgcgctcgat cttggccgta gcttgctgga
     5101 ctatcgagcc gacggactgg aaggtttcgc ggggcgcacg catgacggtg cggcttgcga
     5161 tggtttcggc atcctcggcg gaaaaccccg cgtcgatcag ttcttgcctg tatgccttcc
     5221 ggtcaaacgt ccgattcatt caccctcctt gcgggattgc cccggaatta attccccgga
     5281 tcgatccgtc gatcttgatc ccctgcgcca tcagatcctt ggcggcaaga aagccatcca
     5341 gtttactttg cagggcttcc caaccttacc agagggcgcc ccagctggca attccggttc
     5401 gcttgctgtc cataaaaccg cccagtctag ctatcgccat gtaagcccac tgcaagctac
     5461 ctgctttctc tttgcgcttg cgttttccct tgtccagata gcccagtagc tgacattcat
     5521 ccggggtcag caccgtttct gcggactggc tttctacgtg gctgccattt ttggggtgag
     5581 gccgttcgcg gccgaggggc gcagcccctg gggggatggg aggcccgcgt tagcgggccg
     5641 ggagggttcg agaagggggg gcacccccct tcggcgtgcg cggtcacgcg cacagggcgc
     5701 agccctggtt aaaaacaagg tttataaata ttggtttaaa agcaggttaa aagacaggtt
     5761 agcggtggcc gaaaaacggg cggaaaccct tgcaaatgct ggattttctg cctgtggaca
     5821 gcccctcaaa tgtcaatagg tgcgcccctc atctgtcagc actctgcccc tcaagtgtca
     5881 aggatcgcgc ccctcatctg tcagtagtcg cgcccctcaa gtgtcaatac cgcagggcac
     5941 ttatccccag gcttgtccac atcatctgtg ggaaactcgc gtaaaatcag gcgttttcgc
     6001 cgatttgcga ggctggccag ctccacgtcg ccggccgaaa tcgagcctgc ccctcatctg
     6061 tcaacgccgc gccgggtgag tcggcccctc aagtgtcaac gtccgcccct catctgtcag
     6121 tgagggccaa gttttccgcg aggtatccac aacgccggcg gccctacatg gctctgctgt
     6181 agtgagtggg ttgcgctccg gcagcggtcc tgatcccccg cagaaaaaaa ggatctcaag
     6241 aagatccttt gatcttttct acggcgcgcc gagctcgaat tctagggcgg cggatttgtc
     6301 ctactcagga gagcgttcac cgacaaacaa cagataaaac gaaaggccca gtctttcgac
     6361 tgagcctttc gttttatttg atgcctctag cacgcctgcc aggaattggg gatcggagat
     6421 ctttaagacc cactttcaca tttaagttgt ttttctaatc cgcatataat caattcaagg
     6481 ccgaataaga aggctggctc tgcaccttgg tgatcaaata attcgatagc ttgtcgtaat
     6541 aatggcggca tactatcagt agtaggtgtt tccctttctt ctttagcgac ttgatgctct
     6601 tgatcttcca atacgcaacc taaagtaaaa tgccccacag cgctgagtgc atataatgca
     6661 ttctctagtg aaaaaccttg ttggcataaa aaggctaatt gattttcgag agtttcatac
     6721 tgtttttctg taggccgtgt acctaaatgt acttttgctc catcgcgatg acttagtaaa
     6781 gcacatctaa gacttttagc gttattacgt aaaaaatctt gccagctttc cccttctaaa
     6841 gggcaaaagt gagtatggtg cctatctaac atctcaatgg ctaaggcgtc gagcaaagcc
     6901 cgcttatttt ttacatgcca atacaatgta ggctgctcta cacctagctt ctgggcgagt
     6961 ttacgggttg ttaaaccttc gattccgacc tcattaagca gctctaatgc gctgttaatc
     7021 actttacttt tatctaatct ggacatcatt aattcctaat ttttgttgac actctatcat
     7081 tgatagagtt attttaccac tccctatcag tgatagagaa aagtgaatct agagtcgaca
     7141 agaaggagat atacatatgg caagcatgac cggtggtcag cagatgggtg aagttgaatt
     7201 ttcacatgaa tattggatgc gtcatgcact gaccctggca aaacgtgccc gtgatgaacg
     7261 tgaagttccg gttggtgcag ttctggttct gaataatcgt gttattggtg aaggttggaa
     7321 tcgtgcaatt ggtctgcatg atccgaccgc acatgcagaa attatggcac tgcgtcaagg
     7381 tggtctggtt atgcagaatt atcgtctgat tgatgcaacc ctgtatgtta cctttgaacc
     7441 gtgtgttatg tgtgccggtg caatgattca tagccgtatt ggtcgtgttg tttttggtgt
     7501 tcgtaatgca aaaaccggtg cagcaggtag cctgatggat gttctgcatt atccgggtat
     7561 gaaccatcgt gttgaaatta ccgaaggtat tctggcagat gaatgtgcag cactgctgtg
     7621 ttatttcttt cgtatgcctc gtcaggtttt taacgcacag aaaaaagcac agagcagcac
     7681 cgatctgcag ggtggtggta gcggtggtgg ttcaggtggt ggcagtggc
//
