LOCUS       sequence-335278-        7123 bp    DNA     circular SYN 14-APR-2025
DEFINITION  Expresses T7RNAP in E. coli.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7123)
  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 7123)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 14, 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: pSEVA221T7RNAP
FEATURES             Location/Qualifiers
     source          1..7123
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     114..133
                     /label=Tet-R
                     /note="Tetracycline resistance gene, reverse primer"
     promoter        225..280
                     /gene="tetR"
                     /label=tetR/tetA promoters
                     /note="overlapping promoters for bacterial tetR and tetA"
     protein_bind    231..249
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O1 for the tetR and tetA genes"
     protein_bind    261..279
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     terminator      2997..3091
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     primer_bind     complement(3212..3231)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             3223..4038
                     /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            4205..4313
                     /note="incP origin of transfer"
     CDS             complement(4374..5522)
                     /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      5915..6446
                     /label=oriV
                     /note="incP origin of replication"
     terminator      complement(6577..6663)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     CDS             complement(join(6706..7123,1..209))
                     /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"
ORIGIN
        1 gggcaaaagt gagtatggtg cctatctaac atctcaatgg ctaaggcgtc gagcaaagcc
       61 cgcttatttt ttacatgcca atacaatgta ggctgctcta cacctagctt ctgggcgagt
      121 ttacgggttg ttaaaccttc gattccgacc tcattaagca gctctaatgc gctgttaatc
      181 actttacttt tatctaatct ggacatcatt aattcctaat ttttgttgac actctatcat
      241 tgatagagtt attttaccac tccctatcag tgatagagaa aagtgaatct agagtcgacc
      301 tgcaggtttg caagcttaag aaggagatat acatatgaac acgattaaca tcgctaagaa
      361 cgacttctct gacatcgaac tggctgctat cccgttcaac actctggctg accattacgg
      421 tgagcgttta gctcgcgaac agttggccct tgagcatgag tcttacgaga tgggtgaagc
      481 acgcttccgc aagatgtttg agcgtcaact taaagctggt gaggttgcgg ataacgctgc
      541 cgccaagcct ctcatcacta ccctactccc taagatgatt gcacgcatca acgactggtt
      601 tgaggaagtg aaagctaagc gcggcaagcg cccgacagcc ttccagttcc tgcaagaaat
      661 caagccggaa gccgtagcgt acatcaccat taagaccact ctggcttgcc taaccagtgc
      721 tgacaataca accgttcagg ctgtagcaag cgcaatcggt cgggccattg aggacgaggc
      781 tcgcttcggt cgtatccgtg accttgaagc taagcacttc aagaaaaacg ttgaggaaca
      841 actcaacaag cgcgtagggc acgtctacaa gaaagcattt atgcaagttg tcgaggctga
      901 catgctctct aagggtctac tcggtggcga ggcgtggtct tcgtggcata aggaagactc
      961 tattcatgta ggagtacgct gcatcgagat gctcattgag tcaaccggaa tggttagctt
     1021 acaccgccaa aatgctggcg tagtaggtca agactctgag actatcgaac tcgcacctga
     1081 atacgctgag gctatcgcaa cccgtgcagg tgcgctggct ggcatctctc cgatgttcca
     1141 accttgcgta gttcctccta agccgtggac tggcattact ggtggtggct attgggctaa
     1201 cggtcgtcgt cctctggcgc tggtgcgtac tcacagtaag aaagcactga tgcgctacga
     1261 agacgtttac atgcctgagg tgtacaaagc gattaacatt gcgcaaaaca ccgcatggaa
     1321 aatcaacaag aaagtcctag cggtcgccaa cgtaatcacc aagtggaagc attgtccggt
     1381 cgaggacatc cctgcgattg agcgtgaaga actcccgatg aaaccggaag acatcgacat
     1441 gaatcctgag gctctcaccg cgtggaaacg tgctgccgct gctgtgtacc gcaaggacaa
     1501 ggctcgcaag tctcgccgta tcagccttga gttcatgctt gagcaagcca ataagtttgc
     1561 taaccataag gccatctggt tcccttacaa catggactgg cgcggtcgtg tttacgctgt
     1621 gtcaatgttc aacccgcaag gtaacgatat gaccaaagga ctgcttacgc tggcgaaagg
     1681 taaaccaatc ggtaaggaag gttactactg gctgaaaatc cacggtgcaa actgtgcggg
     1741 tgtcgataag gttccgttcc ctgagcgcat caagttcatt gaggaaaacc acgagaacat
     1801 catggcttgc gctaagtctc cactggagaa cacttggtgg gctgagcaag attctccgtt
     1861 ctgcttcctt gcgttctgct ttgagtacgc tggggtacag caccacggcc tgagctataa
     1921 ctgctccctt ccgctggcgt ttgacgggtc ttgctctggc atccagcact tctccgcgat
     1981 gctccgagat gaggtaggtg gtcgcgcggt taacttgctt cctagtgaaa ccgttcagga
     2041 catctacggg attgttgcta agaaagtcaa cgagattcta caagcagacg caatcaatgg
     2101 gaccgataac gaagtagtta ccgtgaccga tgagaacact ggtgaaatct ctgagaaagt
     2161 caagctgggc actaaggcac tggctggtca atggctggct tacggtgtta ctcgcagtgt
     2221 gactaagcgt tcagtcatga cgctggctta cgggtccaaa gagttcggct tccgtcaaca
     2281 agtgctggaa gataccattc agccagctat tgattccggc aagggtctga tgttcactca
     2341 gccgaatcag gctgctggat acatggctaa gctgatttgg gaatctgtga gcgtgacggt
     2401 ggtagctgcg gttgaagcaa tgaactggct taagtctgct gctaagctgc tggctgctga
     2461 ggtcaaagat aagaagactg gagagattct tcgcaagcgt tgcgctgtgc attgggtaac
     2521 tcctgatggt ttccctgtgt ggcaggaata caagaagcct attcagacgc gcttgaacct
     2581 gatgttcctc ggtcagttcc gcttacagcc taccattaac accaacaaag atagcgagat
     2641 tgatgcacac aaacaggagt ctggtatcgc tcctaacttt gtacacagcc aagacggtag
     2701 ccaccttcgt aagactgtag tgtgggcaca cgagaagtac ggaatcgaat cttttgcact
     2761 gattcacgac tccttcggta ccattccggc tgacgctgcg aacctgttca aagcagtgcg
     2821 cgaaactatg gttgacacat atgagtcttg tgatgtactg gctgatttct acgaccagtt
     2881 cgctgaccag ttgcacgagt ctcaattgga caaaatgcca gcacttccgg ctaaaggtaa
     2941 cttgaacctc cgtgacatct tagagtcgga cttcgcgttc gcgtaaacta gtcttggact
     3001 cctgttgata gatccagtaa tgacctcaga actccatctg gatttgttca gaacgctcgg
     3061 ttgccgccgg gcgtttttta ttggtgagaa tccaggggtc cccaataatt acgatttaaa
     3121 tttgtgtctc aaaatctctg atgttacatt gcacaagata aaaatatatc atcatgaaca
     3181 ataaaactgt ctgcttacat aaacagtaat acaaggggtg ttatgagcca tattcagcgt
     3241 gaaacgagct gtagccgtcc gcgtctgaac agcaacatgg atgcggatct gtatggctat
     3301 aaatgggcgc gtgataacgt gggtcagagc ggcgcgacca tttatcgtct gtatggcaaa
     3361 ccggatgcgc cggaactgtt tctgaaacat ggcaaaggca gcgtggcgaa cgatgtgacc
     3421 gatgaaatgg tgcgtctgaa ctggctgacc gaatttatgc cgctgccgac cattaaacat
     3481 tttattcgca ccccggatga tgcgtggctg ctgaccaccg cgattccggg caaaaccgcg
     3541 tttcaggtgc tggaagaata tccggatagc ggcgaaaaca ttgtggatgc gctggccgtg
     3601 tttctgcgtc gtctgcatag cattccggtg tgcaactgcc cgtttaacag cgatcgtgtg
     3661 tttcgtctgg cccaggcgca gagccgtatg aacaacggcc tggtggatgc gagcgatttt
     3721 gatgatgaac gtaacggctg gccggtggaa caggtgtgga aagaaatgca taaactgctg
     3781 ccgtttagcc cggatagcgt ggtgacccac ggcgatttta gcctggataa cctgattttc
     3841 gatgaaggca aactgattgg ctgcattgat gtgggccgtg tgggcattgc ggatcgttat
     3901 caggatctgg ccattctgtg gaactgcctg ggcgaattta gcccgagcct gcaaaaacgt
     3961 ctgtttcaga aatatggcat tgataatccg gatatgaaca aactgcaatt tcatctgatg
     4021 ctggatgaat ttttctaata attaattgga ccgcggtccg cgcgttgtcc ttttccgctg
     4081 cataaccctg cttcggggtc attatagcga ttttttcggt atatccatcc tttttcgcac
     4141 gatatacagg attttgccaa agggttcgtg tagactttcc ttggtgtatc caacggcgtc
     4201 agccgggcag gataggtgaa gtaggcccac ccgcgagcgg gtgttccttc ttcactgtcc
     4261 cttattcgca cctggcggtg ctcaacggga atcctgctct gcgaggctgg ccgtaggccg
     4321 gccgcgatgc aggtggctgc tgaaccccca gccggaactg accccacaag gccctagcgt
     4381 ttgcaatgca ccaggtcatc attgacccag gcgtgttcca ccaggccgct gcctcgcaac
     4441 tcttcgcagg cttcgccgac ctgctcgcgc cacttcttca cgcgggtgga atccgatccg
     4501 cacatgaggc ggaaggtttc cagcttgagc gggtacggct cccggtgcga gctgaaatag
     4561 tcgaacatcc gtcgggccgt cggcgacagc ttgcggtact tctcccatat gaatttcgtg
     4621 tagtggtcgc cagcaaacag cacgacgatt tcctcgtcga tcaggacctg gcaacgggac
     4681 gttttcttgc cacggtccag gacgcggaag cggtgcagca gcgacaccga ttccaggtgc
     4741 ccaacgcggt cggacgtgaa gcccatcgcc gtcgcctgta ggcgcgacag gcattcctcg
     4801 gccttcgtgt aataccggcc attgatcgac cagcccaggt cctggcaaag ctcgtagaac
     4861 gtgaaggtga tcggctcgcc gataggggtg cgcttcgcgt actccaacac ctgctgccac
     4921 accagttcgt catcgtcggc ccgcagctcg acgccggtgt aggtgatctt cacgtccttg
     4981 ttgacgtgga aaatgacctt gttttgcagc gcctcgcgcg ggattttctt gttgcgcgtg
     5041 gtgaacaggg cagagcgggc cgtgtcgttt ggcatcgctc gcatcgtgtc cggccacggc
     5101 gcaatatcga acaaggaaag ctgcatttcc ttgatctgct gcttcgtgtg tttcagcaac
     5161 gcggcctgct tggcttcgct gacctgtttt gccaggtcct cgccggcggt ttttcgcttc
     5221 ttggtcgtca tagttcctcg cgtgtcgatg gtcatcgact tcgccaaacc tgccgcctcc
     5281 tgttcgagac gacgcgaacg ctccacggcg gccgatggcg cgggcagggc agggggagcc
     5341 agttgcacgc tgtcgcgctc gatcttggcc gtagcttgct ggactatcga gccgacggac
     5401 tggaaggttt cgcggggcgc acgcatgacg gtgcggcttg cgatggtttc ggcatcctcg
     5461 gcggaaaacc ccgcgtcgat cagttcttgc ctgtatgcct tccggtcaaa cgtccgattc
     5521 attcaccctc cttgcgggat tgccccggaa ttaattcccc ggatcgatcc gtcgatcttg
     5581 atcccctgcg ccatcagatc cttggcggca agaaagccat ccagtttact ttgcagggct
     5641 tcccaacctt accagagggc gccccagctg gcaattccgg ttcgcttgct gtccataaaa
     5701 ccgcccagtc tagctatcgc catgtaagcc cactgcaagc tacctgcttt ctctttgcgc
     5761 ttgcgttttc ccttgtccag atagcccagt agctgacatt catccggggt cagcaccgtt
     5821 tctgcggact ggctttctac gtggctgcca tttttggggt gaggccgttc gcggccgagg
     5881 ggcgcagccc ctggggggat gggaggcccg cgttagcggg ccgggagggt tcgagaaggg
     5941 ggggcacccc ccttcggcgt gcgcggtcac gcgcacaggg cgcagccctg gttaaaaaca
     6001 aggtttataa atattggttt aaaagcaggt taaaagacag gttagcggtg gccgaaaaac
     6061 gggcggaaac ccttgcaaat gctggatttt ctgcctgtgg acagcccctc aaatgtcaat
     6121 aggtgcgccc ctcatctgtc agcactctgc ccctcaagtg tcaaggatcg cgcccctcat
     6181 ctgtcagtag tcgcgcccct caagtgtcaa taccgcaggg cacttatccc caggcttgtc
     6241 cacatcatct gtgggaaact cgcgtaaaat caggcgtttt cgccgatttg cgaggctggc
     6301 cagctccacg tcgccggccg aaatcgagcc tgcccctcat ctgtcaacgc cgcgccgggt
     6361 gagtcggccc ctcaagtgtc aacgtccgcc cctcatctgt cagtgagggc caagttttcc
     6421 gcgaggtatc cacaacgccg gcggccctac atggctctgc tgtagtgagt gggttgcgct
     6481 ccggcagcgg tcctgatccc ccgcagaaaa aaaggatctc aagaagatcc tttgatcttt
     6541 tctacggcgc gccgagctcg aattctaggg cggcggattt gtcctactca ggagagcgtt
     6601 caccgacaaa caacagataa aacgaaaggc ccagtctttc gactgagcct ttcgttttat
     6661 ttgatgcctc tagcacgcct gccaggaatt ggggatcgga gatctttaag acccactttc
     6721 acatttaagt tgtttttcta atccgcatat aatcaattca aggccgaata agaaggctgg
     6781 ctctgcacct tggtgatcaa ataattcgat agcttgtcgt aataatggcg gcatactatc
     6841 agtagtaggt gtttcccttt cttctttagc gacttgatgc tcttgatctt ccaatacgca
     6901 acctaaagta aaatgcccca cagcgctgag tgcatataat gcattctcta gtgaaaaacc
     6961 ttgttggcat aaaaaggcta attgattttc gagagtttca tactgttttt ctgtaggccg
     7021 tgtacctaaa tgtacttttg ctccatcgcg atgacttagt aaagcacatc taagactttt
     7081 agcgttatta cgtaaaaaat cttgccagct ttccccttct aaa
//
