LOCUS       sequence-190012-        6739 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses TALE targeted to lac operator with one TEV cut site in its
            N-terminal domain, anhydrotetracycline inducible TEV protease.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6739)
  AUTHORS   Copeland MF, Politz MC, Johnson CB, Markley AL, Pfleger BF
  TITLE     A transcription activator-like effector (TALE) induction system 
            mediated by proteolysis.
  JOURNAL   Nat Chem Biol. 2016 Apr;12(4):254-60. doi: 10.1038/nchembio.2021. 
            Epub 2016 Feb 8.
  PUBMED    26854666
REFERENCE   2  (bases 1 to 6739)
  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"; doi: 
            "10.1038/nchembio.2021"; journalName: "Nat Chem Biol"; date: 
            "2016-04"; volume: "12"; issue: "4"; pages: "254-60"
COMMENT     Sequence Label: p5A3-T
FEATURES             Location/Qualifiers
     source          1..6739
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             1..18
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             436..456
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQS"
     CDS             2545..2568
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     terminator      2606..2637
                     /label=tonB terminator
                     /note="bidirectional E. coli tonB-P14 transcription 
                     terminator"
     CDS             complement(2654..3469)
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNGDRVFRLAQAQSRMNNGLVGA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     3377..3396
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     CDS             complement(3559..4224)
                     /codon_start=1
                     /product="replication protein for the broad-host-range 
                     plasmid pBBR1 from Bordetella bronchiseptica"
                     /label=pBBR1 Rep
                     /translation="MATQSREIGIQAKNKPGHWVQTERKAHEAWAGLIARKPTAAMLLH
                     HLVAQMGHQNAVVVSQKTLSKLIGRSLRTVQYAVKDLVAERWISVVKLNGPGTVSAYVV
                     NDRVAWGQPRDQLRLSVFSAAVVVDHDDQDESLLGHGDLRRIPTLYPGEQQLPTGPGEE
                     PPSQPGIPGMEPDLPALTETEEWERRGQQRLPMPDEPCFLDDGEPLEPPTRVTLPRRT"
     terminator      complement(4884..4955)
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     CDS             complement(5000..5626)
                     /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
                     VKNKRALLDALAIEMLDRHHTHFCPLEGESWQDFLRNNAKSFRCALLSHRDGAKVHLGT
                     RPTEKQYETLENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEDQEHQVAKEERETPT
                     TDSMPPLLRQAIELFDHQGAEPAFLFGLELIICGLEKQLKCESGS"
     primer_bind     5531..5550
                     /label=Tet-R
                     /note="Tetracycline resistance gene, reverse primer"
     promoter        5642..5697
                     /gene="tetR"
                     /label=tetR/tetA promoters
                     /note="overlapping promoters for bacterial tetR and tetA"
     protein_bind    5678..5696
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     CDS             5738..5758
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=7xHis
                     /translation="HHHHHHH"
     CDS             5759..6466
                     /codon_start=1
                     /product="tobacco etch virus protease (Kapust et al., 
                     2001)"
                     /label=TEV protease
                     /translation="GESLFKGPRDYNPISSTICHLTNESDGHTTSLYGIGFGPFIITNK
                     HLFRRNNGTLLVQSLHGVFKVKNTTTLQQHLIDGRDMIIIRMPKDFPPFPQKLKFREPQ
                     REERICLVTTNFQTKSMSSMVSDTSCTFPSSDGIFWKHWIQTKDGQCGSPLVSTRDGFI
                     VGIHSASNFTNTNNYFTSVPKNFMELLTNQEAQQWVSGWRLNADSVLWGGHKVFMVKPE
                     EPFQPVKEATQLMN"
     terminator      6553..6589
                     /label=soxR terminator
                     /note="bidirectional E. coli soxR transcription terminator"
     misc_feature    6640..6661
                     /label=BioBrick prefix
                     /note="BioBrick prefix for parts that do not start with 
                     ""ATG"""
     promoter        6662..6696
                     /label=BBa_J23102
                     /note="Anderson constitutive promoter 
                     (https://parts.igem.org/Promoters/Catalog/Anderson). 
                     Measured strength relative to BBa_J223100: 0.86"
ORIGIN
        1 catcaccatc atcaccatgt tgatctgcgt accctgggtt atagccagca gcagcaagaa
       61 aaaatcaaac cgaaagttcg tagcaccgtt gcacagcatc atgaagcact ggttggtcat
      121 ggttttaccc atgcacatat tgttgcactg agccagcatc cggcagcact gggcaccgtt
      181 gcagttaaat atcaggatat gattgcagca ctgccggaag caacccatga agcaattgtt
      241 ggtgttggta aacagtggtc aggtgcacgt gccctggaag cactgctgac cgttgccggt
      301 gaactgcgtg gtccgcctct gcagctggat accggtcagc tgctgaaaat tgcaaaacgt
      361 ggtggtgtta ccgcagttga agcagttcat gcatggcgta atgcactgac cggtgcaccg
      421 ctgaatggtt ctggcgaaaa cctgtacttc cagtctggct cgggtctgac accggaacag
      481 gttgttgcaa ttgccagcaa tggtggtggc aaacaggcac tggaaaccgt tcagcgtctg
      541 ctgccggttc tgtgtcaggc acatggtctg acccctgaac aggtggtggc cattgcaagc
      601 aataatggcg gtaaacaagc cctggaaaca gtgcagcgcc tgttaccggt gctgtgccag
      661 gcccatggct taactccgga acaggtggta gcgatcgcat caaatggcgg aggtaaacag
      721 gccttagaaa ccgtacagcg cttactgccg gtgttatgcc aggcgcacgg cctgacgcca
      781 gaacaggtag tggcaatcgc ctcaaataat ggtggaaaac aggcgttaga gacagtccag
      841 cgcctgctgc ctgtattatg tcaagcccat ggcctgaccc cagagcaagt tgttgcgatt
      901 gcaagtaaca ttgggggtaa acaggcactt gagacagttc aacgtttact gcctgtactg
      961 tgccaagctc acggtctgac tccggaacaa gtcgtcgcga ttgcgagtaa taacggtggc
     1021 aaacaagcat tagaaacggt gcaacgcctg ctgccagttc tttgccaggc tcacggttta
     1081 acccctgagc aggttgtagc tattgcgagt catgatggtg gtaagcaggc gttggaaact
     1141 gtgcaaagac tgctgcccgt gttgtgccaa gcacatggtt taaccccaga acaagtcgta
     1201 gcaatcgcaa gcaatcatgg tggcaagcaa gcgcttgaaa cagtacagcg tttattaccg
     1261 gtactttgtc aggcccacgg tcttacacca gaacaagttg tggccatagc cagtaacaat
     1321 ggcggaaagc aggctctgga aacggtacaa cgtctgttac ctgttctgtg tcaagcgcac
     1381 ggattaacac ctgaacaagt agttgccatt gcgtcaaata tcggaggcaa gcaggccttg
     1441 gagacagtgc agagattact gccagtgttg tgtcaggctc atggccttac acccgagcag
     1501 gtcgtggcaa ttgcatctaa tggtggcggt aagcaagctt tagagactgt tcagagactg
     1561 cttcctgtcc tgtgccaggc acacggactt acgcctgagc aagtggttgc aatcgcctct
     1621 aacatcggtg gtaagcaagc actggaaact gtccaacgct tacttccggt gctttgtcaa
     1681 gcacacggct taacgccaga gcaggtcgtc gccatagcca gcaatatcgg tggtaaacag
     1741 gcccttgaaa cggtccaaag acttctgccg gtcctttgcc aagcgcatgg gctgacacct
     1801 gagcaggtag tcgcgattgc ctcacatgac ggtgggaagc aggcattaga aacagttcaa
     1861 agattattac cagtcctgtg tcaggcgcat gggttaaccc cagagcaggt agttgcaata
     1921 gcatccaaca ttggcggaaa acaagcgttg gaaacggttc agcggttatt gcctgttttg
     1981 tgccaggcgc atggtttgac acccgagcaa gtggtagcca tagcctcaaa tatagggggt
     2041 aaacaagctt tggagacagt acaacggctg cttccagttt tatgtcaggc ccatggattg
     2101 acgcctgaac aagttgtcgc tatcgcaagt aatgggggtg gtaaacaagc gcttgaaacc
     2161 gttcaacgcc ttctgcctgt gctttgtcag gcacatggat taacacccga acaggttgtc
     2221 gcgatagctt caaacggtgg tggtcgtccg gcactggaaa gcattgttgc acagctgagc
     2281 cgtccggatc cggcactggc agcactgacc aatgatcatc tggttgcact ggcatgtctg
     2341 ggtggtcgcc ctgccctgga tgcagttaaa aaaggtctgc cgcatgcacc ggcactgatt
     2401 aaacgtacca atcgtcgtat tccggaacgt accagccatc gtgttgcaga tcatgcacag
     2461 gttgttcgtg ttctgggttt ttttcagtgt catagccatc cggcacaggc atttgatgat
     2521 gcaatgaccc agtttggtat gagcgattat aaagatgatg atgataaata aggtacccta
     2581 atcatacctg acctccatag cagaaagtca aaaacctccg accggaggct tttgacttga
     2641 gggggatccg ccctcagaaa aactcatcga gcatcaagtg aaactgcaat ttattcatat
     2701 caggattatc aataccatat ttttgaaaaa gccgtttctg taatgaagga gaaaactcac
     2761 cgaggcagtt ccataggatg gcaagatcct ggtatcggtc tgcgattccg actcgtccaa
     2821 catcaataca acctattaat ttcccctcgt caaaaataag gttatcaagt gagaaatcac
     2881 catgagtgac gactgaatcc ggtgagaatg gcaaaagctt atgcatttct ttccagactt
     2941 gttcaacagg ccagccatta cgctcgtcat caaaatcact cgcaccaacc aaaccgttat
     3001 tcattcgtga ttgcgcctga gcgagacgaa atacgcgatc gccgttaaaa ggacaattac
     3061 aaacaggaat cgaatgcaac cggcgcagga acactgccag cgcatcaaca atattttcac
     3121 ctgaatcagg atattcttct aatacctgga atgctgtttt ccctgggatc gcagtggtga
     3181 gtaaccatgc atcatcagga gtacggataa aatgcttgat ggtcggaaga ggcataaatt
     3241 ccgtcagcca gtttagcctg accatctcat ctgtaacatc attggcaacg ctacctttgc
     3301 catgtttcag aaacaactct ggcgcatcgg gcttcccata caatcgatag attgtcgcac
     3361 ctgattgccc gacattatcg cgagcccatt tatacccata taaatcagca tccatgttgg
     3421 aatttaatcg cggcctcgag caagacgttt cccgttgaat atggctcatt ttagcttcct
     3481 tagctcctga aaatctcgat aactcaaaaa atacgcccgg tagtgatctt atttcattat
     3541 ggtgaaagtt ggaacctctt acgtacggcg cggcagcgtg acccgtgtcg gcggctccaa
     3601 cggctcgcca tcgtccagaa aacacggctc atcgggcatc ggcaggcgct gctgcccgcg
     3661 tcgttcccat tcctccgttt cggtcaaggc tggcaggtct ggttccatgc ccggaatgcc
     3721 gggctggctg ggcggctcct cgccggggcc ggtcggtagt tgctgctcgc ccggatacag
     3781 ggtcgggatg cggcgcaggt cgccatgccc caacagcgat tcgtcctggt cgtcgtgatc
     3841 aaccaccacg gcggcactga acaccgacag gcgcaactgg tcgcggggct ggccccacgc
     3901 cacgcggtca ttgaccacgt aggccgacac ggtgccgggg ccgttgagct tcacgacgga
     3961 gatccagcgc tcggccacca agtccttgac tgcgtattgg accgtccgca aagaacgtcc
     4021 gatgagcttg gaaagtgtct tctggctgac caccacggcg ttctggtggc ccatctgcgc
     4081 cacgaggtga tgcagcagca ttgccgccgt gggtttcctc gcaataagcc cggcccacgc
     4141 ctcatgcgct ttgcgttccg tttgcaccca gtgaccgggc ttgttcttgg cttgaatgcc
     4201 gatttctctg gactgcgtgg ccatgcttat ctccatgcgg taggggtgcc gcacggttgc
     4261 ggcaccatgc gcaatcagct gcaacttttc ggcagcgcga caacaattat gcgttgcgta
     4321 aaagtggcag tcaattacag attttcttta acctacgcaa tgagctattg cggggggtgc
     4381 cgcaatgagc tgttgcgtac cccccttttt taagttgttg atttttaagt ctttcgcatt
     4441 tcgccctata tctagttctt tggtgcccaa agaagggcac ccctgcgggg ttcccccacg
     4501 ccttcggcgc ggctccccct ccggcaaaaa gtggcccctc cggggcttgt tgatcgactg
     4561 cgcggccttc ggccttgccc aaggtggcgc tgcccccttg gaacccccgc actcgccgcc
     4621 gtgaggctcg gggggcaggc gggcgggctt cgcccttcga ctgcccccac tcgcataggc
     4681 ttgggtcgtt ccaggcgcgt caaggccaag ccgctgcgcg gtcgctgcgc gagccttgac
     4741 ccgccttcca cttggtgtcc aaccggcaag cgaagcgcgc aggccgcagg ccggaggctt
     4801 ttccccagag aaaattaaaa aaattgatgg ggcaaggccg caggccgcgc agttggagcc
     4861 ggtgggtatg tggtcgaagg ctgggtagcg ttcaccgaca aacaacagat aaaacgaaag
     4921 gcccagtctt tcgactgagc ctttcgtttt atttgatgcc tggctctagt agtgatctac
     4981 actagcacta tcagtgttat taagacccac tttcacattt aagttgtttt tctaatccgc
     5041 atatgatcaa ttcaaggccg aataagaagg ctggctctgc accttggtga tcaaataatt
     5101 cgatagcttg tcgtaataat ggcggcatac tatcagtagt aggtgtttcc ctttcttctt
     5161 tagcgacttg atgctcttga tcttccaata cgcaacctaa agtaaaatgc cccacagcgc
     5221 tgagtgcata taatgcattc tctagtgaaa aaccttgttg gcataaaaag gctaattgat
     5281 tttcgagagt ttcatactgt ttttctgtag gccgtgtacc taaatgtact tttgctccat
     5341 cgcgatgact tagtaaagca catctaaaac ttttagcgtt attacgtaaa aaatcttgcc
     5401 agctttcccc ttctaaaggg caaaagtgag tatggtgcct atctaacatc tcaatggcta
     5461 aggcgtcgag caaagcccgc ttatttttta catgccaata caatgtaggc tgctctacac
     5521 ctagcttctg ggcgagttta cgggttgtta aaccttcgat tccgacctca ttaagcagct
     5581 ctaatgcgct gttaatcact ttacttttat ctaatctaga catcattaat tcctaatttt
     5641 tgttgacact ctatcgttga tagagttatt ttaccactcc ctatcagtga tagagaaaag
     5701 aattcaaaag atcttttaag aaggagatat acatatgcat catcatcatc atcatcatgg
     5761 agaaagcttg tttaaggggc cgcgtgatta caacccgata tcgagcacca tttgtcattt
     5821 gacgaatgaa tctgatgggc acacaacatc gttgtatggt attggatttg gtcccttcat
     5881 cattacaaac aagcacttgt ttagaagaaa taatggaaca ctgttggtcc aatcactaca
     5941 tggtgtattc aaggtcaaga acaccacgac tttgcaacaa cacctcattg atgggaggga
     6001 catgataatt attcgcatgc ctaaggattt cccaccattt cctcaaaagc tgaaatttag
     6061 agagccacaa agggaagagc gcatatgtct tgtgacaacc aacttccaaa ctaagagcat
     6121 gtctagcatg gtgtcagaca ctagttgcac attcccttca tctgatggca tattctggaa
     6181 gcattggatt caaaccaagg atgggcagtg tggcagtcca ttagtatcaa ctagagatgg
     6241 gttcattgtt ggtatacact cagcatcgaa tttcaccaac acaaacaatt atttcacaag
     6301 cgtgccgaaa aacttcatgg aattgttgac aaatcaggag gcgcagcagt gggttagtgg
     6361 ttggcgatta aatgctgact cagtattgtg ggggggccat aaagttttca tggtgaaacc
     6421 tgaagagcct tttcagccag ttaaggaagc gactcaactc atgaatcgtc gtcgccgtcg
     6481 ctaagtccat cagcttgtcc agcagggttg tccacgggcc gagcgaagcg agccagccgg
     6541 tggccgcgga acaaaactaa agcgccacaa gggcgcttta gtttgttttc agtccagtta
     6601 cgctggagtc tgaggctcgt cctgaatgat atcaagcttg aattcgcggc cgcttctaga
     6661 gttgacagct agctcagtcc taggtactgt gctagctact agatatcgct caatactgac
     6721 caaggaggta tctacaatg
//
