LOCUS       sequence-181444-        7147 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses TALE targeted to lysA promoter 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 7147)
  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 7147)
  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: p5B3-T
FEATURES             Location/Qualifiers
     source          1..7147
                     /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             2953..2976
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     terminator      3014..3045
                     /label=tonB terminator
                     /note="bidirectional E. coli tonB-P14 transcription 
                     terminator"
     CDS             complement(3062..3877)
                     /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     3785..3804
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     CDS             complement(3967..4632)
                     /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(5292..5363)
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     CDS             complement(5408..6034)
                     /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     5939..5958
                     /label=Tet-R
                     /note="Tetracycline resistance gene, reverse primer"
     promoter        6050..6105
                     /gene="tetR"
                     /label=tetR/tetA promoters
                     /note="overlapping promoters for bacterial tetR and tetA"
     protein_bind    6086..6104
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     CDS             6146..6166
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=7xHis
                     /translation="HHHHHHH"
     CDS             6167..6874
                     /codon_start=1
                     /product="tobacco etch virus protease (Kapust et al., 
                     2001)"
                     /label=TEV protease
                     /translation="GESLFKGPRDYNPISSTICHLTNESDGHTTSLYGIGFGPFIITNK
                     HLFRRNNGTLLVQSLHGVFKVKNTTTLQQHLIDGRDMIIIRMPKDFPPFPQKLKFREPQ
                     REERICLVTTNFQTKSMSSMVSDTSCTFPSSDGIFWKHWIQTKDGQCGSPLVSTRDGFI
                     VGIHSASNFTNTNNYFTSVPKNFMELLTNQEAQQWVSGWRLNADSVLWGGHKVFMVKPE
                     EPFQPVKEATQLMN"
     terminator      6961..6997
                     /label=soxR terminator
                     /note="bidirectional E. coli soxR transcription terminator"
     misc_feature    7048..7069
                     /label=BioBrick prefix
                     /note="BioBrick prefix for parts that do not start with 
                     ""ATG"""
     promoter        7070..7104
                     /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 ttgccagcca tgatggtggc aaacaggcac tggaaaccgt tcagcgtctg
      541 ctgccggttc tgtgtcaggc acatggtctg acccctgaac aggtggtggc cattgcaagc
      601 aatggtggcg gtaaacaagc cctggaaaca gtgcagcgcc tgttaccggt gctgtgccag
      661 gcccatggct taactccgga acaggtggta gcgatcgcat caaatggcgg aggtaaacag
      721 gccttagaaa ccgtacagcg cttactgccg gtgttatgcc aggcgcacgg cctgacgcca
      781 gaacaggtag tggcaatcgc ctcaaatggc ggtggaaaac aggcgttaga gacagtccag
      841 cgcctgctgc ctgtattatg tcaagcccat ggcctgaccc cagagcaagt tgttgcgatt
      901 gcaagtaatg gtgggggtaa acaggcactt gagacagttc aacgtttact gcctgtactg
      961 tgccaagctc acggtctgac tccggaacaa gtcgtcgcga ttgcgagtaa cggtggtggc
     1021 aaacaagcat tagaaacggt gcaacgcctg ctgccagttc tttgccaggc tcacggttta
     1081 acccctgagc aggttgtagc tattgcgagt aacattggtg gtaagcaggc gttggaaact
     1141 gtgcaaagac tgctgcccgt gttgtgccaa gcacatggtt taaccccaga acaagtcgta
     1201 gcaatcgcaa gcaatggggg 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 aatcatggtg gtaagcaagc actggaaact gtccaacgct tacttccggt gctttgtcaa
     1681 gcacacggct taacgccaga gcaggtcgtc gccatagcca gcaacggtgg tggtaaacag
     1741 gcccttgaaa cggtccaaag acttctgccg gtcctttgcc aagcgcatgg gctgacacct
     1801 gagcaggtag tcgcgattgc ctcaaataac ggtgggaagc aggcattaga aacagttcaa
     1861 agattattac cagtcctgtg tcaggcgcat gggttaaccc cagagcaggt agttgcaata
     1921 gcatccaatc atggcggaaa acaagcgttg gaaacggttc agcggttatt gcctgttttg
     1981 tgccaggcgc atggtttgac acccgagcaa gtggtagcca tagcctcaca tgatgggggt
     2041 aaacaagctt tggagacagt acaacggctg cttccagttt tatgtcaggc ccatggattg
     2101 acgcctgaac aagttgtcgc tatcgcaagt aataatggtg gtaaacaagc gcttgaaacc
     2161 gttcaacgcc ttctgcctgt gctttgtcag gcacatggac ttacgcctga gcaagtggtt
     2221 gcaatcgcct ctaacggtgg tggtaagcaa gcactggaaa ctgtccaacg cttacttccg
     2281 gtgctttgtc aagcacacgg cctgacaccg gaacaggttg ttgcaattgc cagcaatata
     2341 ggtggcaaac aggcactgga aaccgttcag cgtctgctgc cggttctgtg tcaggcacat
     2401 ggtctgacac ctgagcaggt agtcgcgatt gcctcaaaca tcggtgggaa gcaggcatta
     2461 gaaacagttc aaagattatt accagtcctg tgtcaggcgc atgggctgac tccggaacaa
     2521 gtcgtcgcga ttgcgagtaa tggtggtggc aaacaagcat tagaaacggt gcaacgcctg
     2581 ctgccagttc tttgccaggc tcacggttta acacccgaac aggttgtcgc gatagcttca
     2641 catgatggtg gtcgtccggc actggaaagc attgttgcac agctgagccg tccggatccg
     2701 gcactggcag cactgaccaa tgatcatctg gttgcactgg catgtctggg tggtcgccct
     2761 gccctggatg cagttaaaaa aggtctgccg catgcaccgg cactgattaa acgtaccaat
     2821 cgtcgtattc cggaacgtac cagccatcgt gttgcagatc atgcacaggt tgttcgtgtt
     2881 ctgggttttt ttcagtgtca tagccatccg gcacaggcat ttgatgatgc aatgacccag
     2941 tttggtatga gcgattataa agatgatgat gataaataag gtaccctaat catacctgac
     3001 ctccatagca gaaagtcaaa aacctccgac cggaggcttt tgacttgagg gggatccgcc
     3061 ctcagaaaaa ctcatcgagc atcaagtgaa actgcaattt attcatatca ggattatcaa
     3121 taccatattt ttgaaaaagc cgtttctgta atgaaggaga aaactcaccg aggcagttcc
     3181 ataggatggc aagatcctgg tatcggtctg cgattccgac tcgtccaaca tcaatacaac
     3241 ctattaattt cccctcgtca aaaataaggt tatcaagtga gaaatcacca tgagtgacga
     3301 ctgaatccgg tgagaatggc aaaagcttat gcatttcttt ccagacttgt tcaacaggcc
     3361 agccattacg ctcgtcatca aaatcactcg caccaaccaa accgttattc attcgtgatt
     3421 gcgcctgagc gagacgaaat acgcgatcgc cgttaaaagg acaattacaa acaggaatcg
     3481 aatgcaaccg gcgcaggaac actgccagcg catcaacaat attttcacct gaatcaggat
     3541 attcttctaa tacctggaat gctgttttcc ctgggatcgc agtggtgagt aaccatgcat
     3601 catcaggagt acggataaaa tgcttgatgg tcggaagagg cataaattcc gtcagccagt
     3661 ttagcctgac catctcatct gtaacatcat tggcaacgct acctttgcca tgtttcagaa
     3721 acaactctgg cgcatcgggc ttcccataca atcgatagat tgtcgcacct gattgcccga
     3781 cattatcgcg agcccattta tacccatata aatcagcatc catgttggaa tttaatcgcg
     3841 gcctcgagca agacgtttcc cgttgaatat ggctcatttt agcttcctta gctcctgaaa
     3901 atctcgataa ctcaaaaaat acgcccggta gtgatcttat ttcattatgg tgaaagttgg
     3961 aacctcttac gtacggcgcg gcagcgtgac ccgtgtcggc ggctccaacg gctcgccatc
     4021 gtccagaaaa cacggctcat cgggcatcgg caggcgctgc tgcccgcgtc gttcccattc
     4081 ctccgtttcg gtcaaggctg gcaggtctgg ttccatgccc ggaatgccgg gctggctggg
     4141 cggctcctcg ccggggccgg tcggtagttg ctgctcgccc ggatacaggg tcgggatgcg
     4201 gcgcaggtcg ccatgcccca acagcgattc gtcctggtcg tcgtgatcaa ccaccacggc
     4261 ggcactgaac accgacaggc gcaactggtc gcggggctgg ccccacgcca cgcggtcatt
     4321 gaccacgtag gccgacacgg tgccggggcc gttgagcttc acgacggaga tccagcgctc
     4381 ggccaccaag tccttgactg cgtattggac cgtccgcaaa gaacgtccga tgagcttgga
     4441 aagtgtcttc tggctgacca ccacggcgtt ctggtggccc atctgcgcca cgaggtgatg
     4501 cagcagcatt gccgccgtgg gtttcctcgc aataagcccg gcccacgcct catgcgcttt
     4561 gcgttccgtt tgcacccagt gaccgggctt gttcttggct tgaatgccga tttctctgga
     4621 ctgcgtggcc atgcttatct ccatgcggta ggggtgccgc acggttgcgg caccatgcgc
     4681 aatcagctgc aacttttcgg cagcgcgaca acaattatgc gttgcgtaaa agtggcagtc
     4741 aattacagat tttctttaac ctacgcaatg agctattgcg gggggtgccg caatgagctg
     4801 ttgcgtaccc ccctttttta agttgttgat ttttaagtct ttcgcatttc gccctatatc
     4861 tagttctttg gtgcccaaag aagggcaccc ctgcggggtt cccccacgcc ttcggcgcgg
     4921 ctccccctcc ggcaaaaagt ggcccctccg gggcttgttg atcgactgcg cggccttcgg
     4981 ccttgcccaa ggtggcgctg cccccttgga acccccgcac tcgccgccgt gaggctcggg
     5041 gggcaggcgg gcgggcttcg cccttcgact gcccccactc gcataggctt gggtcgttcc
     5101 aggcgcgtca aggccaagcc gctgcgcggt cgctgcgcga gccttgaccc gccttccact
     5161 tggtgtccaa ccggcaagcg aagcgcgcag gccgcaggcc ggaggctttt ccccagagaa
     5221 aattaaaaaa attgatgggg caaggccgca ggccgcgcag ttggagccgg tgggtatgtg
     5281 gtcgaaggct gggtagcgtt caccgacaaa caacagataa aacgaaaggc ccagtctttc
     5341 gactgagcct ttcgttttat ttgatgcctg gctctagtag tgatctacac tagcactatc
     5401 agtgttatta agacccactt tcacatttaa gttgtttttc taatccgcat atgatcaatt
     5461 caaggccgaa taagaaggct ggctctgcac cttggtgatc aaataattcg atagcttgtc
     5521 gtaataatgg cggcatacta tcagtagtag gtgtttccct ttcttcttta gcgacttgat
     5581 gctcttgatc ttccaatacg caacctaaag taaaatgccc cacagcgctg agtgcatata
     5641 atgcattctc tagtgaaaaa ccttgttggc ataaaaaggc taattgattt tcgagagttt
     5701 catactgttt ttctgtaggc cgtgtaccta aatgtacttt tgctccatcg cgatgactta
     5761 gtaaagcaca tctaaaactt ttagcgttat tacgtaaaaa atcttgccag ctttcccctt
     5821 ctaaagggca aaagtgagta tggtgcctat ctaacatctc aatggctaag gcgtcgagca
     5881 aagcccgctt attttttaca tgccaataca atgtaggctg ctctacacct agcttctggg
     5941 cgagtttacg ggttgttaaa ccttcgattc cgacctcatt aagcagctct aatgcgctgt
     6001 taatcacttt acttttatct aatctagaca tcattaattc ctaatttttg ttgacactct
     6061 atcgttgata gagttatttt accactccct atcagtgata gagaaaagaa ttcaaaagat
     6121 cttttaagaa ggagatatac atatgcatca tcatcatcat catcatggag aaagcttgtt
     6181 taaggggccg cgtgattaca acccgatatc gagcaccatt tgtcatttga cgaatgaatc
     6241 tgatgggcac acaacatcgt tgtatggtat tggatttggt cccttcatca ttacaaacaa
     6301 gcacttgttt agaagaaata atggaacact gttggtccaa tcactacatg gtgtattcaa
     6361 ggtcaagaac accacgactt tgcaacaaca cctcattgat gggagggaca tgataattat
     6421 tcgcatgcct aaggatttcc caccatttcc tcaaaagctg aaatttagag agccacaaag
     6481 ggaagagcgc atatgtcttg tgacaaccaa cttccaaact aagagcatgt ctagcatggt
     6541 gtcagacact agttgcacat tcccttcatc tgatggcata ttctggaagc attggattca
     6601 aaccaaggat gggcagtgtg gcagtccatt agtatcaact agagatgggt tcattgttgg
     6661 tatacactca gcatcgaatt tcaccaacac aaacaattat ttcacaagcg tgccgaaaaa
     6721 cttcatggaa ttgttgacaa atcaggaggc gcagcagtgg gttagtggtt ggcgattaaa
     6781 tgctgactca gtattgtggg ggggccataa agttttcatg gtgaaacctg aagagccttt
     6841 tcagccagtt aaggaagcga ctcaactcat gaatcgtcgt cgccgtcgct aagtccatca
     6901 gcttgtccag cagggttgtc cacgggccga gcgaagcgag ccagccggtg gccgcggaac
     6961 aaaactaaag cgccacaagg gcgctttagt ttgttttcag tccagttacg ctggagtctg
     7021 aggctcgtcc tgaatgatat caagcttgaa ttcgcggccg cttctagagt tgacagctag
     7081 ctcagtccta ggtactgtgc tagctactag atatcgctca atactgacca aggaggtatc
     7141 tacaatg
//
