LOCUS       sequence-176811-        6741 bp    DNA     circular SYN 15-APR-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 6741)
  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 6741)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 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..6741
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(1..816)
                     /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     724..743
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     CDS             complement(913..1575)
                     /codon_start=1
                     /product="replication protein for the broad-host-range 
                     plasmid pBBR1 from Bordetella bronchiseptica"
                     /label=pBBR1 Rep
                     /translation="MATQSREIGIQAKNKPGHWVQTERKAHEAWAGLIARKPTAAMLLH
                     HLVAQMGHQNAVVVSQKTLSKLIGRSLRTVQYAVKDLVAERWISVVKLNGPGTVSAYVV
                     NDRVAWGQPRDQLRLSVFSAAVVVDHDDQDESLLGHGDLRRIPTLYPGEQQLPTGPGEE
                     PPSQPGIPGMEPDLPALTETEEWERRGQQRLPMPDEPCFLDDGEPLEPPTRVTLPRR"
     terminator      complement(2233..2304)
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     CDS             complement(2349..2975)
                     /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     2880..2899
                     /label=Tet-R
                     /note="Tetracycline resistance gene, reverse primer"
     promoter        2991..3046
                     /gene="tetR"
                     /label=tetR/tetA promoters
                     /note="overlapping promoters for bacterial tetR and tetA"
     protein_bind    3027..3045
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     CDS             3087..3107
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=7xHis
                     /translation="HHHHHHH"
     CDS             3108..3815
                     /codon_start=1
                     /product="tobacco etch virus protease (Kapust et al., 
                     2001)"
                     /label=TEV protease
                     /translation="GESLFKGPRDYNPISSTICHLTNESDGHTTSLYGIGFGPFIITNK
                     HLFRRNNGTLLVQSLHGVFKVKNTTTLQQHLIDGRDMIIIRMPKDFPPFPQKLKFREPQ
                     REERICLVTTNFQTKSMSSMVSDTSCTFPSSDGIFWKHWIQTKDGQCGSPLVSTRDGFI
                     VGIHSASNFTNTNNYFTSVPKNFMELLTNQEAQQWVSGWRLNADSVLWGGHKVFMVKPE
                     EPFQPVKEATQLMN"
     terminator      3902..3938
                     /label=soxR terminator
                     /note="bidirectional E. coli soxR transcription terminator"
     misc_feature    3989..4010
                     /label=BioBrick prefix
                     /note="BioBrick prefix for parts that do not start with 
                     ""ATG"""
     promoter        4011..4045
                     /label=BBa_J23102
                     /note="Anderson constitutive promoter 
                     (https://parts.igem.org/Promoters/Catalog/Anderson). 
                     Measured strength relative to BBa_J223100: 0.86"
     CDS             4089..4106
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             4524..4544
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQS"
     CDS             6633..6656
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     terminator      6694..6725
                     /label=tonB terminator
                     /note="bidirectional E. coli tonB-P14 transcription 
                     terminator"
ORIGIN
        1 tcagaaaaac tcatcgagca tcaagtgaaa ctgcaattta ttcatatcag gattatcaat
       61 accatatttt tgaaaaagcc gtttctgtaa tgaaggagaa aactcaccga ggcagttcca
      121 taggatggca agatcctggt atcggtctgc gattccgact cgtccaacat caatacaacc
      181 tattaatttc ccctcgtcaa aaataaggtt atcaagtgag aaatcaccat gagtgacgac
      241 tgaatccggt gagaatggca aaagcttatg catttctttc cagacttgtt caacaggcca
      301 gccattacgc tcgtcatcaa aatcactcgc accaaccaaa ccgttattca ttcgtgattg
      361 cgcctgagcg agacgaaata cgcgatcgcc gttaaaagga caattacaaa caggaatcga
      421 atgcaaccgg cgcaggaaca ctgccagcgc atcaacaata ttttcacctg aatcaggata
      481 ttcttctaat acctggaatg ctgttttccc tgggatcgca gtggtgagta accatgcatc
      541 atcaggagta cggataaaat gcttgatggt cggaagaggc ataaattccg tcagccagtt
      601 tagcctgacc atctcatctg taacatcatt ggcaacgcta cctttgccat gtttcagaaa
      661 caactctggc gcatcgggct tcccatacaa tcgatagatt gtcgcacctg attgcccgac
      721 attatcgcga gcccatttat acccatataa atcagcatcc atgttggaat ttaatcgcgg
      781 cctcgagcaa gacgtttccc gttgaatatg gctcatttta gcttccttag ctcctgaaaa
      841 tctcgataac tcaaaaaata cgcccggtag tgatcttatt tcattatggt gaaagttgga
      901 acctcttacg tactaccggc gcggcagcgt gacccgtgtc ggcggctcca acggctcgcc
      961 atcgtccaga aaacacggct catcgggcat cggcaggcgc tgctgcccgc gccgttccca
     1021 ttcctccgtt tcggtcaagg ctggcaggtc tggttccatg cccggaatgc cgggctggct
     1081 gggcggctcc tcgccggggc cggtcggtag ttgctgctcg cccggataca gggtcgggat
     1141 gcggcgcagg tcgccatgcc ccaacagcga ttcgtcctgg tcgtcgtgat caaccaccac
     1201 ggcggcactg aacaccgaca ggcgcaactg gtcgcggggc tggccccacg ccacgcggtc
     1261 attgaccacg taggccgaca cggtgccggg gccgttgagc ttcacgacgg agatccagcg
     1321 ctcggccacc aagtccttga ctgcgtattg gaccgtccgc aaagaacgtc cgatgagctt
     1381 ggaaagtgtc ttctggctga ccaccacggc gttctggtgg cccatctgcg ccacgaggtg
     1441 atgcagcagc attgccgccg tgggtttcct cgcaataagc ccggcccacg cctcatgcgc
     1501 tttgcgttcc gtttgcaccc agtgaccggg cttgttcttg gcttgaatgc cgatttctct
     1561 ggactgcgtg gccatgctta tctccatgcg gtagggtgcc gcacggttgc ggcaccatgc
     1621 gcaatcagct gcaacttttc ggcagcgcga caacaattat gcgttgcgta aaagtggcag
     1681 tcaattacag attttcttta acctacgcaa tgagctattg cggggggtgc cgcaatgagc
     1741 tgttgcgtac cccccttttt taagttgttg atttttaagt ctttcgcatt tcgccctata
     1801 tctagttctt tggtgcccaa agaagggcac ccctgcgggg ttcccccacg ccttcggcgc
     1861 ggctccccct ccggcaaaaa gtggcccctc cggggcttgt tgatcgactg cgcggccttc
     1921 ggccttgccc aaggtggcgc tgcccccttg gaacccccgc actcgccgcc gtgaggctcg
     1981 gggggcaggc gggcgggctt cgccttcgac tgcccccact cgcataggct tgggtcgttc
     2041 caggcgcgtc aaggccaagc cgctgcgcgg tcgctgcgcg agccttgacc cgccttccac
     2101 ttggtgtcca accggcaagc gaagcgcgca ggccgcaggc cggaggcttt tccccagaga
     2161 aaattaaaaa aattgatggg gcaaggccgc aggccgcgca gttggagccg gtgggtatgt
     2221 ggtcgaaggc tgggtagcgt tcaccgacaa acaacagata aaacgaaagg cccagtcttt
     2281 cgactgagcc tttcgtttta tttgatgcct ggctctagta gtgatctaca ctagcactat
     2341 cagtgttatt aagacccact ttcacattta agttgttttt ctaatccgca tatgatcaat
     2401 tcaaggccga ataagaaggc tggctctgca ccttggtgat caaataattc gatagcttgt
     2461 cgtaataatg gcggcatact atcagtagta ggtgtttccc tttcttcttt agcgacttga
     2521 tgctcttgat cttccaatac gcaacctaaa gtaaaatgcc ccacagcgct gagtgcatat
     2581 aatgcattct ctagtgaaaa accttgttgg cataaaaagg ctaattgatt ttcgagagtt
     2641 tcatactgtt tttctgtagg ccgtgtacct aaatgtactt ttgctccatc gcgatgactt
     2701 agtaaagcac atctaaaact tttagcgtta ttacgtaaaa aatcttgcca gctttcccct
     2761 tctaaagggc aaaagtgagt atggtgccta tctaacatct caatggctaa ggcgtcgagc
     2821 aaagcccgct tattttttac atgccaatac aatgtaggct gctctacacc tagcttctgg
     2881 gcgagtttac gggttgttaa accttcgatt ccgacctcat taagcagctc taatgcgctg
     2941 ttaatcactt tacttttatc taatctagac atcattaatt cctaattttt gttgacactc
     3001 tatcgttgat agagttattt taccactccc tatcagtgat agagaaaaga attcaaaaga
     3061 tcttttaaga aggagatata catatgcatc atcatcatca tcatcatgga gaaagcttgt
     3121 ttaaggggcc gcgtgattac aacccgatat cgagcaccat ttgtcatttg acgaatgaat
     3181 ctgatgggca cacaacatcg ttgtatggta ttggatttgg tcccttcatc attacaaaca
     3241 agcacttgtt tagaagaaat aatggaacac tgttggtcca atcactacat ggtgtattca
     3301 aggtcaagaa caccacgact ttgcaacaac acctcattga tgggagggac atgataatta
     3361 ttcgcatgcc taaggatttc ccaccatttc ctcaaaagct gaaatttaga gagccacaaa
     3421 gggaagagcg catatgtctt gtgacaacca acttccaaac taagagcatg tctagcatgg
     3481 tgtcagacac tagttgcaca ttcccttcat ctgatggcat attctggaag cattggattc
     3541 aaaccaagga tgggcagtgt ggcagtccat tagtatcaac tagagatggg ttcattgttg
     3601 gtatacactc agcatcgaat ttcaccaaca caaacaatta tttcacaagc gtgccgaaaa
     3661 acttcatgga attgttgaca aatcaggagg cgcagcagtg ggttagtggt tggcgattaa
     3721 atgctgactc agtattgtgg gggggccata aagttttcat ggtgaaacct gaagagcctt
     3781 ttcagccagt taaggaagcg actcaactca tgaatcgtcg tcgccgtcgc taagtccatc
     3841 agcttgtcca gcagggttgt ccacgggccg agcgaagcga gccagccggt ggccgcggaa
     3901 caaaactaaa gcgccacaag ggcgctttag tttgttttca gtccagttac gctggagtct
     3961 gaggctcgtc ctgaatgata tcaagcttga attcgcggcc gcttctagag ttgacagcta
     4021 gctcagtcct aggtactgtg ctagctacta gatatcgctc aatactgacc aaggaggtat
     4081 ctacaatgca tcaccatcat caccatgttg atctgcgtac cctgggttat agccagcagc
     4141 agcaagaaaa aatcaaaccg aaagttcgta gcaccgttgc acagcatcat gaagcactgg
     4201 ttggtcatgg ttttacccat gcacatattg ttgcactgag ccagcatccg gcagcactgg
     4261 gcaccgttgc agttaaatat caggatatga ttgcagcact gccggaagca acccatgaag
     4321 caattgttgg tgttggtaaa cagtggtcag gtgcacgtgc cctggaagca ctgctgaccg
     4381 ttgccggtga actgcgtggt ccgcctctgc agctggatac cggtcagctg ctgaaaattg
     4441 caaaacgtgg tggtgttacc gcagttgaag cagttcatgc atggcgtaat gcactgaccg
     4501 gtgcaccgct gaatggttct ggcgaaaacc tgtacttcca gtctggctcg ggtctgacac
     4561 cggaacaggt tgttgcaatt gccagcaatg gtggtggcaa acaggcactg gaaaccgttc
     4621 agcgtctgct gccggttctg tgtcaggcac atggtctgac ccctgaacag gtggtggcca
     4681 ttgcaagcaa taatggcggt aaacaagccc tggaaacagt gcagcgcctg ttaccggtgc
     4741 tgtgccaggc ccatggctta actccggaac aggtggtagc gatcgcatca aatggcggag
     4801 gtaaacaggc cttagaaacc gtacagcgct tactgccggt gttatgccag gcgcacggcc
     4861 tgacgccaga acaggtagtg gcaatcgcct caaataatgg tggaaaacag gcgttagaga
     4921 cagtccagcg cctgctgcct gtattatgtc aagcccatgg cctgacccca gagcaagttg
     4981 ttgcgattgc aagtaacatt gggggtaaac aggcacttga gacagttcaa cgtttactgc
     5041 ctgtactgtg ccaagctcac ggtctgactc cggaacaagt cgtcgcgatt gcgagtaata
     5101 acggtggcaa acaagcatta gaaacggtgc aacgcctgct gccagttctt tgccaggctc
     5161 acggtttaac ccctgagcag gttgtagcta ttgcgagtca tgatggtggt aagcaggcgt
     5221 tggaaactgt gcaaagactg ctgcccgtgt tgtgccaagc acatggttta accccagaac
     5281 aagtcgtagc aatcgcaagc aatcatggtg gcaagcaagc gcttgaaaca gtacagcgtt
     5341 tattaccggt actttgtcag gcccacggtc ttacaccaga acaagttgtg gccatagcca
     5401 gtaacaatgg cggaaagcag gctctggaaa cggtacaacg tctgttacct gttctgtgtc
     5461 aagcgcacgg attaacacct gaacaagtag ttgccattgc gtcaaatatc ggaggcaagc
     5521 aggccttgga gacagtgcag agattactgc cagtgttgtg tcaggctcat ggccttacac
     5581 ccgagcaggt cgtggcaatt gcatctaatg gtggcggtaa gcaagcttta gagactgttc
     5641 agagactgct tcctgtcctg tgccaggcac acggacttac gcctgagcaa gtggttgcaa
     5701 tcgcctctaa catcggtggt aagcaagcac tggaaactgt ccaacgctta cttccggtgc
     5761 tttgtcaagc acacggctta acgccagagc aggtcgtcgc catagccagc aatatcggtg
     5821 gtaaacaggc ccttgaaacg gtccaaagac ttctgccggt cctttgccaa gcgcatgggc
     5881 tgacacctga gcaggtagtc gcgattgcct cacatgacgg tgggaagcag gcattagaaa
     5941 cagttcaaag attattacca gtcctgtgtc aggcgcatgg gttaacccca gagcaggtag
     6001 ttgcaatagc atccaacatt ggcggaaaac aagcgttgga aacggttcag cggttattgc
     6061 ctgttttgtg ccaggcgcat ggtttgacac ccgagcaagt ggtagccata gcctcaaata
     6121 tagggggtaa acaagctttg gagacagtac aacggctgct tccagtttta tgtcaggccc
     6181 atggattgac gcctgaacaa gttgtcgcta tcgcaagtaa tgggggtggt aaacaagcgc
     6241 ttgaaaccgt tcaacgcctt ctgcctgtgc tttgtcaggc acatggatta acacccgaac
     6301 aggttgtcgc gatagcttca aacggtggtg gtcgtccggc actggaaagc attgttgcac
     6361 agctgagccg tccggatccg gcactggcag cactgaccaa tgatcatctg gttgcactgg
     6421 catgtctggg tggtcgccct gccctggatg cagttaaaaa aggtctgccg catgcaccgg
     6481 cactgattaa acgtaccaat cgtcgtattc cggaacgtac cagccatcgt gttgcagatc
     6541 atgcacaggt tgttcgtgtt ctgggttttt ttcagtgtca tagccatccg gcacaggcat
     6601 ttgatgatgc aatgacccag tttggtatga gcgattataa agatgatgat gataaataag
     6661 gtaccctaat catacctgac ctccatagca gaaagtcaaa aacctccgac cggaggcttt
     6721 tgacttgagg gggatccgcc c
//
