LOCUS       sequence-173059-        6483 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  mWasabi reporter gene gated by temperature-sensitive TetR A89D.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6483)
  AUTHORS   Piraner DI, Abedi MH, Moser BA, Lee-Gosselin A, Shapiro MG
  TITLE     Tunable thermal bioswitches for in vivo control of microbial 
            therapeutics.
  JOURNAL   Nat Chem Biol. 2017 Jan;13(1):75-80. doi: 10.1038/nchembio.2233. 
            Epub 2016 Nov 14.
  PUBMED    27842069
REFERENCE   2  (bases 1 to 6483)
  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.2233"; journalName: "Nat Chem Biol"; date: 
            "2017-01"; volume: "13"; issue: "1"; pages: "75-80"
COMMENT     Sequence Label: pTetR89-Wasabi
FEATURES             Location/Qualifiers
     source          1..6483
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             1..714
                     /codon_start=1
                     /product="green variant of teal fluorescent protein mTFP1 
                     (Ai et al., 2008)"
                     /label=mWasabi
                     /translation="MVSKGEETTMGVIKPDMKIKLKMEGNVNGHAFVIEGEGEGKPYDG
                     TNTINLEVKEGAPLPFSYDILTTAFSYGNRAFTKYPDDIPNYFKQSFPEGYSWERTMTF
                     EDKGIVKVKSDISMEEDSFIYEIHLKGENFPPNGPVMQKETTGWDASTERMYVRDGVLK
                     GDVKMKLLLEGGGHHRVDFKTIYRAKKAVKLPDYHFVDHRIEILNHDKDYNKVTVYEIA
                     VARNSTDGMDELYKG"
     CDS             727..771
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     primer_bind     complement(809..827)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      823..870
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      907..1362
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(994..1013)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1204..1225
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(join(1477..2268,2269..2337))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     2100..2119
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2338..2430)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      2511..3099
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3000..3019
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3253..3270
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3285..3424
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3350..3369)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3510..3532
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3526..3717)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4289..5371)
                     /codon_start=1
                     /gene="lacI"
                     /product="lac repressor"
                     /label=lacI
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
                     /translation="MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     primer_bind     5333..5352
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5372..5449)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     5655..5674
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     CDS             complement(5740..6363)
                     /codon_start=1
                     /gene="tetR from transposon Tn10"
                     /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="MSRLDKSKVINSALELLNEVGIEGLTTRKLAQKLGVEQPTLYWHV
                     KNKRALLDALAIEMLDRHHTHFCPLEGESWQDFLRNNAKSFRCDLLSHRDGAKVHLGTR
                     PTEKQYETLENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEDQEHQVAKEERETPTT
                     DSMPPLLRQAIELFDHQGAEPAFLFGLELIICGLEKQLKCESGS"
     primer_bind     6271..6290
                     /label=Tet-R
                     /note="Tetracycline resistance gene, reverse primer"
     promoter        6379..6434
                     /gene="tetR"
                     /label=tetR/tetA promoters
                     /note="overlapping promoters for bacterial tetR and tetA"
     protein_bind    6385..6403
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O1 for the tetR and tetA genes"
     protein_bind    6415..6433
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     CDS             6454..6471
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
ORIGIN
        1 atggtgagca agggcgagga gaccacaatg ggcgtaatca agcccgacat gaagatcaag
       61 ctgaagatgg agggcaacgt gaatggccac gccttcgtga tcgagggcga gggcgagggc
      121 aagccctacg acggcaccaa caccatcaac ctggaggtga aggagggagc ccccctgccc
      181 ttctcctacg acattctgac caccgcgttc agttacggca acagggcctt caccaagtac
      241 cccgacgaca tccccaacta cttcaagcag tccttccccg agggctactc ttgggagcgc
      301 accatgacct tcgaggacaa gggcatcgtg aaggtgaagt ccgacatctc catggaggag
      361 gactccttca tctacgagat acacctcaag ggcgagaact tcccccccaa cggccccgtg
      421 atgcagaagg agaccaccgg ctgggacgcc tccaccgaga ggatgtacgt gcgcgacggc
      481 gtgctgaagg gcgacgtcaa gatgaagctg ctgctggagg gcggcggcca ccaccgcgtt
      541 gacttcaaga ccatctacag ggccaagaag gcggtgaagc tgcccgacta tcactttgtg
      601 gaccaccgca tcgagatcct gaaccacgac aaggactaca acaaggtgac cgtttacgag
      661 atcgccgtgg cccgcaactc caccgacggc atggacgagc tgtacaaggg ctgactcgag
      721 tctggtaaag aaaccgctgc tgcgaaattt gaacgccagc acatggactc gtctactagc
      781 gcagcttaat taacctaggc tgctgccacc gctgagcaat aactagcata accccttggg
      841 gcctctaaac gggtcttgag gggttttttg ctgaaaggag gaactatatc cggattggcg
      901 aatgggacgc gccctgtagc ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg
      961 tgaccgctac acttgccagc gccctagcgc ccgctccttt cgctttcttc ccttcctttc
     1021 tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg ggggctccct ttagggttcc
     1081 gatttagtgc tttacggcac ctcgacccca aaaaacttga ttagggtgat ggttcacgta
     1141 gtgggccatc gccctgatag acggtttttc gccctttgac gttggagtcc acgttcttta
     1201 atagtggact cttgttccaa actggaacaa cactcaaccc tatctcggtc tattcttttg
     1261 atttataagg gattttgccg atttcggcct attggttaaa aaatgagctg atttaacaaa
     1321 aatttaacgc gaattttaac aaaatattaa cgtttacaat ttctggcggc acgatggcat
     1381 gagattatca aaaaggatct tcacctagat ccttttaaat taaaaatgaa gttttaaatc
     1441 aatctaaagt atatatgagt aaacttggtc tgacagttac caatgcttaa tcagtgaggc
     1501 acctatctca gcgatctgtc tatttcgttc atccatagtt gcctgactcc ccgtcgtgta
     1561 gataactacg atacgggagg gcttaccatc tggccccagt gctgcaatga taccgcgaga
     1621 cccacgctca ccggctccag atttatcagc aataaaccag ccagccggaa gggccgagcg
     1681 cagaagtggt cctgcaactt tatccgcctc catccagtct attaattgtt gccgggaagc
     1741 tagagtaagt agttcgccag ttaatagttt gcgcaacgtt gttgccattg ctacaggcat
     1801 cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc tccggttccc aacgatcaag
     1861 gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat
     1921 cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg gttatggcag cactgcataa
     1981 ttctcttact gtcatgccat ccgtaagatg cttttctgtg actggtgagt actcaaccaa
     2041 gtcattctga gaatagtgta tgcggcgacc gagttgctct tgcccggcgt caatacggga
     2101 taataccgcg ccacatagca gaactttaaa agtgctcatc attggaaaac gttcttcggg
     2161 gcgaaaactc tcaaggatct taccgctgtt gagatccagt tcgatgtaac ccactcgtgc
     2221 acccaactga tcttcagcat cttttacttt caccagcgtt tctgggtgag caaaaacagg
     2281 aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa tactcatact
     2341 cttccttttt caatcatgat tgaagcattt atcagggtta ttgtctcatg agcggataca
     2401 tatttgaatg tatttagaaa aataaacaaa taggtcatga ccaaaatccc ttaacgtgag
     2461 ttttcgttcc actgagcgtc agaccccgta gaaaagatca aaggatcttc ttgagatcct
     2521 ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt
     2581 tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt cagcagagcg
     2641 cagataccaa atactgtcct tctagtgtag ccgtagttag gccaccactt caagaactct
     2701 gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc tgccagtggc
     2761 gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa ggcgcagcgg
     2821 tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac ctacaccgaa
     2881 ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg
     2941 gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga gcttccaggg
     3001 ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact tgagcgtcga
     3061 tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa cgcggccttt
     3121 ttacggttcc tggccttttg ctggcctttt gctcacatgt tctttcctgc gttatcccct
     3181 gattctgtgg ataaccgtat taccgccttt gagtgagctg ataccgctcg ccgcagccga
     3241 acgaccgagc gcagcgagtc agtgagcgag gaagcggaag agcgcctgat gcggtatttt
     3301 ctccttacgc atctgtgcgg tatttcacac cgcaatggtg cactctcagt acaatctgct
     3361 ctgatgccgc atagttaagc cagtatacac tccgctatcg ctacgtgact gggtcatggc
     3421 tgcgccccga cacccgccaa cacccgctga cgcgccctga cgggcttgtc tgctcccggc
     3481 atccgcttac agacaagctg tgaccgtctc cgggagctgc atgtgtcaga ggttttcacc
     3541 gtcatcaccg aaacgcgcga ggcagctgcg gtaaagctca tcagcgtggt cgtgaagcga
     3601 ttcacagatg tctgcctgtt catccgcgtc cagctcgttg agtttctcca gaagcgttaa
     3661 tgtctggctt ctgataaagc gggccatgtt aagggcggtt ttttcctgtt tggtcactga
     3721 tgcctccgtg taagggggat ttctgttcat gggggtaatg ataccgatga aacgagagag
     3781 gatgctcacg atacgggtta ctgatgatga acatgcccgg ttactggaac gttgtgaggg
     3841 taaacaactg gcggtatgga tgcggcggga ccagagaaaa atcactcagg gtcaatgcca
     3901 gcgcttcgtt aatacagatg taggtgttcc acagggtagc cagcagcatc ctgcgatgca
     3961 gatccggaac ataatggtgc agggcgctga cttccgcgtt tccagacttt acgaaacacg
     4021 gaaaccgaag accattcatg ttgttgctca ggtcgcagac gttttgcagc agcagtcgct
     4081 tcacgttcgc tcgcgtatcg gtgattcatt ctgctaacca gtaaggcaac cccgccagcc
     4141 tagccgggtc ctcaacgaca ggagcacgat catgctagtc atgccccgcg cccaccggaa
     4201 ggagctgact gggttgaagg ctctcaaggg catcggtcga gatcccggtg cctaatgagt
     4261 gagctaactt acattaattg cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc
     4321 gtgccagctg cattaatgaa tcggccaacg cgcggggaga ggcggtttgc gtattgggcg
     4381 ccagggtggt ttttcttttc accagtgaga cgggcaacag ctgattgccc ttcaccgcct
     4441 ggccctgaga gagttgcagc aagcggtcca cgctggtttg ccccagcagg cgaaaatcct
     4501 gtttgatggt ggttaacggc gggatataac atgagctgtc ttcggtatcg tcgtatccca
     4561 ctaccgagat gtccgcacca acgcgcagcc cggactcggt aatggcgcgc attgcgccca
     4621 gcgccatctg atcgttggca accagcatcg cagtgggaac gatgccctca ttcagcattt
     4681 gcatggtttg ttgaaaaccg gacatggcac tccagtcgcc ttcccgttcc gctatcggct
     4741 gaatttgatt gcgagtgaga tatttatgcc aaccagccag acgcagacgc gccgagacag
     4801 aacttaatgg gcccgctaac agcgcgattt gctggtgacc caatgcgacc agatgctcca
     4861 cgcccagtcg cgtaccgtct tcatgggaga aaataatact gttgatgggt gtctggtcag
     4921 agacatcaag aaataacgcc ggaacattag tgcaggcagc ttccacagca atggcatcct
     4981 ggtcatccag cggatagtta atgatcagcc cactgacgcg ttgcgcgaga agattgtgca
     5041 ccgccgcttt acaggcttcg acgccgcttc gttctaccat cgacaccacc acgctggcac
     5101 ccagttgatc ggcgcgagat ttaatcgccg cgacaatttg cgacggcgcg tgcagggcca
     5161 gactggaggt ggcaacgcca atcagcaacg actgtttgcc cgccagttgt tgtgccacgc
     5221 ggttgggaat gtaattcagc tccgccatcg ccgcttccac tttttcccgc gttttcgcag
     5281 aaacgtggct ggcctggttc accacgcggg aaacggtctg ataagagaca ccggcatact
     5341 ctgcgacatc gtataacgtt actggtttca cattcaccac cctgaattga ctctcttccg
     5401 ggcgctatca tgccataccg cgaaaggttt tgcgccattc gatggtgtcc gggatctcga
     5461 cgctctccct tatgcgactc ctgcattagg aagcagccca gtagtaggtt gaggccgttg
     5521 agcaccgccg ccgcaaggaa tggtgcatgc aaggagatgg cgcccaacag tcccccggcc
     5581 acggggcctg ccaccatacc cacgccgaaa caagcgctca tgagcccgaa gtggcgagcc
     5641 cgatcttccc catcggtgat gtcggcgata taggcgccag caaccgcacc tgtggcgccg
     5701 gtgatgccgg ccacgatgcg tccggcgtag aggatcgagt caggacccac tttcacattt
     5761 aagttgtttt tctaatccgc atatgatcaa ttcaaggccg aataagaagg ctggctctgc
     5821 accttggtga tcaaataatt cgatagcttg tcgtaataat ggcggcatac tatcagtagt
     5881 aggtgtttcc ctttcttctt tagcgacttg atgctcttga tcttccaata cgcaacctaa
     5941 agtaaaatgc cccacagcgc tgagtgcata taatgcattc tctagtgaaa aaccttgttg
     6001 gcataaaaag gctaattgat tttcgagagt ttcatactgt ttttctgtag gccgtgtacc
     6061 taaatgtact tttgctccat cgcgatgact tagtaaatca catctaaaac ttttagcgtt
     6121 attacgtaaa aaatcttgcc agctttcccc ttctaaaggg caaaagtgag tatggtgcct
     6181 atctaacatc tcaatggcta aggcgtcgag caaagcccgc ttatttttta catgccaata
     6241 caatgtaggc tgctctacac ctagcttctg ggcgagttta cgggttgtta aaccttcgat
     6301 tccgacctca ttaagcagct ctaatgcgct gttaatcact ttacttttat ctaatctaga
     6361 cattaattcc taatttttgt tgacactcta tcattgatag agttatttta ccactcccta
     6421 tcagtgatag agaaaagtga aatgggcagc agccatcacc atcatcacca cagccaggat
     6481 cca
//
