LOCUS       sequence-173069-        7070 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  mWasabi reporter gene gated by temperature-sensitive Lambda 
            repressor TcI.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7070)
  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 7070)
  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: pTcI-Wasabi
FEATURES             Location/Qualifiers
     source          1..7070
                     /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"
     promoter        768..844
                     /gene="lacI"
                     /label=lacI promoter
     CDS             866..1579
                     /codon_start=1
                     /gene="cIts"
                     /product="temperature-sensitive variant of the phage lambda
                     repressor"
                     /label=lambda repressor (ts)
                     /note="thermosensitivity is conferred by the A67T mutation"
                     /translation="MSTKKKPLTQEQLEDARRLKAIYEKKKNELGLSQESVADKMGMGQ
                     SGVGALFNGINALNAYNAALLTKILKVSVEEFSPSIAREIYEMYEAVSMQPSLRSEYEY
                     PVFSHVQAGMFSPELRTFTKGDAERWVSTTKKASDSAFWLEVEGNSMTAPTGSKPSFPD
                     GMLILVDPEQAVEPGDFCIARLGGDEFTFKKLIRDSGQVFLQPLNPQYPMIPCNESCSV
                     VGKVIASQWPEETFG"
     CDS             1592..1636
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     primer_bind     complement(1674..1692)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1688..1735
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1772..2227
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1859..1878)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2069..2090
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(join(2342..3133,3134..3202))
                     /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     2965..2984
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3203..3295)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      3376..3964
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3865..3884
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4118..4135
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4150..4289
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4215..4234)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4375..4397
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4391..4582)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(5154..6236)
                     /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     6198..6217
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6237..6314)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     6520..6539
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     RBS             6683..6691
                     /label=Shine-Dalgarno sequence
                     /note="full consensus sequence for ribosome-binding sites 
                     upstream of start codons in E. coli; complementary to a 
                     region in the 3' end of the 16S rRNA (Chen et al., 1994)"
     protein_bind    6941..6955
                     /label=boxB
                     /bound_moiety="lambda-N peptide"
                     /note="lambda N peptide binding site consisting of an RNA 
                     hairpin within the N-utliization (nut) site of the lambda 
                     bacteriophage transcript (Tan and Frankel, 1995)"
     CDS             7041..7058
                     /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 ctgaaagctt
      721 gcggccgcat aatgcttaag tcgaacagaa agtaatcgta tgagctcgac accatcgaat
      781 ggcgcaaacc tttcgcggta tggcatgata gcgcccggaa gagagtcaat tcagggtggt
      841 gaatgtataa gaaggagata tacatatgag cacaaaaaag aaaccattaa cacaagagca
      901 gcttgaggac gcacgtcgcc ttaaagcaat ttatgaaaaa aagaaaaatg aacttggctt
      961 atcccaggaa tctgtcgcag acaagatggg gatggggcag tcaggcgttg gtgctttatt
     1021 taatggcatc aatgcattaa atgcttataa cgccgcattg cttacaaaaa ttctcaaagt
     1081 tagcgttgaa gaatttagcc cttcaatcgc cagagaaatc tacgagatgt atgaagcggt
     1141 tagtatgcag ccgtcactta gaagtgagta tgagtaccct gttttttctc atgttcaggc
     1201 agggatgttc tcacctgagc ttagaacctt taccaaaggt gatgcggaga gatgggtaag
     1261 cacaaccaaa aaagccagtg attctgcatt ctggcttgag gttgaaggta attccatgac
     1321 cgcaccaaca ggctccaagc caagctttcc tgacggaatg ttaattctcg ttgaccctga
     1381 gcaggctgtt gagccaggtg atttctgcat agccagactt gggggtgatg agtttacctt
     1441 caagaaactg atcagggata gcggtcaggt gtttttacaa ccactaaacc cacagtaccc
     1501 aatgatccca tgcaatgaga gttgttccgt tgtggggaaa gttatcgcta gtcagtggcc
     1561 tgaagagacg tttggctgac tcgagtctgg taaagaaacc gctgctgcga aatttgaacg
     1621 ccagcacatg gactcgtcta ctagcgcagc ttaattaacc taggctgctg ccaccgctga
     1681 gcaataacta gcataacccc ttggggcctc taaacgggtc ttgaggggtt ttttgctgaa
     1741 aggaggaact atatccggat tggcgaatgg gacgcgccct gtagcggcgc attaagcgcg
     1801 gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg ccagcgccct agcgcccgct
     1861 cctttcgctt tcttcccttc ctttctcgcc acgttcgccg gctttccccg tcaagctcta
     1921 aatcgggggc tccctttagg gttccgattt agtgctttac ggcacctcga ccccaaaaaa
     1981 cttgattagg gtgatggttc acgtagtggg ccatcgccct gatagacggt ttttcgccct
     2041 ttgacgttgg agtccacgtt ctttaatagt ggactcttgt tccaaactgg aacaacactc
     2101 aaccctatct cggtctattc ttttgattta taagggattt tgccgatttc ggcctattgg
     2161 ttaaaaaatg agctgattta acaaaaattt aacgcgaatt ttaacaaaat attaacgttt
     2221 acaatttctg gcggcacgat ggcatgagat tatcaaaaag gatcttcacc tagatccttt
     2281 taaattaaaa atgaagtttt aaatcaatct aaagtatata tgagtaaact tggtctgaca
     2341 gttaccaatg cttaatcagt gaggcaccta tctcagcgat ctgtctattt cgttcatcca
     2401 tagttgcctg actccccgtc gtgtagataa ctacgatacg ggagggctta ccatctggcc
     2461 ccagtgctgc aatgataccg cgagacccac gctcaccggc tccagattta tcagcaataa
     2521 accagccagc cggaagggcc gagcgcagaa gtggtcctgc aactttatcc gcctccatcc
     2581 agtctattaa ttgttgccgg gaagctagag taagtagttc gccagttaat agtttgcgca
     2641 acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat
     2701 tcagctccgg ttcccaacga tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag
     2761 cggttagctc cttcggtcct ccgatcgttg tcagaagtaa gttggccgca gtgttatcac
     2821 tcatggttat ggcagcactg cataattctc ttactgtcat gccatccgta agatgctttt
     2881 ctgtgactgg tgagtactca accaagtcat tctgagaata gtgtatgcgg cgaccgagtt
     2941 gctcttgccc ggcgtcaata cgggataata ccgcgccaca tagcagaact ttaaaagtgc
     3001 tcatcattgg aaaacgttct tcggggcgaa aactctcaag gatcttaccg ctgttgagat
     3061 ccagttcgat gtaacccact cgtgcaccca actgatcttc agcatctttt actttcacca
     3121 gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga ataagggcga
     3181 cacggaaatg ttgaatactc atactcttcc tttttcaatc atgattgaag catttatcag
     3241 ggttattgtc tcatgagcgg atacatattt gaatgtattt agaaaaataa acaaataggt
     3301 catgaccaaa atcccttaac gtgagttttc gttccactga gcgtcagacc ccgtagaaaa
     3361 gatcaaagga tcttcttgag atcctttttt tctgcgcgta atctgctgct tgcaaacaaa
     3421 aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa gagctaccaa ctctttttcc
     3481 gaaggtaact ggcttcagca gagcgcagat accaaatact gtccttctag tgtagccgta
     3541 gttaggccac cacttcaaga actctgtagc accgcctaca tacctcgctc tgctaatcct
     3601 gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt accgggttgg actcaagacg
     3661 atagttaccg gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca cacagcccag
     3721 cttggagcga acgacctaca ccgaactgag atacctacag cgtgagctat gagaaagcgc
     3781 cacgcttccc gaagggagaa aggcggacag gtatccggta agcggcaggg tcggaacagg
     3841 agagcgcacg agggagcttc cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt
     3901 tcgccacctc tgacttgagc gtcgattttt gtgatgctcg tcaggggggc ggagcctatg
     3961 gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc ttttgctggc cttttgctca
     4021 catgttcttt cctgcgttat cccctgattc tgtggataac cgtattaccg cctttgagtg
     4081 agctgatacc gctcgccgca gccgaacgac cgagcgcagc gagtcagtga gcgaggaagc
     4141 ggaagagcgc ctgatgcggt attttctcct tacgcatctg tgcggtattt cacaccgcaa
     4201 tggtgcactc tcagtacaat ctgctctgat gccgcatagt taagccagta tacactccgc
     4261 tatcgctacg tgactgggtc atggctgcgc cccgacaccc gccaacaccc gctgacgcgc
     4321 cctgacgggc ttgtctgctc ccggcatccg cttacagaca agctgtgacc gtctccggga
     4381 gctgcatgtg tcagaggttt tcaccgtcat caccgaaacg cgcgaggcag ctgcggtaaa
     4441 gctcatcagc gtggtcgtga agcgattcac agatgtctgc ctgttcatcc gcgtccagct
     4501 cgttgagttt ctccagaagc gttaatgtct ggcttctgat aaagcgggcc atgttaaggg
     4561 cggttttttc ctgtttggtc actgatgcct ccgtgtaagg gggatttctg ttcatggggg
     4621 taatgatacc gatgaaacga gagaggatgc tcacgatacg ggttactgat gatgaacatg
     4681 cccggttact ggaacgttgt gagggtaaac aactggcggt atggatgcgg cgggaccaga
     4741 gaaaaatcac tcagggtcaa tgccagcgct tcgttaatac agatgtaggt gttccacagg
     4801 gtagccagca gcatcctgcg atgcagatcc ggaacataat ggtgcagggc gctgacttcc
     4861 gcgtttccag actttacgaa acacggaaac cgaagaccat tcatgttgtt gctcaggtcg
     4921 cagacgtttt gcagcagcag tcgcttcacg ttcgctcgcg tatcggtgat tcattctgct
     4981 aaccagtaag gcaaccccgc cagcctagcc gggtcctcaa cgacaggagc acgatcatgc
     5041 tagtcatgcc ccgcgcccac cggaaggagc tgactgggtt gaaggctctc aagggcatcg
     5101 gtcgagatcc cggtgcctaa tgagtgagct aacttacatt aattgcgttg cgctcactgc
     5161 ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg
     5221 ggagaggcgg tttgcgtatt gggcgccagg gtggtttttc ttttcaccag tgagacgggc
     5281 aacagctgat tgcccttcac cgcctggccc tgagagagtt gcagcaagcg gtccacgctg
     5341 gtttgcccca gcaggcgaaa atcctgtttg atggtggtta acggcgggat ataacatgag
     5401 ctgtcttcgg tatcgtcgta tcccactacc gagatgtccg caccaacgcg cagcccggac
     5461 tcggtaatgg cgcgcattgc gcccagcgcc atctgatcgt tggcaaccag catcgcagtg
     5521 ggaacgatgc cctcattcag catttgcatg gtttgttgaa aaccggacat ggcactccag
     5581 tcgccttccc gttccgctat cggctgaatt tgattgcgag tgagatattt atgccaacca
     5641 gccagacgca gacgcgccga gacagaactt aatgggcccg ctaacagcgc gatttgctgg
     5701 tgacccaatg cgaccagatg ctccacgccc agtcgcgtac cgtcttcatg ggagaaaata
     5761 atactgttga tgggtgtctg gtcagagaca tcaagaaata acgccggaac attagtgcag
     5821 gcagcttcca cagcaatggc atcctggtca tccagcggat agttaatgat cagcccactg
     5881 acgcgttgcg cgagaagatt gtgcaccgcc gctttacagg cttcgacgcc gcttcgttct
     5941 accatcgaca ccaccacgct ggcacccagt tgatcggcgc gagatttaat cgccgcgaca
     6001 atttgcgacg gcgcgtgcag ggccagactg gaggtggcaa cgccaatcag caacgactgt
     6061 ttgcccgcca gttgttgtgc cacgcggttg ggaatgtaat tcagctccgc catcgccgct
     6121 tccacttttt cccgcgtttt cgcagaaacg tggctggcct ggttcaccac gcgggaaacg
     6181 gtctgataag agacaccggc atactctgcg acatcgtata acgttactgg tttcacattc
     6241 accaccctga attgactctc ttccgggcgc tatcatgcca taccgcgaaa ggttttgcgc
     6301 cattcgatgg tgtccgggat ctcgacgctc tcccttatgc gactcctgca ttaggaagca
     6361 gcccagtagt aggttgaggc cgttgagcac cgccgccgca aggaatggtg catgcaagga
     6421 gatggcgccc aacagtcccc cggccacggg gcctgccacc atacccacgc cgaaacaagc
     6481 gctcatgagc ccgaagtggc gagcccgatc ttccccatcg gtgatgtcgg cgatataggc
     6541 gccagcaacc gcacctgtgg cgccggtgat gccggccacg atgcgtccgg cgtagaggat
     6601 cgagtatcac cgcaagggat aaatatctaa caccgtgcgt gttgactatt ttacctctgg
     6661 cggtgataat ggttgcatgt actaaggagg ttgtatggaa caacgcataa ccctgaaaga
     6721 ttatgcaatg cgctttgggc aaaccaagac agctaaaaga tctctcacct accaaacaat
     6781 gcccccctgc aaaaaataaa ttcatataaa aaacatacag ataaccatct gcggtgataa
     6841 attatctctg gcggtgttga cataaatacc actggcggtg atactgagca catcagcagg
     6901 acgcactgac caccatgaag gtgacgctct taaaaattaa gccctgaaga agggcagcat
     6961 tcaaagcaga aggctttggg gtgtgtgata cgaaacgaag cattggttaa aaattaagga
     7021 gggaattcat gggcagcagc catcaccatc atcaccacag ccaggatcca
//
