LOCUS       sequence-173197-        8203 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Multiplex temperature-gated expression of mWasabi and mCherry 
            reporters.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8203)
  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 8203)
  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: pCali2
FEATURES             Location/Qualifiers
     source          1..8203
                     /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..845
                     /gene="lacI"
                     /label=lacI promoter
     CDS             2001..2045
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     terminator      2097..2144
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      2181..2636
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2268..2287)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2478..2499
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(join(2751..3542,3543..3611))
                     /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     3374..3393
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3612..3704)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      3785..4373
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4274..4293
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4527..4544
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4559..4698
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4624..4643)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4784..4806
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4800..4991)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     RBS             5641..5649
                     /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    5899..5913
                     /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             5987..6697
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG
                     TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF
                     EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK
                     GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERA
                     EGRHSTGGMDELYK"
     primer_bind     complement(6132..6150)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     6400..6419
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     promoter        6751..6827
                     /gene="lacI"
                     /label=lacI promoter
     CDS             6849..7562
                     /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             7575..7619
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     terminator      7671..7718
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     7960..7979
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     RBS             8133..8155
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             8174..8191
                     /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 ggcgcaaaac ctttcgcggt atggcatgat agcgcccgga agagagtcaa ttcagggtgg
      841 tgaatgtata agaaggagat atacatatgc gtccggcgac atacgaacca gaacagatta
      901 ttgaagcagg gctggccctg caggctgaag gacggaatat caccgggttc gcactacgta
      961 accaggtggg tggcggcaat ccgacacgtc tccgccagat atgggacgaa taccaggctt
     1021 cacagagcac ggtcgtcact gaactcgttg ccgagctgcc agtggaagtg gctgaagaag
     1081 tgaaggccgt ctccgccgcg ctgtccgaac gcatcaccca gctggcgaca gaactgaatg
     1141 acaaggcggt ccgggctgca gaacgccggg ttgcggaagt cacgcgtgct gccggtgaac
     1201 agaccgcaca ggcagagcgg gagctggccg acgccgcgca gacagtcgac gacctggaag
     1261 aaaaactggt tgaactgcag gacagatatg acagtttgac gctggcgctg gagtcagaac
     1321 gttcactgcg tcagcagcat gatgtggaga tggcccagct gaaagagcgt cttgcggccg
     1381 ctgaagagaa tacccgtcag cgagaggaac ggtatcagga gcagaggaca gtgctgcagg
     1441 atgcgcttaa tgcggagcag gcacagcaca taaacacgcg ggaagaccag cagaaacgac
     1501 tggagcaaat ttctgccgaa gctaatgcgc gtacagaaga actgaagtct gaacgcgata
     1561 aagtcaatac tctccttacc cgccttgaat cgcaggaaaa tgcgctggcc tcagaacgtc
     1621 agcagcatct ggccacccgc gaaacgctgc agcaacgcct cgagcaggcc atcgctgaca
     1681 cgcaggcgcg cgccggtgag attgcacttg aacgtgacag agtcagcagc ctcaccgcaa
     1741 ggctggaatc gcaggaaaag gcctcctcgg agcaactggt gcgtatgggc agtgaaatag
     1801 ccagtctgac agagcgttgc acacagctgg aaaaccagcg tgatgatgcc cgtctggaga
     1861 cgatggggga gaaagaaacg gtcgcggcac tgcgtggtga ggctgaagcc ctgaagcgtc
     1921 agaaccagtc actgatggcg gcgctttcag gcaataaaca gaccggtggc cagaatgcgt
     1981 gaggtaccct cgagtctggt aaagaaaccg ctgctgcgaa atttgaacgc cagcacatgg
     2041 actcgtctac tagcgcagct taattaacct aggctgctgc caccgctgag caataactag
     2101 cataacccct tggggcctct aaacgggtct tgaggggttt tttgctgaaa ggaggaacta
     2161 tatccggatt ggcgaatggg acgcgccctg tagcggcgca ttaagcgcgg cgggtgtggt
     2221 ggttacgcgc agcgtgaccg ctacacttgc cagcgcccta gcgcccgctc ctttcgcttt
     2281 cttcccttcc tttctcgcca cgttcgccgg ctttccccgt caagctctaa atcgggggct
     2341 ccctttaggg ttccgattta gtgctttacg gcacctcgac cccaaaaaac ttgattaggg
     2401 tgatggttca cgtagtgggc catcgccctg atagacggtt tttcgccctt tgacgttgga
     2461 gtccacgttc tttaatagtg gactcttgtt ccaaactgga acaacactca accctatctc
     2521 ggtctattct tttgatttat aagggatttt gccgatttcg gcctattggt taaaaaatga
     2581 gctgatttaa caaaaattta acgcgaattt taacaaaata ttaacgttta caatttctgg
     2641 cggcacgatg gcatgagatt atcaaaaagg atcttcacct agatcctttt aaattaaaaa
     2701 tgaagtttta aatcaatcta aagtatatat gagtaaactt ggtctgacag ttaccaatgc
     2761 ttaatcagtg aggcacctat ctcagcgatc tgtctatttc gttcatccat agttgcctga
     2821 ctccccgtcg tgtagataac tacgatacgg gagggcttac catctggccc cagtgctgca
     2881 atgataccgc gagacccacg ctcaccggct ccagatttat cagcaataaa ccagccagcc
     2941 ggaagggccg agcgcagaag tggtcctgca actttatccg cctccatcca gtctattaat
     3001 tgttgccggg aagctagagt aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc
     3061 attgctacag gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt cagctccggt
     3121 tcccaacgat caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc
     3181 ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag tgttatcact catggttatg
     3241 gcagcactgc ataattctct tactgtcatg ccatccgtaa gatgcttttc tgtgactggt
     3301 gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg
     3361 gcgtcaatac gggataatac cgcgccacat agcagaactt taaaagtgct catcattgga
     3421 aaacgttctt cggggcgaaa actctcaagg atcttaccgc tgttgagatc cagttcgatg
     3481 taacccactc gtgcacccaa ctgatcttca gcatctttta ctttcaccag cgtttctggg
     3541 tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa taagggcgac acggaaatgt
     3601 tgaatactca tactcttcct ttttcaatca tgattgaagc atttatcagg gttattgtct
     3661 catgagcgga tacatatttg aatgtattta gaaaaataaa caaataggtc atgaccaaaa
     3721 tcccttaacg tgagttttcg ttccactgag cgtcagaccc cgtagaaaag atcaaaggat
     3781 cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc
     3841 taccagcggt ggtttgtttg ccggatcaag agctaccaac tctttttccg aaggtaactg
     3901 gcttcagcag agcgcagata ccaaatactg tccttctagt gtagccgtag ttaggccacc
     3961 acttcaagaa ctctgtagca ccgcctacat acctcgctct gctaatcctg ttaccagtgg
     4021 ctgctgccag tggcgataag tcgtgtctta ccgggttgga ctcaagacga tagttaccgg
     4081 ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac acagcccagc ttggagcgaa
     4141 cgacctacac cgaactgaga tacctacagc gtgagctatg agaaagcgcc acgcttcccg
     4201 aagggagaaa ggcggacagg tatccggtaa gcggcagggt cggaacagga gagcgcacga
     4261 gggagcttcc agggggaaac gcctggtatc tttatagtcc tgtcgggttt cgccacctct
     4321 gacttgagcg tcgatttttg tgatgctcgt caggggggcg gagcctatgg aaaaacgcca
     4381 gcaacgcggc ctttttacgg ttcctggcct tttgctggcc ttttgctcac atgttctttc
     4441 ctgcgttatc ccctgattct gtggataacc gtattaccgc ctttgagtga gctgataccg
     4501 ctcgccgcag ccgaacgacc gagcgcagcg agtcagtgag cgaggaagcg gaagagcgcc
     4561 tgatgcggta ttttctcctt acgcatctgt gcggtatttc acaccgcaat ggtgcactct
     4621 cagtacaatc tgctctgatg ccgcatagtt aagccagtat acactccgct atcgctacgt
     4681 gactgggtca tggctgcgcc ccgacacccg ccaacacccg ctgacgcgcc ctgacgggct
     4741 tgtctgctcc cggcatccgc ttacagacaa gctgtgaccg tctccgggag ctgcatgtgt
     4801 cagaggtttt caccgtcatc accgaaacgc gcgaggcagc tgcggtaaag ctcatcagcg
     4861 tggtcgtgaa gcgattcaca gatgtctgcc tgttcatccg cgtccagctc gttgagtttc
     4921 tccagaagcg ttaatgtctg gcttctgata aagcgggcca tgttaagggc ggttttttcc
     4981 tgtttggtca ctgatgcctc cgtgtaaggg ggatttctgt tcatgggggt aatgataccg
     5041 atgaaacgag agaggatgct cacgatacgg gttactgatg atgaacatgc ccggttactg
     5101 gaacgttgtg agggtaaaca actggcggta tggatgcggc gggaccagag aaaaatcact
     5161 cagggtcaat gccagcgctt cgttaataca gatgtaggtg ttccacaggg tagccagcag
     5221 catcctgcga tgcagatccg gaacataatg gtgcagggcg ctgacttccg cgtttccaga
     5281 ctttacgaaa cacggaaacc gaagaccatt catgttgttg ctcaggtcgc agacgttttg
     5341 cagcagcagt cgcttcacgt tcgctcgcgt atcggtgatt cattctgcta accagtaagg
     5401 caaccccgcc agcctagccg ggtcctcaac gacaggagca cgatcatgct agtcatgccc
     5461 cgcgcccacc ggaaggagct gactgggttg aaggctctca agggcatcgg tcgagatccc
     5521 ggtgcctaat gagtgagcta acttacatta attgcgttgc gctatcaccg caagggataa
     5581 atatctaaca ccgtgcgtgt tgactatttt acctctggcg gtgataatgg ttgcatgtac
     5641 taaggaggtt gtatggaaca acgcataacc ctgaaagatt atgcaatgcg ctttgggcaa
     5701 accaagacag ctaaaagatc tctcacctac caaacaatgc ccccctgcaa aaaataaatt
     5761 catataaaaa acatacagat aaccatctgc ggtgataaat tatctctggc ggtgttgaca
     5821 taaataccac tggcggtgat actgagcaca tcagcaggac gcactgacca ccatgaaggt
     5881 gacgctctta aaaattaagc cctgaagaag ggcagcattc aaagcagaag gctttggggt
     5941 gtgtgatacg aaacgaagca ttggttaaaa attaaggagg gaattcatgg tgagcaaggg
     6001 cgaggaggat aacatggcca tcatcaagga gttcatgcgc ttcaaggtgc acatggaggg
     6061 ctccgtgaac ggccacgagt tcgagatcga gggcgagggc gagggccgcc cctacgaggg
     6121 cacccagacc gccaagctga aggtgaccaa gggcggcccc ctgcccttcg cctgggacat
     6181 cctgtcccct cagttcatgt acggctccaa ggcctacgtg aagcaccccg ccgacatccc
     6241 cgactacttg aagctgtcct tccccgaggg cttcaagtgg gagcgcgtga tgaacttcga
     6301 ggacggcggc gtggtgaccg tgacccagga ctcctccctg caggacggcg agttcatcta
     6361 caaggtgaag ctgcgcggca ccaacttccc ctccgacggc cccgtaatgc agaagaagac
     6421 catgggctgg gaggcctcct ccgagcggat gtaccccgag gacggcgccc tgaagggcga
     6481 gatcaagcag aggctgaagc tgaaggacgg cggccactac gacgctgagg tcaagaccac
     6541 ctacaaggcc aagaagcccg tgcagctgcc cggcgcctac aacgtcaaca tcaagttgga
     6601 catcacctcc cacaacgagg actacaccat cgtggaacag tacgaacgcg ccgagggccg
     6661 ccactccacc ggcggcatgg acgagctgta caagtaaaag cttgcggccg cataatgctt
     6721 aagtcgaaca gaaagtaatc gtatgagctc gacaccatcg aatggcgcaa acctttcgcg
     6781 gtatggcatg atagcgcccg gaagagagtc aattcagggt ggtgaatgta taagaaggag
     6841 atatacatat gagcacaaaa aagaaaccat taacacaaga gcagcttgag gacgcacgtc
     6901 gccttaaagc aatttatgaa aaaaagaaaa atgaacttgg cttatcccag gaatctgtcg
     6961 cagacaagat ggggatgggg cagtcaggcg ttggtgcttt atttaatggc atcaatgcat
     7021 taaatgctta taacgccgca ttgcttacaa aaattctcaa agttagcgtt gaagaattta
     7081 gcccttcaat cgccagagaa atctacgaga tgtatgaagc ggttagtatg cagccgtcac
     7141 ttagaagtga gtatgagtac cctgtttttt ctcatgttca ggcagggatg ttctcacctg
     7201 agcttagaac ctttaccaaa ggtgatgcgg agagatgggt aagcacaacc aaaaaagcca
     7261 gtgattctgc attctggctt gaggttgaag gtaattccat gaccgcacca acaggctcca
     7321 agccaagctt tcctgacgga atgttaattc tcgttgaccc tgagcaggct gttgagccag
     7381 gtgatttctg catagccaga cttgggggtg atgagtttac cttcaagaaa ctgatcaggg
     7441 atagcggtca ggtgttttta caaccactaa acccacagta cccaatgatc ccatgcaatg
     7501 agagttgttc cgttgtgggg aaagttatcg ctagtcagtg gcctgaagag acgtttggct
     7561 gactcgagtc tggtaaagaa accgctgctg cgaaatttga acgccagcac atggactcgt
     7621 ctactagcgc agcttaatta acctaggctg ctgccaccgc tgagcaataa ctagcataac
     7681 cccttggggc ctctaaacgg gtcttgaggg gttttttgct gaaaggagga actatatccg
     7741 gattggcgaa tgggcgggat ctcgacgctc tcccttatgc gactcctgca ttaggaagca
     7801 gcccagtagt aggttgaggc cgttgagcac cgccgccgca aggaatggtg catgcaagga
     7861 gatggcgccc aacagtcccc cggccacggg gcctgccacc atacccacgc cgaaacaagc
     7921 gctcatgagc ccgaagtggc gagcccgatc ttccccatcg gtgatgtcgg cgatataggc
     7981 gccagcaacc gcacctgtgg cgccggtgat gccggccacg atgcgtccgg cgtagaggat
     8041 cgagatcgat ctcgatcccg cgaaatttta atttgtttgt tagttagttt atttgttggt
     8101 ttgtttgtgt tataatatcc tctagaaata attttgttta actttaagaa ggagatatac
     8161 catgggcagc agccatcacc atcatcacca cagccaggat cca
//
