LOCUS       sequence-426587-        6870 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Temperature state switch expressing low-temperature mWasabi and 
            high-temperature mRFP1.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6870)
  AUTHORS   Xiong LL, Garrett MA, Buss MT, Kornfield JA, Shapiro MG
  TITLE     Tunable Temperature-Sensitive Transcriptional Activation Based on 
            Lambda Repressor.
  JOURNAL   ACS Synth Biol. 2022 Jul 15;11(7):2518-2522. doi: 
            10.1021/acssynbio.2c00093. Epub 2022 Jun 15.
  PUBMED    35708251
REFERENCE   2  (bases 1 to 6870)
  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: 
            "10.1021/acssynbio.2c00093"; journalName: "ACS Synth Biol"; date: 
            "2022-07-15- 15"; volume: "11"; issue: "7"; pages: "2518-2522"
COMMENT     Sequence Label: pTcI39-switch
FEATURES             Location/Qualifiers
     source          1..6870
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      1..589
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     490..509
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     743..760
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    775..914
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(840..859)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     1000..1022
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(1016..1207)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     1984..2003
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     CDS             complement(2159..2836)
                     /codon_start=1
                     /product="monomeric derivative of DsRed (Campbell et al., 
                     2002)"
                     /label=mRFP1
                     /translation="MASSEDVIKEFMRFKVRMEGSVNGHEFEIEGEGEGRPYEGTQTAK
                     LKVTKGGPLPFAWDILSPQFQYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGV
                     VTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASTERMYPEDGALKGEIKM
                     RLKLKDGGHYDAEVKTTYMAKKPVQLPGAYKTDIKLDITSHNEDYTIVEQYERAEGRHS
                     TGA"
     protein_bind    2922..2936
                     /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)"
     RBS             3186..3194
                     /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)"
     RBS             3298..3309
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             3319..3336
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             3349..4056
                     /codon_start=1
                     /product="green variant of teal fluorescent protein mTFP1 
                     (Ai et al., 2008)"
                     /label=mWasabi
                     /translation="MVSKGEETTMGVIKPDMKIKLKMEGNVNGHAFVIEGEGEGKPYDG
                     TNTINLEVKEGAPLPFSYDILTTAFSYGNRAFTKYPDDIPNYFKQSFPEGYSWERTMTF
                     EDKGIVKVKSDISMEEDSFIYEIHLKGENFPPNGPVMQKETTGWDASTERMYVRDGVLK
                     GDVKMKLLLEGGGHHRVDFKTIYRAKKAVKLPDYHFVDHRIEILNHDKDYNKVTVYEIA
                     VARNSTDGMDELYK"
     protein_bind    4136..4150
                     /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)"
     promoter        4263..4339
                     /gene="lacI"
                     /label=lacI promoter
     CDS             4361..5074
                     /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             5087..5131
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     primer_bind     complement(5169..5187)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      5183..5230
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      5267..5722
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5354..5373)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5564..5585
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(join(5837..6628,6629..6697))
                     /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     6460..6479
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6698..6790)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc
       61 agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt
      121 cagcagagcg cagataccaa atactgtcct tctagtgtag ccgtagttag gccaccactt
      181 caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc
      241 tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa
      301 ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac
      361 ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg
      421 gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga
      481 gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact
      541 tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa
      601 cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt tctttcctgc
      661 gttatcccct gattctgtgg ataaccgtat taccgccttt gagtgagctg ataccgctcg
      721 ccgcagccga acgaccgagc gcagcgagtc agtgagcgag gaagcggaag agcgcctgat
      781 gcggtatttt ctccttacgc atctgtgcgg tatttcacac cgcaatggtg cactctcagt
      841 acaatctgct ctgatgccgc atagttaagc cagtatacac tccgctatcg ctacgtgact
      901 gggtcatggc tgcgccccga cacccgccaa cacccgctga cgcgccctga cgggcttgtc
      961 tgctcccggc atccgcttac agacaagctg tgaccgtctc cgggagctgc atgtgtcaga
     1021 ggttttcacc gtcatcaccg aaacgcgcga ggcagctgcg gtaaagctca tcagcgtggt
     1081 cgtgaagcga ttcacagatg tctgcctgtt catccgcgtc cagctcgttg agtttctcca
     1141 gaagcgttaa tgtctggctt ctgataaagc gggccatgtt aagggcggtt ttttcctgtt
     1201 tggtcactga tgcctccgtg taagggggat ttctgttcat gggggtaatg ataccgatga
     1261 aacgagagag gatgctcacg atacgggtta ctgatgatga acatgcccgg ttactggaac
     1321 gttgtgaggg taaacaactg gcggtatgga tgcggcggga ccagagaaaa atcactcagg
     1381 gtcaatgcca gcgcttcgtt aatacagatg taggtgttcc acagggtagc cagcagcatc
     1441 ctgcgatgca gatccggaac ataatggtgc agggcgctga cttccgcgtt tccagacttt
     1501 acgaaacacg gaaaccgaag accattcatg ttgttgctca ggtcgcagac gttttgcagc
     1561 agcagtcgct tcacgttcgc tcgcgtatcg gtgattcatt ctgctaacca gtaaggcaac
     1621 cccgccagcc tagccgggtc ctcaacgaca ggagcacgat catgctagtc atgccccgcg
     1681 cccaccggaa ggagctgact gggttgaagg ctctcaaggg catcggtcga gatcccggtg
     1741 cctaatgagt gagctaactt acattaattg cgttgcgccg ggatctcgac gctctccctt
     1801 atgcgactcc tgcattagga agcagcccag tagtaggttg aggccgttga gcaccgccgc
     1861 cgcaaggaat ggtgcatgca aggagatggc gcccaacagt cccccggcca cggggcctgc
     1921 caccataccc acgccgaaac aagcgctcat gagcccgaag tggcgagccc gatcttcccc
     1981 atcggtgatg tcggcgatat aggcgccagc aaccgcacct gtggcgccgg tgatgccggc
     2041 cacgatgcgt ccggcgtaga ggatcgagtt gagaagagaa aagaaaaccg ccgatcctgt
     2101 ccaccgcatt actgcaaggt agtggacaag accggcggtc ttaagttttt tggctgaatt
     2161 aagcaccggt ggagtgacga ccttcagcac gttcgtactg ttcaacgatg gtgtagtctt
     2221 cgttgtggga ggtgatgtcc agtttgatgt cggttttgta agcacccggc agctgaaccg
     2281 gttttttagc catgtaggtg gttttaactt cagcgtcgta gtgaccaccg tctttcagtt
     2341 tcagacgcat tttgatttca cctttcagag caccgtcttc cgggtacata cgttcggtgg
     2401 aagcttccca acccatggtt tttttctgca taaccggacc gtcggacggg aagttggtac
     2461 cacgcagttt aactttgtag atgaactcac cgtcttgcag ggaggagtcc tgggtaacgg
     2521 taacaacacc accgtcttcg aagttcataa cacgttccca tttgaaacct tccgggaagg
     2581 acagtttcag gtagtccggg atgtcagccg ggtgtttaac gtaagctttg gaaccgtact
     2641 ggaactgcgg ggacaggatg tcccaagcga acggcagcgg accacctttg gtaactttca
     2701 gtttagcggt ctgggtacct tcgtacggac gaccttcacc ttcaccttcg atttcgaact
     2761 cgtgaccgtt aacggaacct tccatacgaa ctttgaaacg catgaactct ttgataacgt
     2821 cttcggagga agccatgctg ctgcccatga attccctcct taatttttaa ccaatgcttc
     2881 gtttcgtatc acacacccca aagccttctg ctttgaatgc tgcccttctt cagggcttaa
     2941 tttttaagag cgtcaccttc atggtggtca gtgcgtcctg ctgatgtgct cagtatcacc
     3001 gccagtggta tttatgtcaa caccgccaga gataatttat caccgcagat ggttatctgt
     3061 atgtttttta tatgaattta ttttttgcag gggggcattg tttggtaggt gagagatctt
     3121 ttagctgtct tggtttgccc aaagcgcatt gcataatctt tcagggttat gcgttgttcc
     3181 atacaacctc cttagtacat gcaaccatta tcaccgccag aggtaaaata gtcaacacgc
     3241 acggtgttag atatttataa atagtggtga tagatttaac gtggatctca attcattaaa
     3301 gaggagaaag gtaccatgca tcaccatcat caccacagcc aggatccaat ggtgagcaag
     3361 ggcgaggaga ccacaatggg cgtaatcaag cccgacatga agatcaagct gaagatggag
     3421 ggcaacgtga atggccacgc cttcgtgatc gagggcgagg gcgagggcaa gccctacgac
     3481 ggcaccaaca ccatcaacct ggaggtgaag gagggagccc ccctgccctt ctcctacgac
     3541 attctgacca ccgcgttcag ttacggcaac agggccttca ccaagtaccc cgacgacatc
     3601 cccaactact tcaagcagtc cttccccgag ggctactctt gggagcgcac catgaccttc
     3661 gaggacaagg gcatcgtgaa ggtgaagtcc gacatctcca tggaggagga ctccttcatc
     3721 tacgagatac acctcaaggg cgagaacttc ccccccaacg gccccgtgat gcagaaggag
     3781 accaccggct gggacgcctc caccgagagg atgtacgtgc gcgacggcgt gctgaagggc
     3841 gacgtcaaga tgaagctgct gctggagggc ggcggccacc accgcgttga cttcaagacc
     3901 atctacaggg ccaagaaggc ggtgaagctg cccgactatc actttgtgga ccaccgcatc
     3961 gagatcctga accacgacaa ggactacaac aaggtgaccg tttacgagat cgccgtggcc
     4021 cgcaactcca ccgacggcat ggacgagctg tacaagggcg ctgctaacga cgaaaactac
     4081 gctgacgctt cttgaacgca ctgaccacca tgaaggtgac gctcttaaaa attaagccct
     4141 gaagaagggc agcattcaaa gcagaaggct ttggggtgtg tgatacgaaa cgaagcattg
     4201 gttaaaaata agcttgcggc cgcataatgc ttaagtcgaa cagaaagtaa tcgtatgagc
     4261 tcgacaccat cgaatggcgc aaacctttcg cggtatggca tgatagcgcc cggaagagag
     4321 tcaattcagg gtggtgaatg tataagaagg agatatacat atgagcacaa aaaagaaacc
     4381 attaacacaa gagcagcttg aggacgcacg tcgccttaaa gcaatttatg aaaaaaagaa
     4441 aaatgaactt ggcttatccc aggaatctgt cgcagacaag atggggatgg ggcagtcagg
     4501 cgttggtgct ttatttaatg gcatcaatgc attaaatgct tataacgccg cattgcttac
     4561 aaaaattctc aaagttagcg ttgaagaatt tagcccttca atcgccagag aaatctacga
     4621 gatgtatgaa gcggttagta tgcagccgtc acttagaagt gagtatgagt accctgtttt
     4681 ttctcatgtt caggcaggga tgttctcacc tgagcttaga acctttacca aaggtgatgc
     4741 ggagagatgg gtaagcacaa ccaaaaaagc cagtgattct gcattctggc ttgaggttga
     4801 aggtaattcc atgaccgcac caacaggctc caagccaagc tttcctgacg gaatgttaat
     4861 tctcgttgac cctgagcagg ctgttgagcc aggtgatttc tgcatagcca gacttggggg
     4921 tgatgagttt accttcaaga aactgatcag ggatagcggt caggtgtttt tacaaccact
     4981 aaacccacag tacccaatga tcccatgcaa tgagagttgt tccgttgtgg ggaaagttat
     5041 cgctagtcag tggcctgaag agacgtttgg ctgactcgag tctggtaaag aaaccgctgc
     5101 tgcgaaattt gaacgccagc acatggactc gtctactagc gcagcttaat taacctaggc
     5161 tgctgccacc gctgagcaat aactagcata accccttggg gcctctaaac gggtcttgag
     5221 gggttttttg ctgaaaggag gaactatatc cggattggcg aatgggacgc gccctgtagc
     5281 ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac acttgccagc
     5341 gccctagcgc ccgctccttt cgctttcttc ccttcctttc tcgccacgtt cgccggcttt
     5401 ccccgtcaag ctctaaatcg ggggctccct ttagggttcc gatttagtgc tttacggcac
     5461 ctcgacccca aaaaacttga ttagggtgat ggttcacgta gtgggccatc gccctgatag
     5521 acggtttttc gccctttgac gttggagtcc acgttcttta atagtggact cttgttccaa
     5581 actggaacaa cactcaaccc tatctcggtc tattcttttg atttataagg gattttgccg
     5641 atttcggcct attggttaaa aaatgagctg atttaacaaa aatttaacgc gaattttaac
     5701 aaaatattaa cgtttacaat ttctggcggc acgatggcat gagattatca aaaaggatct
     5761 tcacctagat ccttttaaat taaaaatgaa gttttaaatc aatctaaagt atatatgagt
     5821 aaacttggtc tgacagttac caatgcttaa tcagtgaggc acctatctca gcgatctgtc
     5881 tatttcgttc atccatagtt gcctgactcc ccgtcgtgta gataactacg atacgggagg
     5941 gcttaccatc tggccccagt gctgcaatga taccgcgaga cccacgctca ccggctccag
     6001 atttatcagc aataaaccag ccagccggaa gggccgagcg cagaagtggt cctgcaactt
     6061 tatccgcctc catccagtct attaattgtt gccgggaagc tagagtaagt agttcgccag
     6121 ttaatagttt gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt
     6181 ttggtatggc ttcattcagc tccggttccc aacgatcaag gcgagttaca tgatccccca
     6241 tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat cgttgtcaga agtaagttgg
     6301 ccgcagtgtt atcactcatg gttatggcag cactgcataa ttctcttact gtcatgccat
     6361 ccgtaagatg cttttctgtg actggtgagt actcaaccaa gtcattctga gaatagtgta
     6421 tgcggcgacc gagttgctct tgcccggcgt caatacggga taataccgcg ccacatagca
     6481 gaactttaaa agtgctcatc attggaaaac gttcttcggg gcgaaaactc tcaaggatct
     6541 taccgctgtt gagatccagt tcgatgtaac ccactcgtgc acccaactga tcttcagcat
     6601 cttttacttt caccagcgtt tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa
     6661 agggaataag ggcgacacgg aaatgttgaa tactcatact cttccttttt caatcatgat
     6721 tgaagcattt atcagggtta ttgtctcatg agcggataca tatttgaatg tatttagaaa
     6781 aataaacaaa taggtcatga ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc
     6841 agaccccgta gaaaagatca aaggatcttc
//
