LOCUS       sequence-394870-        7727 bp    DNA     circular SYN 14-APR-2025
DEFINITION  Fluorescent temperature indicator B-gTEMP fused with mitochondrial 
            targeting sequence.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7727)
  AUTHORS   Lu K, Wazawa T, Sakamoto J, Vu CQ, Nakano M, Kamei Y, Nagai T
  TITLE     Intracellular Heat Transfer and Thermal Property Revealed by 
            Kilohertz Temperature Imaging with a Genetically Encoded 
            Nanothermometer.
  JOURNAL   Nano Lett. 2022 Jul 27;22(14):5698-5707. doi: 
            10.1021/acs.nanolett.2c00608. Epub 2022 Jul 6.
  PUBMED    35792763
REFERENCE   2  (bases 1 to 7727)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 14, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; doi: 
            "10.1021/acs.nanolett.2c00608"; journalName: "Nano Lett"; date: 
            "2022-07-27- 27"; volume: "22"; issue: "14"; pages: "5698-5707"
COMMENT     Sequence Label: mtsCOXVIIIx2-B-gTEMP/pcDNA3
FEATURES             Location/Qualifiers
     source          1..7727
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      complement(1..589)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(81..100)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(760..1551,1552..1620))
                     /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     1383..1402
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(1621..1725)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(1800..1819)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        1991..2370
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        2371..2574
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     2525..2545
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     2619..2638
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        2619..2637
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             2657..2731
                     /codon_start=1
                     /product="mitochondrial presequence of human cytochrome c 
                     oxidase subunit VIII (Rizutto et al., 1989)"
                     /label=COX8 presequence
                     /translation="MSVLTPLLLRGLTGSARRLPVPRAK"
     CDS             2753..2827
                     /codon_start=1
                     /product="mitochondrial presequence of human cytochrome c 
                     oxidase subunit VIII (Rizutto et al., 1989)"
                     /label=COX8 presequence
                     /translation="MSVLTPLLLRGLTGSARRLPVPRAK"
     CDS             3578..5008
                     /codon_start=1
                     /product="tandem dimeric (pseudo-monomeric) derivative of 
                     DsRed (Shaner et al., 2004)"
                     /label=tdTomato
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEEVIKEFMRFKVRMEGSMNGHEFEIEGEGEGRPYEGTQTA
                     KLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYKKLSFPEGFKWERVMNFEDGG
                     LVTVTQDSSLQDGTLIYKVKMRGTNFPPDGPVMQKKTMGWEASTERLYPRDGVLKGEIH
                     QALKLKDGGHYLVEFKTIYMAKKPVQLPGYYYVDTKLDITSHNEDYTIVEQYERSEGRH
                     HLFLGHGTGSTGSGSSGTASSEDNNMAVIKEFMRFKVRMEGSMNGHEFEIEGEGEGRPY
                     EGTQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYKKLSFPEGFKWERVM
                     NFEDGGLVTVTQDSSLQDGTLIYKVKMRGTNFPPDGPVMQKKTMGWEASTERLYPRDGV
                     LKGEIHQALKLKDGGHYLVEFKTIYMAKKPVQLPGYYYVDTKLDITSHNEDYTIVEQYE
                     RSEGRHHLFLYGMDELYK"
     promoter        complement(5036..5054)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(5037..5054)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(5074..5091)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    5080..5304
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      5350..5778
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5437..5456)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5647..5668
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5787..5807)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        5792..6121
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      5972..6107
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     6034..6053
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             6188..6982
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(6242..6261)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     6852..6871
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    7156..7277
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(7193..7212)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     7247..7266
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(7326..7342)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(7326..7342)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(7339..7361)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    7350..7366
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by 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)."
     promoter        complement(join(7374..7380,7381..7398,7399..7404))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    7419..7440
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(7557..7574)
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 tttccatagg ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg
       61 gcgaaacccg acaggactat aaagatacca ggcgtttccc cctggaagct ccctcgtgcg
      121 ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc cttcgggaag
      181 cgtggcgctt tctcatagct cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc
      241 caagctgggc tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct tatccggtaa
      301 ctatcgtctt gagtccaacc cggtaagaca cgacttatcg ccactggcag cagccactgg
      361 taacaggatt agcagagcga ggtatgtagg cggtgctaca gagttcttga agtggtggcc
      421 taactacggc tacactagaa gaacagtatt tggtatctgc gctctgctga agccagttac
      481 cttcggaaaa agagttggta gctcttgatc cggcaaacaa accaccgctg gtagcggtgg
      541 tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag aagatccttt
      601 gatcttttct acggggtctg acgctcagtg gaacgaaaac tcacgttaag ggattttggt
      661 catgagatta tcaaaaagga tcttcaccta gatcctttta aattaaaaat gaagttttaa
      721 atcaatctaa agtatatatg agtaaacttg gtctgacagt taccaatgct taatcagtga
      781 ggcacctatc tcagcgatct gtctatttcg ttcatccata gttgcctgac tccccgtcgt
      841 gtagataact acgatacggg agggcttacc atctggcccc agtgctgcaa tgataccgcg
      901 agacccacgc tcaccggctc cagatttatc agcaataaac cagccagccg gaagggccga
      961 gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag tctattaatt gttgccggga
     1021 agctagagta agtagttcgc cagttaatag tttgcgcaac gttgttgcca ttgctacagg
     1081 catcgtggtg tcacgctcgt cgtttggtat ggcttcattc agctccggtt cccaacgatc
     1141 aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc
     1201 gatcgttgtc agaagtaagt tggccgcagt gttatcactc atggttatgg cagcactgca
     1261 taattctctt actgtcatgc catccgtaag atgcttttct gtgactggtg agtactcaac
     1321 caagtcattc tgagaatagt gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg
     1381 ggataatacc gcgccacata gcagaacttt aaaagtgctc atcattggaa aacgttcttc
     1441 ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc agttcgatgt aacccactcg
     1501 tgcacccaac tgatcttcag catcttttac tttcaccagc gtttctgggt gagcaaaaac
     1561 aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt gaatactcat
     1621 actcttcctt tttcaatatt attgaagcat ttatcagggt tattgtctca tgagcggata
     1681 catatttgaa tgtatttaga aaaataaaca aataggggtt ccgcgcacat ttccccgaaa
     1741 agtgccacct gacgtcgacg gatcgggaga tctcccgatc ccctatggtg cactctcagt
     1801 acaatctgct ctgatgccgc atagttaagc cagtatctgc tccctgcttg tgtgttggag
     1861 gtcgctgagt agtgcgcgag caaaatttaa gctacaacaa ggcaaggctt gaccgacaat
     1921 tgcatgaaga atctgcttag ggttaggcgt tttgcgctgc ttcgcgatgt acgggccaga
     1981 tatacgcgtt gacattgatt attgactagt tattaatagt aatcaattac ggggtcatta
     2041 gttcatagcc catatatgga gttccgcgtt acataactta cggtaaatgg cccgcctggc
     2101 tgaccgccca acgacccccg cccattgacg tcaataatga cgtatgttcc catagtaacg
     2161 ccaataggga ctttccattg acgtcaatgg gtggagtatt tacggtaaac tgcccacttg
     2221 gcagtacatc aagtgtatca tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa
     2281 tggcccgcct ggcattatgc ccagtacatg accttatggg actttcctac ttggcagtac
     2341 atctacgtat tagtcatcgc tattaccatg gtgatgcggt tttggcagta catcaatggg
     2401 cgtggatagc ggtttgactc acggggattt ccaagtctcc accccattga cgtcaatggg
     2461 agtttgtttt ggcaccaaaa tcaacgggac tttccaaaat gtcgtaacaa ctccgcccca
     2521 ttgacgcaaa tgggcggtag gcgtgtacgg tgggaggtct atataagcag agctctctgg
     2581 ctaactagag aacccactgc ttactggctt atcgaaatta atacgactca ctatagggag
     2641 acccaagctt gccaccatgt ctgttctgac tcctctgctg ctccggggtc tcacaggttc
     2701 cgcaagaaga ctccccgtgc ctagggccaa aattcattca ctgggggacc ccatgagcgt
     2761 gctcacccca ctcctgctgc gggggctgac cggcagcgct aggcggctgc cagtccccag
     2821 ggccaagatc cacagtctcg gcgatcccaa gcgcggccgc atcatggtgt ccaagggcga
     2881 agaggacaac atggccagcc tgcctgccac ccacgagctg cacatcttcg gcagcatcaa
     2941 cggcgtggac ttcgacatgg tgggacaggg caccggcaac cccaacgacg gctacgagga
     3001 actgaacctg aagtccacaa agggcgacct gcagttcagc ccctggattc tggtgcccca
     3061 catcggctac ggcttccacc agtacctgcc ctaccccgac ggcatgagcc ctttccaggc
     3121 cgctatggtg gatggcagcg gctaccaggt gcaccggacc atgcagtttg aggacggcgc
     3181 cagcctgacc gtgaactacc ggtacacata cgagggcagc cacatcaagg gcgaggccca
     3241 agtgaagggc acaggctttc cagccgacgg ccccgtgatg accaatagcc tgacagccgc
     3301 cgactggtgc agaagcaaga aaacctaccc caatgacaag accatcatca gcaccttcaa
     3361 gtggtcctac accaccggca atggcaagcg gtacagaagc accgcccgga ccacctacac
     3421 cttcgccaaa cctatggccg ccaactacct gaagaaccag cctatgtacg tgttccgcaa
     3481 gaccgagctg aagcactcca agacagaact gaacttcaaa gagtggcaga aagccttcac
     3541 cgacgtgatg ggcatggacg agctgtacaa gggtaccatg gtgagcaagg gcgaggaggt
     3601 catcaaagag ttcatgcgct tcaaggtgcg catggagggc tccatgaacg gccacgagtt
     3661 cgagatcgag ggcgagggcg agggccgccc ctacgagggc acccagaccg ccaagctgaa
     3721 ggtgaccaag ggcggccccc tgcccttcgc ctgggacatc ctgtcccccc agttcatgta
     3781 cggctccaag gcgtacgtga agcaccccgc cgacatcccc gattacaaga agctgtcctt
     3841 ccccgagggc ttcaagtggg agcgcgtgat gaacttcgag gacggcggtc tggtgaccgt
     3901 gacccaggac tcctccctgc aggacggcac gctgatctac aaggtgaaga tgcgcggcac
     3961 caacttcccc cccgacggcc ccgtaatgca gaagaagacc atgggctggg aggcctccac
     4021 cgagcgcctg tacccccgcg acggcgtgct gaagggcgag atccaccagg ccctgaagct
     4081 gaaggacggc ggccactacc tggtggagtt caagaccatc tacatggcca agaagcccgt
     4141 gcaactgccc ggctactact acgtggacac caagctggac atcacctccc acaacgagga
     4201 ctacaccatc gtggaacagt acgagcgctc cgagggccgc caccacctgt tcctggggca
     4261 tggcaccggc agcaccggca gcggcagctc cggcaccgcc tcctccgagg acaacaacat
     4321 ggccgtcatc aaagagttca tgcgcttcaa ggtgcgcatg gagggctcca tgaacggcca
     4381 cgagttcgag atcgagggcg agggcgaggg ccgcccctac gagggcaccc agaccgccaa
     4441 gctgaaggtg accaagggcg gccccctgcc cttcgcctgg gacatcctgt ccccccagtt
     4501 catgtacggc tccaaggcgt acgtgaagca ccccgccgac atccccgatt acaagaagct
     4561 gtccttcccc gagggcttca agtgggagcg cgtgatgaac ttcgaggacg gcggtctggt
     4621 gaccgtgacc caggactcct ccctgcagga cggcacgctg atctacaagg tgaagatgcg
     4681 cggcaccaac ttcccccccg acggccccgt aatgcagaag aagaccatgg gctgggaggc
     4741 ctccaccgag cgcctgtacc cccgcgacgg cgtgctgaag ggcgagatcc accaggccct
     4801 gaagctgaag gacggcggcc actacctggt ggagttcaag accatctaca tggccaagaa
     4861 gcccgtgcaa ctgcccggct actactacgt ggacaccaag ctggacatca cctcccacaa
     4921 cgaggactac accatcgtgg aacagtacga gcgctccgag ggccgccacc acctgttcct
     4981 gtacggcatg gacgagctgt acaagtgact cgagcatgca tctagagggc cctattctat
     5041 agtgtcacct aaatgctaga gctcgctgat cagcctcgac tgtgccttct agttgccagc
     5101 catctgttgt ttgcccctcc cccgtgcctt ccttgaccct ggaaggtgcc actcccactg
     5161 tcctttccta ataaaatgag gaaattgcat cgcattgtct gagtaggtgt cattctattc
     5221 tggggggtgg ggtggggcag gacagcaagg gggaggattg ggaagacaat agcaggcatg
     5281 ctggggatgc ggtgggctct atggcttctg aggcggaaag aaccagctgg ggctctaggg
     5341 ggtatcccca cgcgccctgt agcggcgcat taagcgcggc gggtgtggtg gttacgcgca
     5401 gcgtgaccgc tacacttgcc agcgccctag cgcccgctcc tttcgctttc ttcccttcct
     5461 ttctcgccac gttcgccggc tttccccgtc aagctctaaa tcgggggctc cctttagggt
     5521 tccgatttag tgctttacgg cacctcgacc ccaaaaaact tgattagggt gatggttcac
     5581 gtagtgggcc atcgccctga tagacggttt ttcgcccttt gacgttggag tccacgttct
     5641 ttaatagtgg actcttgttc caaactggaa caacactcaa ccctatctcg gtctattctt
     5701 ttgatttata agggattttg ccgatttcgg cctattggtt aaaaaatgag ctgatttaac
     5761 aaaaatttaa cgcgaattaa ttctgtggaa tgtgtgtcag ttagggtgtg gaaagtcccc
     5821 aggctcccca gcaggcagaa gtatgcaaag catgcatctc aattagtcag caaccaggtg
     5881 tggaaagtcc ccaggctccc cagcaggcag aagtatgcaa agcatgcatc tcaattagtc
     5941 agcaaccata gtcccgcccc taactccgcc catcccgccc ctaactccgc ccagttccgc
     6001 ccattctccg ccccatggct gactaatttt ttttatttat gcagaggccg aggccgcctc
     6061 tgcctctgag ctattccaga agtagtgagg aggctttttt ggaggcctag gcttttgcaa
     6121 aaagctcccg ggagcttgta tatccatttt cggatctgat caagagacag gatgaggatc
     6181 gtttcgcatg attgaacaag atggattgca cgcaggttct ccggccgctt gggtggagag
     6241 gctattcggc tatgactggg cacaacagac aatcggctgc tctgatgccg ccgtgttccg
     6301 gctgtcagcg caggggcgcc cggttctttt tgtcaagacc gacctgtccg gtgccctgaa
     6361 tgaactgcag gacgaggcag cgcggctatc gtggctggcc acgacgggcg ttccttgcgc
     6421 agctgtgctc gacgttgtca ctgaagcggg aagggactgg ctgctattgg gcgaagtgcc
     6481 ggggcaggat ctcctgtcat ctcaccttgc tcctgccgag aaagtatcca tcatggctga
     6541 tgcaatgcgg cggctgcata cgcttgatcc ggctacctgc ccattcgacc accaagcgaa
     6601 acatcgcatc gagcgagcac gtactcggat ggaagccggt cttgtcgatc aggatgatct
     6661 ggacgaagag catcaggggc tcgcgccagc cgaactgttc gccaggctca aggcgcgcat
     6721 gcccgacggc gaggatctcg tcgtgaccca tggcgatgcc tgcttgccga atatcatggt
     6781 ggaaaatggc cgcttttctg gattcatcga ctgtggccgg ctgggtgtgg cggaccgcta
     6841 tcaggacata gcgttggcta cccgtgatat tgctgaagag cttggcggcg aatgggctga
     6901 ccgcttcctc gtgctttacg gtatcgccgc tcccgattcg cagcgcatcg ccttctatcg
     6961 ccttcttgac gagttcttct gagcgggact ctggggttcg aaatgaccga ccaagcgacg
     7021 cccaacctgc catcacgaga tttcgattcc accgccgcct tctatgaaag gttgggcttc
     7081 ggaatcgttt tccgggacgc cggctggatg atcctccagc gcggggatct catgctggag
     7141 ttcttcgccc accccaactt gtttattgca gcttataatg gttacaaata aagcaatagc
     7201 atcacaaatt tcacaaataa agcatttttt tcactgcatt ctagttgtgg tttgtccaaa
     7261 ctcatcaatg tatcttatca tgtctgtata ccgtcgacct ctagctagag cttggcgtaa
     7321 tcatggtcat agctgtttcc tgtgtgaaat tgttatccgc tcacaattcc acacaacata
     7381 cgagccggaa gcataaagtg taaagcctgg ggtgcctaat gagtgagcta actcacatta
     7441 attgcgttgc gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa
     7501 tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg ggcgctcttc cgcttcctcg
     7561 ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag
     7621 gcggtaatac ggttatccac agaatcaggg gataacgcag gaaagaacat gtgagcaaaa
     7681 ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc tggcgtt
//
