LOCUS       sequence-210139-        7658 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Nucleus targetting gTEMP.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7658)
  AUTHORS   Nakano M, Arai Y, Kotera I, Okabe K, Kamei Y, Nagai T
  TITLE     Genetically encoded ratiometric fluorescent thermometer with wide 
            range and rapid response.
  JOURNAL   PLoS One. 2017 Feb 17;12(2):e0172344. doi: 
            10.1371/journal.pone.0172344. eCollection 2017.
  PUBMED    28212432
REFERENCE   2  (bases 1 to 7658)
  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"; journalName: "PLoS One.";
            date: "2017-02-17"; volume: "12(2):e0172344. doi"; pages: " 
            10.1371/journal.pone.0172344. eCollection 2017"
COMMENT     Sequence Label: nuc-gTEMP_pcDNA3
FEATURES             Location/Qualifiers
     source          1..7658
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        133..512
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        513..716
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     667..687
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     761..780
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        761..779
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      793..802
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1264..1515
                     /codon_start=1
                     /product="C-terminal fragment of mVenus for use in 
                     bimolecular fluorescence complementation (BiFC) (Kodama and
                     Hu, 2010)"
                     /label=VC155
                     /translation="DKQKNGIKAHFKIRHNIEDGGVQLADHYQQNTPIGDGPVLLPDNH
                     YLSVQSKLSKDPNEKRDHMVLLESVTAAGITLGMDEPYK"
     CDS             1918..1971
                     /codon_start=1
                     /product="2A peptide from Thosea asigna virus capsid 
                     protein"
                     /label=T2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="EGRGSLLTCGDVEENPGP"
     CDS             join(1978..1980,1981..1983,1984..2694)
                     /codon_start=1
                     /product="strongly enhanced green fluorescent protein 
                     (Kremers et al., 2007)"
                     /label=SGFP2
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTFSYGVMVFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHNVYIMADKQKNGIK
                     ANFKIRHNIEDGGVQLADHYQQNTPIGDGPVLLPDNHYLSIQSKLSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     promoter        complement(3109..3127)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(3110..3127)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(3147..3164)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3153..3377
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      3423..3851
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3510..3529)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3720..3741
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3860..3880)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3865..4194
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      4045..4180
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     4107..4126
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             4261..5055
                     /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(4315..4334)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4925..4944
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    5229..5350
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5266..5285)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5320..5339
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5399..5415)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5399..5415)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5412..5434)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5423..5439
                     /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(5447..5453,5454..5471,5472..5477))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5492..5513
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5630..5647)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5801..6389)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5881..5900)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6560..7351,7352..7420))
                     /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     7183..7202
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7421..7525)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(7600..7619)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 aggtcgctga gtagtgcgcg agcaaaattt aagctacaac aaggcaaggc ttgaccgaca
       61 attgcatgaa gaatctgctt agggttaggc gttttgcgct gcttcgcgat gtacgggcca
      121 gatatacgcg ttgacattga ttattgacta gttattaata gtaatcaatt acggggtcat
      181 tagttcatag cccatatatg gagttccgcg ttacataact tacggtaaat ggcccgcctg
      241 gctgaccgcc caacgacccc cgcccattga cgtcaataat gacgtatgtt cccatagtaa
      301 cgccaatagg gactttccat tgacgtcaat gggtggagta tttacggtaa actgcccact
      361 tggcagtaca tcaagtgtat catatgccaa gtacgccccc tattgacgtc aatgacggta
      421 aatggcccgc ctggcattat gcccagtaca tgaccttatg ggactttcct acttggcagt
      481 acatctacgt attagtcatc gctattacca tggtgatgcg gttttggcag tacatcaatg
      541 ggcgtggata gcggtttgac tcacggggat ttccaagtct ccaccccatt gacgtcaatg
      601 ggagtttgtt ttggcaccaa aatcaacggg actttccaaa atgtcgtaac aactccgccc
      661 cattgacgca aatgggcggt aggcgtgtac ggtgggaggt ctatataagc agagctctct
      721 ggctaactag agaacccact gcttactggc ttatcgaaat taatacgact cactataggg
      781 agacccaagc ttgccaccat ggtgagcaag ggcgaggagc tgttcaccgg ggtggtgccc
      841 atcctggtcg agctggacgg cgacgtaaac ggccacaggt tcagcgtgtc cggcgagggc
      901 gagggcgatg ccacctacgg caagctgacc ctgaagctca tctgcaccac cggcaagctg
      961 cccgtgccct ggcccaccct cgtgaccacc ctgcaattcg gcgtgctgtg cttcgcccgc
     1021 taccccgacc acatgaagca gcacgacttc ttcaagtccg ccatgcccga aggctacgtc
     1081 caggagcgta ccatcttctt caaggacgac ggcaactaca agacccgcgc cgaggtgaag
     1141 ttcgagggcg acaccctggt gaaccgcatc gagctgaagg gcatcgactt caaggaggac
     1201 ggcaacatcc tggggcacaa gctggagtac aacgggataa gctcaaacgt atatatcacc
     1261 gccgacaagc agaagaacgg catcaaggcc cacttcaaga tccgccacaa catcgaggac
     1321 ggcggcgtgc agctcgccga ccactaccag cagaacaccc ccatcggcga cggccccgtg
     1381 ctgctgcccg acaaccacta cctgagcgtc cagtccaagc tgagcaaaga ccccaacgag
     1441 aagcgcgatc acatggtcct gctggagtcc gtgaccgccg ccgggatcac tctcggcatg
     1501 gacgagccgt acaaggaatt catgccggag ccagcgaagt ctgctcccgc gcccaagaag
     1561 ggctcgaaga aagccgtgac taaggcgcag aagaaggacg gcaagaagcg caagcgcagc
     1621 cgcaaggaga gctactccgt atacgtgtac aaggtgctga agcaggtcca ccccgacacc
     1681 ggcatctcct ctaaggccat gggaatcatg aactccttcg tcaacgacat cttcgaacgc
     1741 atcgcgggtg aggcttcccg cctggcgcat tacaacaagc gctcgaccat cacctccagg
     1801 gagatccaga cggccgtgcg cctgctgctg cccggggagt tggccaagca cgccgtgtcc
     1861 gagggcacca aggccgtcac caagtacacc agcgctaagc gcatgcatgg cagcggcgag
     1921 ggcagaggca gcctgctgac ctgcggcgac gtggaggaga accccggccc cgagctcatg
     1981 gtgagcaagg gcgaggagct gttcaccggg gtggtgccca tcctggtcga gctggacggc
     2041 gacgtaaacg gccacaagtt cagcgtgtcc ggcgagggcg agggcgatgc cacctacggc
     2101 aagctgaccc tgaagttcat ctgcaccacc ggcaagctgc ccgtgccctg gcccaccctc
     2161 gtgaccacct tctcctacgg cgtgatggtg ttcgcccgct accccgacca catgaagcag
     2221 cacgacttct tcaagtccgc catgcccgaa ggctacgtcc aggagcgcac catcttcttc
     2281 aaggacgacg gcaactacaa gacccgcgcc gaggtgaagt tcgagggcga caccctggtg
     2341 aaccgcatcg agctgaaggg catcgacttc aaggaggacg gcaacatcct ggggcacaag
     2401 ctggagtaca acttcaacag ccacaacgtc tatatcatgg ccgacaagca gaagaacggc
     2461 atcaaggcca acttcaagat ccgccacaac atcgaggacg gcggcgtgca gctcgccgac
     2521 cactaccagc agaacacccc catcggcgac ggccccgtgc tgctgcccga caaccactac
     2581 ctgagcatcc agtccaagct gagcaaagac cccaacgaga agcgcgatca catggtcctg
     2641 ctggagttcg tgaccgccgc cgggatcact ctcggcatgg acgagctgta caaggaattc
     2701 atgccggagc cagcgaagtc tgctcccgcg cccaagaagg gctcgaagaa agccgtgact
     2761 aaggcgcaga agaaggacgg caagaagcgc aagcgcagcc gcaaggagag ctactccgta
     2821 tacgtgtaca aggtgctgaa gcaggtccac cccgacaccg gcatctcctc taaggccatg
     2881 ggaatcatga actccttcgt caacgacatc ttcgaacgca tcgcgggtga ggcttcccgc
     2941 ctggcgcatt acaacaagcg ctcgaccatc acctccaggg agatccagac ggccgtgcgc
     3001 ctgctgctgc ccggggagtt ggccaagcac gccgtgtccg agggcaccaa ggccgtcacc
     3061 aagtacacca gcgctaagta actcgagcat gcatctagag ggccctattc tatagtgtca
     3121 cctaaatgct agagctcgct gatcagcctc gactgtgcct tctagttgcc agccatctgt
     3181 tgtttgcccc tcccccgtgc cttccttgac cctggaaggt gccactccca ctgtcctttc
     3241 ctaataaaat gaggaaattg catcgcattg tctgagtagg tgtcattcta ttctgggggg
     3301 tggggtgggg caggacagca agggggagga ttgggaagac aatagcaggc atgctgggga
     3361 tgcggtgggc tctatggctt ctgaggcgga aagaaccagc tggggctcta gggggtatcc
     3421 ccacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac
     3481 cgctacactt gccagcgccc tagcgcccgc tcctttcgct ttcttccctt cctttctcgc
     3541 cacgttcgcc ggctttcccc gtcaagctct aaatcggggg ctccctttag ggttccgatt
     3601 tagtgcttta cggcacctcg accccaaaaa acttgattag ggtgatggtt cacgtagtgg
     3661 gccatcgccc tgatagacgg tttttcgccc tttgacgttg gagtccacgt tctttaatag
     3721 tggactcttg ttccaaactg gaacaacact caaccctatc tcggtctatt cttttgattt
     3781 ataagggatt ttgccgattt cggcctattg gttaaaaaat gagctgattt aacaaaaatt
     3841 taacgcgaat taattctgtg gaatgtgtgt cagttagggt gtggaaagtc cccaggctcc
     3901 ccagcaggca gaagtatgca aagcatgcat ctcaattagt cagcaaccag gtgtggaaag
     3961 tccccaggct ccccagcagg cagaagtatg caaagcatgc atctcaatta gtcagcaacc
     4021 atagtcccgc ccctaactcc gcccatcccg cccctaactc cgcccagttc cgcccattct
     4081 ccgccccatg gctgactaat tttttttatt tatgcagagg ccgaggccgc ctctgcctct
     4141 gagctattcc agaagtagtg aggaggcttt tttggaggcc taggcttttg caaaaagctc
     4201 ccgggagctt gtatatccat tttcggatct gatcaagaga caggatgagg atcgtttcgc
     4261 atgattgaac aagatggatt gcacgcaggt tctccggccg cttgggtgga gaggctattc
     4321 ggctatgact gggcacaaca gacaatcggc tgctctgatg ccgccgtgtt ccggctgtca
     4381 gcgcaggggc gcccggttct ttttgtcaag accgacctgt ccggtgccct gaatgaactg
     4441 caggacgagg cagcgcggct atcgtggctg gccacgacgg gcgttccttg cgcagctgtg
     4501 ctcgacgttg tcactgaagc gggaagggac tggctgctat tgggcgaagt gccggggcag
     4561 gatctcctgt catctcacct tgctcctgcc gagaaagtat ccatcatggc tgatgcaatg
     4621 cggcggctgc atacgcttga tccggctacc tgcccattcg accaccaagc gaaacatcgc
     4681 atcgagcgag cacgtactcg gatggaagcc ggtcttgtcg atcaggatga tctggacgaa
     4741 gagcatcagg ggctcgcgcc agccgaactg ttcgccaggc tcaaggcgcg catgcccgac
     4801 ggcgaggatc tcgtcgtgac ccatggcgat gcctgcttgc cgaatatcat ggtggaaaat
     4861 ggccgctttt ctggattcat cgactgtggc cggctgggtg tggcggaccg ctatcaggac
     4921 atagcgttgg ctacccgtga tattgctgaa gagcttggcg gcgaatgggc tgaccgcttc
     4981 ctcgtgcttt acggtatcgc cgctcccgat tcgcagcgca tcgccttcta tcgccttctt
     5041 gacgagttct tctgagcggg actctggggt tcgaaatgac cgaccaagcg acgcccaacc
     5101 tgccatcacg agatttcgat tccaccgccg ccttctatga aaggttgggc ttcggaatcg
     5161 ttttccggga cgccggctgg atgatcctcc agcgcgggga tctcatgctg gagttcttcg
     5221 cccaccccaa cttgtttatt gcagcttata atggttacaa ataaagcaat agcatcacaa
     5281 atttcacaaa taaagcattt ttttcactgc attctagttg tggtttgtcc aaactcatca
     5341 atgtatctta tcatgtctgt ataccgtcga cctctagcta gagcttggcg taatcatggt
     5401 catagctgtt tcctgtgtga aattgttatc cgctcacaat tccacacaac atacgagccg
     5461 gaagcataaa gtgtaaagcc tggggtgcct aatgagtgag ctaactcaca ttaattgcgt
     5521 tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg
     5581 gccaacgcgc ggggagaggc ggtttgcgta ttgggcgctc ttccgcttcc tcgctcactg
     5641 actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc agctcactca aaggcggtaa
     5701 tacggttatc cacagaatca ggggataacg caggaaagaa catgtgagca aaaggccagc
     5761 aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg ctccgccccc
     5821 ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg acaggactat
     5881 aaagatacca ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc
     5941 cgcttaccgg atacctgtcc gcctttctcc cttcgggaag cgtggcgctt tctcatagct
     6001 cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg
     6061 aaccccccgt tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc
     6121 cggtaagaca cgacttatcg ccactggcag cagccactgg taacaggatt agcagagcga
     6181 ggtatgtagg cggtgctaca gagttcttga agtggtggcc taactacggc tacactagaa
     6241 gaacagtatt tggtatctgc gctctgctga agccagttac cttcggaaaa agagttggta
     6301 gctcttgatc cggcaaacaa accaccgctg gtagcggtgg tttttttgtt tgcaagcagc
     6361 agattacgcg cagaaaaaaa ggatctcaag aagatccttt gatcttttct acggggtctg
     6421 acgctcagtg gaacgaaaac tcacgttaag ggattttggt catgagatta tcaaaaagga
     6481 tcttcaccta gatcctttta aattaaaaat gaagttttaa atcaatctaa agtatatatg
     6541 agtaaacttg gtctgacagt taccaatgct taatcagtga ggcacctatc tcagcgatct
     6601 gtctatttcg ttcatccata gttgcctgac tccccgtcgt gtagataact acgatacggg
     6661 agggcttacc atctggcccc agtgctgcaa tgataccgcg agacccacgc tcaccggctc
     6721 cagatttatc agcaataaac cagccagccg gaagggccga gcgcagaagt ggtcctgcaa
     6781 ctttatccgc ctccatccag tctattaatt gttgccggga agctagagta agtagttcgc
     6841 cagttaatag tttgcgcaac gttgttgcca ttgctacagg catcgtggtg tcacgctcgt
     6901 cgtttggtat ggcttcattc agctccggtt cccaacgatc aaggcgagtt acatgatccc
     6961 ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc agaagtaagt
     7021 tggccgcagt gttatcactc atggttatgg cagcactgca taattctctt actgtcatgc
     7081 catccgtaag atgcttttct gtgactggtg agtactcaac caagtcattc tgagaatagt
     7141 gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg ggataatacc gcgccacata
     7201 gcagaacttt aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa ctctcaagga
     7261 tcttaccgct gttgagatcc agttcgatgt aacccactcg tgcacccaac tgatcttcag
     7321 catcttttac tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa
     7381 aaaagggaat aagggcgaca cggaaatgtt gaatactcat actcttcctt tttcaatatt
     7441 attgaagcat ttatcagggt tattgtctca tgagcggata catatttgaa tgtatttaga
     7501 aaaataaaca aataggggtt ccgcgcacat ttccccgaaa agtgccacct gacgtcgacg
     7561 gatcgggaga tctcccgatc ccctatggtg cactctcagt acaatctgct ctgatgccgc
     7621 atagttaagc cagtatctgc tccctgcttg tgtgttgg
//
