LOCUS       Exported                6939 bp ds-DNA     circular SYN 12-MAY-2021
DEFINITION  Genetically encoded ratiometric fluorescent thermometer.
ACCESSION   .
VERSION     .
KEYWORDS    gTEMP_pcDNA3
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6939)
  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 6939)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 12, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..6939
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     CDS             149..400
                     /codon_start=1
                     /product="C-terminal fragment of mVenus for use in 
                     bimolecular fluorescence complementation (BiFC) (Kodama and
                     Hu, 2010)"
                     /label=VC155
                     /translation="DKQKNGIKAHFKIRHNIEDGGVQLADHYQQNTPIGDGPVLLPDNH
                     YLSVQSKLSKDPNEKRDHMVLLESVTAAGITLGMDELYK"
     CDS             419..472
                     /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             944..1195
                     /codon_start=1
                     /product="C-terminal fragment of mVenus for use in 
                     bimolecular fluorescence complementation (BiFC) (Kodama and
                     Hu, 2010)"
                     /label=VC155
                     /translation="DKQKNGIKANFKIRHNIEDGGVQLADHYQQNTPIGDGPVLLPDNH
                     YLSIQSKLSKDPNEKRDHMVLLEFVTAAGITLGMDELYK"
     promoter        complement(1259..1277)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(1260..1277)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(1297..1314)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1303..1527
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      1573..2001
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1660..1679)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1870..1891
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2010..2030)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2015..2344
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      2195..2330
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     2257..2276
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             2411..3205
                     /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(2465..2484)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3075..3094
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    3379..3500
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3416..3435)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3470..3489
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(3549..3565)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3549..3565)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3562..3584)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3573..3589
                     /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(3597..3627)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3642..3663
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3780..3797)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3951..4539)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4031..4050)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(4710..5570)
                     /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     5333..5352
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5571..5675)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(5750..5769)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        5941..6320
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        6321..6524
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     6475..6495
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     6569..6588
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        6569..6587
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
ORIGIN
        1 ctacaagacc cgcgccgagg tgaagttcga gggcgacacc ctggtgaacc gcatcgagct
       61 gaagggcatc gacttcaagg aggacggcaa catcctgggg cacaagctgg agtacaacgg
      121 gataagctca aacgtatata tcaccgccga caagcagaag aacggcatca aggcccactt
      181 caagatccgc cacaacatcg aggacggcgg cgtgcagctc gccgaccact accagcagaa
      241 cacccccatc ggcgacggcc ccgtgctgct gcccgacaac cactacctga gcgtccagtc
      301 caagctgagc aaagacccca acgagaagcg cgatcacatg gtcctgctgg agtccgtgac
      361 cgccgccggg atcactctcg gcatggacga gctgtacaag cgcatgcatg gcagcggcga
      421 gggcagaggc agcctgctga cctgcggcga cgtggaggag aaccccggcc ccgagctcat
      481 ggtgagcaag ggcgaggagc tgttcaccgg ggtggtgccc atcctggtcg agctggacgg
      541 cgacgtaaac ggccacaagt tcagcgtgtc cggcgagggc gagggcgatg ccacctacgg
      601 caagctgacc ctgaagttca tctgcaccac cggcaagctg cccgtgccct ggcccaccct
      661 cgtgaccacc ttctcctacg gcgtgatggt gttcgcccgc taccccgacc acatgaagca
      721 gcacgacttc ttcaagtccg ccatgcccga aggctacgtc caggagcgca ccatcttctt
      781 caaggacgac ggcaactaca agacccgcgc cgaggtgaag ttcgagggcg acaccctggt
      841 gaaccgcatc gagctgaagg gcatcgactt caaggaggac ggcaacatcc tggggcacaa
      901 gctggagtac aacttcaaca gccacaacgt ctatatcatg gccgacaagc agaagaacgg
      961 catcaaggcc aacttcaaga tccgccacaa catcgaggac ggcggcgtgc agctcgccga
     1021 ccactaccag cagaacaccc ccatcggcga cggccccgtg ctgctgcccg acaaccacta
     1081 cctgagcatc cagtccaagc tgagcaaaga ccccaacgag aagcgcgatc acatggtcct
     1141 gctggagttc gtgaccgccg ccgggatcac tctcggcatg gacgagctgt acaagtaaga
     1201 attctgcaga tatccatcac actggcggcc gctcgagcat gcatctagag ggccctattc
     1261 tatagtgtca cctaaatgct agagctcgct gatcagcctc gactgtgcct tctagttgcc
     1321 agccatctgt tgtttgcccc tcccccgtgc cttccttgac cctggaaggt gccactccca
     1381 ctgtcctttc ctaataaaat gaggaaattg catcgcattg tctgagtagg tgtcattcta
     1441 ttctgggggg tggggtgggg caggacagca agggggagga ttgggaagac aatagcaggc
     1501 atgctgggga tgcggtgggc tctatggctt ctgaggcgga aagaaccagc tggggctcta
     1561 gggggtatcc ccacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc
     1621 gcagcgtgac cgctacactt gccagcgccc tagcgcccgc tcctttcgct ttcttccctt
     1681 cctttctcgc cacgttcgcc ggctttcccc gtcaagctct aaatcggggg ctccctttag
     1741 ggttccgatt tagtgcttta cggcacctcg accccaaaaa acttgattag ggtgatggtt
     1801 cacgtagtgg gccatcgccc tgatagacgg tttttcgccc tttgacgttg gagtccacgt
     1861 tctttaatag tggactcttg ttccaaactg gaacaacact caaccctatc tcggtctatt
     1921 cttttgattt ataagggatt ttgccgattt cggcctattg gttaaaaaat gagctgattt
     1981 aacaaaaatt taacgcgaat taattctgtg gaatgtgtgt cagttagggt gtggaaagtc
     2041 cccaggctcc ccagcaggca gaagtatgca aagcatgcat ctcaattagt cagcaaccag
     2101 gtgtggaaag tccccaggct ccccagcagg cagaagtatg caaagcatgc atctcaatta
     2161 gtcagcaacc atagtcccgc ccctaactcc gcccatcccg cccctaactc cgcccagttc
     2221 cgcccattct ccgccccatg gctgactaat tttttttatt tatgcagagg ccgaggccgc
     2281 ctctgcctct gagctattcc agaagtagtg aggaggcttt tttggaggcc taggcttttg
     2341 caaaaagctc ccgggagctt gtatatccat tttcggatct gatcaagaga caggatgagg
     2401 atcgtttcgc atgattgaac aagatggatt gcacgcaggt tctccggccg cttgggtgga
     2461 gaggctattc ggctatgact gggcacaaca gacaatcggc tgctctgatg ccgccgtgtt
     2521 ccggctgtca gcgcaggggc gcccggttct ttttgtcaag accgacctgt ccggtgccct
     2581 gaatgaactg caggacgagg cagcgcggct atcgtggctg gccacgacgg gcgttccttg
     2641 cgcagctgtg ctcgacgttg tcactgaagc gggaagggac tggctgctat tgggcgaagt
     2701 gccggggcag gatctcctgt catctcacct tgctcctgcc gagaaagtat ccatcatggc
     2761 tgatgcaatg cggcggctgc atacgcttga tccggctacc tgcccattcg accaccaagc
     2821 gaaacatcgc atcgagcgag cacgtactcg gatggaagcc ggtcttgtcg atcaggatga
     2881 tctggacgaa gagcatcagg ggctcgcgcc agccgaactg ttcgccaggc tcaaggcgcg
     2941 catgcccgac ggcgaggatc tcgtcgtgac ccatggcgat gcctgcttgc cgaatatcat
     3001 ggtggaaaat ggccgctttt ctggattcat cgactgtggc cggctgggtg tggcggaccg
     3061 ctatcaggac atagcgttgg ctacccgtga tattgctgaa gagcttggcg gcgaatgggc
     3121 tgaccgcttc ctcgtgcttt acggtatcgc cgctcccgat tcgcagcgca tcgccttcta
     3181 tcgccttctt gacgagttct tctgagcggg actctggggt tcgaaatgac cgaccaagcg
     3241 acgcccaacc tgccatcacg agatttcgat tccaccgccg ccttctatga aaggttgggc
     3301 ttcggaatcg ttttccggga cgccggctgg atgatcctcc agcgcgggga tctcatgctg
     3361 gagttcttcg cccaccccaa cttgtttatt gcagcttata atggttacaa ataaagcaat
     3421 agcatcacaa atttcacaaa taaagcattt ttttcactgc attctagttg tggtttgtcc
     3481 aaactcatca atgtatctta tcatgtctgt ataccgtcga cctctagcta gagcttggcg
     3541 taatcatggt catagctgtt tcctgtgtga aattgttatc cgctcacaat tccacacaac
     3601 atacgagccg gaagcataaa gtgtaaagcc tggggtgcct aatgagtgag ctaactcaca
     3661 ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat
     3721 taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta ttgggcgctc ttccgcttcc
     3781 tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc agctcactca
     3841 aaggcggtaa tacggttatc cacagaatca ggggataacg caggaaagaa catgtgagca
     3901 aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg
     3961 ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg
     4021 acaggactat aaagatacca ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt
     4081 ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc cttcgggaag cgtggcgctt
     4141 tctcatagct cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc caagctgggc
     4201 tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt
     4261 gagtccaacc cggtaagaca cgacttatcg ccactggcag cagccactgg taacaggatt
     4321 agcagagcga ggtatgtagg cggtgctaca gagttcttga agtggtggcc taactacggc
     4381 tacactagaa gaacagtatt tggtatctgc gctctgctga agccagttac cttcggaaaa
     4441 agagttggta gctcttgatc cggcaaacaa accaccgctg gtagcggtgg tttttttgtt
     4501 tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag aagatccttt gatcttttct
     4561 acggggtctg acgctcagtg gaacgaaaac tcacgttaag ggattttggt catgagatta
     4621 tcaaaaagga tcttcaccta gatcctttta aattaaaaat gaagttttaa atcaatctaa
     4681 agtatatatg agtaaacttg gtctgacagt taccaatgct taatcagtga ggcacctatc
     4741 tcagcgatct gtctatttcg ttcatccata gttgcctgac tccccgtcgt gtagataact
     4801 acgatacggg agggcttacc atctggcccc agtgctgcaa tgataccgcg agacccacgc
     4861 tcaccggctc cagatttatc agcaataaac cagccagccg gaagggccga gcgcagaagt
     4921 ggtcctgcaa ctttatccgc ctccatccag tctattaatt gttgccggga agctagagta
     4981 agtagttcgc cagttaatag tttgcgcaac gttgttgcca ttgctacagg catcgtggtg
     5041 tcacgctcgt cgtttggtat ggcttcattc agctccggtt cccaacgatc aaggcgagtt
     5101 acatgatccc ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc
     5161 agaagtaagt tggccgcagt gttatcactc atggttatgg cagcactgca taattctctt
     5221 actgtcatgc catccgtaag atgcttttct gtgactggtg agtactcaac caagtcattc
     5281 tgagaatagt gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg ggataatacc
     5341 gcgccacata gcagaacttt aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa
     5401 ctctcaagga tcttaccgct gttgagatcc agttcgatgt aacccactcg tgcacccaac
     5461 tgatcttcag catcttttac tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa
     5521 aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt gaatactcat actcttcctt
     5581 tttcaatatt attgaagcat ttatcagggt tattgtctca tgagcggata catatttgaa
     5641 tgtatttaga aaaataaaca aataggggtt ccgcgcacat ttccccgaaa agtgccacct
     5701 gacgtcgacg gatcgggaga tctcccgatc ccctatggtg cactctcagt acaatctgct
     5761 ctgatgccgc atagttaagc cagtatctgc tccctgcttg tgtgttggag gtcgctgagt
     5821 agtgcgcgag caaaatttaa gctacaacaa ggcaaggctt gaccgacaat tgcatgaaga
     5881 atctgcttag ggttaggcgt tttgcgctgc ttcgcgatgt acgggccaga tatacgcgtt
     5941 gacattgatt attgactagt tattaatagt aatcaattac ggggtcatta gttcatagcc
     6001 catatatgga gttccgcgtt acataactta cggtaaatgg cccgcctggc tgaccgccca
     6061 acgacccccg cccattgacg tcaataatga cgtatgttcc catagtaacg ccaataggga
     6121 ctttccattg acgtcaatgg gtggagtatt tacggtaaac tgcccacttg gcagtacatc
     6181 aagtgtatca tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct
     6241 ggcattatgc ccagtacatg accttatggg actttcctac ttggcagtac atctacgtat
     6301 tagtcatcgc tattaccatg gtgatgcggt tttggcagta catcaatggg cgtggatagc
     6361 ggtttgactc acggggattt ccaagtctcc accccattga cgtcaatggg agtttgtttt
     6421 ggcaccaaaa tcaacgggac tttccaaaat gtcgtaacaa ctccgcccca ttgacgcaaa
     6481 tgggcggtag gcgtgtacgg tgggaggtct atataagcag agctctctgg ctaactagag
     6541 aacccactgc ttactggctt atcgaaatta atacgactca ctatagggag acccaagctt
     6601 ggtaccgagc tcggatccca ccatggtgag caagggcgag gagctgttca ccggggtggt
     6661 gcccatcctg gtcgagctgg acggcgacgt aaacggccac aggttcagcg tgtccggcga
     6721 gggcgagggc gatgccacct acggcaagct gaccctgaag ctcatctgca ccaccggcaa
     6781 gctgcccgtg ccctggccca ccctcgtgac caccctgcag ttcggcgtgc tgtgcttcgc
     6841 ccgctacccc gaccacatga agcagcacga cttcttcaag tccgccatgc ccgaaggcta
     6901 cgtccaggag cgcaccatct tcttcaagga cgacggcaa
//
