LOCUS       sequence-123359-        8442 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  A FRET sensor for Galphai3 activation, encoded on a single plasmid..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8442)
  AUTHORS   van Unen J, Stumpf AD, Schmid B, Reinhard NR, Hordijk PL, Hoffmann 
            C, Gadella TW Jr, Goedhart J
  TITLE     A New Generation of FRET Sensors for Robust Measurement of Galphai1,
            Galphai2 and Galphai3 Activation Kinetics in Single Cells.
  JOURNAL   PLoS One. 2016 Jan 22;11(1):e0146789. doi: 
            10.1371/journal.pone.0146789. eCollection 2016.
  PUBMED    26799488
REFERENCE   2  (bases 1 to 8442)
  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: "2016-01-22"; volume: "11(1):e0146789. doi"; pages: " 
            10.1371/journal.pone.0146789. eCollection 2016"
COMMENT     Sequence Label: G-beta-2A-cpV-G-gamma-2-IRES-G-alpha-i3-mTq2
FEATURES             Location/Qualifiers
     source          1..8442
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        61..364
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        365..568
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     519..539
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      607..616
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1633..1686
                     /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             1693..2427
                     /codon_start=1
                     /product="circularly permuted form of the Venus yellow 
                     fluorescent protein (Nagai et al., 2004)"
                     /label=cp173Venus
                     /note="efficient FRET acceptor for CFP"
                     /translation="MDGGVQLADHYQQNTPIGDGPVLLPDNHYLSYQSALSKDPNEKRD
                     HMVLLEFVTAAGITLGMDELYKGGSGGMVSKGEELFTGVVPILVELDGDVNGHKFSVSG
                     EGEGDATYGKLTLKLICTTGKLPVPWPTLVTTLGYGLQCFARYPDHMKQHDFFKSAMPE
                     GYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHN
                     VYITADKQKNGIKANFKIRHNIE"
     primer_bind     complement(2727..2744)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     misc_feature    2756..3329
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     misc_feature    2779..3331
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(2923..2940)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     3150..3169
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             join(3682..3684,3685..3687,3688..4200)
                     /codon_start=1
                     /product="N-terminal fragment of mVenus for use in 
                     bimolecular fluorescence complementation (BiFC) (Kodama and
                     Hu, 2010)"
                     /label=VN173
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLSWGVQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYFSDNVYITADKQKNGIK
                     ANFKIRHNIE"
     polyA_signal    5230..5351
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5267..5286)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5321..5340
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(5358..5813)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5495..5516)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5707..5726
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        5840..5944
                     /gene="bla"
                     /label=AmpR promoter
     promoter        5946..6303
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        5946..6137
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(5969..5989)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      6154..6289
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             6338..7132
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     CDS             6338..7132
                     /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
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(6392..6411)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     7002..7021
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(7320..7339)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    7364..7411
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      7740..8328
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     8229..8248
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata tggagttccg
       61 cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc cccgcccatt
      121 gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc attgacgtca
      181 atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt atcatatgcc
      241 aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt atgcccagta
      301 catgacctta tgggactttc ctacttggca gtacatctac gtattagtca tcgctattac
      361 catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg actcacgggg
      421 atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc aaaatcaacg
      481 ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg gtaggcgtgt
      541 acggtgggag gtctatataa gcagagctgg tttagtgaac cgtcagatcc gctagcgcta
      601 ccggtcgcca ccatggggga gcttgaccag ttacggcagg aggccgagca acttaagaac
      661 cagattcgag acgccaggaa agcatgtgca gatgcaactc tctctcagat cacaaacaac
      721 atcgacccag tgggaagaat ccaaatgcgc acgaggagga cactgcgggg gcacctggcc
      781 aagatctacg ccatgcactg gggcacagac tccaggcttc tcgtcagtgc ctcgcaggat
      841 ggtaaactta tcatctggga cagctacacc accaacaagg tccacgccat ccctctgcgc
      901 tcctcctggg tcatgacctg tgcatatgcc ccttctggga actatgtggc ctgcggtggc
      961 ctggataaca tttgctccat ttacaatctg aaaactcgtg aggggaacgt gcgcgtgagt
     1021 cgtgagctgg caggacacac aggttacctg tcctgctgcc gattcctgga tgacaatcag
     1081 atcgtcacca gctctggaga caccacgtgt gccctgtggg acatcgagac cggccagcag
     1141 acgaccacgt ttaccggaca cactggagat gtcatgagcc tttctcttgc tcctgacacc
     1201 agactgttcg tctctggtgc ttgtgatgct tcagccaaac tctgggatgt gcgagaaggc
     1261 atgtgccggc agaccttcac tggccacgag tctgacatca atgccatttg cttctttcca
     1321 aatggcaatg catttgccac tggctcagac gacgccacct gcaggctgtt tgaccttcgt
     1381 gctgaccagg agctcatgac ttactcccat gacaacatca tctgcgggat cacctctgtc
     1441 tccttctcca agagcgggcg cctcctcctt gctgggtacg acgacttcaa ctgcaacgtc
     1501 tgggatgcac tcaaagccga ccgggcaggt gtcttggctg ggcatgacaa ccgcgtcagc
     1561 tgcctgggcg tgactgacga tggcatggct gtggcgacag ggtcctggga tagcttcctc
     1621 aagatctgga acgagggcag aggaagtctt ctaacatgcg gtgacgtgga ggagaatccc
     1681 ggccctaccg gtatggacgg cggcgtgcag ctcgccgacc actaccagca gaacaccccc
     1741 atcggcgacg gccccgtgct gctgcccgac aaccactacc tgagctacca gtccgccctg
     1801 agcaaagacc ccaacgagaa gcgcgatcac atggtcctgc tggagttcgt gaccgccgcc
     1861 gggatcactc tcggcatgga cgagctgtac aagggtggca gcggtggcat ggtgagcaag
     1921 ggcgaggagc tgttcaccgg ggtggtgccc atcctggtcg agctggacgg cgacgtaaac
     1981 ggccacaagt tcagcgtgtc cggcgagggc gagggcgatg ccacctacgg caagctgacc
     2041 ctgaagctga tctgcaccac cggcaagctg cccgtgccct ggcccaccct cgtgaccacc
     2101 ctgggctacg gcctgcagtg cttcgcccgc taccccgacc acatgaagca gcacgacttc
     2161 ttcaagtccg ccatgcccga aggctacgtc caggagcgca ccatcttctt caaggacgac
     2221 ggcaactaca agacccgcgc cgaggtgaag ttcgagggcg acaccctggt gaaccgcatc
     2281 gagctgaagg gcatcgactt caaggaggac ggcaacatcc tggggcacaa gctggagtac
     2341 aactacaaca gccacaacgt ctatatcacc gccgacaagc agaagaacgg catcaaggcc
     2401 aacttcaaga tccgccacaa catcgagtcc ggactcagat ctatggccag caacaacacc
     2461 gccagcatag cacaagccag gaagctggta gagcagctta agatggaagc caatatcgac
     2521 aggataaagg tgtccaaggc agctgcagat ttgatggcct actgtgaagc acatgccaag
     2581 gaagaccccc tcctgacccc tgttccggct tcagaaaacc cgtttaggga gaagaagttt
     2641 ttctgtgcca tcctttaacc gcggcgtacg aggcctgcat gctccggacc tgcaggttcg
     2701 aagtcgacag atctcaattg gggcccctat agtgtcacct aaataattcc gcccccccct
     2761 ctccctcccc cccccctaac gttactggcc gaagccgctt ggaataaggc cggtgtgcgt
     2821 ttgtctatat gttattttcc accatattgc cgtcttttgg caatgtgagg gcccggaaac
     2881 ctggccctgt cttcttgacg agcattccta ggggtctttc ccctctcgcc aaaggaatgc
     2941 aaggtctgtt gaatgtcgtg aaggaagcag ttcctctgga agcttcttga agacaaacaa
     3001 cgtctgtagc gaccctttgc aggcagcgga accccccacc tggcgacagg tgcctctgcg
     3061 gccaaaagcc acgtgtataa gatacacctg caaaggcggc acaaccccag tgccacgttg
     3121 tgagttggat agttgtggaa agagtcaaat ggctctcctc aagcgtattc aacaaggggc
     3181 tgaaggatgc ccagaaggta ccccattgta tgggatctga tctggggcct cggtgcacat
     3241 gctttacatg tgtttagtcg aggttaaaaa aacgtctagg ccccccgaac cacggggacg
     3301 tggttttcct ttgaaaaaca cgatgataat atgggctgca cgttgagcgc cgaagacaag
     3361 gcggcagtgg agcgaagcaa gatgatcgac cgcaacttac gggaggacgg ggaaaaagcg
     3421 gccaaagaag tgaagctgct gctactcggt gctggagaat ctggtaaaag caccattgtg
     3481 aaacagatga aaatcattca tgaggatggc tattcagagg atgaatgtaa acaatataaa
     3541 gtagttgtct acagcaatac tatacagtcc atcattgcaa tcataagagc catgggacgg
     3601 ctaaagattg actttgggga agctgccagg gcagatgatg cccggcaatt atttgtttta
     3661 gctggcagtg ctaccggttc tatggtgagc aagggcgagg agctgttcac cggggtggtg
     3721 cccatcctgg tcgagctgga cggcgacgta aacggccaca agttcagcgt gtccggcgag
     3781 ggcgagggcg atgccaccta cggcaagctg accctgaagt tcatctgcac caccggcaag
     3841 ctgcccgtgc cctggcccac cctcgtgacc accctgtcct ggggcgtgca gtgcttcgcc
     3901 cgctaccccg accacatgaa gcagcacgac ttcttcaagt ccgccatgcc cgaaggctac
     3961 gtccaggagc gcaccatctt cttcaaggac gacggcaact acaagacccg cgccgaggtg
     4021 aagttcgagg gcgacaccct ggtgaaccgc atcgagctga agggcatcga cttcaaggag
     4081 gacggcaaca tcctggggca caagctggag tacaactact ttagcgacaa cgtctatatc
     4141 accgccgaca agcagaagaa cggcatcaag gccaacttca agatccgcca caacatcgag
     4201 gacggcggcg tgcagctcgc cgaccactac cagcagaaca cccccatcgg cgacggcccc
     4261 gtgctgctgc ccgacaacca ctacctgagc acccagtcca agctgagcaa agaccccaac
     4321 gagaagcgcg atcacatggt cctgctggag ttcgtgaccg ccgccgggat caccggtgaa
     4381 gaaggagtca tgactccaga actagcagga gtgattaaac ggttatggcg agatggtggg
     4441 gtacaagctt gcttcagcag atccagggaa tatcagctca atgattctgc ttcatattat
     4501 ctaaatgatc tggatagaat atcccagtct aactacattc caactcagca agatgttctt
     4561 cggacgagag tgaagaccac aggcattgta gaaacacatt tcaccttcaa agacctatac
     4621 ttcaagatgt ttgatgtagg tggccaaaga tcagaacgaa aaaagtggat tcactgtttt
     4681 gagggagtga cagcaattat cttctgtgtg gccctcagtg attatgacct tgttctggct
     4741 gaggacgagg agatgaaccg aatgcatgaa agcatgaaac tgtttgacag catttgtaat
     4801 aacaaatggt ttacagaaac ttcaatcatt ctcttcctta acaagaaaga cctttttgag
     4861 gaaaaaataa agaggagtcc gttaactatc tgttatccag aatacacagg ttccaataca
     4921 tatgaagagg cagctgccta tattcaatgc cagtttgaag atctgaacag aagaaaagat
     4981 accaaggaga tctatactca cttcacctgt gccacagaca cgaagaatgt gcagtttgtt
     5041 tttgatgctg ttacagatgt catcattaaa aacaacttaa aggaatgtgg actttattaa
     5101 gcttctagag ggccgcgact ctagatcata atcagccata ccacatttgt agaggtttta
     5161 cttgctttaa aaaacctccc acacctcccc ctgaacctga aacataaaat gaatgcaatt
     5221 gttgttgtta acttgtttat tgcagcttat aatggttaca aataaagcaa tagcatcaca
     5281 aatttcacaa ataaagcatt tttttcactg cattctagtt gtggtttgtc caaactcatc
     5341 aatgtatctt aaggcgtaaa ttgtaagcgt taatattttg ttaaaattcg cgttaaattt
     5401 ttgttaaatc agctcatttt ttaaccaata ggccgaaatc ggcaaaatcc cttataaatc
     5461 aaaagaatag accgagatag ggttgagtgt tgttccagtt tggaacaaga gtccactatt
     5521 aaagaacgtg gactccaacg tcaaagggcg aaaaaccgtc tatcagggcg atggcccact
     5581 acgtgaacca tcaccctaat caagtttttt ggggtcgagg tgccgtaaag cactaaatcg
     5641 gaaccctaaa gggagccccc gatttagagc ttgacgggga aagccggcga acgtggcgag
     5701 aaaggaaggg aagaaagcga aaggagcggg cgctagggcg ctggcaagtg tagcggtcac
     5761 gctgcgcgta accaccacac ccgccgcgct taatgcgccg ctacagggcg cgtcaggtgg
     5821 cacttttcgg ggaaatgtgc gcggaacccc tatttgttta tttttctaaa tacattcaaa
     5881 tatgtatccg ctcatgagac aataaccctg ataaatgctt caataatatt gaaaaaggaa
     5941 gagtcctgag gcggaaagaa ccagctgtgg aatgtgtgtc agttagggtg tggaaagtcc
     6001 ccaggctccc cagcaggcag aagtatgcaa agcatgcatc tcaattagtc agcaaccagg
     6061 tgtggaaagt ccccaggctc cccagcaggc agaagtatgc aaagcatgca tctcaattag
     6121 tcagcaacca tagtcccgcc cctaactccg cccatcccgc ccctaactcc gcccagttcc
     6181 gcccattctc cgccccatgg ctgactaatt ttttttattt atgcagaggc cgaggccgcc
     6241 tcggcctctg agctattcca gaagtagtga ggaggctttt ttggaggcct aggcttttgc
     6301 aaagatcgat caagagacag gatgaggatc gtttcgcatg attgaacaag atggattgca
     6361 cgcaggttct ccggccgctt gggtggagag gctattcggc tatgactggg cacaacagac
     6421 aatcggctgc tctgatgccg ccgtgttccg gctgtcagcg caggggcgcc cggttctttt
     6481 tgtcaagacc gacctgtccg gtgccctgaa tgaactgcaa gacgaggcag cgcggctatc
     6541 gtggctggcc acgacgggcg ttccttgcgc agctgtgctc gacgttgtca ctgaagcggg
     6601 aagggactgg ctgctattgg gcgaagtgcc ggggcaggat ctcctgtcat ctcaccttgc
     6661 tcctgccgag aaagtatcca tcatggctga tgcaatgcgg cggctgcata cgcttgatcc
     6721 ggctacctgc ccattcgacc accaagcgaa acatcgcatc gagcgagcac gtactcggat
     6781 ggaagccggt cttgtcgatc aggatgatct ggacgaagag catcaggggc tcgcgccagc
     6841 cgaactgttc gccaggctca aggcgagcat gcccgacggc gaggatctcg tcgtgaccca
     6901 tggcgatgcc tgcttgccga atatcatggt ggaaaatggc cgcttttctg gattcatcga
     6961 ctgtggccgg ctgggtgtgg cggaccgcta tcaggacata gcgttggcta cccgtgatat
     7021 tgctgaagag cttggcggcg aatgggctga ccgcttcctc gtgctttacg gtatcgccgc
     7081 tcccgattcg cagcgcatcg ccttctatcg ccttcttgac gagttcttct gagcgggact
     7141 ctggggttcg aaatgaccga ccaagcgacg cccaacctgc catcacgaga tttcgattcc
     7201 accgccgcct tctatgaaag gttgggcttc ggaatcgttt tccgggacgc cggctggatg
     7261 atcctccagc gcggggatct catgctggag ttcttcgccc accctagggg gaggctaact
     7321 gaaacacgga aggagacaat accggaagga acccgcgcta tgacggcaat aaaaagacag
     7381 aataaaacgc acggtgttgg gtcgtttgtt cataaacgcg gggttcggtc ccagggctgg
     7441 cactctgtcg ataccccacc gagaccccat tggggccaat acgcccgcgt ttcttccttt
     7501 tccccacccc accccccaag ttcgggtgaa ggcccagggc tcgcagccaa cgtcggggcg
     7561 gcaggccctg ccatagcctc aggttactca tatatacttt agattgattt aaaacttcat
     7621 ttttaattta aaaggatcta ggtgaagatc ctttttgata atctcatgac caaaatccct
     7681 taacgtgagt tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa aggatcttct
     7741 tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca
     7801 gcggtggttt gtttgccgga tcaagagcta ccaactcttt ttccgaaggt aactggcttc
     7861 agcagagcgc agataccaaa tactgtcctt ctagtgtagc cgtagttagg ccaccacttc
     7921 aagaactctg tagcaccgcc tacatacctc gctctgctaa tcctgttacc agtggctgct
     7981 gccagtggcg ataagtcgtg tcttaccggg ttggactcaa gacgatagtt accggataag
     8041 gcgcagcggt cgggctgaac ggggggttcg tgcacacagc ccagcttgga gcgaacgacc
     8101 tacaccgaac tgagatacct acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg
     8161 agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa caggagagcg cacgagggag
     8221 cttccagggg gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca cctctgactt
     8281 gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac
     8341 gcggcctttt tacggttcct ggccttttgc tggccttttg ctcacatgtt ctttcctgcg
     8401 ttatcccctg attctgtgga taaccgtatt accgccatgc at
//
