LOCUS       sequence-459280-        8445 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  A FRET sensor for Galphai1 activation, encoded on a single plasmid..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8445)
  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 8445)
  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-i1-mTq2
FEATURES             Location/Qualifiers
     source          1..8445
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        72..375
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        376..579
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     530..550
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      618..627
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1644..1697
                     /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             1704..2438
                     /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(2738..2755)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     misc_feature    2767..3340
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     misc_feature    2790..3342
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(2934..2951)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     3161..3180
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             join(3714..3716,3717..3719,3720..4232)
                     /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    5244..5365
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5281..5300)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5335..5354
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(5372..5827)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5509..5530)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5721..5740
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        5854..5958
                     /gene="bla"
                     /label=AmpR promoter
     promoter        5960..6317
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        5960..6151
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(5983..6003)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      6168..6303
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             6352..7146
                     /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             6352..7146
                     /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(6406..6425)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     7016..7035
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(7334..7353)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    7378..7425
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      7754..8342
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     8243..8262
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 ccgccatgca ttagttatta atagtaatca attacggggt cattagttca tagcccatat
       61 atggagttcc gcgttacata acttacggta aatggcccgc ctggctgacc gcccaacgac
      121 ccccgcccat tgacgtcaat aatgacgtat gttcccatag taacgccaat agggactttc
      181 cattgacgtc aatgggtgga gtatttacgg taaactgccc acttggcagt acatcaagtg
      241 tatcatatgc caagtacgcc ccctattgac gtcaatgacg gtaaatggcc cgcctggcat
      301 tatgcccagt acatgacctt atgggacttt cctacttggc agtacatcta cgtattagtc
      361 atcgctatta ccatggtgat gcggttttgg cagtacatca atgggcgtgg atagcggttt
      421 gactcacggg gatttccaag tctccacccc attgacgtca atgggagttt gttttggcac
      481 caaaatcaac gggactttcc aaaatgtcgt aacaactccg ccccattgac gcaaatgggc
      541 ggtaggcgtg tacggtggga ggtctatata agcagagctg gtttagtgaa ccgtcagatc
      601 cgctagcgct accggtcgcc accatggggg agcttgacca gttacggcag gaggccgagc
      661 aacttaagaa ccagattcga gacgccagga aagcatgtgc agatgcaact ctctctcaga
      721 tcacaaacaa catcgaccca gtgggaagaa tccaaatgcg cacgaggagg acactgcggg
      781 ggcacctggc caagatctac gccatgcact ggggcacaga ctccaggctt ctcgtcagtg
      841 cctcgcagga tggtaaactt atcatctggg acagctacac caccaacaag gtccacgcca
      901 tccctctgcg ctcctcctgg gtcatgacct gtgcatatgc cccttctggg aactatgtgg
      961 cctgcggtgg cctggataac atttgctcca tttacaatct gaaaactcgt gaggggaacg
     1021 tgcgcgtgag tcgtgagctg gcaggacaca caggttacct gtcctgctgc cgattcctgg
     1081 atgacaatca gatcgtcacc agctctggag acaccacgtg tgccctgtgg gacatcgaga
     1141 ccggccagca gacgaccacg tttaccggac acactggaga tgtcatgagc ctttctcttg
     1201 ctcctgacac cagactgttc gtctctggtg cttgtgatgc ttcagccaaa ctctgggatg
     1261 tgcgagaagg catgtgccgg cagaccttca ctggccacga gtctgacatc aatgccattt
     1321 gcttctttcc aaatggcaat gcatttgcca ctggctcaga cgacgccacc tgcaggctgt
     1381 ttgaccttcg tgctgaccag gagctcatga cttactccca tgacaacatc atctgcggga
     1441 tcacctctgt ctccttctcc aagagcgggc gcctcctcct tgctgggtac gacgacttca
     1501 actgcaacgt ctgggatgca ctcaaagccg accgggcagg tgtcttggct gggcatgaca
     1561 accgcgtcag ctgcctgggc gtgactgacg atggcatggc tgtggcgaca gggtcctggg
     1621 atagcttcct caagatctgg aacgagggca gaggaagtct tctaacatgc ggtgacgtgg
     1681 aggagaatcc cggccctacc ggtatggacg gcggcgtgca gctcgccgac cactaccagc
     1741 agaacacccc catcggcgac ggccccgtgc tgctgcccga caaccactac ctgagctacc
     1801 agtccgccct gagcaaagac cccaacgaga agcgcgatca catggtcctg ctggagttcg
     1861 tgaccgccgc cgggatcact ctcggcatgg acgagctgta caagggtggc agcggtggca
     1921 tggtgagcaa gggcgaggag ctgttcaccg gggtggtgcc catcctggtc gagctggacg
     1981 gcgacgtaaa cggccacaag ttcagcgtgt ccggcgaggg cgagggcgat gccacctacg
     2041 gcaagctgac cctgaagctg atctgcacca ccggcaagct gcccgtgccc tggcccaccc
     2101 tcgtgaccac cctgggctac ggcctgcagt gcttcgcccg ctaccccgac cacatgaagc
     2161 agcacgactt cttcaagtcc gccatgcccg aaggctacgt ccaggagcgc accatcttct
     2221 tcaaggacga cggcaactac aagacccgcg ccgaggtgaa gttcgagggc gacaccctgg
     2281 tgaaccgcat cgagctgaag ggcatcgact tcaaggagga cggcaacatc ctggggcaca
     2341 agctggagta caactacaac agccacaacg tctatatcac cgccgacaag cagaagaacg
     2401 gcatcaaggc caacttcaag atccgccaca acatcgagtc cggactcaga tctatggcca
     2461 gcaacaacac cgccagcata gcacaagcca ggaagctggt agagcagctt aagatggaag
     2521 ccaatatcga caggataaag gtgtccaagg cagctgcaga tttgatggcc tactgtgaag
     2581 cacatgccaa ggaagacccc ctcctgaccc ctgttccggc ttcagaaaac ccgtttaggg
     2641 agaagaagtt tttctgtgcc atcctttaac cgcggcgtac gaggcctgca tgctccggac
     2701 ctgcaggttc gaagtcgaca gatctcaatt ggggccccta tagtgtcacc taaataattc
     2761 cgcccccccc tctccctccc ccccccctaa cgttactggc cgaagccgct tggaataagg
     2821 ccggtgtgcg tttgtctata tgttattttc caccatattg ccgtcttttg gcaatgtgag
     2881 ggcccggaaa cctggccctg tcttcttgac gagcattcct aggggtcttt cccctctcgc
     2941 caaaggaatg caaggtctgt tgaatgtcgt gaaggaagca gttcctctgg aagcttcttg
     3001 aagacaaaca acgtctgtag cgaccctttg caggcagcgg aaccccccac ctggcgacag
     3061 gtgcctctgc ggccaaaagc cacgtgtata agatacacct gcaaaggcgg cacaacccca
     3121 gtgccacgtt gtgagttgga tagttgtgga aagagtcaaa tggctctcct caagcgtatt
     3181 caacaagggg ctgaaggatg cccagaaggt accccattgt atgggatctg atctggggcc
     3241 tcggtgcaca tgctttacat gtgtttagtc gaggttaaaa aaacgtctag gccccccgaa
     3301 ccacggggac gtggttttcc tttgaaaaac acgatgataa tatgggctgc acactgagcg
     3361 ctgaggacaa ggcggccgtg gagcgcagca agatgatcga ccgcaacctc cgggaggacg
     3421 gagagaaggc agcgcgcgag gtcaagctgc tgctgctggg tgctggtgaa tccgggaaga
     3481 gcacaattgt gaagcagatg aaaattatcc acgaggctgg ctactcagag gaagagtgta
     3541 agcagtacaa agcagtggtc tacagcaaca ccatccagtc catcattgcc atcattagag
     3601 ctatggggag attgaaaatc gactttggag acgctgctcg tgcggatgat gctcgccaac
     3661 tcttcgtgct tgctggggct gcagaggaag gctttatgac cgcgaccggt tctatggtga
     3721 gcaagggcga ggagctgttc accggggtgg tgcccatcct ggtcgagctg gacggcgacg
     3781 taaacggcca caagttcagc gtgtccggcg agggcgaggg cgatgccacc tacggcaagc
     3841 tgaccctgaa gttcatctgc accaccggca agctgcccgt gccctggccc accctcgtga
     3901 ccaccctgtc ctggggcgtg cagtgcttcg cccgctaccc cgaccacatg aagcagcacg
     3961 acttcttcaa gtccgccatg cccgaaggct acgtccagga gcgcaccatc ttcttcaagg
     4021 acgacggcaa ctacaagacc cgcgccgagg tgaagttcga gggcgacacc ctggtgaacc
     4081 gcatcgagct gaagggcatc gacttcaagg aggacggcaa catcctgggg cacaagctgg
     4141 agtacaacta ctttagcgac aacgtctata tcaccgccga caagcagaag aacggcatca
     4201 aggccaactt caagatccgc cacaacatcg aggacggcgg cgtgcagctc gccgaccact
     4261 accagcagaa cacccccatc ggcgacggcc ccgtgctgct gcccgacaac cactacctga
     4321 gcacccagtc caagctgagc aaagacccca acgagaagcg cgatcacatg gtcctgctgg
     4381 agttcgtgac cgccgccggg atcaccggtg aactcgccgg cgtcataaag agactgtgga
     4441 aggacagcgg tgtgcaagcc tgcttcaaca gatcccggga gtaccagctg aacgattcgg
     4501 cggcgtacta cctgaatgac ttggacagaa tagcacaacc aaattacatc ccaacccagc
     4561 aggatgttct cagaactaga gtgaaaacga cgggaattgt ggaaacccac tttactttca
     4621 aagatcttca ttttaaaatg tttgacgtgg gaggccagag atcagagcgg aagaagtgga
     4681 ttcactgctt tgaaggcgtg actgccatca tcttctgtgt ggccctgagt gactatgacc
     4741 tggttcttgc tgaggatgaa gaaatgaacc ggatgcacga aagcatgaag ctgttcgata
     4801 gcatatgtaa caacaagtgg tttacggaca catccatcat ccttttcctg aacaagaagg
     4861 acctcttcga agagaagatc aaaaagagtc ccctcacgat atgctatcca gaatatgcag
     4921 gctcaaacac atatgaagag gcggctgcgt atatccagtg tcagtttgaa gacctcaata
     4981 aaaggaagga cacaaaggaa atttacaccc acttcacttg cgccacggat acgaagaatg
     5041 tgcagtttgt gttcgatgct gtaacggacg tcatcataaa gaataaccta aaagactgtg
     5101 gtctcttcta agctggatcc accggatcta gataactgat cataatcagc cataccacat
     5161 ttgtagaggt tttacttgct ttaaaaaacc tcccacacct ccccctgaac ctgaaacata
     5221 aaatgaatgc aattgttgtt gttaacttgt ttattgcagc ttataatggt tacaaataaa
     5281 gcaatagcat cacaaatttc acaaataaag catttttttc actgcattct agttgtggtt
     5341 tgtccaaact catcaatgta tcttaaggcg taaattgtaa gcgttaatat tttgttaaaa
     5401 ttcgcgttaa atttttgtta aatcagctca ttttttaacc aataggccga aatcggcaaa
     5461 atcccttata aatcaaaaga atagaccgag atagggttga gtgttgttcc agtttggaac
     5521 aagagtccac tattaaagaa cgtggactcc aacgtcaaag ggcgaaaaac cgtctatcag
     5581 ggcgatggcc cactacgtga accatcaccc taatcaagtt ttttggggtc gaggtgccgt
     5641 aaagcactaa atcggaaccc taaagggagc ccccgattta gagcttgacg gggaaagccg
     5701 gcgaacgtgg cgagaaagga agggaagaaa gcgaaaggag cgggcgctag ggcgctggca
     5761 agtgtagcgg tcacgctgcg cgtaaccacc acacccgccg cgcttaatgc gccgctacag
     5821 ggcgcgtcag gtggcacttt tcggggaaat gtgcgcggaa cccctatttg tttatttttc
     5881 taaatacatt caaatatgta tccgctcatg agacaataac cctgataaat gcttcaataa
     5941 tattgaaaaa ggaagagtcc tgaggcggaa agaaccagct gtggaatgtg tgtcagttag
     6001 ggtgtggaaa gtccccaggc tccccagcag gcagaagtat gcaaagcatg catctcaatt
     6061 agtcagcaac caggtgtgga aagtccccag gctccccagc aggcagaagt atgcaaagca
     6121 tgcatctcaa ttagtcagca accatagtcc cgcccctaac tccgcccatc ccgcccctaa
     6181 ctccgcccag ttccgcccat tctccgcccc atggctgact aatttttttt atttatgcag
     6241 aggccgaggc cgcctcggcc tctgagctat tccagaagta gtgaggaggc ttttttggag
     6301 gcctaggctt ttgcaaagat cgatcaagag acaggatgag gatcgtttcg catgattgaa
     6361 caagatggat tgcacgcagg ttctccggcc gcttgggtgg agaggctatt cggctatgac
     6421 tgggcacaac agacaatcgg ctgctctgat gccgccgtgt tccggctgtc agcgcagggg
     6481 cgcccggttc tttttgtcaa gaccgacctg tccggtgccc tgaatgaact gcaagacgag
     6541 gcagcgcggc tatcgtggct ggccacgacg ggcgttcctt gcgcagctgt gctcgacgtt
     6601 gtcactgaag cgggaaggga ctggctgcta ttgggcgaag tgccggggca ggatctcctg
     6661 tcatctcacc ttgctcctgc cgagaaagta tccatcatgg ctgatgcaat gcggcggctg
     6721 catacgcttg atccggctac ctgcccattc gaccaccaag cgaaacatcg catcgagcga
     6781 gcacgtactc ggatggaagc cggtcttgtc gatcaggatg atctggacga agagcatcag
     6841 gggctcgcgc cagccgaact gttcgccagg ctcaaggcga gcatgcccga cggcgaggat
     6901 ctcgtcgtga cccatggcga tgcctgcttg ccgaatatca tggtggaaaa tggccgcttt
     6961 tctggattca tcgactgtgg ccggctgggt gtggcggacc gctatcagga catagcgttg
     7021 gctacccgtg atattgctga agagcttggc ggcgaatggg ctgaccgctt cctcgtgctt
     7081 tacggtatcg ccgctcccga ttcgcagcgc atcgccttct atcgccttct tgacgagttc
     7141 ttctgagcgg gactctgggg ttcgaaatga ccgaccaagc gacgcccaac ctgccatcac
     7201 gagatttcga ttccaccgcc gccttctatg aaaggttggg cttcggaatc gttttccggg
     7261 acgccggctg gatgatcctc cagcgcgggg atctcatgct ggagttcttc gcccacccta
     7321 gggggaggct aactgaaaca cggaaggaga caataccgga aggaacccgc gctatgacgg
     7381 caataaaaag acagaataaa acgcacggtg ttgggtcgtt tgttcataaa cgcggggttc
     7441 ggtcccaggg ctggcactct gtcgataccc caccgagacc ccattggggc caatacgccc
     7501 gcgtttcttc cttttcccca ccccaccccc caagttcggg tgaaggccca gggctcgcag
     7561 ccaacgtcgg ggcggcaggc cctgccatag cctcaggtta ctcatatata ctttagattg
     7621 atttaaaact tcatttttaa tttaaaagga tctaggtgaa gatccttttt gataatctca
     7681 tgaccaaaat cccttaacgt gagttttcgt tccactgagc gtcagacccc gtagaaaaga
     7741 tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa
     7801 aaccaccgct accagcggtg gtttgtttgc cggatcaaga gctaccaact ctttttccga
     7861 aggtaactgg cttcagcaga gcgcagatac caaatactgt tcttctagtg tagccgtagt
     7921 taggccacca cttcaagaac tctgtagcac cgcctacata cctcgctctg ctaatcctgt
     7981 taccagtggc tgctgccagt ggcgataagt cgtgtcttac cgggttggac tcaagacgat
     8041 agttaccgga taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca cagcccagct
     8101 tggagcgaac gacctacacc gaactgagat acctacagcg tgagctatga gaaagcgcca
     8161 cgcttcccga agggagaaag gcggacaggt atccggtaag cggcagggtc ggaacaggag
     8221 agcgcacgag ggagcttcca gggggaaacg cctggtatct ttatagtcct gtcgggtttc
     8281 gccacctctg acttgagcgt cgatttttgt gatgctcgtc aggggggcgg agcctatgga
     8341 aaaacgccag caacgcggcc tttttacggt tcctggcctt ttgctggcct tttgctcaca
     8401 tgttctttcc tgcgttatcc cctgattctg tggataaccg tatta
//
