LOCUS       sequence-123358-        8445 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  A FRET sensor for Galphai2 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-i2-mTq2
FEATURES             Location/Qualifiers
     source          1..8445
                     /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    5233..5354
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5270..5289)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5324..5343
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(5361..5816)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5498..5519)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5710..5729
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        5843..5947
                     /gene="bla"
                     /label=AmpR promoter
     promoter        5949..6306
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        5949..6140
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(5972..5992)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      6157..6292
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             6341..7135
                     /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             6341..7135
                     /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(6395..6414)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     7005..7024
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(7323..7342)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    7367..7414
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      7743..8331
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     8232..8251
                     /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 ccgtgagcgc cgaggacaag
     3361 gcggcggccg agcgctctaa gatgatcgac aagaacctgc gggaggacgg agagaaggcg
     3421 gcgcgggagg tgaagttgct gctgttgggt gctggggagt cagggaagag caccatcgtc
     3481 aagcagatga agatcatcca cgaggatggc tactccgagg aggaatgccg gcagtaccgg
     3541 gcggttgtct acagcaacac catccagtcc atcatggcca ttgtcaaagc catgggcaac
     3601 ctgcagatcg actttgccga cccctccaga gcggacgacg ccaggcagct atttgcactg
     3661 tcctgcaccg ccaccggttc 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 caccggtgag
     4381 gagcaaggcg tgctccctga tgacctgtcc ggcgtcatcc ggaggctctg ggctgaccat
     4441 ggtgtgcagg cctgctttgg ccgctcaagg gaataccagc tcaacgactc agctgcctac
     4501 tacctgaacg acctggagcg tattgcacag agtgactaca tccccacaca gcaagatgtg
     4561 ctacggaccc gcgtaaagac cacggggatc gtggagacac acttcacctt caaggaccta
     4621 cacttcaaga tgtttgatgt gggtggtcag cggtctgagc ggaagaagtg gatccactgc
     4681 tttgagggcg tcacagccat catcttctgc gtagccttga gcgcctatga cttggtgcta
     4741 gctgaggacg aggagatgaa ccgcatgcat gagagcatga agctattcga tagcatctgc
     4801 aacaacaagt ggttcacaga cacgtccatc atcctcttcc tcaacaagaa ggacctgttt
     4861 gaggagaaga tcacacacag tcccctgacc atctgcttcc ctgagtacac aggggccaac
     4921 aaatatgatg aggcagccag ctacatccag agtaagtttg aggacctgaa taagcgcaaa
     4981 gacaccaagg agatctacac gcacttcacg tgcgccaccg acaccaagaa cgtgcagttc
     5041 gtgtttgacg ccgtcaccga tgtcatcatc aagaacaacc tgaaggactg cggcctcttc
     5101 tgagcttcta gagggccgcg actctagatc ataatcagcc ataccacatt tgtagaggtt
     5161 ttacttgctt taaaaaacct cccacacctc cccctgaacc tgaaacataa aatgaatgca
     5221 attgttgttg ttaacttgtt tattgcagct tataatggtt acaaataaag caatagcatc
     5281 acaaatttca caaataaagc atttttttca ctgcattcta gttgtggttt gtccaaactc
     5341 atcaatgtat cttaaggcgt aaattgtaag cgttaatatt ttgttaaaat tcgcgttaaa
     5401 tttttgttaa atcagctcat tttttaacca ataggccgaa atcggcaaaa tcccttataa
     5461 atcaaaagaa tagaccgaga tagggttgag tgttgttcca gtttggaaca agagtccact
     5521 attaaagaac gtggactcca acgtcaaagg gcgaaaaacc gtctatcagg gcgatggccc
     5581 actacgtgaa ccatcaccct aatcaagttt tttggggtcg aggtgccgta aagcactaaa
     5641 tcggaaccct aaagggagcc cccgatttag agcttgacgg ggaaagccgg cgaacgtggc
     5701 gagaaaggaa gggaagaaag cgaaaggagc gggcgctagg gcgctggcaa gtgtagcggt
     5761 cacgctgcgc gtaaccacca cacccgccgc gcttaatgcg ccgctacagg gcgcgtcagg
     5821 tggcactttt cggggaaatg tgcgcggaac ccctatttgt ttatttttct aaatacattc
     5881 aaatatgtat ccgctcatga gacaataacc ctgataaatg cttcaataat attgaaaaag
     5941 gaagagtcct gaggcggaaa gaaccagctg tggaatgtgt gtcagttagg gtgtggaaag
     6001 tccccaggct ccccagcagg cagaagtatg caaagcatgc atctcaatta gtcagcaacc
     6061 aggtgtggaa agtccccagg ctccccagca ggcagaagta tgcaaagcat gcatctcaat
     6121 tagtcagcaa ccatagtccc gcccctaact ccgcccatcc cgcccctaac tccgcccagt
     6181 tccgcccatt ctccgcccca tggctgacta atttttttta tttatgcaga ggccgaggcc
     6241 gcctcggcct ctgagctatt ccagaagtag tgaggaggct tttttggagg cctaggcttt
     6301 tgcaaagatc gatcaagaga caggatgagg atcgtttcgc atgattgaac aagatggatt
     6361 gcacgcaggt tctccggccg cttgggtgga gaggctattc ggctatgact gggcacaaca
     6421 gacaatcggc tgctctgatg ccgccgtgtt ccggctgtca gcgcaggggc gcccggttct
     6481 ttttgtcaag accgacctgt ccggtgccct gaatgaactg caagacgagg cagcgcggct
     6541 atcgtggctg gccacgacgg gcgttccttg cgcagctgtg ctcgacgttg tcactgaagc
     6601 gggaagggac tggctgctat tgggcgaagt gccggggcag gatctcctgt catctcacct
     6661 tgctcctgcc gagaaagtat ccatcatggc tgatgcaatg cggcggctgc atacgcttga
     6721 tccggctacc tgcccattcg accaccaagc gaaacatcgc atcgagcgag cacgtactcg
     6781 gatggaagcc ggtcttgtcg atcaggatga tctggacgaa gagcatcagg ggctcgcgcc
     6841 agccgaactg ttcgccaggc tcaaggcgag catgcccgac ggcgaggatc tcgtcgtgac
     6901 ccatggcgat gcctgcttgc cgaatatcat ggtggaaaat ggccgctttt ctggattcat
     6961 cgactgtggc cggctgggtg tggcggaccg ctatcaggac atagcgttgg ctacccgtga
     7021 tattgctgaa gagcttggcg gcgaatgggc tgaccgcttc ctcgtgcttt acggtatcgc
     7081 cgctcccgat tcgcagcgca tcgccttcta tcgccttctt gacgagttct tctgagcggg
     7141 actctggggt tcgaaatgac cgaccaagcg acgcccaacc tgccatcacg agatttcgat
     7201 tccaccgccg ccttctatga aaggttgggc ttcggaatcg ttttccggga cgccggctgg
     7261 atgatcctcc agcgcgggga tctcatgctg gagttcttcg cccaccctag ggggaggcta
     7321 actgaaacac ggaaggagac aataccggaa ggaacccgcg ctatgacggc aataaaaaga
     7381 cagaataaaa cgcacggtgt tgggtcgttt gttcataaac gcggggttcg gtcccagggc
     7441 tggcactctg tcgatacccc accgagaccc cattggggcc aatacgcccg cgtttcttcc
     7501 ttttccccac cccacccccc aagttcgggt gaaggcccag ggctcgcagc caacgtcggg
     7561 gcggcaggcc ctgccatagc ctcaggttac tcatatatac tttagattga tttaaaactt
     7621 catttttaat ttaaaaggat ctaggtgaag atcctttttg ataatctcat gaccaaaatc
     7681 ccttaacgtg agttttcgtt ccactgagcg tcagaccccg tagaaaagat caaaggatct
     7741 tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta
     7801 ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc tttttccgaa ggtaactggc
     7861 ttcagcagag cgcagatacc aaatactgtc cttctagtgt agccgtagtt aggccaccac
     7921 ttcaagaact ctgtagcacc gcctacatac ctcgctctgc taatcctgtt accagtggct
     7981 gctgccagtg gcgataagtc gtgtcttacc gggttggact caagacgata gttaccggat
     8041 aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac agcccagctt ggagcgaacg
     8101 acctacaccg aactgagata cctacagcgt gagctatgag aaagcgccac gcttcccgaa
     8161 gggagaaagg cggacaggta tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg
     8221 gagcttccag ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg ccacctctga
     8281 cttgagcgtc gatttttgtg atgctcgtca ggggggcgga gcctatggaa aaacgccagc
     8341 aacgcggcct ttttacggtt cctggccttt tgctggcctt ttgctcacat gttctttcct
     8401 gcgttatccc ctgattctgt ggataaccgt attaccgcca tgcat
//
