LOCUS       sequence-223585-        8508 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  A FRET sensor for G13 activation, encoded on a single plasmid..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8508)
  AUTHORS   Mastop M, Reinhard NR, Zuconelli CR, Terwey F, Gadella TWJ Jr, van 
            Unen J, Adjobo-Hermans MJW, Goedhart J
  TITLE     A FRET-based biosensor for measuring Galpha13 activation in single 
            cells.
  JOURNAL   PLoS One. 2018 Mar 5;13(3):e0193705. doi: 
            10.1371/journal.pone.0193705. eCollection 2018.
  PUBMED    29505611
REFERENCE   2  (bases 1 to 8508)
  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: "2018-03-5"; volume: "13(3):e0193705. doi"; pages: " 
            10.1371/journal.pone.0193705. eCollection 2018"
COMMENT     Sequence Label: G13 sensor (Ga13_2)
FEATURES             Location/Qualifiers
     source          1..8508
                     /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(3724..3726,3727..3729,3730..4242)
                     /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    5296..5417
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5333..5352)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5387..5406
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(5424..5879)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5561..5582)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5773..5792
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        5906..6010
                     /gene="bla"
                     /label=AmpR promoter
     promoter        6012..6369
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        6012..6203
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(6035..6055)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      6220..6355
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             6404..7198
                     /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             6404..7198
                     /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(6458..6477)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     7068..7087
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(7386..7405)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    7430..7477
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      7806..8394
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     8295..8314
                     /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 atggcggact tcctgccgtc gcggtccgtg
     3361 ctgtccgtgt gcttccccgg ctgcctgctg acgagtggcg aggccgagca gcaacgcaag
     3421 tccaaggaga tcgacaaatg cctgtctcgg gaaaagacct atgtgaagcg gctggtgaag
     3481 atcctgctgc tgggcgcggg cgagagcggc aagtccacct tcctgaagca gatgcggatc
     3541 atccacgggc aggacttcga ccagcgcgcg cgcgaggagt tccgccccac catctacagc
     3601 aacgtgatca aaggtatgag ggtgctggtt gatgctcgag agaagcttca tattccctgg
     3661 ggagacaact caaaccaaca acatggagat aagatgatgt cgtttgatac ccggaccggt
     3721 tctatggtga gcaagggcga ggagctgttc accggggtgg tgcccatcct ggtcgagctg
     3781 gacggcgacg taaacggcca caagttcagc gtgtccggcg agggcgaggg cgatgccacc
     3841 tacggcaagc tgaccctgaa gttcatctgc accaccggca agctgcccgt gccctggccc
     3901 accctcgtga ccaccctgtc ctggggcgtg cagtgcttcg cccgctaccc cgaccacatg
     3961 aagcagcacg acttcttcaa gtccgccatg cccgaaggct acgtccagga gcgcaccatc
     4021 ttcttcaagg acgacggcaa ctacaagacc cgcgccgagg tgaagttcga gggcgacacc
     4081 ctggtgaacc gcatcgagct gaagggcatc gacttcaagg aggacggcaa catcctgggg
     4141 cacaagctgg agtacaacta ctttagcgac aacgtctata tcaccgccga caagcagaag
     4201 aacggcatca aggccaactt caagatccgc cacaacatcg aggacggcgg cgtgcagctc
     4261 gccgaccact accagcagaa cacccccatc ggcgacggcc ccgtgctgct gcccgacaac
     4321 cactacctga gcacccagtc caagctgagc aaagacccca acgagaagcg cgatcacatg
     4381 gtcctgctgg agttcgtgac cgccgccggg atcaccggtg cccccatggc agcccaagga
     4441 atggtggaaa caagggtttt cttacaatat cttcctgcta taagagcatt atgggcagac
     4501 agcggcatac agaatgccta tgaccggcgt cgagaatttc aactgggtga atctgtaaaa
     4561 tatttcctgg ataacttgga taaacttgga gaaccagatt atattccatc acaacaagat
     4621 attctgcttg ccagaagacc caccaaaggc atccatgaat acgactttga aataaaaaat
     4681 gttcctttca aaatggttga tgtaggtggt cagagatcag aaaggaaacg ttggtttgaa
     4741 tgtttcgaca gtgtgacatc aatacttttc cttgtttcct caagtgaatt tgaccaggtg
     4801 cttatggaag atcgactgac caatcgcctt acagagtctc tgaacatttt tgaaacaatc
     4861 gtcaataacc gggttttcag caatgtctcc ataattctgt tcttaaacaa gacagacttg
     4921 cttgaggaga aggtgcaaat tgtgagcatc aaagactatt tcctagaatt tgaaggggat
     4981 ccccactgct taagagacgt ccaaaaattc ctggtggaat gtttccggaa caaacgccgg
     5041 gaccagcaac agaagccctt ataccaccac ttcaccactg ctatcaacac ggagaacatc
     5101 cgccttgttt tccgtgacgt gaaggatact attctgcatg acaacctcaa gcagcttatg
     5161 ctacagtgat ctagagggcc gcgactctag atcataatca gccataccac atttgtagag
     5221 gttttacttg ctttaaaaaa cctcccacac ctccccctga acctgaaaca taaaatgaat
     5281 gcaattgttg ttgttaactt gtttattgca gcttataatg gttacaaata aagcaatagc
     5341 atcacaaatt tcacaaataa agcatttttt tcactgcatt ctagttgtgg tttgtccaaa
     5401 ctcatcaatg tatcttaagg cgtaaattgt aagcgttaat attttgttaa aattcgcgtt
     5461 aaatttttgt taaatcagct cattttttaa ccaataggcc gaaatcggca aaatccctta
     5521 taaatcaaaa gaatagaccg agatagggtt gagtgttgtt ccagtttgga acaagagtcc
     5581 actattaaag aacgtggact ccaacgtcaa agggcgaaaa accgtctatc agggcgatgg
     5641 cccactacgt gaaccatcac cctaatcaag ttttttgggg tcgaggtgcc gtaaagcact
     5701 aaatcggaac cctaaaggga gcccccgatt tagagcttga cggggaaagc cggcgaacgt
     5761 ggcgagaaag gaagggaaga aagcgaaagg agcgggcgct agggcgctgg caagtgtagc
     5821 ggtcacgctg cgcgtaacca ccacacccgc cgcgcttaat gcgccgctac agggcgcgtc
     5881 aggtggcact tttcggggaa atgtgcgcgg aacccctatt tgtttatttt tctaaataca
     5941 ttcaaatatg tatccgctca tgagacaata accctgataa atgcttcaat aatattgaaa
     6001 aaggaagagt cctgaggcgg aaagaaccag ctgtggaatg tgtgtcagtt agggtgtgga
     6061 aagtccccag gctccccagc aggcagaagt atgcaaagca tgcatctcaa ttagtcagca
     6121 accaggtgtg gaaagtcccc aggctcccca gcaggcagaa gtatgcaaag catgcatctc
     6181 aattagtcag caaccatagt cccgccccta actccgccca tcccgcccct aactccgccc
     6241 agttccgccc attctccgcc ccatggctga ctaatttttt ttatttatgc agaggccgag
     6301 gccgcctcgg cctctgagct attccagaag tagtgaggag gcttttttgg aggcctaggc
     6361 ttttgcaaag atcgatcaag agacaggatg aggatcgttt cgcatgattg aacaagatgg
     6421 attgcacgca ggttctccgg ccgcttgggt ggagaggcta ttcggctatg actgggcaca
     6481 acagacaatc ggctgctctg atgccgccgt gttccggctg tcagcgcagg ggcgcccggt
     6541 tctttttgtc aagaccgacc tgtccggtgc cctgaatgaa ctgcaagacg aggcagcgcg
     6601 gctatcgtgg ctggccacga cgggcgttcc ttgcgcagct gtgctcgacg ttgtcactga
     6661 agcgggaagg gactggctgc tattgggcga agtgccgggg caggatctcc tgtcatctca
     6721 ccttgctcct gccgagaaag tatccatcat ggctgatgca atgcggcggc tgcatacgct
     6781 tgatccggct acctgcccat tcgaccacca agcgaaacat cgcatcgagc gagcacgtac
     6841 tcggatggaa gccggtcttg tcgatcagga tgatctggac gaagagcatc aggggctcgc
     6901 gccagccgaa ctgttcgcca ggctcaaggc gagcatgccc gacggcgagg atctcgtcgt
     6961 gacccatggc gatgcctgct tgccgaatat catggtggaa aatggccgct tttctggatt
     7021 catcgactgt ggccggctgg gtgtggcgga ccgctatcag gacatagcgt tggctacccg
     7081 tgatattgct gaagagcttg gcggcgaatg ggctgaccgc ttcctcgtgc tttacggtat
     7141 cgccgctccc gattcgcagc gcatcgcctt ctatcgcctt cttgacgagt tcttctgagc
     7201 gggactctgg ggttcgaaat gaccgaccaa gcgacgccca acctgccatc acgagatttc
     7261 gattccaccg ccgccttcta tgaaaggttg ggcttcggaa tcgttttccg ggacgccggc
     7321 tggatgatcc tccagcgcgg ggatctcatg ctggagttct tcgcccaccc tagggggagg
     7381 ctaactgaaa cacggaagga gacaataccg gaaggaaccc gcgctatgac ggcaataaaa
     7441 agacagaata aaacgcacgg tgttgggtcg tttgttcata aacgcggggt tcggtcccag
     7501 ggctggcact ctgtcgatac cccaccgaga ccccattggg gccaatacgc ccgcgtttct
     7561 tccttttccc caccccaccc cccaagttcg ggtgaaggcc cagggctcgc agccaacgtc
     7621 ggggcggcag gccctgccat agcctcaggt tactcatata tactttagat tgatttaaaa
     7681 cttcattttt aatttaaaag gatctaggtg aagatccttt ttgataatct catgaccaaa
     7741 atcccttaac gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga
     7801 tcttcttgag atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg
     7861 ctaccagcgg tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact
     7921 ggcttcagca gagcgcagat accaaatact gttcttctag tgtagccgta gttaggccac
     7981 cacttcaaga actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg
     8041 gctgctgcca gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg
     8101 gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga
     8161 acgacctaca ccgaactgag atacctacag cgtgagctat gagaaagcgc cacgcttccc
     8221 gaagggagaa aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg
     8281 agggagcttc cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc
     8341 tgacttgagc gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc
     8401 agcaacgcgg cctttttacg gttcctggcc ttttgctggc cttttgctca catgttcttt
     8461 cctgcgttat cccctgattc tgtggataac cgtattaccg ccatgcat
//
