LOCUS Exported 8292 bp ds-DNA circular SYN 14-MAY-2021 DEFINITION Encodes a BRET-based activity sensor for the heterotrimeric G protein G13. Composed of the subunits G alpha 13 (GNA13) tagged with NanoLuciferase, G beta 3 (GNB3) and Venus-tagged G gamma 9 (GNG9). . ACCESSION . VERSION . KEYWORDS G13-CASE SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 8292) AUTHORS Schihada H, Shekhani R, Schulte G TITLE Quantitative assessment of constitutive G protein-coupled receptor activity with BRET-based G protein biosensors JOURNAL BioRxiv REFERENCE 2 (bases 1 to 8292) AUTHORS . TITLE Direct Submission JOURNAL Exported May 14, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..8292 /organism="synthetic DNA construct" /mol_type="other DNA" enhancer 1..304 /label=CMV enhancer /note="human cytomegalovirus immediate early enhancer" promoter 305..508 /label=CMV promoter /note="human cytomegalovirus (CMV) immediate early promoter" primer_bind 459..479 /label=CMV-F /note="Human CMV immediate early promoter, forward primer" CDS 1557..1610 /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 1617..2351 /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 1754..1775 /label=EGFP-C /note="EGFP, forward primer" primer_bind complement(1878..1899) /label=EGFP-N /note="EGFP, reverse primer" primer_bind complement(2139..2158) /label=EXFP-R /note="For distinguishing EGFP variants, reverse primer" promoter complement(2598..2615) /label=SP6 promoter /note="promoter for bacteriophage SP6 RNA polymerase" primer_bind complement(2598..2615) /label=SP6 /note="SP6 promoter, forward primer" misc_feature 2627..3200 /label=IRES /note="internal ribosome entry site (IRES) of the encephalomyocarditis virus (EMCV)" primer_bind complement(2794..2811) /label=IRES reverse /note="IRES internal ribosome entry site, reverse primer. Also called pCDH-rev" primer_bind 3021..3040 /label=IRES-F /note="IRES internal ribosome entry site, forward primer" CDS 3607..4113 /codon_start=1 /product="NanoLuc(R) luciferase" /label=Nluc /note="human codon-optimized" /translation="VFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQRI VLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGVT PNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGWR LCERIL" polyA_signal 5020..5141 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(5057..5076) /label=SV40pA-R /note="SV40 polyA, reverse primer" primer_bind 5111..5130 /label=EBV-rev /note="SV40 polyA terminator, reverse primer" rep_origin complement(5148..5603) /direction=LEFT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(5285..5306) /label=F1ori-F /note="F1 origin, forward primer" primer_bind 5497..5516 /label=F1ori-R /note="F1 origin, reverse primer" promoter 5630..5734 /gene="bla" /label=AmpR promoter promoter 5736..6093 /label=SV40 promoter /note="SV40 enhancer and early promoter" primer_bind complement(5759..5779) /label=pBABE 3' /note="SV40 enhancer, reverse primer for pBABE vectors" rep_origin 5944..6079 /label=SV40 ori /note="SV40 origin of replication" primer_bind 6006..6025 /label=SV40pro-F /note="SV40 promoter/origin, forward primer" CDS 6128..6922 /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(6182..6201) /label=Neo-R /note="Neomycin resistance gene, reverse primer" primer_bind 6792..6811 /label=Neo-F /note="Neomycin resistance gene, forward primer" primer_bind complement(7110..7129) /label=TK-pA-R /note="Thymidine kinase polyA, reverse primer" polyA_signal 7154..7201 /label=HSV TK poly(A) signal /note="herpes simplex virus thymidine kinase polyadenylation signal (Cole and Stacy, 1985)" rep_origin 7530..8118 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 8019..8038 /label=pBR322ori-F /note="pBR322 origin, forward primer" ORIGIN 1 cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc cccgcccatt 61 gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc attgacgtca 121 atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt atcatatgcc 181 aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt atgcccagta 241 catgacctta tgggactttc ctacttggca gtacatctac gtattagtca tcgctattac 301 catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg actcacgggg 361 atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc aaaatcaacg 421 ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg gtaggcgtgt 481 acggtgggag gtctatataa gcagagctgg tttagtgaac cgtcagatcc gctagcatgg 541 gggagatgga gcaactgcgt caggaagcgg agcagctcaa gaagcagatt gcagatgcca 601 ggaaagcctg tgctgacgtt actctggcag agctggtgtc tggcctagag gtggtgggac 661 gagtccagat gcggacgcgg cggacgttaa ggggacacct ggccaagatt tacgccatgc 721 actgggccac tgattctaag ctgctggtaa gtgcctcgca agatgggaag ctgatcgtgt 781 gggacagcta caccaccaac aaggtgcacg ccatcccact gcgctcctcc tgggtcatga 841 cctgtgccta tgccccatca gggaactttg tggcgtgtgg ggggctggac aacatgtgtt 901 ccatctacaa cctcaaatcc cgtgagggca atgtcaaggt cagccgggag ctttctgctc 961 acacaggtta tctctcctgc tgccgcttcc tggatgacaa caatattgtg accagctcgg 1021 gggacaccac gtgtgccttg tgggacattg agactgggca gcagaagact gtatttgtgg 1081 gacacacggg tgactgcatg agcctggctg tgtctcctga cttcaatctc ttcatttcgg 1141 gggcctgtga tgccagtgcc aagctctggg atgtgcgaga ggggacctgc cgacagactt 1201 tcactggcca cgagtcggac atcaacgcca tctgtttctt ccccaatgga gaggccatct 1261 gcacgggctc ggatgacgct tcctgccgct tgtttgacct gcgggcagac caggagctga 1321 tctgcttctc ccacgagagc atcatctgcg gcatcacgtc tgtggccttc tccctcagtg 1381 gccgcctact attcgctggc tacgacgact tcaactgcaa tgtctgggac tccatgaagt 1441 ctgagcgtgt gggcatcctc tctggccacg ataacagggt gagctgcctg ggagtcacag 1501 ctgacgggat ggctgtggcc acaggttcct gggacagctt cctcaaaatc tggaacgagg 1561 gcagaggaag tcttctaaca tgcggtgacg tggaggagaa tcccggccct accggtatgg 1621 acggcggcgt gcagctcgcc gaccactacc agcagaacac ccccatcggc gacggccccg 1681 tgctgctgcc cgacaaccac tacctgagct accagtccgc cctgagcaaa gaccccaacg 1741 agaagcgcga tcacatggtc ctgctggagt tcgtgaccgc cgccgggatc actctcggca 1801 tggacgagct gtacaagggt ggcagcggtg gcatggtgag caagggcgag gagctgttca 1861 ccggggtggt gcccatcctg gtcgagctgg acggcgacgt aaacggccac aagttcagcg 1921 tgtccggcga gggcgagggc gatgccacct acggcaagct gaccctgaag ctgatctgca 1981 ccaccggcaa gctgcccgtg ccctggccca ccctcgtgac caccctgggc tacggcctgc 2041 agtgcttcgc ccgctacccc gaccacatga agcagcacga cttcttcaag tccgccatgc 2101 ccgaaggcta cgtccaggag cgcaccatct tcttcaagga cgacggcaac tacaagaccc 2161 gcgccgaggt gaagttcgag ggcgacaccc tggtgaaccg catcgagctg aagggcatcg 2221 acttcaagga ggacggcaac atcctggggc acaagctgga gtacaactac aacagccaca 2281 acgtctatat caccgccgac aaacagaaga acggcatcaa ggccaacttc aagatccgcc 2341 acaacatcga gtccggactc agatctgccc aggatctcag cgagaaggac ctgttgaaga 2401 tggaggtgga gcagctgaag aaagaagtga aaaacacaag aattccgatt tccaaagcgg 2461 gaaaggaaat caaggagtac gtggaggccc aagcaggaaa cgatcctttt ctcaaaggca 2521 tccctgagga caagaatccc ttcaaggaga aaggtggctg tctgataagc taagtcgaca 2581 gatctcaatt ggggccccta tagtgtcacc taaataattc cgcccccccc tctccctccc 2641 ccccccctaa cgttactggc cgaagccgct tggaataagg ccggtgtgcg tttgtctata 2701 tgttattttc caccatattg ccgtcttttg gcaatgtgag ggcccggaaa cctggccctg 2761 tcttcttgac gagcattcct aggggtcttt cccctctcgc caaaggaatg caaggtctgt 2821 tgaatgtcgt gaaggaagca gttcctctgg aagcttcttg aagacaaaca acgtctgtag 2881 cgaccctttg caggcagcgg aaccccccac ctggcgacag gtgcctctgc ggccaaaagc 2941 cacgtgtata agatacacct gcaaaggcgg cacaacccca gtgccacgtt gtgagttgga 3001 tagttgtgga aagagtcaaa tggctctcct caagcgtatt caacaagggg ctgaaggatg 3061 cccagaaggt accccattgt atgggatctg atctggggcc tcggtgcaca tgctttacat 3121 gtgtttagtc gaggttaaaa aaacgtctag gccccccgaa ccacggggac gtggttttcc 3181 tttgaaaaac acgatgataa tggatccatg gcggacttcc tgccgtcgcg gtccgtgctg 3241 tccgtgtgct tccccggctg cctgctgacg agtggcgagg ccgagcagca acgcaagtcc 3301 aaggagatcg acaaatgcct gtctcgggaa aagacctatg tgaagcggct ggtgaagatc 3361 ctgctgctgg gcgcgggcga gagcggcaag tccaccttcc tgaagcagat gcggatcatc 3421 cacgggcagg acttcgacca gcgcgcgcgc gaggagttcc gccccaccat ctacagcaac 3481 gtgatcaaag gtatgagggt gctggttgat gctcgagaga agcttcatat tccctgggga 3541 gacaactcaa accaacaaca tggagataag atgatgtcgt ttagcggtgg aggcgggacg 3601 cgttctgtct tcacactcga agatttcgtt ggggactggc gacagacagc cggctacaac 3661 ctggaccaag tccttgaaca gggaggtgtg tccagtttgt ttcagaatct cggggtgtcc 3721 gtaactccga tccaaaggat tgtcctgagc ggtgaaaatg ggctgaagat cgacatccat 3781 gtcatcatcc cgtatgaagg tctgagcggc gaccaaatgg gccagatcga aaaaattttt 3841 aaggtggtgt accctgtgga tgatcatcac tttaaggtga tcctgcacta tggcacactg 3901 gtaatcgacg gggttacgcc gaacatgatc gactatttcg gacggccgta tgaaggcatc 3961 gccgtgttcg acggcaaaaa gatcactgta acagggaccc tgtggaacgg caacaaaatt 4021 atcgacgagc gcctgatcaa ccccgacggc tccctgctgt tccgagtaac catcaacgga 4081 gtgaccggct ggcggctgtg cgaacgcatt ctggccaccg gtggtggcgg atccgatacc 4141 cgggccccca tggcagccca aggaatggtg gaaacaaggg ttttcttaca atatcttcct 4201 gctataagag cattatgggc agacagcggc atacagaatg cctatgaccg gcgtcgagaa 4261 tttcaactgg gtgaatctgt aaaatatttc ctggataact tggataaact tggagaacca 4321 gattatattc catcacaaca agatattctg cttgccagaa gacccaccaa aggcatccat 4381 gaatacgact ttgaaataaa aaatgttcct ttcaaaatgg ttgatgtagg tggtcagaga 4441 tcagaaagga aacgttggtt tgaatgtttc gacagtgtga catcaatact tttccttgtt 4501 tcctcaagtg aatttgacca ggtgcttatg gaagatcgac tgaccaatcg ccttacagag 4561 tctctgaaca tttttgaaac aatcgtcaat aaccgggttt tcagcaatgt ctccataatt 4621 ctgttcttaa acaagacaga cttgcttgag gagaaggtgc aaattgtgag catcaaagac 4681 tatttcctag aatttgaagg ggatccccac tgcttaagag acgtccaaaa attcctggtg 4741 gaatgtttcc ggaacaaacg ccgggaccag caacagaagc ccttatacca ccacttcacc 4801 actgctatca acacggagaa catccgcctt gttttccgtg acgtgaagga tactattctg 4861 catgacaacc tcaagcagct tatgctacag taatctagag ggccgcgact ctagatcata 4921 atcagccata ccacatttgt agaggtttta cttgctttaa aaaacctccc acacctcccc 4981 ctgaacctga aacataaaat gaatgcaatt gttgttgtta acttgtttat tgcagcttat 5041 aatggttaca aataaagcaa tagcatcaca aatttcacaa ataaagcatt tttttcactg 5101 cattctagtt gtggtttgtc caaactcatc aatgtatctt aaggcgtaaa ttgtaagcgt 5161 taatattttg ttaaaattcg cgttaaattt ttgttaaatc agctcatttt ttaaccaata 5221 ggccgaaatc ggcaaaatcc cttataaatc aaaagaatag accgagatag ggttgagtgt 5281 tgttccagtt tggaacaaga gtccactatt aaagaacgtg gactccaacg tcaaagggcg 5341 aaaaaccgtc tatcagggcg atggcccact acgtgaacca tcaccctaat caagtttttt 5401 ggggtcgagg tgccgtaaag cactaaatcg gaaccctaaa gggagccccc gatttagagc 5461 ttgacgggga aagccggcga acgtggcgag aaaggaaggg aagaaagcga aaggagcggg 5521 cgctagggcg ctggcaagtg tagcggtcac gctgcgcgta accaccacac ccgccgcgct 5581 taatgcgccg ctacagggcg cgtcaggtgg cacttttcgg ggaaatgtgc gcggaacccc 5641 tatttgttta tttttctaaa tacattcaaa tatgtatccg ctcatgagac aataaccctg 5701 ataaatgctt caataatatt gaaaaaggaa gagtcctgag gcggaaagaa ccagctgtgg 5761 aatgtgtgtc agttagggtg tggaaagtcc ccaggctccc cagcaggcag aagtatgcaa 5821 agcatgcatc tcaattagtc agcaaccagg tgtggaaagt ccccaggctc cccagcaggc 5881 agaagtatgc aaagcatgca tctcaattag tcagcaacca tagtcccgcc cctaactccg 5941 cccatcccgc ccctaactcc gcccagttcc gcccattctc cgccccatgg ctgactaatt 6001 ttttttattt atgcagaggc cgaggccgcc tcggcctctg agctattcca gaagtagtga 6061 ggaggctttt ttggaggcct aggcttttgc aaagatcgat caagagacag gatgaggatc 6121 gtttcgcatg attgaacaag atggattgca cgcaggttct ccggccgctt gggtggagag 6181 gctattcggc tatgactggg cacaacagac aatcggctgc tctgatgccg ccgtgttccg 6241 gctgtcagcg caggggcgcc cggttctttt tgtcaagacc gacctgtccg gtgccctgaa 6301 tgaactgcaa gacgaggcag cgcggctatc gtggctggcc acgacgggcg ttccttgcgc 6361 agctgtgctc gacgttgtca ctgaagcggg aagggactgg ctgctattgg gcgaagtgcc 6421 ggggcaggat ctcctgtcat ctcaccttgc tcctgccgag aaagtatcca tcatggctga 6481 tgcaatgcgg cggctgcata cgcttgatcc ggctacctgc ccattcgacc accaagcgaa 6541 acatcgcatc gagcgagcac gtactcggat ggaagccggt cttgtcgatc aggatgatct 6601 ggacgaagag catcaggggc tcgcgccagc cgaactgttc gccaggctca aggcgagcat 6661 gcccgacggc gaggatctcg tcgtgaccca tggcgatgcc tgcttgccga atatcatggt 6721 ggaaaatggc cgcttttctg gattcatcga ctgtggccgg ctgggtgtgg cggaccgcta 6781 tcaggacata gcgttggcta cccgtgatat tgctgaagag cttggcggcg aatgggctga 6841 ccgcttcctc gtgctttacg gtatcgccgc tcccgattcg cagcgcatcg ccttctatcg 6901 ccttcttgac gagttcttct gagcgggact ctggggttcg aaatgaccga ccaagcgacg 6961 cccaacctgc catcacgaga tttcgattcc accgccgcct tctatgaaag gttgggcttc 7021 ggaatcgttt tccgggacgc cggctggatg atcctccagc gcggggatct catgctggag 7081 ttcttcgccc accctagggg gaggctaact gaaacacgga aggagacaat accggaagga 7141 acccgcgcta tgacggcaat aaaaagacag aataaaacgc acggtgttgg gtcgtttgtt 7201 cataaacgcg gggttcggtc ccagggctgg cactctgtcg ataccccacc gagaccccat 7261 tggggccaat acgcccgcgt ttcttccttt tccccacccc accccccaag ttcgggtgaa 7321 ggcccagggc tcgcagccaa cgtcggggcg gcaggccctg ccatagcctc aggttactca 7381 tatatacttt agattgattt aaaacttcat ttttaattta aaaggatcta ggtgaagatc 7441 ctttttgata atctcatgac caaaatccct taacgtgagt tttcgttcca ctgagcgtca 7501 gaccccgtag aaaagatcaa aggatcttct tgagatcctt tttttctgcg cgtaatctgc 7561 tgcttgcaaa caaaaaaacc accgctacca gcggtggttt gtttgtcgga tcaagagcta 7621 ccaactcttt ttccgaaggt aactggcttc agcagagcgc agataccaaa tactgttctt 7681 ctagtgtagc cgtagttagg ccaccacttc aagaactctg tagcaccgcc tacatacctc 7741 gctctgctaa tcctgttacc agtggctgct gccagtggcg ataagtcgtg tcttaccggg 7801 ttggactcaa gacgatagtt accggataag gcgcagcggt cgggctgaac ggggggttcg 7861 tgcacacagc ccagcttgga gcgaacgacc tacaccgaac tgagatacct acagcgtgag 7921 ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc 7981 agggtcggaa caggagagcg cacgagggag cttccagggg gaaacgcctg gtatctttat 8041 agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg 8101 gggcggagcc tatggaaaaa cgccagcaac gcggcctttt tacggttcct ggccttttgc 8161 tggccttttg ctcacatgtt ctttcctgcg ttatcccctg attctgtgga taaccgtatt 8221 accgccatgc attagttatt aatagtaatc aattacgggg tcattagttc atagcccata 8281 tatggagttc cg //