LOCUS Exported 7223 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION Encodes a G alpha subunit (GNAl1) containing RLuc8 as an optimal component of a BRET2 biosensor for studying heterotrimeric G protein dissociation. ACCESSION . VERSION . KEYWORDS pcDNA5/FRT/TO-GAlphai1-RLuc8 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 7223) AUTHORS Olsen RHJ, DiBerto JF, English JG, Glaudin AM, Krumm BE, Slocum ST, Che T, Gavin AC, McCorvy JD, Roth BL, Strachan RT TITLE TRUPATH, an open-source biosensor platform for interrogating the GPCR transducerome. JOURNAL Nat Chem Biol. 2020 May 4. pii: 10.1038/s41589-020-0535-8. doi: 10.1038/s41589-020-0535-8. PUBMED 32367019 REFERENCE 2 (bases 1 to 7223) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..7223 /organism="synthetic DNA construct" /mol_type="other DNA" primer_bind complement(44..63) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" enhancer 235..614 /label=CMV enhancer /note="human cytomegalovirus immediate early enhancer" promoter 615..818 /label=CMV promoter /note="human cytomegalovirus (CMV) immediate early promoter" primer_bind 769..789 /label=CMV-F /note="Human CMV immediate early promoter, forward primer" protein_bind 820..838 /gene="tetO" /label=tet operator /bound_moiety="tetracycline repressor TetR" /note="bacterial operator O2 for the tetR and tetA genes" protein_bind 841..859 /gene="tetO" /label=tet operator /bound_moiety="tetracycline repressor TetR" /note="bacterial operator O2 for the tetR and tetA genes" primer_bind 869..893 /label=LNCX /note="Human CMV promoter, forward primer" CDS 1389..2321 /codon_start=1 /product="Renilla luciferase" /label=hRluc /note="human codon-optimized" /translation="MASKVYDPEQRKRMITGPQWWARCKQMNVLDSFINYYDSEKHAEN AVIFLHGNATSSYLWRHVVPHIEPVARCIIPDLIGMGKSGKSGNGSYRLLDHYKYLTAW FELLNLPKKIIFVGHDWGAALAFHYAYEHQDRIKAIVHMESVVDVIESWDEWPDIEEDI ALIKSEEGEKMVLENNFFVETVLPSKIMRKLEPEEFAAYLEPFKEKGEVRRPTLSWPRE IPLVKGGKPDVVQIVRNYNAYLRASDDLPKLFIESDPGFFSNAIVEGAKKFPNTEFVKV KGLHFLQEDAPDEMGKYIKSFVERVLKNEQ" primer_bind complement(3175..3192) /label=BGH-rev /note="Bovine growth hormone terminator, reverse primer. Also called BGH reverse" polyA_signal 3181..3405 /label=bGH poly(A) signal /note="bovine growth hormone polyadenylation signal" primer_bind complement(3538..3557) /label=F1ori-R /note="F1 origin, reverse primer" protein_bind 3689..3736 /label=FRT /bound_moiety="FLP recombinase from the Saccharomyces cerevisiae 2u plasmid" /note="FLP-mediated recombination occurs in the 8-bp core sequence TCTAGAAA (Turan and Bode, 2011)." CDS 3744..4766 /codon_start=1 /gene="aph(4)-Ia" /product="aminoglycoside phosphotransferase from E. coli" /label=HygR /note="confers resistance to hygromycin" /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE " polyA_signal 4896..5017 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(4933..4952) /label=SV40pA-R /note="SV40 polyA, reverse primer" primer_bind 4987..5006 /label=EBV-rev /note="SV40 polyA terminator, reverse primer" primer_bind complement(5066..5082) /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind complement(5066..5082) /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" primer_bind complement(5079..5101) /label=M13/pUC Reverse /note="In lacZ gene" protein_bind 5090..5106 /label=lac operator /bound_moiety="lac repressor encoded by lacI" /note="The lac repressor binds to the lac operator to inhibit transcription in E. coli. This inhibition can be relieved by adding lactose or isopropyl-beta-D-thiogalactopyranoside (IPTG)." promoter complement(5114..5144) /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 5159..5180 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." primer_bind complement(5297..5314) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(5468..6056) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(5548..5567) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS complement(6227..7087) /codon_start=1 /gene="bla" /product="beta-lactamase" /label=AmpR /note="confers resistance to ampicillin, carbenicillin, and related antibiotics" /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS LIKHW" primer_bind 6850..6869 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(7088..7192) /gene="bla" /label=AmpR promoter ORIGIN 1 gacggatcgg gagatctccc gatcccctat ggtgcactct cagtacaatc tgctctgatg 61 ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg 121 cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc 181 ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt 241 gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata 301 tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc 361 cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc 421 attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt 481 atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt 541 atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca 601 tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg 661 actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc 721 aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg 781 gtaggcgtgt acggtgggag gtctatataa gcagagctct ccctatcagt gatagagatc 841 tccctatcag tgatagagat cgtcgacgag ctcgtttagt gaaccgtcag atcgcctgga 901 gacgccatcc acgctgtttt gacctccata gaagacaccg ggaccgatcc agcctccgga 961 ctctagcgtt taaacttaag cttggtaccg agctcggatc cactagtcca gtgtggtgga 1021 attctgcaga tatcaccacc gccagctcct ccctggccaa ggacacttca tcgaccggtg 1081 agaacctgta cttccagcta atgggctgca cgctgagcgc cgaggacaag gcggcggtgg 1141 agcggagtaa gatgatcgac cgcaacctcc gtgaggacgg cgagaaggcg gcgcgcgagg 1201 tcaagctgct gctgctcggt gctggtgaat ctggtaaaag tacaattgtg aagcagatga 1261 aaattatcca tgaagctggt tattcagaag aggagtgtaa acaatacaaa gcagtggtct 1321 acagtaacac catccagtca attattgcta tcattagggc tatggggagg tctggtggtg 1381 gtggatccat ggcttccaag gtgtacgacc ccgagcaacg caaacgcatg atcactgggc 1441 ctcagtggtg ggctcgctgc aagcaaatga acgtgctgga ctccttcatc aactactatg 1501 attccgagaa gcacgccgag aacgccgtga tttttctgca tggtaacgct acctccagct 1561 acctgtggag gcacgtcgtg cctcacatcg agcccgtggc tagatgcatc atccctgatc 1621 tgatcggaat gggtaagtcc ggcaagagcg ggaatggctc atatcgcctc ctggatcact 1681 acaagtacct caccgcttgg ttcgagctgc tgaaccttcc aaagaaaatc atctttgtgg 1741 gccacgactg gggggctgct ctggcctttc actacgccta cgagcaccaa gacaggatca 1801 aggccatcgt ccatatggag agtgtcgtgg acgtgatcga gtcctgggac gagtggcctg 1861 acatcgagga ggatatcgcc ctgatcaaga gcgaagaggg cgagaaaatg gtgcttgaga 1921 ataacttctt cgtcgagacc gtgctcccaa gcaagatcat gcggaaactg gagcctgagg 1981 agttcgctgc ctacctggag ccattcaagg agaagggcga ggttagacgg cctaccctct 2041 cctggcctcg cgagatccct ctcgttaagg gaggcaagcc cgacgtcgtc cagattgtcc 2101 gcaactacaa cgcctacctt cgggccagcg acgatctgcc taagctgttc atcgagtccg 2161 accctgggtt cttttccaac gctattgtcg agggagctaa gaagttccct aacaccgagt 2221 tcgtgaaggt gaagggcctc cacttcctcc aggaggacgc tccagatgaa atgggtaagt 2281 acatcaagag cttcgtggag cgcgtgctga agaacgagca gagcggagga ggcggcagtt 2341 tgaagataga ctttggtgac tcagcccggg cggatgatgc acgccaactc tttgtgctag 2401 ctggagctgc tgaagaaggc tttatgactg cagaacttgc tggagttata aagagattgt 2461 ggaaagatag tggtgtacaa gcctgtttca acagatcccg agagtaccag cttaatgatt 2521 ctgcagcata ctatttgaat gacttggaca gaatagctca accaaattac atcccgactc 2581 aacaagatgt tctcagaact agagtgaaaa ctacaggaat tgttgaaacc cattttactt 2641 tcaaagatct tcattttaaa atgtttgatg tgggaggtca gagatctgag cggaagaagt 2701 ggattcattg cttcgaagga gtgacggcga tcatcttctg tgtagcactg agtgactacg 2761 acctggttct agctgaagat gaagaaatga accgaatgca tgaaagcatg aaattgtttg 2821 acagcatatg taacaacaag tggtttacag atacatccat tatacttttt ctaaacaaga 2881 aggatctctt tgaagaaaaa atcaaaaaga gccctctcac tatatgctat ccagaatatg 2941 caggatcaaa cacatatgaa gaggcagctg catatattca atgtcagttt gaagacctca 3001 ataaaagaaa ggacacaaag gaaatataca cccacttcac atgtgccaca gatactaaga 3061 atgtgcagtt tgtttttgat gctgtaacag atgtcatcat aaaaaataat ctaaaagatt 3121 gtggtctctt ttaatagctc gagtctagag ggcccgttta aacccgctga tcagcctcga 3181 ctgtgccttc tagttgccag ccatctgttg tttgcccctc ccccgtgcct tccttgaccc 3241 tggaaggtgc cactcccact gtcctttcct aataaaatga ggaaattgca tcgcattgtc 3301 tgagtaggtg tcattctatt ctggggggtg gggtggggca ggacagcaag ggggaggatt 3361 gggaagacaa tagcaggcat gctggggatg cggtgggctc tatggcttct gaggcggaaa 3421 gaaccagctg gggctctagg gggtatcccc acgcgccctg tagcggcgca ttaagcgcgg 3481 cgggtgtggt ggttacgcgc agcgtgaccg ctacacttgc cagcgcccta gcgcccgctc 3541 ctttcgcttt cttcccttcc tttctcgcca cgttcgccgg ctttccccgt caagctctaa 3601 atcgggggct ccctttaggg ttccgattta gtgctttacg gcacctcgac cccaaaaaac 3661 ttgattaggg tgatggttca cgtacctaga agttcctatt ccgaagttcc tattctctag 3721 aaagtatagg aacttccttg gccaaaaagc ctgaactcac cgcgacgtct gtcgagaagt 3781 ttctgatcga aaagttcgac agcgtctccg acctgatgca gctctcggag ggcgaagaat 3841 ctcgtgcttt cagcttcgat gtaggagggc gtggatatgt cctgcgggta aatagctgcg 3901 ccgatggttt ctacaaagat cgttatgttt atcggcactt tgcatcggcc gcgctcccga 3961 ttccggaagt gcttgacatt ggggaattca gcgagagcct gacctattgc atctcccgcc 4021 gtgcacaggg tgtcacgttg caagacctgc ctgaaaccga actgcccgct gttctgcagc 4081 cggtcgcgga ggccatggat gcgatcgctg cggccgatct tagccagacg agcgggttcg 4141 gcccattcgg accgcaagga atcggtcaat acactacatg gcgtgatttc atatgcgcga 4201 ttgctgatcc ccatgtgtat cactggcaaa ctgtgatgga cgacaccgtc agtgcgtccg 4261 tcgcgcaggc tctcgatgag ctgatgcttt gggccgagga ctgccccgaa gtccggcacc 4321 tcgtgcacgc ggatttcggc tccaacaatg tcctgacgga caatggccgc ataacagcgg 4381 tcattgactg gagcgaggcg atgttcgggg attcccaata cgaggtcgcc aacatcttct 4441 tctggaggcc gtggttggct tgtatggagc agcagacgcg ctacttcgag cggaggcatc 4501 cggagcttgc aggatcgccg cggctccggg cgtatatgct ccgcattggt cttgaccaac 4561 tctatcagag cttggttgac ggcaatttcg atgatgcagc ttgggcgcag ggtcgatgcg 4621 acgcaatcgt ccgatccgga gccgggactg tcgggcgtac acaaatcgcc cgcagaagcg 4681 cggccgtctg gaccgatggc tgtgtagaag tactcgccga tagtggaaac cgacgcccca 4741 gcactcgtcc gagggcaaag gaatagcacg tactacgaga tttcgattcc accgccgcct 4801 tctatgaaag gttgggcttc ggaatcgttt tccgggacgc cggctggatg atcctccagc 4861 gcggggatct catgctggag ttcttcgccc accccaactt gtttattgca gcttataatg 4921 gttacaaata aagcaatagc atcacaaatt tcacaaataa agcatttttt tcactgcatt 4981 ctagttgtgg tttgtccaaa ctcatcaatg tatcttatca tgtctgtata ccgtcgacct 5041 ctagctagag cttggcgtaa tcatggtcat agctgtttcc tgtgtgaaat tgttatccgc 5101 tcacaattcc acacaacata cgagccggaa gcataaagtg taaagcctgg ggtgcctaat 5161 gagtgagcta actcacatta attgcgttgc gctcactgcc cgctttccag tcgggaaacc 5221 tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg 5281 ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag 5341 cggtatcagc tcactcaaag gcggtaatac ggttatccac agaatcaggg gataacgcag 5401 gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc 5461 tggcgttttt ccataggctc cgcccccctg acgagcatca caaaaatcga cgctcaagtc 5521 agaggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct ggaagctccc 5581 tcgtgcgctc tcctgttccg accctgccgc ttaccggata cctgtccgcc tttctccctt 5641 cgggaagcgt ggcgctttct catagctcac gctgtaggta tctcagttcg gtgtaggtcg 5701 ttcgctccaa gctgggctgt gtgcacgaac cccccgttca gcccgaccgc tgcgccttat 5761 ccggtaacta tcgtcttgag tccaacccgg taagacacga cttatcgcca ctggcagcag 5821 ccactggtaa caggattagc agagcgaggt atgtaggcgg tgctacagag ttcttgaagt 5881 ggtggcctaa ctacggctac actagaagaa cagtatttgg tatctgcgct ctgctgaagc 5941 cagttacctt cggaaaaaga gttggtagct cttgatccgg caaacaaacc accgctggta 6001 gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag 6061 atcctttgat cttttctacg gggtctgacg ctcagtggaa cgaaaactca cgttaaggga 6121 ttttggtcat gagattatca aaaaggatct tcacctagat ccttttaaat taaaaatgaa 6181 gttttaaatc aatctaaagt atatatgagt aaacttggtc tgacagttac caatgcttaa 6241 tcagtgaggc acctatctca gcgatctgtc tatttcgttc atccatagtt gcctgactcc 6301 ccgtcgtgta gataactacg atacgggagg gcttaccatc tggccccagt gctgcaatga 6361 taccgcgaga cccacgctca ccggctccag atttatcagc aataaaccag ccagccggaa 6421 gggccgagcg cagaagtggt cctgcaactt tatccgcctc catccagtct attaattgtt 6481 gccgggaagc tagagtaagt agttcgccag ttaatagttt gcgcaacgtt gttgccattg 6541 ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc tccggttccc 6601 aacgatcaag gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt agctccttcg 6661 gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg gttatggcag 6721 cactgcataa ttctcttact gtcatgccat ccgtaagatg cttttctgtg actggtgagt 6781 actcaaccaa gtcattctga gaatagtgta tgcggcgacc gagttgctct tgcccggcgt 6841 caatacggga taataccgcg ccacatagca gaactttaaa agtgctcatc attggaaaac 6901 gttcttcggg gcgaaaactc tcaaggatct taccgctgtt gagatccagt tcgatgtaac 6961 ccactcgtgc acccaactga tcttcagcat cttttacttt caccagcgtt tctgggtgag 7021 caaaaacagg aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa 7081 tactcatact cttccttttt caatattatt gaagcattta tcagggttat tgtctcatga 7141 gcggatacat atttgaatgt atttagaaaa ataaacaaat aggggttccg cgcacatttc 7201 cccgaaaagt gccacctgac gtc //