pcDNA5/FRT/TO-GAlphaGustducin-RLuc8

LOCUS       pcDNA5/FRT/TO-GAlphaGustducin-RLuc8               7168 bp ds-DNA     circular SYN 12-MAY-2021
DEFINITION  Encodes a G alpha subunit (GNAT3) containing RLuc8 as an optimal 
            component of a BRET2 biosensor for studying heterotrimeric G protein
            dissociation.
ACCESSION   .
VERSION     .
KEYWORDS    pcDNA5/FRT/TO-GAlphaGustducin-RLuc8
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7168)
  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 7168)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 12, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..7168
                     /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             1398..2330
                     /codon_start=1
                     /product="Renilla luciferase"
                     /label=hRluc
                     /note="human codon-optimized"
                     /translation="MASKVYDPEQRKRMITGPQWWARCKQMNVLDSFINYYDSEKHAEN
                     AVIFLHGNATSSYLWRHVVPHIEPVARCIIPDLIGMGKSGKSGNGSYRLLDHYKYLTAW
                     FELLNLPKKIIFVGHDWGAALAFHYAYEHQDRIKAIVHMESVVDVIESWDEWPDIEEDI
                     ALIKSEEGEKMVLENNFFVETVLPSKIMRKLEPEEFAAYLEPFKEKGEVRRPTLSWPRE
                     IPLVKGGKPDVVQIVRNYNAYLRASDDLPKLFIESDPGFFSNAIVEGAKKFPNTEFVKV
                     KGLHFLQEDAPDEMGKYIKSFVERVLKNEQ"
     primer_bind     complement(3120..3137)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3126..3350
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(3483..3502)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    3634..3681
                     /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             3689..4711
                     /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    4841..4962
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4878..4897)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4932..4951
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5011..5027)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5011..5027)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5024..5046)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5035..5051
                     /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(5059..5089)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5104..5125
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5242..5259)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5413..6001)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5493..5512)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(6172..7032)
                     /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     6795..6814
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7033..7137)
                     /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 tatgggtagc gggatttcgt ccgagtctaa agagtccgca aaacgctcaa
     1081 aggagctgga gaagaagctc caagaggatg cggaaagaga tgccaggaca gttaaactcc
     1141 tgttgctcgg cgcaggggaa tccggtaaga gcaccatcgt caagcagatg aagatcatac
     1201 ataagaatgg atactctgag caagaatgta tggagtttaa ggcagttata tactcaaata
     1261 ctcttcaatc tattttggca attgttaaag caatgacaac gcttggtata gactatgtga
     1321 acccccggtc cgctgaggac caacgccaat tgtatgcaat ggccaatacc ctggaagatt
     1381 ctggtggtgg tggatccatg gcttccaagg tgtacgaccc cgagcaacgc aaacgcatga
     1441 tcactgggcc tcagtggtgg gctcgctgca agcaaatgaa cgtgctggac tccttcatca
     1501 actactatga ttccgagaag cacgccgaga acgccgtgat ttttctgcat ggtaacgcta
     1561 cctccagcta cctgtggagg cacgtcgtgc ctcacatcga gcccgtggct agatgcatca
     1621 tccctgatct gatcggaatg ggtaagtccg gcaagagcgg gaatggctca tatcgcctcc
     1681 tggatcacta caagtacctc accgcttggt tcgagctgct gaaccttcca aagaaaatca
     1741 tctttgtggg ccacgactgg ggggctgctc tggcctttca ctacgcctac gagcaccaag
     1801 acaggatcaa ggccatcgtc catatggaga gtgtcgtgga cgtgatcgag tcctgggacg
     1861 agtggcctga catcgaggag gatatcgccc tgatcaagag cgaagagggc gagaaaatgg
     1921 tgcttgagaa taacttcttc gtcgagaccg tgctcccaag caagatcatg cggaaactgg
     1981 agcctgagga gttcgctgcc tacctggagc cattcaagga gaagggcgag gttagacggc
     2041 ctaccctctc ctggcctcgc gagatccctc tcgttaaggg aggcaagccc gacgtcgtcc
     2101 agattgtccg caactacaac gcctaccttc gggccagcga cgatctgcct aagctgttca
     2161 tcgagtccga ccctgggttc ttttccaacg ctattgtcga gggagctaag aagttcccta
     2221 acaccgagtt cgtgaaggtg aagggcctcc acttcctcca ggaggacgct ccagatgaaa
     2281 tgggtaagta catcaagagc ttcgtggagc gcgtgctgaa gaacgagcag agcggaggag
     2341 gcggcagtgg tgggatgacg ccccagttgg cagaagtaat aaaaaggttg tggcgggacc
     2401 ccggcattca ggcgtgtttt gagcgagctt cagaatacca attgaacgat tccgcagcct
     2461 attacctcaa cgatctggat cgaataacag cctctgggta cgtcccgaac gagcaagatg
     2521 tcttgcactc aagagtaaaa accaccggga ttattgagac acaatttagt ttcaaagatc
     2581 ttcattttcg gatgttcgac gttggcgggc aacggtccga acgaaaaaaa tggatacatt
     2641 gctttgaagg agtaacttgt attatatttt gcgctgcgct tagtgcatac gatatggtac
     2701 ttgtggagga cgaagaggta aatcggatgc acgagagttt gcatctgttt aactctatat
     2761 gcaatcataa gtatttctct acaaccagca tagtactctt cctgaacaaa aaggatatat
     2821 ttcaggaaaa agttacgaag gtccacttgt caatatgctt tccagagtac actggtccca
     2881 acactttcga ggatgccggc aattatatca agaatcaatt tcttgatttg aatttgaaaa
     2941 aggaggataa ggagatatac tctcatatga cctgcgctac agacacccaa aatgtaaagt
     3001 ttgtatttga cgctgtgacc gatataataa ttaaggaaaa cctgaaggat tgcggtctgt
     3061 tttagcagca cagtggcggc cgctcgagtc tagagggccc gtttaaaccc gctgatcagc
     3121 ctcgactgtg ccttctagtt gccagccatc tgttgtttgc ccctcccccg tgccttcctt
     3181 gaccctggaa ggtgccactc ccactgtcct ttcctaataa aatgaggaaa ttgcatcgca
     3241 ttgtctgagt aggtgtcatt ctattctggg gggtggggtg gggcaggaca gcaaggggga
     3301 ggattgggaa gacaatagca ggcatgctgg ggatgcggtg ggctctatgg cttctgaggc
     3361 ggaaagaacc agctggggct ctagggggta tccccacgcg ccctgtagcg gcgcattaag
     3421 cgcggcgggt gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg ccctagcgcc
     3481 cgctcctttc gctttcttcc cttcctttct cgccacgttc gccggctttc cccgtcaagc
     3541 tctaaatcgg gggctccctt tagggttccg atttagtgct ttacggcacc tcgaccccaa
     3601 aaaacttgat tagggtgatg gttcacgtac ctagaagttc ctattccgaa gttcctattc
     3661 tctagaaagt ataggaactt ccttggccaa aaagcctgaa ctcaccgcga cgtctgtcga
     3721 gaagtttctg atcgaaaagt tcgacagcgt ctccgacctg atgcagctct cggagggcga
     3781 agaatctcgt gctttcagct tcgatgtagg agggcgtgga tatgtcctgc gggtaaatag
     3841 ctgcgccgat ggtttctaca aagatcgtta tgtttatcgg cactttgcat cggccgcgct
     3901 cccgattccg gaagtgcttg acattgggga attcagcgag agcctgacct attgcatctc
     3961 ccgccgtgca cagggtgtca cgttgcaaga cctgcctgaa accgaactgc ccgctgttct
     4021 gcagccggtc gcggaggcca tggatgcgat cgctgcggcc gatcttagcc agacgagcgg
     4081 gttcggccca ttcggaccgc aaggaatcgg tcaatacact acatggcgtg atttcatatg
     4141 cgcgattgct gatccccatg tgtatcactg gcaaactgtg atggacgaca ccgtcagtgc
     4201 gtccgtcgcg caggctctcg atgagctgat gctttgggcc gaggactgcc ccgaagtccg
     4261 gcacctcgtg cacgcggatt tcggctccaa caatgtcctg acggacaatg gccgcataac
     4321 agcggtcatt gactggagcg aggcgatgtt cggggattcc caatacgagg tcgccaacat
     4381 cttcttctgg aggccgtggt tggcttgtat ggagcagcag acgcgctact tcgagcggag
     4441 gcatccggag cttgcaggat cgccgcggct ccgggcgtat atgctccgca ttggtcttga
     4501 ccaactctat cagagcttgg ttgacggcaa tttcgatgat gcagcttggg cgcagggtcg
     4561 atgcgacgca atcgtccgat ccggagccgg gactgtcggg cgtacacaaa tcgcccgcag
     4621 aagcgcggcc gtctggaccg atggctgtgt agaagtactc gccgatagtg gaaaccgacg
     4681 ccccagcact cgtccgaggg caaaggaata gcacgtacta cgagatttcg attccaccgc
     4741 cgccttctat gaaaggttgg gcttcggaat cgttttccgg gacgccggct ggatgatcct
     4801 ccagcgcggg gatctcatgc tggagttctt cgcccacccc aacttgttta ttgcagctta
     4861 taatggttac aaataaagca atagcatcac aaatttcaca aataaagcat ttttttcact
     4921 gcattctagt tgtggtttgt ccaaactcat caatgtatct tatcatgtct gtataccgtc
     4981 gacctctagc tagagcttgg cgtaatcatg gtcatagctg tttcctgtgt gaaattgtta
     5041 tccgctcaca attccacaca acatacgagc cggaagcata aagtgtaaag cctggggtgc
     5101 ctaatgagtg agctaactca cattaattgc gttgcgctca ctgcccgctt tccagtcggg
     5161 aaacctgtcg tgccagctgc attaatgaat cggccaacgc gcggggagag gcggtttgcg
     5221 tattgggcgc tcttccgctt cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg
     5281 gcgagcggta tcagctcact caaaggcggt aatacggtta tccacagaat caggggataa
     5341 cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc
     5401 gttgctggcg tttttccata ggctccgccc ccctgacgag catcacaaaa atcgacgctc
     5461 aagtcagagg tggcgaaacc cgacaggact ataaagatac caggcgtttc cccctggaag
     5521 ctccctcgtg cgctctcctg ttccgaccct gccgcttacc ggatacctgt ccgcctttct
     5581 cccttcggga agcgtggcgc tttctcatag ctcacgctgt aggtatctca gttcggtgta
     5641 ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc gttcagcccg accgctgcgc
     5701 cttatccggt aactatcgtc ttgagtccaa cccggtaaga cacgacttat cgccactggc
     5761 agcagccact ggtaacagga ttagcagagc gaggtatgta ggcggtgcta cagagttctt
     5821 gaagtggtgg cctaactacg gctacactag aagaacagta tttggtatct gcgctctgct
     5881 gaagccagtt accttcggaa aaagagttgg tagctcttga tccggcaaac aaaccaccgc
     5941 tggtagcggt ggtttttttg tttgcaagca gcagattacg cgcagaaaaa aaggatctca
     6001 agaagatcct ttgatctttt ctacggggtc tgacgctcag tggaacgaaa actcacgtta
     6061 agggattttg gtcatgagat tatcaaaaag gatcttcacc tagatccttt taaattaaaa
     6121 atgaagtttt aaatcaatct aaagtatata tgagtaaact tggtctgaca gttaccaatg
     6181 cttaatcagt gaggcaccta tctcagcgat ctgtctattt cgttcatcca tagttgcctg
     6241 actccccgtc gtgtagataa ctacgatacg ggagggctta ccatctggcc ccagtgctgc
     6301 aatgataccg cgagacccac gctcaccggc tccagattta tcagcaataa accagccagc
     6361 cggaagggcc gagcgcagaa gtggtcctgc aactttatcc gcctccatcc agtctattaa
     6421 ttgttgccgg gaagctagag taagtagttc gccagttaat agtttgcgca acgttgttgc
     6481 cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat tcagctccgg
     6541 ttcccaacga tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag cggttagctc
     6601 cttcggtcct ccgatcgttg tcagaagtaa gttggccgca gtgttatcac tcatggttat
     6661 ggcagcactg cataattctc ttactgtcat gccatccgta agatgctttt ctgtgactgg
     6721 tgagtactca accaagtcat tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc
     6781 ggcgtcaata cgggataata ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg
     6841 aaaacgttct tcggggcgaa aactctcaag gatcttaccg ctgttgagat ccagttcgat
     6901 gtaacccact cgtgcaccca actgatcttc agcatctttt actttcacca gcgtttctgg
     6961 gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga ataagggcga cacggaaatg
     7021 ttgaatactc atactcttcc tttttcaata ttattgaagc atttatcagg gttattgtct
     7081 catgagcgga tacatatttg aatgtattta gaaaaataaa caaatagggg ttccgcgcac
     7141 atttccccga aaagtgccac ctgacgtc
//