LOCUS       Exported                6459 bp ds-DNA     circular SYN 12-MAY-2021
DEFINITION  Encodes a G gamma subunit (GNG8) containing GFP2 as an optimal 
            component of a BRET2 biosensor for studying heterotrimeric G protein
            dissociation.
ACCESSION   .
VERSION     .
KEYWORDS    pcDNA3.1-GGamma8-GFP2
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6459)
  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 6459)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 12, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..6459
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     rep_origin      complement(66..654)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(146..165)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(825..1685)
                     /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     1448..1467
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(1686..1790)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(1865..1884)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        2056..2435
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        2436..2639
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     2590..2610
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     2684..2703
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        2684..2702
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      2720..2729
                     /regulatory_class="other"
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
     CDS             2726..3442
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLSYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNPHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLFTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(2771..2792)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(3032..3051)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     3379..3400
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(3702..3719)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3708..3932
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      3978..4406
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4065..4084)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4275..4296
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4415..4435)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        4420..4749
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      4600..4735
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     4662..4681
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             4798..5823
                     /codon_start=1
                     /gene="aph(4)-Ia"
                     /product="aminoglycoside phosphotransferase from E. coli"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
                     YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
                     ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
                     TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
                     GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
                     NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
                     E"
     polyA_signal    5953..6074
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5990..6009)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     6044..6063
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(6123..6139)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6123..6139)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(6136..6158)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    6147..6163
                     /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(6171..6201)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6216..6237
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(6354..6371)
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg
       61 gcgtttttcc ataggctccg cccccctgac gagcatcaca aaaatcgacg ctcaagtcag
      121 aggtggcgaa acccgacagg actataaaga taccaggcgt ttccccctgg aagctccctc
      181 gtgcgctctc ctgttccgac cctgccgctt accggatacc tgtccgcctt tctcccttcg
      241 ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt
      301 cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc
      361 ggtaactatc gtcttgagtc caacccggta agacacgact tatcgccact ggcagcagcc
      421 actggtaaca ggattagcag agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg
      481 tggcctaact acggctacac tagaagaaca gtatttggta tctgcgctct gctgaagcca
      541 gttaccttcg gaaaaagagt tggtagctct tgatccggca aacaaaccac cgctggtagc
      601 ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat
      661 cctttgatct tttctacggg gtctgacgct cagtggaacg aaaactcacg ttaagggatt
      721 ttggtcatga gattatcaaa aaggatcttc acctagatcc ttttaaatta aaaatgaagt
      781 tttaaatcaa tctaaagtat atatgagtaa acttggtctg acagttacca atgcttaatc
      841 agtgaggcac ctatctcagc gatctgtcta tttcgttcat ccatagttgc ctgactcccc
      901 gtcgtgtaga taactacgat acgggagggc ttaccatctg gccccagtgc tgcaatgata
      961 ccgcgagacc cacgctcacc ggctccagat ttatcagcaa taaaccagcc agccggaagg
     1021 gccgagcgca gaagtggtcc tgcaacttta tccgcctcca tccagtctat taattgttgc
     1081 cgggaagcta gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt tgccattgct
     1141 acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc cggttcccaa
     1201 cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa aagcggttag ctccttcggt
     1261 cctccgatcg ttgtcagaag taagttggcc gcagtgttat cactcatggt tatggcagca
     1321 ctgcataatt ctcttactgt catgccatcc gtaagatgct tttctgtgac tggtgagtac
     1381 tcaaccaagt cattctgaga atagtgtatg cggcgaccga gttgctcttg cccggcgtca
     1441 atacgggata ataccgcgcc acatagcaga actttaaaag tgctcatcat tggaaaacgt
     1501 tcttcggggc gaaaactctc aaggatctta ccgctgttga gatccagttc gatgtaaccc
     1561 actcgtgcac ccaactgatc ttcagcatct tttactttca ccagcgtttc tgggtgagca
     1621 aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa atgttgaata
     1681 ctcatactct tcctttttca atattattga agcatttatc agggttattg tctcatgagc
     1741 ggatacatat ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg cacatttccc
     1801 cgaaaagtgc cacctgacgt cgacggatcg ggagatctcc cgatccccta tggtgcactc
     1861 tcagtacaat ctgctctgat gccgcatagt taagccagta tctgctccct gcttgtgtgt
     1921 tggaggtcgc tgagtagtgc gcgagcaaaa tttaagctac aacaaggcaa ggcttgaccg
     1981 acaattgcat gaagaatctg cttagggtta ggcgttttgc gctgcttcgc gatgtacggg
     2041 ccagatatac gcgttgacat tgattattga ctagttatta atagtaatca attacggggt
     2101 cattagttca tagcccatat atggagttcc gcgttacata acttacggta aatggcccgc
     2161 ctggctgacc gcccaacgac ccccgcccat tgacgtcaat aatgacgtat gttcccatag
     2221 taacgccaat agggactttc cattgacgtc aatgggtgga gtatttacgg taaactgccc
     2281 acttggcagt acatcaagtg tatcatatgc caagtacgcc ccctattgac gtcaatgacg
     2341 gtaaatggcc cgcctggcat tatgcccagt acatgacctt atgggacttt cctacttggc
     2401 agtacatcta cgtattagtc atcgctatta ccatggtgat gcggttttgg cagtacatca
     2461 atgggcgtgg atagcggttt gactcacggg gatttccaag tctccacccc attgacgtca
     2521 atgggagttt gttttggcac caaaatcaac gggactttcc aaaatgtcgt aacaactccg
     2581 ccccattgac gcaaatgggc ggtaggcgtg tacggtggga ggtctatata agcagagctc
     2641 tctggctaac tagagaaccc actgcttact ggcttatcga aattaatacg actcactata
     2701 gggagaccca agctggctag ccaccatggt gagcaagggc gaggagctgt tcaccggggt
     2761 ggtgcccatc ctggtcgagc tggacggcga cgtaaacggc cacaagttca gcgtgtccgg
     2821 cgagggcgag ggcgatgcca cctacggcaa gctgaccctg aagttcatct gcaccaccgg
     2881 caagctgccc gtgccctggc ccaccctcgt gaccaccctg agctacggcg tgcagtgctt
     2941 cagccgctac cccgaccaca tgaagcagca cgacttcttc aagtccgcca tgcccgaagg
     3001 ctacgtccag gagcgcacca tcttcttcaa ggacgacggc aactacaaga cccgcgccga
     3061 ggtgaagttc gagggcgaca ccctggtgaa ccgcatcgag ctgaagggca tcgacttcaa
     3121 ggaggacggc aacatcctgg ggcacaagct ggagtacaac tacaaccccc acaacgtcta
     3181 tatcatggcc gacaagcaga agaacggcat caaggtgaac ttcaagatcc gccacaacat
     3241 cgaggacggc agcgtgcagc tcgccgacca ctaccagcag aacaccccca tcggcgacgg
     3301 ccccgtgctg ctgcccgaca accactacct gttcacccag tccgccctga gcaaagaccc
     3361 caacgagaag cgcgatcaca tggtcctgct ggagttcgtg accgccgccg ggatcactct
     3421 cggcatggac gagctgtaca agggatccgc cggtaccatg tccaacaaca tggccaagat
     3481 tgccgaggcc cgcaagacgg tggaacagct gaagctggag gtgaacatcg accgcatgaa
     3541 ggtgtcgcag gcagcagcgg aactcctggc tttctgcgag acgcatgcca aagatgaccc
     3601 gctggtgacg ccagtacccg ccgcggagaa ccccttccgc gacaagcgcc tcttttgtgt
     3661 tctgctctga tctagagggc ccgtttaaac ccgctgatca gcctcgactg tgccttctag
     3721 ttgccagcca tctgttgttt gcccctcccc cgtgccttcc ttgaccctgg aaggtgccac
     3781 tcccactgtc ctttcctaat aaaatgagga aattgcatcg cattgtctga gtaggtgtca
     3841 ttctattctg gggggtgggg tggggcagga cagcaagggg gaggattggg aagacaatag
     3901 caggcatgct ggggatgcgg tgggctctat ggcttctgag gcggaaagaa ccagctgggg
     3961 ctctaggggg tatccccacg cgccctgtag cggcgcatta agcgcggcgg gtgtggtggt
     4021 tacgcgcagc gtgaccgcta cacttgccag cgccctagcg cccgctcctt tcgctttctt
     4081 cccttccttt ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc gggggctccc
     4141 tttagggttc cgatttagtg ctttacggca cctcgacccc aaaaaacttg attagggtga
     4201 tggttcacgt agtgggccat cgccctgata gacggttttt cgccctttga cgttggagtc
     4261 cacgttcttt aatagtggac tcttgttcca aactggaaca acactcaacc ctatctcggt
     4321 ctattctttt gatttataag ggattttgcc gatttcggcc tattggttaa aaaatgagct
     4381 gatttaacaa aaatttaacg cgaattaatt ctgtggaatg tgtgtcagtt agggtgtgga
     4441 aagtccccag gctccccagc aggcagaagt atgcaaagca tgcatctcaa ttagtcagca
     4501 accaggtgtg gaaagtcccc aggctcccca gcaggcagaa gtatgcaaag catgcatctc
     4561 aattagtcag caaccatagt cccgccccta actccgccca tcccgcccct aactccgccc
     4621 agttccgccc attctccgcc ccatggctga ctaatttttt ttatttatgc agaggccgag
     4681 gccgcctctg cctctgagct attccagaag tagtgaggag gcttttttgg aggcctaggc
     4741 ttttgcaaaa agctcccggg agcttgtata tccattttcg gatctgatca gcacgtgatg
     4801 aaaaagcctg aactcaccgc gacgtctgtc gagaagtttc tgatcgaaaa gttcgacagc
     4861 gtctccgacc tgatgcagct ctcggagggc gaagaatctc gtgctttcag cttcgatgta
     4921 ggagggcgtg gatatgtcct gcgggtaaat agctgcgccg atggtttcta caaagatcgt
     4981 tatgtttatc ggcactttgc atcggccgcg ctcccgattc cggaagtgct tgacattggg
     5041 gaattcagcg agagcctgac ctattgcatc tcccgccgtg cacagggtgt cacgttgcaa
     5101 gacctgcctg aaaccgaact gcccgctgtt ctgcagccgg tcgcggaggc catggatgcg
     5161 atcgctgcgg ccgatcttag ccagacgagc gggttcggcc cattcggacc gcaaggaatc
     5221 ggtcaataca ctacatggcg tgatttcata tgcgcgattg ctgatcccca tgtgtatcac
     5281 tggcaaactg tgatggacga caccgtcagt gcgtccgtcg cgcaggctct cgatgagctg
     5341 atgctttggg ccgaggactg ccccgaagtc cggcacctcg tgcacgcgga tttcggctcc
     5401 aacaatgtcc tgacggacaa tggccgcata acagcggtca ttgactggag cgaggcgatg
     5461 ttcggggatt cccaatacga ggtcgccaac atcttcttct ggaggccgtg gttggcttgt
     5521 atggagcagc agacgcgcta cttcgagcgg aggcatccgg agcttgcagg atcgccgcgg
     5581 ctccgggcgt atatgctccg cattggtctt gaccaactct atcagagctt ggttgacggc
     5641 aatttcgatg atgcagcttg ggcgcagggt cgatgcgacg caatcgtccg atccggagcc
     5701 gggactgtcg ggcgtacaca aatcgcccgc agaagcgcgg ccgtctggac cgatggctgt
     5761 gtagaagtac tcgccgatag tggaaaccga cgccccagca ctcgtccgag ggcaaaggaa
     5821 tagcacgtgc tacgagattt cgattccacc gccgccttct atgaaaggtt gggcttcgga
     5881 atcgttttcc gggacgccgg ctggatgatc ctccagcgcg gggatctcat gctggagttc
     5941 ttcgcccacc ccaacttgtt tattgcagct tataatggtt acaaataaag caatagcatc
     6001 acaaatttca caaataaagc atttttttca ctgcattcta gttgtggttt gtccaaactc
     6061 atcaatgtat cttatcatgt ctgtataccg tcgacctcta gctagagctt ggcgtaatca
     6121 tggtcatagc tgtttcctgt gtgaaattgt tatccgctca caattccaca caacatacga
     6181 gccggaagca taaagtgtaa agcctggggt gcctaatgag tgagctaact cacattaatt
     6241 gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt cgtgccagct gcattaatga
     6301 atcggccaac gcgcggggag aggcggtttg cgtattgggc gctcttccgc ttcctcgctc
     6361 actgactcgc tgcgctcggt cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg
     6421 gtaatacggt tatccacaga atcaggggat aacgcagga
//
