LOCUS       Exported                8223 bp ds-DNA     circular SYN 14-MAY-2021
DEFINITION  Encodes a BRET-based activity sensor for the heterotrimeric G 
            protein Go1. Composed of the subunits G alpha o1 (GNAO1) tagged with
            NanoLuciferase, G beta 3 (GNB3) and Venus-tagged G gamma 9 (GNG9). .
ACCESSION   .
VERSION     .
KEYWORDS    Go1-CASE
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8223)
  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 8223)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 14, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..8223
                     /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             3505..4011
                     /codon_start=1
                     /product="NanoLuc(R) luciferase"
                     /label=Nluc
                     /note="human codon-optimized"
                     /translation="VFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQRI
                     VLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGVT
                     PNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGWR
                     LCERIL"
     polyA_signal    4951..5072
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4988..5007)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5042..5061
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(5079..5534)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5216..5237)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5428..5447
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        5561..5665
                     /gene="bla"
                     /label=AmpR promoter
     promoter        5667..6024
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     primer_bind     complement(5690..5710)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      5875..6010
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     5937..5956
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             6059..6853
                     /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(6113..6132)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     6723..6742
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(7041..7060)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    7085..7132
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      7461..8049
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7950..7969
                     /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 ggatgtactc tgagcgcaga ggagagagcc
     3241 gccctcgagc ggagcaaggc gattgagaaa aacctcaaag aggatggcat cagcgccgcc
     3301 aaagacgtga aattactcct gctcggtgct ggagaatcag gaaaaagcac cattgtgaag
     3361 cagatgaaaa taatccatga agatggcttc tccggagaag acgtgaaaca gtacaagcct
     3421 gttgtctaca gcaacactat ccagtccctg gcagccatcg tccgggccat ggacactttg
     3481 agcggtggag gcgggacgcg ttctgtcttc acactcgaag atttcgttgg ggactggcga
     3541 cagacagccg gctacaacct ggaccaagtc cttgaacagg gaggtgtgtc cagtttgttt
     3601 cagaatctcg gggtgtccgt aactccgatc caaaggattg tcctgagcgg tgaaaatggg
     3661 ctgaagatcg acatccatgt catcatcccg tatgaaggtc tgagcggcga ccaaatgggc
     3721 cagatcgaaa aaatttttaa ggtggtgtac cctgtggatg atcatcactt taaggtgatc
     3781 ctgcactatg gcacactggt aatcgacggg gttacgccga acatgatcga ctatttcgga
     3841 cggccgtatg aaggcatcgc cgtgttcgac ggcaaaaaga tcactgtaac agggaccctg
     3901 tggaacggca acaaaattat cgacgagcgc ctgatcaacc ccgacggctc cctgctgttc
     3961 cgagtaacca tcaacggagt gaccggctgg cggctgtgcg aacgcattct ggccaccggt
     4021 ggtggcggat ccggcatcga atatggtgat aaggagagaa aggctgacgc caagatggtg
     4081 tgtgatgtgg tgagtcggat ggaagacacc gagcccttct ctgcagagct gctttctgcc
     4141 atgatgcggc tctggggcga ctcaggaatc caagagtgct tcaaccggtc ccgggagtat
     4201 cagctcaacg actctgccaa atactacctg gacagcctgg atcggattgg ggccgccgac
     4261 taccagccca ccgagcagga catcctccga accagggtca aaaccactgg catcgtagaa
     4321 acccacttca cattcaagaa cctccacttc aggctgtttg acgtcggagg ccagcgatct
     4381 gaacgcaaga agtggatcca ttgcttcgag gacgtcacgg ccatcatttt ctgtgtcgcg
     4441 ctcagcggct atgaccaggt gctccacgaa gacgaaacca cgaaccgcat gcacgagtct
     4501 ctcatgctct tcgactccat ctgtaacaac aagttcttca tcgatacctc catcattctc
     4561 ttcctcaaca agaaagatct ctttggcgag aagatcaaga agtcaccttt gaccatctgc
     4621 tttcctgaat acacaggtcc caatacctat gaagacgcag ccgcctacat ccaagcacaa
     4681 tttgaaagca aaaaccgctc acccaacaaa gaaatatatt gtcacatgac ttgtgccaca
     4741 gacacgaata acatccaggt ggtgttcgac gccgtcaccg acatcatcat tgccaacaac
     4801 ctccggggct gcggcttgta ctaatctaga gggccgcgac tctagatcat aatcagccat
     4861 accacatttg tagaggtttt acttgcttta aaaaacctcc cacacctccc cctgaacctg
     4921 aaacataaaa tgaatgcaat tgttgttgtt aacttgttta ttgcagctta taatggttac
     4981 aaataaagca atagcatcac aaatttcaca aataaagcat ttttttcact gcattctagt
     5041 tgtggtttgt ccaaactcat caatgtatct taaggcgtaa attgtaagcg ttaatatttt
     5101 gttaaaattc gcgttaaatt tttgttaaat cagctcattt tttaaccaat aggccgaaat
     5161 cggcaaaatc ccttataaat caaaagaata gaccgagata gggttgagtg ttgttccagt
     5221 ttggaacaag agtccactat taaagaacgt ggactccaac gtcaaagggc gaaaaaccgt
     5281 ctatcagggc gatggcccac tacgtgaacc atcaccctaa tcaagttttt tggggtcgag
     5341 gtgccgtaaa gcactaaatc ggaaccctaa agggagcccc cgatttagag cttgacgggg
     5401 aaagccggcg aacgtggcga gaaaggaagg gaagaaagcg aaaggagcgg gcgctagggc
     5461 gctggcaagt gtagcggtca cgctgcgcgt aaccaccaca cccgccgcgc ttaatgcgcc
     5521 gctacagggc gcgtcaggtg gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt
     5581 atttttctaa atacattcaa atatgtatcc gctcatgaga caataaccct gataaatgct
     5641 tcaataatat tgaaaaagga agagtcctga ggcggaaaga accagctgtg gaatgtgtgt
     5701 cagttagggt gtggaaagtc cccaggctcc ccagcaggca gaagtatgca aagcatgcat
     5761 ctcaattagt cagcaaccag gtgtggaaag tccccaggct ccccagcagg cagaagtatg
     5821 caaagcatgc atctcaatta gtcagcaacc atagtcccgc ccctaactcc gcccatcccg
     5881 cccctaactc cgcccagttc cgcccattct ccgccccatg gctgactaat tttttttatt
     5941 tatgcagagg ccgaggccgc ctcggcctct gagctattcc agaagtagtg aggaggcttt
     6001 tttggaggcc taggcttttg caaagatcga tcaagagaca ggatgaggat cgtttcgcat
     6061 gattgaacaa gatggattgc acgcaggttc tccggccgct tgggtggaga ggctattcgg
     6121 ctatgactgg gcacaacaga caatcggctg ctctgatgcc gccgtgttcc ggctgtcagc
     6181 gcaggggcgc ccggttcttt ttgtcaagac cgacctgtcc ggtgccctga atgaactgca
     6241 agacgaggca gcgcggctat cgtggctggc cacgacgggc gttccttgcg cagctgtgct
     6301 cgacgttgtc actgaagcgg gaagggactg gctgctattg ggcgaagtgc cggggcagga
     6361 tctcctgtca tctcaccttg ctcctgccga gaaagtatcc atcatggctg atgcaatgcg
     6421 gcggctgcat acgcttgatc cggctacctg cccattcgac caccaagcga aacatcgcat
     6481 cgagcgagca cgtactcgga tggaagccgg tcttgtcgat caggatgatc tggacgaaga
     6541 gcatcagggg ctcgcgccag ccgaactgtt cgccaggctc aaggcgagca tgcccgacgg
     6601 cgaggatctc gtcgtgaccc atggcgatgc ctgcttgccg aatatcatgg tggaaaatgg
     6661 ccgcttttct ggattcatcg actgtggccg gctgggtgtg gcggaccgct atcaggacat
     6721 agcgttggct acccgtgata ttgctgaaga gcttggcggc gaatgggctg accgcttcct
     6781 cgtgctttac ggtatcgccg ctcccgattc gcagcgcatc gccttctatc gccttcttga
     6841 cgagttcttc tgagcgggac tctggggttc gaaatgaccg accaagcgac gcccaacctg
     6901 ccatcacgag atttcgattc caccgccgcc ttctatgaaa ggttgggctt cggaatcgtt
     6961 ttccgggacg ccggctggat gatcctccag cgcggggatc tcatgctgga gttcttcgcc
     7021 caccctaggg ggaggctaac tgaaacacgg aaggagacaa taccggaagg aacccgcgct
     7081 atgacggcaa taaaaagaca gaataaaacg cacggtgttg ggtcgtttgt tcataaacgc
     7141 ggggttcggt cccagggctg gcactctgtc gataccccac cgagacccca ttggggccaa
     7201 tacgcccgcg tttcttcctt ttccccaccc caccccccaa gttcgggtga aggcccaggg
     7261 ctcgcagcca acgtcggggc ggcaggccct gccatagcct caggttactc atatatactt
     7321 tagattgatt taaaacttca tttttaattt aaaaggatct aggtgaagat cctttttgat
     7381 aatctcatga ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc agaccccgta
     7441 gaaaagatca aaggatcttc ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa
     7501 acaaaaaaac caccgctacc agcggtggtt tgtttgccgg atcaagagct accaactctt
     7561 tttccgaagg taactggctt cagcagagcg cagataccaa atactgttct tctagtgtag
     7621 ccgtagttag gccaccactt caagaactct gtagcaccgc ctacatacct cgctctgcta
     7681 atcctgttac cagtggctgc tgccagtggc gataagtcgt gtcttaccgg gttggactca
     7741 agacgatagt taccggataa ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag
     7801 cccagcttgg agcgaacgac ctacaccgaa ctgagatacc tacagcgtga gctatgagaa
     7861 agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga
     7921 acaggagagc gcacgaggga gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc
     7981 gggtttcgcc acctctgact tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc
     8041 ctatggaaaa acgccagcaa cgcggccttt ttacggttcc tggccttttg ctggcctttt
     8101 gctcacatgt tctttcctgc gttatcccct gattctgtgg ataaccgtat taccgccatg
     8161 cattagttat taatagtaat caattacggg gtcattagtt catagcccat atatggagtt
     8221 ccg
//
