LOCUS       sequence-336171-        6319 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Plasmid expressing GRK2(RH)-Nluc L118A in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6319)
  AUTHORS   Maziarz M, Park JC, Leyme A, Marivin A, Garcia-Lopez A, Patel PP, 
            Garcia-Marcos M
  TITLE     Revealing the Activity of Trimeric G-proteins in Live Cells with a 
            Versatile Biosensor Design.
  JOURNAL   Cell. 2020 Jun 27. pii: S0092-8674(20)30752-2. doi: 
            10.1016/j.cell.2020.06.020.
  PUBMED    32634377
REFERENCE   2  (bases 1 to 6319)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Cell. 2020 
            Jun 27. pii: S0092-8674(20)30752-2. doi: 
            10.1016/j.cell.2020.06.020."
COMMENT     Sequence Label: pcDNA3.1-mas-GRK2RH-Nluc L118A
FEATURES             Location/Qualifiers
     source          1..6319
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        1..380
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        381..584
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     535..555
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     629..648
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        629..647
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             670..690
                     /codon_start=1
                     /product="N-myristoylation signal from Src kinase (Pellman 
                     et al., 1985; Kaplan et al., 1988)"
                     /label=myr
                     /translation="MGSSKSK"
     CDS             1123..1638
                     /codon_start=1
                     /product="NanoLuc(R) luciferase"
                     /label=Nluc
                     /note="human codon-optimized"
                     /translation="MVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQR
                     IVLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGV
                     TPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGW
                     RLCERILA"
     primer_bind     complement(1676..1693)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1682..1906
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      1952..2380
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2039..2058)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2249..2270
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2389..2409)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2394..2723
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      2574..2709
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     2636..2655
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             2790..3584
                     /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
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(2844..2863)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3454..3473
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    3758..3879
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3795..3814)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3849..3868
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(3928..3944)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3928..3944)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3941..3963)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3952..3968
                     /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(join(3976..3982,3983..4000,4001..4006))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4021..4042
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4159..4176)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4330..4918)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4410..4429)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5089..5880,5881..5949))
                     /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     5712..5731
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5950..6054)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(6129..6148)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 gacattgatt attgactagt tattaatagt aatcaattac ggggtcatta gttcatagcc
       61 catatatgga gttccgcgtt acataactta cggtaaatgg cccgcctggc tgaccgccca
      121 acgacccccg cccattgacg tcaataatga cgtatgttcc catagtaacg ccaataggga
      181 ctttccattg acgtcaatgg gtggagtatt tacggtaaac tgcccacttg gcagtacatc
      241 aagtgtatca tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct
      301 ggcattatgc ccagtacatg accttatggg actttcctac ttggcagtac atctacgtat
      361 tagtcatcgc tattaccatg gtgatgcggt tttggcagta catcaatggg cgtggatagc
      421 ggtttgactc acggggattt ccaagtctcc accccattga cgtcaatggg agtttgtttt
      481 ggcaccaaaa tcaacgggac tttccaaaat gtcgtaacaa ctccgcccca ttgacgcaaa
      541 tgggcggtag gcgtgtacgg tgggaggtct atataagcag agctctctgg ctaactagag
      601 aacccactgc ttactggctt atcgaaatta atacgactca ctatagggag acccaagctg
      661 gctagcacca tggggagtag caagagcaag acgtcgaatt ccggaatcaa gcttaagtac
      721 ctggaggacc ggggcgaggt gacttttgag aagatcttct cccagaagct ggggtacctg
      781 cttttccgag acttctgcct gaagcacctg gaggaggcca agcccttggt agagttctac
      841 gaggagatca agaaatacga gaagctggag acagaggagg agcgcctggt ctgcagccga
      901 gagatcttcg acacgtacat catgaaggag ctggcggcct gctcacatcc tttctcgaag
      961 agcgccattg agcacgtcca gggccatctg gtgaagaagc aggtgcctcc ggatctcttc
     1021 cagccatata ttgaagaaat ttgccagaac ctccgaggag acgtgttcca gaaattcatc
     1081 gagagcgata aattcacacg gttttgccag tggaagggat ccatggtctt cacactcgaa
     1141 gatttcgttg gggactggcg acagacagcc ggctacaacc tggaccaagt ccttgaacag
     1201 ggaggtgtgt ccagtttgtt tcagaatctc ggggtgtccg taactccgat ccaaaggatt
     1261 gtcctgagcg gtgaaaatgg gctgaagatc gacatccatg tcatcatccc gtatgaaggt
     1321 ctgagcggcg accaaatggg ccagatcgaa aaaattttta aggtggtgta ccctgtggat
     1381 gatcatcact ttaaggtgat cctgcactat ggcacactgg taatcgacgg ggttacgccg
     1441 aacatgatcg actatttcgg acggccgtat gaaggcatcg ccgtgttcga cggcaaaaag
     1501 atcactgtaa cagggaccct gtggaacggc aacaaaatta tcgacgagcg cctgatcaac
     1561 cccgacggct ccctgctgtt ccgagtaacc atcaacggag tgaccggctg gcggctgtgc
     1621 gaacgcattc tggcgtaact cgagtctaga gggcccgttt aaacccgctg atcagcctcg
     1681 actgtgcctt ctagttgcca gccatctgtt gtttgcccct cccccgtgcc ttccttgacc
     1741 ctggaaggtg ccactcccac tgtcctttcc taataaaatg aggaaattgc atcgcattgt
     1801 ctgagtaggt gtcattctat tctggggggt ggggtggggc aggacagcaa gggggaggat
     1861 tgggaagaca atagcaggca tgctggggat gcggtgggct ctatggcttc tgaggcggaa
     1921 agaaccagct ggggctctag ggggtatccc cacgcgccct gtagcggcgc attaagcgcg
     1981 gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg ccagcgccct agcgcccgct
     2041 cctttcgctt tcttcccttc ctttctcgcc acgttcgccg gctttccccg tcaagctcta
     2101 aatcgggggc tccctttagg gttccgattt agtgctttac ggcacctcga ccccaaaaaa
     2161 cttgattagg gtgatggttc acgtagtggg ccatcgccct gatagacggt ttttcgccct
     2221 ttgacgttgg agtccacgtt ctttaatagt ggactcttgt tccaaactgg aacaacactc
     2281 aaccctatct cggtctattc ttttgattta taagggattt tgccgatttc ggcctattgg
     2341 ttaaaaaatg agctgattta acaaaaattt aacgcgaatt aattctgtgg aatgtgtgtc
     2401 agttagggtg tggaaagtcc ccaggctccc cagcaggcag aagtatgcaa agcatgcatc
     2461 tcaattagtc agcaaccagg tgtggaaagt ccccaggctc cccagcaggc agaagtatgc
     2521 aaagcatgca tctcaattag tcagcaacca tagtcccgcc cctaactccg cccatcccgc
     2581 ccctaactcc gcccagttcc gcccattctc cgccccatgg ctgactaatt ttttttattt
     2641 atgcagaggc cgaggccgcc tctgcctctg agctattcca gaagtagtga ggaggctttt
     2701 ttggaggcct aggcttttgc aaaaagctcc cgggagcttg tatatccatt ttcggatctg
     2761 atcaagagac aggatgagga tcgtttcgca tgattgaaca agatggattg cacgcaggtt
     2821 ctccggccgc ttgggtggag aggctattcg gctatgactg ggcacaacag acaatcggct
     2881 gctctgatgc cgccgtgttc cggctgtcag cgcaggggcg cccggttctt tttgtcaaga
     2941 ccgacctgtc cggtgccctg aatgaactgc aggacgaggc agcgcggcta tcgtggctgg
     3001 ccacgacggg cgttccttgc gcagctgtgc tcgacgttgt cactgaagcg ggaagggact
     3061 ggctgctatt gggcgaagtg ccggggcagg atctcctgtc atctcacctt gctcctgccg
     3121 agaaagtatc catcatggct gatgcaatgc ggcggctgca tacgcttgat ccggctacct
     3181 gcccattcga ccaccaagcg aaacatcgca tcgagcgagc acgtactcgg atggaagccg
     3241 gtcttgtcga tcaggatgat ctggacgaag agcatcaggg gctcgcgcca gccgaactgt
     3301 tcgccaggct caaggcgcgc atgcccgacg gcgaggatct cgtcgtgacc catggcgatg
     3361 cctgcttgcc gaatatcatg gtggaaaatg gccgcttttc tggattcatc gactgtggcc
     3421 ggctgggtgt ggcggaccgc tatcaggaca tagcgttggc tacccgtgat attgctgaag
     3481 agcttggcgg cgaatgggct gaccgcttcc tcgtgcttta cggtatcgcc gctcccgatt
     3541 cgcagcgcat cgccttctat cgccttcttg acgagttctt ctgagcggga ctctggggtt
     3601 cgaaatgacc gaccaagcga cgcccaacct gccatcacga gatttcgatt ccaccgccgc
     3661 cttctatgaa aggttgggct tcggaatcgt tttccgggac gccggctgga tgatcctcca
     3721 gcgcggggat ctcatgctgg agttcttcgc ccaccccaac ttgtttattg cagcttataa
     3781 tggttacaaa taaagcaata gcatcacaaa tttcacaaat aaagcatttt tttcactgca
     3841 ttctagttgt ggtttgtcca aactcatcaa tgtatcttat catgtctgta taccgtcgac
     3901 ctctagctag agcttggcgt aatcatggtc atagctgttt cctgtgtgaa attgttatcc
     3961 gctcacaatt ccacacaaca tacgagccgg aagcataaag tgtaaagcct ggggtgccta
     4021 atgagtgagc taactcacat taattgcgtt gcgctcactg cccgctttcc agtcgggaaa
     4081 cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat
     4141 tgggcgctct tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg
     4201 agcggtatca gctcactcaa aggcggtaat acggttatcc acagaatcag gggataacgc
     4261 aggaaagaac atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt
     4321 gctggcgttt ttccataggc tccgcccccc tgacgagcat cacaaaaatc gacgctcaag
     4381 tcagaggtgg cgaaacccga caggactata aagataccag gcgtttcccc ctggaagctc
     4441 cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga tacctgtccg cctttctccc
     4501 ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg tatctcagtt cggtgtaggt
     4561 cgttcgctcc aagctgggct gtgtgcacga accccccgtt cagcccgacc gctgcgcctt
     4621 atccggtaac tatcgtcttg agtccaaccc ggtaagacac gacttatcgc cactggcagc
     4681 agccactggt aacaggatta gcagagcgag gtatgtaggc ggtgctacag agttcttgaa
     4741 gtggtggcct aactacggct acactagaag aacagtattt ggtatctgcg ctctgctgaa
     4801 gccagttacc ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg
     4861 tagcggtggt ttttttgttt gcaagcagca gattacgcgc agaaaaaaag gatctcaaga
     4921 agatcctttg atcttttcta cggggtctga cgctcagtgg aacgaaaact cacgttaagg
     4981 gattttggtc atgagattat caaaaaggat cttcacctag atccttttaa attaaaaatg
     5041 aagttttaaa tcaatctaaa gtatatatga gtaaacttgg tctgacagtt accaatgctt
     5101 aatcagtgag gcacctatct cagcgatctg tctatttcgt tcatccatag ttgcctgact
     5161 ccccgtcgtg tagataacta cgatacggga gggcttacca tctggcccca gtgctgcaat
     5221 gataccgcga gacccacgct caccggctcc agatttatca gcaataaacc agccagccgg
     5281 aagggccgag cgcagaagtg gtcctgcaac tttatccgcc tccatccagt ctattaattg
     5341 ttgccgggaa gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat
     5401 tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg gcttcattca gctccggttc
     5461 ccaacgatca aggcgagtta catgatcccc catgttgtgc aaaaaagcgg ttagctcctt
     5521 cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg ttatcactca tggttatggc
     5581 agcactgcat aattctctta ctgtcatgcc atccgtaaga tgcttttctg tgactggtga
     5641 gtactcaacc aagtcattct gagaatagtg tatgcggcga ccgagttgct cttgcccggc
     5701 gtcaatacgg gataataccg cgccacatag cagaacttta aaagtgctca tcattggaaa
     5761 acgttcttcg gggcgaaaac tctcaaggat cttaccgctg ttgagatcca gttcgatgta
     5821 acccactcgt gcacccaact gatcttcagc atcttttact ttcaccagcg tttctgggtg
     5881 agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata agggcgacac ggaaatgttg
     5941 aatactcata ctcttccttt ttcaatatta ttgaagcatt tatcagggtt attgtctcat
     6001 gagcggatac atatttgaat gtatttagaa aaataaacaa ataggggttc cgcgcacatt
     6061 tccccgaaaa gtgccacctg acgtcgacgg atcgggagat ctcccgatcc cctatggtgc
     6121 actctcagta caatctgctc tgatgccgca tagttaagcc agtatctgct ccctgcttgt
     6181 gtgttggagg tcgctgagta gtgcgcgagc aaaatttaag ctacaacaag gcaaggcttg
     6241 accgacaatt gcatgaagaa tctgcttagg gttaggcgtt ttgcgctgct tcgcgatgta
     6301 cgggccagat atacgcgtt
//
