LOCUS       sequence-443646-        7998 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Plasmid expressing GINIP-Nluc W139A in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7998)
  AUTHORS   Luebbers A, Janicot R, Zhao J, Philibert CE, Garcia-Marcos M
  TITLE     A sensitive biosensor of endogenous Galpha(i) activity enables the 
            accurate characterization of endogenous GPCR agonist responses.
  JOURNAL   Sci Signal. 2025 Mar 25;18(879):eadp6457. doi: 
            10.1126/scisignal.adp6457. Epub 2025 Mar 25.
  PUBMED    40132053
REFERENCE   2  (bases 1 to 7998)
  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: "Sci 
            Signal."; date: "2025-03-25"; volume: "18(879):eadp6457. doi"; 
            pages: " 10.1126/scisignal.adp6457. Epub 2025 Mar 25"
COMMENT     Sequence Label: p3xFLAG-CMV-14-GINIP-Nluc W139A
FEATURES             Location/Qualifiers
     source          1..7998
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     112..132
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     CDS             1416..1928
                     /codon_start=1
                     /product="NanoLuc(R) luciferase"
                     /label=Nluc
                     /note="human codon-optimized"
                     /translation="MVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQR
                     IVLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGV
                     TPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGW
                     RLCERILA"
     CDS             1941..2006
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     polyA_signal    2017..2639
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(2216..2235)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     primer_bind     complement(2663..2683)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2681..2978
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     CDS             3032..3826
                     /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(3086..3105)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3696..3715
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4478..4612
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4515..4534)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4569..4588
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4617..4636)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4618..4636)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4650..4666)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4650..4666)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4663..4685)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4674..4690
                     /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(4698..4704,4705..4722,4723..4728))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4743..4764
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4881..4898)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5052..5640)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5132..5151)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5811..6602,6603..6671))
                     /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     6434..6453
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6672..6776)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      6803..7258
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6890..6909)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     7100..7121
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     7384..7406
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     7398..7415
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     7399..7415
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     enhancer        7576..7955
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        join(7956..7998,1..161)
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
ORIGIN
        1 ttgactcacg gggatttcca agtctccacc ccattgacgt caatgggagt ttgttttggc
       61 accaaaatca acgggacttt ccaaaatgtc gtaataaccc cgccccgttg acgcaaatgg
      121 gcggtaggcg tgtacggtgg gaggtctata taagcagagc tcgtttagtg aaccgtcaga
      181 attaagcttg cggccgcgaa ttcatggggg tgttgatgtc caagcggcag acagtggagc
      241 aggtgcagaa ggtgagtctg gctgtgtctg ctttcaagga tgggctgcgg gacaggcctt
      301 ccatccgacg cacaggtgag ctgccagggt cccgccgtgg cactgtagag ggctccgtcc
      361 aggaggtaca ggaggagaag gaagcagaag caggaacctc ggtggtccag gaagagagta
      421 gtgccggccg cgcagcctgg gagcggctcc gagatgggcg cggcgtggag cctgaggagt
      481 ttgacaggac aagtcgattc acaccccctg cgttcatccg ccccacccgg aagctggatg
      541 atgacaaacc tccagaaatc tgcctggagc ccagagagcc tgttgtcaac gatgagatgt
      601 gtgatgtttg tgaggtcgcg acagctgaga gcctcttccc gtgcagggtc tgcaccaggg
      661 ttttccatga tggctgcctg cgccgcatgg gctacatcca aggagacagt gcagcggagg
      721 tgacggagat ggcccacaca gaaacaggct ggagctgcca ctactgtgac aacatcaact
      781 tgctgcttac tgaggaggaa atgtatagcc tcacggagac ctttcagcgg tgtaaagtca
      841 tccctgattg ctccctgaca ctggaggact ttctgcgtta ccgccaccaa gcagccaagc
      901 ggggggaccg tgacagggcc ctgagtgagg agcaagaaga gcaggcggcc cgccagtttg
      961 ctgccctgga ccctgaacat cgaggccaca tagagtggcc tgacttcttg tcccatgagt
     1021 ccctcctgct tctgcagcag ttgcgtcccc agaactctct gttgaggctt ctgacagtga
     1081 aggagcggga gcgagcccga gccgccttcc tggctcgggg cagtgggagc accgtcagtg
     1141 aggcagagtg ccgccgggcc cagcactctt ggttttgcaa acggttccca gaggctcctt
     1201 cctgcagtgt cagcatcagc catgtgggtc ccatcgcaga tagcagccca gccagcagca
     1261 gcagcaagag tcaggacaag accctgctgc ccacagagca ggagtccaga tttgtggact
     1321 ggcctacctt cctgcaagag aatgtcctct acatcctggc tgctcgcccc aacagcgcag
     1381 ccattcacct gaagccccca ggaggtggtg gttctatggt cttcacactc gaagatttcg
     1441 ttggggactg gcgacagaca gccggctaca acctggacca agtccttgaa cagggaggtg
     1501 tgtccagttt gtttcagaat ctcggggtgt ccgtaactcc gatccaaagg attgtcctga
     1561 gcggtgaaaa tgggctgaag atcgacatcc atgtcatcat cccgtatgaa ggtctgagcg
     1621 gcgaccaaat gggccagatc gaaaaaattt ttaaggtggt gtaccctgtg gatgatcatc
     1681 actttaaggt gatcctgcac tatggcacac tggtaatcga cggggttacg ccgaacatga
     1741 tcgactattt cggacggccg tatgaaggca tcgccgtgtt cgacggcaaa aagatcactg
     1801 taacagggac cctgtggaac ggcaacaaaa ttatcgacga gcgcctgatc aaccccgacg
     1861 gctccctgct gttccgagta accatcaacg gagtgaccgg ctggcggctg tgcgaacgca
     1921 ttctggcggg atcccgggct gactacaaag accatgacgg tgattataaa gatcatgaca
     1981 tcgactacaa ggatgacgat gacaagtagt gatcccgggt ggcatccctg tgacccctcc
     2041 ccagtgcctc tcctggccct ggaagttgcc actccagtgc ccaccagcct tgtcctaata
     2101 aaattaagtt gcatcatttt gtctgactag gtgtccttct ataatattat ggggtggagg
     2161 ggggtggtat ggagcaaggg gcaagttggg aagacaacct gtagggcctg cggggtctat
     2221 tgggaaccaa gctggagtgc agtggcacaa tcttggctca ctgcaatctc cgcctcctgg
     2281 gttcaagcga ttctcctgcc tcagcctccc gagttgttgg gattccaggc atgcatgacc
     2341 aggctcagct aatttttgtt tttttggtag agacggggtt tcaccatatt ggccaggctg
     2401 gtctccaact cctaatctca ggtgatctac ccaccttggc ctcccaaatt gctgggatta
     2461 caggcgtgaa ccactgctcc cttccctgtc cttctgattt taaaataact ataccagcag
     2521 gaggacgtcc agacacagca taggctacct ggccatgccc aaccggtggg acatttgagt
     2581 tgcttgcttg gcactgtcct ctcatgcgtt gggtccactc agtagatgcc tgttgaattg
     2641 ggtacgcggc cagcttggct gtggaatgtg tgtcagttag ggtgtggaaa gtccccaggc
     2701 tccccagcag gcagaagtat gcaaagcatg catctcaatt agtcagcaac caggtgtgga
     2761 aagtccccag gctccccagc aggcagaagt atgcaaagca tgcatctcaa ttagtcagca
     2821 accatagtcc cgcccctaac tccgcccatc ccgcccctaa ctccgcccag ttccgcccat
     2881 tctccgcccc atggctgact aatttttttt atttatgcag aggccgaggc cgcctcggcc
     2941 tctgagctat tccagaagta gtgaggaggc ttttttggag gaattgatca gcttgggatc
     3001 tgatcaagag acaggatgag gatcgtttcg catgattgaa caagatggat tgcacgcagg
     3061 ttctccggcc gcttgggtgg agaggctatt cggctatgac tgggcacaac agacaatcgg
     3121 ctgctctgat gccgccgtgt tccggctgtc agcgcagggg cgcccggttc tttttgtcaa
     3181 gaccgacctg tccggtgccc tgaatgaact gcaggacgag gcagcgcggc tatcgtggct
     3241 ggccacgacg ggcgttcctt gcgcagctgt gctcgacgtt gtcactgaag cgggaaggga
     3301 ctggctgcta ttgggcgaag tgccggggca ggatctcctg tcatctcacc ttgctcctgc
     3361 cgagaaagta tccatcatgg ctgatgcaat gcggcggctg catacgcttg atccggctac
     3421 ctgcccattc gaccaccaag cgaaacatcg catcgagcga gcacgtactc ggatggaagc
     3481 cggtcttgtc gatcaggatg atctggacga agagcatcag gggctcgcgc cagccgaact
     3541 gttcgccagg ctcaaggcgc gcatgcccga cggcgaggat ctcgtcgtga cccatggcga
     3601 tgcctgcttg ccgaatatca tggtggaaaa tggccgcttt tctggattca tcgactgtgg
     3661 ccggctgggt gtggcggacc gctatcagga catagcgttg gctacccgtg atattgctga
     3721 agagcttggc ggcgaatggg ctgaccgctt cctcgtgctt tacggtatcg ccgctcccga
     3781 ttcgcagcgc atcgccttct atcgccttct tgacgagttc ttctgagcgg gactctgggg
     3841 ttcgaaatga ccgaccaagc gacgcccaac ctgccatcac gagatttcga ttccaccgcc
     3901 gccttctatg aaaggttggg cttcggaatc gttttccggg acgccggctg gatgatcctc
     3961 cagcgcgggg atctcatgct ggagttcttc gcccaccccg ggctcgatcc cctcgcgagt
     4021 tggttcagct gctgcctgag gctggacgac ctcgcggagt tctaccggca gtgcaaatcc
     4081 gtcggcatcc aggaaaccag cagcggctat ccgcgcatcc atgcccccga actgcaggag
     4141 tggggaggca cgatggccgc tttggtcgac ccggacggga cgctcctgcg cctgatacag
     4201 aacgaattgc ttgcaggcat ctcatgagtg tgtcttcccg ttttccgcct gaggtcactg
     4261 cgtggatgga gcgctggcgc ctgctgcgcg acggcgagct gctcaccacc cactcgccaa
     4321 gctggaaccg taaaaaggcc gcgttgctgg cgtttttcca taggctccgc cgatcataat
     4381 cagccatacc acatttgtag aggttttact tgctttaaaa aacctcccac acctccccct
     4441 gaacctgaaa cataaaatga atgcaattgt tgttgttaac ttgtttattg cagcttataa
     4501 tggttacaaa taaagcaata gcatcacaaa tttcacaaat aaagcatttt tttcactgca
     4561 ttctagttgt ggtttgtcca aactcatcaa tgtatcttat catgtctgga tcaattccct
     4621 atagtgagtc gtattaaatt cgtaatcatg tcatagctgt ttcctgtgtg aaattgttat
     4681 ccgctcacaa ttccacacaa catacgagcc ggaagcataa agtgtaaagc ctggggtgcc
     4741 taatgagtga gctaactcac attaattgcg ttgcgctcac tgcccgcttt ccagtcggga
     4801 aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt
     4861 attgggcgct cttccgcttc ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg
     4921 cgagcggtat cagctcactc aaaggcggta atacggttat ccacagaatc aggggataac
     4981 gcaggaaaga acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg
     5041 ttgctggcgt ttttccatag gctccgcccc cctgacgagc atcacaaaaa tcgacgctca
     5101 agtcagaggt ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggaagc
     5161 tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc
     5221 ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta ggtatctcag ttcggtgtag
     5281 gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc
     5341 ttatccggta actatcgtct tgagtccaac ccggtaagac acgacttatc gccactggca
     5401 gcagccactg gtaacaggat tagcagagcg aggtatgtag gcggtgctac agagttcttg
     5461 aagtggtggc ctaactacgg ctacactaga agaacagtat ttggtatctg cgctctgctg
     5521 aagccagtta ccttcggaaa aagagttggt agctcttgat ccggcaaaca aaccaccgct
     5581 ggtagcggtg gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa
     5641 gaagatcctt tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa
     5701 gggattttgg tcatgagatt atcaaaaagg atcttcacct agatcctttt aaattaaaaa
     5761 tgaagtttta aatcaatcta aagtatatat gagtaaactt ggtctgacag ttaccaatgc
     5821 ttaatcagtg aggcacctat ctcagcgatc tgtctatttc gttcatccat agttgcctga
     5881 ctccccgtcg tgtagataac tacgatacgg gagggcttac catctggccc cagtgctgca
     5941 atgataccgc gagacccacg ctcaccggct ccagatttat cagcaataaa ccagccagcc
     6001 ggaagggccg agcgcagaag tggtcctgca actttatccg cctccatcca gtctattaat
     6061 tgttgccggg aagctagagt aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc
     6121 attgctacag gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt cagctccggt
     6181 tcccaacgat caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc
     6241 ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag tgttatcact catggttatg
     6301 gcagcactgc ataattctct tactgtcatg ccatccgtaa gatgcttttc tgtgactggt
     6361 gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg
     6421 gcgtcaatac gggataatac cgcgccacat agcagaactt taaaagtgct catcattgga
     6481 aaacgttctt cggggcgaaa actctcaagg atcttaccgc tgttgagatc cagttcgatg
     6541 taacccactc gtgcacccaa ctgatcttca gcatctttta ctttcaccag cgtttctggg
     6601 tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa taagggcgac acggaaatgt
     6661 tgaatactca tactcttcct ttttcaatat tattgaagca tttatcaggg ttattgtctc
     6721 atgagcggat acatatttga atgtatttag aaaaataaac aaataggggt tccgcgcaca
     6781 tttccccgaa aagtgccacc tgacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg
     6841 gtggttacgc gcagcgtgac cgctacactt gccagcgccc tagcgcccgc tcctttcgct
     6901 ttcttccctt cctttctcgc cacgttcgcc ggctttcccc gtcaagctct aaatcggggg
     6961 ctccctttag ggttccgatt tagtgcttta cggcacctcg accccaaaaa acttgattag
     7021 ggtgatggtt cacgtagtgg gccatcgccc tgatagacgg tttttcgccc tttgacgttg
     7081 gagtccacgt tctttaatag tggactcttg ttccaaactg gaacaacact caaccctatc
     7141 tcggtctatt cttttgattt ataagggatt ttgccgattt cggcctattg gttaaaaaat
     7201 gagctgattt aacaaaaatt taacgcgaat tttaacaaaa tattaacgct tacaatttcc
     7261 attcgccatt caggctgcgc aactgttggg aagggcgatc ggtgcgggcc tcttcgctat
     7321 tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta acgccagggt
     7381 tttcccagtc acgacgttgt aaaacgacgg ccagtgccaa gctgatctat acattgaatc
     7441 aatattggca attagccata ttagtcattg gttatatagc ataaatcaat attggctatt
     7501 ggccattgca tacgttgtat ctatatcata atatgtacat ttatattggc tcatgtccaa
     7561 tatgaccgcc atgttgacat tgattattga ctagttatta atagtaatca attacggggt
     7621 cattagttca tagcccatat atggagttcc gcgttacata acttacggta aatggcccgc
     7681 ctggctgacc gcccaacgac ccccgcccat tgacgtcaat aatgacgtat gttcccatag
     7741 taacgccaat agggactttc cattgacgtc aatgggtgga gtatttacgg taaactgccc
     7801 acttggcagt acatcaagtg tatcatatgc caagtccgcc ccctattgac gtcaatgacg
     7861 gtaaatggcc cgcctggcat tatgcccagt acatgacctt acgggacttt cctacttggc
     7921 agtacatcta cgtattagtc atcgctatta ccatggtgat gcggttttgg cagtacacca
     7981 atgggcgtgg atagcggt
//
