LOCUS       sequence-443647-        7998 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Plasmid expressing GINIP-Nluc 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
FEATURES             Location/Qualifiers
     source          1..7998
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             226..1020
                     /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(280..299)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     890..909
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    1672..1806
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1709..1728)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     1763..1782
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(1811..1830)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1812..1830)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1844..1860)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1844..1860)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1857..1879)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    1868..1884
                     /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(1892..1898,1899..1916,1917..1922))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1937..1958
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2075..2092)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2246..2834)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2326..2345)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3005..3796,3797..3865))
                     /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     3628..3647
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3866..3970)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      3997..4452
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4084..4103)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4294..4315
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     4578..4600
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     4592..4609
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     4593..4609
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     enhancer        4770..5149
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        5150..5353
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     5304..5324
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     CDS             6608..7120
                     /codon_start=1
                     /product="NanoLuc(R) luciferase"
                     /label=Nluc
                     /note="human codon-optimized"
                     /translation="MVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQR
                     IVLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGV
                     TPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGW
                     RLCERILA"
     CDS             7133..7198
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     polyA_signal    7209..7831
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(7408..7427)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     primer_bind     complement(7855..7875)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        join(7873..7998,1..172)
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
ORIGIN
        1 tcaattagtc agcaaccata gtcccgcccc taactccgcc catcccgccc ctaactccgc
       61 ccagttccgc ccattctccg ccccatggct gactaatttt ttttatttat gcagaggccg
      121 aggccgcctc ggcctctgag ctattccaga agtagtgagg aggctttttt ggaggaattg
      181 atcagcttgg gatctgatca agagacagga tgaggatcgt ttcgcatgat tgaacaagat
      241 ggattgcacg caggttctcc ggccgcttgg gtggagaggc tattcggcta tgactgggca
      301 caacagacaa tcggctgctc tgatgccgcc gtgttccggc tgtcagcgca ggggcgcccg
      361 gttctttttg tcaagaccga cctgtccggt gccctgaatg aactgcagga cgaggcagcg
      421 cggctatcgt ggctggccac gacgggcgtt ccttgcgcag ctgtgctcga cgttgtcact
      481 gaagcgggaa gggactggct gctattgggc gaagtgccgg ggcaggatct cctgtcatct
      541 caccttgctc ctgccgagaa agtatccatc atggctgatg caatgcggcg gctgcatacg
      601 cttgatccgg ctacctgccc attcgaccac caagcgaaac atcgcatcga gcgagcacgt
      661 actcggatgg aagccggtct tgtcgatcag gatgatctgg acgaagagca tcaggggctc
      721 gcgccagccg aactgttcgc caggctcaag gcgcgcatgc ccgacggcga ggatctcgtc
      781 gtgacccatg gcgatgcctg cttgccgaat atcatggtgg aaaatggccg cttttctgga
      841 ttcatcgact gtggccggct gggtgtggcg gaccgctatc aggacatagc gttggctacc
      901 cgtgatattg ctgaagagct tggcggcgaa tgggctgacc gcttcctcgt gctttacggt
      961 atcgccgctc ccgattcgca gcgcatcgcc ttctatcgcc ttcttgacga gttcttctga
     1021 gcgggactct ggggttcgaa atgaccgacc aagcgacgcc caacctgcca tcacgagatt
     1081 tcgattccac cgccgccttc tatgaaaggt tgggcttcgg aatcgttttc cgggacgccg
     1141 gctggatgat cctccagcgc ggggatctca tgctggagtt cttcgcccac cccgggctcg
     1201 atcccctcgc gagttggttc agctgctgcc tgaggctgga cgacctcgcg gagttctacc
     1261 ggcagtgcaa atccgtcggc atccaggaaa ccagcagcgg ctatccgcgc atccatgccc
     1321 ccgaactgca ggagtgggga ggcacgatgg ccgctttggt cgacccggac gggacgctcc
     1381 tgcgcctgat acagaacgaa ttgcttgcag gcatctcatg agtgtgtctt cccgttttcc
     1441 gcctgaggtc actgcgtgga tggagcgctg gcgcctgctg cgcgacggcg agctgctcac
     1501 cacccactcg ccaagctgga accgtaaaaa ggccgcgttg ctggcgtttt tccataggct
     1561 ccgccgatca taatcagcca taccacattt gtagaggttt tacttgcttt aaaaaacctc
     1621 ccacacctcc ccctgaacct gaaacataaa atgaatgcaa ttgttgttgt taacttgttt
     1681 attgcagctt ataatggtta caaataaagc aatagcatca caaatttcac aaataaagca
     1741 tttttttcac tgcattctag ttgtggtttg tccaaactca tcaatgtatc ttatcatgtc
     1801 tggatcaatt ccctatagtg agtcgtatta aattcgtaat catgtcatag ctgtttcctg
     1861 tgtgaaattg ttatccgctc acaattccac acaacatacg agccggaagc ataaagtgta
     1921 aagcctgggg tgcctaatga gtgagctaac tcacattaat tgcgttgcgc tcactgcccg
     1981 ctttccagtc gggaaacctg tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga
     2041 gaggcggttt gcgtattggg cgctcttccg cttcctcgct cactgactcg ctgcgctcgg
     2101 tcgttcggct gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg ttatccacag
     2161 aatcagggga taacgcagga aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc
     2221 gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg cccccctgac gagcatcaca
     2281 aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg actataaaga taccaggcgt
     2341 ttccccctgg aagctccctc gtgcgctctc ctgttccgac cctgccgctt accggatacc
     2401 tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc
     2461 tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc
     2521 ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc caacccggta agacacgact
     2581 tatcgccact ggcagcagcc actggtaaca ggattagcag agcgaggtat gtaggcggtg
     2641 ctacagagtt cttgaagtgg tggcctaact acggctacac tagaagaaca gtatttggta
     2701 tctgcgctct gctgaagcca gttaccttcg gaaaaagagt tggtagctct tgatccggca
     2761 aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa
     2821 aaaaaggatc tcaagaagat cctttgatct tttctacggg gtctgacgct cagtggaacg
     2881 aaaactcacg ttaagggatt ttggtcatga gattatcaaa aaggatcttc acctagatcc
     2941 ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa acttggtctg
     3001 acagttacca atgcttaatc agtgaggcac ctatctcagc gatctgtcta tttcgttcat
     3061 ccatagttgc ctgactcccc gtcgtgtaga taactacgat acgggagggc ttaccatctg
     3121 gccccagtgc tgcaatgata ccgcgagacc cacgctcacc ggctccagat ttatcagcaa
     3181 taaaccagcc agccggaagg gccgagcgca gaagtggtcc tgcaacttta tccgcctcca
     3241 tccagtctat taattgttgc cgggaagcta gagtaagtag ttcgccagtt aatagtttgc
     3301 gcaacgttgt tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt
     3361 cattcagctc cggttcccaa cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa
     3421 aagcggttag ctccttcggt cctccgatcg ttgtcagaag taagttggcc gcagtgttat
     3481 cactcatggt tatggcagca ctgcataatt ctcttactgt catgccatcc gtaagatgct
     3541 tttctgtgac tggtgagtac tcaaccaagt cattctgaga atagtgtatg cggcgaccga
     3601 gttgctcttg cccggcgtca atacgggata ataccgcgcc acatagcaga actttaaaag
     3661 tgctcatcat tggaaaacgt tcttcggggc gaaaactctc aaggatctta ccgctgttga
     3721 gatccagttc gatgtaaccc actcgtgcac ccaactgatc ttcagcatct tttactttca
     3781 ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg
     3841 cgacacggaa atgttgaata ctcatactct tcctttttca atattattga agcatttatc
     3901 agggttattg tctcatgagc ggatacatat ttgaatgtat ttagaaaaat aaacaaatag
     3961 gggttccgcg cacatttccc cgaaaagtgc cacctgacgc gccctgtagc ggcgcattaa
     4021 gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac acttgccagc gccctagcgc
     4081 ccgctccttt cgctttcttc ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag
     4141 ctctaaatcg ggggctccct ttagggttcc gatttagtgc tttacggcac ctcgacccca
     4201 aaaaacttga ttagggtgat ggttcacgta gtgggccatc gccctgatag acggtttttc
     4261 gccctttgac gttggagtcc acgttcttta atagtggact cttgttccaa actggaacaa
     4321 cactcaaccc tatctcggtc tattcttttg atttataagg gattttgccg atttcggcct
     4381 attggttaaa aaatgagctg atttaacaaa aatttaacgc gaattttaac aaaatattaa
     4441 cgcttacaat ttccattcgc cattcaggct gcgcaactgt tgggaagggc gatcggtgcg
     4501 ggcctcttcg ctattacgcc agctggcgaa agggggatgt gctgcaaggc gattaagttg
     4561 ggtaacgcca gggttttccc agtcacgacg ttgtaaaacg acggccagtg ccaagctgat
     4621 ctatacattg aatcaatatt ggcaattagc catattagtc attggttata tagcataaat
     4681 caatattggc tattggccat tgcatacgtt gtatctatat cataatatgt acatttatat
     4741 tggctcatgt ccaatatgac cgccatgttg acattgatta ttgactagtt attaatagta
     4801 atcaattacg gggtcattag ttcatagccc atatatggag ttccgcgtta cataacttac
     4861 ggtaaatggc ccgcctggct gaccgcccaa cgacccccgc ccattgacgt caataatgac
     4921 gtatgttccc atagtaacgc caatagggac tttccattga cgtcaatggg tggagtattt
     4981 acggtaaact gcccacttgg cagtacatca agtgtatcat atgccaagtc cgccccctat
     5041 tgacgtcaat gacggtaaat ggcccgcctg gcattatgcc cagtacatga ccttacggga
     5101 ctttcctact tggcagtaca tctacgtatt agtcatcgct attaccatgg tgatgcggtt
     5161 ttggcagtac accaatgggc gtggatagcg gtttgactca cggggatttc caagtctcca
     5221 ccccattgac gtcaatggga gtttgttttg gcaccaaaat caacgggact ttccaaaatg
     5281 tcgtaataac cccgccccgt tgacgcaaat gggcggtagg cgtgtacggt gggaggtcta
     5341 tataagcaga gctcgtttag tgaaccgtca gaattaagct tgcggccgcg aattcatggg
     5401 ggtgttgatg tccaagcggc agacagtgga gcaggtgcag aaggtgagtc tggctgtgtc
     5461 tgctttcaag gatgggctgc gggacaggcc ttccatccga cgcacaggtg agctgccagg
     5521 gtcccgccgt ggcactgtag agggctccgt ccaggaggta caggaggaga aggaagcaga
     5581 agcaggaacc tcggtggtcc aggaagagag tagtgccggc cgcgcagcct gggagcggct
     5641 ccgagatggg cgcggcgtgg agcctgagga gtttgacagg acaagtcgat tcacaccccc
     5701 tgcgttcatc cgccccaccc ggaagctgga tgatgacaaa cctccagaaa tctgcctgga
     5761 gcccagagag cctgttgtca acgatgagat gtgtgatgtt tgtgaggtct ggacagctga
     5821 gagcctcttc ccgtgcaggg tctgcaccag ggttttccat gatggctgcc tgcgccgcat
     5881 gggctacatc caaggagaca gtgcagcgga ggtgacggag atggcccaca cagaaacagg
     5941 ctggagctgc cactactgtg acaacatcaa cttgctgctt actgaggagg aaatgtatag
     6001 cctcacggag acctttcagc ggtgtaaagt catccctgat tgctccctga cactggagga
     6061 ctttctgcgt taccgccacc aagcagccaa gcggggggac cgtgacaggg ccctgagtga
     6121 ggagcaagaa gagcaggcgg cccgccagtt tgctgccctg gaccctgaac atcgaggcca
     6181 catagagtgg cctgacttct tgtcccatga gtccctcctg cttctgcagc agttgcgtcc
     6241 ccagaactct ctgttgaggc ttctgacagt gaaggagcgg gagcgagccc gagccgcctt
     6301 cctggctcgg ggcagtggga gcaccgtcag tgaggcagag tgccgccggg cccagcactc
     6361 ttggttttgc aaacggttcc cagaggctcc ttcctgcagt gtcagcatca gccatgtggg
     6421 tcccatcgca gatagcagcc cagccagcag cagcagcaag agtcaggaca agaccctgct
     6481 gcccacagag caggagtcca gatttgtgga ctggcctacc ttcctgcaag agaatgtcct
     6541 ctacatcctg gctgctcgcc ccaacagcgc agccattcac ctgaagcccc caggaggtgg
     6601 tggttctatg gtcttcacac tcgaagattt cgttggggac tggcgacaga cagccggcta
     6661 caacctggac caagtccttg aacagggagg tgtgtccagt ttgtttcaga atctcggggt
     6721 gtccgtaact ccgatccaaa ggattgtcct gagcggtgaa aatgggctga agatcgacat
     6781 ccatgtcatc atcccgtatg aaggtctgag cggcgaccaa atgggccaga tcgaaaaaat
     6841 ttttaaggtg gtgtaccctg tggatgatca tcactttaag gtgatcctgc actatggcac
     6901 actggtaatc gacggggtta cgccgaacat gatcgactat ttcggacggc cgtatgaagg
     6961 catcgccgtg ttcgacggca aaaagatcac tgtaacaggg accctgtgga acggcaacaa
     7021 aattatcgac gagcgcctga tcaaccccga cggctccctg ctgttccgag taaccatcaa
     7081 cggagtgacc ggctggcggc tgtgcgaacg cattctggcg ggatcccggg ctgactacaa
     7141 agaccatgac ggtgattata aagatcatga catcgactac aaggatgacg atgacaagta
     7201 gtgatcccgg gtggcatccc tgtgacccct ccccagtgcc tctcctggcc ctggaagttg
     7261 ccactccagt gcccaccagc cttgtcctaa taaaattaag ttgcatcatt ttgtctgact
     7321 aggtgtcctt ctataatatt atggggtgga ggggggtggt atggagcaag gggcaagttg
     7381 ggaagacaac ctgtagggcc tgcggggtct attgggaacc aagctggagt gcagtggcac
     7441 aatcttggct cactgcaatc tccgcctcct gggttcaagc gattctcctg cctcagcctc
     7501 ccgagttgtt gggattccag gcatgcatga ccaggctcag ctaatttttg tttttttggt
     7561 agagacgggg tttcaccata ttggccaggc tggtctccaa ctcctaatct caggtgatct
     7621 acccaccttg gcctcccaaa ttgctgggat tacaggcgtg aaccactgct cccttccctg
     7681 tccttctgat tttaaaataa ctataccagc aggaggacgt ccagacacag cataggctac
     7741 ctggccatgc ccaaccggtg ggacatttga gttgcttgct tggcactgtc ctctcatgcg
     7801 ttgggtccac tcagtagatg cctgttgaat tgggtacgcg gccagcttgg ctgtggaatg
     7861 tgtgtcagtt agggtgtgga aagtccccag gctccccagc aggcagaagt atgcaaagca
     7921 tgcatctcaa ttagtcagca accaggtgtg gaaagtcccc aggctcccca gcaggcagaa
     7981 gtatgcaaag catgcatc
//
