LOCUS       sequence-410273-        6986 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Part of REGA-SIGN, a NanoBRET-based G protein biosensor. It encodes 
            GNAI2 tagged with NanoLuciferase and is meant to be paired with 
            Ggamma2 labeled with LSS-mKATE2.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6986)
  AUTHORS   Boon K, Vanalken N, Meyen E, Schols D, Van Loy T
  TITLE     REGA-SIGN: Development of a Novel Set of NanoBRET-Based G Protein 
            Biosensors.
  JOURNAL   Biosensors (Basel). 2023 Jul 28;13(8):767. doi: 
            10.3390/bios13080767.
  PUBMED    37622853
REFERENCE   2  (bases 1 to 6986)
  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: "Biosensors 
            (Basel)."; date: "2023-07-28"; volume: "13(8):767. doi"; pages: " 
            10.3390/bios13080767"
COMMENT     Sequence Label: pcDNA3.1(+)G-alpha-i2-91-NLuc
FEATURES             Location/Qualifiers
     source          1..6986
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(44..63)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        235..614
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        615..818
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     769..789
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     863..882
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        863..881
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             1217..1726
                     /codon_start=1
                     /product="NanoLuc(R) luciferase"
                     /label=Nluc
                     /note="human codon-optimized"
                     /translation="VFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQRI
                     VLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGVT
                     PNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGWR
                     LCERILA"
     primer_bind     complement(2577..2594)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2583..2807
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2853..3281
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2940..2959)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3150..3171
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3290..3310)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3295..3624
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3475..3610
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3537..3556
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3691..4485
                     /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(3745..3764)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4355..4374
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4659..4780
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4696..4715)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4750..4769
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4829..4845)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4829..4845)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4842..4864)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4853..4869
                     /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(4877..4883,4884..4901,4902..4907))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4922..4943
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5060..5077)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5231..5819)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5311..5330)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5990..6781,6782..6850))
                     /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     6613..6632
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6851..6955)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gacggatcgg gagatctccc gatcccctat ggtgcactct cagtacaatc tgctctgatg
       61 ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg
      121 cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc
      181 ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt
      241 gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata
      301 tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc
      361 cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc
      421 attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt
      481 atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt
      541 atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca
      601 tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg
      661 actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc
      721 aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg
      781 gtaggcgtgt acggtgggag gtctatataa gcagagctct ctggctaact agagaaccca
      841 ctgcttactg gcttatcgaa attaatacga ctcactatag ggagacccaa gctggctagc
      901 gtttaaactt aagcttggta ccaccatggg ctgcaccgtg agcgccgagg acaaggcggc
      961 ggccgagcgc tctaagatga tcgacaagaa cctgcgggag gacggagaga aggcggcgcg
     1021 ggaggtgaag ttgctgctgt tgggtgctgg ggagtcaggg aagagcacca tcgtcaagca
     1081 gatgaagatc atccacgagg atggctactc cgaggaggaa tgccggcagt accgggcggt
     1141 tgtctacagc aacaccatcc agtccatcat ggccattgtc aaagccatgg gcaacctgag
     1201 cggcggcggc ggcagcgtct tcacactcga agatttcgtt ggggactggc gacagacagc
     1261 cggctacaac ctggaccaag tccttgaaca gggaggtgtg tccagtttgt ttcagaatct
     1321 cggggtgtcc gtaactccga tccaaaggat tgtcctgagc ggtgaaaatg ggctgaagat
     1381 cgacatccat gtcatcatcc cgtatgaagg tctgagcggc gaccaaatgg gccagatcga
     1441 aaaaattttt aaggtggtgt accctgtgga tgatcatcac tttaaggtga tcctgcacta
     1501 tggcacactg gtaatcgacg gggttacgcc gaacatgatc gactatttcg gacggccgta
     1561 tgaaggcatc gccgtgttcg acggcaaaaa gatcactgta acagggaccc tgtggaacgg
     1621 caacaaaatt atcgacgagc gcctgatcaa ccccgacggc tccctgctgt tccgagtaac
     1681 catcaacgga gtgaccggct ggcggctgtg cgaacgcatt ctggcgagcg gcggcggcgg
     1741 cagccagatc gactttgccg acccctccag agcggacgac gccaggcagc tatttgcact
     1801 gtcctgcacc gccgaggagc aaggcgtgct ccctgatgac ctgtccggcg tcatccggag
     1861 gctctgggct gaccatggtg tgcaggcctg ctttggccgc tcaagggaat accagctcaa
     1921 cgactcagct gcctactacc tgaacgacct ggagcgtatt gcacagagtg actacatccc
     1981 cacacagcaa gatgtgctac ggacccgcgt aaagaccacg gggatcgtgg agacacactt
     2041 caccttcaag gacctacact tcaagatgtt tgatgtgggt ggtcagcggt ctgagcggaa
     2101 gaagtggatc cactgctttg agggcgtcac agccatcatc ttctgcgtag ccttgagcgc
     2161 ctatgacttg gtgctagctg aggacgagga gatgaaccgc atgcatgaga gcatgaagct
     2221 attcgatagc atctgcaaca acaagtggtt cacagacacg tccatcatcc tcttcctcaa
     2281 caagaaggac ctgtttgagg agaagatcac acacagtccc ctgaccatct gcttccctga
     2341 gtacacaggg gccaacaaat atgatgaggc agccagctac atccagagta agtttgagga
     2401 cctgaataag cgcaaagaca ccaaggagat ctacacgcac ttcacgtgcg ccaccgacac
     2461 caagaacgtg cagttcgtgt ttgacgccgt caccgatgtc atcatcaaga acaacctgaa
     2521 ggactgcggc ctcttctgac tcgagtctag agggcccgtt taaacccgct gatcagcctc
     2581 gactgtgcct tctagttgcc agccatctgt tgtttgcccc tcccccgtgc cttccttgac
     2641 cctggaaggt gccactccca ctgtcctttc ctaataaaat gaggaaattg catcgcattg
     2701 tctgagtagg tgtcattcta ttctgggggg tggggtgggg caggacagca agggggagga
     2761 ttgggaagac aatagcaggc atgctgggga tgcggtgggc tctatggctt ctgaggcgga
     2821 aagaaccagc tggggctcta gggggtatcc ccacgcgccc tgtagcggcg cattaagcgc
     2881 ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt gccagcgccc tagcgcccgc
     2941 tcctttcgct ttcttccctt cctttctcgc cacgttcgcc ggctttcccc gtcaagctct
     3001 aaatcggggg ctccctttag ggttccgatt tagtgcttta cggcacctcg accccaaaaa
     3061 acttgattag ggtgatggtt cacgtagtgg gccatcgccc tgatagacgg tttttcgccc
     3121 tttgacgttg gagtccacgt tctttaatag tggactcttg ttccaaactg gaacaacact
     3181 caaccctatc tcggtctatt cttttgattt ataagggatt ttgccgattt cggcctattg
     3241 gttaaaaaat gagctgattt aacaaaaatt taacgcgaat taattctgtg gaatgtgtgt
     3301 cagttagggt gtggaaagtc cccaggctcc ccagcaggca gaagtatgca aagcatgcat
     3361 ctcaattagt cagcaaccag gtgtggaaag tccccaggct ccccagcagg cagaagtatg
     3421 caaagcatgc atctcaatta gtcagcaacc atagtcccgc ccctaactcc gcccatcccg
     3481 cccctaactc cgcccagttc cgcccattct ccgccccatg gctgactaat tttttttatt
     3541 tatgcagagg ccgaggccgc ctctgcctct gagctattcc agaagtagtg aggaggcttt
     3601 tttggaggcc taggcttttg caaaaagctc ccgggagctt gtatatccat tttcggatct
     3661 gatcaagaga caggatgagg atcgtttcgc atgattgaac aagatggatt gcacgcaggt
     3721 tctccggccg cttgggtgga gaggctattc ggctatgact gggcacaaca gacaatcggc
     3781 tgctctgatg ccgccgtgtt ccggctgtca gcgcaggggc gcccggttct ttttgtcaag
     3841 accgacctgt ccggtgccct gaatgaactg caggacgagg cagcgcggct atcgtggctg
     3901 gccacgacgg gcgttccttg cgcagctgtg ctcgacgttg tcactgaagc gggaagggac
     3961 tggctgctat tgggcgaagt gccggggcag gatctcctgt catctcacct tgctcctgcc
     4021 gagaaagtat ccatcatggc tgatgcaatg cggcggctgc atacgcttga tccggctacc
     4081 tgcccattcg accaccaagc gaaacatcgc atcgagcgag cacgtactcg gatggaagcc
     4141 ggtcttgtcg atcaggatga tctggacgaa gagcatcagg ggctcgcgcc agccgaactg
     4201 ttcgccaggc tcaaggcgcg catgcccgac ggcgaggatc tcgtcgtgac ccatggcgat
     4261 gcctgcttgc cgaatatcat ggtggaaaat ggccgctttt ctggattcat cgactgtggc
     4321 cggctgggtg tggcggaccg ctatcaggac atagcgttgg ctacccgtga tattgctgaa
     4381 gagcttggcg gcgaatgggc tgaccgcttc ctcgtgcttt acggtatcgc cgctcccgat
     4441 tcgcagcgca tcgccttcta tcgccttctt gacgagttct tctgagcggg actctggggt
     4501 tcgaaatgac cgaccaagcg acgcccaacc tgccatcacg agatttcgat tccaccgccg
     4561 ccttctatga aaggttgggc ttcggaatcg ttttccggga cgccggctgg atgatcctcc
     4621 agcgcgggga tctcatgctg gagttcttcg cccaccccaa cttgtttatt gcagcttata
     4681 atggttacaa ataaagcaat agcatcacaa atttcacaaa taaagcattt ttttcactgc
     4741 attctagttg tggtttgtcc aaactcatca atgtatctta tcatgtctgt ataccgtcga
     4801 cctctagcta gagcttggcg taatcatggt catagctgtt tcctgtgtga aattgttatc
     4861 cgctcacaat tccacacaac atacgagccg gaagcataaa gtgtaaagcc tggggtgcct
     4921 aatgagtgag ctaactcaca ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa
     4981 acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta
     5041 ttgggcgctc ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc
     5101 gagcggtatc agctcactca aaggcggtaa tacggttatc cacagaatca ggggataacg
     5161 caggaaagaa catgtgagca aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt
     5221 tgctggcgtt tttccatagg ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa
     5281 gtcagaggtg gcgaaacccg acaggactat aaagatacca ggcgtttccc cctggaagct
     5341 ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc
     5401 cttcgggaag cgtggcgctt tctcatagct cacgctgtag gtatctcagt tcggtgtagg
     5461 tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct
     5521 tatccggtaa ctatcgtctt gagtccaacc cggtaagaca cgacttatcg ccactggcag
     5581 cagccactgg taacaggatt agcagagcga ggtatgtagg cggtgctaca gagttcttga
     5641 agtggtggcc taactacggc tacactagaa gaacagtatt tggtatctgc gctctgctga
     5701 agccagttac cttcggaaaa agagttggta gctcttgatc cggcaaacaa accaccgctg
     5761 gtagcggtgg tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag
     5821 aagatccttt gatcttttct acggggtctg acgctcagtg gaacgaaaac tcacgttaag
     5881 ggattttggt catgagatta tcaaaaagga tcttcaccta gatcctttta aattaaaaat
     5941 gaagttttaa atcaatctaa agtatatatg agtaaacttg gtctgacagt taccaatgct
     6001 taatcagtga ggcacctatc tcagcgatct gtctatttcg ttcatccata gttgcctgac
     6061 tccccgtcgt gtagataact acgatacggg agggcttacc atctggcccc agtgctgcaa
     6121 tgataccgcg agacccacgc tcaccggctc cagatttatc agcaataaac cagccagccg
     6181 gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag tctattaatt
     6241 gttgccggga agctagagta agtagttcgc cagttaatag tttgcgcaac gttgttgcca
     6301 ttgctacagg catcgtggtg tcacgctcgt cgtttggtat ggcttcattc agctccggtt
     6361 cccaacgatc aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg gttagctcct
     6421 tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt gttatcactc atggttatgg
     6481 cagcactgca taattctctt actgtcatgc catccgtaag atgcttttct gtgactggtg
     6541 agtactcaac caagtcattc tgagaatagt gtatgcggcg accgagttgc tcttgcccgg
     6601 cgtcaatacg ggataatacc gcgccacata gcagaacttt aaaagtgctc atcattggaa
     6661 aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc agttcgatgt
     6721 aacccactcg tgcacccaac tgatcttcag catcttttac tttcaccagc gtttctgggt
     6781 gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt
     6841 gaatactcat actcttcctt tttcaatatt attgaagcat ttatcagggt tattgtctca
     6901 tgagcggata catatttgaa tgtatttaga aaaataaaca aataggggtt ccgcgcacat
     6961 ttccccgaaa agtgccacct gacgtc
//
