LOCUS       sequence-410277-        6983 bp    DNA     circular SYN 14-APR-2025
DEFINITION  Part of REGA-SIGN, a NanoBRET-based G protein biosensor. It encodes 
            GNAI1 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 6983)
  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 6983)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 14, 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-i1-91-NLuc
FEATURES             Location/Qualifiers
     source          1..6983
                     /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(2574..2591)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2580..2804
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2850..3278
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2937..2956)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3147..3168
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3287..3307)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3292..3621
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3472..3607
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3534..3553
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3688..4482
                     /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(3742..3761)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4352..4371
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4656..4777
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4693..4712)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4747..4766
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4826..4842)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4826..4842)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4839..4861)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4850..4866
                     /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(4874..4880,4881..4898,4899..4904))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4919..4940
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5057..5074)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5228..5816)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5308..5327)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5987..6778,6779..6847))
                     /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     6610..6629
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6848..6952)
                     /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 ctgcacgctg agcgccgagg acaaggcggc
      961 ggtggagcgg agtaagatga tcgaccgcaa cctccgtgag gacggcgaga aggcggcgcg
     1021 cgaggtcaag ctgctgctgc tcggtgctgg tgaatctggt aaaagtacaa ttgtgaagca
     1081 gatgaaaatt atccatgaag ctggttattc agaagaggag tgtaaacaat acaaagcagt
     1141 ggtctacagt aacaccatcc agtcaattat tgctatcatt agggctatgg ggaggttgag
     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 cagcaagata gactttggtg actcagcccg ggcggatgat gcacgccaac tctttgtgct
     1801 agctggagct gctgaagaag gctttatgac tgcagaactt gctggagtta taaagagatt
     1861 gtggaaagat agtggtgtac aagcctgttt caacagatcc cgagagtacc agcttaatga
     1921 ttctgcagca tactatttga atgacttgga cagaatagct caaccaaatt acatcccgac
     1981 tcaacaagat gttctcagaa ctagagtgaa aactacagga attgttgaaa cccattttac
     2041 tttcaaagat cttcatttta aaatgtttga tgtgggaggt cagagatctg agcggaagaa
     2101 gtggattcat tgcttcgaag gagtgacggc gatcatcttc tgtgtagcac tgagtgacta
     2161 cgacctggtt ctagctgaag atgaagaaat gaaccgaatg catgaaagca tgaaattgtt
     2221 tgacagcata tgtaacaaca agtggtttac agatacatcc attatacttt ttctaaacaa
     2281 gaaggatctc tttgaagaaa aaatcaaaaa gagccctctc actatatgct atccagaata
     2341 tgcaggatca aacacatatg aagaggcagc tgcatatatt caatgtcagt ttgaagacct
     2401 caataaaaga aaggacacaa aggaaatata cacccacttc acatgtgcca cagatactaa
     2461 gaatgtgcag tttgtttttg atgctgtaac agatgtcatc ataaaaaata atctaaaaga
     2521 ttgtggtctc ttttaactcg agtctagagg gcccgtttaa acccgctgat cagcctcgac
     2581 tgtgccttct agttgccagc catctgttgt ttgcccctcc cccgtgcctt ccttgaccct
     2641 ggaaggtgcc actcccactg tcctttccta ataaaatgag gaaattgcat cgcattgtct
     2701 gagtaggtgt cattctattc tggggggtgg ggtggggcag gacagcaagg gggaggattg
     2761 ggaagacaat agcaggcatg ctggggatgc ggtgggctct atggcttctg aggcggaaag
     2821 aaccagctgg ggctctaggg ggtatcccca cgcgccctgt agcggcgcat taagcgcggc
     2881 gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc agcgccctag cgcccgctcc
     2941 tttcgctttc ttcccttcct ttctcgccac gttcgccggc tttccccgtc aagctctaaa
     3001 tcgggggctc cctttagggt tccgatttag tgctttacgg cacctcgacc ccaaaaaact
     3061 tgattagggt gatggttcac gtagtgggcc atcgccctga tagacggttt ttcgcccttt
     3121 gacgttggag tccacgttct ttaatagtgg actcttgttc caaactggaa caacactcaa
     3181 ccctatctcg gtctattctt ttgatttata agggattttg ccgatttcgg cctattggtt
     3241 aaaaaatgag ctgatttaac aaaaatttaa cgcgaattaa ttctgtggaa tgtgtgtcag
     3301 ttagggtgtg gaaagtcccc aggctcccca gcaggcagaa gtatgcaaag catgcatctc
     3361 aattagtcag caaccaggtg tggaaagtcc ccaggctccc cagcaggcag aagtatgcaa
     3421 agcatgcatc tcaattagtc agcaaccata gtcccgcccc taactccgcc catcccgccc
     3481 ctaactccgc ccagttccgc ccattctccg ccccatggct gactaatttt ttttatttat
     3541 gcagaggccg aggccgcctc tgcctctgag ctattccaga agtagtgagg aggctttttt
     3601 ggaggcctag gcttttgcaa aaagctcccg ggagcttgta tatccatttt cggatctgat
     3661 caagagacag gatgaggatc gtttcgcatg attgaacaag atggattgca cgcaggttct
     3721 ccggccgctt gggtggagag gctattcggc tatgactggg cacaacagac aatcggctgc
     3781 tctgatgccg ccgtgttccg gctgtcagcg caggggcgcc cggttctttt tgtcaagacc
     3841 gacctgtccg gtgccctgaa tgaactgcag gacgaggcag cgcggctatc gtggctggcc
     3901 acgacgggcg ttccttgcgc agctgtgctc gacgttgtca ctgaagcggg aagggactgg
     3961 ctgctattgg gcgaagtgcc ggggcaggat ctcctgtcat ctcaccttgc tcctgccgag
     4021 aaagtatcca tcatggctga tgcaatgcgg cggctgcata cgcttgatcc ggctacctgc
     4081 ccattcgacc accaagcgaa acatcgcatc gagcgagcac gtactcggat ggaagccggt
     4141 cttgtcgatc aggatgatct ggacgaagag catcaggggc tcgcgccagc cgaactgttc
     4201 gccaggctca aggcgcgcat gcccgacggc gaggatctcg tcgtgaccca tggcgatgcc
     4261 tgcttgccga atatcatggt ggaaaatggc cgcttttctg gattcatcga ctgtggccgg
     4321 ctgggtgtgg cggaccgcta tcaggacata gcgttggcta cccgtgatat tgctgaagag
     4381 cttggcggcg aatgggctga ccgcttcctc gtgctttacg gtatcgccgc tcccgattcg
     4441 cagcgcatcg ccttctatcg ccttcttgac gagttcttct gagcgggact ctggggttcg
     4501 aaatgaccga ccaagcgacg cccaacctgc catcacgaga tttcgattcc accgccgcct
     4561 tctatgaaag gttgggcttc ggaatcgttt tccgggacgc cggctggatg atcctccagc
     4621 gcggggatct catgctggag ttcttcgccc accccaactt gtttattgca gcttataatg
     4681 gttacaaata aagcaatagc atcacaaatt tcacaaataa agcatttttt tcactgcatt
     4741 ctagttgtgg tttgtccaaa ctcatcaatg tatcttatca tgtctgtata ccgtcgacct
     4801 ctagctagag cttggcgtaa tcatggtcat agctgtttcc tgtgtgaaat tgttatccgc
     4861 tcacaattcc acacaacata cgagccggaa gcataaagtg taaagcctgg ggtgcctaat
     4921 gagtgagcta actcacatta attgcgttgc gctcactgcc cgctttccag tcgggaaacc
     4981 tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg
     5041 ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag
     5101 cggtatcagc tcactcaaag gcggtaatac ggttatccac agaatcaggg gataacgcag
     5161 gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc
     5221 tggcgttttt ccataggctc cgcccccctg acgagcatca caaaaatcga cgctcaagtc
     5281 agaggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct ggaagctccc
     5341 tcgtgcgctc tcctgttccg accctgccgc ttaccggata cctgtccgcc tttctccctt
     5401 cgggaagcgt ggcgctttct catagctcac gctgtaggta tctcagttcg gtgtaggtcg
     5461 ttcgctccaa gctgggctgt gtgcacgaac cccccgttca gcccgaccgc tgcgccttat
     5521 ccggtaacta tcgtcttgag tccaacccgg taagacacga cttatcgcca ctggcagcag
     5581 ccactggtaa caggattagc agagcgaggt atgtaggcgg tgctacagag ttcttgaagt
     5641 ggtggcctaa ctacggctac actagaagaa cagtatttgg tatctgcgct ctgctgaagc
     5701 cagttacctt cggaaaaaga gttggtagct cttgatccgg caaacaaacc accgctggta
     5761 gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag
     5821 atcctttgat cttttctacg gggtctgacg ctcagtggaa cgaaaactca cgttaaggga
     5881 ttttggtcat gagattatca aaaaggatct tcacctagat ccttttaaat taaaaatgaa
     5941 gttttaaatc aatctaaagt atatatgagt aaacttggtc tgacagttac caatgcttaa
     6001 tcagtgaggc acctatctca gcgatctgtc tatttcgttc atccatagtt gcctgactcc
     6061 ccgtcgtgta gataactacg atacgggagg gcttaccatc tggccccagt gctgcaatga
     6121 taccgcgaga cccacgctca ccggctccag atttatcagc aataaaccag ccagccggaa
     6181 gggccgagcg cagaagtggt cctgcaactt tatccgcctc catccagtct attaattgtt
     6241 gccgggaagc tagagtaagt agttcgccag ttaatagttt gcgcaacgtt gttgccattg
     6301 ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc tccggttccc
     6361 aacgatcaag gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt agctccttcg
     6421 gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg gttatggcag
     6481 cactgcataa ttctcttact gtcatgccat ccgtaagatg cttttctgtg actggtgagt
     6541 actcaaccaa gtcattctga gaatagtgta tgcggcgacc gagttgctct tgcccggcgt
     6601 caatacggga taataccgcg ccacatagca gaactttaaa agtgctcatc attggaaaac
     6661 gttcttcggg gcgaaaactc tcaaggatct taccgctgtt gagatccagt tcgatgtaac
     6721 ccactcgtgc acccaactga tcttcagcat cttttacttt caccagcgtt tctgggtgag
     6781 caaaaacagg aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa
     6841 tactcatact cttccttttt caatattatt gaagcattta tcagggttat tgtctcatga
     6901 gcggatacat atttgaatgt atttagaaaa ataaacaaat aggggttccg cgcacatttc
     6961 cccgaaaagt gccacctgac gtc
//
