LOCUS       sequence-379753-        8304 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Encodes a BRET-based activity sensor for the heterotrimeric G 
            protein G12. Composed of the subunits G alpha 12 (GNA12) tagged with
            NanoLuciferase, G beta 3 (GNB3) and Venus-tagged G gamma 9 (GNG9)..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8304)
  AUTHORS   Schihada H, Klompstra TM, Humphrys LJ, Cervenka I, Dadvar S, Kolb P,
            Ruas JL, Schulte G
  TITLE     Isoforms of GPR35 have distinct extracellular N-termini that 
            allosterically modify receptor-transducer coupling and mediate 
            intracellular pathway bias.
  JOURNAL   J Biol Chem. 2022 Aug 3:102328. doi: 10.1016/j.jbc.2022.102328.
  PUBMED    35933013
REFERENCE   2  (bases 1 to 8304)
  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: "J Biol 
            Chem. 2022 Aug 3:102328. doi: 10.1016/j.jbc.2022.102328."
COMMENT     Sequence Label: G12-CASE
FEATURES             Location/Qualifiers
     source          1..8304
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        61..364
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        365..568
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     519..539
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     CDS             1617..1670
                     /codon_start=1
                     /product="2A peptide from Thosea asigna virus capsid 
                     protein"
                     /label=T2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="EGRGSLLTCGDVEENPGP"
     CDS             1677..2411
                     /codon_start=1
                     /product="circularly permuted form of the Venus yellow 
                     fluorescent protein (Nagai et al., 2004)"
                     /label=cp173Venus
                     /note="efficient FRET acceptor for CFP"
                     /translation="MDGGVQLADHYQQNTPIGDGPVLLPDNHYLSYQSALSKDPNEKRD
                     HMVLLEFVTAAGITLGMDELYKGGSGGMVSKGEELFTGVVPILVELDGDVNGHKFSVSG
                     EGEGDATYGKLTLKLICTTGKLPVPWPTLVTTLGYGLQCFARYPDHMKQHDFFKSAMPE
                     GYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHN
                     VYITADKQKNGIKANFKIRHNIE"
     primer_bind     1814..1835
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(1938..1959)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2199..2218)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     complement(2658..2675)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     misc_feature    2687..3260
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(2854..2871)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     3081..3100
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             3691..4200
                     /codon_start=1
                     /product="NanoLuc(R) luciferase"
                     /label=Nluc
                     /note="human codon-optimized"
                     /translation="VFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQRI
                     VLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGVT
                     PNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGWR
                     LCERILA"
     polyA_signal    5092..5213
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5129..5148)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5183..5202
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(5220..5675)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5357..5378)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5569..5588
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        5702..5806
                     /gene="bla"
                     /label=AmpR promoter
     promoter        5808..6165
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        5808..5999
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(5831..5851)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      6016..6151
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             6200..6994
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     CDS             6200..6994
                     /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
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(6254..6273)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     6864..6883
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(7182..7201)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    7226..7273
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      7602..8190
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     8091..8110
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata tggagttccg
       61 cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc cccgcccatt
      121 gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc attgacgtca
      181 atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt atcatatgcc
      241 aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt atgcccagta
      301 catgacctta tgggactttc ctacttggca gtacatctac gtattagtca tcgctattac
      361 catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg actcacgggg
      421 atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc aaaatcaacg
      481 ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg gtaggcgtgt
      541 acggtgggag gtctatataa gcagagctgg tttagtgaac cgtcagatcc gctagcatgg
      601 gggagatgga gcaactgcgt caggaagcgg agcagctcaa gaagcagatt gcagatgcca
      661 ggaaagcctg tgctgacgtt actctggcag agctggtgtc tggcctagag gtggtgggac
      721 gagtccagat gcggacgcgg cggacgttaa ggggacacct ggccaagatt tacgccatgc
      781 actgggccac tgattctaag ctgctggtaa gtgcctcgca agatgggaag ctgatcgtgt
      841 gggacagcta caccaccaac aaggtgcacg ccatcccact gcgctcctcc tgggtcatga
      901 cctgtgccta tgccccatca gggaactttg tggcgtgtgg ggggctggac aacatgtgtt
      961 ccatctacaa cctcaaatcc cgtgagggca atgtcaaggt cagccgggag ctttctgctc
     1021 acacaggtta tctctcctgc tgccgcttcc tggatgacaa caatattgtg accagctcgg
     1081 gggacaccac gtgtgccttg tgggacattg agactgggca gcagaagact gtatttgtgg
     1141 gacacacggg tgactgcatg agcctggctg tgtctcctga cttcaatctc ttcatttcgg
     1201 gggcctgtga tgccagtgcc aagctctggg atgtgcgaga ggggacctgc cgacagactt
     1261 tcactggcca cgagtcggac atcaacgcca tctgtttctt ccccaatgga gaggccatct
     1321 gcacgggctc ggatgacgct tcctgccgct tgtttgacct gcgggcagac caggagctga
     1381 tctgcttctc ccacgagagc atcatctgcg gcatcacgtc tgtggccttc tccctcagtg
     1441 gccgcctact attcgctggc tacgacgact tcaactgcaa tgtctgggac tccatgaagt
     1501 ctgagcgtgt gggcatcctc tctggccacg ataacagggt gagctgcctg ggagtcacag
     1561 ctgacgggat ggctgtggcc acaggttcct gggacagctt cctcaaaatc tggaacgagg
     1621 gcagaggaag tcttctaaca tgcggtgacg tggaggagaa tcccggccct accggtatgg
     1681 acggcggcgt gcagctcgcc gaccactacc agcagaacac ccccatcggc gacggccccg
     1741 tgctgctgcc cgacaaccac tacctgagct accagtccgc cctgagcaaa gaccccaacg
     1801 agaagcgcga tcacatggtc ctgctggagt tcgtgaccgc cgccgggatc actctcggca
     1861 tggacgagct gtacaagggt ggcagcggtg gcatggtgag caagggcgag gagctgttca
     1921 ccggggtggt gcccatcctg gtcgagctgg acggcgacgt aaacggccac aagttcagcg
     1981 tgtccggcga gggcgagggc gatgccacct acggcaagct gaccctgaag ctgatctgca
     2041 ccaccggcaa gctgcccgtg ccctggccca ccctcgtgac caccctgggc tacggcctgc
     2101 agtgcttcgc ccgctacccc gaccacatga agcagcacga cttcttcaag tccgccatgc
     2161 ccgaaggcta cgtccaggag cgcaccatct tcttcaagga cgacggcaac tacaagaccc
     2221 gcgccgaggt gaagttcgag ggcgacaccc tggtgaaccg catcgagctg aagggcatcg
     2281 acttcaagga ggacggcaac atcctggggc acaagctgga gtacaactac aacagccaca
     2341 acgtctatat caccgccgac aaacagaaga acggcatcaa ggccaacttc aagatccgcc
     2401 acaacatcga gtccggactc agatctgccc aggatctcag cgagaaggac ctgttgaaga
     2461 tggaggtgga gcagctgaag aaagaagtga aaaacacaag aattccgatt tccaaagcgg
     2521 gaaaggaaat caaggagtac gtggaggccc aagcaggaaa cgatcctttt ctcaaaggca
     2581 tccctgagga caagaatccc ttcaaggaga aaggtggctg tctgataagc taagtcgaca
     2641 gatctcaatt ggggccccta tagtgtcacc taaataattc cgcccccccc tctccctccc
     2701 ccccccctaa cgttactggc cgaagccgct tggaataagg ccggtgtgcg tttgtctata
     2761 tgttattttc caccatattg ccgtcttttg gcaatgtgag ggcccggaaa cctggccctg
     2821 tcttcttgac gagcattcct aggggtcttt cccctctcgc caaaggaatg caaggtctgt
     2881 tgaatgtcgt gaaggaagca gttcctctgg aagcttcttg aagacaaaca acgtctgtag
     2941 cgaccctttg caggcagcgg aaccccccac ctggcgacag gtgcctctgc ggccaaaagc
     3001 cacgtgtata agatacacct gcaaaggcgg cacaacccca gtgccacgtt gtgagttgga
     3061 tagttgtgga aagagtcaaa tggctctcct caagcgtatt caacaagggg ctgaaggatg
     3121 cccagaaggt accccattgt atgggatctg atctggggcc tcggtgcaca tgctttacat
     3181 gtgtttagtc gaggttaaaa aaacgtctag gccccccgaa ccacggggac gtggttttcc
     3241 tttgaaaaac acgatgataa tggatccatg tccggggtgg tgcggaccct cagccgctgc
     3301 ctgctgccgg ccgaggccgg cggggcccgc gagcgcaggg cgggcagcgg cgcgcgcgac
     3361 gcggagcgcg aggcccggag gcgtagccgc gacatcgacg cgctgctggc ccgcgagcgg
     3421 cgcgcggtcc ggcgcctggt gaagatcctg ctgctgggcg cgggcgagag cggcaagtcc
     3481 acgttcctca agcagatgcg catcatccac ggccgcgagt tcgaccagaa ggcgctgctg
     3541 gagttccgcg acaccatctt cgacaacatc ctcaagggct caagggttct tgttgatgca
     3601 cgagataagc ttggcattcc ttggcagtat tctgaaaatg agaagcatgg gatgttcctg
     3661 atggccagcg gtggaggcgg gacgcgttct gtcttcacac tcgaagattt cgttggggac
     3721 tggcgacaga cagccggcta caacctggac caagtccttg aacagggagg tgtgtccagt
     3781 ttgtttcaga atctcggggt gtccgtaact ccgatccaaa ggattgtcct gagcggtgaa
     3841 aatgggctga agatcgacat ccatgtcatc atcccgtatg aaggtctgag cggcgaccaa
     3901 atgggccaga tcgaaaaaat ttttaaggtg gtgtaccctg tggatgatca tcactttaag
     3961 gtgatcctgc actatggcac actggtaatc gacggggtta cgccgaacat gatcgactat
     4021 ttcggacggc cgtatgaagg catcgccgtg ttcgacggca aaaagatcac tgtaacaggg
     4081 accctgtgga acggcaacaa aattatcgac gagcgcctga tcaaccccga cggctccctg
     4141 ctgttccgag taaccatcaa cggagtgacc ggctggcggc tgtgcgaacg cattctggcc
     4201 accggtggtg gcggatcctt cgagaacaag gcggggctgc ctgtggagcc ggccaccttc
     4261 cagctgtacg tcccggccct gagcgcactc tggagggatt ctggcatcag ggaggctttc
     4321 agccggagaa gcgagtttca gctgggggag tcggtgaagt acttcctgga caacttggac
     4381 cggatcggcc agctgaatta ctttcctagt aagcaagata tcctgctggc taggaaagcc
     4441 accaagggaa ttgtggagca tgacttcgtt attaagaaga tcccctttaa gatggtggat
     4501 gtgggcggcc agcggtccca gcgccagaag tggttccagt gcttcgacgg gatcacgtcc
     4561 atcctgttca tggtctcctc cagcgagtac gaccaggtcc tcatggagga caggcgcacc
     4621 aaccggctgg tggagtccat gaacatcttc gagaccatcg tcaacaacaa gctcttcttc
     4681 aacgtctcca tcattctctt cctcaacaag atggacctcc tggtggagaa ggtgaagacc
     4741 gtgagcatca agaagcactt cccggacttc aggggcgacc cgcacaggct ggaggacgtc
     4801 cagcgctacc tggtccagtg cttcgacagg aagagacgga accgcagcaa gccactcttc
     4861 caccacttca ccaccgccat cgacaccgag aacgtccgct tcgtgttcca tgctgtgaaa
     4921 gacaccatcc tgcaggagaa cctgaaggac atcatgctgc agtaatctag agggccgcga
     4981 ctctagatca taatcagcca taccacattt gtagaggttt tacttgcttt aaaaaacctc
     5041 ccacacctcc ccctgaacct gaaacataaa atgaatgcaa ttgttgttgt taacttgttt
     5101 attgcagctt ataatggtta caaataaagc aatagcatca caaatttcac aaataaagca
     5161 tttttttcac tgcattctag ttgtggtttg tccaaactca tcaatgtatc ttaaggcgta
     5221 aattgtaagc gttaatattt tgttaaaatt cgcgttaaat ttttgttaaa tcagctcatt
     5281 ttttaaccaa taggccgaaa tcggcaaaat cccttataaa tcaaaagaat agaccgagat
     5341 agggttgagt gttgttccag tttggaacaa gagtccacta ttaaagaacg tggactccaa
     5401 cgtcaaaggg cgaaaaaccg tctatcaggg cgatggccca ctacgtgaac catcacccta
     5461 atcaagtttt ttggggtcga ggtgccgtaa agcactaaat cggaacccta aagggagccc
     5521 ccgatttaga gcttgacggg gaaagccggc gaacgtggcg agaaaggaag ggaagaaagc
     5581 gaaaggagcg ggcgctaggg cgctggcaag tgtagcggtc acgctgcgcg taaccaccac
     5641 acccgccgcg cttaatgcgc cgctacaggg cgcgtcaggt ggcacttttc ggggaaatgt
     5701 gcgcggaacc cctatttgtt tatttttcta aatacattca aatatgtatc cgctcatgag
     5761 acaataaccc tgataaatgc ttcaataata ttgaaaaagg aagagtcctg aggcggaaag
     5821 aaccagctgt ggaatgtgtg tcagttaggg tgtggaaagt ccccaggctc cccagcaggc
     5881 agaagtatgc aaagcatgca tctcaattag tcagcaacca ggtgtggaaa gtccccaggc
     5941 tccccagcag gcagaagtat gcaaagcatg catctcaatt agtcagcaac catagtcccg
     6001 cccctaactc cgcccatccc gcccctaact ccgcccagtt ccgcccattc tccgccccat
     6061 ggctgactaa ttttttttat ttatgcagag gccgaggccg cctcggcctc tgagctattc
     6121 cagaagtagt gaggaggctt ttttggaggc ctaggctttt gcaaagatcg atcaagagac
     6181 aggatgagga tcgtttcgca tgattgaaca agatggattg cacgcaggtt ctccggccgc
     6241 ttgggtggag aggctattcg gctatgactg ggcacaacag acaatcggct gctctgatgc
     6301 cgccgtgttc cggctgtcag cgcaggggcg cccggttctt tttgtcaaga ccgacctgtc
     6361 cggtgccctg aatgaactgc aagacgaggc agcgcggcta tcgtggctgg ccacgacggg
     6421 cgttccttgc gcagctgtgc tcgacgttgt cactgaagcg ggaagggact ggctgctatt
     6481 gggcgaagtg ccggggcagg atctcctgtc atctcacctt gctcctgccg agaaagtatc
     6541 catcatggct gatgcaatgc ggcggctgca tacgcttgat ccggctacct gcccattcga
     6601 ccaccaagcg aaacatcgca tcgagcgagc acgtactcgg atggaagccg gtcttgtcga
     6661 tcaggatgat ctggacgaag agcatcaggg gctcgcgcca gccgaactgt tcgccaggct
     6721 caaggcgagc atgcccgacg gcgaggatct cgtcgtgacc catggcgatg cctgcttgcc
     6781 gaatatcatg gtggaaaatg gccgcttttc tggattcatc gactgtggcc ggctgggtgt
     6841 ggcggaccgc tatcaggaca tagcgttggc tacccgtgat attgctgaag agcttggcgg
     6901 cgaatgggct gaccgcttcc tcgtgcttta cggtatcgcc gctcccgatt cgcagcgcat
     6961 cgccttctat cgccttcttg acgagttctt ctgagcggga ctctggggtt cgaaatgacc
     7021 gaccaagcga cgcccaacct gccatcacga gatttcgatt ccaccgccgc cttctatgaa
     7081 aggttgggct tcggaatcgt tttccgggac gccggctgga tgatcctcca gcgcggggat
     7141 ctcatgctgg agttcttcgc ccaccctagg gggaggctaa ctgaaacacg gaaggagaca
     7201 ataccggaag gaacccgcgc tatgacggca ataaaaagac agaataaaac gcacggtgtt
     7261 gggtcgtttg ttcataaacg cggggttcgg tcccagggct ggcactctgt cgatacccca
     7321 ccgagacccc attggggcca atacgcccgc gtttcttcct tttccccacc ccacccccca
     7381 agttcgggtg aaggcccagg gctcgcagcc aacgtcgggg cggcaggccc tgccatagcc
     7441 tcaggttact catatatact ttagattgat ttaaaacttc atttttaatt taaaaggatc
     7501 taggtgaaga tcctttttga taatctcatg accaaaatcc cttaacgtga gttttcgttc
     7561 cactgagcgt cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg
     7621 cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg
     7681 gatcaagagc taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca
     7741 aatactgttc ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg
     7801 cctacatacc tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg
     7861 tgtcttaccg ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga
     7921 acggggggtt cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac
     7981 ctacagcgtg agctatgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat
     8041 ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc
     8101 tggtatcttt atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga
     8161 tgctcgtcag gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc
     8221 ctggcctttt gctggccttt tgctcacatg ttctttcctg cgttatcccc tgattctgtg
     8281 gataaccgta ttaccgccat gcat
//
