LOCUS       sequence-306391-        6873 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Nanoluc complementation assay. Expression of dopamine receptor D2 
            fused at C terminus with Natural peptide (NP) of NanoLuc. Addition 
            of the signal sequence and Flag epitope at N terminus of D2..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6873)
  AUTHORS   Laschet C, Dupuis N, Hanson J
  TITLE     A dynamic and screening-compatible nanoluciferase-based 
            complementation assay enables profiling of individual GPCR-G protein
            interactions.
  JOURNAL   J Biol Chem. 2019 Mar 15;294(11):4079-4090. doi: 
            10.1074/jbc.RA118.006231. Epub 2018 Dec 28.
  PUBMED    30593506
REFERENCE   2  (bases 1 to 6873)
  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"; doi: 
            "10.1074/jbc.RA118.006231"; journalName: "J Biol Chem"; date: 
            "2019-03-15- 15"; volume: "294"; issue: "11"; pages: "4079-4090"
COMMENT     Sequence Label: pcDNA3.1.D2-NP
FEATURES             Location/Qualifiers
     source          1..6873
                     /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             986..1009
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     primer_bind     complement(2464..2481)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2470..2694
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2740..3168
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2827..2846)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3037..3058
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3177..3197)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3182..3511
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3362..3497
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3424..3443
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3578..4372
                     /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(3632..3651)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4242..4261
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4546..4667
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4583..4602)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4637..4656
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4716..4732)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4716..4732)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4729..4751)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4740..4756
                     /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(4764..4770,4771..4788,4789..4794))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4809..4830
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4947..4964)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5118..5706)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5198..5217)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5877..6668,6669..6737))
                     /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     6500..6519
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6738..6842)
                     /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 ccgagctcgg atccaccatg aagacgatca tcgccctgag
      961 ctacatcttc tgcctggtat tcgccgatta taaagatgat gatgataaag atccactgaa
     1021 tctgtcctgg tatgatgatg atctggagag gcagaactgg agccggccct tcaacgggtc
     1081 agacgggaag gcggacagac cccactacaa ctactatgcc acactgctca ccctgctcat
     1141 cgctgtcatc gtcttcggca acgtgctggt gtgcatggct gtgtcccgcg agaaggcgct
     1201 gcagaccacc accaactacc tgatcgtcag cctcgcagtg gccgacctcc tcgtcgccac
     1261 actggtcatg ccctgggttg tctacctgga ggtggtaggt gagtggaaat tcagcaggat
     1321 tcactgtgac atcttcgtca ctctggacgt catgatgtgc acggcgagca tcctgaactt
     1381 gtgtgccatc agcatcgaca ggtacacagc tgtggccatg cccatgctgt acaatacgcg
     1441 ctacagctcc aagcgccggg tcaccgtcat gatctccatc gtctgggtcc tgtccttcac
     1501 catctcctgc ccactcctct tcggactcaa taacgcagac cagaacgagt gcatcattgc
     1561 caacccggcc ttcgtggtct actcctccat cgtctccttc tacgtgccct tcattgtcac
     1621 cctgctggtc tacatcaaga tctacattgt cctccgcaga cgccgcaagc gagtcaacac
     1681 caaacgcagc agccgagctt tcagggccca cctgagggct ccactaaagg gcaactgtac
     1741 tcaccccgag gacatgaaac tctgcaccgt tatcatgaag tctaatggga gtttcccagt
     1801 gaacaggcgg agagtggagg ctgcccggcg agcccaggag ctggagatgg agatgctctc
     1861 cagcaccagc ccacccgaga ggacccggta cagccccatc ccacccagcc accaccagct
     1921 gactctcccc gacccgtccc accatggtct ccacagcact cccgacagcc ccgccaaacc
     1981 agagaagaat gggcatgcca aagaccaccc caagattgcc aagatctttg agatccagac
     2041 catgcccaat ggcaaaaccc ggacctccct caagaccatg agccgtagga agctctccca
     2101 gcagaaggag aagaaagcca ctcagatgct cgccattgtt ctcggcgtgt tcatcatctg
     2161 ctggctgccc ttcttcatca cacacatcct gaacatacac tgtgactgca acatcccgcc
     2221 tgtcctgtac agcgccttca cgtggctggg ctatgtcaac agcgccgtga accccatcat
     2281 ctacaccacc ttcaacattg agttccgcaa ggccttcctg aagatcctcc actgcgggaa
     2341 ttctggctcg agcggtggtg gcgggagcgg aggtggaggg tcgtcaggtg gtgtgaccgg
     2401 ctggcggctg tgtgagcgaa ttctggcata actctagagg gcccgtttaa acccgctgat
     2461 cagcctcgac tgtgccttct agttgccagc catctgttgt ttgcccctcc cccgtgcctt
     2521 ccttgaccct ggaaggtgcc actcccactg tcctttccta ataaaatgag gaaattgcat
     2581 cgcattgtct gagtaggtgt cattctattc tggggggtgg ggtggggcag gacagcaagg
     2641 gggaggattg ggaagacaat agcaggcatg ctggggatgc ggtgggctct atggcttctg
     2701 aggcggaaag aaccagctgg ggctctaggg ggtatcccca cgcgccctgt agcggcgcat
     2761 taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc agcgccctag
     2821 cgcccgctcc tttcgctttc ttcccttcct ttctcgccac gttcgccggc tttccccgtc
     2881 aagctctaaa tcgggggctc cctttagggt tccgatttag tgctttacgg cacctcgacc
     2941 ccaaaaaact tgattagggt gatggttcac gtagtgggcc atcgccctga tagacggttt
     3001 ttcgcccttt gacgttggag tccacgttct ttaatagtgg actcttgttc caaactggaa
     3061 caacactcaa ccctatctcg gtctattctt ttgatttata agggattttg ccgatttcgg
     3121 cctattggtt aaaaaatgag ctgatttaac aaaaatttaa cgcgaattaa ttctgtggaa
     3181 tgtgtgtcag ttagggtgtg gaaagtcccc aggctcccca gcaggcagaa gtatgcaaag
     3241 catgcatctc aattagtcag caaccaggtg tggaaagtcc ccaggctccc cagcaggcag
     3301 aagtatgcaa agcatgcatc tcaattagtc agcaaccata gtcccgcccc taactccgcc
     3361 catcccgccc ctaactccgc ccagttccgc ccattctccg ccccatggct gactaatttt
     3421 ttttatttat gcagaggccg aggccgcctc tgcctctgag ctattccaga agtagtgagg
     3481 aggctttttt ggaggcctag gcttttgcaa aaagctcccg ggagcttgta tatccatttt
     3541 cggatctgat caagagacag gatgaggatc gtttcgcatg attgaacaag atggattgca
     3601 cgcaggttct ccggccgctt gggtggagag gctattcggc tatgactggg cacaacagac
     3661 aatcggctgc tctgatgccg ccgtgttccg gctgtcagcg caggggcgcc cggttctttt
     3721 tgtcaagacc gacctgtccg gtgccctgaa tgaactgcag gacgaggcag cgcggctatc
     3781 gtggctggcc acgacgggcg ttccttgcgc agctgtgctc gacgttgtca ctgaagcggg
     3841 aagggactgg ctgctattgg gcgaagtgcc ggggcaggat ctcctgtcat ctcaccttgc
     3901 tcctgccgag aaagtatcca tcatggctga tgcaatgcgg cggctgcata cgcttgatcc
     3961 ggctacctgc ccattcgacc accaagcgaa acatcgcatc gagcgagcac gtactcggat
     4021 ggaagccggt cttgtcgatc aggatgatct ggacgaagag catcaggggc tcgcgccagc
     4081 cgaactgttc gccaggctca aggcgcgcat gcccgacggc gaggatctcg tcgtgaccca
     4141 tggcgatgcc tgcttgccga atatcatggt ggaaaatggc cgcttttctg gattcatcga
     4201 ctgtggccgg ctgggtgtgg cggaccgcta tcaggacata gcgttggcta cccgtgatat
     4261 tgctgaagag cttggcggcg aatgggctga ccgcttcctc gtgctttacg gtatcgccgc
     4321 tcccgattcg cagcgcatcg ccttctatcg ccttcttgac gagttcttct gagcgggact
     4381 ctggggttcg aaatgaccga ccaagcgacg cccaacctgc catcacgaga tttcgattcc
     4441 accgccgcct tctatgaaag gttgggcttc ggaatcgttt tccgggacgc cggctggatg
     4501 atcctccagc gcggggatct catgctggag ttcttcgccc accccaactt gtttattgca
     4561 gcttataatg gttacaaata aagcaatagc atcacaaatt tcacaaataa agcatttttt
     4621 tcactgcatt ctagttgtgg tttgtccaaa ctcatcaatg tatcttatca tgtctgtata
     4681 ccgtcgacct ctagctagag cttggcgtaa tcatggtcat agctgtttcc tgtgtgaaat
     4741 tgttatccgc tcacaattcc acacaacata cgagccggaa gcataaagtg taaagcctgg
     4801 ggtgcctaat gagtgagcta actcacatta attgcgttgc gctcactgcc cgctttccag
     4861 tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt
     4921 ttgcgtattg ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg
     4981 ctgcggcgag cggtatcagc tcactcaaag gcggtaatac ggttatccac agaatcaggg
     5041 gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag
     5101 gccgcgttgc tggcgttttt ccataggctc cgcccccctg acgagcatca caaaaatcga
     5161 cgctcaagtc agaggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct
     5221 ggaagctccc tcgtgcgctc tcctgttccg accctgccgc ttaccggata cctgtccgcc
     5281 tttctccctt cgggaagcgt ggcgctttct catagctcac gctgtaggta tctcagttcg
     5341 gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac cccccgttca gcccgaccgc
     5401 tgcgccttat ccggtaacta tcgtcttgag tccaacccgg taagacacga cttatcgcca
     5461 ctggcagcag ccactggtaa caggattagc agagcgaggt atgtaggcgg tgctacagag
     5521 ttcttgaagt ggtggcctaa ctacggctac actagaagaa cagtatttgg tatctgcgct
     5581 ctgctgaagc cagttacctt cggaaaaaga gttggtagct cttgatccgg caaacaaacc
     5641 accgctggta gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga
     5701 tctcaagaag atcctttgat cttttctacg gggtctgacg ctcagtggaa cgaaaactca
     5761 cgttaaggga ttttggtcat gagattatca aaaaggatct tcacctagat ccttttaaat
     5821 taaaaatgaa gttttaaatc aatctaaagt atatatgagt aaacttggtc tgacagttac
     5881 caatgcttaa tcagtgaggc acctatctca gcgatctgtc tatttcgttc atccatagtt
     5941 gcctgactcc ccgtcgtgta gataactacg atacgggagg gcttaccatc tggccccagt
     6001 gctgcaatga taccgcgaga cccacgctca ccggctccag atttatcagc aataaaccag
     6061 ccagccggaa gggccgagcg cagaagtggt cctgcaactt tatccgcctc catccagtct
     6121 attaattgtt gccgggaagc tagagtaagt agttcgccag ttaatagttt gcgcaacgtt
     6181 gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc
     6241 tccggttccc aacgatcaag gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt
     6301 agctccttcg gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg
     6361 gttatggcag cactgcataa ttctcttact gtcatgccat ccgtaagatg cttttctgtg
     6421 actggtgagt actcaaccaa gtcattctga gaatagtgta tgcggcgacc gagttgctct
     6481 tgcccggcgt caatacggga taataccgcg ccacatagca gaactttaaa agtgctcatc
     6541 attggaaaac gttcttcggg gcgaaaactc tcaaggatct taccgctgtt gagatccagt
     6601 tcgatgtaac ccactcgtgc acccaactga tcttcagcat cttttacttt caccagcgtt
     6661 tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg
     6721 aaatgttgaa tactcatact cttccttttt caatattatt gaagcattta tcagggttat
     6781 tgtctcatga gcggatacat atttgaatgt atttagaaaa ataaacaaat aggggttccg
     6841 cgcacatttc cccgaaaagt gccacctgac gtc
//
