LOCUS       sequence-306386-        6942 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Nanoluc complementation assay. Expression of histamine receptor H1 
            fused at C termimus with Natural peptide (NP) of NanoLuc. Addition 
            of the Flag epitope at N terminus of H1 receptor..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6942)
  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 6942)
  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.H1-NP
FEATURES             Location/Qualifiers
     source          1..6942
                     /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             923..946
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     primer_bind     complement(2533..2550)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2539..2763
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2809..3237
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2896..2915)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3106..3127
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3246..3266)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3251..3580
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3431..3566
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3493..3512
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3647..4441
                     /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(3701..3720)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4311..4330
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4615..4736
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4652..4671)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4706..4725
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4785..4801)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4785..4801)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4798..4820)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4809..4825
                     /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(4833..4839,4840..4857,4858..4863))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4878..4899
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5016..5033)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5187..5775)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5267..5286)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5946..6737,6738..6806))
                     /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     6569..6588
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6807..6911)
                     /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 aagcttacca tggattataa agatgatgat gataaaagcc tccccaattc
      961 ctcctgcctc ttagaagaca agatgtgtga gggcaacaag accactatgg ccagccccca
     1021 gctgatgccc ctggtggtgg tcctgagcac tatctgcttg gtcacagtag ggctcaacct
     1081 gctggtgctg tatgccgtac ggagtgagcg gaagctccac actgtgggga acctgtacat
     1141 cgtcagcctc tcggtggcgg acttgatcgt gggtgccgtc gtcatgccta tgaacatcct
     1201 ctacctgctc atgtccaagt ggtcactggg ccgtcctctc tgcctctttt ggctttccat
     1261 ggactatgtg gccagcacag cgtccatttt cagtgtcttc atcctgtgca ttgatcgcta
     1321 ccgctctgtc cagcagcccc tcaggtacct taagtatcgt accaagaccc gagcctcggc
     1381 caccattctg ggggcctggt ttctctcttt tctgtgggtt attcccattc taggctggaa
     1441 tcacttcatg cagcagacct cggtgcgccg agaggacaag tgtgagacag acttctatga
     1501 tgtcacctgg ttcaaggtca tgactgccat catcaacttc tacctgccca ccttgctcat
     1561 gctctggttc tatgccaaga tctacaaggc cgtacgacaa cactgccagc accgggagct
     1621 catcaatagg tccctccctt ccttctcaga aattaagctg aggccagaga accccaaggg
     1681 ggatgccaag aaaccaggga aggagtctcc ctgggaggtt ctgaaaagga agccaaaaga
     1741 tgctggtggt ggatctgtct tgaagtcacc atcccaaacc cccaaggaga tgaaatcccc
     1801 agttgtcttc agccaagagg atgatagaga agtagacaaa ctctactgct ttccacttga
     1861 tattgtgcac atgcaggctg cggcagaggg gagtagcagg gactatgtag ccgtcaaccg
     1921 gagccatggc cagctcaaga cagatgagca gggcctgaac acacatgggg ccagcgagat
     1981 atcagaggat cagatgttag gtgatagcca gtccttctct cgaacggact cagataccac
     2041 cacagagaca gcaccaggca aaggcaaatt gaggagtggg tctaacacag gcctggatta
     2101 catcaagttt acttggaaga ggctccgctc gcattcaaga cagtatgtat ctgggttgca
     2161 catgaaccgc gaaaggaagg ccgccaaaca gttgggtttt atcatggcag ccttcatcct
     2221 ctgctggatc ccttatttca tcttcttcat ggtcattgcc ttctgcaaga actgttgcaa
     2281 tgaacatttg cacatgttca ccatctggct gggctacatc aactccacac tgaaccccct
     2341 catctacccc ttgtgcaatg agaacttcaa gaagacattc aagagaattc tgcatattcg
     2401 ctccgggaat tctggctcga gcggtggtgg cgggagcgga ggtggagggt cgtcaggtgg
     2461 tgtgaccggc tggcggctgt gtgagcgaat tctggcataa ctctagaggg cccgtttaaa
     2521 cccgctgatc agcctcgact gtgccttcta gttgccagcc atctgttgtt tgcccctccc
     2581 ccgtgccttc cttgaccctg gaaggtgcca ctcccactgt cctttcctaa taaaatgagg
     2641 aaattgcatc gcattgtctg agtaggtgtc attctattct ggggggtggg gtggggcagg
     2701 acagcaaggg ggaggattgg gaagacaata gcaggcatgc tggggatgcg gtgggctcta
     2761 tggcttctga ggcggaaaga accagctggg gctctagggg gtatccccac gcgccctgta
     2821 gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca
     2881 gcgccctagc gcccgctcct ttcgctttct tcccttcctt tctcgccacg ttcgccggct
     2941 ttccccgtca agctctaaat cgggggctcc ctttagggtt ccgatttagt gctttacggc
     3001 acctcgaccc caaaaaactt gattagggtg atggttcacg tagtgggcca tcgccctgat
     3061 agacggtttt tcgccctttg acgttggagt ccacgttctt taatagtgga ctcttgttcc
     3121 aaactggaac aacactcaac cctatctcgg tctattcttt tgatttataa gggattttgc
     3181 cgatttcggc ctattggtta aaaaatgagc tgatttaaca aaaatttaac gcgaattaat
     3241 tctgtggaat gtgtgtcagt tagggtgtgg aaagtcccca ggctccccag caggcagaag
     3301 tatgcaaagc atgcatctca attagtcagc aaccaggtgt ggaaagtccc caggctcccc
     3361 agcaggcaga agtatgcaaa gcatgcatct caattagtca gcaaccatag tcccgcccct
     3421 aactccgccc atcccgcccc taactccgcc cagttccgcc cattctccgc cccatggctg
     3481 actaattttt tttatttatg cagaggccga ggccgcctct gcctctgagc tattccagaa
     3541 gtagtgagga ggcttttttg gaggcctagg cttttgcaaa aagctcccgg gagcttgtat
     3601 atccattttc ggatctgatc aagagacagg atgaggatcg tttcgcatga ttgaacaaga
     3661 tggattgcac gcaggttctc cggccgcttg ggtggagagg ctattcggct atgactgggc
     3721 acaacagaca atcggctgct ctgatgccgc cgtgttccgg ctgtcagcgc aggggcgccc
     3781 ggttcttttt gtcaagaccg acctgtccgg tgccctgaat gaactgcagg acgaggcagc
     3841 gcggctatcg tggctggcca cgacgggcgt tccttgcgca gctgtgctcg acgttgtcac
     3901 tgaagcggga agggactggc tgctattggg cgaagtgccg gggcaggatc tcctgtcatc
     3961 tcaccttgct cctgccgaga aagtatccat catggctgat gcaatgcggc ggctgcatac
     4021 gcttgatccg gctacctgcc cattcgacca ccaagcgaaa catcgcatcg agcgagcacg
     4081 tactcggatg gaagccggtc ttgtcgatca ggatgatctg gacgaagagc atcaggggct
     4141 cgcgccagcc gaactgttcg ccaggctcaa ggcgcgcatg cccgacggcg aggatctcgt
     4201 cgtgacccat ggcgatgcct gcttgccgaa tatcatggtg gaaaatggcc gcttttctgg
     4261 attcatcgac tgtggccggc tgggtgtggc ggaccgctat caggacatag cgttggctac
     4321 ccgtgatatt gctgaagagc ttggcggcga atgggctgac cgcttcctcg tgctttacgg
     4381 tatcgccgct cccgattcgc agcgcatcgc cttctatcgc cttcttgacg agttcttctg
     4441 agcgggactc tggggttcga aatgaccgac caagcgacgc ccaacctgcc atcacgagat
     4501 ttcgattcca ccgccgcctt ctatgaaagg ttgggcttcg gaatcgtttt ccgggacgcc
     4561 ggctggatga tcctccagcg cggggatctc atgctggagt tcttcgccca ccccaacttg
     4621 tttattgcag cttataatgg ttacaaataa agcaatagca tcacaaattt cacaaataaa
     4681 gcattttttt cactgcattc tagttgtggt ttgtccaaac tcatcaatgt atcttatcat
     4741 gtctgtatac cgtcgacctc tagctagagc ttggcgtaat catggtcata gctgtttcct
     4801 gtgtgaaatt gttatccgct cacaattcca cacaacatac gagccggaag cataaagtgt
     4861 aaagcctggg gtgcctaatg agtgagctaa ctcacattaa ttgcgttgcg ctcactgccc
     4921 gctttccagt cgggaaacct gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg
     4981 agaggcggtt tgcgtattgg gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg
     5041 gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg cggtaatacg gttatccaca
     5101 gaatcagggg ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac
     5161 cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc gcccccctga cgagcatcac
     5221 aaaaatcgac gctcaagtca gaggtggcga aacccgacag gactataaag ataccaggcg
     5281 tttccccctg gaagctccct cgtgcgctct cctgttccga ccctgccgct taccggatac
     5341 ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc atagctcacg ctgtaggtat
     5401 ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag
     5461 cccgaccgct gcgccttatc cggtaactat cgtcttgagt ccaacccggt aagacacgac
     5521 ttatcgccac tggcagcagc cactggtaac aggattagca gagcgaggta tgtaggcggt
     5581 gctacagagt tcttgaagtg gtggcctaac tacggctaca ctagaagaac agtatttggt
     5641 atctgcgctc tgctgaagcc agttaccttc ggaaaaagag ttggtagctc ttgatccggc
     5701 aaacaaacca ccgctggtag cggtggtttt tttgtttgca agcagcagat tacgcgcaga
     5761 aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc tcagtggaac
     5821 gaaaactcac gttaagggat tttggtcatg agattatcaa aaaggatctt cacctagatc
     5881 cttttaaatt aaaaatgaag ttttaaatca atctaaagta tatatgagta aacttggtct
     5941 gacagttacc aatgcttaat cagtgaggca cctatctcag cgatctgtct atttcgttca
     6001 tccatagttg cctgactccc cgtcgtgtag ataactacga tacgggaggg cttaccatct
     6061 ggccccagtg ctgcaatgat accgcgagac ccacgctcac cggctccaga tttatcagca
     6121 ataaaccagc cagccggaag ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc
     6181 atccagtcta ttaattgttg ccgggaagct agagtaagta gttcgccagt taatagtttg
     6241 cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct
     6301 tcattcagct ccggttccca acgatcaagg cgagttacat gatcccccat gttgtgcaaa
     6361 aaagcggtta gctccttcgg tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta
     6421 tcactcatgg ttatggcagc actgcataat tctcttactg tcatgccatc cgtaagatgc
     6481 ttttctgtga ctggtgagta ctcaaccaag tcattctgag aatagtgtat gcggcgaccg
     6541 agttgctctt gcccggcgtc aatacgggat aataccgcgc cacatagcag aactttaaaa
     6601 gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct caaggatctt accgctgttg
     6661 agatccagtt cgatgtaacc cactcgtgca cccaactgat cttcagcatc ttttactttc
     6721 accagcgttt ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg
     6781 gcgacacgga aatgttgaat actcatactc ttcctttttc aatattattg aagcatttat
     6841 cagggttatt gtctcatgag cggatacata tttgaatgta tttagaaaaa taaacaaata
     6901 ggggttccgc gcacatttcc ccgaaaagtg ccacctgacg tc
//
