LOCUS       sequence-471657-        7816 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian expression of human muscarinic M1 receptor; N-terminal HA 
            tag, C-terminal Rluc8.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7816)
  AUTHORS   Gimenez LE, Vishnivetskiy SA, Baameur F, Gurevich VV
  TITLE     Manipulation of very few receptor discriminator residues greatly 
            enhances receptor specificity of non-visual arrestins.
  JOURNAL   J Biol Chem. 2012 Aug 24;287(35):29495-505. doi: 
            10.1074/jbc.M112.366674. Epub 2012 Jul 11.
  PUBMED    22787152
REFERENCE   2  (bases 1 to 7816)
  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.M112.366674"; journalName: "J Biol Chem"; date: 
            "2012-08-24- 24"; volume: "287"; issue: "35"; pages: "29495-505"
COMMENT     Sequence Label: p3HM1Luc4
FEATURES             Location/Qualifiers
     source          1..7816
                     /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             922..1002
                     /codon_start=1
                     /product="three tandem HA epitope tags"
                     /label=3xHA
                     /translation="YPYDVPDYAYPYDVPDYAYPYDVPDYA"
     CDS             2395..3330
                     /codon_start=1
                     /product="Renilla luciferase"
                     /label=hRluc
                     /note="human codon-optimized"
                     /translation="MASKVYDPEQRKRMITGPQWWARCKQMNVLDSFINYYDSEKHAEN
                     AVIFLHGNATSSYLWRHVVPHIEPVARCIIPDLIGMGKSGKSGNGSYRLLDHYKYLTAW
                     FELLNLPKKIIFVGHDWGAALAFHYAYEHQDRIKAIVHMESVVDVIESWDEWPDIEEDI
                     ALIKSEEGEKMVLENNFFVETVLPSKIMRKLEPEEFAAYLEPFKEKGEVRRPTLSWPRE
                     IPLVKGGKPDVVQIVRNYNAYLRASDDLPKLFIESDPGFFSNAIVEGAKKFPNTEFVKV
                     KGLHFLQEDAPDEMGKYIKSFVERVLKNEQ"
     promoter        complement(3369..3387)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(3370..3387)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(3407..3424)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3413..3637
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      3683..4111
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3770..3789)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3980..4001
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4120..4140)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        4125..4454
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      4305..4440
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     4367..4386
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             4521..5315
                     /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(4575..4594)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     5185..5204
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    5489..5610
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5526..5545)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5580..5599
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5659..5675)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5659..5675)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5672..5694)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5683..5699
                     /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(5707..5713,5714..5731,5732..5737))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5752..5773
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5890..5907)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6061..6649)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6141..6160)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6820..7611,7612..7680))
                     /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     7443..7462
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7681..7785)
                     /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 gctggaattc
      901 tgcgatcgca ccaccatggc gtacccatac gatgttccag attacgctta cccatacgat
      961 gttccagatt acgcttaccc atacgatgtt ccagattacg ctgataacac ttcagcccca
     1021 cctgctgtca gccccaacat caccgtcctg gcaccaggaa agggtccctg gcaagtggcc
     1081 ttcattggga tcaccacggg cctcctgtcg ctagccacag tgacaggcaa cctgctggta
     1141 ctcatctctt tcaaggtcaa cacggagctc aagacagtca ataactactt cctgctgagc
     1201 ctggcctgtg ctgacctcat catcggtacc ttctccatga acctctatac cacgtacctg
     1261 ctcatgggcc actgggctct gggcacgctg gcttgtgacc tctggctggc cctggactat
     1321 gtggccagca atgcctccgt catgaatctg ctgctcatca gctttgaccg ctacttctcc
     1381 gtgactcggc ccctgagcta ccgtgccaag cgcacacccc gccgggcagc tctgatgatc
     1441 ggcctggcct ggctggtttc ctttgtgctc tgggccccag ccatcctctt ctggcagtac
     1501 ctggtagggg agcggacagt gctagctggg cagtgctaca tccagttcct ctcccagccc
     1561 atcatcacct ttggcacagc catggctgcc ttctacctcc ctgtcacagt catgtgcacg
     1621 ctctactggc gcatctaccg ggagacagag aaccgagcac gggagctggc agcccttcag
     1681 ggctccgaga cgccaggcaa agggggtggc agcagcagca gctcagagag gtctcagcca
     1741 ggggctgagg gctcaccaga gactcctcca ggccgctgct gtcgctgctg ccgggccccc
     1801 aggctgctgc aggcctacag ctggaaggaa gaagaggaag aggacgaagg ctccatggag
     1861 tccctcacat cctcagaggg agaggagcct ggctccgaag tggtgatcaa gatgccaatg
     1921 gtggaccccg aggcacaggc ccccaccaag cagcccccac ggagctcccc aaatacagtc
     1981 aagaggccga ctaagaaagg gcgtgatcga gctggcaagg gccagaagcc ccgtggaaag
     2041 gagcagctgg ccaagcggaa gaccttctcg ctggtcaagg agaagaaggc ggctcggacc
     2101 ctgagtgcca tcctcctggc cttcatcctc acctggacac cgtacaacat catggtgctg
     2161 gtgtccacct tctgcaagga ctgtgttccc gagaccctgt gggagctggg ctactggctg
     2221 tgctacgtca acagcaccat caaccccatg tgctacgcac tctgcaacaa agccttccgg
     2281 gacacctttc gcctgctgct gctttgccgc tgggacaaga gacgctggcg caagatcccc
     2341 aagcgccctg gctccgtgca ccgcactccc tcccgccaat gcgcacctgc aggcatggct
     2401 tccaaggtgt acgaccccga gcaacgcaaa cgcatgatca ctgggcctca gtggtgggct
     2461 cgctgcaagc aaatgaacgt gctggactcc ttcatcaact actatgattc cgagaagcac
     2521 gccgagaacg ccgtgatttt tctgcatggt aacgctacct ccagctacct gtggaggcac
     2581 gtcgtgcctc acatcgagcc cgtggctaga tgcatcatcc ctgatctgat cggaatgggt
     2641 aagtccggca agagcgggaa tggctcatat cgcctcctgg atcactacaa gtacctcacc
     2701 gcttggttcg agctgctgaa ccttccaaag aaaatcatct ttgtgggcca cgactggggg
     2761 gctgctctgg cctttcacta cgcctacgag caccaagaca ggatcaaggc catcgtccat
     2821 atggagagtg tcgtggacgt gatcgagtcc tgggacgagt ggcctgacat cgaggaggat
     2881 atcgccctga tcaagagcga agagggcgag aaaatggtgc ttgagaataa cttcttcgtc
     2941 gagaccgtgc tcccaagcaa gatcatgcgg aaactggagc ctgaggagtt cgctgcctac
     3001 ctggagccat tcaaggagaa gggcgaggtt agacggccta ccctctcctg gcctcgcgag
     3061 atccctctcg ttaagggagg caagcccgac gtcgtccaga ttgtccgcaa ctacaacgcc
     3121 taccttcggg ccagcgacga tctgcctaag ctgttcatcg agtccgaccc tgggttcttt
     3181 tccaacgcta ttgtcgaggg agctaagaag ttccctaaca ccgagttcgt gaaggtgaag
     3241 ggcctccact tcctccagga ggacgctcca gatgaaatgg gtaagtacat caagagcttc
     3301 gtggagcgcg tgctgaagaa cgagcagtaa tcggcgcgcc tctagattaa ttaaaagctt
     3361 ggccctattc tatagtgtca cctaaatgct agagctcgct gatcagcctc gactgtgcct
     3421 tctagttgcc agccatctgt tgtttgcccc tcccccgtgc cttccttgac cctggaaggt
     3481 gccactccca ctgtcctttc ctaataaaat gaggaaattg catcgcattg tctgagtagg
     3541 tgtcattcta ttctgggggg tggggtgggg caggacagca agggggagga ttgggaagac
     3601 aatagcaggc atgctgggga tgcggtgggc tctatggctt ctgaggcgga aagaaccagc
     3661 tggggctcta gggggtatcc ccacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg
     3721 gtggttacgc gcagcgtgac cgctacactt gccagcgccc tagcgcccgc tcctttcgct
     3781 ttcttccctt cctttctcgc cacgttcgcc ggctttcccc gtcaagctct aaatcggggg
     3841 ctccctttag ggttccgatt tagtgcttta cggcacctcg accccaaaaa acttgattag
     3901 ggtgatggtt cacgtagtgg gccatcgccc tgatagacgg tttttcgccc tttgacgttg
     3961 gagtccacgt tctttaatag tggactcttg ttccaaactg gaacaacact caaccctatc
     4021 tcggtctatt cttttgattt ataagggatt ttgccgattt cggcctattg gttaaaaaat
     4081 gagctgattt aacaaaaatt taacgcgaat taattctgtg gaatgtgtgt cagttagggt
     4141 gtggaaagtc cccaggctcc ccagcaggca gaagtatgca aagcatgcat ctcaattagt
     4201 cagcaaccag gtgtggaaag tccccaggct ccccagcagg cagaagtatg caaagcatgc
     4261 atctcaatta gtcagcaacc atagtcccgc ccctaactcc gcccatcccg cccctaactc
     4321 cgcccagttc cgcccattct ccgccccatg gctgactaat tttttttatt tatgcagagg
     4381 ccgaggccgc ctctgcctct gagctattcc agaagtagtg aggaggcttt tttggaggcc
     4441 taggcttttg caaaaagctc ccgggagctt gtatatccat tttcggatct gatcaagaga
     4501 caggatgagg atcgtttcgc atgattgaac aagatggatt gcacgcaggt tctccggccg
     4561 cttgggtgga gaggctattc ggctatgact gggcacaaca gacaatcggc tgctctgatg
     4621 ccgccgtgtt ccggctgtca gcgcaggggc gcccggttct ttttgtcaag accgacctgt
     4681 ccggtgccct gaatgaactg caggacgagg cagcgcggct atcgtggctg gccacgacgg
     4741 gcgttccttg cgcagctgtg ctcgacgttg tcactgaagc gggaagggac tggctgctat
     4801 tgggcgaagt gccggggcag gatctcctgt catctcacct tgctcctgcc gagaaagtat
     4861 ccatcatggc tgatgcaatg cggcggctgc atacgcttga tccggctacc tgcccattcg
     4921 accaccaagc gaaacatcgc atcgagcgag cacgtactcg gatggaagcc ggtcttgtcg
     4981 atcaggatga tctggacgaa gagcatcagg ggctcgcgcc agccgaactg ttcgccaggc
     5041 tcaaggcgcg catgcccgac ggcgaggatc tcgtcgtgac ccatggcgat gcctgcttgc
     5101 cgaatatcat ggtggaaaat ggccgctttt ctggattcat cgactgtggc cggctgggtg
     5161 tggcggaccg ctatcaggac atagcgttgg ctacccgtga tattgctgaa gagcttggcg
     5221 gcgaatgggc tgaccgcttc ctcgtgcttt acggtatcgc cgctcccgat tcgcagcgca
     5281 tcgccttcta tcgccttctt gacgagttct tctgagcggg actctggggt tcgaaatgac
     5341 cgaccaagcg acgcccaacc tgccatcacg agatttcgat tccaccgccg ccttctatga
     5401 aaggttgggc ttcggaatcg ttttccggga cgccggctgg atgatcctcc agcgcgggga
     5461 tctcatgctg gagttcttcg cccaccccaa cttgtttatt gcagcttata atggttacaa
     5521 ataaagcaat agcatcacaa atttcacaaa taaagcattt ttttcactgc attctagttg
     5581 tggtttgtcc aaactcatca atgtatctta tcatgtctgt ataccgtcga cctctagcta
     5641 gagcttggcg taatcatggt catagctgtt tcctgtgtga aattgttatc cgctcacaat
     5701 tccacacaac atacgagccg gaagcataaa gtgtaaagcc tggggtgcct aatgagtgag
     5761 ctaactcaca ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg
     5821 ccagctgcat taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta ttgggcgctc
     5881 ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc
     5941 agctcactca aaggcggtaa tacggttatc cacagaatca ggggataacg caggaaagaa
     6001 catgtgagca aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt
     6061 tttccatagg ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg
     6121 gcgaaacccg acaggactat aaagatacca ggcgtttccc cctggaagct ccctcgtgcg
     6181 ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc cttcgggaag
     6241 cgtggcgctt tctcatagct cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc
     6301 caagctgggc tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct tatccggtaa
     6361 ctatcgtctt gagtccaacc cggtaagaca cgacttatcg ccactggcag cagccactgg
     6421 taacaggatt agcagagcga ggtatgtagg cggtgctaca gagttcttga agtggtggcc
     6481 taactacggc tacactagaa gaacagtatt tggtatctgc gctctgctga agccagttac
     6541 cttcggaaaa agagttggta gctcttgatc cggcaaacaa accaccgctg gtagcggtgg
     6601 tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag aagatccttt
     6661 gatcttttct acggggtctg acgctcagtg gaacgaaaac tcacgttaag ggattttggt
     6721 catgagatta tcaaaaagga tcttcaccta gatcctttta aattaaaaat gaagttttaa
     6781 atcaatctaa agtatatatg agtaaacttg gtctgacagt taccaatgct taatcagtga
     6841 ggcacctatc tcagcgatct gtctatttcg ttcatccata gttgcctgac tccccgtcgt
     6901 gtagataact acgatacggg agggcttacc atctggcccc agtgctgcaa tgataccgcg
     6961 agacccacgc tcaccggctc cagatttatc agcaataaac cagccagccg gaagggccga
     7021 gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag tctattaatt gttgccggga
     7081 agctagagta agtagttcgc cagttaatag tttgcgcaac gttgttgcca ttgctacagg
     7141 catcgtggtg tcacgctcgt cgtttggtat ggcttcattc agctccggtt cccaacgatc
     7201 aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc
     7261 gatcgttgtc agaagtaagt tggccgcagt gttatcactc atggttatgg cagcactgca
     7321 taattctctt actgtcatgc catccgtaag atgcttttct gtgactggtg agtactcaac
     7381 caagtcattc tgagaatagt gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg
     7441 ggataatacc gcgccacata gcagaacttt aaaagtgctc atcattggaa aacgttcttc
     7501 ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc agttcgatgt aacccactcg
     7561 tgcacccaac tgatcttcag catcttttac tttcaccagc gtttctgggt gagcaaaaac
     7621 aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt gaatactcat
     7681 actcttcctt tttcaatatt attgaagcat ttatcagggt tattgtctca tgagcggata
     7741 catatttgaa tgtatttaga aaaataaaca aataggggtt ccgcgcacat ttccccgaaa
     7801 agtgccacct gacgtc
//
