LOCUS       sequence-251141-        6964 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses N-terminally NanoLuc-Tagged GLP-1R in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6964)
  AUTHORS   Cary BP, Deganutti G, Zhao P, Truong TT, Piper SJ, Liu X, Belousoff 
            MJ, Danev R, Sexton PM, Wootten D, Gellman SH
  TITLE     Structural and functional diversity among agonist-bound states of 
            the GLP-1 receptor.
  JOURNAL   Nat Chem Biol. 2022 Mar;18(3):256-263. doi: 
            10.1038/s41589-021-00945-w. Epub 2021 Dec 22.
  PUBMED    34937906
REFERENCE   2  (bases 1 to 6964)
  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: 
            "10.1038/s41589-021-00945-w"; journalName: "Nat Chem Biol"; date: 
            "2022-03"; volume: "18"; issue: "3"; pages: "256-263"
COMMENT     Sequence Label: nLuc-GLP-1R-His-FLAG
FEATURES             Location/Qualifiers
     source          1..6964
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(12..31)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        203..582
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        583..786
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     737..757
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     831..850
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        831..849
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      867..876
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             939..1451
                     /codon_start=1
                     /product="NanoLuc(R) luciferase"
                     /label=Nluc
                     /note="human codon-optimized"
                     /translation="MVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQR
                     IVLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGV
                     TPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGW
                     RLCERILA"
     CDS             2787..2804
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             2805..2828
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             2855..2884
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     CDS             2900..2917
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(2940..2957)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2946..3170
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      3216..3644
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3303..3322)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3513..3534
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3653..3673)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3658..3987
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3838..3973
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3900..3919
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     promoter        4035..4082
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             4101..4475
                     /codon_start=1
                     /gene="Sh ble from Streptoalloteichus hindustanus"
                     /product="antibiotic-binding protein"
                     /label=BleoR
                     /note="confers resistance to bleomycin, phleomycin, and 
                     Zeocin(TM)"
                     /translation="MAKLTSAVPVLTARDVAGAVEFWTDRLGFSRDFVEDDFAGVVRDD
                     VTLFISAVQDQVVPDNTLAWVWVRGLDELYAEWSEVVSTNFRDASGPAMTEIGEQPWGR
                     EFALRDPAGNCVHFVAEEQD"
     polyA_signal    4605..4726
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4642..4661)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4696..4715
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4775..4791)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4775..4791)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4788..4810)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4799..4815
                     /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(4823..4829,4830..4847,4848..4853))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     primer_bind     complement(5006..5023)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5177..5765)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5257..5276)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5936..6727,6728..6796))
                     /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     6559..6578
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6797..6901)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 tgcactctca gtacaatctg ctctgatgcc gcatagttaa gccagtatct gctccctgct
       61 tgtgtgttgg aggtcgctga gtagtgcgcg agcaaaattt aagctacaac aaggcaaggc
      121 ttgaccgaca attgcatgaa gaatctgctt agggttaggc gttttgcgct gcttcgcgat
      181 gtacgggcca gatatacgcg ttgacattga ttattgacta gttattaata gtaatcaatt
      241 acggggtcat tagttcatag cccatatatg gagttccgcg ttacataact tacggtaaat
      301 ggcccgcctg gctgaccgcc caacgacccc cgcccattga cgtcaataat gacgtatgtt
      361 cccatagtaa cgccaatagg gactttccat tgacgtcaat gggtggagta tttacggtaa
      421 actgcccact tggcagtaca tcaagtgtat catatgccaa gtacgccccc tattgacgtc
      481 aatgacggta aatggcccgc ctggcattat gcccagtaca tgaccttatg ggactttcct
      541 acttggcagt acatctacgt attagtcatc gctattacca tggtgatgcg gttttggcag
      601 tacatcaatg ggcgtggata gcggtttgac tcacggggat ttccaagtct ccaccccatt
      661 gacgtcaatg ggagtttgtt ttggcaccaa aatcaacggg actttccaaa atgtcgtaac
      721 aactccgccc cattgacgca aatgggcggt aggcgtgtac ggtgggaggt ctatataagc
      781 agagctctct ggctaactag agaacccact gcttactggc ttatcgaaat taatacgact
      841 cactataggg agacccaagc tggctagcca ccatggagac agacacactc ctgctatggg
      901 tactgctgct ctgggttcca ggttccactg gggatggtat ggtcttcaca ctcgaagatt
      961 tcgttgggga ctggcgacag acagccggct acaacctgga ccaagtcctt gaacagggag
     1021 gtgtgtccag tttgtttcag aatctcgggg tgtccgtaac tccgatccaa aggattgtcc
     1081 tgagcggtga aaatgggctg aagatcgaca tccatgtcat catcccgtat gaaggtctga
     1141 gcggcgacca aatgggccag atcgaaaaaa tttttaaggt ggtgtaccct gtggatgatc
     1201 atcactttaa ggtgatcctg cactatggca cactggtaat cgacggggtt acgccgaaca
     1261 tgatcgacta tttcggacgg ccgtatgaag gcatcgccgt gttcgacggc aaaaagatca
     1321 ctgtaacagg gaccctgtgg aacggcaaca aaattatcga cgagcgcctg atcaaccccg
     1381 acggctccct gctgttccga gtaaccatca acggagtgac cggctggcgg ctgtgcgaac
     1441 gtattctggc gggtggtcct cgccctcagg gcgctaccgt gtccctctgg gaaaccgtcc
     1501 agaagtggag ggagtaccgt agacaatgcc agcgtagctt gaccgaggac cctcctcccg
     1561 cgacagacct cttctgtaac aggacgttcg atgaatacgc atgctggcct gatggagagc
     1621 cgggctcatt cgttaacgtc agctgtccgt ggtatctgcc ttgggcttcc tctgttcccc
     1681 agggtcatgt ctaccgcttc tgtacggcag aaggtctgtg gctccaaaaa gacaacagtt
     1741 cgttgccttg gcgcgacctg tccgaatgcg aggaatcaaa aagaggcgaa cgctcgtctc
     1801 cagaggaaca actgctgttc ctttacatta tctacacggt aggatacgcc ctttctttct
     1861 cggcgctcgt aatcgcctcg gcgatcctgc ttggctttcg ccacctgcac tgcactagga
     1921 actacattca cctgaatttg ttcgcttcat tcatcttgcg tgccctctca gttttcatta
     1981 aggacgccgc tcttaagtgg atgtacagca cagctgctca acagcatcag tgggacggtc
     2041 tgttgtccta ccaggacagt ctgagttgtc gtctggtctt cctgctgatg cagtattgtg
     2101 tcgcagcaaa ctactattgg ctcttggttg agggtgtcta cctgtacact cttttggcct
     2161 tcagcgtgtt ttctgagcag tggatcttca gattgtacgt ttccatcggc tggggagtgc
     2221 cccttctctt tgttgtgcct tggggcatag ttaagtacct ttacgaggat gaaggttgct
     2281 ggacaagaaa ttctaatatg aactactggc tgattatccg tctgcctatt ctctttgcca
     2341 ttggtgtaaa ctttttgatc ttcgtaaggg taatctgcat agtcgtatct aagttgaagg
     2401 ctaacttgat gtgtaaaacc gatatcaagt gtcgcctggc caagtcaaca ctcactttga
     2461 tcccccttct gggtacgcac gaggtaatct ttgcgtttgt catggacgaa cacgcccgtg
     2521 gtactctccg cttcatcaag ttgtttaccg agttgtcctt taccagtttc caaggactca
     2581 tggtggccat tctgtactgc ttcgtcaata acgaggtcca gttggagttc cgcaagtcat
     2641 gggaacgctg gcgcctggaa cacctgcata tccagcgcga ctcctctatg aaaccgctga
     2701 agtgtccaac cagttcattg tcatccggag ctactgcggg atcttccatg tataccgcca
     2761 cttgtcaggc ttcatgttct ctcgagcatc atcaccacca ccacgactat aaggacgatg
     2821 atgacaagta atagcggccg ctcgaggagg gcccgaacaa aaactcatct cagaagagga
     2881 tctgaatagc gccgtcgacc atcatcatca tcatcattga gtttaaaccc gctgatcagc
     2941 ctcgactgtg ccttctagtt gccagccatc tgttgtttgc ccctcccccg tgccttcctt
     3001 gaccctggaa ggtgccactc ccactgtcct ttcctaataa aatgaggaaa ttgcatcgca
     3061 ttgtctgagt aggtgtcatt ctattctggg gggtggggtg gggcaggaca gcaaggggga
     3121 ggattgggaa gacaatagca ggcatgctgg ggatgcggtg ggctctatgg cttctgaggc
     3181 ggaaagaacc agctggggct ctagggggta tccccacgcg ccctgtagcg gcgcattaag
     3241 cgcggcgggt gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg ccctagcgcc
     3301 cgctcctttc gctttcttcc cttcctttct cgccacgttc gccggctttc cccgtcaagc
     3361 tctaaatcgg gggctccctt tagggttccg atttagtgct ttacggcacc tcgaccccaa
     3421 aaaacttgat tagggtgatg gttcacgtag tgggccatcg ccctgataga cggtttttcg
     3481 ccctttgacg ttggagtcca cgttctttaa tagtggactc ttgttccaaa ctggaacaac
     3541 actcaaccct atctcggtct attcttttga tttataaggg attttgccga tttcggccta
     3601 ttggttaaaa aatgagctga tttaacaaaa atttaacgcg aattaattct gtggaatgtg
     3661 tgtcagttag ggtgtggaaa gtccccaggc tccccagcag gcagaagtat gcaaagcatg
     3721 catctcaatt agtcagcaac caggtgtgga aagtccccag gctccccagc aggcagaagt
     3781 atgcaaagca tgcatctcaa ttagtcagca accatagtcc cgcccctaac tccgcccatc
     3841 ccgcccctaa ctccgcccag ttccgcccat tctccgcccc atggctgact aatttttttt
     3901 atttatgcag aggccgaggc cgcctctgcc tctgagctat tccagaagta gtgaggaggc
     3961 ttttttggag gcctaggctt ttgcaaaaag ctcccgggag cttgtatatc cattttcgga
     4021 tctgatcagc acgtgttgac aattaatcat cggcatagta tatcggcata gtataatacg
     4081 acaaggtgag gaactaaacc atggccaagt tgaccagtgc cgttccggtg ctcaccgcgc
     4141 gcgacgtcgc cggagcggtc gagttctgga ccgaccggct cgggttctcc cgggacttcg
     4201 tggaggacga cttcgccggt gtggtccggg acgacgtgac cctgttcatc agcgcggtcc
     4261 aggaccaggt ggtgccggac aacaccctgg cctgggtgtg ggtgcgcggc ctggacgagc
     4321 tgtacgccga gtggtcggag gtcgtgtcca cgaacttccg ggacgcctcc gggccggcca
     4381 tgaccgagat cggcgagcag ccgtgggggc gggagttcgc cctgcgcgac ccggccggca
     4441 actgcgtgca cttcgtggcc gaggagcagg actgacacgt gctacgagat ttcgattcca
     4501 ccgccgcctt ctatgaaagg ttgggcttcg gaatcgtttt ccgggacgcc ggctggatga
     4561 tcctccagcg cggggatctc atgctggagt tcttcgccca ccccaacttg tttattgcag
     4621 cttataatgg ttacaaataa agcaatagca tcacaaattt cacaaataaa gcattttttt
     4681 cactgcattc tagttgtggt ttgtccaaac tcatcaatgt atcttatcat gtctgtatac
     4741 cgtcgacctc tagctagagc ttggcgtaat catggtcata gctgtttcct gtgtgaaatt
     4801 gttatccgct cacaattcca cacaacatac gagccggaag cataaagtgt aaagcctggg
     4861 gtgcctaatg agtgagctaa cttacattaa ttgcgttgcg ctcactgccc gctttccagt
     4921 cgggaaacct gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg agaggcggtt
     4981 tgcgtattgg gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc
     5041 tgcggcgagc ggtatcagct cactcaaagg cggtaatacg gttatccaca gaatcagggg
     5101 ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg
     5161 ccgcgttgct ggcgtttttc cataggctcc gcccccctga cgagcatcac aaaaatcgac
     5221 gctcaagtca gaggtggcga aacccgacag gactataaag ataccaggcg tttccccctg
     5281 gaagctccct cgtgcgctct cctgttccga ccctgccgct taccggatac ctgtccgcct
     5341 ttctcccttc gggaagcgtg gcgctttctc atagctcacg ctgtaggtat ctcagttcgg
     5401 tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct
     5461 gcgccttatc cggtaactat cgtcttgagt ccaacccggt aagacacgac ttatcgccac
     5521 tggcagcagc cactggtaac aggattagca gagcgaggta tgtaggcggt gctacagagt
     5581 tcttgaagtg gtggcctaac tacggctaca ctagaagaac agtatttggt atctgcgctc
     5641 tgctgaagcc agttaccttc ggaaaaagag ttggtagctc ttgatccggc aaacaaacca
     5701 ccgctggtag cggtggtttt tttgtttgca agcagcagat tacgcgcaga aaaaaaggat
     5761 ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc tcagtggaac gaaaactcac
     5821 gttaagggat tttggtcatg agattatcaa aaaggatctt cacctagatc cttttaaatt
     5881 aaaaatgaag ttttaaatca atctaaagta tatatgagta aacttggtct gacagttacc
     5941 aatgcttaat cagtgaggca cctatctcag cgatctgtct atttcgttca tccatagttg
     6001 cctgactccc cgtcgtgtag ataactacga tacgggaggg cttaccatct ggccccagtg
     6061 ctgcaatgat accgcgagac ccacgctcac cggctccaga tttatcagca ataaaccagc
     6121 cagccggaag ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc atccagtcta
     6181 ttaattgttg ccgggaagct agagtaagta gttcgccagt taatagtttg cgcaacgttg
     6241 ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct tcattcagct
     6301 ccggttccca acgatcaagg cgagttacat gatcccccat gttgtgcaaa aaagcggtta
     6361 gctccttcgg tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg
     6421 ttatggcagc actgcataat tctcttactg tcatgccatc cgtaagatgc ttttctgtga
     6481 ctggtgagta ctcaaccaag tcattctgag aatagtgtat gcggcgaccg agttgctctt
     6541 gcccggcgtc aatacgggat aataccgcgc cacatagcag aactttaaaa gtgctcatca
     6601 ttggaaaacg ttcttcgggg cgaaaactct caaggatctt accgctgttg agatccagtt
     6661 cgatgtaacc cactcgtgca cccaactgat cttcagcatc ttttactttc accagcgttt
     6721 ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga
     6781 aatgttgaat actcatactc ttcctttttc aatattattg aagcatttat cagggttatt
     6841 gtctcatgag cggatacata tttgaatgta tttagaaaaa taaacaaata ggggttccgc
     6901 gcacatttcc ccgaaaagtg ccacctgacg tcgacggatc gggagatctc ccgatcccct
     6961 atgg
//
