LOCUS       sequence-324482-        7299 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses N-terminally NanoLuc-Tagged PTHR1 in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7299)
  AUTHORS   Yu Z, Cary BP, Kim TW, Nguyen KD, Gardella TJ, Gellman SH
  TITLE     Kinetic and Thermodynamic Insights into Agonist Interactions with 
            the Parathyroid Hormone Receptor-1 from a New NanoBRET Assay.
  JOURNAL   ACS Chem Biol. 2022 Nov 18;17(11):3148-3158. doi: 
            10.1021/acschembio.2c00595. Epub 2022 Oct 25.
  PUBMED    36282520
REFERENCE   2  (bases 1 to 7299)
  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: 
            "10.1021/acschembio.2c00595"; journalName: "ACS Chem Biol"; date: 
            "2022-11-18- 18"; volume: "17"; issue: "11"; pages: "3148-3158"
COMMENT     Sequence Label: nLuc-PTHR1
FEATURES             Location/Qualifiers
     source          1..7299
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        1..380
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        381..584
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     535..555
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     629..648
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        629..647
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      665..674
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             737..1249
                     /codon_start=1
                     /product="NanoLuc(R) luciferase"
                     /label=Nluc
                     /note="human codon-optimized"
                     /translation="MVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQR
                     IVLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGV
                     TPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGW
                     RLCERILA"
     CDS             2988..3017
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     CDS             3033..3050
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(3073..3090)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3079..3303
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      3349..3777
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3436..3455)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3646..3667
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3786..3806)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3791..4120
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3971..4106
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     4033..4052
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     promoter        4168..4215
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             4234..4608
                     /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    4738..4859
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4775..4794)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4829..4848
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4908..4924)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4908..4924)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4921..4943)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4932..4948
                     /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(4956..4962,4963..4980,4981..4986))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     primer_bind     complement(5139..5156)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5310..5898)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5390..5409)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6069..6860,6861..6929))
                     /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     6692..6711
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6930..7034)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(7109..7128)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 gacattgatt attgactagt tattaatagt aatcaattac ggggtcatta gttcatagcc
       61 catatatgga gttccgcgtt acataactta cggtaaatgg cccgcctggc tgaccgccca
      121 acgacccccg cccattgacg tcaataatga cgtatgttcc catagtaacg ccaataggga
      181 ctttccattg acgtcaatgg gtggagtatt tacggtaaac tgcccacttg gcagtacatc
      241 aagtgtatca tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct
      301 ggcattatgc ccagtacatg accttatggg actttcctac ttggcagtac atctacgtat
      361 tagtcatcgc tattaccatg gtgatgcggt tttggcagta catcaatggg cgtggatagc
      421 ggtttgactc acggggattt ccaagtctcc accccattga cgtcaatggg agtttgtttt
      481 ggcaccaaaa tcaacgggac tttccaaaat gtcgtaacaa ctccgcccca ttgacgcaaa
      541 tgggcggtag gcgtgtacgg tgggaggtct atataagcag agctctctgg ctaactagag
      601 aacccactgc ttactggctt atcgaaatta atacgactca ctatagggag acccaagctg
      661 gctagccacc atggagacag acacactcct gctatgggta ctgctgctct gggttccagg
      721 ttccactggg gatggtatgg tcttcacact cgaagatttc gttggggact ggcgacagac
      781 agccggctac aacctggacc aagtccttga acagggaggt gtgtccagtt tgtttcagaa
      841 tctcggggtg tccgtaactc cgatccaaag gattgtcctg agcggtgaaa atgggctgaa
      901 gatcgacatc catgtcatca tcccgtatga aggtctgagc ggcgaccaaa tgggccagat
      961 cgaaaaaatt tttaaggtgg tgtaccctgt ggatgatcat cactttaagg tgatcctgca
     1021 ctatggcaca ctggtaatcg acggggttac gccgaacatg atcgactatt tcggacggcc
     1081 gtatgaaggc atcgccgtgt tcgacggcaa aaagatcact gtaacaggga ccctgtggaa
     1141 cggcaacaaa attatcgacg agcgcctgat caaccccgac ggctccctgc tgttccgagt
     1201 aaccatcaac ggagtgaccg gctggcggct gtgcgaacgc attctggcgg gtggttctgg
     1261 agatgcagat gacgtcatga ctaaagagga acagatcttc ctgctgcacc gtgctcaggc
     1321 ccagtgcgaa aaacggctca aggaggtcct gcagaggcca gccagcataa tggaatcaga
     1381 caagggatgg acatctgcgt ccacatcagg gaagcccagg aaagataagg catctgggaa
     1441 gctctaccct gagtctgagg aggacaagga ggcacccact ggcagcaggt accgagggcg
     1501 cccctgtctg ccggaatggg accacatcct gtgctggccg ctgggggcac caggtgaggt
     1561 ggtggctgtg ccctgtccgg actacattta tgacttcaat cacaaaggcc atgcctaccg
     1621 acgctgtgac cgcaatggca gctgggagct ggtgcctggg cacaacagga cgtgggccaa
     1681 ctacagcgag tgtgtcaaat ttctcaccaa tgagactcgt gaacgggagg tgtttgaccg
     1741 cctgggcatg atttacaccg tgggctactc cgtgtccctg gcgtccctca ccgtagctgt
     1801 gctcatcctg gcctacttta ggcggctgca ctgcacgcgc aactacatcc acatgcacct
     1861 gttcctgtcc ttcatgctgc gcgccgtgag catcttcgtc aaggacgctg tgctctactc
     1921 tggcgccacg cttgatgagg ctgagcgcct caccgaggag gagctgcgcg ccatcgccca
     1981 ggcgcccccg ccgcctgcca ccgccgctgc cggctacgcg ggctgcaggg tggctgtgac
     2041 cttcttcctt tacttcctgg ccaccaacta ctactggatt ctggtggagg ggctgtacct
     2101 gcacagcctc atcttcatgg ccttcttctc agagaagaag tacctgtggg gcttcacagt
     2161 cttcggctgg ggtctgcccg ctgtcttcgt ggctgtgtgg gtcagtgtca gagctaccct
     2221 ggccaacacc gggtgctggg acttgagctc cgggaacaaa aagtggatca tccaggtgcc
     2281 catcctggcc tccattgtgc tcaacttcat cctcttcatc aatatcgtcc gggtgctcgc
     2341 caccaagctg cgggagacca acgccggccg gtgtgacaca cggcagcagt accggaagct
     2401 gctcaaatcc acgctggtgc tcatgcccct ctttggcgtc cactacattg tcttcatggc
     2461 cacaccatac accgaggtct cagggacgct ctggcaagtc cagatgcact atgagatgct
     2521 cttcaactcc ttccagggat tttttgtcgc aatcatatac tgtttctgca acggcgaggt
     2581 acaagctgag atcaagaaat cttggagccg ctggacactg gcactggact tcaagcgaaa
     2641 ggcacgcagc gggagcagca gctatagcta cggccccatg gtgtcccaca caagtgtgac
     2701 caatgtcggc ccccgtgtgg gactcggcct gcccctcagc ccccgcctac tgcccactgc
     2761 caccaccaac ggccaccctc agctgcctgg ccatgccaag ccagggaccc cagccctgga
     2821 gaccctcgag accacaccac ctgccatggc tgctcccaag gacgatgggt tcctcaacgg
     2881 ctcctgctca ggcctggacg aggaggcctc tgggcctgag cggccacctg ccctgctaca
     2941 ggaagagtgg gagacagtca tgtgaagcgg ccgctcgagg agggcccgaa caaaaactca
     3001 tctcagaaga ggatctgaat agcgccgtcg accatcatca tcatcatcat tgagtttaaa
     3061 cccgctgatc agcctcgact gtgccttcta gttgccagcc atctgttgtt tgcccctccc
     3121 ccgtgccttc cttgaccctg gaaggtgcca ctcccactgt cctttcctaa taaaatgagg
     3181 aaattgcatc gcattgtctg agtaggtgtc attctattct ggggggtggg gtggggcagg
     3241 acagcaaggg ggaggattgg gaagacaata gcaggcatgc tggggatgcg gtgggctcta
     3301 tggcttctga ggcggaaaga accagctggg gctctagggg gtatccccac gcgccctgta
     3361 gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca
     3421 gcgccctagc gcccgctcct ttcgctttct tcccttcctt tctcgccacg ttcgccggct
     3481 ttccccgtca agctctaaat cgggggctcc ctttagggtt ccgatttagt gctttacggc
     3541 acctcgaccc caaaaaactt gattagggtg atggttcacg tagtgggcca tcgccctgat
     3601 agacggtttt tcgccctttg acgttggagt ccacgttctt taatagtgga ctcttgttcc
     3661 aaactggaac aacactcaac cctatctcgg tctattcttt tgatttataa gggattttgc
     3721 cgatttcggc ctattggtta aaaaatgagc tgatttaaca aaaatttaac gcgaattaat
     3781 tctgtggaat gtgtgtcagt tagggtgtgg aaagtcccca ggctccccag caggcagaag
     3841 tatgcaaagc atgcatctca attagtcagc aaccaggtgt ggaaagtccc caggctcccc
     3901 agcaggcaga agtatgcaaa gcatgcatct caattagtca gcaaccatag tcccgcccct
     3961 aactccgccc atcccgcccc taactccgcc cagttccgcc cattctccgc cccatggctg
     4021 actaattttt tttatttatg cagaggccga ggccgcctct gcctctgagc tattccagaa
     4081 gtagtgagga ggcttttttg gaggcctagg cttttgcaaa aagctcccgg gagcttgtat
     4141 atccattttc ggatctgatc agcacgtgtt gacaattaat catcggcata gtatatcggc
     4201 atagtataat acgacaaggt gaggaactaa accatggcca agttgaccag tgccgttccg
     4261 gtgctcaccg cgcgcgacgt cgccggagcg gtcgagttct ggaccgaccg gctcgggttc
     4321 tcccgggact tcgtggagga cgacttcgcc ggtgtggtcc gggacgacgt gaccctgttc
     4381 atcagcgcgg tccaggacca ggtggtgccg gacaacaccc tggcctgggt gtgggtgcgc
     4441 ggcctggacg agctgtacgc cgagtggtcg gaggtcgtgt ccacgaactt ccgggacgcc
     4501 tccgggccgg ccatgaccga gatcggcgag cagccgtggg ggcgggagtt cgccctgcgc
     4561 gacccggccg gcaactgcgt gcacttcgtg gccgaggagc aggactgaca cgtgctacga
     4621 gatttcgatt ccaccgccgc cttctatgaa aggttgggct tcggaatcgt tttccgggac
     4681 gccggctgga tgatcctcca gcgcggggat ctcatgctgg agttcttcgc ccaccccaac
     4741 ttgtttattg cagcttataa tggttacaaa taaagcaata gcatcacaaa tttcacaaat
     4801 aaagcatttt tttcactgca ttctagttgt ggtttgtcca aactcatcaa tgtatcttat
     4861 catgtctgta taccgtcgac ctctagctag agcttggcgt aatcatggtc atagctgttt
     4921 cctgtgtgaa attgttatcc gctcacaatt ccacacaaca tacgagccgg aagcataaag
     4981 tgtaaagcct ggggtgccta atgagtgagc taacttacat taattgcgtt gcgctcactg
     5041 cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg
     5101 gggagaggcg gtttgcgtat tgggcgctct tccgcttcct cgctcactga ctcgctgcgc
     5161 tcggtcgttc ggctgcggcg agcggtatca gctcactcaa aggcggtaat acggttatcc
     5221 acagaatcag gggataacgc aggaaagaac atgtgagcaa aaggccagca aaaggccagg
     5281 aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc tccgcccccc tgacgagcat
     5341 cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag
     5401 gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga
     5461 tacctgtccg cctttctccc ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg
     5521 tatctcagtt cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt
     5581 cagcccgacc gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac
     5641 gacttatcgc cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc
     5701 ggtgctacag agttcttgaa gtggtggcct aactacggct acactagaag aacagtattt
     5761 ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa gagttggtag ctcttgatcc
     5821 ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt gcaagcagca gattacgcgc
     5881 agaaaaaaag gatctcaaga agatcctttg atcttttcta cggggtctga cgctcagtgg
     5941 aacgaaaact cacgttaagg gattttggtc atgagattat caaaaaggat cttcacctag
     6001 atccttttaa attaaaaatg aagttttaaa tcaatctaaa gtatatatga gtaaacttgg
     6061 tctgacagtt accaatgctt aatcagtgag gcacctatct cagcgatctg tctatttcgt
     6121 tcatccatag ttgcctgact ccccgtcgtg tagataacta cgatacggga gggcttacca
     6181 tctggcccca gtgctgcaat gataccgcga gacccacgct caccggctcc agatttatca
     6241 gcaataaacc agccagccgg aagggccgag cgcagaagtg gtcctgcaac tttatccgcc
     6301 tccatccagt ctattaattg ttgccgggaa gctagagtaa gtagttcgcc agttaatagt
     6361 ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg
     6421 gcttcattca gctccggttc ccaacgatca aggcgagtta catgatcccc catgttgtgc
     6481 aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg
     6541 ttatcactca tggttatggc agcactgcat aattctctta ctgtcatgcc atccgtaaga
     6601 tgcttttctg tgactggtga gtactcaacc aagtcattct gagaatagtg tatgcggcga
     6661 ccgagttgct cttgcccggc gtcaatacgg gataataccg cgccacatag cagaacttta
     6721 aaagtgctca tcattggaaa acgttcttcg gggcgaaaac tctcaaggat cttaccgctg
     6781 ttgagatcca gttcgatgta acccactcgt gcacccaact gatcttcagc atcttttact
     6841 ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata
     6901 agggcgacac ggaaatgttg aatactcata ctcttccttt ttcaatatta ttgaagcatt
     6961 tatcagggtt attgtctcat gagcggatac atatttgaat gtatttagaa aaataaacaa
     7021 ataggggttc cgcgcacatt tccccgaaaa gtgccacctg acgtcgacgg atcgggagat
     7081 ctcccgatcc cctatggtgc actctcagta caatctgctc tgatgccgca tagttaagcc
     7141 agtatctgct ccctgcttgt gtgttggagg tcgctgagta gtgcgcgagc aaaatttaag
     7201 ctacaacaag gcaaggcttg accgacaatt gcatgaagaa tctgcttagg gttaggcgtt
     7261 ttgcgctgct tcgcgatgta cgggccagat atacgcgtt
//
