LOCUS       sequence-430803-        6860 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  The DuET construct encodes the named human GPCR engineered with a 
            cleaved hemagglutinin signal peptide, a 5' FLAG and a 3' 1D4 epitope
            tag optimized for expression in mammalian cells and proteomics 
            studies..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6860)
  AUTHORS   Dahl L, Kotliar IB, Bendes A, Dodig-Crnkovic T, Fromm S, Elofsson A,
            Uhlen M, Sakmar TP, Schwenk JM
  TITLE     Multiplexed selectivity screening of anti-GPCR antibodies.
  JOURNAL   Sci Adv. 2023 May 3;9(18):eadf9297. doi: 10.1126/sciadv.adf9297. 
            Epub 2023 May 3.
  PUBMED    37134173
REFERENCE   2  (bases 1 to 6860)
  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"; journalName: "Sci Adv."; 
            date: "2023-05-3"; volume: "9(18):eadf9297. doi"; pages: " 
            10.1126/sciadv.adf9297. Epub 2023 May 3"
COMMENT     Sequence Label: GLP1R-DuET
FEATURES             Location/Qualifiers
     source          1..6860
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     332..351
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(570..674)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(749..768)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        940..1319
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        1320..1523
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     1474..1494
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     1568..1587
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1568..1586
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(3156..3173)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3162..3386
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      3432..3860
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3519..3538)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3729..3750
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3869..3889)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3874..4203
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      4054..4189
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     4116..4135
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             4270..5064
                     /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(4324..4343)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4934..4953
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    5238..5359
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5275..5294)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5329..5348
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5408..5424)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5408..5424)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5421..5443)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5432..5448
                     /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(5456..5462,5463..5480,5481..5486))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5501..5522
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5639..5656)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5810..6398)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5890..5909)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6569..6860,1..500,501..569))
                     /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"
ORIGIN
        1 ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg
       61 gcttcattca gctccggttc ccaacgatca aggcgagtta catgatcccc catgttgtgc
      121 aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg
      181 ttatcactca tggttatggc agcactgcat aattctctta ctgtcatgcc atccgtaaga
      241 tgcttttctg tgactggtga gtactcaacc aagtcattct gagaatagtg tatgcggcga
      301 ccgagttgct cttgcccggc gtcaatacgg gataataccg cgccacatag cagaacttta
      361 aaagtgctca tcattggaaa acgttcttcg gggcgaaaac tctcaaggat cttaccgctg
      421 ttgagatcca gttcgatgta acccactcgt gcacccaact gatcttcagc atcttttact
      481 ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata
      541 agggcgacac ggaaatgttg aatactcata ctcttccttt ttcaatatta ttgaagcatt
      601 tatcagggtt attgtctcat gagcggatac atatttgaat gtatttagaa aaataaacaa
      661 ataggggttc cgcgcacatt tccccgaaaa gtgccacctg acgtcgacgg atcgggagat
      721 ctcccgatcc cctatggtgc actctcagta caatctgctc tgatgccgca tagttaagcc
      781 agtatctgct ccctgcttgt gtgttggagg tcgctgagta gtgcgcgagc aaaatttaag
      841 ctacaacaag gcaaggcttg accgacattt gcatgaagaa tctgcttagg gttaggcgtt
      901 ttgcgctgct tcgcgatgta cgggccagat atacgcgttg acattgatta ttgactagtt
      961 attaatagta atcaattacg gggtcattag ttcatagccc atatatggag ttccgcgtta
     1021 cataacttac ggtaaatggc ccgcctggct gaccgcccaa cgacccccgc ccattgacgt
     1081 caataatgac gtatgttccc atagtaacgc caatagggac tttccattga cgtcaatggg
     1141 tggagtattt acggtaaact gcccacttgg cagtacatca agtgtatcat atgccaagta
     1201 cgccccctat tgacgtcaat gacggtaaat ggcccgcctg gcattatgcc cagtacatga
     1261 ccttatggga ctttcctact tggcagtaca tctacgtatt agtcatcgct attaccatgg
     1321 tgatgcggtt ttggcagtac atcaatgggc gtggatagcg gtttgactca cggggatttc
     1381 caagtctcca ccccattgac gtcaatggga gtttgttttg gcaccaaaat caacgggact
     1441 ttccaaaatg tcgtaacaac tccgccccat tgacgcaaat gggcggtagg cgtgtacggt
     1501 gggaggtcta tataagcaga gctctctggc taactagaga acccactgct tactggctta
     1561 tcgaaattaa tacgactcac tatagggaga cccaagctgg ctagcgttta aacttaagct
     1621 tagcgccacc atgaagacga tcatcgccct gagctacatc ttctgcctgg tattcgccga
     1681 ctacaaggac gatgatgacg ccagcatcga tagacctcag ggcgctaccg tgagtctttg
     1741 ggagaccgtc cagaagtggc gggagtatcg ccgccagtgt caacgcagcc tgacagaaga
     1801 tcctcctcca gccaccgact tgttttgcaa taggacattt gatgagtatg cctgctggcc
     1861 agacggcgag cccggctcat tcgtgaatgt gtcatgtcca tggtacctgc catgggcatc
     1921 atctgtgcct caaggacacg tctacaggtt ctgcacagct gagggcctct ggctgcaaaa
     1981 agataacagt tcccttccat ggcgcgatct tagcgagtgc gaggaatcta agaggggcga
     2041 gcgcagttcc cccgaggaac agctgctgtt tctgtacatt atctacaccg tcggctacgc
     2101 gctgtccttt tccgctctgg ttatcgctag tgccatcctt ttggggtttc gccaccttca
     2161 ctgcacaaga aattacatcc acttgaatct ctttgcaagt tttattctgc gggccttgag
     2221 tgtgttcatt aaggacgctg ccctgaagtg gatgtactct accgcggccc aacagcacca
     2281 atgggatgga ctcctcagct atcaggattc tctttcctgt cgactcgtgt ttctgcttat
     2341 gcagtactgt gtggcagcca attattattg gctgctggtc gaaggagtat atctttacac
     2401 actgctcgca ttctccgtgc tgagtgaaca gtggattttc aggctttacg tgtccattgg
     2461 atggggtgtg cctctgctgt tcgtggtgcc ttggggcatc gtcaagtacc tgtatgaaga
     2521 tgaaggctgt tggaccagaa actctaacat gaactactgg ctcatcatac gcctgcccat
     2581 actcttcgcg attggagtca actttcttat atttgtgaga gtgatctgta tcgtcgtgtc
     2641 aaagctgaaa gctaacctta tgtgcaaaac cgacattaag tgcagactgg ctaagtccac
     2701 tctcaccctc atcccgctgc tcggaacaca tgaagtgata tttgccttcg tgatggacga
     2761 gcatgcgaga ggcacactgc ggtttatcaa actgttcaca gagctgtctt tcactagttt
     2821 ccaaggattg atggtggcta ttctctactg cttcgtgaac aatgaggtcc aacttgaatt
     2881 tcggaagtca tgggaacggt ggcggcttga acacctccat attcagcgcg atagttctat
     2941 gaagccactt aaatgtccca cttccagcct gagttccgga gccaccgccg gctcttcaat
     3001 gtatacagcc acttgtcagg cgtcctgctc tatcgatacc ggtggacgca ccggtgaatt
     3061 cgatgaggcc agcaccaccg tgtctaagac cgagacttct caggtggccc ctgcctaact
     3121 cgagtctaga gggcccgttt aaacccgctg atcagcctcg actgtgcctt ctagttgcca
     3181 gccatctgtt gtttgcccct cccccgtgcc ttccttgacc ctggaaggtg ccactcccac
     3241 tgtcctttcc taataaaatg aggaaattgc atcgcattgt ctgagtaggt gtcattctat
     3301 tctggggggt ggggtggggc aggacagcaa gggggaggat tgggaagaca atagcaggca
     3361 tgctggggat gcggtgggct ctatggcttc tgaggcggaa agaaccagct ggggctctag
     3421 ggggtatccc cacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg
     3481 cagcgtgacc gctacacttg ccagcgccct agcgcccgct cctttcgctt tcttcccttc
     3541 ctttctcgcc acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg
     3601 gttccgattt agtgctttac ggcacctcga ccccaaaaaa cttgattagg gtgatggttc
     3661 acgtagtggg ccatcgccct gatagacggt ttttcgccct ttgacgttgg agtccacgtt
     3721 ctttaatagt ggactcttgt tccaaactgg aacaacactc aaccctatct cggtctattc
     3781 ttttgattta taagggattt tgccgatttc ggcctattgg ttaaaaaatg agctgattta
     3841 acaaaaattt aacgcgaatt aattctgtgg aatgtgtgtc agttagggtg tggaaagtcc
     3901 ccaggctccc cagcaggcag aagtatgcaa agcatgcatc tcaattagtc agcaaccagg
     3961 tgtggaaagt ccccaggctc cccagcaggc agaagtatgc aaagcatgca tctcaattag
     4021 tcagcaacca tagtcccgcc cctaactccg cccatcccgc ccctaactcc gcccagttcc
     4081 gcccattctc cgccccatgg ctgactaatt ttttttattt atgcagaggc cgaggccgcc
     4141 tctgcctctg agctattcca gaagtagtga ggaggctttt ttggaggcct aggcttttgc
     4201 aaaaagctcc cgggagcttg tatatccatt ttcggatctg atcaagagac aggatgagga
     4261 tcgtttcgca tgattgaaca agatggattg cacgcaggtt ctccggccgc ttgggtggag
     4321 aggctattcg gctatgactg ggcacaacag acaatcggct gctctgatgc cgccgtgttc
     4381 cggctgtcag cgcaggggcg cccggttctt tttgtcaaga ccgacctgtc cggtgccctg
     4441 aatgaactgc aggacgaggc agcgcggcta tcgtggctgg ccacgacggg cgttccttgc
     4501 gcagctgtgc tcgacgttgt cactgaagcg ggaagggact ggctgctatt gggcgaagtg
     4561 ccggggcagg atctcctgtc atctcacctt gctcctgccg agaaagtatc catcatggct
     4621 gatgcaatgc ggcggctgca tacgcttgat ccggctacct gcccattcga ccaccaagcg
     4681 aaacatcgca tcgagcgagc acgtactcgg atggaagccg gtcttgtcga tcaggatgat
     4741 ctggacgaag agcatcaggg gctcgcgcca gccgaactgt tcgccaggct caaggcgcgc
     4801 atgcccgacg gcgaggatct cgtcgtgacc catggcgatg cctgcttgcc gaatatcatg
     4861 gtggaaaatg gccgcttttc tggattcatc gactgtggcc ggctgggtgt ggcggaccgc
     4921 tatcaggaca tagcgttggc tacccgtgat attgctgaag agcttggcgg cgaatgggct
     4981 gaccgcttcc tcgtgcttta cggtatcgcc gctcccgatt cgcagcgcat cgccttctat
     5041 cgccttcttg acgagttctt ctgagcggga ctctggggtt cgaaatgacc gaccaagcga
     5101 cgcccaacct gccatcacga gatttcgatt ccaccgccgc cttctatgaa aggttgggct
     5161 tcggaatcgt tttccgggac gccggctgga tgatcctcca gcgcggggat ctcatgctgg
     5221 agttcttcgc ccaccccaac ttgtttattg cagcttataa tggttacaaa taaagcaata
     5281 gcatcacaaa tttcacaaat aaagcatttt tttcactgca ttctagttgt ggtttgtcca
     5341 aactcatcaa tgtatcttat catgtctgta taccgtcgac ctctagctag agcttggcgt
     5401 aatcatggtc atagctgttt cctgtgtgaa attgttatcc gctcacaatt ccacacaaca
     5461 tacgagccgg aagcataaag tgtaaagcct ggggtgccta atgagtgagc taactcacat
     5521 taattgcgtt gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt
     5581 aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat tgggcgctct tccgcttcct
     5641 cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca gctcactcaa
     5701 aggcggtaat acggttatcc acagaatcag gggataacgc aggaaagaac atgtgagcaa
     5761 aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc
     5821 tccgcccccc tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga
     5881 caggactata aagataccag gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc
     5941 cgaccctgcc gcttaccgga tacctgtccg cctttctccc ttcgggaagc gtggcgcttt
     6001 ctcatagctc acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc aagctgggct
     6061 gtgtgcacga accccccgtt cagcccgacc gctgcgcctt atccggtaac tatcgtcttg
     6121 agtccaaccc ggtaagacac gacttatcgc cactggcagc agccactggt aacaggatta
     6181 gcagagcgag gtatgtaggc ggtgctacag agttcttgaa gtggtggcct aactacggct
     6241 acactagaag aacggtattt ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa
     6301 gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt
     6361 gcaagcagca gattacgcgc agaaaaaaag gatctcaaga agatcctttg atcttttcta
     6421 cggggtctga cgctcagtgg aacgaaaact cacgttaagg gattttggtc atgagattat
     6481 caaaaaggat cttcacctag atccttttaa attaaaaatg aagttttaaa tcaatctaaa
     6541 gtatatatga gtaaacttgg tctgacagtt accaatgctt aatcagtgag gcacctatct
     6601 cagcgatctg tctatttcgt tcatccatag ttgcctgact ccccgtcgtg tagataacta
     6661 cgatacggga gggcttacca tctggcccca gtgctgcaat gataccgcga gacccacgct
     6721 caccggctcc agatttatca gcaataaacc agccagccgg aagggccgag cgcagaagtg
     6781 gtcctgcaac tttatccgcc tccatccagt ctattaattg ttgccgggaa gctagagtaa
     6841 gtagttcgcc agttaatagt
//
