LOCUS       sequence-430867-        6560 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 6560)
  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 6560)
  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: MCHR2-DuET
FEATURES             Location/Qualifiers
     source          1..6560
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     475..494
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(713..817)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(892..911)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        1083..1462
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        1463..1666
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     1617..1637
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     1711..1730
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1711..1729
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2999..3016)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3005..3229
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      3275..3703
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3362..3381)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3572..3593
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3712..3732)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3717..4046
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3897..4032
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3959..3978
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             4113..4907
                     /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(4167..4186)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4777..4796
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    5081..5202
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5118..5137)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5172..5191
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5251..5267)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5251..5267)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5264..5286)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5275..5291
                     /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(5299..5305,5306..5323,5324..5329))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5344..5365
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5482..5499)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5653..6241)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5733..5752)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6412..6560,1..643,644..712))
                     /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 gctcaccggc tccagattta tcagcaataa accagccagc cggaagggcc gagcgcagaa
       61 gtggtcctgc aactttatcc gcctccatcc agtctattaa ttgttgccgg gaagctagag
      121 taagtagttc gccagttaat agtttgcgca acgttgttgc cattgctaca ggcatcgtgg
      181 tgtcacgctc gtcgtttggt atggcttcat tcagctccgg ttcccaacga tcaaggcgag
      241 ttacatgatc ccccatgttg tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg
      301 tcagaagtaa gttggccgca gtgttatcac tcatggttat ggcagcactg cataattctc
      361 ttactgtcat gccatccgta agatgctttt ctgtgactgg tgagtactca accaagtcat
      421 tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata cgggataata
      481 ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa
      541 aactctcaag gatcttaccg ctgttgagat ccagttcgat gtaacccact cgtgcaccca
      601 actgatcttc agcatctttt actttcacca gcgtttctgg gtgagcaaaa acaggaaggc
      661 aaaatgccgc aaaaaaggga ataagggcga cacggaaatg ttgaatactc atactcttcc
      721 tttttcaata ttattgaagc atttatcagg gttattgtct catgagcgga tacatatttg
      781 aatgtattta gaaaaataaa caaatagggg ttccgcgcac atttccccga aaagtgccac
      841 ctgacgtcga cggatcggga gatctcccga tcccctatgg tgcactctca gtacaatctg
      901 ctctgatgcc gcatagttaa gccagtatct gctccctgct tgtgtgttgg aggtcgctga
      961 gtagtgcgcg agcaaaattt aagctacaac aaggcaaggc ttgaccgaca tttgcatgaa
     1021 gaatctgctt agggttaggc gttttgcgct gcttcgcgat gtacgggcca gatatacgcg
     1081 ttgacattga ttattgacta gttattaata gtaatcaatt acggggtcat tagttcatag
     1141 cccatatatg gagttccgcg ttacataact tacggtaaat ggcccgcctg gctgaccgcc
     1201 caacgacccc cgcccattga cgtcaataat gacgtatgtt cccatagtaa cgccaatagg
     1261 gactttccat tgacgtcaat gggtggagta tttacggtaa actgcccact tggcagtaca
     1321 tcaagtgtat catatgccaa gtacgccccc tattgacgtc aatgacggta aatggcccgc
     1381 ctggcattat gcccagtaca tgaccttatg ggactttcct acttggcagt acatctacgt
     1441 attagtcatc gctattacca tggtgatgcg gttttggcag tacatcaatg ggcgtggata
     1501 gcggtttgac tcacggggat ttccaagtct ccaccccatt gacgtcaatg ggagtttgtt
     1561 ttggcaccaa aatcaacggg actttccaaa atgtcgtaac aactccgccc cattgacgca
     1621 aatgggcggt aggcgtgtac ggtgggaggt ctatataagc agagctctct ggctaactag
     1681 agaacccact gcttactggc ttatcgaaat taatacgact cactataggg agacccaagc
     1741 tggctagcgt ttaaacttaa gcttagcgcc accatgaaga cgatcatcgc cctgagctac
     1801 atcttctgcc tggtattcgc cgactacaag gacgatgatg acgccagcat cgatatgaat
     1861 ccattccatg cgagctgctg gaacacctct gctgaacttc ttaataagtc ttggaacaaa
     1921 gagtttgctt accagactgc ttccgtcgtg gatacagtga tactccccag catgatcggt
     1981 atcatatgct ccactggact cgtgggcaat atcttgatcg tgtttaccat tatccggtca
     2041 cgcaaaaaga ccgtacccga tatctacatc tgcaatcttg ccgtggccga cctggtgcac
     2101 atagtcggta tgccattctt gatacaccag tgggcccgag gaggagaatg ggtattcggc
     2161 ggacccctgt gcacgattat cacctcattg gacacctgca atcagttcgc ttgcagcgct
     2221 atcatgaccg ttatgagcgt ggatcgctac ttcgcacttg tacagccgtt ccgcttgact
     2281 cgatggcgga cccggtacaa gacgattcgg attaaccttg gtctctgggc tgcctctttc
     2341 attttggccc tccccgtctg ggtatattcc aaagtgatca agttcaaaga tggagtggag
     2401 agctgcgcgt ttgatcttac ctcacccgat gacgttcttt ggtacacgtt gtacctcact
     2461 atcacaacgt tcttttttcc actgcccctg atcctggtat gttatatcct tatcctgtgc
     2521 tacacatggg aaatgtatca gcagaacaaa gatgcgcggt gttgcaatcc gagcgtccca
     2581 aaacagaggg tgatgaagct taccaagatg gtgctcgtgc tggttgtggt tttcatactg
     2641 agcgcggcgc cttaccatgt tatccaactg gtcaatcttc agatggagca gcccaccctc
     2701 gctttctatg tcggctacta tttgagtatt tgcctctctt atgcatcctc ttcaataaat
     2761 cctttcctgt acattcttct tagcggcaac ttccagaagc ggctccccca gatccagcgg
     2821 agggccacgg aaaaagaaat taacaacatg ggtaataccc tgaagtctca ctttatcgat
     2881 accggtggac gcaccggtga attcgatgag gccagcacca ccgtgtctaa gaccgagact
     2941 tctcaggtgg cccctgccta actcgagtct agagggcccg tttaaacccg ctgatcagcc
     3001 tcgactgtgc cttctagttg ccagccatct gttgtttgcc cctcccccgt gccttccttg
     3061 accctggaag gtgccactcc cactgtcctt tcctaataaa atgaggaaat tgcatcgcat
     3121 tgtctgagta ggtgtcattc tattctgggg ggtggggtgg ggcaggacag caagggggag
     3181 gattgggaag acaatagcag gcatgctggg gatgcggtgg gctctatggc ttctgaggcg
     3241 gaaagaacca gctggggctc tagggggtat ccccacgcgc cctgtagcgg cgcattaagc
     3301 gcggcgggtg tggtggttac gcgcagcgtg accgctacac ttgccagcgc cctagcgccc
     3361 gctcctttcg ctttcttccc ttcctttctc gccacgttcg ccggctttcc ccgtcaagct
     3421 ctaaatcggg ggctcccttt agggttccga tttagtgctt tacggcacct cgaccccaaa
     3481 aaacttgatt agggtgatgg ttcacgtagt gggccatcgc cctgatagac ggtttttcgc
     3541 cctttgacgt tggagtccac gttctttaat agtggactct tgttccaaac tggaacaaca
     3601 ctcaacccta tctcggtcta ttcttttgat ttataaggga ttttgccgat ttcggcctat
     3661 tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga attaattctg tggaatgtgt
     3721 gtcagttagg gtgtggaaag tccccaggct ccccagcagg cagaagtatg caaagcatgc
     3781 atctcaatta gtcagcaacc aggtgtggaa agtccccagg ctccccagca ggcagaagta
     3841 tgcaaagcat gcatctcaat tagtcagcaa ccatagtccc gcccctaact ccgcccatcc
     3901 cgcccctaac tccgcccagt tccgcccatt ctccgcccca tggctgacta atttttttta
     3961 tttatgcaga ggccgaggcc gcctctgcct ctgagctatt ccagaagtag tgaggaggct
     4021 tttttggagg cctaggcttt tgcaaaaagc tcccgggagc ttgtatatcc attttcggat
     4081 ctgatcaaga gacaggatga ggatcgtttc gcatgattga acaagatgga ttgcacgcag
     4141 gttctccggc cgcttgggtg gagaggctat tcggctatga ctgggcacaa cagacaatcg
     4201 gctgctctga tgccgccgtg ttccggctgt cagcgcaggg gcgcccggtt ctttttgtca
     4261 agaccgacct gtccggtgcc ctgaatgaac tgcaggacga ggcagcgcgg ctatcgtggc
     4321 tggccacgac gggcgttcct tgcgcagctg tgctcgacgt tgtcactgaa gcgggaaggg
     4381 actggctgct attgggcgaa gtgccggggc aggatctcct gtcatctcac cttgctcctg
     4441 ccgagaaagt atccatcatg gctgatgcaa tgcggcggct gcatacgctt gatccggcta
     4501 cctgcccatt cgaccaccaa gcgaaacatc gcatcgagcg agcacgtact cggatggaag
     4561 ccggtcttgt cgatcaggat gatctggacg aagagcatca ggggctcgcg ccagccgaac
     4621 tgttcgccag gctcaaggcg cgcatgcccg acggcgagga tctcgtcgtg acccatggcg
     4681 atgcctgctt gccgaatatc atggtggaaa atggccgctt ttctggattc atcgactgtg
     4741 gccggctggg tgtggcggac cgctatcagg acatagcgtt ggctacccgt gatattgctg
     4801 aagagcttgg cggcgaatgg gctgaccgct tcctcgtgct ttacggtatc gccgctcccg
     4861 attcgcagcg catcgccttc tatcgccttc ttgacgagtt cttctgagcg ggactctggg
     4921 gttcgaaatg accgaccaag cgacgcccaa cctgccatca cgagatttcg attccaccgc
     4981 cgccttctat gaaaggttgg gcttcggaat cgttttccgg gacgccggct ggatgatcct
     5041 ccagcgcggg gatctcatgc tggagttctt cgcccacccc aacttgttta ttgcagctta
     5101 taatggttac aaataaagca atagcatcac aaatttcaca aataaagcat ttttttcact
     5161 gcattctagt tgtggtttgt ccaaactcat caatgtatct tatcatgtct gtataccgtc
     5221 gacctctagc tagagcttgg cgtaatcatg gtcatagctg tttcctgtgt gaaattgtta
     5281 tccgctcaca attccacaca acatacgagc cggaagcata aagtgtaaag cctggggtgc
     5341 ctaatgagtg agctaactca cattaattgc gttgcgctca ctgcccgctt tccagtcggg
     5401 aaacctgtcg tgccagctgc attaatgaat cggccaacgc gcggggagag gcggtttgcg
     5461 tattgggcgc tcttccgctt cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg
     5521 gcgagcggta tcagctcact caaaggcggt aatacggtta tccacagaat caggggataa
     5581 cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc
     5641 gttgctggcg tttttccata ggctccgccc ccctgacgag catcacaaaa atcgacgctc
     5701 aagtcagagg tggcgaaacc cgacaggact ataaagatac caggcgtttc cccctggaag
     5761 ctccctcgtg cgctctcctg ttccgaccct gccgcttacc ggatacctgt ccgcctttct
     5821 cccttcggga agcgtggcgc tttctcatag ctcacgctgt aggtatctca gttcggtgta
     5881 ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc gttcagcccg accgctgcgc
     5941 cttatccggt aactatcgtc ttgagtccaa cccggtaaga cacgacttat cgccactggc
     6001 agcagccact ggtaacagga ttagcagagc gaggtatgta ggcggtgcta cagagttctt
     6061 gaagtggtgg cctaactacg gctacactag aagaacggta tttggtatct gcgctctgct
     6121 gaagccagtt accttcggaa aaagagttgg tagctcttga tccggcaaac aaaccaccgc
     6181 tggtagcggt ggtttttttg tttgcaagca gcagattacg cgcagaaaaa aaggatctca
     6241 agaagatcct ttgatctttt ctacggggtc tgacgctcag tggaacgaaa actcacgtta
     6301 agggattttg gtcatgagat tatcaaaaag gatcttcacc tagatccttt taaattaaaa
     6361 atgaagtttt aaatcaatct aaagtatata tgagtaaact tggtctgaca gttaccaatg
     6421 cttaatcagt gaggcaccta tctcagcgat ctgtctattt cgttcatcca tagttgcctg
     6481 actccccgtc gtgtagataa ctacgatacg ggagggctta ccatctggcc ccagtgctgc
     6541 aatgataccg cgagacccac
//
