LOCUS       sequence-430881-        6680 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 6680)
  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 6680)
  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: OPRK1-DuET
FEATURES             Location/Qualifiers
     source          1..6680
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     422..441
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(660..764)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(839..858)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        1030..1409
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        1410..1613
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     1564..1584
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     1658..1677
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1658..1676
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(3066..3083)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3072..3296
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      3342..3770
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3429..3448)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3639..3660
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3779..3799)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3784..4113
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3964..4099
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     4026..4045
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             4180..4974
                     /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(4234..4253)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4844..4863
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    5148..5269
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5185..5204)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5239..5258
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5318..5334)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5318..5334)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5331..5353)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5342..5358
                     /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(5366..5372,5373..5390,5391..5396))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5411..5432
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5549..5566)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5720..6308)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5800..5819)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6479..6680,1..590,591..659))
                     /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 cgcagaagtg gtcctgcaac tttatccgcc tccatccagt ctattaattg ttgccgggaa
       61 gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat tgctacaggc
      121 atcgtggtgt cacgctcgtc gtttggtatg gcttcattca gctccggttc ccaacgatca
      181 aggcgagtta catgatcccc catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg
      241 atcgttgtca gaagtaagtt ggccgcagtg ttatcactca tggttatggc agcactgcat
      301 aattctctta ctgtcatgcc atccgtaaga tgcttttctg tgactggtga gtactcaacc
      361 aagtcattct gagaatagtg tatgcggcga ccgagttgct cttgcccggc gtcaatacgg
      421 gataataccg cgccacatag cagaacttta aaagtgctca tcattggaaa acgttcttcg
      481 gggcgaaaac tctcaaggat cttaccgctg ttgagatcca gttcgatgta acccactcgt
      541 gcacccaact gatcttcagc atcttttact ttcaccagcg tttctgggtg agcaaaaaca
      601 ggaaggcaaa atgccgcaaa aaagggaata agggcgacac ggaaatgttg aatactcata
      661 ctcttccttt ttcaatatta ttgaagcatt tatcagggtt attgtctcat gagcggatac
      721 atatttgaat gtatttagaa aaataaacaa ataggggttc cgcgcacatt tccccgaaaa
      781 gtgccacctg acgtcgacgg atcgggagat ctcccgatcc cctatggtgc actctcagta
      841 caatctgctc tgatgccgca tagttaagcc agtatctgct ccctgcttgt gtgttggagg
      901 tcgctgagta gtgcgcgagc aaaatttaag ctacaacaag gcaaggcttg accgacattt
      961 gcatgaagaa tctgcttagg gttaggcgtt ttgcgctgct tcgcgatgta cgggccagat
     1021 atacgcgttg acattgatta ttgactagtt attaatagta atcaattacg gggtcattag
     1081 ttcatagccc atatatggag ttccgcgtta cataacttac ggtaaatggc ccgcctggct
     1141 gaccgcccaa cgacccccgc ccattgacgt caataatgac gtatgttccc atagtaacgc
     1201 caatagggac tttccattga cgtcaatggg tggagtattt acggtaaact gcccacttgg
     1261 cagtacatca agtgtatcat atgccaagta cgccccctat tgacgtcaat gacggtaaat
     1321 ggcccgcctg gcattatgcc cagtacatga ccttatggga ctttcctact tggcagtaca
     1381 tctacgtatt agtcatcgct attaccatgg tgatgcggtt ttggcagtac atcaatgggc
     1441 gtggatagcg gtttgactca cggggatttc caagtctcca ccccattgac gtcaatggga
     1501 gtttgttttg gcaccaaaat caacgggact ttccaaaatg tcgtaacaac tccgccccat
     1561 tgacgcaaat gggcggtagg cgtgtacggt gggaggtcta tataagcaga gctctctggc
     1621 taactagaga acccactgct tactggctta tcgaaattaa tacgactcac tatagggaga
     1681 cccaagctgg ctagcgttta aacttaagct tagcgccacc atgaagacga tcatcgccct
     1741 gagctacatc ttctgcctgg tattcgccga ctacaaggac gatgatgacg ccagcatcga
     1801 tatggacagc cccattcaaa tcttccgggg cgagcccgga ccaacctgcg ctccaagcgc
     1861 ttgcctgcca ccgaattcta gtgcatggtt cccagggtgg gcagagcccg atagcaatgg
     1921 ctcagccggg tccgaggatg cgcagcttga acccgctcac atctcacccg ctatccctgt
     1981 cattattacg gcagtctact cagttgtttt cgttgtgggc ctggttggaa atagcctcgt
     2041 gatgtttgtg attatcaggt atactaaaat gaagaccgcc acgaatatct acatctttaa
     2101 cctggctctg gctgacgcct tggtgaccac cactatgcca tttcagtcca ccgtctatct
     2161 tatgaactct tggccattcg gcgacgttct gtgcaagatt gtcatttcca ttgactatta
     2221 taatatgttt acatcaatct ttacgcttac aatgatgagc gtggaccgat acattgctgt
     2281 ttgccatccg gtgaaagcac tggattttcg cacccccttg aaggccaaga ttatcaatat
     2341 ctgtatttgg ctcctgtcct catccgtcgg aatttccgcc attgtcttgg gtggtacaaa
     2401 ggtgagggag gacgtggacg ttatagagtg cagcctgcag ttccctgacg atgactacag
     2461 ctggtgggac ctttttatga agatctgtgt cttcattttt gctttcgtca tccccgtgct
     2521 gattatcata gtgtgctata cactgatgat attgcggctg aaatctgtgc gcctgctgag
     2581 cggcagccgc gaaaaggatc ggaatctgcg ccgaatcaca cgcctcgtgc tggttgtagt
     2641 cgctgtcttc gtggtatgct ggaccccaat ccacattttc atcctggtgg aagctctggg
     2701 atccacctct cactccactg ccgcacttag ctcatattac ttctgcattg ccttgggcta
     2761 cactaattcc tcactgaatc ccattctgta cgcatttctt gacgagaact tcaaacggtg
     2821 ctttagagac ttttgcttcc ccctcaagat gcgcatggag cggcagtcaa ccagccgagt
     2881 ccggaatacg gtccaggatc ccgcctatct gcgggacatc gacggcatga acaagcctgt
     2941 gatcgatacc ggtggacgca ccggtgaatt cgatgaggcc agcaccaccg tgtctaagac
     3001 cgagacttct caggtggccc ctgcctaact cgagtctaga gggcccgttt aaacccgctg
     3061 atcagcctcg actgtgcctt ctagttgcca gccatctgtt gtttgcccct cccccgtgcc
     3121 ttccttgacc ctggaaggtg ccactcccac tgtcctttcc taataaaatg aggaaattgc
     3181 atcgcattgt ctgagtaggt gtcattctat tctggggggt ggggtggggc aggacagcaa
     3241 gggggaggat tgggaagaca atagcaggca tgctggggat gcggtgggct ctatggcttc
     3301 tgaggcggaa agaaccagct ggggctctag ggggtatccc cacgcgccct gtagcggcgc
     3361 attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg ccagcgccct
     3421 agcgcccgct cctttcgctt tcttcccttc ctttctcgcc acgttcgccg gctttccccg
     3481 tcaagctcta aatcgggggc tccctttagg gttccgattt agtgctttac ggcacctcga
     3541 ccccaaaaaa cttgattagg gtgatggttc acgtagtggg ccatcgccct gatagacggt
     3601 ttttcgccct ttgacgttgg agtccacgtt ctttaatagt ggactcttgt tccaaactgg
     3661 aacaacactc aaccctatct cggtctattc ttttgattta taagggattt tgccgatttc
     3721 ggcctattgg ttaaaaaatg agctgattta acaaaaattt aacgcgaatt aattctgtgg
     3781 aatgtgtgtc agttagggtg tggaaagtcc ccaggctccc cagcaggcag aagtatgcaa
     3841 agcatgcatc tcaattagtc agcaaccagg tgtggaaagt ccccaggctc cccagcaggc
     3901 agaagtatgc aaagcatgca tctcaattag tcagcaacca tagtcccgcc cctaactccg
     3961 cccatcccgc ccctaactcc gcccagttcc gcccattctc cgccccatgg ctgactaatt
     4021 ttttttattt atgcagaggc cgaggccgcc tctgcctctg agctattcca gaagtagtga
     4081 ggaggctttt ttggaggcct aggcttttgc aaaaagctcc cgggagcttg tatatccatt
     4141 ttcggatctg atcaagagac aggatgagga tcgtttcgca tgattgaaca agatggattg
     4201 cacgcaggtt ctccggccgc ttgggtggag aggctattcg gctatgactg ggcacaacag
     4261 acaatcggct gctctgatgc cgccgtgttc cggctgtcag cgcaggggcg cccggttctt
     4321 tttgtcaaga ccgacctgtc cggtgccctg aatgaactgc aggacgaggc agcgcggcta
     4381 tcgtggctgg ccacgacggg cgttccttgc gcagctgtgc tcgacgttgt cactgaagcg
     4441 ggaagggact ggctgctatt gggcgaagtg ccggggcagg atctcctgtc atctcacctt
     4501 gctcctgccg agaaagtatc catcatggct gatgcaatgc ggcggctgca tacgcttgat
     4561 ccggctacct gcccattcga ccaccaagcg aaacatcgca tcgagcgagc acgtactcgg
     4621 atggaagccg gtcttgtcga tcaggatgat ctggacgaag agcatcaggg gctcgcgcca
     4681 gccgaactgt tcgccaggct caaggcgcgc atgcccgacg gcgaggatct cgtcgtgacc
     4741 catggcgatg cctgcttgcc gaatatcatg gtggaaaatg gccgcttttc tggattcatc
     4801 gactgtggcc ggctgggtgt ggcggaccgc tatcaggaca tagcgttggc tacccgtgat
     4861 attgctgaag agcttggcgg cgaatgggct gaccgcttcc tcgtgcttta cggtatcgcc
     4921 gctcccgatt cgcagcgcat cgccttctat cgccttcttg acgagttctt ctgagcggga
     4981 ctctggggtt cgaaatgacc gaccaagcga cgcccaacct gccatcacga gatttcgatt
     5041 ccaccgccgc cttctatgaa aggttgggct tcggaatcgt tttccgggac gccggctgga
     5101 tgatcctcca gcgcggggat ctcatgctgg agttcttcgc ccaccccaac ttgtttattg
     5161 cagcttataa tggttacaaa taaagcaata gcatcacaaa tttcacaaat aaagcatttt
     5221 tttcactgca ttctagttgt ggtttgtcca aactcatcaa tgtatcttat catgtctgta
     5281 taccgtcgac ctctagctag agcttggcgt aatcatggtc atagctgttt cctgtgtgaa
     5341 attgttatcc gctcacaatt ccacacaaca tacgagccgg aagcataaag tgtaaagcct
     5401 ggggtgccta atgagtgagc taactcacat taattgcgtt gcgctcactg cccgctttcc
     5461 agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg
     5521 gtttgcgtat tgggcgctct tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc
     5581 ggctgcggcg agcggtatca gctcactcaa aggcggtaat acggttatcc acagaatcag
     5641 gggataacgc aggaaagaac atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa
     5701 aggccgcgtt gctggcgttt ttccataggc tccgcccccc tgacgagcat cacaaaaatc
     5761 gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag gcgtttcccc
     5821 ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga tacctgtccg
     5881 cctttctccc ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg tatctcagtt
     5941 cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt cagcccgacc
     6001 gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac gacttatcgc
     6061 cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc ggtgctacag
     6121 agttcttgaa gtggtggcct aactacggct acactagaag aacggtattt ggtatctgcg
     6181 ctctgctgaa gccagttacc ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa
     6241 ccaccgctgg tagcggtggt ttttttgttt gcaagcagca gattacgcgc agaaaaaaag
     6301 gatctcaaga agatcctttg atcttttcta cggggtctga cgctcagtgg aacgaaaact
     6361 cacgttaagg gattttggtc atgagattat caaaaaggat cttcacctag atccttttaa
     6421 attaaaaatg aagttttaaa tcaatctaaa gtatatatga gtaaacttgg tctgacagtt
     6481 accaatgctt aatcagtgag gcacctatct cagcgatctg tctatttcgt tcatccatag
     6541 ttgcctgact ccccgtcgtg tagataacta cgatacggga gggcttacca tctggcccca
     6601 gtgctgcaat gataccgcga gacccacgct caccggctcc agatttatca gcaataaacc
     6661 agccagccgg aagggccgag
//
