LOCUS       sequence-430787-        6551 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 6551)
  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 6551)
  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: CYSLTR1-DuET
FEATURES             Location/Qualifiers
     source          1..6551
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     551..570
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(789..893)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(968..987)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        1159..1538
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        1539..1742
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     1693..1713
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     1787..1806
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1787..1805
                     /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..6551,1..719,720..788))
                     /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 cccgtcgtgt agataactac gatacgggag ggcttaccat ctggccccag tgctgcaatg
       61 ataccgcgag acccacgctc accggctcca gatttatcag caataaacca gccagccgga
      121 agggccgagc gcagaagtgg tcctgcaact ttatccgcct ccatccagtc tattaattgt
      181 tgccgggaag ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt
      241 gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag ctccggttcc
      301 caacgatcaa ggcgagttac atgatccccc atgttgtgca aaaaagcggt tagctccttc
      361 ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt tatcactcat ggttatggca
      421 gcactgcata attctcttac tgtcatgcca tccgtaagat gcttttctgt gactggtgag
      481 tactcaacca agtcattctg agaatagtgt atgcggcgac cgagttgctc ttgcccggcg
      541 tcaatacggg ataataccgc gccacatagc agaactttaa aagtgctcat cattggaaaa
      601 cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa
      661 cccactcgtg cacccaactg atcttcagca tcttttactt tcaccagcgt ttctgggtga
      721 gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg gaaatgttga
      781 atactcatac tcttcctttt tcaatattat tgaagcattt atcagggtta ttgtctcatg
      841 agcggataca tatttgaatg tatttagaaa aataaacaaa taggggttcc gcgcacattt
      901 ccccgaaaag tgccacctga cgtcgacgga tcgggagatc tcccgatccc ctatggtgca
      961 ctctcagtac aatctgctct gatgccgcat agttaagcca gtatctgctc cctgcttgtg
     1021 tgttggaggt cgctgagtag tgcgcgagca aaatttaagc tacaacaagg caaggcttga
     1081 ccgacatttg catgaagaat ctgcttaggg ttaggcgttt tgcgctgctt cgcgatgtac
     1141 gggccagata tacgcgttga cattgattat tgactagtta ttaatagtaa tcaattacgg
     1201 ggtcattagt tcatagccca tatatggagt tccgcgttac ataacttacg gtaaatggcc
     1261 cgcctggctg accgcccaac gacccccgcc cattgacgtc aataatgacg tatgttccca
     1321 tagtaacgcc aatagggact ttccattgac gtcaatgggt ggagtattta cggtaaactg
     1381 cccacttggc agtacatcaa gtgtatcata tgccaagtac gccccctatt gacgtcaatg
     1441 acggtaaatg gcccgcctgg cattatgccc agtacatgac cttatgggac tttcctactt
     1501 ggcagtacat ctacgtatta gtcatcgcta ttaccatggt gatgcggttt tggcagtaca
     1561 tcaatgggcg tggatagcgg tttgactcac ggggatttcc aagtctccac cccattgacg
     1621 tcaatgggag tttgttttgg caccaaaatc aacgggactt tccaaaatgt cgtaacaact
     1681 ccgccccatt gacgcaaatg ggcggtaggc gtgtacggtg ggaggtctat ataagcagag
     1741 ctctctggct aactagagaa cccactgctt actggcttat cgaaattaat acgactcact
     1801 atagggagac ccaagctggc tagcgtttaa acttaagctt agcgccacca tgaagacgat
     1861 catcgccctg agctacatct tctgcctggt attcgccgac tacaaggacg atgatgacgc
     1921 cagcatcgat atggacgaaa cggggaatct cacagtgtca agcgccactt gccacgacac
     1981 aatcgacgat tttcggaatc aggtgtacag cactctgtac agtatgatca gtgtagtggg
     2041 tttttttggc aatgggttcg tactgtatgt actcatcaag acctaccata agaaatctgc
     2101 atttcaagtg tacatgatca acttggctgt ggctgacctg ctttgcgtgt gtaccctccc
     2161 cctccgggtg gtttattacg tccacaaagg tatctggctg ttcggagact ttctgtgtag
     2221 gctgtccaca tacgccctct acgtgaacct gtactgctca atttttttca tgactgccat
     2281 gtctttcttc aggtgcatcg ccatcgtatt tccagtccaa aacattaact tggtgacgca
     2341 gaaaaaagcg agattcgttt gcgttgggat ttggatcttc gtcattctga cttcctcccc
     2401 attcctgatg gctaagcccc aaaaggacga aaaaaacaac acaaaatgtt ttgagcctcc
     2461 tcaggacaac cagacgaaaa accacgtgct cgtgctccac tatgtgtccc tgtttgtcgg
     2521 gtttattatc cccttcgtga tcataattgt ctgttacact atgataatcc tgacactgtt
     2581 gaagaagagc atgaagaaaa acctgagctc ccataaaaaa gcaataggca tgatcatggt
     2641 tgtcactgca gcctttctcg tgagcttcat gccgtatcac atccaacgca ctattcacct
     2701 gcacttcctc cacaatgaga cgaagccctg cgattccgtg ctgagaatgc agaaaagcgt
     2761 agtaataacc ctgagcttgg cagctagcaa ttgctgcttc gatccattgc tctatttctt
     2821 ttctggtggg aattttagga agcggcttag caccttccga aagcactccc tgagtagcgt
     2881 cacgtacgtc cctagaaaga aagcatccct gcccgagaaa ggtgaggaaa tttgtaaggt
     2941 catcgatacc 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 c
//
