LOCUS       sequence-431752-        6662 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 6662)
  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 6662)
  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: CCR2-DuET
FEATURES             Location/Qualifiers
     source          1..6662
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     603..622
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(841..945)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(1020..1039)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        1211..1590
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        1591..1794
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     1745..1765
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     1839..1858
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1839..1857
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(3229..3246)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3235..3459
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      3505..3933
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3592..3611)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3802..3823
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3942..3962)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3947..4276
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      4127..4262
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     4189..4208
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             4343..5137
                     /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(4397..4416)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     5007..5026
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    5311..5432
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5348..5367)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5402..5421
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5481..5497)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5481..5497)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5494..5516)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5505..5521
                     /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(5529..5535,5536..5553,5554..5559))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5574..5595
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5712..5729)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5883..6471)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5963..5982)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6642..6662,1..771,772..840))
                     /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 ggcacctatc tcagcgatct gtctatttcg ttcatccata gttgcctgac tccccgtcgt
       61 gtagataact acgatacggg agggcttacc atctggcccc agtgctgcaa tgataccgcg
      121 agacccacgc tcaccggctc cagatttatc agcaataaac cagccagccg gaagggccga
      181 gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag tctattaatt gttgccggga
      241 agctagagta agtagttcgc cagttaatag tttgcgcaac gttgttgcca ttgctacagg
      301 catcgtggtg tcacgctcgt cgtttggtat ggcttcattc agctccggtt cccaacgatc
      361 aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc
      421 gatcgttgtc agaagtaagt tggccgcagt gttatcactc atggttatgg cagcactgca
      481 taattctctt actgtcatgc catccgtaag atgcttttct gtgactggtg agtactcaac
      541 caagtcattc tgagaatagt gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg
      601 ggataatacc gcgccacata gcagaacttt aaaagtgctc atcattggaa aacgttcttc
      661 ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc agttcgatgt aacccactcg
      721 tgcacccaac tgatcttcag catcttttac tttcaccagc gtttctgggt gagcaaaaac
      781 aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt gaatactcat
      841 actcttcctt tttcaatatt attgaagcat ttatcagggt tattgtctca tgagcggata
      901 catatttgaa tgtatttaga aaaataaaca aataggggtt ccgcgcacat ttccccgaaa
      961 agtgccacct gacgtcgacg gatcgggaga tctcccgatc ccctatggtg cactctcagt
     1021 acaatctgct ctgatgccgc atagttaagc cagtatctgc tccctgcttg tgtgttggag
     1081 gtcgctgagt agtgcgcgag caaaatttaa gctacaacaa ggcaaggctt gaccgacatt
     1141 tgcatgaaga atctgcttag ggttaggcgt tttgcgctgc ttcgcgatgt acgggccaga
     1201 tatacgcgtt gacattgatt attgactagt tattaatagt aatcaattac ggggtcatta
     1261 gttcatagcc catatatgga gttccgcgtt acataactta cggtaaatgg cccgcctggc
     1321 tgaccgccca acgacccccg cccattgacg tcaataatga cgtatgttcc catagtaacg
     1381 ccaataggga ctttccattg acgtcaatgg gtggagtatt tacggtaaac tgcccacttg
     1441 gcagtacatc aagtgtatca tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa
     1501 tggcccgcct ggcattatgc ccagtacatg accttatggg actttcctac ttggcagtac
     1561 atctacgtat tagtcatcgc tattaccatg gtgatgcggt tttggcagta catcaatggg
     1621 cgtggatagc ggtttgactc acggggattt ccaagtctcc accccattga cgtcaatggg
     1681 agtttgtttt ggcaccaaaa tcaacgggac tttccaaaat gtcgtaacaa ctccgcccca
     1741 ttgacgcaaa tgggcggtag gcgtgtacgg tgggaggtct atataagcag agctctctgg
     1801 ctaactagag aacccactgc ttactggctt atcgaaatta atacgactca ctatagggag
     1861 acccaagctg gctagcgttt aaacttaagc ttagcgccac catgaagacg atcatcgccc
     1921 tgagctacat cttctgcctg gtattcgccg actacaagga cgatgatgac gccagcatcg
     1981 atatgttgtc caccagcagg agcagattca ttcgcaatac caatgaaagt ggcgaagagg
     2041 ttacaacctt ttttgattac gattacggtg cgccctgtca taagttcgac gtgaagcaga
     2101 tcggagcaca gctcctgccc cctctgtact ctctggtgtt tatcttcggg ttcgttggca
     2161 acatgcttgt tgtccttatc ttgatcaact gcaaaaagct gaagtgtctg actgatattt
     2221 acctcttgaa cctggcaatc tctgacttgc tctttctgat taccctcccc ctttgggccc
     2281 acagtgcggc caacgagtgg gtgtttggaa atgccatgtg taagctcttt acgggactgt
     2341 atcacatcgg gtactttgga gggatcttct ttatcatact gttgaccata gatagatacc
     2401 ttgccattgt ccacgctgtt ttcgcgctca aggccaggac cgtgaccttt ggcgtggtta
     2461 catctgtcat tacatggttg gtcgcggttt tcgcatccgt gccaggaata atttttacta
     2521 agtgtcaaaa ggaagactct gtgtacgttt gcggtcccta cttccccaga ggatggaata
     2581 atttccacac tattatgaga aacatactcg ggttggtgct ccctctgctt attatggtta
     2641 tttgctattc cggaatcctt aaaaccctcc tgcgctgcag aaatgagaag aagcgccaca
     2701 gagctgtacg cgtcatcttt acaatcatga ttgtgtattt tttgttctgg acaccttaca
     2761 acatcgtgat cctgttgaat acgtttcagg agttctttgg actctctaac tgtgaatcaa
     2821 cttcccagtt ggatcaggcc acgcaggtga ctgaaacact gggcatgacc cattgctgca
     2881 taaacccaat catttacgca ttcgttggtg agaagttccg cagccttttc catatcgccc
     2941 tgggttgccg catcgcgcct ttgcaaaagc ctgtgtgcgg gggccccggc gtgagaccgg
     3001 ggaagaacgt aaaagttacc acccaggggc tcctggacgg tcgcggtaaa ggcaaaagta
     3061 tcggacgcgc tcctgaagcc tccttgcagg acaaggaggg tgctatcgat accggtggaa
     3121 gcaccggtga attcgatgag gccagcacca ccgtgtctaa gaccgagact tctcaggtgg
     3181 cccctgccta actcgagtct agagggcccg tttaaacccg ctgatcagcc tcgactgtgc
     3241 cttctagttg ccagccatct gttgtttgcc cctcccccgt gccttccttg accctggaag
     3301 gtgccactcc cactgtcctt tcctaataaa atgaggaaat tgcatcgcat tgtctgagta
     3361 ggtgtcattc tattctgggg ggtggggtgg ggcaggacag caagggggag gattgggaag
     3421 acaatagcag gcatgctggg gatgcggtgg gctctatggc ttctgaggcg gaaagaacca
     3481 gctggggctc tagggggtat ccccacgcgc cctgtagcgg cgcattaagc gcggcgggtg
     3541 tggtggttac gcgcagcgtg accgctacac ttgccagcgc cctagcgccc gctcctttcg
     3601 ctttcttccc ttcctttctc gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg
     3661 ggctcccttt agggttccga tttagtgctt tacggcacct cgaccccaaa aaacttgatt
     3721 agggtgatgg ttcacgtagt gggccatcgc cctgatagac ggtttttcgc cctttgacgt
     3781 tggagtccac gttctttaat agtggactct tgttccaaac tggaacaaca ctcaacccta
     3841 tctcggtcta ttcttttgat ttataaggga ttttgccgat ttcggcctat tggttaaaaa
     3901 atgagctgat ttaacaaaaa tttaacgcga attaattctg tggaatgtgt gtcagttagg
     3961 gtgtggaaag tccccaggct ccccagcagg cagaagtatg caaagcatgc atctcaatta
     4021 gtcagcaacc aggtgtggaa agtccccagg ctccccagca ggcagaagta tgcaaagcat
     4081 gcatctcaat tagtcagcaa ccatagtccc gcccctaact ccgcccatcc cgcccctaac
     4141 tccgcccagt tccgcccatt ctccgcccca tggctgacta atttttttta tttatgcaga
     4201 ggccgaggcc gcctctgcct ctgagctatt ccagaagtag tgaggaggct tttttggagg
     4261 cctaggcttt tgcaaaaagc tcccgggagc ttgtatatcc attttcggat ctgatcaaga
     4321 gacaggatga ggatcgtttc gcatgattga acaagatgga ttgcacgcag gttctccggc
     4381 cgcttgggtg gagaggctat tcggctatga ctgggcacaa cagacaatcg gctgctctga
     4441 tgccgccgtg ttccggctgt cagcgcaggg gcgcccggtt ctttttgtca agaccgacct
     4501 gtccggtgcc ctgaatgaac tgcaggacga ggcagcgcgg ctatcgtggc tggccacgac
     4561 gggcgttcct tgcgcagctg tgctcgacgt tgtcactgaa gcgggaaggg actggctgct
     4621 attgggcgaa gtgccggggc aggatctcct gtcatctcac cttgctcctg ccgagaaagt
     4681 atccatcatg gctgatgcaa tgcggcggct gcatacgctt gatccggcta cctgcccatt
     4741 cgaccaccaa gcgaaacatc gcatcgagcg agcacgtact cggatggaag ccggtcttgt
     4801 cgatcaggat gatctggacg aagagcatca ggggctcgcg ccagccgaac tgttcgccag
     4861 gctcaaggcg cgcatgcccg acggcgagga tctcgtcgtg acccatggcg atgcctgctt
     4921 gccgaatatc atggtggaaa atggccgctt ttctggattc atcgactgtg gccggctggg
     4981 tgtggcggac cgctatcagg acatagcgtt ggctacccgt gatattgctg aagagcttgg
     5041 cggcgaatgg gctgaccgct tcctcgtgct ttacggtatc gccgctcccg attcgcagcg
     5101 catcgccttc tatcgccttc ttgacgagtt cttctgagcg ggactctggg gttcgaaatg
     5161 accgaccaag cgacgcccaa cctgccatca cgagatttcg attccaccgc cgccttctat
     5221 gaaaggttgg gcttcggaat cgttttccgg gacgccggct ggatgatcct ccagcgcggg
     5281 gatctcatgc tggagttctt cgcccacccc aacttgttta ttgcagctta taatggttac
     5341 aaataaagca atagcatcac aaatttcaca aataaagcat ttttttcact gcattctagt
     5401 tgtggtttgt ccaaactcat caatgtatct tatcatgtct gtataccgtc gacctctagc
     5461 tagagcttgg cgtaatcatg gtcatagctg tttcctgtgt gaaattgtta tccgctcaca
     5521 attccacaca acatacgagc cggaagcata aagtgtaaag cctggggtgc ctaatgagtg
     5581 agctaactca cattaattgc gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg
     5641 tgccagctgc attaatgaat cggccaacgc gcggggagag gcggtttgcg tattgggcgc
     5701 tcttccgctt cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta
     5761 tcagctcact caaaggcggt aatacggtta tccacagaat caggggataa cgcaggaaag
     5821 aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg
     5881 tttttccata ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg
     5941 tggcgaaacc cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg
     6001 cgctctcctg ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga
     6061 agcgtggcgc tttctcatag ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc
     6121 tccaagctgg gctgtgtgca cgaacccccc gttcagcccg accgctgcgc cttatccggt
     6181 aactatcgtc ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact
     6241 ggtaacagga ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg
     6301 cctaactacg gctacactag aagaacggta tttggtatct gcgctctgct gaagccagtt
     6361 accttcggaa aaagagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt
     6421 ggtttttttg tttgcaagca gcagattacg cgcagaaaaa aaggatctca agaagatcct
     6481 ttgatctttt ctacggggtc tgacgctcag tggaacgaaa actcacgtta agggattttg
     6541 gtcatgagat tatcaaaaag gatcttcacc tagatccttt taaattaaaa atgaagtttt
     6601 aaatcaatct aaagtatata tgagtaaact tggtctgaca gttaccaatg cttaatcagt
     6661 ga
//
