LOCUS       sequence-430758-        7064 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 7064)
  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 7064)
  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: CALCRb-DuET
FEATURES             Location/Qualifiers
     source          1..7064
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      438..1026
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     927..946
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     1180..1197
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    1314..1335
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(1350..1355,1356..1373,1374..1380)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1388..1404
                     /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)."
     primer_bind     1393..1415
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     1412..1428
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     1412..1428
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     polyA_signal    1477..1598
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1488..1507)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     1542..1561
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     CDS             complement(1772..2566)
                     /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(1883..1902)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     2493..2512
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     promoter        complement(2633..2962)
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      2647..2782
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(2701..2720)
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     primer_bind     2947..2967
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      complement(2976..3404)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3086..3107)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3298..3317
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     polyA_signal    3450..3674
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     3663..3680
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     primer_bind     complement(5453..5472)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(5454..5472)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        complement(5517..5720)
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     complement(5546..5566)
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     enhancer        5721..6100
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     primer_bind     6272..6291
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        6366..6470
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(6471..6539,6540..7064,1..267)
                     /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"
     primer_bind     complement(6689..6708)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 ctagcttccc ggcaacaatt aatagactgg atggaggcgg ataaagttgc aggaccactt
       61 ctgcgctcgg cccttccggc tggctggttt attgctgata aatctggagc cggtgagcgt
      121 gggtctcgcg gtatcattgc agcactgggg ccagatggta agccctcccg tatcgtagtt
      181 atctacacga cggggagtca ggcaactatg gatgaacgaa atagacagat cgctgagata
      241 ggtgcctcac tgattaagca ttggtaactg tcagaccaag tttactcata tatactttag
      301 attgatttaa aacttcattt ttaatttaaa aggatctagg tgaagatcct ttttgataat
      361 ctcatgacca aaatccctta acgtgagttt tcgttccact gagcgtcaga ccccgtagaa
      421 aagatcaaag gatcttcttg agatcctttt tttctgcgcg taatctgctg cttgcaaaca
      481 aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc aagagctacc aactcttttt
      541 ccgaaggtaa ctggcttcag cagagcgcag ataccaaata ccgttcttct agtgtagccg
      601 tagttaggcc accacttcaa gaactctgta gcaccgccta catacctcgc tctgctaatc
      661 ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga
      721 cgatagttac cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc
      781 agcttggagc gaacgaccta caccgaactg agatacctac agcgtgagct atgagaaagc
      841 gccacgcttc ccgaagggag aaaggcggac aggtatccgg taagcggcag ggtcggaaca
      901 ggagagcgca cgagggagct tccaggggga aacgcctggt atctttatag tcctgtcggg
      961 tttcgccacc tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta
     1021 tggaaaaacg ccagcaacgc ggccttttta cggttcctgg ccttttgctg gccttttgct
     1081 cacatgttct ttcctgcgtt atcccctgat tctgtggata accgtattac cgcctttgag
     1141 tgagctgata ccgctcgccg cagccgaacg accgagcgca gcgagtcagt gagcgaggaa
     1201 gcggaagagc gcccaatacg caaaccgcct ctccccgcgc gttggccgat tcattaatgc
     1261 agctggcacg acaggtttcc cgactggaaa gcgggcagtg agcgcaacgc aattaatgtg
     1321 agttagctca ctcattaggc accccaggct ttacacttta tgcttccggc tcgtatgttg
     1381 tgtggaattg tgagcggata acaatttcac acaggaaaca gctatgacca tgattacgcc
     1441 aagctctagc tagaggtcga cggtatacag acatgataag atacattgat gagtttggac
     1501 aaaccacaac tagaatgcag tgaaaaaaat gctttatttg tgaaatttgt gatgctattg
     1561 ctttatttgt aaccattata agctgcaata aacaagttgg ggtgggcgaa gaactccagc
     1621 atgagatccc cgcgctggag gatcatccag ccggcgtccc ggaaaacgat tccgaagccc
     1681 aacctttcat agaaggcggc ggtggaatcg aaatctcgtg atggcaggtt gggcgtcgct
     1741 tggtcggtca tttcgaaccc cagagtcccg ctcagaagaa ctcgtcaaga aggcgataga
     1801 aggcgatgcg ctgcgaatcg ggagcggcga taccgtaaag cacgaggaag cggtcagccc
     1861 attcgccgcc aagctcttca gcaatatcac gggtagccaa cgctatgtcc tgatagcggt
     1921 ccgccacacc cagccggcca cagtcgatga atccagaaaa gcggccattt tccaccatga
     1981 tattcggcaa gcaggcatcg ccatgggtca cgacgagatc ctcgccgtcg ggcatgcgcg
     2041 ccttgagcct ggcgaacagt tcggctggcg cgagcccctg atgctcttcg tccagatcat
     2101 cctgatcgac aagaccggct tccatccgag tacgtgctcg ctcgatgcga tgtttcgctt
     2161 ggtggtcgaa tgggcaggta gccggatcaa gcgtatgcag ccgccgcatt gcatcagcca
     2221 tgatggatac tttctcggca ggagcaaggt gagatgacag gagatcctgc cccggcactt
     2281 cgcccaatag cagccagtcc cttcccgctt cagtgacaac gtcgagcaca gctgcgcaag
     2341 gaacgcccgt cgtggccagc cacgatagcc gcgctgcctc gtcctgcagt tcattcaggg
     2401 caccggacag gtcggtcttg acaaaaagaa ccgggcgccc ctgcgctgac agccggaaca
     2461 cggcggcatc agagcagccg attgtctgtt gtgcccagtc atagccgaat agcctctcca
     2521 cccaagcggc cggagaacct gcgtgcaatc catcttgttc aatcatgcga aacgatcctc
     2581 atcctgtctc ttgatcagat ccgaaaatgg atatacaagc tcccgggagc tttttgcaaa
     2641 agcctaggcc tccaaaaaag cctcctcact acttctggaa tagctcagag gcagaggcgg
     2701 cctcggcctc tgcataaata aaaaaaatta gtcagccatg gggcggagaa tgggcggaac
     2761 tgggcggagt taggggcggg atgggcggag ttaggggcgg gactatggtt gctgactaat
     2821 tgagatgcat gctttgcata cttctgcctg ctggggagcc tggggacttt ccacacctgg
     2881 ttgctgacta attgagatgc atgctttgca tacttctgcc tgctggggag cctggggact
     2941 ttccacaccc taactgacac acattccaca gaattaattc gcgttaaatt tttgttaaat
     3001 cagctcattt tttaaccaat aggccgaaat cggcaaaatc ccttataaat caaaagaata
     3061 gaccgagata gggttgagtg ttgttccagt ttggaacaag agtccactat taaagaacgt
     3121 ggactccaac gtcaaagggc gaaaaaccgt ctatcagggc gatggcccac tacgtgaacc
     3181 atcaccctaa tcaagttttt tggggtcgag gtgccgtaaa gcactaaatc ggaaccctaa
     3241 agggagcccc cgatttagag cttgacgggg aaagccggcg aacgtggcga gaaaggaagg
     3301 gaagaaagcg aaaggagcgg gcgctagggc gctggcaagt gtagcggtca cgctgcgcgt
     3361 aaccaccaca cccgccgcgc ttaatgcgcc gctacagggc gcgtggggat accccctaga
     3421 gccccagctg gttctttccg cctcagaagc catagagccc accgcatccc cagcatgcct
     3481 gctattgtct tcccaatcct cccccttgct gtcctgcccc accccacccc ccagaataga
     3541 atgacaccta ctcagacaat gcgatgcaat ttcctcattt tattaggaaa ggacagtggg
     3601 agtggcacct tccagggtca aggaaggcac gggggagggg caaacaacag atggctggca
     3661 actagaaggc acagtcgagg ctgatcagcg ggtttaaacg ggccctctag actcgagtta
     3721 ggcaggggcc acctgagaag tctcggtctt agacacggtg gtgctggcct catcgaattc
     3781 accggtgcgt ccaccggtat cgatggcgga agattcctgc tcaatgatat tcagggggat
     3841 aatctcggca gactcttcgc cttgattgtt agcgggctcg ttcctcagtt cttgatggca
     3901 gatatatatg gggatgtcac cggcctccgc tgccgcggct gcggctctag ctgacctgtt
     3961 ggatggccgt ctgccccagc gttggttcca ctggatctta aattgagccc actgccgctt
     4021 aactgtggtt tgcacttcat tattacaaaa acaataaatg gttgctacaa aaaacccttg
     4081 aaaatggatc aggctatgca tcacgtaatc ataaatcttg cccagcatct tgttgctagg
     4141 ccgccacgga aacaccacaa attggatccc aaggagagga accaggatca tcgtagcctt
     4201 aacagccttc agatacatgt gggactccgc ttcatgagtc tctctcatct ttgtcacaag
     4261 cactctcacg atattaagga ggaaaaagaa gttcaccacc agagctgcca tcacggggcc
     4321 gtggattatg tacagcaaat gggtttccac agacagccaa cagttgtcat tgaaatatac
     4381 agccctggta atggcgtgaa ttgtggttgg caccagagga aagccccatc cgagcaggta
     4441 gtaccatcgg aggcgctgct tttcggtgaa cactgccaca acgataagag tgtgcaggta
     4501 gatcccttcg cacagcatcc agaagtaatt gcatgccatc atatactggt gaaagaaatg
     4561 cagaatcttg caggacaccg gatccctgcg caccagttca ccatttggca cgacttccac
     4621 cagatgaata ataatgatca tggagttaag gatataggta aggaacatat tcttgtgaag
     4681 ggttactctc tgacacccca ggctcagggc ttttcgatac ttccagttca gtggaaatat
     4741 agtggtaagt ttcctgaaga agacgaatat gcccaagcta atcacgagtg tgaaaatact
     4801 cagagaatgt cccacaatag ccagataata aaggacgtaa gcgtttttaa gcttttcggg
     4861 ggtaaacgcg ttgcacatgg tatagtttga ccacgtccgg ttattctccg ggtgtttaaa
     4921 ccagacgcct ttttcgtcgc aatatttggt cactttctcg cttgggtcaa agtccgggaa
     4981 ataatcaggg cagaattggt aggaaaggac accagcggga gtatcgtccc agcacagcca
     5041 cccgtcccag gttctgttac aatatgggcc ctctccctgg tacgccggca gttgttgcat
     5101 cctatcatag cacttgtact gagcgtccat catctttttt cgtcccacaa catacaaaaa
     5161 gggttttggt tcgatggtag ggtatgtctg atttgaaaag gcaggcagga tgggtgtggg
     5221 atgattaaga aggaggaaca gtgcgagaca tctggatgta aatgtaaagc gcattttgga
     5281 tttctgcaag tctcgctgtc ctgaaatttt ttcgcctgag aactgcatat cgatgctggc
     5341 gtcatcatcg tccttgtagt cggcgaatac caggcagaag atgtagctca gggcgatgat
     5401 cgtcttcatg gtggcgctaa gcttaagttt aaacgctagc cagcttgggt ctccctatag
     5461 tgagtcgtat taatttcgat aagccagtaa gcagtgggtt ctctagttag ccagagagct
     5521 ctgcttatat agacctccca ccgtacacgc ctaccgccca tttgcgtcaa tggggcggag
     5581 ttgttacgac attttggaaa gtcccgttga ttttggtgcc aaaacaaact cccattgacg
     5641 tcaatggggt ggagacttgg aaatccccgt gagtcaaacc gctatccacg cccattgatg
     5701 tactgccaaa accgcatcac catggtaata gcgatgacta atacgtagat gtactgccaa
     5761 gtaggaaagt cccataaggt catgtactgg gcataatgcc aggcgggcca tttaccgtca
     5821 ttgacgtcaa tagggggcgt acttggcata tgatacactt gatgtactgc caagtgggca
     5881 gtttaccgta aatactccac ccattgacgt caatggaaag tccctattgg cgttactatg
     5941 ggaacatacg tcattattga cgtcaatggg cgggggtcgt tgggcggtca gccaggcggg
     6001 ccatttaccg taagttatgt aacgcggaac tccatatatg ggctatgaac taatgacccc
     6061 gtaattgatt actattaata actagtcaat aatcaatgtc aacgcgtata tctggcccgt
     6121 acatcgcgaa gcagcgcaaa acgcctaacc ctaagcagat tcttcatgca aatgtcggtc
     6181 aagccttgcc ttgttgtagc ttaaattttg ctcgcgcact actcagcgac ctccaacaca
     6241 caagcaggga gcagatactg gcttaactat gcggcatcag agcagattgt actgagagtg
     6301 caccataggg gatcgggaga tctcccgatc cgtcgacgtc aggtggcact tttcggggaa
     6361 atgtgcgcgg aacccctatt tgtttatttt tctaaataca ttcaaatatg tatccgctca
     6421 tgagacaata accctgataa atgcttcaat aatattgaaa aaggaagagt atgagtattc
     6481 aacatttccg tgtcgccctt attccctttt ttgcggcatt ttgccttcct gtttttgctc
     6541 acccagaaac gctggtgaaa gtaaaagatg ctgaagatca gttgggtgca cgagtgggtt
     6601 acatcgaact ggatctcaac agcggtaaga tccttgagag ttttcgcccc gaagaacgtt
     6661 ttccaatgat gagcactttt aaagttctgc tatgtggcgc ggtattatcc cgtattgacg
     6721 ccgggcaaga gcaactcggt cgccgcatac actattctca gaatgacttg gttgagtact
     6781 caccagtcac agaaaagcat cttacggatg gcatgacagt aagagaatta tgcagtgctg
     6841 ccataaccat gagtgataac actgcggcca acttacttct gacaacgatc ggaggaccga
     6901 aggagctaac cgcttttttg cacaacatgg gggatcatgt aactcgcctt gatcgttggg
     6961 aaccggagct gaatgaagcc ataccaaacg acgagcgtga caccacgatg cctgtagcaa
     7021 tggcaacaac gttgcgcaaa ctattaactg gcgaactact tact
//
