LOCUS       sequence-430794-        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: F2RL2-DuET
FEATURES             Location/Qualifiers
     source          1..6662
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      237..825
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     726..745
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     979..996
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    1113..1134
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(1149..1154,1155..1172,1173..1179)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1187..1203
                     /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     1192..1214
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     1211..1227
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     1211..1227
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     polyA_signal    1276..1397
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1287..1306)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     1341..1360
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     CDS             complement(1571..2365)
                     /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(1682..1701)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     2292..2311
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     promoter        complement(2432..2761)
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      2446..2581
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(2500..2519)
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     primer_bind     2746..2766
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      complement(2775..3203)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2885..2906)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3097..3116
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     polyA_signal    3249..3473
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     3462..3479
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     primer_bind     complement(4850..4869)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4851..4869)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        complement(4914..5117)
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     complement(4943..4963)
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     enhancer        5118..5497
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     primer_bind     5669..5688
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        5763..5867
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5868..5936,5937..6662,1..66)
                     /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(6086..6105)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 gcaactatgg atgaacgaaa tagacagatc gctgagatag gtgcctcact gattaagcat
       61 tggtaactgt cagaccaagt ttactcatat atactttaga ttgatttaaa acttcatttt
      121 taatttaaaa ggatctaggt gaagatcctt tttgataatc tcatgaccaa aatcccttaa
      181 cgtgagtttt cgttccactg agcgtcagac cccgtagaaa agatcaaagg atcttcttga
      241 gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg
      301 gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac tggcttcagc
      361 agagcgcaga taccaaatac cgttcttcta gtgtagccgt agttaggcca ccacttcaag
      421 aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt ggctgctgcc
      481 agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttacc ggataaggcg
      541 cagcggtcgg gctgaacggg gggttcgtgc acacagccca gcttggagcg aacgacctac
      601 accgaactga gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga
      661 aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcac gagggagctt
      721 ccagggggaa acgcctggta tctttatagt cctgtcgggt ttcgccacct ctgacttgag
      781 cgtcgatttt tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg
      841 gcctttttac ggttcctggc cttttgctgg ccttttgctc acatgttctt tcctgcgtta
      901 tcccctgatt ctgtggataa ccgtattacc gcctttgagt gagctgatac cgctcgccgc
      961 agccgaacga ccgagcgcag cgagtcagtg agcgaggaag cggaagagcg cccaatacgc
     1021 aaaccgcctc tccccgcgcg ttggccgatt cattaatgca gctggcacga caggtttccc
     1081 gactggaaag cgggcagtga gcgcaacgca attaatgtga gttagctcac tcattaggca
     1141 ccccaggctt tacactttat gcttccggct cgtatgttgt gtggaattgt gagcggataa
     1201 caatttcaca caggaaacag ctatgaccat gattacgcca agctctagct agaggtcgac
     1261 ggtatacaga catgataaga tacattgatg agtttggaca aaccacaact agaatgcagt
     1321 gaaaaaaatg ctttatttgt gaaatttgtg atgctattgc tttatttgta accattataa
     1381 gctgcaataa acaagttggg gtgggcgaag aactccagca tgagatcccc gcgctggagg
     1441 atcatccagc cggcgtcccg gaaaacgatt ccgaagccca acctttcata gaaggcggcg
     1501 gtggaatcga aatctcgtga tggcaggttg ggcgtcgctt ggtcggtcat ttcgaacccc
     1561 agagtcccgc tcagaagaac tcgtcaagaa ggcgatagaa ggcgatgcgc tgcgaatcgg
     1621 gagcggcgat accgtaaagc acgaggaagc ggtcagccca ttcgccgcca agctcttcag
     1681 caatatcacg ggtagccaac gctatgtcct gatagcggtc cgccacaccc agccggccac
     1741 agtcgatgaa tccagaaaag cggccatttt ccaccatgat attcggcaag caggcatcgc
     1801 catgggtcac gacgagatcc tcgccgtcgg gcatgcgcgc cttgagcctg gcgaacagtt
     1861 cggctggcgc gagcccctga tgctcttcgt ccagatcatc ctgatcgaca agaccggctt
     1921 ccatccgagt acgtgctcgc tcgatgcgat gtttcgcttg gtggtcgaat gggcaggtag
     1981 ccggatcaag cgtatgcagc cgccgcattg catcagccat gatggatact ttctcggcag
     2041 gagcaaggtg agatgacagg agatcctgcc ccggcacttc gcccaatagc agccagtccc
     2101 ttcccgcttc agtgacaacg tcgagcacag ctgcgcaagg aacgcccgtc gtggccagcc
     2161 acgatagccg cgctgcctcg tcctgcagtt cattcagggc accggacagg tcggtcttga
     2221 caaaaagaac cgggcgcccc tgcgctgaca gccggaacac ggcggcatca gagcagccga
     2281 ttgtctgttg tgcccagtca tagccgaata gcctctccac ccaagcggcc ggagaacctg
     2341 cgtgcaatcc atcttgttca atcatgcgaa acgatcctca tcctgtctct tgatcagatc
     2401 cgaaaatgga tatacaagct cccgggagct ttttgcaaaa gcctaggcct ccaaaaaagc
     2461 ctcctcacta cttctggaat agctcagagg cagaggcggc ctcggcctct gcataaataa
     2521 aaaaaattag tcagccatgg ggcggagaat gggcggaact gggcggagtt aggggcggga
     2581 tgggcggagt taggggcggg actatggttg ctgactaatt gagatgcatg ctttgcatac
     2641 ttctgcctgc tggggagcct ggggactttc cacacctggt tgctgactaa ttgagatgca
     2701 tgctttgcat acttctgcct gctggggagc ctggggactt tccacaccct aactgacaca
     2761 cattccacag aattaattcg cgttaaattt ttgttaaatc agctcatttt ttaaccaata
     2821 ggccgaaatc ggcaaaatcc cttataaatc aaaagaatag accgagatag ggttgagtgt
     2881 tgttccagtt tggaacaaga gtccactatt aaagaacgtg gactccaacg tcaaagggcg
     2941 aaaaaccgtc tatcagggcg atggcccact acgtgaacca tcaccctaat caagtttttt
     3001 ggggtcgagg tgccgtaaag cactaaatcg gaaccctaaa gggagccccc gatttagagc
     3061 ttgacgggga aagccggcga acgtggcgag aaaggaaggg aagaaagcga aaggagcggg
     3121 cgctagggcg ctggcaagtg tagcggtcac gctgcgcgta accaccacac ccgccgcgct
     3181 taatgcgccg ctacagggcg cgtggggata ccccctagag ccccagctgg ttctttccgc
     3241 ctcagaagcc atagagccca ccgcatcccc agcatgcctg ctattgtctt cccaatcctc
     3301 ccccttgctg tcctgcccca ccccaccccc cagaatagaa tgacacctac tcagacaatg
     3361 cgatgcaatt tcctcatttt attaggaaag gacagtggga gtggcacctt ccagggtcaa
     3421 ggaaggcacg ggggaggggc aaacaacaga tggctggcaa ctagaaggca cagtcgaggc
     3481 tgatcagcgg gtttaaacgg gccctctaga ctcgagttag gcaggggcca cctgagaagt
     3541 ctcggtctta gacacggtgg tgctggcctc atcgaattca ccggtgcgtc caccggtatc
     3601 gatcttggtc aggtaagctg tggaatggtt cctggtcttg gacatgagga agtacaaaaa
     3661 tgggtcgaga caagaattaa gactgcccag acacagtgct atgagataaa taaaataaag
     3721 cccgtcagtg ttgttgtagt agtagttggc gtgatggatg atcaagatga tgttggaggg
     3781 agcaaagcag atggtgaaaa taaccagaat cagcagggat gccttcacat accagagcca
     3841 tctgtggtca tatgcattca aggttctgat tatagcggca tagcagtaaa ttatgagcac
     3901 gaacgggatc agaaagccga agaaggccag gctaataaag taatacagct ggaacgggct
     3961 gctgctctcg catgtgttgt gcacgtcgtg gcaggtagtg atatcgggct gaacaagata
     4021 atattcctgc ttgagaataa aaaaaggcaa catatacagg aagacggtgg cccacaccag
     4081 cccgcaggta accagcgcgt acgtatgctt tgggaggccc ctgtaggtga aagggtggac
     4141 gatagccaga tatctgttaa tagatatgca ggccagcaaa agaatagagc agtacatgtt
     4201 tccgtagaag ataacagttg tggcccgaca cagcacttcg ccaaagaccc agttgttgcc
     4261 gttcagatgg tacgctatct tgaagggcaa agtgacgcaa aagaggaagt cagcaatcgc
     4321 gaggtttgtg taaaaaactg tcgtacaaat actgcgggtg cgaaaaaaca gcatccagag
     4381 tgtcacagca ttagcgggaa caccgaccac aaataccagg aggtagattg cggggatcag
     4441 tttggtagac aggctagagg tgaggtatcc catagttgca ttcttcacat gcagatgtga
     4501 ggcagactct tccgggcatt ttattttcac agttatagta gcaccagtcc aaccttccag
     4561 tgcggagaaa gggaattcct cgaaagagtt cggcggagct ccccgaaagg tcttgatagg
     4621 aagggtcggt tttgccagat tgttagtatc attctccatg ccgctctggc aaaaagttgg
     4681 aagaagcaag agcagtccag cagcggcaaa gatcagggct ttcatatcga tgctggcgtc
     4741 atcatcgtcc ttgtagtcgg cgaataccag gcagaagatg tagctcaggg cgatgatcgt
     4801 cttcatggtg gcgctaagct taagtttaaa cgctagccag cttgggtctc cctatagtga
     4861 gtcgtattaa tttcgataag ccagtaagca gtgggttctc tagttagcca gagagctctg
     4921 cttatataga cctcccaccg tacacgccta ccgcccattt gcgtcaatgg ggcggagttg
     4981 ttacgacatt ttggaaagtc ccgttgattt tggtgccaaa acaaactccc attgacgtca
     5041 atggggtgga gacttggaaa tccccgtgag tcaaaccgct atccacgccc attgatgtac
     5101 tgccaaaacc gcatcaccat ggtaatagcg atgactaata cgtagatgta ctgccaagta
     5161 ggaaagtccc ataaggtcat gtactgggca taatgccagg cgggccattt accgtcattg
     5221 acgtcaatag ggggcgtact tggcatatga tacacttgat gtactgccaa gtgggcagtt
     5281 taccgtaaat actccaccca ttgacgtcaa tggaaagtcc ctattggcgt tactatggga
     5341 acatacgtca ttattgacgt caatgggcgg gggtcgttgg gcggtcagcc aggcgggcca
     5401 tttaccgtaa gttatgtaac gcggaactcc atatatgggc tatgaactaa tgaccccgta
     5461 attgattact attaataact agtcaataat caatgtcaac gcgtatatct ggcccgtaca
     5521 tcgcgaagca gcgcaaaacg cctaacccta agcagattct tcatgcaaat gtcggtcaag
     5581 ccttgccttg ttgtagctta aattttgctc gcgcactact cagcgacctc caacacacaa
     5641 gcagggagca gatactggct taactatgcg gcatcagagc agattgtact gagagtgcac
     5701 cataggggat cgggagatct cccgatccgt cgacgtcagg tggcactttt cggggaaatg
     5761 tgcgcggaac ccctatttgt ttatttttct aaatacattc aaatatgtat ccgctcatga
     5821 gacaataacc ctgataaatg cttcaataat attgaaaaag gaagagtatg agtattcaac
     5881 atttccgtgt cgcccttatt cccttttttg cggcattttg ccttcctgtt tttgctcacc
     5941 cagaaacgct ggtgaaagta aaagatgctg aagatcagtt gggtgcacga gtgggttaca
     6001 tcgaactgga tctcaacagc ggtaagatcc ttgagagttt tcgccccgaa gaacgttttc
     6061 caatgatgag cacttttaaa gttctgctat gtggcgcggt attatcccgt attgacgccg
     6121 ggcaagagca actcggtcgc cgcatacact attctcagaa tgacttggtt gagtactcac
     6181 cagtcacaga aaagcatctt acggatggca tgacagtaag agaattatgc agtgctgcca
     6241 taaccatgag tgataacact gcggccaact tacttctgac aacgatcgga ggaccgaagg
     6301 agctaaccgc ttttttgcac aacatggggg atcatgtaac tcgccttgat cgttgggaac
     6361 cggagctgaa tgaagccata ccaaacgacg agcgtgacac cacgatgcct gtagcaatgg
     6421 caacaacgtt gcgcaaacta ttaactggcg aactacttac tctagcttcc cggcaacaat
     6481 taatagactg gatggaggcg gataaagttg caggaccact tctgcgctcg gcccttccgg
     6541 ctggctggtt tattgctgat aaatctggag ccggtgagcg tgggtctcgc ggtatcattg
     6601 cagcactggg gccagatggt aagccctccc gtatcgtagt tatctacacg acggggagtc
     6661 ag
//
