LOCUS       sequence-430796-        6530 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 6530)
  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 6530)
  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: FFA2-DuET
FEATURES             Location/Qualifiers
     source          1..6530
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     429..448
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     682..699
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    816..837
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(852..857,858..875,876..882)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    890..906
                     /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     895..917
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     914..930
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     914..930
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     polyA_signal    979..1100
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(990..1009)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     1044..1063
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     CDS             complement(1274..2068)
                     /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(1385..1404)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     1995..2014
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     promoter        complement(2135..2464)
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      2149..2284
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(2203..2222)
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     primer_bind     2449..2469
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      complement(2478..2906)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2588..2609)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2800..2819
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     polyA_signal    2952..3176
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     3165..3182
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     regulatory      3534..3543
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     primer_bind     complement(4421..4440)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4422..4440)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        complement(4485..4688)
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     complement(4514..4534)
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     enhancer        4689..5068
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     primer_bind     5240..5259
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        5334..5438
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5439..5507,5508..6299)
                     /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(5657..5676)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      join(6470..6530,1..528)
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
ORIGIN
        1 gcggtggttt gtttgccgga tcaagagcta ccaactcttt ttccgaaggt aactggcttc
       61 agcagagcgc agataccaaa taccgttctt ctagtgtagc cgtagttagg ccaccacttc
      121 aagaactctg tagcaccgcc tacatacctc gctctgctaa tcctgttacc agtggctgct
      181 gccagtggcg ataagtcgtg tcttaccggg ttggactcaa gacgatagtt accggataag
      241 gcgcagcggt cgggctgaac ggggggttcg tgcacacagc ccagcttgga gcgaacgacc
      301 tacaccgaac tgagatacct acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg
      361 agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa caggagagcg cacgagggag
      421 cttccagggg gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca cctctgactt
      481 gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac
      541 gcggcctttt tacggttcct ggccttttgc tggccttttg ctcacatgtt ctttcctgcg
      601 ttatcccctg attctgtgga taaccgtatt accgcctttg agtgagctga taccgctcgc
      661 cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga gcgcccaata
      721 cgcaaaccgc ctctccccgc gcgttggccg attcattaat gcagctggca cgacaggttt
      781 cccgactgga aagcgggcag tgagcgcaac gcaattaatg tgagttagct cactcattag
      841 gcaccccagg ctttacactt tatgcttccg gctcgtatgt tgtgtggaat tgtgagcgga
      901 taacaatttc acacaggaaa cagctatgac catgattacg ccaagctcta gctagaggtc
      961 gacggtatac agacatgata agatacattg atgagtttgg acaaaccaca actagaatgc
     1021 agtgaaaaaa atgctttatt tgtgaaattt gtgatgctat tgctttattt gtaaccatta
     1081 taagctgcaa taaacaagtt ggggtgggcg aagaactcca gcatgagatc cccgcgctgg
     1141 aggatcatcc agccggcgtc ccggaaaacg attccgaagc ccaacctttc atagaaggcg
     1201 gcggtggaat cgaaatctcg tgatggcagg ttgggcgtcg cttggtcggt catttcgaac
     1261 cccagagtcc cgctcagaag aactcgtcaa gaaggcgata gaaggcgatg cgctgcgaat
     1321 cgggagcggc gataccgtaa agcacgagga agcggtcagc ccattcgccg ccaagctctt
     1381 cagcaatatc acgggtagcc aacgctatgt cctgatagcg gtccgccaca cccagccggc
     1441 cacagtcgat gaatccagaa aagcggccat tttccaccat gatattcggc aagcaggcat
     1501 cgccatgggt cacgacgaga tcctcgccgt cgggcatgcg cgccttgagc ctggcgaaca
     1561 gttcggctgg cgcgagcccc tgatgctctt cgtccagatc atcctgatcg acaagaccgg
     1621 cttccatccg agtacgtgct cgctcgatgc gatgtttcgc ttggtggtcg aatgggcagg
     1681 tagccggatc aagcgtatgc agccgccgca ttgcatcagc catgatggat actttctcgg
     1741 caggagcaag gtgagatgac aggagatcct gccccggcac ttcgcccaat agcagccagt
     1801 cccttcccgc ttcagtgaca acgtcgagca cagctgcgca aggaacgccc gtcgtggcca
     1861 gccacgatag ccgcgctgcc tcgtcctgca gttcattcag ggcaccggac aggtcggtct
     1921 tgacaaaaag aaccgggcgc ccctgcgctg acagccggaa cacggcggca tcagagcagc
     1981 cgattgtctg ttgtgcccag tcatagccga atagcctctc cacccaagcg gccggagaac
     2041 ctgcgtgcaa tccatcttgt tcaatcatgc gaaacgatcc tcatcctgtc tcttgatcag
     2101 atccgaaaat ggatatacaa gctcccggga gctttttgca aaagcctagg cctccaaaaa
     2161 agcctcctca ctacttctgg aatagctcag aggcagaggc ggcctcggcc tctgcataaa
     2221 taaaaaaaat tagtcagcca tggggcggag aatgggcgga actgggcgga gttaggggcg
     2281 ggatgggcgg agttaggggc gggactatgg ttgctgacta attgagatgc atgctttgca
     2341 tacttctgcc tgctggggag cctggggact ttccacacct ggttgctgac taattgagat
     2401 gcatgctttg catacttctg cctgctgggg agcctgggga ctttccacac cctaactgac
     2461 acacattcca cagaattaat tcgcgttaaa tttttgttaa atcagctcat tttttaacca
     2521 ataggccgaa atcggcaaaa tcccttataa atcaaaagaa tagaccgaga tagggttgag
     2581 tgttgttcca gtttggaaca agagtccact attaaagaac gtggactcca acgtcaaagg
     2641 gcgaaaaacc gtctatcagg gcgatggccc actacgtgaa ccatcaccct aatcaagttt
     2701 tttggggtcg aggtgccgta aagcactaaa tcggaaccct aaagggagcc cccgatttag
     2761 agcttgacgg ggaaagccgg cgaacgtggc gagaaaggaa gggaagaaag cgaaaggagc
     2821 gggcgctagg gcgctggcaa gtgtagcggt cacgctgcgc gtaaccacca cacccgccgc
     2881 gcttaatgcg ccgctacagg gcgcgtgggg atacccccta gagccccagc tggttctttc
     2941 cgcctcagaa gccatagagc ccaccgcatc cccagcatgc ctgctattgt cttcccaatc
     3001 ctcccccttg ctgtcctgcc ccaccccacc ccccagaata gaatgacacc tactcagaca
     3061 atgcgatgca atttcctcat tttattagga aaggacagtg ggagtggcac cttccagggt
     3121 caaggaaggc acgggggagg ggcaaacaac agatggctgg caactagaag gcacagtcga
     3181 ggctgatcag cgggtttaaa cgggccctct agactcgagt taggcagggg ccacctgaga
     3241 agtctcggtc ttagacacgg tggtgctggc ctcatcgaat tcaccggtgc gtccaccggt
     3301 atcgatctca gttgtgaagt cggatgatgg catcccctcg ccctgaccga ctcctcgatc
     3361 ctcgtttgtg ccttctgcgg tatcctttcc ccgcctgccg agcaagctgc taccctggtt
     3421 cctcagtacc tggagtcctc ttccaaaagc ccggcgcacg acgctactgc tgaagtagaa
     3481 caggagagga tccaggcttg cattgaggct ggaaaacacc acggctatgc tgcgccacca
     3541 tggggacttg cgctgatgat agccaaccag gtggctcaca ttatatgggc caaagcacac
     3601 gaggaaattc agcagggtaa caacagccaa tccaacagct cggcgccttc tctgtgcccc
     3661 taccaaaggt tgggaaagca tgatccacac aaatctccaa taacaaaaga tggtaactgc
     3721 cattgggatg aagaacagca ccaagcacag ttccagccga accggcaaaa cgacgtcgag
     3781 ctgattatcc gtaaaattct cgtaacatgt tatttcgttc ccgctcctca cctgctcggt
     3841 ggtattcaga tactgtacga taatcacgat ggtgcaatgg ccgaaactca taacccaagc
     3901 cacgagtgca gctatcactc catagagagg gcggcgtgag agcttgtact gcacggggaa
     3961 tgctacgccg agatagcgct caatggagat tcctgccaaa agccaagtgg agcagtagat
     4021 gctactgtag aagccaaagg aggtaagggc gcataccacc ttgggcaagt accacctgaa
     4081 attggaagcg gcttcaataa tcttaaaagg gagaagaagc agcaacagga gatccgccag
     4141 ggtcagggac agcaagagga tgtggacagg ggcgggttgg ggctggcgga tcctgccgac
     4201 aaaagccctc agggccagca gattagcagg gagacctgta aggaaaataa ttatgtaggc
     4261 catcagtatc agggaagatt tccagtctgg aagcatatcg atgctggcgt catcatcgtc
     4321 cttgtagtcg gcgaatacca ggcagaagat gtagctcagg gcgatgatcg tcttcatggt
     4381 ggcgctaagc ttaagtttaa acgctagcca gcttgggtct ccctatagtg agtcgtatta
     4441 atttcgataa gccagtaagc agtgggttct ctagttagcc agagagctct gcttatatag
     4501 acctcccacc gtacacgcct accgcccatt tgcgtcaatg gggcggagtt gttacgacat
     4561 tttggaaagt cccgttgatt ttggtgccaa aacaaactcc cattgacgtc aatggggtgg
     4621 agacttggaa atccccgtga gtcaaaccgc tatccacgcc cattgatgta ctgccaaaac
     4681 cgcatcacca tggtaatagc gatgactaat acgtagatgt actgccaagt aggaaagtcc
     4741 cataaggtca tgtactgggc ataatgccag gcgggccatt taccgtcatt gacgtcaata
     4801 gggggcgtac ttggcatatg atacacttga tgtactgcca agtgggcagt ttaccgtaaa
     4861 tactccaccc attgacgtca atggaaagtc cctattggcg ttactatggg aacatacgtc
     4921 attattgacg tcaatgggcg ggggtcgttg ggcggtcagc caggcgggcc atttaccgta
     4981 agttatgtaa cgcggaactc catatatggg ctatgaacta atgaccccgt aattgattac
     5041 tattaataac tagtcaataa tcaatgtcaa cgcgtatatc tggcccgtac atcgcgaagc
     5101 agcgcaaaac gcctaaccct aagcagattc ttcatgcaaa tgtcggtcaa gccttgcctt
     5161 gttgtagctt aaattttgct cgcgcactac tcagcgacct ccaacacaca agcagggagc
     5221 agatactggc ttaactatgc ggcatcagag cagattgtac tgagagtgca ccatagggga
     5281 tcgggagatc tcccgatccg tcgacgtcag gtggcacttt tcggggaaat gtgcgcggaa
     5341 cccctatttg tttatttttc taaatacatt caaatatgta tccgctcatg agacaataac
     5401 cctgataaat gcttcaataa tattgaaaaa ggaagagtat gagtattcaa catttccgtg
     5461 tcgcccttat tccctttttt gcggcatttt gccttcctgt ttttgctcac ccagaaacgc
     5521 tggtgaaagt aaaagatgct gaagatcagt tgggtgcacg agtgggttac atcgaactgg
     5581 atctcaacag cggtaagatc cttgagagtt ttcgccccga agaacgtttt ccaatgatga
     5641 gcacttttaa agttctgcta tgtggcgcgg tattatcccg tattgacgcc gggcaagagc
     5701 aactcggtcg ccgcatacac tattctcaga atgacttggt tgagtactca ccagtcacag
     5761 aaaagcatct tacggatggc atgacagtaa gagaattatg cagtgctgcc ataaccatga
     5821 gtgataacac tgcggccaac ttacttctga caacgatcgg aggaccgaag gagctaaccg
     5881 cttttttgca caacatgggg gatcatgtaa ctcgccttga tcgttgggaa ccggagctga
     5941 atgaagccat accaaacgac gagcgtgaca ccacgatgcc tgtagcaatg gcaacaacgt
     6001 tgcgcaaact attaactggc gaactactta ctctagcttc ccggcaacaa ttaatagact
     6061 ggatggaggc ggataaagtt gcaggaccac ttctgcgctc ggcccttccg gctggctggt
     6121 ttattgctga taaatctgga gccggtgagc gtgggtctcg cggtatcatt gcagcactgg
     6181 ggccagatgg taagccctcc cgtatcgtag ttatctacac gacggggagt caggcaacta
     6241 tggatgaacg aaatagacag atcgctgaga taggtgcctc actgattaag cattggtaac
     6301 tgtcagacca agtttactca tatatacttt agattgattt aaaacttcat ttttaattta
     6361 aaaggatcta ggtgaagatc ctttttgata atctcatgac caaaatccct taacgtgagt
     6421 tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa aggatcttct tgagatcctt
     6481 tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca
//
