LOCUS       sequence-430858-        6983 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 6983)
  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 6983)
  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: HTR2B-DuET
FEATURES             Location/Qualifiers
     source          1..6983
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      167..755
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     656..675
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     909..926
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    1043..1064
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(1079..1084,1085..1102,1103..1109)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1117..1133
                     /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     1122..1144
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     1141..1157
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     1141..1157
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     polyA_signal    1206..1327
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1217..1236)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     1271..1290
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     CDS             complement(1501..2295)
                     /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(1612..1631)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     2222..2241
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     promoter        complement(2362..2691)
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      2376..2511
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(2430..2449)
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     primer_bind     2676..2696
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      complement(2705..3133)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2815..2836)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3027..3046
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     polyA_signal    3179..3403
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     3392..3409
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     primer_bind     complement(5101..5120)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(5102..5120)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        complement(5165..5368)
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     complement(5194..5214)
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     enhancer        5369..5748
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     primer_bind     5920..5939
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        6014..6118
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(6119..6187,6188..6979)
                     /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(6337..6356)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 cagaccaagt ttactcatat atactttaga ttgatttaaa acttcatttt taatttaaaa
       61 ggatctaggt gaagatcctt tttgataatc tcatgaccaa aatcccttaa cgtgagtttt
      121 cgttccactg agcgtcagac cccgtagaaa agatcaaagg atcttcttga gatccttttt
      181 ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt
      241 tgccggatca agagctacca actctttttc cgaaggtaac tggcttcagc agagcgcaga
      301 taccaaatac cgttcttcta gtgtagccgt agttaggcca ccacttcaag aactctgtag
      361 caccgcctac atacctcgct ctgctaatcc tgttaccagt ggctgctgcc agtggcgata
      421 agtcgtgtct taccgggttg gactcaagac gatagttacc ggataaggcg cagcggtcgg
      481 gctgaacggg gggttcgtgc acacagccca gcttggagcg aacgacctac accgaactga
      541 gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca
      601 ggtatccggt aagcggcagg gtcggaacag gagagcgcac gagggagctt ccagggggaa
      661 acgcctggta tctttatagt cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt
      721 tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg gcctttttac
      781 ggttcctggc cttttgctgg ccttttgctc acatgttctt tcctgcgtta tcccctgatt
      841 ctgtggataa ccgtattacc gcctttgagt gagctgatac cgctcgccgc agccgaacga
      901 ccgagcgcag cgagtcagtg agcgaggaag cggaagagcg cccaatacgc aaaccgcctc
      961 tccccgcgcg ttggccgatt cattaatgca gctggcacga caggtttccc gactggaaag
     1021 cgggcagtga gcgcaacgca attaatgtga gttagctcac tcattaggca ccccaggctt
     1081 tacactttat gcttccggct cgtatgttgt gtggaattgt gagcggataa caatttcaca
     1141 caggaaacag ctatgaccat gattacgcca agctctagct agaggtcgac ggtatacaga
     1201 catgataaga tacattgatg agtttggaca aaccacaact agaatgcagt gaaaaaaatg
     1261 ctttatttgt gaaatttgtg atgctattgc tttatttgta accattataa gctgcaataa
     1321 acaagttggg gtgggcgaag aactccagca tgagatcccc gcgctggagg atcatccagc
     1381 cggcgtcccg gaaaacgatt ccgaagccca acctttcata gaaggcggcg gtggaatcga
     1441 aatctcgtga tggcaggttg ggcgtcgctt ggtcggtcat ttcgaacccc agagtcccgc
     1501 tcagaagaac tcgtcaagaa ggcgatagaa ggcgatgcgc tgcgaatcgg gagcggcgat
     1561 accgtaaagc acgaggaagc ggtcagccca ttcgccgcca agctcttcag caatatcacg
     1621 ggtagccaac gctatgtcct gatagcggtc cgccacaccc agccggccac agtcgatgaa
     1681 tccagaaaag cggccatttt ccaccatgat attcggcaag caggcatcgc catgggtcac
     1741 gacgagatcc tcgccgtcgg gcatgcgcgc cttgagcctg gcgaacagtt cggctggcgc
     1801 gagcccctga tgctcttcgt ccagatcatc ctgatcgaca agaccggctt ccatccgagt
     1861 acgtgctcgc tcgatgcgat gtttcgcttg gtggtcgaat gggcaggtag ccggatcaag
     1921 cgtatgcagc cgccgcattg catcagccat gatggatact ttctcggcag gagcaaggtg
     1981 agatgacagg agatcctgcc ccggcacttc gcccaatagc agccagtccc ttcccgcttc
     2041 agtgacaacg tcgagcacag ctgcgcaagg aacgcccgtc gtggccagcc acgatagccg
     2101 cgctgcctcg tcctgcagtt cattcagggc accggacagg tcggtcttga caaaaagaac
     2161 cgggcgcccc tgcgctgaca gccggaacac ggcggcatca gagcagccga ttgtctgttg
     2221 tgcccagtca tagccgaata gcctctccac ccaagcggcc ggagaacctg cgtgcaatcc
     2281 atcttgttca atcatgcgaa acgatcctca tcctgtctct tgatcagatc cgaaaatgga
     2341 tatacaagct cccgggagct ttttgcaaaa gcctaggcct ccaaaaaagc ctcctcacta
     2401 cttctggaat agctcagagg cagaggcggc ctcggcctct gcataaataa aaaaaattag
     2461 tcagccatgg ggcggagaat gggcggaact gggcggagtt aggggcggga tgggcggagt
     2521 taggggcggg actatggttg ctgactaatt gagatgcatg ctttgcatac ttctgcctgc
     2581 tggggagcct ggggactttc cacacctggt tgctgactaa ttgagatgca tgctttgcat
     2641 acttctgcct gctggggagc ctggggactt tccacaccct aactgacaca cattccacag
     2701 aattaattcg cgttaaattt ttgttaaatc agctcatttt ttaaccaata ggccgaaatc
     2761 ggcaaaatcc cttataaatc aaaagaatag accgagatag ggttgagtgt tgttccagtt
     2821 tggaacaaga gtccactatt aaagaacgtg gactccaacg tcaaagggcg aaaaaccgtc
     2881 tatcagggcg atggcccact acgtgaacca tcaccctaat caagtttttt ggggtcgagg
     2941 tgccgtaaag cactaaatcg gaaccctaaa gggagccccc gatttagagc ttgacgggga
     3001 aagccggcga acgtggcgag aaaggaaggg aagaaagcga aaggagcggg cgctagggcg
     3061 ctggcaagtg tagcggtcac gctgcgcgta accaccacac ccgccgcgct taatgcgccg
     3121 ctacagggcg cgtggggata ccccctagag ccccagctgg ttctttccgc ctcagaagcc
     3181 atagagccca ccgcatcccc agcatgcctg ctattgtctt cccaatcctc ccccttgctg
     3241 tcctgcccca ccccaccccc cagaatagaa tgacacctac tcagacaatg cgatgcaatt
     3301 tcctcatttt attaggaaag gacagtggga gtggcacctt ccagggtcaa ggaaggcacg
     3361 ggggaggggc aaacaacaga tggctggcaa ctagaaggca cagtcgaggc tgatcagcgg
     3421 gtttaaacgg gccctctaga ctcgagttag gcaggggcca cctgagaagt ctcggtctta
     3481 gacacggtgg tgctggcctc atcgaattca ccggtgcgtc caccggtatc gatcacatag
     3541 gagacctgct cctcggtttt atctccttca ttctcagtga gcagcagagt atcaagcagg
     3601 atgatgctgc tggactgtat tgtagaggac cgaagcctca ttggagactg gtacatcgcg
     3661 gggtttattc cgttccgtat tccgtgcttt ttaaagaact tggaattttc tgccatagga
     3721 tttctaaaat agattttgga tgaccgcttc cgcaaggtct tcacggactt tgtggctctg
     3781 tagttgcagg tgatatatct cccaaaggcg tcgcggaaag tcttattgaa cagagtgtac
     3841 acaagggggt tcactccgct gctcacgtag ccgatccaca cgaatatttc gagcagcatc
     3901 tgcaatgtgg tctggttgca agagtcgcaa agcaccaggg ttatgtttgt gatgaagaaa
     3961 gggcaccaca tcagcagaaa caggaagaac acaataccca gaactttgga cgcccgctgt
     4021 tcgttggaga ttgtctgaac agactttttg ccaatcgtag aggttcggcg cataagtgtc
     4081 tcatcgccag aattaggaag ggctttgtcc tttcgggaac cgtcgagcat ggccactttt
     4141 tcaggggaac tacacggggt ctcgtcgcgc tggaagacgg tggacactgt cagccaagtc
     4201 agccgttggg gcggtttgtt cttaaccaga taggcttttt tctggagcgc atgtatggtc
     4261 agaaaatatg tcacaatcat gatggcaaga ggtgtaaaga atgcggcaag gctcccgaac
     4321 agcataaagt ctccgaacct ctccttggtg agtacgcatg tgatattgtt tggattgtcc
     4381 acatccgtct cgatcccctt gatcggcacg gggatggcga taccgatgct gataagccac
     4441 acaacggtta tcttgataaa ggcggtggcc cggctgttgt attgattggc ctgaatgggt
     4501 tttttgatag caatgtatcg gtccacggag atggcacaca aatgcatgat gcttgctgtg
     4561 ctgaacagca catcaaggaa caaccatgca gggcacagca caagagggag gggccacata
     4621 gcttcgaaca tgatggtgag aagtgcgata ggcataacaa acaaccccac caggagatcg
     4681 gcgacggcga ggctcatgag aaagtaattg gttgcgtact gcagtttctt ctccaggcta
     4741 accgcaagga tcacgagagt attgcctcca attgtcggga tgatcaccat cagtatgagc
     4801 agggctgccc agtgcagttt gttaccctgc tcctccacga tttgcttcat ctcctctggg
     4861 atagattcag tctggagccc agaccaattg gaactaatga catgtacgaa agtgctctgc
     4921 aaaatgtgct ctgggatggt agattgaagc tcgctcaccc gataagacag ggccatatcg
     4981 atgctggcgt catcatcgtc cttgtagtcg gcgaatacca ggcagaagat gtagctcagg
     5041 gcgatgatcg tcttcatggt ggcgctaagc ttaagtttaa acgctagcca gcttgggtct
     5101 ccctatagtg agtcgtatta atttcgataa gccagtaagc agtgggttct ctagttagcc
     5161 agagagctct gcttatatag acctcccacc gtacacgcct accgcccatt tgcgtcaatg
     5221 gggcggagtt gttacgacat tttggaaagt cccgttgatt ttggtgccaa aacaaactcc
     5281 cattgacgtc aatggggtgg agacttggaa atccccgtga gtcaaaccgc tatccacgcc
     5341 cattgatgta ctgccaaaac cgcatcacca tggtaatagc gatgactaat acgtagatgt
     5401 actgccaagt aggaaagtcc cataaggtca tgtactgggc ataatgccag gcgggccatt
     5461 taccgtcatt gacgtcaata gggggcgtac ttggcatatg atacacttga tgtactgcca
     5521 agtgggcagt ttaccgtaaa tactccaccc attgacgtca atggaaagtc cctattggcg
     5581 ttactatggg aacatacgtc attattgacg tcaatgggcg ggggtcgttg ggcggtcagc
     5641 caggcgggcc atttaccgta agttatgtaa cgcggaactc catatatggg ctatgaacta
     5701 atgaccccgt aattgattac tattaataac tagtcaataa tcaatgtcaa cgcgtatatc
     5761 tggcccgtac atcgcgaagc agcgcaaaac gcctaaccct aagcagattc ttcatgcaaa
     5821 tgtcggtcaa gccttgcctt gttgtagctt aaattttgct cgcgcactac tcagcgacct
     5881 ccaacacaca agcagggagc agatactggc ttaactatgc ggcatcagag cagattgtac
     5941 tgagagtgca ccatagggga tcgggagatc tcccgatccg tcgacgtcag gtggcacttt
     6001 tcggggaaat gtgcgcggaa cccctatttg tttatttttc taaatacatt caaatatgta
     6061 tccgctcatg agacaataac cctgataaat gcttcaataa tattgaaaaa ggaagagtat
     6121 gagtattcaa catttccgtg tcgcccttat tccctttttt gcggcatttt gccttcctgt
     6181 ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt tgggtgcacg
     6241 agtgggttac atcgaactgg atctcaacag cggtaagatc cttgagagtt ttcgccccga
     6301 agaacgtttt ccaatgatga gcacttttaa agttctgcta tgtggcgcgg tattatcccg
     6361 tattgacgcc gggcaagagc aactcggtcg ccgcatacac tattctcaga atgacttggt
     6421 tgagtactca ccagtcacag aaaagcatct tacggatggc atgacagtaa gagaattatg
     6481 cagtgctgcc ataaccatga gtgataacac tgcggccaac ttacttctga caacgatcgg
     6541 aggaccgaag gagctaaccg cttttttgca caacatgggg gatcatgtaa ctcgccttga
     6601 tcgttgggaa ccggagctga atgaagccat accaaacgac gagcgtgaca ccacgatgcc
     6661 tgtagcaatg gcaacaacgt tgcgcaaact attaactggc gaactactta ctctagcttc
     6721 ccggcaacaa ttaatagact ggatggaggc ggataaagtt gcaggaccac ttctgcgctc
     6781 ggcccttccg gctggctggt ttattgctga taaatctgga gccggtgagc gtgggtctcg
     6841 cggtatcatt gcagcactgg ggccagatgg taagccctcc cgtatcgtag ttatctacac
     6901 gacggggagt caggcaacta tggatgaacg aaatagacag atcgctgaga taggtgcctc
     6961 actgattaag cattggtaac tgt
//
