LOCUS       sequence-430853-        6806 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 6806)
  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 6806)
  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: HTR1A-DuET
FEATURES             Location/Qualifiers
     source          1..6806
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      78..666
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     567..586
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     820..837
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    954..975
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(990..995,996..1013,1014..1020)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1028..1044
                     /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     1033..1055
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     1052..1068
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     1052..1068
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     polyA_signal    1117..1238
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1128..1147)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     1182..1201
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     CDS             complement(1412..2206)
                     /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(1523..1542)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     2133..2152
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     promoter        complement(2273..2602)
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      2287..2422
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(2341..2360)
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     primer_bind     2587..2607
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      complement(2616..3044)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2726..2747)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2938..2957
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     polyA_signal    3090..3314
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     3303..3320
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     primer_bind     complement(4835..4854)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4836..4854)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        complement(4899..5102)
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     complement(4928..4948)
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     enhancer        5103..5482
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     primer_bind     5654..5673
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        5748..5852
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5853..5921,5922..6713)
                     /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(6071..6090)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 ctcatgacca aaatccctta acgtgagttt tcgttccact gagcgtcaga ccccgtagaa
       61 aagatcaaag gatcttcttg agatcctttt tttctgcgcg taatctgctg cttgcaaaca
      121 aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc aagagctacc aactcttttt
      181 ccgaaggtaa ctggcttcag cagagcgcag ataccaaata ccgttcttct agtgtagccg
      241 tagttaggcc accacttcaa gaactctgta gcaccgccta catacctcgc tctgctaatc
      301 ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga
      361 cgatagttac cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc
      421 agcttggagc gaacgaccta caccgaactg agatacctac agcgtgagct atgagaaagc
      481 gccacgcttc ccgaagggag aaaggcggac aggtatccgg taagcggcag ggtcggaaca
      541 ggagagcgca cgagggagct tccaggggga aacgcctggt atctttatag tcctgtcggg
      601 tttcgccacc tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta
      661 tggaaaaacg ccagcaacgc ggccttttta cggttcctgg ccttttgctg gccttttgct
      721 cacatgttct ttcctgcgtt atcccctgat tctgtggata accgtattac cgcctttgag
      781 tgagctgata ccgctcgccg cagccgaacg accgagcgca gcgagtcagt gagcgaggaa
      841 gcggaagagc gcccaatacg caaaccgcct ctccccgcgc gttggccgat tcattaatgc
      901 agctggcacg acaggtttcc cgactggaaa gcgggcagtg agcgcaacgc aattaatgtg
      961 agttagctca ctcattaggc accccaggct ttacacttta tgcttccggc tcgtatgttg
     1021 tgtggaattg tgagcggata acaatttcac acaggaaaca gctatgacca tgattacgcc
     1081 aagctctagc tagaggtcga cggtatacag acatgataag atacattgat gagtttggac
     1141 aaaccacaac tagaatgcag tgaaaaaaat gctttatttg tgaaatttgt gatgctattg
     1201 ctttatttgt aaccattata agctgcaata aacaagttgg ggtgggcgaa gaactccagc
     1261 atgagatccc cgcgctggag gatcatccag ccggcgtccc ggaaaacgat tccgaagccc
     1321 aacctttcat agaaggcggc ggtggaatcg aaatctcgtg atggcaggtt gggcgtcgct
     1381 tggtcggtca tttcgaaccc cagagtcccg ctcagaagaa ctcgtcaaga aggcgataga
     1441 aggcgatgcg ctgcgaatcg ggagcggcga taccgtaaag cacgaggaag cggtcagccc
     1501 attcgccgcc aagctcttca gcaatatcac gggtagccaa cgctatgtcc tgatagcggt
     1561 ccgccacacc cagccggcca cagtcgatga atccagaaaa gcggccattt tccaccatga
     1621 tattcggcaa gcaggcatcg ccatgggtca cgacgagatc ctcgccgtcg ggcatgcgcg
     1681 ccttgagcct ggcgaacagt tcggctggcg cgagcccctg atgctcttcg tccagatcat
     1741 cctgatcgac aagaccggct tccatccgag tacgtgctcg ctcgatgcga tgtttcgctt
     1801 ggtggtcgaa tgggcaggta gccggatcaa gcgtatgcag ccgccgcatt gcatcagcca
     1861 tgatggatac tttctcggca ggagcaaggt gagatgacag gagatcctgc cccggcactt
     1921 cgcccaatag cagccagtcc cttcccgctt cagtgacaac gtcgagcaca gctgcgcaag
     1981 gaacgcccgt cgtggccagc cacgatagcc gcgctgcctc gtcctgcagt tcattcaggg
     2041 caccggacag gtcggtcttg acaaaaagaa ccgggcgccc ctgcgctgac agccggaaca
     2101 cggcggcatc agagcagccg attgtctgtt gtgcccagtc atagccgaat agcctctcca
     2161 cccaagcggc cggagaacct gcgtgcaatc catcttgttc aatcatgcga aacgatcctc
     2221 atcctgtctc ttgatcagat ccgaaaatgg atatacaagc tcccgggagc tttttgcaaa
     2281 agcctaggcc tccaaaaaag cctcctcact acttctggaa tagctcagag gcagaggcgg
     2341 cctcggcctc tgcataaata aaaaaaatta gtcagccatg gggcggagaa tgggcggaac
     2401 tgggcggagt taggggcggg atgggcggag ttaggggcgg gactatggtt gctgactaat
     2461 tgagatgcat gctttgcata cttctgcctg ctggggagcc tggggacttt ccacacctgg
     2521 ttgctgacta attgagatgc atgctttgca tacttctgcc tgctggggag cctggggact
     2581 ttccacaccc taactgacac acattccaca gaattaattc gcgttaaatt tttgttaaat
     2641 cagctcattt tttaaccaat aggccgaaat cggcaaaatc ccttataaat caaaagaata
     2701 gaccgagata gggttgagtg ttgttccagt ttggaacaag agtccactat taaagaacgt
     2761 ggactccaac gtcaaagggc gaaaaaccgt ctatcagggc gatggcccac tacgtgaacc
     2821 atcaccctaa tcaagttttt tggggtcgag gtgccgtaaa gcactaaatc ggaaccctaa
     2881 agggagcccc cgatttagag cttgacgggg aaagccggcg aacgtggcga gaaaggaagg
     2941 gaagaaagcg aaaggagcgg gcgctagggc gctggcaagt gtagcggtca cgctgcgcgt
     3001 aaccaccaca cccgccgcgc ttaatgcgcc gctacagggc gcgtggggat accccctaga
     3061 gccccagctg gttctttccg cctcagaagc catagagccc accgcatccc cagcatgcct
     3121 gctattgtct tcccaatcct cccccttgct gtcctgcccc accccacccc ccagaataga
     3181 atgacaccta ctcagacaat gcgatgcaat ttcctcattt tattaggaaa ggacagtggg
     3241 agtggcacct tccagggtca aggaaggcac gggggagggg caaacaacag atggctggca
     3301 actagaaggc acagtcgagg ctgatcagcg ggtttaaacg ggccctctag actcgagtta
     3361 ggcaggggcc acctgagaag tctcggtctt agacacggtg gtgctggcct catcgaattc
     3421 accggtgcgt ccaccggtat cgatttgtct acagaacttg cacttaatta tctttttaaa
     3481 ggcattctgg aaatccttgt tgaagtaagc gtagatgacg ggattcagca ggctattgct
     3541 gtaacccagc caattaatga tggccccgag aagcgtgggc atatgacatg atgattcgca
     3601 aaatggcagc acgagtgcca cgatgaaaaa gggcagccaa cacaggatga atgtccccat
     3661 gataatcccc agggttttta ctgttttccg ttcccgggca agggccatct tccgctttgc
     3721 ctcagcattt ctctcgtttt tccgctcaaa agaggccggg gcgcatggag ttggtccggc
     3781 ctcggagggg agaggcaggt gttctttact attccccact ctatgcacct cgattacttc
     3841 aagtgcagca ccgtcgtctc cttgtcttac tgcaccgttg gcacacagtg cccctccggc
     3901 cttagattcc actcccaatc tccagttcct gctcccactc tccccgttca cggacttctt
     3961 gggctgtgga gcgggggagg caccatgccg ggtatccgcc ccagtctttt ccactttctt
     4021 tacagtcttc cggatcctga accgcgcggc gcggaatatc cgtccataca acaccaacat
     4081 caacaacagt ggtatataga atgctccgaa ggtggagtag atggtgtagc cgtgatcctt
     4141 tgaaatggta cacgcgtcag ggtcgcttct atcctcaggt gtcctccacc caagcatggg
     4201 aggtatacta atcaaaaagc caatcagcca cgtcaagctg atcagagcgg cagcccgccg
     4261 aggtgtcctc ttgttcacgt agtcgatcgg atctgtgata gcccagtacc tatccaatgc
     4321 tatggcacac agatgcaaaa tggaagaggt gcaacaaagc acgtcgagtg cgataaacag
     4381 gtcacaggtc acttggccca gggtccactt attgagaacc tgatagagag ctgccattgg
     4441 aagaacgagc acgctcacca tgaggtccgt cactgccagt gagccaatca aatagtttgc
     4501 cacattctga aggctgcgct ccagggcaat ggctgctacc acacatgcgt ttcccaatac
     4561 ggcacagaag atcagagtac caagcagcag gctagtgatc acttgataac tgacggtaac
     4621 atcagagatg ccggttgtat taccgccagt ttcgaacgga gccggtggag aagtggtgtt
     4681 gttgccctgg ccgggactca gcacgtccat atcgatgctg gcgtcatcat cgtccttgta
     4741 gtcggcgaat accaggcaga agatgtagct cagggcgatg atcgtcttca tggtggcgct
     4801 aagcttaagt ttaaacgcta gccagcttgg gtctccctat agtgagtcgt attaatttcg
     4861 ataagccagt aagcagtggg ttctctagtt agccagagag ctctgcttat atagacctcc
     4921 caccgtacac gcctaccgcc catttgcgtc aatggggcgg agttgttacg acattttgga
     4981 aagtcccgtt gattttggtg ccaaaacaaa ctcccattga cgtcaatggg gtggagactt
     5041 ggaaatcccc gtgagtcaaa ccgctatcca cgcccattga tgtactgcca aaaccgcatc
     5101 accatggtaa tagcgatgac taatacgtag atgtactgcc aagtaggaaa gtcccataag
     5161 gtcatgtact gggcataatg ccaggcgggc catttaccgt cattgacgtc aatagggggc
     5221 gtacttggca tatgatacac ttgatgtact gccaagtggg cagtttaccg taaatactcc
     5281 acccattgac gtcaatggaa agtccctatt ggcgttacta tgggaacata cgtcattatt
     5341 gacgtcaatg ggcgggggtc gttgggcggt cagccaggcg ggccatttac cgtaagttat
     5401 gtaacgcgga actccatata tgggctatga actaatgacc ccgtaattga ttactattaa
     5461 taactagtca ataatcaatg tcaacgcgta tatctggccc gtacatcgcg aagcagcgca
     5521 aaacgcctaa ccctaagcag attcttcatg caaatgtcgg tcaagccttg ccttgttgta
     5581 gcttaaattt tgctcgcgca ctactcagcg acctccaaca cacaagcagg gagcagatac
     5641 tggcttaact atgcggcatc agagcagatt gtactgagag tgcaccatag gggatcggga
     5701 gatctcccga tccgtcgacg tcaggtggca cttttcgggg aaatgtgcgc ggaaccccta
     5761 tttgtttatt tttctaaata cattcaaata tgtatccgct catgagacaa taaccctgat
     5821 aaatgcttca ataatattga aaaaggaaga gtatgagtat tcaacatttc cgtgtcgccc
     5881 ttattccctt ttttgcggca ttttgccttc ctgtttttgc tcacccagaa acgctggtga
     5941 aagtaaaaga tgctgaagat cagttgggtg cacgagtggg ttacatcgaa ctggatctca
     6001 acagcggtaa gatccttgag agttttcgcc ccgaagaacg ttttccaatg atgagcactt
     6061 ttaaagttct gctatgtggc gcggtattat cccgtattga cgccgggcaa gagcaactcg
     6121 gtcgccgcat acactattct cagaatgact tggttgagta ctcaccagtc acagaaaagc
     6181 atcttacgga tggcatgaca gtaagagaat tatgcagtgc tgccataacc atgagtgata
     6241 acactgcggc caacttactt ctgacaacga tcggaggacc gaaggagcta accgcttttt
     6301 tgcacaacat gggggatcat gtaactcgcc ttgatcgttg ggaaccggag ctgaatgaag
     6361 ccataccaaa cgacgagcgt gacaccacga tgcctgtagc aatggcaaca acgttgcgca
     6421 aactattaac tggcgaacta cttactctag cttcccggca acaattaata gactggatgg
     6481 aggcggataa agttgcagga ccacttctgc gctcggccct tccggctggc tggtttattg
     6541 ctgataaatc tggagccggt gagcgtgggt ctcgcggtat cattgcagca ctggggccag
     6601 atggtaagcc ctcccgtatc gtagttatct acacgacggg gagtcaggca actatggatg
     6661 aacgaaatag acagatcgct gagataggtg cctcactgat taagcattgg taactgtcag
     6721 accaagttta ctcatatata ctttagattg atttaaaact tcatttttaa tttaaaagga
     6781 tctaggtgaa gatccttttt gataat
//
