LOCUS       sequence-430866-        6515 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 6515)
  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 6515)
  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: MAS1-DuET
FEATURES             Location/Qualifiers
     source          1..6515
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             55..849
                     /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(109..128)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     719..738
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    1023..1144
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1060..1079)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     1114..1133
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(1193..1209)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1193..1209)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1206..1228)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    1217..1233
                     /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)."
     promoter        complement(join(1241..1247,1248..1265,1266..1271))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1286..1307
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(1424..1441)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1595..2183)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1675..1694)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2354..3145,3146..3214))
                     /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     2977..2996
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3215..3319)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(3394..3413)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        3585..3964
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        3965..4168
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     4119..4139
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     4213..4232
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        4213..4231
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(5456..5473)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    5462..5686
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      5732..6160
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5819..5838)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     6029..6050
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(6169..6189)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        6174..6503
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      6354..6489
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     6416..6435
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
ORIGIN
        1 gcttgtatat ccattttcgg atctgatcaa gagacaggat gaggatcgtt tcgcatgatt
       61 gaacaagatg gattgcacgc aggttctccg gccgcttggg tggagaggct attcggctat
      121 gactgggcac aacagacaat cggctgctct gatgccgccg tgttccggct gtcagcgcag
      181 gggcgcccgg ttctttttgt caagaccgac ctgtccggtg ccctgaatga actgcaggac
      241 gaggcagcgc ggctatcgtg gctggccacg acgggcgttc cttgcgcagc tgtgctcgac
      301 gttgtcactg aagcgggaag ggactggctg ctattgggcg aagtgccggg gcaggatctc
      361 ctgtcatctc accttgctcc tgccgagaaa gtatccatca tggctgatgc aatgcggcgg
      421 ctgcatacgc ttgatccggc tacctgccca ttcgaccacc aagcgaaaca tcgcatcgag
      481 cgagcacgta ctcggatgga agccggtctt gtcgatcagg atgatctgga cgaagagcat
      541 caggggctcg cgccagccga actgttcgcc aggctcaagg cgcgcatgcc cgacggcgag
      601 gatctcgtcg tgacccatgg cgatgcctgc ttgccgaata tcatggtgga aaatggccgc
      661 ttttctggat tcatcgactg tggccggctg ggtgtggcgg accgctatca ggacatagcg
      721 ttggctaccc gtgatattgc tgaagagctt ggcggcgaat gggctgaccg cttcctcgtg
      781 ctttacggta tcgccgctcc cgattcgcag cgcatcgcct tctatcgcct tcttgacgag
      841 ttcttctgag cgggactctg gggttcgaaa tgaccgacca agcgacgccc aacctgccat
      901 cacgagattt cgattccacc gccgccttct atgaaaggtt gggcttcgga atcgttttcc
      961 gggacgccgg ctggatgatc ctccagcgcg gggatctcat gctggagttc ttcgcccacc
     1021 ccaacttgtt tattgcagct tataatggtt acaaataaag caatagcatc acaaatttca
     1081 caaataaagc atttttttca ctgcattcta gttgtggttt gtccaaactc atcaatgtat
     1141 cttatcatgt ctgtataccg tcgacctcta gctagagctt ggcgtaatca tggtcatagc
     1201 tgtttcctgt gtgaaattgt tatccgctca caattccaca caacatacga gccggaagca
     1261 taaagtgtaa agcctggggt gcctaatgag tgagctaact cacattaatt gcgttgcgct
     1321 cactgcccgc tttccagtcg ggaaacctgt cgtgccagct gcattaatga atcggccaac
     1381 gcgcggggag aggcggtttg cgtattgggc gctcttccgc ttcctcgctc actgactcgc
     1441 tgcgctcggt cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt
     1501 tatccacaga atcaggggat aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg
     1561 ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca taggctccgc ccccctgacg
     1621 agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga ctataaagat
     1681 accaggcgtt tccccctgga agctccctcg tgcgctctcc tgttccgacc ctgccgctta
     1741 ccggatacct gtccgccttt ctcccttcgg gaagcgtggc gctttctcat agctcacgct
     1801 gtaggtatct cagttcggtg taggtcgttc gctccaagct gggctgtgtg cacgaacccc
     1861 ccgttcagcc cgaccgctgc gccttatccg gtaactatcg tcttgagtcc aacccggtaa
     1921 gacacgactt atcgccactg gcagcagcca ctggtaacag gattagcaga gcgaggtatg
     1981 taggcggtgc tacagagttc ttgaagtggt ggcctaacta cggctacact agaagaacgg
     2041 tatttggtat ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt
     2101 gatccggcaa acaaaccacc gctggtagcg gtggtttttt tgtttgcaag cagcagatta
     2161 cgcgcagaaa aaaaggatct caagaagatc ctttgatctt ttctacgggg tctgacgctc
     2221 agtggaacga aaactcacgt taagggattt tggtcatgag attatcaaaa aggatcttca
     2281 cctagatcct tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa
     2341 cttggtctga cagttaccaa tgcttaatca gtgaggcacc tatctcagcg atctgtctat
     2401 ttcgttcatc catagttgcc tgactccccg tcgtgtagat aactacgata cgggagggct
     2461 taccatctgg ccccagtgct gcaatgatac cgcgagaccc acgctcaccg gctccagatt
     2521 tatcagcaat aaaccagcca gccggaaggg ccgagcgcag aagtggtcct gcaactttat
     2581 ccgcctccat ccagtctatt aattgttgcc gggaagctag agtaagtagt tcgccagtta
     2641 atagtttgcg caacgttgtt gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg
     2701 gtatggcttc attcagctcc ggttcccaac gatcaaggcg agttacatga tcccccatgt
     2761 tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt tgtcagaagt aagttggccg
     2821 cagtgttatc actcatggtt atggcagcac tgcataattc tcttactgtc atgccatccg
     2881 taagatgctt ttctgtgact ggtgagtact caaccaagtc attctgagaa tagtgtatgc
     2941 ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca catagcagaa
     3001 ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg aaaactctca aggatcttac
     3061 cgctgttgag atccagttcg atgtaaccca ctcgtgcacc caactgatct tcagcatctt
     3121 ttactttcac cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg
     3181 gaataagggc gacacggaaa tgttgaatac tcatactctt cctttttcaa tattattgaa
     3241 gcatttatca gggttattgt ctcatgagcg gatacatatt tgaatgtatt tagaaaaata
     3301 aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc acctgacgtc gacggatcgg
     3361 gagatctccc gatcccctat ggtgcactct cagtacaatc tgctctgatg ccgcatagtt
     3421 aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg cgagcaaaat
     3481 ttaagctaca acaaggcaag gcttgaccga catttgcatg aagaatctgc ttagggttag
     3541 gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt gattattgac
     3601 tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata tggagttccg
     3661 cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc cccgcccatt
     3721 gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc attgacgtca
     3781 atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt atcatatgcc
     3841 aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt atgcccagta
     3901 catgacctta tgggactttc ctacttggca gtacatctac gtattagtca tcgctattac
     3961 catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg actcacgggg
     4021 atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc aaaatcaacg
     4081 ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg gtaggcgtgt
     4141 acggtgggag gtctatataa gcagagctct ctggctaact agagaaccca ctgcttactg
     4201 gcttatcgaa attaatacga ctcactatag ggagacccaa gctggctagc gtttaaactt
     4261 aagcttagcg ccaccatgaa gacgatcatc gccctgagct acatcttctg cctggtattc
     4321 gccgactaca aggacgatga tgacgccagc atcgatatgg acggatccaa cgtgacctcc
     4381 ttcgttgtgg aggagcccac aaacatcagc actgggagaa acgcctccgt tggtaacgcc
     4441 catcgccaga tcccaatagt ccactgggtt atcatgtcca tttctccagt aggattcgtg
     4501 gagaatggga tattgctctg gtttttgtgt tttcgcatgc gccgaaatcc ttttacagtt
     4561 tacatcactc acctgtctat cgccgacatc tccttgttgt tctgtatctt cattctgtcc
     4621 atcgactacg cactggatta cgagctgtct tccggccact attatactat tgtaaccttg
     4681 tccgtgactt ttctgtttgg ttacaacaca ggactctatc ttctgaccgc aatttccgtc
     4741 gaacggtgcc tgagtgtcct gtatcctatt tggtatcggt gccatagacc aaagtatcag
     4801 agcgctcttg tctgtgcact gctgtgggct ctgtcttgtt tggtgacaac tatggaatac
     4861 gtgatgtgta tagacaggga ggaggagtca cacagcagaa atgactgtag agctgtaatc
     4921 atattcattg ctatcctgag cttcctcgtt ttcaccccgc tcatgctcgt gagttcaacg
     4981 atactggtgg tgaagatcag gaagaacacg tgggcgagtc actcttctaa actgtacatc
     5041 gtgatcatgg tgacgattat catatttctg atttttgcca tgcccatgag attgctctac
     5101 ctgctgtact atgagtactg gtccaccttt ggaaatctgc accacatctc actgctgttc
     5161 tccaccataa acagtagcgc caatcccttt atatatttct tcgttggctc ctcaaagaaa
     5221 aagcggttta aagagagcct caaggttgtt ctgacaagag ccttcaaaga cgaaatgcaa
     5281 ccacgaaggc agaaagataa ttgcaataca gttacagtgg agaccgtggt gatcgatacc
     5341 ggtggacgca ccggtgaatt cgatgaggcc agcaccaccg tgtctaagac cgagacttct
     5401 caggtggccc ctgcctaact cgagtctaga gggcccgttt aaacccgctg atcagcctcg
     5461 actgtgcctt ctagttgcca gccatctgtt gtttgcccct cccccgtgcc ttccttgacc
     5521 ctggaaggtg ccactcccac tgtcctttcc taataaaatg aggaaattgc atcgcattgt
     5581 ctgagtaggt gtcattctat tctggggggt ggggtggggc aggacagcaa gggggaggat
     5641 tgggaagaca atagcaggca tgctggggat gcggtgggct ctatggcttc tgaggcggaa
     5701 agaaccagct ggggctctag ggggtatccc cacgcgccct gtagcggcgc attaagcgcg
     5761 gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg ccagcgccct agcgcccgct
     5821 cctttcgctt tcttcccttc ctttctcgcc acgttcgccg gctttccccg tcaagctcta
     5881 aatcgggggc tccctttagg gttccgattt agtgctttac ggcacctcga ccccaaaaaa
     5941 cttgattagg gtgatggttc acgtagtggg ccatcgccct gatagacggt ttttcgccct
     6001 ttgacgttgg agtccacgtt ctttaatagt ggactcttgt tccaaactgg aacaacactc
     6061 aaccctatct cggtctattc ttttgattta taagggattt tgccgatttc ggcctattgg
     6121 ttaaaaaatg agctgattta acaaaaattt aacgcgaatt aattctgtgg aatgtgtgtc
     6181 agttagggtg tggaaagtcc ccaggctccc cagcaggcag aagtatgcaa agcatgcatc
     6241 tcaattagtc agcaaccagg tgtggaaagt ccccaggctc cccagcaggc agaagtatgc
     6301 aaagcatgca tctcaattag tcagcaacca tagtcccgcc cctaactccg cccatcccgc
     6361 ccctaactcc gcccagttcc gcccattctc cgccccatgg ctgactaatt ttttttattt
     6421 atgcagaggc cgaggccgcc tctgcctctg agctattcca gaagtagtga ggaggctttt
     6481 ttggaggcct aggcttttgc aaaaagctcc cggga
//
