LOCUS       sequence-430900-        6587 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 6587)
  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 6587)
  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: GAL1-DuET
FEATURES             Location/Qualifiers
     source          1..6587
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     572..591
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(810..914)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(989..1008)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        1180..1559
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        1560..1763
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     1714..1734
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     1808..1827
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1808..1826
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(3123..3140)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3129..3353
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      3399..3827
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3486..3505)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3696..3717
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3836..3856)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3841..4170
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      4021..4156
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     4083..4102
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             4237..5031
                     /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(4291..4310)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4901..4920
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    5205..5326
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5242..5261)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5296..5315
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5375..5391)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5375..5391)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5388..5410)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5399..5415
                     /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(5423..5429,5430..5447,5448..5453))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5468..5489
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5606..5623)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5777..6365)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5857..5876)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6536..6587,1..740,741..809))
                     /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"
ORIGIN
        1 tcatccatag ttgcctgact ccccgtcgtg tagataacta cgatacggga gggcttacca
       61 tctggcccca gtgctgcaat gataccgcga gacccacgct caccggctcc agatttatca
      121 gcaataaacc agccagccgg aagggccgag cgcagaagtg gtcctgcaac tttatccgcc
      181 tccatccagt ctattaattg ttgccgggaa gctagagtaa gtagttcgcc agttaatagt
      241 ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg
      301 gcttcattca gctccggttc ccaacgatca aggcgagtta catgatcccc catgttgtgc
      361 aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg
      421 ttatcactca tggttatggc agcactgcat aattctctta ctgtcatgcc atccgtaaga
      481 tgcttttctg tgactggtga gtactcaacc aagtcattct gagaatagtg tatgcggcga
      541 ccgagttgct cttgcccggc gtcaatacgg gataataccg cgccacatag cagaacttta
      601 aaagtgctca tcattggaaa acgttcttcg gggcgaaaac tctcaaggat cttaccgctg
      661 ttgagatcca gttcgatgta acccactcgt gcacccaact gatcttcagc atcttttact
      721 ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata
      781 agggcgacac ggaaatgttg aatactcata ctcttccttt ttcaatatta ttgaagcatt
      841 tatcagggtt attgtctcat gagcggatac atatttgaat gtatttagaa aaataaacaa
      901 ataggggttc cgcgcacatt tccccgaaaa gtgccacctg acgtcgacgg atcgggagat
      961 ctcccgatcc cctatggtgc actctcagta caatctgctc tgatgccgca tagttaagcc
     1021 agtatctgct ccctgcttgt gtgttggagg tcgctgagta gtgcgcgagc aaaatttaag
     1081 ctacaacaag gcaaggcttg accgacattt gcatgaagaa tctgcttagg gttaggcgtt
     1141 ttgcgctgct tcgcgatgta cgggccagat atacgcgttg acattgatta ttgactagtt
     1201 attaatagta atcaattacg gggtcattag ttcatagccc atatatggag ttccgcgtta
     1261 cataacttac ggtaaatggc ccgcctggct gaccgcccaa cgacccccgc ccattgacgt
     1321 caataatgac gtatgttccc atagtaacgc caatagggac tttccattga cgtcaatggg
     1381 tggagtattt acggtaaact gcccacttgg cagtacatca agtgtatcat atgccaagta
     1441 cgccccctat tgacgtcaat gacggtaaat ggcccgcctg gcattatgcc cagtacatga
     1501 ccttatggga ctttcctact tggcagtaca tctacgtatt agtcatcgct attaccatgg
     1561 tgatgcggtt ttggcagtac atcaatgggc gtggatagcg gtttgactca cggggatttc
     1621 caagtctcca ccccattgac gtcaatggga gtttgttttg gcaccaaaat caacgggact
     1681 ttccaaaatg tcgtaacaac tccgccccat tgacgcaaat gggcggtagg cgtgtacggt
     1741 gggaggtcta tataagcaga gctctctggc taactagaga acccactgct tactggctta
     1801 tcgaaattaa tacgactcac tatagggaga cccaagctgg ctagcgttta aacttaagct
     1861 tagcggccgc gccaccatga agacgatcat cgccctgagc tacatcttct gcctggtatt
     1921 cgccgactac aaggacgatg atgacgccag catcgatatg gaactggcgg tcggcaatct
     1981 tagcgaaggc aacgcttcct ggccagagcc cccagcaccc gagcctgggc cgctgtttgg
     2041 cattggcgtt gaaaattttg tgactctggt cgtctttggg ctgatattcg ctctgggggt
     2101 gcttggaaac tctctggtca ttacagtgct tgctaggagt aaaccaggga agccaagatc
     2161 tactacaaac ctctttatac ttaacctgtc catcgcagat ctggcttatc tgctgttctg
     2221 catccctttc caggccacag tctacgccct tccgacttgg gtcctggggg ctttcatctg
     2281 caagtttatt cactattttt tcacggtctc aatgttggtt tccattttta ccctcgctgc
     2341 aatgagcgta gatcgctacg tagctatcgt gcactctagg agatctagct ctctgcgcgt
     2401 atcccggaat gctcttctgg gtgtcggctg catttgggca ctcagcattg cgatggcttc
     2461 acctgtggcc tatcaccagg gcctgtttca cccacgggcc tctaatcaaa cattctgttg
     2521 ggaacagtgg ccagacccca ggcacaagaa agcatatgtg gtttgcactt ttgtgtttgg
     2581 ctatctcctg ccactcctgc tcatctgttt ctgttatgct aaagtattga atcacttgca
     2641 caaaaagctc aaaaatatgt ccaagaagtc tgaagcatcc aagaagaaaa cagcacagac
     2701 ggtgctggtt gtagtggtcg tcttcggaat atcatggctg cctcaccaca tcatacacct
     2761 ctgggctgag ttcggagtgt tcccgctcac tcccgcttct tttctcttta gaatcacagc
     2821 ccactgcctc gcgtactcaa actcttcagt gaaccccata atctatgctt ttctgagcga
     2881 gaactttaga aaggcctaca aacaggtttt caagtgtcac atacgcaaag actctcacct
     2941 tagtgatact aaggagtcca agtccaggat tgatacacct ccttcaacaa actgcaccca
     3001 cgtaatcgat accggtggac gcaccggtga attcgatgag gccagcacca ccgtgtctaa
     3061 gaccgagact tctcaggtgg cccctgccta atctagaggg cccgtttaaa cccgctgatc
     3121 agcctcgact gtgccttcta gttgccagcc atctgttgtt tgcccctccc ccgtgccttc
     3181 cttgaccctg gaaggtgcca ctcccactgt cctttcctaa taaaatgagg aaattgcatc
     3241 gcattgtctg agtaggtgtc attctattct ggggggtggg gtggggcagg acagcaaggg
     3301 ggaggattgg gaagacaata gcaggcatgc tggggatgcg gtgggctcta tggcttctga
     3361 ggcggaaaga accagctggg gctctagggg gtatccccac gcgccctgta gcggcgcatt
     3421 aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca gcgccctagc
     3481 gcccgctcct ttcgctttct tcccttcctt tctcgccacg ttcgccggct ttccccgtca
     3541 agctctaaat cgggggctcc ctttagggtt ccgatttagt gctttacggc acctcgaccc
     3601 caaaaaactt gattagggtg atggttcacg tagtgggcca tcgccctgat agacggtttt
     3661 tcgccctttg acgttggagt ccacgttctt taatagtgga ctcttgttcc aaactggaac
     3721 aacactcaac cctatctcgg tctattcttt tgatttataa gggattttgc cgatttcggc
     3781 ctattggtta aaaaatgagc tgatttaaca aaaatttaac gcgaattaat tctgtggaat
     3841 gtgtgtcagt tagggtgtgg aaagtcccca ggctccccag caggcagaag tatgcaaagc
     3901 atgcatctca attagtcagc aaccaggtgt ggaaagtccc caggctcccc agcaggcaga
     3961 agtatgcaaa gcatgcatct caattagtca gcaaccatag tcccgcccct aactccgccc
     4021 atcccgcccc taactccgcc cagttccgcc cattctccgc cccatggctg actaattttt
     4081 tttatttatg cagaggccga ggccgcctct gcctctgagc tattccagaa gtagtgagga
     4141 ggcttttttg gaggcctagg cttttgcaaa aagctcccgg gagcttgtat atccattttc
     4201 ggatctgatc aagagacagg atgaggatcg tttcgcatga ttgaacaaga tggattgcac
     4261 gcaggttctc cggccgcttg ggtggagagg ctattcggct atgactgggc acaacagaca
     4321 atcggctgct ctgatgccgc cgtgttccgg ctgtcagcgc aggggcgccc ggttcttttt
     4381 gtcaagaccg acctgtccgg tgccctgaat gaactgcagg acgaggcagc gcggctatcg
     4441 tggctggcca cgacgggcgt tccttgcgca gctgtgctcg acgttgtcac tgaagcggga
     4501 agggactggc tgctattggg cgaagtgccg gggcaggatc tcctgtcatc tcaccttgct
     4561 cctgccgaga aagtatccat catggctgat gcaatgcggc ggctgcatac gcttgatccg
     4621 gctacctgcc cattcgacca ccaagcgaaa catcgcatcg agcgagcacg tactcggatg
     4681 gaagccggtc ttgtcgatca ggatgatctg gacgaagagc atcaggggct cgcgccagcc
     4741 gaactgttcg ccaggctcaa ggcgcgcatg cccgacggcg aggatctcgt cgtgacccat
     4801 ggcgatgcct gcttgccgaa tatcatggtg gaaaatggcc gcttttctgg attcatcgac
     4861 tgtggccggc tgggtgtggc ggaccgctat caggacatag cgttggctac ccgtgatatt
     4921 gctgaagagc ttggcggcga atgggctgac cgcttcctcg tgctttacgg tatcgccgct
     4981 cccgattcgc agcgcatcgc cttctatcgc cttcttgacg agttcttctg agcgggactc
     5041 tggggttcga aatgaccgac caagcgacgc ccaacctgcc atcacgagat ttcgattcca
     5101 ccgccgcctt ctatgaaagg ttgggcttcg gaatcgtttt ccgggacgcc ggctggatga
     5161 tcctccagcg cggggatctc atgctggagt tcttcgccca ccccaacttg tttattgcag
     5221 cttataatgg ttacaaataa agcaatagca tcacaaattt cacaaataaa gcattttttt
     5281 cactgcattc tagttgtggt ttgtccaaac tcatcaatgt atcttatcat gtctgtatac
     5341 cgtcgacctc tagctagagc ttggcgtaat catggtcata gctgtttcct gtgtgaaatt
     5401 gttatccgct cacaattcca cacaacatac gagccggaag cataaagtgt aaagcctggg
     5461 gtgcctaatg agtgagctaa ctcacattaa ttgcgttgcg ctcactgccc gctttccagt
     5521 cgggaaacct gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg agaggcggtt
     5581 tgcgtattgg gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc
     5641 tgcggcgagc ggtatcagct cactcaaagg cggtaatacg gttatccaca gaatcagggg
     5701 ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg
     5761 ccgcgttgct ggcgtttttc cataggctcc gcccccctga cgagcatcac aaaaatcgac
     5821 gctcaagtca gaggtggcga aacccgacag gactataaag ataccaggcg tttccccctg
     5881 gaagctccct cgtgcgctct cctgttccga ccctgccgct taccggatac ctgtccgcct
     5941 ttctcccttc gggaagcgtg gcgctttctc atagctcacg ctgtaggtat ctcagttcgg
     6001 tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct
     6061 gcgccttatc cggtaactat cgtcttgagt ccaacccggt aagacacgac ttatcgccac
     6121 tggcagcagc cactggtaac aggattagca gagcgaggta tgtaggcggt gctacagagt
     6181 tcttgaagtg gtggcctaac tacggctaca ctagaagaac ggtatttggt atctgcgctc
     6241 tgctgaagcc agttaccttc ggaaaaagag ttggtagctc ttgatccggc aaacaaacca
     6301 ccgctggtag cggtggtttt tttgtttgca agcagcagat tacgcgcaga aaaaaaggat
     6361 ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc tcagtggaac gaaaactcac
     6421 gttaagggat tttggtcatg agattatcaa aaaggatctt cacctagatc cttttaaatt
     6481 aaaaatgaag ttttaaatca atctaaagta tatatgagta aacttggtct gacagttacc
     6541 aatgcttaat cagtgaggca cctatctcag cgatctgtct atttcgt
//
