LOCUS       sequence-430818-        6539 bp    DNA     circular SYN 15-APR-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 6539)
  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 6539)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 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: GPR174-DuET
FEATURES             Location/Qualifiers
     source          1..6539
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      183..771
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     672..691
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     925..942
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    1059..1080
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(1095..1100,1101..1118,1119..1125)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1133..1149
                     /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     1138..1160
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     1157..1173
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     1157..1173
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     polyA_signal    1222..1343
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1233..1252)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     1287..1306
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     CDS             complement(1517..2311)
                     /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(1628..1647)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     2238..2257
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     promoter        complement(2378..2707)
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      2392..2527
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(2446..2465)
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     primer_bind     2692..2712
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      complement(2721..3149)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2831..2852)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3043..3062
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     polyA_signal    3195..3419
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     3408..3425
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     primer_bind     complement(4673..4692)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4674..4692)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        complement(4737..4940)
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     complement(4766..4786)
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     enhancer        4941..5320
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     primer_bind     5492..5511
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        5586..5690
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5691..5759,5760..6539,1..12)
                     /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(5909..5928)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 aagcattggt aactgtcaga ccaagtttac tcatatatac tttagattga tttaaaactt
       61 catttttaat ttaaaaggat ctaggtgaag atcctttttg ataatctcat gaccaaaatc
      121 ccttaacgtg agttttcgtt ccactgagcg tcagaccccg tagaaaagat caaaggatct
      181 tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta
      241 ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc tttttccgaa ggtaactggc
      301 ttcagcagag cgcagatacc aaataccgtt cttctagtgt agccgtagtt aggccaccac
      361 ttcaagaact ctgtagcacc gcctacatac ctcgctctgc taatcctgtt accagtggct
      421 gctgccagtg gcgataagtc gtgtcttacc gggttggact caagacgata gttaccggat
      481 aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac agcccagctt ggagcgaacg
      541 acctacaccg aactgagata cctacagcgt gagctatgag aaagcgccac gcttcccgaa
      601 gggagaaagg cggacaggta tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg
      661 gagcttccag ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg ccacctctga
      721 cttgagcgtc gatttttgtg atgctcgtca ggggggcgga gcctatggaa aaacgccagc
      781 aacgcggcct ttttacggtt cctggccttt tgctggcctt ttgctcacat gttctttcct
      841 gcgttatccc ctgattctgt ggataaccgt attaccgcct ttgagtgagc tgataccgct
      901 cgccgcagcc gaacgaccga gcgcagcgag tcagtgagcg aggaagcgga agagcgccca
      961 atacgcaaac cgcctctccc cgcgcgttgg ccgattcatt aatgcagctg gcacgacagg
     1021 tttcccgact ggaaagcggg cagtgagcgc aacgcaatta atgtgagtta gctcactcat
     1081 taggcacccc aggctttaca ctttatgctt ccggctcgta tgttgtgtgg aattgtgagc
     1141 ggataacaat ttcacacagg aaacagctat gaccatgatt acgccaagct ctagctagag
     1201 gtcgacggta tacagacatg ataagataca ttgatgagtt tggacaaacc acaactagaa
     1261 tgcagtgaaa aaaatgcttt atttgtgaaa tttgtgatgc tattgcttta tttgtaacca
     1321 ttataagctg caataaacaa gttggggtgg gcgaagaact ccagcatgag atccccgcgc
     1381 tggaggatca tccagccggc gtcccggaaa acgattccga agcccaacct ttcatagaag
     1441 gcggcggtgg aatcgaaatc tcgtgatggc aggttgggcg tcgcttggtc ggtcatttcg
     1501 aaccccagag tcccgctcag aagaactcgt caagaaggcg atagaaggcg atgcgctgcg
     1561 aatcgggagc ggcgataccg taaagcacga ggaagcggtc agcccattcg ccgccaagct
     1621 cttcagcaat atcacgggta gccaacgcta tgtcctgata gcggtccgcc acacccagcc
     1681 ggccacagtc gatgaatcca gaaaagcggc cattttccac catgatattc ggcaagcagg
     1741 catcgccatg ggtcacgacg agatcctcgc cgtcgggcat gcgcgccttg agcctggcga
     1801 acagttcggc tggcgcgagc ccctgatgct cttcgtccag atcatcctga tcgacaagac
     1861 cggcttccat ccgagtacgt gctcgctcga tgcgatgttt cgcttggtgg tcgaatgggc
     1921 aggtagccgg atcaagcgta tgcagccgcc gcattgcatc agccatgatg gatactttct
     1981 cggcaggagc aaggtgagat gacaggagat cctgccccgg cacttcgccc aatagcagcc
     2041 agtcccttcc cgcttcagtg acaacgtcga gcacagctgc gcaaggaacg cccgtcgtgg
     2101 ccagccacga tagccgcgct gcctcgtcct gcagttcatt cagggcaccg gacaggtcgg
     2161 tcttgacaaa aagaaccggg cgcccctgcg ctgacagccg gaacacggcg gcatcagagc
     2221 agccgattgt ctgttgtgcc cagtcatagc cgaatagcct ctccacccaa gcggccggag
     2281 aacctgcgtg caatccatct tgttcaatca tgcgaaacga tcctcatcct gtctcttgat
     2341 cagatccgaa aatggatata caagctcccg ggagcttttt gcaaaagcct aggcctccaa
     2401 aaaagcctcc tcactacttc tggaatagct cagaggcaga ggcggcctcg gcctctgcat
     2461 aaataaaaaa aattagtcag ccatggggcg gagaatgggc ggaactgggc ggagttaggg
     2521 gcgggatggg cggagttagg ggcgggacta tggttgctga ctaattgaga tgcatgcttt
     2581 gcatacttct gcctgctggg gagcctgggg actttccaca cctggttgct gactaattga
     2641 gatgcatgct ttgcatactt ctgcctgctg gggagcctgg ggactttcca caccctaact
     2701 gacacacatt ccacagaatt aattcgcgtt aaatttttgt taaatcagct cattttttaa
     2761 ccaataggcc gaaatcggca aaatccctta taaatcaaaa gaatagaccg agatagggtt
     2821 gagtgttgtt ccagtttgga acaagagtcc actattaaag aacgtggact ccaacgtcaa
     2881 agggcgaaaa accgtctatc agggcgatgg cccactacgt gaaccatcac cctaatcaag
     2941 ttttttgggg tcgaggtgcc gtaaagcact aaatcggaac cctaaaggga gcccccgatt
     3001 tagagcttga cggggaaagc cggcgaacgt ggcgagaaag gaagggaaga aagcgaaagg
     3061 agcgggcgct agggcgctgg caagtgtagc ggtcacgctg cgcgtaacca ccacacccgc
     3121 cgcgcttaat gcgccgctac agggcgcgtg gggatacccc ctagagcccc agctggttct
     3181 ttccgcctca gaagccatag agcccaccgc atccccagca tgcctgctat tgtcttccca
     3241 atcctccccc ttgctgtcct gccccacccc accccccaga atagaatgac acctactcag
     3301 acaatgcgat gcaatttcct cattttatta ggaaaggaca gtgggagtgg caccttccag
     3361 ggtcaaggaa ggcacggggg aggggcaaac aacagatggc tggcaactag aaggcacagt
     3421 cgaggctgat cagcgggttt aaacgggccc tctagactcg agttaggcag gggccacctg
     3481 agaagtctcg gtcttagaca cggtggtgct ggcctcatcg aattcaccgg tgcgtccacc
     3541 ggtatcgatg cacagctcag gggtcatagt ggaggcggtg tggttggaaa cgaaggactt
     3601 ggcatgcagc tgaatgctat catggagatc ctgcctactg agtctgcggc gaaattcgtt
     3661 ggtgctaaaa tagtaaatca ctgggtcgag acagctgtta agagacgcca gacaaagagc
     3721 cacagaatga aaaatcagga tcacgcgccg ggccaagcag cttttaattt cattgctctt
     3781 aaccaggaag tcgagtggaa aggagaaatg gtagggtgcg aaacatatca ggaagacacc
     3841 tgcacatgtc aagatcatct tcagtgcctt ctgcttctcg ccgagatcct gagccatggg
     3901 atatttgtcc tggagactga gcactgtttt ccaagtgcag tacaagacta tcagcagtgg
     3961 cgtcacgaaa ccaatcagct cgccgattgt catcatcacg actgactggg ccaggttaac
     4021 attgcgggtg gggaggtcga cgaaacactt ggtgcggtta ccactagtgt cgtcggaagt
     4081 gcggagcaga gggaagagca cacatgccag gcagattata agccatcctg cgatactgat
     4141 gtacagatca tacttctgct tgcagtcatg gaatcgaaaa ggatacatga ggaaccaaaa
     4201 tctgcgcacg ctaatacaaa ccagaaaata tatggatgcg tacatgttca catatttaag
     4261 gtaaaagcag aacatgcaga ggccggggcc aaagggccag tcgtggttaa gataataaaa
     4321 tatgcgcagg ggcaagctca gcacttgcag gaggtctgcg atagcgaggt ttatcataaa
     4381 gatgacggcc cgcttggtct ccttcatgta tccgtaaaac acccacagag ccaggatatt
     4441 gccgatcaat ccaggtacga gaatcacggt gtaagtcaca gcgtaaataa agtacctgaa
     4501 atctgtatta tcgccgtcag ggcgggtaca ggtataattg gctggcatat cgatgctggc
     4561 gtcatcatcg tccttgtagt cggcgaatac caggcagaag atgtagctca gggcgatgat
     4621 cgtcttcatg gtggcgctaa gcttaagttt aaacgctagc cagcttgggt ctccctatag
     4681 tgagtcgtat taatttcgat aagccagtaa gcagtgggtt ctctagttag ccagagagct
     4741 ctgcttatat agacctccca ccgtacacgc ctaccgccca tttgcgtcaa tggggcggag
     4801 ttgttacgac attttggaaa gtcccgttga ttttggtgcc aaaacaaact cccattgacg
     4861 tcaatggggt ggagacttgg aaatccccgt gagtcaaacc gctatccacg cccattgatg
     4921 tactgccaaa accgcatcac catggtaata gcgatgacta atacgtagat gtactgccaa
     4981 gtaggaaagt cccataaggt catgtactgg gcataatgcc aggcgggcca tttaccgtca
     5041 ttgacgtcaa tagggggcgt acttggcata tgatacactt gatgtactgc caagtgggca
     5101 gtttaccgta aatactccac ccattgacgt caatggaaag tccctattgg cgttactatg
     5161 ggaacatacg tcattattga cgtcaatggg cgggggtcgt tgggcggtca gccaggcggg
     5221 ccatttaccg taagttatgt aacgcggaac tccatatatg ggctatgaac taatgacccc
     5281 gtaattgatt actattaata actagtcaat aatcaatgtc aacgcgtata tctggcccgt
     5341 acatcgcgaa gcagcgcaaa acgcctaacc ctaagcagat tcttcatgca aatgtcggtc
     5401 aagccttgcc ttgttgtagc ttaaattttg ctcgcgcact actcagcgac ctccaacaca
     5461 caagcaggga gcagatactg gcttaactat gcggcatcag agcagattgt actgagagtg
     5521 caccataggg gatcgggaga tctcccgatc cgtcgacgtc aggtggcact tttcggggaa
     5581 atgtgcgcgg aacccctatt tgtttatttt tctaaataca ttcaaatatg tatccgctca
     5641 tgagacaata accctgataa atgcttcaat aatattgaaa aaggaagagt atgagtattc
     5701 aacatttccg tgtcgccctt attccctttt ttgcggcatt ttgccttcct gtttttgctc
     5761 acccagaaac gctggtgaaa gtaaaagatg ctgaagatca gttgggtgca cgagtgggtt
     5821 acatcgaact ggatctcaac agcggtaaga tccttgagag ttttcgcccc gaagaacgtt
     5881 ttccaatgat gagcactttt aaagttctgc tatgtggcgc ggtattatcc cgtattgacg
     5941 ccgggcaaga gcaactcggt cgccgcatac actattctca gaatgacttg gttgagtact
     6001 caccagtcac agaaaagcat cttacggatg gcatgacagt aagagaatta tgcagtgctg
     6061 ccataaccat gagtgataac actgcggcca acttacttct gacaacgatc ggaggaccga
     6121 aggagctaac cgcttttttg cacaacatgg gggatcatgt aactcgcctt gatcgttggg
     6181 aaccggagct gaatgaagcc ataccaaacg acgagcgtga caccacgatg cctgtagcaa
     6241 tggcaacaac gttgcgcaaa ctattaactg gcgaactact tactctagct tcccggcaac
     6301 aattaataga ctggatggag gcggataaag ttgcaggacc acttctgcgc tcggcccttc
     6361 cggctggctg gtttattgct gataaatctg gagccggtga gcgtgggtct cgcggtatca
     6421 ttgcagcact ggggccagat ggtaagccct cccgtatcgt agttatctac acgacgggga
     6481 gtcaggcaac tatggatgaa cgaaatagac agatcgctga gataggtgcc tcactgatt
//
