LOCUS       sequence-430852-        6710 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 6710)
  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 6710)
  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: HRH4-DuET
FEATURES             Location/Qualifiers
     source          1..6710
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(235..252)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    241..465
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      511..939
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(598..617)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     808..829
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(948..968)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        953..1282
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      1133..1268
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     1195..1214
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             1349..2143
                     /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(1403..1422)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     2013..2032
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    2317..2438
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2354..2373)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2408..2427
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(2487..2503)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2487..2503)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2500..2522)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2511..2527
                     /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(2535..2541,2542..2559,2560..2565))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2580..2601
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2718..2735)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2889..3477)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2969..2988)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3648..4439,4440..4508))
                     /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     4271..4290
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4509..4613)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(4688..4707)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        4879..5258
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        5259..5462
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     5413..5433
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     5507..5526
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        5507..5525
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
ORIGIN
        1 atcctctcct gtaccccctg tgtcacaagc gctttcagaa ggccttcttg aagatcttct
       61 gcatcaagaa acagccactg cccagtcagc actcaagatc agtctctagc atcgataccg
      121 gtggacgcac cggtgaattc gatgaggcca gcaccaccgt gtctaagacc gagacttctc
      181 aggtggcccc tgcctaactc gagtctagag ggcccgttta aacccgctga tcagcctcga
      241 ctgtgccttc tagttgccag ccatctgttg tttgcccctc ccccgtgcct tccttgaccc
      301 tggaaggtgc cactcccact gtcctttcct aataaaatga ggaaattgca tcgcattgtc
      361 tgagtaggtg tcattctatt ctggggggtg gggtggggca ggacagcaag ggggaggatt
      421 gggaagacaa tagcaggcat gctggggatg cggtgggctc tatggcttct gaggcggaaa
      481 gaaccagctg gggctctagg gggtatcccc acgcgccctg tagcggcgca ttaagcgcgg
      541 cgggtgtggt ggttacgcgc agcgtgaccg ctacacttgc cagcgcccta gcgcccgctc
      601 ctttcgcttt cttcccttcc tttctcgcca cgttcgccgg ctttccccgt caagctctaa
      661 atcgggggct ccctttaggg ttccgattta gtgctttacg gcacctcgac cccaaaaaac
      721 ttgattaggg tgatggttca cgtagtgggc catcgccctg atagacggtt tttcgccctt
      781 tgacgttgga gtccacgttc tttaatagtg gactcttgtt ccaaactgga acaacactca
      841 accctatctc ggtctattct tttgatttat aagggatttt gccgatttcg gcctattggt
      901 taaaaaatga gctgatttaa caaaaattta acgcgaatta attctgtgga atgtgtgtca
      961 gttagggtgt ggaaagtccc caggctcccc agcaggcaga agtatgcaaa gcatgcatct
     1021 caattagtca gcaaccaggt gtggaaagtc cccaggctcc ccagcaggca gaagtatgca
     1081 aagcatgcat ctcaattagt cagcaaccat agtcccgccc ctaactccgc ccatcccgcc
     1141 cctaactccg cccagttccg cccattctcc gccccatggc tgactaattt tttttattta
     1201 tgcagaggcc gaggccgcct ctgcctctga gctattccag aagtagtgag gaggcttttt
     1261 tggaggccta ggcttttgca aaaagctccc gggagcttgt atatccattt tcggatctga
     1321 tcaagagaca ggatgaggat cgtttcgcat gattgaacaa gatggattgc acgcaggttc
     1381 tccggccgct tgggtggaga ggctattcgg ctatgactgg gcacaacaga caatcggctg
     1441 ctctgatgcc gccgtgttcc ggctgtcagc gcaggggcgc ccggttcttt ttgtcaagac
     1501 cgacctgtcc ggtgccctga atgaactgca ggacgaggca gcgcggctat cgtggctggc
     1561 cacgacgggc gttccttgcg cagctgtgct cgacgttgtc actgaagcgg gaagggactg
     1621 gctgctattg ggcgaagtgc cggggcagga tctcctgtca tctcaccttg ctcctgccga
     1681 gaaagtatcc atcatggctg atgcaatgcg gcggctgcat acgcttgatc cggctacctg
     1741 cccattcgac caccaagcga aacatcgcat cgagcgagca cgtactcgga tggaagccgg
     1801 tcttgtcgat caggatgatc tggacgaaga gcatcagggg ctcgcgccag ccgaactgtt
     1861 cgccaggctc aaggcgcgca tgcccgacgg cgaggatctc gtcgtgaccc atggcgatgc
     1921 ctgcttgccg aatatcatgg tggaaaatgg ccgcttttct ggattcatcg actgtggccg
     1981 gctgggtgtg gcggaccgct atcaggacat agcgttggct acccgtgata ttgctgaaga
     2041 gcttggcggc gaatgggctg accgcttcct cgtgctttac ggtatcgccg ctcccgattc
     2101 gcagcgcatc gccttctatc gccttcttga cgagttcttc tgagcgggac tctggggttc
     2161 gaaatgaccg accaagcgac gcccaacctg ccatcacgag atttcgattc caccgccgcc
     2221 ttctatgaaa ggttgggctt cggaatcgtt ttccgggacg ccggctggat gatcctccag
     2281 cgcggggatc tcatgctgga gttcttcgcc caccccaact tgtttattgc agcttataat
     2341 ggttacaaat aaagcaatag catcacaaat ttcacaaata aagcattttt ttcactgcat
     2401 tctagttgtg gtttgtccaa actcatcaat gtatcttatc atgtctgtat accgtcgacc
     2461 tctagctaga gcttggcgta atcatggtca tagctgtttc ctgtgtgaaa ttgttatccg
     2521 ctcacaattc cacacaacat acgagccgga agcataaagt gtaaagcctg gggtgcctaa
     2581 tgagtgagct aactcacatt aattgcgttg cgctcactgc ccgctttcca gtcgggaaac
     2641 ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt
     2701 gggcgctctt ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg gctgcggcga
     2761 gcggtatcag ctcactcaaa ggcggtaata cggttatcca cagaatcagg ggataacgca
     2821 ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa ggccgcgttg
     2881 ctggcgtttt tccataggct ccgcccccct gacgagcatc acaaaaatcg acgctcaagt
     2941 cagaggtggc gaaacccgac aggactataa agataccagg cgtttccccc tggaagctcc
     3001 ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat acctgtccgc ctttctccct
     3061 tcgggaagcg tggcgctttc tcatagctca cgctgtaggt atctcagttc ggtgtaggtc
     3121 gttcgctcca agctgggctg tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta
     3181 tccggtaact atcgtcttga gtccaacccg gtaagacacg acttatcgcc actggcagca
     3241 gccactggta acaggattag cagagcgagg tatgtaggcg gtgctacaga gttcttgaag
     3301 tggtggccta actacggcta cactagaaga acggtatttg gtatctgcgc tctgctgaag
     3361 ccagttacct tcggaaaaag agttggtagc tcttgatccg gcaaacaaac caccgctggt
     3421 agcggtggtt tttttgtttg caagcagcag attacgcgca gaaaaaaagg atctcaagaa
     3481 gatcctttga tcttttctac ggggtctgac gctcagtgga acgaaaactc acgttaaggg
     3541 attttggtca tgagattatc aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga
     3601 agttttaaat caatctaaag tatatatgag taaacttggt ctgacagtta ccaatgctta
     3661 atcagtgagg cacctatctc agcgatctgt ctatttcgtt catccatagt tgcctgactc
     3721 cccgtcgtgt agataactac gatacgggag ggcttaccat ctggccccag tgctgcaatg
     3781 ataccgcgag acccacgctc accggctcca gatttatcag caataaacca gccagccgga
     3841 agggccgagc gcagaagtgg tcctgcaact ttatccgcct ccatccagtc tattaattgt
     3901 tgccgggaag ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt
     3961 gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag ctccggttcc
     4021 caacgatcaa ggcgagttac atgatccccc atgttgtgca aaaaagcggt tagctccttc
     4081 ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt tatcactcat ggttatggca
     4141 gcactgcata attctcttac tgtcatgcca tccgtaagat gcttttctgt gactggtgag
     4201 tactcaacca agtcattctg agaatagtgt atgcggcgac cgagttgctc ttgcccggcg
     4261 tcaatacggg ataataccgc gccacatagc agaactttaa aagtgctcat cattggaaaa
     4321 cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa
     4381 cccactcgtg cacccaactg atcttcagca tcttttactt tcaccagcgt ttctgggtga
     4441 gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg gaaatgttga
     4501 atactcatac tcttcctttt tcaatattat tgaagcattt atcagggtta ttgtctcatg
     4561 agcggataca tatttgaatg tatttagaaa aataaacaaa taggggttcc gcgcacattt
     4621 ccccgaaaag tgccacctga cgtcgacgga tcgggagatc tcccgatccc ctatggtgca
     4681 ctctcagtac aatctgctct gatgccgcat agttaagcca gtatctgctc cctgcttgtg
     4741 tgttggaggt cgctgagtag tgcgcgagca aaatttaagc tacaacaagg caaggcttga
     4801 ccgacatttg catgaagaat ctgcttaggg ttaggcgttt tgcgctgctt cgcgatgtac
     4861 gggccagata tacgcgttga cattgattat tgactagtta ttaatagtaa tcaattacgg
     4921 ggtcattagt tcatagccca tatatggagt tccgcgttac ataacttacg gtaaatggcc
     4981 cgcctggctg accgcccaac gacccccgcc cattgacgtc aataatgacg tatgttccca
     5041 tagtaacgcc aatagggact ttccattgac gtcaatgggt ggagtattta cggtaaactg
     5101 cccacttggc agtacatcaa gtgtatcata tgccaagtac gccccctatt gacgtcaatg
     5161 acggtaaatg gcccgcctgg cattatgccc agtacatgac cttatgggac tttcctactt
     5221 ggcagtacat ctacgtatta gtcatcgcta ttaccatggt gatgcggttt tggcagtaca
     5281 tcaatgggcg tggatagcgg tttgactcac ggggatttcc aagtctccac cccattgacg
     5341 tcaatgggag tttgttttgg caccaaaatc aacgggactt tccaaaatgt cgtaacaact
     5401 ccgccccatt gacgcaaatg ggcggtaggc gtgtacggtg ggaggtctat ataagcagag
     5461 ctctctggct aactagagaa cccactgctt actggcttat cgaaattaat acgactcact
     5521 atagggagac ccaagctggc tagcgtttaa acttaagctt agcgccacca tgaagacgat
     5581 catcgccctg agctacatct tctgcctggt attcgccgac tacaaggacg atgatgacgc
     5641 cagcatcgat atgccagata ctaatagcac aatcaacctg tccctgtcca ctcgggtgac
     5701 cctggcgttc tttatgagtc tggtagcatt cgctatcatg ctgggaaatg ccctggtcat
     5761 tctggccttc gttgtcgaca aaaacctgcg acatcgaagt agttactttt ttttgaattt
     5821 ggcaatcagc gatttcttcg tgggcgtgat cagcatccct ttgtatattc ctcatacact
     5881 gttcgaatgg gattttggca aggaaatttg tgttttttgg ctgaccacag attatctttt
     5941 gtgtactgcc tcagtgtaca acatcgtgct tatctcttat gacaggtatt tgtccgtctc
     6001 taatgcggtc agttacagaa cccagcacac cggcgtgttg aaaatcgtga ctttgatggt
     6061 agccgtgtgg gtgctggcct tcctggttaa tgggccaatg atcctggtga gtgagtcttg
     6121 gaaagatgag ggatccgaat gcgagccagg cttcttttct gagtggtaca ttctggccat
     6181 cacgagtttt cttgaatttg taattcccgt catcttggtg gcttacttta atatgaacat
     6241 ttactggagt ctgtggaaga gagaccacct gagtcgctgt caaagtcacc ctggccttac
     6301 agcggtttcc tctaacatct gcggccattc ttttcgcggc aggctgtcat ctaggcggtc
     6361 cctcagcgcc agtaccgagg tgccagccag ctttcattcc gaaaggcaga gacgaaagag
     6421 tagcctgatg ttcagttctc ggactaagat gaatagtaac accatagcct ccaagatggg
     6481 cagcttctcc caatccgact ctgtcgcact tcatcaaaga gagcacgtgg agttgctccg
     6541 ggccagacgc ttggccaaat ccttggcgat cctgttggga gttttcgcag tgtgctgggc
     6601 accatatagt ctgttcacaa ttgtacttag cttctatagc agcgccaccg ggccaaaaag
     6661 tgtttggtac agaatcgcat tctggttgca gtggttcaac tcctttgtga
//
