LOCUS       sequence-430752-        6812 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 6812)
  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 6812)
  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: AVPR1B-DuET
FEATURES             Location/Qualifiers
     source          1..6812
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     polyA_signal    26..147
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(37..56)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     91..110
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     CDS             complement(321..1115)
                     /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(432..451)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     1042..1061
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     promoter        complement(1182..1511)
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      1196..1331
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(1250..1269)
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     primer_bind     1496..1516
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      complement(1525..1953)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1635..1656)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     1847..1866
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     polyA_signal    1999..2223
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     2212..2229
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     primer_bind     complement(3750..3769)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(3751..3769)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        complement(3814..4017)
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     complement(3843..3863)
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     enhancer        4018..4397
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     primer_bind     4569..4588
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        4663..4767
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4768..4836,4837..5628)
                     /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(4986..5005)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5799..6387
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6288..6307
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6541..6558
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    6675..6696
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(6711..6716,6717..6734,6735..6741)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6749..6765
                     /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     6754..6776
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     6773..6789
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     6773..6789
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
ORIGIN
        1 agaggtcgac ggtatacaga catgataaga tacattgatg agtttggaca aaccacaact
       61 agaatgcagt gaaaaaaatg ctttatttgt gaaatttgtg atgctattgc tttatttgta
      121 accattataa gctgcaataa acaagttggg gtgggcgaag aactccagca tgagatcccc
      181 gcgctggagg atcatccagc cggcgtcccg gaaaacgatt ccgaagccca acctttcata
      241 gaaggcggcg gtggaatcga aatctcgtga tggcaggttg ggcgtcgctt ggtcggtcat
      301 ttcgaacccc agagtcccgc tcagaagaac tcgtcaagaa ggcgatagaa ggcgatgcgc
      361 tgcgaatcgg gagcggcgat accgtaaagc acgaggaagc ggtcagccca ttcgccgcca
      421 agctcttcag caatatcacg ggtagccaac gctatgtcct gatagcggtc cgccacaccc
      481 agccggccac agtcgatgaa tccagaaaag cggccatttt ccaccatgat attcggcaag
      541 caggcatcgc catgggtcac gacgagatcc tcgccgtcgg gcatgcgcgc cttgagcctg
      601 gcgaacagtt cggctggcgc gagcccctga tgctcttcgt ccagatcatc ctgatcgaca
      661 agaccggctt ccatccgagt acgtgctcgc tcgatgcgat gtttcgcttg gtggtcgaat
      721 gggcaggtag ccggatcaag cgtatgcagc cgccgcattg catcagccat gatggatact
      781 ttctcggcag gagcaaggtg agatgacagg agatcctgcc ccggcacttc gcccaatagc
      841 agccagtccc ttcccgcttc agtgacaacg tcgagcacag ctgcgcaagg aacgcccgtc
      901 gtggccagcc acgatagccg cgctgcctcg tcctgcagtt cattcagggc accggacagg
      961 tcggtcttga caaaaagaac cgggcgcccc tgcgctgaca gccggaacac ggcggcatca
     1021 gagcagccga ttgtctgttg tgcccagtca tagccgaata gcctctccac ccaagcggcc
     1081 ggagaacctg cgtgcaatcc atcttgttca atcatgcgaa acgatcctca tcctgtctct
     1141 tgatcagatc cgaaaatgga tatacaagct cccgggagct ttttgcaaaa gcctaggcct
     1201 ccaaaaaagc ctcctcacta cttctggaat agctcagagg cagaggcggc ctcggcctct
     1261 gcataaataa aaaaaattag tcagccatgg ggcggagaat gggcggaact gggcggagtt
     1321 aggggcggga tgggcggagt taggggcggg actatggttg ctgactaatt gagatgcatg
     1381 ctttgcatac ttctgcctgc tggggagcct ggggactttc cacacctggt tgctgactaa
     1441 ttgagatgca tgctttgcat acttctgcct gctggggagc ctggggactt tccacaccct
     1501 aactgacaca cattccacag aattaattcg cgttaaattt ttgttaaatc agctcatttt
     1561 ttaaccaata ggccgaaatc ggcaaaatcc cttataaatc aaaagaatag accgagatag
     1621 ggttgagtgt tgttccagtt tggaacaaga gtccactatt aaagaacgtg gactccaacg
     1681 tcaaagggcg aaaaaccgtc tatcagggcg atggcccact acgtgaacca tcaccctaat
     1741 caagtttttt ggggtcgagg tgccgtaaag cactaaatcg gaaccctaaa gggagccccc
     1801 gatttagagc ttgacgggga aagccggcga acgtggcgag aaaggaaggg aagaaagcga
     1861 aaggagcggg cgctagggcg ctggcaagtg tagcggtcac gctgcgcgta accaccacac
     1921 ccgccgcgct taatgcgccg ctacagggcg cgtggggata ccccctagag ccccagctgg
     1981 ttctttccgc ctcagaagcc atagagccca ccgcatcccc agcatgcctg ctattgtctt
     2041 cccaatcctc ccccttgctg tcctgcccca ccccaccccc cagaatagaa tgacacctac
     2101 tcagacaatg cgatgcaatt tcctcatttt attaggaaag gacagtggga gtggcacctt
     2161 ccagggtcaa ggaaggcacg ggggaggggc aaacaacaga tggctggcaa ctagaaggca
     2221 cagtcgaggc tgatcagcgg gtttaaacgg gccctctaga ctcgagttag gcaggggcca
     2281 cctgagaagt ctcggtctta gacacggtgg tgctggcctc atcgaattca ccggtgcgtc
     2341 caccggtatc gataaaaatg attgtctctg ccgtgccctc tccatctgca agctccaggt
     2401 ctctcgggga ctcttcgggg cgcggcctgc cactcagggt aagggacaga ctcagggaga
     2461 gggtagcggg acaggatgag cgggtcagca aagtggtgtg gcgagaggac aggcttccgt
     2521 cgctgaggcg tctccgcatc ctaggctgtg gcccaccgca gcatgccaga tgtcggaggg
     2581 gtctggggag cagatgagaa ttaaatccca tataaatcca tgggttacaa caactattca
     2641 ggtttcccaa cagcatggag attgtgaagg cgacattggt actgtcctcg tccggagcat
     2701 tcttgtccca cacagaccac atctgcacgg agaaaaaggg ggcccagcaa gcaatgtagg
     2761 ccagtacgat cacgaaggtc atcttcacgg tgcgaatttt ggccctggag atagtgttaa
     2821 tggaggacac cctggacggc agtccccgtg tggtagcagc cagggtggat gggcttgggc
     2881 gatcccatgt ccgccaacct ccaccaccta cgcgccacgc ttgtgttttc actttcagat
     2941 tcttgcaaat ttcatggcag atcagactgt aacatgcggt cagcatggtc acaggcagaa
     3001 cgaatatggc gagtgtagtc caggtcaggt aggcccgcgg gccccacgga aacccaaaat
     3061 cagcccagca gtccagaacg cctgaaccct gaatgacctc cctcaaagag aagatgaaga
     3121 cctgaggcag agagaaaatg gccgccagca accagggcgc agcgatcagg agatacgtgc
     3181 tctgcccggg ttgctgcagg ctgcgcaggg ggtggcatac ggcaaggtac cgatcgaggg
     3241 tcatagccaa gagcatgtag gtggaggcaa acatggaaag gacctgcaga tactttacag
     3301 cccggcagag cagatcaggt ccctggaacc gatatgttat gtcccacagc agttggggca
     3361 gcacctggaa gagggccact gcgagatctg tcagtgccag gtgcagcacg aaaaggtgca
     3421 ttcgtgacct ctttctcccc agttggccaa gagtcagaag aacggccaga ttcccgccag
     3481 tagccaacac cagaacagtg gcaagaaccc cgatctctac ttttgccagt tcctcatcgc
     3541 ggcccagcca tggggtagtt gcattgggcg cgctcagggt cccccgcggg gtagggtttg
     3601 catcccagag agggccagag tccatatcga tgctggcgtc atcatcgtcc ttgtagtcgg
     3661 cgaataccag gcagaagatg tagctcaggg cgatgatcgt cttcatggtg gcgctaagct
     3721 taagtttaaa cgctagccag cttgggtctc cctatagtga gtcgtattaa tttcgataag
     3781 ccagtaagca gtgggttctc tagttagcca gagagctctg cttatataga cctcccaccg
     3841 tacacgccta ccgcccattt gcgtcaatgg ggcggagttg ttacgacatt ttggaaagtc
     3901 ccgttgattt tggtgccaaa acaaactccc attgacgtca atggggtgga gacttggaaa
     3961 tccccgtgag tcaaaccgct atccacgccc attgatgtac tgccaaaacc gcatcaccat
     4021 ggtaatagcg atgactaata cgtagatgta ctgccaagta ggaaagtccc ataaggtcat
     4081 gtactgggca taatgccagg cgggccattt accgtcattg acgtcaatag ggggcgtact
     4141 tggcatatga tacacttgat gtactgccaa gtgggcagtt taccgtaaat actccaccca
     4201 ttgacgtcaa tggaaagtcc ctattggcgt tactatggga acatacgtca ttattgacgt
     4261 caatgggcgg gggtcgttgg gcggtcagcc aggcgggcca tttaccgtaa gttatgtaac
     4321 gcggaactcc atatatgggc tatgaactaa tgaccccgta attgattact attaataact
     4381 agtcaataat caatgtcaac gcgtatatct ggcccgtaca tcgcgaagca gcgcaaaacg
     4441 cctaacccta agcagattct tcatgcaaat gtcggtcaag ccttgccttg ttgtagctta
     4501 aattttgctc gcgcactact cagcgacctc caacacacaa gcagggagca gatactggct
     4561 taactatgcg gcatcagagc agattgtact gagagtgcac cataggggat cgggagatct
     4621 cccgatccgt cgacgtcagg tggcactttt cggggaaatg tgcgcggaac ccctatttgt
     4681 ttatttttct aaatacattc aaatatgtat ccgctcatga gacaataacc ctgataaatg
     4741 cttcaataat attgaaaaag gaagagtatg agtattcaac atttccgtgt cgcccttatt
     4801 cccttttttg cggcattttg ccttcctgtt tttgctcacc cagaaacgct ggtgaaagta
     4861 aaagatgctg aagatcagtt gggtgcacga gtgggttaca tcgaactgga tctcaacagc
     4921 ggtaagatcc ttgagagttt tcgccccgaa gaacgttttc caatgatgag cacttttaaa
     4981 gttctgctat gtggcgcggt attatcccgt attgacgccg ggcaagagca actcggtcgc
     5041 cgcatacact attctcagaa tgacttggtt gagtactcac cagtcacaga aaagcatctt
     5101 acggatggca tgacagtaag agaattatgc agtgctgcca taaccatgag tgataacact
     5161 gcggccaact tacttctgac aacgatcgga ggaccgaagg agctaaccgc ttttttgcac
     5221 aacatggggg atcatgtaac tcgccttgat cgttgggaac cggagctgaa tgaagccata
     5281 ccaaacgacg agcgtgacac cacgatgcct gtagcaatgg caacaacgtt gcgcaaacta
     5341 ttaactggcg aactacttac tctagcttcc cggcaacaat taatagactg gatggaggcg
     5401 gataaagttg caggaccact tctgcgctcg gcccttccgg ctggctggtt tattgctgat
     5461 aaatctggag ccggtgagcg tgggtctcgc ggtatcattg cagcactggg gccagatggt
     5521 aagccctccc gtatcgtagt tatctacacg acggggagtc aggcaactat ggatgaacga
     5581 aatagacaga tcgctgagat aggtgcctca ctgattaagc attggtaact gtcagaccaa
     5641 gtttactcat atatacttta gattgattta aaacttcatt tttaatttaa aaggatctag
     5701 gtgaagatcc tttttgataa tctcatgacc aaaatccctt aacgtgagtt ttcgttccac
     5761 tgagcgtcag accccgtaga aaagatcaaa ggatcttctt gagatccttt ttttctgcgc
     5821 gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg tttgccggat
     5881 caagagctac caactctttt tccgaaggta actggcttca gcagagcgca gataccaaat
     5941 accgttcttc tagtgtagcc gtagttaggc caccacttca agaactctgt agcaccgcct
     6001 acatacctcg ctctgctaat cctgttacca gtggctgctg ccagtggcga taagtcgtgt
     6061 cttaccgggt tggactcaag acgatagtta ccggataagg cgcagcggtc gggctgaacg
     6121 gggggttcgt gcacacagcc cagcttggag cgaacgacct acaccgaact gagataccta
     6181 cagcgtgagc tatgagaaag cgccacgctt cccgaaggga gaaaggcgga caggtatccg
     6241 gtaagcggca gggtcggaac aggagagcgc acgagggagc ttccaggggg aaacgcctgg
     6301 tatctttata gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc
     6361 tcgtcagggg ggcggagcct atggaaaaac gccagcaacg cggccttttt acggttcctg
     6421 gccttttgct ggccttttgc tcacatgttc tttcctgcgt tatcccctga ttctgtggat
     6481 aaccgtatta ccgcctttga gtgagctgat accgctcgcc gcagccgaac gaccgagcgc
     6541 agcgagtcag tgagcgagga agcggaagag cgcccaatac gcaaaccgcc tctccccgcg
     6601 cgttggccga ttcattaatg cagctggcac gacaggtttc ccgactggaa agcgggcagt
     6661 gagcgcaacg caattaatgt gagttagctc actcattagg caccccaggc tttacacttt
     6721 atgcttccgg ctcgtatgtt gtgtggaatt gtgagcggat aacaatttca cacaggaaac
     6781 agctatgacc atgattacgc caagctctag ct
//
