LOCUS       sequence-430737-        6557 bp    DNA     circular SYN 14-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 6557)
  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 6557)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 14, 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: TA1-DuET
FEATURES             Location/Qualifiers
     source          1..6557
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(542..561)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(543..561)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        complement(606..809)
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     complement(635..655)
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     enhancer        810..1189
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     primer_bind     1361..1380
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        1455..1559
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1560..1628,1629..2420)
                     /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(1778..1797)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2591..3179
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3080..3099
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3333..3350
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    3467..3488
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(3503..3508,3509..3526,3527..3533)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3541..3557
                     /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     3546..3568
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     3565..3581
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     3565..3581
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     polyA_signal    3630..3751
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3641..3660)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     3695..3714
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     CDS             complement(3925..4719)
                     /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(4036..4055)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     4646..4665
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     promoter        complement(4786..5115)
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      4800..4935
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(4854..4873)
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     primer_bind     5100..5120
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      complement(5129..5557)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5239..5260)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5451..5470
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     polyA_signal    5603..5827
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     5816..5833
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
ORIGIN
        1 cacaaggata ttcatctttg ctttgtatcg cagagggtcg caaacggcgt agtatctgtc
       61 tatactgata aaactcaggt gaaagattga agcgctgctg agcatgatat ccgtagacgt
      121 gtgaattttg cagaacactt cgccgaaata ccaacagtgc tcggcagagc ggaccatgct
      181 gtagggcatg acgaggcagc caagaaggaa atcgacggtg gccatggagt ggatgagcca
      241 atttgttgga gtatgcagct gcttgaaatg agagatgctg acaatcacaa tgagattgcc
      301 aaccaaagtg gtaagaatta tcaagaccat gaggctgtac agggaagccc tcacgtcatt
      361 agaccaattg tttttcacgc aggaaatgtt gatgatatta tgacaaaaag gcatcatatc
      421 gatgctggcg tcatcatcgt ccttgtagtc ggcgaatacc aggcagaaga tgtagctcag
      481 ggcgatgatc gtcttcatgg tggcgctaag cttaagttta aacgctagcc agcttgggtc
      541 tccctatagt gagtcgtatt aatttcgata agccagtaag cagtgggttc tctagttagc
      601 cagagagctc tgcttatata gacctcccac cgtacacgcc taccgcccat ttgcgtcaat
      661 ggggcggagt tgttacgaca ttttggaaag tcccgttgat tttggtgcca aaacaaactc
      721 ccattgacgt caatggggtg gagacttgga aatccccgtg agtcaaaccg ctatccacgc
      781 ccattgatgt actgccaaaa ccgcatcacc atggtaatag cgatgactaa tacgtagatg
      841 tactgccaag taggaaagtc ccataaggtc atgtactggg cataatgcca ggcgggccat
      901 ttaccgtcat tgacgtcaat agggggcgta cttggcatat gatacacttg atgtactgcc
      961 aagtgggcag tttaccgtaa atactccacc cattgacgtc aatggaaagt ccctattggc
     1021 gttactatgg gaacatacgt cattattgac gtcaatgggc gggggtcgtt gggcggtcag
     1081 ccaggcgggc catttaccgt aagttatgta acgcggaact ccatatatgg gctatgaact
     1141 aatgaccccg taattgatta ctattaataa ctagtcaata atcaatgtca acgcgtatat
     1201 ctggcccgta catcgcgaag cagcgcaaaa cgcctaaccc taagcagatt cttcatgcaa
     1261 atgtcggtca agccttgcct tgttgtagct taaattttgc tcgcgcacta ctcagcgacc
     1321 tccaacacac aagcagggag cagatactgg cttaactatg cggcatcaga gcagattgta
     1381 ctgagagtgc accatagggg atcgggagat ctcccgatcc gtcgacgtca ggtggcactt
     1441 ttcggggaaa tgtgcgcgga acccctattt gtttattttt ctaaatacat tcaaatatgt
     1501 atccgctcat gagacaataa ccctgataaa tgcttcaata atattgaaaa aggaagagta
     1561 tgagtattca acatttccgt gtcgccctta ttcccttttt tgcggcattt tgccttcctg
     1621 tttttgctca cccagaaacg ctggtgaaag taaaagatgc tgaagatcag ttgggtgcac
     1681 gagtgggtta catcgaactg gatctcaaca gcggtaagat ccttgagagt tttcgccccg
     1741 aagaacgttt tccaatgatg agcactttta aagttctgct atgtggcgcg gtattatccc
     1801 gtattgacgc cgggcaagag caactcggtc gccgcataca ctattctcag aatgacttgg
     1861 ttgagtactc accagtcaca gaaaagcatc ttacggatgg catgacagta agagaattat
     1921 gcagtgctgc cataaccatg agtgataaca ctgcggccaa cttacttctg acaacgatcg
     1981 gaggaccgaa ggagctaacc gcttttttgc acaacatggg ggatcatgta actcgccttg
     2041 atcgttggga accggagctg aatgaagcca taccaaacga cgagcgtgac accacgatgc
     2101 ctgtagcaat ggcaacaacg ttgcgcaaac tattaactgg cgaactactt actctagctt
     2161 cccggcaaca attaatagac tggatggagg cggataaagt tgcaggacca cttctgcgct
     2221 cggcccttcc ggctggctgg tttattgctg ataaatctgg agccggtgag cgtgggtctc
     2281 gcggtatcat tgcagcactg gggccagatg gtaagccctc ccgtatcgta gttatctaca
     2341 cgacggggag tcaggcaact atggatgaac gaaatagaca gatcgctgag ataggtgcct
     2401 cactgattaa gcattggtaa ctgtcagacc aagtttactc atatatactt tagattgatt
     2461 taaaacttca tttttaattt aaaaggatct aggtgaagat cctttttgat aatctcatga
     2521 ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc agaccccgta gaaaagatca
     2581 aaggatcttc ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac
     2641 caccgctacc agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg
     2701 taactggctt cagcagagcg cagataccaa ataccgttct tctagtgtag ccgtagttag
     2761 gccaccactt caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac
     2821 cagtggctgc tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt
     2881 taccggataa ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg
     2941 agcgaacgac ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc
     3001 ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc
     3061 gcacgaggga gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc
     3121 acctctgact tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa
     3181 acgccagcaa cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt
     3241 tctttcctgc gttatcccct gattctgtgg ataaccgtat taccgccttt gagtgagctg
     3301 ataccgctcg ccgcagccga acgaccgagc gcagcgagtc agtgagcgag gaagcggaag
     3361 agcgcccaat acgcaaaccg cctctccccg cgcgttggcc gattcattaa tgcagctggc
     3421 acgacaggtt tcccgactgg aaagcgggca gtgagcgcaa cgcaattaat gtgagttagc
     3481 tcactcatta ggcaccccag gctttacact ttatgcttcc ggctcgtatg ttgtgtggaa
     3541 ttgtgagcgg ataacaattt cacacaggaa acagctatga ccatgattac gccaagctct
     3601 agctagaggt cgacggtata cagacatgat aagatacatt gatgagtttg gacaaaccac
     3661 aactagaatg cagtgaaaaa aatgctttat ttgtgaaatt tgtgatgcta ttgctttatt
     3721 tgtaaccatt ataagctgca ataaacaagt tggggtgggc gaagaactcc agcatgagat
     3781 ccccgcgctg gaggatcatc cagccggcgt cccggaaaac gattccgaag cccaaccttt
     3841 catagaaggc ggcggtggaa tcgaaatctc gtgatggcag gttgggcgtc gcttggtcgg
     3901 tcatttcgaa ccccagagtc ccgctcagaa gaactcgtca agaaggcgat agaaggcgat
     3961 gcgctgcgaa tcgggagcgg cgataccgta aagcacgagg aagcggtcag cccattcgcc
     4021 gccaagctct tcagcaatat cacgggtagc caacgctatg tcctgatagc ggtccgccac
     4081 acccagccgg ccacagtcga tgaatccaga aaagcggcca ttttccacca tgatattcgg
     4141 caagcaggca tcgccatggg tcacgacgag atcctcgccg tcgggcatgc gcgccttgag
     4201 cctggcgaac agttcggctg gcgcgagccc ctgatgctct tcgtccagat catcctgatc
     4261 gacaagaccg gcttccatcc gagtacgtgc tcgctcgatg cgatgtttcg cttggtggtc
     4321 gaatgggcag gtagccggat caagcgtatg cagccgccgc attgcatcag ccatgatgga
     4381 tactttctcg gcaggagcaa ggtgagatga caggagatcc tgccccggca cttcgcccaa
     4441 tagcagccag tcccttcccg cttcagtgac aacgtcgagc acagctgcgc aaggaacgcc
     4501 cgtcgtggcc agccacgata gccgcgctgc ctcgtcctgc agttcattca gggcaccgga
     4561 caggtcggtc ttgacaaaaa gaaccgggcg cccctgcgct gacagccgga acacggcggc
     4621 atcagagcag ccgattgtct gttgtgccca gtcatagccg aatagcctct ccacccaagc
     4681 ggccggagaa cctgcgtgca atccatcttg ttcaatcatg cgaaacgatc ctcatcctgt
     4741 ctcttgatca gatccgaaaa tggatataca agctcccggg agctttttgc aaaagcctag
     4801 gcctccaaaa aagcctcctc actacttctg gaatagctca gaggcagagg cggcctcggc
     4861 ctctgcataa ataaaaaaaa ttagtcagcc atggggcgga gaatgggcgg aactgggcgg
     4921 agttaggggc gggatgggcg gagttagggg cgggactatg gttgctgact aattgagatg
     4981 catgctttgc atacttctgc ctgctgggga gcctggggac tttccacacc tggttgctga
     5041 ctaattgaga tgcatgcttt gcatacttct gcctgctggg gagcctgggg actttccaca
     5101 ccctaactga cacacattcc acagaattaa ttcgcgttaa atttttgtta aatcagctca
     5161 ttttttaacc aataggccga aatcggcaaa atcccttata aatcaaaaga atagaccgag
     5221 atagggttga gtgttgttcc agtttggaac aagagtccac tattaaagaa cgtggactcc
     5281 aacgtcaaag ggcgaaaaac cgtctatcag ggcgatggcc cactacgtga accatcaccc
     5341 taatcaagtt ttttggggtc gaggtgccgt aaagcactaa atcggaaccc taaagggagc
     5401 ccccgattta gagcttgacg gggaaagccg gcgaacgtgg cgagaaagga agggaagaaa
     5461 gcgaaaggag cgggcgctag ggcgctggca agtgtagcgg tcacgctgcg cgtaaccacc
     5521 acacccgccg cgcttaatgc gccgctacag ggcgcgtggg gataccccct agagccccag
     5581 ctggttcttt ccgcctcaga agccatagag cccaccgcat ccccagcatg cctgctattg
     5641 tcttcccaat cctccccctt gctgtcctgc cccaccccac cccccagaat agaatgacac
     5701 ctactcagac aatgcgatgc aatttcctca ttttattagg aaaggacagt gggagtggca
     5761 ccttccaggg tcaaggaagg cacgggggag gggcaaacaa cagatggctg gcaactagaa
     5821 ggcacagtcg aggctgatca gcgggtttaa acgggccctc tagactcgag ttaggcaggg
     5881 gccacctgag aagtctcggt cttagacacg gtggtgctgg cctcatcgaa ttcaccggtg
     5941 cgtccaccgg tatcgatgct agacaattcg agaaagagtt tacaccgact agaatccttc
     6001 tggaaaatct tgccgaacag catcattttc agcgcctttc gaaaccaagg gtagaagaaa
     6061 gcgtacacca taggattgaa cgtagaattc agatagccga accagataag cacgtcgttc
     6121 agggtgggcg gtataatgta gtgcaggaaa gggtccatga cagtacaaat gaagaaagga
     6181 caccaacata tcagaaagac gcccatgacg atgcccagcg ttttaacagc ttttctctcc
     6241 ttagactggg agatgccgtt tttcatttcc aaaccaattt gcagcttctg gttagcgtcg
     6301 ctgatcaacc gtgcctgctc cttagctatg agatagatcc tataatagac gcacagcatg
     6361 attgagcccg ggatgtaaaa ggaagtcatg aacgtcagga cgccgctgat tttactaaaa
     6421 aacacggagc atccaccccg acagtggaca tgcttatagt aaatctcctc ggctccttta
     6481 aagttgagtt ccaggaagat catgccaaat gcgaataccg ctgggacgct ccaggagata
     6541 aatatcatca cacaaat
//
