LOCUS       sequence-430766-        6674 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 6674)
  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 6674)
  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: CCR7-DuET
FEATURES             Location/Qualifiers
     source          1..6674
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(56..75)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(57..75)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        complement(120..323)
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     complement(149..169)
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     enhancer        324..703
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     primer_bind     875..894
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        969..1073
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1074..1142,1143..1934)
                     /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(1292..1311)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2105..2693
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2594..2613
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2847..2864
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    2981..3002
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(3017..3022,3023..3040,3041..3047)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3055..3071
                     /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     3060..3082
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     3079..3095
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     3079..3095
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     polyA_signal    3144..3265
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3155..3174)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     3209..3228
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     CDS             complement(3439..4233)
                     /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(3550..3569)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     4160..4179
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     promoter        complement(4300..4629)
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      4314..4449
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(4368..4387)
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     primer_bind     4614..4634
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      complement(4643..5071)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4753..4774)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     4965..4984
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     polyA_signal    5117..5341
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     5330..5347
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
ORIGIN
        1 gatcgtcttc atggtggcgc taagcttaag tttaaacgct agccagcttg ggtctcccta
       61 tagtgagtcg tattaatttc gataagccag taagcagtgg gttctctagt tagccagaga
      121 gctctgctta tatagacctc ccaccgtaca cgcctaccgc ccatttgcgt caatggggcg
      181 gagttgttac gacattttgg aaagtcccgt tgattttggt gccaaaacaa actcccattg
      241 acgtcaatgg ggtggagact tggaaatccc cgtgagtcaa accgctatcc acgcccattg
      301 atgtactgcc aaaaccgcat caccatggta atagcgatga ctaatacgta gatgtactgc
      361 caagtaggaa agtcccataa ggtcatgtac tgggcataat gccaggcggg ccatttaccg
      421 tcattgacgt caataggggg cgtacttggc atatgataca cttgatgtac tgccaagtgg
      481 gcagtttacc gtaaatactc cacccattga cgtcaatgga aagtccctat tggcgttact
      541 atgggaacat acgtcattat tgacgtcaat gggcgggggt cgttgggcgg tcagccaggc
      601 gggccattta ccgtaagtta tgtaacgcgg aactccatat atgggctatg aactaatgac
      661 cccgtaattg attactatta ataactagtc aataatcaat gtcaacgcgt atatctggcc
      721 cgtacatcgc gaagcagcgc aaaacgccta accctaagca gattcttcat gcaaatgtcg
      781 gtcaagcctt gccttgttgt agcttaaatt ttgctcgcgc actactcagc gacctccaac
      841 acacaagcag ggagcagata ctggcttaac tatgcggcat cagagcagat tgtactgaga
      901 gtgcaccata ggggatcggg agatctcccg atccgtcgac gtcaggtggc acttttcggg
      961 gaaatgtgcg cggaacccct atttgtttat ttttctaaat acattcaaat atgtatccgc
     1021 tcatgagaca ataaccctga taaatgcttc aataatattg aaaaaggaag agtatgagta
     1081 ttcaacattt ccgtgtcgcc cttattccct tttttgcggc attttgcctt cctgtttttg
     1141 ctcacccaga aacgctggtg aaagtaaaag atgctgaaga tcagttgggt gcacgagtgg
     1201 gttacatcga actggatctc aacagcggta agatccttga gagttttcgc cccgaagaac
     1261 gttttccaat gatgagcact tttaaagttc tgctatgtgg cgcggtatta tcccgtattg
     1321 acgccgggca agagcaactc ggtcgccgca tacactattc tcagaatgac ttggttgagt
     1381 actcaccagt cacagaaaag catcttacgg atggcatgac agtaagagaa ttatgcagtg
     1441 ctgccataac catgagtgat aacactgcgg ccaacttact tctgacaacg atcggaggac
     1501 cgaaggagct aaccgctttt ttgcacaaca tgggggatca tgtaactcgc cttgatcgtt
     1561 gggaaccgga gctgaatgaa gccataccaa acgacgagcg tgacaccacg atgcctgtag
     1621 caatggcaac aacgttgcgc aaactattaa ctggcgaact acttactcta gcttcccggc
     1681 aacaattaat agactggatg gaggcggata aagttgcagg accacttctg cgctcggccc
     1741 ttccggctgg ctggtttatt gctgataaat ctggagccgg tgagcgtggg tctcgcggta
     1801 tcattgcagc actggggcca gatggtaagc cctcccgtat cgtagttatc tacacgacgg
     1861 ggagtcaggc aactatggat gaacgaaata gacagatcgc tgagataggt gcctcactga
     1921 ttaagcattg gtaactgtca gaccaagttt actcatatat actttagatt gatttaaaac
     1981 ttcattttta atttaaaagg atctaggtga agatcctttt tgataatctc atgaccaaaa
     2041 tcccttaacg tgagttttcg ttccactgag cgtcagaccc cgtagaaaag atcaaaggat
     2101 cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc
     2161 taccagcggt ggtttgtttg ccggatcaag agctaccaac tctttttccg aaggtaactg
     2221 gcttcagcag agcgcagata ccaaataccg ttcttctagt gtagccgtag ttaggccacc
     2281 acttcaagaa ctctgtagca ccgcctacat acctcgctct gctaatcctg ttaccagtgg
     2341 ctgctgccag tggcgataag tcgtgtctta ccgggttgga ctcaagacga tagttaccgg
     2401 ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac acagcccagc ttggagcgaa
     2461 cgacctacac cgaactgaga tacctacagc gtgagctatg agaaagcgcc acgcttcccg
     2521 aagggagaaa ggcggacagg tatccggtaa gcggcagggt cggaacagga gagcgcacga
     2581 gggagcttcc agggggaaac gcctggtatc tttatagtcc tgtcgggttt cgccacctct
     2641 gacttgagcg tcgatttttg tgatgctcgt caggggggcg gagcctatgg aaaaacgcca
     2701 gcaacgcggc ctttttacgg ttcctggcct tttgctggcc ttttgctcac atgttctttc
     2761 ctgcgttatc ccctgattct gtggataacc gtattaccgc ctttgagtga gctgataccg
     2821 ctcgccgcag ccgaacgacc gagcgcagcg agtcagtgag cgaggaagcg gaagagcgcc
     2881 caatacgcaa accgcctctc cccgcgcgtt ggccgattca ttaatgcagc tggcacgaca
     2941 ggtttcccga ctggaaagcg ggcagtgagc gcaacgcaat taatgtgagt tagctcactc
     3001 attaggcacc ccaggcttta cactttatgc ttccggctcg tatgttgtgt ggaattgtga
     3061 gcggataaca atttcacaca ggaaacagct atgaccatga ttacgccaag ctctagctag
     3121 aggtcgacgg tatacagaca tgataagata cattgatgag tttggacaaa ccacaactag
     3181 aatgcagtga aaaaaatgct ttatttgtga aatttgtgat gctattgctt tatttgtaac
     3241 cattataagc tgcaataaac aagttggggt gggcgaagaa ctccagcatg agatccccgc
     3301 gctggaggat catccagccg gcgtcccgga aaacgattcc gaagcccaac ctttcataga
     3361 aggcggcggt ggaatcgaaa tctcgtgatg gcaggttggg cgtcgcttgg tcggtcattt
     3421 cgaaccccag agtcccgctc agaagaactc gtcaagaagg cgatagaagg cgatgcgctg
     3481 cgaatcggga gcggcgatac cgtaaagcac gaggaagcgg tcagcccatt cgccgccaag
     3541 ctcttcagca atatcacggg tagccaacgc tatgtcctga tagcggtccg ccacacccag
     3601 ccggccacag tcgatgaatc cagaaaagcg gccattttcc accatgatat tcggcaagca
     3661 ggcatcgcca tgggtcacga cgagatcctc gccgtcgggc atgcgcgcct tgagcctggc
     3721 gaacagttcg gctggcgcga gcccctgatg ctcttcgtcc agatcatcct gatcgacaag
     3781 accggcttcc atccgagtac gtgctcgctc gatgcgatgt ttcgcttggt ggtcgaatgg
     3841 gcaggtagcc ggatcaagcg tatgcagccg ccgcattgca tcagccatga tggatacttt
     3901 ctcggcagga gcaaggtgag atgacaggag atcctgcccc ggcacttcgc ccaatagcag
     3961 ccagtccctt cccgcttcag tgacaacgtc gagcacagct gcgcaaggaa cgcccgtcgt
     4021 ggccagccac gatagccgcg ctgcctcgtc ctgcagttca ttcagggcac cggacaggtc
     4081 ggtcttgaca aaaagaaccg ggcgcccctg cgctgacagc cggaacacgg cggcatcaga
     4141 gcagccgatt gtctgttgtg cccagtcata gccgaatagc ctctccaccc aagcggccgg
     4201 agaacctgcg tgcaatccat cttgttcaat catgcgaaac gatcctcatc ctgtctcttg
     4261 atcagatccg aaaatggata tacaagctcc cgggagcttt ttgcaaaagc ctaggcctcc
     4321 aaaaaagcct cctcactact tctggaatag ctcagaggca gaggcggcct cggcctctgc
     4381 ataaataaaa aaaattagtc agccatgggg cggagaatgg gcggaactgg gcggagttag
     4441 gggcgggatg ggcggagtta ggggcgggac tatggttgct gactaattga gatgcatgct
     4501 ttgcatactt ctgcctgctg gggagcctgg ggactttcca cacctggttg ctgactaatt
     4561 gagatgcatg ctttgcatac ttctgcctgc tggggagcct ggggactttc cacaccctaa
     4621 ctgacacaca ttccacagaa ttaattcgcg ttaaattttt gttaaatcag ctcatttttt
     4681 aaccaatagg ccgaaatcgg caaaatccct tataaatcaa aagaatagac cgagataggg
     4741 ttgagtgttg ttccagtttg gaacaagagt ccactattaa agaacgtgga ctccaacgtc
     4801 aaagggcgaa aaaccgtcta tcagggcgat ggcccactac gtgaaccatc accctaatca
     4861 agttttttgg ggtcgaggtg ccgtaaagca ctaaatcgga accctaaagg gagcccccga
     4921 tttagagctt gacggggaaa gccggcgaac gtggcgagaa aggaagggaa gaaagcgaaa
     4981 ggagcgggcg ctagggcgct ggcaagtgta gcggtcacgc tgcgcgtaac caccacaccc
     5041 gccgcgctta atgcgccgct acagggcgcg tggggatacc ccctagagcc ccagctggtt
     5101 ctttccgcct cagaagccat agagcccacc gcatccccag catgcctgct attgtcttcc
     5161 caatcctccc ccttgctgtc ctgccccacc ccacccccca gaatagaatg acacctactc
     5221 agacaatgcg atgcaatttc ctcattttat taggaaagga cagtgggagt ggcaccttcc
     5281 agggtcaagg aaggcacggg ggaggggcaa acaacagatg gctggcaact agaaggcaca
     5341 gtcgaggctg atcagcgggt ttaaacgggc cctctagact cgagttaggc aggggccacc
     5401 tgagaagtct cggtcttaga cacggtggtg ctggcctcat cgaattcacc ggtgcgtcca
     5461 ccggtatcga taggggagaa cgtagtagtg gtctctgcct ccacactcat actggagcgc
     5521 ctaatgtgtc tgcatgatga ccactggcga agctgctcct gagacagaca gcccaagtct
     5581 ttgaaaagtt taaacaggtc atttcgaaac ttcacaccaa taaaagcgta caggaaagga
     5641 ttcacgcaac acctgacgca cgccaaggag tatgtcacat cgtatgcgat attcagctgc
     5701 tttgacagct cacatgttga agaagtgatg ttaaagttag caactgtttg ggccaaaact
     5761 accccattat aaggcagctg aaagacaatg aatacaacca ctacggcaat tatcacctta
     5821 atagccttgt tccgctcaaa attccgggcc tgcaggagtg tgcgtataat tacgaggtag
     5881 caaaagctca tagcaagcaa aggaacaagg aagccaatca ccatttgggc gacttggatc
     5941 gtaatgaacg cttccacatg ctcagtgatc agtgaacatc gcatagcttg ctcagaggaa
     6001 ctcctctgca aatctgagta caacagttca ggaatggaca agactgtggc caaaatccag
     6061 atcccaacgc agctcaattt acttatcaga agcaccctag cccggtgtct atgtgcagag
     6121 accgcctgca caatggccac atatctatct atgctgatgc acagcagcag cagcatccca
     6181 gagaaaaagc tcatcttgta tatagcaaag atcagcttgc aaaagtggac tccgaagacc
     6241 catgactttg ctgcggaata tgcccagaaa gggagagtga gaagaaacag aatatcggcc
     6301 acggcgaggt tcaaaaggta ggtatctgtc attgtcttca ggcgtttgaa gtaaatatac
     6361 gtcaagacga ccaaaccgtt ccccagcagc ccgacaaagc agattatgga atacataata
     6421 ggcagaaacc atgccttgaa attcctcacg tccttcttgc tgcagaggga ctcgaaaagg
     6481 gtgtaatcta ctgtggtatt gtcgcctatg tagtcgtctg taacttcatc ctgacacaga
     6541 catacctgga atatcaccag caaagccaca accagtacgc ttttcatagg cttcccgaga
     6601 tccatatcga tgctggcgtc atcatcgtcc ttgtagtcgg cgaataccag gcagaagatg
     6661 tagctcaggg cgat
//
