LOCUS       sequence-430770-        6938 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 6938)
  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 6938)
  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: CHRM2-DuET
FEATURES             Location/Qualifiers
     source          1..6938
                     /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(5072..5091)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(5073..5091)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        complement(5136..5339)
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     complement(5165..5185)
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     enhancer        5340..5719
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     primer_bind     5891..5910
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        5985..6089
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(6090..6158,6159..6938,1..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(6308..6327)
                     /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 cgtgtcgcgc ctatgttttt gtagtgacac atcaggaggt gcttaaatgt
     3601 ttttttgaat gtggcattac acagggcata gcatgcaggg ttgattgtac tgttaatata
     3661 acacagccag tagccgatag tccaaacagt gttggggata cagggagcgc agaaagtgtt
     3721 gataagtacc ataacattgt atggggccca tgtgataatg aatgccagga ggatagcgag
     3781 gatagttcta gtcacctttt tttcgcgact ggggggaggc tttttcttag cgggctgttt
     3841 ggtcatcttc acgatcttcc gggcaacaat gttctgcttc tcgtctccgt tctggcctga
     3901 ggaacccacc acttcgacag ttgtgtttgt cggagtgcaa ctgtcggatt taggtgtctt
     3961 ggtcccaatg cgaatgcatg tttgcttgga gttttcgtcc ttgctgtggc ccaaggatgt
     4021 ggaaacagta ttttcatcct gagtaatttc atcatcccgc atattggaag ccacggcgct
     4081 gacagaggta gagtcgttag aactctcttt ctcctccccc tgcacgcaat tctcggtcac
     4141 ggggtccctt ggtgctttcc cattctgaat tttgttatgt tccaggccgt cgtcggagct
     4201 cggcatattg ttattatttg gtttgacgat tctcccctgc acaagggaag gtgagacggg
     4261 gtcttgattt gcaactggct ctttcttgtc cttttttatc cgggacttgc tagctcttga
     4321 gatatgccaa tacagcactg tcataatgat gacgggcagg tagaaggcag cgatggcagt
     4381 cccgaaggtt acagctgcgt tagagaagaa ctggatatag cactcgccgt cttccacagt
     4441 ccttacaccc acaataaact gccagaacag aatagcggga gcccacagta taaaagaaag
     4501 cacccaagct gctgcaatca tcattccggc catctttgta gtcctcttca ctgggtaagt
     4561 caggggcttg gtaacgcaga agtaccgatc gaatgagata atcagcaggt tcattacaga
     4621 tgcattggaa acaacgtagt cgagtgcaag ccaaaggtcg cagaccacgg ggcccaatgg
     4681 ccaatatcca atgacagtat acagtgtata aaggttcatg gagaatacgc ctattatcaa
     4741 atctgcgcat gccaatgaga acaggaaata gttgttgact gtctgcaagt gcctattgac
     4801 ctttatagac accatcacaa ggatattgcc gatgatggtt accagggaca gtgatccagc
     4861 caccagcaca atgaagacaa cctcaaaggt cttgtaaggg cttgtgaggg ccaatgagtt
     4921 gtttgagcta ttagtactat tgttcatatc gatgctggcg tcatcatcgt ccttgtagtc
     4981 ggcgaatacc aggcagaaga tgtagctcag ggcgatgatc gtcttcatgg tggcgctaag
     5041 cttaagttta aacgctagcc agcttgggtc tccctatagt gagtcgtatt aatttcgata
     5101 agccagtaag cagtgggttc tctagttagc cagagagctc tgcttatata gacctcccac
     5161 cgtacacgcc taccgcccat ttgcgtcaat ggggcggagt tgttacgaca ttttggaaag
     5221 tcccgttgat tttggtgcca aaacaaactc ccattgacgt caatggggtg gagacttgga
     5281 aatccccgtg agtcaaaccg ctatccacgc ccattgatgt actgccaaaa ccgcatcacc
     5341 atggtaatag cgatgactaa tacgtagatg tactgccaag taggaaagtc ccataaggtc
     5401 atgtactggg cataatgcca ggcgggccat ttaccgtcat tgacgtcaat agggggcgta
     5461 cttggcatat gatacacttg atgtactgcc aagtgggcag tttaccgtaa atactccacc
     5521 cattgacgtc aatggaaagt ccctattggc gttactatgg gaacatacgt cattattgac
     5581 gtcaatgggc gggggtcgtt gggcggtcag ccaggcgggc catttaccgt aagttatgta
     5641 acgcggaact ccatatatgg gctatgaact aatgaccccg taattgatta ctattaataa
     5701 ctagtcaata atcaatgtca acgcgtatat ctggcccgta catcgcgaag cagcgcaaaa
     5761 cgcctaaccc taagcagatt cttcatgcaa atgtcggtca agccttgcct tgttgtagct
     5821 taaattttgc tcgcgcacta ctcagcgacc tccaacacac aagcagggag cagatactgg
     5881 cttaactatg cggcatcaga gcagattgta ctgagagtgc accatagggg atcgggagat
     5941 ctcccgatcc gtcgacgtca ggtggcactt ttcggggaaa tgtgcgcgga acccctattt
     6001 gtttattttt ctaaatacat tcaaatatgt atccgctcat gagacaataa ccctgataaa
     6061 tgcttcaata atattgaaaa aggaagagta tgagtattca acatttccgt gtcgccctta
     6121 ttcccttttt tgcggcattt tgccttcctg tttttgctca cccagaaacg ctggtgaaag
     6181 taaaagatgc tgaagatcag ttgggtgcac gagtgggtta catcgaactg gatctcaaca
     6241 gcggtaagat ccttgagagt tttcgccccg aagaacgttt tccaatgatg agcactttta
     6301 aagttctgct atgtggcgcg gtattatccc gtattgacgc cgggcaagag caactcggtc
     6361 gccgcataca ctattctcag aatgacttgg ttgagtactc accagtcaca gaaaagcatc
     6421 ttacggatgg catgacagta agagaattat gcagtgctgc cataaccatg agtgataaca
     6481 ctgcggccaa cttacttctg acaacgatcg gaggaccgaa ggagctaacc gcttttttgc
     6541 acaacatggg ggatcatgta actcgccttg atcgttggga accggagctg aatgaagcca
     6601 taccaaacga cgagcgtgac accacgatgc ctgtagcaat ggcaacaacg ttgcgcaaac
     6661 tattaactgg cgaactactt actctagctt cccggcaaca attaatagac tggatggagg
     6721 cggataaagt tgcaggacca cttctgcgct cggcccttcc ggctggctgg tttattgctg
     6781 ataaatctgg agccggtgag cgtgggtctc gcggtatcat tgcagcactg gggccagatg
     6841 gtaagccctc ccgtatcgta gttatctaca cgacggggag tcaggcaact atggatgaac
     6901 gaaatagaca gatcgctgag ataggtgcct cactgatt
//
