LOCUS       sequence-430862-        6734 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 6734)
  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 6734)
  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: KISSPEPTIN-R-DuET
FEATURES             Location/Qualifiers
     source          1..6734
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      60..648
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     549..568
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     802..819
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    936..957
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(972..977,978..995,996..1002)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1010..1026
                     /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     1015..1037
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     1034..1050
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     1034..1050
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     polyA_signal    1099..1220
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1110..1129)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     1164..1183
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     CDS             complement(1394..2188)
                     /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(1505..1524)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     2115..2134
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     promoter        complement(2255..2584)
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      2269..2404
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(2323..2342)
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     primer_bind     2569..2589
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      complement(2598..3026)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2708..2729)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2920..2939
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     polyA_signal    3072..3296
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     3285..3302
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     primer_bind     complement(4745..4764)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4746..4764)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        complement(4809..5012)
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     complement(4838..4858)
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     enhancer        5013..5392
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     primer_bind     5564..5583
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        5658..5762
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5763..5831,5832..6623)
                     /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(5981..6000)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 taacgtgagt tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa aggatcttct
       61 tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca
      121 gcggtggttt gtttgccgga tcaagagcta ccaactcttt ttccgaaggt aactggcttc
      181 agcagagcgc agataccaaa taccgttctt ctagtgtagc cgtagttagg ccaccacttc
      241 aagaactctg tagcaccgcc tacatacctc gctctgctaa tcctgttacc agtggctgct
      301 gccagtggcg ataagtcgtg tcttaccggg ttggactcaa gacgatagtt accggataag
      361 gcgcagcggt cgggctgaac ggggggttcg tgcacacagc ccagcttgga gcgaacgacc
      421 tacaccgaac tgagatacct acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg
      481 agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa caggagagcg cacgagggag
      541 cttccagggg gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca cctctgactt
      601 gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac
      661 gcggcctttt tacggttcct ggccttttgc tggccttttg ctcacatgtt ctttcctgcg
      721 ttatcccctg attctgtgga taaccgtatt accgcctttg agtgagctga taccgctcgc
      781 cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga gcgcccaata
      841 cgcaaaccgc ctctccccgc gcgttggccg attcattaat gcagctggca cgacaggttt
      901 cccgactgga aagcgggcag tgagcgcaac gcaattaatg tgagttagct cactcattag
      961 gcaccccagg ctttacactt tatgcttccg gctcgtatgt tgtgtggaat tgtgagcgga
     1021 taacaatttc acacaggaaa cagctatgac catgattacg ccaagctcta gctagaggtc
     1081 gacggtatac agacatgata agatacattg atgagtttgg acaaaccaca actagaatgc
     1141 agtgaaaaaa atgctttatt tgtgaaattt gtgatgctat tgctttattt gtaaccatta
     1201 taagctgcaa taaacaagtt ggggtgggcg aagaactcca gcatgagatc cccgcgctgg
     1261 aggatcatcc agccggcgtc ccggaaaacg attccgaagc ccaacctttc atagaaggcg
     1321 gcggtggaat cgaaatctcg tgatggcagg ttgggcgtcg cttggtcggt catttcgaac
     1381 cccagagtcc cgctcagaag aactcgtcaa gaaggcgata gaaggcgatg cgctgcgaat
     1441 cgggagcggc gataccgtaa agcacgagga agcggtcagc ccattcgccg ccaagctctt
     1501 cagcaatatc acgggtagcc aacgctatgt cctgatagcg gtccgccaca cccagccggc
     1561 cacagtcgat gaatccagaa aagcggccat tttccaccat gatattcggc aagcaggcat
     1621 cgccatgggt cacgacgaga tcctcgccgt cgggcatgcg cgccttgagc ctggcgaaca
     1681 gttcggctgg cgcgagcccc tgatgctctt cgtccagatc atcctgatcg acaagaccgg
     1741 cttccatccg agtacgtgct cgctcgatgc gatgtttcgc ttggtggtcg aatgggcagg
     1801 tagccggatc aagcgtatgc agccgccgca ttgcatcagc catgatggat actttctcgg
     1861 caggagcaag gtgagatgac aggagatcct gccccggcac ttcgcccaat agcagccagt
     1921 cccttcccgc ttcagtgaca acgtcgagca cagctgcgca aggaacgccc gtcgtggcca
     1981 gccacgatag ccgcgctgcc tcgtcctgca gttcattcag ggcaccggac aggtcggtct
     2041 tgacaaaaag aaccgggcgc ccctgcgctg acagccggaa cacggcggca tcagagcagc
     2101 cgattgtctg ttgtgcccag tcatagccga atagcctctc cacccaagcg gccggagaac
     2161 ctgcgtgcaa tccatcttgt tcaatcatgc gaaacgatcc tcatcctgtc tcttgatcag
     2221 atccgaaaat ggatatacaa gctcccggga gctttttgca aaagcctagg cctccaaaaa
     2281 agcctcctca ctacttctgg aatagctcag aggcagaggc ggcctcggcc tctgcataaa
     2341 taaaaaaaat tagtcagcca tggggcggag aatgggcgga actgggcgga gttaggggcg
     2401 ggatgggcgg agttaggggc gggactatgg ttgctgacta attgagatgc atgctttgca
     2461 tacttctgcc tgctggggag cctggggact ttccacacct ggttgctgac taattgagat
     2521 gcatgctttg catacttctg cctgctgggg agcctgggga ctttccacac cctaactgac
     2581 acacattcca cagaattaat tcgcgttaaa tttttgttaa atcagctcat tttttaacca
     2641 ataggccgaa atcggcaaaa tcccttataa atcaaaagaa tagaccgaga tagggttgag
     2701 tgttgttcca gtttggaaca agagtccact attaaagaac gtggactcca acgtcaaagg
     2761 gcgaaaaacc gtctatcagg gcgatggccc actacgtgaa ccatcaccct aatcaagttt
     2821 tttggggtcg aggtgccgta aagcactaaa tcggaaccct aaagggagcc cccgatttag
     2881 agcttgacgg ggaaagccgg cgaacgtggc gagaaaggaa gggaagaaag cgaaaggagc
     2941 gggcgctagg gcgctggcaa gtgtagcggt cacgctgcgc gtaaccacca cacccgccgc
     3001 gcttaatgcg ccgctacagg gcgcgtgggg atacccccta gagccccagc tggttctttc
     3061 cgcctcagaa gccatagagc ccaccgcatc cccagcatgc ctgctattgt cttcccaatc
     3121 ctcccccttg ctgtcctgcc ccaccccacc ccccagaata gaatgacacc tactcagaca
     3181 atgcgatgca atttcctcat tttattagga aaggacagtg ggagtggcac cttccagggt
     3241 caaggaaggc acgggggagg ggcaaacaac agatggctgg caactagaag gcacagtcga
     3301 ggctgatcag cgggtttaaa cgggccctct agactcgagt taggcagggg ccacctgaga
     3361 agtctcggtc ttagacacgg tggtgctggc ctcatcgaat tcaccggtgc gtccaccggt
     3421 atcgatcaga ggagcattgt cctctcccag cacgcaaaga cctctagcag cgagccctga
     3481 tgatcctggt ttctgcgccc tggcgggtgc cggatgactg cccaacctca ggagttccgc
     3541 atgaggggcg gcagggtcac ttggtcctgg tctccgaggc cgtctgggtc gcctgggagc
     3601 gcaagggcat actcttctaa aagcttggcg gaaatgtgac cccagaaagg cataaaggag
     3661 tgggttcaaa gcactgttgc tgtaagacat acagtgggcc catgttttca aggcgtatgc
     3721 ggcgtagctc ctgggatgcc agctgccggc cggacccagg gcttgcagca ccaaaaagag
     3781 ctgtatggga ccccagcatg ccgcgaaaag gagcacaact gcagcgacca gcctagagac
     3841 tttcgcccgg accgcgccag cccgctcggc caagacctgt ccctgaagag cggaatcggc
     3901 aggagctggc ctgactgcga ccctcccgag gtgtctgagc atggcggcgt aacaagcaca
     3961 cgtggccagg agtgggagca ggtacaatgc aagcaggtta tagagagcga aggccctctc
     4021 cagggcgcga gatgggaagg cttcgctgca ataagctctg gggcctgggg acagcctatg
     4081 aagagcgagc acgggagcag acacggcggc gcttcccacc catattgaca gagagaccgc
     4141 gagggccagc ctaggtgtcc gcctgtgcag tgctcgaagc gggaagactg tcacgtacca
     4201 cctatccacg ctcattgcgg tcaatgtggc acatgttgct tgcacggaga cctgctggat
     4261 gtagttcaca aatttgcaca tgaagtcccc aagcacccaa ccgggcagag ggtacaacag
     4321 ggctgtaaat ggcacacaac acaggaggaa tgtcacatcc gtggcggcga ggttagcaat
     4381 atagaaatta gtcacggtcc tcattggctt atgtcgacaa atgacataga tcacgagaga
     4441 atttcccacg agtccgagga gcatcaaagc agcgaaaaac aagggtacaa gccaggcgtc
     4501 taccgcccgg ggacttggaa ctggcccatc gctggcattg gctccgcagc ctggacaccc
     4561 actggcatta gcgggagctc cccaactggc gttgggacct gatgttgcga ccgtgtgcat
     4621 atcgatgctg gcgtcatcat cgtccttgta gtcggcgaat accaggcaga agatgtagct
     4681 cagggcgatg atcgtcttca tggtggcgct aagcttaagt ttaaacgcta gccagcttgg
     4741 gtctccctat agtgagtcgt attaatttcg ataagccagt aagcagtggg ttctctagtt
     4801 agccagagag ctctgcttat atagacctcc caccgtacac gcctaccgcc catttgcgtc
     4861 aatggggcgg agttgttacg acattttgga aagtcccgtt gattttggtg ccaaaacaaa
     4921 ctcccattga cgtcaatggg gtggagactt ggaaatcccc gtgagtcaaa ccgctatcca
     4981 cgcccattga tgtactgcca aaaccgcatc accatggtaa tagcgatgac taatacgtag
     5041 atgtactgcc aagtaggaaa gtcccataag gtcatgtact gggcataatg ccaggcgggc
     5101 catttaccgt cattgacgtc aatagggggc gtacttggca tatgatacac ttgatgtact
     5161 gccaagtggg cagtttaccg taaatactcc acccattgac gtcaatggaa agtccctatt
     5221 ggcgttacta tgggaacata cgtcattatt gacgtcaatg ggcgggggtc gttgggcggt
     5281 cagccaggcg ggccatttac cgtaagttat gtaacgcgga actccatata tgggctatga
     5341 actaatgacc ccgtaattga ttactattaa taactagtca ataatcaatg tcaacgcgta
     5401 tatctggccc gtacatcgcg aagcagcgca aaacgcctaa ccctaagcag attcttcatg
     5461 caaatgtcgg tcaagccttg ccttgttgta gcttaaattt tgctcgcgca ctactcagcg
     5521 acctccaaca cacaagcagg gagcagatac tggcttaact atgcggcatc agagcagatt
     5581 gtactgagag tgcaccatag gggatcggga gatctcccga tccgtcgacg tcaggtggca
     5641 cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt tttctaaata cattcaaata
     5701 tgtatccgct catgagacaa taaccctgat aaatgcttca ataatattga aaaaggaaga
     5761 gtatgagtat tcaacatttc cgtgtcgccc ttattccctt ttttgcggca ttttgccttc
     5821 ctgtttttgc tcacccagaa acgctggtga aagtaaaaga tgctgaagat cagttgggtg
     5881 cacgagtggg ttacatcgaa ctggatctca acagcggtaa gatccttgag agttttcgcc
     5941 ccgaagaacg ttttccaatg atgagcactt ttaaagttct gctatgtggc gcggtattat
     6001 cccgtattga cgccgggcaa gagcaactcg gtcgccgcat acactattct cagaatgact
     6061 tggttgagta ctcaccagtc acagaaaagc atcttacgga tggcatgaca gtaagagaat
     6121 tatgcagtgc tgccataacc atgagtgata acactgcggc caacttactt ctgacaacga
     6181 tcggaggacc gaaggagcta accgcttttt tgcacaacat gggggatcat gtaactcgcc
     6241 ttgatcgttg ggaaccggag ctgaatgaag ccataccaaa cgacgagcgt gacaccacga
     6301 tgcctgtagc aatggcaaca acgttgcgca aactattaac tggcgaacta cttactctag
     6361 cttcccggca acaattaata gactggatgg aggcggataa agttgcagga ccacttctgc
     6421 gctcggccct tccggctggc tggtttattg ctgataaatc tggagccggt gagcgtgggt
     6481 ctcgcggtat cattgcagca ctggggccag atggtaagcc ctcccgtatc gtagttatct
     6541 acacgacggg gagtcaggca actatggatg aacgaaatag acagatcgct gagataggtg
     6601 cctcactgat taagcattgg taactgtcag accaagttta ctcatatata ctttagattg
     6661 atttaaaact tcatttttaa tttaaaagga tctaggtgaa gatccttttt gataatctca
     6721 tgaccaaaat ccct
//
