LOCUS       sequence-430778-        6773 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 6773)
  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 6773)
  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: CRHR2-DuET
FEATURES             Location/Qualifiers
     source          1..6773
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     556..575
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(794..898)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(973..992)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        1164..1543
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        1544..1747
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     1698..1718
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     1792..1811
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1792..1810
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(3293..3310)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3299..3523
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      3569..3997
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3656..3675)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3866..3887
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4006..4026)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        4011..4340
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      4191..4326
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     4253..4272
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             4407..5201
                     /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(4461..4480)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     5071..5090
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    5375..5496
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5412..5431)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5466..5485
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5545..5561)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5545..5561)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5558..5580)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5569..5585
                     /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)."
     promoter        complement(join(5593..5599,5600..5617,5618..5623))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5638..5659
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5776..5793)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5947..6535)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6027..6046)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6706..6773,1..724,725..793))
                     /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"
ORIGIN
        1 gactccccgt cgtgtagata actacgatac gggagggctt accatctggc cccagtgctg
       61 caatgatacc gcgagaccca cgctcaccgg ctccagattt atcagcaata aaccagccag
      121 ccggaagggc cgagcgcaga agtggtcctg caactttatc cgcctccatc cagtctatta
      181 attgttgccg ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg
      241 ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg
      301 gttcccaacg atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa gcggttagct
      361 ccttcggtcc tccgatcgtt gtcagaagta agttggccgc agtgttatca ctcatggtta
      421 tggcagcact gcataattct cttactgtca tgccatccgt aagatgcttt tctgtgactg
      481 gtgagtactc aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc
      541 cggcgtcaat acgggataat accgcgccac atagcagaac tttaaaagtg ctcatcattg
      601 gaaaacgttc ttcggggcga aaactctcaa ggatcttacc gctgttgaga tccagttcga
      661 tgtaacccac tcgtgcaccc aactgatctt cagcatcttt tactttcacc agcgtttctg
      721 ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg aataagggcg acacggaaat
      781 gttgaatact catactcttc ctttttcaat attattgaag catttatcag ggttattgtc
      841 tcatgagcgg atacatattt gaatgtattt agaaaaataa acaaataggg gttccgcgca
      901 catttccccg aaaagtgcca cctgacgtcg acggatcggg agatctcccg atcccctatg
      961 gtgcactctc agtacaatct gctctgatgc cgcatagtta agccagtatc tgctccctgc
     1021 ttgtgtgttg gaggtcgctg agtagtgcgc gagcaaaatt taagctacaa caaggcaagg
     1081 cttgaccgac atttgcatga agaatctgct tagggttagg cgttttgcgc tgcttcgcga
     1141 tgtacgggcc agatatacgc gttgacattg attattgact agttattaat agtaatcaat
     1201 tacggggtca ttagttcata gcccatatat ggagttccgc gttacataac ttacggtaaa
     1261 tggcccgcct ggctgaccgc ccaacgaccc ccgcccattg acgtcaataa tgacgtatgt
     1321 tcccatagta acgccaatag ggactttcca ttgacgtcaa tgggtggagt atttacggta
     1381 aactgcccac ttggcagtac atcaagtgta tcatatgcca agtacgcccc ctattgacgt
     1441 caatgacggt aaatggcccg cctggcatta tgcccagtac atgaccttat gggactttcc
     1501 tacttggcag tacatctacg tattagtcat cgctattacc atggtgatgc ggttttggca
     1561 gtacatcaat gggcgtggat agcggtttga ctcacgggga tttccaagtc tccaccccat
     1621 tgacgtcaat gggagtttgt tttggcacca aaatcaacgg gactttccaa aatgtcgtaa
     1681 caactccgcc ccattgacgc aaatgggcgg taggcgtgta cggtgggagg tctatataag
     1741 cagagctctc tggctaacta gagaacccac tgcttactgg cttatcgaaa ttaatacgac
     1801 tcactatagg gagacccaag ctggctagcg tttaaactta agcttagcgc caccatgaag
     1861 acgatcatcg ccctgagcta catcttctgc ctggtattcg ccgactacaa ggacgatgat
     1921 gacgccagca tcgatatgga cgccgctttg ctgcactctc tgctggaggc aaattgcagc
     1981 ctggcccttg cggaggaatt gctcctggac ggttggggcc cccctctgga tccggaaggc
     2041 ccctactcat attgcaacac caccctcgac cagatcggca cctgttggcc tcggagtgcc
     2101 gccggagcct tggttgagag gccctgtcca gagtacttta atggagtgaa atataataca
     2161 acacgcaacg cttacaggga atgcctggaa aacggaacat gggcatccaa aatcaactat
     2221 agccaatgtg agccaattct tgacgacaaa cagagaaaat atgatcttca ctacaggatt
     2281 gcactcgttg tcaactactt gggtcattgc gtctctgttg ccgccttggt ggctgcattc
     2341 ctgctgttcc tggcactgcg ctccatccgg tgtcttcgga atgtcatcca ctggaatctt
     2401 attactacct ttatactgcg caatgttatg tggttcctcc ttcagctggt tgatcacgaa
     2461 gtgcacgaaa gcaacgaggt atggtgccgg tgtattacta cgatatttaa ttactttgta
     2521 gttactaatt tcttttggat gtttgttgaa ggatgctatc tgcacaccgc catcgtcatg
     2581 acatattcta ccgaaagact gcgcaagtgc ctgtttttgt tcatcggctg gtgtatcccc
     2641 ttcccaatca tagtcgcttg ggctattggc aaactgtatt acgagaacga gcagtgctgg
     2701 ttcgggaaag agcccggcga ccttgtcgac tacatttacc agggacctat cattcttgtg
     2761 cttcttataa acttcgtgtt cctcttcaac attgtgcgca ttctgatgac aaagctgcga
     2821 gccagcacaa catcagagac tatacagtac cggaaggcag ttaaggccac tctggtcctt
     2881 ttgccactgc tggggatcac ttacatgctg tttttcgtta accccggcga ggatgacctg
     2941 tctcagatca tgtttatcta cttcaacagc ttcctgcaga gtttccaagg cttttttgta
     3001 tcagtgttct attgcttctt taatggggag gtgcggtctg cagtgcggaa aagatggcac
     3061 aggtggcaag accaccattc tctccgagtt cccatggctc gcgctatgtc aatccccacg
     3121 tctcccaccc ggatcagctt ccacagcatt aagcagactg ctgcggtgat cgataccggt
     3181 ggacgcaccg gtgaattcga tgaggccagc accaccgtgt ctaagaccga gacttctcag
     3241 gtggcccctg cctaactcga gtctagaggg cccgtttaaa cccgctgatc agcctcgact
     3301 gtgccttcta gttgccagcc atctgttgtt tgcccctccc ccgtgccttc cttgaccctg
     3361 gaaggtgcca ctcccactgt cctttcctaa taaaatgagg aaattgcatc gcattgtctg
     3421 agtaggtgtc attctattct ggggggtggg gtggggcagg acagcaaggg ggaggattgg
     3481 gaagacaata gcaggcatgc tggggatgcg gtgggctcta tggcttctga ggcggaaaga
     3541 accagctggg gctctagggg gtatccccac gcgccctgta gcggcgcatt aagcgcggcg
     3601 ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca gcgccctagc gcccgctcct
     3661 ttcgctttct tcccttcctt tctcgccacg ttcgccggct ttccccgtca agctctaaat
     3721 cgggggctcc ctttagggtt ccgatttagt gctttacggc acctcgaccc caaaaaactt
     3781 gattagggtg atggttcacg tagtgggcca tcgccctgat agacggtttt tcgccctttg
     3841 acgttggagt ccacgttctt taatagtgga ctcttgttcc aaactggaac aacactcaac
     3901 cctatctcgg tctattcttt tgatttataa gggattttgc cgatttcggc ctattggtta
     3961 aaaaatgagc tgatttaaca aaaatttaac gcgaattaat tctgtggaat gtgtgtcagt
     4021 tagggtgtgg aaagtcccca ggctccccag caggcagaag tatgcaaagc atgcatctca
     4081 attagtcagc aaccaggtgt ggaaagtccc caggctcccc agcaggcaga agtatgcaaa
     4141 gcatgcatct caattagtca gcaaccatag tcccgcccct aactccgccc atcccgcccc
     4201 taactccgcc cagttccgcc cattctccgc cccatggctg actaattttt tttatttatg
     4261 cagaggccga ggccgcctct gcctctgagc tattccagaa gtagtgagga ggcttttttg
     4321 gaggcctagg cttttgcaaa aagctcccgg gagcttgtat atccattttc ggatctgatc
     4381 aagagacagg atgaggatcg tttcgcatga ttgaacaaga tggattgcac gcaggttctc
     4441 cggccgcttg ggtggagagg ctattcggct atgactgggc acaacagaca atcggctgct
     4501 ctgatgccgc cgtgttccgg ctgtcagcgc aggggcgccc ggttcttttt gtcaagaccg
     4561 acctgtccgg tgccctgaat gaactgcagg acgaggcagc gcggctatcg tggctggcca
     4621 cgacgggcgt tccttgcgca gctgtgctcg acgttgtcac tgaagcggga agggactggc
     4681 tgctattggg cgaagtgccg gggcaggatc tcctgtcatc tcaccttgct cctgccgaga
     4741 aagtatccat catggctgat gcaatgcggc ggctgcatac gcttgatccg gctacctgcc
     4801 cattcgacca ccaagcgaaa catcgcatcg agcgagcacg tactcggatg gaagccggtc
     4861 ttgtcgatca ggatgatctg gacgaagagc atcaggggct cgcgccagcc gaactgttcg
     4921 ccaggctcaa ggcgcgcatg cccgacggcg aggatctcgt cgtgacccat ggcgatgcct
     4981 gcttgccgaa tatcatggtg gaaaatggcc gcttttctgg attcatcgac tgtggccggc
     5041 tgggtgtggc ggaccgctat caggacatag cgttggctac ccgtgatatt gctgaagagc
     5101 ttggcggcga atgggctgac cgcttcctcg tgctttacgg tatcgccgct cccgattcgc
     5161 agcgcatcgc cttctatcgc cttcttgacg agttcttctg agcgggactc tggggttcga
     5221 aatgaccgac caagcgacgc ccaacctgcc atcacgagat ttcgattcca ccgccgcctt
     5281 ctatgaaagg ttgggcttcg gaatcgtttt ccgggacgcc ggctggatga tcctccagcg
     5341 cggggatctc atgctggagt tcttcgccca ccccaacttg tttattgcag cttataatgg
     5401 ttacaaataa agcaatagca tcacaaattt cacaaataaa gcattttttt cactgcattc
     5461 tagttgtggt ttgtccaaac tcatcaatgt atcttatcat gtctgtatac cgtcgacctc
     5521 tagctagagc ttggcgtaat catggtcata gctgtttcct gtgtgaaatt gttatccgct
     5581 cacaattcca cacaacatac gagccggaag cataaagtgt aaagcctggg gtgcctaatg
     5641 agtgagctaa ctcacattaa ttgcgttgcg ctcactgccc gctttccagt cgggaaacct
     5701 gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg
     5761 gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc
     5821 ggtatcagct cactcaaagg cggtaatacg gttatccaca gaatcagggg ataacgcagg
     5881 aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct
     5941 ggcgtttttc cataggctcc gcccccctga cgagcatcac aaaaatcgac gctcaagtca
     6001 gaggtggcga aacccgacag gactataaag ataccaggcg tttccccctg gaagctccct
     6061 cgtgcgctct cctgttccga ccctgccgct taccggatac ctgtccgcct ttctcccttc
     6121 gggaagcgtg gcgctttctc atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt
     6181 tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct gcgccttatc
     6241 cggtaactat cgtcttgagt ccaacccggt aagacacgac ttatcgccac tggcagcagc
     6301 cactggtaac aggattagca gagcgaggta tgtaggcggt gctacagagt tcttgaagtg
     6361 gtggcctaac tacggctaca ctagaagaac ggtatttggt atctgcgctc tgctgaagcc
     6421 agttaccttc ggaaaaagag ttggtagctc ttgatccggc aaacaaacca ccgctggtag
     6481 cggtggtttt tttgtttgca agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga
     6541 tcctttgatc ttttctacgg ggtctgacgc tcagtggaac gaaaactcac gttaagggat
     6601 tttggtcatg agattatcaa aaaggatctt cacctagatc cttttaaatt aaaaatgaag
     6661 ttttaaatca atctaaagta tatatgagta aacttggtct gacagttacc aatgcttaat
     6721 cagtgaggca cctatctcag cgatctgtct atttcgttca tccatagttg cct
//
