LOCUS       sequence-430893-        6707 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 6707)
  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 6707)
  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: UTS2R-DuET
FEATURES             Location/Qualifiers
     source          1..6707
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     447..466
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    751..872
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(788..807)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     842..861
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(921..937)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(921..937)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(934..956)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    945..961
                     /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(969..975,976..993,994..999))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1014..1035
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(1152..1169)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1323..1911)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1403..1422)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2082..2873,2874..2942))
                     /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     2705..2724
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2943..3047)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(3122..3141)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        3313..3692
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        3693..3896
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     3847..3867
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     3941..3960
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        3941..3959
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(5376..5393)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    5382..5606
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      5652..6080
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5739..5758)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5949..5970
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(6089..6109)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        6094..6423
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      6274..6409
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     6336..6355
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             join(6490..6707,1..577)
                     /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(6544..6563)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
ORIGIN
        1 gggcgttcct tgcgcagctg tgctcgacgt tgtcactgaa gcgggaaggg actggctgct
       61 attgggcgaa gtgccggggc aggatctcct gtcatctcac cttgctcctg ccgagaaagt
      121 atccatcatg gctgatgcaa tgcggcggct gcatacgctt gatccggcta cctgcccatt
      181 cgaccaccaa gcgaaacatc gcatcgagcg agcacgtact cggatggaag ccggtcttgt
      241 cgatcaggat gatctggacg aagagcatca ggggctcgcg ccagccgaac tgttcgccag
      301 gctcaaggcg cgcatgcccg acggcgagga tctcgtcgtg acccatggcg atgcctgctt
      361 gccgaatatc atggtggaaa atggccgctt ttctggattc atcgactgtg gccggctggg
      421 tgtggcggac cgctatcagg acatagcgtt ggctacccgt gatattgctg aagagcttgg
      481 cggcgaatgg gctgaccgct tcctcgtgct ttacggtatc gccgctcccg attcgcagcg
      541 catcgccttc tatcgccttc ttgacgagtt cttctgagcg ggactctggg gttcgaaatg
      601 accgaccaag cgacgcccaa cctgccatca cgagatttcg attccaccgc cgccttctat
      661 gaaaggttgg gcttcggaat cgttttccgg gacgccggct ggatgatcct ccagcgcggg
      721 gatctcatgc tggagttctt cgcccacccc aacttgttta ttgcagctta taatggttac
      781 aaataaagca atagcatcac aaatttcaca aataaagcat ttttttcact gcattctagt
      841 tgtggtttgt ccaaactcat caatgtatct tatcatgtct gtataccgtc gacctctagc
      901 tagagcttgg cgtaatcatg gtcatagctg tttcctgtgt gaaattgtta tccgctcaca
      961 attccacaca acatacgagc cggaagcata aagtgtaaag cctggggtgc ctaatgagtg
     1021 agctaactca cattaattgc gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg
     1081 tgccagctgc attaatgaat cggccaacgc gcggggagag gcggtttgcg tattgggcgc
     1141 tcttccgctt cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta
     1201 tcagctcact caaaggcggt aatacggtta tccacagaat caggggataa cgcaggaaag
     1261 aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg
     1321 tttttccata ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg
     1381 tggcgaaacc cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg
     1441 cgctctcctg ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga
     1501 agcgtggcgc tttctcatag ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc
     1561 tccaagctgg gctgtgtgca cgaacccccc gttcagcccg accgctgcgc cttatccggt
     1621 aactatcgtc ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact
     1681 ggtaacagga ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg
     1741 cctaactacg gctacactag aagaacggta tttggtatct gcgctctgct gaagccagtt
     1801 accttcggaa aaagagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt
     1861 ggtttttttg tttgcaagca gcagattacg cgcagaaaaa aaggatctca agaagatcct
     1921 ttgatctttt ctacggggtc tgacgctcag tggaacgaaa actcacgtta agggattttg
     1981 gtcatgagat tatcaaaaag gatcttcacc tagatccttt taaattaaaa atgaagtttt
     2041 aaatcaatct aaagtatata tgagtaaact tggtctgaca gttaccaatg cttaatcagt
     2101 gaggcaccta tctcagcgat ctgtctattt cgttcatcca tagttgcctg actccccgtc
     2161 gtgtagataa ctacgatacg ggagggctta ccatctggcc ccagtgctgc aatgataccg
     2221 cgagacccac gctcaccggc tccagattta tcagcaataa accagccagc cggaagggcc
     2281 gagcgcagaa gtggtcctgc aactttatcc gcctccatcc agtctattaa ttgttgccgg
     2341 gaagctagag taagtagttc gccagttaat agtttgcgca acgttgttgc cattgctaca
     2401 ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat tcagctccgg ttcccaacga
     2461 tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag cggttagctc cttcggtcct
     2521 ccgatcgttg tcagaagtaa gttggccgca gtgttatcac tcatggttat ggcagcactg
     2581 cataattctc ttactgtcat gccatccgta agatgctttt ctgtgactgg tgagtactca
     2641 accaagtcat tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata
     2701 cgggataata ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg aaaacgttct
     2761 tcggggcgaa aactctcaag gatcttaccg ctgttgagat ccagttcgat gtaacccact
     2821 cgtgcaccca actgatcttc agcatctttt actttcacca gcgtttctgg gtgagcaaaa
     2881 acaggaaggc aaaatgccgc aaaaaaggga ataagggcga cacggaaatg ttgaatactc
     2941 atactcttcc tttttcaata ttattgaagc atttatcagg gttattgtct catgagcgga
     3001 tacatatttg aatgtattta gaaaaataaa caaatagggg ttccgcgcac atttccccga
     3061 aaagtgccac ctgacgtcga cggatcggga gatctcccga tcccctatgg tgcactctca
     3121 gtacaatctg ctctgatgcc gcatagttaa gccagtatct gctccctgct tgtgtgttgg
     3181 aggtcgctga gtagtgcgcg agcaaaattt aagctacaac aaggcaaggc ttgaccgaca
     3241 tttgcatgaa gaatctgctt agggttaggc gttttgcgct gcttcgcgat gtacgggcca
     3301 gatatacgcg ttgacattga ttattgacta gttattaata gtaatcaatt acggggtcat
     3361 tagttcatag cccatatatg gagttccgcg ttacataact tacggtaaat ggcccgcctg
     3421 gctgaccgcc caacgacccc cgcccattga cgtcaataat gacgtatgtt cccatagtaa
     3481 cgccaatagg gactttccat tgacgtcaat gggtggagta tttacggtaa actgcccact
     3541 tggcagtaca tcaagtgtat catatgccaa gtacgccccc tattgacgtc aatgacggta
     3601 aatggcccgc ctggcattat gcccagtaca tgaccttatg ggactttcct acttggcagt
     3661 acatctacgt attagtcatc gctattacca tggtgatgcg gttttggcag tacatcaatg
     3721 ggcgtggata gcggtttgac tcacggggat ttccaagtct ccaccccatt gacgtcaatg
     3781 ggagtttgtt ttggcaccaa aatcaacggg actttccaaa atgtcgtaac aactccgccc
     3841 cattgacgca aatgggcggt aggcgtgtac ggtgggaggt ctatataagc agagctctct
     3901 ggctaactag agaacccact gcttactggc ttatcgaaat taatacgact cactataggg
     3961 agacccaagc tggctagcgt ttaaacttaa gcttagcggc cgcgccacca tgaagacgat
     4021 catcgccctg agctacatct tctgcctggt attcgccgac tacaaggacg atgatgacgc
     4081 cagcatcgat atggctctca cacctgaaag tccttccagt ttccctggcc tcgccgccac
     4141 cggctctagc gtcccggagc cccctggcgg ccctaacgcc acactcaata gctcctgggc
     4201 aagtcccact gaaccgtctt ccctggagga tctcgtcgca acggggacca tcgggaccct
     4261 cctttccgca atgggtgtcg taggcgtagt cgggaatgct tatacactgg tcgtgacctg
     4321 caggtccctg agagcagtcg catccatgta tgtctacgtg gttaacctcg ctctcgctga
     4381 cttgctgtat ctgctctcta ttccctttat tgtggctacc tacgtgacta aagagtggca
     4441 tttcggagat gtcggctgca gggttctgtt cggccttgac tttctgacta tgcacgcttc
     4501 catcttcacg ttgaccgtaa tgagctctga acggtacgca gccgtgcttc ggcccctcga
     4561 cacggttcag aggccgaaag gttatagaaa gctcctggcc ctcggaacct ggctgttggc
     4621 tctgttgctg accctccctg tgatgctggc tatgcggctc gttagacgag gacctaaaag
     4681 cttgtgtttg cctgcctggg gccccagggc gcatcgggcc tacctgaccc tgctgttcgc
     4741 cacttccata gctgggcccg gactgctgat tggcctgctg tacgcaaggc tcgctcgggc
     4801 ctatagacgc tcccaaaggg cctcattcaa aagagccagg cggccggggg ccagagcact
     4861 ccggctggtt ctgggcatag ttctgctgtt ctgggcatgt ttcttgccct tttggctttg
     4921 gcaactgctg gcacagtacc atcaggcacc actggctccg cggaccgctc gcatcgtgaa
     4981 ttacttgaca acttgtttga cgtacggaaa ttcttgtgcc aaccctttct tgtacacact
     5041 cctcactcgc aattatcgag atcacttgag aggcagagtc cggggccccg gatctggagg
     5101 cgggaggggc cccgtgccaa gtctccagcc tagagcaagg ttccagcgct gttcaggcag
     5161 gagtttgagc tcttgttccc cacagcctac agatagtctg gtgttggccc cggcagcacc
     5221 ggcccgcccc gcccccgaag gtcctagagc tccagccatc gataccggtg gacgcaccgg
     5281 tgaattcgat gaggccagca ccaccgtgtc taagaccgag acttctcagg tggcccctgc
     5341 ctaatctaga gggcccgttt aaacccgctg atcagcctcg actgtgcctt ctagttgcca
     5401 gccatctgtt gtttgcccct cccccgtgcc ttccttgacc ctggaaggtg ccactcccac
     5461 tgtcctttcc taataaaatg aggaaattgc atcgcattgt ctgagtaggt gtcattctat
     5521 tctggggggt ggggtggggc aggacagcaa gggggaggat tgggaagaca atagcaggca
     5581 tgctggggat gcggtgggct ctatggcttc tgaggcggaa agaaccagct ggggctctag
     5641 ggggtatccc cacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg
     5701 cagcgtgacc gctacacttg ccagcgccct agcgcccgct cctttcgctt tcttcccttc
     5761 ctttctcgcc acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg
     5821 gttccgattt agtgctttac ggcacctcga ccccaaaaaa cttgattagg gtgatggttc
     5881 acgtagtggg ccatcgccct gatagacggt ttttcgccct ttgacgttgg agtccacgtt
     5941 ctttaatagt ggactcttgt tccaaactgg aacaacactc aaccctatct cggtctattc
     6001 ttttgattta taagggattt tgccgatttc ggcctattgg ttaaaaaatg agctgattta
     6061 acaaaaattt aacgcgaatt aattctgtgg aatgtgtgtc agttagggtg tggaaagtcc
     6121 ccaggctccc cagcaggcag aagtatgcaa agcatgcatc tcaattagtc agcaaccagg
     6181 tgtggaaagt ccccaggctc cccagcaggc agaagtatgc aaagcatgca tctcaattag
     6241 tcagcaacca tagtcccgcc cctaactccg cccatcccgc ccctaactcc gcccagttcc
     6301 gcccattctc cgccccatgg ctgactaatt ttttttattt atgcagaggc cgaggccgcc
     6361 tctgcctctg agctattcca gaagtagtga ggaggctttt ttggaggcct aggcttttgc
     6421 aaaaagctcc cgggagcttg tatatccatt ttcggatctg atcaagagac aggatgagga
     6481 tcgtttcgca tgattgaaca agatggattg cacgcaggtt ctccggccgc ttgggtggag
     6541 aggctattcg gctatgactg ggcacaacag acaatcggct gctctgatgc cgccgtgttc
     6601 cggctgtcag cgcaggggcg cccggttctt tttgtcaaga ccgacctgtc cggtgccctg
     6661 aatgaactgc aggacgaggc agcgcggcta tcgtggctgg ccacgac
//
