LOCUS       sequence-334261-        8285 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian expression of SARS-CoV-2 spike receptor-binding domain 
            (RBD), E484K mutant.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8285)
  AUTHORS   Mehalko J, Drew M, Snead K, Denson JP, Wall V, Taylor T, Sadtler K, 
            Messing S, Gillette W, Esposito D
  TITLE     Improved production of SARS-CoV-2 spike receptor-binding domain 
            (RBD) for serology assays.
  JOURNAL   Protein Expr Purif. 2021 Mar;179:105802. doi: 
            10.1016/j.pep.2020.105802. Epub 2020 Nov 25.
  PUBMED    33248226
REFERENCE   2  (bases 1 to 8285)
  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: "Protein 
            Expr Purif."; date: "2021-03"; volume: "179:105802. doi"; pages: " 
            10.1016/j.pep.2020.105802. Epub 2020 Nov 25"
COMMENT     Sequence Label: R620-M08-303: CMV51p> TPAss-SARS-CoV-2
            S-RBD(3...
FEATURES             Location/Qualifiers
     source          1..8285
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(413..432)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     protein_bind    582..602
                     /gene="mutant version of attB"
                     /label=attB4
                     /bound_moiety="BP Clonase(TM)"
                     /note="core recombination site for the Gateway(R) BP 
                     reaction"
     enhancer        613..916
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        917..1120
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     1071..1091
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     1117..1141
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     protein_bind    1186..1210
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             1939..1962
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     CDS             1978..2001
                     /codon_start=1
                     /product="8xHis affinity tag"
                     /label=8xHis
                     /translation="HHHHHHHH"
     CDS             2020..2133
                     /codon_start=1
                     /product="streptavidin-binding peptide"
                     /label=SBP
                     /note="selected from a peptide library; binds streptavidin 
                     with nanomolar affinity (Keefe et al., 2001)"
                     /translation="MDEKTTGWRGGHVVEGLAGELEQLRARLEHHPQGQREP"
     protein_bind    complement(2135..2159)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     promoter        complement(2221..2239)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(2222..2239)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(2259..2276)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2265..2489
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2535..2963
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2622..2641)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2832..2853
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2972..2992)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2977..3306
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3157..3292
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3219..3238
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3373..4167
                     /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(3427..3446)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4037..4056
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4341..4462
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4378..4397)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4432..4451
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4511..4527)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4511..4527)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4524..4546)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4535..4551
                     /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(4559..4565,4566..4583,4584..4589))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4604..4625
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4742..4759)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4913..5501)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4993..5012)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5672..6463,6464..6532))
                     /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     6295..6314
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6533..6637)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      join(6872..8285,1..376)
                     /label=oriP
                     /note="Epstein-Barr virus oriP replication origin (Yates et
                     al., 2000)"
ORIGIN
        1 tatctggata gcatatgcta tcctaatcta tatctgggta gcatatgcta tcctaatcta
       61 tatctgggta gcataggcta tcctaatcta tatctgggta gcatatgcta tcctaatcta
      121 tatctgggta gtatatgcta tcctaattta tatctgggta gcataggcta tcctaatcta
      181 tatctgggta gcatatgcta tcctaatcta tatctgggta gtatatgcta tcctaatctg
      241 tatccgggta gcatatgcta tcctcatgca tatacagtca gcatatgata cccagtagta
      301 gagtgggagt gctatccttt gcatatgccg ccacctccca agggggcgtg aattttcgct
      361 gcttgtcctt ttcctgctgg ggatctcccg atcccctatg gtgcactctc agtacaatct
      421 gctctgatgc cgcatagtta agccagtatc tgctccctgc ttgtgtgttg gaggtcgctg
      481 agtagtgcgc gagcaaaatt taagctacaa caaggcaagg cttgaccgac aattgcatga
      541 agaatctgct tagggttagg cgttttgcgc tgcttcgatc acaactttgt atagaaaagt
      601 tggccctgca ggcgttacat aacttacggt aaatggcccg cctggctgac cgcccaacga
      661 cccccgccca ttgacgtcaa taatgacgta tgttcccata gtaacgccaa tagggacttt
      721 ccattgacgt caatgggtgg agtatttacg gtaaactgcc cacttggcag tacatcaagt
      781 gtatcatatg ccaagtccgc cccctattga cgtcaatgac ggtaaatggc ccgcctggca
      841 ttatgcccag tacatgacct tacgggactt tcctacttgg cagtacatct acgtattagt
      901 catcgctatt accatggtga tgcggttttg gcagtacacc aatgggcgtg gatagcggtt
      961 tgactcacgg ggatttccaa gtctccaccc cattgacgtc aatgggagtt tgttttggca
     1021 ccaaaatcaa cgggactttc caaaatgtcg taataacccc gccccgttga cgcaaatggg
     1081 cggtaggcgt gtacggtggg aggtctatat aagcagagct cgtttagtga accgtcagat
     1141 cgcctggaga cgccatccac gctgttttga cctccataga agacaacaac tttgtacaaa
     1201 aaagttggca ccatggatgc catgaagcgg ggcctctgtt gcgtgctcct cctgtgcgga
     1261 gctgtgttcg tgagcccttc ggcatcgttc agagtgcagc ccaccgagtc catcgtgcgc
     1321 ttcccgaaca ttaccaacct ctgcccgttt ggggaggtgt tcaatgccac tagattcgcc
     1381 tccgtgtacg cgtggaatcg caagcggatt tccaactgcg tcgccgacta cagcgtgctg
     1441 tacaactccg cctccttttc gaccttcaag tgctacggag tgtcgcccac taagctgaac
     1501 gacctctgct tcaccaacgt gtacgcagac agcttcgtga ttagggggga tgaggtccgg
     1561 cagatcgctc cagggcagac ggggaagatc gccgattaca actacaagct gcccgacgac
     1621 ttcactggtt gcgtcatcgc ctggaactcc aacaacctgg acagcaaagt cggcggcaac
     1681 tacaactacc tgtaccggct gttccgcaag tccaacttga agccgttcga gcgggacatc
     1741 agcaccgaga tctaccaggc cggatcaacc ccctgtaacg gcgtcaaggg attcaactgt
     1801 tacttcccgc tgcaatccta tgggtttcag ccgaccaatg gcgtgggata tcagccctac
     1861 cgggtggtgg tgctgtcctt cgaacttctg cacgcgccag ccacagtgtg cggtcctaaa
     1921 aagggagcgg gttcctccct ggaagtgctg ttccaaggac ctggctcagg atcatcccac
     1981 caccaccatc atcaccacca cggaggcagc ggatcgtcta tggacgaaaa gactaccggt
     2041 tggagaggcg gccatgtcgt ggaaggcttg gccggagaac tggagcagct tagagccagg
     2101 ctggagcacc acccgcaagg acagcgcgaa ccttaatcaa ctttcttgta caaagttgtg
     2161 aaattctgca gatatccatc acactggcgg ccgctcgagc atgcatctag agggccctat
     2221 tctatagtgt cacctaaatg ctagagctcg ctgatcagcc tcgactgtgc cttctagttg
     2281 ccagccatct gttgtttgcc cctcccccgt gccttccttg accctggaag gtgccactcc
     2341 cactgtcctt tcctaataaa atgaggaaat tgcatcgcat tgtctgagta ggtgtcattc
     2401 tattctgggg ggtggggtgg ggcaggacag caagggggag gattgggaag acaatagcag
     2461 gcatgctggg gatgcggtgg gctctatggc ttctgaggcg gaaagaacca gctggggctc
     2521 tagggggtat ccccacgcgc cctgtagcgg cgcattaagc gcggcgggtg tggtggttac
     2581 gcgcagcgtg accgctacac ttgccagcgc cctagcgccc gctcctttcg ctttcttccc
     2641 ttcctttctc gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt
     2701 agggttccga tttagtgctt tacggcacct cgaccccaaa aaacttgatt agggtgatgg
     2761 ttcacgtagt gggccatcgc cctgatagac ggtttttcgc cctttgacgt tggagtccac
     2821 gttctttaat agtggactct tgttccaaac tggaacaaca ctcaacccta tctcggtcta
     2881 ttcttttgat ttataaggga ttttgccgat ttcggcctat tggttaaaaa atgagctgat
     2941 ttaacaaaaa tttaacgcga attaattctg tggaatgtgt gtcagttagg gtgtggaaag
     3001 tccccaggct ccccagcagg cagaagtatg caaagcatgc atctcaatta gtcagcaacc
     3061 aggtgtggaa agtccctagg ctccccagca ggcagaagta tgcaaagcat gcatctcaat
     3121 tagtcagcaa ccatagtccc gcccctaact ccgcccatcc cgcccctaac tccgcccagt
     3181 tccgcccatt ctccgcccca tggctgacta atttttttta tttatgcaga ggccgaggcc
     3241 gcctctgcct ctgagctatt ccagaagtag tgaggaggct tttttggagg cctaggcttt
     3301 tgcaaaaagc tcccgggagc ttgtatatcc attttcggat ctgatcaaga gacaggatga
     3361 ggatcgtttc gcatgattga acaagatgga ttgcacgcag gttctccggc cgcttgggtg
     3421 gagaggctat tcggctatga ctgggcacaa cagacaatcg gctgctctga tgccgccgtg
     3481 ttccggctgt cagcgcaggg gcgcccggtt ctttttgtca agaccgacct gtccggtgcc
     3541 ctgaatgaac tgcaggacga ggcagcgcgg ctatcgtggc tggccacgac gggcgttcct
     3601 tgcgcagctg tgctcgacgt tgtcactgaa gcgggaaggg actggctgct attgggcgaa
     3661 gtgccggggc aggatctcct gtcatctcac cttgctcctg ccgagaaagt atccatcatg
     3721 gctgatgcaa tgcggcggct gcatacgctt gatccggcta cctgcccatt cgaccaccaa
     3781 gcgaaacatc gcatcgagcg agcacgtact cggatggaag ccggtcttgt cgatcaggat
     3841 gatctggacg aagagcatca ggggctcgcg ccagccgaac tgttcgccag gctcaaggcg
     3901 cgcatgcccg acggcgagga tctcgtcgtg acccatggcg atgcctgctt gccgaatatc
     3961 atggtggaaa atggccgctt ttctggattc atcgactgtg gccggctggg tgtggcggac
     4021 cgctatcagg acatagcgtt ggctacccgt gatattgctg aagagcttgg cggcgaatgg
     4081 gctgaccgct tcctcgtgct ttacggtatc gccgctcccg attcgcagcg catcgccttc
     4141 tatcgccttc ttgacgagtt cttctgagcg ggactctggg gttcgaaatg accgaccaag
     4201 cgacgcccaa cctgccatca cgagatttcg attccaccgc cgccttctat gaaaggttgg
     4261 gcttcggaat cgttttccgg gacgccggct ggatgatcct ccagcgcggg gatctcatgc
     4321 tggagttctt cgcccacccc aacttgttta ttgcagctta taatggttac aaataaagca
     4381 atagcatcac aaatttcaca aataaagcat ttttttcact gcattctagt tgtggtttgt
     4441 ccaaactcat caatgtatct tatcatgtct gtataccgtc gacctctagc tagagcttgg
     4501 cgtaatcatg gtcatagctg tttcctgtgt gaaattgtta tccgctcaca attccacaca
     4561 acatacgagc cggaagcata aagtgtaaag cctggggtgc ctaatgagtg agctaactca
     4621 cattaattgc gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc
     4681 attaatgaat cggccaacgc gcggggagag gcggtttgcg tattgggcgc tcttccgctt
     4741 cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta tcagctcact
     4801 caaaggcggt aatacggtta tccacagaat caggggataa cgcaggaaag aacatgtgag
     4861 caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg tttttccata
     4921 ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg tggcgaaacc
     4981 cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg cgctctcctg
     5041 ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga agcgtggcgc
     5101 tttctcatag ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc tccaagctgg
     5161 gctgtgtgca cgaacccccc gttcagcccg accgctgcgc cttatccggt aactatcgtc
     5221 ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact ggtaacagga
     5281 ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg cctaactacg
     5341 gctacactag aagaacagta tttggtatct gcgctctgct gaagccagtt accttcggaa
     5401 aaagagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt ggtttttttg
     5461 tttgcaagca gcagattacg cgcagaaaaa aaggatctca agaagatcct ttgatctttt
     5521 ctacggggtc tgacgctcag tggaacgaaa actcacgtta agggattttg gtcatgagat
     5581 tatcaaaaag gatcttcacc tagatccttt taaattaaaa atgaagtttt aaatcaatct
     5641 aaagtatata tgagtaaact tggtctgaca gttaccaatg cttaatcagt gaggcaccta
     5701 tctcagcgat ctgtctattt cgttcatcca tagttgcctg actccccgtc gtgtagataa
     5761 ctacgatacg ggagggctta ccatctggcc ccagtgctgc aatgataccg cgagacccac
     5821 gctcaccggc tccagattta tcagcaataa accagccagc cggaagggcc gagcgcagaa
     5881 gtggtcctgc aactttatcc gcctccatcc agtctattaa ttgttgccgg gaagctagag
     5941 taagtagttc gccagttaat agtttgcgca acgttgttgc cattgctaca ggcatcgtgg
     6001 tgtcacgctc gtcgtttggt atggcttcat tcagctccgg ttcccaacga tcaaggcgag
     6061 ttacatgatc ccccatgttg tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg
     6121 tcagaagtaa gttggccgca gtgttatcac tcatggttat ggcagcactg cataattctc
     6181 ttactgtcat gccatccgta agatgctttt ctgtgactgg tgagtactca accaagtcat
     6241 tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata cgggataata
     6301 ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa
     6361 aactctcaag gatcttaccg ctgttgagat ccagttcgat gtaacccact cgtgcaccca
     6421 actgatcttc agcatctttt actttcacca gcgtttctgg gtgagcaaaa acaggaaggc
     6481 aaaatgccgc aaaaaaggga ataagggcga cacggaaatg ttgaatactc atactcttcc
     6541 tttttcaata ttattgaagc atttatcagg gttattgtct catgagcgga tacatatttg
     6601 aatgtattta gaaaaataaa caaatagggg ttccgcgcac atttccccga aaagtgccac
     6661 ctgacgtcga cggatcggga gatccgcctt tatgtgtaac tcttggctga agctcttaca
     6721 ccaatgctgg gggacatgta cctcccaggg gcccaggaag actacgggag gctacaccaa
     6781 cgtcaatcag aggggcctgt gtagctaccg ataagcggac cctcaagagg gcattagcaa
     6841 tagtgtttat aaggccccct tgttaaccct aaacgggtag catatgcttc ccgggtagta
     6901 gtatatacta tccagactaa ccctaattca atagcatatg ttacccaacg ggaagcatat
     6961 gctatcgaat tagggttagt aaaagggtcc taaggaacag cgatatctcc caccccatga
     7021 gctgtcacgg ttttatttac atggggtcag gattccacga gggtagtgaa ccattttagt
     7081 cacaagggca gtggctgaag atcaaggagc gggcagtgaa ctctcctgaa tcttcgcctg
     7141 cttcttcatt ctccttcgtt tagctaatag aataactgct gagttgtgaa cagtaaggtg
     7201 tatgtgaggt gctcgaaaac aaggtttcag gtgacgcccc cagaataaaa tttggacggg
     7261 gggttcagtg gtggcattgt gctatgacac caatataacc ctcacaaacc ccttgggcaa
     7321 taaatactag tgtaggaatg aaacattctg aatatcttta acaatagaaa tccatggggt
     7381 ggggacaagc cgtaaagact ggatgtccat ctcacacgaa tttatggcta tgggcaacac
     7441 ataatcctag tgcaatatga tactggggtt attaagatgt gtcccaggca gggaccaaga
     7501 caggtgaacc atgttgttac actctatttg taacaagggg aaagagagtg gacgccgaca
     7561 gcagcggact ccactggttg tctctaacac ccccgaaaat taaacggggc tccacgccaa
     7621 tggggcccat aaacaaagac aagtggccac tctttttttt gaaattgtgg agtgggggca
     7681 cgcgtcagcc cccacacgcc gccctgcggt tttggactgt aaaataaggg tgtaataact
     7741 tggctgattg taaccccgct aaccactgcg gtcaaaccac ttgcccacaa aaccactaat
     7801 ggcaccccgg ggaatacctg cataagtagg tgggcgggcc aagatagggg cgcgattgct
     7861 gcgatctgga ggacaaatta cacacacttg cgcctgagcg ccaagcacag ggttgttggt
     7921 cctcatattc acgaggtcgc tgagagcacg gtgggctaat gttgccatgg gtagcatata
     7981 ctacccaaat atctggatag catatgctat cctaatctat atctgggtag cataggctat
     8041 cctaatctat atctgggtag catatgctat cctaatctat atctgggtag tatatgctat
     8101 cctaatttat atctgggtag cataggctat cctaatctat atctgggtag catatgctat
     8161 cctaatctat atctgggtag tatatgctat cctaatctgt atccgggtag catatgctat
     8221 cctaatagag attagggtag tatatgctat cctaatttat atctgggtag catatactac
     8281 ccaaa
//
