LOCUS       sequence-334254-        8093 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian expression of SARS-CoV-2 spike receptor-binding domain 
            (RBD), triple mutant from B.1.351 variant.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8093)
  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 8093)
  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-M05-303: CMV51p> TPAss-SARS-CoV-2
            S-RBD(3...
FEATURES             Location/Qualifiers
     source          1..8093
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(570..589)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     protein_bind    739..759
                     /gene="mutant version of attB"
                     /label=attB4
                     /bound_moiety="BP Clonase(TM)"
                     /note="core recombination site for the Gateway(R) BP 
                     reaction"
     enhancer        770..1073
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        1074..1277
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     1228..1248
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     1274..1298
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     protein_bind    1343..1367
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             2081..2098
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     protein_bind    complement(2100..2124)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     promoter        complement(2186..2204)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(2187..2204)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(2224..2241)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2230..2454
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2500..2928
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2587..2606)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2797..2818
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2937..2957)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2942..3271
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3122..3257
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3184..3203
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3338..4132
                     /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(3392..3411)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4002..4021
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4306..4427
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4343..4362)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4397..4416
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4476..4492)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4476..4492)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4489..4511)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4500..4516
                     /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(4524..4530,4531..4548,4549..4554))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4569..4590
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4707..4724)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4878..5466)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4958..4977)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5637..6428,6429..6497))
                     /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     6260..6279
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6498..6602)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      join(6837..8093,1..533)
                     /label=oriP
                     /note="Epstein-Barr virus oriP replication origin (Yates et
                     al., 2000)"
ORIGIN
        1 atcctaatct atatctgggt agcatatgct atcctaatct atatctgggt agtatatgct
       61 atcctaatct gtatccgggt agcatatgct atcctaatag agattagggt agtatatgct
      121 atcctaattt atatctgggt agcatatact acccaaatat ctggatagca tatgctatcc
      181 taatctatat ctgggtagca tatgctatcc taatctatat ctgggtagca taggctatcc
      241 taatctatat ctgggtagca tatgctatcc taatctatat ctgggtagta tatgctatcc
      301 taatttatat ctgggtagca taggctatcc taatctatat ctgggtagca tatgctatcc
      361 taatctatat ctgggtagta tatgctatcc taatctgtat ccgggtagca tatgctatcc
      421 tcatgcatat acagtcagca tatgataccc agtagtagag tgggagtgct atcctttgca
      481 tatgccgcca cctcccaagg gggcgtgaat tttcgctgct tgtccttttc ctgctgggga
      541 tctcccgatc ccctatggtg cactctcagt acaatctgct ctgatgccgc atagttaagc
      601 cagtatctgc tccctgcttg tgtgttggag gtcgctgagt agtgcgcgag caaaatttaa
      661 gctacaacaa ggcaaggctt gaccgacaat tgcatgaaga atctgcttag ggttaggcgt
      721 tttgcgctgc ttcgatcaca actttgtata gaaaagttgg ccctgcaggc gttacataac
      781 ttacggtaaa tggcccgcct ggctgaccgc ccaacgaccc ccgcccattg acgtcaataa
      841 tgacgtatgt tcccatagta acgccaatag ggactttcca ttgacgtcaa tgggtggagt
      901 atttacggta aactgcccac ttggcagtac atcaagtgta tcatatgcca agtccgcccc
      961 ctattgacgt caatgacggt aaatggcccg cctggcatta tgcccagtac atgaccttac
     1021 gggactttcc tacttggcag tacatctacg tattagtcat cgctattacc atggtgatgc
     1081 ggttttggca gtacaccaat gggcgtggat agcggtttga ctcacgggga tttccaagtc
     1141 tccaccccat tgacgtcaat gggagtttgt tttggcacca aaatcaacgg gactttccaa
     1201 aatgtcgtaa taaccccgcc ccgttgacgc aaatgggcgg taggcgtgta cggtgggagg
     1261 tctatataag cagagctcgt ttagtgaacc gtcagatcgc ctggagacgc catccacgct
     1321 gttttgacct ccatagaaga caacaacttt gtacaaaaaa gttggcacca tgttcgtgtt
     1381 tctcgtcctg ctgccactcg tgtccagcca gagggtgcag ccgaccgaat ccatcgtgcg
     1441 cttccctaac atcaccaatc tgtgcccgtt cggggaggtg ttcaacgcta cccggttcgc
     1501 ctcggtctac gcctggaacc ggaagcggat ttccaactgc gtcgcggact actcagtgtt
     1561 gtacaactcc gcctcttttt ccactttcaa atgctacggg gtgtcgccca caaagctgaa
     1621 tgatctgtgc tttaccaacg tgtacgccga ctcgttcgtg atcagaggag atgaggtccg
     1681 gcagattgca cctggccaga ccggaaacat cgcggattac aactataagc tgcccgacga
     1741 cttcaccggc tgcgtgattg cttggaactc caacaacctg gacagcaaag tcggcggaaa
     1801 ctacaactac ctgtaccggc tgttccgcaa gtccaacctg aagccgttcg agcgggacat
     1861 ctccacggaa atctaccagg ccggatcgac cccatgcaac ggcgtgaagg ggttcaactg
     1921 ttatttcccc ttgcaatcct acggattcca gccaacttac ggggtcggct accagcctta
     1981 ccgcgtggtg gtgctttcgt ttgaactgct ccatgcaccc gccactgtgt gcggcccgaa
     2041 gaagtccacc aatctggtca agaacaaatg cgtgaacttc caccatcatc accaccacta
     2101 atcaactttc ttgtacaaag ttgtgaaatt ctgcagatat ccatcacact ggcggccgct
     2161 cgagcatgca tctagagggc cctattctat agtgtcacct aaatgctaga gctcgctgat
     2221 cagcctcgac tgtgccttct agttgccagc catctgttgt ttgcccctcc cccgtgcctt
     2281 ccttgaccct ggaaggtgcc actcccactg tcctttccta ataaaatgag gaaattgcat
     2341 cgcattgtct gagtaggtgt cattctattc tggggggtgg ggtggggcag gacagcaagg
     2401 gggaggattg ggaagacaat agcaggcatg ctggggatgc ggtgggctct atggcttctg
     2461 aggcggaaag aaccagctgg ggctctaggg ggtatcccca cgcgccctgt agcggcgcat
     2521 taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc agcgccctag
     2581 cgcccgctcc tttcgctttc ttcccttcct ttctcgccac gttcgccggc tttccccgtc
     2641 aagctctaaa tcgggggctc cctttagggt tccgatttag tgctttacgg cacctcgacc
     2701 ccaaaaaact tgattagggt gatggttcac gtagtgggcc atcgccctga tagacggttt
     2761 ttcgcccttt gacgttggag tccacgttct ttaatagtgg actcttgttc caaactggaa
     2821 caacactcaa ccctatctcg gtctattctt ttgatttata agggattttg ccgatttcgg
     2881 cctattggtt aaaaaatgag ctgatttaac aaaaatttaa cgcgaattaa ttctgtggaa
     2941 tgtgtgtcag ttagggtgtg gaaagtcccc aggctcccca gcaggcagaa gtatgcaaag
     3001 catgcatctc aattagtcag caaccaggtg tggaaagtcc ctaggctccc cagcaggcag
     3061 aagtatgcaa agcatgcatc tcaattagtc agcaaccata gtcccgcccc taactccgcc
     3121 catcccgccc ctaactccgc ccagttccgc ccattctccg ccccatggct gactaatttt
     3181 ttttatttat gcagaggccg aggccgcctc tgcctctgag ctattccaga agtagtgagg
     3241 aggctttttt ggaggcctag gcttttgcaa aaagctcccg ggagcttgta tatccatttt
     3301 cggatctgat caagagacag gatgaggatc gtttcgcatg attgaacaag atggattgca
     3361 cgcaggttct ccggccgctt gggtggagag gctattcggc tatgactggg cacaacagac
     3421 aatcggctgc tctgatgccg ccgtgttccg gctgtcagcg caggggcgcc cggttctttt
     3481 tgtcaagacc gacctgtccg gtgccctgaa tgaactgcag gacgaggcag cgcggctatc
     3541 gtggctggcc acgacgggcg ttccttgcgc agctgtgctc gacgttgtca ctgaagcggg
     3601 aagggactgg ctgctattgg gcgaagtgcc ggggcaggat ctcctgtcat ctcaccttgc
     3661 tcctgccgag aaagtatcca tcatggctga tgcaatgcgg cggctgcata cgcttgatcc
     3721 ggctacctgc ccattcgacc accaagcgaa acatcgcatc gagcgagcac gtactcggat
     3781 ggaagccggt cttgtcgatc aggatgatct ggacgaagag catcaggggc tcgcgccagc
     3841 cgaactgttc gccaggctca aggcgcgcat gcccgacggc gaggatctcg tcgtgaccca
     3901 tggcgatgcc tgcttgccga atatcatggt ggaaaatggc cgcttttctg gattcatcga
     3961 ctgtggccgg ctgggtgtgg cggaccgcta tcaggacata gcgttggcta cccgtgatat
     4021 tgctgaagag cttggcggcg aatgggctga ccgcttcctc gtgctttacg gtatcgccgc
     4081 tcccgattcg cagcgcatcg ccttctatcg ccttcttgac gagttcttct gagcgggact
     4141 ctggggttcg aaatgaccga ccaagcgacg cccaacctgc catcacgaga tttcgattcc
     4201 accgccgcct tctatgaaag gttgggcttc ggaatcgttt tccgggacgc cggctggatg
     4261 atcctccagc gcggggatct catgctggag ttcttcgccc accccaactt gtttattgca
     4321 gcttataatg gttacaaata aagcaatagc atcacaaatt tcacaaataa agcatttttt
     4381 tcactgcatt ctagttgtgg tttgtccaaa ctcatcaatg tatcttatca tgtctgtata
     4441 ccgtcgacct ctagctagag cttggcgtaa tcatggtcat agctgtttcc tgtgtgaaat
     4501 tgttatccgc tcacaattcc acacaacata cgagccggaa gcataaagtg taaagcctgg
     4561 ggtgcctaat gagtgagcta actcacatta attgcgttgc gctcactgcc cgctttccag
     4621 tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt
     4681 ttgcgtattg ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg
     4741 ctgcggcgag cggtatcagc tcactcaaag gcggtaatac ggttatccac agaatcaggg
     4801 gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag
     4861 gccgcgttgc tggcgttttt ccataggctc cgcccccctg acgagcatca caaaaatcga
     4921 cgctcaagtc agaggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct
     4981 ggaagctccc tcgtgcgctc tcctgttccg accctgccgc ttaccggata cctgtccgcc
     5041 tttctccctt cgggaagcgt ggcgctttct catagctcac gctgtaggta tctcagttcg
     5101 gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac cccccgttca gcccgaccgc
     5161 tgcgccttat ccggtaacta tcgtcttgag tccaacccgg taagacacga cttatcgcca
     5221 ctggcagcag ccactggtaa caggattagc agagcgaggt atgtaggcgg tgctacagag
     5281 ttcttgaagt ggtggcctaa ctacggctac actagaagaa cagtatttgg tatctgcgct
     5341 ctgctgaagc cagttacctt cggaaaaaga gttggtagct cttgatccgg caaacaaacc
     5401 accgctggta gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga
     5461 tctcaagaag atcctttgat cttttctacg gggtctgacg ctcagtggaa cgaaaactca
     5521 cgttaaggga ttttggtcat gagattatca aaaaggatct tcacctagat ccttttaaat
     5581 taaaaatgaa gttttaaatc aatctaaagt atatatgagt aaacttggtc tgacagttac
     5641 caatgcttaa tcagtgaggc acctatctca gcgatctgtc tatttcgttc atccatagtt
     5701 gcctgactcc ccgtcgtgta gataactacg atacgggagg gcttaccatc tggccccagt
     5761 gctgcaatga taccgcgaga cccacgctca ccggctccag atttatcagc aataaaccag
     5821 ccagccggaa gggccgagcg cagaagtggt cctgcaactt tatccgcctc catccagtct
     5881 attaattgtt gccgggaagc tagagtaagt agttcgccag ttaatagttt gcgcaacgtt
     5941 gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc
     6001 tccggttccc aacgatcaag gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt
     6061 agctccttcg gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg
     6121 gttatggcag cactgcataa ttctcttact gtcatgccat ccgtaagatg cttttctgtg
     6181 actggtgagt actcaaccaa gtcattctga gaatagtgta tgcggcgacc gagttgctct
     6241 tgcccggcgt caatacggga taataccgcg ccacatagca gaactttaaa agtgctcatc
     6301 attggaaaac gttcttcggg gcgaaaactc tcaaggatct taccgctgtt gagatccagt
     6361 tcgatgtaac ccactcgtgc acccaactga tcttcagcat cttttacttt caccagcgtt
     6421 tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg
     6481 aaatgttgaa tactcatact cttccttttt caatattatt gaagcattta tcagggttat
     6541 tgtctcatga gcggatacat atttgaatgt atttagaaaa ataaacaaat aggggttccg
     6601 cgcacatttc cccgaaaagt gccacctgac gtcgacggat cgggagatcc gcctttatgt
     6661 gtaactcttg gctgaagctc ttacaccaat gctgggggac atgtacctcc caggggccca
     6721 ggaagactac gggaggctac accaacgtca atcagagggg cctgtgtagc taccgataag
     6781 cggaccctca agagggcatt agcaatagtg tttataaggc ccccttgtta accctaaacg
     6841 ggtagcatat gcttcccggg tagtagtata tactatccag actaacccta attcaatagc
     6901 atatgttacc caacgggaag catatgctat cgaattaggg ttagtaaaag ggtcctaagg
     6961 aacagcgata tctcccaccc catgagctgt cacggtttta tttacatggg gtcaggattc
     7021 cacgagggta gtgaaccatt ttagtcacaa gggcagtggc tgaagatcaa ggagcgggca
     7081 gtgaactctc ctgaatcttc gcctgcttct tcattctcct tcgtttagct aatagaataa
     7141 ctgctgagtt gtgaacagta aggtgtatgt gaggtgctcg aaaacaaggt ttcaggtgac
     7201 gcccccagaa taaaatttgg acggggggtt cagtggtggc attgtgctat gacaccaata
     7261 taaccctcac aaaccccttg ggcaataaat actagtgtag gaatgaaaca ttctgaatat
     7321 ctttaacaat agaaatccat ggggtgggga caagccgtaa agactggatg tccatctcac
     7381 acgaatttat ggctatgggc aacacataat cctagtgcaa tatgatactg gggttattaa
     7441 gatgtgtccc aggcagggac caagacaggt gaaccatgtt gttacactct atttgtaaca
     7501 aggggaaaga gagtggacgc cgacagcagc ggactccact ggttgtctct aacacccccg
     7561 aaaattaaac ggggctccac gccaatgggg cccataaaca aagacaagtg gccactcttt
     7621 tttttgaaat tgtggagtgg gggcacgcgt cagcccccac acgccgccct gcggttttgg
     7681 actgtaaaat aagggtgtaa taacttggct gattgtaacc ccgctaacca ctgcggtcaa
     7741 accacttgcc cacaaaacca ctaatggcac cccggggaat acctgcataa gtaggtgggc
     7801 gggccaagat aggggcgcga ttgctgcgat ctggaggaca aattacacac acttgcgcct
     7861 gagcgccaag cacagggttg ttggtcctca tattcacgag gtcgctgaga gcacggtggg
     7921 ctaatgttgc catgggtagc atatactacc caaatatctg gatagcatat gctatcctaa
     7981 tctatatctg ggtagcatag gctatcctaa tctatatctg ggtagcatat gctatcctaa
     8041 tctatatctg ggtagtatat gctatcctaa tttatatctg ggtagcatag gct
//
