LOCUS       sequence-334271-        8093 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian expression of SARS-CoV-2 spike receptor-binding domain 
            (RBD), N501Y mutant from B.1.1.28 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-M18-303: CMV51p> SARS-CoV-2 S-RBD(319-541...
FEATURES             Location/Qualifiers
     source          1..8093
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(403..422)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     protein_bind    572..592
                     /gene="mutant version of attB"
                     /label=attB4
                     /bound_moiety="BP Clonase(TM)"
                     /note="core recombination site for the Gateway(R) BP 
                     reaction"
     enhancer        603..906
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        907..1110
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     1061..1081
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     1107..1131
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     protein_bind    1176..1200
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             1914..1931
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     protein_bind    complement(1933..1957)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     promoter        complement(2019..2037)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(2020..2037)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(2057..2074)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2063..2287
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2333..2761
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2420..2439)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2630..2651
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2770..2790)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2775..3104
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      2955..3090
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3017..3036
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3171..3965
                     /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(3225..3244)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3835..3854
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4139..4260
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4176..4195)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4230..4249
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4309..4325)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4309..4325)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4322..4344)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4333..4349
                     /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(4357..4363,4364..4381,4382..4387))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4402..4423
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4540..4557)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4711..5299)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4791..4810)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5470..6261,6262..6330))
                     /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     6093..6112
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6331..6435)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      join(6670..8093,1..366)
                     /label=oriP
                     /note="Epstein-Barr virus oriP replication origin (Yates et
                     al., 2000)"
ORIGIN
        1 gcatatgcta tcctaatcta tatctgggta gcatatgcta tcctaatcta tatctgggta
       61 gcataggcta tcctaatcta tatctgggta gcatatgcta tcctaatcta tatctgggta
      121 gtatatgcta tcctaattta tatctgggta gcataggcta tcctaatcta tatctgggta
      181 gcatatgcta tcctaatcta tatctgggta gtatatgcta tcctaatctg tatccgggta
      241 gcatatgcta tcctcatgca tatacagtca gcatatgata cccagtagta gagtgggagt
      301 gctatccttt gcatatgccg ccacctccca agggggcgtg aattttcgct gcttgtcctt
      361 ttcctgctgg ggatctcccg atcccctatg gtgcactctc agtacaatct gctctgatgc
      421 cgcatagtta agccagtatc tgctccctgc ttgtgtgttg gaggtcgctg agtagtgcgc
      481 gagcaaaatt taagctacaa caaggcaagg cttgaccgac aattgcatga agaatctgct
      541 tagggttagg cgttttgcgc tgcttcgatc acaactttgt atagaaaagt tggccctgca
      601 ggcgttacat aacttacggt aaatggcccg cctggctgac cgcccaacga cccccgccca
      661 ttgacgtcaa taatgacgta tgttcccata gtaacgccaa tagggacttt ccattgacgt
      721 caatgggtgg agtatttacg gtaaactgcc cacttggcag tacatcaagt gtatcatatg
      781 ccaagtccgc cccctattga cgtcaatgac ggtaaatggc ccgcctggca ttatgcccag
      841 tacatgacct tacgggactt tcctacttgg cagtacatct acgtattagt catcgctatt
      901 accatggtga tgcggttttg gcagtacacc aatgggcgtg gatagcggtt tgactcacgg
      961 ggatttccaa gtctccaccc cattgacgtc aatgggagtt tgttttggca ccaaaatcaa
     1021 cgggactttc caaaatgtcg taataacccc gccccgttga cgcaaatggg cggtaggcgt
     1081 gtacggtggg aggtctatat aagcagagct cgtttagtga accgtcagat cgcctggaga
     1141 cgccatccac gctgttttga cctccataga agacaacaac tttgtacaaa aaagttggca
     1201 ccatgttcgt gtttctcgtc ctgctgccac tcgtgtccag ccagagggtg cagccgaccg
     1261 aatccatcgt gcgcttccct aacatcacca atctgtgccc gttcggggag gtgttcaacg
     1321 ctacccggtt cgcctcggtc tacgcctgga accggaagcg gatttccaac tgcgtcgcgg
     1381 actactcagt gttgtacaac tccgcctctt tttccacttt caaatgctac ggggtgtcgc
     1441 ccacaaagct gaatgatctg tgctttacca acgtgtacgc cgactcgttc gtgatcagag
     1501 gagatgaggt ccggcagatt gcacctggcc agaccggaac tatcgcggat tacaactata
     1561 agctgcccga cgacttcacc ggctgcgtga ttgcttggaa ctccaacaac ctggacagca
     1621 aagtcggcgg aaactacaac tacctgtacc ggctgttccg caagtccaac ctgaagccgt
     1681 tcgagcggga catctccacg gaaatctacc aggccggatc gaccccatgc aacggcgtga
     1741 aggggttcaa ctgttatttc cccttgcaat cctacggatt ccagccaact tacggggtcg
     1801 gctaccagcc ttaccgcgtg gtggtgcttt cgtttgaact gctccatgca cccgccactg
     1861 tgtgcggccc gaagaagtcc accaatctgg tcaagaacaa atgcgtgaac ttccaccatc
     1921 atcaccacca ctaatcaact ttcttgtaca aagttgtgaa attctgcaga tatccatcac
     1981 actggcggcc gctcgagcat gcatctagag ggccctattc tatagtgtca cctaaatgct
     2041 agagctcgct gatcagcctc gactgtgcct tctagttgcc agccatctgt tgtttgcccc
     2101 tcccccgtgc cttccttgac cctggaaggt gccactccca ctgtcctttc ctaataaaat
     2161 gaggaaattg catcgcattg tctgagtagg tgtcattcta ttctgggggg tggggtgggg
     2221 caggacagca agggggagga ttgggaagac aatagcaggc atgctgggga tgcggtgggc
     2281 tctatggctt ctgaggcgga aagaaccagc tggggctcta gggggtatcc ccacgcgccc
     2341 tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt
     2401 gccagcgccc tagcgcccgc tcctttcgct ttcttccctt cctttctcgc cacgttcgcc
     2461 ggctttcccc gtcaagctct aaatcggggg ctccctttag ggttccgatt tagtgcttta
     2521 cggcacctcg accccaaaaa acttgattag ggtgatggtt cacgtagtgg gccatcgccc
     2581 tgatagacgg tttttcgccc tttgacgttg gagtccacgt tctttaatag tggactcttg
     2641 ttccaaactg gaacaacact caaccctatc tcggtctatt cttttgattt ataagggatt
     2701 ttgccgattt cggcctattg gttaaaaaat gagctgattt aacaaaaatt taacgcgaat
     2761 taattctgtg gaatgtgtgt cagttagggt gtggaaagtc cccaggctcc ccagcaggca
     2821 gaagtatgca aagcatgcat ctcaattagt cagcaaccag gtgtggaaag tccctaggct
     2881 ccccagcagg cagaagtatg caaagcatgc atctcaatta gtcagcaacc atagtcccgc
     2941 ccctaactcc gcccatcccg cccctaactc cgcccagttc cgcccattct ccgccccatg
     3001 gctgactaat tttttttatt tatgcagagg ccgaggccgc ctctgcctct gagctattcc
     3061 agaagtagtg aggaggcttt tttggaggcc taggcttttg caaaaagctc ccgggagctt
     3121 gtatatccat tttcggatct gatcaagaga caggatgagg atcgtttcgc atgattgaac
     3181 aagatggatt gcacgcaggt tctccggccg cttgggtgga gaggctattc ggctatgact
     3241 gggcacaaca gacaatcggc tgctctgatg ccgccgtgtt ccggctgtca gcgcaggggc
     3301 gcccggttct ttttgtcaag accgacctgt ccggtgccct gaatgaactg caggacgagg
     3361 cagcgcggct atcgtggctg gccacgacgg gcgttccttg cgcagctgtg ctcgacgttg
     3421 tcactgaagc gggaagggac tggctgctat tgggcgaagt gccggggcag gatctcctgt
     3481 catctcacct tgctcctgcc gagaaagtat ccatcatggc tgatgcaatg cggcggctgc
     3541 atacgcttga tccggctacc tgcccattcg accaccaagc gaaacatcgc atcgagcgag
     3601 cacgtactcg gatggaagcc ggtcttgtcg atcaggatga tctggacgaa gagcatcagg
     3661 ggctcgcgcc agccgaactg ttcgccaggc tcaaggcgcg catgcccgac ggcgaggatc
     3721 tcgtcgtgac ccatggcgat gcctgcttgc cgaatatcat ggtggaaaat ggccgctttt
     3781 ctggattcat cgactgtggc cggctgggtg tggcggaccg ctatcaggac atagcgttgg
     3841 ctacccgtga tattgctgaa gagcttggcg gcgaatgggc tgaccgcttc ctcgtgcttt
     3901 acggtatcgc cgctcccgat tcgcagcgca tcgccttcta tcgccttctt gacgagttct
     3961 tctgagcggg actctggggt tcgaaatgac cgaccaagcg acgcccaacc tgccatcacg
     4021 agatttcgat tccaccgccg ccttctatga aaggttgggc ttcggaatcg ttttccggga
     4081 cgccggctgg atgatcctcc agcgcgggga tctcatgctg gagttcttcg cccaccccaa
     4141 cttgtttatt gcagcttata atggttacaa ataaagcaat agcatcacaa atttcacaaa
     4201 taaagcattt ttttcactgc attctagttg tggtttgtcc aaactcatca atgtatctta
     4261 tcatgtctgt ataccgtcga cctctagcta gagcttggcg taatcatggt catagctgtt
     4321 tcctgtgtga aattgttatc cgctcacaat tccacacaac atacgagccg gaagcataaa
     4381 gtgtaaagcc tggggtgcct aatgagtgag ctaactcaca ttaattgcgt tgcgctcact
     4441 gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc
     4501 ggggagaggc ggtttgcgta ttgggcgctc ttccgcttcc tcgctcactg actcgctgcg
     4561 ctcggtcgtt cggctgcggc gagcggtatc agctcactca aaggcggtaa tacggttatc
     4621 cacagaatca ggggataacg caggaaagaa catgtgagca aaaggccagc aaaaggccag
     4681 gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg ctccgccccc ctgacgagca
     4741 tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg acaggactat aaagatacca
     4801 ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg
     4861 atacctgtcc gcctttctcc cttcgggaag cgtggcgctt tctcatagct cacgctgtag
     4921 gtatctcagt tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt
     4981 tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc cggtaagaca
     5041 cgacttatcg ccactggcag cagccactgg taacaggatt agcagagcga ggtatgtagg
     5101 cggtgctaca gagttcttga agtggtggcc taactacggc tacactagaa gaacagtatt
     5161 tggtatctgc gctctgctga agccagttac cttcggaaaa agagttggta gctcttgatc
     5221 cggcaaacaa accaccgctg gtagcggtgg tttttttgtt tgcaagcagc agattacgcg
     5281 cagaaaaaaa ggatctcaag aagatccttt gatcttttct acggggtctg acgctcagtg
     5341 gaacgaaaac tcacgttaag ggattttggt catgagatta tcaaaaagga tcttcaccta
     5401 gatcctttta aattaaaaat gaagttttaa atcaatctaa agtatatatg agtaaacttg
     5461 gtctgacagt taccaatgct taatcagtga ggcacctatc tcagcgatct gtctatttcg
     5521 ttcatccata gttgcctgac tccccgtcgt gtagataact acgatacggg agggcttacc
     5581 atctggcccc agtgctgcaa tgataccgcg agacccacgc tcaccggctc cagatttatc
     5641 agcaataaac cagccagccg gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc
     5701 ctccatccag tctattaatt gttgccggga agctagagta agtagttcgc cagttaatag
     5761 tttgcgcaac gttgttgcca ttgctacagg catcgtggtg tcacgctcgt cgtttggtat
     5821 ggcttcattc agctccggtt cccaacgatc aaggcgagtt acatgatccc ccatgttgtg
     5881 caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt
     5941 gttatcactc atggttatgg cagcactgca taattctctt actgtcatgc catccgtaag
     6001 atgcttttct gtgactggtg agtactcaac caagtcattc tgagaatagt gtatgcggcg
     6061 accgagttgc tcttgcccgg cgtcaatacg ggataatacc gcgccacata gcagaacttt
     6121 aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct
     6181 gttgagatcc agttcgatgt aacccactcg tgcacccaac tgatcttcag catcttttac
     6241 tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat
     6301 aagggcgaca cggaaatgtt gaatactcat actcttcctt tttcaatatt attgaagcat
     6361 ttatcagggt tattgtctca tgagcggata catatttgaa tgtatttaga aaaataaaca
     6421 aataggggtt ccgcgcacat ttccccgaaa agtgccacct gacgtcgacg gatcgggaga
     6481 tccgccttta tgtgtaactc ttggctgaag ctcttacacc aatgctgggg gacatgtacc
     6541 tcccaggggc ccaggaagac tacgggaggc tacaccaacg tcaatcagag gggcctgtgt
     6601 agctaccgat aagcggaccc tcaagagggc attagcaata gtgtttataa ggcccccttg
     6661 ttaaccctaa acgggtagca tatgcttccc gggtagtagt atatactatc cagactaacc
     6721 ctaattcaat agcatatgtt acccaacggg aagcatatgc tatcgaatta gggttagtaa
     6781 aagggtccta aggaacagcg atatctccca ccccatgagc tgtcacggtt ttatttacat
     6841 ggggtcagga ttccacgagg gtagtgaacc attttagtca caagggcagt ggctgaagat
     6901 caaggagcgg gcagtgaact ctcctgaatc ttcgcctgct tcttcattct ccttcgttta
     6961 gctaatagaa taactgctga gttgtgaaca gtaaggtgta tgtgaggtgc tcgaaaacaa
     7021 ggtttcaggt gacgccccca gaataaaatt tggacggggg gttcagtggt ggcattgtgc
     7081 tatgacacca atataaccct cacaaacccc ttgggcaata aatactagtg taggaatgaa
     7141 acattctgaa tatctttaac aatagaaatc catggggtgg ggacaagccg taaagactgg
     7201 atgtccatct cacacgaatt tatggctatg ggcaacacat aatcctagtg caatatgata
     7261 ctggggttat taagatgtgt cccaggcagg gaccaagaca ggtgaaccat gttgttacac
     7321 tctatttgta acaaggggaa agagagtgga cgccgacagc agcggactcc actggttgtc
     7381 tctaacaccc ccgaaaatta aacggggctc cacgccaatg gggcccataa acaaagacaa
     7441 gtggccactc ttttttttga aattgtggag tgggggcacg cgtcagcccc cacacgccgc
     7501 cctgcggttt tggactgtaa aataagggtg taataacttg gctgattgta accccgctaa
     7561 ccactgcggt caaaccactt gcccacaaaa ccactaatgg caccccgggg aatacctgca
     7621 taagtaggtg ggcgggccaa gataggggcg cgattgctgc gatctggagg acaaattaca
     7681 cacacttgcg cctgagcgcc aagcacaggg ttgttggtcc tcatattcac gaggtcgctg
     7741 agagcacggt gggctaatgt tgccatgggt agcatatact acccaaatat ctggatagca
     7801 tatgctatcc taatctatat ctgggtagca taggctatcc taatctatat ctgggtagca
     7861 tatgctatcc taatctatat ctgggtagta tatgctatcc taatttatat ctgggtagca
     7921 taggctatcc taatctatat ctgggtagca tatgctatcc taatctatat ctgggtagta
     7981 tatgctatcc taatctgtat ccgggtagca tatgctatcc taatagagat tagggtagta
     8041 tatgctatcc taatttatat ctgggtagca tatactaccc aaatatctgg ata
//
