LOCUS       sequence-334252-        8285 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 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-M06-303: 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(579..598)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     protein_bind    748..768
                     /gene="mutant version of attB"
                     /label=attB4
                     /bound_moiety="BP Clonase(TM)"
                     /note="core recombination site for the Gateway(R) BP 
                     reaction"
     enhancer        779..1082
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        1083..1286
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     1237..1257
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     1283..1307
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     protein_bind    1352..1376
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             2105..2128
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     CDS             2144..2167
                     /codon_start=1
                     /product="8xHis affinity tag"
                     /label=8xHis
                     /translation="HHHHHHHH"
     CDS             2186..2299
                     /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(2301..2325)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     promoter        complement(2387..2405)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(2388..2405)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(2425..2442)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2431..2655
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2701..3129
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2788..2807)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2998..3019
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3138..3158)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3143..3472
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3323..3458
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3385..3404
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3539..4333
                     /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(3593..3612)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4203..4222
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4507..4628
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4544..4563)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4598..4617
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4677..4693)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4677..4693)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4690..4712)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4701..4717
                     /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(4725..4731,4732..4749,4750..4755))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4770..4791
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4908..4925)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5079..5667)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5159..5178)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5838..6629,6630..6698))
                     /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     6461..6480
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6699..6803)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      join(7038..8285,1..542)
                     /label=oriP
                     /note="Epstein-Barr virus oriP replication origin (Yates et
                     al., 2000)"
ORIGIN
        1 gcataggcta tcctaatcta tatctgggta gcatatgcta tcctaatcta tatctgggta
       61 gtatatgcta tcctaatctg tatccgggta gcatatgcta tcctaataga gattagggta
      121 gtatatgcta tcctaattta tatctgggta gcatatacta cccaaatatc tggatagcat
      181 atgctatcct aatctatatc tgggtagcat atgctatcct aatctatatc tgggtagcat
      241 aggctatcct aatctatatc tgggtagcat atgctatcct aatctatatc tgggtagtat
      301 atgctatcct aatttatatc tgggtagcat aggctatcct aatctatatc tgggtagcat
      361 atgctatcct aatctatatc tgggtagtat atgctatcct aatctgtatc cgggtagcat
      421 atgctatcct catgcatata cagtcagcat atgataccca gtagtagagt gggagtgcta
      481 tcctttgcat atgccgccac ctcccaaggg ggcgtgaatt ttcgctgctt gtccttttcc
      541 tgctggggat ctcccgatcc cctatggtgc actctcagta caatctgctc tgatgccgca
      601 tagttaagcc agtatctgct ccctgcttgt gtgttggagg tcgctgagta gtgcgcgagc
      661 aaaatttaag ctacaacaag gcaaggcttg accgacaatt gcatgaagaa tctgcttagg
      721 gttaggcgtt ttgcgctgct tcgatcacaa ctttgtatag aaaagttggc cctgcaggcg
      781 ttacataact tacggtaaat ggcccgcctg gctgaccgcc caacgacccc cgcccattga
      841 cgtcaataat gacgtatgtt cccatagtaa cgccaatagg gactttccat tgacgtcaat
      901 gggtggagta tttacggtaa actgcccact tggcagtaca tcaagtgtat catatgccaa
      961 gtccgccccc tattgacgtc aatgacggta aatggcccgc ctggcattat gcccagtaca
     1021 tgaccttacg ggactttcct acttggcagt acatctacgt attagtcatc gctattacca
     1081 tggtgatgcg gttttggcag tacaccaatg ggcgtggata gcggtttgac tcacggggat
     1141 ttccaagtct ccaccccatt gacgtcaatg ggagtttgtt ttggcaccaa aatcaacggg
     1201 actttccaaa atgtcgtaat aaccccgccc cgttgacgca aatgggcggt aggcgtgtac
     1261 ggtgggaggt ctatataagc agagctcgtt tagtgaaccg tcagatcgcc tggagacgcc
     1321 atccacgctg ttttgacctc catagaagac aacaactttg tacaaaaaag ttggcaccat
     1381 ggatgccatg aagcggggcc tctgttgcgt gctcctcctg tgcggagctg tgttcgtgag
     1441 cccttcggca tcgttcagag tgcagcccac cgagtccatc gtgcgcttcc cgaacattac
     1501 caacctctgc ccgtttgggg aggtgttcaa tgccactaga ttcgcctccg tgtacgcgtg
     1561 gaatcgcaag cggatttcca actgcgtcgc cgactacagc gtgctgtaca actccgcctc
     1621 cttttcgacc ttcaagtgct acggagtgtc gcccactaag ctgaacgacc tctgcttcac
     1681 caacgtgtac gcagacagct tcgtgattag gggggatgag gtccggcaga tcgctccagg
     1741 gcagacgggg aatatcgccg attacaacta caagctgccc gacgacttca ctggttgcgt
     1801 catcgcctgg aactccaaca acctggacag caaagtcggc ggcaactaca actacctgta
     1861 ccggctgttc cgcaagtcca acttgaagcc gttcgagcgg gacatcagca ccgagatcta
     1921 ccaggccgga tcaaccccct gtaacggcgt caagggattc aactgttact tcccgctgca
     1981 atcctatggg tttcagccga cctacggcgt gggatatcag ccctaccggg tggtggtgct
     2041 gtccttcgaa cttctgcacg cgccagccac agtgtgcggt cctaaaaagg gagcgggttc
     2101 ctccctggaa gtgctgttcc aaggacctgg ctcaggatca tcccaccacc accatcatca
     2161 ccaccacgga ggcagcggat cgtctatgga cgaaaagact accggttgga gaggcggcca
     2221 tgtcgtggaa ggcttggccg gagaactgga gcagcttaga gccaggctgg agcaccaccc
     2281 gcaaggacag cgcgaacctt aatcaacttt cttgtacaaa gttgtgaaat tctgcagata
     2341 tccatcacac tggcggccgc tcgagcatgc atctagaggg ccctattcta tagtgtcacc
     2401 taaatgctag agctcgctga tcagcctcga ctgtgccttc tagttgccag ccatctgttg
     2461 tttgcccctc ccccgtgcct tccttgaccc tggaaggtgc cactcccact gtcctttcct
     2521 aataaaatga ggaaattgca tcgcattgtc tgagtaggtg tcattctatt ctggggggtg
     2581 gggtggggca ggacagcaag ggggaggatt gggaagacaa tagcaggcat gctggggatg
     2641 cggtgggctc tatggcttct gaggcggaaa gaaccagctg gggctctagg gggtatcccc
     2701 acgcgccctg tagcggcgca ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg
     2761 ctacacttgc cagcgcccta gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca
     2821 cgttcgccgg ctttccccgt caagctctaa atcgggggct ccctttaggg ttccgattta
     2881 gtgctttacg gcacctcgac cccaaaaaac ttgattaggg tgatggttca cgtagtgggc
     2941 catcgccctg atagacggtt tttcgccctt tgacgttgga gtccacgttc tttaatagtg
     3001 gactcttgtt ccaaactgga acaacactca accctatctc ggtctattct tttgatttat
     3061 aagggatttt gccgatttcg gcctattggt taaaaaatga gctgatttaa caaaaattta
     3121 acgcgaatta attctgtgga atgtgtgtca gttagggtgt ggaaagtccc caggctcccc
     3181 agcaggcaga agtatgcaaa gcatgcatct caattagtca gcaaccaggt gtggaaagtc
     3241 cctaggctcc ccagcaggca gaagtatgca aagcatgcat ctcaattagt cagcaaccat
     3301 agtcccgccc ctaactccgc ccatcccgcc cctaactccg cccagttccg cccattctcc
     3361 gccccatggc tgactaattt tttttattta tgcagaggcc gaggccgcct ctgcctctga
     3421 gctattccag aagtagtgag gaggcttttt tggaggccta ggcttttgca aaaagctccc
     3481 gggagcttgt atatccattt tcggatctga tcaagagaca ggatgaggat cgtttcgcat
     3541 gattgaacaa gatggattgc acgcaggttc tccggccgct tgggtggaga ggctattcgg
     3601 ctatgactgg gcacaacaga caatcggctg ctctgatgcc gccgtgttcc ggctgtcagc
     3661 gcaggggcgc ccggttcttt ttgtcaagac cgacctgtcc ggtgccctga atgaactgca
     3721 ggacgaggca gcgcggctat cgtggctggc cacgacgggc gttccttgcg cagctgtgct
     3781 cgacgttgtc actgaagcgg gaagggactg gctgctattg ggcgaagtgc cggggcagga
     3841 tctcctgtca tctcaccttg ctcctgccga gaaagtatcc atcatggctg atgcaatgcg
     3901 gcggctgcat acgcttgatc cggctacctg cccattcgac caccaagcga aacatcgcat
     3961 cgagcgagca cgtactcgga tggaagccgg tcttgtcgat caggatgatc tggacgaaga
     4021 gcatcagggg ctcgcgccag ccgaactgtt cgccaggctc aaggcgcgca tgcccgacgg
     4081 cgaggatctc gtcgtgaccc atggcgatgc ctgcttgccg aatatcatgg tggaaaatgg
     4141 ccgcttttct ggattcatcg actgtggccg gctgggtgtg gcggaccgct atcaggacat
     4201 agcgttggct acccgtgata ttgctgaaga gcttggcggc gaatgggctg accgcttcct
     4261 cgtgctttac ggtatcgccg ctcccgattc gcagcgcatc gccttctatc gccttcttga
     4321 cgagttcttc tgagcgggac tctggggttc gaaatgaccg accaagcgac gcccaacctg
     4381 ccatcacgag atttcgattc caccgccgcc ttctatgaaa ggttgggctt cggaatcgtt
     4441 ttccgggacg ccggctggat gatcctccag cgcggggatc tcatgctgga gttcttcgcc
     4501 caccccaact tgtttattgc agcttataat ggttacaaat aaagcaatag catcacaaat
     4561 ttcacaaata aagcattttt ttcactgcat tctagttgtg gtttgtccaa actcatcaat
     4621 gtatcttatc atgtctgtat accgtcgacc tctagctaga gcttggcgta atcatggtca
     4681 tagctgtttc ctgtgtgaaa ttgttatccg ctcacaattc cacacaacat acgagccgga
     4741 agcataaagt gtaaagcctg gggtgcctaa tgagtgagct aactcacatt aattgcgttg
     4801 cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc
     4861 caacgcgcgg ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc gctcactgac
     4921 tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa ggcggtaata
     4981 cggttatcca cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa
     5041 aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt tccataggct ccgcccccct
     5101 gacgagcatc acaaaaatcg acgctcaagt cagaggtggc gaaacccgac aggactataa
     5161 agataccagg cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg
     5221 cttaccggat acctgtccgc ctttctccct tcgggaagcg tggcgctttc tcatagctca
     5281 cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa
     5341 ccccccgttc agcccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg
     5401 gtaagacacg acttatcgcc actggcagca gccactggta acaggattag cagagcgagg
     5461 tatgtaggcg gtgctacaga gttcttgaag tggtggccta actacggcta cactagaaga
     5521 acagtatttg gtatctgcgc tctgctgaag ccagttacct tcggaaaaag agttggtagc
     5581 tcttgatccg gcaaacaaac caccgctggt agcggtggtt tttttgtttg caagcagcag
     5641 attacgcgca gaaaaaaagg atctcaagaa gatcctttga tcttttctac ggggtctgac
     5701 gctcagtgga acgaaaactc acgttaaggg attttggtca tgagattatc aaaaaggatc
     5761 ttcacctaga tccttttaaa ttaaaaatga agttttaaat caatctaaag tatatatgag
     5821 taaacttggt ctgacagtta ccaatgctta atcagtgagg cacctatctc agcgatctgt
     5881 ctatttcgtt catccatagt tgcctgactc cccgtcgtgt agataactac gatacgggag
     5941 ggcttaccat ctggccccag tgctgcaatg ataccgcgag acccacgctc accggctcca
     6001 gatttatcag caataaacca gccagccgga agggccgagc gcagaagtgg tcctgcaact
     6061 ttatccgcct ccatccagtc tattaattgt tgccgggaag ctagagtaag tagttcgcca
     6121 gttaatagtt tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg
     6181 tttggtatgg cttcattcag ctccggttcc caacgatcaa ggcgagttac atgatccccc
     6241 atgttgtgca aaaaagcggt tagctccttc ggtcctccga tcgttgtcag aagtaagttg
     6301 gccgcagtgt tatcactcat ggttatggca gcactgcata attctcttac tgtcatgcca
     6361 tccgtaagat gcttttctgt gactggtgag tactcaacca agtcattctg agaatagtgt
     6421 atgcggcgac cgagttgctc ttgcccggcg tcaatacggg ataataccgc gccacatagc
     6481 agaactttaa aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc
     6541 ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg cacccaactg atcttcagca
     6601 tcttttactt tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa
     6661 aagggaataa gggcgacacg gaaatgttga atactcatac tcttcctttt tcaatattat
     6721 tgaagcattt atcagggtta ttgtctcatg agcggataca tatttgaatg tatttagaaa
     6781 aataaacaaa taggggttcc gcgcacattt ccccgaaaag tgccacctga cgtcgacgga
     6841 tcgggagatc cgcctttatg tgtaactctt ggctgaagct cttacaccaa tgctggggga
     6901 catgtacctc ccaggggccc aggaagacta cgggaggcta caccaacgtc aatcagaggg
     6961 gcctgtgtag ctaccgataa gcggaccctc aagagggcat tagcaatagt gtttataagg
     7021 cccccttgtt aaccctaaac gggtagcata tgcttcccgg gtagtagtat atactatcca
     7081 gactaaccct aattcaatag catatgttac ccaacgggaa gcatatgcta tcgaattagg
     7141 gttagtaaaa gggtcctaag gaacagcgat atctcccacc ccatgagctg tcacggtttt
     7201 atttacatgg ggtcaggatt ccacgagggt agtgaaccat tttagtcaca agggcagtgg
     7261 ctgaagatca aggagcgggc agtgaactct cctgaatctt cgcctgcttc ttcattctcc
     7321 ttcgtttagc taatagaata actgctgagt tgtgaacagt aaggtgtatg tgaggtgctc
     7381 gaaaacaagg tttcaggtga cgcccccaga ataaaatttg gacggggggt tcagtggtgg
     7441 cattgtgcta tgacaccaat ataaccctca caaacccctt gggcaataaa tactagtgta
     7501 ggaatgaaac attctgaata tctttaacaa tagaaatcca tggggtgggg acaagccgta
     7561 aagactggat gtccatctca cacgaattta tggctatggg caacacataa tcctagtgca
     7621 atatgatact ggggttatta agatgtgtcc caggcaggga ccaagacagg tgaaccatgt
     7681 tgttacactc tatttgtaac aaggggaaag agagtggacg ccgacagcag cggactccac
     7741 tggttgtctc taacaccccc gaaaattaaa cggggctcca cgccaatggg gcccataaac
     7801 aaagacaagt ggccactctt ttttttgaaa ttgtggagtg ggggcacgcg tcagccccca
     7861 cacgccgccc tgcggttttg gactgtaaaa taagggtgta ataacttggc tgattgtaac
     7921 cccgctaacc actgcggtca aaccacttgc ccacaaaacc actaatggca ccccggggaa
     7981 tacctgcata agtaggtggg cgggccaaga taggggcgcg attgctgcga tctggaggac
     8041 aaattacaca cacttgcgcc tgagcgccaa gcacagggtt gttggtcctc atattcacga
     8101 ggtcgctgag agcacggtgg gctaatgttg ccatgggtag catatactac ccaaatatct
     8161 ggatagcata tgctatccta atctatatct gggtagcata ggctatccta atctatatct
     8221 gggtagcata tgctatccta atctatatct gggtagtata tgctatccta atttatatct
     8281 gggta
//
