LOCUS       sequence-329958-        8286 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 8286)
  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 8286)
  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..8286
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      204..1993
                     /label=oriP
                     /note="Epstein-Barr virus oriP replication origin (Yates et
                     al., 2000)"
     primer_bind     complement(2030..2049)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     protein_bind    2199..2219
                     /gene="mutant version of attB"
                     /label=attB4
                     /bound_moiety="BP Clonase(TM)"
                     /note="core recombination site for the Gateway(R) BP 
                     reaction"
     enhancer        2230..2533
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        2534..2737
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     2688..2708
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     2734..2758
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     protein_bind    2803..2827
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             3556..3579
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     CDS             3595..3618
                     /codon_start=1
                     /product="8xHis affinity tag"
                     /label=8xHis
                     /translation="HHHHHHHH"
     CDS             3637..3750
                     /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(3752..3776)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     promoter        complement(3838..3856)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(3839..3856)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(3876..3893)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3882..4106
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      4152..4580
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4239..4258)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4449..4470
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4589..4609)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        4594..4924
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      4775..4910
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     4837..4856
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             4991..5785
                     /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(5045..5064)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     5655..5674
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    5959..6080
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5996..6015)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     6050..6069
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(6129..6145)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6129..6145)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(6142..6164)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    6153..6169
                     /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(6177..6183,6184..6201,6202..6207))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6222..6243
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(6360..6377)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6531..7119)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6611..6630)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(7290..8081,8082..8150))
                     /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     7913..7932
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(8151..8255)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gacggatcgg gagatccgcc tttatgtgta actcttggct gaagctctta caccaatgct
       61 gggggacatg tacctcccag gggcccagga agactacggg aggctacacc aacgtcaatc
      121 agaggggcct gtgtagctac cgataagcgg accctcaaga gggcattagc aatagtgttt
      181 ataaggcccc cttgttaacc ctaaacgggt agcatatgct tcccgggtag tagtatatac
      241 tatccagact aaccctaatt caatagcata tgttacccaa cgggaagcat atgctatcga
      301 attagggtta gtaaaagggt cctaaggaac agcgatatct cccaccccat gagctgtcac
      361 ggttttattt acatggggtc aggattccac gagggtagtg aaccatttta gtcacaaggg
      421 cagtggctga agatcaagga gcgggcagtg aactctcctg aatcttcgcc tgcttcttca
      481 ttctccttcg tttagctaat agaataactg ctgagttgtg aacagtaagg tgtatgtgag
      541 gtgctcgaaa acaaggtttc aggtgacgcc cccagaataa aatttggacg gggggttcag
      601 tggtggcatt gtgctatgac accaatataa ccctcacaaa ccccttgggc aataaatact
      661 agtgtaggaa tgaaacattc tgaatatctt taacaataga aatccatggg gtggggacaa
      721 gccgtaaaga ctggatgtcc atctcacacg aatttatggc tatgggcaac acataatcct
      781 agtgcaatat gatactgggg ttattaagat gtgtcccagg cagggaccaa gacaggtgaa
      841 ccatgttgtt acactctatt tgtaacaagg ggaaagagag tggacgccga cagcagcgga
      901 ctccactggt tgtctctaac acccccgaaa attaaacggg gctccacgcc aatggggccc
      961 ataaacaaag acaagtggcc actctttttt ttgaaattgt ggagtggggg cacgcgtcag
     1021 cccccacacg ccgccctgcg gttttggact gtaaaataag ggtgtaataa cttggctgat
     1081 tgtaaccccg ctaaccactg cggtcaaacc acttgcccac aaaaccacta atggcacccc
     1141 ggggaatacc tgcataagta ggtgggcggg ccaagatagg ggcgcgattg ctgcgatctg
     1201 gaggacaaat tacacacact tgcgcctgag cgccaagcac agggttgttg gtcctcatat
     1261 tcacgaggtc gctgagagca cggtgggcta atgttgccat gggtagcata tactacccaa
     1321 atatctggat agcatatgct atcctaatct atatctgggt agcataggct atcctaatct
     1381 atatctgggt agcatatgct atcctaatct atatctgggt agtatatgct atcctaattt
     1441 atatctgggt agcataggct atcctaatct atatctgggt agcatatgct atcctaatct
     1501 atatctgggt agtatatgct atcctaatct gtatccgggt agcatatgct atcctaatag
     1561 agattagggt agtatatgct atcctaattt atatctgggt agcatatact acccaaatat
     1621 ctggatagca tatgctatcc taatctatat ctgggtagca tatgctatcc taatctatat
     1681 ctgggtagca taggctatcc taatctatat ctgggtagca tatgctatcc taatctatat
     1741 ctgggtagta tatgctatcc taatttatat ctgggtagca taggctatcc taatctatat
     1801 ctgggtagca tatgctatcc taatctatat ctgggtagta tatgctatcc taatctgtat
     1861 ccgggtagca tatgctatcc tcatgcatat acagtcagca tatgataccc agtagtagag
     1921 tgggagtgct atcctttgca tatgccgcca cctcccaagg gggcgtgaat tttcgctgct
     1981 tgtccttttc ctgctgggga tctcccgatc ccctatggtg cactctcagt acaatctgct
     2041 ctgatgccgc atagttaagc cagtatctgc tccctgcttg tgtgttggag gtcgctgagt
     2101 agtgcgcgag caaaatttaa gctacaacaa ggcaaggctt gaccgacaat tgcatgaaga
     2161 atctgcttag ggttaggcgt tttgcgctgc ttcgatcaca actttgtata gaaaagttgg
     2221 ccctgcaggc gttacataac ttacggtaaa tggcccgcct ggctgaccgc ccaacgaccc
     2281 ccgcccattg acgtcaataa tgacgtatgt tcccatagta acgccaatag ggactttcca
     2341 ttgacgtcaa tgggtggagt atttacggta aactgcccac ttggcagtac atcaagtgta
     2401 tcatatgcca agtccgcccc ctattgacgt caatgacggt aaatggcccg cctggcatta
     2461 tgcccagtac atgaccttac gggactttcc tacttggcag tacatctacg tattagtcat
     2521 cgctattacc atggtgatgc ggttttggca gtacaccaat gggcgtggat agcggtttga
     2581 ctcacgggga tttccaagtc tccaccccat tgacgtcaat gggagtttgt tttggcacca
     2641 aaatcaacgg gactttccaa aatgtcgtaa taaccccgcc ccgttgacgc aaatgggcgg
     2701 taggcgtgta cggtgggagg tctatataag cagagctcgt ttagtgaacc gtcagatcgc
     2761 ctggagacgc catccacgct gttttgacct ccatagaaga caacaacttt gtacaaaaaa
     2821 gttggcacca tggatgccat gaagcggggc ctctgttgcg tgctcctcct gtgcggagct
     2881 gtgttcgtga gcccttcggc atcgttcaga gtgcagccca ccgagtccat cgtgcgcttc
     2941 ccgaacatta ccaacctctg cccgtttggg gaggtgttca atgccactag attcgcctcc
     3001 gtgtacgcgt ggaatcgcaa gcggatttcc aactgcgtcg ccgactacag cgtgctgtac
     3061 aactccgcct ccttttcgac cttcaagtgc tacggagtgt cgcccactaa gctgaacgac
     3121 ctctgcttca ccaacgtgta cgcagacagc ttcgtgatta ggggggatga ggtccggcag
     3181 atcgctccag ggcagacggg gaatatcgcc gattacaact acaagctgcc cgacgacttc
     3241 actggttgcg tcatcgcctg gaactccaac aacctggaca gcaaagtcgg cggcaactac
     3301 aactacctgt accggctgtt ccgcaagtcc aacttgaagc cgttcgagcg ggacatcagc
     3361 accgagatct accaggccgg atcaaccccc tgtaacggcg tcaagggatt caactgttac
     3421 ttcccgctgc aatcctatgg gtttcagccg acctacggcg tgggatatca gccctaccgg
     3481 gtggtggtgc tgtccttcga acttctgcac gcgccagcca cagtgtgcgg tcctaaaaag
     3541 ggagcgggtt cctccctgga agtgctgttc caaggacctg gctcaggatc atcccaccac
     3601 caccatcatc accaccacgg aggcagcgga tcgtctatgg acgaaaagac taccggttgg
     3661 agaggcggcc atgtcgtgga aggcttggcc ggagaactgg agcagcttag agccaggctg
     3721 gagcaccacc cgcaaggaca gcgcgaacct taatcaactt tcttgtacaa agttgtgaaa
     3781 ttctgcagat atccatcaca ctggcggccg ctcgagcatg catctagagg gccctattct
     3841 atagtgtcac ctaaatgcta gagctcgctg atcagcctcg actgtgcctt ctagttgcca
     3901 gccatctgtt gtttgcccct cccccgtgcc ttccttgacc ctggaaggtg ccactcccac
     3961 tgtcctttcc taataaaatg aggaaattgc atcgcattgt ctgagtaggt gtcattctat
     4021 tctggggggt ggggtggggc aggacagcaa gggggaggat tgggaagaca atagcaggca
     4081 tgctggggat gcggtgggct ctatggcttc tgaggcggaa agaaccagct ggggctctag
     4141 ggggtatccc cacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg
     4201 cagcgtgacc gctacacttg ccagcgccct agcgcccgct cctttcgctt tcttcccttc
     4261 ctttctcgcc acgttcgccg gctttccccg tcaagctcta aatcggggca tccctttagg
     4321 gttccgattt agtgctttac ggcacctcga ccccaaaaaa cttgattagg gtgatggttc
     4381 acgtagtggg ccatcgccct gatagacggt ttttcgccct ttgacgttgg agtccacgtt
     4441 ctttaatagt ggactcttgt tccaaactgg aacaacactc aaccctatct cggtctattc
     4501 ttttgattta taagggattt tggggatttc ggcctattgg ttaaaaaatg agctgattta
     4561 acaaaaattt aacgcgaatt aattctgtgg aatgtgtgtc agttagggtg tggaaagtcc
     4621 ccaggctccc caggcaggca gaagtatgca aagcatgcat ctcaattagt cagcaaccag
     4681 gtgtggaaag tccccaggct ccccagcagg cagaagtatg caaagcatgc atctcaatta
     4741 gtcagcaacc atagtcccgc ccctaactcc gcccatcccg cccctaactc cgcccagttc
     4801 cgcccattct ccgccccatg gctgactaat tttttttatt tatgcagagg ccgaggccgc
     4861 ctctgcctct gagctattcc agaagtagtg aggaggcttt tttggaggcc taggcttttg
     4921 caaaaagctc ccgggagctt gtatatccat tttcggatct gatcaagaga caggatgagg
     4981 atcgtttcgc atgattgaac aagatggatt gcacgcaggt tctccggccg cttgggtgga
     5041 gaggctattc ggctatgact gggcacaaca gacaatcggc tgctctgatg ccgccgtgtt
     5101 ccggctgtca gcgcaggggc gcccggttct ttttgtcaag accgacctgt ccggtgccct
     5161 gaatgaactg caggacgagg cagcgcggct atcgtggctg gccacgacgg gcgttccttg
     5221 cgcagctgtg ctcgacgttg tcactgaagc gggaagggac tggctgctat tgggcgaagt
     5281 gccggggcag gatctcctgt catctcacct tgctcctgcc gagaaagtat ccatcatggc
     5341 tgatgcaatg cggcggctgc atacgcttga tccggctacc tgcccattcg accaccaagc
     5401 gaaacatcgc atcgagcgag cacgtactcg gatggaagcc ggtcttgtcg atcaggatga
     5461 tctggacgaa gagcatcagg ggctcgcgcc agccgaactg ttcgccaggc tcaaggcgcg
     5521 catgcccgac ggcgaggatc tcgtcgtgac ccatggcgat gcctgcttgc cgaatatcat
     5581 ggtggaaaat ggccgctttt ctggattcat cgactgtggc cggctgggtg tggcggaccg
     5641 ctatcaggac atagcgttgg ctacccgtga tattgctgaa gagcttggcg gcgaatgggc
     5701 tgaccgcttc ctcgtgcttt acggtatcgc cgctcccgat tcgcagcgca tcgccttcta
     5761 tcgccttctt gacgagttct tctgagcggg actctggggt tcgaaatgac cgaccaagcg
     5821 acgcccaacc tgccatcacg agatttcgat tccaccgccg ccttctatga aaggttgggc
     5881 ttcggaatcg ttttccggga cgccggctgg atgatcctcc agcgcgggga tctcatgctg
     5941 gagttcttcg cccaccccaa cttgtttatt gcagcttata atggttacaa ataaagcaat
     6001 agcatcacaa atttcacaaa taaagcattt ttttcactgc attctagttg tggtttgtcc
     6061 aaactcatca atgtatctta tcatgtctgt ataccgtcga cctctagcta gagcttggcg
     6121 taatcatggt catagctgtt tcctgtgtga aattgttatc cgctcacaat tccacacaac
     6181 atacgagccg gaagcataaa gtgtaaagcc tggggtgcct aatgagtgag ctaactcaca
     6241 ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat
     6301 taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta ttgggcgctc ttccgcttcc
     6361 tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc agctcactca
     6421 aaggcggtaa tacggttatc cacagaatca ggggataacg caggaaagaa catgtgagca
     6481 aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg
     6541 ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg
     6601 acaggactat aaagatacca ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt
     6661 ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc cttcgggaag cgtggcgctt
     6721 tctcaatgct cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc caagctgggc
     6781 tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt
     6841 gagtccaacc cggtaagaca cgacttatcg ccactggcag cagccactgg taacaggatt
     6901 agcagagcga ggtatgtagg cggtgctaca gagttcttga agtggtggcc taactacggc
     6961 tacactagaa ggacagtatt tggtatctgc gctctgctga agccagttac cttcggaaaa
     7021 agagttggta gctcttgatc cggcaaacaa accaccgctg gtagcggtgg tttttttgtt
     7081 tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag aagatccttt gatcttttct
     7141 acggggtctg acgctcagtg gaacgaaaac tcacgttaag ggattttggt catgagatta
     7201 tcaaaaagga tcttcaccta gatcctttta aattaaaaat gaagttttaa atcaatctaa
     7261 agtatatatg agtaaacttg gtctgacagt taccaatgct taatcagtga ggcacctatc
     7321 tcagcgatct gtctatttcg ttcatccata gttgcctgac tccccgtcgt gtagataact
     7381 acgatacggg agggcttacc atctggcccc agtgctgcaa tgataccgcg agacccacgc
     7441 tcaccggctc cagatttatc agcaataaac cagccagccg gaagggccga gcgcagaagt
     7501 ggtcctgcaa ctttatccgc ctccatccag tctattaatt gttgccggga agctagagta
     7561 agtagttcgc cagttaatag tttgcgcaac gttgttgcca ttgctacagg catcgtggtg
     7621 tcacgctcgt cgtttggtat ggcttcattc agctccggtt cccaacgatc aaggcgagtt
     7681 acatgatccc ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc
     7741 agaagtaagt tggccgcagt gttatcactc atggttatgg cagcactgca taattctctt
     7801 actgtcatgc catccgtaag atgcttttct gtgactggtg agtactcaac caagtcattc
     7861 tgagaatagt gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg ggataatacc
     7921 gcgccacata gcagaacttt aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa
     7981 ctctcaagga tcttaccgct gttgagatcc agttcgatgt aacccactcg tgcacccaac
     8041 tgatcttcag catcttttac tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa
     8101 aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt gaatactcat actcttcctt
     8161 tttcaatatt attgaagcat ttatcagggt tattgtctca tgagcggata catatttgaa
     8221 tgtatttaga aaaataaaca aataggggtt ccgcgcacat ttccccgaaa agtgccacct
     8281 gacgtc
//
