LOCUS       sequence-334219-        8285 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 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-M19-303: CMV51p> TPAss-SARS-CoV-2
            S-RBD(3...
FEATURES             Location/Qualifiers
     source          1..8285
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        1646..1750
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1751..1819,1820..2611)
                     /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     complement(1969..1988)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2782..3370
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3271..3290
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3524..3541
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    3658..3679
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(3694..3699,3700..3717,3718..3724)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3732..3748
                     /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)."
     primer_bind     3737..3759
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     3756..3772
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     3756..3772
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     polyA_signal    3821..3942
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3832..3851)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     3886..3905
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     CDS             complement(4116..4910)
                     /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(4227..4246)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     4837..4856
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     promoter        complement(4977..5306)
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      4991..5126
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(5045..5064)
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     primer_bind     5291..5311
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      complement(5320..5748)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5430..5451)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5642..5661
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     polyA_signal    5794..6018
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     6007..6024
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     promoter        6044..6062
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     6044..6061
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     protein_bind    6124..6148
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             complement(6150..6263)
                     /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"
     CDS             complement(6282..6305)
                     /codon_start=1
                     /product="8xHis affinity tag"
                     /label=8xHis
                     /translation="HHHHHHHH"
     CDS             complement(6321..6344)
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     protein_bind    complement(7073..7097)
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     primer_bind     complement(7142..7166)
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     promoter        complement(7163..7366)
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     complement(7192..7212)
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     enhancer        7367..7670
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     protein_bind    complement(7681..7701)
                     /gene="mutant version of attB"
                     /label=attB4
                     /bound_moiety="BP Clonase(TM)"
                     /note="core recombination site for the Gateway(R) BP 
                     reaction"
     primer_bind     7851..7870
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     rep_origin      join(7907..8285,1..1411)
                     /label=oriP
                     /note="Epstein-Barr virus oriP replication origin (Yates et
                     al., 2000)"
ORIGIN
        1 gggtagtata tgctacccag atataaatta ggatagcata tactacccta atctctatta
       61 ggatagcata tgctacccgg atacagatta ggatagcata tactacccag atatagatta
      121 ggatagcata tgctacccag atatagatta ggatagccta tgctacccag atataaatta
      181 ggatagcata tactacccag atatagatta ggatagcata tgctacccag atatagatta
      241 ggatagccta tgctacccag atatagatta ggatagcata tgctatccag atatttgggt
      301 agtatatgct acccatggca acattagccc accgtgctct cagcgacctc gtgaatatga
      361 ggaccaacaa ccctgtgctt ggcgctcagg cgcaagtgtg tgtaatttgt cctccagatc
      421 gcagcaatcg cgcccctatc ttggcccgcc cacctactta tgcaggtatt ccccggggtg
      481 ccattagtgg ttttgtgggc aagtggtttg accgcagtgg ttagcggggt tacaatcagc
      541 caagttatta cacccttatt ttacagtcca aaaccgcagg gcggcgtgtg ggggctgacg
      601 cgtgccccca ctccacaatt tcaaaaaaaa gagtggccac ttgtctttgt ttatgggccc
      661 cattggcgtg gagccccgtt taattttcgg gggtgttaga gacaaccagt ggagtccgct
      721 gctgtcggcg tccactctct ttccccttgt tacaaataga gtgtaacaac atggttcacc
      781 tgtcttggtc cctgcctggg acacatctta ataaccccag tatcatattg cactaggatt
      841 atgtgttgcc catagccata aattcgtgtg agatggacat ccagtcttta cggcttgtcc
      901 ccaccccatg gatttctatt gttaaagata ttcagaatgt ttcattccta cactagtatt
      961 tattgcccaa ggggtttgtg agggttatat tggtgtcata gcacaatgcc accactgaac
     1021 cccccgtcca aattttattc tgggggcgtc acctgaaacc ttgttttcga gcacctcaca
     1081 tacaccttac tgttcacaac tcagcagtta ttctattagc taaacgaagg agaatgaaga
     1141 agcaggcgaa gattcaggag agttcactgc ccgctccttg atcttcagcc actgcccttg
     1201 tgactaaaat ggttcactac cctcgtggaa tcctgacccc atgtaaataa aaccgtgaca
     1261 gctcatgggg tgggagatat cgctgttcct taggaccctt ttactaaccc taattcgata
     1321 gcatatgctt cccgttgggt aacatatgct attgaattag ggttagtctg gatagtatat
     1381 actactaccc gggaagcata tgctacccgt ttagggttaa caagggggcc ttataaacac
     1441 tattgctaat gccctcttga gggtccgctt atcggtagct acacaggccc ctctgattga
     1501 cgttggtgta gcctcccgta gtcttcctgg gcccctggga ggtacatgtc ccccagcatt
     1561 ggtgtaagag cttcagccaa gagttacaca taaaggcgga tctcccgatc cgtcgacgtc
     1621 aggtggcact tttcggggaa atgtgcgcgg aacccctatt tgtttatttt tctaaataca
     1681 ttcaaatatg tatccgctca tgagacaata accctgataa atgcttcaat aatattgaaa
     1741 aaggaagagt atgagtattc aacatttccg tgtcgccctt attccctttt ttgcggcatt
     1801 ttgccttcct gtttttgctc acccagaaac gctggtgaaa gtaaaagatg ctgaagatca
     1861 gttgggtgca cgagtgggtt acatcgaact ggatctcaac agcggtaaga tccttgagag
     1921 ttttcgcccc gaagaacgtt ttccaatgat gagcactttt aaagttctgc tatgtggcgc
     1981 ggtattatcc cgtattgacg ccgggcaaga gcaactcggt cgccgcatac actattctca
     2041 gaatgacttg gttgagtact caccagtcac agaaaagcat cttacggatg gcatgacagt
     2101 aagagaatta tgcagtgctg ccataaccat gagtgataac actgcggcca acttacttct
     2161 gacaacgatc ggaggaccga aggagctaac cgcttttttg cacaacatgg gggatcatgt
     2221 aactcgcctt gatcgttggg aaccggagct gaatgaagcc ataccaaacg acgagcgtga
     2281 caccacgatg cctgtagcaa tggcaacaac gttgcgcaaa ctattaactg gcgaactact
     2341 tactctagct tcccggcaac aattaataga ctggatggag gcggataaag ttgcaggacc
     2401 acttctgcgc tcggcccttc cggctggctg gtttattgct gataaatctg gagccggtga
     2461 gcgtgggtct cgcggtatca ttgcagcact ggggccagat ggtaagccct cccgtatcgt
     2521 agttatctac acgacgggga gtcaggcaac tatggatgaa cgaaatagac agatcgctga
     2581 gataggtgcc tcactgatta agcattggta actgtcagac caagtttact catatatact
     2641 ttagattgat ttaaaacttc atttttaatt taaaaggatc taggtgaaga tcctttttga
     2701 taatctcatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt cagaccccgt
     2761 agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct gctgcttgca
     2821 aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc taccaactct
     2881 ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgttc ttctagtgta
     2941 gccgtagtta ggccaccact tcaagaactc tgtagcaccg cctacatacc tcgctctgct
     3001 aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg ggttggactc
     3061 aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt cgtgcacaca
     3121 gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg agctatgaga
     3181 aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg
     3241 aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt atagtcctgt
     3301 cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag gggggcggag
     3361 cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt gctggccttt
     3421 tgctcacatg ttctttcctg cgttatcccc tgattctgtg gataaccgta ttaccgcctt
     3481 tgagtgagct gataccgctc gccgcagccg aacgaccgag cgcagcgagt cagtgagcga
     3541 ggaagcggaa gagcgcccaa tacgcaaacc gcctctcccc gcgcgttggc cgattcatta
     3601 atgcagctgg cacgacaggt ttcccgactg gaaagcgggc agtgagcgca acgcaattaa
     3661 tgtgagttag ctcactcatt aggcacccca ggctttacac tttatgcttc cggctcgtat
     3721 gttgtgtgga attgtgagcg gataacaatt tcacacagga aacagctatg accatgatta
     3781 cgccaagctc tagctagagg tcgacggtat acagacatga taagatacat tgatgagttt
     3841 ggacaaacca caactagaat gcagtgaaaa aaatgcttta tttgtgaaat ttgtgatgct
     3901 attgctttat ttgtaaccat tataagctgc aataaacaag ttggggtggg cgaagaactc
     3961 cagcatgaga tccccgcgct ggaggatcat ccagccggcg tcccggaaaa cgattccgaa
     4021 gcccaacctt tcatagaagg cggcggtgga atcgaaatct cgtgatggca ggttgggcgt
     4081 cgcttggtcg gtcatttcga accccagagt cccgctcaga agaactcgtc aagaaggcga
     4141 tagaaggcga tgcgctgcga atcgggagcg gcgataccgt aaagcacgag gaagcggtca
     4201 gcccattcgc cgccaagctc ttcagcaata tcacgggtag ccaacgctat gtcctgatag
     4261 cggtccgcca cacccagccg gccacagtcg atgaatccag aaaagcggcc attttccacc
     4321 atgatattcg gcaagcaggc atcgccatgg gtcacgacga gatcctcgcc gtcgggcatg
     4381 cgcgccttga gcctggcgaa cagttcggct ggcgcgagcc cctgatgctc ttcgtccaga
     4441 tcatcctgat cgacaagacc ggcttccatc cgagtacgtg ctcgctcgat gcgatgtttc
     4501 gcttggtggt cgaatgggca ggtagccgga tcaagcgtat gcagccgccg cattgcatca
     4561 gccatgatgg atactttctc ggcaggagca aggtgagatg acaggagatc ctgccccggc
     4621 acttcgccca atagcagcca gtcccttccc gcttcagtga caacgtcgag cacagctgcg
     4681 caaggaacgc ccgtcgtggc cagccacgat agccgcgctg cctcgtcctg cagttcattc
     4741 agggcaccgg acaggtcggt cttgacaaaa agaaccgggc gcccctgcgc tgacagccgg
     4801 aacacggcgg catcagagca gccgattgtc tgttgtgccc agtcatagcc gaatagcctc
     4861 tccacccaag cggccggaga acctgcgtgc aatccatctt gttcaatcat gcgaaacgat
     4921 cctcatcctg tctcttgatc agatccgaaa atggatatac aagctcccgg gagctttttg
     4981 caaaagccta ggcctccaaa aaagcctcct cactacttct ggaatagctc agaggcagag
     5041 gcggcctcgg cctctgcata aataaaaaaa attagtcagc catggggcgg agaatgggcg
     5101 gaactgggcg gagttagggg cgggatgggc ggagttaggg gcgggactat ggttgctgac
     5161 taattgagat gcatgctttg catacttctg cctgctgggg agcctaggga ctttccacac
     5221 ctggttgctg actaattgag atgcatgctt tgcatacttc tgcctgctgg ggagcctggg
     5281 gactttccac accctaactg acacacattc cacagaatta attcgcgtta aatttttgtt
     5341 aaatcagctc attttttaac caataggccg aaatcggcaa aatcccttat aaatcaaaag
     5401 aatagaccga gatagggttg agtgttgttc cagtttggaa caagagtcca ctattaaaga
     5461 acgtggactc caacgtcaaa gggcgaaaaa ccgtctatca gggcgatggc ccactacgtg
     5521 aaccatcacc ctaatcaagt tttttggggt cgaggtgccg taaagcacta aatcggaacc
     5581 ctaaagggag cccccgattt agagcttgac ggggaaagcc ggcgaacgtg gcgagaaagg
     5641 aagggaagaa agcgaaagga gcgggcgcta gggcgctggc aagtgtagcg gtcacgctgc
     5701 gcgtaaccac cacacccgcc gcgcttaatg cgccgctaca gggcgcgtgg ggataccccc
     5761 tagagcccca gctggttctt tccgcctcag aagccataga gcccaccgca tccccagcat
     5821 gcctgctatt gtcttcccaa tcctccccct tgctgtcctg ccccacccca ccccccagaa
     5881 tagaatgaca cctactcaga caatgcgatg caatttcctc attttattag gaaaggacag
     5941 tgggagtggc accttccagg gtcaaggaag gcacggggga ggggcaaaca acagatggct
     6001 ggcaactaga aggcacagtc gaggctgatc agcgagctct agcatttagg tgacactata
     6061 gaatagggcc ctctagatgc atgctcgagc ggccgccagt gtgatggata tctgcagaat
     6121 ttcacaactt tgtacaagaa agttgattaa ggttcgcgct gtccttgcgg gtggtgctcc
     6181 agcctggctc taagctgctc cagttctccg gccaagcctt ccacgacatg gccgcctctc
     6241 caaccggtag tcttttcgtc catagacgat ccgctgcctc cgtggtggtg atgatggtgg
     6301 tggtgggatg atcctgagcc aggtccttgg aacagcactt ccagggagga acccgctccc
     6361 tttttaggac cgcacactgt ggctggcgcg tgcagaagtt cgaaggacag caccaccacc
     6421 cggtagggct gatatcccac gccgtaggtc ggctgaaacc cataggattg cagcgggaag
     6481 taacagttga atcccttgac gccgttacag ggggttgatc cggcctggta gatctcggtg
     6541 ctgatgtccc gctcgaacgg cttcaagttg gacttgcgga acagccggta caggtagttg
     6601 tagttgccgc cgactttgct gtccaggttg ttggagttcc aggcgatgac gcaaccagtg
     6661 aagtcgtcgg gcagcttgta gttgtaatcg gcgatggtcc ccgtctgccc tggagcgatc
     6721 tgccggacct catcccccct aatcacgaag ctgtctgcgt acacgttggt gaagcagagg
     6781 tcgttcagct tagtgggcga cactccgtag cacttgaagg tcgaaaagga ggcggagttg
     6841 tacagcacgc tgtagtcggc gacgcagttg gaaatccgct tgcgattcca cgcgtacacg
     6901 gaggcgaatc tagtggcatt gaacacctcc ccaaacgggc agaggttggt aatgttcggg
     6961 aagcgcacga tggactcggt gggctgcact ctgaacgatg ccgaagggct cacgaacaca
     7021 gctccgcaca ggaggagcac gcaacagagg ccccgcttca tggcatccat ggtgccaact
     7081 tttttgtaca aagttgttgt cttctatgga ggtcaaaaca gcgtggatgg cgtctccagg
     7141 cgatctgacg gttcactaaa cgagctctgc ttatatagac ctcccaccgt acacgcctac
     7201 cgcccatttg cgtcaacggg gcggggttat tacgacattt tggaaagtcc cgttgatttt
     7261 ggtgccaaaa caaactccca ttgacgtcaa tggggtggag acttggaaat ccccgtgagt
     7321 caaaccgcta tccacgccca ttggtgtact gccaaaaccg catcaccatg gtaatagcga
     7381 tgactaatac gtagatgtac tgccaagtag gaaagtcccg taaggtcatg tactgggcat
     7441 aatgccaggc gggccattta ccgtcattga cgtcaatagg gggcggactt ggcatatgat
     7501 acacttgatg tactgccaag tgggcagttt accgtaaata ctccacccat tgacgtcaat
     7561 ggaaagtccc tattggcgtt actatgggaa catacgtcat tattgacgtc aatgggcggg
     7621 ggtcgttggg cggtcagcca ggcgggccat ttaccgtaag ttatgtaacg cctgcagggc
     7681 caacttttct atacaaagtt gtgatcgaag cagcgcaaaa cgcctaaccc taagcagatt
     7741 cttcatgcaa ttgtcggtca agccttgcct tgttgtagct taaattttgc tcgcgcacta
     7801 ctcagcgacc tccaacacac aagcagggag cagatactgg cttaactatg cggcatcaga
     7861 gcagattgta ctgagagtgc accatagggg atcgggagat ccccagcagg aaaaggacaa
     7921 gcagcgaaaa ttcacgcccc cttgggaggt ggcggcatat gcaaaggata gcactcccac
     7981 tctactactg ggtatcatat gctgactgta tatgcatgag gatagcatat gctacccgga
     8041 tacagattag gatagcatat actacccaga tatagattag gatagcatat gctacccaga
     8101 tatagattag gatagcctat gctacccaga tataaattag gatagcatat actacccaga
     8161 tatagattag gatagcatat gctacccaga tatagattag gatagcctat gctacccaga
     8221 tatagattag gatagcatat gctacccaga tatagattag gatagcatat gctatccaga
     8281 tattt
//
