LOCUS       sequence-334266-        8285 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  ammalian expression of SARS-CoV-2 spike receptor-binding domain 
            (RBD), N501Y mutant from B.1.1.7 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-M04-303: 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(490..509)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     protein_bind    659..679
                     /gene="mutant version of attB"
                     /label=attB4
                     /bound_moiety="BP Clonase(TM)"
                     /note="core recombination site for the Gateway(R) BP 
                     reaction"
     enhancer        690..993
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        994..1197
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     1148..1168
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     1194..1218
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     protein_bind    1263..1287
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             2016..2039
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     CDS             2055..2078
                     /codon_start=1
                     /product="8xHis affinity tag"
                     /label=8xHis
                     /translation="HHHHHHHH"
     CDS             2097..2210
                     /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(2212..2236)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     promoter        complement(2298..2316)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(2299..2316)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(2336..2353)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2342..2566
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2612..3040
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2699..2718)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2909..2930
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3049..3069)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3054..3383
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3234..3369
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3296..3315
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3450..4244
                     /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(3504..3523)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4114..4133
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4418..4539
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4455..4474)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4509..4528
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4588..4604)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4588..4604)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4601..4623)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4612..4628
                     /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(4636..4642,4643..4660,4661..4666))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4681..4702
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4819..4836)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4990..5578)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5070..5089)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5749..6540,6541..6609))
                     /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     6372..6391
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6610..6714)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      join(6949..8285,1..453)
                     /label=oriP
                     /note="Epstein-Barr virus oriP replication origin (Yates et
                     al., 2000)"
ORIGIN
        1 agcatatgct atcctaatag agattagggt agtatatgct atcctaattt atatctgggt
       61 agcatatact acccaaatat ctggatagca tatgctatcc taatctatat ctgggtagca
      121 tatgctatcc taatctatat ctgggtagca taggctatcc taatctatat ctgggtagca
      181 tatgctatcc taatctatat ctgggtagta tatgctatcc taatttatat ctgggtagca
      241 taggctatcc taatctatat ctgggtagca tatgctatcc taatctatat ctgggtagta
      301 tatgctatcc taatctgtat ccgggtagca tatgctatcc tcatgcatat acagtcagca
      361 tatgataccc agtagtagag tgggagtgct atcctttgca tatgccgcca cctcccaagg
      421 gggcgtgaat tttcgctgct tgtccttttc ctgctgggga tctcccgatc ccctatggtg
      481 cactctcagt acaatctgct ctgatgccgc atagttaagc cagtatctgc tccctgcttg
      541 tgtgttggag gtcgctgagt agtgcgcgag caaaatttaa gctacaacaa ggcaaggctt
      601 gaccgacaat tgcatgaaga atctgcttag ggttaggcgt tttgcgctgc ttcgatcaca
      661 actttgtata gaaaagttgg ccctgcaggc gttacataac ttacggtaaa tggcccgcct
      721 ggctgaccgc ccaacgaccc ccgcccattg acgtcaataa tgacgtatgt tcccatagta
      781 acgccaatag ggactttcca ttgacgtcaa tgggtggagt atttacggta aactgcccac
      841 ttggcagtac atcaagtgta tcatatgcca agtccgcccc ctattgacgt caatgacggt
      901 aaatggcccg cctggcatta tgcccagtac atgaccttac gggactttcc tacttggcag
      961 tacatctacg tattagtcat cgctattacc atggtgatgc ggttttggca gtacaccaat
     1021 gggcgtggat agcggtttga ctcacgggga tttccaagtc tccaccccat tgacgtcaat
     1081 gggagtttgt tttggcacca aaatcaacgg gactttccaa aatgtcgtaa taaccccgcc
     1141 ccgttgacgc aaatgggcgg taggcgtgta cggtgggagg tctatataag cagagctcgt
     1201 ttagtgaacc gtcagatcgc ctggagacgc catccacgct gttttgacct ccatagaaga
     1261 caacaacttt gtacaaaaaa gttggcacca tggatgccat gaagcggggc ctctgttgcg
     1321 tgctcctcct gtgcggagct gtgttcgtga gcccttcggc atcgttcaga gtgcagccca
     1381 ccgagtccat cgtgcgcttc ccgaacatta ccaacctctg cccgtttggg gaggtgttca
     1441 atgccactag attcgcctcc gtgtacgcgt ggaatcgcaa gcggatttcc aactgcgtcg
     1501 ccgactacag cgtgctgtac aactccgcct ccttttcgac cttcaagtgc tacggagtgt
     1561 cgcccactaa gctgaacgac ctctgcttca ccaacgtgta cgcagacagc ttcgtgatta
     1621 ggggggatga ggtccggcag atcgctccag ggcagacggg gaagatcgcc gattacaact
     1681 acaagctgcc cgacgacttc actggttgcg tcatcgcctg gaactccaac aacctggaca
     1741 gcaaagtcgg cggcaactac aactacctgt accggctgtt ccgcaagtcc aacttgaagc
     1801 cgttcgagcg ggacatcagc accgagatct accaggccgg atcaaccccc tgtaacggcg
     1861 tcgaaggatt caactgttac ttcccgctgc aatcctatgg gtttcagccg acctacggcg
     1921 tgggatatca gccctaccgg gtggtggtgc tgtccttcga acttctgcac gcgccagcca
     1981 cagtgtgcgg tcctaaaaag ggagcgggtt cctccctgga agtgctgttc caaggacctg
     2041 gctcaggatc atcccaccac caccatcatc accaccacgg aggcagcgga tcgtctatgg
     2101 acgaaaagac taccggttgg agaggcggcc atgtcgtgga aggcttggcc ggagaactgg
     2161 agcagcttag agccaggctg gagcaccacc cgcaaggaca gcgcgaacct taatcaactt
     2221 tcttgtacaa agttgtgaaa ttctgcagat atccatcaca ctggcggccg ctcgagcatg
     2281 catctagagg gccctattct atagtgtcac ctaaatgcta gagctcgctg atcagcctcg
     2341 actgtgcctt ctagttgcca gccatctgtt gtttgcccct cccccgtgcc ttccttgacc
     2401 ctggaaggtg ccactcccac tgtcctttcc taataaaatg aggaaattgc atcgcattgt
     2461 ctgagtaggt gtcattctat tctggggggt ggggtggggc aggacagcaa gggggaggat
     2521 tgggaagaca atagcaggca tgctggggat gcggtgggct ctatggcttc tgaggcggaa
     2581 agaaccagct ggggctctag ggggtatccc cacgcgccct gtagcggcgc attaagcgcg
     2641 gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg ccagcgccct agcgcccgct
     2701 cctttcgctt tcttcccttc ctttctcgcc acgttcgccg gctttccccg tcaagctcta
     2761 aatcgggggc tccctttagg gttccgattt agtgctttac ggcacctcga ccccaaaaaa
     2821 cttgattagg gtgatggttc acgtagtggg ccatcgccct gatagacggt ttttcgccct
     2881 ttgacgttgg agtccacgtt ctttaatagt ggactcttgt tccaaactgg aacaacactc
     2941 aaccctatct cggtctattc ttttgattta taagggattt tgccgatttc ggcctattgg
     3001 ttaaaaaatg agctgattta acaaaaattt aacgcgaatt aattctgtgg aatgtgtgtc
     3061 agttagggtg tggaaagtcc ccaggctccc cagcaggcag aagtatgcaa agcatgcatc
     3121 tcaattagtc agcaaccagg tgtggaaagt ccctaggctc cccagcaggc agaagtatgc
     3181 aaagcatgca tctcaattag tcagcaacca tagtcccgcc cctaactccg cccatcccgc
     3241 ccctaactcc gcccagttcc gcccattctc cgccccatgg ctgactaatt ttttttattt
     3301 atgcagaggc cgaggccgcc tctgcctctg agctattcca gaagtagtga ggaggctttt
     3361 ttggaggcct aggcttttgc aaaaagctcc cgggagcttg tatatccatt ttcggatctg
     3421 atcaagagac aggatgagga tcgtttcgca tgattgaaca agatggattg cacgcaggtt
     3481 ctccggccgc ttgggtggag aggctattcg gctatgactg ggcacaacag acaatcggct
     3541 gctctgatgc cgccgtgttc cggctgtcag cgcaggggcg cccggttctt tttgtcaaga
     3601 ccgacctgtc cggtgccctg aatgaactgc aggacgaggc agcgcggcta tcgtggctgg
     3661 ccacgacggg cgttccttgc gcagctgtgc tcgacgttgt cactgaagcg ggaagggact
     3721 ggctgctatt gggcgaagtg ccggggcagg atctcctgtc atctcacctt gctcctgccg
     3781 agaaagtatc catcatggct gatgcaatgc ggcggctgca tacgcttgat ccggctacct
     3841 gcccattcga ccaccaagcg aaacatcgca tcgagcgagc acgtactcgg atggaagccg
     3901 gtcttgtcga tcaggatgat ctggacgaag agcatcaggg gctcgcgcca gccgaactgt
     3961 tcgccaggct caaggcgcgc atgcccgacg gcgaggatct cgtcgtgacc catggcgatg
     4021 cctgcttgcc gaatatcatg gtggaaaatg gccgcttttc tggattcatc gactgtggcc
     4081 ggctgggtgt ggcggaccgc tatcaggaca tagcgttggc tacccgtgat attgctgaag
     4141 agcttggcgg cgaatgggct gaccgcttcc tcgtgcttta cggtatcgcc gctcccgatt
     4201 cgcagcgcat cgccttctat cgccttcttg acgagttctt ctgagcggga ctctggggtt
     4261 cgaaatgacc gaccaagcga cgcccaacct gccatcacga gatttcgatt ccaccgccgc
     4321 cttctatgaa aggttgggct tcggaatcgt tttccgggac gccggctgga tgatcctcca
     4381 gcgcggggat ctcatgctgg agttcttcgc ccaccccaac ttgtttattg cagcttataa
     4441 tggttacaaa taaagcaata gcatcacaaa tttcacaaat aaagcatttt tttcactgca
     4501 ttctagttgt ggtttgtcca aactcatcaa tgtatcttat catgtctgta taccgtcgac
     4561 ctctagctag agcttggcgt aatcatggtc atagctgttt cctgtgtgaa attgttatcc
     4621 gctcacaatt ccacacaaca tacgagccgg aagcataaag tgtaaagcct ggggtgccta
     4681 atgagtgagc taactcacat taattgcgtt gcgctcactg cccgctttcc agtcgggaaa
     4741 cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat
     4801 tgggcgctct tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg
     4861 agcggtatca gctcactcaa aggcggtaat acggttatcc acagaatcag gggataacgc
     4921 aggaaagaac atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt
     4981 gctggcgttt ttccataggc tccgcccccc tgacgagcat cacaaaaatc gacgctcaag
     5041 tcagaggtgg cgaaacccga caggactata aagataccag gcgtttcccc ctggaagctc
     5101 cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga tacctgtccg cctttctccc
     5161 ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg tatctcagtt cggtgtaggt
     5221 cgttcgctcc aagctgggct gtgtgcacga accccccgtt cagcccgacc gctgcgcctt
     5281 atccggtaac tatcgtcttg agtccaaccc ggtaagacac gacttatcgc cactggcagc
     5341 agccactggt aacaggatta gcagagcgag gtatgtaggc ggtgctacag agttcttgaa
     5401 gtggtggcct aactacggct acactagaag aacagtattt ggtatctgcg ctctgctgaa
     5461 gccagttacc ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg
     5521 tagcggtggt ttttttgttt gcaagcagca gattacgcgc agaaaaaaag gatctcaaga
     5581 agatcctttg atcttttcta cggggtctga cgctcagtgg aacgaaaact cacgttaagg
     5641 gattttggtc atgagattat caaaaaggat cttcacctag atccttttaa attaaaaatg
     5701 aagttttaaa tcaatctaaa gtatatatga gtaaacttgg tctgacagtt accaatgctt
     5761 aatcagtgag gcacctatct cagcgatctg tctatttcgt tcatccatag ttgcctgact
     5821 ccccgtcgtg tagataacta cgatacggga gggcttacca tctggcccca gtgctgcaat
     5881 gataccgcga gacccacgct caccggctcc agatttatca gcaataaacc agccagccgg
     5941 aagggccgag cgcagaagtg gtcctgcaac tttatccgcc tccatccagt ctattaattg
     6001 ttgccgggaa gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat
     6061 tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg gcttcattca gctccggttc
     6121 ccaacgatca aggcgagtta catgatcccc catgttgtgc aaaaaagcgg ttagctcctt
     6181 cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg ttatcactca tggttatggc
     6241 agcactgcat aattctctta ctgtcatgcc atccgtaaga tgcttttctg tgactggtga
     6301 gtactcaacc aagtcattct gagaatagtg tatgcggcga ccgagttgct cttgcccggc
     6361 gtcaatacgg gataataccg cgccacatag cagaacttta aaagtgctca tcattggaaa
     6421 acgttcttcg gggcgaaaac tctcaaggat cttaccgctg ttgagatcca gttcgatgta
     6481 acccactcgt gcacccaact gatcttcagc atcttttact ttcaccagcg tttctgggtg
     6541 agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata agggcgacac ggaaatgttg
     6601 aatactcata ctcttccttt ttcaatatta ttgaagcatt tatcagggtt attgtctcat
     6661 gagcggatac atatttgaat gtatttagaa aaataaacaa ataggggttc cgcgcacatt
     6721 tccccgaaaa gtgccacctg acgtcgacgg atcgggagat ccgcctttat gtgtaactct
     6781 tggctgaagc tcttacacca atgctggggg acatgtacct cccaggggcc caggaagact
     6841 acgggaggct acaccaacgt caatcagagg ggcctgtgta gctaccgata agcggaccct
     6901 caagagggca ttagcaatag tgtttataag gcccccttgt taaccctaaa cgggtagcat
     6961 atgcttcccg ggtagtagta tatactatcc agactaaccc taattcaata gcatatgtta
     7021 cccaacggga agcatatgct atcgaattag ggttagtaaa agggtcctaa ggaacagcga
     7081 tatctcccac cccatgagct gtcacggttt tatttacatg gggtcaggat tccacgaggg
     7141 tagtgaacca ttttagtcac aagggcagtg gctgaagatc aaggagcggg cagtgaactc
     7201 tcctgaatct tcgcctgctt cttcattctc cttcgtttag ctaatagaat aactgctgag
     7261 ttgtgaacag taaggtgtat gtgaggtgct cgaaaacaag gtttcaggtg acgcccccag
     7321 aataaaattt ggacgggggg ttcagtggtg gcattgtgct atgacaccaa tataaccctc
     7381 acaaacccct tgggcaataa atactagtgt aggaatgaaa cattctgaat atctttaaca
     7441 atagaaatcc atggggtggg gacaagccgt aaagactgga tgtccatctc acacgaattt
     7501 atggctatgg gcaacacata atcctagtgc aatatgatac tggggttatt aagatgtgtc
     7561 ccaggcaggg accaagacag gtgaaccatg ttgttacact ctatttgtaa caaggggaaa
     7621 gagagtggac gccgacagca gcggactcca ctggttgtct ctaacacccc cgaaaattaa
     7681 acggggctcc acgccaatgg ggcccataaa caaagacaag tggccactct tttttttgaa
     7741 attgtggagt gggggcacgc gtcagccccc acacgccgcc ctgcggtttt ggactgtaaa
     7801 ataagggtgt aataacttgg ctgattgtaa ccccgctaac cactgcggtc aaaccacttg
     7861 cccacaaaac cactaatggc accccgggga atacctgcat aagtaggtgg gcgggccaag
     7921 ataggggcgc gattgctgcg atctggagga caaattacac acacttgcgc ctgagcgcca
     7981 agcacagggt tgttggtcct catattcacg aggtcgctga gagcacggtg ggctaatgtt
     8041 gccatgggta gcatatacta cccaaatatc tggatagcat atgctatcct aatctatatc
     8101 tgggtagcat aggctatcct aatctatatc tgggtagcat atgctatcct aatctatatc
     8161 tgggtagtat atgctatcct aatttatatc tgggtagcat aggctatcct aatctatatc
     8221 tgggtagcat atgctatcct aatctatatc tgggtagtat atgctatcct aatctgtatc
     8281 cgggt
//
