LOCUS       sequence-334255-        8093 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian expression of SARS-CoV-2 spike receptor-binding domain 
            (RBD), N501Y mutant from B.1.1.7 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-M03-303: CMV51p> TPAss-SARS-CoV-2
            S-RBD(3...
FEATURES             Location/Qualifiers
     source          1..8093
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(534..553)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     protein_bind    703..723
                     /gene="mutant version of attB"
                     /label=attB4
                     /bound_moiety="BP Clonase(TM)"
                     /note="core recombination site for the Gateway(R) BP 
                     reaction"
     enhancer        734..1037
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        1038..1241
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     1192..1212
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     1238..1262
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     protein_bind    1307..1331
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             2045..2062
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     protein_bind    complement(2064..2088)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     promoter        complement(2150..2168)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(2151..2168)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(2188..2205)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2194..2418
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2464..2892
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2551..2570)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2761..2782
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2901..2921)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2906..3235
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3086..3221
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3148..3167
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3302..4096
                     /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(3356..3375)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3966..3985
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4270..4391
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4307..4326)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4361..4380
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4440..4456)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4440..4456)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4453..4475)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4464..4480
                     /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(4488..4494,4495..4512,4513..4518))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4533..4554
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4671..4688)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4842..5430)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4922..4941)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5601..6392,6393..6461))
                     /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     6224..6243
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6462..6566)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      join(6801..8093,1..497)
                     /label=oriP
                     /note="Epstein-Barr virus oriP replication origin (Yates et
                     al., 2000)"
ORIGIN
        1 atctatatct gggtagtata tgctatccta atctgtatcc gggtagcata tgctatccta
       61 atagagatta gggtagtata tgctatccta atttatatct gggtagcata tactacccaa
      121 atatctggat agcatatgct atcctaatct atatctgggt agcatatgct atcctaatct
      181 atatctgggt agcataggct atcctaatct atatctgggt agcatatgct atcctaatct
      241 atatctgggt agtatatgct atcctaattt atatctgggt agcataggct atcctaatct
      301 atatctgggt agcatatgct atcctaatct atatctgggt agtatatgct atcctaatct
      361 gtatccgggt agcatatgct atcctcatgc atatacagtc agcatatgat acccagtagt
      421 agagtgggag tgctatcctt tgcatatgcc gccacctccc aagggggcgt gaattttcgc
      481 tgcttgtcct tttcctgctg gggatctccc gatcccctat ggtgcactct cagtacaatc
      541 tgctctgatg ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct
      601 gagtagtgcg cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg
      661 aagaatctgc ttagggttag gcgttttgcg ctgcttcgat cacaactttg tatagaaaag
      721 ttggccctgc aggcgttaca taacttacgg taaatggccc gcctggctga ccgcccaacg
      781 acccccgccc attgacgtca ataatgacgt atgttcccat agtaacgcca atagggactt
      841 tccattgacg tcaatgggtg gagtatttac ggtaaactgc ccacttggca gtacatcaag
      901 tgtatcatat gccaagtccg ccccctattg acgtcaatga cggtaaatgg cccgcctggc
      961 attatgccca gtacatgacc ttacgggact ttcctacttg gcagtacatc tacgtattag
     1021 tcatcgctat taccatggtg atgcggtttt ggcagtacac caatgggcgt ggatagcggt
     1081 ttgactcacg gggatttcca agtctccacc ccattgacgt caatgggagt ttgttttggc
     1141 accaaaatca acgggacttt ccaaaatgtc gtaataaccc cgccccgttg acgcaaatgg
     1201 gcggtaggcg tgtacggtgg gaggtctata taagcagagc tcgtttagtg aaccgtcaga
     1261 tcgcctggag acgccatcca cgctgttttg acctccatag aagacaacaa ctttgtacaa
     1321 aaaagttggc accatgttcg tgtttctcgt cctgctgcca ctcgtgtcca gccagagggt
     1381 gcagccgacc gaatccatcg tgcgcttccc taacatcacc aatctgtgcc cgttcgggga
     1441 ggtgttcaac gctacccggt tcgcctcggt ctacgcctgg aaccggaagc ggatttccaa
     1501 ctgcgtcgcg gactactcag tgttgtacaa ctccgcctct ttttccactt tcaaatgcta
     1561 cggggtgtcg cccacaaagc tgaatgatct gtgctttacc aacgtgtacg ccgactcgtt
     1621 cgtgatcaga ggagatgagg tccggcagat tgcacctggc cagaccggaa agatcgcgga
     1681 ttacaactat aagctgcccg acgacttcac cggctgcgtg attgcttgga actccaacaa
     1741 cctggacagc aaagtcggcg gaaactacaa ctacctgtac cggctgttcc gcaagtccaa
     1801 cctgaagccg ttcgagcggg acatctccac ggaaatctac caggccggat cgaccccatg
     1861 caacggcgtg gaggggttca actgttattt ccccttgcaa tcctacggat tccagccaac
     1921 ttacggggtc ggctaccagc cttaccgcgt ggtggtgctt tcgtttgaac tgctccatgc
     1981 acccgccact gtgtgcggcc cgaagaagtc caccaatctg gtcaagaaca aatgcgtgaa
     2041 cttccaccat catcaccacc actaatcaac tttcttgtac aaagttgtga aattctgcag
     2101 atatccatca cactggcggc cgctcgagca tgcatctaga gggccctatt ctatagtgtc
     2161 acctaaatgc tagagctcgc tgatcagcct cgactgtgcc ttctagttgc cagccatctg
     2221 ttgtttgccc ctcccccgtg ccttccttga ccctggaagg tgccactccc actgtccttt
     2281 cctaataaaa tgaggaaatt gcatcgcatt gtctgagtag gtgtcattct attctggggg
     2341 gtggggtggg gcaggacagc aagggggagg attgggaaga caatagcagg catgctgggg
     2401 atgcggtggg ctctatggct tctgaggcgg aaagaaccag ctggggctct agggggtatc
     2461 cccacgcgcc ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga
     2521 ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct tcctttctcg
     2581 ccacgttcgc cggctttccc cgtcaagctc taaatcgggg gctcccttta gggttccgat
     2641 ttagtgcttt acggcacctc gaccccaaaa aacttgatta gggtgatggt tcacgtagtg
     2701 ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg ttctttaata
     2761 gtggactctt gttccaaact ggaacaacac tcaaccctat ctcggtctat tcttttgatt
     2821 tataagggat tttgccgatt tcggcctatt ggttaaaaaa tgagctgatt taacaaaaat
     2881 ttaacgcgaa ttaattctgt ggaatgtgtg tcagttaggg tgtggaaagt ccccaggctc
     2941 cccagcaggc agaagtatgc aaagcatgca tctcaattag tcagcaacca ggtgtggaaa
     3001 gtccctaggc tccccagcag gcagaagtat gcaaagcatg catctcaatt agtcagcaac
     3061 catagtcccg cccctaactc cgcccatccc gcccctaact ccgcccagtt ccgcccattc
     3121 tccgccccat ggctgactaa ttttttttat ttatgcagag gccgaggccg cctctgcctc
     3181 tgagctattc cagaagtagt gaggaggctt ttttggaggc ctaggctttt gcaaaaagct
     3241 cccgggagct tgtatatcca ttttcggatc tgatcaagag acaggatgag gatcgtttcg
     3301 catgattgaa caagatggat tgcacgcagg ttctccggcc gcttgggtgg agaggctatt
     3361 cggctatgac tgggcacaac agacaatcgg ctgctctgat gccgccgtgt tccggctgtc
     3421 agcgcagggg cgcccggttc tttttgtcaa gaccgacctg tccggtgccc tgaatgaact
     3481 gcaggacgag gcagcgcggc tatcgtggct ggccacgacg ggcgttcctt gcgcagctgt
     3541 gctcgacgtt gtcactgaag cgggaaggga ctggctgcta ttgggcgaag tgccggggca
     3601 ggatctcctg tcatctcacc ttgctcctgc cgagaaagta tccatcatgg ctgatgcaat
     3661 gcggcggctg catacgcttg atccggctac ctgcccattc gaccaccaag cgaaacatcg
     3721 catcgagcga gcacgtactc ggatggaagc cggtcttgtc gatcaggatg atctggacga
     3781 agagcatcag gggctcgcgc cagccgaact gttcgccagg ctcaaggcgc gcatgcccga
     3841 cggcgaggat ctcgtcgtga cccatggcga tgcctgcttg ccgaatatca tggtggaaaa
     3901 tggccgcttt tctggattca tcgactgtgg ccggctgggt gtggcggacc gctatcagga
     3961 catagcgttg gctacccgtg atattgctga agagcttggc ggcgaatggg ctgaccgctt
     4021 cctcgtgctt tacggtatcg ccgctcccga ttcgcagcgc atcgccttct atcgccttct
     4081 tgacgagttc ttctgagcgg gactctgggg ttcgaaatga ccgaccaagc gacgcccaac
     4141 ctgccatcac gagatttcga ttccaccgcc gccttctatg aaaggttggg cttcggaatc
     4201 gttttccggg acgccggctg gatgatcctc cagcgcgggg atctcatgct ggagttcttc
     4261 gcccacccca acttgtttat tgcagcttat aatggttaca aataaagcaa tagcatcaca
     4321 aatttcacaa ataaagcatt tttttcactg cattctagtt gtggtttgtc caaactcatc
     4381 aatgtatctt atcatgtctg tataccgtcg acctctagct agagcttggc gtaatcatgg
     4441 tcatagctgt ttcctgtgtg aaattgttat ccgctcacaa ttccacacaa catacgagcc
     4501 ggaagcataa agtgtaaagc ctggggtgcc taatgagtga gctaactcac attaattgcg
     4561 ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt gccagctgca ttaatgaatc
     4621 ggccaacgcg cggggagagg cggtttgcgt attgggcgct cttccgcttc ctcgctcact
     4681 gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat cagctcactc aaaggcggta
     4741 atacggttat ccacagaatc aggggataac gcaggaaaga acatgtgagc aaaaggccag
     4801 caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag gctccgcccc
     4861 cctgacgagc atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc gacaggacta
     4921 taaagatacc aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt tccgaccctg
     4981 ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc
     5041 tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac
     5101 gaaccccccg ttcagcccga ccgctgcgcc ttatccggta actatcgtct tgagtccaac
     5161 ccggtaagac acgacttatc gccactggca gcagccactg gtaacaggat tagcagagcg
     5221 aggtatgtag gcggtgctac agagttcttg aagtggtggc ctaactacgg ctacactaga
     5281 agaacagtat ttggtatctg cgctctgctg aagccagtta ccttcggaaa aagagttggt
     5341 agctcttgat ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag
     5401 cagattacgc gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc tacggggtct
     5461 gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt atcaaaaagg
     5521 atcttcacct agatcctttt aaattaaaaa tgaagtttta aatcaatcta aagtatatat
     5581 gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc
     5641 tgtctatttc gttcatccat agttgcctga ctccccgtcg tgtagataac tacgatacgg
     5701 gagggcttac catctggccc cagtgctgca atgataccgc gagacccacg ctcaccggct
     5761 ccagatttat cagcaataaa ccagccagcc ggaagggccg agcgcagaag tggtcctgca
     5821 actttatccg cctccatcca gtctattaat tgttgccggg aagctagagt aagtagttcg
     5881 ccagttaata gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt gtcacgctcg
     5941 tcgtttggta tggcttcatt cagctccggt tcccaacgat caaggcgagt tacatgatcc
     6001 cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag
     6061 ttggccgcag tgttatcact catggttatg gcagcactgc ataattctct tactgtcatg
     6121 ccatccgtaa gatgcttttc tgtgactggt gagtactcaa ccaagtcatt ctgagaatag
     6181 tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac gggataatac cgcgccacat
     6241 agcagaactt taaaagtgct catcattgga aaacgttctt cggggcgaaa actctcaagg
     6301 atcttaccgc tgttgagatc cagttcgatg taacccactc gtgcacccaa ctgatcttca
     6361 gcatctttta ctttcaccag cgtttctggg tgagcaaaaa caggaaggca aaatgccgca
     6421 aaaaagggaa taagggcgac acggaaatgt tgaatactca tactcttcct ttttcaatat
     6481 tattgaagca tttatcaggg ttattgtctc atgagcggat acatatttga atgtatttag
     6541 aaaaataaac aaataggggt tccgcgcaca tttccccgaa aagtgccacc tgacgtcgac
     6601 ggatcgggag atccgccttt atgtgtaact cttggctgaa gctcttacac caatgctggg
     6661 ggacatgtac ctcccagggg cccaggaaga ctacgggagg ctacaccaac gtcaatcaga
     6721 ggggcctgtg tagctaccga taagcggacc ctcaagaggg cattagcaat agtgtttata
     6781 aggccccctt gttaacccta aacgggtagc atatgcttcc cgggtagtag tatatactat
     6841 ccagactaac cctaattcaa tagcatatgt tacccaacgg gaagcatatg ctatcgaatt
     6901 agggttagta aaagggtcct aaggaacagc gatatctccc accccatgag ctgtcacggt
     6961 tttatttaca tggggtcagg attccacgag ggtagtgaac cattttagtc acaagggcag
     7021 tggctgaaga tcaaggagcg ggcagtgaac tctcctgaat cttcgcctgc ttcttcattc
     7081 tccttcgttt agctaataga ataactgctg agttgtgaac agtaaggtgt atgtgaggtg
     7141 ctcgaaaaca aggtttcagg tgacgccccc agaataaaat ttggacgggg ggttcagtgg
     7201 tggcattgtg ctatgacacc aatataaccc tcacaaaccc cttgggcaat aaatactagt
     7261 gtaggaatga aacattctga atatctttaa caatagaaat ccatggggtg gggacaagcc
     7321 gtaaagactg gatgtccatc tcacacgaat ttatggctat gggcaacaca taatcctagt
     7381 gcaatatgat actggggtta ttaagatgtg tcccaggcag ggaccaagac aggtgaacca
     7441 tgttgttaca ctctatttgt aacaagggga aagagagtgg acgccgacag cagcggactc
     7501 cactggttgt ctctaacacc cccgaaaatt aaacggggct ccacgccaat ggggcccata
     7561 aacaaagaca agtggccact cttttttttg aaattgtgga gtgggggcac gcgtcagccc
     7621 ccacacgccg ccctgcggtt ttggactgta aaataagggt gtaataactt ggctgattgt
     7681 aaccccgcta accactgcgg tcaaaccact tgcccacaaa accactaatg gcaccccggg
     7741 gaatacctgc ataagtaggt gggcgggcca agataggggc gcgattgctg cgatctggag
     7801 gacaaattac acacacttgc gcctgagcgc caagcacagg gttgttggtc ctcatattca
     7861 cgaggtcgct gagagcacgg tgggctaatg ttgccatggg tagcatatac tacccaaata
     7921 tctggatagc atatgctatc ctaatctata tctgggtagc ataggctatc ctaatctata
     7981 tctgggtagc atatgctatc ctaatctata tctgggtagt atatgctatc ctaatttata
     8041 tctgggtagc ataggctatc ctaatctata tctgggtagc atatgctatc cta
//
