LOCUS       sequence-329957-        8094 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 8094)
  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 8094)
  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-M05-303: CMV51p> TPAss-SARS-CoV-2
            S-RBD(3...
FEATURES             Location/Qualifiers
     source          1..8094
                     /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             3541..3558
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     protein_bind    complement(3560..3584)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     promoter        complement(3646..3664)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(3647..3664)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(3684..3701)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3690..3914
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      3960..4388
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4047..4066)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4257..4278
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4397..4417)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        4402..4732
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      4583..4718
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     4645..4664
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             4799..5593
                     /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(4853..4872)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     5463..5482
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    5767..5888
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5804..5823)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5858..5877
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5937..5953)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5937..5953)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5950..5972)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5961..5977
                     /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(5985..5991,5992..6009,6010..6015))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6030..6051
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(6168..6185)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6339..6927)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6419..6438)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(7098..7889,7890..7958))
                     /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     7721..7740
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7959..8063)
                     /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 tgttcgtgtt tctcgtcctg ctgccactcg tgtccagcca gagggtgcag
     2881 ccgaccgaat ccatcgtgcg cttccctaac atcaccaatc tgtgcccgtt cggggaggtg
     2941 ttcaacgcta cccggttcgc ctcggtctac gcctggaacc ggaagcggat ttccaactgc
     3001 gtcgcggact actcagtgtt gtacaactcc gcctcttttt ccactttcaa atgctacggg
     3061 gtgtcgccca caaagctgaa tgatctgtgc tttaccaacg tgtacgccga ctcgttcgtg
     3121 atcagaggag atgaggtccg gcagattgca cctggccaga ccggaaacat cgcggattac
     3181 aactataagc tgcccgacga cttcaccggc tgcgtgattg cttggaactc caacaacctg
     3241 gacagcaaag tcggcggaaa ctacaactac ctgtaccggc tgttccgcaa gtccaacctg
     3301 aagccgttcg agcgggacat ctccacggaa atctaccagg ccggatcgac cccatgcaac
     3361 ggcgtgaagg ggttcaactg ttatttcccc ttgcaatcct acggattcca gccaacttac
     3421 ggggtcggct accagcctta ccgcgtggtg gtgctttcgt ttgaactgct ccatgcaccc
     3481 gccactgtgt gcggcccgaa gaagtccacc aatctggtca agaacaaatg cgtgaacttc
     3541 caccatcatc accaccacta atcaactttc ttgtacaaag ttgtgaaatt ctgcagatat
     3601 ccatcacact ggcggccgct cgagcatgca tctagagggc cctattctat agtgtcacct
     3661 aaatgctaga gctcgctgat cagcctcgac tgtgccttct agttgccagc catctgttgt
     3721 ttgcccctcc cccgtgcctt ccttgaccct ggaaggtgcc actcccactg tcctttccta
     3781 ataaaatgag gaaattgcat cgcattgtct gagtaggtgt cattctattc tggggggtgg
     3841 ggtggggcag gacagcaagg gggaggattg ggaagacaat agcaggcatg ctggggatgc
     3901 ggtgggctct atggcttctg aggcggaaag aaccagctgg ggctctaggg ggtatcccca
     3961 cgcgccctgt agcggcgcat taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc
     4021 tacacttgcc agcgccctag cgcccgctcc tttcgctttc ttcccttcct ttctcgccac
     4081 gttcgccggc tttccccgtc aagctctaaa tcggggcatc cctttagggt tccgatttag
     4141 tgctttacgg cacctcgacc ccaaaaaact tgattagggt gatggttcac gtagtgggcc
     4201 atcgccctga tagacggttt ttcgcccttt gacgttggag tccacgttct ttaatagtgg
     4261 actcttgttc caaactggaa caacactcaa ccctatctcg gtctattctt ttgatttata
     4321 agggattttg gggatttcgg cctattggtt aaaaaatgag ctgatttaac aaaaatttaa
     4381 cgcgaattaa ttctgtggaa tgtgtgtcag ttagggtgtg gaaagtcccc aggctcccca
     4441 ggcaggcaga agtatgcaaa gcatgcatct caattagtca gcaaccaggt gtggaaagtc
     4501 cccaggctcc ccagcaggca gaagtatgca aagcatgcat ctcaattagt cagcaaccat
     4561 agtcccgccc ctaactccgc ccatcccgcc cctaactccg cccagttccg cccattctcc
     4621 gccccatggc tgactaattt tttttattta tgcagaggcc gaggccgcct ctgcctctga
     4681 gctattccag aagtagtgag gaggcttttt tggaggccta ggcttttgca aaaagctccc
     4741 gggagcttgt atatccattt tcggatctga tcaagagaca ggatgaggat cgtttcgcat
     4801 gattgaacaa gatggattgc acgcaggttc tccggccgct tgggtggaga ggctattcgg
     4861 ctatgactgg gcacaacaga caatcggctg ctctgatgcc gccgtgttcc ggctgtcagc
     4921 gcaggggcgc ccggttcttt ttgtcaagac cgacctgtcc ggtgccctga atgaactgca
     4981 ggacgaggca gcgcggctat cgtggctggc cacgacgggc gttccttgcg cagctgtgct
     5041 cgacgttgtc actgaagcgg gaagggactg gctgctattg ggcgaagtgc cggggcagga
     5101 tctcctgtca tctcaccttg ctcctgccga gaaagtatcc atcatggctg atgcaatgcg
     5161 gcggctgcat acgcttgatc cggctacctg cccattcgac caccaagcga aacatcgcat
     5221 cgagcgagca cgtactcgga tggaagccgg tcttgtcgat caggatgatc tggacgaaga
     5281 gcatcagggg ctcgcgccag ccgaactgtt cgccaggctc aaggcgcgca tgcccgacgg
     5341 cgaggatctc gtcgtgaccc atggcgatgc ctgcttgccg aatatcatgg tggaaaatgg
     5401 ccgcttttct ggattcatcg actgtggccg gctgggtgtg gcggaccgct atcaggacat
     5461 agcgttggct acccgtgata ttgctgaaga gcttggcggc gaatgggctg accgcttcct
     5521 cgtgctttac ggtatcgccg ctcccgattc gcagcgcatc gccttctatc gccttcttga
     5581 cgagttcttc tgagcgggac tctggggttc gaaatgaccg accaagcgac gcccaacctg
     5641 ccatcacgag atttcgattc caccgccgcc ttctatgaaa ggttgggctt cggaatcgtt
     5701 ttccgggacg ccggctggat gatcctccag cgcggggatc tcatgctgga gttcttcgcc
     5761 caccccaact tgtttattgc agcttataat ggttacaaat aaagcaatag catcacaaat
     5821 ttcacaaata aagcattttt ttcactgcat tctagttgtg gtttgtccaa actcatcaat
     5881 gtatcttatc atgtctgtat accgtcgacc tctagctaga gcttggcgta atcatggtca
     5941 tagctgtttc ctgtgtgaaa ttgttatccg ctcacaattc cacacaacat acgagccgga
     6001 agcataaagt gtaaagcctg gggtgcctaa tgagtgagct aactcacatt aattgcgttg
     6061 cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc
     6121 caacgcgcgg ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc gctcactgac
     6181 tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa ggcggtaata
     6241 cggttatcca cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa
     6301 aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt tccataggct ccgcccccct
     6361 gacgagcatc acaaaaatcg acgctcaagt cagaggtggc gaaacccgac aggactataa
     6421 agataccagg cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg
     6481 cttaccggat acctgtccgc ctttctccct tcgggaagcg tggcgctttc tcaatgctca
     6541 cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa
     6601 ccccccgttc agcccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg
     6661 gtaagacacg acttatcgcc actggcagca gccactggta acaggattag cagagcgagg
     6721 tatgtaggcg gtgctacaga gttcttgaag tggtggccta actacggcta cactagaagg
     6781 acagtatttg gtatctgcgc tctgctgaag ccagttacct tcggaaaaag agttggtagc
     6841 tcttgatccg gcaaacaaac caccgctggt agcggtggtt tttttgtttg caagcagcag
     6901 attacgcgca gaaaaaaagg atctcaagaa gatcctttga tcttttctac ggggtctgac
     6961 gctcagtgga acgaaaactc acgttaaggg attttggtca tgagattatc aaaaaggatc
     7021 ttcacctaga tccttttaaa ttaaaaatga agttttaaat caatctaaag tatatatgag
     7081 taaacttggt ctgacagtta ccaatgctta atcagtgagg cacctatctc agcgatctgt
     7141 ctatttcgtt catccatagt tgcctgactc cccgtcgtgt agataactac gatacgggag
     7201 ggcttaccat ctggccccag tgctgcaatg ataccgcgag acccacgctc accggctcca
     7261 gatttatcag caataaacca gccagccgga agggccgagc gcagaagtgg tcctgcaact
     7321 ttatccgcct ccatccagtc tattaattgt tgccgggaag ctagagtaag tagttcgcca
     7381 gttaatagtt tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg
     7441 tttggtatgg cttcattcag ctccggttcc caacgatcaa ggcgagttac atgatccccc
     7501 atgttgtgca aaaaagcggt tagctccttc ggtcctccga tcgttgtcag aagtaagttg
     7561 gccgcagtgt tatcactcat ggttatggca gcactgcata attctcttac tgtcatgcca
     7621 tccgtaagat gcttttctgt gactggtgag tactcaacca agtcattctg agaatagtgt
     7681 atgcggcgac cgagttgctc ttgcccggcg tcaatacggg ataataccgc gccacatagc
     7741 agaactttaa aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc
     7801 ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg cacccaactg atcttcagca
     7861 tcttttactt tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa
     7921 aagggaataa gggcgacacg gaaatgttga atactcatac tcttcctttt tcaatattat
     7981 tgaagcattt atcagggtta ttgtctcatg agcggataca tatttgaatg tatttagaaa
     8041 aataaacaaa taggggttcc gcgcacattt ccccgaaaag tgccacctga cgtc
//
