LOCUS       sequence-327088-        8282 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  mammalian expression of SARS-CoV-2 spike receptor-binding domain 
            (RBD) optimized for serology.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8282)
  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 8282)
  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: R619-M68-303: CMV51p> TPAss-SARS-CoV-2
            S-RBD(3...
FEATURES             Location/Qualifiers
     source          1..8282
                     /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             3553..3576
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     CDS             3592..3615
                     /codon_start=1
                     /product="8xHis affinity tag"
                     /label=8xHis
                     /translation="HHHHHHHH"
     CDS             3634..3747
                     /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(3749..3773)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     promoter        complement(3835..3853)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(3836..3853)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(3873..3890)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3879..4103
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      4149..4577
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4236..4255)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4446..4467
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4586..4606)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        4591..4920
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      4771..4906
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     4833..4852
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             4987..5781
                     /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(5041..5060)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     5651..5670
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    5955..6076
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5992..6011)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     6046..6065
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(6125..6141)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6125..6141)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(6138..6160)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    6149..6165
                     /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(6173..6179,6180..6197,6198..6203))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6218..6239
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(6356..6373)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6527..7115)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6607..6626)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(7286..8077,8078..8146))
                     /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"
     promoter        complement(8147..8251)
                     /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 tggacgccat gaagcgcggt ctgtgctgtg tcctcctgct gtgtggcgcg
     2881 gtgttcgtgt ccccttccgc gtcaagggtg cagccgaccg aatccatcgt gcgcttccct
     2941 aacatcacca atctgtgccc gttcggggag gtgttcaacg ctacccggtt cgcctcggtc
     3001 tacgcctgga accggaagcg gatttccaac tgcgtcgcgg actactcagt gttgtacaac
     3061 tccgcctctt tttccacttt caaatgctac ggggtgtcgc ccacaaagct gaatgatctg
     3121 tgctttacca acgtgtacgc cgactcgttc gtgatcagag gagatgaggt ccggcagatt
     3181 gcacctggcc agaccggaaa gatcgcggat tacaactata agctgcccga cgacttcacc
     3241 ggctgcgtga ttgcttggaa ctccaacaac ctggacagca aagtcggcgg aaactacaac
     3301 tacctgtacc ggctgttccg caagtccaac ctgaagccgt tcgagcggga catctccacg
     3361 gaaatctacc aggccggatc gaccccatgc aacggcgtgg aggggttcaa ctgttatttc
     3421 cccttgcaat cctacggatt ccagccaact aatggggtcg gctaccagcc ttaccgcgtg
     3481 gtggtgcttt cgtttgaact gctccatgca cccgccactg tgtgcggccc gaagaagggt
     3541 gccgggagct cactcgaggt gctgttccaa ggacccggtt ccggaagcag ccaccaccac
     3601 catcatcacc accacggagg cagcggatcc agcatggacg aaaagaccac tggatggaga
     3661 ggcggtcatg tggtggaagg ccttgccgga gaactcgagc agctgagagc ccgcctcgaa
     3721 caccacccgc aaggacagag ggagccgtaa tcaactttct tgtacaaagt tgtgaaattc
     3781 tgcagatatc catcacactg gcggccgctc gagcatgcat ctagagggcc ctattctata
     3841 gtgtcaccta aatgctagag ctcgctgatc agcctcgact gtgccttcta gttgccagcc
     3901 atctgttgtt tgcccctccc ccgtgccttc cttgaccctg gaaggtgcca ctcccactgt
     3961 cctttcctaa taaaatgagg aaattgcatc gcattgtctg agtaggtgtc attctattct
     4021 ggggggtggg gtggggcagg acagcaaggg ggaggattgg gaagacaata gcaggcatgc
     4081 tggggatgcg gtgggctcta tggcttctga ggcggaaaga accagctggg gctctagggg
     4141 gtatccccac gcgccctgta gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcag
     4201 cgtgaccgct acacttgcca gcgccctagc gcccgctcct ttcgctttct tcccttcctt
     4261 tctcgccacg ttcgccggct ttccccgtca agctctaaat cgggggctcc ctttagggtt
     4321 ccgatttagt gctttacggc acctcgaccc caaaaaactt gattagggtg atggttcacg
     4381 tagtgggcca tcgccctgat agacggtttt tcgccctttg acgttggagt ccacgttctt
     4441 taatagtgga ctcttgttcc aaactggaac aacactcaac cctatctcgg tctattcttt
     4501 tgatttataa gggattttgc cgatttcggc ctattggtta aaaaatgagc tgatttaaca
     4561 aaaatttaac gcgaattaat tctgtggaat gtgtgtcagt tagggtgtgg aaagtcccca
     4621 ggctccccag caggcagaag tatgcaaagc atgcatctca attagtcagc aaccaggtgt
     4681 ggaaagtccc taggctcccc agcaggcaga agtatgcaaa gcatgcatct caattagtca
     4741 gcaaccatag tcccgcccct aactccgccc atcccgcccc taactccgcc cagttccgcc
     4801 cattctccgc cccatggctg actaattttt tttatttatg cagaggccga ggccgcctct
     4861 gcctctgagc tattccagaa gtagtgagga ggcttttttg gaggcctagg cttttgcaaa
     4921 aagctcccgg gagcttgtat atccattttc ggatctgatc aagagacagg atgaggatcg
     4981 tttcgcatga ttgaacaaga tggattgcac gcaggttctc cggccgcttg ggtggagagg
     5041 ctattcggct atgactgggc acaacagaca atcggctgct ctgatgccgc cgtgttccgg
     5101 ctgtcagcgc aggggcgccc ggttcttttt gtcaagaccg acctgtccgg tgccctgaat
     5161 gaactgcagg acgaggcagc gcggctatcg tggctggcca cgacgggcgt tccttgcgca
     5221 gctgtgctcg acgttgtcac tgaagcggga agggactggc tgctattggg cgaagtgccg
     5281 gggcaggatc tcctgtcatc tcaccttgct cctgccgaga aagtatccat catggctgat
     5341 gcaatgcggc ggctgcatac gcttgatccg gctacctgcc cattcgacca ccaagcgaaa
     5401 catcgcatcg agcgagcacg tactcggatg gaagccggtc ttgtcgatca ggatgatctg
     5461 gacgaagagc atcaggggct cgcgccagcc gaactgttcg ccaggctcaa ggcgcgcatg
     5521 cccgacggcg aggatctcgt cgtgacccat ggcgatgcct gcttgccgaa tatcatggtg
     5581 gaaaatggcc gcttttctgg attcatcgac tgtggccggc tgggtgtggc ggaccgctat
     5641 caggacatag cgttggctac ccgtgatatt gctgaagagc ttggcggcga atgggctgac
     5701 cgcttcctcg tgctttacgg tatcgccgct cccgattcgc agcgcatcgc cttctatcgc
     5761 cttcttgacg agttcttctg agcgggactc tggggttcga aatgaccgac caagcgacgc
     5821 ccaacctgcc atcacgagat ttcgattcca ccgccgcctt ctatgaaagg ttgggcttcg
     5881 gaatcgtttt ccgggacgcc ggctggatga tcctccagcg cggggatctc atgctggagt
     5941 tcttcgccca ccccaacttg tttattgcag cttataatgg ttacaaataa agcaatagca
     6001 tcacaaattt cacaaataaa gcattttttt cactgcattc tagttgtggt ttgtccaaac
     6061 tcatcaatgt atcttatcat gtctgtatac cgtcgacctc tagctagagc ttggcgtaat
     6121 catggtcata gctgtttcct gtgtgaaatt gttatccgct cacaattcca cacaacatac
     6181 gagccggaag cataaagtgt aaagcctggg gtgcctaatg agtgagctaa ctcacattaa
     6241 ttgcgttgcg ctcactgccc gctttccagt cgggaaacct gtcgtgccag ctgcattaat
     6301 gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg gcgctcttcc gcttcctcgc
     6361 tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg
     6421 cggtaatacg gttatccaca gaatcagggg ataacgcagg aaagaacatg tgagcaaaag
     6481 gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc
     6541 gcccccctga cgagcatcac aaaaatcgac gctcaagtca gaggtggcga aacccgacag
     6601 gactataaag ataccaggcg tttccccctg gaagctccct cgtgcgctct cctgttccga
     6661 ccctgccgct taccggatac ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc
     6721 atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg
     6781 tgcacgaacc ccccgttcag cccgaccgct gcgccttatc cggtaactat cgtcttgagt
     6841 ccaacccggt aagacacgac ttatcgccac tggcagcagc cactggtaac aggattagca
     6901 gagcgaggta tgtaggcggt gctacagagt tcttgaagtg gtggcctaac tacggctaca
     6961 ctagaagaac agtatttggt atctgcgctc tgctgaagcc agttaccttc ggaaaaagag
     7021 ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggtggtttt tttgtttgca
     7081 agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg
     7141 ggtctgacgc tcagtggaac gaaaactcac gttaagggat tttggtcatg agattatcaa
     7201 aaaggatctt cacctagatc cttttaaatt aaaaatgaag ttttaaatca atctaaagta
     7261 tatatgagta aacttggtct gacagttacc aatgcttaat cagtgaggca cctatctcag
     7321 cgatctgtct atttcgttca tccatagttg cctgactccc cgtcgtgtag ataactacga
     7381 tacgggaggg cttaccatct ggccccagtg ctgcaatgat accgcgagac ccacgctcac
     7441 cggctccaga tttatcagca ataaaccagc cagccggaag ggccgagcgc agaagtggtc
     7501 ctgcaacttt atccgcctcc atccagtcta ttaattgttg ccgggaagct agagtaagta
     7561 gttcgccagt taatagtttg cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac
     7621 gctcgtcgtt tggtatggct tcattcagct ccggttccca acgatcaagg cgagttacat
     7681 gatcccccat gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc gttgtcagaa
     7741 gtaagttggc cgcagtgtta tcactcatgg ttatggcagc actgcataat tctcttactg
     7801 tcatgccatc cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag tcattctgag
     7861 aatagtgtat gcggcgaccg agttgctctt gcccggcgtc aatacgggat aataccgcgc
     7921 cacatagcag aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct
     7981 caaggatctt accgctgttg agatccagtt cgatgtaacc cactcgtgca cccaactgat
     8041 cttcagcatc ttttactttc accagcgttt ctgggtgagc aaaaacagga aggcaaaatg
     8101 ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat actcatactc ttcctttttc
     8161 aatattattg aagcatttat cagggttatt gtctcatgag cggatacata tttgaatgta
     8221 tttagaaaaa taaacaaata ggggttccgc gcacatttcc ccgaaaagtg ccacctgacg
     8281 tc
//
