LOCUS       sequence-342193-        3488 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Encodes the SARS-CoV-2 Spike Receptor Binding Domain (RBD) with the 
            E484K mutation and can be combined with pJS697 (Addgene plasmid # 
            168778) through homologous recombination.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3488)
  AUTHORS   Francino-Urdaniz IM, Steiner PJ, Kirby MB, Zhao F, Haas CM, Barman 
            S, Rhodes ER, Leonard AC, Peng L, Sprenger KG, Jardine JG, Whitehead
            TA
  TITLE     One-shot identification of SARS-CoV-2 S RBD escape mutants using 
            yeast screening.
  JOURNAL   Cell Rep. 2021 Aug 31;36(9):109627. doi: 
            10.1016/j.celrep.2021.109627. Epub 2021 Aug 10.
  PUBMED    34416153
REFERENCE   2  (bases 1 to 3488)
  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: "Cell Rep.";
            date: "2021-08-31"; volume: "36(9):109627. doi"; pages: " 
            10.1016/j.celrep.2021.109627. Epub 2021 Aug 10"
COMMENT     Sequence Label: pIFU001
FEATURES             Location/Qualifiers
     source          1..3488
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             31..42
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     CDS             43..69
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             754..783
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     primer_bind     complement(888..905)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(888..904)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(897..919)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     complement(1113..1132)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     1232..1254
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(1292..1310)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        1378..1482
                     /gene="bla"
                     /label=AmpR promoter
     CDS             1483..2298
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     CDS             1483..2298
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     complement(1556..1575)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      2478..3066
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2967..2986
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3220..3237
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    3354..3375
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(3390..3395,3396..3413,3414..3420)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3428..3444
                     /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     3433..3455
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     3452..3468
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     3452..3468
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
ORIGIN
        1 atgcctgcag gtcgacgcgg ccgctcgacg attgaaggta gatacccata cgacgttcca
       61 gactacgctc tgcaggctag tggtggagga ggctctggtg gaggcggtag cggaggcgga
      121 gggtcgacaa acttgtgccc ttttggtgaa gtttttcaag ccaccagatt tgcatctgtt
      181 tatgcttgga acaggaagag aatcagcaac tgtgttgctg attattctgt cctatataat
      241 tccgcatcat tttccacttt taagtgttat ggagtgtctc ctactaaatt aaatgatctc
      301 tgctttacta atgtctatgc agattcattt gtaattagag gtgatgaagt cagacaaatc
      361 gctccagggc aaactggaaa gattgctgat tataattata aattaccaga tgattttaca
      421 ggctgcgtta tagcttggaa ttctaacaat cttgattcta aggttggtgg taattataat
      481 tacctgtata gattgtttag gaagtctaat ctcaaacctt ttgagagaga tatttcaact
      541 gaaatctatc aggccggtag cacaccttgt aatggtgtta aaggttttaa ttgttacttt
      601 cctttacaat catatggttt ccaacccact aatggtgttg gttaccaacc atacagagta
      661 gtagtacttt cttttgaact tctacatgca ccagcaactg tttgtggacc taaaaagtct
      721 actaatttgg ttaaaaacaa agggggcgga tccgaacaaa agcttatttc tgaagaggac
      781 ttgtaataga gatctgataa caacagtgta gatgtaacaa aatcgacttt gttcccactg
      841 tacttttagc ggccgccctc agcgatatcg gtaccgagct cgaattcact ggccgtcgtt
      901 ttacaacgtc gtgactggga aaaccctggc gttacccaac ttaatcgcct tgcagcacat
      961 ccccctttcg ccagctggcg taatagcgaa gaggcccgca ccgatcgccc ttcccaacag
     1021 ttgcgcagcc tgaatggcga atggcgcctg atgcggtatt ttctccttac gcatctgtgc
     1081 ggtatttcac accgcatatg gtgcactctc agtacaatct gctctgatgc cgcatagtta
     1141 agccagcccc gacacccgcc aacacccgct gacgcgccct gacgggcttg tctgctcccg
     1201 gcatccgctt acagacaagc tgtgaccgtc tccgggagct gcatgtgtca gaggttttca
     1261 ccgtcatcac cgaaacgcgc gagacgaaag ggcctcgtga tacgcctatt tttataggtt
     1321 aatgtcatga taataatggt ttcttagacg tcaggtggca cttttcgggg aaatgtgcgc
     1381 ggaaccccta tttgtttatt tttctaaata cattcaaata tgtatccgct catgagacaa
     1441 taaccctgat aaatgcttca ataatattga aaaaggaaga gtatgagcca tattcaacgg
     1501 gaaacgtctt gctctaggcc gcgattaaat tccaacatgg atgctgattt atatgggtat
     1561 aaatgggctc gcgataatgt cgggcaatca ggtgcgacaa tctatcgatt gtatgggaag
     1621 cccgatgcgc cagagttgtt tctgaaacat ggcaaaggta gcgttgccaa tgatgttaca
     1681 gatgagatgg tcagactaaa ctggctgacg gaatttatgc ctcttccgac catcaagcat
     1741 tttatccgta ctcctgatga tgcatggtta ctcaccactg cgatccccgg gaaaacagca
     1801 ttccaggtat tagaagaata tcctgattca ggtgaaaata ttgttgatgc gctggcagtg
     1861 ttcctgcgcc ggttgcattc gattcctgtt tgtaattgtc cttttaacag cgatcgcgta
     1921 tttcgtctcg ctcaggcgca atcacgaatg aataacggtt tggttgatgc gagtgatttt
     1981 gatgacgagc gtaatggctg gcctgttgaa caagtctgga aagaaatgca taaacttttg
     2041 ccattctcac cggattcagt cgtcactcat ggtgatttct cacttgataa ccttattttt
     2101 gacgagggga aattaatagg ttgtattgat gttggacgag tcggaatcgc agaccgatac
     2161 caggatcttg ccatcctatg gaactgcctc ggtgagtttt ctccttcatt acagaaacgg
     2221 ctttttcaaa aatatggtat tgataatcct gatatgaata aattgcagtt tcatttgatg
     2281 ctcgatgagt ttttctaacc tcagcaactg tcagaccaag tttactcata tatactttag
     2341 attgatttaa aacttcattt ttaatttaaa aggatctagg tgaagatcct ttttgataat
     2401 ctcatgacca aaatccctta acgtgagttt tcgttccact gagcgtcaga ccccgtagaa
     2461 aagatcaaag gatcttcttg agatcctttt tttctgcgcg taatctgctg cttgcaaaca
     2521 aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc aagagctacc aactcttttt
     2581 ccgaaggtaa ctggcttcag cagagcgcag ataccaaata ctgttcttct agtgtagccg
     2641 tagttaggcc accacttcaa gaactctgta gcaccgccta catacctcgc tctgctaatc
     2701 ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga
     2761 cgatagttac cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc
     2821 agcttggagc gaacgaccta caccgaactg agatacctac agcgtgagct atgagaaagc
     2881 gccacgcttc ccgaagggag aaaggcggac aggtatccgg taagcggcag ggtcggaaca
     2941 ggagagcgca cgagggagct tccaggggga aacgcctggt atctttatag tcctgtcggg
     3001 tttcgccacc tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta
     3061 tggaaaaacg ccagcaacgc ggccttttta cggttcctgg ccttttgctg gccttttgct
     3121 cacatgttct ttcctgcgtt atcccctgat tctgtggata accgtattac cgcctttgag
     3181 tgagctgata ccgctcgccg cagccgaacg accgagcgca gcgagtcagt gagcgaggaa
     3241 gcggaagagc gcccaatacg caaaccgcct ctccccgcgc gttggccgat tcattaatgc
     3301 agctggcacg acaggtttcc cgactggaaa gcgggcagtg agcgcaacgc aattaatgtg
     3361 agttagctca ctcattaggc accccaggct ttacacttta tgcttccggc tcgtatgttg
     3421 tgtggaattg tgagcggata acaatttcac acaggaaaca gctatgacca tgattacgcc
     3481 aagcttgc
//
