LOCUS       sequence-362506-        6774 bp    DNA     circular SYN 15-APR-2025
DEFINITION  yeast surface display of the SARS-CoV-2 Beta variant RBD.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6774)
  AUTHORS   Starr TN, Greaney AJ, Hannon WW, Loes AN, Hauser K, Dillen JR, Ferri
            E, Farrell AG, Dadonaite B, McCallum M, Matreyek KA, Corti D, 
            Veesler D, Snell G, Bloom JD
  TITLE     Shifting mutational constraints in the SARS-CoV-2 receptor-binding 
            domain during viral evolution.
  JOURNAL   Science. 2022 Jun 28:eabo7896. doi: 10.1126/science.abo7896.
  PUBMED    35762884
REFERENCE   2  (bases 1 to 6774)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Science. 
            2022 Jun 28:eabo7896. doi: 10.1126/science.abo7896."
COMMENT     Sequence Label: 3021_pETcon-SARS-CoV-2-RBD_K417N_E484K_N501Y
FEATURES             Location/Qualifiers
     source          1..6774
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    30..51
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(66..71,72..89,90..96)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    104..120
                     /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     109..131
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     128..144
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     128..144
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     promoter        165..183
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        213..865
                     /gene="GAL1 and GAL10 genes of S. cerevisiae"
                     /label=GAL1,10 promoter
                     /note="divergent inducible promoter, regulated by Gal4"
     protein_bind    417..534
                     /label=UAS
                     /bound_moiety="Gal4"
                     /note="upstream activating sequence mediating 
                     Gal4-dependent induction"
     primer_bind     829..852
                     /label=GAL1
                     /note="S. cerevisiae GAL1 promoter, forward primer"
     CDS             1184..1195
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     CDS             1196..1222
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             1913..1942
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     primer_bind     complement(2271..2290)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2272..2290)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     rep_origin      2480..2935
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2567..2586)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2777..2798
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(3038..3712)
                     /codon_start=1
                     /gene="S. cerevisiae TRP1"
                     /product="phosphoribosylanthranilate isomerase, required 
                     for tryptophan biosynthesis"
                     /label=TRP1
                     /note="yeast auxotrophic marker"
                     /translation="MSVINFTGSSGPLVKVCGLQSTEAAECALDSDADLLGIICVPNRK
                     RTIDPVIARKISSLVKAYKNSSGTPKYLVGVFRNQPKEDVLALVNDYGIDIVQLHGDES
                     WQEYQEFLGLPVIKRLVFPKDCNILLSAASQKPHSFIPLFDSEAGGTGELLDWNSISDW
                     VGRQESPESLHFMLAGGLTPENVGDALRLNGVIGVDVSGGVETNGVKDSNKIANFVKNA
                     KK"
     promoter        complement(3713..3993)
                     /gene="S. cerevisiae TRP1"
                     /label=TRP1 promoter
     primer_bind     complement(4014..4033)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4133..4155
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4193..4211)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     misc_feature    4252..4755
                     /label=CEN/ARS
                     /note="S. cerevisiae CEN6 centromere fused to an 
                     autonomously replicating sequence"
     promoter        4792..4896
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4897..4965,4966..5757)
                     /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     complement(5115..5134)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5928..6516
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6417..6436
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6670..6687
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 tggaaagcgg gcagtgagcg caacgcaatt aatgtgagtt agctcactca ttaggcaccc
       61 caggctttac actttatgct tccggctcgt atgttgtgtg gaattgtgag cggataacaa
      121 tttcacacag gaaacagcta tgaccatgat tacgccaagc tcgaaattaa ccctcactaa
      181 agggaacaaa agctggtacc aattccttga attttcaaaa attcttactt tttttttgga
      241 tggacgcaaa gaagtttaat aatcatatta catggcatta ccaccatata catatccata
      301 tctaatctta cttatatgtt gtggaaatgt aaagagcccc attatcttag cctaaaaaaa
      361 ccttctcttt ggaactttca gtaatacgct taactgctca ttgctatatt gaagtacgga
      421 ttagaagccg ccgagcgggt gacagccctc cgaaggaaga ctctcctccg tgcgtcctcg
      481 tcttcaccgg tcgcgttcct gaaacgcaga tgtgcctcgc gccgcactgc tccgaacaat
      541 aaagattcta caatactagc ttttatggtt atgaagagga aaaattggca gtaacctggc
      601 cccacaaacc ttcaaatgaa cgaatcaaat taacaaccat aggatgataa tgcgattagt
      661 tttttagcct tatttctggg gtaattaatc agcgaagcga tgatttttga tctattaaca
      721 gatatataaa tgcaaaaact gcataaccac tttaactaat actttcaaca ttttcggttt
      781 gtattacttc ttattcaaat gtaataaaag tatcaacaaa aaattgttaa tatacctcta
      841 tactttaacg tcaaggagaa aaaaccccgg atcgaattcc ctacttcata cattttcaat
      901 taagatgcag ttacttcgct gtttttcaat attttctgtt attgcttcag ttttagcaca
      961 ggaactgaca actatatgcg agcaaatccc ctcaccaact ttagaatcga cgccgtactc
     1021 tttgtcaacg actactattt tggccaacgg gaaggcaatg caaggagttt ttgaatatta
     1081 caaatcagta acgtttgtca gtaattgcgg ttctcacccc tcaacaacta gcaaaggcag
     1141 ccccataaac acacagtatg tttttaagga caatagctcg acgattgaag gtagataccc
     1201 atacgacgtt ccagactacg ctctgcaggc tagtggtgga ggaggctctg gtggaggcgg
     1261 ccgcggaggc ggagggtcgg ctagccatat gaatatcacg aacctttgtc ctttcggtga
     1321 ggtcttcaat gctactagat tcgcatccgt gtatgcatgg aatagaaaga gaattagtaa
     1381 ttgtgtagcg gactactctg tactttataa ctccgcctcc ttctccacat tcaagtgtta
     1441 cggtgtatct cccaccaagt tgaatgatct atgctttaca aacgtttacg ccgatagttt
     1501 cgtaattaga ggcgatgaag tgcgtcagat cgcaccaggc cagacgggca acatagcaga
     1561 ctataattat aagctgcctg atgacttcac cggctgtgtg atagcttgga actcaaataa
     1621 tctagattcc aaggtgggag gcaattacaa ttatttgtac cgtctgttcc gtaaaagcaa
     1681 tttgaaacca tttgaaagag acattagcac tgaaatttat caagcagggt ccaccccgtg
     1741 caacggcgta aagggcttta actgttattt cccattacag tcttatggtt tccaacctac
     1801 gtacggagtc gggtatcagc cgtacagggt tgtggttctt tcatttgaac tgctgcacgc
     1861 gcccgcaacc gtatgcgggc cgaagaaatc aacgctcgag gggggcggtt ccgaacaaaa
     1921 gcttatttct gaagaggact tgtaatagag atctgataac aacagtgtag atgtaacaaa
     1981 atcgactttg ttcccactgt acttttagct cgtacaaaat acaatatact tttcatttct
     2041 ccgtaaacaa catgttttcc catgtaatat ccttttctat ttttcgttcc gttaccaact
     2101 ttacacatac tttatatagc tattcacttc tatacactaa aaaactaaga caattttaat
     2161 tttgctgcct gccatatttc aatttgttat aaattcctat aatttatcct attagtagct
     2221 aaaaaaagat gaatgtgaat cgaatcctaa gagaattgag ctccaattcg ccctatagtg
     2281 agtcgtatta caattcactg gccgtcgtta gatcggaaga gcacactcca gtcacttaca
     2341 acgtcgtgac tgggaaaacc ctggcgttac ccaacttaat cgccttgcag cacatccccc
     2401 tttcgccagc tggcgtaata gcgaagaggc ccgcaccgat cgcctttccc aacagttgcg
     2461 cagcctgaat ggcgaatgga cgcgccctgt agcggcgcat taagcgcggc gggtgtggtg
     2521 gttacgcgca gcgtgaccgc tacacttgcc agcgccctag cgcccgctcc tttcgctttc
     2581 ttcccttcct ttctcgccac gttcgccggc tttccccgtc aagctctaaa tcgggggctc
     2641 cctttagggt tccgatttag tgctttacgg cacctcgacc ccaaaaaact tgattagggt
     2701 gatggttcac gtagtgggcc atcgccctga tagacggttt ttcgcccttt gacgttggag
     2761 tccacgttct ttaatagtgg actcttgttc caaactggaa caacactcaa ccctatctcg
     2821 gtctattctt ttgatttata agggattttg ccgatttcgg cctattggtt aaaaaatgag
     2881 ctgatttaac aaaaatttaa cgcgaatttt aacaaaatat taacgcttac aatttcctga
     2941 tgcggtattt tctccttacg catctgtgcg gtatttcaca ccgcatagat cggcaagtgc
     3001 acaaacaata cttaaataaa tactactcag taataaccta tttcttagca tttttgacga
     3061 aatttgctat tttgttagag tcttttacac catttgtctc cacacctccg cttacatcaa
     3121 caccaataac gccatttaat ctaagcgcat caccaacatt ttctggcgtc agtccaccag
     3181 ctaacataaa atgtaagctt tcggggctct cttgccttcc aacccagtca gaaatcgagt
     3241 tccaatccaa aagttcacct gtcccacctg cttctgaatc aaacaaggga ataaacgaat
     3301 gaggtttctg tgaagctgca ctgagtagta tgttgcagtc ttttggaaat acgagtcttt
     3361 taataactgg caaaccgagg aactcttggt attcttgcca cgactcatct ccatgcagtt
     3421 ggacgatatc aatgccgtaa tcattgacca gagccaaaac atcctcctta ggttgattac
     3481 gaaacacgcc aaccaagtat ttcggagtgc ctgaactatt tttatatgct tttacaagac
     3541 ttgaaatttt ccttgcaata accgggtcaa ttgttctctt tctattgggc acacatataa
     3601 tacccagcaa gtcagcatcg gaatctagag cacattctgc ggcctctgtg ctctgcaagc
     3661 cgcaaacttt caccaatgga ccagaactac ctgtgaaatt aataacagac atactccaag
     3721 ctgcctttgt gtgcttaatc acgtatactc acgtgctcaa tagtcaccaa tgccctccct
     3781 cttggccctc tccttttctt ttttcgaccg aattaattct taatcggcaa aaaaagaaaa
     3841 gctccggatc aagattgtac gtaaggtgac aagctatttt tcaataaaga atatcttcca
     3901 ctactgccat ctggcgtcat aactgcaaag tacacatata ttacgatgct gttctattaa
     3961 atgcttccta tattatatat atagtaatgt cgtgatctat ggtgcactct cagtacaatc
     4021 tgctctgatg ccgcatagtt aagccagccc cgacacccgc caacacccgc tgacgcgccc
     4081 tgacgggctt gtctgctccc ggcatccgct tacagacaag ctgtgaccgt ctccgggagc
     4141 tgcatgtgtc agaggttttc accgtcatca ccgaaacgcg cgagacgaaa gggcctcgtg
     4201 atacgcctat ttttataggt taatgtcatg ataataatgg tttcttagac ggatcgcttg
     4261 cctgtaactt acacgcgcct cgtatctttt aatgatggaa taatttggga atttactctg
     4321 tgtttattta tttttatgtt ttgtatttgg attttagaaa gtaaataaag aaggtagaag
     4381 agttacggaa tgaagaaaaa aaaataaaca aaggtttaaa aaatttcaac aaaaagcgta
     4441 ctttacatat atatttatta gacaagaaaa gcagattaaa tagatataca ttcgattaac
     4501 gataagtaaa atgtaaaatc acaggatttt cgtgtgtggt cttctacaca gacaagatga
     4561 aacaattcgg cattaatacc tgagagcagg aagagcaaga taaaaggtag tatttgttgg
     4621 cgatccccct agagtctttt acatcttcgg aaaacaaaaa ctattttttc tttaatttct
     4681 ttttttactt tctattttta atttatatat ttatattaaa aaatttaaat tataattatt
     4741 tttatagcac gtgatgaaaa ggacccaggt ggcacttttc ggggaaatgt gcgcggaacc
     4801 cctatttgtt tatttttcta aatacattca aatatgtatc cgctcatgag acaataaccc
     4861 tgataaatgc ttcaataata ttgaaaaagg aagagtatga gtattcaaca tttccgtgtc
     4921 gcccttattc ccttttttgc ggcattttgc cttcctgttt ttgctcaccc agaaacgctg
     4981 gtgaaagtaa aagatgctga agatcagttg ggtgcacgag tgggttacat cgaactggat
     5041 ctcaacagcg gtaagatcct tgagagtttt cgccccgaag aacgttttcc aatgatgagc
     5101 acttttaaag ttctgctatg tggcgcggta ttatcccgta ttgacgccgg gcaagagcaa
     5161 ctcggtcgcc gcatacacta ttctcagaat gacttggttg agtactcacc agtcacagaa
     5221 aagcatctta cggatggcat gacagtaaga gaattatgca gtgctgccat aaccatgagt
     5281 gataacactg cggccaactt acttctgaca acgatcggag gaccgaagga gctaaccgct
     5341 tttttgcaca acatggggga tcatgtaact cgccttgatc gttgggaacc ggagctgaat
     5401 gaagccatac caaacgacga gcgtgacacc acgatgcctg tagcaatggc aacaacgttg
     5461 cgcaaactat taactggcga actacttact ctagcttccc ggcaacaatt aatagactgg
     5521 atggaggcgg ataaagttgc aggaccactt ctgcgctcgg cccttccggc tggctggttt
     5581 attgctgata aatctggagc cggtgagcgt gggtctcgcg gtatcattgc agcactgggg
     5641 ccagatggta agccctcccg tatcgtagtt atctacacga cggggagtca ggcaactatg
     5701 gatgaacgaa atagacagat cgctgagata ggtgcctcac tgattaagca ttggtaactg
     5761 tcagaccaag tttactcata tatactttag attgatttaa aacttcattt ttaatttaaa
     5821 aggatctagg tgaagatcct ttttgataat ctcatgacca aaatccctta acgtgagttt
     5881 tcgttccact gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg agatcctttt
     5941 tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt
     6001 ttgccggatc aagagctacc aactcttttt ccgaaggtaa ctggcttcag cagagcgcag
     6061 ataccaaata ctgttcttct agtgtagccg tagttaggcc accacttcaa gaactctgta
     6121 gcaccgccta catacctcgc tctgctaatc ctgttaccag tggctgctgc cagtggcgat
     6181 aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac cggataaggc gcagcggtcg
     6241 ggctgaacgg ggggttcgtg cacacagccc agcttggagc gaacgaccta caccgaactg
     6301 agatacctac agcgtgagct atgagaaagc gccacgcttc ccgaagggag aaaggcggac
     6361 aggtatccgg taagcggcag ggtcggaaca ggagagcgca cgagggagct tccaggggga
     6421 aacgcctggt atctttatag tcctgtcggg tttcgccacc tctgacttga gcgtcgattt
     6481 ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc ggccttttta
     6541 cggttcctgg ccttttgctg gccttttgct cacatgttct ttcctgcgtt atcccctgat
     6601 tctgtggata accgtattac cgcctttgag tgagctgata ccgctcgccg cagccgaacg
     6661 accgagcgca gcgagtcagt gagcgaggaa gcggaagagc gcccaatacg caaaccgcct
     6721 ctccccgcgc gttggccgat tcattaatgc agctggcacg acaggtttcc cgac
//
