LOCUS       sequence-444396-        6782 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  yeast surface display of the SARS-CoV-2 FLip variant RBD.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6782)
  AUTHORS   Taylor AL, Starr TN
  TITLE     Deep mutational scanning of SARS-CoV-2 Omicron BA.2.86 and epistatic
            emergence of the KP.3 variant.
  JOURNAL   Virus Evol. 2024 Sep 2;10(1):veae067. doi: 10.1093/ve/veae067. 
            eCollection 2024.
  PUBMED    39310091
REFERENCE   2  (bases 1 to 6782)
  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: "Virus 
            Evol."; date: "2024-09-2"; volume: "10(1):veae067. doi"; pages: " 
            10.1093/ve/veae067. eCollection 2024"
COMMENT     Sequence Label: 300_pETcon_SARS2_FLip
FEATURES             Location/Qualifiers
     source          1..6782
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    2..18
                     /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     7..29
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     26..42
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     26..42
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     promoter        63..81
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        111..763
                     /gene="GAL1 and GAL10 genes of S. cerevisiae"
                     /label=GAL1,10 promoter
                     /note="divergent inducible promoter, regulated by Gal4"
     protein_bind    315..432
                     /label=UAS
                     /bound_moiety="Gal4"
                     /note="upstream activating sequence mediating 
                     Gal4-dependent induction"
     primer_bind     727..750
                     /label=GAL1
                     /note="S. cerevisiae GAL1 promoter, forward primer"
     CDS             1082..1093
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     CDS             1094..1120
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             1811..1840
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     primer_bind     complement(2169..2188)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2170..2188)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     rep_origin      2386..2841
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2473..2492)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2683..2704
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(2944..3618)
                     /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(3619..3899)
                     /gene="S. cerevisiae TRP1"
                     /label=TRP1 promoter
     primer_bind     complement(3920..3939)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4039..4061
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4099..4117)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     misc_feature    4158..4661
                     /label=CEN/ARS
                     /note="S. cerevisiae CEN6 centromere fused to an 
                     autonomously replicating sequence"
     promoter        4698..4802
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4803..4871,4872..5663)
                     /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(5021..5040)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5834..6422
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6323..6342
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6576..6593
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    6710..6731
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(6746..6751,6752..6769,6770..6776)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
ORIGIN
        1 attgtgagcg gataacaatt tcacacagga aacagctatg accatgatta cgccaagctc
       61 gaaattaacc ctcactaaag ggaacaaaag ctggtaccaa ttccttgaat tttcaaaaat
      121 tcttactttt tttttggatg gacgcaaaga agtttaataa tcatattaca tggcattacc
      181 accatataca tatccatatc taatcttact tatatgttgt ggaaatgtaa agagccccat
      241 tatcttagcc taaaaaaacc ttctctttgg aactttcagt aatacgctta actgctcatt
      301 gctatattga agtacggatt agaagccgcc gagcgggtga cagccctccg aaggaagact
      361 ctcctccgtg cgtcctcgtc ttcaccggtc gcgttcctga aacgcagatg tgcctcgcgc
      421 cgcactgctc cgaacaataa agattctaca atactagctt ttatggttat gaagaggaaa
      481 aattggcagt aacctggccc cacaaacctt caaatgaacg aatcaaatta acaaccatag
      541 gatgataatg cgattagttt tttagcctta tttctggggt aattaatcag cgaagcgatg
      601 atttttgatc tattaacaga tatataaatg caaaaactgc ataaccactt taactaatac
      661 tttcaacatt ttcggtttgt attacttctt attcaaatgt aataaaagta tcaacaaaaa
      721 attgttaata tacctctata ctttaacgtc aaggagaaaa aaccccggat cgaattccct
      781 acttcataca ttttcaatta agatgcagtt acttcgctgt ttttcaatat tttctgttat
      841 tgcttcagtt ttagcacagg aactgacaac tatatgcgag caaatcccct caccaacttt
      901 agaatcgacg ccgtactctt tgtcaacgac tactattttg gccaacggga aggcaatgca
      961 aggagttttt gaatattaca aatcagtaac gtttgtcagt aattgcggtt ctcacccctc
     1021 aacaactagc aaaggcagcc ccataaacac acagtatgtt tttaaggaca atagctcgac
     1081 gattgaaggt agatacccat acgacgttcc agactacgct ctgcaggcta gtggtggagg
     1141 aggctctggt ggaggcggcc gcggaggcgg agggtcggct agccatatga acatcaccaa
     1201 cttgtgtcca ttccatgaag ttttcaatgc tactactttc gcttctgttt acgcttggaa
     1261 tagaaagaga atctctaact gcgttgctga ctattctgtc atttacaatt ttgctccatt
     1321 cttcgctttc aagtgctatg gtgtttctcc aactaagttg aacgatttgt gtttcaccaa
     1381 cgtttacgcc gattcctttg ttattagagg taacgaagtc tcccaaattg ctccaggtca
     1441 aactggtaat attgccgatt acaattacaa gttgccagat gatttcaccg gttgtgttat
     1501 tgcttggaac tctaacaagt tggattctaa gccttctggc aactacaatt acttgtacag
     1561 gttcttgcgt aagtccaaat tgaagccatt cgaaagagat atttccaccg aaatctatca
     1621 agctggtaac aagccatgta atggtgttgc tggtcctaac tgttactctc cattgcaatc
     1681 ttacggtttc agaccaactt atggtgttgg tcatcaacca tacagagttg ttgttttgtc
     1741 tttcgagttg ttgcatgctc cagctactgt ttgtggtcca aagaaatcta ctctcgaggg
     1801 gggcggttcc gaacaaaagc ttatttctga agaggacttg taatagagat ctgataacaa
     1861 cagtgtagat gtaacaaaat cgactttgtt cccactgtac ttttagctcg tacaaaatac
     1921 aatatacttt tcatttctcc gtaaacaaca tgttttccca tgtaatatcc ttttctattt
     1981 ttcgttccgt taccaacttt acacatactt tatatagcta ttcacttcta tacactaaaa
     2041 aactaagaca attttaattt tgctgcctgc catatttcaa tttgttataa attcctataa
     2101 tttatcctat tagtagctaa aaaaagatga atgtgaatcg aatcctaaga gaattgagct
     2161 ccaattcgcc ctatagtgag tcgtattaca attcactggc cgtcgttaga tcggaagagc
     2221 acacgtctga actccagtca cttacaacgt cgtgactggg aaaaccctgg cgttacccaa
     2281 cttaatcgcc ttgcagcaca tccccctttc gccagctggc gtaatagcga agaggcccgc
     2341 accgatcgcc tttcccaaca gttgcgcagc ctgaatggcg aatggacgcg ccctgtagcg
     2401 gcgcattaag cgcggcgggt gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg
     2461 ccctagcgcc cgctcctttc gctttcttcc cttcctttct cgccacgttc gccggctttc
     2521 cccgtcaagc tctaaatcgg gggctccctt tagggttccg atttagtgct ttacggcacc
     2581 tcgaccccaa aaaacttgat tagggtgatg gttcacgtag tgggccatcg ccctgataga
     2641 cggtttttcg ccctttgacg ttggagtcca cgttctttaa tagtggactc ttgttccaaa
     2701 ctggaacaac actcaaccct atctcggtct attcttttga tttataaggg attttgccga
     2761 tttcggccta ttggttaaaa aatgagctga tttaacaaaa atttaacgcg aattttaaca
     2821 aaatattaac gcttacaatt tcctgatgcg gtattttctc cttacgcatc tgtgcggtat
     2881 ttcacaccgc atagatcggc aagtgcacaa acaatactta aataaatact actcagtaat
     2941 aacctatttc ttagcatttt tgacgaaatt tgctattttg ttagagtctt ttacaccatt
     3001 tgtctccaca cctccgctta catcaacacc aataacgcca tttaatctaa gcgcatcacc
     3061 aacattttct ggcgtcagtc caccagctaa cataaaatgt aagctttcgg ggctctcttg
     3121 ccttccaacc cagtcagaaa tcgagttcca atccaaaagt tcacctgtcc cacctgcttc
     3181 tgaatcaaac aagggaataa acgaatgagg tttctgtgaa gctgcactga gtagtatgtt
     3241 gcagtctttt ggaaatacga gtcttttaat aactggcaaa ccgaggaact cttggtattc
     3301 ttgccacgac tcatctccat gcagttggac gatatcaatg ccgtaatcat tgaccagagc
     3361 caaaacatcc tccttaggtt gattacgaaa cacgccaacc aagtatttcg gagtgcctga
     3421 actattttta tatgctttta caagacttga aattttcctt gcaataaccg ggtcaattgt
     3481 tctctttcta ttgggcacac atataatacc cagcaagtca gcatcggaat ctagagcaca
     3541 ttctgcggcc tctgtgctct gcaagccgca aactttcacc aatggaccag aactacctgt
     3601 gaaattaata acagacatac tccaagctgc ctttgtgtgc ttaatcacgt atactcacgt
     3661 gctcaatagt caccaatgcc ctccctcttg gccctctcct tttctttttt cgaccgaatt
     3721 aattcttaat cggcaaaaaa agaaaagctc cggatcaaga ttgtacgtaa ggtgacaagc
     3781 tatttttcaa taaagaatat cttccactac tgccatctgg cgtcataact gcaaagtaca
     3841 catatattac gatgctgttc tattaaatgc ttcctatatt atatatatag taatgtcgtg
     3901 atctatggtg cactctcagt acaatctgct ctgatgccgc atagttaagc cagccccgac
     3961 acccgccaac acccgctgac gcgccctgac gggcttgtct gctcccggca tccgcttaca
     4021 gacaagctgt gaccgtctcc gggagctgca tgtgtcagag gttttcaccg tcatcaccga
     4081 aacgcgcgag acgaaagggc ctcgtgatac gcctattttt ataggttaat gtcatgataa
     4141 taatggtttc ttagacggat cgcttgcctg taacttacac gcgcctcgta tcttttaatg
     4201 atggaataat ttgggaattt actctgtgtt tatttatttt tatgttttgt atttggattt
     4261 tagaaagtaa ataaagaagg tagaagagtt acggaatgaa gaaaaaaaaa taaacaaagg
     4321 tttaaaaaat ttcaacaaaa agcgtacttt acatatatat ttattagaca agaaaagcag
     4381 attaaataga tatacattcg attaacgata agtaaaatgt aaaatcacag gattttcgtg
     4441 tgtggtcttc tacacagaca agatgaaaca attcggcatt aatacctgag agcaggaaga
     4501 gcaagataaa aggtagtatt tgttggcgat ccccctagag tcttttacat cttcggaaaa
     4561 caaaaactat tttttcttta atttcttttt ttactttcta tttttaattt atatatttat
     4621 attaaaaaat ttaaattata attattttta tagcacgtga tgaaaaggac ccaggtggca
     4681 cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt tttctaaata cattcaaata
     4741 tgtatccgct catgagacaa taaccctgat aaatgcttca ataatattga aaaaggaaga
     4801 gtatgagtat tcaacatttc cgtgtcgccc ttattccctt ttttgcggca ttttgccttc
     4861 ctgtttttgc tcacccagaa acgctggtga aagtaaaaga tgctgaagat cagttgggtg
     4921 cacgagtggg ttacatcgaa ctggatctca acagcggtaa gatccttgag agttttcgcc
     4981 ccgaagaacg ttttccaatg atgagcactt ttaaagttct gctatgtggc gcggtattat
     5041 cccgtattga cgccgggcaa gagcaactcg gtcgccgcat acactattct cagaatgact
     5101 tggttgagta ctcaccagtc acagaaaagc atcttacgga tggcatgaca gtaagagaat
     5161 tatgcagtgc tgccataacc atgagtgata acactgcggc caacttactt ctgacaacga
     5221 tcggaggacc gaaggagcta accgcttttt tgcacaacat gggggatcat gtaactcgcc
     5281 ttgatcgttg ggaaccggag ctgaatgaag ccataccaaa cgacgagcgt gacaccacga
     5341 tgcctgtagc aatggcaaca acgttgcgca aactattaac tggcgaacta cttactctag
     5401 cttcccggca acaattaata gactggatgg aggcggataa agttgcagga ccacttctgc
     5461 gctcggccct tccggctggc tggtttattg ctgataaatc tggagccggt gagcgtgggt
     5521 ctcgcggtat cattgcagca ctggggccag atggtaagcc ctcccgtatc gtagttatct
     5581 acacgacggg gagtcaggca actatggatg aacgaaatag acagatcgct gagataggtg
     5641 cctcactgat taagcattgg taactgtcag accaagttta ctcatatata ctttagattg
     5701 atttaaaact tcatttttaa tttaaaagga tctaggtgaa gatccttttt gataatctca
     5761 tgaccaaaat cccttaacgt gagttttcgt tccactgagc gtcagacccc gtagaaaaga
     5821 tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa
     5881 aaccaccgct accagcggtg gtttgtttgc cggatcaaga gctaccaact ctttttccga
     5941 aggtaactgg cttcagcaga gcgcagatac caaatactgt tcttctagtg tagccgtagt
     6001 taggccacca cttcaagaac tctgtagcac cgcctacata cctcgctctg ctaatcctgt
     6061 taccagtggc tgctgccagt ggcgataagt cgtgtcttac cgggttggac tcaagacgat
     6121 agttaccgga taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca cagcccagct
     6181 tggagcgaac gacctacacc gaactgagat acctacagcg tgagctatga gaaagcgcca
     6241 cgcttcccga agggagaaag gcggacaggt atccggtaag cggcagggtc ggaacaggag
     6301 agcgcacgag ggagcttcca gggggaaacg cctggtatct ttatagtcct gtcgggtttc
     6361 gccacctctg acttgagcgt cgatttttgt gatgctcgtc aggggggcgg agcctatgga
     6421 aaaacgccag caacgcggcc tttttacggt tcctggcctt ttgctggcct tttgctcaca
     6481 tgttctttcc tgcgttatcc cctgattctg tggataaccg tattaccgcc tttgagtgag
     6541 ctgataccgc tcgccgcagc cgaacgaccg agcgcagcga gtcagtgagc gaggaagcgg
     6601 aagagcgccc aatacgcaaa ccgcctctcc ccgcgcgttg gccgattcat taatgcagct
     6661 ggcacgacag gtttcccgac tggaaagcgg gcagtgagcg caacgcaatt aatgtgagtt
     6721 agctcactca ttaggcaccc caggctttac actttatgct tccggctcgt atgttgtgtg
     6781 ga
//
