LOCUS       sequence-436202-        6634 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial expression plasmid for Saccharomyces cerevisiae 
            beta′-COPI-WD40-Tyr33Ala mutant (residues 1-301).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6634)
  AUTHORS   Dey D, Qing E, He Y, Chen Y, Jennings B, Cohn W, Singh S, Gakhar L, 
            Schnicker NJ, Pierce BG, Whitelegge JP, Doray B, Orban J, Gallagher 
            T, Hasan SS
  TITLE     A single C-terminal residue controls SARS-CoV-2 spike trafficking 
            and incorporation into VLPs.
  JOURNAL   Nat Commun. 2023 Dec 15;14(1):8358. doi: 10.1038/s41467-023-44076-3.
  PUBMED    38102143
REFERENCE   2  (bases 1 to 6634)
  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: "Nat 
            Commun."; date: "2023-12-15"; volume: "14(1):8358. doi"; pages: " 
            10.1038/s41467-023-44076-3"
COMMENT     Sequence Label: SSH-pSUMO-beta′-COPI-WD40-Tyr33Ala
FEATURES             Location/Qualifiers
     source          1..6634
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             483..674
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(668..690)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    776..915
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     831..850
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(930..947)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1101..1689)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1181..1200)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(1860..2651,2652..2720))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     2483..2502
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2721..2825)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(2851..3306)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2988..3009)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3200..3219
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     terminator      complement(3343..3390)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     3386..3404
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     CDS             complement(3457..3474)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             complement(4405..4695)
                     /codon_start=1
                     /gene="S. cerevisiae SMT3 (truncated)"
                     /product="cleavable ubiquitin-like protein tag"
                     /label=SUMO
                     /translation="SDSEVNQEAKPEVKPEVKPETHINLKVSDGSSEIFFKIKKTTPLR
                     RLMEAFAKRQGKEMDSLRFLYDGIRIQADQTPEDLDMEDNDIIEAHREQIGG"
     CDS             complement(4699..4716)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             complement(4726..4749)
                     /codon_start=1
                     /product="peptide that binds Strep-Tactin(R), an engineered
                     form of streptavidin"
                     /label=Strep-Tag II
                     /translation="WSHPQFEK"
     RBS             4759..4781
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    4796..4820
                     /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     complement(4820..4839)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4821..4839)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4923..4942)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        5148..5225
                     /gene="lacI"
                     /label=lacI promoter
     CDS             5226..6308
                     /codon_start=1
                     /gene="lacI"
                     /product="lac repressor"
                     /label=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)."
                     /translation="MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     primer_bind     complement(5245..5264)
                     /label=LacI-R
                     /note="LacI, reverse primer"
ORIGIN
        1 ccggcatggc ggccccacgg gtgcgcatga tcgtgctcct gtcgttgagg acccggctag
       61 gctggcgggg ttgccttact ggttagcaga atgaatcacc gatacgcgag cgaacgtgaa
      121 gcgactgctg ctgcaaaacg tctgcgacct gagcaacaac atgaatggtc ttcggtttcc
      181 gtgtttcgta aagtctggaa acgcggaagt cagcgccctg caccattatg ttccggatct
      241 gcatcgcagg atgctgctgg ctaccctgtg gaacacctac atctgtatta acgaagcgct
      301 ggcattgacc ctgagtgatt tttctctggt cccgccgcat ccataccgcc agttgtttac
      361 cctcacaacg ttccagtaac cgggcatgtt catcatcagt aacccgtatc gtgagcatcc
      421 tctctcgttt catcggtatc attaccccca tgaacagaaa tcccccttac acggaggcat
      481 cagtgaccaa acaggaaaaa accgccctta acatggcccg ctttatcaga agccagacat
      541 taacgcttct ggagaaactc aacgagctgg acgcggatga acaggcagac atctgtgaat
      601 cgcttcacga ccacgctgat gagctttacc gcagctgcct cgcgcgtttc ggtgatgacg
      661 gtgaaaacct ctgacacatg cagctcccgg agacggtcac agcttgtctg taagcggatg
      721 ccgggagcag acaagcccgt cagggcgcgt cagcgggtgt tggcgggtgt cggggcgcag
      781 ccatgaccca gtcacgtagc gatagcggag tgtatactgg cttaactatg cggcatcaga
      841 gcagattgta ctgagagtgc accattgcgg tgtgaaatac cgcacagatg cgtaaggaga
      901 aaataccgca tcaggcgctc ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt
      961 cggctgcggc gagcggtatc agctcactca aaggcggtaa tacggttatc cacagaatca
     1021 ggggataacg caggaaagaa catgtgagca aaaggccagc aaaaggccag gaaccgtaaa
     1081 aaggccgcgt tgctggcgtt tttccatagg ctccgccccc ctgacgagca tcacaaaaat
     1141 cgacgctcaa gtcagaggtg gcgaaacccg acaggactat aaagatacca ggcgtttccc
     1201 cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc
     1261 gcctttctcc cttcgggaag cgtggcgctt tctcatagct cacgctgtag gtatctcagt
     1321 tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt tcagcccgac
     1381 cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc cggtaagaca cgacttatcg
     1441 ccactggcag cagccactgg taacaggatt agcagagcga ggtatgtagg cggtgctaca
     1501 gagttcttga agtggtggcc taactacggc tacactagaa ggacagtatt tggtatctgc
     1561 gctctgctga agccagttac cttcggaaaa agagttggta gctcttgatc cggcaaacaa
     1621 accaccgctg gtagcggtgg tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa
     1681 ggatctcaag aagatccttt gatcttttct acggggtctg acgctcagtg gaacgaaaac
     1741 tcacgttaag ggattttggt catgagatta tcaaaaagga tcttcaccta gatcctttta
     1801 aattaaaaat gaagttttaa atcaatctaa agtatatatg agtaaacttg gtctgacagt
     1861 taccaatgct taatcagtga ggcacctatc tcagcgatct gtctatttcg ttcatccata
     1921 gttgcctgac tccccgtcgt gtagataact acgatacggg agggcttacc atctggcccc
     1981 agtgctgcaa tgataccgcg agaaccacgc tcaccggctc cagatttatc agcaataaac
     2041 cagccagccg gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag
     2101 tctattaatt gttgccggga agctagagta agtagttcgc cagttaatag tttgcgcaac
     2161 gttgttgcca ttgctgcagg catcgtggtg tcacgctcgt cgtttggtat ggcttcattc
     2221 agctccggtt cccaacgatc aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg
     2281 gttagctcct tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt gttatcactc
     2341 atggttatgg cagcactgca taattctctt actgtcatgc catccgtaag atgcttttct
     2401 gtgactggtg agtactcaac caagtcattc tgagaatagt gtatgcggcg accgagttgc
     2461 tcttgcccgg cgtcaatacg ggataatacc gcgccacata gcagaacttt aaaagtgctc
     2521 atcattggaa aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc
     2581 agttcgatgt aacccactcg tgcacccaac tgatcttcag catcttttac tttcaccagc
     2641 gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca
     2701 cggaaatgtt gaatactcat actcttcctt tttcaatatt attgaagcat ttatcagggt
     2761 tattgtctca tgagcggata catatttgaa tgtatttaga aaaataaaca aataggggtt
     2821 ccgcgcacat ttccccgaaa agtgccacct aaattgtaag cgttaatatt ttgttaaaat
     2881 tcgcgttaaa tttttgttaa atcagctcat tttttaacca ataggccgaa atcggcaaaa
     2941 tcccttataa atcaaaagaa tagaccgaga tagggttgag tgttgttcca gtttggaaca
     3001 agagtccact attaaagaac gtggactcca acgtcaaagg gcgaaaaacc gtctatcagg
     3061 gcgatggccc actacgtgaa ccatcaccct aatcaagttt tttggggtcg aggtgccgta
     3121 aagcactaaa tcggaaccct aaagggagcc cccgatttag agcttgacgg ggaaagccgg
     3181 cgaacgtggc gagaaaggaa gggaagaaag cgaaaggagc gggcgctagg gcgctggcaa
     3241 gtgtagcggt cacgctgcgc gtaaccacca cacccgccgc gcttaatgcg ccgctacagg
     3301 gcgcgtccca ttcgccaatc cggatatagt tcctcctttc agcaaaaaac ccctcaagac
     3361 ccgtttagag gccccaaggg gttatgctag ttattgctca gcggtggcag cagccaactc
     3421 agcttccttt cgggctttgt tagcagccgg atctcagtgg tggtggtggt ggtgctcgag
     3481 tgcggccgca agctttcatc ccaaggaaag tacggtaaac ccattatcaa acccggatgc
     3541 aatgtagttt ttacgtcctg ttggatgggt agcgatacac caacttcttt ctaaaccaac
     3601 gtttaaagtc ttttctacct tgtaagtgga agaattccat atctttaaag tgccatcttc
     3661 ggaaccagaa attataatgg gtaaagttgg atggaaaaca gcaaaagaaa cattggacat
     3721 gtgaccttct agagtggcta cacatgactt cgtttggtaa tcccaaattt tgatggttaa
     3781 gtcgtccgac gcagtaatca tataaggctt gtcaggtagc gggtaataat caacataatt
     3841 cacacctctt tcttgtcctg tggttaacgt gaaatttggt gtcgattgtc ccaatgacca
     3901 aactttaacg gttcggtcca aacacccaga tgcaaacgta ctagggtcct ttggattgaa
     3961 tgcgacgcac atgacaaagt gttcgtgacc ttcaaaagtt tgttctaagg cccagttatt
     4021 ttcccaattc catagtttga ctgtcaaatc atcactgccg gataaaacat aaggctttgt
     4081 tggatgtaca gcaatggaac ggatatagtc tggatgagct tcgaaatcaa caactttttc
     4141 acctgtattg tagttgaaga ctctgatcct gaaatcatca ctcccaacaa ttatccaatt
     4201 ctttcttgca atgaatttac ctgctctaac aggagtttcc gtaacttgga tggatctaac
     4261 ttcaacttgt gtttcgtaat tccagatctc aacacgacca gaagccaagg tagtcaaaac
     4321 ccatggctca gtaggatgga aatcgatacc tttaactcga tcggatctat tactaaaagt
     4381 tttctttata tccaacttca tcgaacctcc aatctgttcg cggtgagcct caataatatc
     4441 gttatcctcc atgtccaaat cttcaggggt ctgatcagct tgaattctaa taccgtcgta
     4501 caagaatctt aaggagtcca tttccttacc ctgtctttta gcgaacgctt ccatcagcct
     4561 tcttaaagga gtggtctttt tgatcttgaa gaagatctct gaagatccat cggacacctt
     4621 taaattgatg tgagtctcag gcttgacttc tggcttgacc tctggcttag cttcttgatt
     4681 gacttctgag tccgacccgt gatgatgatg gtgatgacca gcactttttt caaattgggg
     4741 gtgtgaccac atggtatatc tccttcttaa agttaaacaa aattatttct agaggggaat
     4801 tgttatccgc tcacaattcc cctatagtga gtcgtattaa tttcgcggga tcgagatctc
     4861 gatcctctac gccggacgca tcgtggccgg catcaccggc gccacaggtg cggttgctgg
     4921 cgcctatatc gccgacatca ccgatgggga agatcgggct cgccacttcg ggctcatgag
     4981 cgcttgtttc ggcgtgggta tggtggcagg ccccgtggcc gggggactgt tgggcgccat
     5041 ctccttgcat gcaccattcc ttgcggcggc ggtgctcaac ggcctcaacc tactactggg
     5101 ctgcttccta atgcaggagt cgcataaggg agagcgtcga gatcccggac accatcgaat
     5161 ggcgcaaaac ctttcgcggt atggcatgat agcgcccgga agagagtcaa ttcagggtgg
     5221 tgaatgtgaa accagtaacg ttatacgatg tcgcagagta tgccggtgtc tcttatcaga
     5281 ccgtttcccg cgtggtgaac caggccagcc acgtttctgc gaaaacgcgg gaaaaagtgg
     5341 aagcggcgat ggcggagctg aattacattc ccaaccgcgt ggcacaacaa ctggcgggca
     5401 aacagtcgtt gctgattggc gttgccacct ccagtctggc cctgcacgcg ccgtcgcaaa
     5461 ttgtcgcggc gattaaatct cgcgccgatc aactgggtgc cagcgtggtg gtgtcgatgg
     5521 tagaacgaag cggcgtcgaa gcctgtaaag cggcggtgca caatcttctc gcgcaacgcg
     5581 tcagtgggct gatcattaac tatccgctgg atgaccagga tgccattgct gtggaagctg
     5641 cctgcactaa tgttccggcg ttatttcttg atgtctctga ccagacaccc atcaacagta
     5701 ttattttctc ccatgaagac ggtacgcgac tgggcgtgga gcatctggtc gcattgggtc
     5761 accagcaaat cgcgctgtta gcgggcccat taagttctgt ctcggcgcgt ctgcgtctgg
     5821 ctggctggca taaatatctc actcgcaatc aaattcagcc gatagcggaa cgggaaggcg
     5881 actggagtgc catgtccggt tttcaacaaa ccatgcaaat gctgaatgag ggcatcgttc
     5941 ccactgcgat gctggttgcc aacgatcaga tggcgctggg cgcaatgcgc gccattaccg
     6001 agtccgggct gcgcgttggt gcggatatct cggtagtggg atacgacgat accgaagaca
     6061 gctcatgtta tatcccgccg ttaaccacca tcaaacagga ttttcgcctg ctggggcaaa
     6121 ccagcgtgga ccgcttgctg caactctctc agggccaggc ggtgaagggc aatcagctgt
     6181 tgcccgtctc actggtgaaa agaaaaacca ccctggcgcc caatacgcaa accgcctctc
     6241 cccgcgcgtt ggccgattca ttaatgcagc tggcacgaca ggtttcccga ctggaaagcg
     6301 ggcagtgagc gcaacgcaat taatgtaagt tagctcactc attaggcacc gggatctcga
     6361 ccgatgccct tgagagcctt caacccagtc agctccttcc ggtgggcgcg gggcatgact
     6421 atcgtcgccg cacttatgac tgtcttcttt atcatgcaac tcgtaggaca ggtgccggca
     6481 gcgctctggg tcattttcgg cgaggaccgc tttcgctgga gcgcgacgat gatcggcctg
     6541 tcgcttgcgg tattcggaat cttgcacgcc ctcgctcaag ccttcgtcac tggtcccgcc
     6601 accaaacgtt tcggcgagaa gcaggccatt atcg
//
