LOCUS       Exported                6348 bp ds-DNA     circular SYN 13-MAY-2021
DEFINITION  Yeast Expression plasmid for SARS-CoV-2 nsp4.
ACCESSION   .
VERSION     .
KEYWORDS    pGBW-m4046460
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6348)
  TITLE     SARS-CoV-2 protein Yeast expression constructs, tagged, for protein 
            expression and/or purification
REFERENCE   2  (bases 1 to 6348)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 13, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..6348
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     promoter        25..466
                     /gene="S. cerevisiae GAL1"
                     /label=GAL1 promoter
                     /note="inducible promoter, regulated by Gal4"
     protein_bind    25..142
                     /label=UAS
                     /bound_moiety="Gal4"
                     /note="upstream activating sequence mediating 
                     Gal4-dependent induction"
     primer_bind     437..460
                     /label=GAL1
                     /note="S. cerevisiae GAL1 promoter, forward primer"
     CDS             1975..1992
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             1993..2058
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     primer_bind     complement(2103..2121)
                     /label=CYC1
                     /note="CYC1 transcription termination signal, reverse 
                     primer"
     CDS             2472..3275
                     /codon_start=1
                     /gene="S. cerevisiae URA3"
                     /product="orotidine-5'-phosphate decarboxylase, required 
                     for uracil biosynthesis"
                     /label=URA3
                     /note="yeast auxotrophic marker, counterselectable with 
                     5-fluoroorotic acid (5-FOA)"
                     /translation="MSKATYKERAATHPSPVAAKLFNIMHEKQTNLCASLDVRTTKELL
                     ELVEALGPKICLLKTHVDILTDFSMEGTVKPLKALSAKYNFLLFEDRKFADIGNTVKLQ
                     YSAGVYRIAEWADITNAHGVVGPGIVSGLKQAAEEVTKEPRGLLMLAELSCKGSLSTGE
                     YTKGTVDIAKSDKDFVIGFIAQRDMGGRDEGYDWLIMTPGVGLDDKGDALGQQYRTVDD
                     VVSTGSDIIIVGRGLFAKGRDAKVEGERYRKAGWEAYLRRCGQQN"
     rep_origin      3358..4340
                     /label=2u ori
                     /note="yeast 2u plasmid origin of replication"
     terminator      4418..4447
                     /label=T3Te terminator
                     /note="phage T3 early transcription terminator"
     rep_origin      4469..5056
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     terminator      5068..5095
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     CDS             complement(5119..5310)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(5311..6171)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESLRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLATIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGSLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     5934..5953
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6172..6274)
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     terminator      6275..6306
                     /label=tonB terminator
                     /note="bidirectional E. coli tonB-P14 transcription 
                     terminator"
ORIGIN
        1 gcggccgctt gctatattga agtacggatt agaagccgcc gagcgggcga cagccctccg
       61 acggaagact ctcctccgtg cgtcctcgtc ttcaccggtc gcgttcctga aacgcagatg
      121 tgcctcgcgc cgcactgctc cgaacaataa agattctaca atactagctt ttatggttat
      181 gaagaggaaa aattggcagt aacctggccc cacaaacctt caaattaacg aatcaaatta
      241 acaaccatag gatgataatg cgattagttt tttagcctta tttctggggt aattaatcag
      301 cgaagcgatg atttttgatc tattaacaga tatataaatg gaaaagctgc ataaccactt
      361 taactaatac tttcaacatt ttcagtttgt attacttctt attcaaatgt cataaaagta
      421 tcaacaaaaa attgttaata tacctctata ctttaacgtc aaggagaaaa aactataatg
      481 attgttaata attggttgaa gcagttaatt aaagttacac ttgtgttcct ttttgttgct
      541 gctattttct atttaataac acctgttcat gtcatgtcta aacatactga cttttcaagt
      601 gaaatcatag gatacaaggc tattgatggt ggtgtcactc gtgacatagc atctacagat
      661 acttgttttg ctaacaaaca tgctgatttt gacacatggt ttagccagcg tggtggtagt
      721 tatactaatg acaaagcttg cccattgatt gctgcagtca taacaagaga agtgggtttt
      781 gtcgtgcctg gtttgcctgg cacgatatta cgcacaacta atggtgactt tttgcatttc
      841 ttacctagag tttttagtgc agttggtaac atctgttaca caccatcaaa acttatagag
      901 tacactgact ttgcaacatc agcttgtgtt ttggctgctg aatgtacaat ttttaaagat
      961 gcttctggta agccagtacc atattgttat gataccaatg tactagaagg ttctgttgct
     1021 tatgaaagtt tacgccctga cacacgttat gtgctcatgg atggctctat tattcaattt
     1081 cctaacacct accttgaagg ttctgttaga gtggtaacaa cttttgattc tgagtactgt
     1141 aggcacggca cttgtgaaag atcagaagct ggtgtttgtg tatctactag tggtagatgg
     1201 gtacttaaca atgattatta cagatcttta ccaggagttt tctgtggtgt agatgctgta
     1261 aatttactta ctaatatgtt tacaccacta attcaaccta ttggtgcttt ggacatatca
     1321 gcatctatag tagctggtgg tattgtagct atcgtagtaa catgccttgc ctactatttt
     1381 atgaggttta gaagagcttt tggtgaatac agtcatgtag ttgcctttaa tactttacta
     1441 ttccttatgt cattcactgt actctgttta acaccagttt actcattctt acctggtgtt
     1501 tattctgtta tttacttgta cttgacattt tatcttacta atgatgtttc ttttttagca
     1561 catattcagt ggatggttat gttcacacct ttagtacctt tctggataac aattgcttat
     1621 atcatttgta tttccacaaa gcatttctat tggttcttta gtaattacct aaagagacgt
     1681 gtagtcttta atggtgtttc ctttagtact tttgaagaag ctgcgctgtg cacctttttg
     1741 ttaaataaag aaatgtatct aaagttgcgt agtgatgtgc tattacctct tacgcaatat
     1801 aatagatact tagctcttta taataagtac aagtatttta gtggagcaat ggatacaact
     1861 agctacagag aagctgcttg ttgtcatctc gcaaaggctc tcaatgactt cagtaactca
     1921 ggttctgatg ttctttacca accaccacaa acctctatca cctcagctgt tttgctcgtg
     1981 ccgagaggtt ctgattataa agatcatgat ggtgattata aagatcatga tattgattat
     2041 aaagatgatg atgataaata aacaggcccc ttttcctttg tcgatatcat gtaattagtt
     2101 atgtcacgct tacattcacg ccctcctccc acatccgctc taaccgaaaa ggaaggagtt
     2161 agacaacctg aagtctaggt ccctatttat tttttttaat agttatgtta gtattaagaa
     2221 cgttatttat atttcaaatt tttctttttt ttctgtacaa agcggccgcc gaaccactag
     2281 tgcactgcag tacacggttt ccttgaaatt tttttgattc ggtaatctcc gaacagaagg
     2341 aagaacgaag gaaggagcac agacttagat tggtatatat acgcatatgt agtgttgaag
     2401 aaacatgaaa ttgcccagta ttcttaaccc aactgcacag aacaaaaacg tgcaggaaac
     2461 gaagataaat catgtcgaaa gctacatata aggaacgtgc tgctactcat cctagtcctg
     2521 ttgctgccaa gctatttaat atcatgcacg aaaagcaaac aaacttgtgt gcttcattgg
     2581 atgttcgtac caccaaggaa ttactggagt tagttgaagc attaggtccc aaaatttgtt
     2641 tactaaaaac acatgtggat atcttgactg atttttccat ggagggcaca gttaagccgc
     2701 taaaggcatt atccgccaag tacaattttt tactcttcga ggacagaaaa tttgctgaca
     2761 ttggtaatac agtcaaattg cagtactctg cgggtgtata cagaatagca gaatgggcag
     2821 acattacgaa tgcacacggt gtggtgggcc caggtattgt tagcggtttg aagcaggcgg
     2881 cagaagaagt aacaaaggaa cctagaggcc ttttgatgtt agcagaattg tcatgcaagg
     2941 gctccctatc tactggagaa tatactaagg gtactgttga cattgcgaag agcgacaaag
     3001 attttgttat cggctttatt gctcaaagag acatgggtgg aagagatgaa ggttacgatt
     3061 ggttgattat gacacccggt gtgggtttag atgacaaggg agatgcattg ggtcaacagt
     3121 atagaaccgt ggatgatgtg gtttctacag gatctgacat tattattgtt ggaagaggac
     3181 tatttgcaaa gggaagggat gctaaggtag agggtgaacg ttacagaaaa gcaggctggg
     3241 aagcatattt gagaagatgc ggccagcaaa actaaaaaac tgtattataa gtaaatgcat
     3301 gtatactaaa ctcacaaatt agagcttcaa tttaattata tcagttatta cccgagtaac
     3361 gaagcatctg tgcttcattt tgtagaacaa aaatgcaacg cgagagcgct aatttttcaa
     3421 acaaagaatc tgagctgcat ttttacagaa cagaaatgca acgcgaaagc gctattttac
     3481 caacgaagaa tctgtgcttc atttttgtaa aacaaaaatg caacgcgaga gcgctaattt
     3541 ttcaaacaaa gaatctgagc tgcattttta cagaacagaa atgcaacgcg agagcgctat
     3601 tttaccaaca aagaatctat acttcttttt tgttctacaa aaatgcatcc cgagagcgct
     3661 atttttctaa caaagcatct tagattactt tttttctcct ttgtgcgctc tataatgcag
     3721 tctcttgata actttttgca ctgtaggtcc gttaaggtta gaagaaggct actttggtgt
     3781 ctattttctc ttccataaaa aaagcctgac tccacttccc gcgtttactg attactagcg
     3841 aagctgcggg tgcatttttt caagataaag gcatccccga ttatattcta taccgatgtg
     3901 gattgcgcat actttgtgaa cagaaagtga tagcgttgat gattcttcat tggtcagaaa
     3961 attatgaacg gtttcttcta ttttgtctct atatactacg tataggaaat gtttacattt
     4021 tcgtattgtt ttcgattcac tctatgaata gttcttacta caattttttt gtctaaagag
     4081 taatactaga gataaacata aaaaatgtag aggtcgagtt tagatgcaag ttcaaggagc
     4141 gaaaggtgga tgggtaggtt atatagggat atagcacaga gatatatagc aaagagatac
     4201 ttttgagcaa tgtttgtgga agcggtattc gcaatatttt agtagctcgt tacagtccgg
     4261 tgcgtttttg gttttttgaa agtgcgtcat cagagcgctt ttggttttca aaagcgctct
     4321 gaagttccta tactttctag ctagagaata ggaacttccc gagcggccgc gtgttacaag
     4381 cttgactcca gcgtaactgg actgcaatca actcactggc tcaccttcac gggtgggcct
     4441 ttcttcggta gaaaatcaaa ggatcttctt gagatccttt ttttctgcgc gtaatctgct
     4501 gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg tttgccggat caagagctac
     4561 caactctttt tccgaggtaa ctggcttcag cagagcgcag ataccaaata ctgttcttct
     4621 agtgtagccg tagttaggcc accacttcaa gaactctgta gcaccgccta catacctcgc
     4681 tctgctaatc ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt
     4741 ggactcaaga cgatagttac cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg
     4801 cacacagccc agcttggagc gaacgaccta caccgaactg agatacctac agcgtgagct
     4861 atgagaaagc gccacgcttc ccgaagggag aaaggcggac aggtatccgg taagcggcag
     4921 ggtcggaaca ggagagcgca cgagggagct tccaggggta aacgcctggt atctttatag
     4981 tcctgtcggg tttcgccacc tctgacttga gcatcgattt ttgtgatgct cgtcaggggg
     5041 gcggagccta tggaaaaacg ccagcaacgc agaaaggccc acccgaaggt gagccaggtg
     5101 attacatttg ggccctcatc agaggttttc accgtcatca ccgaaacgcg cgaggcagct
     5161 gcggtaaagc tcatcagcgt ggtcgtgaag cgattcacag atgtctgcct gttcatccgc
     5221 gtccagctcg ttgagtttct ccagaagcgt taatgtctgg cttctgataa agcgggccat
     5281 gttaagggcg gttttttcct gtttggtcac ttaccaatgc ttaatcagtg aggcacctat
     5341 ctcagcgatc tgtctatttc gttcatccat agttgcctga ctccccgtcg tgtagataac
     5401 tacgatgcgg gagggcttac catctggccc cagtgctgca atgataccgc gagacccacg
     5461 ctcaccggct ccagatttat cagcaataaa ccagccagcc gggagggccg agcgcagaag
     5521 tgatcctgca actttatccg cctccatcca gtctattaat tgttgccggg aagctagagt
     5581 aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt
     5641 gtcacgctcg tcgtttggta tggcttcatt cagctccggt tcccaacgat caaggcgagt
     5701 tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgc
     5761 cagaagtaag ttggccgcag tgttatcact catggttatg gcagcactgc ataattctct
     5821 tactgtcatg ccatccgtga gatgcttttc tgtgactggt gagtactcaa ccaagtcatt
     5881 ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac gggataatac
     5941 cgcgccacat agcagaactt taaaagtgct catcattgga aaacgttctt cggggcgtaa
     6001 actctcaagg atcttaccgc tgttgagatc cagttcgatg taacccactc gtgcacccaa
     6061 ctgatcttca gcatctttta ctttcaccag cgtttctggg tgagcaaaaa caggaaggca
     6121 aaatgccgca aaaaagggaa taagggcgac acggaaatgt tgaatactca ttttagcttc
     6181 cttagctcct gaaaatctcg ataactcaaa aaatacgccc ggtagtgatc ttatttcatt
     6241 atggtgaaag ttggaacctc ttacgtgccg atcaagtcaa aagcctccgg tcggaggctt
     6301 ttgactttct gctatggagg tcaggtatga tttaaatggt cagttaga
//
