LOCUS       sequence-295240-        7633 bp    DNA     circular SYN 15-APR-2025
DEFINITION  Mammalian expression plasmid for SARS-CoV-2 nsp4.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7633)
  TITLE     SARS-CoV-2 protein Mammalian expression constructs, tagged, for 
            protein expression and/or purification
REFERENCE   2  (bases 1 to 7633)
  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"
COMMENT     Sequence Label: pGBW-m4133898
FEATURES             Location/Qualifiers
     source          1..7633
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             1502..1522
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQS"
     CDS             1523..1549
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     polyA_signal    1602..1723
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1613..1632)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     1667..1686
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     terminator      1838..1884
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     terminator      1914..2050
                     /label=rrnG terminator
                     /note="transcription terminator from the E. coli ribosomal 
                     RNA rrnG operon (Albrechtsen et al., 1991)"
     misc_feature    2095..2166
                     /label=3' beta-globin insulator
                     /note="insulator downstream of the human beta-globin locus 
                     (Farrell et al., 2002)"
     rep_origin      complement(2392..2980)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2472..2491)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     promoter        3480..4689
                     /label=UbC promoter
                     /note="human ubiquitin C promoter"
     primer_bind     4526..4545
                     /label=hUBCpro-F
                     /note="Human Ubiquitin C (UbC) promoter, forward primer"
     CDS             4693..5292
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
                     VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
                     AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
                     APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     primer_bind     complement(4693..4712)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     5189..5209
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     polyA_signal    5319..5367
                     /note="synthetic polyadenylation signal"
     CDS             join(5633..5701,5702..6493)
                     /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(5851..5870)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     terminator      6506..6642
                     /label=rrnG terminator
                     /note="transcription terminator from the E. coli ribosomal 
                     RNA rrnG operon (Albrechtsen et al., 1991)"
     misc_feature    6787..6858
                     /label=5' beta-globin insulator
                     /note="insulator upstream of the human beta-globin locus 
                     (Farrell et al., 2002)"
     terminator      complement(6863..6909)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     polyA_signal    6927..6975
                     /note="synthetic polyadenylation signal"
     enhancer        7056..7359
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        7361..7563
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     7514..7534
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
ORIGIN
        1 caccatgatt gttaataatt ggttgaagca gttaattaaa gttacacttg tgttcctttt
       61 tgttgctgct attttctatt taataacacc tgttcatgtc atgtctaaac atactgactt
      121 ttcaagtgaa atcataggat acaaggctat tgatggtggt gtcactcgtg acatagcatc
      181 tacagatact tgttttgcta acaaacatgc tgattttgac acatggttta gccagcgtgg
      241 tggtagttat actaatgaca aagcttgccc attgattgct gcagtcataa caagagaagt
      301 gggttttgtc gtgcctggtt tgcctggcac gatattacgc acaactaatg gtgacttttt
      361 gcatttctta cctagagttt ttagtgcagt tggtaacatc tgttacacac catcaaaact
      421 tatagagtac actgactttg caacatcagc ttgtgttttg gctgctgaat gtacaatttt
      481 taaagatgct tctggtaagc cagtaccata ttgttatgat accaatgtac tagaaggttc
      541 tgttgcttat gaaagtttac gccctgacac acgttatgtg ctcatggatg gctctattat
      601 tcaatttcct aacacctacc ttgaaggttc tgttagagtg gtaacaactt ttgattctga
      661 gtactgtagg cacggcactt gtgaaagatc agaagctggt gtttgtgtat ctactagtgg
      721 tagatgggta cttaacaatg attattacag atctttacca ggagttttct gtggtgtaga
      781 tgctgtaaat ttacttacta atatgtttac accactaatt caacctattg gtgctttgga
      841 catatcagca tctatagtag ctggtggtat tgtagctatc gtagtaacat gccttgccta
      901 ctattttatg aggtttagaa gagcttttgg tgaatacagt catgtagttg cctttaatac
      961 tttactattc cttatgtcat tcactgtact ctgtttaaca ccagtttact cattcttacc
     1021 tggtgtttat tctgttattt acttgtactt gacattttat cttactaatg atgtttcttt
     1081 tttagcacat attcagtgga tggttatgtt cacaccttta gtacctttct ggataacaat
     1141 tgcttatatc atttgtattt ccacaaagca tttctattgg ttctttagta attacctaaa
     1201 gagacgtgta gtctttaatg gtgtttcctt tagtactttt gaagaagctg cgctgtgcac
     1261 ctttttgtta aataaagaaa tgtatctaaa gttgcgtagt gatgtgctat tacctcttac
     1321 gcaatataat agatacttag ctctttataa taagtacaag tattttagtg gagcaatgga
     1381 tacaactagc tacagagaag ctgcttgttg tcatctcgca aaggctctca atgacttcag
     1441 taactcaggt tctgatgttc tttaccaacc accacaaacc tctatcacct cagctgtttt
     1501 ggaaaacctt tactttcagt cgtaccccta tgacgtacca gattatgcgt aagctagctt
     1561 gactgactga gatacagcgt accttcagct cacagacatg ataagataca ttgatgagtt
     1621 tggacaaacc acaactagaa tgcagtgaaa aaaatgcttt atttgtgaaa tttgtgatgc
     1681 tattgcttta tttgtaacca ttataagctg caataaacaa gttaacaaca acaattgcat
     1741 tcattttatg tttcaggttc agggggaggt gtgggaggtt ttttaaagca agtaaaacct
     1801 ctacaaatgt ggtattggcc catctctatc ggtatcgtag cataacccct tggggcctct
     1861 aaacgggtct tgaggggttt tttgtgcccc tcgggccgga ttgctatcta ccggcattgg
     1921 cgcagaaaaa aatgcctgat gcgacgctgc gcgtcttata ctcccacata tgccagattc
     1981 agcaacggat acggcttccc caacttgccc acttccatac gtgtcctcct taccagaaat
     2041 ttatccttaa ggtcgtcagc tatcctgcag gcgatctctc gatttcgatc aagacattcc
     2101 tttaatggtc ttttctggac accactaggg gtcagaagta gttcatcaaa ctttcttccc
     2161 tccctaatct cattggttac cttgggctat cgaaacttaa ttaaccagtc aagtcagcta
     2221 cttggcgaga tcgacttgtc tgggtttcga ctacgctcag aattgcgtca gtcaagttcg
     2281 atctggtcct tgctattgca cccgttctcc gattacgagt ttcatttaaa tcatgtgagc
     2341 aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag
     2401 gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc
     2461 gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt
     2521 tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct
     2581 ttctcatagc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg
     2641 ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta actatcgtct
     2701 tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg gtaacaggat
     2761 tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggc ctaactacgg
     2821 ctacactaga agaacagtat ttggtatctg cgctctgctg aagccagtta ccttcggaaa
     2881 aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt
     2941 ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc
     3001 tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt
     3061 atcaaaaagg atcttcacct agatcctttt aaattaaaaa tgaagtttta aatcaatcta
     3121 aagtatatat gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg aggcacctat
     3181 ctcagcgatc tgtctatttc gttcatccat agttgcattt aaatttccga actctccaag
     3241 gccctcgtcg gaaaatcttc aaacctttcg tccgatccat cttgcaggct acctctcgaa
     3301 cgaactatcg caagtctctt ggccggcctt gcgccttggc tattgcttgg cagcgcctat
     3361 cgccaggtat tactccaatc ccgaatatcc gagatcggga tcacccgaga gaagttcaac
     3421 ctacatcctc aatcccgatc tatccgagat ccgaggaata tcgaaatcgg ggcgcgcctg
     3481 gcctccgcgc cgggttttgg cgcctcccgc gggcgccccc ctcgtcacgg cgagcgctgc
     3541 cacgtcagac gaagggcgca ggagcgtcct gatccttccg cccggacgct caggacagcg
     3601 gcccgctgct cataagactc ggccttagaa ccccagtatc agcagaagga cattttagga
     3661 cgggacttgg gtgactctag ggcactggtt ttctttccag agagcggaac aggcgaggaa
     3721 aagtagtccc ttctcggcga ttctgcggag ggatctccgt ggggcggtga acgccgatga
     3781 ttatataagg acgcgccggg tgtggcacag ctagttccgt cgcagccggg atttgggtcg
     3841 cggttcttgt ttgtggatcg ctgtgatcgt cacttggtga gtagcgggct gctgggctgg
     3901 ccggggcttt cgtggccgcc gggccgctcg gtgggacgga agcgtgtgga gagaccgcca
     3961 agggctgtag tctgggtccg cgagcaaggt tgccctgaac tgggggttgg ggggagcgca
     4021 gcaaaatggc ggctgttccc gagtcttgaa tggaagacgc ttgtgaggcg ggctgtgagg
     4081 tcgttgaaac aaggtggggg gcatggtggg cggcaagaac ccaaggtctt gagcccttcg
     4141 ctaatgcggg aaagctctta ttcgggtgag atgggctggg caccatctgg ggaccctgac
     4201 gtgaagtttg tcactgactg gagaactcgg tttgtcgtct gttgcggggg cggcagttat
     4261 ggcggtgccg ttgggcagtg cacccgtacc tttgggagcg cgcgccctcg tcgtgtcgtg
     4321 acgtcacccg ttctgttggc ttataatgca gggtggggcc acctgccggt aggtgtgcgg
     4381 taggcttttc tccgtcgcag gacgcagggt tcgggcctag ggtaggctct cctgaatcga
     4441 caggcgccgg acctctggtg aggggaggga taagtgaggc gtcagtttct ttggtcggtt
     4501 ttatgtacct atcttcttaa gtagctgaag ctccggtttt gaactatgcg ctcggggttg
     4561 gcgagtgtgt tttgtgaagt tttttaggca ccttttgaaa tgtaatcatt tgggtcaata
     4621 tgtaattttc agtgttagac ttgtaaattg tccgctaaat tctggccgtt tttggctttt
     4681 ttgttagaca acatgaccga gtacaagccc acggtgcgcc tcgccacccg cgacgacgtc
     4741 cccagggccg tacgcaccct cgccgccgcg ttcgccgact accccgccac gcgccacacc
     4801 gtcgatccgg accgccacat cgagcgggtc accgagctgc aagaactctt cctcacgcgc
     4861 gtcgggctcg acatcggcaa ggtgtgggtc gcggacgacg gcgccgcggt ggcggtctgg
     4921 accacgccgg agagcgtcga agcgggggcg gtgttcgccg agatcggccc gcgcatggcc
     4981 gagttgagcg gttcccggct ggccgcgcag caacagatgg agggcctcct ggcgccgcac
     5041 cggcccaagg agcccgcgtg gttcctggcc accgtcggcg tctcgcccga ccaccagggc
     5101 aagggtctgg gcagcgccgt cgtgctcccc ggagtggagg ccgccgagcg cgccggggtg
     5161 cccgccttcc tggagacctc cgcgccccgc aacctcccct tctacgagcg gctcggcttc
     5221 accgtcaccg ccgacgtcga ggtgcccgaa ggaccgcgca cctggtgcat gacccgcaag
     5281 cccggtgcct aggctagtat gtaagcctag tcttagataa taaaatcgct atccatcgaa
     5341 gatggatgtg tgttggtttt ttgtgtgtgt aacgctaggc gcgcctggtg taccgagaac
     5401 gatcctctca gtgcgagtct cgacgatcca tatcgttgct tggcagtcag ccagtcggaa
     5461 tccagcttgg gacccaggaa gtccaatcgt cagatattgt actcaagcct ggtcacggca
     5521 gcgtaccgat ctgtttaaac ctagatattg atagtctgat cggtcaacgt ataatcgagt
     5581 cctagctttt gcaaacatct atcaagagac aggatcagca ggaggctttc gcatgagtat
     5641 tcaacatttc cgtgtcgccc ttattccctt ttttgcggca ttttgccttc ctgtttttgc
     5701 tcacccagaa acgctggtga aagtaaaaga tgctgaagat cagttgggtg cgcgagtggg
     5761 ttacatcgaa ctggatctca acagcggtaa gatccttgag agttttcgcc ccgaagaacg
     5821 ctttccaatg atgagcactt ttaaagttct gctatgtggc gcggtattat cccgtattga
     5881 cgccgggcaa gagcaactcg gtcgccgcat acactattct cagaatgact tggttgagta
     5941 ttcaccagtc acagaaaagc atcttacgga tggcatgaca gtaagagaat tatgcagtgc
     6001 tgccataacc atgagtgata acactgcggc caacttactt ctgacaacga ttggaggacc
     6061 gaaggagcta accgcttttt tgcacaacat gggggatcat gtaactcgcc ttgatcgttg
     6121 ggaaccggag ctgaatgaag ccataccaaa cgacgagcgt gacaccacga tgcctgtagc
     6181 aatggcaaca accttgcgta aactattaac tggcgaacta cttactctag cttcccggca
     6241 acagttgata gactggatgg aggcggataa agttgcagga ccacttctgc gctcggccct
     6301 tccggctggc tggtttattg ctgataaatc tggagccggt gagcgtgggt ctcgcggtat
     6361 cattgcagca ctggggccag atggtaagcc ctcccgtatc gtagttatct acacgacggg
     6421 gagtcaggca actatggatg aacgaaatag acagatcgct gagataggtg cctcactgat
     6481 taagcattgg taaccgattc taggtgcatt ggcgcagaaa aaaatgcctg atgcgacgct
     6541 gcgcgtctta tactcccaca tatgccagat tcagcaacgg atacggcttc cccaacttgc
     6601 ccacttccat acgtgtcctc cttaccagaa atttatcctt aagatcgttt aaactcgact
     6661 ctggctctat cgaatctccg tcgtttcgag cttacgcgaa cagccgtggc gctcatttgc
     6721 tcgtcgggca tcgaatctcg tcagctatcg tcagcttacc tttttggcag cgatcgcggc
     6781 tcccgacatc ttggaccatt agctccacag gtatcttctt ccctctagtg gtcataacag
     6841 cagcttcagc tacctctcaa ttcaaaaaac ccctcaagac ccgtttagag gccccaaggg
     6901 gttatgctat caatcgttgc gttacacaca caaaaaacca acacacatcc atcttcgatg
     6961 gatagcgatt ttattatcta actgctgatc gagtgtagcc agatctagta atcaattacg
     7021 gggtcattag ttcatagccc atatatggag ttccgcgtta cataacttac ggtaaatggc
     7081 ccgcctggct gaccgcccaa cgacccccgc ccattgacgt caataatgac gtatgttccc
     7141 atagtaacgc caatagggac tttccattga cgtcaatggg tggagtattt acggtaaact
     7201 gcccacttgg cagtacatca agtgtatcat atgccaagta cgccccctat tgacgtcaat
     7261 gacggtaaat ggcccgcctg gcattatgcc cagtacatga ccttatggga ctttcctact
     7321 tggcagtaca tctacgtatt agtcatcgct attaccatgc tgatgcggtt ttggcagtac
     7381 atcaatgggc gtggatagcg gtttgactca cggggatttc caagtctcca ccccattgac
     7441 gtcaatggga gtttgttttg gcaccaaaat caacgggact ttccaaaatg tcgtaacaac
     7501 tccgccccat tgacgcaaat gggcggtagg cgtgtacggt gggaggtcta tataagcaga
     7561 gctggtttag tgaaccgtca gatcagatct ttgtcgatcc taccatccac tcgacacacc
     7621 cgccagcggc cgc
//
