LOCUS       sequence-300227-        6851 bp    DNA     circular SYN 14-APR-2025
DEFINITION  Mammalian expression plasmid for SARS-CoV-2 M (membrane).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6851)
  TITLE     SARS-CoV-2 protein Mammalian expression constructs, tagged, for 
            protein expression and/or purification
REFERENCE   2  (bases 1 to 6851)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 14, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"
COMMENT     Sequence Label: pGBW-m4134388
FEATURES             Location/Qualifiers
     source          1..6851
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        2..204
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     155..175
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      273..282
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             945..965
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQS"
     CDS             969..1034
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     polyA_signal    1087..1208
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1098..1117)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     1152..1171
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     terminator      1323..1369
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     terminator      1399..1535
                     /label=rrnG terminator
                     /note="transcription terminator from the E. coli ribosomal 
                     RNA rrnG operon (Albrechtsen et al., 1991)"
     misc_feature    1580..1651
                     /label=3' beta-globin insulator
                     /note="insulator downstream of the human beta-globin locus 
                     (Farrell et al., 2002)"
     rep_origin      complement(1877..2465)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1957..1976)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     promoter        2965..4174
                     /label=UbC promoter
                     /note="human ubiquitin C promoter"
     primer_bind     4011..4030
                     /label=hUBCpro-F
                     /note="Human Ubiquitin C (UbC) promoter, forward primer"
     CDS             4178..4777
                     /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(4178..4197)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     4674..4694
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     polyA_signal    4804..4852
                     /note="synthetic polyadenylation signal"
     CDS             join(5118..5186,5187..5978)
                     /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(5336..5355)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     terminator      5991..6127
                     /label=rrnG terminator
                     /note="transcription terminator from the E. coli ribosomal 
                     RNA rrnG operon (Albrechtsen et al., 1991)"
     misc_feature    6279..6350
                     /label=5' beta-globin insulator
                     /note="insulator upstream of the human beta-globin locus 
                     (Farrell et al., 2002)"
     terminator      complement(6355..6401)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     polyA_signal    6419..6467
                     /note="synthetic polyadenylation signal"
     enhancer        6548..6851
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
ORIGIN
        1 ctgatgcggt tttggcagta catcaatggg cgtggatagc ggtttgactc acggggattt
       61 ccaagtctcc accccattga cgtcaatggg agtttgtttt ggcaccaaaa tcaacgggac
      121 tttccaaaat gtcgtaacaa ctccgcccca ttgacgcaaa tgggcggtag gcgtgtacgg
      181 tgggaggtct atataagcag agctggttta gtgaaccgtc agatcagatc tttgtcgatc
      241 ctaccatcca ctcgacacac ccgccagcgg ccgccaccat ggcggacagc aacggcacta
      301 ttacagtcga agagcttaag aaattactcg agcagtggaa tttggtaata ggattcctgt
      361 ttctaacgtg gatctgtctg ctgcaattcg catatgccaa ccggaataga tttctctaca
      421 tcattaagtt gatcttcctg tggctgcttt ggcctgtgac cttggcttgc tttgttctgg
      481 ccgccgtgta ccgcataaac tggattaccg gtgggatcgc tatcgcaatg gcgtgcctcg
      541 tgggccttat gtggctgtcc tattttatag cctcattccg actctttgct cgtactagga
      601 gtatgtggtc tttcaatccc gaaacaaata ttttactaaa cgtcccgctg catgggacca
      661 ttttaacaag accattgcta gagagcgagc tcgtgatcgg cgcagttata ctgcggggac
      721 acctcaggat cgccggacac catctgggtc gctgtgatat taaagacctg cccaaggaaa
      781 taacagtggc tacctcgcgt actctttcat actataaact gggagccagc cagagagtgg
      841 ccggggattc tggcttcgca gcatatagtc gatacaggat cgggaactac aagctgaata
      901 cggaccactc ctcctctagt gataatattg cattgctcgt acaggaaaac ctttactttc
      961 agtcgatgga ttataaggac cacgatggag actacaaaga tcatgacatt gattacaagg
     1021 acgacgatga caaataagct agcttgactg actgagatac agcgtacctt cagctcacag
     1081 acatgataag atacattgat gagtttggac aaaccacaac tagaatgcag tgaaaaaaat
     1141 gctttatttg tgaaatttgt gatgctattg ctttatttgt aaccattata agctgcaata
     1201 aacaagttaa caacaacaat tgcattcatt ttatgtttca ggttcagggg gaggtgtggg
     1261 aggtttttta aagcaagtaa aacctctaca aatgtggtat tggcccatct ctatcggtat
     1321 cgtagcataa ccccttgggg cctctaaacg ggtcttgagg ggttttttgt gcccctcggg
     1381 ccggattgct atctaccggc attggcgcag aaaaaaatgc ctgatgcgac gctgcgcgtc
     1441 ttatactccc acatatgcca gattcagcaa cggatacggc ttccccaact tgcccacttc
     1501 catacgtgtc ctccttacca gaaatttatc cttaaggtcg tcagctatcc tgcaggcgat
     1561 ctctcgattt cgatcaagac attcctttaa tggtcttttc tggacaccac taggggtcag
     1621 aagtagttca tcaaactttc ttccctccct aatctcattg gttaccttgg gctatcgaaa
     1681 cttaattaac cagtcaagtc agctacttgg cgagatcgac ttgtctgggt ttcgactacg
     1741 ctcagaattg cgtcagtcaa gttcgatctg gtccttgcta ttgcacccgt tctccgatta
     1801 cgagtttcat ttaaatcatg tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg
     1861 ccgcgttgct ggcgtttttc cataggctcc gcccccctga cgagcatcac aaaaatcgac
     1921 gctcaagtca gaggtggcga aacccgacag gactataaag ataccaggcg tttccccctg
     1981 gaagctccct cgtgcgctct cctgttccga ccctgccgct taccggatac ctgtccgcct
     2041 ttctcccttc gggaagcgtg gcgctttctc atagctcacg ctgtaggtat ctcagttcgg
     2101 tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct
     2161 gcgccttatc cggtaactat cgtcttgagt ccaacccggt aagacacgac ttatcgccac
     2221 tggcagcagc cactggtaac aggattagca gagcgaggta tgtaggcggt gctacagagt
     2281 tcttgaagtg gtggcctaac tacggctaca ctagaagaac agtatttggt atctgcgctc
     2341 tgctgaagcc agttaccttc ggaaaaagag ttggtagctc ttgatccggc aaacaaacca
     2401 ccgctggtag cggtggtttt tttgtttgca agcagcagat tacgcgcaga aaaaaaggat
     2461 ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc tcagtggaac gaaaactcac
     2521 gttaagggat tttggtcatg agattatcaa aaaggatctt cacctagatc cttttaaatt
     2581 aaaaatgaag ttttaaatca atctaaagta tatatgagta aacttggtct gacagttacc
     2641 aatgcttaat cagtgaggca cctatctcag cgatctgtct atttcgttca tccatagttg
     2701 catttaaatt tccgaactct ccaaggccct cgtcggaaaa tcttcaaacc tttcgtccga
     2761 tccatcttgc aggctacctc tcgaacgaac tatcgcaagt ctcttggccg gccttgcgcc
     2821 ttggctattg cttggcagcg cctatcgcca ggtattactc caatcccgaa tatccgagat
     2881 cgggatcacc cgagagaagt tcaacctaca tcctcaatcc cgatctatcc gagatccgag
     2941 gaatatcgaa atcggggcgc gcctggcctc cgcgccgggt tttggcgcct cccgcgggcg
     3001 cccccctcgt cacggcgagc gctgccacgt cagacgaagg gcgcaggagc gtcctgatcc
     3061 ttccgcccgg acgctcagga cagcggcccg ctgctcataa gactcggcct tagaacccca
     3121 gtatcagcag aaggacattt taggacggga cttgggtgac tctagggcac tggttttctt
     3181 tccagagagc ggaacaggcg aggaaaagta gtcccttctc ggcgattctg cggagggatc
     3241 tccgtggggc ggtgaacgcc gatgattata taaggacgcg ccgggtgtgg cacagctagt
     3301 tccgtcgcag ccgggatttg ggtcgcggtt cttgtttgtg gatcgctgtg atcgtcactt
     3361 ggtgagtagc gggctgctgg gctggccggg gctttcgtgg ccgccgggcc gctcggtggg
     3421 acggaagcgt gtggagagac cgccaagggc tgtagtctgg gtccgcgagc aaggttgccc
     3481 tgaactgggg gttgggggga gcgcagcaaa atggcggctg ttcccgagtc ttgaatggaa
     3541 gacgcttgtg aggcgggctg tgaggtcgtt gaaacaaggt ggggggcatg gtgggcggca
     3601 agaacccaag gtcttgagcc cttcgctaat gcgggaaagc tcttattcgg gtgagatggg
     3661 ctgggcacca tctggggacc ctgacgtgaa gtttgtcact gactggagaa ctcggtttgt
     3721 cgtctgttgc gggggcggca gttatggcgg tgccgttggg cagtgcaccc gtacctttgg
     3781 gagcgcgcgc cctcgtcgtg tcgtgacgtc acccgttctg ttggcttata atgcagggtg
     3841 gggccacctg ccggtaggtg tgcggtaggc ttttctccgt cgcaggacgc agggttcggg
     3901 cctagggtag gctctcctga atcgacaggc gccggacctc tggtgagggg agggataagt
     3961 gaggcgtcag tttctttggt cggttttatg tacctatctt cttaagtagc tgaagctccg
     4021 gttttgaact atgcgctcgg ggttggcgag tgtgttttgt gaagtttttt aggcaccttt
     4081 tgaaatgtaa tcatttgggt caatatgtaa ttttcagtgt tagacttgta aattgtccgc
     4141 taaattctgg ccgtttttgg cttttttgtt agacaacatg accgagtaca agcccacggt
     4201 gcgcctcgcc acccgcgacg acgtccccag ggccgtacgc accctcgccg ccgcgttcgc
     4261 cgactacccc gccacgcgcc acaccgtcga tccggaccgc cacatcgagc gggtcaccga
     4321 gctgcaagaa ctcttcctca cgcgcgtcgg gctcgacatc ggcaaggtgt gggtcgcgga
     4381 cgacggcgcc gcggtggcgg tctggaccac gccggagagc gtcgaagcgg gggcggtgtt
     4441 cgccgagatc ggcccgcgca tggccgagtt gagcggttcc cggctggccg cgcagcaaca
     4501 gatggagggc ctcctggcgc cgcaccggcc caaggagccc gcgtggttcc tggccaccgt
     4561 cggcgtctcg cccgaccacc agggcaaggg tctgggcagc gccgtcgtgc tccccggagt
     4621 ggaggccgcc gagcgcgccg gggtgcccgc cttcctggag acctccgcgc cccgcaacct
     4681 ccccttctac gagcggctcg gcttcaccgt caccgccgac gtcgaggtgc ccgaaggacc
     4741 gcgcacctgg tgcatgaccc gcaagcccgg tgcctaggct agtatgtaag cctagtctta
     4801 gataataaaa tcgctatcca tcgaagatgg atgtgtgttg gttttttgtg tgtgtaacgc
     4861 taggcgcgcc tggtgtaccg agaacgatcc tctcagtgcg agtctcgacg atccatatcg
     4921 ttgcttggca gtcagccagt cggaatccag cttgggaccc aggaagtcca atcgtcagat
     4981 attgtactca agcctggtca cggcagcgta ccgatctgtt taaacctaga tattgatagt
     5041 ctgatcggtc aacgtataat cgagtcctag cttttgcaaa catctatcaa gagacaggat
     5101 cagcaggagg ctttcgcatg agtattcaac atttccgtgt cgcccttatt cccttttttg
     5161 cggcattttg ccttcctgtt tttgctcacc cagaaacgct ggtgaaagta aaagatgctg
     5221 aagatcagtt gggtgcgcga gtgggttaca tcgaactgga tctcaacagc ggtaagatcc
     5281 ttgagagttt tcgccccgaa gaacgctttc caatgatgag cacttttaaa gttctgctat
     5341 gtggcgcggt attatcccgt attgacgccg ggcaagagca actcggtcgc cgcatacact
     5401 attctcagaa tgacttggtt gagtattcac cagtcacaga aaagcatctt acggatggca
     5461 tgacagtaag agaattatgc agtgctgcca taaccatgag tgataacact gcggccaact
     5521 tacttctgac aacgattgga ggaccgaagg agctaaccgc ttttttgcac aacatggggg
     5581 atcatgtaac tcgccttgat cgttgggaac cggagctgaa tgaagccata ccaaacgacg
     5641 agcgtgacac cacgatgcct gtagcaatgg caacaacctt gcgtaaacta ttaactggcg
     5701 aactacttac tctagcttcc cggcaacagt tgatagactg gatggaggcg gataaagttg
     5761 caggaccact tctgcgctcg gcccttccgg ctggctggtt tattgctgat aaatctggag
     5821 ccggtgagcg tgggtctcgc ggtatcattg cagcactggg gccagatggt aagccctccc
     5881 gtatcgtagt tatctacacg acggggagtc aggcaactat ggatgaacga aatagacaga
     5941 tcgctgagat aggtgcctca ctgattaagc attggtaacc gattctaggt gcattggcgc
     6001 agaaaaaaat gcctgatgcg acgctgcgcg tcttatactc ccacatatgc cagattcagc
     6061 aacggatacg gcttccccaa cttgcccact tccatacgtg tcctccttac cagaaattta
     6121 tccttaagat cccgaatcgt ttaaactcga ctctggctct atcgaatctc cgtcgtttcg
     6181 agcttacgcg aacagccgtg gcgctcattt gctcgtcggg catcgaatct cgtcagctat
     6241 cgtcagctta cctttttggc agcgatcgcg gctcccgaca tcttggacca ttagctccac
     6301 aggtatcttc ttccctctag tggtcataac agcagcttca gctacctctc aattcaaaaa
     6361 acccctcaag acccgtttag aggccccaag gggttatgct atcaatcgtt gcgttacaca
     6421 cacaaaaaac caacacacat ccatcttcga tggatagcga ttttattatc taactgctga
     6481 tcgagtgtag ccagatctag taatcaatta cggggtcatt agttcatagc ccatatatgg
     6541 agttccgcgt tacataactt acggtaaatg gcccgcctgg ctgaccgccc aacgaccccc
     6601 gcccattgac gtcaataatg acgtatgttc ccatagtaac gccaataggg actttccatt
     6661 gacgtcaatg ggtggagtat ttacggtaaa ctgcccactt ggcagtacat caagtgtatc
     6721 atatgccaag tacgccccct attgacgtca atgacggtaa atggcccgcc tggcattatg
     6781 cccagtacat gaccttatgg gactttccta cttggcagta catctacgta ttagtcatcg
     6841 ctattaccat g
//
