LOCUS       sequence-300165-        6419 bp    DNA     circular SYN 15-APR-2025
DEFINITION  Mammalian expression plasmid for SARS-CoV-2 E (envelope).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6419)
  TITLE     SARS-CoV-2 protein Mammalian expression constructs, tagged, for 
            protein expression and/or purification
REFERENCE   2  (bases 1 to 6419)
  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-m4252858
FEATURES             Location/Qualifiers
     source          1..6419
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             230..250
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQS"
     CDS             251..328
                     /codon_start=1
                     /product="calmodulin-binding peptide"
                     /label=CBP
                     /note="derived from skeletal muscle myosin light chain 
                     kinase; binds calmodulin with nanomolar affinity in the 
                     presence of calcium"
                     /translation="KRRWKKNFIAVSAANRFKKISSSGAL"
     polyA_signal    381..502
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(392..411)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     446..465
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     terminator      617..663
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     terminator      693..829
                     /label=rrnG terminator
                     /note="transcription terminator from the E. coli ribosomal 
                     RNA rrnG operon (Albrechtsen et al., 1991)"
     misc_feature    874..945
                     /label=3' beta-globin insulator
                     /note="insulator downstream of the human beta-globin locus 
                     (Farrell et al., 2002)"
     rep_origin      complement(1171..1759)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1251..1270)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     promoter        2259..3468
                     /label=UbC promoter
                     /note="human ubiquitin C promoter"
     primer_bind     3305..3324
                     /label=hUBCpro-F
                     /note="Human Ubiquitin C (UbC) promoter, forward primer"
     CDS             3472..4071
                     /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(3472..3491)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     3968..3988
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     polyA_signal    4098..4146
                     /note="synthetic polyadenylation signal"
     CDS             join(4412..4480,4481..5272)
                     /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(4630..4649)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     terminator      5285..5421
                     /label=rrnG terminator
                     /note="transcription terminator from the E. coli ribosomal 
                     RNA rrnG operon (Albrechtsen et al., 1991)"
     misc_feature    5573..5644
                     /label=5' beta-globin insulator
                     /note="insulator upstream of the human beta-globin locus 
                     (Farrell et al., 2002)"
     terminator      complement(5649..5695)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     polyA_signal    5713..5761
                     /note="synthetic polyadenylation signal"
     enhancer        5842..6145
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        6147..6349
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     6300..6320
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
ORIGIN
        1 caccatgtac agctttgtat ctgaagagac aggcactctc atcgttaatt cagtgttgct
       61 gttcctggca tttgtggtct tccttttagt gaccctagcg atactgacgg ccctccggct
      121 gtgcgcttat tgttgtaaca ttgtcaacgt tagtctcgtg aagccctcgt tctacgttta
      181 ttcccgcgtg aaaaatctaa acagctcccg tgtgcctgat cttcttgtcg aaaaccttta
      241 ctttcagtcg aagaggcgat ggaaaaagaa ttttatcgcg gtgagtgcag ccaacagatt
      301 caaaaagata tcctcgtctg gtgccctcta agctagcttg actgactgag atacagcgta
      361 ccttcagctc acagacatga taagatacat tgatgagttt ggacaaacca caactagaat
      421 gcagtgaaaa aaatgcttta tttgtgaaat ttgtgatgct attgctttat ttgtaaccat
      481 tataagctgc aataaacaag ttaacaacaa caattgcatt cattttatgt ttcaggttca
      541 gggggaggtg tgggaggttt tttaaagcaa gtaaaacctc tacaaatgtg gtattggccc
      601 atctctatcg gtatcgtagc ataacccctt ggggcctcta aacgggtctt gaggggtttt
      661 ttgtgcccct cgggccggat tgctatctac cggcattggc gcagaaaaaa atgcctgatg
      721 cgacgctgcg cgtcttatac tcccacatat gccagattca gcaacggata cggcttcccc
      781 aacttgccca cttccatacg tgtcctcctt accagaaatt tatccttaag gtcgtcagct
      841 atcctgcagg cgatctctcg atttcgatca agacattcct ttaatggtct tttctggaca
      901 ccactagggg tcagaagtag ttcatcaaac tttcttccct ccctaatctc attggttacc
      961 ttgggctatc gaaacttaat taaccagtca agtcagctac ttggcgagat cgacttgtct
     1021 gggtttcgac tacgctcaga attgcgtcag tcaagttcga tctggtcctt gctattgcac
     1081 ccgttctccg attacgagtt tcatttaaat catgtgagca aaaggccagc aaaaggccag
     1141 gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg ctccgccccc ctgacgagca
     1201 tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg acaggactat aaagatacca
     1261 ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg
     1321 atacctgtcc gcctttctcc cttcgggaag cgtggcgctt tctcatagct cacgctgtag
     1381 gtatctcagt tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt
     1441 tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc cggtaagaca
     1501 cgacttatcg ccactggcag cagccactgg taacaggatt agcagagcga ggtatgtagg
     1561 cggtgctaca gagttcttga agtggtggcc taactacggc tacactagaa gaacagtatt
     1621 tggtatctgc gctctgctga agccagttac cttcggaaaa agagttggta gctcttgatc
     1681 cggcaaacaa accaccgctg gtagcggtgg tttttttgtt tgcaagcagc agattacgcg
     1741 cagaaaaaaa ggatctcaag aagatccttt gatcttttct acggggtctg acgctcagtg
     1801 gaacgaaaac tcacgttaag ggattttggt catgagatta tcaaaaagga tcttcaccta
     1861 gatcctttta aattaaaaat gaagttttaa atcaatctaa agtatatatg agtaaacttg
     1921 gtctgacagt taccaatgct taatcagtga ggcacctatc tcagcgatct gtctatttcg
     1981 ttcatccata gttgcattta aatttccgaa ctctccaagg ccctcgtcgg aaaatcttca
     2041 aacctttcgt ccgatccatc ttgcaggcta cctctcgaac gaactatcgc aagtctcttg
     2101 gccggccttg cgccttggct attgcttggc agcgcctatc gccaggtatt actccaatcc
     2161 cgaatatccg agatcgggat cacccgagag aagttcaacc tacatcctca atcccgatct
     2221 atccgagatc cgaggaatat cgaaatcggg gcgcgcctgg cctccgcgcc gggttttggc
     2281 gcctcccgcg ggcgcccccc tcgtcacggc gagcgctgcc acgtcagacg aagggcgcag
     2341 gagcgtcctg atccttccgc ccggacgctc aggacagcgg cccgctgctc ataagactcg
     2401 gccttagaac cccagtatca gcagaaggac attttaggac gggacttggg tgactctagg
     2461 gcactggttt tctttccaga gagcggaaca ggcgaggaaa agtagtccct tctcggcgat
     2521 tctgcggagg gatctccgtg gggcggtgaa cgccgatgat tatataagga cgcgccgggt
     2581 gtggcacagc tagttccgtc gcagccggga tttgggtcgc ggttcttgtt tgtggatcgc
     2641 tgtgatcgtc acttggtgag tagcgggctg ctgggctggc cggggctttc gtggccgccg
     2701 ggccgctcgg tgggacggaa gcgtgtggag agaccgccaa gggctgtagt ctgggtccgc
     2761 gagcaaggtt gccctgaact gggggttggg gggagcgcag caaaatggcg gctgttcccg
     2821 agtcttgaat ggaagacgct tgtgaggcgg gctgtgaggt cgttgaaaca aggtgggggg
     2881 catggtgggc ggcaagaacc caaggtcttg agcccttcgc taatgcggga aagctcttat
     2941 tcgggtgaga tgggctgggc accatctggg gaccctgacg tgaagtttgt cactgactgg
     3001 agaactcggt ttgtcgtctg ttgcgggggc ggcagttatg gcggtgccgt tgggcagtgc
     3061 acccgtacct ttgggagcgc gcgccctcgt cgtgtcgtga cgtcacccgt tctgttggct
     3121 tataatgcag ggtggggcca ccagccggta ggtgtgcggt aggcttttct ccgtcgcagg
     3181 acgcagggtt cgggcctagg gtaggctctc ctgaatcgac aggcgccgga cctctggtga
     3241 ggggagggat aagtgaggcg tcagtttctt tggtcggttt tatgtaccta tcttcttaag
     3301 tagctgaagc tccggttttg aactatgcgc tcggggttgg cgagtgtgtt ttgtgaagtt
     3361 ttttaggcac cttttgaaat gtaatcattt gggtcaatat gtaattttca gtgttagact
     3421 tgtaaattgt ccgctaaatt ctggccgttt ttggcttttt tgttagacaa catgaccgag
     3481 tacaagccca cggtgcgcct cgccacccgc gacgacgtcc ccagggccgt acgcaccctc
     3541 gccgccgcgt tcgccgacta ccccgccacg cgccacaccg tcgatccgga ccgccacatc
     3601 gagcgggtca ccgagctgca agaactcttc ctcacgcgcg tcgggctcga catcggcaag
     3661 gtgtgggtcg cggacgacgg cgccgcggtg gcggtctgga ccacgccgga gagcgtcgaa
     3721 gcgggggcgg tgttcgccga gatcggcccg cgcatggccg agttgagcgg ttcccggctg
     3781 gccgcgcagc aacagatgga gggcctcctg gcgccgcacc ggcccaagga gcccgcgtgg
     3841 ttcctggcca ccgtcggcgt ctcgcccgac caccagggca agggtctggg cagcgccgtc
     3901 gtgctccccg gagtggaggc cgccgagcgc gccggggtgc ccgccttcct ggagacctcc
     3961 gcgccccgca acctcccctt ctacgagcgg ctcggcttca ccgtcaccgc cgacgtcgag
     4021 gtgcccgaag gaccgcgcac ctggtgcatg acccgcaagc ccggtgccta ggctagtatg
     4081 taagcctagt cttagataat aaaatcgcta tccatcgaag atggatgtgt gttggttttt
     4141 tgtgtgtgta acgctaggcg cgcctggtgt accgagaacg atcctctcag tgcgagtctc
     4201 gacgatccat atcgttgctt ggcagtcagc cagtcggaat ccagcttggg acccaggaag
     4261 tccaatcgtc agatattgta ctcaagcctg gtcacggcag cgtaccgatc tgtttaaacc
     4321 tagatattga tagtctgatc ggtcaacgta taatcgagtc ctagcttttg caaacatcta
     4381 tcaagagaca ggatcagcag gaggctttcg catgagtatt caacatttcc gtgtcgccct
     4441 tattcccttt tttgcggcat tttgccttcc tgtttttgct cacccagaaa cgctggtgaa
     4501 agtaaaagat gctgaagatc agttgggtgc gcgagtgggt tacatcgaac tggatctcaa
     4561 cagcggtaag atccttgaga gttttcgccc cgaagaacgc tttccaatga tgagcacttt
     4621 taaagttctg ctatgtggcg cggtattatc ccgtattgac gccgggcaag agcaactcgg
     4681 tcgccgcata cactattctc agaatgactt ggttgagtat tcaccagtca cagaaaagca
     4741 tcttacggat ggcatgacag taagagaatt atgcagtgct gccataacca tgagtgataa
     4801 cactgcggcc aacttacttc tgacaacgat tggaggaccg aaggagctaa ccgctttttt
     4861 gcacaacatg ggggatcatg taactcgcct tgatcgttgg gaaccggagc tgaatgaagc
     4921 cataccaaac gacgagcgtg acaccacgat gcctgtagca atggcaacaa ccttgcgtaa
     4981 actattaact ggcgaactac ttactctagc ttcccggcaa cagttgatag actggatgga
     5041 ggcggataaa gttgcaggac cacttctgcg ctcggccctt ccggctggct ggtttattgc
     5101 tgataaatct ggagccggtg agcgtgggtc tcgcggtatc attgcagcac tggggccaga
     5161 tggtaagccc tcccgtatcg tagttatcta cacgacgggg agtcaggcaa ctatggatga
     5221 acgaaataga cagatcgctg agataggtgc ctcactgatt aagcattggt aaccgattct
     5281 aggtgcattg gcgcagaaaa aaatgcctga tgcgacgctg cgcgtcttat actcccacat
     5341 atgccagatt cagcaacgga tacggcttcc ccaacttgcc cacttccata cgtgtcctcc
     5401 ttaccagaaa tttatcctta agatcccgaa tcgtttaaac tcgactctgg ctctatcgaa
     5461 tctccgtcgt ttcgagctta cgcgaacagc cgtggcgctc atttgctcgt cgggcatcga
     5521 atctcgtcag ctatcgtcag cttacctttt tggcagcgat cgcggctccc gacatcttgg
     5581 accattagct ccacaggtat cttcttccct ctagtggtca taacagcagc ttcagctacc
     5641 tctcaattca aaaaacccct caagacccgt ttagaggccc caaggggtta tgctatcaat
     5701 cgttgcgtta cacacacaaa aaaccaacac acatccatct tcgatggata gcgattttat
     5761 tatctaactg ctgatcgagt gtagccagat ctagtaatca attacggggt cattagttca
     5821 tagcccatat atggagttcc gcgttacata acttacggta aatggcccgc ctggctgacc
     5881 gcccaacgac ccccgcccat tgacgtcaat aatgacgtat gttcccatag taacgccaat
     5941 agggactttc cattgacgtc aatgggtgga gtatttacgg taaactgccc acttggcagt
     6001 acatcaagtg tatcatatgc caagtacgcc ccctattgac gtcaatgacg gtaaatggcc
     6061 cgcctggcat tatgcccagt acatgacctt atgggacttt cctacttggc agtacatcta
     6121 cgtattagtc atcgctatta ccatgctgat gcggttttgg cagtacatca atgggcgtgg
     6181 atagcggttt gactcacggg gatttccaag tctccacccc attgacgtca atgggagttt
     6241 gttttggcac caaaatcaac gggactttcc aaaatgtcgt aacaactccg ccccattgac
     6301 gcaaatgggc ggtaggcgtg tacggtggga ggtctatata agcagagctg gtttagtgaa
     6361 ccgtcagatc agatctttgt cgatcctacc atccactcga cacacccgcc agcggccgc
//
