LOCUS       sequence-295663-        7324 bp    DNA     circular SYN 15-APR-2025
DEFINITION  Mammalian expression plasmid for SARS-CoV-2 ORF3a protein.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7324)
  TITLE     SARS-CoV-2 protein Mammalian expression constructs, tagged, for 
            protein expression and/or purification
REFERENCE   2  (bases 1 to 7324)
  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-m4134543
FEATURES             Location/Qualifiers
     source          1..7324
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             830..850
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQS"
     CDS             1199..1216
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     polyA_signal    1293..1414
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1304..1323)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     1358..1377
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     terminator      1529..1575
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     terminator      1605..1741
                     /label=rrnG terminator
                     /note="transcription terminator from the E. coli ribosomal 
                     RNA rrnG operon (Albrechtsen et al., 1991)"
     misc_feature    1786..1857
                     /label=3' beta-globin insulator
                     /note="insulator downstream of the human beta-globin locus 
                     (Farrell et al., 2002)"
     rep_origin      complement(2083..2671)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2163..2182)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     promoter        3171..4380
                     /label=UbC promoter
                     /note="human ubiquitin C promoter"
     primer_bind     4217..4236
                     /label=hUBCpro-F
                     /note="Human Ubiquitin C (UbC) promoter, forward primer"
     CDS             4384..4983
                     /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(4384..4403)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     4880..4900
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     polyA_signal    5010..5058
                     /note="synthetic polyadenylation signal"
     CDS             join(5324..5392,5393..6184)
                     /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(5542..5561)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     terminator      6197..6333
                     /label=rrnG terminator
                     /note="transcription terminator from the E. coli ribosomal 
                     RNA rrnG operon (Albrechtsen et al., 1991)"
     misc_feature    6478..6549
                     /label=5' beta-globin insulator
                     /note="insulator upstream of the human beta-globin locus 
                     (Farrell et al., 2002)"
     terminator      complement(6554..6600)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     polyA_signal    6618..6666
                     /note="synthetic polyadenylation signal"
     enhancer        6747..7050
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        7052..7254
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     7205..7225
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      join(7323..7324,1..8)
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
ORIGIN
        1 caccatggat ctgtttatgc gtatattcac catcggcacg gttacattga agcaagggga
       61 aattaaagac gcaactccca gtgacttcgt acgggcgacc gctacaattc cgatccaggc
      121 cagcctccct tttggatggc ttatcgtcgg cgtggcccta ctggctgtgt ttcagtcggc
      181 atcaaagata atcactctga aaaagcgatg gcagttagcc cttagcaaag gtgtgcactt
      241 cgtatgcaat ttgctgctcc tgttcgtcac cgtttattcc catctgctcc tggtggctgc
      301 cggattggag gcaccatttt tatacctata cgccctcgtg tattttctgc aatctattaa
      361 cttcgtccgc attatcatga gactctggct ctgttggaag tgcaggtcta aaaatccctt
      421 gttatatgat gccaactact tcctgtgttg gcacactaac tgctatgact actgtattcc
      481 atacaattca gttacctcca gtatagtgat cacatctggg gatggtacga cttcgcctat
      541 ttccgagcat gactatcaga tcggcgggta cacagaaaag tgggagagcg gagtcaagga
      601 ttgcgtggtg cttcactcct atttcaccag tgactactac cagctttata gcacacaact
      661 ttcaacagac accggcgtgg agcatgtgac ttttttcatt tataataaaa tcgttgatga
      721 accggaggaa cacgttcaga tacacactat cgacggcagc tctggtgtcg tcaaccctgt
      781 tatggagccc atttacgatg aaccaacgac cacaacatct gtacctctgg aaaaccttta
      841 ctttcagtcg atgagtgaca aaatcattca tctaaccgat gactcctttg atacagacgt
      901 gttgaaggcc gatggtgcga tactggtaga tttctgggct gaatggtgtg gcccgtgcaa
      961 gatgatcgca cccattctgg acgagatcgc cgacgagtat caaggaaaat taactgtcgc
     1021 taagcttaat atagatcaga acccagggac ggcacctaaa tacggcatca ggggaatacc
     1081 taccctcctg ctctttaaga atggggaagt tgccgcgaca aaagtgggag ccctgtctaa
     1141 gggccagcta aaagagttct tggacgctaa cctggcaggt tcaggctcgg ggcacatgca
     1201 ccatcaccat caccatagca gcgggctcgt gccacgcggc taagctagct tgactgactg
     1261 agatacagcg taccttcagc tcacagacat gataagatac attgatgagt ttggacaaac
     1321 cacaactaga atgcagtgaa aaaaatgctt tatttgtgaa atttgtgatg ctattgcttt
     1381 atttgtaacc attataagct gcaataaaca agttaacaac aacaattgca ttcattttat
     1441 gtttcaggtt cagggggagg tgtgggaggt tttttaaagc aagtaaaacc tctacaaatg
     1501 tggtattggc ccatctctat cggtatcgta gcataacccc ttggggcctc taaacgggtc
     1561 ttgaggggtt ttttgtgccc ctcgggccgg attgctatct accggcattg gcgcagaaaa
     1621 aaatgcctga tgcgacgctg cgcgtcttat actcccacat atgccagatt cagcaacgga
     1681 tacggcttcc ccaacttgcc cacttccata cgtgtcctcc ttaccagaaa tttatcctta
     1741 aggtcgtcag ctatcctgca ggcgatctct cgatttcgat caagacattc ctttaatggt
     1801 cttttctgga caccactagg ggtcagaagt agttcatcaa actttcttcc ctccctaatc
     1861 tcattggtta ccttgggcta tcgaaactta attaaccagt caagtcagct acttggcgag
     1921 atcgacttgt ctgggtttcg actacgctca gaattgcgtc agtcaagttc gatctggtcc
     1981 ttgctattgc acccgttctc cgattacgag tttcatttaa atcatgtgag caaaaggcca
     2041 gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg tttttccata ggctccgccc
     2101 ccctgacgag catcacaaaa atcgacgctc aagtcagagg tggcgaaacc cgacaggact
     2161 ataaagatac caggcgtttc cccctggaag ctccctcgtg cgctctcctg ttccgaccct
     2221 gccgcttacc ggatacctgt ccgcctttct cccttcggga agcgtggcgc tttctcatag
     2281 ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca
     2341 cgaacccccc gttcagcccg accgctgcgc cttatccggt aactatcgtc ttgagtccaa
     2401 cccggtaaga cacgacttat cgccactggc agcagccact ggtaacagga ttagcagagc
     2461 gaggtatgta ggcggtgcta cagagttctt gaagtggtgg cctaactacg gctacactag
     2521 aagaacagta tttggtatct gcgctctgct gaagccagtt accttcggaa aaagagttgg
     2581 tagctcttga tccggcaaac aaaccaccgc tggtagcggt ggtttttttg tttgcaagca
     2641 gcagattacg cgcagaaaaa aaggatctca agaagatcct ttgatctttt ctacggggtc
     2701 tgacgctcag tggaacgaaa actcacgtta agggattttg gtcatgagat tatcaaaaag
     2761 gatcttcacc tagatccttt taaattaaaa atgaagtttt aaatcaatct aaagtatata
     2821 tgagtaaact tggtctgaca gttaccaatg cttaatcagt gaggcaccta tctcagcgat
     2881 ctgtctattt cgttcatcca tagttgcatt taaatttccg aactctccaa ggccctcgtc
     2941 ggaaaatctt caaacctttc gtccgatcca tcttgcaggc tacctctcga acgaactatc
     3001 gcaagtctct tggccggcct tgcgccttgg ctattgcttg gcagcgccta tcgccaggta
     3061 ttactccaat cccgaatatc cgagatcggg atcacccgag agaagttcaa cctacatcct
     3121 caatcccgat ctatccgaga tccgaggaat atcgaaatcg gggcgcgcct ggcctccgcg
     3181 ccgggttttg gcgcctcccg cgggcgcccc cctcgtcacg gcgagcgctg ccacgtcaga
     3241 cgaagggcgc aggagcgtcc tgatccttcc gcccggacgc tcaggacagc ggcccgctgc
     3301 tcataagact cggccttaga accccagtat cagcagaagg acattttagg acgggacttg
     3361 ggtgactcta gggcactggt tttctttcca gagagcggaa caggcgagga aaagtagtcc
     3421 cttctcggcg attctgcgga gggatctccg tggggcggtg aacgccgatg attatataag
     3481 gacgcgccgg gtgtggcaca gctagttccg tcgcagccgg gatttgggtc gcggttcttg
     3541 tttgtggatc gctgtgatcg tcacttggtg agtagcgggc tgctgggctg gccggggctt
     3601 tcgtggccgc cgggccgctc ggtgggacgg aagcgtgtgg agagaccgcc aagggctgta
     3661 gtctgggtcc gcgagcaagg ttgccctgaa ctgggggttg gggggagcgc agcaaaatgg
     3721 cggctgttcc cgagtcttga atggaagacg cttgtgaggc gggctgtgag gtcgttgaaa
     3781 caaggtgggg ggcatggtgg gcggcaagaa cccaaggtct tgagcccttc gctaatgcgg
     3841 gaaagctctt attcgggtga gatgggctgg gcaccatctg gggaccctga cgtgaagttt
     3901 gtcactgact ggagaactcg gtttgtcgtc tgttgcgggg gcggcagtta tggcggtgcc
     3961 gttgggcagt gcacccgtac ctttgggagc gcgcgccctc gtcgtgtcgt gacgtcaccc
     4021 gttctgttgg cttataatgc agggtggggc cacctgccgg taggtgtgcg gtaggctttt
     4081 ctccgtcgca ggacgcaggg ttcgggccta gggtaggctc tcctgaatcg acaggcgccg
     4141 gacctctggt gaggggaggg ataagtgagg cgtcagtttc tttggtcggt tttatgtacc
     4201 tatcttctta agtagctgaa gctccggttt tgaactatgc gctcggggtt ggcgagtgtg
     4261 ttttgtgaag ttttttaggc accttttgaa atgtaatcat ttgggtcaat atgtaatttt
     4321 cagtgttaga cttgtaaatt gtccgctaaa ttctggccgt ttttggcttt tttgttagac
     4381 aacatgaccg agtacaagcc cacggtgcgc ctcgccaccc gcgacgacgt ccccagggcc
     4441 gtacgcaccc tcgccgccgc gttcgccgac taccccgcca cgcgccacac cgtcgatccg
     4501 gaccgccaca tcgagcgggt caccgagctg caagaactct tcctcacgcg cgtcgggctc
     4561 gacatcggca aggtgtgggt cgcggacgac ggcgccgcgg tggcggtctg gaccacgccg
     4621 gagagcgtcg aagcgggggc ggtgttcgcc gagatcggcc cgcgcatggc cgagttgagc
     4681 ggttcccggc tggccgcgca gcaacagatg gagggcctcc tggcgccgca ccggcccaag
     4741 gagcccgcgt ggttcctggc caccgtcggc gtctcgcccg accaccaggg caagggtctg
     4801 ggcagcgccg tcgtgctccc cggagtggag gccgccgagc gcgccggggt gcccgccttc
     4861 ctggagacct ccgcgccccg caacctcccc ttctacgagc ggctcggctt caccgtcacc
     4921 gccgacgtcg aggtgcccga aggaccgcgc acctggtgca tgacccgcaa gcccggtgcc
     4981 taggctagta tgtaagccta gtcttagata ataaaatcgc tatccatcga agatggatgt
     5041 gtgttggttt tttgtgtgtg taacgctagg cgcgcctggt gtaccgagaa cgatcctctc
     5101 agtgcgagtc tcgacgatcc atatcgttgc ttggcagtca gccagtcgga atccagcttg
     5161 ggacccagga agtccaatcg tcagatattg tactcaagcc tggtcacggc agcgtaccga
     5221 tctgtttaaa cctagatatt gatagtctga tcggtcaacg tataatcgag tcctagcttt
     5281 tgcaaacatc tatcaagaga caggatcagc aggaggcttt cgcatgagta ttcaacattt
     5341 ccgtgtcgcc cttattccct tttttgcggc attttgcctt cctgtttttg ctcacccaga
     5401 aacgctggtg aaagtaaaag atgctgaaga tcagttgggt gcgcgagtgg gttacatcga
     5461 actggatctc aacagcggta agatccttga gagttttcgc cccgaagaac gctttccaat
     5521 gatgagcact tttaaagttc tgctatgtgg cgcggtatta tcccgtattg acgccgggca
     5581 agagcaactc ggtcgccgca tacactattc tcagaatgac ttggttgagt attcaccagt
     5641 cacagaaaag catcttacgg atggcatgac agtaagagaa ttatgcagtg ctgccataac
     5701 catgagtgat aacactgcgg ccaacttact tctgacaacg attggaggac cgaaggagct
     5761 aaccgctttt ttgcacaaca tgggggatca tgtaactcgc cttgatcgtt gggaaccgga
     5821 gctgaatgaa gccataccaa acgacgagcg tgacaccacg atgcctgtag caatggcaac
     5881 aaccttgcgt aaactattaa ctggcgaact acttactcta gcttcccggc aacagttgat
     5941 agactggatg gaggcggata aagttgcagg accacttctg cgctcggccc ttccggctgg
     6001 ctggtttatt gctgataaat ctggagccgg tgagcgtggg tctcgcggta tcattgcagc
     6061 actggggcca gatggtaagc cctcccgtat cgtagttatc tacacgacgg ggagtcaggc
     6121 aactatggat gaacgaaata gacagatcgc tgagataggt gcctcactga ttaagcattg
     6181 gtaaccgatt ctaggtgcat tggcgcagaa aaaaatgcct gatgcgacgc tgcgcgtctt
     6241 atactcccac atatgccaga ttcagcaacg gatacggctt ccccaacttg cccacttcca
     6301 tacgtgtcct ccttaccaga aatttatcct taagatcgtt taaactcgac tctggctcta
     6361 tcgaatctcc gtcgtttcga gcttacgcga acagccgtgg cgctcatttg ctcgtcgggc
     6421 atcgaatctc gtcagctatc gtcagcttac ctttttggca gcgatcgcgg ctcccgacat
     6481 cttggaccat tagctccaca ggtatcttct tccctctagt ggtcataaca gcagcttcag
     6541 ctacctctca attcaaaaaa cccctcaaga cccgtttaga ggccccaagg ggttatgcta
     6601 tcaatcgttg cgttacacac acaaaaaacc aacacacatc catcttcgat ggatagcgat
     6661 tttattatct aactgctgat cgagtgtagc cagatctagt aatcaattac ggggtcatta
     6721 gttcatagcc catatatgga gttccgcgtt acataactta cggtaaatgg cccgcctggc
     6781 tgaccgccca acgacccccg cccattgacg tcaataatga cgtatgttcc catagtaacg
     6841 ccaataggga ctttccattg acgtcaatgg gtggagtatt tacggtaaac tgcccacttg
     6901 gcagtacatc aagtgtatca tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa
     6961 tggcccgcct ggcattatgc ccagtacatg accttatggg actttcctac ttggcagtac
     7021 atctacgtat tagtcatcgc tattaccatg ctgatgcggt tttggcagta catcaatggg
     7081 cgtggatagc ggtttgactc acggggattt ccaagtctcc accccattga cgtcaatggg
     7141 agtttgtttt ggcaccaaaa tcaacgggac tttccaaaat gtcgtaacaa ctccgcccca
     7201 ttgacgcaaa tgggcggtag gcgtgtacgg tgggaggtct atataagcag agctggttta
     7261 gtgaaccgtc agatcagatc tttgtcgatc ctaccatcca ctcgacacac ccgccagcgg
     7321 ccgc
//
