LOCUS       sequence-295944-        6952 bp    DNA     circular SYN 15-DEC-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 6952)
  TITLE     SARS-CoV-2 protein Mammalian expression constructs, tagged, for 
            protein expression and/or purification
REFERENCE   2  (bases 1 to 6952)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"
COMMENT     Sequence Label: pGBW-m4133661
FEATURES             Location/Qualifiers
     source          1..6952
                     /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             851..868
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     polyA_signal    921..1042
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(932..951)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     986..1005
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     terminator      1157..1203
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     terminator      1233..1369
                     /label=rrnG terminator
                     /note="transcription terminator from the E. coli ribosomal 
                     RNA rrnG operon (Albrechtsen et al., 1991)"
     misc_feature    1414..1485
                     /label=3' beta-globin insulator
                     /note="insulator downstream of the human beta-globin locus 
                     (Farrell et al., 2002)"
     rep_origin      complement(1711..2299)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1791..1810)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     promoter        2799..4008
                     /label=UbC promoter
                     /note="human ubiquitin C promoter"
     primer_bind     3845..3864
                     /label=hUBCpro-F
                     /note="Human Ubiquitin C (UbC) promoter, forward primer"
     CDS             4012..4611
                     /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(4012..4031)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     4508..4528
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     polyA_signal    4638..4686
                     /note="synthetic polyadenylation signal"
     CDS             join(4952..5020,5021..5812)
                     /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(5170..5189)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     terminator      5825..5961
                     /label=rrnG terminator
                     /note="transcription terminator from the E. coli ribosomal 
                     RNA rrnG operon (Albrechtsen et al., 1991)"
     misc_feature    6106..6177
                     /label=5' beta-globin insulator
                     /note="insulator upstream of the human beta-globin locus 
                     (Farrell et al., 2002)"
     terminator      complement(6182..6228)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     polyA_signal    6246..6294
                     /note="synthetic polyadenylation signal"
     enhancer        6375..6678
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        6680..6882
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     6833..6853
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      join(6951..6952,1..8)
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
ORIGIN
        1 caccatggat ttgtttatga gaatcttcac aattggaact gtaactttga agcaaggtga
       61 aatcaaggat gctactcctt cagattttgt tcgcgctact gcaacgatac cgatacaagc
      121 ctcactccct ttcggatggc ttattgttgg cgttgcactt cttgctgttt ttcagagcgc
      181 ttccaaaatc ataaccctca aaaagagatg gcaactagca ctctccaagg gtgttcactt
      241 tgtttgcaac ttgctgttgt tgtttgtaac agtttactca caccttttgc tcgttgctgc
      301 tggccttgaa gccccttttc tctatcttta tgctttagtc tacttcttgc agagtataaa
      361 ctttgtaaga ataataatga ggctttggct ttgctggaaa tgccgttcca aaaacccatt
      421 actttatgat gccaactatt ttctttgctg gcatactaat tgttacgact attgtatacc
      481 ttacaatagt gtaacttctt caattgtcat tacttcaggt gatggcacaa caagtcctat
      541 ttctgaacat gactaccaga ttggtggtta tactgaaaaa tgggaatctg gagtaaaaga
      601 ctgtgttgta ttacacagtt acttcacttc agactattac cagctgtact caactcaatt
      661 gagtacagac actggtgttg aacatgttac cttcttcatc tacaataaaa ttgttgatga
      721 gcctgaagaa catgtccaaa ttcacacaat cgacggttca tccggagttg ttaatccagt
      781 aatggaacca atttatgatg aaccgacgac gactactagc gtgcctttgg aaaaccttta
      841 ctttcagtcg caccatcacc atcaccatta agctagcttg actgactgag atacagcgta
      901 ccttcagctc acagacatga taagatacat tgatgagttt ggacaaacca caactagaat
      961 gcagtgaaaa aaatgcttta tttgtgaaat ttgtgatgct attgctttat ttgtaaccat
     1021 tataagctgc aataaacaag ttaacaacaa caattgcatt cattttatgt ttcaggttca
     1081 gggggaggtg tgggaggttt tttaaagcaa gtaaaacctc tacaaatgtg gtattggccc
     1141 atctctatcg gtatcgtagc ataacccctt ggggcctcta aacgggtctt gaggggtttt
     1201 ttgtgcccct cgggccggat tgctatctac cggcattggc gcagaaaaaa atgcctgatg
     1261 cgacgctgcg cgtcttatac tcccacatat gccagattca gcaacggata cggcttcccc
     1321 aacttgccca cttccatacg tgtcctcctt accagaaatt tatccttaag gtcgtcagct
     1381 atcctgcagg cgatctctcg atttcgatca agacattcct ttaatggtct tttctggaca
     1441 ccactagggg tcagaagtag ttcatcaaac tttcttccct ccctaatctc attggttacc
     1501 ttgggctatc gaaacttaat taaccagtca agtcagctac ttggcgagat cgacttgtct
     1561 gggtttcgac tacgctcaga attgcgtcag tcaagttcga tctggtcctt gctattgcac
     1621 ccgttctccg attacgagtt tcatttaaat catgtgagca aaaggccagc aaaaggccag
     1681 gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg ctccgccccc ctgacgagca
     1741 tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg acaggactat aaagatacca
     1801 ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg
     1861 atacctgtcc gcctttctcc cttcgggaag cgtggcgctt tctcatagct cacgctgtag
     1921 gtatctcagt tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt
     1981 tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc cggtaagaca
     2041 cgacttatcg ccactggcag cagccactgg taacaggatt agcagagcga ggtatgtagg
     2101 cggtgctaca gagttcttga agtggtggcc taactacggc tacactagaa gaacagtatt
     2161 tggtatctgc gctctgctga agccagttac cttcggaaaa agagttggta gctcttgatc
     2221 cggcaaacaa accaccgctg gtagcggtgg tttttttgtt tgcaagcagc agattacgcg
     2281 cagaaaaaaa ggatctcaag aagatccttt gatcttttct acggggtctg acgctcagtg
     2341 gaacgaaaac tcacgttaag ggattttggt catgagatta tcaaaaagga tcttcaccta
     2401 gatcctttta aattaaaaat gaagttttaa atcaatctaa agtatatatg agtaaacttg
     2461 gtctgacagt taccaatgct taatcagtga ggcacctatc tcagcgatct gtctatttcg
     2521 ttcatccata gttgcattta aatttccgaa ctctccaagg ccctcgtcgg aaaatcttca
     2581 aacctttcgt ccgatccatc ttgcaggcta cctctcgaac gaactatcgc aagtctcttg
     2641 gccggccttg cgccttggct attgcttggc agcgcctatc gccaggtatt actccaatcc
     2701 cgaatatccg agatcgggat cacccgagag aagttcaacc tacatcctca atcccgatct
     2761 atccgagatc cgaggaatat cgaaatcggg gcgcgcctgg cctccgcgcc gggttttggc
     2821 gcctcccgcg ggcgcccccc tcgtcacggc gagcgctgcc acgtcagacg aagggcgcag
     2881 gagcgtcctg atccttccgc ccggacgctc aggacagcgg cccgctgctc ataagactcg
     2941 gccttagaac cccagtatca gcagaaggac attttaggac gggacttggg tgactctagg
     3001 gcactggttt tctttccaga gagcggaaca ggcgaggaaa agtagtccct tctcggcgat
     3061 tctgcggagg gatctccgtg gggcggtgaa cgccgatgat tatataagga cgcgccgggt
     3121 gtggcacagc tagttccgtc gcagccggga tttgggtcgc ggttcttgtt tgtggatcgc
     3181 tgtgatcgtc acttggtgag tagcgggctg ctgggctggc cggggctttc gtggccgccg
     3241 ggccgctcgg tgggacggaa gcgtgtggag agaccgccaa gggctgtagt ctgggtccgc
     3301 gagcaaggtt gccctgaact gggggttggg gggagcgcag caaaatggcg gctgttcccg
     3361 agtcttgaat ggaagacgct tgtgaggcgg gctgtgaggt cgttgaaaca aggtgggggg
     3421 catggtgggc ggcaagaacc caaggtcttg agcccttcgc taatgcggga aagctcttat
     3481 tcgggtgaga tgggctgggc accatctggg gaccctgacg tgaagtttgt cactgactgg
     3541 agaactcggt ttgtcgtctg ttgcgggggc ggcagttatg gcggtgccgt tgggcagtgc
     3601 acccgtacct ttgggagcgc gcgccctcgt cgtgtcgtga cgtcacccgt tctgttggct
     3661 tataatgcag ggtggggcca cctgccggta ggtgtgcggt aggcttttct ccgtcgcagg
     3721 acgcagggtt cgggcctagg gtaggctctc ctgaatcgac aggcgccgga cctctggtga
     3781 ggggagggat aagtgaggcg tcagtttctt tggtcggttt tatgtaccta tcttcttaag
     3841 tagctgaagc tccggttttg aactatgcgc tcggggttgg cgagtgtgtt ttgtgaagtt
     3901 ttttaggcac cttttgaaat gtaatcattt gggtcaatat gtaattttca gtgttagact
     3961 tgtaaattgt ccgctaaatt ctggccgttt ttggcttttt tgttagacaa catgaccgag
     4021 tacaagccca cggtgcgcct cgccacccgc gacgacgtcc ccagggccgt acgcaccctc
     4081 gccgccgcgt tcgccgacta ccccgccacg cgccacaccg tcgatccgga ccgccacatc
     4141 gagcgggtca ccgagctgca agaactcttc ctcacgcgcg tcgggctcga catcggcaag
     4201 gtgtgggtcg cggacgacgg cgccgcggtg gcggtctgga ccacgccgga gagcgtcgaa
     4261 gcgggggcgg tgttcgccga gatcggcccg cgcatggccg agttgagcgg ttcccggctg
     4321 gccgcgcagc aacagatgga gggcctcctg gcgccgcacc ggcccaagga gcccgcgtgg
     4381 ttcctggcca ccgtcggcgt ctcgcccgac caccagggca agggtctggg cagcgccgtc
     4441 gtgctccccg gagtggaggc cgccgagcgc gccggggtgc ccgccttcct ggagacctcc
     4501 gcgccccgca acctcccctt ctacgagcgg ctcggcttca ccgtcaccgc cgacgtcgag
     4561 gtgcccgaag gaccgcgcac ctggtgcatg acccgcaagc ccggtgccta ggctagtatg
     4621 taagcctagt cttagataat aaaatcgcta tccatcgaag atggatgtgt gttggttttt
     4681 tgtgtgtgta acgctaggcg cgcctggtgt accgagaacg atcctctcag tgcgagtctc
     4741 gacgatccat atcgttgctt ggcagtcagc cagtcggaat ccagcttggg acccaggaag
     4801 tccaatcgtc agatattgta ctcaagcctg gtcacggcag cgtaccgatc tgtttaaacc
     4861 tagatattga tagtctgatc ggtcaacgta taatcgagtc ctagcttttg caaacatcta
     4921 tcaagagaca ggatcagcag gaggctttcg catgagtatt caacatttcc gtgtcgccct
     4981 tattcccttt tttgcggcat tttgccttcc tgtttttgct cacccagaaa cgctggtgaa
     5041 agtaaaagat gctgaagatc agttgggtgc gcgagtgggt tacatcgaac tggatctcaa
     5101 cagcggtaag atccttgaga gttttcgccc cgaagaacgc tttccaatga tgagcacttt
     5161 taaagttctg ctatgtggcg cggtattatc ccgtattgac gccgggcaag agcaactcgg
     5221 tcgccgcata cactattctc agaatgactt ggttgagtat tcaccagtca cagaaaagca
     5281 tcttacggat ggcatgacag taagagaatt atgcagtgct gccataacca tgagtgataa
     5341 cactgcggcc aacttacttc tgacaacgat tggaggaccg aaggagctaa ccgctttttt
     5401 gcacaacatg ggggatcatg taactcgcct tgatcgttgg gaaccggagc tgaatgaagc
     5461 cataccaaac gacgagcgtg acaccacgat gcctgtagca atggcaacaa ccttgcgtaa
     5521 actattaact ggcgaactac ttactctagc ttcccggcaa cagttgatag actggatgga
     5581 ggcggataaa gttgcaggac cacttctgcg ctcggccctt ccggctggct ggtttattgc
     5641 tgataaatct ggagccggtg agcgtgggtc tcgcggtatc attgcagcac tggggccaga
     5701 tggtaagccc tcccgtatcg tagttatcta cacgacgggg agtcaggcaa ctatggatga
     5761 acgaaataga cagatcgctg agataggtgc ctcactgatt aagcattggt aaccgattct
     5821 aggtgcattg gcgcagaaaa aaatgcctga tgcgacgctg cgcgtcttat actcccacat
     5881 atgccagatt cagcaacgga tacggcttcc ccaacttgcc cacttccata cgtgtcctcc
     5941 ttaccagaaa tttatcctta agatcgttta aactcgactc tggctctatc gaatctccgt
     6001 cgtttcgagc ttacgcgaac agccgtggcg ctcatttgct cgtcgggcat cgaatctcgt
     6061 cagctatcgt cagcttacct ttttggcagc gatcgcggct cccgacatct tggaccatta
     6121 gctccacagg tatcttcttc cctctagtgg tcataacagc agcttcagct acctctcaat
     6181 tcaaaaaacc cctcaagacc cgtttagagg ccccaagggg ttatgctatc aatcgttgcg
     6241 ttacacacac aaaaaaccaa cacacatcca tcttcgatgg atagcgattt tattatctaa
     6301 ctgctgatcg agtgtagcca gatctagtaa tcaattacgg ggtcattagt tcatagccca
     6361 tatatggagt tccgcgttac ataacttacg gtaaatggcc cgcctggctg accgcccaac
     6421 gacccccgcc cattgacgtc aataatgacg tatgttccca tagtaacgcc aatagggact
     6481 ttccattgac gtcaatgggt ggagtattta cggtaaactg cccacttggc agtacatcaa
     6541 gtgtatcata tgccaagtac gccccctatt gacgtcaatg acggtaaatg gcccgcctgg
     6601 cattatgccc agtacatgac cttatgggac tttcctactt ggcagtacat ctacgtatta
     6661 gtcatcgcta ttaccatgct gatgcggttt tggcagtaca tcaatgggcg tggatagcgg
     6721 tttgactcac ggggatttcc aagtctccac cccattgacg tcaatgggag tttgttttgg
     6781 caccaaaatc aacgggactt tccaaaatgt cgtaacaact ccgccccatt gacgcaaatg
     6841 ggcggtaggc gtgtacggtg ggaggtctat ataagcagag ctggtttagt gaaccgtcag
     6901 atcagatctt tgtcgatcct accatccact cgacacaccc gccagcggcc gc
//
