LOCUS       sequence-325525-        6968 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 6968)
  TITLE     SARS-CoV-2 protein Mammalian expression constructs, tagged, for 
            protein expression and/or purification
REFERENCE   2  (bases 1 to 6968)
  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-m4133786
FEATURES             Location/Qualifiers
     source          1..6968
                     /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..877
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     polyA_signal    930..1051
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(941..960)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     995..1014
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     terminator      1166..1212
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     terminator      1242..1378
                     /label=rrnG terminator
                     /note="transcription terminator from the E. coli ribosomal 
                     RNA rrnG operon (Albrechtsen et al., 1991)"
     misc_feature    1423..1494
                     /label=3' beta-globin insulator
                     /note="insulator downstream of the human beta-globin locus 
                     (Farrell et al., 2002)"
     rep_origin      complement(1720..2308)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1800..1819)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     promoter        2808..4017
                     /label=UbC promoter
                     /note="human ubiquitin C promoter"
     primer_bind     3854..3873
                     /label=hUBCpro-F
                     /note="Human Ubiquitin C (UbC) promoter, forward primer"
     CDS             4021..4620
                     /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(4021..4040)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     4517..4537
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     polyA_signal    4647..4695
                     /note="synthetic polyadenylation signal"
     CDS             join(4961..5029,5030..5821)
                     /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(5179..5198)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     terminator      5834..5970
                     /label=rrnG terminator
                     /note="transcription terminator from the E. coli ribosomal 
                     RNA rrnG operon (Albrechtsen et al., 1991)"
     misc_feature    6122..6193
                     /label=5' beta-globin insulator
                     /note="insulator upstream of the human beta-globin locus 
                     (Farrell et al., 2002)"
     terminator      complement(6198..6244)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     polyA_signal    6262..6310
                     /note="synthetic polyadenylation signal"
     enhancer        6391..6694
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        6696..6898
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     6849..6869
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      join(6967..6968,1..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 tacccctatg acgtaccaga ttatgcgtaa gctagcttga ctgactgaga
      901 tacagcgtac cttcagctca cagacatgat aagatacatt gatgagtttg gacaaaccac
      961 aactagaatg cagtgaaaaa aatgctttat ttgtgaaatt tgtgatgcta ttgctttatt
     1021 tgtaaccatt ataagctgca ataaacaagt taacaacaac aattgcattc attttatgtt
     1081 tcaggttcag ggggaggtgt gggaggtttt ttaaagcaag taaaacctct acaaatgtgg
     1141 tattggccca tctctatcgg tatcgtagca taaccccttg gggcctctaa acgggtcttg
     1201 aggggttttt tgtgcccctc gggccggatt gctatctacc ggcattggcg cagaaaaaaa
     1261 tgcctgatgc gacgctgcgc gtcttatact cccacatatg ccagattcag caacggatac
     1321 ggcttcccca acttgcccac ttccatacgt gtcctcctta ccagaaattt atccttaagg
     1381 tcgtcagcta tcctgcaggc gatctctcga tttcgatcaa gacattcctt taatggtctt
     1441 ttctggacac cactaggggt cagaagtagt tcatcaaact ttcttccctc cctaatctca
     1501 ttggttacct tgggctatcg aaacttaatt aaccagtcaa gtcagctact tggcgagatc
     1561 gacttgtctg ggtttcgact acgctcagaa ttgcgtcagt caagttcgat ctggtccttg
     1621 ctattgcacc cgttctccga ttacgagttt catttaaatc atgtgagcaa aaggccagca
     1681 aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc tccgcccccc
     1741 tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga caggactata
     1801 aagataccag gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc
     1861 gcttaccgga tacctgtccg cctttctccc ttcgggaagc gtggcgcttt ctcatagctc
     1921 acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga
     1981 accccccgtt cagcccgacc gctgcgcctt atccggtaac tatcgtcttg agtccaaccc
     2041 ggtaagacac gacttatcgc cactggcagc agccactggt aacaggatta gcagagcgag
     2101 gtatgtaggc ggtgctacag agttcttgaa gtggtggcct aactacggct acactagaag
     2161 aacagtattt ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa gagttggtag
     2221 ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt gcaagcagca
     2281 gattacgcgc agaaaaaaag gatctcaaga agatcctttg atcttttcta cggggtctga
     2341 cgctcagtgg aacgaaaact cacgttaagg gattttggtc atgagattat caaaaaggat
     2401 cttcacctag atccttttaa attaaaaatg aagttttaaa tcaatctaaa gtatatatga
     2461 gtaaacttgg tctgacagtt accaatgctt aatcagtgag gcacctatct cagcgatctg
     2521 tctatttcgt tcatccatag ttgcatttaa atttccgaac tctccaaggc cctcgtcgga
     2581 aaatcttcaa acctttcgtc cgatccatct tgcaggctac ctctcgaacg aactatcgca
     2641 agtctcttgg ccggccttgc gccttggcta ttgcttggca gcgcctatcg ccaggtatta
     2701 ctccaatccc gaatatccga gatcgggatc acccgagaga agttcaacct acatcctcaa
     2761 tcccgatcta tccgagatcc gaggaatatc gaaatcgggg cgcgcctggc ctccgcgccg
     2821 ggttttggcg cctcccgcgg gcgcccccct cgtcacggcg agcgctgcca cgtcagacga
     2881 agggcgcagg agcgtcctga tccttccgcc cggacgctca ggacagcggc ccgctgctca
     2941 taagactcgg ccttagaacc ccagtatcag cagaaggaca ttttaggacg ggacttgggt
     3001 gactctaggg cactggtttt ctttccagag agcggaacag gcgaggaaaa gtagtccctt
     3061 ctcggcgatt ctgcggaggg atctccgtgg ggcggtgaac gccgatgatt atataaggac
     3121 gcgccgggtg tggcacagct agttccgtcg cagccgggat ttgggtcgcg gttcttgttt
     3181 gtggatcgct gtgatcgtca cttggtgagt agcgggctgc tgggctggcc ggggctttcg
     3241 tggccgccgg gccgctcggt gggacggaag cgtgtggaga gaccgccaag ggctgtagtc
     3301 tgggtccgcg agcaaggttg ccctgaactg ggggttgggg ggagcgcagc aaaatggcgg
     3361 ctgttcccga gtcttgaatg gaagacgctt gtgaggcggg ctgtgaggtc gttgaaacaa
     3421 ggtggggggc atggtgggcg gcaagaaccc aaggtcttga gcccttcgct aatgcgggaa
     3481 agctcttatt cgggtgagat gggctgggca ccatctgggg accctgacgt gaagtttgtc
     3541 actgactgga gaactcggtt tgtcgtctgt tgcgggggcg gcagttatgg cggtgccgtt
     3601 gggcagtgca cccgtacctt tgggagcgcg cgccctcgtc gtgtcgtgac gtcacccgtt
     3661 ctgttggctt ataatgcagg gtggggccac ctgccggtag gtgtgcggta ggcttttctc
     3721 cgtcgcagga cgcagggttc gggcctaggg taggctctcc tgaatcgaca ggcgccggac
     3781 ctctggtgag gggagggata agtgaggcgt cagtttcttt ggtcggtttt atgtacctat
     3841 cttcttaagt agctgaagct ccggttttga actatgcgct cggggttggc gagtgtgttt
     3901 tgtgaagttt tttaggcacc ttttgaaatg taatcatttg ggtcaatatg taattttcag
     3961 tgttagactt gtaaattgtc cgctaaattc tggccgtttt tggctttttt gttagacaac
     4021 atgaccgagt acaagcccac ggtgcgcctc gccacccgcg acgacgtccc cagggccgta
     4081 cgcaccctcg ccgccgcgtt cgccgactac cccgccacgc gccacaccgt cgatccggac
     4141 cgccacatcg agcgggtcac cgagctgcaa gaactcttcc tcacgcgcgt cgggctcgac
     4201 atcggcaagg tgtgggtcgc ggacgacggc gccgcggtgg cggtctggac cacgccggag
     4261 agcgtcgaag cgggggcggt gttcgccgag atcggcccgc gcatggccga gttgagcggt
     4321 tcccggctgg ccgcgcagca acagatggag ggcctcctgg cgccgcaccg gcccaaggag
     4381 cccgcgtggt tcctggccac cgtcggcgtc tcgcccgacc accagggcaa gggtctgggc
     4441 agcgccgtcg tgctccccgg agtggaggcc gccgagcgcg ccggggtgcc cgccttcctg
     4501 gagacctccg cgccccgcaa cctccccttc tacgagcggc tcggcttcac cgtcaccgcc
     4561 gacgtcgagg tgcccgaagg accgcgcacc tggtgcatga cccgcaagcc cggtgcctag
     4621 gctagtatgt aagcctagtc ttagataata aaatcgctat ccatcgaaga tggatgtgtg
     4681 ttggtttttt gtgtgtgtaa cgctaggcgc gcctggtgta ccgagaacga tcctctcagt
     4741 gcgagtctcg acgatccata tcgttgcttg gcagtcagcc agtcggaatc cagcttggga
     4801 cccaggaagt ccaatcgtca gatattgtac tcaagcctgg tcacggcagc gtaccgatct
     4861 gtttaaacct agatattgat agtctgatcg gtcaacgtat aatcgagtcc tagcttttgc
     4921 aaacatctat caagagacag gatcagcagg aggctttcgc atgagtattc aacatttccg
     4981 tgtcgccctt attccctttt ttgcggcatt ttgccttcct gtttttgctc acccagaaac
     5041 gctggtgaaa gtaaaagatg ctgaagatca gttgggtgcg cgagtgggtt acatcgaact
     5101 ggatctcaac agcggtaaga tccttgagag ttttcgcccc gaagaacgct ttccaatgat
     5161 gagcactttt aaagttctgc tatgtggcgc ggtattatcc cgtattgacg ccgggcaaga
     5221 gcaactcggt cgccgcatac actattctca gaatgacttg gttgagtatt caccagtcac
     5281 agaaaagcat cttacggatg gcatgacagt aagagaatta tgcagtgctg ccataaccat
     5341 gagtgataac actgcggcca acttacttct gacaacgatt ggaggaccga aggagctaac
     5401 cgcttttttg cacaacatgg gggatcatgt aactcgcctt gatcgttggg aaccggagct
     5461 gaatgaagcc ataccaaacg acgagcgtga caccacgatg cctgtagcaa tggcaacaac
     5521 cttgcgtaaa ctattaactg gcgaactact tactctagct tcccggcaac agttgataga
     5581 ctggatggag gcggataaag ttgcaggacc acttctgcgc tcggcccttc cggctggctg
     5641 gtttattgct gataaatctg gagccggtga gcgtgggtct cgcggtatca ttgcagcact
     5701 ggggccagat ggtaagccct cccgtatcgt agttatctac acgacgggga gtcaggcaac
     5761 tatggatgaa cgaaatagac agatcgctga gataggtgcc tcactgatta agcattggta
     5821 accgattcta ggtgcattgg cgcagaaaaa aatgcctgat gcgacgctgc gcgtcttata
     5881 ctcccacata tgccagattc agcaacggat acggcttccc caacttgccc acttccatac
     5941 gtgtcctcct taccagaaat ttatccttaa gatcccgaat cgtttaaact cgactctggc
     6001 tctatcgaat ctccgtcgtt tcgagcttac gcgaacagcc gtggcgctca tttgctcgtc
     6061 gggcatcgaa tctcgtcagc tatcgtcagc ttaccttttt ggcagcgatc gcggctcccg
     6121 acatcttgga ccattagctc cacaggtatc ttcttccctc tagtggtcat aacagcagct
     6181 tcagctacct ctcaattcaa aaaacccctc aagacccgtt tagaggcccc aaggggttat
     6241 gctatcaatc gttgcgttac acacacaaaa aaccaacaca catccatctt cgatggatag
     6301 cgattttatt atctaactgc tgatcgagtg tagccagatc tagtaatcaa ttacggggtc
     6361 attagttcat agcccatata tggagttccg cgttacataa cttacggtaa atggcccgcc
     6421 tggctgaccg cccaacgacc cccgcccatt gacgtcaata atgacgtatg ttcccatagt
     6481 aacgccaata gggactttcc attgacgtca atgggtggag tatttacggt aaactgccca
     6541 cttggcagta catcaagtgt atcatatgcc aagtacgccc cctattgacg tcaatgacgg
     6601 taaatggccc gcctggcatt atgcccagta catgacctta tgggactttc ctacttggca
     6661 gtacatctac gtattagtca tcgctattac catgctgatg cggttttggc agtacatcaa
     6721 tgggcgtgga tagcggtttg actcacgggg atttccaagt ctccacccca ttgacgtcaa
     6781 tgggagtttg ttttggcacc aaaatcaacg ggactttcca aaatgtcgta acaactccgc
     6841 cccattgacg caaatgggcg gtaggcgtgt acggtgggag gtctatataa gcagagctgg
     6901 tttagtgaac cgtcagatca gatctttgtc gatcctacca tccactcgac acacccgcca
     6961 gcggccgc
//
