LOCUS       sequence-295168-        7003 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 7003)
  TITLE     SARS-CoV-2 protein Mammalian expression constructs, tagged, for 
            protein expression and/or purification
REFERENCE   2  (bases 1 to 7003)
  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-m4133648
FEATURES             Location/Qualifiers
     source          1..7003
                     /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             854..919
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     polyA_signal    972..1093
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(983..1002)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     1037..1056
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     terminator      1208..1254
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     terminator      1284..1420
                     /label=rrnG terminator
                     /note="transcription terminator from the E. coli ribosomal 
                     RNA rrnG operon (Albrechtsen et al., 1991)"
     misc_feature    1465..1536
                     /label=3' beta-globin insulator
                     /note="insulator downstream of the human beta-globin locus 
                     (Farrell et al., 2002)"
     rep_origin      complement(1762..2350)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1842..1861)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     promoter        2850..4059
                     /label=UbC promoter
                     /note="human ubiquitin C promoter"
     primer_bind     3896..3915
                     /label=hUBCpro-F
                     /note="Human Ubiquitin C (UbC) promoter, forward primer"
     CDS             4063..4662
                     /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(4063..4082)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     4559..4579
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     polyA_signal    4689..4737
                     /note="synthetic polyadenylation signal"
     CDS             join(5003..5071,5072..5863)
                     /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(5221..5240)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     terminator      5876..6012
                     /label=rrnG terminator
                     /note="transcription terminator from the E. coli ribosomal 
                     RNA rrnG operon (Albrechtsen et al., 1991)"
     misc_feature    6157..6228
                     /label=5' beta-globin insulator
                     /note="insulator upstream of the human beta-globin locus 
                     (Farrell et al., 2002)"
     terminator      complement(6233..6279)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     polyA_signal    6297..6345
                     /note="synthetic polyadenylation signal"
     enhancer        6426..6729
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        6731..6933
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     6884..6904
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      join(7002..7003,1..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 atggattata aggaccacga tggagactac aaagatcatg acattgatta
      901 caaggacgac gatgacaaat aagctagctt gactgactga gatacagcgt accttcagct
      961 cacagacatg ataagataca ttgatgagtt tggacaaacc acaactagaa tgcagtgaaa
     1021 aaaatgcttt atttgtgaaa tttgtgatgc tattgcttta tttgtaacca ttataagctg
     1081 caataaacaa gttaacaaca acaattgcat tcattttatg tttcaggttc agggggaggt
     1141 gtgggaggtt ttttaaagca agtaaaacct ctacaaatgt ggtattggcc catctctatc
     1201 ggtatcgtag cataacccct tggggcctct aaacgggtct tgaggggttt tttgtgcccc
     1261 tcgggccgga ttgctatcta ccggcattgg cgcagaaaaa aatgcctgat gcgacgctgc
     1321 gcgtcttata ctcccacata tgccagattc agcaacggat acggcttccc caacttgccc
     1381 acttccatac gtgtcctcct taccagaaat ttatccttaa ggtcgtcagc tatcctgcag
     1441 gcgatctctc gatttcgatc aagacattcc tttaatggtc ttttctggac accactaggg
     1501 gtcagaagta gttcatcaaa ctttcttccc tccctaatct cattggttac cttgggctat
     1561 cgaaacttaa ttaaccagtc aagtcagcta cttggcgaga tcgacttgtc tgggtttcga
     1621 ctacgctcag aattgcgtca gtcaagttcg atctggtcct tgctattgca cccgttctcc
     1681 gattacgagt ttcatttaaa tcatgtgagc aaaaggccag caaaaggcca ggaaccgtaa
     1741 aaaggccgcg ttgctggcgt ttttccatag gctccgcccc cctgacgagc atcacaaaaa
     1801 tcgacgctca agtcagaggt ggcgaaaccc gacaggacta taaagatacc aggcgtttcc
     1861 ccctggaagc tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg gatacctgtc
     1921 cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta ggtatctcag
     1981 ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga
     2041 ccgctgcgcc ttatccggta actatcgtct tgagtccaac ccggtaagac acgacttatc
     2101 gccactggca gcagccactg gtaacaggat tagcagagcg aggtatgtag gcggtgctac
     2161 agagttcttg aagtggtggc ctaactacgg ctacactaga agaacagtat ttggtatctg
     2221 cgctctgctg aagccagtta ccttcggaaa aagagttggt agctcttgat ccggcaaaca
     2281 aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa
     2341 aggatctcaa gaagatcctt tgatcttttc tacggggtct gacgctcagt ggaacgaaaa
     2401 ctcacgttaa gggattttgg tcatgagatt atcaaaaagg atcttcacct agatcctttt
     2461 aaattaaaaa tgaagtttta aatcaatcta aagtatatat gagtaaactt ggtctgacag
     2521 ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc tgtctatttc gttcatccat
     2581 agttgcattt aaatttccga actctccaag gccctcgtcg gaaaatcttc aaacctttcg
     2641 tccgatccat cttgcaggct acctctcgaa cgaactatcg caagtctctt ggccggcctt
     2701 gcgccttggc tattgcttgg cagcgcctat cgccaggtat tactccaatc ccgaatatcc
     2761 gagatcggga tcacccgaga gaagttcaac ctacatcctc aatcccgatc tatccgagat
     2821 ccgaggaata tcgaaatcgg ggcgcgcctg gcctccgcgc cgggttttgg cgcctcccgc
     2881 gggcgccccc ctcgtcacgg cgagcgctgc cacgtcagac gaagggcgca ggagcgtcct
     2941 gatccttccg cccggacgct caggacagcg gcccgctgct cataagactc ggccttagaa
     3001 ccccagtatc agcagaagga cattttagga cgggacttgg gtgactctag ggcactggtt
     3061 ttctttccag agagcggaac aggcgaggaa aagtagtccc ttctcggcga ttctgcggag
     3121 ggatctccgt ggggcggtga acgccgatga ttatataagg acgcgccggg tgtggcacag
     3181 ctagttccgt cgcagccggg atttgggtcg cggttcttgt ttgtggatcg ctgtgatcgt
     3241 cacttggtga gtagcgggct gctgggctgg ccggggcttt cgtggccgcc gggccgctcg
     3301 gtgggacgga agcgtgtgga gagaccgcca agggctgtag tctgggtccg cgagcaaggt
     3361 tgccctgaac tgggggttgg ggggagcgca gcaaaatggc ggctgttccc gagtcttgaa
     3421 tggaagacgc ttgtgaggcg ggctgtgagg tcgttgaaac aaggtggggg gcatggtggg
     3481 cggcaagaac ccaaggtctt gagcccttcg ctaatgcggg aaagctctta ttcgggtgag
     3541 atgggctggg caccatctgg ggaccctgac gtgaagtttg tcactgactg gagaactcgg
     3601 tttgtcgtct gttgcggggg cggcagttat ggcggtgccg ttgggcagtg cacccgtacc
     3661 tttgggagcg cgcgccctcg tcgtgtcgtg acgtcacccg ttctgttggc ttataatgca
     3721 gggtggggcc acctgccggt aggtgtgcgg taggcttttc tccgtcgcag gacgcagggt
     3781 tcgggcctag ggtaggctct cctgaatcga caggcgccgg acctctggtg aggggaggga
     3841 taagtgaggc gtcagtttct ttggtcggtt ttatgtacct atcttcttaa gtagctgaag
     3901 ctccggtttt gaactatgcg ctcggggttg gcgagtgtgt tttgtgaagt tttttaggca
     3961 ccttttgaaa tgtaatcatt tgggtcaata tgtaattttc agtgttagac ttgtaaattg
     4021 tccgctaaat tctggccgtt tttggctttt ttgttagaca acatgaccga gtacaagccc
     4081 acggtgcgcc tcgccacccg cgacgacgtc cccagggccg tacgcaccct cgccgccgcg
     4141 ttcgccgact accccgccac gcgccacacc gtcgatccgg accgccacat cgagcgggtc
     4201 accgagctgc aagaactctt cctcacgcgc gtcgggctcg acatcggcaa ggtgtgggtc
     4261 gcggacgacg gcgccgcggt ggcggtctgg accacgccgg agagcgtcga agcgggggcg
     4321 gtgttcgccg agatcggccc gcgcatggcc gagttgagcg gttcccggct ggccgcgcag
     4381 caacagatgg agggcctcct ggcgccgcac cggcccaagg agcccgcgtg gttcctggcc
     4441 accgtcggcg tctcgcccga ccaccagggc aagggtctgg gcagcgccgt cgtgctcccc
     4501 ggagtggagg ccgccgagcg cgccggggtg cccgccttcc tggagacctc cgcgccccgc
     4561 aacctcccct tctacgagcg gctcggcttc accgtcaccg ccgacgtcga ggtgcccgaa
     4621 ggaccgcgca cctggtgcat gacccgcaag cccggtgcct aggctagtat gtaagcctag
     4681 tcttagataa taaaatcgct atccatcgaa gatggatgtg tgttggtttt ttgtgtgtgt
     4741 aacgctaggc gcgcctggtg taccgagaac gatcctctca gtgcgagtct cgacgatcca
     4801 tatcgttgct tggcagtcag ccagtcggaa tccagcttgg gacccaggaa gtccaatcgt
     4861 cagatattgt actcaagcct ggtcacggca gcgtaccgat ctgtttaaac ctagatattg
     4921 atagtctgat cggtcaacgt ataatcgagt cctagctttt gcaaacatct atcaagagac
     4981 aggatcagca ggaggctttc gcatgagtat tcaacatttc cgtgtcgccc ttattccctt
     5041 ttttgcggca ttttgccttc ctgtttttgc tcacccagaa acgctggtga aagtaaaaga
     5101 tgctgaagat cagttgggtg cgcgagtggg ttacatcgaa ctggatctca acagcggtaa
     5161 gatccttgag agttttcgcc ccgaagaacg ctttccaatg atgagcactt ttaaagttct
     5221 gctatgtggc gcggtattat cccgtattga cgccgggcaa gagcaactcg gtcgccgcat
     5281 acactattct cagaatgact tggttgagta ttcaccagtc acagaaaagc atcttacgga
     5341 tggcatgaca gtaagagaat tatgcagtgc tgccataacc atgagtgata acactgcggc
     5401 caacttactt ctgacaacga ttggaggacc gaaggagcta accgcttttt tgcacaacat
     5461 gggggatcat gtaactcgcc ttgatcgttg ggaaccggag ctgaatgaag ccataccaaa
     5521 cgacgagcgt gacaccacga tgcctgtagc aatggcaaca accttgcgta aactattaac
     5581 tggcgaacta cttactctag cttcccggca acagttgata gactggatgg aggcggataa
     5641 agttgcagga ccacttctgc gctcggccct tccggctggc tggtttattg ctgataaatc
     5701 tggagccggt gagcgtgggt ctcgcggtat cattgcagca ctggggccag atggtaagcc
     5761 ctcccgtatc gtagttatct acacgacggg gagtcaggca actatggatg aacgaaatag
     5821 acagatcgct gagataggtg cctcactgat taagcattgg taaccgattc taggtgcatt
     5881 ggcgcagaaa aaaatgcctg atgcgacgct gcgcgtctta tactcccaca tatgccagat
     5941 tcagcaacgg atacggcttc cccaacttgc ccacttccat acgtgtcctc cttaccagaa
     6001 atttatcctt aagatcgttt aaactcgact ctggctctat cgaatctccg tcgtttcgag
     6061 cttacgcgaa cagccgtggc gctcatttgc tcgtcgggca tcgaatctcg tcagctatcg
     6121 tcagcttacc tttttggcag cgatcgcggc tcccgacatc ttggaccatt agctccacag
     6181 gtatcttctt ccctctagtg gtcataacag cagcttcagc tacctctcaa ttcaaaaaac
     6241 ccctcaagac ccgtttagag gccccaaggg gttatgctat caatcgttgc gttacacaca
     6301 caaaaaacca acacacatcc atcttcgatg gatagcgatt ttattatcta actgctgatc
     6361 gagtgtagcc agatctagta atcaattacg gggtcattag ttcatagccc atatatggag
     6421 ttccgcgtta cataacttac ggtaaatggc ccgcctggct gaccgcccaa cgacccccgc
     6481 ccattgacgt caataatgac gtatgttccc atagtaacgc caatagggac tttccattga
     6541 cgtcaatggg tggagtattt acggtaaact gcccacttgg cagtacatca agtgtatcat
     6601 atgccaagta cgccccctat tgacgtcaat gacggtaaat ggcccgcctg gcattatgcc
     6661 cagtacatga ccttatggga ctttcctact tggcagtaca tctacgtatt agtcatcgct
     6721 attaccatgc tgatgcggtt ttggcagtac atcaatgggc gtggatagcg gtttgactca
     6781 cggggatttc caagtctcca ccccattgac gtcaatggga gtttgttttg gcaccaaaat
     6841 caacgggact ttccaaaatg tcgtaacaac tccgccccat tgacgcaaat gggcggtagg
     6901 cgtgtacggt gggaggtcta tataagcaga gctggtttag tgaaccgtca gatcagatct
     6961 ttgtcgatcc taccatccac tcgacacacc cgccagcggc cgc
//
