LOCUS       sequence-299795-        7496 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 7496)
  TITLE     SARS-CoV-2 protein Mammalian expression constructs, tagged, for 
            protein expression and/or purification
REFERENCE   2  (bases 1 to 7496)
  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-m4134116
FEATURES             Location/Qualifiers
     source          1..7496
                     /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             884..961
                     /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"
     CDS             989..1009
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     polyA_signal    1458..1579
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1469..1488)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     1523..1542
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     terminator      1694..1740
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     terminator      1770..1906
                     /label=rrnG terminator
                     /note="transcription terminator from the E. coli ribosomal 
                     RNA rrnG operon (Albrechtsen et al., 1991)"
     misc_feature    1951..2022
                     /label=3' beta-globin insulator
                     /note="insulator downstream of the human beta-globin locus 
                     (Farrell et al., 2002)"
     rep_origin      complement(2248..2836)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2328..2347)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     promoter        3336..4545
                     /label=UbC promoter
                     /note="human ubiquitin C promoter"
     primer_bind     4382..4401
                     /label=hUBCpro-F
                     /note="Human Ubiquitin C (UbC) promoter, forward primer"
     CDS             4549..5148
                     /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(4549..4568)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     5045..5065
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     polyA_signal    5175..5223
                     /note="synthetic polyadenylation signal"
     CDS             join(5489..5557,5558..6349)
                     /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(5707..5726)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     terminator      6362..6498
                     /label=rrnG terminator
                     /note="transcription terminator from the E. coli ribosomal 
                     RNA rrnG operon (Albrechtsen et al., 1991)"
     misc_feature    6650..6721
                     /label=5' beta-globin insulator
                     /note="insulator upstream of the human beta-globin locus 
                     (Farrell et al., 2002)"
     terminator      complement(6726..6772)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     polyA_signal    6790..6838
                     /note="synthetic polyadenylation signal"
     enhancer        6919..7222
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        7224..7426
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     7377..7397
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      join(7495..7496,1..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 ggacggcgaa tcccggggct gataaatccc tggaaaagga gatggaagaa
      901 gaactttatt gcagtgtctg cggctaaccg gttcaaaaag atcagttcaa gcggcgccct
      961 agattacgac attcctacga ccgcctccga gaatctctat ttccagggtg aatttggctt
     1021 ggcacaacat gacgaggccg tcgataacaa gtttaataaa gaacagcaga acgctttcta
     1081 tgagatcctt cacttaccaa atctgaacga agagcaacgc aacgctttca ttcagagcct
     1141 gaaagacgat ccctcgcagt ctgcaaatct cctggccgaa gccaagaagc tgaatgacgc
     1201 acaggctcca aaagttgata acaagtttaa caaggagcag cagaatgctt tctacgagat
     1261 attgcacctt cctaatctga atgaggaaca aagaaatgca tttatccagt ccctaaaaga
     1321 cgatccatca cagagtgcca acctgcttgc ggaagccaaa aagctcaatg atgcccaggc
     1381 cccgaaagta gacgcgaacc atcaataagc tagcttgact gactgagata cagcgtacct
     1441 tcagctcaca gacatgataa gatacattga tgagtttgga caaaccacaa ctagaatgca
     1501 gtgaaaaaaa tgctttattt gtgaaatttg tgatgctatt gctttatttg taaccattat
     1561 aagctgcaat aaacaagtta acaacaacaa ttgcattcat tttatgtttc aggttcaggg
     1621 ggaggtgtgg gaggtttttt aaagcaagta aaacctctac aaatgtggta ttggcccatc
     1681 tctatcggta tcgtagcata accccttggg gcctctaaac gggtcttgag gggttttttg
     1741 tgcccctcgg gccggattgc tatctaccgg cattggcgca gaaaaaaatg cctgatgcga
     1801 cgctgcgcgt cttatactcc cacatatgcc agattcagca acggatacgg cttccccaac
     1861 ttgcccactt ccatacgtgt cctccttacc agaaatttat ccttaaggtc gtcagctatc
     1921 ctgcaggcga tctctcgatt tcgatcaaga cattccttta atggtctttt ctggacacca
     1981 ctaggggtca gaagtagttc atcaaacttt cttccctccc taatctcatt ggttaccttg
     2041 ggctatcgaa acttaattaa ccagtcaagt cagctacttg gcgagatcga cttgtctggg
     2101 tttcgactac gctcagaatt gcgtcagtca agttcgatct ggtccttgct attgcacccg
     2161 ttctccgatt acgagtttca tttaaatcat gtgagcaaaa ggccagcaaa aggccaggaa
     2221 ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc cgcccccctg acgagcatca
     2281 caaaaatcga cgctcaagtc agaggtggcg aaacccgaca ggactataaa gataccaggc
     2341 gtttccccct ggaagctccc tcgtgcgctc tcctgttccg accctgccgc ttaccggata
     2401 cctgtccgcc tttctccctt cgggaagcgt ggcgctttct catagctcac gctgtaggta
     2461 tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac cccccgttca
     2521 gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag tccaacccgg taagacacga
     2581 cttatcgcca ctggcagcag ccactggtaa caggattagc agagcgaggt atgtaggcgg
     2641 tgctacagag ttcttgaagt ggtggcctaa ctacggctac actagaagaa cagtatttgg
     2701 tatctgcgct ctgctgaagc cagttacctt cggaaaaaga gttggtagct cttgatccgg
     2761 caaacaaacc accgctggta gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag
     2821 aaaaaaagga tctcaagaag atcctttgat cttttctacg gggtctgacg ctcagtggaa
     2881 cgaaaactca cgttaaggga ttttggtcat gagattatca aaaaggatct tcacctagat
     2941 ccttttaaat taaaaatgaa gttttaaatc aatctaaagt atatatgagt aaacttggtc
     3001 tgacagttac caatgcttaa tcagtgaggc acctatctca gcgatctgtc tatttcgttc
     3061 atccatagtt gcatttaaat ttccgaactc tccaaggccc tcgtcggaaa atcttcaaac
     3121 ctttcgtccg atccatcttg caggctacct ctcgaacgaa ctatcgcaag tctcttggcc
     3181 ggccttgcgc cttggctatt gcttggcagc gcctatcgcc aggtattact ccaatcccga
     3241 atatccgaga tcgggatcac ccgagagaag ttcaacctac atcctcaatc ccgatctatc
     3301 cgagatccga ggaatatcga aatcggggcg cgcctggcct ccgcgccggg ttttggcgcc
     3361 tcccgcgggc gcccccctcg tcacggcgag cgctgccacg tcagacgaag ggcgcaggag
     3421 cgtcctgatc cttccgcccg gacgctcagg acagcggccc gctgctcata agactcggcc
     3481 ttagaacccc agtatcagca gaaggacatt ttaggacggg acttgggtga ctctagggca
     3541 ctggttttct ttccagagag cggaacaggc gaggaaaagt agtcccttct cggcgattct
     3601 gcggagggat ctccgtgggg cggtgaacgc cgatgattat ataaggacgc gccgggtgtg
     3661 gcacagctag ttccgtcgca gccgggattt gggtcgcggt tcttgtttgt ggatcgctgt
     3721 gatcgtcact tggtgagtag cgggctgctg ggctggccgg ggctttcgtg gccgccgggc
     3781 cgctcggtgg gacggaagcg tgtggagaga ccgccaaggg ctgtagtctg ggtccgcgag
     3841 caaggttgcc ctgaactggg ggttgggggg agcgcagcaa aatggcggct gttcccgagt
     3901 cttgaatgga agacgcttgt gaggcgggct gtgaggtcgt tgaaacaagg tggggggcat
     3961 ggtgggcggc aagaacccaa ggtcttgagc ccttcgctaa tgcgggaaag ctcttattcg
     4021 ggtgagatgg gctgggcacc atctggggac cctgacgtga agtttgtcac tgactggaga
     4081 actcggtttg tcgtctgttg cgggggcggc agttatggcg gtgccgttgg gcagtgcacc
     4141 cgtacctttg ggagcgcgcg ccctcgtcgt gtcgtgacgt cacccgttct gttggcttat
     4201 aatgcagggt ggggccacct gccggtaggt gtgcggtagg cttttctccg tcgcaggacg
     4261 cagggttcgg gcctagggta ggctctcctg aatcgacagg cgccggacct ctggtgaggg
     4321 gagggataag tgaggcgtca gtttctttgg tcggttttat gtacctatct tcttaagtag
     4381 ctgaagctcc ggttttgaac tatgcgctcg gggttggcga gtgtgttttg tgaagttttt
     4441 taggcacctt ttgaaatgta atcatttggg tcaatatgta attttcagtg ttagacttgt
     4501 aaattgtccg ctaaattctg gccgtttttg gcttttttgt tagacaacat gaccgagtac
     4561 aagcccacgg tgcgcctcgc cacccgcgac gacgtcccca gggccgtacg caccctcgcc
     4621 gccgcgttcg ccgactaccc cgccacgcgc cacaccgtcg atccggaccg ccacatcgag
     4681 cgggtcaccg agctgcaaga actcttcctc acgcgcgtcg ggctcgacat cggcaaggtg
     4741 tgggtcgcgg acgacggcgc cgcggtggcg gtctggacca cgccggagag cgtcgaagcg
     4801 ggggcggtgt tcgccgagat cggcccgcgc atggccgagt tgagcggttc ccggctggcc
     4861 gcgcagcaac agatggaggg cctcctggcg ccgcaccggc ccaaggagcc cgcgtggttc
     4921 ctggccaccg tcggcgtctc gcccgaccac cagggcaagg gtctgggcag cgccgtcgtg
     4981 ctccccggag tggaggccgc cgagcgcgcc ggggtgcccg ccttcctgga gacctccgcg
     5041 ccccgcaacc tccccttcta cgagcggctc ggcttcaccg tcaccgccga cgtcgaggtg
     5101 cccgaaggac cgcgcacctg gtgcatgacc cgcaagcccg gtgcctaggc tagtatgtaa
     5161 gcctagtctt agataataaa atcgctatcc atcgaagatg gatgtgtgtt ggttttttgt
     5221 gtgtgtaacg ctaggcgcgc ctggtgtacc gagaacgatc ctctcagtgc gagtctcgac
     5281 gatccatatc gttgcttggc agtcagccag tcggaatcca gcttgggacc caggaagtcc
     5341 aatcgtcaga tattgtactc aagcctggtc acggcagcgt accgatctgt ttaaacctag
     5401 atattgatag tctgatcggt caacgtataa tcgagtccta gcttttgcaa acatctatca
     5461 agagacagga tcagcaggag gctttcgcat gagtattcaa catttccgtg tcgcccttat
     5521 tccctttttt gcggcatttt gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt
     5581 aaaagatgct gaagatcagt tgggtgcgcg agtgggttac atcgaactgg atctcaacag
     5641 cggtaagatc cttgagagtt ttcgccccga agaacgcttt ccaatgatga gcacttttaa
     5701 agttctgcta tgtggcgcgg tattatcccg tattgacgcc gggcaagagc aactcggtcg
     5761 ccgcatacac tattctcaga atgacttggt tgagtattca ccagtcacag aaaagcatct
     5821 tacggatggc atgacagtaa gagaattatg cagtgctgcc ataaccatga gtgataacac
     5881 tgcggccaac ttacttctga caacgattgg aggaccgaag gagctaaccg cttttttgca
     5941 caacatgggg gatcatgtaa ctcgccttga tcgttgggaa ccggagctga atgaagccat
     6001 accaaacgac gagcgtgaca ccacgatgcc tgtagcaatg gcaacaacct tgcgtaaact
     6061 attaactggc gaactactta ctctagcttc ccggcaacag ttgatagact ggatggaggc
     6121 ggataaagtt gcaggaccac ttctgcgctc ggcccttccg gctggctggt ttattgctga
     6181 taaatctgga gccggtgagc gtgggtctcg cggtatcatt gcagcactgg ggccagatgg
     6241 taagccctcc cgtatcgtag ttatctacac gacggggagt caggcaacta tggatgaacg
     6301 aaatagacag atcgctgaga taggtgcctc actgattaag cattggtaac cgattctagg
     6361 tgcattggcg cagaaaaaaa tgcctgatgc gacgctgcgc gtcttatact cccacatatg
     6421 ccagattcag caacggatac ggcttcccca acttgcccac ttccatacgt gtcctcctta
     6481 ccagaaattt atccttaaga tcccgaatcg tttaaactcg actctggctc tatcgaatct
     6541 ccgtcgtttc gagcttacgc gaacagccgt ggcgctcatt tgctcgtcgg gcatcgaatc
     6601 tcgtcagcta tcgtcagctt acctttttgg cagcgatcgc ggctcccgac atcttggacc
     6661 attagctcca caggtatctt cttccctcta gtggtcataa cagcagcttc agctacctct
     6721 caattcaaaa aacccctcaa gacccgttta gaggccccaa ggggttatgc tatcaatcgt
     6781 tgcgttacac acacaaaaaa ccaacacaca tccatcttcg atggatagcg attttattat
     6841 ctaactgctg atcgagtgta gccagatcta gtaatcaatt acggggtcat tagttcatag
     6901 cccatatatg gagttccgcg ttacataact tacggtaaat ggcccgcctg gctgaccgcc
     6961 caacgacccc cgcccattga cgtcaataat gacgtatgtt cccatagtaa cgccaatagg
     7021 gactttccat tgacgtcaat gggtggagta tttacggtaa actgcccact tggcagtaca
     7081 tcaagtgtat catatgccaa gtacgccccc tattgacgtc aatgacggta aatggcccgc
     7141 ctggcattat gcccagtaca tgaccttatg ggactttcct acttggcagt acatctacgt
     7201 attagtcatc gctattacca tgctgatgcg gttttggcag tacatcaatg ggcgtggata
     7261 gcggtttgac tcacggggat ttccaagtct ccaccccatt gacgtcaatg ggagtttgtt
     7321 ttggcaccaa aatcaacggg actttccaaa atgtcgtaac aactccgccc cattgacgca
     7381 aatgggcggt aggcgtgtac ggtgggaggt ctatataagc agagctggtt tagtgaaccg
     7441 tcagatcaga tctttgtcga tcctaccatc cactcgacac acccgccagc ggccgc
//
