LOCUS       sequence-295638-        7012 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 7012)
  TITLE     SARS-CoV-2 protein Mammalian expression constructs, tagged, for 
            protein expression and/or purification
REFERENCE   2  (bases 1 to 7012)
  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-m4134523
FEATURES             Location/Qualifiers
     source          1..7012
                     /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..928
                     /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"
     polyA_signal    981..1102
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(992..1011)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     1046..1065
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     terminator      1217..1263
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     terminator      1293..1429
                     /label=rrnG terminator
                     /note="transcription terminator from the E. coli ribosomal 
                     RNA rrnG operon (Albrechtsen et al., 1991)"
     misc_feature    1474..1545
                     /label=3' beta-globin insulator
                     /note="insulator downstream of the human beta-globin locus 
                     (Farrell et al., 2002)"
     rep_origin      complement(1771..2359)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1851..1870)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     promoter        2859..4068
                     /label=UbC promoter
                     /note="human ubiquitin C promoter"
     primer_bind     3905..3924
                     /label=hUBCpro-F
                     /note="Human Ubiquitin C (UbC) promoter, forward primer"
     CDS             4072..4671
                     /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(4072..4091)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     4568..4588
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     polyA_signal    4698..4746
                     /note="synthetic polyadenylation signal"
     CDS             join(5012..5080,5081..5872)
                     /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(5230..5249)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     terminator      5885..6021
                     /label=rrnG terminator
                     /note="transcription terminator from the E. coli ribosomal 
                     RNA rrnG operon (Albrechtsen et al., 1991)"
     misc_feature    6166..6237
                     /label=5' beta-globin insulator
                     /note="insulator upstream of the human beta-globin locus 
                     (Farrell et al., 2002)"
     terminator      complement(6242..6288)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     polyA_signal    6306..6354
                     /note="synthetic polyadenylation signal"
     enhancer        6435..6738
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        6740..6942
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     6893..6913
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      join(7011..7012,1..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 aagaggcgat ggaaaaagaa ttttatcgcg gtgagtgcag ccaacagatt
      901 caaaaagata tcctcgtctg gtgccctcta agctagcttg actgactgag atacagcgta
      961 ccttcagctc acagacatga taagatacat tgatgagttt ggacaaacca caactagaat
     1021 gcagtgaaaa aaatgcttta tttgtgaaat ttgtgatgct attgctttat ttgtaaccat
     1081 tataagctgc aataaacaag ttaacaacaa caattgcatt cattttatgt ttcaggttca
     1141 gggggaggtg tgggaggttt tttaaagcaa gtaaaacctc tacaaatgtg gtattggccc
     1201 atctctatcg gtatcgtagc ataacccctt ggggcctcta aacgggtctt gaggggtttt
     1261 ttgtgcccct cgggccggat tgctatctac cggcattggc gcagaaaaaa atgcctgatg
     1321 cgacgctgcg cgtcttatac tcccacatat gccagattca gcaacggata cggcttcccc
     1381 aacttgccca cttccatacg tgtcctcctt accagaaatt tatccttaag gtcgtcagct
     1441 atcctgcagg cgatctctcg atttcgatca agacattcct ttaatggtct tttctggaca
     1501 ccactagggg tcagaagtag ttcatcaaac tttcttccct ccctaatctc attggttacc
     1561 ttgggctatc gaaacttaat taaccagtca agtcagctac ttggcgagat cgacttgtct
     1621 gggtttcgac tacgctcaga attgcgtcag tcaagttcga tctggtcctt gctattgcac
     1681 ccgttctccg attacgagtt tcatttaaat catgtgagca aaaggccagc aaaaggccag
     1741 gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg ctccgccccc ctgacgagca
     1801 tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg acaggactat aaagatacca
     1861 ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg
     1921 atacctgtcc gcctttctcc cttcgggaag cgtggcgctt tctcatagct cacgctgtag
     1981 gtatctcagt tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt
     2041 tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc cggtaagaca
     2101 cgacttatcg ccactggcag cagccactgg taacaggatt agcagagcga ggtatgtagg
     2161 cggtgctaca gagttcttga agtggtggcc taactacggc tacactagaa gaacagtatt
     2221 tggtatctgc gctctgctga agccagttac cttcggaaaa agagttggta gctcttgatc
     2281 cggcaaacaa accaccgctg gtagcggtgg tttttttgtt tgcaagcagc agattacgcg
     2341 cagaaaaaaa ggatctcaag aagatccttt gatcttttct acggggtctg acgctcagtg
     2401 gaacgaaaac tcacgttaag ggattttggt catgagatta tcaaaaagga tcttcaccta
     2461 gatcctttta aattaaaaat gaagttttaa atcaatctaa agtatatatg agtaaacttg
     2521 gtctgacagt taccaatgct taatcagtga ggcacctatc tcagcgatct gtctatttcg
     2581 ttcatccata gttgcattta aatttccgaa ctctccaagg ccctcgtcgg aaaatcttca
     2641 aacctttcgt ccgatccatc ttgcaggcta cctctcgaac gaactatcgc aagtctcttg
     2701 gccggccttg cgccttggct attgcttggc agcgcctatc gccaggtatt actccaatcc
     2761 cgaatatccg agatcgggat cacccgagag aagttcaacc tacatcctca atcccgatct
     2821 atccgagatc cgaggaatat cgaaatcggg gcgcgcctgg cctccgcgcc gggttttggc
     2881 gcctcccgcg ggcgcccccc tcgtcacggc gagcgctgcc acgtcagacg aagggcgcag
     2941 gagcgtcctg atccttccgc ccggacgctc aggacagcgg cccgctgctc ataagactcg
     3001 gccttagaac cccagtatca gcagaaggac attttaggac gggacttggg tgactctagg
     3061 gcactggttt tctttccaga gagcggaaca ggcgaggaaa agtagtccct tctcggcgat
     3121 tctgcggagg gatctccgtg gggcggtgaa cgccgatgat tatataagga cgcgccgggt
     3181 gtggcacagc tagttccgtc gcagccggga tttgggtcgc ggttcttgtt tgtggatcgc
     3241 tgtgatcgtc acttggtgag tagcgggctg ctgggctggc cggggctttc gtggccgccg
     3301 ggccgctcgg tgggacggaa gcgtgtggag agaccgccaa gggctgtagt ctgggtccgc
     3361 gagcaaggtt gccctgaact gggggttggg gggagcgcag caaaatggcg gctgttcccg
     3421 agtcttgaat ggaagacgct tgtgaggcgg gctgtgaggt cgttgaaaca aggtgggggg
     3481 catggtgggc ggcaagaacc caaggtcttg agcccttcgc taatgcggga aagctcttat
     3541 tcgggtgaga tgggctgggc accatctggg gaccctgacg tgaagtttgt cactgactgg
     3601 agaactcggt ttgtcgtctg ttgcgggggc ggcagttatg gcggtgccgt tgggcagtgc
     3661 acccgtacct ttgggagcgc gcgccctcgt cgtgtcgtga cgtcacccgt tctgttggct
     3721 tataatgcag ggtggggcca cctgccggta ggtgtgcggt aggcttttct ccgtcgcagg
     3781 acgcagggtt cgggcctagg gtaggctctc ctgaatcgac aggcgccgga cctctggtga
     3841 ggggagggat aagtgaggcg tcagtttctt tggtcggttt tatgtaccta tcttcttaag
     3901 tagctgaagc tccggttttg aactatgcgc tcggggttgg cgagtgtgtt ttgtgaagtt
     3961 ttttaggcac cttttgaaat gtaatcattt gggtcaatat gtaattttca gtgttagact
     4021 tgtaaattgt ccgctaaatt ctggccgttt ttggcttttt tgttagacaa catgaccgag
     4081 tacaagccca cggtgcgcct cgccacccgc gacgacgtcc ccagggccgt acgcaccctc
     4141 gccgccgcgt tcgccgacta ccccgccacg cgccacaccg tcgatccgga ccgccacatc
     4201 gagcgggtca ccgagctgca agaactcttc ctcacgcgcg tcgggctcga catcggcaag
     4261 gtgtgggtcg cggacgacgg cgccgcggtg gcggtctgga ccacgccgga gagcgtcgaa
     4321 gcgggggcgg tgttcgccga gatcggcccg cgcatggccg agttgagcgg ttcccggctg
     4381 gccgcgcagc aacagatgga gggcctcctg gcgccgcacc ggcccaagga gcccgcgtgg
     4441 ttcctggcca ccgtcggcgt ctcgcccgac caccagggca agggtctggg cagcgccgtc
     4501 gtgctccccg gagtggaggc cgccgagcgc gccggggtgc ccgccttcct ggagacctcc
     4561 gcgccccgca acctcccctt ctacgagcgg ctcggcttca ccgtcaccgc cgacgtcgag
     4621 gtgcccgaag gaccgcgcac ctggtgcatg acccgcaagc ccggtgccta ggctagtatg
     4681 taagcctagt cttagataat aaaatcgcta tccatcgaag atggatgtgt gttggttttt
     4741 tgtgtgtgta acgctaggcg cgcctggtgt accgagaacg atcctctcag tgcgagtctc
     4801 gacgatccat atcgttgctt ggcagtcagc cagtcggaat ccagcttggg acccaggaag
     4861 tccaatcgtc agatattgta ctcaagcctg gtcacggcag cgtaccgatc tgtttaaacc
     4921 tagatattga tagtctgatc ggtcaacgta taatcgagtc ctagcttttg caaacatcta
     4981 tcaagagaca ggatcagcag gaggctttcg catgagtatt caacatttcc gtgtcgccct
     5041 tattcccttt tttgcggcat tttgccttcc tgtttttgct cacccagaaa cgctggtgaa
     5101 agtaaaagat gctgaagatc agttgggtgc gcgagtgggt tacatcgaac tggatctcaa
     5161 cagcggtaag atccttgaga gttttcgccc cgaagaacgc tttccaatga tgagcacttt
     5221 taaagttctg ctatgtggcg cggtattatc ccgtattgac gccgggcaag agcaactcgg
     5281 tcgccgcata cactattctc agaatgactt ggttgagtat tcaccagtca cagaaaagca
     5341 tcttacggat ggcatgacag taagagaatt atgcagtgct gccataacca tgagtgataa
     5401 cactgcggcc aacttacttc tgacaacgat tggaggaccg aaggagctaa ccgctttttt
     5461 gcacaacatg ggggatcatg taactcgcct tgatcgttgg gaaccggagc tgaatgaagc
     5521 cataccaaac gacgagcgtg acaccacgat gcctgtagca atggcaacaa ccttgcgtaa
     5581 actattaact ggcgaactac ttactctagc ttcccggcaa cagttgatag actggatgga
     5641 ggcggataaa gttgcaggac cacttctgcg ctcggccctt ccggctggct ggtttattgc
     5701 tgataaatct ggagccggtg agcgtgggtc tcgcggtatc attgcagcac tggggccaga
     5761 tggtaagccc tcccgtatcg tagttatcta cacgacgggg agtcaggcaa ctatggatga
     5821 acgaaataga cagatcgctg agataggtgc ctcactgatt aagcattggt aaccgattct
     5881 aggtgcattg gcgcagaaaa aaatgcctga tgcgacgctg cgcgtcttat actcccacat
     5941 atgccagatt cagcaacgga tacggcttcc ccaacttgcc cacttccata cgtgtcctcc
     6001 ttaccagaaa tttatcctta agatcgttta aactcgactc tggctctatc gaatctccgt
     6061 cgtttcgagc ttacgcgaac agccgtggcg ctcatttgct cgtcgggcat cgaatctcgt
     6121 cagctatcgt cagcttacct ttttggcagc gatcgcggct cccgacatct tggaccatta
     6181 gctccacagg tatcttcttc cctctagtgg tcataacagc agcttcagct acctctcaat
     6241 tcaaaaaacc cctcaagacc cgtttagagg ccccaagggg ttatgctatc aatcgttgcg
     6301 ttacacacac aaaaaaccaa cacacatcca tcttcgatgg atagcgattt tattatctaa
     6361 ctgctgatcg agtgtagcca gatctagtaa tcaattacgg ggtcattagt tcatagccca
     6421 tatatggagt tccgcgttac ataacttacg gtaaatggcc cgcctggctg accgcccaac
     6481 gacccccgcc cattgacgtc aataatgacg tatgttccca tagtaacgcc aatagggact
     6541 ttccattgac gtcaatgggt ggagtattta cggtaaactg cccacttggc agtacatcaa
     6601 gtgtatcata tgccaagtac gccccctatt gacgtcaatg acggtaaatg gcccgcctgg
     6661 cattatgccc agtacatgac cttatgggac tttcctactt ggcagtacat ctacgtatta
     6721 gtcatcgcta ttaccatgct gatgcggttt tggcagtaca tcaatgggcg tggatagcgg
     6781 tttgactcac ggggatttcc aagtctccac cccattgacg tcaatgggag tttgttttgg
     6841 caccaaaatc aacgggactt tccaaaatgt cgtaacaact ccgccccatt gacgcaaatg
     6901 ggcggtaggc gtgtacggtg ggaggtctat ataagcagag ctggtttagt gaaccgtcag
     6961 atcagatctt tgtcgatcct accatccact cgacacaccc gccagcggcc gc
//
