LOCUS       sequence-295194-        7448 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 7448)
  TITLE     SARS-CoV-2 protein Mammalian expression constructs, tagged, for 
            protein expression and/or purification
REFERENCE   2  (bases 1 to 7448)
  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-m4133618
FEATURES             Location/Qualifiers
     source          1..7448
                     /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    988..1109
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(999..1018)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     1053..1072
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     terminator      1224..1270
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     terminator      1300..1436
                     /label=rrnG terminator
                     /note="transcription terminator from the E. coli ribosomal 
                     RNA rrnG operon (Albrechtsen et al., 1991)"
     misc_feature    1481..1552
                     /label=3' beta-globin insulator
                     /note="insulator downstream of the human beta-globin locus 
                     (Farrell et al., 2002)"
     rep_origin      complement(1778..2366)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1858..1877)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     promoter        2866..4075
                     /label=UbC promoter
                     /note="human ubiquitin C promoter"
     primer_bind     3912..3931
                     /label=hUBCpro-F
                     /note="Human Ubiquitin C (UbC) promoter, forward primer"
     CDS             4079..5107
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MGKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGR
                     GYVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDL
                     PETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHW
                     QTVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAM
                     FGDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVD
                     GNFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRA
                     KE"
     polyA_signal    5134..5182
                     /note="synthetic polyadenylation signal"
     CDS             join(5448..5516,5517..6308)
                     /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(5666..5685)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     terminator      6321..6457
                     /label=rrnG terminator
                     /note="transcription terminator from the E. coli ribosomal 
                     RNA rrnG operon (Albrechtsen et al., 1991)"
     misc_feature    6602..6673
                     /label=5' beta-globin insulator
                     /note="insulator upstream of the human beta-globin locus 
                     (Farrell et al., 2002)"
     terminator      complement(6678..6724)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     polyA_signal    6742..6790
                     /note="synthetic polyadenylation signal"
     enhancer        6871..7174
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        7176..7378
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     7329..7349
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      join(7447..7448,1..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 aaggatccgc aggcctctgc tagcttgact gactgagata
      961 cagcgtacct tcagctcaca gacatgataa gatacattga tgagtttgga caaaccacaa
     1021 ctagaatgca gtgaaaaaaa tgctttattt gtgaaatttg tgatgctatt gctttatttg
     1081 taaccattat aagctgcaat aaacaagtta acaacaacaa ttgcattcat tttatgtttc
     1141 aggttcaggg ggaggtgtgg gaggtttttt aaagcaagta aaacctctac aaatgtggta
     1201 ttggcccatc tctatcggta tcgtagcata accccttggg gcctctaaac gggtcttgag
     1261 gggttttttg tgcccctcgg gccggattgc tatctaccgg cattggcgca gaaaaaaatg
     1321 cctgatgcga cgctgcgcgt cttatactcc cacatatgcc agattcagca acggatacgg
     1381 cttccccaac ttgcccactt ccatacgtgt cctccttacc agaaatttat ccttaaggtc
     1441 gtcagctatc ctgcaggcga tctctcgatt tcgatcaaga cattccttta atggtctttt
     1501 ctggacacca ctaggggtca gaagtagttc atcaaacttt cttccctccc taatctcatt
     1561 ggttaccttg ggctatcgaa acttaattaa ccagtcaagt cagctacttg gcgagatcga
     1621 cttgtctggg tttcgactac gctcagaatt gcgtcagtca agttcgatct ggtccttgct
     1681 attgcacccg ttctccgatt acgagtttca tttaaatcat gtgagcaaaa ggccagcaaa
     1741 aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc cgcccccctg
     1801 acgagcatca caaaaatcga cgctcaagtc agaggtggcg aaacccgaca ggactataaa
     1861 gataccaggc gtttccccct ggaagctccc tcgtgcgctc tcctgttccg accctgccgc
     1921 ttaccggata cctgtccgcc tttctccctt cgggaagcgt ggcgctttct catagctcac
     1981 gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac
     2041 cccccgttca gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag tccaacccgg
     2101 taagacacga cttatcgcca ctggcagcag ccactggtaa caggattagc agagcgaggt
     2161 atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa ctacggctac actagaagaa
     2221 cagtatttgg tatctgcgct ctgctgaagc cagttacctt cggaaaaaga gttggtagct
     2281 cttgatccgg caaacaaacc accgctggta gcggtggttt ttttgtttgc aagcagcaga
     2341 ttacgcgcag aaaaaaagga tctcaagaag atcctttgat cttttctacg gggtctgacg
     2401 ctcagtggaa cgaaaactca cgttaaggga ttttggtcat gagattatca aaaaggatct
     2461 tcacctagat ccttttaaat taaaaatgaa gttttaaatc aatctaaagt atatatgagt
     2521 aaacttggtc tgacagttac caatgcttaa tcagtgaggc acctatctca gcgatctgtc
     2581 tatttcgttc atccatagtt gcatttaaat ttccgaactc tccaaggccc tcgtcggaaa
     2641 atcttcaaac ctttcgtccg atccatcttg caggctacct ctcgaacgaa ctatcgcaag
     2701 tctcttggcc ggccttgcgc cttggctatt gcttggcagc gcctatcgcc aggtattact
     2761 ccaatcccga atatccgaga tcgggatcac ccgagagaag ttcaacctac atcctcaatc
     2821 ccgatctatc cgagatccga ggaatatcga aatcggggcg cgcctggcct ccgcgccggg
     2881 ttttggcgcc tcccgcgggc gcccccctcg tcacggcgag cgctgccacg tcagacgaag
     2941 ggcgcaggag cgtcctgatc cttccgcccg gacgctcagg acagcggccc gctgctcata
     3001 agactcggcc ttagaacccc agtatcagca gaaggacatt ttaggacggg acttgggtga
     3061 ctctagggca ctggttttct ttccagagag cggaacaggc gaggaaaagt agtcccttct
     3121 cggcgattct gcggagggat ctccgtgggg cggtgaacgc cgatgattat ataaggacgc
     3181 gccgggtgtg gcacagctag ttccgtcgca gccgggattt gggtcgcggt tcttgtttgt
     3241 ggatcgctgt gatcgtcact tggtgagtag cgggctgctg ggctggccgg ggctttcgtg
     3301 gccgccgggc cgctcggtgg gacggaagcg tgtggagaga ccgccaaggg ctgtagtctg
     3361 ggtccgcgag caaggttgcc ctgaactggg ggttgggggg agcgcagcaa aatggcggct
     3421 gttcccgagt cttgaatgga agacgcttgt gaggcgggct gtgaggtcgt tgaaacaagg
     3481 tggggggcat ggtgggcggc aagaacccaa ggtcttgagc ccttcgctaa tgcgggaaag
     3541 ctcttattcg ggtgagatgg gctgggcacc atctggggac cctgacgtga agtttgtcac
     3601 tgactggaga actcggtttg tcgtctgttg cgggggcggc agttatggcg gtgccgttgg
     3661 gcagtgcacc cgtacctttg ggagcgcgcg ccctcgtcgt gtcgtgacgt cacccgttct
     3721 gttggcttat aatgcagggt ggggccacct gccggtaggt gtgcggtagg cttttctccg
     3781 tcgcaggacg cagggttcgg gcctagggta ggctctcctg aatcgacagg cgccggacct
     3841 ctggtgaggg gagggataag tgaggcgtca gtttctttgg tcggttttat gtacctatct
     3901 tcttaagtag ctgaagctcc ggttttgaac tatgcgctcg gggttggcga gtgtgttttg
     3961 tgaagttttt taggcacctt ttgaaatgta atcatttggg tcaatatgta attttcagtg
     4021 ttagacttgt aaattgtccg ctaaattctg gccgtttttg gcttttttgt tagacaacat
     4081 gggtaaaaag cctgaactca ccgcgacgtc tgtcgagaag tttctgatcg aaaagttcga
     4141 cagcgtctcc gacctgatgc agctctcgga gggcgaagaa tctcgtgctt tcagcttcga
     4201 tgtaggaggg cgtggatatg tcctgcgggt aaatagctgc gccgatggtt tctacaaaga
     4261 tcgttatgtt tatcggcact ttgcatccgc cgcgctcccg attccggaag tgcttgacat
     4321 tggggagttc agcgagagcc tgacctattg catctcccgc cgtgcacagg gtgtcacgtt
     4381 gcaagacctg cctgaaaccg aactgcccgc tgttctgcag ccggtcgcgg aggcaatgga
     4441 tgccatcgct gccgccgatc ttagccagac gagcgggttc ggcccattcg gaccgcaagg
     4501 aatcggtcaa tacactacat ggcgtgattt catatgcgcg attgctgatc cccatgtgta
     4561 tcactggcaa actgtgatgg acgacaccgt cagtgcgtcc gtcgcgcagg ctctcgatga
     4621 gctgatgctt tgggccgagg actgccccga agtccggcac ctcgtgcacg cggatttcgg
     4681 ctccaacaat gtcctgacgg acaatggccg cataacagcg gtcattgact ggagcgaggc
     4741 gatgttcggg gattcccaat acgaggtcgc caacatcttc ttctggaggc cgtggttggc
     4801 ttgtatggag cagcagacgc gctacttcga gcggaggcat ccggagcttg caggatcgcc
     4861 ccggctccgg gcgtatatgc tccgcattgg tcttgaccaa ctctatcaga gcttggttga
     4921 cggcaatttc gatgatgcag cttgggcgca gggtcgatgc gacgcaatcg tccgatccgg
     4981 agccgggact gtcgggcgta cacaaatcgc ccgcagaagc gccgccgtct ggaccgatgg
     5041 ctgtgtagaa gtactcgccg atagtggaaa ccgacgcccc agcactcgtc cgagggcaaa
     5101 ggaataagct agtatgtaag cctagtctta gataataaaa tcgctatcca tcgaagatgg
     5161 atgtgtgttg gttttttgtg tgtgtaacgc taggcgcgcc tggtgtaccg agaacgatcc
     5221 tctcagtgcg agtctcgacg atccatatcg ttgcttggca gtcagccagt cggaatccag
     5281 cttgggaccc aggaagtcca atcgtcagat attgtactca agcctggtca cggcagcgta
     5341 ccgatctgtt taaacctaga tattgatagt ctgatcggtc aacgtataat cgagtcctag
     5401 cttttgcaaa catctatcaa gagacaggat cagcaggagg ctttcgcatg agtattcaac
     5461 atttccgtgt cgcccttatt cccttttttg cggcattttg ccttcctgtt tttgctcacc
     5521 cagaaacgct ggtgaaagta aaagatgctg aagatcagtt gggtgcgcga gtgggttaca
     5581 tcgaactgga tctcaacagc ggtaagatcc ttgagagttt tcgccccgaa gaacgctttc
     5641 caatgatgag cacttttaaa gttctgctat gtggcgcggt attatcccgt attgacgccg
     5701 ggcaagagca actcggtcgc cgcatacact attctcagaa tgacttggtt gagtattcac
     5761 cagtcacaga aaagcatctt acggatggca tgacagtaag agaattatgc agtgctgcca
     5821 taaccatgag tgataacact gcggccaact tacttctgac aacgattgga ggaccgaagg
     5881 agctaaccgc ttttttgcac aacatggggg atcatgtaac tcgccttgat cgttgggaac
     5941 cggagctgaa tgaagccata ccaaacgacg agcgtgacac cacgatgcct gtagcaatgg
     6001 caacaacctt gcgtaaacta ttaactggcg aactacttac tctagcttcc cggcaacagt
     6061 tgatagactg gatggaggcg gataaagttg caggaccact tctgcgctcg gcccttccgg
     6121 ctggctggtt tattgctgat aaatctggag ccggtgagcg tgggtctcgc ggtatcattg
     6181 cagcactggg gccagatggt aagccctccc gtatcgtagt tatctacacg acggggagtc
     6241 aggcaactat ggatgaacga aatagacaga tcgctgagat aggtgcctca ctgattaagc
     6301 attggtaacc gattctaggt gcattggcgc agaaaaaaat gcctgatgcg acgctgcgcg
     6361 tcttatactc ccacatatgc cagattcagc aacggatacg gcttccccaa cttgcccact
     6421 tccatacgtg tcctccttac cagaaattta tccttaagat cgtttaaact cgactctggc
     6481 tctatcgaat ctccgtcgtt tcgagcttac gcgaacagcc gtggcgctca tttgctcgtc
     6541 gggcatcgaa tctcgtcagc tatcgtcagc ttaccttttt ggcagcgatc gcggctcccg
     6601 acatcttgga ccattagctc cacaggtatc ttcttccctc tagtggtcat aacagcagct
     6661 tcagctacct ctcaattcaa aaaacccctc aagacccgtt tagaggcccc aaggggttat
     6721 gctatcaatc gttgcgttac acacacaaaa aaccaacaca catccatctt cgatggatag
     6781 cgattttatt atctaactgc tgatcgagtg tagccagatc tagtaatcaa ttacggggtc
     6841 attagttcat agcccatata tggagttccg cgttacataa cttacggtaa atggcccgcc
     6901 tggctgaccg cccaacgacc cccgcccatt gacgtcaata atgacgtatg ttcccatagt
     6961 aacgccaata gggactttcc attgacgtca atgggtggag tatttacggt aaactgccca
     7021 cttggcagta catcaagtgt atcatatgcc aagtacgccc cctattgacg tcaatgacgg
     7081 taaatggccc gcctggcatt atgcccagta catgacctta tgggactttc ctacttggca
     7141 gtacatctac gtattagtca tcgctattac catgctgatg cggttttggc agtacatcaa
     7201 tgggcgtgga tagcggtttg actcacgggg atttccaagt ctccacccca ttgacgtcaa
     7261 tgggagtttg ttttggcacc aaaatcaacg ggactttcca aaatgtcgta acaactccgc
     7321 cccattgacg caaatgggcg gtaggcgtgt acggtgggag gtctatataa gcagagctgg
     7381 tttagtgaac cgtcagatca gatctttgtc gatcctacca tccactcgac acacccgcca
     7441 gcggccgc
//
