LOCUS       sequence-295037-        6715 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian expression plasmid for SARS-CoV-2 leader protein.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6715)
  TITLE     SARS-CoV-2 protein Mammalian expression constructs, tagged, for 
            protein expression and/or purification
REFERENCE   2  (bases 1 to 6715)
  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-m4133278
FEATURES             Location/Qualifiers
     source          1..6715
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             542..562
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQS"
     CDS             566..631
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     polyA_signal    684..805
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(695..714)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     749..768
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     terminator      920..966
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     terminator      996..1132
                     /label=rrnG terminator
                     /note="transcription terminator from the E. coli ribosomal 
                     RNA rrnG operon (Albrechtsen et al., 1991)"
     misc_feature    1177..1248
                     /label=3' beta-globin insulator
                     /note="insulator downstream of the human beta-globin locus 
                     (Farrell et al., 2002)"
     rep_origin      complement(1474..2062)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1554..1573)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     promoter        2562..3771
                     /label=UbC promoter
                     /note="human ubiquitin C promoter"
     primer_bind     3608..3627
                     /label=hUBCpro-F
                     /note="Human Ubiquitin C (UbC) promoter, forward primer"
     CDS             3775..4374
                     /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(3775..3794)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     4271..4291
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     polyA_signal    4401..4449
                     /note="synthetic polyadenylation signal"
     CDS             join(4715..4783,4784..5575)
                     /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(4933..4952)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     terminator      5588..5724
                     /label=rrnG terminator
                     /note="transcription terminator from the E. coli ribosomal 
                     RNA rrnG operon (Albrechtsen et al., 1991)"
     misc_feature    5869..5940
                     /label=5' beta-globin insulator
                     /note="insulator upstream of the human beta-globin locus 
                     (Farrell et al., 2002)"
     terminator      complement(5945..5991)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     polyA_signal    6009..6057
                     /note="synthetic polyadenylation signal"
     enhancer        6138..6441
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        6443..6645
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     6596..6616
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      join(6714..6715,1..8)
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
ORIGIN
        1 caccatggag agccttgtcc ctggtttcaa cgagaaaaca cacgtccaac tcagtttgcc
       61 tgttttacag gttcgcgacg tgctcgtacg tggctttgga gactccgtgg aggaggtctt
      121 atcagaggca cgtcaacatc ttaaagatgg cacttgtggc ttagtagaag ttgaaaaagg
      181 cgttttgcct caacttgaac agccctatgt gttcatcaaa cgttcggatg ctcgaactgc
      241 acctcatggt catgttatgg ttgagctggt agcagaactc gaaggcattc agtacggtcg
      301 tagtggtgag acacttggtg tccttgtccc tcatgtgggc gaaataccag tggcttaccg
      361 caaggttctt cttcgtaaga acggtaataa aggagctggt ggccatagtt acggcgccga
      421 tctaaagtca tttgacttag gcgacgagct tggcactgat ccttatgaag attttcaaga
      481 aaactggaac actaaacata gcagtggtgt tacccgtgaa ctcatgcgtg agcttaacgg
      541 agaaaacctt tactttcagt cgatggatta taaggaccac gatggagact acaaagatca
      601 tgacattgat tacaaggacg acgatgacaa ataagctagc ttgactgact gagatacagc
      661 gtaccttcag ctcacagaca tgataagata cattgatgag tttggacaaa ccacaactag
      721 aatgcagtga aaaaaatgct ttatttgtga aatttgtgat gctattgctt tatttgtaac
      781 cattataagc tgcaataaac aagttaacaa caacaattgc attcatttta tgtttcaggt
      841 tcagggggag gtgtgggagg ttttttaaag caagtaaaac ctctacaaat gtggtattgg
      901 cccatctcta tcggtatcgt agcataaccc cttggggcct ctaaacgggt cttgaggggt
      961 tttttgtgcc cctcgggccg gattgctatc taccggcatt ggcgcagaaa aaaatgcctg
     1021 atgcgacgct gcgcgtctta tactcccaca tatgccagat tcagcaacgg atacggcttc
     1081 cccaacttgc ccacttccat acgtgtcctc cttaccagaa atttatcctt aaggtcgtca
     1141 gctatcctgc aggcgatctc tcgatttcga tcaagacatt cctttaatgg tcttttctgg
     1201 acaccactag gggtcagaag tagttcatca aactttcttc cctccctaat ctcattggtt
     1261 accttgggct atcgaaactt aattaaccag tcaagtcagc tacttggcga gatcgacttg
     1321 tctgggtttc gactacgctc agaattgcgt cagtcaagtt cgatctggtc cttgctattg
     1381 cacccgttct ccgattacga gtttcattta aatcatgtga gcaaaaggcc agcaaaaggc
     1441 caggaaccgt aaaaaggccg cgttgctggc gtttttccat aggctccgcc cccctgacga
     1501 gcatcacaaa aatcgacgct caagtcagag gtggcgaaac ccgacaggac tataaagata
     1561 ccaggcgttt ccccctggaa gctccctcgt gcgctctcct gttccgaccc tgccgcttac
     1621 cggatacctg tccgcctttc tcccttcggg aagcgtggcg ctttctcata gctcacgctg
     1681 taggtatctc agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc
     1741 cgttcagccc gaccgctgcg ccttatccgg taactatcgt cttgagtcca acccggtaag
     1801 acacgactta tcgccactgg cagcagccac tggtaacagg attagcagag cgaggtatgt
     1861 aggcggtgct acagagttct tgaagtggtg gcctaactac ggctacacta gaagaacagt
     1921 atttggtatc tgcgctctgc tgaagccagt taccttcgga aaaagagttg gtagctcttg
     1981 atccggcaaa caaaccaccg ctggtagcgg tggttttttt gtttgcaagc agcagattac
     2041 gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt tctacggggt ctgacgctca
     2101 gtggaacgaa aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac
     2161 ctagatcctt ttaaattaaa aatgaagttt taaatcaatc taaagtatat atgagtaaac
     2221 ttggtctgac agttaccaat gcttaatcag tgaggcacct atctcagcga tctgtctatt
     2281 tcgttcatcc atagttgcat ttaaatttcc gaactctcca aggccctcgt cggaaaatct
     2341 tcaaaccttt cgtccgatcc atcttgcagg ctacctctcg aacgaactat cgcaagtctc
     2401 ttggccggcc ttgcgccttg gctattgctt ggcagcgcct atcgccaggt attactccaa
     2461 tcccgaatat ccgagatcgg gatcacccga gagaagttca acctacatcc tcaatcccga
     2521 tctatccgag atccgaggaa tatcgaaatc ggggcgcgcc tggcctccgc gccgggtttt
     2581 ggcgcctccc gcgggcgccc ccctcgtcac ggcgagcgct gccacgtcag acgaagggcg
     2641 caggagcgtc ctgatccttc cgcccggacg ctcaggacag cggcccgctg ctcataagac
     2701 tcggccttag aaccccagta tcagcagaag gacattttag gacgggactt gggtgactct
     2761 agggcactgg ttttctttcc agagagcgga acaggcgagg aaaagtagtc ccttctcggc
     2821 gattctgcgg agggatctcc gtggggcggt gaacgccgat gattatataa ggacgcgccg
     2881 ggtgtggcac agctagttcc gtcgcagccg ggatttgggt cgcggttctt gtttgtggat
     2941 cgctgtgatc gtcacttggt gagtagcggg ctgctgggct ggccggggct ttcgtggccg
     3001 ccgggccgct cggtgggacg gaagcgtgtg gagagaccgc caagggctgt agtctgggtc
     3061 cgcgagcaag gttgccctga actgggggtt ggggggagcg cagcaaaatg gcggctgttc
     3121 ccgagtcttg aatggaagac gcttgtgagg cgggctgtga ggtcgttgaa acaaggtggg
     3181 gggcatggtg ggcggcaaga acccaaggtc ttgagccctt cgctaatgcg ggaaagctct
     3241 tattcgggtg agatgggctg ggcaccatct ggggaccctg acgtgaagtt tgtcactgac
     3301 tggagaactc ggtttgtcgt ctgttgcggg ggcggcagtt atggcggtgc cgttgggcag
     3361 tgcacccgta cctttgggag cgcgcgccct cgtcgtgtcg tgacgtcacc cgttctgttg
     3421 gcttataatg cagggtgggg ccacctgccg gtaggtgtgc ggtaggcttt tctccgtcgc
     3481 aggacgcagg gttcgggcct agggtaggct ctcctgaatc gacaggcgcc ggacctctgg
     3541 tgaggggagg gataagtgag gcgtcagttt ctttggtcgg ttttatgtac ctatcttctt
     3601 aagtagctga agctccggtt ttgaactatg cgctcggggt tggcgagtgt gttttgtgaa
     3661 gttttttagg caccttttga aatgtaatca tttgggtcaa tatgtaattt tcagtgttag
     3721 acttgtaaat tgtccgctaa attctggccg tttttggctt ttttgttaga caacatgacc
     3781 gagtacaagc ccacggtgcg cctcgccacc cgcgacgacg tccccagggc cgtacgcacc
     3841 ctcgccgccg cgttcgccga ctaccccgcc acgcgccaca ccgtcgatcc ggaccgccac
     3901 atcgagcggg tcaccgagct gcaagaactc ttcctcacgc gcgtcgggct cgacatcggc
     3961 aaggtgtggg tcgcggacga cggcgccgcg gtggcggtct ggaccacgcc ggagagcgtc
     4021 gaagcggggg cggtgttcgc cgagatcggc ccgcgcatgg ccgagttgag cggttcccgg
     4081 ctggccgcgc agcaacagat ggagggcctc ctggcgccgc accggcccaa ggagcccgcg
     4141 tggttcctgg ccaccgtcgg cgtctcgccc gaccaccagg gcaagggtct gggcagcgcc
     4201 gtcgtgctcc ccggagtgga ggccgccgag cgcgccgggg tgcccgcctt cctggagacc
     4261 tccgcgcccc gcaacctccc cttctacgag cggctcggct tcaccgtcac cgccgacgtc
     4321 gaggtgcccg aaggaccgcg cacctggtgc atgacccgca agcccggtgc ctaggctagt
     4381 atgtaagcct agtcttagat aataaaatcg ctatccatcg aagatggatg tgtgttggtt
     4441 ttttgtgtgt gtaacgctag gcgcgcctgg tgtaccgaga acgatcctct cagtgcgagt
     4501 ctcgacgatc catatcgttg cttggcagtc agccagtcgg aatccagctt gggacccagg
     4561 aagtccaatc gtcagatatt gtactcaagc ctggtcacgg cagcgtaccg atctgtttaa
     4621 acctagatat tgatagtctg atcggtcaac gtataatcga gtcctagctt ttgcaaacat
     4681 ctatcaagag acaggatcag caggaggctt tcgcatgagt attcaacatt tccgtgtcgc
     4741 ccttattccc ttttttgcgg cattttgcct tcctgttttt gctcacccag aaacgctggt
     4801 gaaagtaaaa gatgctgaag atcagttggg tgcgcgagtg ggttacatcg aactggatct
     4861 caacagcggt aagatccttg agagttttcg ccccgaagaa cgctttccaa tgatgagcac
     4921 ttttaaagtt ctgctatgtg gcgcggtatt atcccgtatt gacgccgggc aagagcaact
     4981 cggtcgccgc atacactatt ctcagaatga cttggttgag tattcaccag tcacagaaaa
     5041 gcatcttacg gatggcatga cagtaagaga attatgcagt gctgccataa ccatgagtga
     5101 taacactgcg gccaacttac ttctgacaac gattggagga ccgaaggagc taaccgcttt
     5161 tttgcacaac atgggggatc atgtaactcg ccttgatcgt tgggaaccgg agctgaatga
     5221 agccatacca aacgacgagc gtgacaccac gatgcctgta gcaatggcaa caaccttgcg
     5281 taaactatta actggcgaac tacttactct agcttcccgg caacagttga tagactggat
     5341 ggaggcggat aaagttgcag gaccacttct gcgctcggcc cttccggctg gctggtttat
     5401 tgctgataaa tctggagccg gtgagcgtgg gtctcgcggt atcattgcag cactggggcc
     5461 agatggtaag ccctcccgta tcgtagttat ctacacgacg gggagtcagg caactatgga
     5521 tgaacgaaat agacagatcg ctgagatagg tgcctcactg attaagcatt ggtaaccgat
     5581 tctaggtgca ttggcgcaga aaaaaatgcc tgatgcgacg ctgcgcgtct tatactccca
     5641 catatgccag attcagcaac ggatacggct tccccaactt gcccacttcc atacgtgtcc
     5701 tccttaccag aaatttatcc ttaagatcgt ttaaactcga ctctggctct atcgaatctc
     5761 cgtcgtttcg agcttacgcg aacagccgtg gcgctcattt gctcgtcggg catcgaatct
     5821 cgtcagctat cgtcagctta cctttttggc agcgatcgcg gctcccgaca tcttggacca
     5881 ttagctccac aggtatcttc ttccctctag tggtcataac agcagcttca gctacctctc
     5941 aattcaaaaa acccctcaag acccgtttag aggccccaag gggttatgct atcaatcgtt
     6001 gcgttacaca cacaaaaaac caacacacat ccatcttcga tggatagcga ttttattatc
     6061 taactgctga tcgagtgtag ccagatctag taatcaatta cggggtcatt agttcatagc
     6121 ccatatatgg agttccgcgt tacataactt acggtaaatg gcccgcctgg ctgaccgccc
     6181 aacgaccccc gcccattgac gtcaataatg acgtatgttc ccatagtaac gccaataggg
     6241 actttccatt gacgtcaatg ggtggagtat ttacggtaaa ctgcccactt ggcagtacat
     6301 caagtgtatc atatgccaag tacgccccct attgacgtca atgacggtaa atggcccgcc
     6361 tggcattatg cccagtacat gaccttatgg gactttccta cttggcagta catctacgta
     6421 ttagtcatcg ctattaccat gctgatgcgg ttttggcagt acatcaatgg gcgtggatag
     6481 cggtttgact cacggggatt tccaagtctc caccccattg acgtcaatgg gagtttgttt
     6541 tggcaccaaa atcaacggga ctttccaaaa tgtcgtaaca actccgcccc attgacgcaa
     6601 atgggcggta ggcgtgtacg gtgggaggtc tatataagca gagctggttt agtgaaccgt
     6661 cagatcagat ctttgtcgat cctaccatcc actcgacaca cccgccagcg gccgc
//
