LOCUS       sequence-320328-        8978 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian expression plasmid for SARS-CoV-2 RNA-dependent RNA 
            polymerase.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8978)
  TITLE     SARS-CoV-2 protein Mammalian expression constructs, tagged, for 
            protein expression and/or purification
REFERENCE   2  (bases 1 to 8978)
  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-m4133590
FEATURES             Location/Qualifiers
     source          1..8978
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        1..304
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        306..508
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     459..479
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      577..586
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             3376..3396
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQS"
     CDS             3400..3465
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     polyA_signal    3518..3639
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3529..3548)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     3583..3602
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     terminator      3754..3800
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     terminator      3830..3966
                     /label=rrnG terminator
                     /note="transcription terminator from the E. coli ribosomal 
                     RNA rrnG operon (Albrechtsen et al., 1991)"
     misc_feature    4011..4082
                     /label=3' beta-globin insulator
                     /note="insulator downstream of the human beta-globin locus 
                     (Farrell et al., 2002)"
     rep_origin      complement(4308..4896)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4388..4407)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     promoter        5396..6605
                     /label=UbC promoter
                     /note="human ubiquitin C promoter"
     primer_bind     6442..6461
                     /label=hUBCpro-F
                     /note="Human Ubiquitin C (UbC) promoter, forward primer"
     CDS             6609..7208
                     /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(6609..6628)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     7105..7125
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     polyA_signal    7235..7283
                     /note="synthetic polyadenylation signal"
     CDS             join(7549..7617,7618..8409)
                     /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(7767..7786)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     terminator      8422..8558
                     /label=rrnG terminator
                     /note="transcription terminator from the E. coli ribosomal 
                     RNA rrnG operon (Albrechtsen et al., 1991)"
     misc_feature    8710..8781
                     /label=5' beta-globin insulator
                     /note="insulator upstream of the human beta-globin locus 
                     (Farrell et al., 2002)"
     terminator      complement(8786..8832)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     polyA_signal    8850..8898
                     /note="synthetic polyadenylation signal"
ORIGIN
        1 cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc cccgcccatt
       61 gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc attgacgtca
      121 atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt atcatatgcc
      181 aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt atgcccagta
      241 catgacctta tgggactttc ctacttggca gtacatctac gtattagtca tcgctattac
      301 catgctgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg actcacgggg
      361 atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc aaaatcaacg
      421 ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg gtaggcgtgt
      481 acggtgggag gtctatataa gcagagctgg tttagtgaac cgtcagatca gatctttgtc
      541 gatcctacca tccactcgac acacccgcca gcggccgcca ccatggctga tgcacaatcg
      601 tttttaaacc gggtttgcgg tgtaagtgca gcccgtctta caccgtgcgg cacaggcact
      661 agtactgatg tcgtatacag ggcttttgac atctacaatg ataaagtagc tggttttgct
      721 aaattcctaa aaactaattg ttgtcgcttc caagaaaagg acgaagatga caatttaatt
      781 gattcttact ttgtagttaa gagacacact ttctctaact accaacatga agaaacaatt
      841 tataatttac ttaaggattg tccagctgtt gctaaacatg acttctttaa gtttagaata
      901 gacggtgaca tggtaccaca tatatcacgt caacgtctta ctaaatacac aatggcagac
      961 ctcgtctatg ctttaaggca ttttgatgaa ggtaattgtg acacattaaa agaaatactt
     1021 gtcacataca attgttgtga tgatgattat ttcaataaaa aggactggta tgattttgta
     1081 gaaaacccag atatattacg cgtatacgcc aacttaggtg aacgtgtacg ccaagctttg
     1141 ttaaaaacag tacaattctg tgatgccatg cgaaatgctg gtattgttgg tgtactgaca
     1201 ttagataatc aagatctcaa tggtaactgg tatgatttcg gtgatttcat acaaaccacg
     1261 ccaggtagtg gagttcctgt tgtagattct tattattcat tgttaatgcc tatattaacc
     1321 ttgaccaggg ctttaactgc agagtcacat gttgacactg acttaacaaa gccttacatt
     1381 aagtgggatt tgttaaaata tgacttcacg gaagagaggt taaaactctt tgaccgttat
     1441 tttaaatatt gggatcagac ataccaccca aattgtgtta actgtttgga tgacagatgc
     1501 attctgcatt gtgcaaactt taatgtttta ttctctacag tgttcccacc tacaagtttt
     1561 ggaccactag tgagaaaaat atttgttgat ggtgttccat ttgtagtttc aactggatac
     1621 cacttcagag agctaggtgt tgtacataat caggatgtaa acttacatag ctctagactt
     1681 agttttaagg aattacttgt gtatgctgct gaccctgcta tgcacgctgc ttctggtaat
     1741 ctattactag ataaacgcac tacgtgcttt tcagtagctg cacttactaa caatgttgct
     1801 tttcaaactg tcaaacccgg taattttaac aaagacttct atgactttgc tgtgtctaag
     1861 ggtttcttta aggaaggaag ttctgttgaa ttaaaacact tcttctttgc tcaggatggt
     1921 aatgctgcta tcagcgatta tgactactat cgttataatc taccaacaat gtgtgatatc
     1981 agacaactac tatttgtagt tgaagttgtt gataagtact ttgattgtta cgatggtggc
     2041 tgtattaatg ctaaccaagt catcgtcaac aacctagaca aatcagctgg ttttccattt
     2101 aataaatggg gtaaggctag actttattat gattcaatga gttatgagga tcaagatgca
     2161 cttttcgcat atacaaaacg taatgtcatc cctactataa ctcaaatgaa tcttaagtat
     2221 gccattagtg caaagaatag agctcgcacc gtagctggtg tctctatctg tagtactatg
     2281 accaatagac agtttcatca aaaattattg aaatcaatag ccgccactag aggagctact
     2341 gtagtaattg gaacaagcaa attctatggt ggttggcaca acatgttaaa aactgtttat
     2401 agtgatgtag aaaaccctca ccttatgggt tgggattatc ctaaatgtga tagagccatg
     2461 cctaacatgc ttagaattat ggcctcactt gttcttgctc gcaaacatac aacgtgttgt
     2521 agcttgtcac accgtttcta tagattagct aatgagtgtg ctcaagtatt gagtgaaatg
     2581 gtcatgtgtg gcggttcact atatgttaaa ccaggtggaa cctcatcagg agatgccaca
     2641 actgcttatg ctaatagtgt ttttaacatt tgtcaagctg tcacggccaa tgttaatgca
     2701 cttttatcta ctgatggtaa caaaattgcc gataagtatg tccgcaattt acaacacaga
     2761 ctttatgagt gtctctatag aaatagagat gttgacacag actttgtgaa tgagttttac
     2821 gcatatttgc gtaaacattt ctcaatgatg atactctctg acgatgctgt tgtgtgtttc
     2881 aatagcactt atgcatctca aggtctagtg gctagcataa agaactttaa gtcagttctt
     2941 tattatcaaa acaatgtttt tatgtctgaa gcaaaatgtt ggactgagac tgaccttact
     3001 aaaggacctc atgaattttg ctctcaacat acaatgctag ttaaacaggg tgatgattat
     3061 gtgtaccttc cttacccaga tccatcaaga atcctagggg ccggctgttt tgtagatgat
     3121 atcgtaaaaa cagatggtac acttatgatt gaacggttcg tgtctttagc tatagatgct
     3181 tacccactta ctaaacatcc taatcaggag tatgctgatg tctttcattt gtacttacaa
     3241 tacataagaa agctacatga tgagttaaca ggacacatgt tagacatgta ttctgttatg
     3301 cttactaatg ataacacttc aaggtattgg gaacctgagt tttatgaggc tatgtacaca
     3361 ccgcatacag tcttagaaaa cctttacttt cagtcgatgg attataagga ccacgatgga
     3421 gactacaaag atcatgacat tgattacaag gacgacgatg acaaataagc tagcttgact
     3481 gactgagata cagcgtacct tcagctcaca gacatgataa gatacattga tgagtttgga
     3541 caaaccacaa ctagaatgca gtgaaaaaaa tgctttattt gtgaaatttg tgatgctatt
     3601 gctttatttg taaccattat aagctgcaat aaacaagtta acaacaacaa ttgcattcat
     3661 tttatgtttc aggttcaggg ggaggtgtgg gaggtttttt aaagcaagta aaacctctac
     3721 aaatgtggta ttggcccatc tctatcggta tcgtagcata accccttggg gcctctaaac
     3781 gggtcttgag gggttttttg tgcccctcgg gccggattgc tatctaccgg cattggcgca
     3841 gaaaaaaatg cctgatgcga cgctgcgcgt cttatactcc cacatatgcc agattcagca
     3901 acggatacgg cttccccaac ttgcccactt ccatacgtgt cctccttacc agaaatttat
     3961 ccttaaggtc gtcagctatc ctgcaggcga tctctcgatt tcgatcaaga cattccttta
     4021 atggtctttt ctggacacca ctaggggtca gaagtagttc atcaaacttt cttccctccc
     4081 taatctcatt ggttaccttg ggctatcgaa acttaattaa ccagtcaagt cagctacttg
     4141 gcgagatcga cttgtctggg tttcgactac gctcagaatt gcgtcagtca agttcgatct
     4201 ggtccttgct attgcacccg ttctccgatt acgagtttca tttaaatcat gtgagcaaaa
     4261 ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc
     4321 cgcccccctg acgagcatca caaaaatcga cgctcaagtc agaggtggcg aaacccgaca
     4381 ggactataaa gataccaggc gtttccccct ggaagctccc tcgtgcgctc tcctgttccg
     4441 accctgccgc ttaccggata cctgtccgcc tttctccctt cgggaagcgt ggcgctttct
     4501 catagctcac gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt
     4561 gtgcacgaac cccccgttca gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag
     4621 tccaacccgg taagacacga cttatcgcca ctggcagcag ccactggtaa caggattagc
     4681 agagcgaggt atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa ctacggctac
     4741 actagaagaa cagtatttgg tatctgcgct ctgctgaagc cagttacctt cggaaaaaga
     4801 gttggtagct cttgatccgg caaacaaacc accgctggta gcggtggttt ttttgtttgc
     4861 aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag atcctttgat cttttctacg
     4921 gggtctgacg ctcagtggaa cgaaaactca cgttaaggga ttttggtcat gagattatca
     4981 aaaaggatct tcacctagat ccttttaaat taaaaatgaa gttttaaatc aatctaaagt
     5041 atatatgagt aaacttggtc tgacagttac caatgcttaa tcagtgaggc acctatctca
     5101 gcgatctgtc tatttcgttc atccatagtt gcatttaaat ttccgaactc tccaaggccc
     5161 tcgtcggaaa atcttcaaac ctttcgtccg atccatcttg caggctacct ctcgaacgaa
     5221 ctatcgcaag tctcttggcc ggccttgcgc cttggctatt gcttggcagc gcctatcgcc
     5281 aggtattact ccaatcccga atatccgaga tcgggatcac ccgagagaag ttcaacctac
     5341 atcctcaatc ccgatctatc cgagatccga ggaatatcga aatcggggcg cgcctggcct
     5401 ccgcgccggg ttttggcgcc tcccgcgggc gcccccctcg tcacggcgag cgctgccacg
     5461 tcagacgaag ggcgcaggag cgtcctgatc cttccgcccg gacgctcagg acagcggccc
     5521 gctgctcata agactcggcc ttagaacccc agtatcagca gaaggacatt ttaggacggg
     5581 acttgggtga ctctagggca ctggttttct ttccagagag cggaacaggc gaggaaaagt
     5641 agtcccttct cggcgattct gcggagggat ctccgtgggg cggtgaacgc cgatgattat
     5701 ataaggacgc gccgggtgtg gcacagctag ttccgtcgca gccgggattt gggtcgcggt
     5761 tcttgtttgt ggatcgctgt gatcgtcact tggtgagtag cgggctgctg ggctggccgg
     5821 ggctttcgtg gccgccgggc cgctcggtgg gacggaagcg tgtggagaga ccgccaaggg
     5881 ctgtagtctg ggtccgcgag caaggttgcc ctgaactggg ggttgggggg agcgcagcaa
     5941 aatggcggct gttcccgagt cttgaatgga agacgcttgt gaggcgggct gtgaggtcgt
     6001 tgaaacaagg tggggggcat ggtgggcggc aagaacccaa ggtcttgagc ccttcgctaa
     6061 tgcgggaaag ctcttattcg ggtgagatgg gctgggcacc atctggggac cctgacgtga
     6121 agtttgtcac tgactggaga actcggtttg tcgtctgttg cgggggcggc agttatggcg
     6181 gtgccgttgg gcagtgcacc cgtacctttg ggagcgcgcg ccctcgtcgt gtcgtgacgt
     6241 cacccgttct gttggcttat aatgcagggt ggggccacct gccggtaggt gtgcggtagg
     6301 cttttctccg tcgcaggacg cagggttcgg gcctagggta ggctctcctg aatcgacagg
     6361 cgccggacct ctggtgaggg gagggataag tgaggcgtca gtttctttgg tcggttttat
     6421 gtacctatct tcttaagtag ctgaagctcc ggttttgaac tatgcgctcg gggttggcga
     6481 gtgtgttttg tgaagttttt taggcacctt ttgaaatgta atcatttggg tcaatatgta
     6541 attttcagtg ttagacttgt aaattgtccg ctaaattctg gccgtttttg gcttttttgt
     6601 tagacaacat gaccgagtac aagcccacgg tgcgcctcgc cacccgcgac gacgtcccca
     6661 gggccgtacg caccctcgcc gccgcgttcg ccgactaccc cgccacgcgc cacaccgtcg
     6721 atccggaccg ccacatcgag cgggtcaccg agctgcaaga actcttcctc acgcgcgtcg
     6781 ggctcgacat cggcaaggtg tgggtcgcgg acgacggcgc cgcggtggcg gtctggacca
     6841 cgccggagag cgtcgaagcg ggggcggtgt tcgccgagat cggcccgcgc atggccgagt
     6901 tgagcggttc ccggctggcc gcgcagcaac agatggaggg cctcctggcg ccgcaccggc
     6961 ccaaggagcc cgcgtggttc ctggccaccg tcggcgtctc gcccgaccac cagggcaagg
     7021 gtctgggcag cgccgtcgtg ctccccggag tggaggccgc cgagcgcgcc ggggtgcccg
     7081 ccttcctgga gacctccgcg ccccgcaacc tccccttcta cgagcggctc ggcttcaccg
     7141 tcaccgccga cgtcgaggtg cccgaaggac cgcgcacctg gtgcatgacc cgcaagcccg
     7201 gtgcctaggc tagtatgtaa gcctagtctt agataataaa atcgctatcc atcgaagatg
     7261 gatgtgtgtt ggttttttgt gtgtgtaacg ctaggcgcgc ctggtgtacc gagaacgatc
     7321 ctctcagtgc gagtctcgac gatccatatc gttgcttggc agtcagccag tcggaatcca
     7381 gcttgggacc caggaagtcc aatcgtcaga tattgtactc aagcctggtc acggcagcgt
     7441 accgatctgt ttaaacctag atattgatag tctgatcggt caacgtataa tcgagtccta
     7501 gcttttgcaa acatctatca agagacagga tcagcaggag gctttcgcat gagtattcaa
     7561 catttccgtg tcgcccttat tccctttttt gcggcatttt gccttcctgt ttttgctcac
     7621 ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt tgggtgcgcg agtgggttac
     7681 atcgaactgg atctcaacag cggtaagatc cttgagagtt ttcgccccga agaacgcttt
     7741 ccaatgatga gcacttttaa agttctgcta tgtggcgcgg tattatcccg tattgacgcc
     7801 gggcaagagc aactcggtcg ccgcatacac tattctcaga atgacttggt tgagtattca
     7861 ccagtcacag aaaagcatct tacggatggc atgacagtaa gagaattatg cagtgctgcc
     7921 ataaccatga gtgataacac tgcggccaac ttacttctga caacgattgg aggaccgaag
     7981 gagctaaccg cttttttgca caacatgggg gatcatgtaa ctcgccttga tcgttgggaa
     8041 ccggagctga atgaagccat accaaacgac gagcgtgaca ccacgatgcc tgtagcaatg
     8101 gcaacaacct tgcgtaaact attaactggc gaactactta ctctagcttc ccggcaacag
     8161 ttgatagact ggatggaggc ggataaagtt gcaggaccac ttctgcgctc ggcccttccg
     8221 gctggctggt ttattgctga taaatctgga gccggtgagc gtgggtctcg cggtatcatt
     8281 gcagcactgg ggccagatgg taagccctcc cgtatcgtag ttatctacac gacggggagt
     8341 caggcaacta tggatgaacg aaatagacag atcgctgaga taggtgcctc actgattaag
     8401 cattggtaac cgattctagg tgcattggcg cagaaaaaaa tgcctgatgc gacgctgcgc
     8461 gtcttatact cccacatatg ccagattcag caacggatac ggcttcccca acttgcccac
     8521 ttccatacgt gtcctcctta ccagaaattt atccttaaga tcccgaatcg tttaaactcg
     8581 actctggctc tatcgaatct ccgtcgtttc gagcttacgc gaacagccgt ggcgctcatt
     8641 tgctcgtcgg gcatcgaatc tcgtcagcta tcgtcagctt acctttttgg cagcgatcgc
     8701 ggctcccgac atcttggacc attagctcca caggtatctt cttccctcta gtggtcataa
     8761 cagcagcttc agctacctct caattcaaaa aacccctcaa gacccgttta gaggccccaa
     8821 ggggttatgc tatcaatcgt tgcgttacac acacaaaaaa ccaacacaca tccatcttcg
     8881 atggatagcg attttattat ctaactgctg atcgagtgta gccagatcta gtaatcaatt
     8941 acggggtcat tagttcatag cccatatatg gagttccg
//
