LOCUS       sequence-295429-        8920 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 8920)
  TITLE     SARS-CoV-2 protein Mammalian expression constructs, tagged, for 
            protein expression and/or purification
REFERENCE   2  (bases 1 to 8920)
  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-m4134140
FEATURES             Location/Qualifiers
     source          1..8920
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             2798..2818
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQS"
     CDS             2819..2836
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     polyA_signal    2889..3010
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2900..2919)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     2954..2973
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     terminator      3125..3171
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     terminator      3201..3337
                     /label=rrnG terminator
                     /note="transcription terminator from the E. coli ribosomal 
                     RNA rrnG operon (Albrechtsen et al., 1991)"
     misc_feature    3382..3453
                     /label=3' beta-globin insulator
                     /note="insulator downstream of the human beta-globin locus 
                     (Farrell et al., 2002)"
     rep_origin      complement(3679..4267)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3759..3778)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     promoter        4767..5976
                     /label=UbC promoter
                     /note="human ubiquitin C promoter"
     primer_bind     5813..5832
                     /label=hUBCpro-F
                     /note="Human Ubiquitin C (UbC) promoter, forward primer"
     CDS             5980..6579
                     /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(5980..5999)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     6476..6496
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     polyA_signal    6606..6654
                     /note="synthetic polyadenylation signal"
     CDS             join(6920..6988,6989..7780)
                     /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(7138..7157)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     terminator      7793..7929
                     /label=rrnG terminator
                     /note="transcription terminator from the E. coli ribosomal 
                     RNA rrnG operon (Albrechtsen et al., 1991)"
     misc_feature    8074..8145
                     /label=5' beta-globin insulator
                     /note="insulator upstream of the human beta-globin locus 
                     (Farrell et al., 2002)"
     terminator      complement(8150..8196)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     polyA_signal    8214..8262
                     /note="synthetic polyadenylation signal"
     enhancer        8343..8646
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        8648..8850
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     8801..8821
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      join(8919..8920,1..8)
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
ORIGIN
        1 caccatggct gatgcacaat cgtttttaaa ccgggtttgc ggtgtaagtg cagcccgtct
       61 tacaccgtgc ggcacaggca ctagtactga tgtcgtatac agggcttttg acatctacaa
      121 tgataaagta gctggttttg ctaaattcct aaaaactaat tgttgtcgct tccaagaaaa
      181 ggacgaagat gacaatttaa ttgattctta ctttgtagtt aagagacaca ctttctctaa
      241 ctaccaacat gaagaaacaa tttataattt acttaaggat tgtccagctg ttgctaaaca
      301 tgacttcttt aagtttagaa tagacggtga catggtacca catatatcac gtcaacgtct
      361 tactaaatac acaatggcag acctcgtcta tgctttaagg cattttgatg aaggtaattg
      421 tgacacatta aaagaaatac ttgtcacata caattgttgt gatgatgatt atttcaataa
      481 aaaggactgg tatgattttg tagaaaaccc agatatatta cgcgtatacg ccaacttagg
      541 tgaacgtgta cgccaagctt tgttaaaaac agtacaattc tgtgatgcca tgcgaaatgc
      601 tggtattgtt ggtgtactga cattagataa tcaagatctc aatggtaact ggtatgattt
      661 cggtgatttc atacaaacca cgccaggtag tggagttcct gttgtagatt cttattattc
      721 attgttaatg cctatattaa ccttgaccag ggctttaact gcagagtcac atgttgacac
      781 tgacttaaca aagccttaca ttaagtggga tttgttaaaa tatgacttca cggaagagag
      841 gttaaaactc tttgaccgtt attttaaata ttgggatcag acataccacc caaattgtgt
      901 taactgtttg gatgacagat gcattctgca ttgtgcaaac tttaatgttt tattctctac
      961 agtgttccca cctacaagtt ttggaccact agtgagaaaa atatttgttg atggtgttcc
     1021 atttgtagtt tcaactggat accacttcag agagctaggt gttgtacata atcaggatgt
     1081 aaacttacat agctctagac ttagttttaa ggaattactt gtgtatgctg ctgaccctgc
     1141 tatgcacgct gcttctggta atctattact agataaacgc actacgtgct tttcagtagc
     1201 tgcacttact aacaatgttg cttttcaaac tgtcaaaccc ggtaatttta acaaagactt
     1261 ctatgacttt gctgtgtcta agggtttctt taaggaagga agttctgttg aattaaaaca
     1321 cttcttcttt gctcaggatg gtaatgctgc tatcagcgat tatgactact atcgttataa
     1381 tctaccaaca atgtgtgata tcagacaact actatttgta gttgaagttg ttgataagta
     1441 ctttgattgt tacgatggtg gctgtattaa tgctaaccaa gtcatcgtca acaacctaga
     1501 caaatcagct ggttttccat ttaataaatg gggtaaggct agactttatt atgattcaat
     1561 gagttatgag gatcaagatg cacttttcgc atatacaaaa cgtaatgtca tccctactat
     1621 aactcaaatg aatcttaagt atgccattag tgcaaagaat agagctcgca ccgtagctgg
     1681 tgtctctatc tgtagtacta tgaccaatag acagtttcat caaaaattat tgaaatcaat
     1741 agccgccact agaggagcta ctgtagtaat tggaacaagc aaattctatg gtggttggca
     1801 caacatgtta aaaactgttt atagtgatgt agaaaaccct caccttatgg gttgggatta
     1861 tcctaaatgt gatagagcca tgcctaacat gcttagaatt atggcctcac ttgttcttgc
     1921 tcgcaaacat acaacgtgtt gtagcttgtc acaccgtttc tatagattag ctaatgagtg
     1981 tgctcaagta ttgagtgaaa tggtcatgtg tggcggttca ctatatgtta aaccaggtgg
     2041 aacctcatca ggagatgcca caactgctta tgctaatagt gtttttaaca tttgtcaagc
     2101 tgtcacggcc aatgttaatg cacttttatc tactgatggt aacaaaattg ccgataagta
     2161 tgtccgcaat ttacaacaca gactttatga gtgtctctat agaaatagag atgttgacac
     2221 agactttgtg aatgagtttt acgcatattt gcgtaaacat ttctcaatga tgatactctc
     2281 tgacgatgct gttgtgtgtt tcaatagcac ttatgcatct caaggtctag tggctagcat
     2341 aaagaacttt aagtcagttc tttattatca aaacaatgtt tttatgtctg aagcaaaatg
     2401 ttggactgag actgacctta ctaaaggacc tcatgaattt tgctctcaac atacaatgct
     2461 agttaaacag ggtgatgatt atgtgtacct tccttaccca gatccatcaa gaatcctagg
     2521 ggccggctgt tttgtagatg atatcgtaaa aacagatggt acacttatga ttgaacggtt
     2581 cgtgtcttta gctatagatg cttacccact tactaaacat cctaatcagg agtatgctga
     2641 tgtctttcat ttgtacttac aatacataag aaagctacat gatgagttaa caggacacat
     2701 gttagacatg tattctgtta tgcttactaa tgataacact tcaaggtatt gggaacctga
     2761 gttttatgag gctatgtaca caccgcatac agtcttagaa aacctttact ttcagtcgca
     2821 ccatcaccat caccattaag ctagcttgac tgactgagat acagcgtacc ttcagctcac
     2881 agacatgata agatacattg atgagtttgg acaaaccaca actagaatgc agtgaaaaaa
     2941 atgctttatt tgtgaaattt gtgatgctat tgctttattt gtaaccatta taagctgcaa
     3001 taaacaagtt aacaacaaca attgcattca ttttatgttt caggttcagg gggaggtgtg
     3061 ggaggttttt taaagcaagt aaaacctcta caaatgtggt attggcccat ctctatcggt
     3121 atcgtagcat aaccccttgg ggcctctaaa cgggtcttga ggggtttttt gtgcccctcg
     3181 ggccggattg ctatctaccg gcattggcgc agaaaaaaat gcctgatgcg acgctgcgcg
     3241 tcttatactc ccacatatgc cagattcagc aacggatacg gcttccccaa cttgcccact
     3301 tccatacgtg tcctccttac cagaaattta tccttaaggt cgtcagctat cctgcaggcg
     3361 atctctcgat ttcgatcaag acattccttt aatggtcttt tctggacacc actaggggtc
     3421 agaagtagtt catcaaactt tcttccctcc ctaatctcat tggttacctt gggctatcga
     3481 aacttaatta accagtcaag tcagctactt ggcgagatcg acttgtctgg gtttcgacta
     3541 cgctcagaat tgcgtcagtc aagttcgatc tggtccttgc tattgcaccc gttctccgat
     3601 tacgagtttc atttaaatca tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa
     3661 ggccgcgttg ctggcgtttt tccataggct ccgcccccct gacgagcatc acaaaaatcg
     3721 acgctcaagt cagaggtggc gaaacccgac aggactataa agataccagg cgtttccccc
     3781 tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat acctgtccgc
     3841 ctttctccct tcgggaagcg tggcgctttc tcatagctca cgctgtaggt atctcagttc
     3901 ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa ccccccgttc agcccgaccg
     3961 ctgcgcctta tccggtaact atcgtcttga gtccaacccg gtaagacacg acttatcgcc
     4021 actggcagca gccactggta acaggattag cagagcgagg tatgtaggcg gtgctacaga
     4081 gttcttgaag tggtggccta actacggcta cactagaaga acagtatttg gtatctgcgc
     4141 tctgctgaag ccagttacct tcggaaaaag agttggtagc tcttgatccg gcaaacaaac
     4201 caccgctggt agcggtggtt tttttgtttg caagcagcag attacgcgca gaaaaaaagg
     4261 atctcaagaa gatcctttga tcttttctac ggggtctgac gctcagtgga acgaaaactc
     4321 acgttaaggg attttggtca tgagattatc aaaaaggatc ttcacctaga tccttttaaa
     4381 ttaaaaatga agttttaaat caatctaaag tatatatgag taaacttggt ctgacagtta
     4441 ccaatgctta atcagtgagg cacctatctc agcgatctgt ctatttcgtt catccatagt
     4501 tgcatttaaa tttccgaact ctccaaggcc ctcgtcggaa aatcttcaaa cctttcgtcc
     4561 gatccatctt gcaggctacc tctcgaacga actatcgcaa gtctcttggc cggccttgcg
     4621 ccttggctat tgcttggcag cgcctatcgc caggtattac tccaatcccg aatatccgag
     4681 atcgggatca cccgagagaa gttcaaccta catcctcaat cccgatctat ccgagatccg
     4741 aggaatatcg aaatcggggc gcgcctggcc tccgcgccgg gttttggcgc ctcccgcggg
     4801 cgcccccctc gtcacggcga gcgctgccac gtcagacgaa gggcgcagga gcgtcctgat
     4861 ccttccgccc ggacgctcag gacagcggcc cgctgctcat aagactcggc cttagaaccc
     4921 cagtatcagc agaaggacat tttaggacgg gacttgggtg actctagggc actggttttc
     4981 tttccagaga gcggaacagg cgaggaaaag tagtcccttc tcggcgattc tgcggaggga
     5041 tctccgtggg gcggtgaacg ccgatgatta tataaggacg cgccgggtgt ggcacagcta
     5101 gttccgtcgc agccgggatt tgggtcgcgg ttcttgtttg tggatcgctg tgatcgtcac
     5161 ttggtgagta gcgggctgct gggctggccg gggctttcgt ggccgccggg ccgctcggtg
     5221 ggacggaagc gtgtggagag accgccaagg gctgtagtct gggtccgcga gcaaggttgc
     5281 cctgaactgg gggttggggg gagcgcagca aaatggcggc tgttcccgag tcttgaatgg
     5341 aagacgcttg tgaggcgggc tgtgaggtcg ttgaaacaag gtggggggca tggtgggcgg
     5401 caagaaccca aggtcttgag cccttcgcta atgcgggaaa gctcttattc gggtgagatg
     5461 ggctgggcac catctgggga ccctgacgtg aagtttgtca ctgactggag aactcggttt
     5521 gtcgtctgtt gcgggggcgg cagttatggc ggtgccgttg ggcagtgcac ccgtaccttt
     5581 gggagcgcgc gccctcgtcg tgtcgtgacg tcacccgttc tgttggctta taatgcaggg
     5641 tggggccacc tgccggtagg tgtgcggtag gcttttctcc gtcgcaggac gcagggttcg
     5701 ggcctagggt aggctctcct gaatcgacag gcgccggacc tctggtgagg ggagggataa
     5761 gtgaggcgtc agtttctttg gtcggtttta tgtacctatc ttcttaagta gctgaagctc
     5821 cggttttgaa ctatgcgctc ggggttggcg agtgtgtttt gtgaagtttt ttaggcacct
     5881 tttgaaatgt aatcatttgg gtcaatatgt aattttcagt gttagacttg taaattgtcc
     5941 gctaaattct ggccgttttt ggcttttttg ttagacaaca tgaccgagta caagcccacg
     6001 gtgcgcctcg ccacccgcga cgacgtcccc agggccgtac gcaccctcgc cgccgcgttc
     6061 gccgactacc ccgccacgcg ccacaccgtc gatccggacc gccacatcga gcgggtcacc
     6121 gagctgcaag aactcttcct cacgcgcgtc gggctcgaca tcggcaaggt gtgggtcgcg
     6181 gacgacggcg ccgcggtggc ggtctggacc acgccggaga gcgtcgaagc gggggcggtg
     6241 ttcgccgaga tcggcccgcg catggccgag ttgagcggtt cccggctggc cgcgcagcaa
     6301 cagatggagg gcctcctggc gccgcaccgg cccaaggagc ccgcgtggtt cctggccacc
     6361 gtcggcgtct cgcccgacca ccagggcaag ggtctgggca gcgccgtcgt gctccccgga
     6421 gtggaggccg ccgagcgcgc cggggtgccc gccttcctgg agacctccgc gccccgcaac
     6481 ctccccttct acgagcggct cggcttcacc gtcaccgccg acgtcgaggt gcccgaagga
     6541 ccgcgcacct ggtgcatgac ccgcaagccc ggtgcctagg ctagtatgta agcctagtct
     6601 tagataataa aatcgctatc catcgaagat ggatgtgtgt tggttttttg tgtgtgtaac
     6661 gctaggcgcg cctggtgtac cgagaacgat cctctcagtg cgagtctcga cgatccatat
     6721 cgttgcttgg cagtcagcca gtcggaatcc agcttgggac ccaggaagtc caatcgtcag
     6781 atattgtact caagcctggt cacggcagcg taccgatctg tttaaaccta gatattgata
     6841 gtctgatcgg tcaacgtata atcgagtcct agcttttgca aacatctatc aagagacagg
     6901 atcagcagga ggctttcgca tgagtattca acatttccgt gtcgccctta ttcccttttt
     6961 tgcggcattt tgccttcctg tttttgctca cccagaaacg ctggtgaaag taaaagatgc
     7021 tgaagatcag ttgggtgcgc gagtgggtta catcgaactg gatctcaaca gcggtaagat
     7081 ccttgagagt tttcgccccg aagaacgctt tccaatgatg agcactttta aagttctgct
     7141 atgtggcgcg gtattatccc gtattgacgc cgggcaagag caactcggtc gccgcataca
     7201 ctattctcag aatgacttgg ttgagtattc accagtcaca gaaaagcatc ttacggatgg
     7261 catgacagta agagaattat gcagtgctgc cataaccatg agtgataaca ctgcggccaa
     7321 cttacttctg acaacgattg gaggaccgaa ggagctaacc gcttttttgc acaacatggg
     7381 ggatcatgta actcgccttg atcgttggga accggagctg aatgaagcca taccaaacga
     7441 cgagcgtgac accacgatgc ctgtagcaat ggcaacaacc ttgcgtaaac tattaactgg
     7501 cgaactactt actctagctt cccggcaaca gttgatagac tggatggagg cggataaagt
     7561 tgcaggacca cttctgcgct cggcccttcc ggctggctgg tttattgctg ataaatctgg
     7621 agccggtgag cgtgggtctc gcggtatcat tgcagcactg gggccagatg gtaagccctc
     7681 ccgtatcgta gttatctaca cgacggggag tcaggcaact atggatgaac gaaatagaca
     7741 gatcgctgag ataggtgcct cactgattaa gcattggtaa ccgattctag gtgcattggc
     7801 gcagaaaaaa atgcctgatg cgacgctgcg cgtcttatac tcccacatat gccagattca
     7861 gcaacggata cggcttcccc aacttgccca cttccatacg tgtcctcctt accagaaatt
     7921 tatccttaag atcgtttaaa ctcgactctg gctctatcga atctccgtcg tttcgagctt
     7981 acgcgaacag ccgtggcgct catttgctcg tcgggcatcg aatctcgtca gctatcgtca
     8041 gcttaccttt ttggcagcga tcgcggctcc cgacatcttg gaccattagc tccacaggta
     8101 tcttcttccc tctagtggtc ataacagcag cttcagctac ctctcaattc aaaaaacccc
     8161 tcaagacccg tttagaggcc ccaaggggtt atgctatcaa tcgttgcgtt acacacacaa
     8221 aaaaccaaca cacatccatc ttcgatggat agcgatttta ttatctaact gctgatcgag
     8281 tgtagccaga tctagtaatc aattacgggg tcattagttc atagcccata tatggagttc
     8341 cgcgttacat aacttacggt aaatggcccg cctggctgac cgcccaacga cccccgccca
     8401 ttgacgtcaa taatgacgta tgttcccata gtaacgccaa tagggacttt ccattgacgt
     8461 caatgggtgg agtatttacg gtaaactgcc cacttggcag tacatcaagt gtatcatatg
     8521 ccaagtacgc cccctattga cgtcaatgac ggtaaatggc ccgcctggca ttatgcccag
     8581 tacatgacct tatgggactt tcctacttgg cagtacatct acgtattagt catcgctatt
     8641 accatgctga tgcggttttg gcagtacatc aatgggcgtg gatagcggtt tgactcacgg
     8701 ggatttccaa gtctccaccc cattgacgtc aatgggagtt tgttttggca ccaaaatcaa
     8761 cgggactttc caaaatgtcg taacaactcc gccccattga cgcaaatggg cggtaggcgt
     8821 gtacggtggg aggtctatat aagcagagct ggtttagtga accgtcagat cagatctttg
     8881 tcgatcctac catccactcg acacacccgc cagcggccgc
//
