LOCUS       sequence-307252-        8933 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 8933)
  TITLE     SARS-CoV-2 protein Mammalian expression constructs, tagged, for 
            protein expression and/or purification
REFERENCE   2  (bases 1 to 8933)
  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-m4133875
FEATURES             Location/Qualifiers
     source          1..8933
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(96..115)
                     /label=hUBCpro-F
                     /note="Human Ubiquitin C (UbC) promoter, forward primer"
     rep_origin      1661..2249
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2150..2169
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     misc_feature    2475..2546
                     /label=3' beta-globin insulator
                     /note="insulator downstream of the human beta-globin locus 
                     (Farrell et al., 2002)"
     terminator      complement(2591..2727)
                     /label=rrnG terminator
                     /note="transcription terminator from the E. coli ribosomal 
                     RNA rrnG operon (Albrechtsen et al., 1991)"
     terminator      complement(2757..2803)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     polyA_signal    2918..3039
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2955..2974)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3009..3028
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     CDS             complement(3092..3109)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             complement(3110..3130)
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQS"
     promoter        complement(6004..6206)
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     complement(6033..6053)
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     enhancer        6208..6511
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     polyA_signal    6592..6640
                     /note="synthetic polyadenylation signal"
     terminator      6658..6704
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     misc_feature    6709..6780
                     /label=5' beta-globin insulator
                     /note="insulator upstream of the human beta-globin locus 
                     (Farrell et al., 2002)"
     terminator      complement(6932..7068)
                     /label=rrnG terminator
                     /note="transcription terminator from the E. coli ribosomal 
                     RNA rrnG operon (Albrechtsen et al., 1991)"
     CDS             complement(join(7081..7872,7873..7941))
                     /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     7704..7723
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     polyA_signal    8207..8255
                     /note="synthetic polyadenylation signal"
     CDS             complement(8282..8881)
                     /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(8365..8385)
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     primer_bind     8862..8881
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     promoter        complement(join(8885..8933,1..1161))
                     /label=UbC promoter
                     /note="human ubiquitin C promoter"
ORIGIN
        1 gtctaacact gaaaattaca tattgaccca aatgattaca tttcaaaagg tgcctaaaaa
       61 acttcacaaa acacactcgc caaccccgag cgcatagttc aaaaccggag cttcagctac
      121 ttaagaagat aggtacataa aaccgaccaa agaaactgac gcctcactta tccctcccct
      181 caccagaggt ccggcgcctg tcgattcagg agagcctacc ctaggcccga accctgcgtc
      241 ctgcgacgga gaaaagccta ccgcacacct accggcaggt ggccccaccc tgcattataa
      301 gccaacagaa cgggtgacgt cacgacacga cgagggcgcg cgctcccaaa ggtacgggtg
      361 cactgcccaa cggcaccgcc ataactgccg cccccgcaac agacgacaaa ccgagttctc
      421 cagtcagtga caaacttcac gtcagggtcc ccagatggtg cccagcccat ctcacccgaa
      481 taagagcttt cccgcattag cgaagggctc aagaccttgg gttcttgccg cccaccatgc
      541 cccccacctt gtttcaacga cctcacagcc cgcctcacaa gcgtcttcca ttcaagactc
      601 gggaacagcc gccattttgc tgcgctcccc ccaaccccca gttcagggca accttgctcg
      661 cggacccaga ctacagccct tggcggtctc tccacacgct tccgtcccac cgagcggccc
      721 ggcggccacg aaagccccgg ccagcccagc agcccgctac tcaccaagtg acgatcacag
      781 cgatccacaa acaagaaccg cgacccaaat cccggctgcg acggaactag ctgtgccaca
      841 cccggcgcgt ccttatataa tcatcggcgt tcaccgcccc acggagatcc ctccgcagaa
      901 tcgccgagaa gggactactt ttcctcgcct gttccgctct ctggaaagaa aaccagtgcc
      961 ctagagtcac ccaagtcccg tcctaaaatg tccttctgct gatactgggg ttctaaggcc
     1021 gagtcttatg agcagcgggc cgctgtcctg agcgtccggg cggaaggatc aggacgctcc
     1081 tgcgcccttc gtctgacgtg gcagcgctcg ccgtgacgag gggggcgccc gcgggaggcg
     1141 ccaaaacccg gcgcggaggc caggcgcgcc ccgatttcga tattcctcgg atctcggata
     1201 gatcgggatt gaggatgtag gttgaacttc tctcgggtga tcccgatctc ggatattcgg
     1261 gattggagta atacctggcg ataggcgctg ccaagcaata gccaaggcgc aaggccggcc
     1321 aagagacttg cgatagttcg ttcgagaggt agcctgcaag atggatcgga cgaaaggttt
     1381 gaagattttc cgacgagggc cttggagagt tcggaaattt aaatgcaact atggatgaac
     1441 gaaatagaca gatcgctgag ataggtgcct cactgattaa gcattggtaa ctgtcagacc
     1501 aagtttactc atatatactt tagattgatt taaaacttca tttttaattt aaaaggatct
     1561 aggtgaagat cctttttgat aatctcatga ccaaaatccc ttaacgtgag ttttcgttcc
     1621 actgagcgtc agaccccgta gaaaagatca aaggatcttc ttgagatcct ttttttctgc
     1681 gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt tgtttgccgg
     1741 atcaagagct accaactctt tttccgaagg taactggctt cagcagagcg cagataccaa
     1801 atactgttct tctagtgtag ccgtagttag gccaccactt caagaactct gtagcaccgc
     1861 ctacatacct cgctctgcta atcctgttac cagtggctgc tgccagtggc gataagtcgt
     1921 gtcttaccgg gttggactca agacgatagt taccggataa ggcgcagcgg tcgggctgaa
     1981 cggggggttc gtgcacacag cccagcttgg agcgaacgac ctacaccgaa ctgagatacc
     2041 tacagcgtga gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc
     2101 cggtaagcgg cagggtcgga acaggagagc gcacgaggga gcttccaggg ggaaacgcct
     2161 ggtatcttta tagtcctgtc gggtttcgcc acctctgact tgagcgtcga tttttgtgat
     2221 gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa cgcggccttt ttacggttcc
     2281 tggccttttg ctggcctttt gctcacatga tttaaatgaa actcgtaatc ggagaacggg
     2341 tgcaatagca aggaccagat cgaacttgac tgacgcaatt ctgagcgtag tcgaaaccca
     2401 gacaagtcga tctcgccaag tagctgactt gactggttaa ttaagtttcg atagcccaag
     2461 gtaaccaatg agattaggga gggaagaaag tttgatgaac tacttctgac ccctagtggt
     2521 gtccagaaaa gaccattaaa ggaatgtctt gatcgaaatc gagagatcgc ctgcaggata
     2581 gctgacgacc ttaaggataa atttctggta aggaggacac gtatggaagt gggcaagttg
     2641 gggaagccgt atccgttgct gaatctggca tatgtgggag tataagacgc gcagcgtcgc
     2701 atcaggcatt tttttctgcg ccaatgccgg tagatagcaa tccggcccga ggggcacaaa
     2761 aaacccctca agacccgttt agaggcccca aggggttatg ctacgatacc gatagagatg
     2821 ggccaatacc acatttgtag aggttttact tgctttaaaa aacctcccac acctccccct
     2881 gaacctgaaa cataaaatga atgcaattgt tgttgttaac ttgtttattg cagcttataa
     2941 tggttacaaa taaagcaata gcatcacaaa tttcacaaat aaagcatttt tttcactgca
     3001 ttctagttgt ggtttgtcca aactcatcaa tgtatcttat catgtctgtg agctgaaggt
     3061 acgctgtatc tcagtcagtc aagctagctt aatggtgatg gtgatggtgc gactgaaagt
     3121 aaaggttttc ttgcaggaca gtatgtgggg tgtacatggc ttcataaaac tccggttccc
     3181 agtaccgtga ggtattgtca tttgtaagca ttacagaata catgtcgagc atatgtccag
     3241 ttagttcatc atgaagtttc cttatgtatt gaaggtacag gtgaaacaca tccgcatatt
     3301 cctgatttgg atgtttggta agggggtaag cgtcgattgc caaactgacg aagcgctcaa
     3361 tcatgagtgt accgtcagtc ttcacaatgt catccacaaa acaaccggct cccaagatcc
     3421 tactcggatc tgggtatggt aaatagacat agtcatcacc ctgcttgacc agcatggtgt
     3481 gctgggaaca gaactcgtgc gggcctttgg tcagatcagt ctcggtccaa cacttagcct
     3541 cgctcataaa cacgttattc tgataataaa gaaccgactt aaaatttttg atactggcca
     3601 ccaagccctg agaggcgtaa gttgaattaa agcacacaac tgcatcgtcg gagaggatca
     3661 tcatggagaa atgtttccgt aagtatgcgt aaaattcgtt cacgaagtcg gtgtctacat
     3721 ctcgattacg atacagacac tcgtacagcc tgtgttgcag gttccggacg tacttatcgg
     3781 cgatcttgtt cccatcagta gacagcagag cattcacgtt tgctgtaacg gcctgacata
     3841 tgttgaatac tgaatttgcg taggcagttg tggcgtcgcc tgaggacgtt cctccgggct
     3901 tcacgtaaag tgaaccccca cacatcacca tctcgctaag cacttgggcg cattcattag
     3961 ccaatctgta aaagcgatgt gacagactac agcacgtggt gtgctttcgc gccaggacta
     4021 atgacgccat aattctcagc atgtttggca tcgcccggtc gcacttagga taatcccaac
     4081 ccatcaggtg agggttttcc acgtcggagt acacagtttt taacatatta tgccaccctc
     4141 cgtagaattt ggaggtgcca atcacgacgg tagcccctcg tgtagcggca atagatttca
     4201 acagcttctg gtgaaactgc ctgttagtca ttgtcgaaca tatactcacc ccagcaactg
     4261 tcctcgctct gttctttgcg ctgattgcat atttgaggtt catctgggta attgtgggga
     4321 taacgttgcg cttggtatac gcgaagagag cgtcctgatc ctcataggac atagagtcat
     4381 aatatagcct tgcctttccc catttgttaa aggggaagcc ggcgctcttg tccagattgt
     4441 tcacgatgac ttgattggcg ttaatgcatc cgccatcgta gcaatcaaag tatttatcaa
     4501 cgacctccac cacgaacaac agctgccgaa tgtcgcacat agttggtagg ttataccggt
     4561 agtagtcgta gtccgagata gcggcattac cgtcctgagc aaagaaaaaa tgtttcaatt
     4621 ctacgcttga tccctcctta aaaaaccctt tactgacggc gaaatcgtag aaatctttat
     4681 tgaagttgcc aggcttgacg gtttggaagg caacgttatt tgttaaagcc gccacgctga
     4741 agcacgtggt ccgcttatcc agcaacaaat taccgcttgc agcgtgcatc gcagggtctg
     4801 cggcatacac cagaagttcc ttaaaactta acctggaaga atgcaggttg acatcttggt
     4861 tgtgaactac gccgagctca cgaaagtgat agcctgtgga aacaacgaag ggcaccccat
     4921 ccacaaagat tttgcgcact agaggaccaa acgaagttgg tgggaacact gtgctaaaca
     4981 gtacattgaa attggcacaa tgaagtatgc atctgtcgtc cagacaattc acacagttgg
     5041 gatgataagt ctggtcccag tatttgaaat aacgatcaaa gagctttaac ctctcttccg
     5101 tgaaatcgta cttcagcaaa tcccacttga tatacggttt tgtgaggtcg gtgtccacgt
     5161 gactctcagc tgtgagagct cgggttaagg ttaggattgg catcagaagt gagtagtaag
     5221 agtcaacgac aggtactccg gacccgggcg tggtctgaat gaaatctcca aagtcatacc
     5281 agttgccatt caggtcctgg ttatcaagtg tcaggacacc cacaatgccg gcattccgca
     5341 tcgcgtcgca aaattggact gtcttcaata gtgcctggcg aaccctttcc cccaggttag
     5401 cgtacactct gagtatatcg ggattctcga caaaatcgta ccaatctttc ttgttgaaat
     5461 aatcgtcatc gcaacaatta taagtcacga ggatttcttt taacgtgtca cagtttccct
     5521 cgtcgaagtg tcgcagggcg tacaccaggt ctgccatagt gtacttggtt aaccgctgtc
     5581 tgctaatatg cgggaccata tcgccatcga tgcggaattt aaagaagtcg tgttttgcta
     5641 cggcagggca gtccttcagc aagttataaa ttgtttcctc atgctgataa ttactaaaag
     5701 tgtgacgctt aaccacaaag tatgagtcta tgagattatc atcttcgtct ttctcttgga
     5761 acctgcaaca gttggtctta agaaacttgg cgaagccagc gactttgtcg ttatagatat
     5821 caaaagctcg gtacaccaca tctgtgctcg tcccggttcc gcatggagtt agccgggccg
     5881 cggatacacc acaaactcta ttcaggaacg actgtgcgtc ggcagacatg gtggcggccg
     5941 ctggcgggtg tgtcgagtgg atggtaggat cgacaaagat ctgatctgac ggttcactaa
     6001 accagctctg cttatataga cctcccaccg tacacgccta ccgcccattt gcgtcaatgg
     6061 ggcggagttg ttacgacatt ttggaaagtc ccgttgattt tggtgccaaa acaaactccc
     6121 attgacgtca atggggtgga gacttggaaa tccccgtgag tcaaaccgct atccacgccc
     6181 attgatgtac tgccaaaacc gcatcagcat ggtaatagcg atgactaata cgtagatgta
     6241 ctgccaagta ggaaagtccc ataaggtcat gtactgggca taatgccagg cgggccattt
     6301 accgtcattg acgtcaatag ggggcgtact tggcatatga tacacttgat gtactgccaa
     6361 gtgggcagtt taccgtaaat actccaccca ttgacgtcaa tggaaagtcc ctattggcgt
     6421 tactatggga acatacgtca ttattgacgt caatgggcgg gggtcgttgg gcggtcagcc
     6481 aggcgggcca tttaccgtaa gttatgtaac gcggaactcc atatatgggc tatgaactaa
     6541 tgaccccgta attgattact agatctggct acactcgatc agcagttaga taataaaatc
     6601 gctatccatc gaagatggat gtgtgttggt tttttgtgtg tgtaacgcaa cgattgatag
     6661 cataacccct tggggcctct aaacgggtct tgaggggttt tttgaattga gaggtagctg
     6721 aagctgctgt tatgaccact agagggaaga agatacctgt ggagctaatg gtccaagatg
     6781 tcgggagccg cgatcgctgc caaaaaggta agctgacgat agctgacgag attcgatgcc
     6841 cgacgagcaa atgagcgcca cggctgttcg cgtaagctcg aaacgacgga gattcgatag
     6901 agccagagtc gagtttaaac gattcgggat cttaaggata aatttctggt aaggaggaca
     6961 cgtatggaag tgggcaagtt ggggaagccg tatccgttgc tgaatctggc atatgtggga
     7021 gtataagacg cgcagcgtcg catcaggcat ttttttctgc gccaatgcac ctagaatcgg
     7081 ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc tgtctatttc gttcatccat
     7141 agttgcctga ctccccgtcg tgtagataac tacgatacgg gagggcttac catctggccc
     7201 cagtgctgca atgataccgc gagacccacg ctcaccggct ccagatttat cagcaataaa
     7261 ccagccagcc ggaagggccg agcgcagaag tggtcctgca actttatccg cctccatcca
     7321 gtctatcaac tgttgccggg aagctagagt aagtagttcg ccagttaata gtttacgcaa
     7381 ggttgttgcc attgctacag gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt
     7441 cagctccggt tcccaacgat caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc
     7501 ggttagctcc ttcggtcctc caatcgttgt cagaagtaag ttggccgcag tgttatcact
     7561 catggttatg gcagcactgc ataattctct tactgtcatg ccatccgtaa gatgcttttc
     7621 tgtgactggt gaatactcaa ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg
     7681 ctcttgcccg gcgtcaatac gggataatac cgcgccacat agcagaactt taaaagtgct
     7741 catcattgga aagcgttctt cggggcgaaa actctcaagg atcttaccgc tgttgagatc
     7801 cagttcgatg taacccactc gcgcacccaa ctgatcttca gcatctttta ctttcaccag
     7861 cgtttctggg tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa taagggcgac
     7921 acggaaatgt tgaatactca tgcgaaagcc tcctgctgat cctgtctctt gatagatgtt
     7981 tgcaaaagct aggactcgat tatacgttga ccgatcagac tatcaatatc taggtttaaa
     8041 cagatcggta cgctgccgtg accaggcttg agtacaatat ctgacgattg gacttcctgg
     8101 gtcccaagct ggattccgac tggctgactg ccaagcaacg atatggatcg tcgagactcg
     8161 cactgagagg atcgttctcg gtacaccagg cgcgcctagc gttacacaca caaaaaacca
     8221 acacacatcc atcttcgatg gatagcgatt ttattatcta agactaggct tacatactag
     8281 cctaggcacc gggcttgcgg gtcatgcacc aggtgcgcgg tccttcgggc acctcgacgt
     8341 cggcggtgac ggtgaagccg agccgctcgt agaaggggag gttgcggggc gcggaggtct
     8401 ccaggaaggc gggcaccccg gcgcgctcgg cggcctccac tccggggagc acgacggcgc
     8461 tgcccagacc cttgccctgg tggtcgggcg agacgccgac ggtggccagg aaccacgcgg
     8521 gctccttggg ccggtgcggc gccaggaggc cctccatctg ttgctgcgcg gccagccggg
     8581 aaccgctcaa ctcggccatg cgcgggccga tctcggcgaa caccgccccc gcttcgacgc
     8641 tctccggcgt ggtccagacc gccaccgcgg cgccgtcgtc cgcgacccac accttgccga
     8701 tgtcgagccc gacgcgcgtg aggaagagtt cttgcagctc ggtgacccgc tcgatgtggc
     8761 ggtccggatc gacggtgtgg cgcgtggcgg ggtagtcggc gaacgcggcg gcgagggtgc
     8821 gtacggccct ggggacgtcg tcgcgggtgg cgaggcgcac cgtgggcttg tactcggtca
     8881 tgttgtctaa caaaaaagcc aaaaacggcc agaatttagc ggacaattta caa
//
