LOCUS       sequence-295162-        9292 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 9292)
  TITLE     SARS-CoV-2 protein Mammalian expression constructs, tagged, for 
            protein expression and/or purification
REFERENCE   2  (bases 1 to 9292)
  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-m4133389
FEATURES             Location/Qualifiers
     source          1..9292
                     /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             3167..3184
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     polyA_signal    3261..3382
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3272..3291)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     3326..3345
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     terminator      3497..3543
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     terminator      3573..3709
                     /label=rrnG terminator
                     /note="transcription terminator from the E. coli ribosomal 
                     RNA rrnG operon (Albrechtsen et al., 1991)"
     misc_feature    3754..3825
                     /label=3' beta-globin insulator
                     /note="insulator downstream of the human beta-globin locus 
                     (Farrell et al., 2002)"
     rep_origin      complement(4051..4639)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4131..4150)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     promoter        5139..6348
                     /label=UbC promoter
                     /note="human ubiquitin C promoter"
     primer_bind     6185..6204
                     /label=hUBCpro-F
                     /note="Human Ubiquitin C (UbC) promoter, forward primer"
     CDS             6352..6951
                     /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(6352..6371)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     6848..6868
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     polyA_signal    6978..7026
                     /note="synthetic polyadenylation signal"
     CDS             join(7292..7360,7361..8152)
                     /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(7510..7529)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     terminator      8165..8301
                     /label=rrnG terminator
                     /note="transcription terminator from the E. coli ribosomal 
                     RNA rrnG operon (Albrechtsen et al., 1991)"
     misc_feature    8446..8517
                     /label=5' beta-globin insulator
                     /note="insulator upstream of the human beta-globin locus 
                     (Farrell et al., 2002)"
     terminator      complement(8522..8568)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     polyA_signal    8586..8634
                     /note="synthetic polyadenylation signal"
     enhancer        8715..9018
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        9020..9222
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     9173..9193
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      join(9291..9292,1..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 ttcagtcgat
     2821 gagtgacaaa atcattcatc taaccgatga ctcctttgat acagacgtgt tgaaggccga
     2881 tggtgcgata ctggtagatt tctgggctga atggtgtggc ccgtgcaaga tgatcgcacc
     2941 cattctggac gagatcgccg acgagtatca aggaaaatta actgtcgcta agcttaatat
     3001 agatcagaac ccagggacgg cacctaaata cggcatcagg ggaataccta ccctcctgct
     3061 ctttaagaat ggggaagttg ccgcgacaaa agtgggagcc ctgtctaagg gccagctaaa
     3121 agagttcttg gacgctaacc tggcaggttc aggctcgggg cacatgcacc atcaccatca
     3181 ccatagcagc gggctcgtgc cacgcggcta agctagcttg actgactgag atacagcgta
     3241 ccttcagctc acagacatga taagatacat tgatgagttt ggacaaacca caactagaat
     3301 gcagtgaaaa aaatgcttta tttgtgaaat ttgtgatgct attgctttat ttgtaaccat
     3361 tataagctgc aataaacaag ttaacaacaa caattgcatt cattttatgt ttcaggttca
     3421 gggggaggtg tgggaggttt tttaaagcaa gtaaaacctc tacaaatgtg gtattggccc
     3481 atctctatcg gtatcgtagc ataacccctt ggggcctcta aacgggtctt gaggggtttt
     3541 ttgtgcccct cgggccggat tgctatctac cggcattggc gcagaaaaaa atgcctgatg
     3601 cgacgctgcg cgtcttatac tcccacatat gccagattca gcaacggata cggcttcccc
     3661 aacttgccca cttccatacg tgtcctcctt accagaaatt tatccttaag gtcgtcagct
     3721 atcctgcagg cgatctctcg atttcgatca agacattcct ttaatggtct tttctggaca
     3781 ccactagggg tcagaagtag ttcatcaaac tttcttccct ccctaatctc attggttacc
     3841 ttgggctatc gaaacttaat taaccagtca agtcagctac ttggcgagat cgacttgtct
     3901 gggtttcgac tacgctcaga attgcgtcag tcaagttcga tctggtcctt gctattgcac
     3961 ccgttctccg attacgagtt tcatttaaat catgtgagca aaaggccagc aaaaggccag
     4021 gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg ctccgccccc ctgacgagca
     4081 tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg acaggactat aaagatacca
     4141 ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg
     4201 atacctgtcc gcctttctcc cttcgggaag cgtggcgctt tctcatagct cacgctgtag
     4261 gtatctcagt tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt
     4321 tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc cggtaagaca
     4381 cgacttatcg ccactggcag cagccactgg taacaggatt agcagagcga ggtatgtagg
     4441 cggtgctaca gagttcttga agtggtggcc taactacggc tacactagaa gaacagtatt
     4501 tggtatctgc gctctgctga agccagttac cttcggaaaa agagttggta gctcttgatc
     4561 cggcaaacaa accaccgctg gtagcggtgg tttttttgtt tgcaagcagc agattacgcg
     4621 cagaaaaaaa ggatctcaag aagatccttt gatcttttct acggggtctg acgctcagtg
     4681 gaacgaaaac tcacgttaag ggattttggt catgagatta tcaaaaagga tcttcaccta
     4741 gatcctttta aattaaaaat gaagttttaa atcaatctaa agtatatatg agtaaacttg
     4801 gtctgacagt taccaatgct taatcagtga ggcacctatc tcagcgatct gtctatttcg
     4861 ttcatccata gttgcattta aatttccgaa ctctccaagg ccctcgtcgg aaaatcttca
     4921 aacctttcgt ccgatccatc ttgcaggcta cctctcgaac gaactatcgc aagtctcttg
     4981 gccggccttg cgccttggct attgcttggc agcgcctatc gccaggtatt actccaatcc
     5041 cgaatatccg agatcgggat cacccgagag aagttcaacc tacatcctca atcccgatct
     5101 atccgagatc cgaggaatat cgaaatcggg gcgcgcctgg cctccgcgcc gggttttggc
     5161 gcctcccgcg ggcgcccccc tcgtcacggc gagcgctgcc acgtcagacg aagggcgcag
     5221 gagcgtcctg atccttccgc ccggacgctc aggacagcgg cccgctgctc ataagactcg
     5281 gccttagaac cccagtatca gcagaaggac attttaggac gggacttggg tgactctagg
     5341 gcactggttt tctttccaga gagcggaaca ggcgaggaaa agtagtccct tctcggcgat
     5401 tctgcggagg gatctccgtg gggcggtgaa cgccgatgat tatataagga cgcgccgggt
     5461 gtggcacagc tagttccgtc gcagccggga tttgggtcgc ggttcttgtt tgtggatcgc
     5521 tgtgatcgtc acttggtgag tagcgggctg ctgggctggc cggggctttc gtggccgccg
     5581 ggccgctcgg tgggacggaa gcgtgtggag agaccgccaa gggctgtagt ctgggtccgc
     5641 gagcaaggtt gccctgaact gggggttggg gggagcgcag caaaatggcg gctgttcccg
     5701 agtcttgaat ggaagacgct tgtgaggcgg gctgtgaggt cgttgaaaca aggtgggggg
     5761 catggtgggc ggcaagaacc caaggtcttg agcccttcgc taatgcggga aagctcttat
     5821 tcgggtgaga tgggctgggc accatctggg gaccctgacg tgaagtttgt cactgactgg
     5881 agaactcggt ttgtcgtctg ttgcgggggc ggcagttatg gcggtgccgt tgggcagtgc
     5941 acccgtacct ttgggagcgc gcgccctcgt cgtgtcgtga cgtcacccgt tctgttggct
     6001 tataatgcag ggtggggcca cctgccggta ggtgtgcggt aggcttttct ccgtcgcagg
     6061 acgcagggtt cgggcctagg gtaggctctc ctgaatcgac aggcgccgga cctctggtga
     6121 ggggagggat aagtgaggcg tcagtttctt tggtcggttt tatgtaccta tcttcttaag
     6181 tagctgaagc tccggttttg aactatgcgc tcggggttgg cgagtgtgtt ttgtgaagtt
     6241 ttttaggcac cttttgaaat gtaatcattt gggtcaatat gtaattttca gtgttagact
     6301 tgtaaattgt ccgctaaatt ctggccgttt ttggcttttt tgttagacaa catgaccgag
     6361 tacaagccca cggtgcgcct cgccacccgc gacgacgtcc ccagggccgt acgcaccctc
     6421 gccgccgcgt tcgccgacta ccccgccacg cgccacaccg tcgatccgga ccgccacatc
     6481 gagcgggtca ccgagctgca agaactcttc ctcacgcgcg tcgggctcga catcggcaag
     6541 gtgtgggtcg cggacgacgg cgccgcggtg gcggtctgga ccacgccgga gagcgtcgaa
     6601 gcgggggcgg tgttcgccga gatcggcccg cgcatggccg agttgagcgg ttcccggctg
     6661 gccgcgcagc aacagatgga gggcctcctg gcgccgcacc ggcccaagga gcccgcgtgg
     6721 ttcctggcca ccgtcggcgt ctcgcccgac caccagggca agggtctggg cagcgccgtc
     6781 gtgctccccg gagtggaggc cgccgagcgc gccggggtgc ccgccttcct ggagacctcc
     6841 gcgccccgca acctcccctt ctacgagcgg ctcggcttca ccgtcaccgc cgacgtcgag
     6901 gtgcccgaag gaccgcgcac ctggtgcatg acccgcaagc ccggtgccta ggctagtatg
     6961 taagcctagt cttagataat aaaatcgcta tccatcgaag atggatgtgt gttggttttt
     7021 tgtgtgtgta acgctaggcg cgcctggtgt accgagaacg atcctctcag tgcgagtctc
     7081 gacgatccat atcgttgctt ggcagtcagc cagtcggaat ccagcttggg acccaggaag
     7141 tccaatcgtc agatattgta ctcaagcctg gtcacggcag cgtaccgatc tgtttaaacc
     7201 tagatattga tagtctgatc ggtcaacgta taatcgagtc ctagcttttg caaacatcta
     7261 tcaagagaca ggatcagcag gaggctttcg catgagtatt caacatttcc gtgtcgccct
     7321 tattcccttt tttgcggcat tttgccttcc tgtttttgct cacccagaaa cgctggtgaa
     7381 agtaaaagat gctgaagatc agttgggtgc gcgagtgggt tacatcgaac tggatctcaa
     7441 cagcggtaag atccttgaga gttttcgccc cgaagaacgc tttccaatga tgagcacttt
     7501 taaagttctg ctatgtggcg cggtattatc ccgtattgac gccgggcaag agcaactcgg
     7561 tcgccgcata cactattctc agaatgactt ggttgagtat tcaccagtca cagaaaagca
     7621 tcttacggat ggcatgacag taagagaatt atgcagtgct gccataacca tgagtgataa
     7681 cactgcggcc aacttacttc tgacaacgat tggaggaccg aaggagctaa ccgctttttt
     7741 gcacaacatg ggggatcatg taactcgcct tgatcgttgg gaaccggagc tgaatgaagc
     7801 cataccaaac gacgagcgtg acaccacgat gcctgtagca atggcaacaa ccttgcgtaa
     7861 actattaact ggcgaactac ttactctagc ttcccggcaa cagttgatag actggatgga
     7921 ggcggataaa gttgcaggac cacttctgcg ctcggccctt ccggctggct ggtttattgc
     7981 tgataaatct ggagccggtg agcgtgggtc tcgcggtatc attgcagcac tggggccaga
     8041 tggtaagccc tcccgtatcg tagttatcta cacgacgggg agtcaggcaa ctatggatga
     8101 acgaaataga cagatcgctg agataggtgc ctcactgatt aagcattggt aaccgattct
     8161 aggtgcattg gcgcagaaaa aaatgcctga tgcgacgctg cgcgtcttat actcccacat
     8221 atgccagatt cagcaacgga tacggcttcc ccaacttgcc cacttccata cgtgtcctcc
     8281 ttaccagaaa tttatcctta agatcgttta aactcgactc tggctctatc gaatctccgt
     8341 cgtttcgagc ttacgcgaac agccgtggcg ctcatttgct cgtcgggcat cgaatctcgt
     8401 cagctatcgt cagcttacct ttttggcagc gatcgcggct cccgacatct tggaccatta
     8461 gctccacagg tatcttcttc cctctagtgg tcataacagc agcttcagct acctctcaat
     8521 tcaaaaaacc cctcaagacc cgtttagagg ccccaagggg ttatgctatc aatcgttgcg
     8581 ttacacacac aaaaaaccaa cacacatcca tcttcgatgg atagcgattt tattatctaa
     8641 ctgctgatcg agtgtagcca gatctagtaa tcaattacgg ggtcattagt tcatagccca
     8701 tatatggagt tccgcgttac ataacttacg gtaaatggcc cgcctggctg accgcccaac
     8761 gacccccgcc cattgacgtc aataatgacg tatgttccca tagtaacgcc aatagggact
     8821 ttccattgac gtcaatgggt ggagtattta cggtaaactg cccacttggc agtacatcaa
     8881 gtgtatcata tgccaagtac gccccctatt gacgtcaatg acggtaaatg gcccgcctgg
     8941 cattatgccc agtacatgac cttatgggac tttcctactt ggcagtacat ctacgtatta
     9001 gtcatcgcta ttaccatgct gatgcggttt tggcagtaca tcaatgggcg tggatagcgg
     9061 tttgactcac ggggatttcc aagtctccac cccattgacg tcaatgggag tttgttttgg
     9121 caccaaaatc aacgggactt tccaaaatgt cgtaacaact ccgccccatt gacgcaaatg
     9181 ggcggtaggc gtgtacggtg ggaggtctat ataagcagag ctggtttagt gaaccgtcag
     9241 atcagatctt tgtcgatcct accatccact cgacacaccc gccagcggcc gc
//
