LOCUS       sequence-295744-        9457 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 9457)
  TITLE     SARS-CoV-2 protein Mammalian expression constructs, tagged, for 
            protein expression and/or purification
REFERENCE   2  (bases 1 to 9457)
  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-m4133722
FEATURES             Location/Qualifiers
     source          1..9457
                     /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             2852..2929
                     /codon_start=1
                     /product="calmodulin-binding peptide"
                     /label=CBP
                     /note="derived from skeletal muscle myosin light chain 
                     kinase; binds calmodulin with nanomolar affinity in the 
                     presence of calcium"
                     /translation="KRRWKKNFIAVSAANRFKKISSSGAL"
     CDS             2957..2977
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     polyA_signal    3426..3547
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3437..3456)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     3491..3510
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     terminator      3662..3708
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     terminator      3738..3874
                     /label=rrnG terminator
                     /note="transcription terminator from the E. coli ribosomal 
                     RNA rrnG operon (Albrechtsen et al., 1991)"
     misc_feature    3919..3990
                     /label=3' beta-globin insulator
                     /note="insulator downstream of the human beta-globin locus 
                     (Farrell et al., 2002)"
     rep_origin      complement(4216..4804)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4296..4315)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     promoter        5304..6513
                     /label=UbC promoter
                     /note="human ubiquitin C promoter"
     primer_bind     6350..6369
                     /label=hUBCpro-F
                     /note="Human Ubiquitin C (UbC) promoter, forward primer"
     CDS             6517..7116
                     /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(6517..6536)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     7013..7033
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     polyA_signal    7143..7191
                     /note="synthetic polyadenylation signal"
     CDS             join(7457..7525,7526..8317)
                     /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(7675..7694)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     terminator      8330..8466
                     /label=rrnG terminator
                     /note="transcription terminator from the E. coli ribosomal 
                     RNA rrnG operon (Albrechtsen et al., 1991)"
     misc_feature    8611..8682
                     /label=5' beta-globin insulator
                     /note="insulator upstream of the human beta-globin locus 
                     (Farrell et al., 2002)"
     terminator      complement(8687..8733)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     polyA_signal    8751..8799
                     /note="synthetic polyadenylation signal"
     enhancer        8880..9183
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        9185..9387
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     9338..9358
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      join(9456..9457,1..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 ttcagtcggg
     2821 acggcgaatc ccggggctga taaatccctg gaaaaggaga tggaagaaga actttattgc
     2881 agtgtctgcg gctaaccggt tcaaaaagat cagttcaagc ggcgccctag attacgacat
     2941 tcctacgacc gcctccgaga atctctattt ccagggtgaa tttggcttgg cacaacatga
     3001 cgaggccgtc gataacaagt ttaataaaga acagcagaac gctttctatg agatccttca
     3061 cttaccaaat ctgaacgaag agcaacgcaa cgctttcatt cagagcctga aagacgatcc
     3121 ctcgcagtct gcaaatctcc tggccgaagc caagaagctg aatgacgcac aggctccaaa
     3181 agttgataac aagtttaaca aggagcagca gaatgctttc tacgagatat tgcaccttcc
     3241 taatctgaat gaggaacaaa gaaatgcatt tatccagtcc ctaaaagacg atccatcaca
     3301 gagtgccaac ctgcttgcgg aagccaaaaa gctcaatgat gcccaggccc cgaaagtaga
     3361 cgcgaaccat caataagcta gcttgactga ctgagataca gcgtaccttc agctcacaga
     3421 catgataaga tacattgatg agtttggaca aaccacaact agaatgcagt gaaaaaaatg
     3481 ctttatttgt gaaatttgtg atgctattgc tttatttgta accattataa gctgcaataa
     3541 acaagttaac aacaacaatt gcattcattt tatgtttcag gttcaggggg aggtgtggga
     3601 ggttttttaa agcaagtaaa acctctacaa atgtggtatt ggcccatctc tatcggtatc
     3661 gtagcataac cccttggggc ctctaaacgg gtcttgaggg gttttttgtg cccctcgggc
     3721 cggattgcta tctaccggca ttggcgcaga aaaaaatgcc tgatgcgacg ctgcgcgtct
     3781 tatactccca catatgccag attcagcaac ggatacggct tccccaactt gcccacttcc
     3841 atacgtgtcc tccttaccag aaatttatcc ttaaggtcgt cagctatcct gcaggcgatc
     3901 tctcgatttc gatcaagaca ttcctttaat ggtcttttct ggacaccact aggggtcaga
     3961 agtagttcat caaactttct tccctcccta atctcattgg ttaccttggg ctatcgaaac
     4021 ttaattaacc agtcaagtca gctacttggc gagatcgact tgtctgggtt tcgactacgc
     4081 tcagaattgc gtcagtcaag ttcgatctgg tccttgctat tgcacccgtt ctccgattac
     4141 gagtttcatt taaatcatgt gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc
     4201 cgcgttgctg gcgtttttcc ataggctccg cccccctgac gagcatcaca aaaatcgacg
     4261 ctcaagtcag aggtggcgaa acccgacagg actataaaga taccaggcgt ttccccctgg
     4321 aagctccctc gtgcgctctc ctgttccgac cctgccgctt accggatacc tgtccgcctt
     4381 tctcccttcg ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc tcagttcggt
     4441 gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg
     4501 cgccttatcc ggtaactatc gtcttgagtc caacccggta agacacgact tatcgccact
     4561 ggcagcagcc actggtaaca ggattagcag agcgaggtat gtaggcggtg ctacagagtt
     4621 cttgaagtgg tggcctaact acggctacac tagaagaaca gtatttggta tctgcgctct
     4681 gctgaagcca gttaccttcg gaaaaagagt tggtagctct tgatccggca aacaaaccac
     4741 cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc
     4801 tcaagaagat cctttgatct tttctacggg gtctgacgct cagtggaacg aaaactcacg
     4861 ttaagggatt ttggtcatga gattatcaaa aaggatcttc acctagatcc ttttaaatta
     4921 aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa acttggtctg acagttacca
     4981 atgcttaatc agtgaggcac ctatctcagc gatctgtcta tttcgttcat ccatagttgc
     5041 atttaaattt ccgaactctc caaggccctc gtcggaaaat cttcaaacct ttcgtccgat
     5101 ccatcttgca ggctacctct cgaacgaact atcgcaagtc tcttggccgg ccttgcgcct
     5161 tggctattgc ttggcagcgc ctatcgccag gtattactcc aatcccgaat atccgagatc
     5221 gggatcaccc gagagaagtt caacctacat cctcaatccc gatctatccg agatccgagg
     5281 aatatcgaaa tcggggcgcg cctggcctcc gcgccgggtt ttggcgcctc ccgcgggcgc
     5341 ccccctcgtc acggcgagcg ctgccacgtc agacgaaggg cgcaggagcg tcctgatcct
     5401 tccgcccgga cgctcaggac agcggcccgc tgctcataag actcggcctt agaaccccag
     5461 tatcagcaga aggacatttt aggacgggac ttgggtgact ctagggcact ggttttcttt
     5521 ccagagagcg gaacaggcga ggaaaagtag tcccttctcg gcgattctgc ggagggatct
     5581 ccgtggggcg gtgaacgccg atgattatat aaggacgcgc cgggtgtggc acagctagtt
     5641 ccgtcgcagc cgggatttgg gtcgcggttc ttgtttgtgg atcgctgtga tcgtcacttg
     5701 gtgagtagcg ggctgctggg ctggccgggg ctttcgtggc cgccgggccg ctcggtggga
     5761 cggaagcgtg tggagagacc gccaagggct gtagtctggg tccgcgagca aggttgccct
     5821 gaactggggg ttggggggag cgcagcaaaa tggcggctgt tcccgagtct tgaatggaag
     5881 acgcttgtga ggcgggctgt gaggtcgttg aaacaaggtg gggggcatgg tgggcggcaa
     5941 gaacccaagg tcttgagccc ttcgctaatg cgggaaagct cttattcggg tgagatgggc
     6001 tgggcaccat ctggggaccc tgacgtgaag tttgtcactg actggagaac tcggtttgtc
     6061 gtctgttgcg ggggcggcag ttatggcggt gccgttgggc agtgcacccg tacctttggg
     6121 agcgcgcgcc ctcgtcgtgt cgtgacgtca cccgttctgt tggcttataa tgcagggtgg
     6181 ggccacctgc cggtaggtgt gcggtaggct tttctccgtc gcaggacgca gggttcgggc
     6241 ctagggtagg ctctcctgaa tcgacaggcg ccggacctct ggtgagggga gggataagtg
     6301 aggcgtcagt ttctttggtc ggttttatgt acctatcttc ttaagtagct gaagctccgg
     6361 ttttgaacta tgcgctcggg gttggcgagt gtgttttgtg aagtttttta ggcacctttt
     6421 gaaatgtaat catttgggtc aatatgtaat tttcagtgtt agacttgtaa attgtccgct
     6481 aaattctggc cgtttttggc ttttttgtta gacaacatga ccgagtacaa gcccacggtg
     6541 cgcctcgcca cccgcgacga cgtccccagg gccgtacgca ccctcgccgc cgcgttcgcc
     6601 gactaccccg ccacgcgcca caccgtcgat ccggaccgcc acatcgagcg ggtcaccgag
     6661 ctgcaagaac tcttcctcac gcgcgtcggg ctcgacatcg gcaaggtgtg ggtcgcggac
     6721 gacggcgccg cggtggcggt ctggaccacg ccggagagcg tcgaagcggg ggcggtgttc
     6781 gccgagatcg gcccgcgcat ggccgagttg agcggttccc ggctggccgc gcagcaacag
     6841 atggagggcc tcctggcgcc gcaccggccc aaggagcccg cgtggttcct ggccaccgtc
     6901 ggcgtctcgc ccgaccacca gggcaagggt ctgggcagcg ccgtcgtgct ccccggagtg
     6961 gaggccgccg agcgcgccgg ggtgcccgcc ttcctggaga cctccgcgcc ccgcaacctc
     7021 cccttctacg agcggctcgg cttcaccgtc accgccgacg tcgaggtgcc cgaaggaccg
     7081 cgcacctggt gcatgacccg caagcccggt gcctaggcta gtatgtaagc ctagtcttag
     7141 ataataaaat cgctatccat cgaagatgga tgtgtgttgg ttttttgtgt gtgtaacgct
     7201 aggcgcgcct ggtgtaccga gaacgatcct ctcagtgcga gtctcgacga tccatatcgt
     7261 tgcttggcag tcagccagtc ggaatccagc ttgggaccca ggaagtccaa tcgtcagata
     7321 ttgtactcaa gcctggtcac ggcagcgtac cgatctgttt aaacctagat attgatagtc
     7381 tgatcggtca acgtataatc gagtcctagc ttttgcaaac atctatcaag agacaggatc
     7441 agcaggaggc tttcgcatga gtattcaaca tttccgtgtc gcccttattc ccttttttgc
     7501 ggcattttgc cttcctgttt ttgctcaccc agaaacgctg gtgaaagtaa aagatgctga
     7561 agatcagttg ggtgcgcgag tgggttacat cgaactggat ctcaacagcg gtaagatcct
     7621 tgagagtttt cgccccgaag aacgctttcc aatgatgagc acttttaaag ttctgctatg
     7681 tggcgcggta ttatcccgta ttgacgccgg gcaagagcaa ctcggtcgcc gcatacacta
     7741 ttctcagaat gacttggttg agtattcacc agtcacagaa aagcatctta cggatggcat
     7801 gacagtaaga gaattatgca gtgctgccat aaccatgagt gataacactg cggccaactt
     7861 acttctgaca acgattggag gaccgaagga gctaaccgct tttttgcaca acatggggga
     7921 tcatgtaact cgccttgatc gttgggaacc ggagctgaat gaagccatac caaacgacga
     7981 gcgtgacacc acgatgcctg tagcaatggc aacaaccttg cgtaaactat taactggcga
     8041 actacttact ctagcttccc ggcaacagtt gatagactgg atggaggcgg ataaagttgc
     8101 aggaccactt ctgcgctcgg cccttccggc tggctggttt attgctgata aatctggagc
     8161 cggtgagcgt gggtctcgcg gtatcattgc agcactgggg ccagatggta agccctcccg
     8221 tatcgtagtt atctacacga cggggagtca ggcaactatg gatgaacgaa atagacagat
     8281 cgctgagata ggtgcctcac tgattaagca ttggtaaccg attctaggtg cattggcgca
     8341 gaaaaaaatg cctgatgcga cgctgcgcgt cttatactcc cacatatgcc agattcagca
     8401 acggatacgg cttccccaac ttgcccactt ccatacgtgt cctccttacc agaaatttat
     8461 ccttaagatc gtttaaactc gactctggct ctatcgaatc tccgtcgttt cgagcttacg
     8521 cgaacagccg tggcgctcat ttgctcgtcg ggcatcgaat ctcgtcagct atcgtcagct
     8581 tacctttttg gcagcgatcg cggctcccga catcttggac cattagctcc acaggtatct
     8641 tcttccctct agtggtcata acagcagctt cagctacctc tcaattcaaa aaacccctca
     8701 agacccgttt agaggcccca aggggttatg ctatcaatcg ttgcgttaca cacacaaaaa
     8761 accaacacac atccatcttc gatggatagc gattttatta tctaactgct gatcgagtgt
     8821 agccagatct agtaatcaat tacggggtca ttagttcata gcccatatat ggagttccgc
     8881 gttacataac ttacggtaaa tggcccgcct ggctgaccgc ccaacgaccc ccgcccattg
     8941 acgtcaataa tgacgtatgt tcccatagta acgccaatag ggactttcca ttgacgtcaa
     9001 tgggtggagt atttacggta aactgcccac ttggcagtac atcaagtgta tcatatgcca
     9061 agtacgcccc ctattgacgt caatgacggt aaatggcccg cctggcatta tgcccagtac
     9121 atgaccttat gggactttcc tacttggcag tacatctacg tattagtcat cgctattacc
     9181 atgctgatgc ggttttggca gtacatcaat gggcgtggat agcggtttga ctcacgggga
     9241 tttccaagtc tccaccccat tgacgtcaat gggagtttgt tttggcacca aaatcaacgg
     9301 gactttccaa aatgtcgtaa caactccgcc ccattgacgc aaatgggcgg taggcgtgta
     9361 cggtgggagg tctatataag cagagctggt ttagtgaacc gtcagatcag atctttgtcg
     9421 atcctaccat ccactcgaca cacccgccag cggccgc
//
