LOCUS       sequence-332025-        8987 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 8987)
  TITLE     SARS-CoV-2 protein Mammalian expression constructs, tagged, for 
            protein expression and/or purification
REFERENCE   2  (bases 1 to 8987)
  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-m4133654
FEATURES             Location/Qualifiers
     source          1..8987
                     /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..2896
                     /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"
     polyA_signal    2949..3070
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2960..2979)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     3014..3033
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     terminator      3185..3231
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     terminator      3261..3397
                     /label=rrnG terminator
                     /note="transcription terminator from the E. coli ribosomal 
                     RNA rrnG operon (Albrechtsen et al., 1991)"
     misc_feature    3442..3513
                     /label=3' beta-globin insulator
                     /note="insulator downstream of the human beta-globin locus 
                     (Farrell et al., 2002)"
     rep_origin      complement(3739..4327)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3819..3838)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     promoter        4827..6036
                     /label=UbC promoter
                     /note="human ubiquitin C promoter"
     primer_bind     5873..5892
                     /label=hUBCpro-F
                     /note="Human Ubiquitin C (UbC) promoter, forward primer"
     CDS             6040..6639
                     /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(6040..6059)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     6536..6556
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     polyA_signal    6666..6714
                     /note="synthetic polyadenylation signal"
     CDS             join(6980..7048,7049..7840)
                     /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(7198..7217)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     terminator      7853..7989
                     /label=rrnG terminator
                     /note="transcription terminator from the E. coli ribosomal 
                     RNA rrnG operon (Albrechtsen et al., 1991)"
     misc_feature    8141..8212
                     /label=5' beta-globin insulator
                     /note="insulator upstream of the human beta-globin locus 
                     (Farrell et al., 2002)"
     terminator      complement(8217..8263)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     polyA_signal    8281..8329
                     /note="synthetic polyadenylation signal"
     enhancer        8410..8713
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        8715..8917
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     8868..8888
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      join(8986..8987,1..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 ttcagtcgaa
     2821 gaggcgatgg aaaaagaatt ttatcgcggt gagtgcagcc aacagattca aaaagatatc
     2881 ctcgtctggt gccctctaag ctagcttgac tgactgagat acagcgtacc ttcagctcac
     2941 agacatgata agatacattg atgagtttgg acaaaccaca actagaatgc agtgaaaaaa
     3001 atgctttatt tgtgaaattt gtgatgctat tgctttattt gtaaccatta taagctgcaa
     3061 taaacaagtt aacaacaaca attgcattca ttttatgttt caggttcagg gggaggtgtg
     3121 ggaggttttt taaagcaagt aaaacctcta caaatgtggt attggcccat ctctatcggt
     3181 atcgtagcat aaccccttgg ggcctctaaa cgggtcttga ggggtttttt gtgcccctcg
     3241 ggccggattg ctatctaccg gcattggcgc agaaaaaaat gcctgatgcg acgctgcgcg
     3301 tcttatactc ccacatatgc cagattcagc aacggatacg gcttccccaa cttgcccact
     3361 tccatacgtg tcctccttac cagaaattta tccttaaggt cgtcagctat cctgcaggcg
     3421 atctctcgat ttcgatcaag acattccttt aatggtcttt tctggacacc actaggggtc
     3481 agaagtagtt catcaaactt tcttccctcc ctaatctcat tggttacctt gggctatcga
     3541 aacttaatta accagtcaag tcagctactt ggcgagatcg acttgtctgg gtttcgacta
     3601 cgctcagaat tgcgtcagtc aagttcgatc tggtccttgc tattgcaccc gttctccgat
     3661 tacgagtttc atttaaatca tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa
     3721 ggccgcgttg ctggcgtttt tccataggct ccgcccccct gacgagcatc acaaaaatcg
     3781 acgctcaagt cagaggtggc gaaacccgac aggactataa agataccagg cgtttccccc
     3841 tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat acctgtccgc
     3901 ctttctccct tcgggaagcg tggcgctttc tcatagctca cgctgtaggt atctcagttc
     3961 ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa ccccccgttc agcccgaccg
     4021 ctgcgcctta tccggtaact atcgtcttga gtccaacccg gtaagacacg acttatcgcc
     4081 actggcagca gccactggta acaggattag cagagcgagg tatgtaggcg gtgctacaga
     4141 gttcttgaag tggtggccta actacggcta cactagaaga acagtatttg gtatctgcgc
     4201 tctgctgaag ccagttacct tcggaaaaag agttggtagc tcttgatccg gcaaacaaac
     4261 caccgctggt agcggtggtt tttttgtttg caagcagcag attacgcgca gaaaaaaagg
     4321 atctcaagaa gatcctttga tcttttctac ggggtctgac gctcagtgga acgaaaactc
     4381 acgttaaggg attttggtca tgagattatc aaaaaggatc ttcacctaga tccttttaaa
     4441 ttaaaaatga agttttaaat caatctaaag tatatatgag taaacttggt ctgacagtta
     4501 ccaatgctta atcagtgagg cacctatctc agcgatctgt ctatttcgtt catccatagt
     4561 tgcatttaaa tttccgaact ctccaaggcc ctcgtcggaa aatcttcaaa cctttcgtcc
     4621 gatccatctt gcaggctacc tctcgaacga actatcgcaa gtctcttggc cggccttgcg
     4681 ccttggctat tgcttggcag cgcctatcgc caggtattac tccaatcccg aatatccgag
     4741 atcgggatca cccgagagaa gttcaaccta catcctcaat cccgatctat ccgagatccg
     4801 aggaatatcg aaatcggggc gcgcctggcc tccgcgccgg gttttggcgc ctcccgcggg
     4861 cgcccccctc gtcacggcga gcgctgccac gtcagacgaa gggcgcagga gcgtcctgat
     4921 ccttccgccc ggacgctcag gacagcggcc cgctgctcat aagactcggc cttagaaccc
     4981 cagtatcagc agaaggacat tttaggacgg gacttgggtg actctagggc actggttttc
     5041 tttccagaga gcggaacagg cgaggaaaag tagtcccttc tcggcgattc tgcggaggga
     5101 tctccgtggg gcggtgaacg ccgatgatta tataaggacg cgccgggtgt ggcacagcta
     5161 gttccgtcgc agccgggatt tgggtcgcgg ttcttgtttg tggatcgctg tgatcgtcac
     5221 ttggtgagta gcgggctgct gggctggccg gggctttcgt ggccgccggg ccgctcggtg
     5281 ggacggaagc gtgtggagag accgccaagg gctgtagtct gggtccgcga gcaaggttgc
     5341 cctgaactgg gggttggggg gagcgcagca aaatggcggc tgttcccgag tcttgaatgg
     5401 aagacgcttg tgaggcgggc tgtgaggtcg ttgaaacaag gtggggggca tggtgggcgg
     5461 caagaaccca aggtcttgag cccttcgcta atgcgggaaa gctcttattc gggtgagatg
     5521 ggctgggcac catctgggga ccctgacgtg aagtttgtca ctgactggag aactcggttt
     5581 gtcgtctgtt gcgggggcgg cagttatggc ggtgccgttg ggcagtgcac ccgtaccttt
     5641 gggagcgcgc gccctcgtcg tgtcgtgacg tcacccgttc tgttggctta taatgcaggg
     5701 tggggccacc tgccggtagg tgtgcggtag gcttttctcc gtcgcaggac gcagggttcg
     5761 ggcctagggt aggctctcct gaatcgacag gcgccggacc tctggtgagg ggagggataa
     5821 gtgaggcgtc agtttctttg gtcggtttta tgtacctatc ttcttaagta gctgaagctc
     5881 cggttttgaa ctatgcgctc ggggttggcg agtgtgtttt gtgaagtttt ttaggcacct
     5941 tttgaaatgt aatcatttgg gtcaatatgt aattttcagt gttagacttg taaattgtcc
     6001 gctaaattct ggccgttttt ggcttttttg ttagacaaca tgaccgagta caagcccacg
     6061 gtgcgcctcg ccacccgcga cgacgtcccc agggccgtac gcaccctcgc cgccgcgttc
     6121 gccgactacc ccgccacgcg ccacaccgtc gatccggacc gccacatcga gcgggtcacc
     6181 gagctgcaag aactcttcct cacgcgcgtc gggctcgaca tcggcaaggt gtgggtcgcg
     6241 gacgacggcg ccgcggtggc ggtctggacc acgccggaga gcgtcgaagc gggggcggtg
     6301 ttcgccgaga tcggcccgcg catggccgag ttgagcggtt cccggctggc cgcgcagcaa
     6361 cagatggagg gcctcctggc gccgcaccgg cccaaggagc ccgcgtggtt cctggccacc
     6421 gtcggcgtct cgcccgacca ccagggcaag ggtctgggca gcgccgtcgt gctccccgga
     6481 gtggaggccg ccgagcgcgc cggggtgccc gccttcctgg agacctccgc gccccgcaac
     6541 ctccccttct acgagcggct cggcttcacc gtcaccgccg acgtcgaggt gcccgaagga
     6601 ccgcgcacct ggtgcatgac ccgcaagccc ggtgcctagg ctagtatgta agcctagtct
     6661 tagataataa aatcgctatc catcgaagat ggatgtgtgt tggttttttg tgtgtgtaac
     6721 gctaggcgcg cctggtgtac cgagaacgat cctctcagtg cgagtctcga cgatccatat
     6781 cgttgcttgg cagtcagcca gtcggaatcc agcttgggac ccaggaagtc caatcgtcag
     6841 atattgtact caagcctggt cacggcagcg taccgatctg tttaaaccta gatattgata
     6901 gtctgatcgg tcaacgtata atcgagtcct agcttttgca aacatctatc aagagacagg
     6961 atcagcagga ggctttcgca tgagtattca acatttccgt gtcgccctta ttcccttttt
     7021 tgcggcattt tgccttcctg tttttgctca cccagaaacg ctggtgaaag taaaagatgc
     7081 tgaagatcag ttgggtgcgc gagtgggtta catcgaactg gatctcaaca gcggtaagat
     7141 ccttgagagt tttcgccccg aagaacgctt tccaatgatg agcactttta aagttctgct
     7201 atgtggcgcg gtattatccc gtattgacgc cgggcaagag caactcggtc gccgcataca
     7261 ctattctcag aatgacttgg ttgagtattc accagtcaca gaaaagcatc ttacggatgg
     7321 catgacagta agagaattat gcagtgctgc cataaccatg agtgataaca ctgcggccaa
     7381 cttacttctg acaacgattg gaggaccgaa ggagctaacc gcttttttgc acaacatggg
     7441 ggatcatgta actcgccttg atcgttggga accggagctg aatgaagcca taccaaacga
     7501 cgagcgtgac accacgatgc ctgtagcaat ggcaacaacc ttgcgtaaac tattaactgg
     7561 cgaactactt actctagctt cccggcaaca gttgatagac tggatggagg cggataaagt
     7621 tgcaggacca cttctgcgct cggcccttcc ggctggctgg tttattgctg ataaatctgg
     7681 agccggtgag cgtgggtctc gcggtatcat tgcagcactg gggccagatg gtaagccctc
     7741 ccgtatcgta gttatctaca cgacggggag tcaggcaact atggatgaac gaaatagaca
     7801 gatcgctgag ataggtgcct cactgattaa gcattggtaa ccgattctag gtgcattggc
     7861 gcagaaaaaa atgcctgatg cgacgctgcg cgtcttatac tcccacatat gccagattca
     7921 gcaacggata cggcttcccc aacttgccca cttccatacg tgtcctcctt accagaaatt
     7981 tatccttaag atcccgaatc gtttaaactc gactctggct ctatcgaatc tccgtcgttt
     8041 cgagcttacg cgaacagccg tggcgctcat ttgctcgtcg ggcatcgaat ctcgtcagct
     8101 atcgtcagct tacctttttg gcagcgatcg cggctcccga catcttggac cattagctcc
     8161 acaggtatct tcttccctct agtggtcata acagcagctt cagctacctc tcaattcaaa
     8221 aaacccctca agacccgttt agaggcccca aggggttatg ctatcaatcg ttgcgttaca
     8281 cacacaaaaa accaacacac atccatcttc gatggatagc gattttatta tctaactgct
     8341 gatcgagtgt agccagatct agtaatcaat tacggggtca ttagttcata gcccatatat
     8401 ggagttccgc gttacataac ttacggtaaa tggcccgcct ggctgaccgc ccaacgaccc
     8461 ccgcccattg acgtcaataa tgacgtatgt tcccatagta acgccaatag ggactttcca
     8521 ttgacgtcaa tgggtggagt atttacggta aactgcccac ttggcagtac atcaagtgta
     8581 tcatatgcca agtacgcccc ctattgacgt caatgacggt aaatggcccg cctggcatta
     8641 tgcccagtac atgaccttat gggactttcc tacttggcag tacatctacg tattagtcat
     8701 cgctattacc atgctgatgc ggttttggca gtacatcaat gggcgtggat agcggtttga
     8761 ctcacgggga tttccaagtc tccaccccat tgacgtcaat gggagtttgt tttggcacca
     8821 aaatcaacgg gactttccaa aatgtcgtaa caactccgcc ccattgacgc aaatgggcgg
     8881 taggcgtgta cggtgggagg tctatataag cagagctggt ttagtgaacc gtcagatcag
     8941 atctttgtcg atcctaccat ccactcgaca cacccgccag cggccgc
//
