LOCUS       sequence-306275-        7226 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian expression plasmid for Human coronavirus NL63 
            nucleocapsid.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7226)
  TITLE     Human coronavirus mammalian expression constructs
REFERENCE   2  (bases 1 to 7226)
  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-m4134908
FEATURES             Location/Qualifiers
     source          1..7226
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     polyA_signal    1188..1309
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1199..1218)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     1253..1272
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     terminator      1424..1470
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     terminator      1500..1636
                     /label=rrnG terminator
                     /note="transcription terminator from the E. coli ribosomal 
                     RNA rrnG operon (Albrechtsen et al., 1991)"
     misc_feature    1681..1752
                     /label=3' beta-globin insulator
                     /note="insulator downstream of the human beta-globin locus 
                     (Farrell et al., 2002)"
     rep_origin      complement(1978..2566)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2058..2077)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     promoter        3066..4275
                     /label=UbC promoter
                     /note="human ubiquitin C promoter"
     primer_bind     4112..4131
                     /label=hUBCpro-F
                     /note="Human Ubiquitin C (UbC) promoter, forward primer"
     CDS             4279..4878
                     /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(4279..4298)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     4775..4795
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     polyA_signal    4905..4953
                     /note="synthetic polyadenylation signal"
     CDS             join(5219..5287,5288..6079)
                     /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(5437..5456)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     terminator      6092..6228
                     /label=rrnG terminator
                     /note="transcription terminator from the E. coli ribosomal 
                     RNA rrnG operon (Albrechtsen et al., 1991)"
     misc_feature    6380..6451
                     /label=5' beta-globin insulator
                     /note="insulator upstream of the human beta-globin locus 
                     (Farrell et al., 2002)"
     terminator      complement(6456..6502)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     polyA_signal    6520..6568
                     /note="synthetic polyadenylation signal"
     enhancer        6649..6952
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        6954..7156
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     7107..7127
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      join(7225..7226,1..8)
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
ORIGIN
        1 caccatggct agtgtaaatt gggccgatga cagagctgct aggaagaaat ttcctcctcc
       61 ttcattttac atgcctcttt tggttagttc tgataaggca ccatataggg tcattcccag
      121 gaatcttgtc cctattggta agggtaataa agatgagcag attggttatt ggaatgttca
      181 agagcgttgg cgtatgcgca gggggcaacg tgttgatttg cctcctaaag ttcattttta
      241 ttacctaggt actggacctc ataaggacct taaattcaga caacgttctg atggtgttgt
      301 ttgggttgct aaggaaggtg ctaaaactgt taataccagt cttggtaatc gcaaacgtaa
      361 tcagaaacct ttggaaccaa agttctctat tgctttgcct ccagagctct ctgttgttga
      421 gtttgaggat cgctctaata actcatctcg tgctagcagt cgttcttcaa ctcgtaacaa
      481 ctcacgagac tcttctcgta gcacttcaag acaacagtct cgcactcgtt ctgattctaa
      541 ccagtcttct tcagatcttg ttgctgctgt tactttggct ttaaagaact taggttttga
      601 taaccagtcg aagtcaccta gttcttctgg tacttccact cctaagaaac ctaataagcc
      661 tctttctcaa cccagggctg ataagccttc tcagttgaag aaacctcgtt ggaagcgtgt
      721 tcctaccaga gaggaaaatg ttattcagtg ctttggtcct cgtgatttta atcacaatat
      781 gggggattca gatcttgttc agaatggtgt tgatgccaaa ggttttccac agcttgctga
      841 attgattcct aatcaggctg cgttattctt tgatagtgag gttagcactg atgaagtggg
      901 tgataatgtt cagattacct acacctacaa aatgcttgta gctaaggata ataagaacct
      961 tcctaagttc attgagcaga ttagtgcttt tactaaaccc agttctatca aagaaatgca
     1021 gtcacaatca tctcatgttg ctcagaacac agtacttaat gcttctattc cagaatctaa
     1081 accattggct gatgatgatt cagccattat agaaattgtc aacgaggttt tgcattaagc
     1141 tagcttgact gactgagata cagcgtacct tcagctcaca gacatgataa gatacattga
     1201 tgagtttgga caaaccacaa ctagaatgca gtgaaaaaaa tgctttattt gtgaaatttg
     1261 tgatgctatt gctttatttg taaccattat aagctgcaat aaacaagtta acaacaacaa
     1321 ttgcattcat tttatgtttc aggttcaggg ggaggtgtgg gaggtttttt aaagcaagta
     1381 aaacctctac aaatgtggta ttggcccatc tctatcggta tcgtagcata accccttggg
     1441 gcctctaaac gggtcttgag gggttttttg tgcccctcgg gccggattgc tatctaccgg
     1501 cattggcgca gaaaaaaatg cctgatgcga cgctgcgcgt cttatactcc cacatatgcc
     1561 agattcagca acggatacgg cttccccaac ttgcccactt ccatacgtgt cctccttacc
     1621 agaaatttat ccttaaggtc gtcagctatc ctgcaggcga tctctcgatt tcgatcaaga
     1681 cattccttta atggtctttt ctggacacca ctaggggtca gaagtagttc atcaaacttt
     1741 cttccctccc taatctcatt ggttaccttg ggctatcgaa acttaattaa ccagtcaagt
     1801 cagctacttg gcgagatcga cttgtctggg tttcgactac gctcagaatt gcgtcagtca
     1861 agttcgatct ggtccttgct attgcacccg ttctccgatt acgagtttca tttaaatcat
     1921 gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt
     1981 ccataggctc cgcccccctg acgagcatca caaaaatcga cgctcaagtc agaggtggcg
     2041 aaacccgaca ggactataaa gataccaggc gtttccccct ggaagctccc tcgtgcgctc
     2101 tcctgttccg accctgccgc ttaccggata cctgtccgcc tttctccctt cgggaagcgt
     2161 ggcgctttct catagctcac gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa
     2221 gctgggctgt gtgcacgaac cccccgttca gcccgaccgc tgcgccttat ccggtaacta
     2281 tcgtcttgag tccaacccgg taagacacga cttatcgcca ctggcagcag ccactggtaa
     2341 caggattagc agagcgaggt atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa
     2401 ctacggctac actagaagaa cagtatttgg tatctgcgct ctgctgaagc cagttacctt
     2461 cggaaaaaga gttggtagct cttgatccgg caaacaaacc accgctggta gcggtggttt
     2521 ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag atcctttgat
     2581 cttttctacg gggtctgacg ctcagtggaa cgaaaactca cgttaaggga ttttggtcat
     2641 gagattatca aaaaggatct tcacctagat ccttttaaat taaaaatgaa gttttaaatc
     2701 aatctaaagt atatatgagt aaacttggtc tgacagttac caatgcttaa tcagtgaggc
     2761 acctatctca gcgatctgtc tatttcgttc atccatagtt gcatttaaat ttccgaactc
     2821 tccaaggccc tcgtcggaaa atcttcaaac ctttcgtccg atccatcttg caggctacct
     2881 ctcgaacgaa ctatcgcaag tctcttggcc ggccttgcgc cttggctatt gcttggcagc
     2941 gcctatcgcc aggtattact ccaatcccga atatccgaga tcgggatcac ccgagagaag
     3001 ttcaacctac atcctcaatc ccgatctatc cgagatccga ggaatatcga aatcggggcg
     3061 cgcctggcct ccgcgccggg ttttggcgcc tcccgcgggc gcccccctcg tcacggcgag
     3121 cgctgccacg tcagacgaag ggcgcaggag cgtcctgatc cttccgcccg gacgctcagg
     3181 acagcggccc gctgctcata agactcggcc ttagaacccc agtatcagca gaaggacatt
     3241 ttaggacggg acttgggtga ctctagggca ctggttttct ttccagagag cggaacaggc
     3301 gaggaaaagt agtcccttct cggcgattct gcggagggat ctccgtgggg cggtgaacgc
     3361 cgatgattat ataaggacgc gccgggtgtg gcacagctag ttccgtcgca gccgggattt
     3421 gggtcgcggt tcttgtttgt ggatcgctgt gatcgtcact tggtgagtag cgggctgctg
     3481 ggctggccgg ggctttcgtg gccgccgggc cgctcggtgg gacggaagcg tgtggagaga
     3541 ccgccaaggg ctgtagtctg ggtccgcgag caaggttgcc ctgaactggg ggttgggggg
     3601 agcgcagcaa aatggcggct gttcccgagt cttgaatgga agacgcttgt gaggcgggct
     3661 gtgaggtcgt tgaaacaagg tggggggcat ggtgggcggc aagaacccaa ggtcttgagc
     3721 ccttcgctaa tgcgggaaag ctcttattcg ggtgagatgg gctgggcacc atctggggac
     3781 cctgacgtga agtttgtcac tgactggaga actcggtttg tcgtctgttg cgggggcggc
     3841 agttatggcg gtgccgttgg gcagtgcacc cgtacctttg ggagcgcgcg ccctcgtcgt
     3901 gtcgtgacgt cacccgttct gttggcttat aatgcagggt ggggccacct gccggtaggt
     3961 gtgcggtagg cttttctccg tcgcaggacg cagggttcgg gcctagggta ggctctcctg
     4021 aatcgacagg cgccggacct ctggtgaggg gagggataag tgaggcgtca gtttctttgg
     4081 tcggttttat gtacctatct tcttaagtag ctgaagctcc ggttttgaac tatgcgctcg
     4141 gggttggcga gtgtgttttg tgaagttttt taggcacctt ttgaaatgta atcatttggg
     4201 tcaatatgta attttcagtg ttagacttgt aaattgtccg ctaaattctg gccgtttttg
     4261 gcttttttgt tagacaacat gaccgagtac aagcccacgg tgcgcctcgc cacccgcgac
     4321 gacgtcccca gggccgtacg caccctcgcc gccgcgttcg ccgactaccc cgccacgcgc
     4381 cacaccgtcg atccggaccg ccacatcgag cgggtcaccg agctgcaaga actcttcctc
     4441 acgcgcgtcg ggctcgacat cggcaaggtg tgggtcgcgg acgacggcgc cgcggtggcg
     4501 gtctggacca cgccggagag cgtcgaagcg ggggcggtgt tcgccgagat cggcccgcgc
     4561 atggccgagt tgagcggttc ccggctggcc gcgcagcaac agatggaggg cctcctggcg
     4621 ccgcaccggc ccaaggagcc cgcgtggttc ctggccaccg tcggcgtctc gcccgaccac
     4681 cagggcaagg gtctgggcag cgccgtcgtg ctccccggag tggaggccgc cgagcgcgcc
     4741 ggggtgcccg ccttcctgga gacctccgcg ccccgcaacc tccccttcta cgagcggctc
     4801 ggcttcaccg tcaccgccga cgtcgaggtg cccgaaggac cgcgcacctg gtgcatgacc
     4861 cgcaagcccg gtgcctaggc tagtatgtaa gcctagtctt agataataaa atcgctatcc
     4921 atcgaagatg gatgtgtgtt ggttttttgt gtgtgtaacg ctaggcgcgc ctggtgtacc
     4981 gagaacgatc ctctcagtgc gagtctcgac gatccatatc gttgcttggc agtcagccag
     5041 tcggaatcca gcttgggacc caggaagtcc aatcgtcaga tattgtactc aagcctggtc
     5101 acggcagcgt accgatctgt ttaaacctag atattgatag tctgatcggt caacgtataa
     5161 tcgagtccta gcttttgcaa acatctatca agagacagga tcagcaggag gctttcgcat
     5221 gagtattcaa catttccgtg tcgcccttat tccctttttt gcggcatttt gccttcctgt
     5281 ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt tgggtgcgcg
     5341 agtgggttac atcgaactgg atctcaacag cggtaagatc cttgagagtt ttcgccccga
     5401 agaacgcttt ccaatgatga gcacttttaa agttctgcta tgtggcgcgg tattatcccg
     5461 tattgacgcc gggcaagagc aactcggtcg ccgcatacac tattctcaga atgacttggt
     5521 tgagtattca ccagtcacag aaaagcatct tacggatggc atgacagtaa gagaattatg
     5581 cagtgctgcc ataaccatga gtgataacac tgcggccaac ttacttctga caacgattgg
     5641 aggaccgaag gagctaaccg cttttttgca caacatgggg gatcatgtaa ctcgccttga
     5701 tcgttgggaa ccggagctga atgaagccat accaaacgac gagcgtgaca ccacgatgcc
     5761 tgtagcaatg gcaacaacct tgcgtaaact attaactggc gaactactta ctctagcttc
     5821 ccggcaacag ttgatagact ggatggaggc ggataaagtt gcaggaccac ttctgcgctc
     5881 ggcccttccg gctggctggt ttattgctga taaatctgga gccggtgagc gtgggtctcg
     5941 cggtatcatt gcagcactgg ggccagatgg taagccctcc cgtatcgtag ttatctacac
     6001 gacggggagt caggcaacta tggatgaacg aaatagacag atcgctgaga taggtgcctc
     6061 actgattaag cattggtaac cgattctagg tgcattggcg cagaaaaaaa tgcctgatgc
     6121 gacgctgcgc gtcttatact cccacatatg ccagattcag caacggatac ggcttcccca
     6181 acttgcccac ttccatacgt gtcctcctta ccagaaattt atccttaaga tcccgaatcg
     6241 tttaaactcg actctggctc tatcgaatct ccgtcgtttc gagcttacgc gaacagccgt
     6301 ggcgctcatt tgctcgtcgg gcatcgaatc tcgtcagcta tcgtcagctt acctttttgg
     6361 cagcgatcgc ggctcccgac atcttggacc attagctcca caggtatctt cttccctcta
     6421 gtggtcataa cagcagcttc agctacctct caattcaaaa aacccctcaa gacccgttta
     6481 gaggccccaa ggggttatgc tatcaatcgt tgcgttacac acacaaaaaa ccaacacaca
     6541 tccatcttcg atggatagcg attttattat ctaactgctg atcgagtgta gccagatcta
     6601 gtaatcaatt acggggtcat tagttcatag cccatatatg gagttccgcg ttacataact
     6661 tacggtaaat ggcccgcctg gctgaccgcc caacgacccc cgcccattga cgtcaataat
     6721 gacgtatgtt cccatagtaa cgccaatagg gactttccat tgacgtcaat gggtggagta
     6781 tttacggtaa actgcccact tggcagtaca tcaagtgtat catatgccaa gtacgccccc
     6841 tattgacgtc aatgacggta aatggcccgc ctggcattat gcccagtaca tgaccttatg
     6901 ggactttcct acttggcagt acatctacgt attagtcatc gctattacca tgctgatgcg
     6961 gttttggcag tacatcaatg ggcgtggata gcggtttgac tcacggggat ttccaagtct
     7021 ccaccccatt gacgtcaatg ggagtttgtt ttggcaccaa aatcaacggg actttccaaa
     7081 atgtcgtaac aactccgccc cattgacgca aatgggcggt aggcgtgtac ggtgggaggt
     7141 ctatataagc agagctggtt tagtgaaccg tcagatcaga tctttgtcga tcctaccatc
     7201 cactcgacac acccgccagc ggccgc
//
