LOCUS       sequence-481548-        6181 bp    DNA     circular SYN 30-JUL-2025
DEFINITION  Express VHL-NanoLuc Fusion Protein in Mammalian Cells under a CMV 
            promoter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6181)
  AUTHORS   Promega
  TITLE     Promega Plasmid Collection
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 6181)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Jul 30, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: 
            "Unpublished"
COMMENT     Sequence Label: VHL-NanoLuc Vector
FEATURES             Location/Qualifiers
     source          1..6181
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     polyA_signal    17..98
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     42..61
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     CDS             complement(275..787)
                     /codon_start=1
                     /product="NanoLuc(R) luciferase"
                     /label=Nluc
                     /note="human codon-optimized"
                     /translation="VFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQRI
                     VLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGVT
                     PNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGWR
                     LCERILA"
     promoter        complement(1486..1504)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(1487..1504)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     enhancer        1624..1927
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     primer_bind     2082..2102
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     promoter        2135..2182
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             2214..2873
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     complement(2280..2299)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     rep_origin      complement(3045..3633)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3125..3144)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     polyA_signal    3759..3807
                     /note="synthetic polyadenylation signal"
     CDS             complement(3831..4625)
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(3942..3961)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     4552..4571
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     promoter        complement(5003..6181)
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          5012..5950
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     complement(5035..5055)
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
ORIGIN
        1 aagacgcgtg acgtcggcag tgaaaaaaat gctttatttg tgaaatttgt gatgctattg
       61 ctttatttgt aaccattata agctgcaata aacaagttaa caacaacaat tgcattcatt
      121 ttatgtttca ggttcagggg gaggtgtggg aggtttttta aagcaagtaa aacctctaca
      181 aatgtggtat ggctgattat gatccgcggc cgcaatacgt cgacgttatc agctgacttc
      241 gtacgagagc ctaggtgttt ctagagtcgc ggccttacgc cagaatgcgt tcgcacagcc
      301 gccagccggt cactccgttg atggttactc ggaacagcag ggagccgtcg gggttgatca
      361 ggcgctcgtc gataattttg ttgccgttcc acagggtccc tgttacagtg atctttttgc
      421 cgtcgaacac ggcgatgcct tcatacggcc gtccgaaata gtcgatcatg ttcggcgtaa
      481 ccccgtcgat taccagtgtg ccatagtgca ggatcacctt aaagtgatga tcatccacag
      541 ggtacaccac cttaaaaatt ttttcgatct ggcccatttg gtcgccgctc agaccttcat
      601 acgggatgat gacatggatg tcgatcttca gcccattttc accgctcagg acaatccttt
      661 ggatcggagt tacggacacc ccgagattct gaaacaaact ggacacacct ccctgttcaa
      721 ggacttggtc caggttgtag ccggctgtct gtcgccagtc cccaacgaaa tcttcgagtg
      781 tgaagacgcc gctcgagccg agagaaacat ctcccatccg ttgatgtgca atgcgctcct
      841 gtgtcagccg ctccaggtct ttctgcacat ttgggtggtc ttccagatct tcgtagagcg
      901 acctgacgat gtccagtctc ctgtaattct caggcttgac taggctccgg acaacctgga
      961 ggcatcgctc tttcagagta tacactggca gtgtgatatt ggcaaaaata ggctgtccgt
     1021 caacattgag agatggcaca aataattcag tttggttaac cagaagccca tcgtgtgtcc
     1081 ctgcatctct gaagagccaa aggtgacctc ggtagctgtg gatgcggcgg cccgtgccag
     1141 gcggcagcgt tgggtagggc tgcggctcgc cgtcgaagtt gagccatacg ggcagcacga
     1201 cgcgcggact gcgattgcag aagatgacct gggagggctc gcgcgagttc accgagcgca
     1261 gcacgggccg cggccgcccg gcctccatct cctcctcggc gcccagttcc tccgggccgg
     1321 actcttccgg gccggactcc tcggcgcccg actcctcccc gccgtcttct tcagggccgt
     1381 actcttcgac gcctgcctcc tccgcgccta cctcggcctc gtcccagttc tccgccctcc
     1441 ggggcatggc gatcgctagc tgcaaaaaga acaagtagct tgtattctat agtgtcacct
     1501 aaatgggcga tctgacggtt cactaaacca gctctgctta tataggcctc cctggcgcgc
     1561 cactagttat taatagtaat caattacggg gtcattagtt catagcccat atatggagtt
     1621 ccgcgttaca taacttacgg taaatggccc gcctggctga ccgcccaacg acccccgccc
     1681 attgacgtca ataatgacgt atgttcccat agtaacgcca atagggactt tccattgacg
     1741 tcaatgggtg gagtatttac ggtaaactgc ccacttggca gtacatcaag tgtatcatat
     1801 gccaagtccg ccccctattg acgtcaatga cggtaaatgg cccgcctggc attatgccca
     1861 gtacatgacc ttacgggact ttcctacttg gcagtacatc tacgtattag tcatcgctat
     1921 taccatggtg atgcggtttt ggcagtacac caatgggcgt ggatagcggt ttgactcacg
     1981 gggatttcca agtctccacc ccattgacgt caatgggagt ttgttttggc accaaaatca
     2041 acgggacttt ccaaaatgtc gtaataaccc cgccccgttg acgcaaatgg gcggtaggcg
     2101 tgtacggtgg gaggtcaaat cgaattctaa tactgttgac aattaatcat cggcatagta
     2161 tatcggcata gtataatacg acaaggtgag gaactaaacc caggaggcag atcatggaga
     2221 aaaaaatcac tggatatacc accgttgata tatcccaatg gcatcgtaaa gaacattttg
     2281 aggcatttca gtcagttgct caatgtacct ataaccagac cgttcagctg gatattacgg
     2341 cctttttaaa gaccgtaaag aaaaataagc acaagtttta tccggccttt attcacattc
     2401 ttgcccgcct gatgaatgct catccggaat tccgtatggc aatgaaagac ggtgagctgg
     2461 tgatatggga tagtgttcac ccttgttaca ccgttttcca tgagcaaact gaaacgtttt
     2521 catcgctctg gagtgaatac cacgacgatt tccggcagtt tctacacata tattcgcaag
     2581 atgtggcgtg ttacggtgaa aacctggcct atttccctaa agggtttatt gagaatatgt
     2641 ttttcgtctc agccaatccc tgggtgagtt tcaccagttt tgatttaaac gtggccaata
     2701 tggacaactt cttcgccccc gttttcacta tgggcaaata ttatacgcaa ggcgacaagg
     2761 tgctgatgcc gctggcgatt caggttcatc atgccgtctg tgatggcttc catgtcggca
     2821 gaatgcttaa tgaattacaa cagtactgcg atgagtggca gggcggggcg taagcgggac
     2881 tctggggttc gaaatgaccg accaagcgac gcccaaccgg tatcagctca ctcaaaggcg
     2941 gtaatacggt tatccacaga atcaggggat aacgcaggaa agaacatgtg agcaaaaggc
     3001 cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca taggctccgc
     3061 ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga
     3121 ctataaagat accaggcgtt tccccctgga agctccctcg tgcgctctcc tgttccgacc
     3181 ctgccgctta ccggatacct gtccgccttt ctcccttcgg gaagcgtggc gctttctcat
     3241 agctcacgct gtaggtatct cagttcggtg taggtcgttc gctccaagct gggctgtgtg
     3301 cacgaacccc ccgttcagcc cgaccgctgc gccttatccg gtaactatcg tcttgagtcc
     3361 aacccggtaa gacacgactt atcgccactg gcagcagcca ctggtaacag gattagcaga
     3421 gcgaggtatg taggcggtgc tacagagttc ttgaagtggt ggcctaacta cggctacact
     3481 agaaggacag tatttggtat ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt
     3541 ggtagctctt gatccggcaa acaaaccacc gctggtagcg gtggtttttt tgtttgcaag
     3601 cagcagatta cgcgcagaaa aaaaggattt caagaagatc ctttgatctt ttctacgggg
     3661 tctgacgctc agtggaacga aaactcacgt taagggattt tggtcatgag attatcaaaa
     3721 aggatcttca cctagatcct tttatagtcc ggaatcgtca cacaaaaaac caacacacag
     3781 atgtaatgaa aataaagata ttttattgcg gccatcgtga tggctagagt tcagaagaac
     3841 tcgtcaagaa ggcgatagaa ggcgatgcgc tgcgaatcgg gagcggcgat accgtaaagc
     3901 acgaggaagc ggtcagccca ttcgccgcca agctcttcag caatatcacg ggtagccaac
     3961 gctatgtcct gatagcggtc cgccacaccc agccggccac agtcgatgaa tccagaaaag
     4021 cggccatttt ccaccatgat attcggcaag caggcatcgc cgtgggtcac gacgagatcc
     4081 tcgccgtcgg gcatgcgcgc cttgagcctg gcgaacagtt cggctggcgc gagcccctga
     4141 tgctcttcgt ccagatcatc ctgatcgaca agaccggctt ccatccgagt acgtgctcgc
     4201 tcgatgcgat gtttcgcttg gtggtcgaat gggcaggtag ccggatcaag cgtatgcagc
     4261 cgccgcattg catcagccat gatggatact ttctcggcag gagcaaggtg agatgacagg
     4321 agatcctgcc ccggcacttc gcccaatagc agccagtccc ttcccgcttc agtgacaacg
     4381 tcgagcacag ctgcgcaagg aacgcccgtc gtggccagcc acgatagccg cgctgcctcg
     4441 tcctgcagtt cattcagggc accggacagg tcggtcttga caaaaagaac cgggcgcccc
     4501 tgcgctgaca gccggaacac ggcggcatca gagcagccga ttgtctgttg tgcccagtca
     4561 tagccgaata gcctctccac ccaagcggcc ggagaacctg cgtgcaatcc atcttgttca
     4621 atcatggtgg catcgattcc cggtcaacat ccagtacacc tccggcacac cggcttcccc
     4681 aagggcgcca cgctctccca ccacaacatc ctcaacaacg gctactgggt gggccgcacg
     4741 gtcggctaca cggagcagga ccgggtgtgt ctgccggtgc ccttctacca ctgcttcggc
     4801 atggtcatgg gcaacttggg cgccacctcc cacgccgcct gcatcgtcat cccggccccg
     4861 tccttcgagc cggcggcgac gctggaggcg gtgcagcggg agaggtgcac gtccctgtac
     4921 ggcgtcccca cgaagccgaa ttccaccaca ctggactaga tgcatgctcg acagtgtgat
     4981 ggatatctgc agaattcggc tttcacgaca cctgaaatgg aagaaaaaaa ctttgaacca
     5041 ctgtctgagg cttgagaatg aaccaagatc caaactcaaa aagggcaaat tccaaggaga
     5101 attacatcaa gtgccaagct ggcctaactt cagtctccac ccactcagtg tggggaaact
     5161 ccatcgcata aaacccctcc ccccaaccta aagacgacgt actccaaaag ctcgagaact
     5221 aatcgaggtg cctggacggc gcccggtact ccgtggagtc acatgaagcg acggctgagg
     5281 acggaaaggc ccttttcctt tgtgtgggtg actcacccgc ccgctctccc gagcgccgcg
     5341 tcctccattt tgagctgcct gcagcagggc cgggaagcgg ccatctttcc gctcacgcaa
     5401 ctggtgccga ccgggccagc cttgccgccc agggcggggc gatacacggc ggcgcgaggc
     5461 caggcaccag agcaggccgg ccagcttgag actacccccg tccgattctc ggtggccgcg
     5521 ctcgcaggcc ccgcctcgcc gaacatgtgc gctgggacgc acgggccccg tcgccgcccg
     5581 cggccccaaa aaccgaaata ccagtgtgca gatcttggcc cgcatttaca agactatctt
     5641 gccagaaaaa aagcgtcgca gcaggtcatc aaaaatttta aatggctaga gacttatcga
     5701 aagcagcgag acaggcgcga aggtgccacc agattcgcac gcggcggccc cagcgcccag
     5761 gccaggcctc aactcaagca cgaggcgaag gggctcctta agcgcaaggc ctcgaactct
     5821 cccacccact tccaacccga agctcgggat caagaatcac gtactgcagc caggtggaag
     5881 taattcaagg cacgcaaggg ccataacccg taaagaggcc aggcccgcgg gaaccacaca
     5941 cggcacttac ctgtgttctg gcggcaaacc cgttgcgaaa aagaacgttc acggcgacta
     6001 ctgcacttat atacggttct cccccaccct cgggaaaaag gcggagccag tacacgacat
     6061 cactttccca gtttaccccg cgccaccttc tctaggcacc ggttcaattg ccgacccctc
     6121 cccccaactt ctcggggact gtgggcgatg tgcgctctgc ccactgacgg gcaccggagc
     6181 c
//
