The pSP-luc+NF Fusion Vector is a luciferase cassette vector containing the engineered firefly luciferase gene, luc+NF. The luc+NF gene is related to the luc+ gene found in Promega's pGL3 family of eukaryotic reporter vectors (1), but has been further modified for maximum flexibility in constructing N-terminal fusions (NF) with luciferase. The pSP-luc+NF Fusion Vector is not itself intended for the expression of luciferase in eukaryotic cells because it does not contain eukaryotic promoters, enhancers or polyadenylation signals. For applications other than generating N-terminal fusions, we recommend using the pSP-luc+ Vector.

The luc+NF gene is positioned downstream of an SP6 promoter and ribosome binding site. An opposing T7 promoter* is located immediately downstream of luc+NF. Thus, the pSP-luc+NF Fusion Vector provides a convenient template for the in vitro synthesis of both sense and anti-sense luciferase transcripts for studies involving in situ hybridization, RNA processing, RNA transfection or coupled in vitro transcription/translation and protein folding. Multiple cloning regions containing recognition sequences for commonly used restriction enzymes are positioned at the 5 and 3 ends of luc+NF to provide maximum flexibility in cloning. Subcloning luc+NF into expression vectors provides a useful genetic reporter with exceptional sensitivity. Luciferase enzymatic activity can be assayed most efficiently using Promega's Luciferase Assay System.

*U.S. Patent No. 4,766,072 has been issued to Promega Corporation for transcription vectors having two different promoter sequences separated by a series of unique restriction sites into which foreign DNA can be inserted.

Luciferase is a 62kDa monomeric protein that does not require post-translational modifications for enzymatic activity. Thus, it can function as a genetic reporter immediately upon translation (2-4). Luciferase synthesized by in vitro translation can be labeled with 35S, as the protein contains 4 cysteine and 14 methionine residues. To ensure full enzymatic activity of luciferase, no more than 5 codons can be deleted from either the 5 or 3 end of the coding region.

 back Return to this vector's summary.


LOCUS       CVU47123     4103 bp    DNA             SYN       20-FEB-1996
DEFINITION  Cloning vector pSP-luc+NF, luciferase cassette fusion vector,
            complete sequence.
ACCESSION   U47123
NID         g1197682
KEYWORDS    .
SOURCE      Cloning vector pSP-luc+NF.
  ORGANISM  Cloning vector pSP-luc+NF
            artificial sequence; cloning vectors.
REFERENCE   1  (bases 1 to 4103)
  AUTHORS   Brondyk,W.H. and Groskreutz,D.J.
  TITLE     Direct Submission
  JOURNAL   Submitted (25-JAN-1996) Melinda Brady, R&D, Promega Corporation,
            5445 East Cheryl Parkway, Madison, WI 53711, USA
COMMENT
            NCBI gi: 1197682
FEATURES               Location/Qualifiers
     source          1..4103
                     /organism="Cloning vector pSP-luc+NF"
     misc_feature    1
                     /note="SP6 RNA polymerase initiation site"
     misc_feature    11..53
                     /note="upstream multiple cloning site"
     CDS             45..1700
                     /gene="luc+"
                     /note="firefly luciferase;  NCBI gi: 1197683"
                     /codon_start=1
                     /product="luciferase"
                     /db_xref="PID:g1197683"
                     /translation="MVTDAKNIKKGPAPFYPLEDGTAGEQLHKAMKRYALVPGTIAFT
                     DAHIEVDITYAEYFEMSVRLAEAMKRYGLNTNHRIVVCSENSLQFFMPVLGALFIGVA
                     VAPANDIYNERELLNSMGISQPTVVFVSKKGLQKILNVQKKLPIIQKIIIMDSKTDYQ
                     GFQSMYTFVTSHLPPGFNEYDFVPESFDRDKTIALIMNSSGSTGLPKGVALPHRTACV
                     RFSHARDPIFGNQIIPDTAILSVVPFHHGFGMFTTLGYLICGFRVVLMYRFEEELFLR
                     SLQDYKIQSALLVPTLFSFFAKSTLIDKYDLSNLHEIASGGAPLSKEVGEAVAKRFHL
                     PGIRQGYGLTETTSAILITPEGDDKPGAVGKVVPFFEAKVVDLDTGKTLGVNQRGELC
                     VRGPMIMSGYVNNPEATNALIDKDGWLHSGDIAYWDEDEHFFIVDRLKSLIKYKGYQV
                     APAELESILLQHPNIFDAGVAGLPDDDAGELPAAVVVLEHGKTMTEKEIVDYVASQVT
                     TAKKLRGGVVFVDEVPKGLTGKLDARKIREILIKAKKGGKIAV"
     misc_feature    1702..1739
                     /note="downstream multiple cloning site"
     promoter        complement(1736..1759)
                     /note="T7 RNA polymerase promoter"
     misc_feature    1742
                     /note="T7 RNA polymerase initiation site"
     rep_origin      2014
                     /note="ColE1-derived replication origin"
     CDS             complement(2776..3636)
                     /gene="ampR"
                     /note="NCBI gi: 1197684"
                     /codon_start=1
                     /function="ampicillin resistance"
                     /product="beta-lactamase"
                     /db_xref="PID:g1197684"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGY
                     IELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVE
                     YSPVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRL
                     DRWEPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPL
                     LRSALPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIA
                     EIGASLIKHW"
     promoter        join(4087..4103,1..6)
                     /note="SP6 RNA polymerase promoter"
BASE COUNT     1075 a    975 c   1008 g   1045 t
ORIGIN      
        1 gaaccagatc ggtaccgcta gcaagatctc ctaggaagct ttccatggtc accgacgcca
       61 aaaacataaa gaaaggcccg gcgccattct atccgctgga agatggaacc gctggagagc
      121 aactgcataa ggctatgaag agatacgccc tggttcctgg aacaattgct tttacagatg
      181 cacatatcga ggtggacatc acttacgctg agtacttcga aatgtccgtt cggttggcag
      241 aagctatgaa acgatatggg ctgaatacaa atcacagaat cgtcgtatgc agtgaaaact
      301 ctcttcaatt ctttatgccg gtgttgggcg cgttatttat cggagttgca gttgcgcccg
      361 cgaacgacat ttataatgaa cgtgaattgc tcaacagtat gggcatttcg cagcctaccg
      421 tggtgttcgt ttccaaaaag gggttgcaaa aaattttgaa cgtgcaaaaa aagctcccaa
      481 tcatccaaaa aattattatc atggattcta aaacggatta ccagggattt cagtcgatgt
      541 acacgttcgt cacatctcat ctacctcccg gttttaatga atacgatttt gtgccagagt

      601 ccttcgatag ggacaagaca attgcactga tcatgaactc ctctggatct actggtctgc
      661 ctaaaggtgt cgctctgcct catagaactg cctgcgtgag attctcgcat gccagagatc
      721 ctatttttgg caatcaaatc attccggata ctgcgatttt aagtgttgtt ccattccatc
      781 acggttttgg aatgtttact acactcggat atttgatatg tggatttcga gtcgtcttaa
      841 tgtatagatt tgaagaagag ctgtttctga ggagccttca ggattacaag attcaaagtg
      901 cgctgctggt gccaacccta ttctccttct tcgccaaaag cactctgatt gacaaatacg
      961 atttatctaa tttacacgaa attgcttctg gtggcgctcc cctctctaag gaagtcgggg
     1021 aagcggttgc caagaggttc catctgccag gtatcaggca aggatatggg ctcactgaga
     1081 ctacatcagc tattctgatt acacccgagg gggatgataa accgggcgcg gtcggtaaag
     1141 ttgttccatt ttttgaagcg aaggttgtgg atctggatac cgggaaaacg ctgggcgtta
     1201 atcaaagagg cgaactgtgt gtgagaggtc ctatgattat gtccggttat gtaaacaatc
     1261 cggaagcgac caacgccttg attgacaagg atggatggct acattctgga gacatagctt
     1321 actgggacga agacgaacac ttcttcatcg ttgaccgcct gaagtctctg attaagtaca
     1381 aaggctatca ggtggctccc gctgaattgg aatccatctt gctccaacac cccaacatct
     1441 tcgacgcagg tgtcgcaggt cttcccgacg atgacgccgg tgaacttccc gccgccgttg
     1501 ttgttttgga gcacggaaag acgatgacgg aaaaagagat cgtggattac gtcgccagtc
     1561 aagtaacaac cgcgaaaaag ttgcgcggag gagttgtgtt tgtggacgaa gtaccgaaag
     1621 gtcttaccgg aaaactcgac gcaagaaaaa tcagagagat cctcataaag gccaagaagg
     1681 gcggaaagat cgccgtgtaa ttctagagaa ttcgatatca tcgatctcga ggccggtctc
     1741 cctatagtga gtcgtattaa tttcgataag ccaggttaac ctgcattaat gaatcggcca
     1801 acgcgcgggg agaggcggtt tgcgtattgg gcgctcttcc gcttcctcgc tcactgactc
     1861 gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg cggtaatacg
     1921 gttatccaca gaatcagggg ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa
     1981 ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc gcccccctga
     2041 cgagcatcac aaaaatcgac gctcaagtca gaggtggcga aacccgacag gactataaag
     2101 ataccaggcg tttccccctg gaagctccct cgtgcgctct cctgttccga ccctgccgct
     2161 taccggatac ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc aatgctcacg
     2221 ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc
     2281 ccccgttcag cccgaccgct gcgccttatc cggtaactat cgtcttgagt ccaacccggt
     2341 aagacacgac ttatcgccac tggcagcagc cactggtaac aggattagca gagcgaggta
     2401 tgtaggcggt gctacagagt tcttgaagtg gtggcctaac tacggctaca ctagaaggac
     2461 agtatttggt atctgcgctc tgctgaagcc agttaccttc ggaaaaagag ttggtagctc
     2521 ttgatccggc aaacaaacca ccgctggtag cggtggtttt tttgtttgca agcagcagat
     2581 tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc
     2641 tcagtggaac gaaaactcac gttaagggat tttggtcatg agattatcaa aaaggatctt
     2701 cacctagatc cttttaaatt aaaaatgaag ttttaaatca atctaaagta tatatgagta
     2761 aacttggtct gacagttacc aatgcttaat cagtgaggca cctatctcag cgatctgtct
     2821 atttcgttca tccatagttg cctgactccc cgtcgtgtag ataactacga tacgggaggg
     2881 cttaccatct ggccccagtg ctgcaatgat accgcgagac ccacgctcac cggctccaga
     2941 tttatcagca ataaaccagc cagccggaag ggccgagcgc agaagtggtc ctgcaacttt
     3001 atccgcctcc atccagtcta ttaattgttg ccgggaagct agagtaagta gttcgccagt
     3061 taatagtttg cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt
     3121 tggtatggct tcattcagct ccggttccca acgatcaagg cgagttacat gatcccccat
     3181 gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc gttgtcagaa gtaagttggc
     3241 cgcagtgtta tcactcatgg ttatggcagc actgcataat tctcttactg tcatgccatc
     3301 cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag tcattctgag aatagtgtat
     3361 gcggcgaccg agttgctctt gcccggcgtc aatacgggat aataccgcgc cacatagcag
     3421 aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct caaggatctt
     3481 accgctgttg agatccagtt cgatgtaacc cactcgtgca cccaactgat cttcagcatc
     3541 ttttactttc accagcgttt ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa
     3601 gggaataagg gcgacacgga aatgttgaat actcatactc ttcctttttc aatattattg
     3661 aagcatttat cagggttatt gtctcatgag cggatacata tttgaatgta tttagaaaaa
     3721 taaacaaata ggggttccgc gcacatttcc ccgaaaagtg ccacctgacg tctaagaaac
     3781 cattattatc atgacattaa cctataaaaa taggcgtatc acgaggccct ttcgtctcgc
     3841 gcgtttcggt gatgacggtg aaaacctctg acacatgcag ctcccggaga cggtcacagc
     3901 ttgtctgtaa gcggatgccg ggagcagaca agcccgtcag ggcgcgtcag cgggtgttgg
     3961 cgggtgtcgg ggctggctta actatgcggc atcagagcag attgtactga gagtgcacca
     4021 tatggacata ttgtcgttag aacgcggcta caattaatac ataaccttat gtatcataca
     4081 catacgattt aggtgacact ata
//