LOCUS       sequence-479846-        7067 bp    DNA     circular SYN 07-AUG-2025
DEFINITION  Express NanoLuc-bRaf 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 7067)
  AUTHORS   Promega
  TITLE     Promega Plasmid Collection
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 7067)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Aug 7, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: 
            "Unpublished"
COMMENT     Sequence Label: NanoLuc-bRaf Vector
FEATURES             Location/Qualifiers
     source          1..7067
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        138..517
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        518..721
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     672..692
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     intron          857..989
                     /label=chimeric intron
                     /note="chimera between introns from human beta-globin and 
                     immunoglobulin heavy chain genes"
     primer_bind     972..991
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     primer_bind     1033..1052
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1033..1051
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             1065..1577
                     /codon_start=1
                     /product="NanoLuc(R) luciferase"
                     /label=Nluc
                     /note="human codon-optimized"
                     /translation="MVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQR
                     IVLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGV
                     TPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGW
                     RLCERILA"
     regulatory      1597..1606
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     primer_bind     complement(4010..4028)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     polyA_signal    4065..4186
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4076..4095)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     4130..4149
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     promoter        4431..4788
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        4431..4622
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(4454..4474)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      4639..4774
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     promoter        4803..4850
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             4882..5676
                     /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(4936..4955)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     5546..5565
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    5740..5788
                     /note="synthetic polyadenylation signal"
     rep_origin      complement(6027..6615)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6107..6126)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     misc_feature    6731..7014
                     /label=cer region
                     /note="ColE1-derived recombination site that helps to 
                     maintain plasmids as monomers"
ORIGIN
        1 tcaatattgg ccattagcca tattattcat tggttatata gcataaatca atattggcta
       61 ttggccattg catacgttgt atctatatca taatatgtac atttatattg gctcatgtcc
      121 aatatgaccg ccatgttggc attgattatt gactagttat taatagtaat caattacggg
      181 gtcattagtt catagcccat atatggagtt ccgcgttaca taacttacgg taaatggccc
      241 gcctggctga ccgcccaacg acccccgccc attgacgtca ataatgacgt atgttcccat
      301 agtaacgcca atagggactt tccattgacg tcaatgggtg gagtatttac ggtaaactgc
      361 ccacttggca gtacatcaag tgtatcatat gccaagtccg ccccctattg acgtcaatga
      421 cggtaaatgg cccgcctggc attatgccca gtacatgacc ttacgggact ttcctacttg
      481 gcagtacatc tacgtattag tcatcgctat taccatggtg atgcggtttt ggcagtacac
      541 caatgggcgt ggatagcggt ttgactcacg gggatttcca agtctccacc ccattgacgt
      601 caatgggagt ttgttttggc accaaaatca acgggacttt ccaaaatgtc gtaataaccc
      661 cgccccgttg acgcaaatgg gcggtaggcg tgtacggtgg gaggtctata taagcagagc
      721 tggtttagtg aaccgtcaga tcactagaag ctttattgcg gtagtttatc acagttaaat
      781 tgctaacgca gtcagtgctt ctgacacaac agtctcgaac ttaagctgca gaagttggtc
      841 gtgaggcact gggcaggtaa gtatcaaggt tacaagacag gtttaaggag accaatagaa
      901 actgggcttg tcgagacaga gaagactctt gcgtttctga taggcaccta ttggtcttac
      961 tgacatccac tttgcctttc tctccacagg tgtccactcc cagttcaatt acagctctta
     1021 aggctagagt attaatacga ctcactatag ggctagcgct caccatggtc ttcacactcg
     1081 aagatttcgt tggggactgg cgacagacag ccggctacaa cctggaccaa gtccttgaac
     1141 agggaggtgt gtccagtttg tttcagaatc tcggggtgtc cgtaactccg atccaaagga
     1201 ttgtcctgag cggtgaaaat gggctgaaga tcgacatcca tgtcatcatc ccgtatgaag
     1261 gtctgagcgg cgaccaaatg ggccagatcg aaaaaatttt taaggtggtg taccctgtgg
     1321 atgatcatca ctttaaggtg atcctgcact atggcacact ggtaatcgac ggggttacgc
     1381 cgaacatgat cgactatttc ggacggccgt atgaaggcat cgccgtgttc gacggcaaaa
     1441 agatcactgt aacagggacc ctgtggaacg gcaacaaaat tatcgacgag cgcctgatca
     1501 accccgacgg ctccctgctg ttccgagtaa ccatcaacgg agtgaccggc tggcggctgt
     1561 gcgaacgcat tctggcgggc tcgagcggcg cgatcgccat ggcggcgctg agcggtggcg
     1621 gtggtggcgg cgcggagccg ggccaggctc tgttcaacgg ggacatggag cccgaggccg
     1681 gcgccggcgc cggcgccgcg gcctcttcgg ctgcggaccc tgccattccg gaggaggtgt
     1741 ggaatatcaa acaaatgatt aagttgacac aggaacatat agaggcccta ttggacaaat
     1801 ttggtgggga gcataatcca ccatcaatat atctggaggc ctatgaagaa tacaccagca
     1861 agctagatgc actccaacaa agagaacaac agttattgga atctctgggg aacggaactg
     1921 atttttctgt ttctagctct gcatcaatgg ataccgttac atcttcttcc tcttctagcc
     1981 tttcagtgct accttcatct ctttcagttt ttcaaaatcc cacagatgtg gcacggagca
     2041 accccaagtc accacaaaaa cctatcgtta gagtcttcct gcccaacaaa cagaggacag
     2101 tggtacctgc aaggtgtgga gttacagtcc gagacagtct aaagaaagca ctgatgatga
     2161 gaggtctaat cccagagtgc tgtgctgttt acagaattca ggatggagag aagaaaccaa
     2221 ttggttggga cactgatatt tcctggctta ctggagaaga attgcatgtg gaagtgttgg
     2281 agaatgttcc acttacaaca cacaactttg tacgaaaaac gtttttcacc ttagcatttt
     2341 gtgacttttg tcgaaagctg cttttccagg gtttccgctg tcaaacatgt ggttataaat
     2401 ttcaccagcg ttgtagtaca gaagttccac tgatgtgtgt taattatgac caacttgatt
     2461 tgctgtttgt ctccaagttc tttgaacacc acccaatacc acaggaagag gcgtccttag
     2521 cagagactgc cctaacatct ggatcatccc cttccgcacc cgcctcggac tctattgggc
     2581 cccaaattct caccagtccg tctccttcaa aatccattcc aattccacag cccttccgac
     2641 cagcagatga agatcatcga aatcaatttg ggcaacgaga ccgatcctca tcagctccca
     2701 atgtgcatat aaacacaata gaacctgtca atattgatga cttgattaga gaccaaggat
     2761 ttcgtggtga tggaggatca accacaggtt tgtctgctac cccccctgcc tcattacctg
     2821 gctcactaac taacgtgaaa gccttacaga aatctccagg acctcagcga gaaaggaagt
     2881 catcttcatc ctcagaagac aggaatcgaa tgaaaacact tggtagacgg gactcgagtg
     2941 atgattggga gattcctgat gggcagatta cagtgggaca aagaattgga tctggatcat
     3001 ttggaacagt ctacaaggga aagtggcatg gtgatgtggc agtgaaaatg ttgaatgtga
     3061 cagcacctac acctcagcag ttacaagcct tcaaaaatga agtaggagta ctcaggaaaa
     3121 cacgacatgt gaatatccta ctcttcatgg gctattccac aaagccacaa ctggctattg
     3181 ttacccagtg gtgtgagggc tccagcttgt atcaccatct ccatatcatt gagaccaaat
     3241 ttgagatgat caaacttata gatattgcac gacagactgc acagggcatg gattacttac
     3301 acgccaagtc aatcatccac agagacctca agagtaataa tatatttctt catgaagacc
     3361 tcacagtaaa aataggtgat tttggtctag ctacagtgaa atctcgatgg agtgggtccc
     3421 atcagtttga acagttgtct ggatccattt tgtggatggc accagaagtc atcagaatgc
     3481 aagataaaaa tccatacagc tttcagtcag atgtatatgc atttggaatt gttctgtatg
     3541 aattgatgac tggacagtta ccttattcaa acatcaacaa cagggaccag ataattttta
     3601 tggtgggacg aggatacctg tctccagatc tcagtaaggt acggagtaac tgtccaaaag
     3661 ccatgaagag attaatggca gagtgcctca aaaagaaaag agatgagaga ccactctttc
     3721 cccaaattct cgcctctatt gagctgctgg cccgctcatt gccaaaaatt caccgcagtg
     3781 catcagaacc ctccttgaat cgggctggtt tccaaacaga ggattttagt ctatatgctt
     3841 gtgcttctcc aaaaacaccc atccaggcag ggggatatgg tgcgtttcct gtccacgttt
     3901 aaacgaattc gggctcggta cccggggatc ctctagagtc gacctgcagg catgcaagct
     3961 gatccggctg ctaacaaagc ccgaaaggaa gctgagttgg ctgctgccac cgctgagcaa
     4021 taactagcat aaccccttgg ggcggccgct tcgagcagac atgataagat acattgatga
     4081 gtttggacaa accacaacta gaatgcagtg aaaaaaatgc tttatttgtg aaatttgtga
     4141 tgctattgct ttatttgtaa ccattataag ctgcaataaa caagttaaca acaacaattg
     4201 cattcatttt atgtttcagg ttcaggggga gatgtgggag gtttttttaa gcaagtaaaa
     4261 cctctacaaa tgtggtaaaa tcgaatttta acaaaatatt aacgcttaca atttcctgat
     4321 gcggtatttt ctccttacgc atctgtgcgg tatttcacac cgcatacgcg gatctgcgca
     4381 gcaccatggc ctgaaataac ctctgaaaga ggaacttggt taggtacctt ctgaggcgga
     4441 aagaaccagc tgtggaatgt gtgtcagtta gggtgtggaa agtccccagg ctccccagca
     4501 ggcagaagta tgcaaagcat gcatctcaat tagtcagcaa ccaggtgtgg aaagtcccca
     4561 ggctccccag caggcagaag tatgcaaagc atgcatctca attagtcagc aaccatagtc
     4621 ccgcccctaa ctccgcccat cccgccccta actccgccca gttccgccca ttctccgccc
     4681 catggctgac taattttttt tatttatgca gaggccgagg ccgcctcggc ctctgagcta
     4741 ttccagaagt agtgaggagg cttttttgga ggcctaggct tttgcaaaaa gcttaattaa
     4801 ctgttgacaa ttaatcatcg gcatagtata tcggcatagt ataatacgac aaggtgagga
     4861 actaaaccca ggaggcagat catgattgaa caagatggat tgcacgcagg ttctccggcc
     4921 gcttgggtgg agaggctatt cggctatgac tgggcacaac agacaatcgg ctgctctgat
     4981 gccgccgtgt tccggctgtc agcgcagggg cgcccggttc tttttgtcaa gaccgacctg
     5041 tccggtgccc tgaatgaact gcaggacgag gcagcgcggc tatcgtggct ggccacgacg
     5101 ggcgttcctt gcgcagctgt gctcgacgtt gtcactgaag cgggaaggga ctggctgcta
     5161 ttgggcgaag tgccggggca ggatctcctg tcatctcacc ttgctcctgc cgagaaagta
     5221 tccatcatgg ctgatgcaat gcggcggctg catacgcttg atccggctac ctgcccattc
     5281 gaccaccaag cgaaacatcg catcgagcga gcacgtactc ggatggaagc cggtcttgtc
     5341 gatcaggatg atctggacga agagcatcag gggctcgcgc cagccgaact gttcgccagg
     5401 ctcaaggcgc gcatgcccga cggcgaggat ctcgtcgtga cccatggcga tgcctgcttg
     5461 ccgaatatca tggtggaaaa tggccgcttt tctggattca tcgactgtgg ccggctgggt
     5521 gtggcggacc gctatcagga catagcgttg gctacccgtg atattgctga agagcttggc
     5581 ggcgaatggg ctgaccgctt cctcgtgctt tacggtatcg ccgctcccga ttcgcagcgc
     5641 atcgccttct atcgccttct tgacgagttc ttctgagcgg gactctgggg ttcgaaatga
     5701 ccgaccaagc gacgcccaac ctgccatcac gatggccgca ataaaatatc tttattttca
     5761 ttacatctgt gtgttggttt tttgtgtgaa tcgatagcga taaggatcct ctttgcgctt
     5821 gcgttttccc ttgtccagat agcccagtag ctgacattca tccggggtca gcaccgtttc
     5881 tgcggactgg ctttctaccc ggtatcagct cactcaaagg cggtaatacg gttatccaca
     5941 gaatcagggg ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac
     6001 cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc gcccccctga cgagcatcac
     6061 aaaaatcgac gctcaagtca gaggtggcga aacccgacag gactataaag ataccaggcg
     6121 tttccccctg gaagctccct cgtgcgctct cctgttccga ccctgccgct taccggatac
     6181 ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc atagctcacg ctgtaggtat
     6241 ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag
     6301 cccgaccgct gcgccttatc cggtaactat cgtcttgagt ccaacccggt aagacacgac
     6361 ttatcgccac tggcagcagc cactggtaac aggattagca gagcgaggta tgtaggcggt
     6421 gctacagagt tcttgaagtg gtggcctaac tacggctaca ctagaaggac agtatttggt
     6481 atctgcgctc tgctgaagcc agttaccttc ggaaaaagag ttggtagctc ttgatccggc
     6541 aaacaaacca ccgctggtag cggtggtttt tttgtttgca agcagcagat tacgcgcaga
     6601 aaaaaaggat ttcaagaaga tcctttgatc ttttctacgg ggtctgacgc tcagtggaac
     6661 gaaaactcac gttaagggat tttggtcatg agattatcaa aaaggatctt cacctagatc
     6721 cttttatagt ccggaaatac aggaacgcac gctggatggc ccttcgctgg gatggtgaaa
     6781 ccatgaaaaa tggcagcttc agtggattaa gtgggggtaa tgtggcctgt accctctggt
     6841 tgcataggta ttcatacggt taaaatttat caggcgcgat tgcggcagtt tttcgggtgg
     6901 tttgttgcca tttttacctg tctgctgccg tgatcgcgct gaacgcgttt tagcggtgcg
     6961 tacaattaag ggattatggt aaatccactt actgtctgcc ctcgtagcca tcgagataaa
     7021 ccgcagtact ccggccacga tgcgtccggc gtagaggatc gagatct
//
