LOCUS       sequence-475284-        6713 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Express NanoLuc-cRaf 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 6713)
  AUTHORS   Promega
  TITLE     Promega Plasmid Collection
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 6713)
  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"; journalName: 
            "Unpublished"
COMMENT     Sequence Label: NanoBRET Assay Vector, NanoLuc-cRaf
FEATURES             Location/Qualifiers
     source          1..6713
                     /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"
     primer_bind     complement(3656..3674)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     polyA_signal    3711..3832
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3722..3741)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     3776..3795
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     promoter        4077..4434
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        4077..4268
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(4100..4120)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      4285..4420
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     promoter        4449..4496
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             4528..5322
                     /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(4582..4601)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     5192..5211
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    5386..5434
                     /note="synthetic polyadenylation signal"
     rep_origin      complement(5673..6261)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5753..5772)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     misc_feature    6377..6660
                     /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 ggagcacata cagggagctt
     1621 ggaagacgat cagcaatggt tttggattca aagatgccgt gtttgatggc tccagctgca
     1681 tctctcctac aatagttcag cagtttggct atcagcgccg ggcatcagat gatggcaaac
     1741 tcacagatcc ttctaagaca agcaacacta tccgtgtttt cttgccgaac aagcaaagaa
     1801 cagtggtcaa tgtgcgaaat ggaatgagct tgcatgactg ccttatgaaa gcactcaagg
     1861 tgaggggcct gcaaccagag tgctgtgcag tgttcagact tctccacgaa cacaaaggta
     1921 aaaaagcacg cttagattgg aatactgatg ctgcgtcttt gattggagaa gaacttcaag
     1981 tagatttcct ggatcatgtt cccctcacaa cacacaactt tgctcggaag acgttcctga
     2041 agcttgcctt ctgtgacatc tgtcagaaat tcctgctcaa tggatttcga tgtcagactt
     2101 gtggctacaa atttcatgag cactgtagca ccaaagtacc tactatgtgt gtggactgga
     2161 gtaacatcag acaactctta ttgtttccaa attccactat tggtgatagt ggagtcccag
     2221 cactaccttc tttgactatg cgtcgtatgc gagagtctgt ttccaggatg cctgttagtt
     2281 ctcagcacag atattctaca cctcacgcct tcacctttaa cacctccagt ccctcatctg
     2341 aaggttccct ctcccagagg cagaggtcga catccacacc taatgtccac atggtcagca
     2401 ccaccctgcc tgtggacagc aggatgattg aggatgcaat tcgaagtcac agcgaatcag
     2461 cctcaccttc agccctgtcc agtagcccca acaatctgag cccaacaggc tggtcacagc
     2521 cgaaaacccc cgtgccagca caaagagagc gggcaccagt atctgggacc caggagaaaa
     2581 acaaaattag gcctcgtgga cagagagatt caagctatta ttgggaaata gaagccagtg
     2641 aagtgatgct gtccactcgg attgggtcag gctcttttgg aactgtttat aagggtaaat
     2701 ggcacggaga tgttgcagta aagatcctaa aggttgtcga cccaacccca gagcaattcc
     2761 aggccttcag gaatgaggtg gctgttctgc gcaaaacacg gcatgtgaac attctgcttt
     2821 tcatggggta catgacaaag gacaacctgg caattgtgac ccagtggtgc gagggcagca
     2881 gcctctacaa acacctgcat gtccaggaga ccaagtttca gatgttccag ctaattgaca
     2941 ttgcccggca gacggctcag ggaatggact atttgcatgc aaagaacatc atccatagag
     3001 acatgaaatc caacaatata tttctccatg aaggcttaac agtgaaaatt ggagattttg
     3061 gtttggcaac agtaaagtca cgctggagtg gttctcagca ggttgaacaa cctactggct
     3121 ctgtcctctg gatggcccca gaggtgatcc gaatgcagga taacaaccca ttcagtttcc
     3181 agtcggatgt ctactcctat ggcatcgtat tgtatgaact gatgacgggg gagcttcctt
     3241 attctcacat caacaaccga gatcagatca tcttcatggt gggccgagga tatgcctccc
     3301 cagatcttag taagctatat aagaactgcc ccaaagcaat gaagaggctg gtagctgact
     3361 gtgtgaagaa agtaaaggaa gagaggcctc tttttcccca gatcctgtct tccattgagc
     3421 tgctccaaca ctctctaccg aagatcaacc ggagcgcttc cgagccatcc ttgcatcggg
     3481 cagcccacac tgaggatatc aatgcttgca cgctgaccac gtccccgagg ctgcctgtct
     3541 tcgtttaaac gaattcgggc tcggtacccg gggatcctct agagtcgacc tgcaggcatg
     3601 caagctgatc cggctgctaa caaagcccga aaggaagctg agttggctgc tgccaccgct
     3661 gagcaataac tagcataacc ccttggggcg gccgcttcga gcagacatga taagatacat
     3721 tgatgagttt ggacaaacca caactagaat gcagtgaaaa aaatgcttta tttgtgaaat
     3781 ttgtgatgct attgctttat ttgtaaccat tataagctgc aataaacaag ttaacaacaa
     3841 caattgcatt cattttatgt ttcaggttca gggggagatg tgggaggttt ttttaagcaa
     3901 gtaaaacctc tacaaatgtg gtaaaatcga attttaacaa aatattaacg cttacaattt
     3961 cctgatgcgg tattttctcc ttacgcatct gtgcggtatt tcacaccgca tacgcggatc
     4021 tgcgcagcac catggcctga aataacctct gaaagaggaa cttggttagg taccttctga
     4081 ggcggaaaga accagctgtg gaatgtgtgt cagttagggt gtggaaagtc cccaggctcc
     4141 ccagcaggca gaagtatgca aagcatgcat ctcaattagt cagcaaccag gtgtggaaag
     4201 tccccaggct ccccagcagg cagaagtatg caaagcatgc atctcaatta gtcagcaacc
     4261 atagtcccgc ccctaactcc gcccatcccg cccctaactc cgcccagttc cgcccattct
     4321 ccgccccatg gctgactaat tttttttatt tatgcagagg ccgaggccgc ctcggcctct
     4381 gagctattcc agaagtagtg aggaggcttt tttggaggcc taggcttttg caaaaagctt
     4441 aattaactgt tgacaattaa tcatcggcat agtatatcgg catagtataa tacgacaagg
     4501 tgaggaacta aacccaggag gcagatcatg attgaacaag atggattgca cgcaggttct
     4561 ccggccgctt gggtggagag gctattcggc tatgactggg cacaacagac aatcggctgc
     4621 tctgatgccg ccgtgttccg gctgtcagcg caggggcgcc cggttctttt tgtcaagacc
     4681 gacctgtccg gtgccctgaa tgaactgcag gacgaggcag cgcggctatc gtggctggcc
     4741 acgacgggcg ttccttgcgc agctgtgctc gacgttgtca ctgaagcggg aagggactgg
     4801 ctgctattgg gcgaagtgcc ggggcaggat ctcctgtcat ctcaccttgc tcctgccgag
     4861 aaagtatcca tcatggctga tgcaatgcgg cggctgcata cgcttgatcc ggctacctgc
     4921 ccattcgacc accaagcgaa acatcgcatc gagcgagcac gtactcggat ggaagccggt
     4981 cttgtcgatc aggatgatct ggacgaagag catcaggggc tcgcgccagc cgaactgttc
     5041 gccaggctca aggcgcgcat gcccgacggc gaggatctcg tcgtgaccca tggcgatgcc
     5101 tgcttgccga atatcatggt ggaaaatggc cgcttttctg gattcatcga ctgtggccgg
     5161 ctgggtgtgg cggaccgcta tcaggacata gcgttggcta cccgtgatat tgctgaagag
     5221 cttggcggcg aatgggctga ccgcttcctc gtgctttacg gtatcgccgc tcccgattcg
     5281 cagcgcatcg ccttctatcg ccttcttgac gagttcttct gagcgggact ctggggttcg
     5341 aaatgaccga ccaagcgacg cccaacctgc catcacgatg gccgcaataa aatatcttta
     5401 ttttcattac atctgtgtgt tggttttttg tgtgaatcga tagcgataag gatcctcttt
     5461 gcgcttgcgt tttcccttgt ccagatagcc cagtagctga cattcatccg gggtcagcac
     5521 cgtttctgcg gactggcttt ctacccggta tcagctcact caaaggcggt aatacggtta
     5581 tccacagaat caggggataa cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc
     5641 aggaaccgta aaaaggccgc gttgctggcg tttttccata ggctccgccc ccctgacgag
     5701 catcacaaaa atcgacgctc aagtcagagg tggcgaaacc cgacaggact ataaagatac
     5761 caggcgtttc cccctggaag ctccctcgtg cgctctcctg ttccgaccct gccgcttacc
     5821 ggatacctgt ccgcctttct cccttcggga agcgtggcgc tttctcatag ctcacgctgt
     5881 aggtatctca gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc
     5941 gttcagcccg accgctgcgc cttatccggt aactatcgtc ttgagtccaa cccggtaaga
     6001 cacgacttat cgccactggc agcagccact ggtaacagga ttagcagagc gaggtatgta
     6061 ggcggtgcta cagagttctt gaagtggtgg cctaactacg gctacactag aaggacagta
     6121 tttggtatct gcgctctgct gaagccagtt accttcggaa aaagagttgg tagctcttga
     6181 tccggcaaac aaaccaccgc tggtagcggt ggtttttttg tttgcaagca gcagattacg
     6241 cgcagaaaaa aaggatttca agaagatcct ttgatctttt ctacggggtc tgacgctcag
     6301 tggaacgaaa actcacgtta agggattttg gtcatgagat tatcaaaaag gatcttcacc
     6361 tagatccttt tatagtccgg aaatacagga acgcacgctg gatggccctt cgctgggatg
     6421 gtgaaaccat gaaaaatggc agcttcagtg gattaagtgg gggtaatgtg gcctgtaccc
     6481 tctggttgca taggtattca tacggttaaa atttatcagg cgcgattgcg gcagtttttc
     6541 gggtggtttg ttgccatttt tacctgtctg ctgccgtgat cgcgctgaac gcgttttagc
     6601 ggtgcgtaca attaagggat tatggtaaat ccacttactg tctgccctcg tagccatcga
     6661 gataaaccgc agtactccgg ccacgatgcg tccggcgtag aggatcgaga tct
//
