LOCUS       sequence-477412-        6138 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Express NanoLuc(R)-NRAS(Q61K) in Mammalian Cells under a CMV 
            promoter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6138)
  AUTHORS   Promega
  TITLE     Promega Plasmid Collection
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 6138)
  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            "Unpublished"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-NRAS(Q61K)
FEATURES             Location/Qualifiers
     source          1..6138
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      complement(621..1209)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(701..720)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     polyA_signal    1335..1383
                     /note="synthetic polyadenylation signal"
     CDS             complement(1407..2201)
                     /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(1518..1537)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     2128..2147
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     promoter        complement(2579..3757)
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          2588..3526
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     complement(2611..2631)
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     polyA_signal    3774..3855
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     3799..3818
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     CDS             complement(4649..5161)
                     /codon_start=1
                     /product="NanoLuc(R) luciferase"
                     /label=Nluc
                     /note="human codon-optimized"
                     /translation="MVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQR
                     IVLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGV
                     TPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGW
                     RLCERILA"
     promoter        complement(5200..5218)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(5201..5218)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     enhancer        5338..5641
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     primer_bind     5796..5816
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     promoter        5849..5896
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             join(5928..6138,1..449)
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     complement(5994..6013)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
ORIGIN
        1 cggaattccg tatggcaatg aaagacggtg agctggtgat atgggatagt gttcaccctt
       61 gttacaccgt tttccatgag caaactgaaa cgttttcatc gctctggagt gaataccacg
      121 acgatttccg gcagtttcta cacatatatt cgcaagatgt ggcgtgttac ggtgaaaacc
      181 tggcctattt ccctaaaggg tttattgaga atatgttttt cgtctcagcc aatccctggg
      241 tgagtttcac cagttttgat ttaaacgtgg ccaatatgga caacttcttc gcccccgttt
      301 tcactatggg caaatattat acgcaaggcg acaaggtgct gatgccgctg gcgattcagg
      361 ttcatcatgc cgtctgtgat ggcttccatg tcggcagaat gcttaatgaa ttacaacagt
      421 actgcgatga gtggcagggc ggggcgtaag cgggactctg gggttcgaaa tgaccgacca
      481 agcgacgccc aaccggtatc agctcactca aaggcggtaa tacggttatc cacagaatca
      541 ggggataacg caggaaagaa catgtgagca aaaggccagc aaaaggccag gaaccgtaaa
      601 aaggccgcgt tgctggcgtt tttccatagg ctccgccccc ctgacgagca tcacaaaaat
      661 cgacgctcaa gtcagaggtg gcgaaacccg acaggactat aaagatacca ggcgtttccc
      721 cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc
      781 gcctttctcc cttcgggaag cgtggcgctt tctcatagct cacgctgtag gtatctcagt
      841 tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt tcagcccgac
      901 cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc cggtaagaca cgacttatcg
      961 ccactggcag cagccactgg taacaggatt agcagagcga ggtatgtagg cggtgctaca
     1021 gagttcttga agtggtggcc taactacggc tacactagaa ggacagtatt tggtatctgc
     1081 gctctgctga agccagttac cttcggaaaa agagttggta gctcttgatc cggcaaacaa
     1141 accaccgctg gtagcggtgg tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa
     1201 ggatttcaag aagatccttt gatcttttct acggggtctg acgctcagtg gaacgaaaac
     1261 tcacgttaag ggattttggt catgagatta tcaaaaagga tcttcaccta gatcctttta
     1321 tagtccggaa tcgtcacaca aaaaaccaac acacagatgt aatgaaaata aagatatttt
     1381 attgcggcca tcgtgatggc tagagttcag aagaactcgt caagaaggcg atagaaggcg
     1441 atgcgctgcg aatcgggagc ggcgataccg taaagcacga ggaagcggtc agcccattcg
     1501 ccgccaagct cttcagcaat atcacgggta gccaacgcta tgtcctgata gcggtccgcc
     1561 acacccagcc ggccacagtc gatgaatcca gaaaagcggc cattttccac catgatattc
     1621 ggcaagcagg catcgccgtg ggtcacgacg agatcctcgc cgtcgggcat gcgcgccttg
     1681 agcctggcga acagttcggc tggcgcgagc ccctgatgct cttcgtccag atcatcctga
     1741 tcgacaagac cggcttccat ccgagtacgt gctcgctcga tgcgatgttt cgcttggtgg
     1801 tcgaatgggc aggtagccgg atcaagcgta tgcagccgcc gcattgcatc agccatgatg
     1861 gatactttct cggcaggagc aaggtgagat gacaggagat cctgccccgg cacttcgccc
     1921 aatagcagcc agtcccttcc cgcttcagtg acaacgtcga gcacagctgc gcaaggaacg
     1981 cccgtcgtgg ccagccacga tagccgcgct gcctcgtcct gcagttcatt cagggcaccg
     2041 gacaggtcgg tcttgacaaa aagaaccggg cgcccctgcg ctgacagccg gaacacggcg
     2101 gcatcagagc agccgattgt ctgttgtgcc cagtcatagc cgaatagcct ctccacccaa
     2161 gcggccggag aacctgcgtg caatccatct tgttcaatca tggtggcatc gattcccggt
     2221 caacatccag tacacctccg gcacaccggc ttccccaagg gcgccacgct ctcccaccac
     2281 aacatcctca acaacggcta ctgggtgggc cgcacggtcg gctacacgga gcaggaccgg
     2341 gtgtgtctgc cggtgccctt ctaccactgc ttcggcatgg tcatgggcaa cttgggcgcc
     2401 acctcccacg ccgcctgcat cgtcatcccg gccccgtcct tcgagccggc ggcgacgctg
     2461 gaggcggtgc agcgggagag gtgcacgtcc ctgtacggcg tccccacgaa gccgaattcc
     2521 accacactgg actagatgca tgctcgacag tgtgatggat atctgcagaa ttcggctttc
     2581 acgacacctg aaatggaaga aaaaaacttt gaaccactgt ctgaggcttg agaatgaacc
     2641 aagatccaaa ctcaaaaagg gcaaattcca aggagaatta catcaagtgc caagctggcc
     2701 taacttcagt ctccacccac tcagtgtggg gaaactccat cgcataaaac ccctcccccc
     2761 aacctaaaga cgacgtactc caaaagctcg agaactaatc gaggtgcctg gacggcgccc
     2821 ggtactccgt ggagtcacat gaagcgacgg ctgaggacgg aaaggccctt ttcctttgtg
     2881 tgggtgactc acccgcccgc tctcccgagc gccgcgtcct ccattttgag ctgcctgcag
     2941 cagggccggg aagcggccat ctttccgctc acgcaactgg tgccgaccgg gccagccttg
     3001 ccgcccaggg cggggcgata cacggcggcg cgaggccagg caccagagca ggccggccag
     3061 cttgagacta cccccgtccg attctcggtg gccgcgctcg caggccccgc ctcgccgaac
     3121 atgtgcgctg ggacgcacgg gccccgtcgc cgcccgcggc cccaaaaacc gaaataccag
     3181 tgtgcagatc ttggcccgca tttacaagac tatcttgcca gaaaaaaagc gtcgcagcag
     3241 gtcatcaaaa attttaaatg gctagagact tatcgaaagc agcgagacag gcgcgaaggt
     3301 gccaccagat tcgcacgcgg cggccccagc gcccaggcca ggcctcaact caagcacgag
     3361 gcgaaggggc tccttaagcg caaggcctcg aactctccca cccacttcca acccgaagct
     3421 cgggatcaag aatcacgtac tgcagccagg tggaagtaat tcaaggcacg caagggccat
     3481 aacccgtaaa gaggccaggc ccgcgggaac cacacacggc acttacctgt gttctggcgg
     3541 caaacccgtt gcgaaaaaga acgttcacgg cgactactgc acttatatac ggttctcccc
     3601 caccctcggg aaaaaggcgg agccagtaca cgacatcact ttcccagttt accccgcgcc
     3661 accttctcta ggcaccggtt caattgccga cccctccccc caacttctcg gggactgtgg
     3721 gcgatgtgcg ctctgcccac tgacgggcac cggagccaag acgcgtgacg tcggcagtga
     3781 aaaaaatgct ttatttgtga aatttgtgat gctattgctt tatttgtaac cattataagc
     3841 tgcaataaac aagttaacaa caacaattgc attcatttta tgtttcaggt tcagggggag
     3901 gtgtgggagg ttttttaaag caagtaaaac ctctacaaat gtggtatggc tgattatgat
     3961 ccgcggccgc aatacgtcga cgttatcagc tgacttcgta cgagagccta ggtgtttcta
     4021 gaggatcccc gggtaccgag cccgaattcg tttaaactta catcaccaca catggcaatc
     4081 ccatacaacc ctgagtccca tcatcactgc tgttgagttt tttcattcgg tactggcgta
     4141 tttctcttac cagtgtgtaa aaagcatctt caacaccctg tctggtcttg gctgaggttt
     4201 caatgaatgg aatcccgtaa ctcttggcca gttcgtgggc ttgttttgta tcaactgtcc
     4261 ttgttggcaa atcacacttg tttcccacta gcaccatagg tacatcatcc gagtctttta
     4321 ctcgcttaat ctgctccctg tagaggttaa tatccgcaaa tgacttgcta ttattgatgg
     4381 caaatacaca gaggaagcct tcgcctgtcc tcatgtattg gtctctcatg gcactgtact
     4441 cttcttttcc agctgtatcc agtatgtcca acaaacaggt ttcaccatct ataaccactt
     4501 gttttctgta agaatcctct atggtgggat catattcatc tacaaagtgg ttctggatta
     4561 gctggattgt cagtgcgctt ttcccaacac cacctgctcc aaccaccacc agtttgtact
     4621 cagtcatggc gatcgcgccg ctcgagcccg ccagaatgcg ttcgcacagc cgccagccgg
     4681 tcactccgtt gatggttact cggaacagca gggagccgtc ggggttgatc aggcgctcgt
     4741 cgataatttt gttgccgttc cacagggtcc ctgttacagt gatctttttg ccgtcgaaca
     4801 cggcgatgcc ttcatacggc cgtccgaaat agtcgatcat gttcggcgta accccgtcga
     4861 ttaccagtgt gccatagtgc aggatcacct taaagtgatg atcatccaca gggtacacca
     4921 ccttaaaaat tttttcgatc tggcccattt ggtcgccgct cagaccttca tacgggatga
     4981 tgacatggat gtcgatcttc agcccatttt caccgctcag gacaatcctt tggatcggag
     5041 ttacggacac cccgagattc tgaaacaaac tggacacacc tccctgttca aggacttggt
     5101 ccaggttgta gccggctgtc tgtcgccagt ccccaacgaa atcttcgagt gtgaagacca
     5161 tggtgagcgc tagctgcaaa aagaacaagt agcttgtatt ctatagtgtc acctaaatgg
     5221 gcgatctgac ggttcactaa accagctctg cttatatagg cctccctggc gcgccactag
     5281 ttattaatag taatcaatta cggggtcatt agttcatagc ccatatatgg agttccgcgt
     5341 tacataactt acggtaaatg gcccgcctgg ctgaccgccc aacgaccccc gcccattgac
     5401 gtcaataatg acgtatgttc ccatagtaac gccaataggg actttccatt gacgtcaatg
     5461 ggtggagtat ttacggtaaa ctgcccactt ggcagtacat caagtgtatc atatgccaag
     5521 tccgccccct attgacgtca atgacggtaa atggcccgcc tggcattatg cccagtacat
     5581 gaccttacgg gactttccta cttggcagta catctacgta ttagtcatcg ctattaccat
     5641 ggtgatgcgg ttttggcagt acaccaatgg gcgtggatag cggtttgact cacggggatt
     5701 tccaagtctc caccccattg acgtcaatgg gagtttgttt tggcaccaaa atcaacggga
     5761 ctttccaaaa tgtcgtaata accccgcccc gttgacgcaa atgggcggta ggcgtgtacg
     5821 gtgggaggtc aaatcgaatt ctaatactgt tgacaattaa tcatcggcat agtatatcgg
     5881 catagtataa tacgacaagg tgaggaacta aacccaggag gcagatcatg gagaaaaaaa
     5941 tcactggata taccaccgtt gatatatccc aatggcatcg taaagaacat tttgaggcat
     6001 ttcagtcagt tgctcaatgt acctataacc agaccgttca gctggatatt acggcctttt
     6061 taaagaccgt aaagaaaaat aagcacaagt tttatccggc ctttattcac attcttgccc
     6121 gcctgatgaa tgctcatc
//
