LOCUS       sequence-472476-        6138 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Express NanoLuc(R)-HRAS WT 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  JOURNAL   Unpublished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            "Unpublished"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-HRAS WT
FEATURES             Location/Qualifiers
     source          1..6138
                     /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(301..870)
                     /codon_start=1
                     /product="human small GTPase proto-oncoprotein"
                     /label=H-Ras
                     /translation="MTEYKLVVVGAGGVGKSALTIQLIQNHFVDEYDPTIEDSYRKQVV
                     IDGETCLLDILDTAGQEEYSAMRDQYMRTGEGFLCVFAINNTKSFEDIHQYREQIKRVK
                     DSDDVPMVLVGNKCDLAARTVESRQAQDLARSYGIPYIETSAKTRQGVEDAFYTLVREI
                     RQHKLRKLNPPDESGPGCMSCKCVLS"
     CDS             complement(892..1404)
                     /codon_start=1
                     /product="NanoLuc(R) luciferase"
                     /label=Nluc
                     /note="human codon-optimized"
                     /translation="MVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQR
                     IVLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGV
                     TPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGW
                     RLCERILA"
     promoter        complement(1443..1461)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(1444..1461)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     enhancer        1581..1884
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     primer_bind     2039..2059
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     promoter        2092..2139
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             2171..2830
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     complement(2237..2256)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     rep_origin      complement(3002..3590)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3082..3101)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     polyA_signal    3716..3764
                     /note="synthetic polyadenylation signal"
     CDS             complement(3788..4582)
                     /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(3899..3918)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     4509..4528
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     promoter        complement(4960..6138)
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          4969..5907
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     complement(4992..5012)
                     /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 ctagaggatc cccgggtacc gagcccgaat tcgtttaaac
      301 tcaggagagc acacacttgc agctcatgca gccggggcca ctctcatcag gagggttcag
      361 cttccgcagc ttgtgctgcc ggatctcacg caccaacgtg tagaaggcat cctccactcc
      421 ctgccgggtc ttggccgagg tctcgatgta ggggatgccg tagcttcggg cgaggtcctg
      481 agcctgccga gattccacag tgcgtgcagc caggtcacac ttgttcccca ccagcaccat
      541 gggcacgtca tccgagtcct tcacccgttt gatctgctcc ctgtactggt ggatgtcctc
      601 aaaagacttg gtgttgttga tggcaaacac acacaggaag ccctccccgg tgcgcatgta
      661 ctggtcccgc atggcgctgt actcctcctg gccggcggta tccaggatgt ccaacaggca
      721 cgtctcccca tcaatgacca cctgcttccg gtaggaatcc tctatagtgg ggtcgtattc
      781 gtccacaaaa tggttctgga tcagctggat ggtcagcgca ctcttgccca caccgccggc
      841 gcccaccacc accagcttat attccgtcat ggcgatcgcg ccgctcgagc ccgccagaat
      901 gcgttcgcac agccgccagc cggtcactcc gttgatggtt actcggaaca gcagggagcc
      961 gtcggggttg atcaggcgct cgtcgataat tttgttgccg ttccacaggg tccctgttac
     1021 agtgatcttt ttgccgtcga acacggcgat gccttcatac ggccgtccga aatagtcgat
     1081 catgttcggc gtaaccccgt cgattaccag tgtgccatag tgcaggatca ccttaaagtg
     1141 atgatcatcc acagggtaca ccaccttaaa aattttttcg atctggccca tttggtcgcc
     1201 gctcagacct tcatacggga tgatgacatg gatgtcgatc ttcagcccat tttcaccgct
     1261 caggacaatc ctttggatcg gagttacgga caccccgaga ttctgaaaca aactggacac
     1321 acctccctgt tcaaggactt ggtccaggtt gtagccggct gtctgtcgcc agtccccaac
     1381 gaaatcttcg agtgtgaaga ccatggtgag cgctagctgc aaaaagaaca agtagcttgt
     1441 attctatagt gtcacctaaa tgggcgatct gacggttcac taaaccagct ctgcttatat
     1501 aggcctccct ggcgcgccac tagttattaa tagtaatcaa ttacggggtc attagttcat
     1561 agcccatata tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg
     1621 cccaacgacc cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata
     1681 gggactttcc attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta
     1741 catcaagtgt atcatatgcc aagtccgccc cctattgacg tcaatgacgg taaatggccc
     1801 gcctggcatt atgcccagta catgacctta cgggactttc ctacttggca gtacatctac
     1861 gtattagtca tcgctattac catggtgatg cggttttggc agtacaccaa tgggcgtgga
     1921 tagcggtttg actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg
     1981 ttttggcacc aaaatcaacg ggactttcca aaatgtcgta ataaccccgc cccgttgacg
     2041 caaatgggcg gtaggcgtgt acggtgggag gtcaaatcga attctaatac tgttgacaat
     2101 taatcatcgg catagtatat cggcatagta taatacgaca aggtgaggaa ctaaacccag
     2161 gaggcagatc atggagaaaa aaatcactgg atataccacc gttgatatat cccaatggca
     2221 tcgtaaagaa cattttgagg catttcagtc agttgctcaa tgtacctata accagaccgt
     2281 tcagctggat attacggcct ttttaaagac cgtaaagaaa aataagcaca agttttatcc
     2341 ggcctttatt cacattcttg cccgcctgat gaatgctcat ccggaattcc gtatggcaat
     2401 gaaagacggt gagctggtga tatgggatag tgttcaccct tgttacaccg ttttccatga
     2461 gcaaactgaa acgttttcat cgctctggag tgaataccac gacgatttcc ggcagtttct
     2521 acacatatat tcgcaagatg tggcgtgtta cggtgaaaac ctggcctatt tccctaaagg
     2581 gtttattgag aatatgtttt tcgtctcagc caatccctgg gtgagtttca ccagttttga
     2641 tttaaacgtg gccaatatgg acaacttctt cgcccccgtt ttcactatgg gcaaatatta
     2701 tacgcaaggc gacaaggtgc tgatgccgct ggcgattcag gttcatcatg ccgtctgtga
     2761 tggcttccat gtcggcagaa tgcttaatga attacaacag tactgcgatg agtggcaggg
     2821 cggggcgtaa gcgggactct ggggttcgaa atgaccgacc aagcgacgcc caaccggtat
     2881 cagctcactc aaaggcggta atacggttat ccacagaatc aggggataac gcaggaaaga
     2941 acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt
     3001 ttttccatag gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt
     3061 ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc
     3121 gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa
     3181 gcgtggcgct ttctcatagc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct
     3241 ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta
     3301 actatcgtct tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg
     3361 gtaacaggat tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggc
     3421 ctaactacgg ctacactaga aggacagtat ttggtatctg cgctctgctg aagccagtta
     3481 ccttcggaaa aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtg
     3541 gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatttcaa gaagatcctt
     3601 tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg
     3661 tcatgagatt atcaaaaagg atcttcacct agatcctttt atagtccgga atcgtcacac
     3721 aaaaaaccaa cacacagatg taatgaaaat aaagatattt tattgcggcc atcgtgatgg
     3781 ctagagttca gaagaactcg tcaagaaggc gatagaaggc gatgcgctgc gaatcgggag
     3841 cggcgatacc gtaaagcacg aggaagcggt cagcccattc gccgccaagc tcttcagcaa
     3901 tatcacgggt agccaacgct atgtcctgat agcggtccgc cacacccagc cggccacagt
     3961 cgatgaatcc agaaaagcgg ccattttcca ccatgatatt cggcaagcag gcatcgccgt
     4021 gggtcacgac gagatcctcg ccgtcgggca tgcgcgcctt gagcctggcg aacagttcgg
     4081 ctggcgcgag cccctgatgc tcttcgtcca gatcatcctg atcgacaaga ccggcttcca
     4141 tccgagtacg tgctcgctcg atgcgatgtt tcgcttggtg gtcgaatggg caggtagccg
     4201 gatcaagcgt atgcagccgc cgcattgcat cagccatgat ggatactttc tcggcaggag
     4261 caaggtgaga tgacaggaga tcctgccccg gcacttcgcc caatagcagc cagtcccttc
     4321 ccgcttcagt gacaacgtcg agcacagctg cgcaaggaac gcccgtcgtg gccagccacg
     4381 atagccgcgc tgcctcgtcc tgcagttcat tcagggcacc ggacaggtcg gtcttgacaa
     4441 aaagaaccgg gcgcccctgc gctgacagcc ggaacacggc ggcatcagag cagccgattg
     4501 tctgttgtgc ccagtcatag ccgaatagcc tctccaccca agcggccgga gaacctgcgt
     4561 gcaatccatc ttgttcaatc atggtggcat cgattcccgg tcaacatcca gtacacctcc
     4621 ggcacaccgg cttccccaag ggcgccacgc tctcccacca caacatcctc aacaacggct
     4681 actgggtggg ccgcacggtc ggctacacgg agcaggaccg ggtgtgtctg ccggtgccct
     4741 tctaccactg cttcggcatg gtcatgggca acttgggcgc cacctcccac gccgcctgca
     4801 tcgtcatccc ggccccgtcc ttcgagccgg cggcgacgct ggaggcggtg cagcgggaga
     4861 ggtgcacgtc cctgtacggc gtccccacga agccgaattc caccacactg gactagatgc
     4921 atgctcgaca gtgtgatgga tatctgcaga attcggcttt cacgacacct gaaatggaag
     4981 aaaaaaactt tgaaccactg tctgaggctt gagaatgaac caagatccaa actcaaaaag
     5041 ggcaaattcc aaggagaatt acatcaagtg ccaagctggc ctaacttcag tctccaccca
     5101 ctcagtgtgg ggaaactcca tcgcataaaa cccctccccc caacctaaag acgacgtact
     5161 ccaaaagctc gagaactaat cgaggtgcct ggacggcgcc cggtactccg tggagtcaca
     5221 tgaagcgacg gctgaggacg gaaaggccct tttcctttgt gtgggtgact cacccgcccg
     5281 ctctcccgag cgccgcgtcc tccattttga gctgcctgca gcagggccgg gaagcggcca
     5341 tctttccgct cacgcaactg gtgccgaccg ggccagcctt gccgcccagg gcggggcgat
     5401 acacggcggc gcgaggccag gcaccagagc aggccggcca gcttgagact acccccgtcc
     5461 gattctcggt ggccgcgctc gcaggccccg cctcgccgaa catgtgcgct gggacgcacg
     5521 ggccccgtcg ccgcccgcgg ccccaaaaac cgaaatacca gtgtgcagat cttggcccgc
     5581 atttacaaga ctatcttgcc agaaaaaaag cgtcgcagca ggtcatcaaa aattttaaat
     5641 ggctagagac ttatcgaaag cagcgagaca ggcgcgaagg tgccaccaga ttcgcacgcg
     5701 gcggccccag cgcccaggcc aggcctcaac tcaagcacga ggcgaagggg ctccttaagc
     5761 gcaaggcctc gaactctccc acccacttcc aacccgaagc tcgggatcaa gaatcacgta
     5821 ctgcagccag gtggaagtaa ttcaaggcac gcaagggcca taacccgtaa agaggccagg
     5881 cccgcgggaa ccacacacgg cacttacctg tgttctggcg gcaaacccgt tgcgaaaaag
     5941 aacgttcacg gcgactactg cacttatata cggttctccc ccaccctcgg gaaaaaggcg
     6001 gagccagtac acgacatcac tttcccagtt taccccgcgc caccttctct aggcaccggt
     6061 tcaattgccg acccctcccc ccaacttctc ggggactgtg ggcgatgtgc gctctgccca
     6121 ctgacgggca ccggagcc
//
