LOCUS       sequence-486819-        5336 bp    DNA     circular SYN 22-SEP-2025
DEFINITION  Express NanoLuc(R)-KRAS(G12V) in Mammalian Cells under a CMV 
            promoter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5336)
  AUTHORS   Promega
  TITLE     Promega Plasmid Collection
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 5336)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Sep 22, 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            "Unpublished"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-KRAS(G12V)
FEATURES             Location/Qualifiers
     source          1..5336
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_feature    496..779
                     /label=cer region
                     /note="ColE1-derived recombination site that helps to 
                     maintain plasmids as monomers"
     rep_origin      895..1483
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1384..1403
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     polyA_signal    1722..1770
                     /note="synthetic polyadenylation signal"
     CDS             complement(1834..2628)
                     /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(1945..1964)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     2555..2574
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     promoter        complement(2660..2707)
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     promoter        complement(2722..3079)
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      2736..2871
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     enhancer        2888..3079
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     3036..3056
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     polyA_signal    3324..3445
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3361..3380)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3415..3434
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     3482..3500
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     CDS             complement(join(3614..4144,4145..4147,4148..4180))
                     /codon_start=1
                     /product="human oncoprotein generated by the G12V mutation 
                     in the small GTPase K-Ras"
                     /label=K-Ras (G12V)
                     /translation="MTEYKLVVVGAVGVGKSALTIQLIQNHFVDEYDPTIEDSYRKQVV
                     IDGETCLLDILDTAGQEEYSAMRDQYMRTGEGFLCVFAINNTKSFEDIHHYREQIKRVK
                     DSEDVPMVLVGNKCDLPSRTVDTKQAQDLARSYGIPFIETSAKTRQGVDDAFYTLVREI
                     RKHKEKMSKDGKKKKKKSKTKCVIM"
     CDS             complement(4202..4714)
                     /codon_start=1
                     /product="NanoLuc(R) luciferase"
                     /label=Nluc
                     /note="human codon-optimized"
                     /translation="MVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQR
                     IVLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGV
                     TPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGW
                     RLCERILA"
     primer_bind     complement(4727..4746)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4728..4746)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4788..4807)
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     intron          4790..4922
                     /label=chimeric intron
                     /note="chimera between introns from human beta-globin and 
                     immunoglobulin heavy chain genes"
     promoter        complement(5058..5261)
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     complement(5087..5107)
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     enhancer        join(5262..5336,1..305)
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
ORIGIN
        1 atgccaggcg ggccatttac cgtcattgac gtcaataggg ggcggacttg gcatatgata
       61 cacttgatgt actgccaagt gggcagttta ccgtaaatac tccacccatt gacgtcaatg
      121 gaaagtccct attggcgtta ctatgggaac atacgtcatt attgacgtca atgggcgggg
      181 gtcgttgggc ggtcagccag gcgggccatt taccgtaagt tatgtaacgc ggaactccat
      241 atatgggcta tgaactaatg accccgtaat tgattactat taataactag tcaataatca
      301 atgccaacat ggcggtcata ttggacatga gccaatataa atgtacatat tatgatatag
      361 atacaacgta tgcaatggcc aatagccaat attgatttat gctatataac caatgaataa
      421 tatggctaat ggccaatatt gaagatctcg atcctctacg ccggacgcat cgtggccgga
      481 gtactgcggt ttatctcgat ggctacgagg gcagacagta agtggattta ccataatccc
      541 ttaattgtac gcaccgctaa aacgcgttca gcgcgatcac ggcagcagac aggtaaaaat
      601 ggcaacaaac cacccgaaaa actgccgcaa tcgcgcctga taaattttaa ccgtatgaat
      661 acctatgcaa ccagagggta caggccacat tacccccact taatccactg aagctgccat
      721 ttttcatggt ttcaccatcc cagcgaaggg ccatccagcg tgcgttcctg tatttccgga
      781 ctataaaagg atctaggtga agatcctttt tgataatctc atgaccaaaa tcccttaacg
      841 tgagttttcg ttccactgag cgtcagaccc cgtagaaaag atcaaaggat cttcttgaaa
      901 tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt
      961 ggtttgtttg ccggatcaag agctaccaac tctttttccg aaggtaactg gcttcagcag
     1021 agcgcagata ccaaatactg tccttctagt gtagccgtag ttaggccacc acttcaagaa
     1081 ctctgtagca ccgcctacat acctcgctct gctaatcctg ttaccagtgg ctgctgccag
     1141 tggcgataag tcgtgtctta ccgggttgga ctcaagacga tagttaccgg ataaggcgca
     1201 gcggtcgggc tgaacggggg gttcgtgcac acagcccagc ttggagcgaa cgacctacac
     1261 cgaactgaga tacctacagc gtgagctatg agaaagcgcc acgcttcccg aagggagaaa
     1321 ggcggacagg tatccggtaa gcggcagggt cggaacagga gagcgcacga gggagcttcc
     1381 agggggaaac gcctggtatc tttatagtcc tgtcgggttt cgccacctct gacttgagcg
     1441 tcgatttttg tgatgctcgt caggggggcg gagcctatgg aaaaacgcca gcaacgcggc
     1501 ctttttacgg ttcctggcct tttgctggcc ttttgctcac atgttctttc ctgcgttatc
     1561 ccctgattct gtggataacc gtattaccgc ctttgagtga gctgataccg ggtagaaagc
     1621 cagtccgcag aaacggtgct gaccccggat gaatgtcagc tactgggcta tctggacaag
     1681 ggaaaacgca agcgcaaaga ggatccttat cgctatcgat tcacacaaaa aaccaacaca
     1741 cagatgtaat gaaaataaag atattttatt gcggccatcg tgatggcagg ttgggcgtcg
     1801 cttggtcggt catttcgaac cccagagtcc cgctcagaag aactcgtcaa gaaggcgata
     1861 gaaggcgatg cgctgcgaat cgggagcggc gataccgtaa agcacgagga agcggtcagc
     1921 ccattcgccg ccaagctctt cagcaatatc acgggtagcc aacgctatgt cctgatagcg
     1981 gtccgccaca cccagccggc cacagtcgat gaatccagaa aagcggccat tttccaccat
     2041 gatattcggc aagcaggcat cgccatgggt cacgacgaga tcctcgccgt cgggcatgcg
     2101 cgccttgagc ctggcgaaca gttcggctgg cgcgagcccc tgatgctctt cgtccagatc
     2161 atcctgatcg acaagaccgg cttccatccg agtacgtgct cgctcgatgc gatgtttcgc
     2221 ttggtggtcg aatgggcagg tagccggatc aagcgtatgc agccgccgca ttgcatcagc
     2281 catgatggat actttctcgg caggagcaag gtgagatgac aggagatcct gccccggcac
     2341 ttcgcccaat agcagccagt cccttcccgc ttcagtgaca acgtcgagca cagctgcgca
     2401 aggaacgccc gtcgtggcca gccacgatag ccgcgctgcc tcgtcctgca gttcattcag
     2461 ggcaccggac aggtcggtct tgacaaaaag aaccgggcgc ccctgcgctg acagccggaa
     2521 cacggcggca tcagagcagc cgattgtctg ttgtgcccag tcatagccga atagcctctc
     2581 cacccaagcg gccggagaac ctgcgtgcaa tccatcttgt tcaatcatga tctgcctcct
     2641 gggtttagtt cctcaccttg tcgtattata ctatgccgat atactatgcc gatgattaat
     2701 tgtcaacagt taattaagct ttttgcaaaa gcctaggcct ccaaaaaagc ctcctcacta
     2761 cttctggaat agctcagagg ccgaggcggc ctcggcctct gcataaataa aaaaaattag
     2821 tcagccatgg ggcggagaat gggcggaact gggcggagtt aggggcggga tgggcggagt
     2881 taggggcggg actatggttg ctgactaatt gagatgcatg ctttgcatac ttctgcctgc
     2941 tggggagcct ggggactttc cacacctggt tgctgactaa ttgagatgca tgctttgcat
     3001 acttctgcct gctggggagc ctggggactt tccacaccct aactgacaca cattccacag
     3061 ctggttcttt ccgcctcaga aggtacctaa ccaagttcct ctttcagagg ttatttcagg
     3121 ccatggtgct gcgcagatcc gcgtatgcgg tgtgaaatac cgcacagatg cgtaaggaga
     3181 aaataccgca tcaggaaatt gtaagcgtta atattttgtt aaaattcgat tttaccacat
     3241 ttgtagaggt tttacttgct taaaaaaacc tcccacatct ccccctgaac ctgaaacata
     3301 aaatgaatgc aattgttgtt gttaacttgt ttattgcagc ttataatggt tacaaataaa
     3361 gcaatagcat cacaaatttc acaaataaag catttttttc actgcattct agttgtggtt
     3421 tgtccaaact catcaatgta tcttatcatg tctgctcgaa gcggccgccc caaggggtta
     3481 tgctagttat tgctcagcgg tggcagcagc caactcagct tcctttcggg ctttgttagc
     3541 agccggatca gcttgcatgc ctgcaggtcg actctagagg atccccgggt accgagcccg
     3601 aattcgttta aacttacata attacacact ttgtctttga cttctttttc ttctttttac
     3661 catctttgct catcttttct ttatgttttc gaatttctcg aactaatgta tagaaggcat
     3721 catcaacacc ctgtcttgtc tttgctgatg tttcaataaa aggaattcca taacttcttg
     3781 ctaagtcctg agcctgtttt gtgtctactg ttctagaagg caaatcacat ttatttccta
     3841 ctaggaccat aggtacatct tcagagtcct taactctttt aatttgttct ctataatggt
     3901 gaatatcttc aaatgattta gtattattta tggcaaatac acaaagaaag ccctccccag
     3961 tcctcatgta ctggtccctc attgcactgt actcctcttg acctgctgtg tcgagaatat
     4021 ccaagagaca ggtttctcca tcaattacta cttgcttcct gtaggaatcc tctattgttg
     4081 gatcatattc gtccacaaaa tgattctgaa ttagctgtat cgtcaaggca ctcttgccta
     4141 cgccaacagc tccaactacc acaagtttat attcagtcat ggcgatcgcg ccgctcgagc
     4201 ccgccagaat gcgttcgcac agccgccagc cggtcactcc gttgatggtt actcggaaca
     4261 gcagggagcc gtcggggttg atcaggcgct cgtcgataat tttgttgccg ttccacaggg
     4321 tccctgttac agtgatcttt ttgccgtcga acacggcgat gccttcatac ggccgtccga
     4381 aatagtcgat catgttcggc gtaaccccgt cgattaccag tgtgccatag tgcaggatca
     4441 ccttaaagtg atgatcatcc acagggtaca ccaccttaaa aattttttcg atctggccca
     4501 tttggtcgcc gctcagacct tcatacggga tgatgacatg gatgtcgatc ttcagcccat
     4561 tttcaccgct caggacaatc ctttggatcg gagttacgga caccccgaga ttctgaaaca
     4621 aactggacac acctccctgt tcaaggactt ggtccaggtt gtagccggct gtctgtcgcc
     4681 agtccccaac gaaatcttcg agtgtgaaga ccatggtgag cgctagccct atagtgagtc
     4741 gtattaatac tctagcctta agagctgtaa ttgaactggg agtggacacc tgtggagaga
     4801 aaggcaaagt ggatgtcagt aagaccaata ggtgcctatc agaaacgcaa gagtcttctc
     4861 tgtctcgaca agcccagttt ctattggtct ccttaaacct gtcttgtaac cttgatactt
     4921 acctgcccag tgcctcacga ccaacttctg cagcttaagt tcgagactgt tgtgtcagaa
     4981 gcactgactg cgttagcaat ttaactgtga taaactaccg caataaagct tctagtgatc
     5041 tgacggttca ctaaaccagc tctgcttata tagacctccc accgtacacg cctaccgccc
     5101 atttgcgtca acggggcggg gttattacga cattttggaa agtcccgttg attttggtgc
     5161 caaaacaaac tcccattgac gtcaatgggg tggagacttg gaaatccccg tgagtcaaac
     5221 cgctatccac gcccattggt gtactgccaa aaccgcatca ccatggtaat agcgatgact
     5281 aatacgtaga tgtactgcca agtaggaaag tcccgtaagg tcatgtactg ggcata
//
