LOCUS       sequence-481209-        5323 bp    DNA     circular SYN 30-JUL-2025
DEFINITION  Express NanoLuc-KRas(G12D) 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 5323)
  AUTHORS   Promega
  TITLE     Promega Plasmid Collection
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 5323)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Jul 30, 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            "Unpublished"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(G12D)
FEATURES             Location/Qualifiers
     source          1..5323
                     /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"
     CDS             join(1599..1631,1632..1634,1635..2165)
                     /codon_start=1
                     /product="human oncoprotein generated by the G12V mutation 
                     in the small GTPase K-Ras"
                     /label=K-Ras (G12V)
                     /translation="MTEYKLVVVGADGVGKSALTIQLIQNHFVDEYDPTIEDSYRKQVV
                     IDGETCLLDILDTAGQEEYSAMRDQYMRTGEGFLCVFAINNTKSFEDIHHYREQIKRVK
                     DSEDVPMVLVGNKCDLPSRTVDTKQAQDLARSYGIPFIETSAKTRQGVDDAFYTLVREI
                     RKHKEKMSKDGKKKKKKSKTKCVIM"
     CDS             1599..2165
                     /codon_start=1
                     /product="human small GTPase proto-oncoprotein"
                     /label=K-Ras
                     /translation="MTEYKLVVVGADGVGKSALTIQLIQNHFVDEYDPTIEDSYRKQVV
                     IDGETCLLDILDTAGQEEYSAMRDQYMRTGEGFLCVFAINNTKSFEDIHHYREQIKRVK
                     DSEDVPMVLVGNKCDLPSRTVDTKQAQDLARSYGIPFIETSAKTRQGVDDAFYTLVREI
                     RKHKEKMSKDGKKKKKKSKTKCVIM"
     primer_bind     complement(2279..2297)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     polyA_signal    2334..2455
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2345..2364)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     2399..2418
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     promoter        2700..3057
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        2700..2891
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(2723..2743)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      2908..3043
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             3138..3932
                     /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(3192..3211)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3802..3821
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    3996..4044
                     /note="synthetic polyadenylation signal"
     rep_origin      complement(4283..4871)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4363..4382)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     misc_feature    4987..5270
                     /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 gactgaatat aaacttgtgg
     1621 tagttggagc tgacggcgta ggcaagagtg ccttgacgat acagctaatt cagaatcatt
     1681 ttgtggacga atatgatcca acaatagagg attcctacag gaagcaagta gtaattgatg
     1741 gagaaacctg tctcttggat attctcgaca cagcaggtca agaggagtac agtgcaatga
     1801 gggaccagta catgaggact ggggagggct ttctttgtgt atttgccata aataatacta
     1861 aatcatttga agatattcac cattatagag aacaaattaa aagagttaag gactctgaag
     1921 atgtacctat ggtcctagta ggaaataaat gtgatttgcc ttctagaaca gtagacacaa
     1981 aacaggctca ggacttagca agaagttatg gaattccttt tattgaaaca tcagcaaaga
     2041 caagacaggg tgttgatgat gccttctata cattagttcg agaaattcga aaacataaag
     2101 aaaagatgag caaagatggt aaaaagaaga aaaagaagtc aaagacaaag tgtgtaatta
     2161 tgtaagttta aacgaattcg ggctcggtac ccggggatcc tctagagtcg acctgcaggc
     2221 atgcaagctg atccggctgc taacaaagcc cgaaaggaag ctgagttggc tgctgccacc
     2281 gctgagcaat aactagcata accccttggg gcggccgctt cgagcagaca tgataagata
     2341 cattgatgag tttggacaaa ccacaactag aatgcagtga aaaaaatgct ttatttgtga
     2401 aatttgtgat gctattgctt tatttgtaac cattataagc tgcaataaac aagttaacaa
     2461 caacaattgc attcatttta tgtttcaggt tcagggggag atgtgggagg tttttttaag
     2521 caagtaaaac ctctacaaat gtggtaaaat cgaattttaa caaaatatta acgcttacaa
     2581 tttcctgatg cggtattttc tccttacgca tctgtgcggt atttcacacc gcatacgcgg
     2641 atctgcgcag caccatggcc tgaaataacc tctgaaagag gaacttggtt aggtaccttc
     2701 tgaggcggaa agaaccagct gtggaatgtg tgtcagttag ggtgtggaaa gtccccaggc
     2761 tccccagcag gcagaagtat gcaaagcatg catctcaatt agtcagcaac caggtgtgga
     2821 aagtccccag gctccccagc aggcagaagt atgcaaagca tgcatctcaa ttagtcagca
     2881 accatagtcc cgcccctaac tccgcccatc ccgcccctaa ctccgcccag ttccgcccat
     2941 tctccgcccc atggctgact aatttttttt atttatgcag aggccgaggc cgcctcggcc
     3001 tctgagctat tccagaagta gtgaggaggc ttttttggag gcctaggctt ttgcaaaaag
     3061 cttaattaac tgttgacaat taatcatcgg catagtataa tacgacaagg tgaggaacta
     3121 aacccaggag gcagatcatg attgaacaag atggattgca cgcaggttct ccggccgctt
     3181 gggtggagag gctattcggc tatgactggg cacaacagac aatcggctgc tctgatgccg
     3241 ccgtgttccg gctgtcagcg caggggcgcc cggttctttt tgtcaagacc gacctgtccg
     3301 gtgccctgaa tgaactgcag gacgaggcag cgcggctatc gtggctggcc acgacgggcg
     3361 ttccttgcgc agctgtgctc gacgttgtca ctgaagcggg aagggactgg ctgctattgg
     3421 gcgaagtgcc ggggcaggat ctcctgtcat ctcaccttgc tcctgccgag aaagtatcca
     3481 tcatggctga tgcaatgcgg cggctgcata cgcttgatcc ggctacctgc ccattcgacc
     3541 accaagcgaa acatcgcatc gagcgagcac gtactcggat ggaagccggt cttgtcgatc
     3601 aggatgatct ggacgaagag catcaggggc tcgcgccagc cgaactgttc gccaggctca
     3661 aggcgcgcat gcccgacggc gaggatctcg tcgtgaccca tggcgatgcc tgcttgccga
     3721 atatcatggt ggaaaatggc cgcttttctg gattcatcga ctgtggccgg ctgggtgtgg
     3781 cggaccgcta tcaggacata gcgttggcta cccgtgatat tgctgaagag cttggcggcg
     3841 aatgggctga ccgcttcctc gtgctttacg gtatcgccgc tcccgattcg cagcgcatcg
     3901 ccttctatcg ccttcttgac gagttcttct gagcgggact ctggggttcg aaatgaccga
     3961 ccaagcgacg cccaacctgc catcacgatg gccgcaataa aatatcttta ttttcattac
     4021 atctgtgtgt tggttttttg tgtgaatcga tagcgataag gatcctcttt gcgcttgcgt
     4081 tttcccttgt ccagatagcc cagtagctga cattcatccg gggtcagcac cgtttctgcg
     4141 gactggcttt ctacccggta tcagctcact caaaggcggt aatacggtta tccacagaat
     4201 caggggataa cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta
     4261 aaaaggccgc gttgctggcg tttttccata ggctccgccc ccctgacgag catcacaaaa
     4321 atcgacgctc aagtcagagg tggcgaaacc cgacaggact ataaagatac caggcgtttc
     4381 cccctggaag ctccctcgtg cgctctcctg ttccgaccct gccgcttacc ggatacctgt
     4441 ccgcctttct cccttcggga agcgtggcgc tttctcatag ctcacgctgt aggtatctca
     4501 gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc gttcagcccg
     4561 accgctgcgc cttatccggt aactatcgtc ttgagtccaa cccggtaaga cacgacttat
     4621 cgccactggc agcagccact ggtaacagga ttagcagagc gaggtatgta ggcggtgcta
     4681 cagagttctt gaagtggtgg cctaactacg gctacactag aaggacagta tttggtatct
     4741 gcgctctgct gaagccagtt accttcggaa aaagagttgg tagctcttga tccggcaaac
     4801 aaaccaccgc tggtagcggt ggtttttttg tttgcaagca gcagattacg cgcagaaaaa
     4861 aaggatttca agaagatcct ttgatctttt ctacggggtc tgacgctcag tggaacgaaa
     4921 actcacgtta agggattttg gtcatgagat tatcaaaaag gatcttcacc tagatccttt
     4981 tatagtccgg aaatacagga acgcacgctg gatggccctt cgctgggatg gtgaaaccat
     5041 gaaaaatggc agcttcagtg gattaagtgg gggtaatgtg gcctgtaccc tctggttgca
     5101 taggtattca tacggttaaa atttatcagg cgcgattgcg gcagtttttc gggtggtttg
     5161 ttgccatttt tacctgtctg ctgccgtgat cgcgctgaac gcgttttagc ggtgcgtaca
     5221 attaagggat tatggtaaat ccacttactg tctgccctcg tagccatcga gataaaccgc
     5281 agtactccgg ccacgatgcg tccggcgtag aggatcgaga tct
//
