LOCUS       sequence-470713-        5336 bp    DNA     circular SYN 01-MAY-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)
  TITLE     Promega Early Access Collection
REFERENCE   2  (bases 1 to 5336)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 1, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"
COMMENT     Sequence Label: NanoBRET Assay Vector, NanoLuc-KRAS(G12V)
FEATURES             Location/Qualifiers
     source          1..5336
                     /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="MTEYKLVVVGAVGVGKSALTIQLIQNHFVDEYDPTIEDSYRKQVV
                     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"
     promoter        3072..3119
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             3151..3945
                     /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(3205..3224)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3815..3834
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4009..4057
                     /note="synthetic polyadenylation signal"
     rep_origin      complement(4296..4884)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4376..4395)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     misc_feature    5000..5283
                     /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 tgttggcgta 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 catagtatat cggcatagta taatacgaca
     3121 aggtgaggaa ctaaacccag gaggcagatc atgattgaac aagatggatt gcacgcaggt
     3181 tctccggccg cttgggtgga gaggctattc ggctatgact gggcacaaca gacaatcggc
     3241 tgctctgatg ccgccgtgtt ccggctgtca gcgcaggggc gcccggttct ttttgtcaag
     3301 accgacctgt ccggtgccct gaatgaactg caggacgagg cagcgcggct atcgtggctg
     3361 gccacgacgg gcgttccttg cgcagctgtg ctcgacgttg tcactgaagc gggaagggac
     3421 tggctgctat tgggcgaagt gccggggcag gatctcctgt catctcacct tgctcctgcc
     3481 gagaaagtat ccatcatggc tgatgcaatg cggcggctgc atacgcttga tccggctacc
     3541 tgcccattcg accaccaagc gaaacatcgc atcgagcgag cacgtactcg gatggaagcc
     3601 ggtcttgtcg atcaggatga tctggacgaa gagcatcagg ggctcgcgcc agccgaactg
     3661 ttcgccaggc tcaaggcgcg catgcccgac ggcgaggatc tcgtcgtgac ccatggcgat
     3721 gcctgcttgc cgaatatcat ggtggaaaat ggccgctttt ctggattcat cgactgtggc
     3781 cggctgggtg tggcggaccg ctatcaggac atagcgttgg ctacccgtga tattgctgaa
     3841 gagcttggcg gcgaatgggc tgaccgcttc ctcgtgcttt acggtatcgc cgctcccgat
     3901 tcgcagcgca tcgccttcta tcgccttctt gacgagttct tctgagcggg actctggggt
     3961 tcgaaatgac cgaccaagcg acgcccaacc tgccatcacg atggccgcaa taaaatatct
     4021 ttattttcat tacatctgtg tgttggtttt ttgtgtgaat cgatagcgat aaggatcctc
     4081 tttgcgcttg cgttttccct tgtccagata gcccagtagc tgacattcat ccggggtcag
     4141 caccgtttct gcggactggc tttctacccg gtatcagctc actcaaaggc ggtaatacgg
     4201 ttatccacag aatcagggga taacgcagga aagaacatgt gagcaaaagg ccagcaaaag
     4261 gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg cccccctgac
     4321 gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg actataaaga
     4381 taccaggcgt ttccccctgg aagctccctc gtgcgctctc ctgttccgac cctgccgctt
     4441 accggatacc tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca tagctcacgc
     4501 tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc
     4561 cccgttcagc ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc caacccggta
     4621 agacacgact tatcgccact ggcagcagcc actggtaaca ggattagcag agcgaggtat
     4681 gtaggcggtg ctacagagtt cttgaagtgg tggcctaact acggctacac tagaaggaca
     4741 gtatttggta tctgcgctct gctgaagcca gttaccttcg gaaaaagagt tggtagctct
     4801 tgatccggca aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt
     4861 acgcgcagaa aaaaaggatt tcaagaagat cctttgatct tttctacggg gtctgacgct
     4921 cagtggaacg aaaactcacg ttaagggatt ttggtcatga gattatcaaa aaggatcttc
     4981 acctagatcc ttttatagtc cggaaataca ggaacgcacg ctggatggcc cttcgctggg
     5041 atggtgaaac catgaaaaat ggcagcttca gtggattaag tgggggtaat gtggcctgta
     5101 ccctctggtt gcataggtat tcatacggtt aaaatttatc aggcgcgatt gcggcagttt
     5161 ttcgggtggt ttgttgccat ttttacctgt ctgctgccgt gatcgcgctg aacgcgtttt
     5221 agcggtgcgt acaattaagg gattatggta aatccactta ctgtctgccc tcgtagccat
     5281 cgagataaac cgcagtactc cggccacgat gcgtccggcg tagaggatcg agatct
//
