LOCUS       sequence-479514-        8345 bp    DNA     circular SYN 23-JUL-2025
DEFINITION  Express NanoLuc-KRAS(G12D)_CRAF(FL)-HaloTag 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 8345)
  AUTHORS   Promega
  TITLE     Promega Plasmid Collection
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 8345)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Jul 23, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: 
            "Unpublished"
COMMENT     Sequence Label: NanoLuc-KRAS(G12D)_CRAF(FL)-HaloTag BiBRET Vector
FEATURES             Location/Qualifiers
     source          1..8345
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     polyA_signal    61..182
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(72..91)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     protein_bind    complement(297..330)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     promoter        340..387
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             419..1213
                     /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(473..492)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     1083..1102
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     rep_origin      complement(1371..1959)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1451..1470)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     polyA_signal    2085..2133
                     /note="synthetic polyadenylation signal"
     CDS             complement(2157..2555)
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
                     VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
                     KAIVKDSDGQPTAVGIRELLPSGYVWEG"
     regulatory      2552..2561
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     promoter        complement(2586..2943)
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      2600..2735
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     enhancer        2752..2943
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     2900..2920
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     polyA_signal    3022..3103
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     CDS             complement(join(3306..3836,3837..3839,3840..3872))
                     /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             complement(3306..3872)
                     /codon_start=1
                     /product="human small GTPase proto-oncoprotein"
                     /label=K-Ras
                     /translation="MTEYKLVVVGADGVGKSALTIQLIQNHFVDEYDPTIEDSYRKQVV
                     IDGETCLLDILDTAGQEEYSAMRDQYMRTGEGFLCVFAINNTKSFEDIHHYREQIKRVK
                     DSEDVPMVLVGNKCDLPSRTVDTKQAQDLARSYGIPFIETSAKTRQGVDDAFYTLVREI
                     RKHKEKMSKDGKKKKKKSKTKCVIM"
     CDS             complement(3894..4406)
                     /codon_start=1
                     /product="NanoLuc(R) luciferase"
                     /label=Nluc
                     /note="human codon-optimized"
                     /translation="MVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQR
                     IVLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGV
                     TPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGW
                     RLCERILA"
     promoter        complement(4445..4463)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(4446..4463)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     enhancer        4583..4886
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        4887..5090
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     5041..5061
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     intron          5226..5358
                     /label=chimeric intron
                     /note="chimera between introns from human beta-globin and 
                     immunoglobulin heavy chain genes"
     primer_bind     5341..5360
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     primer_bind     5402..5421
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        5402..5420
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             7399..7419
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="EDLYFQS"
     CDS             7435..8319
                     /codon_start=1
                     /product="modified bacterial dehalogenase that forms 
                     covalent bonds with chloroalkane derivatives"
                     /label=HaloTag(R)
                     /translation="EIGTGFPFDPHYVEVLGERMHYVDVGPRDGTPVLFLHGNPTSSYV
                     WRNIIPHVAPTHRCIAPDLIGMGKSDKPDLGYFFDDHVRFMDAFIEALGLEEVVLVIHD
                     WGSALGFHWAKRNPERVKGIAFMEFIRPIPTWDEWPEFARETFQAFRTTDVGRKLIIDQ
                     NVFIEGTLPMGVVRPLTEVEMDHYREPFLNPVDREPLWRFPNELPIAGEPANIVALVEE
                     YMDWLHQSPVPKLLFWGTPGVLIPPAEAARLAKSLPNCKAVDIGPGLNLLQEDNPDLIG
                     SEIARWLSTLEISG"
ORIGIN
        1 ggcatgcaag ctgatttggc tgctgccacc gctgagcaag gccgcttcga gcagacatga
       61 taagatacat tgatgagttt ggacaaacca caactagaat gcagtgaaaa aaatgcttta
      121 tttgtgaaat ttgtgatgct attgctttat ttgtaaccat tataagctgc aataaacaag
      181 ttaacaacaa caattgcatt cattttatgt ttcaggttca gggggagatg tgggaggttt
      241 ttttaagcaa gtaaaacctc tacaaatgtg gtaaaatctt aattaagaat tctaatataa
      301 cttcgtatag catacattat acgaagttat ggatccactg ttgacaatta atcatcggca
      361 tagtatatcg gcatagtata atacgacaag gtgaggaact aaacccagga ggcagatcat
      421 gattgaacaa gatggattgc acgcaggttc tccggccgct tgggtggaga ggctattcgg
      481 ctatgactgg gcacaacaga caatcggctg ctctgatgcc gccgtgttcc ggctgtcagc
      541 gcaggggcgc ccggttcttt ttgtcaagac cgacctgtcc ggtgccctga atgaactgca
      601 ggacgaggca gcgcggctat cgtggctggc cacgacgggc gttccttgcg cagctgtgct
      661 cgacgttgtc actgaagcgg gaagggactg gctgctattg ggcgaagtgc cggggcagga
      721 tctcctgtca tctcaccttg ctcctgccga gaaagtatcc atcatggctg atgcaatgcg
      781 gcggctgcat acgcttgatc cggctacctg cccattcgac caccaagcga aacatcgcat
      841 cgagcgagca cgtactcgga tggaagccgg tcttgtcgat caggatgatc tggacgaaga
      901 gcatcagggg ctcgcgccag ccgaactgtt cgccaggctc aaggcgcgca tgcccgacgg
      961 cgaggatctc gtcgtgaccc atggcgatgc ctgcttgccg aatatcatgg tggaaaatgg
     1021 ccgcttttct ggattcatcg actgtggccg gctgggtgtg gcggaccgct atcaggacat
     1081 agcgttggct acccgtgata ttgctgaaga gcttggcggc gaatgggctg accgcttcct
     1141 cgtgctttac ggtatcgccg ctcccgattc gcagcgcatc gccttctatc gccttcttga
     1201 cgagttcttc tgattcgaaa tgaccgacca agcgacgccc aaccggtatc agctcactca
     1261 aaggcggtaa tacggttatc cacagaatca ggggataacg caggaaagaa catgtgagca
     1321 aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg
     1381 ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg
     1441 acaggactat aaagatacca ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt
     1501 ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc cttcgggaag cgtggcgctt
     1561 tctcatagct cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc caagctgggc
     1621 tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt
     1681 gagtccaacc cggtaagaca cgacttatcg ccactggcag cagccactgg taacaggatt
     1741 agcagagcga ggtatgtagg cggtgctaca gagttcttga agtggtggcc taactacggc
     1801 tacactagaa ggacagtatt tggtatctgc gctctgctga agccagttac cttcggaaaa
     1861 agagttggta gctcttgatc cggcaaacaa accaccgctg gtagcggtgg tttttttgtt
     1921 tgcaagcagc agattacgcg cagaaaaaaa ggatttcaag aagatccttt gatcttttct
     1981 acggggtctg acgctcagtg gaacgaaaac tcacgttaag ggattttggt catgagatta
     2041 tcaaaaagga tcttcaccta gatcctttta tagtccggaa tcgtcacaca aaaaaccaac
     2101 acacagatgt aatgaaaata aagatatttt attgcggcca tcgtgatggc tagagtttag
     2161 ccctcccaca cataaccaga gggcagcaat tcacgaatcc caactgccgt cggctgtcca
     2221 tcactgtcct tcactatggc tttgatccca ggatgcagat cgagaagcac ctgtcggcac
     2281 cgtccgcagg ggctcaagat gcccctgttc tcatttccga tcgcgacgat acaagtcagg
     2341 ttgccagctg ccgcagcagc agcagtgccc agcaccacga gttctgcaca aggtccccca
     2401 gtaaaatgat atacattgac accagtgaag atgcggccgt cgctagagag agctgcgctg
     2461 gcgacgctgt agtcttcaga gatggggatg ctgttgattg tagccgttgc tctttcaatg
     2521 agggtggatt cttcttgaga caaaggcttg gccatggtgg cgctagtgtc agaagaatcg
     2581 agctttttgc aaaagcctag gcctccaaaa aagcctcctc actacttctg gaatagctca
     2641 gaggccgagg cggcctcggc ctctgcataa ataaaaaaaa ttagtcagcc atggggcgga
     2701 gaatgggcgg aactgggcgg agttaggggc gggatgggcg gagttagggg cgggactatg
     2761 gttgctgact aattgagatg catgctttgc atacttctgc ctgctgggga gcctggggac
     2821 tttccacacc tggttgctga ctaattgaga tgcatgcttt gcatacttct gcctgctggg
     2881 gagcctgggg actttccaca ccctaactga cacacattcc acagctggtt ctttccgcct
     2941 cagaaggtac ctaaccaagt tcctctttca gaggttattt caggccatgg tgctgcgcaa
     3001 gacgcgttaa ttaagacgtc ggcagtgaaa aaaatgcttt atttgtgaaa tttgtgatgc
     3061 tattgcttta tttgtaacca ttataagctg caataaacaa gttaacaaca acaattgcat
     3121 tcattttatg tttcaggttc agggggaggt gtgggaggtt ttttaaagca agtaaaacct
     3181 ctacaaatgt ggtatggctg attatgatcc gcggccgcaa tacgtcgacg ttatcagctg
     3241 acttcgtacg agagcctagg tgtttctaga ggatccccgg gtaccgagcc cgaattcgtt
     3301 taaacttaca taattacaca ctttgtcttt gacttctttt tcttcttttt accatctttg
     3361 ctcatctttt ctttatgttt tcgaatttct cgaactaatg tatagaaggc atcatcaaca
     3421 ccctgtcttg tctttgctga tgtttcaata aaaggaattc cataacttct tgctaagtcc
     3481 tgagcctgtt ttgtgtctac tgttctagaa ggcaaatcac atttatttcc tactaggacc
     3541 ataggtacat cttcagagtc cttaactctt ttaatttgtt ctctataatg gtgaatatct
     3601 tcaaatgatt tagtattatt tatggcaaat acacaaagaa agccctcccc agtcctcatg
     3661 tactggtccc tcattgcact gtactcctct tgacctgctg tgtcgagaat atccaagaga
     3721 caggtttctc catcaattac tacttgcttc ctgtaggaat cctctattgt tggatcatat
     3781 tcgtccacaa aatgattctg aattagctgt atcgtcaagg cactcttgcc tacgccgtca
     3841 gctccaacta ccacaagttt atattcagtc atggcgatcg cgccgctcga gcccgccaga
     3901 atgcgttcgc acagccgcca gccggtcact ccgttgatgg ttactcggaa cagcagggag
     3961 ccgtcggggt tgatcaggcg ctcgtcgata attttgttgc cgttccacag ggtccctgtt
     4021 acagtgatct ttttgccgtc gaacacggcg atgccttcat acggccgtcc gaaatagtcg
     4081 atcatgttcg gcgtaacccc gtcgattacc agtgtgccat agtgcaggat caccttaaag
     4141 tgatgatcat ccacagggta caccacctta aaaatttttt cgatctggcc catttggtcg
     4201 ccgctcagac cttcatacgg gatgatgaca tggatgtcga tcttcagccc attttcaccg
     4261 ctcaggacaa tcctttggat cggagttacg gacaccccga gattctgaaa caaactggac
     4321 acacctccct gttcaaggac ttggtccagg ttgtagccgg ctgtctgtcg ccagtcccca
     4381 acgaaatctt cgagtgtgaa gaccatggtg agcgctagct gcaaaaagaa caagtagctt
     4441 gtattctata gtgtcaccta aatgggcgat ctgacggttc actaaaccag ctctgcttat
     4501 ataggcctcc ctggcgcgcc actagttatt aatagtaatc aattacgggg tcattagttc
     4561 atagcccata tatggagttc cgcgttacat aacttacggt aaatggcccg cctggctgac
     4621 cgcccaacga cccccgccca ttgacgtcaa taatgacgta tgttcccata gtaacgccaa
     4681 tagggacttt ccattgacgt caatgggtgg agtatttacg gtaaactgcc cacttggcag
     4741 tacatcaagt gtatcatatg ccaagtccgc cccctattga cgtcaatgac ggtaaatggc
     4801 ccgcctggca ttatgcccag tacatgacct tacgggactt tcctacttgg cagtacatct
     4861 acgtattagt catcgctatt accatggtga tgcggttttg gcagtacacc aatgggcgtg
     4921 gatagcggtt tgactcacgg ggatttccaa gtctccaccc cattgacgtc aatgggagtt
     4981 tgttttggca ccaaaatcaa cgggactttc caaaatgtcg taataacccc gccccgttga
     5041 cgcaaatggg cggtaggcgt gtacggtggg aggtctatat aagcagagct ggtttagtga
     5101 accgtcagat cactagaagc tttattgcgg tagtttatca cagttaaatt gctaacgcag
     5161 tcagtgcttc tgacacaaca gtctcgaact taagctgcag aagttggtcg tgaggcactg
     5221 ggcaggtaag tatcaaggtt acaagacagg tttaaggaga ccaatagaaa ctgggcttgt
     5281 cgagacagag aagactcttg cgtttctgat aggcacctat tggtcttact gacatccact
     5341 ttgcctttct ctccacaggt gtccactccc agttcaatta cagctcttaa ggctagagta
     5401 ttaatacgac tcactatagg gctagcgatc gccatggagc acatacaggg agcttggaag
     5461 acgatcagca atggttttgg attcaaagat gccgtgtttg atggctccag ctgcatctct
     5521 cctacaatag ttcagcagtt tggctatcag cgccgggcat cagatgatgg caaactcaca
     5581 gatccttcta agacaagcaa cactatccgt gttttcttgc cgaacaagca aagaacagtg
     5641 gtcaatgtgc gaaatggaat gagcttgcat gactgcctta tgaaagcact caaggtgagg
     5701 ggcctgcaac cagagtgctg tgcagtgttc agacttctcc acgaacacaa aggtaaaaaa
     5761 gcacgcttag attggaatac tgatgctgcg tctttgattg gagaagaact tcaagtagat
     5821 ttcctggatc atgttcccct cacaacacac aactttgctc ggaagacgtt cctgaagctt
     5881 gccttctgtg acatctgtca gaaattcctg ctcaatggat ttcgatgtca gacttgtggc
     5941 tacaaatttc atgagcactg tagcaccaaa gtacctacta tgtgtgtgga ctggagtaac
     6001 atcagacaac tcttattgtt tccaaattcc actattggtg atagtggagt cccagcacta
     6061 ccttctttga ctatgcgtcg tatgcgagag tctgtttcca ggatgcctgt tagttctcag
     6121 cacagatatt ctacacctca cgccttcacc tttaacacct ccagtccctc atctgaaggt
     6181 tccctctccc agaggcagag gtcgacatcc acacctaatg tccacatggt cagcaccacc
     6241 ctgcctgtgg acagcaggat gattgaggat gcaattcgaa gtcacagcga atcagcctca
     6301 ccttcagccc tgtccagtag ccccaacaat ctgagcccaa caggctggtc acagccgaaa
     6361 acccccgtgc cagcacaaag agagcgggca ccagtatctg ggacccagga gaaaaacaaa
     6421 attaggcctc gtggacagag agattcaagc tattattggg aaatagaagc cagtgaagtg
     6481 atgctgtcca ctcggattgg gtcaggctct tttggaactg tttataaggg taaatggcac
     6541 ggagatgttg cagtaaagat cctaaaggtt gtcgacccaa ccccagagca attccaggcc
     6601 ttcaggaatg aggtggctgt tctgcgcaaa acacggcatg tgaacattct gcttttcatg
     6661 gggtacatga caaaggacaa cctggcaatt gtgacccagt ggtgcgaggg cagcagcctc
     6721 tacaaacacc tgcatgtcca ggagaccaag tttcagatgt tccagctaat tgacattgcc
     6781 cggcagacgg ctcagggaat ggactatttg catgcaaaga acatcatcca tagagacatg
     6841 aaatccaaca atatatttct ccatgaaggc ttaacagtga aaattggaga ttttggtttg
     6901 gcaacagtaa agtcacgctg gagtggttct cagcaggttg aacaacctac tggctctgtc
     6961 ctctggatgg ccccagaggt gatccgaatg caggataaca acccattcag tttccagtcg
     7021 gatgtctact cctatggcat cgtattgtat gaactgatga cgggggagct tccttattct
     7081 cacatcaaca accgagatca gatcatcttc atggtgggcc gaggatatgc ctccccagat
     7141 cttagtaagc tatataagaa ctgccccaaa gcaatgaaga ggctggtagc tgactgtgtg
     7201 aagaaagtaa aggaagagag gcctcttttt ccccagatcc tgtcttccat tgagctgctc
     7261 caacactctc taccgaagat caaccggagc gcttccgagc catccttgca tcgggcagcc
     7321 cacactgagg atatcaatgc ttgcacgctg accacgtccc cgaggctgcc tgtcttcgtt
     7381 tctctcgagc caaccactga ggatctgtac tttcagagcg ataacgatgg atccgaaatc
     7441 ggtactggct ttccattcga cccccattat gtggaagtcc tgggcgagcg catgcactac
     7501 gtcgatgttg gtccgcgcga tggcacccct gtgctgttcc tgcacggtaa cccgacctcc
     7561 tcctacgtgt ggcgcaacat catcccgcat gttgcaccga cccatcgctg cattgctcca
     7621 gacctgatcg gtatgggcaa atccgacaaa ccagacctgg gttatttctt cgacgaccac
     7681 gtccgcttca tggatgcctt catcgaagcc ctgggtctgg aagaggtcgt cctggtcatt
     7741 cacgactggg gctccgctct gggtttccac tgggccaagc gcaatccaga gcgcgtcaaa
     7801 ggtattgcat ttatggagtt catccgccct atcccgacct gggacgaatg gccagaattt
     7861 gcccgcgaga ccttccaggc cttccgcacc accgacgtcg gccgcaagct gatcatcgat
     7921 cagaacgttt ttatcgaggg tacgctgccg atgggtgtcg tccgcccgct gactgaagtc
     7981 gagatggacc attaccgcga gccgttcctg aatcctgttg accgcgagcc actgtggcgc
     8041 ttcccaaacg agctgccaat cgccggtgag ccagcgaaca tcgtcgcgct ggtcgaagaa
     8101 tacatggact ggctgcacca gtcccctgtc ccgaagctgc tgttctgggg caccccaggc
     8161 gttctgatcc caccggccga agccgctcgc ctggccaaaa gcctgcctaa ctgcaaggct
     8221 gtggacatcg gcccgggtct gaatctgctg caagaagaca acccggacct gatcggcagc
     8281 gagatcgcgc gctggctgtc tactctggag atttccggtt aatagaattc tagagtcgac
     8341 ctgca
//
