LOCUS       sequence-478457-        8698 bp    DNA     circular SYN 07-AUG-2025
DEFINITION  Express HaloTag-KRAS_BRAF-NanoLuc 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 8698)
  AUTHORS   Promega
  TITLE     Promega Plasmid Collection
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 8698)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Aug 7, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: 
            "Unpublished"
COMMENT     Sequence Label: NanoBRET BiBRET Vector HaloTag-KRAS_BRAF-NanoLuc
FEATURES             Location/Qualifiers
     source          1..8698
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     polyA_signal    3..84
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     CDS             complement(261..773)
                     /codon_start=1
                     /product="NanoLuc(R) luciferase"
                     /label=Nluc
                     /note="human codon-optimized"
                     /translation="VFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQRI
                     VLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGVT
                     PNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGWR
                     LCERILA"
     regulatory      3085..3094
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     promoter        complement(3131..3149)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(3132..3149)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     enhancer        3269..3572
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        3573..3776
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     3727..3747
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     intron          3912..4044
                     /label=chimeric intron
                     /note="chimera between introns from human beta-globin and 
                     immunoglobulin heavy chain genes"
     primer_bind     4027..4046
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     primer_bind     4088..4107
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        4088..4106
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      4116..4125
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             4122..5012
                     /codon_start=1
                     /product="modified bacterial dehalogenase that forms 
                     covalent bonds with chloroalkane derivatives"
                     /label=HaloTag(R)
                     /translation="MAEIGTGFPFDPHYVEVLGERMHYVDVGPRDGTPVLFLHGNPTSS
                     YVWRNIIPHVAPTHRCIAPDLIGMGKSDKPDLGYFFDDHVRFMDAFIEALGLEEVVLVI
                     HDWGSALGFHWAKRNPERVKGIAFMEFIRPIPTWDEWPEFARETFQAFRTTDVGRKLII
                     DQNVFIEGTLPMGVVRPLTEVEMDHYREPFLNPVDREPLWRFPNELPIAGEPANIVALV
                     EEYMDWLHQSPVPKLLFWGTPGVLIPPAEAARLAKSLPNCKAVDIGPGLNLLQEDNPDL
                     IGSEIARWLSTLEISG"
     CDS             5025..5045
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="EDLYFQS"
     CDS             5061..5627
                     /codon_start=1
                     /product="human small GTPase proto-oncoprotein"
                     /label=K-Ras
                     /translation="MTEYKLVVVGAGGVGKSALTIQLIQNHFVDEYDPTIEDSYRKQVV
                     IDGETCLLDILDTAGQEEYSAMRDQYMRTGEGFLCVFAINNTKSFEDIHHYREQIKRVK
                     DSEDVPMVLVGNKCDLPSRTVDTKQAQDLARSYGIPFIETSAKTRQGVDDAFYTLVREI
                     RKHKEKMSKDGKKKKKKSKTKCVIM"
     polyA_signal    5740..5861
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5751..5770)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     protein_bind    complement(5976..6009)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     promoter        6019..6066
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             6098..6892
                     /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(6152..6171)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     6762..6781
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     rep_origin      complement(7050..7638)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(7130..7149)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     polyA_signal    7764..7812
                     /note="synthetic polyadenylation signal"
     CDS             complement(7836..8234)
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
                     VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
                     KAIVKDSDGQPTAVGIRELLPSGYVWEG"
     regulatory      8231..8240
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     promoter        complement(8265..8622)
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      8279..8414
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     enhancer        8431..8622
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     8579..8599
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
ORIGIN
        1 cggcagtgaa aaaaatgctt tatttgtgaa atttgtgatg ctattgcttt atttgtaacc
       61 attataagct gcaataaaca agttaacaac aacaattgca ttcattttat gtttcaggtt
      121 cagggggagg tgtgggaggt tttttaaagc aagtaaaacc tctacaaatg tggtatggct
      181 gattatgatc cgcggccgca atacgtcgac gttatcagct gacttcgtac gagagcctag
      241 gtgtttctag agtcgcggcc ttacgccaga atgcgttcgc acagccgcca gccggtcact
      301 ccgttgatgg ttactcggaa cagcagggag ccgtcggggt tgatcaggcg ctcgtcgata
      361 attttgttgc cgttccacag ggtccctgtt acagtgatct ttttgccgtc gaacacggcg
      421 atgccttcat acggccgtcc gaaatagtcg atcatgttcg gcgtaacccc gtcgattacc
      481 agtgtgccat agtgcaggat caccttaaag tgatgatcat ccacagggta caccacctta
      541 aaaatttttt cgatctggcc catttggtcg ccgctcagac cttcatacgg gatgatgaca
      601 tggatgtcga tcttcagccc attttcaccg ctcaggacaa tcctttggat cggagttacg
      661 gacaccccga gattctgaaa caaactggac acacctccct gttcaaggac ttggtccagg
      721 ttgtagccgg ctgtctgtcg ccagtcccca acgaaatctt cgagtgtgaa gacgccgctc
      781 gagccgagag aaacgtggac aggaaacgca ccatatcccc ctgcctggat gggtgttttt
      841 ggagaagcac aagcatatag actaaaatcc tctgtttgga aaccagcccg attcaaggag
      901 ggttctgatg cactgcggtg aatttttggc aatgagcggg ccagcagctc aatagaggcg
      961 agaatttggg gaaagagtgg tctctcatct cttttctttt tgaggcactc tgccattaat
     1021 ctcttcatgg cttttggaca gttactccgt accttactga gatctggaga caggtatcct
     1081 cgtcccacca taaaaattat ctggtccctg ttgttgatgt ttgaataagg taactgtcca
     1141 gtcatcaatt catacagaac aattccaaat gcatatacat ctgactgaaa gctgtatgga
     1201 tttttatctt gcattctgat gacttctggt gccatccaca aaatggatcc agacaactgt
     1261 tcaaactgat gggacccact ccatcgagat ttcactgtag ctagaccaaa atcacctatt
     1321 tttactgtga ggtcttcatg aagaaatata ttattactct tgaggtctct gtggatgatt
     1381 gacttggcgt gtaagtaatc catgccctgt gcagtctgtc gtgcaatatc tataagtttg
     1441 atcatctcaa atttggtctc aatgatatgg agatggtgat acaagctgga gccctcacac
     1501 cactgggtaa caatagccag ttgtggcttt gtggaatagc ccatgaagag taggatattc
     1561 acatgtcgtg ttttcctgag tactcctact tcatttttga aggcttgtaa ctgctgaggt
     1621 gtaggtgctg tcacattcaa cattttcact gccacatcac catgccactt tcccttgtag
     1681 actgttccaa atgatccaga tccaattctt tgtcccactg taatctgccc atcaggaatc
     1741 tcccaatcat cactcgagtc ccgtctacca agtgttttca ttcgattcct gtcttctgag
     1801 gatgaagatg acttcctttc tcgctgaggt cctggagatt tctgtaaggc tttcacgtta
     1861 gttagtgagc caggtaatga ggcagggggg gtagcagaca aacctgtggt tgatcctcca
     1921 tcaccacgaa atccttggtc tctaatcaag tcatcaatat tgacaggttc tattgtgttt
     1981 atatgcacat tgggagctga tgaggatcgg tctcgttgcc caaattgatt tcgatgatct
     2041 tcatctgctg gtcggaaggg ctgtggaatt ggaatggatt ttgaaggaga cggactggtg
     2101 agaatttggg gcccaataga gtccgaggcg ggtgcggaag gggatgatcc agatgttagg
     2161 gcagtctctg ctaaggacgc ctcttcctgt ggtattgggt ggtgttcaaa gaacttggag
     2221 acaaacagca aatcaagttg gtcataatta acacacatca gtggaacttc tgtactacaa
     2281 cgctggtgaa atttataacc acatgtttga cagcggaaac cctggaaaag cagctttcga
     2341 caaaagtcac aaaatgctaa ggtgaaaaac gtttttcgta caaagttgtg tgttgtaagt
     2401 ggaacattct ccaacacttc cacatgcaat tcttctccag taagccagga aatatcagtg
     2461 tcccaaccaa ttggtttctt ctctccatcc tgaattctgt aaacagcaca gcactctggg
     2521 attagacctc tcatcatcag tgctttcttt agactgtctc ggactgtaac tccacacctt
     2581 gcaggtacca ctgtcctctg tttgttgggc aggaagactc taacgatagg tttttgtggt
     2641 gacttggggt tgctccgtgc cacatctgtg ggattttgaa aaactgaaag agatgaaggt
     2701 agcactgaaa ggctagaaga ggaagaagat gtaacggtat ccattgatgc agagctagaa
     2761 acagaaaaat cagttccgtt ccccagagat tccaataact gttgttctct ttgttggagt
     2821 gcatctagct tgctggtgta ttcttcatag gcctccagat atattgatgg tggattatgc
     2881 tccccaccaa atttgtccaa tagggcctct atatgttcct gtgtcaactt aatcatttgt
     2941 ttgatattcc acacctcctc cggaatggca gggtccgcag ccgaagaggc cgcggcgccg
     3001 gcgccggcgc cggcctcggg ctccatgtcc ccgttgaaca gagcctggcc cggctccgcg
     3061 ccgccaccac cgccaccgct cagcgccgcc atggcgatcg ctagctgcaa aaagaacaag
     3121 tagcttgtat tctatagtgt cacctaaatg ggcgatctga cggttcacta aaccagctct
     3181 gcttatatag gcctccctgg cgcgccacta gttattaata gtaatcaatt acggggtcat
     3241 tagttcatag cccatatatg gagttccgcg ttacataact tacggtaaat ggcccgcctg
     3301 gctgaccgcc caacgacccc cgcccattga cgtcaataat gacgtatgtt cccatagtaa
     3361 cgccaatagg gactttccat tgacgtcaat gggtggagta tttacggtaa actgcccact
     3421 tggcagtaca tcaagtgtat catatgccaa gtccgccccc tattgacgtc aatgacggta
     3481 aatggcccgc ctggcattat gcccagtaca tgaccttacg ggactttcct acttggcagt
     3541 acatctacgt attagtcatc gctattacca tggtgatgcg gttttggcag tacaccaatg
     3601 ggcgtggata gcggtttgac tcacggggat ttccaagtct ccaccccatt gacgtcaatg
     3661 ggagtttgtt ttggcaccaa aatcaacggg actttccaaa atgtcgtaat aaccccgccc
     3721 cgttgacgca aatgggcggt aggcgtgtac ggtgggaggt ctatataagc agagctggtt
     3781 tagtgaaccg tcagatcact agaagcttta ttgcggtagt ttatcacagt taaattgcta
     3841 acgcagtcag tgcttctgac acaacagtct cgaacttaag ctgcagaagt tggtcgtgag
     3901 gcactgggca ggtaagtatc aaggttacaa gacaggttta aggagaccaa tagaaactgg
     3961 gcttgtcgag acagagaaga ctcttgcgtt tctgataggc acctattggt cttactgaca
     4021 tccactttgc ctttctctcc acaggtgtcc actcccagtt caattacagc tcttaaggct
     4081 agagtattaa tacgactcac tatagggcta gcaaagccac catggcagaa atcggtactg
     4141 gctttccatt cgacccccat tatgtggaag tcctgggcga gcgcatgcac tacgtcgatg
     4201 ttggtccgcg cgatggcacc cctgtgctgt tcctgcacgg taacccgacc tcctcctacg
     4261 tgtggcgcaa catcatcccg catgttgcac cgacccatcg ctgcattgct ccagacctga
     4321 tcggtatggg caaatccgac aaaccagacc tgggttattt cttcgacgac cacgtccgct
     4381 tcatggatgc cttcatcgaa gccctgggtc tggaagaggt cgtcctggtc attcacgact
     4441 ggggctccgc tctgggtttc cactgggcca agcgcaatcc agagcgcgtc aaaggtattg
     4501 catttatgga gttcatccgc cctatcccga cctgggacga atggccagaa tttgcccgcg
     4561 agaccttcca ggccttccgc accaccgacg tcggccgcaa gctgatcatc gatcagaacg
     4621 tttttatcga gggtacgctg ccgatgggtg tcgtccgccc gctgactgaa gtcgagatgg
     4681 accattaccg cgagccgttc ctgaatcctg ttgaccgcga gccactgtgg cgcttcccaa
     4741 acgagctgcc aatcgccggt gagccagcga acatcgtcgc gctggtcgaa gaatacatgg
     4801 actggctgca ccagtcccct gtcccgaagc tgctgttctg gggcacccca ggcgttctga
     4861 tcccaccggc cgaagccgct cgcctggcca aaagcctgcc taactgcaag gctgtggaca
     4921 tcggcccggg tctgaatctg ctgcaagaag acaacccgga cctgatcggc agcgagatcg
     4981 cgcgctggct gtcgacgctc gagatttccg gcgagccaac cactgaggat ctgtactttc
     5041 agagcgataa cgcgatcgcc atgactgaat ataaacttgt ggtagttgga gctggtggcg
     5101 taggcaagag tgccttgacg atacagctaa ttcagaatca ttttgtggac gaatatgatc
     5161 caacaataga ggattcctac aggaagcaag tagtaattga tggagaaacc tgtctcttgg
     5221 atattctcga cacagcaggt caagaggagt acagtgcaat gagggaccag tacatgagga
     5281 ctggggaggg ctttctttgt gtatttgcca taaataatac taaatcattt gaagatattc
     5341 accattatag agaacaaatt aaaagagtta aggactctga agatgtacct atggtcctag
     5401 taggaaataa atgtgatttg ccttctagaa cagtagacac aaaacaggct caggacttag
     5461 caagaagtta tggaattcct tttattgaaa catcagcaaa gacaagacag ggtgttgatg
     5521 atgccttcta tacattagtt cgagaaattc gaaaacataa agaaaagatg agcaaagatg
     5581 gtaaaaagaa gaaaaagaag tcaaagacaa agtgtgtaat tatgtaagtt taaacgaatt
     5641 cgggctcggt acccggggat cctctagagt cgacctgcag gcatgcaagc tgatttggct
     5701 gctgccaccg ctgagcaagg ccgcttcgag cagacatgat aagatacatt gatgagtttg
     5761 gacaaaccac aactagaatg cagtgaaaaa aatgctttat ttgtgaaatt tgtgatgcta
     5821 ttgctttatt tgtaaccatt ataagctgca ataaacaagt taacaacaac aattgcattc
     5881 attttatgtt tcaggttcag ggggagatgt gggaggtttt tttaagcaag taaaacctct
     5941 acaaatgtgg taaaatctta attaagaatt ctaatataac ttcgtatagc atacattata
     6001 cgaagttatg gatccactgt tgacaattaa tcatcggcat agtatatcgg catagtataa
     6061 tacgacaagg tgaggaacta aacccaggag gcagatcatg attgaacaag atggattgca
     6121 cgcaggttct ccggccgctt gggtggagag gctattcggc tatgactggg cacaacagac
     6181 aatcggctgc tctgatgccg ccgtgttccg gctgtcagcg caggggcgcc cggttctttt
     6241 tgtcaagacc gacctgtccg gtgccctgaa tgaactgcag gacgaggcag cgcggctatc
     6301 gtggctggcc acgacgggcg ttccttgcgc agctgtgctc gacgttgtca ctgaagcggg
     6361 aagggactgg ctgctattgg gcgaagtgcc ggggcaggat ctcctgtcat ctcaccttgc
     6421 tcctgccgag aaagtatcca tcatggctga tgcaatgcgg cggctgcata cgcttgatcc
     6481 ggctacctgc ccattcgacc accaagcgaa acatcgcatc gagcgagcac gtactcggat
     6541 ggaagccggt cttgtcgatc aggatgatct ggacgaagag catcaggggc tcgcgccagc
     6601 cgaactgttc gccaggctca aggcgcgcat gcccgacggc gaggatctcg tcgtgaccca
     6661 tggcgatgcc tgcttgccga atatcatggt ggaaaatggc cgcttttctg gattcatcga
     6721 ctgtggccgg ctgggtgtgg cggaccgcta tcaggacata gcgttggcta cccgtgatat
     6781 tgctgaagag cttggcggcg aatgggctga ccgcttcctc gtgctttacg gtatcgccgc
     6841 tcccgattcg cagcgcatcg ccttctatcg ccttcttgac gagttcttct gattcgaaat
     6901 gaccgaccaa gcgacgccca accggtatca gctcactcaa aggcggtaat acggttatcc
     6961 acagaatcag gggataacgc aggaaagaac atgtgagcaa aaggccagca aaaggccagg
     7021 aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc tccgcccccc tgacgagcat
     7081 cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag
     7141 gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga
     7201 tacctgtccg cctttctccc ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg
     7261 tatctcagtt cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt
     7321 cagcccgacc gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac
     7381 gacttatcgc cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc
     7441 ggtgctacag agttcttgaa gtggtggcct aactacggct acactagaag gacagtattt
     7501 ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa gagttggtag ctcttgatcc
     7561 ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt gcaagcagca gattacgcgc
     7621 agaaaaaaag gatttcaaga agatcctttg atcttttcta cggggtctga cgctcagtgg
     7681 aacgaaaact cacgttaagg gattttggtc atgagattat caaaaaggat cttcacctag
     7741 atccttttat agtccggaat cgtcacacaa aaaaccaaca cacagatgta atgaaaataa
     7801 agatatttta ttgcggccat cgtgatggct agagtttagc cctcccacac ataaccagag
     7861 ggcagcaatt cacgaatccc aactgccgtc ggctgtccat cactgtcctt cactatggct
     7921 ttgatcccag gatgcagatc gagaagcacc tgtcggcacc gtccgcaggg gctcaagatg
     7981 cccctgttct catttccgat cgcgacgata caagtcaggt tgccagctgc cgcagcagca
     8041 gcagtgccca gcaccacgag ttctgcacaa ggtcccccag taaaatgata tacattgaca
     8101 ccagtgaaga tgcggccgtc gctagagaga gctgcgctgg cgacgctgta gtcttcagag
     8161 atggggatgc tgttgattgt agccgttgct ctttcaatga gggtggattc ttcttgagac
     8221 aaaggcttgg ccatggtggc gctagtgtca gaagaatcga gctttttgca aaagcctagg
     8281 cctccaaaaa agcctcctca ctacttctgg aatagctcag aggccgaggc ggcctcggcc
     8341 tctgcataaa taaaaaaaat tagtcagcca tggggcggag aatgggcgga actgggcgga
     8401 gttaggggcg ggatgggcgg agttaggggc gggactatgg ttgctgacta attgagatgc
     8461 atgctttgca tacttctgcc tgctggggag cctggggact ttccacacct ggttgctgac
     8521 taattgagat gcatgctttg catacttctg cctgctgggg agcctgggga ctttccacac
     8581 cctaactgac acacattcca cagctggttc tttccgcctc agaaggtacc taaccaagtt
     8641 cctctttcag aggttatttc aggccatggt gctgcgcaag acgcgttaat taagacgt
//
