LOCUS       sequence-471851-        6682 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Express HaloTag(R)-KRAS 2B WT-BRAF RBD-NanoLuc(R) in Mammalian Cells
            under a CMV promoter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6682)
  AUTHORS   Promega
  TITLE     Promega Plasmid Collection
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 6682)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 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            "Unpublished"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 2B WT-BRAF...
FEATURES             Location/Qualifiers
     source          1..6682
                     /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"
     promoter        complement(1115..1133)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(1116..1133)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     enhancer        1253..1556
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        1557..1760
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     1711..1731
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     intron          1896..2028
                     /label=chimeric intron
                     /note="chimera between introns from human beta-globin and 
                     immunoglobulin heavy chain genes"
     primer_bind     2011..2030
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     primer_bind     2072..2091
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        2072..2090
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      2100..2109
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2106..2996
                     /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             3009..3029
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="EDLYFQS"
     CDS             3045..3611
                     /codon_start=1
                     /product="human small GTPase proto-oncoprotein"
                     /label=K-Ras
                     /translation="MTEYKLVVVGAGGVGKSALTIQLIQNHFVDEYDPTIEDSYRKQVV
                     IDGETCLLDILDTAGQEEYSAMRDQYMRTGEGFLCVFAINNTKSFEDIHHYREQIKRVK
                     DSEDVPMVLVGNKCDLPSRTVDTKQAQDLARSYGIPFIETSAKTRQGVDDAFYTLVREI
                     RKHKEKMSKDGKKKKKKSKTKCVIM"
     polyA_signal    3724..3845
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3735..3754)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     protein_bind    complement(3960..3993)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     promoter        4003..4050
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             4082..4876
                     /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(4136..4155)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4746..4765
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     rep_origin      complement(5034..5622)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5114..5133)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     polyA_signal    5748..5796
                     /note="synthetic polyadenylation signal"
     CDS             complement(5820..6218)
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
                     VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
                     KAIVKDSDGQPTAVGIRELLPSGYVWEG"
     regulatory      6215..6224
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     promoter        complement(6249..6606)
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      6263..6398
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     enhancer        6415..6606
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     6563..6583
                     /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 aaacaaagtt gtgtgttgta agtggaacat tctccaacac ttccacatgc
      841 aattcttctc cagtaagcca ggaaatatca gtgtcccaac caattggttt cttctctcca
      901 tcctgaattc tgtaaacagc acagcactct gggattagac ctctcatcat cagtgctttc
      961 tttagactgt ctcggactgt aactccacac cttgcaggta ccactgtcct ctgtttgttg
     1021 ggcaggaaga ctctaacgat aggtttttgt ggtgacttgg ggttgctccg tgccatggcg
     1081 atcgctagct gcaaaaagaa caagtagctt gtattctata gtgtcaccta aatgggcgat
     1141 ctgacggttc actaaaccag ctctgcttat ataggcctcc ctggcgcgcc actagttatt
     1201 aatagtaatc aattacgggg tcattagttc atagcccata tatggagttc cgcgttacat
     1261 aacttacggt aaatggcccg cctggctgac cgcccaacga cccccgccca ttgacgtcaa
     1321 taatgacgta tgttcccata gtaacgccaa tagggacttt ccattgacgt caatgggtgg
     1381 agtatttacg gtaaactgcc cacttggcag tacatcaagt gtatcatatg ccaagtccgc
     1441 cccctattga cgtcaatgac ggtaaatggc ccgcctggca ttatgcccag tacatgacct
     1501 tacgggactt tcctacttgg cagtacatct acgtattagt catcgctatt accatggtga
     1561 tgcggttttg gcagtacacc aatgggcgtg gatagcggtt tgactcacgg ggatttccaa
     1621 gtctccaccc cattgacgtc aatgggagtt tgttttggca ccaaaatcaa cgggactttc
     1681 caaaatgtcg taataacccc gccccgttga cgcaaatggg cggtaggcgt gtacggtggg
     1741 aggtctatat aagcagagct ggtttagtga accgtcagat cactagaagc tttattgcgg
     1801 tagtttatca cagttaaatt gctaacgcag tcagtgcttc tgacacaaca gtctcgaact
     1861 taagctgcag aagttggtcg tgaggcactg ggcaggtaag tatcaaggtt acaagacagg
     1921 tttaaggaga ccaatagaaa ctgggcttgt cgagacagag aagactcttg cgtttctgat
     1981 aggcacctat tggtcttact gacatccact ttgcctttct ctccacaggt gtccactccc
     2041 agttcaatta cagctcttaa ggctagagta ttaatacgac tcactatagg gctagcaaag
     2101 ccaccatggc agaaatcggt actggctttc cattcgaccc ccattatgtg gaagtcctgg
     2161 gcgagcgcat gcactacgtc gatgttggtc cgcgcgatgg cacccctgtg ctgttcctgc
     2221 acggtaaccc gacctcctcc tacgtgtggc gcaacatcat cccgcatgtt gcaccgaccc
     2281 atcgctgcat tgctccagac ctgatcggta tgggcaaatc cgacaaacca gacctgggtt
     2341 atttcttcga cgaccacgtc cgcttcatgg atgccttcat cgaagccctg ggtctggaag
     2401 aggtcgtcct ggtcattcac gactggggct ccgctctggg tttccactgg gccaagcgca
     2461 atccagagcg cgtcaaaggt attgcattta tggagttcat ccgccctatc ccgacctggg
     2521 acgaatggcc agaatttgcc cgcgagacct tccaggcctt ccgcaccacc gacgtcggcc
     2581 gcaagctgat catcgatcag aacgttttta tcgagggtac gctgccgatg ggtgtcgtcc
     2641 gcccgctgac tgaagtcgag atggaccatt accgcgagcc gttcctgaat cctgttgacc
     2701 gcgagccact gtggcgcttc ccaaacgagc tgccaatcgc cggtgagcca gcgaacatcg
     2761 tcgcgctggt cgaagaatac atggactggc tgcaccagtc ccctgtcccg aagctgctgt
     2821 tctggggcac cccaggcgtt ctgatcccac cggccgaagc cgctcgcctg gccaaaagcc
     2881 tgcctaactg caaggctgtg gacatcggcc cgggtctgaa tctgctgcaa gaagacaacc
     2941 cggacctgat cggcagcgag atcgcgcgct ggctgtcgac gctcgagatt tccggcgagc
     3001 caaccactga ggatctgtac tttcagagcg ataacgcgat cgccatgact gaatataaac
     3061 ttgtggtagt tggagctggt ggcgtaggca agagtgcctt gacgatacag ctaattcaga
     3121 atcattttgt ggacgaatat gatccaacaa tagaggattc ctacaggaag caagtagtaa
     3181 ttgatggaga aacctgtctc ttggatattc tcgacacagc aggtcaagag gagtacagtg
     3241 caatgaggga ccagtacatg aggactgggg agggctttct ttgtgtattt gccataaata
     3301 atactaaatc atttgaagat attcaccatt atagagaaca aattaaaaga gttaaggact
     3361 ctgaagatgt acctatggtc ctagtaggaa ataaatgtga tttgccttct agaacagtag
     3421 acacaaaaca ggctcaggac ttagcaagaa gttatggaat tccttttatt gaaacatcag
     3481 caaagacaag acagggtgtt gatgatgcct tctatacatt agttcgagaa attcgaaaac
     3541 ataaagaaaa gatgagcaaa gatggtaaaa agaagaaaaa gaagtcaaag acaaagtgtg
     3601 taattatgta agtttaaacg aattcgggct cggtacccgg ggatcctcta gagtcgacct
     3661 gcaggcatgc aagctgattt ggctgctgcc accgctgagc aaggccgctt cgagcagaca
     3721 tgataagata cattgatgag tttggacaaa ccacaactag aatgcagtga aaaaaatgct
     3781 ttatttgtga aatttgtgat gctattgctt tatttgtaac cattataagc tgcaataaac
     3841 aagttaacaa caacaattgc attcatttta tgtttcaggt tcagggggag atgtgggagg
     3901 tttttttaag caagtaaaac ctctacaaat gtggtaaaat cttaattaag aattctaata
     3961 taacttcgta tagcatacat tatacgaagt tatggatcca ctgttgacaa ttaatcatcg
     4021 gcatagtata tcggcatagt ataatacgac aaggtgagga actaaaccca ggaggcagat
     4081 catgattgaa caagatggat tgcacgcagg ttctccggcc gcttgggtgg agaggctatt
     4141 cggctatgac tgggcacaac agacaatcgg ctgctctgat gccgccgtgt tccggctgtc
     4201 agcgcagggg cgcccggttc tttttgtcaa gaccgacctg tccggtgccc tgaatgaact
     4261 gcaggacgag gcagcgcggc tatcgtggct ggccacgacg ggcgttcctt gcgcagctgt
     4321 gctcgacgtt gtcactgaag cgggaaggga ctggctgcta ttgggcgaag tgccggggca
     4381 ggatctcctg tcatctcacc ttgctcctgc cgagaaagta tccatcatgg ctgatgcaat
     4441 gcggcggctg catacgcttg atccggctac ctgcccattc gaccaccaag cgaaacatcg
     4501 catcgagcga gcacgtactc ggatggaagc cggtcttgtc gatcaggatg atctggacga
     4561 agagcatcag gggctcgcgc cagccgaact gttcgccagg ctcaaggcgc gcatgcccga
     4621 cggcgaggat ctcgtcgtga cccatggcga tgcctgcttg ccgaatatca tggtggaaaa
     4681 tggccgcttt tctggattca tcgactgtgg ccggctgggt gtggcggacc gctatcagga
     4741 catagcgttg gctacccgtg atattgctga agagcttggc ggcgaatggg ctgaccgctt
     4801 cctcgtgctt tacggtatcg ccgctcccga ttcgcagcgc atcgccttct atcgccttct
     4861 tgacgagttc ttctgattcg aaatgaccga ccaagcgacg cccaaccggt atcagctcac
     4921 tcaaaggcgg taatacggtt atccacagaa tcaggggata acgcaggaaa gaacatgtga
     4981 gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc gtttttccat
     5041 aggctccgcc cccctgacga gcatcacaaa aatcgacgct caagtcagag gtggcgaaac
     5101 ccgacaggac tataaagata ccaggcgttt ccccctggaa gctccctcgt gcgctctcct
     5161 gttccgaccc tgccgcttac cggatacctg tccgcctttc tcccttcggg aagcgtggcg
     5221 ctttctcata gctcacgctg taggtatctc agttcggtgt aggtcgttcg ctccaagctg
     5281 ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg ccttatccgg taactatcgt
     5341 cttgagtcca acccggtaag acacgactta tcgccactgg cagcagccac tggtaacagg
     5401 attagcagag cgaggtatgt aggcggtgct acagagttct tgaagtggtg gcctaactac
     5461 ggctacacta gaaggacagt atttggtatc tgcgctctgc tgaagccagt taccttcgga
     5521 aaaagagttg gtagctcttg atccggcaaa caaaccaccg ctggtagcgg tggttttttt
     5581 gtttgcaagc agcagattac gcgcagaaaa aaaggatttc aagaagatcc tttgatcttt
     5641 tctacggggt ctgacgctca gtggaacgaa aactcacgtt aagggatttt ggtcatgaga
     5701 ttatcaaaaa ggatcttcac ctagatcctt ttatagtccg gaatcgtcac acaaaaaacc
     5761 aacacacaga tgtaatgaaa ataaagatat tttattgcgg ccatcgtgat ggctagagtt
     5821 tagccctccc acacataacc agagggcagc aattcacgaa tcccaactgc cgtcggctgt
     5881 ccatcactgt ccttcactat ggctttgatc ccaggatgca gatcgagaag cacctgtcgg
     5941 caccgtccgc aggggctcaa gatgcccctg ttctcatttc cgatcgcgac gatacaagtc
     6001 aggttgccag ctgccgcagc agcagcagtg cccagcacca cgagttctgc acaaggtccc
     6061 ccagtaaaat gatatacatt gacaccagtg aagatgcggc cgtcgctaga gagagctgcg
     6121 ctggcgacgc tgtagtcttc agagatgggg atgctgttga ttgtagccgt tgctctttca
     6181 atgagggtgg attcttcttg agacaaaggc ttggccatgg tggcgctagt gtcagaagaa
     6241 tcgagctttt tgcaaaagcc taggcctcca aaaaagcctc ctcactactt ctggaatagc
     6301 tcagaggccg aggcggcctc ggcctctgca taaataaaaa aaattagtca gccatggggc
     6361 ggagaatggg cggaactggg cggagttagg ggcgggatgg gcggagttag gggcgggact
     6421 atggttgctg actaattgag atgcatgctt tgcatacttc tgcctgctgg ggagcctggg
     6481 gactttccac acctggttgc tgactaattg agatgcatgc tttgcatact tctgcctgct
     6541 ggggagcctg gggactttcc acaccctaac tgacacacat tccacagctg gttctttccg
     6601 cctcagaagg tacctaacca agttcctctt tcagaggtta tttcaggcca tggtgctgcg
     6661 caagacgcgt taattaagac gt
//
