LOCUS       sequence-470698-        5746 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Express HaloTag(R)-KRAS(G12D) in Mammalian Cells under a CMV 
            promoter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5746)
  AUTHORS   Promega
  TITLE     Promega Plasmid Collection
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 5746)
  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            "Unpublished"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 Assay Vector, HaloTag-KRAS(G12D)
FEATURES             Location/Qualifiers
     source          1..5746
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     polyA_signal    3..84
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     enhancer        317..620
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        621..824
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     775..795
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     intron          960..1092
                     /label=chimeric intron
                     /note="chimera between introns from human beta-globin and 
                     immunoglobulin heavy chain genes"
     primer_bind     1075..1094
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     primer_bind     1136..1155
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1136..1154
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      1164..1173
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1170..2060
                     /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             2073..2093
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="EDLYFQS"
     CDS             join(2109..2141,2142..2144,2145..2675)
                     /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             2109..2675
                     /codon_start=1
                     /product="human small GTPase proto-oncoprotein"
                     /label=K-Ras
                     /translation="MTEYKLVVVGADGVGKSALTIQLIQNHFVDEYDPTIEDSYRKQVV
                     IDGETCLLDILDTAGQEEYSAMRDQYMRTGEGFLCVFAINNTKSFEDIHHYREQIKRVK
                     DSEDVPMVLVGNKCDLPSRTVDTKQAQDLARSYGIPFIETSAKTRQGVDDAFYTLVREI
                     RKHKEKMSKDGKKKKKKSKTKCVIM"
     polyA_signal    2788..2909
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2799..2818)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     protein_bind    complement(3024..3057)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     promoter        3067..3114
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             3146..3940
                     /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(3200..3219)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3810..3829
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     rep_origin      complement(4098..4686)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4178..4197)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     polyA_signal    4812..4860
                     /note="synthetic polyadenylation signal"
     CDS             complement(4884..5282)
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
                     VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
                     KAIVKDSDGQPTAVGIRELLPSGYVWEG"
     regulatory      5279..5288
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     promoter        complement(5313..5670)
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      5327..5462
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     enhancer        5479..5670
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     5627..5647
                     /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 gattatggcg cgccactagt tattaatagt aatcaattac ggggtcatta gttcatagcc
      301 catatatgga gttccgcgtt acataactta cggtaaatgg cccgcctggc tgaccgccca
      361 acgacccccg cccattgacg tcaataatga cgtatgttcc catagtaacg ccaataggga
      421 ctttccattg acgtcaatgg gtggagtatt tacggtaaac tgcccacttg gcagtacatc
      481 aagtgtatca tatgccaagt ccgcccccta ttgacgtcaa tgacggtaaa tggcccgcct
      541 ggcattatgc ccagtacatg accttacggg actttcctac ttggcagtac atctacgtat
      601 tagtcatcgc tattaccatg gtgatgcggt tttggcagta caccaatggg cgtggatagc
      661 ggtttgactc acggggattt ccaagtctcc accccattga cgtcaatggg agtttgtttt
      721 ggcaccaaaa tcaacgggac tttccaaaat gtcgtaataa ccccgccccg ttgacgcaaa
      781 tgggcggtag gcgtgtacgg tgggaggtct atataagcag agctggttta gtgaaccgtc
      841 agatcactag aagctttatt gcggtagttt atcacagtta aattgctaac gcagtcagtg
      901 cttctgacac aacagtctcg aacttaagct gcagaagttg gtcgtgaggc actgggcagg
      961 taagtatcaa ggttacaaga caggtttaag gagaccaata gaaactgggc ttgtcgagac
     1021 agagaagact cttgcgtttc tgataggcac ctattggtct tactgacatc cactttgcct
     1081 ttctctccac aggtgtccac tcccagttca attacagctc ttaaggctag agtattaata
     1141 cgactcacta tagggctagc aaagccacca tggcagaaat cggtactggc tttccattcg
     1201 acccccatta tgtggaagtc ctgggcgagc gcatgcacta cgtcgatgtt ggtccgcgcg
     1261 atggcacccc tgtgctgttc ctgcacggta acccgacctc ctcctacgtg tggcgcaaca
     1321 tcatcccgca tgttgcaccg acccatcgct gcattgctcc agacctgatc ggtatgggca
     1381 aatccgacaa accagacctg ggttatttct tcgacgacca cgtccgcttc atggatgcct
     1441 tcatcgaagc cctgggtctg gaagaggtcg tcctggtcat tcacgactgg ggctccgctc
     1501 tgggtttcca ctgggccaag cgcaatccag agcgcgtcaa aggtattgca tttatggagt
     1561 tcatccgccc tatcccgacc tgggacgaat ggccagaatt tgcccgcgag accttccagg
     1621 ccttccgcac caccgacgtc ggccgcaagc tgatcatcga tcagaacgtt tttatcgagg
     1681 gtacgctgcc gatgggtgtc gtccgcccgc tgactgaagt cgagatggac cattaccgcg
     1741 agccgttcct gaatcctgtt gaccgcgagc cactgtggcg cttcccaaac gagctgccaa
     1801 tcgccggtga gccagcgaac atcgtcgcgc tggtcgaaga atacatggac tggctgcacc
     1861 agtcccctgt cccgaagctg ctgttctggg gcaccccagg cgttctgatc ccaccggccg
     1921 aagccgctcg cctggccaaa agcctgccta actgcaaggc tgtggacatc ggcccgggtc
     1981 tgaatctgct gcaagaagac aacccggacc tgatcggcag cgagatcgcg cgctggctgt
     2041 cgacgctcga gatttccggc gagccaacca ctgaggatct gtactttcag agcgataacg
     2101 cgatcgccat gactgaatat aaacttgtgg tagttggagc tgacggcgta ggcaagagtg
     2161 ccttgacgat acagctaatt cagaatcatt ttgtggacga atatgatcca acaatagagg
     2221 attcctacag gaagcaagta gtaattgatg gagaaacctg tctcttggat attctcgaca
     2281 cagcaggtca agaggagtac agtgcaatga gggaccagta catgaggact ggggagggct
     2341 ttctttgtgt atttgccata aataatacta aatcatttga agatattcac cattatagag
     2401 aacaaattaa aagagttaag gactctgaag atgtacctat ggtcctagta ggaaataaat
     2461 gtgatttgcc ttctagaaca gtagacacaa aacaggctca ggacttagca agaagttatg
     2521 gaattccttt tattgaaaca tcagcaaaga caagacaggg tgttgatgat gccttctata
     2581 cattagttcg agaaattcga aaacataaag aaaagatgag caaagatggt aaaaagaaga
     2641 aaaagaagtc aaagacaaag tgtgtaatta tgtaagttta aacgaattcg ggctcggtac
     2701 ccggggatcc tctagagtcg acctgcaggc atgcaagctg atttggctgc tgccaccgct
     2761 gagcaaggcc gcttcgagca gacatgataa gatacattga tgagtttgga caaaccacaa
     2821 ctagaatgca gtgaaaaaaa tgctttattt gtgaaatttg tgatgctatt gctttatttg
     2881 taaccattat aagctgcaat aaacaagtta acaacaacaa ttgcattcat tttatgtttc
     2941 aggttcaggg ggagatgtgg gaggtttttt taagcaagta aaacctctac aaatgtggta
     3001 aaatcttaat taagaattct aatataactt cgtatagcat acattatacg aagttatgga
     3061 tccactgttg acaattaatc atcggcatag tatatcggca tagtataata cgacaaggtg
     3121 aggaactaaa cccaggaggc agatcatgat tgaacaagat ggattgcacg caggttctcc
     3181 ggccgcttgg gtggagaggc tattcggcta tgactgggca caacagacaa tcggctgctc
     3241 tgatgccgcc gtgttccggc tgtcagcgca ggggcgcccg gttctttttg tcaagaccga
     3301 cctgtccggt gccctgaatg aactgcagga cgaggcagcg cggctatcgt ggctggccac
     3361 gacgggcgtt ccttgcgcag ctgtgctcga cgttgtcact gaagcgggaa gggactggct
     3421 gctattgggc gaagtgccgg ggcaggatct cctgtcatct caccttgctc ctgccgagaa
     3481 agtatccatc atggctgatg caatgcggcg gctgcatacg cttgatccgg ctacctgccc
     3541 attcgaccac caagcgaaac atcgcatcga gcgagcacgt actcggatgg aagccggtct
     3601 tgtcgatcag gatgatctgg acgaagagca tcaggggctc gcgccagccg aactgttcgc
     3661 caggctcaag gcgcgcatgc ccgacggcga ggatctcgtc gtgacccatg gcgatgcctg
     3721 cttgccgaat atcatggtgg aaaatggccg cttttctgga ttcatcgact gtggccggct
     3781 gggtgtggcg gaccgctatc aggacatagc gttggctacc cgtgatattg ctgaagagct
     3841 tggcggcgaa tgggctgacc gcttcctcgt gctttacggt atcgccgctc ccgattcgca
     3901 gcgcatcgcc ttctatcgcc ttcttgacga gttcttctga ttcgaaatga ccgaccaagc
     3961 gacgcccaac cggtatcagc tcactcaaag gcggtaatac ggttatccac agaatcaggg
     4021 gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag
     4081 gccgcgttgc tggcgttttt ccataggctc cgcccccctg acgagcatca caaaaatcga
     4141 cgctcaagtc agaggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct
     4201 ggaagctccc tcgtgcgctc tcctgttccg accctgccgc ttaccggata cctgtccgcc
     4261 tttctccctt cgggaagcgt ggcgctttct catagctcac gctgtaggta tctcagttcg
     4321 gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac cccccgttca gcccgaccgc
     4381 tgcgccttat ccggtaacta tcgtcttgag tccaacccgg taagacacga cttatcgcca
     4441 ctggcagcag ccactggtaa caggattagc agagcgaggt atgtaggcgg tgctacagag
     4501 ttcttgaagt ggtggcctaa ctacggctac actagaagga cagtatttgg tatctgcgct
     4561 ctgctgaagc cagttacctt cggaaaaaga gttggtagct cttgatccgg caaacaaacc
     4621 accgctggta gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga
     4681 tttcaagaag atcctttgat cttttctacg gggtctgacg ctcagtggaa cgaaaactca
     4741 cgttaaggga ttttggtcat gagattatca aaaaggatct tcacctagat ccttttatag
     4801 tccggaatcg tcacacaaaa aaccaacaca cagatgtaat gaaaataaag atattttatt
     4861 gcggccatcg tgatggctag agtttagccc tcccacacat aaccagaggg cagcaattca
     4921 cgaatcccaa ctgccgtcgg ctgtccatca ctgtccttca ctatggcttt gatcccagga
     4981 tgcagatcga gaagcacctg tcggcaccgt ccgcaggggc tcaagatgcc cctgttctca
     5041 tttccgatcg cgacgataca agtcaggttg ccagctgccg cagcagcagc agtgcccagc
     5101 accacgagtt ctgcacaagg tcccccagta aaatgatata cattgacacc agtgaagatg
     5161 cggccgtcgc tagagagagc tgcgctggcg acgctgtagt cttcagagat ggggatgctg
     5221 ttgattgtag ccgttgctct ttcaatgagg gtggattctt cttgagacaa aggcttggcc
     5281 atggtggcgc tagtgtcaga agaatcgagc tttttgcaaa agcctaggcc tccaaaaaag
     5341 cctcctcact acttctggaa tagctcagag gccgaggcgg cctcggcctc tgcataaata
     5401 aaaaaaatta gtcagccatg gggcggagaa tgggcggaac tgggcggagt taggggcggg
     5461 atgggcggag ttaggggcgg gactatggtt gctgactaat tgagatgcat gctttgcata
     5521 cttctgcctg ctggggagcc tggggacttt ccacacctgg ttgctgacta attgagatgc
     5581 atgctttgca tacttctgcc tgctggggag cctggggact ttccacaccc taactgacac
     5641 acattccaca gctggttctt tccgcctcag aaggtaccta accaagttcc tctttcagag
     5701 gttatttcag gccatggtgc tgcgcaagac gcgttaatta agacgt
//
