LOCUS       sequence-471848-        6685 bp    DNA     circular SYN 12-JUN-2025
DEFINITION  Express HaloTag(R)-HRAS 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 6685)
  TITLE     Promega Early Access Collection
REFERENCE   2  (bases 1 to 6685)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Jun 12, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"
COMMENT     Sequence Label: NanoBRET BiBRET Vector HaloTag-HRAS WT-BRAF
            RB...
FEATURES             Location/Qualifiers
     source          1..6685
                     /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..3614
                     /codon_start=1
                     /product="human small GTPase proto-oncoprotein"
                     /label=H-Ras
                     /translation="MTEYKLVVVGAGGVGKSALTIQLIQNHFVDEYDPTIEDSYRKQVV
                     IDGETCLLDILDTAGQEEYSAMRDQYMRTGEGFLCVFAINNTKSFEDIHQYREQIKRVK
                     DSDDVPMVLVGNKCDLAARTVESRQAQDLARSYGIPYIETSAKTRQGVEDAFYTLVREI
                     RQHKLRKLNPPDESGPGCMSCKCVLS"
     polyA_signal    3727..3848
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3738..3757)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     protein_bind    complement(3963..3996)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     promoter        4006..4053
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             4085..4879
                     /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(4139..4158)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4749..4768
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     rep_origin      complement(5037..5625)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5117..5136)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     polyA_signal    5751..5799
                     /note="synthetic polyadenylation signal"
     CDS             complement(5823..6221)
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
                     VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
                     KAIVKDSDGQPTAVGIRELLPSGYVWEG"
     regulatory      6218..6227
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     promoter        complement(6252..6609)
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      6266..6401
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     enhancer        6418..6609
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     6566..6586
                     /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 cgccatgacg gaatataagc
     3061 tggtggtggt gggcgccggc ggtgtgggca agagtgcgct gaccatccag ctgatccaga
     3121 accattttgt ggacgaatac gaccccacta tagaggattc ctaccggaag caggtggtca
     3181 ttgatgggga gacgtgcctg ttggacatcc tggataccgc cggccaggag gagtacagcg
     3241 ccatgcggga ccagtacatg cgcaccgggg agggcttcct gtgtgtgttt gccatcaaca
     3301 acaccaagtc ttttgaggac atccaccagt acagggagca gatcaaacgg gtgaaggact
     3361 cggatgacgt gcccatggtg ctggtgggga acaagtgtga cctggctgca cgcactgtgg
     3421 aatctcggca ggctcaggac ctcgcccgaa gctacggcat cccctacatc gagacctcgg
     3481 ccaagacccg gcagggagtg gaggatgcct tctacacgtt ggtgcgtgag atccggcagc
     3541 acaagctgcg gaagctgaac cctcctgatg agagtggccc cggctgcatg agctgcaagt
     3601 gtgtgctctc ctgagtttaa acgaattcgg gctcggtacc cggggatcct ctagagtcga
     3661 cctgcaggca tgcaagctga tttggctgct gccaccgctg agcaaggccg cttcgagcag
     3721 acatgataag atacattgat gagtttggac aaaccacaac tagaatgcag tgaaaaaaat
     3781 gctttatttg tgaaatttgt gatgctattg ctttatttgt aaccattata agctgcaata
     3841 aacaagttaa caacaacaat tgcattcatt ttatgtttca ggttcagggg gagatgtggg
     3901 aggttttttt aagcaagtaa aacctctaca aatgtggtaa aatcttaatt aagaattcta
     3961 atataacttc gtatagcata cattatacga agttatggat ccactgttga caattaatca
     4021 tcggcatagt atatcggcat agtataatac gacaaggtga ggaactaaac ccaggaggca
     4081 gatcatgatt gaacaagatg gattgcacgc aggttctccg gccgcttggg tggagaggct
     4141 attcggctat gactgggcac aacagacaat cggctgctct gatgccgccg tgttccggct
     4201 gtcagcgcag gggcgcccgg ttctttttgt caagaccgac ctgtccggtg ccctgaatga
     4261 actgcaggac gaggcagcgc ggctatcgtg gctggccacg acgggcgttc cttgcgcagc
     4321 tgtgctcgac gttgtcactg aagcgggaag ggactggctg ctattgggcg aagtgccggg
     4381 gcaggatctc ctgtcatctc accttgctcc tgccgagaaa gtatccatca tggctgatgc
     4441 aatgcggcgg ctgcatacgc ttgatccggc tacctgccca ttcgaccacc aagcgaaaca
     4501 tcgcatcgag cgagcacgta ctcggatgga agccggtctt gtcgatcagg atgatctgga
     4561 cgaagagcat caggggctcg cgccagccga actgttcgcc aggctcaagg cgcgcatgcc
     4621 cgacggcgag gatctcgtcg tgacccatgg cgatgcctgc ttgccgaata tcatggtgga
     4681 aaatggccgc ttttctggat tcatcgactg tggccggctg ggtgtggcgg accgctatca
     4741 ggacatagcg ttggctaccc gtgatattgc tgaagagctt ggcggcgaat gggctgaccg
     4801 cttcctcgtg ctttacggta tcgccgctcc cgattcgcag cgcatcgcct tctatcgcct
     4861 tcttgacgag ttcttctgat tcgaaatgac cgaccaagcg acgcccaacc ggtatcagct
     4921 cactcaaagg cggtaatacg gttatccaca gaatcagggg ataacgcagg aaagaacatg
     4981 tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc
     5041 cataggctcc gcccccctga cgagcatcac aaaaatcgac gctcaagtca gaggtggcga
     5101 aacccgacag gactataaag ataccaggcg tttccccctg gaagctccct cgtgcgctct
     5161 cctgttccga ccctgccgct taccggatac ctgtccgcct ttctcccttc gggaagcgtg
     5221 gcgctttctc atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag
     5281 ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct gcgccttatc cggtaactat
     5341 cgtcttgagt ccaacccggt aagacacgac ttatcgccac tggcagcagc cactggtaac
     5401 aggattagca gagcgaggta tgtaggcggt gctacagagt tcttgaagtg gtggcctaac
     5461 tacggctaca ctagaaggac agtatttggt atctgcgctc tgctgaagcc agttaccttc
     5521 ggaaaaagag ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggtggtttt
     5581 tttgtttgca agcagcagat tacgcgcaga aaaaaaggat ttcaagaaga tcctttgatc
     5641 ttttctacgg ggtctgacgc tcagtggaac gaaaactcac gttaagggat tttggtcatg
     5701 agattatcaa aaaggatctt cacctagatc cttttatagt ccggaatcgt cacacaaaaa
     5761 accaacacac agatgtaatg aaaataaaga tattttattg cggccatcgt gatggctaga
     5821 gtttagccct cccacacata accagagggc agcaattcac gaatcccaac tgccgtcggc
     5881 tgtccatcac tgtccttcac tatggctttg atcccaggat gcagatcgag aagcacctgt
     5941 cggcaccgtc cgcaggggct caagatgccc ctgttctcat ttccgatcgc gacgatacaa
     6001 gtcaggttgc cagctgccgc agcagcagca gtgcccagca ccacgagttc tgcacaaggt
     6061 cccccagtaa aatgatatac attgacacca gtgaagatgc ggccgtcgct agagagagct
     6121 gcgctggcga cgctgtagtc ttcagagatg gggatgctgt tgattgtagc cgttgctctt
     6181 tcaatgaggg tggattcttc ttgagacaaa ggcttggcca tggtggcgct agtgtcagaa
     6241 gaatcgagct ttttgcaaaa gcctaggcct ccaaaaaagc ctcctcacta cttctggaat
     6301 agctcagagg ccgaggcggc ctcggcctct gcataaataa aaaaaattag tcagccatgg
     6361 ggcggagaat gggcggaact gggcggagtt aggggcggga tgggcggagt taggggcggg
     6421 actatggttg ctgactaatt gagatgcatg ctttgcatac ttctgcctgc tggggagcct
     6481 ggggactttc cacacctggt tgctgactaa ttgagatgca tgctttgcat acttctgcct
     6541 gctggggagc ctggggactt tccacaccct aactgacaca cattccacag ctggttcttt
     6601 ccgcctcaga aggtacctaa ccaagttcct ctttcagagg ttatttcagg ccatggtgct
     6661 gcgcaagacg cgttaattaa gacgt
//
