LOCUS       sequence-474106-        6656 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Express NanoLuc(R)-HRAS WT-CRAF RBD-HaloTag(R) in Mammalian Cells 
            under a CMV promoter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6656)
  AUTHORS   Promega
  TITLE     Promega Plasmid Collection
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 6656)
  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            "Unpublished"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, NanoLuc-HRAS WT-CRAF
            R...
FEATURES             Location/Qualifiers
     source          1..6656
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     490..509
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(747..1541)
                     /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(858..877)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     1468..1487
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     promoter        complement(1573..1620)
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     protein_bind    1630..1663
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     polyA_signal    1778..1899
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     1869..1888
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     CDS             complement(1986..2870)
                     /codon_start=1
                     /product="modified bacterial dehalogenase that forms 
                     covalent bonds with chloroalkane derivatives"
                     /label=HaloTag(R)
                     /translation="EIGTGFPFDPHYVEVLGERMHYVDVGPRDGTPVLFLHGNPTSSYV
                     WRNIIPHVAPTHRCIAPDLIGMGKSDKPDLGYFFDDHVRFMDAFIEALGLEEVVLVIHD
                     WGSALGFHWAKRNPERVKGIAFMEFIRPIPTWDEWPEFARETFQAFRTTDVGRKLIIDQ
                     NVFIEGTLPMGVVRPLTEVEMDHYREPFLNPVDREPLWRFPNELPIAGEPANIVALVEE
                     YMDWLHQSPVPKLLFWGTPGVLIPPAEAARLAKSLPNCKAVDIGPGLNLLQEDNPDLIG
                     SEIARWLSTLEISG"
     CDS             complement(2886..2906)
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="EDLYFQS"
     primer_bind     complement(3192..3211)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(3193..3211)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(3253..3272)
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     intron          3255..3387
                     /label=chimeric intron
                     /note="chimera between introns from human beta-globin and 
                     immunoglobulin heavy chain genes"
     promoter        complement(3523..3726)
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     complement(3552..3572)
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     enhancer        3727..4030
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        4150..4168
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     4150..4167
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     CDS             4207..4719
                     /codon_start=1
                     /product="NanoLuc(R) luciferase"
                     /label=Nluc
                     /note="human codon-optimized"
                     /translation="MVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQR
                     IVLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGV
                     TPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGW
                     RLCERILA"
     CDS             4741..5310
                     /codon_start=1
                     /product="human small GTPase proto-oncoprotein"
                     /label=H-Ras
                     /translation="MTEYKLVVVGAGGVGKSALTIQLIQNHFVDEYDPTIEDSYRKQVV
                     IDGETCLLDILDTAGQEEYSAMRDQYMRTGEGFLCVFAINNTKSFEDIHQYREQIKRVK
                     DSDDVPMVLVGNKCDLAARTVESRQAQDLARSYGIPYIETSAKTRQGVEDAFYTLVREI
                     RQHKLRKLNPPDESGPGCMSCKCVLS"
     polyA_signal    5513..5594
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     promoter        5673..6030
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        5673..5864
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(5696..5716)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      5881..6016
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     regulatory      6055..6064
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             6061..6459
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
                     VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
                     KAIVKDSDGQPTAVGIRELLPSGYVWEG"
     polyA_signal    6483..6531
                     /note="synthetic polyadenylation signal"
ORIGIN
        1 ttgaaatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc
       61 agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt
      121 cagcagagcg cagataccaa atactgtcct tctagtgtag ccgtagttag gccaccactt
      181 caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc
      241 tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa
      301 ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac
      361 ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg
      421 gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga
      481 gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact
      541 tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa
      601 cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt tctttcctgc
      661 gttatcccct gattctgtgg ataaccgtat taccgccttt gagtgagctg ataccggttg
      721 ggcgtcgctt ggtcggtcat ttcgaatcag aagaactcgt caagaaggcg atagaaggcg
      781 atgcgctgcg aatcgggagc ggcgataccg taaagcacga ggaagcggtc agcccattcg
      841 ccgccaagct cttcagcaat atcacgggta gccaacgcta tgtcctgata gcggtccgcc
      901 acacccagcc ggccacagtc gatgaatcca gaaaagcggc cattttccac catgatattc
      961 ggcaagcagg catcgccatg ggtcacgacg agatcctcgc cgtcgggcat gcgcgccttg
     1021 agcctggcga acagttcggc tggcgcgagc ccctgatgct cttcgtccag atcatcctga
     1081 tcgacaagac cggcttccat ccgagtacgt gctcgctcga tgcgatgttt cgcttggtgg
     1141 tcgaatgggc aggtagccgg atcaagcgta tgcagccgcc gcattgcatc agccatgatg
     1201 gatactttct cggcaggagc aaggtgagat gacaggagat cctgccccgg cacttcgccc
     1261 aatagcagcc agtcccttcc cgcttcagtg acaacgtcga gcacagctgc gcaaggaacg
     1321 cccgtcgtgg ccagccacga tagccgcgct gcctcgtcct gcagttcatt cagggcaccg
     1381 gacaggtcgg tcttgacaaa aagaaccggg cgcccctgcg ctgacagccg gaacacggcg
     1441 gcatcagagc agccgattgt ctgttgtgcc cagtcatagc cgaatagcct ctccacccaa
     1501 gcggccggag aacctgcgtg caatccatct tgttcaatca tgatctgcct cctgggttta
     1561 gttcctcacc ttgtcgtatt atactatgcc gatatactat gccgatgatt aattgtcaac
     1621 agtggatcca taacttcgta taatgtatgc tatacgaagt tatattagaa ttcttaatta
     1681 agattttacc acatttgtag aggttttact tgcttaaaaa aacctcccac atctccccct
     1741 gaacctgaaa cataaaatga atgcaattgt tgttgttaac ttgtttattg cagcttataa
     1801 tggttacaaa taaagcaata gcatcacaaa tttcacaaat aaagcatttt tttcactgca
     1861 ttctagttgt ggtttgtcca aactcatcaa tgtatcttat catgtctgct cgaagcggcc
     1921 ttgctcagcg gtggcagcag ccaaatcagc ttgcatgcct gcaggtcgac tctagaattc
     1981 tattaaccgg aaatctccag agtagacagc cagcgcgcga tctcgctgcc gatcaggtcc
     2041 gggttgtctt cttgcagcag attcagaccc gggccgatgt ccacagcctt gcagttaggc
     2101 aggcttttgg ccaggcgagc ggcttcggcc ggtgggatca gaacgcctgg ggtgccccag
     2161 aacagcagct tcgggacagg ggactggtgc agccagtcca tgtattcttc gaccagcgcg
     2221 acgatgttcg ctggctcacc ggcgattggc agctcgtttg ggaagcgcca cagtggctcg
     2281 cggtcaacag gattcaggaa cggctcgcgg taatggtcca tctcgacttc agtcagcggg
     2341 cggacgacac ccatcggcag cgtaccctcg ataaaaacgt tctgatcgat gatcagcttg
     2401 cggccgacgt cggtggtgcg gaaggcctgg aaggtctcgc gggcaaattc tggccattcg
     2461 tcccaggtcg ggatagggcg gatgaactcc ataaatgcaa tacctttgac gcgctctgga
     2521 ttgcgcttgg cccagtggaa acccagagcg gagccccagt cgtgaatgac caggacgacc
     2581 tcttccagac ccagggcttc gatgaaggca tccatgaagc ggacgtggtc gtcgaagaaa
     2641 taacccaggt ctggtttgtc ggatttgccc ataccgatca ggtctggagc aatgcagcga
     2701 tgggtcggtg caacatgcgg gatgatgttg cgccacacgt aggaggaggt cgggttaccg
     2761 tgcaggaaca gcacaggggt gccatcgcgc ggaccaacat cgacgtagtg catgcgctcg
     2821 cccaggactt ccacataatg ggggtcgaat ggaaagccag taccgatttc ggatccatcg
     2881 ttatcgctct gaaagtacag atcctcagtg gttggctcga gagaaacatg atccaggaaa
     2941 tctacttgaa gttcttctcc aatcaaagac gcagcatcag tattccaatc taagcgtgct
     3001 tttttacctt tgtgttcgtg gagaagtctg aacactgcac agcactctgg ttgcaggccc
     3061 ctcaccttga gtgctttcat aaggcagtca tgcaagctca ttccatttcg cacattgacc
     3121 actgttcttt gcttgttcgg caagaaaaca cggatagtgt tgcttgtctt agaaggcatg
     3181 gcgatcgcta gccctatagt gagtcgtatt aatactctag ccttaagagc tgtaattgaa
     3241 ctgggagtgg acacctgtgg agagaaaggc aaagtggatg tcagtaagac caataggtgc
     3301 ctatcagaaa cgcaagagtc ttctctgtct cgacaagccc agtttctatt ggtctcctta
     3361 aacctgtctt gtaaccttga tacttacctg cccagtgcct cacgaccaac ttctgcagct
     3421 taagttcgag actgttgtgt cagaagcact gactgcgtta gcaatttaac tgtgataaac
     3481 taccgcaata aagcttctag tgatctgacg gttcactaaa ccagctctgc ttatatagac
     3541 ctcccaccgt acacgcctac cgcccatttg cgtcaacggg gcggggttat tacgacattt
     3601 tggaaagtcc cgttgatttt ggtgccaaaa caaactccca ttgacgtcaa tggggtggag
     3661 acttggaaat ccccgtgagt caaaccgcta tccacgccca ttggtgtact gccaaaaccg
     3721 catcaccatg gtaatagcga tgactaatac gtagatgtac tgccaagtag gaaagtcccg
     3781 taaggtcatg tactgggcat aatgccaggc gggccattta ccgtcattga cgtcaatagg
     3841 gggcggactt ggcatatgat acacttgatg tactgccaag tgggcagttt accgtaaata
     3901 ctccacccat tgacgtcaat ggaaagtccc tattggcgtt actatgggaa catacgtcat
     3961 tattgacgtc aatgggcggg ggtcgttggg cggtcagcca ggcgggccat ttaccgtaag
     4021 ttatgtaacg cggaactcca tatatgggct atgaactaat gaccccgtaa ttgattacta
     4081 ttaataacta gtggcgcgcc agggaggcct atataagcag agctggttta gtgaaccgtc
     4141 agatcgccca tttaggtgac actatagaat acaagctact tgttcttttt gcagctagcg
     4201 ctcaccatgg tcttcacact cgaagatttc gttggggact ggcgacagac agccggctac
     4261 aacctggacc aagtccttga acagggaggt gtgtccagtt tgtttcagaa tctcggggtg
     4321 tccgtaactc cgatccaaag gattgtcctg agcggtgaaa atgggctgaa gatcgacatc
     4381 catgtcatca tcccgtatga aggtctgagc ggcgaccaaa tgggccagat cgaaaaaatt
     4441 tttaaggtgg tgtaccctgt ggatgatcat cactttaagg tgatcctgca ctatggcaca
     4501 ctggtaatcg acggggttac gccgaacatg atcgactatt tcggacggcc gtatgaaggc
     4561 atcgccgtgt tcgacggcaa aaagatcact gtaacaggga ccctgtggaa cggcaacaaa
     4621 attatcgacg agcgcctgat caaccccgac ggctccctgc tgttccgagt aaccatcaac
     4681 ggagtgaccg gctggcggct gtgcgaacgc attctggcgg gctcgagcgg cgcgatcgcc
     4741 atgacggaat ataagctggt ggtggtgggc gccggcggtg tgggcaagag tgcgctgacc
     4801 atccagctga tccagaacca ttttgtggac gaatacgacc ccactataga ggattcctac
     4861 cggaagcagg tggtcattga tggggagacg tgcctgttgg acatcctgga taccgccggc
     4921 caggaggagt acagcgccat gcgggaccag tacatgcgca ccggggaggg cttcctgtgt
     4981 gtgtttgcca tcaacaacac caagtctttt gaggacatcc accagtacag ggagcagatc
     5041 aaacgggtga aggactcgga tgacgtgccc atggtgctgg tggggaacaa gtgtgacctg
     5101 gctgcacgca ctgtggaatc tcggcaggct caggacctcg cccgaagcta cggcatcccc
     5161 tacatcgaga cctcggccaa gacccggcag ggagtggagg atgccttcta cacgttggtg
     5221 cgtgagatcc ggcagcacaa gctgcggaag ctgaaccctc ctgatgagag tggccccggc
     5281 tgcatgagct gcaagtgtgt gctctcctga gtttaaacga attcgggctc ggtacccggg
     5341 gatcctctag aaacacctag gctctcgtac gaagtcagct gataacgtcg acgtattgcg
     5401 gccgcggatc ataatcagcc ataccacatt tgtagaggtt ttacttgctt taaaaaacct
     5461 cccacacctc cccctgaacc tgaaacataa aatgaatgca attgttgttg ttaacttgtt
     5521 tattgcagct tataatggtt acaaataaag caatagcatc acaaatttca caaataaagc
     5581 atttttttca ctgccgacgt cttaattaac gcgtcttgcg cagcaccatg gcctgaaata
     5641 acctctgaaa gaggaacttg gttaggtacc ttctgaggcg gaaagaacca gctgtggaat
     5701 gtgtgtcagt tagggtgtgg aaagtcccca ggctccccag caggcagaag tatgcaaagc
     5761 atgcatctca attagtcagc aaccaggtgt ggaaagtccc caggctcccc agcaggcaga
     5821 agtatgcaaa gcatgcatct caattagtca gcaaccatag tcccgcccct aactccgccc
     5881 atcccgcccc taactccgcc cagttccgcc cattctccgc cccatggctg actaattttt
     5941 tttatttatg cagaggccga ggccgcctcg gcctctgagc tattccagaa gtagtgagga
     6001 ggcttttttg gaggcctagg cttttgcaaa aagctcgatt cttctgacac tagcgccacc
     6061 atggccaagc ctttgtctca agaagaatcc accctcattg aaagagcaac ggctacaatc
     6121 aacagcatcc ccatctctga agactacagc gtcgccagcg cagctctctc tagcgacggc
     6181 cgcatcttca ctggtgtcaa tgtatatcat tttactgggg gaccttgtgc agaactcgtg
     6241 gtgctgggca ctgctgctgc tgcggcagct ggcaacctga cttgtatcgt cgcgatcgga
     6301 aatgagaaca ggggcatctt gagcccctgc ggacggtgcc gacaggtgct tctcgatctg
     6361 catcctggga tcaaagccat agtgaaggac agtgatggac agccgacggc agttgggatt
     6421 cgtgaattgc tgccctctgg ttatgtgtgg gagggctaaa ctctagccat cacgatggcc
     6481 gcaataaaat atctttattt tcattacatc tgtgtgttgg ttttttgtgt gacgattccg
     6541 gactataaaa ggatctaggt gaagatcctt tttgataatc tcatgaccaa aatcccttaa
     6601 cgtgagtttt cgttccactg agcgtcagac cccgtagaaa agatcaaagg atcttc
//
