LOCUS       sequence-481206-        6653 bp    DNA     circular SYN 30-JUL-2025
DEFINITION  Express NanoLuc-KRAS_CRAF(RBD)-HaloTag 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 6653)
  AUTHORS   Promega
  TITLE     Promega Plasmid Collection
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 6653)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Jul 30, 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            "Unpublished"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-KRAS_CRAF(RBD)...
FEATURES             Location/Qualifiers
     source          1..6653
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     polyA_signal    21..102
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     CDS             complement(305..871)
                     /codon_start=1
                     /product="human small GTPase proto-oncoprotein"
                     /label=K-Ras
                     /translation="MTEYKLVVVGAGGVGKSALTIQLIQNHFVDEYDPTIEDSYRKQVV
                     IDGETCLLDILDTAGQEEYSAMRDQYMRTGEGFLCVFAINNTKSFEDIHHYREQIKRVK
                     DSEDVPMVLVGNKCDLPSRTVDTKQAQDLARSYGIPFIETSAKTRQGVDDAFYTLVREI
                     RKHKEKMSKDGKKKKKKSKTKCVIM"
     CDS             complement(893..1405)
                     /codon_start=1
                     /product="NanoLuc(R) luciferase"
                     /label=Nluc
                     /note="human codon-optimized"
                     /translation="MVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQR
                     IVLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGV
                     TPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGW
                     RLCERILA"
     promoter        complement(1444..1462)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(1445..1462)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     enhancer        1582..1885
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        1886..2089
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     2040..2060
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     intron          2225..2357
                     /label=chimeric intron
                     /note="chimera between introns from human beta-globin and 
                     immunoglobulin heavy chain genes"
     primer_bind     2340..2359
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     primer_bind     2401..2420
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        2401..2419
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             2706..2726
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="EDLYFQS"
     CDS             2742..3626
                     /codon_start=1
                     /product="modified bacterial dehalogenase that forms 
                     covalent bonds with chloroalkane derivatives"
                     /label=HaloTag(R)
                     /translation="EIGTGFPFDPHYVEVLGERMHYVDVGPRDGTPVLFLHGNPTSSYV
                     WRNIIPHVAPTHRCIAPDLIGMGKSDKPDLGYFFDDHVRFMDAFIEALGLEEVVLVIHD
                     WGSALGFHWAKRNPERVKGIAFMEFIRPIPTWDEWPEFARETFQAFRTTDVGRKLIIDQ
                     NVFIEGTLPMGVVRPLTEVEMDHYREPFLNPVDREPLWRFPNELPIAGEPANIVALVEE
                     YMDWLHQSPVPKLLFWGTPGVLIPPAEAARLAKSLPNCKAVDIGPGLNLLQEDNPDLIG
                     SEIARWLSTLEISG"
     polyA_signal    3713..3834
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     protein_bind    complement(3949..3982)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     promoter        3992..4039
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             4071..4865
                     /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
                     GLAPAELFARLKARMPDGEDLAVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(4125..4144)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4735..4754
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     rep_origin      complement(5023..5611)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5103..5122)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     polyA_signal    5737..5785
                     /note="synthetic polyadenylation signal"
     CDS             complement(5809..6207)
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
                     VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
                     KAIVKDSDGQPTAVGIRELLPSGYVWEG"
     regulatory      6204..6213
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     promoter        complement(6238..6595)
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      6252..6387
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     enhancer        6404..6595
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     6552..6572
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
ORIGIN
        1 acgcgttaat taagacgtcg gcagtgaaaa aaatgcttta tttgtgaaat ttgtgatgct
       61 attgctttat ttgtaaccat tataagctgc aataaacaag ttaacaacaa caattgcatt
      121 cattttatgt ttcaggttca gggggaggtg tgggaggttt tttaaagcaa gtaaaacctc
      181 tacaaatgtg gtatggctga ttatgatccg cggccgcaat acgtcgacgt tatcagctga
      241 cttcgtacga gagcctaggt gtttctagag gatccccggg taccgagccc gaattcgttt
      301 aaacttacat aattacacac tttgtctttg acttcttttt cttcttttta ccatctttgc
      361 tcatcttttc tttatgtttt cgaatttctc gaactaatgt atagaaggca tcatcaacac
      421 cctgtcttgt ctttgctgat gtttcaataa aaggaattcc ataacttctt gctaagtcct
      481 gagcctgttt tgtgtctact gttctagaag gcaaatcaca tttatttcct actaggacca
      541 taggtacatc ttcagagtcc ttaactcttt taatttgttc tctataatgg tgaatatctt
      601 caaatgattt agtattattt atggcaaata cacaaagaaa gccctcccca gtcctcatgt
      661 actggtccct cattgcactg tactcctctt gacctgctgt gtcgagaata tccaagagac
      721 aggtttctcc atcaattact acttgcttcc tgtaggaatc ctctattgtt ggatcatatt
      781 cgtccacaaa atgattctga attagctgta tcgtcaaggc actcttgcct acgccaccag
      841 ctccaactac cacaagttta tattcagtca tggcgatcgc gccgctcgag cccgccagaa
      901 tgcgttcgca cagccgccag ccggtcactc cgttgatggt tactcggaac agcagggagc
      961 cgtcggggtt gatcaggcgc tcgtcgataa ttttgttgcc gttccacagg gtccctgtta
     1021 cagtgatctt tttgccgtcg aacacggcga tgccttcata cggccgtccg aaatagtcga
     1081 tcatgttcgg cgtaaccccg tcgattacca gtgtgccata gtgcaggatc accttaaagt
     1141 gatgatcatc cacagggtac accaccttaa aaattttttc gatctggccc atttggtcgc
     1201 cgctcagacc ttcatacggg atgatgacat ggatgtcgat cttcagccca ttttcaccgc
     1261 tcaggacaat cctttggatc ggagttacgg acaccccgag attctgaaac aaactggaca
     1321 cacctccctg ttcaaggact tggtccaggt tgtagccggc tgtctgtcgc cagtccccaa
     1381 cgaaatcttc gagtgtgaag accatggtga gcgctagctg caaaaagaac aagtagcttg
     1441 tattctatag tgtcacctaa atgggcgatc tgacggttca ctaaaccagc tctgcttata
     1501 taggcctccc tggcgcgcca ctagttatta atagtaatca attacggggt cattagttca
     1561 tagcccatat atggagttcc gcgttacata acttacggta aatggcccgc ctggctgacc
     1621 gcccaacgat ccccgcccat tgacgtcaat aatgacgtat gttcccatag taacgccaat
     1681 agggactttc cattgacgtc aatgggtgga gtatttacgg taaactgccc acttggcagt
     1741 acatcaagtg tatcatatgc caagtccgcc ccctattgac gtcaatgacg gtaaatggcc
     1801 cgcctggcat tatgcccagt acatgacctt acgggacttt cctacttggc agtacatcta
     1861 cgtattagtc atcgctatta ccatggtgat gcggttttgg cagtacacca atgggcgtgg
     1921 atagcggttt gactcacggg gatttccaag tctccacccc attgacgtca atgggagttt
     1981 gttttggcac caaaatcaac gggactttcc aaaatgtcgt aataaccccg ccccgttgac
     2041 gcaaatgggc ggtaggcgtg tacggtggga ggtctatata agcagagctg gtttagtgaa
     2101 ccgtcagatc actagaagct ttattgcggt agtttatcac agttaaattg ctaacgcagt
     2161 cagtgcttct gacacaacag tctcgaactt aagctgcaga agttggtcgt gaggcactgg
     2221 gcaggtaagt atcaaggtta caagacaggt ttaaggagac caatagaaac tgggcttgtc
     2281 gagacagaga agactcttgc gtttctgata ggcacctatt ggtcttactg acatccactt
     2341 tgcctttctc tccacaggtg tccactccca gttcaattac agctcttaag gctagagtat
     2401 taatacgact cactataggg ctagcgatcg ccatgccttc taagacaagc aacactatcc
     2461 gtgttttctt gccgaacaag caaagaacag tggtcaatgt gcgaaatgga atgagcttgc
     2521 atgactgcct tatgaaagca ctcaaggtga ggggcctgca accagagtgc tgtgcagtgt
     2581 tcagacttct ccacgaacac aaaggtaaaa aagcacgctt agattggaat actgatgctg
     2641 cgtctttgat tggagaagaa cttcaagtag atttcctgga tcatgtttct ctcgagccaa
     2701 ccactgagga tctgtacttt cagagcgata acgatggatc cgaaatcggt actggctttc
     2761 cattcgaccc ccattatgtg gaagtcctgg gcgagcgcat gcactacgtc gatgttggtc
     2821 cgcgcgatgg cacccctgtg ctgttcctgc acggtaaccc gacctcctcc tacgtgtggc
     2881 gcaacatcat cccgcatgtt gcaccgaccc atcgctgcat tgctccagac ctgatcggta
     2941 tgggcaaatc cgacaaacca gacctgggtt atttcttcga cgaccacgtc cgcttcatgg
     3001 atgccttcat cgaagccctg ggtctggaag aggtcgtcct ggtcattcac gactggggct
     3061 ccgctctggg tttccactgg gccaagcgca atccagagcg cgtcaaaggt attgcattta
     3121 tggagttcat ccgccctatc ccgacctggg acgaatggcc agaatttgcc cgcgagacct
     3181 tccaggcctt ccgcaccacc gacgtcggcc gcaagctgat catcgatcag aacgttttta
     3241 tcgagggtac gctgccgatg ggtgtcgtcc gcccgctgac tgaagtcgag atggaccatt
     3301 accgcgagcc gttcctgaat cctgttgacc gcgagccact gtggcgcttc ccaaacgagc
     3361 tgccaatcgc cggtgagcca gcgaacatcg tcgcgctggt cgaagaatac atggactggc
     3421 tgcaccagtc ccctgtcccg aagctgctgt tctggggcac cccaggcgtt ctgatcccac
     3481 cggccgaagc cgctcgcctg gccaaaagcc tgcctaactg caaggctgtg gacatcggcc
     3541 cgggtctgaa tctgctgcaa gaagacaacc cggacctgat cggcagcgag atcgcgcgct
     3601 ggctgtctac tctggagatt tccggttaat agaattctag agtcgacctg caggcatgca
     3661 agctgatttg gctgctgcca ccgctgagca aggccgcttc gagcagacat gataagatac
     3721 attgataagt ttggacaaac cacaactaga atgcagtgaa aaaaatgctt tatttgtgaa
     3781 atttgtgatg ctattgcttt atttgtaacc attataagct gcaataaaca agttaacaac
     3841 aacaattgca ttcattttat gtttcaggtt cagggggaga tgtgggaggt ttttttaagc
     3901 aagtaaaacc tctacaaatg tggtaaaatc ttaattaaga attctaatat aacttcgtat
     3961 agcatacatt atacgaagtt atggatccac tgttgacaat taatcatcgg catagtatat
     4021 cggcatagta taatacgaca aggtgaggaa ctaaacccag gaggcagatc atgattgaac
     4081 aagatggatt gcacgcaggt tctccggccg cttgggtgga gaggctattc ggctatgact
     4141 gggcacaaca gacaatcggc tgctctgatg ccgccgtgtt ccggctgtca gcgcaggggc
     4201 gcccggttct ttttgtcaag accgacctgt ccggtgccct gaatgaactg caggacgagg
     4261 cagcgcggct atcgtggctg gccacgacgg gcgttccttg cgcagctgtg ctcgacgttg
     4321 tcactgaagc gggaagggac tggctgctat tgggcgaagt gccggggcag gatctcctgt
     4381 catctcacct tgctcctgcc gagaaagtat ccatcatggc tgatgcaatg cggcggctgc
     4441 atacgcttga tccggctacc tgcccattcg accaccaagc gaaacatcgc atcgagcgag
     4501 cacgtactcg gatggaagcc ggtcttgtcg atcaggatga tctggacgaa gagcatcagg
     4561 ggctcgcgcc agccgaactg ttcgccaggc tcaaggcgcg catgcccgac ggcgaggatc
     4621 tcgccgtgac ccatggcgat gcctgcttgc cgaatatcat ggtggaaaat ggccgctttt
     4681 ctggattcat cgactgtggc cggctgggtg tggcggaccg ctatcaggac atagcgttgg
     4741 ctacccgtga tattgctgaa gagcttggcg gcgaatgggc tgaccgcttc ctcgtgcttt
     4801 acggtatcgc cgctcccgat tcgcagcgca tcgccttcta tcgccttctt gacgagttct
     4861 tctgattcga aatgaccgac caagcgacgc ccaaccggta tcagctcact caaaggcggt
     4921 aatacggtta tccacagaat caggggataa cgcaggaaag aacatgtgag caaaaggcca
     4981 gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg tttttccata ggctccgccc
     5041 ccctgacgag catcacaaaa atcgacgctc aagtcagagg tggcgaaacc cgacaggact
     5101 ataaagatac caggcgtttc cccctggaag ctccctcgtg cgctctcctg ttccgaccct
     5161 gccgcttacc ggatacctgt ccgcctttct cccttcggga agcgtggcgc tttctcatag
     5221 ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca
     5281 cgaacccccc gttcagcccg accgctgcgc cttatccggt aactatcgtc ttgagtccaa
     5341 cccggtaaga cacgacttat cgccactggc agcagccact ggtaacagga ttagcagagc
     5401 gaggtatgta ggcggtgcta cagagttctt gaagtggtgg cctaactacg gctacactag
     5461 aaggacagta tttggtatct gcgctctgct gaagccagtt accttcggaa aaagagttgg
     5521 tagctcttga tccggcaaac aaaccaccgc tggtagcggt ggtttttttg tttgcaagca
     5581 gcagattacg cgcagaaaaa aaggatttca agaagatcct ttgatctttt ctacggggtc
     5641 tgacgctcag tggaacgaaa actcacgtta agggattttg gtcatgagat tatcaaaaag
     5701 gatcttcacc tagatccttt tatagtccgg aatcgtcaca caaaaaacca acacacagat
     5761 gtaatgaaaa taaagatatt ttattgcggc catcgtgatg gctagagttt agccctccca
     5821 cacataacca gagggcagca attcacgaat cccaactgcc gtcggctgtc catcactgtc
     5881 cttcactatg gctttgatcc caggatgcag atcgagaagc acctgtcggc accgtccgca
     5941 ggggctcaag atgcccctgt tctcatttcc gatcgcgacg atacaagtca ggttgccagc
     6001 tgccgcagca gcagcagtgc ccagcaccac gagttctgca caaggtcccc cagtaaaatg
     6061 atatacattg acaccagtga agatgcggcc gtcgctagag agagctgcgc tggcgacgct
     6121 gtagtcttca gagatgggga tgctgttgat tgtagccgtt gctctttcaa tgagggtgga
     6181 ttcttcttga gacaaaggct tggccatggt ggcgctagtg tcagaagaat cgagcttttt
     6241 gcaaaagcct aggcctccaa aaaagcctcc tcactacttc tggaatagct cagaggccga
     6301 ggcggcctcg gcctctgcat aaataaaaaa aattagtcag ccatggggcg gagaatgggc
     6361 ggaactgggc ggagttaggg gcgggatggg cggagttagg ggcgggacta tggttgctga
     6421 ctaattgaga tgcatgcttt gcatacttct gcctgctggg gagcctgggg actttccaca
     6481 cctggttgct gactaattga gatgcatgct ttgcatactt ctgcctgctg gggagcctgg
     6541 ggactttcca caccctaact gacacacatt ccacagctgg ttctttccgc ctcagaaggt
     6601 acctaaccaa gttcctcttt cagaggttat ttcaggccat ggtgctgcgc aag
//
