LOCUS       sequence-470796-        6656 bp    DNA     circular SYN 13-MAY-2025
DEFINITION  Express NanoLuc(R)-KRAS4A-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)
  TITLE     Promega Early Access Collection
REFERENCE   2  (bases 1 to 6656)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 13, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"
COMMENT     Sequence Label: NanoLuc-KRAS4A-cRAF(RBD)-HaloTag BiBRET Vector
FEATURES             Location/Qualifiers
     source          1..6656
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     polyA_signal    21..102
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     CDS             complement(896..1408)
                     /codon_start=1
                     /product="NanoLuc(R) luciferase"
                     /label=Nluc
                     /note="human codon-optimized"
                     /translation="MVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQR
                     IVLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGV
                     TPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGW
                     RLCERILA"
     promoter        complement(1447..1465)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(1448..1465)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     enhancer        1585..1888
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        1889..2092
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     2043..2063
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     intron          2228..2360
                     /label=chimeric intron
                     /note="chimera between introns from human beta-globin and 
                     immunoglobulin heavy chain genes"
     primer_bind     2343..2362
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     primer_bind     2404..2423
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        2404..2422
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             2709..2729
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="EDLYFQS"
     CDS             2745..3629
                     /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    3716..3837
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3727..3746)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     protein_bind    complement(3952..3985)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     promoter        3995..4042
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             4074..4868
                     /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(4128..4147)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4738..4757
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     rep_origin      complement(5026..5614)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5106..5125)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     polyA_signal    5740..5788
                     /note="synthetic polyadenylation signal"
     CDS             complement(5812..6210)
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
                     VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
                     KAIVKDSDGQPTAVGIRELLPSGYVWEG"
     regulatory      6207..6216
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     promoter        complement(6241..6598)
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      6255..6390
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     enhancer        6407..6598
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     6555..6575
                     /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 tataatgcat tttttaattt tcacacagcc aggagtcttt tcttctttgc
      361 tgattttttt caatctgtat tgtcggatct ccctcaccaa tgtataaaaa gcatcctcca
      421 ctctctgtct tgtctttgct gatgtttcaa taaaaggaat tccataactt cttgctaagt
      481 cctgagcctg ttttgtgtct actgttctag aaggcaaatc acatttattt cctactagga
      541 ccataggtac atcttcagag tccttaactc ttttaatttg ttctctataa tggtgaatat
      601 cttcaaatga tttagtatta tttatggcaa atacacaaag aaagccctcc ccagtcctca
      661 tgtactggtc cctcattgca ctgtactcct cttgacctgc tgtgtcgaga atatccaaga
      721 gacaggtttc tccatcaatt actacttgct tcctgtagga atcctctatt gttggatcat
      781 attcgtccac aaaatgattc tgaattagct gtatcgtcaa ggcactcttg cctacgccac
      841 cagctccaac taccacaagt ttatattcag tcatggcgat cgcgccgctc gagcccgcca
      901 gaatgcgttc gcacagccgc cagccggtca ctccgttgat ggttactcgg aacagcaggg
      961 agccgtcggg gttgatcagg cgctcgtcga taattttgtt gccgttccac agggtccctg
     1021 ttacagtgat ctttttgccg tcgaacacgg cgatgccttc atacggccgt ccgaaatagt
     1081 cgatcatgtt cggcgtaacc ccgtcgatta ccagtgtgcc atagtgcagg atcaccttaa
     1141 agtgatgatc atccacaggg tacaccacct taaaaatttt ttcgatctgg cccatttggt
     1201 cgccgctcag accttcatac gggatgatga catggatgtc gatcttcagc ccattttcac
     1261 cgctcaggac aatcctttgg atcggagtta cggacacccc gagattctga aacaaactgg
     1321 acacacctcc ctgttcaagg acttggtcca ggttgtagcc ggctgtctgt cgccagtccc
     1381 caacgaaatc ttcgagtgtg aagaccatgg tgagcgctag ctgcaaaaag aacaagtagc
     1441 ttgtattcta tagtgtcacc taaatgggcg atctgacggt tcactaaacc agctctgctt
     1501 atataggcct ccctggcgcg ccactagtta ttaatagtaa tcaattacgg ggtcattagt
     1561 tcatagccca tatatggagt tccgcgttac ataacttacg gtaaatggcc cgcctggctg
     1621 accgcccaac gacccccgcc cattgacgtc aataatgacg tatgttccca tagtaacgcc
     1681 aatagggact ttccattgac gtcaatgggt ggagtattta cggtaaactg cccacttggc
     1741 agtacatcaa gtgtatcata tgccaagtcc gccccctatt gacgtcaatg acggtaaatg
     1801 gcccgcctgg cattatgccc agtacatgac cttacgggac tttcctactt ggcagtacat
     1861 ctacgtatta gtcatcgcta ttaccatggt gatgcggttt tggcagtaca ccaatgggcg
     1921 tggatagcgg tttgactcac ggggatttcc aagtctccac cccattgacg tcaatgggag
     1981 tttgttttgg caccaaaatc aacgggactt tccaaaatgt cgtaataacc ccgccccgtt
     2041 gacgcaaatg ggcggtaggc gtgtacggtg ggaggtctat ataagcagag ctggtttagt
     2101 gaaccgtcag atcactagaa gctttattgc ggtagtttat cacagttaaa ttgctaacgc
     2161 agtcagtgct tctgacacaa cagtctcgaa cttaagctgc agaagttggt cgtgaggcac
     2221 tgggcaggta agtatcaagg ttacaagaca ggtttaagga gaccaataga aactgggctt
     2281 gtcgagacag agaagactct tgcgtttctg ataggcacct attggtctta ctgacatcca
     2341 ctttgccttt ctctccacag gtgtccactc ccagttcaat tacagctctt aaggctagag
     2401 tattaatacg actcactata gggctagcga tcgccatgcc ttctaagaca agcaacacta
     2461 tccgtgtttt cttgccgaac aagcaaagaa cagtggtcaa tgtgcgaaat ggaatgagct
     2521 tgcatgactg ccttatgaaa gcactcaagg tgaggggcct gcaaccagag tgctgtgcag
     2581 tgttcagact tctccacgaa cacaaaggta aaaaagcacg cttagattgg aatactgatg
     2641 ctgcgtcttt gattggagaa gaacttcaag tagatttcct ggatcatgtt tctctcgagc
     2701 caaccactga ggatctgtac tttcagagcg ataacgatgg atccgaaatc ggtactggct
     2761 ttccattcga cccccattat gtggaagtcc tgggcgagcg catgcactac gtcgatgttg
     2821 gtccgcgcga tggcacccct gtgctgttcc tgcacggtaa cccgacctcc tcctacgtgt
     2881 ggcgcaacat catcccgcat gttgcaccga cccatcgctg cattgctcca gacctgatcg
     2941 gtatgggcaa atccgacaaa ccagacctgg gttatttctt cgacgaccac gtccgcttca
     3001 tggatgcctt catcgaagcc ctgggtctgg aagaggtcgt cctggtcatt cacgactggg
     3061 gctccgctct gggtttccac tgggccaagc gcaatccaga gcgcgtcaaa ggtattgcat
     3121 ttatggagtt catccgccct atcccgacct gggacgaatg gccagaattt gcccgcgaga
     3181 ccttccaggc cttccgcacc accgacgtcg gccgcaagct gatcatcgat cagaacgttt
     3241 ttatcgaggg tacgctgccg atgggtgtcg tccgcccgct gactgaagtc gagatggacc
     3301 attaccgcga gccgttcctg aatcctgttg accgcgagcc actgtggcgc ttcccaaacg
     3361 agctgccaat cgccggtgag ccagcgaaca tcgtcgcgct ggtcgaagaa tacatggact
     3421 ggctgcacca gtcccctgtc ccgaagctgc tgttctgggg caccccaggc gttctgatcc
     3481 caccggccga agccgctcgc ctggccaaaa gcctgcctaa ctgcaaggct gtggacatcg
     3541 gcccgggtct gaatctgctg caagaagaca acccggacct gatcggcagc gagatcgcgc
     3601 gctggctgtc tactctggag atttccggtt aatagaattc tagagtcgac ctgcaggcat
     3661 gcaagctgat ttggctgctg ccaccgctga gcaaggccgc ttcgagcaga catgataaga
     3721 tacattgatg agtttggaca aaccacaact agaatgcagt gaaaaaaatg ctttatttgt
     3781 gaaatttgtg atgctattgc tttatttgta accattataa gctgcaataa acaagttaac
     3841 aacaacaatt gcattcattt tatgtttcag gttcaggggg agatgtggga ggttttttta
     3901 agcaagtaaa acctctacaa atgtggtaaa atcttaatta agaattctaa tataacttcg
     3961 tatagcatac attatacgaa gttatggatc cactgttgac aattaatcat cggcatagta
     4021 tatcggcata gtataatacg acaaggtgag gaactaaacc caggaggcag atcatgattg
     4081 aacaagatgg attgcacgca ggttctccgg ccgcttgggt ggagaggcta ttcggctatg
     4141 actgggcaca acagacaatc ggctgctctg atgccgccgt gttccggctg tcagcgcagg
     4201 ggcgcccggt tctttttgtc aagaccgacc tgtccggtgc cctgaatgaa ctgcaggacg
     4261 aggcagcgcg gctatcgtgg ctggccacga cgggcgttcc ttgcgcagct gtgctcgacg
     4321 ttgtcactga agcgggaagg gactggctgc tattgggcga agtgccgggg caggatctcc
     4381 tgtcatctca ccttgctcct gccgagaaag tatccatcat ggctgatgca atgcggcggc
     4441 tgcatacgct tgatccggct acctgcccat tcgaccacca agcgaaacat cgcatcgagc
     4501 gagcacgtac tcggatggaa gccggtcttg tcgatcagga tgatctggac gaagagcatc
     4561 aggggctcgc gccagccgaa ctgttcgcca ggctcaaggc gcgcatgccc gacggcgagg
     4621 atctcgtcgt gacccatggc gatgcctgct tgccgaatat catggtggaa aatggccgct
     4681 tttctggatt catcgactgt ggccggctgg gtgtggcgga ccgctatcag gacatagcgt
     4741 tggctacccg tgatattgct gaagagcttg gcggcgaatg ggctgaccgc ttcctcgtgc
     4801 tttacggtat cgccgctccc gattcgcagc gcatcgcctt ctatcgcctt cttgacgagt
     4861 tcttctgatt cgaaatgacc gaccaagcga cgcccaaccg gtatcagctc actcaaaggc
     4921 ggtaatacgg ttatccacag aatcagggga taacgcagga aagaacatgt gagcaaaagg
     4981 ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg
     5041 cccccctgac gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg
     5101 actataaaga taccaggcgt ttccccctgg aagctccctc gtgcgctctc ctgttccgac
     5161 cctgccgctt accggatacc tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca
     5221 tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt
     5281 gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc
     5341 caacccggta agacacgact tatcgccact ggcagcagcc actggtaaca ggattagcag
     5401 agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg tggcctaact acggctacac
     5461 tagaaggaca gtatttggta tctgcgctct gctgaagcca gttaccttcg gaaaaagagt
     5521 tggtagctct tgatccggca aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa
     5581 gcagcagatt acgcgcagaa aaaaaggatt tcaagaagat cctttgatct tttctacggg
     5641 gtctgacgct cagtggaacg aaaactcacg ttaagggatt ttggtcatga gattatcaaa
     5701 aaggatcttc acctagatcc ttttatagtc cggaatcgtc acacaaaaaa ccaacacaca
     5761 gatgtaatga aaataaagat attttattgc ggccatcgtg atggctagag tttagccctc
     5821 ccacacataa ccagagggca gcaattcacg aatcccaact gccgtcggct gtccatcact
     5881 gtccttcact atggctttga tcccaggatg cagatcgaga agcacctgtc ggcaccgtcc
     5941 gcaggggctc aagatgcccc tgttctcatt tccgatcgcg acgatacaag tcaggttgcc
     6001 agctgccgca gcagcagcag tgcccagcac cacgagttct gcacaaggtc ccccagtaaa
     6061 atgatataca ttgacaccag tgaagatgcg gccgtcgcta gagagagctg cgctggcgac
     6121 gctgtagtct tcagagatgg ggatgctgtt gattgtagcc gttgctcttt caatgagggt
     6181 ggattcttct tgagacaaag gcttggccat ggtggcgcta gtgtcagaag aatcgagctt
     6241 tttgcaaaag cctaggcctc caaaaaagcc tcctcactac ttctggaata gctcagaggc
     6301 cgaggcggcc tcggcctctg cataaataaa aaaaattagt cagccatggg gcggagaatg
     6361 ggcggaactg ggcggagtta ggggcgggat gggcggagtt aggggcggga ctatggttgc
     6421 tgactaattg agatgcatgc tttgcatact tctgcctgct ggggagcctg gggactttcc
     6481 acacctggtt gctgactaat tgagatgcat gctttgcata cttctgcctg ctggggagcc
     6541 tggggacttt ccacacccta actgacacac attccacagc tggttctttc cgcctcagaa
     6601 ggtacctaac caagttcctc tttcagaggt tatttcaggc catggtgctg cgcaag
//
