LOCUS       sequence-470804-        6656 bp    DNA     circular SYN 09-MAY-2025
DEFINITION  Express NanoLuc(R)-NRAS-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 9, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"
COMMENT     Sequence Label: NanoLuc-NRAS-CRAF(RBD)-HaloTag BiBRET Vector
FEATURES             Location/Qualifiers
     source          1..6656
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     polyA_signal    61..182
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(72..91)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     protein_bind    complement(297..330)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     promoter        340..387
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             419..1213
                     /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(473..492)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     1083..1102
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     rep_origin      complement(1371..1959)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1451..1470)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     polyA_signal    2085..2133
                     /note="synthetic polyadenylation signal"
     CDS             complement(2157..2555)
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
                     VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
                     KAIVKDSDGQPTAVGIRELLPSGYVWEG"
     regulatory      2552..2561
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     promoter        complement(2586..2943)
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      2600..2735
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     enhancer        2752..2943
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     2900..2920
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     polyA_signal    3022..3103
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     CDS             complement(3306..3875)
                     /codon_start=1
                     /product="human small GTPase proto-oncoprotein"
                     /label=H-Ras
                     /translation="MTEYKLVVVGAGGVGKSALTIQLIQNHFVDEYDPTIEDSYRKQVV
                     IDGETCLLDILDTAGQEEYSAMRDQYMRTGEGFLCVFAINNTKSFEDIHQYREQIKRVK
                     DSDDVPMVLVGNKCDLAARTVESRQAQDLARSYGIPYIETSAKTRQGVEDAFYTLVREI
                     RQHKLRKLNPPDESGPGCMSCKCVLS"
     CDS             complement(3897..4409)
                     /codon_start=1
                     /product="NanoLuc(R) luciferase"
                     /label=Nluc
                     /note="human codon-optimized"
                     /translation="MVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQR
                     IVLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGV
                     TPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGW
                     RLCERILA"
     promoter        complement(4448..4466)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(4449..4466)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     enhancer        4586..4889
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        4890..5093
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     5044..5064
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     intron          5229..5361
                     /label=chimeric intron
                     /note="chimera between introns from human beta-globin and 
                     immunoglobulin heavy chain genes"
     primer_bind     5344..5363
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     primer_bind     5405..5424
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        5405..5423
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             5710..5730
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="EDLYFQS"
     CDS             5746..6630
                     /codon_start=1
                     /product="modified bacterial dehalogenase that forms 
                     covalent bonds with chloroalkane derivatives"
                     /label=HaloTag(R)
                     /translation="EIGTGFPFDPHYVEVLGERMHYVDVGPRDGTPVLFLHGNPTSSYV
                     WRNIIPHVAPTHRCIAPDLIGMGKSDKPDLGYFFDDHVRFMDAFIEALGLEEVVLVIHD
                     WGSALGFHWAKRNPERVKGIAFMEFIRPIPTWDEWPEFARETFQAFRTTDVGRKLIIDQ
                     NVFIEGTLPMGVVRPLTEVEMDHYREPFLNPVDREPLWRFPNELPIAGEPANIVALVEE
                     YMDWLHQSPVPKLLFWGTPGVLIPPAEAARLAKSLPNCKAVDIGPGLNLLQEDNPDLIG
                     SEIARWLSTLEISG"
ORIGIN
        1 ggcatgcaag ctgatttggc tgctgccacc gctgagcaag gccgcttcga gcagacatga
       61 taagatacat tgatgagttt ggacaaacca caactagaat gcagtgaaaa aaatgcttta
      121 tttgtgaaat ttgtgatgct attgctttat ttgtaaccat tataagctgc aataaacaag
      181 ttaacaacaa caattgcatt cattttatgt ttcaggttca gggggagatg tgggaggttt
      241 ttttaagcaa gtaaaacctc tacaaatgtg gtaaaatctt aattaagaat tctaatataa
      301 cttcgtatag catacattat acgaagttat ggatccactg ttgacaatta atcatcggca
      361 tagtatatcg gcatagtata atacgacaag gtgaggaact aaacccagga ggcagatcat
      421 gattgaacaa gatggattgc acgcaggttc tccggccgct tgggtggaga ggctattcgg
      481 ctatgactgg gcacaacaga caatcggctg ctctgatgcc gccgtgttcc ggctgtcagc
      541 gcaggggcgc ccggttcttt ttgtcaagac cgacctgtcc ggtgccctga atgaactgca
      601 ggacgaggca gcgcggctat cgtggctggc cacgacgggc gttccttgcg cagctgtgct
      661 cgacgttgtc actgaagcgg gaagggactg gctgctattg ggcgaagtgc cggggcagga
      721 tctcctgtca tctcaccttg ctcctgccga gaaagtatcc atcatggctg atgcaatgcg
      781 gcggctgcat acgcttgatc cggctacctg cccattcgac caccaagcga aacatcgcat
      841 cgagcgagca cgtactcgga tggaagccgg tcttgtcgat caggatgatc tggacgaaga
      901 gcatcagggg ctcgcgccag ccgaactgtt cgccaggctc aaggcgcgca tgcccgacgg
      961 cgaggatctc gtcgtgaccc atggcgatgc ctgcttgccg aatatcatgg tggaaaatgg
     1021 ccgcttttct ggattcatcg actgtggccg gctgggtgtg gcggaccgct atcaggacat
     1081 agcgttggct acccgtgata ttgctgaaga gcttggcggc gaatgggctg accgcttcct
     1141 cgtgctttac ggtatcgccg ctcccgattc gcagcgcatc gccttctatc gccttcttga
     1201 cgagttcttc tgattcgaaa tgaccgacca agcgacgccc aaccggtatc agctcactca
     1261 aaggcggtaa tacggttatc cacagaatca ggggataacg caggaaagaa catgtgagca
     1321 aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg
     1381 ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg
     1441 acaggactat aaagatacca ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt
     1501 ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc cttcgggaag cgtggcgctt
     1561 tctcatagct cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc caagctgggc
     1621 tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt
     1681 gagtccaacc cggtaagaca cgacttatcg ccactggcag cagccactgg taacaggatt
     1741 agcagagcga ggtatgtagg cggtgctaca gagttcttga agtggtggcc taactacggc
     1801 tacactagaa ggacagtatt tggtatctgc gctctgctga agccagttac cttcggaaaa
     1861 agagttggta gctcttgatc cggcaaacaa accaccgctg gtagcggtgg tttttttgtt
     1921 tgcaagcagc agattacgcg cagaaaaaaa ggatttcaag aagatccttt gatcttttct
     1981 acggggtctg acgctcagtg gaacgaaaac tcacgttaag ggattttggt catgagatta
     2041 tcaaaaagga tcttcaccta gatcctttta tagtccggaa tcgtcacaca aaaaaccaac
     2101 acacagatgt aatgaaaata aagatatttt attgcggcca tcgtgatggc tagagtttag
     2161 ccctcccaca cataaccaga gggcagcaat tcacgaatcc caactgccgt cggctgtcca
     2221 tcactgtcct tcactatggc tttgatccca ggatgcagat cgagaagcac ctgtcggcac
     2281 cgtccgcagg ggctcaagat gcccctgttc tcatttccga tcgcgacgat acaagtcagg
     2341 ttgccagctg ccgcagcagc agcagtgccc agcaccacga gttctgcaca aggtccccca
     2401 gtaaaatgat atacattgac accagtgaag atgcggccgt cgctagagag agctgcgctg
     2461 gcgacgctgt agtcttcaga gatggggatg ctgttgattg tagccgttgc tctttcaatg
     2521 agggtggatt cttcttgaga caaaggcttg gccatggtgg cgctagtgtc agaagaatcg
     2581 agctttttgc aaaagcctag gcctccaaaa aagcctcctc actacttctg gaatagctca
     2641 gaggccgagg cggcctcggc ctctgcataa ataaaaaaaa ttagtcagcc atggggcgga
     2701 gaatgggcgg aactgggcgg agttaggggc gggatgggcg gagttagggg cgggactatg
     2761 gttgctgact aattgagatg catgctttgc atacttctgc ctgctgggga gcctggggac
     2821 tttccacacc tggttgctga ctaattgaga tgcatgcttt gcatacttct gcctgctggg
     2881 gagcctgggg actttccaca ccctaactga cacacattcc acagctggtt ctttccgcct
     2941 cagaaggtac ctaaccaagt tcctctttca gaggttattt caggccatgg tgctgcgcaa
     3001 gacgcgttaa ttaagacgtc ggcagtgaaa aaaatgcttt atttgtgaaa tttgtgatgc
     3061 tattgcttta tttgtaacca ttataagctg caataaacaa gttaacaaca acaattgcat
     3121 tcattttatg tttcaggttc agggggaggt gtgggaggtt ttttaaagca agtaaaacct
     3181 ctacaaatgt ggtatggctg attatgatcc gcggccgcaa tacgtcgacg ttatcagctg
     3241 acttcgtacg agagcctagg tgtttctaga ggatccccgg gtaccgagcc cgaattcgtt
     3301 taaactcagg agagcacaca cttgcagctc atgcagccgg ggccactctc atcaggaggg
     3361 ttcagcttcc gcagcttgtg ctgccggatc tcacgcacca acgtgtagaa ggcatcctcc
     3421 actccctgcc gggtcttggc cgaggtctcg atgtagggga tgccgtagct tcgggcgagg
     3481 tcctgagcct gccgagattc cacagtgcgt gcagccaggt cacacttgtt ccccaccagc
     3541 accatgggca cgtcatccga gtccttcacc cgtttgatct gctccctgta ctggtggatg
     3601 tcctcaaaag acttggtgtt gttgatggca aacacacaca ggaagccctc cccggtgcgc
     3661 atgtactggt cccgcatggc gctgtactcc tcctggccgg cggtatccag gatgtccaac
     3721 aggcacgtct ccccatcaat gaccacctgc ttccggtagg aatcctctat agtggggtcg
     3781 tattcgtcca caaaatggtt ctggatcagc tggatggtca gcgcactctt gcccacaccg
     3841 ccggcgccca ccaccaccag cttatattcc gtcatggcga tcgcgccgct cgagcccgcc
     3901 agaatgcgtt cgcacagccg ccagccggtc actccgttga tggttactcg gaacagcagg
     3961 gagccgtcgg ggttgatcag gcgctcgtcg ataattttgt tgccgttcca cagggtccct
     4021 gttacagtga tctttttgcc gtcgaacacg gcgatgcctt catacggccg tccgaaatag
     4081 tcgatcatgt tcggcgtaac cccgtcgatt accagtgtgc catagtgcag gatcacctta
     4141 aagtgatgat catccacagg gtacaccacc ttaaaaattt tttcgatctg gcccatttgg
     4201 tcgccgctca gaccttcata cgggatgatg acatggatgt cgatcttcag cccattttca
     4261 ccgctcagga caatcctttg gatcggagtt acggacaccc cgagattctg aaacaaactg
     4321 gacacacctc cctgttcaag gacttggtcc aggttgtagc cggctgtctg tcgccagtcc
     4381 ccaacgaaat cttcgagtgt gaagaccatg gtgagcgcta gctgcaaaaa gaacaagtag
     4441 cttgtattct atagtgtcac ctaaatgggc gatctgacgg ttcactaaac cagctctgct
     4501 tatataggcc tccctggcgc gccactagtt attaatagta atcaattacg gggtcattag
     4561 ttcatagccc atatatggag ttccgcgtta cataacttac ggtaaatggc ccgcctggct
     4621 gaccgcccaa cgacccccgc ccattgacgt caataatgac gtatgttccc atagtaacgc
     4681 caatagggac tttccattga cgtcaatggg tggagtattt acggtaaact gcccacttgg
     4741 cagtacatca agtgtatcat atgccaagtc cgccccctat tgacgtcaat gacggtaaat
     4801 ggcccgcctg gcattatgcc cagtacatga ccttacggga ctttcctact tggcagtaca
     4861 tctacgtatt agtcatcgct attaccatgg tgatgcggtt ttggcagtac accaatgggc
     4921 gtggatagcg gtttgactca cggggatttc caagtctcca ccccattgac gtcaatggga
     4981 gtttgttttg gcaccaaaat caacgggact ttccaaaatg tcgtaataac cccgccccgt
     5041 tgacgcaaat gggcggtagg cgtgtacggt gggaggtcta tataagcaga gctggtttag
     5101 tgaaccgtca gatcactaga agctttattg cggtagttta tcacagttaa attgctaacg
     5161 cagtcagtgc ttctgacaca acagtctcga acttaagctg cagaagttgg tcgtgaggca
     5221 ctgggcaggt aagtatcaag gttacaagac aggtttaagg agaccaatag aaactgggct
     5281 tgtcgagaca gagaagactc ttgcgtttct gataggcacc tattggtctt actgacatcc
     5341 actttgcctt tctctccaca ggtgtccact cccagttcaa ttacagctct taaggctaga
     5401 gtattaatac gactcactat agggctagcg atcgccatgc cttctaagac aagcaacact
     5461 atccgtgttt tcttgccgaa caagcaaaga acagtggtca atgtgcgaaa tggaatgagc
     5521 ttgcatgact gccttatgaa agcactcaag gtgaggggcc tgcaaccaga gtgctgtgca
     5581 gtgttcagac ttctccacga acacaaaggt aaaaaagcac gcttagattg gaatactgat
     5641 gctgcgtctt tgattggaga agaacttcaa gtagatttcc tggatcatgt ttctctcgag
     5701 ccaaccactg aggatctgta ctttcagagc gataacgatg gatccgaaat cggtactggc
     5761 tttccattcg acccccatta tgtggaagtc ctgggcgagc gcatgcacta cgtcgatgtt
     5821 ggtccgcgcg atggcacccc tgtgctgttc ctgcacggta acccgacctc ctcctacgtg
     5881 tggcgcaaca tcatcccgca tgttgcaccg acccatcgct gcattgctcc agacctgatc
     5941 ggtatgggca aatccgacaa accagacctg ggttatttct tcgacgacca cgtccgcttc
     6001 atggatgcct tcatcgaagc cctgggtctg gaagaggtcg tcctggtcat tcacgactgg
     6061 ggctccgctc tgggtttcca ctgggccaag cgcaatccag agcgcgtcaa aggtattgca
     6121 tttatggagt tcatccgccc tatcccgacc tgggacgaat ggccagaatt tgcccgcgag
     6181 accttccagg ccttccgcac caccgacgtc ggccgcaagc tgatcatcga tcagaacgtt
     6241 tttatcgagg gtacgctgcc gatgggtgtc gtccgcccgc tgactgaagt cgagatggac
     6301 cattaccgcg agccgttcct gaatcctgtt gaccgcgagc cactgtggcg cttcccaaac
     6361 gagctgccaa tcgccggtga gccagcgaac atcgtcgcgc tggtcgaaga atacatggac
     6421 tggctgcacc agtcccctgt cccgaagctg ctgttctggg gcaccccagg cgttctgatc
     6481 ccaccggccg aagccgctcg cctggccaaa agcctgccta actgcaaggc tgtggacatc
     6541 ggcccgggtc tgaatctgct gcaagaagac aacccggacc tgatcggcag cgagatcgcg
     6601 cgctggctgt ctactctgga gatttccggt taatagaatt ctagagtcga cctgca
//
