LOCUS       sequence-471915-        5857 bp    DNA     circular SYN 13-JUN-2025
DEFINITION  Express LgBiT-KRAS(G12V)_SmBiT-CRAF-RBD 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 5857)
  TITLE     Promega Early Access Collection
REFERENCE   2  (bases 1 to 5857)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Jun 13, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"
COMMENT     Sequence Label: LgBiT-KRAS(G12V)_SmBiT-CRAF-RBD Bidirectional ...
FEATURES             Location/Qualifiers
     source          1..5857
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     polyA_signal    24..105
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     CDS             complement(join(308..838,839..841,842..874))
                     /codon_start=1
                     /product="human oncoprotein generated by the G12V mutation 
                     in the small GTPase K-Ras"
                     /label=K-Ras (G12V)
                     /translation="MTEYKLVVVGAVGVGKSALTIQLIQNHFVDEYDPTIEDSYRKQVV
                     IDGETCLLDILDTAGQEEYSAMRDQYMRTGEGFLCVFAINNTKSFEDIHHYREQIKRVK
                     DSEDVPMVLVGNKCDLPSRTVDTKQAQDLARSYGIPFIETSAKTRQGVDDAFYTLVREI
                     RKHKEKMSKDGKKKKKKSKTKCVIM"
     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"
     polyA_signal    2920..3041
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2931..2950)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     protein_bind    complement(3156..3189)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     promoter        3199..3246
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             3278..4072
                     /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(3332..3351)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3942..3961
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     rep_origin      complement(4230..4818)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4310..4329)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     polyA_signal    4944..4992
                     /note="synthetic polyadenylation signal"
     CDS             complement(5016..5414)
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
                     VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
                     KAIVKDSDGQPTAVGIRELLPSGYVWEG"
     regulatory      5411..5420
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     promoter        complement(5445..5802)
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      5459..5594
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     enhancer        5611..5802
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     5759..5779
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
ORIGIN
        1 aagacgcgtt aattaagacg tcggcagtga aaaaaatgct ttatttgtga aatttgtgat
       61 gctattgctt tatttgtaac cattataagc tgcaataaac aagttaacaa caacaattgc
      121 attcatttta tgtttcaggt tcagggggag gtgtgggagg ttttttaaag caagtaaaac
      181 ctctacaaat gtggtatggc tgattatgat ccgcggccgc aatacgtcga cgttatcagc
      241 tgacttcgta cgagagccta ggtgtttcta gaggatcccc gggtaccgag cccgaattcg
      301 tttaaactta cataattaca cactttgtct ttgacttctt tttcttcttt ttaccatctt
      361 tgctcatctt ttctttatgt tttcgaattt ctcgaactaa tgtatagaag gcatcatcaa
      421 caccctgtct 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 cctacgccaa
      841 cagctccaac taccacaagt ttatattcag tcatggcgat cgcaccgctc gagcctccac
      901 ctccgctccc gccaccaccg gagctcccac tgttgatggt tactcggaac agcatggagc
      961 cgtcgggggt gatcaggcgc tcgtcgataa ttttgttgcc gttccacagg gtccctgtta
     1021 cagtgatctt tttgccgtcg aacacggcga tgccttcata cggccgtccg aaatagttca
     1081 gcatgttcgg cgtaaccccg tcgattacca gtgtgccata gggcaggatc accttaaagt
     1141 gatgatcatc cacagggtac accaccttaa acacctcttc gatctgggcc atttggtcgg
     1201 cgctcagacc ttcatacggg atgatgacat ggatgtcgat cttcagggca ttttcaccgc
     1261 tccggacaat cctttggatc ggagttacgg acacggcgag attctgcagc aaactggaca
     1321 cacctccctg ttcaaggact tggtccaggt tgtaggcggc tgtctgttcc 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 gcccaacgac 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 ctagcgctca ccatggtgac cggctaccgg ctgttcgagg
     2461 agattctcgg gagctccggt ggtggcggga gcggaggtgg aggctcgagc ggtgcgatcg
     2521 ccatgccttc taagacaagc aacactatcc gtgttttctt gccgaacaag caaagaacag
     2581 tggtcaatgt gcgaaatgga atgagcttgc atgactgcct tatgaaagca ctcaaggtga
     2641 ggggcctgca accagagtgc tgtgcagtgt tcagacttct ccacgaacac aaaggtaaaa
     2701 aagcacgctt agattggaat actgatgctg cgtctttgat tggagaagaa cttcaagtag
     2761 atttcctgga tcatgtttaa acgaattcgg gctcggtacc cggggatcct ctagagtcga
     2821 cctgcaggca tgcaagctga tccggctgct aacaaagccc gaaaggaagc tgagttggct
     2881 gctgccaccg ctgagcaagg ccgcttcgag cagacatgat aagatacatt gatgagtttg
     2941 gacaaaccac aactagaatg cagtgaaaaa aatgctttat ttgtgaaatt tgtgatgcta
     3001 ttgctttatt tgtaaccatt ataagctgca ataaacaagt taacaacaac aattgcattc
     3061 attttatgtt tcaggttcag ggggagatgt gggaggtttt tttaagcaag taaaacctct
     3121 acaaatgtgg taaaatctta attaagaatt ctaatataac ttcgtatagc atacattata
     3181 cgaagttatg gatccactgt tgacaattaa tcatcggcat agtatatcgg catagtataa
     3241 tacgacaagg tgaggaacta aacccaggag gcagatcatg attgaacaag atggattgca
     3301 cgcaggttct ccggccgctt gggtggagag gctattcggc tatgactggg cacaacagac
     3361 aatcggctgc tctgatgccg ccgtgttccg gctgtcagcg caggggcgcc cggttctttt
     3421 tgtcaagacc gacctgtccg gtgccctgaa tgaactgcag gacgaggcag cgcggctatc
     3481 gtggctggcc acgacgggcg ttccttgcgc agctgtgctc gacgttgtca ctgaagcggg
     3541 aagggactgg ctgctattgg gcgaagtgcc ggggcaggat ctcctgtcat ctcaccttgc
     3601 tcctgccgag aaagtatcca tcatggctga tgcaatgcgg cggctgcata cgcttgatcc
     3661 ggctacctgc ccattcgacc accaagcgaa acatcgcatc gagcgagcac gtactcggat
     3721 ggaagccggt cttgtcgatc aggatgatct ggacgaagag catcaggggc tcgcgccagc
     3781 cgaactgttc gccaggctca aggcgcgcat gcccgacggc gaggatctcg tcgtgaccca
     3841 tggcgatgcc tgcttgccga atatcatggt ggaaaatggc cgcttttctg gattcatcga
     3901 ctgtggccgg ctgggtgtgg cggaccgcta tcaggacata gcgttggcta cccgtgatat
     3961 tgctgaagag cttggcggcg aatgggctga ccgcttcctc gtgctttacg gtatcgccgc
     4021 tcccgattcg cagcgcatcg ccttctatcg ccttcttgac gagttcttct gattcgaaat
     4081 gaccgaccaa gcgacgccca accggtatca gctcactcaa aggcggtaat acggttatcc
     4141 acagaatcag gggataacgc aggaaagaac atgtgagcaa aaggccagca aaaggccagg
     4201 aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc tccgcccccc tgacgagcat
     4261 cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag
     4321 gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga
     4381 tacctgtccg cctttctccc ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg
     4441 tatctcagtt cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt
     4501 cagcccgacc gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac
     4561 gacttatcgc cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc
     4621 ggtgctacag agttcttgaa gtggtggcct aactacggct acactagaag gacagtattt
     4681 ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa gagttggtag ctcttgatcc
     4741 ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt gcaagcagca gattacgcgc
     4801 agaaaaaaag gatttcaaga agatcctttg atcttttcta cggggtctga cgctcagtgg
     4861 aacgaaaact cacgttaagg gattttggtc atgagattat caaaaaggat cttcacctag
     4921 atccttttat agtccggaat cgtcacacaa aaaaccaaca cacagatgta atgaaaataa
     4981 agatatttta ttgcggccat cgtgatggct agagtttagc cctcccacac ataaccagag
     5041 ggcagcaatt cacgaatccc aactgccgtc ggctgtccat cactgtcctt cactatggct
     5101 ttgatcccag gatgcagatc gagaagcacc tgtcggcacc gtccgcaggg gctcaagatg
     5161 cccctgttct catttccgat cgcgacgata caagtcaggt tgccagctgc cgcagcagca
     5221 gcagtgccca gcaccacgag ttctgcacaa ggtcccccag taaaatgata tacattgaca
     5281 ccagtgaaga tgcggccgtc gctagagaga gctgcgctgg cgacgctgta gtcttcagag
     5341 atggggatgc tgttgattgt agccgttgct ctttcaatga gggtggattc ttcttgagac
     5401 aaaggcttgg ccatggtggc gctagtgtca gaagaatcga gctttttgca aaagcctagg
     5461 cctccaaaaa agcctcctca ctacttctgg aatagctcag aggccgaggc ggcctcggcc
     5521 tctgcataaa taaaaaaaat tagtcagcca tggggcggag aatgggcgga actgggcgga
     5581 gttaggggcg ggatgggcgg agttaggggc gggactatgg ttgctgacta attgagatgc
     5641 atgctttgca tacttctgcc tgctggggag cctggggact ttccacacct ggttgctgac
     5701 taattgagat gcatgctttg catacttctg cctgctgggg agcctgggga ctttccacac
     5761 cctaactgac acacattcca cagctggttc tttccgcctc agaaggtacc taaccaagtt
     5821 cctctttcag aggttatttc aggccatggt gctgcgc
//
