LOCUS       sequence-473530-        5102 bp    DNA     circular SYN 21-MAY-2025
DEFINITION  Express SmBiT-KRAS4B(G12D) 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 5102)
  TITLE     Promega Early Access Collection
REFERENCE   2  (bases 1 to 5102)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 21, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"
COMMENT     Sequence Label: SmBiT-KRAS4B(G12D) BiBiT-Ready Vector
FEATURES             Location/Qualifiers
     source          1..5102
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      1..546
                     /direction=RIGHT
                     /label=p15A ori
                     /note="Plasmids containing the medium-copy-number p15A 
                     origin of replication can be propagated in E. coli cells 
                     that contain a second plasmid with the ColE1 origin.
                     "
     primer_bind     447..466
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     663..680
                     /label=L4440
                     /note="L4440 vector, forward primer"
     polyA_signal    830..878
                     /note="synthetic polyadenylation signal"
     CDS             complement(902..1300)
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
                     VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
                     KAIVKDSDGQPTAVGIRELLPSGYVWEG"
     regulatory      1297..1306
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     promoter        complement(1331..1688)
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      1345..1480
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     enhancer        1497..1688
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     1645..1665
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     polyA_signal    1767..1848
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     enhancer        2081..2384
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        2385..2588
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     2539..2559
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     intron          2724..2856
                     /label=chimeric intron
                     /note="chimera between introns from human beta-globin and 
                     immunoglobulin heavy chain genes"
     primer_bind     2839..2858
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     primer_bind     2900..2919
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        2900..2918
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             join(3022..3054,3055..3057,3058..3588)
                     /codon_start=1
                     /product="human oncoprotein generated by the G12V mutation 
                     in the small GTPase K-Ras"
                     /label=K-Ras (G12V)
                     /translation="MTEYKLVVVGADGVGKSALTIQLIQNHFVDEYDPTIEDSYRKQVV
                     IDGETCLLDILDTAGQEEYSAMRDQYMRTGEGFLCVFAINNTKSFEDIHHYREQIKRVK
                     DSEDVPMVLVGNKCDLPSRTVDTKQAQDLARSYGIPFIETSAKTRQGVDDAFYTLVREI
                     RKHKEKMSKDGKKKKKKSKTKCVIM"
     CDS             3022..3588
                     /codon_start=1
                     /product="human small GTPase proto-oncoprotein"
                     /label=K-Ras
                     /translation="MTEYKLVVVGADGVGKSALTIQLIQNHFVDEYDPTIEDSYRKQVV
                     IDGETCLLDILDTAGQEEYSAMRDQYMRTGEGFLCVFAINNTKSFEDIHHYREQIKRVK
                     DSEDVPMVLVGNKCDLPSRTVDTKQAQDLARSYGIPFIETSAKTRQGVDDAFYTLVREI
                     RKHKEKMSKDGKKKKKKSKTKCVIM"
     polyA_signal    3734..3855
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3745..3764)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     protein_bind    complement(3970..4003)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     promoter        4013..4060
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             4092..4886
                     /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(4146..4165)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4756..4775
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
ORIGIN
        1 ttgagatcgt tttggtctgc gcgtaatctc ttgctctgaa aacgaaaaaa ccgccttgca
       61 gggcggtttt tcgaaggttc tctgagctac caactctttg aaccgaggta actggcttgg
      121 aggagcgcag tcaccaaaac ttgtcctttc agtttagcct taaccggcgc atgacttcaa
      181 gactaactcc tctaaatcaa ttaccagtgg ctgctgccag tggtgctttt gcatgtcttt
      241 ccgggttgga ctcaagacga tagttaccgg ataaggcgca gcggtcggac tgaacggggg
      301 gttcgtgcat acagtccagc ttggagcgaa ctgcctaccc ggaactgagt gtcaggcgtg
      361 gaatgagaca aacgcggcca taacagcgga atgacaccgg taaaccgaaa ggcaggaaca
      421 ggagagcgca cgagggagcc gccaggggga aacgcctggt atctttatag tcctgtcggg
      481 tttcgccacc actgatttga gcgtcagatt tcgtgatgct tgtcaggggg gcggagccta
      541 tggaaaaacg gctttgccgc ggccctctca cttccctgtt aagtatcttc ctggcatctt
      601 ccaggaaatc tccgccccgt tcgtaagcca tttccgctcg ccgcagtcga acgaccgagc
      661 gtagcgagtc agtgagcgag gaagcggaat atatcctgta tcacatattc tgctgacgca
      721 ccggtgcagc cttttttctc ctgccacatg aagcacttca ctgacaccct catcagtgcc
      781 aacatagtaa gccagtatac actccgctag cgctgatgtc cggaatcgtc acacaaaaaa
      841 ccaacacaca gatgtaatga aaataaagat attttattgc ggccatcgtg atggctagag
      901 tttagccctc ccacacataa ccagagggca gcaattcacg aatcccaact gccgtcggct
      961 gtccatcact gtccttcact atggctttga tcccaggatg cagatcgaga agcacctgtc
     1021 ggcaccgtcc gcaggggctc aagatgcccc tgttctcatt tccgatcgcg acgatacaag
     1081 tcaggttgcc agctgccgca gcagcagcag tgcccagcac cacgagttct gcacaaggtc
     1141 ccccagtaaa atgatataca ttgacaccag tgaagatgcg gccgtcgcta gagagagctg
     1201 cgctggcgac gctgtagtct tcagagatgg ggatgctgtt gattgtagcc gttgctcttt
     1261 caatgagggt ggattcttct tgagacaaag gcttggccat ggtggcgcta gtgtcagaag
     1321 aatcgagctt tttgcaaaag cctaggcctc caaaaaagcc tcctcactac ttctggaata
     1381 gctcagaggc cgaggcggcc tcggcctctg cataaataaa aaaaattagt cagccatggg
     1441 gcggagaatg ggcggaactg ggcggagtta ggggcgggat gggcggagtt aggggcggga
     1501 ctatggttgc tgactaattg agatgcatgc tttgcatact tctgcctgct ggggagcctg
     1561 gggactttcc acacctggtt gctgactaat tgagatgcat gctttgcata cttctgcctg
     1621 ctggggagcc tggggacttt ccacacccta actgacacac attccacagc tggttctttc
     1681 cgcctcagaa ggtacctaac caagttcctc tttcagaggt tatttcaggc catggtgctg
     1741 cgcaagacgc gttaattaag acgtcggcag tgaaaaaaat gctttatttg tgaaatttgt
     1801 gatgctattg ctttatttgt aaccattata agctgcaata aacaagttaa caacaacaat
     1861 tgcattcatt ttatgtttca ggttcagggg gaggtgtggg aggtttttta aagcaagtaa
     1921 aacctctaca aatgtggtat ggctgattat gatccgcggc cgcaatacgt cgacgttatc
     1981 agctgacttc gtacgagagc ctaggattat ggcgcgccac tagttattaa tagtaatcaa
     2041 ttacggggtc attagttcat agcccatata tggagttccg cgttacataa cttacggtaa
     2101 atggcccgcc tggctgaccg cccaacgacc cccgcccatt gacgtcaata atgacgtatg
     2161 ttcccatagt aacgccaata gggactttcc attgacgtca atgggtggag tatttacggt
     2221 aaactgccca cttggcagta catcaagtgt atcatatgcc aagtccgccc cctattgacg
     2281 tcaatgacgg taaatggccc gcctggcatt atgcccagta catgacctta cgggactttc
     2341 ctacttggca gtacatctac gtattagtca tcgctattac catggtgatg cggttttggc
     2401 agtacaccaa tgggcgtgga tagcggtttg actcacgggg atttccaagt ctccacccca
     2461 ttgacgtcaa tgggagtttg ttttggcacc aaaatcaacg ggactttcca aaatgtcgta
     2521 ataaccccgc cccgttgacg caaatgggcg gtaggcgtgt acggtgggag gtctatataa
     2581 gcagagctgg tttagtgaac cgtcagatca ctagaagctt tattgcggta gtttatcaca
     2641 gttaaattgc taacgcagtc agtgcttctg acacaacagt ctcgaactta agctgcagaa
     2701 gttggtcgtg aggcactggg caggtaagta tcaaggttac aagacaggtt taaggagacc
     2761 aatagaaact gggcttgtcg agacagagaa gactcttgcg tttctgatag gcacctattg
     2821 gtcttactga catccacttt gcctttctct ccacaggtgt ccactcccag ttcaattaca
     2881 gctcttaagg ctagagtatt aatacgactc actatagggc tagcgctcac catggtgacc
     2941 ggctaccggc tgttcgagga gattctcggg agctccggtg gtggcgggag cggaggtgga
     3001 ggctcgagcg gtgcgatcgc catgactgaa tataaacttg tggtagttgg agctgacggc
     3061 gtaggcaaga gtgccttgac gatacagcta attcagaatc attttgtgga cgaatatgat
     3121 ccaacaatag aggattccta caggaagcaa gtagtaattg atggagaaac ctgtctcttg
     3181 gatattctcg acacagcagg tcaagaggag tacagtgcaa tgagggacca gtacatgagg
     3241 actggggagg gctttctttg tgtatttgcc ataaataata ctaaatcatt tgaagatatt
     3301 caccattata gagaacaaat taaaagagtt aaggactctg aagatgtacc tatggtccta
     3361 gtaggaaata aatgtgattt gccttctaga acagtagaca caaaacaggc tcaggactta
     3421 gcaagaagtt atggaattcc ttttattgaa acatcagcaa agacaagaca gggtgttgat
     3481 gatgccttct atacattagt tcgagaaatt cgaaaacata aagaaaagat gagcaaagat
     3541 ggtaaaaaga agaaaaagaa gtcaaagaca aagtgtgtaa ttatgtaagt ttaaacgaat
     3601 tcgggctcgg tacccgggga tcctctagag tcgacctgca ggcatgcaag ctgatccggc
     3661 tgctaacaaa gcccgaaagg aagctgagtt ggctgctgcc accgctgagc aaggccgctt
     3721 cgagcagaca tgataagata cattgatgag tttggacaaa ccacaactag aatgcagtga
     3781 aaaaaatgct ttatttgtga aatttgtgat gctattgctt tatttgtaac cattataagc
     3841 tgcaataaac aagttaacaa caacaattgc attcatttta tgtttcaggt tcagggggag
     3901 atgtgggagg tttttttaag caagtaaaac ctctacaaat gtggtaaaat cttaattaag
     3961 aattctaata taacttcgta tagcatacat tatacgaagt tatggatcca ctgttgacaa
     4021 ttaatcatcg gcatagtata tcggcatagt ataatacgac aaggtgagga actaaaccca
     4081 ggaggcagat catgattgaa caagatggat tgcacgcagg ttctccggcc gcttgggtgg
     4141 agaggctatt cggctatgac tgggcacaac agacaatcgg ctgctctgat gccgccgtgt
     4201 tccggctgtc agcgcagggg cgcccggttc tttttgtcaa gaccgacctg tccggtgccc
     4261 tgaatgaact gcaggacgag gcagcgcggc tatcgtggct ggccacgacg ggcgttcctt
     4321 gcgcagctgt gctcgacgtt gtcactgaag cgggaaggga ctggctgcta ttgggcgaag
     4381 tgccggggca ggatctcctg tcatctcacc ttgctcctgc cgagaaagta tccatcatgg
     4441 ctgatgcaat gcggcggctg catacgcttg atccggctac ctgcccattc gaccaccaag
     4501 cgaaacatcg catcgagcga gcacgtactc ggatggaagc cggtcttgtc gatcaggatg
     4561 atctggacga agagcatcag gggctcgcgc cagccgaact gttcgccagg ctcaaggcgc
     4621 gcatgcccga cggcgaggat ctcgtcgtga cccatggcga tgcctgcttg ccgaatatca
     4681 tggtggaaaa tggccgcttt tctggattca tcgactgtgg ccggctgggt gtggcggacc
     4741 gctatcagga catagcgttg gctacccgtg atattgctga agagcttggc ggcgaatggg
     4801 ctgaccgctt cctcgtgctt tacggtatcg ccgctcccga ttcgcagcgc atcgccttct
     4861 atcgccttct tgacgagttc ttctgattcg aaatgaccga ccaagcgacg cccaaccggt
     4921 atcagctcac tcaaaggcgg taatacggtt atccacagaa tcaggggata acgcaggaaa
     4981 gaacatgtca aacatgagaa ttacaactta tatcgtatgg ggctgacttc aggtgctaca
     5041 tttgaagaga taaattgcac tgaaatctag aaatatttta tctgattaat aagatgatct
     5101 tc
//
