LOCUS       sequence-479923-        5102 bp    DNA     circular SYN 07-AUG-2025
DEFINITION  Express SmBiT-KRAS(G12V) 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)
  AUTHORS   Promega
  TITLE     Promega Plasmid Collection
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 5102)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Aug 7, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: 
            "Unpublished"
COMMENT     Sequence Label: SmBiT-KRAS(G12V) BiBiT Ready Vector
FEATURES             Location/Qualifiers
     source          1..5102
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     polyA_signal    24..105
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     enhancer        338..641
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        642..845
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     796..816
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     intron          981..1113
                     /label=chimeric intron
                     /note="chimera between introns from human beta-globin and 
                     immunoglobulin heavy chain genes"
     primer_bind     1096..1115
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     primer_bind     1157..1176
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1157..1175
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             join(1279..1311,1312..1314,1315..1845)
                     /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"
     polyA_signal    1991..2112
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2002..2021)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     protein_bind    complement(2227..2260)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     promoter        2270..2317
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             2349..3143
                     /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(2403..2422)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3013..3032
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     rep_origin      3360..3905
                     /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     3806..3825
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4022..4039
                     /label=L4440
                     /note="L4440 vector, forward primer"
     polyA_signal    4189..4237
                     /note="synthetic polyadenylation signal"
     CDS             complement(4261..4659)
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
                     VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
                     KAIVKDSDGQPTAVGIRELLPSGYVWEG"
     regulatory      4656..4665
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     promoter        complement(4690..5047)
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      4704..4839
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     enhancer        4856..5047
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     5004..5024
                     /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 ggattatggc gcgccactag ttattaatag taatcaatta
      301 cggggtcatt agttcatagc ccatatatgg agttccgcgt tacataactt acggtaaatg
      361 gcccgcctgg ctgaccgccc aacgaccccc gcccattgac gtcaataatg acgtatgttc
      421 ccatagtaac gccaataggg actttccatt gacgtcaatg ggtggagtat ttacggtaaa
      481 ctgcccactt ggcagtacat caagtgtatc atatgccaag tccgccccct attgacgtca
      541 atgacggtaa atggcccgcc tggcattatg cccagtacat gaccttacgg gactttccta
      601 cttggcagta catctacgta ttagtcatcg ctattaccat ggtgatgcgg ttttggcagt
      661 acaccaatgg gcgtggatag cggtttgact cacggggatt tccaagtctc caccccattg
      721 acgtcaatgg gagtttgttt tggcaccaaa atcaacggga ctttccaaaa tgtcgtaata
      781 accccgcccc gttgacgcaa atgggcggta ggcgtgtacg gtgggaggtc tatataagca
      841 gagctggttt agtgaaccgt cagatcacta gaagctttat tgcggtagtt tatcacagtt
      901 aaattgctaa cgcagtcagt gcttctgaca caacagtctc gaacttaagc tgcagaagtt
      961 ggtcgtgagg cactgggcag gtaagtatca aggttacaag acaggtttaa ggagaccaat
     1021 agaaactggg cttgtcgaga cagagaagac tcttgcgttt ctgataggca cctattggtc
     1081 ttactgacat ccactttgcc tttctctcca caggtgtcca ctcccagttc aattacagct
     1141 cttaaggcta gagtattaat acgactcact atagggctag cgctcaccat ggtgaccggc
     1201 taccggctgt tcgaggagat tctcgggagc tccggtggtg gcgggagcgg aggtggaggc
     1261 tcgagcggtg cgatcgccat gactgaatat aaacttgtgg tagttggagc tgttggcgta
     1321 ggcaagagtg ccttgacgat acagctaatt cagaatcatt ttgtggacga atatgatcca
     1381 acaatagagg attcctacag gaagcaagta gtaattgatg gagaaacctg tctcttggat
     1441 attctcgaca cagcaggtca agaggagtac agtgcaatga gggaccagta catgaggact
     1501 ggggagggct ttctttgtgt atttgccata aataatacta aatcatttga agatattcac
     1561 cattatagag aacaaattaa aagagttaag gactctgaag atgtacctat ggtcctagta
     1621 ggaaataaat gtgatttgcc ttctagaaca gtagacacaa aacaggctca ggacttagca
     1681 agaagttatg gaattccttt tattgaaaca tcagcaaaga caagacaggg tgttgatgat
     1741 gccttctata cattagttcg agaaattcga aaacataaag aaaagatgag caaagatggt
     1801 aaaaagaaga aaaagaagtc aaagacaaag tgtgtaatta tgtaagttta aacgaattcg
     1861 ggctcggtac ccggggatcc tctagagtcg acctgcaggc atgcaagctg atccggctgc
     1921 taacaaagcc cgaaaggaag ctgagttggc tgctgccacc gctgagcaag gccgcttcga
     1981 gcagacatga taagatacat tgatgagttt ggacaaacca caactagaat gcagtgaaaa
     2041 aaatgcttta tttgtgaaat ttgtgatgct attgctttat ttgtaaccat tataagctgc
     2101 aataaacaag ttaacaacaa caattgcatt cattttatgt ttcaggttca gggggagatg
     2161 tgggaggttt ttttaagcaa gtaaaacctc tacaaatgtg gtaaaatctt aattaagaat
     2221 tctaatataa cttcgtatag catacattat acgaagttat ggatccactg ttgacaatta
     2281 atcatcggca tagtatatcg gcatagtata atacgacaag gtgaggaact aaacccagga
     2341 ggcagatcat gattgaacaa gatggattgc acgcaggttc tccggccgct tgggtggaga
     2401 ggctattcgg ctatgactgg gcacaacaga caatcggctg ctctgatgcc gccgtgttcc
     2461 ggctgtcagc gcaggggcgc ccggttcttt ttgtcaagac cgacctgtcc ggtgccctga
     2521 atgaactgca ggacgaggca gcgcggctat cgtggctggc cacgacgggc gttccttgcg
     2581 cagctgtgct cgacgttgtc actgaagcgg gaagggactg gctgctattg ggcgaagtgc
     2641 cggggcagga tctcctgtca tctcaccttg ctcctgccga gaaagtatcc atcatggctg
     2701 atgcaatgcg gcggctgcat acgcttgatc cggctacctg cccattcgac caccaagcga
     2761 aacatcgcat cgagcgagca cgtactcgga tggaagccgg tcttgtcgat caggatgatc
     2821 tggacgaaga gcatcagggg ctcgcgccag ccgaactgtt cgccaggctc aaggcgcgca
     2881 tgcccgacgg cgaggatctc gtcgtgaccc atggcgatgc ctgcttgccg aatatcatgg
     2941 tggaaaatgg ccgcttttct ggattcatcg actgtggccg gctgggtgtg gcggaccgct
     3001 atcaggacat agcgttggct acccgtgata ttgctgaaga gcttggcggc gaatgggctg
     3061 accgcttcct cgtgctttac ggtatcgccg ctcccgattc gcagcgcatc gccttctatc
     3121 gccttcttga cgagttcttc tgattcgaaa tgaccgacca agcgacgccc aaccggtatc
     3181 agctcactca aaggcggtaa tacggttatc cacagaatca ggggataacg caggaaagaa
     3241 catgtcaaac atgagaatta caacttatat cgtatggggc tgacttcagg tgctacattt
     3301 gaagagataa attgcactga aatctagaaa tattttatct gattaataag atgatcttct
     3361 tgagatcgtt ttggtctgcg cgtaatctct tgctctgaaa acgaaaaaac cgccttgcag
     3421 ggcggttttt cgaaggttct ctgagctacc aactctttga accgaggtaa ctggcttgga
     3481 ggagcgcagt caccaaaact tgtcctttca gtttagcctt aaccggcgca tgacttcaag
     3541 actaactcct ctaaatcaat taccagtggc tgctgccagt ggtgcttttg catgtctttc
     3601 cgggttggac tcaagacgat agttaccgga taaggcgcag cggtcggact gaacgggggg
     3661 ttcgtgcata cagtccagct tggagcgaac tgcctacccg gaactgagtg tcaggcgtgg
     3721 aatgagacaa acgcggccat aacagcggaa tgacaccggt aaaccgaaag gcaggaacag
     3781 gagagcgcac gagggagccg ccagggggaa acgcctggta tctttatagt cctgtcgggt
     3841 ttcgccacca ctgatttgag cgtcagattt cgtgatgctt gtcagggggg cggagcctat
     3901 ggaaaaacgg ctttgccgcg gccctctcac ttccctgtta agtatcttcc tggcatcttc
     3961 caggaaatct ccgccccgtt cgtaagccat ttccgctcgc cgcagtcgaa cgaccgagcg
     4021 tagcgagtca gtgagcgagg aagcggaata tatcctgtat cacatattct gctgacgcac
     4081 cggtgcagcc ttttttctcc tgccacatga agcacttcac tgacaccctc atcagtgcca
     4141 acatagtaag ccagtataca ctccgctagc gctgatgtcc ggaatcgtca cacaaaaaac
     4201 caacacacag atgtaatgaa aataaagata ttttattgcg gccatcgtga tggctagagt
     4261 ttagccctcc cacacataac cagagggcag caattcacga atcccaactg ccgtcggctg
     4321 tccatcactg tccttcacta tggctttgat cccaggatgc agatcgagaa gcacctgtcg
     4381 gcaccgtccg caggggctca agatgcccct gttctcattt ccgatcgcga cgatacaagt
     4441 caggttgcca gctgccgcag cagcagcagt gcccagcacc acgagttctg cacaaggtcc
     4501 cccagtaaaa tgatatacat tgacaccagt gaagatgcgg ccgtcgctag agagagctgc
     4561 gctggcgacg ctgtagtctt cagagatggg gatgctgttg attgtagccg ttgctctttc
     4621 aatgagggtg gattcttctt gagacaaagg cttggccatg gtggcgctag tgtcagaaga
     4681 atcgagcttt ttgcaaaagc ctaggcctcc aaaaaagcct cctcactact tctggaatag
     4741 ctcagaggcc gaggcggcct cggcctctgc ataaataaaa aaaattagtc agccatgggg
     4801 cggagaatgg gcggaactgg gcggagttag gggcgggatg ggcggagtta ggggcgggac
     4861 tatggttgct gactaattga gatgcatgct ttgcatactt ctgcctgctg gggagcctgg
     4921 ggactttcca cacctggttg ctgactaatt gagatgcatg ctttgcatac ttctgcctgc
     4981 tggggagcct ggggactttc cacaccctaa ctgacacaca ttccacagct ggttctttcc
     5041 gcctcagaag gtacctaacc aagttcctct ttcagaggtt atttcaggcc atggtgctgc
     5101 gc
//
