LOCUS       sequence-471890-        5383 bp    DNA     circular SYN 14-JUL-2025
DEFINITION  Express LgBiT-KRAS(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 5383)
  TITLE     Promega Early Access Collection
REFERENCE   2  (bases 1 to 5383)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Jul 14, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"
COMMENT     Sequence Label: LgBiT-KRAS(G12D) Vector
FEATURES             Location/Qualifiers
     source          1..5383
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     polyA_signal    17..98
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     42..61
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     CDS             complement(join(301..831,832..834,835..867))
                     /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             complement(301..867)
                     /codon_start=1
                     /product="human small GTPase proto-oncoprotein"
                     /label=K-Ras
                     /translation="MTEYKLVVVGADGVGKSALTIQLIQNHFVDEYDPTIEDSYRKQVV
                     IDGETCLLDILDTAGQEEYSAMRDQYMRTGEGFLCVFAINNTKSFEDIHHYREQIKRVK
                     DSEDVPMVLVGNKCDLPSRTVDTKQAQDLARSYGIPFIETSAKTRQGVDDAFYTLVREI
                     RKHKEKMSKDGKKKKKKSKTKCVIM"
     promoter        complement(1437..1455)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(1438..1455)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     enhancer        1575..1878
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     primer_bind     2033..2053
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     promoter        2086..2133
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             2165..2959
                     /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(2219..2238)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     2829..2848
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     rep_origin      complement(3117..3705)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3197..3216)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     polyA_signal    3831..3879
                     /note="synthetic polyadenylation signal"
     CDS             complement(3903..4940)
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
                     YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
                     ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
                     TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
                     GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
                     NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
                     EVGRV"
     RBS             3916..3924
                     /label=Shine-Dalgarno sequence
                     /note="full consensus sequence for ribosome-binding sites 
                     upstream of start codons in E. coli; complementary to a 
                     region in the 3' end of the 16S rRNA (Chen et al., 1994)"
     promoter        complement(4971..5328)
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      4985..5120
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     enhancer        5137..5328
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     5285..5305
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
ORIGIN
        1 aagacgcgtg acgtcggcag tgaaaaaaat gctttatttg tgaaatttgt gatgctattg
       61 ctttatttgt aaccattata agctgcaata aacaagttaa caacaacaat tgcattcatt
      121 ttatgtttca ggttcagggg gaggtgtggg aggtttttta aagcaagtaa aacctctaca
      181 aatgtggtat ggctgattat gatccgcggc cgcaatacgt cgacgttatc agctgacttc
      241 gtacgagagc ctaggtgttt ctagaggatc cccgggtacc gagcccgaat tcgtttaaac
      301 ttacataatt acacactttg tctttgactt ctttttcttc tttttaccat ctttgctcat
      361 cttttcttta tgttttcgaa tttctcgaac taatgtatag aaggcatcat caacaccctg
      421 tcttgtcttt gctgatgttt caataaaagg aattccataa cttcttgcta agtcctgagc
      481 ctgttttgtg tctactgttc tagaaggcaa atcacattta tttcctacta ggaccatagg
      541 tacatcttca gagtccttaa ctcttttaat ttgttctcta taatggtgaa tatcttcaaa
      601 tgatttagta ttatttatgg caaatacaca aagaaagccc tccccagtcc tcatgtactg
      661 gtccctcatt gcactgtact cctcttgacc tgctgtgtcg agaatatcca agagacaggt
      721 ttctccatca attactactt gcttcctgta ggaatcctct attgttggat catattcgtc
      781 cacaaaatga ttctgaatta gctgtatcgt caaggcactc ttgcctacgc cgtcagctcc
      841 aactaccaca agtttatatt cagtcatggc gatcgcaccg ctcgagcctc cacctccgct
      901 cccgccacca ccggagctcc cactgttgat ggttactcgg aacagcatgg agccgtcggg
      961 ggtgatcagg cgctcgtcga taattttgtt gccgttccac agggtccctg ttacagtgat
     1021 ctttttgccg tcgaacacgg cgatgccttc atacggccgt ccgaaatagt tcagcatgtt
     1081 cggcgtaacc ccgtcgatta ccagtgtgcc atagggcagg atcaccttaa agtgatgatc
     1141 atccacaggg tacaccacct taaacacctc ttcgatctgg gccatttggt cggcgctcag
     1201 accttcatac gggatgatga catggatgtc gatcttcagg gcattttcac cgctccggac
     1261 aatcctttgg atcggagtta cggacacggc gagattctgc agcaaactgg acacacctcc
     1321 ctgttcaagg acttggtcca ggttgtaggc ggctgtctgt tcccagtccc caacgaaatc
     1381 ttcgagtgtg aagaccatgg tgagcgctag ctgcaaaaag aacaagtagc ttgtattcta
     1441 tagtgtcacc taaatgggcg atctgacggt tcactaaacc agctctgctt atataggcct
     1501 ccctggcgcg ccactagtta ttaatagtaa tcaattacgg ggtcattagt tcatagccca
     1561 tatatggagt tccgcgttac ataacttacg gtaaatggcc cgcctggctg accgcccaac
     1621 gacccccgcc cattgacgtc aataatgacg tatgttccca tagtaacgcc aatagggact
     1681 ttccattgac gtcaatgggt ggagtattta cggtaaactg cccacttggc agtacatcaa
     1741 gtgtatcata tgccaagtcc gccccctatt gacgtcaatg acggtaaatg gcccgcctgg
     1801 cattatgccc agtacatgac cttacgggac tttcctactt ggcagtacat ctacgtatta
     1861 gtcatcgcta ttaccatggt gatgcggttt tggcagtaca ccaatgggcg tggatagcgg
     1921 tttgactcac ggggatttcc aagtctccac cccattgacg tcaatgggag tttgttttgg
     1981 caccaaaatc aacgggactt tccaaaatgt cgtaataacc ccgccccgtt gacgcaaatg
     2041 ggcggtaggc gtgtacggtg ggaggtcaaa tcgaattcta atactgttga caattaatca
     2101 tcggcatagt atatcggcat agtataatac gacaaggtga ggaactaaac ccaggaggca
     2161 gatcatgatt gaacaagatg gattgcacgc aggttctccg gccgcttggg tggagaggct
     2221 attcggctat gactgggcac aacagacaat cggctgctct gatgccgccg tgttccggct
     2281 gtcagcgcag gggcgcccgg ttctttttgt caagaccgac ctgtccggtg ccctgaatga
     2341 actgcaggac gaggcagcgc ggctatcgtg gctggccacg acgggcgttc cttgcgcagc
     2401 tgtgctcgac gttgtcactg aagcgggaag ggactggctg ctattgggcg aagtgccggg
     2461 gcaggatctc ctgtcatctc accttgctcc tgccgagaaa gtatccatca tggctgatgc
     2521 aatgcggcgg ctgcatacgc ttgatccggc tacctgccca ttcgaccacc aagcgaaaca
     2581 tcgcatcgag cgagcacgta ctcggatgga agccggtctt gtcgatcagg atgatctgga
     2641 cgaagagcat caggggctcg cgccagccga actgttcgcc aggctcaagg cgcgcatgcc
     2701 cgacggcgag gatctcgtcg tgacccatgg cgatgcctgc ttgccgaata tcatggtgga
     2761 aaatggccgc ttttctggat tcatcgactg tggccggctg ggtgtggcgg accgctatca
     2821 ggacatagcg ttggctaccc gtgatattgc tgaagagctt ggcggcgaat gggctgaccg
     2881 cttcctcgtg ctttacggta tcgccgctcc cgattcgcag cgcatcgcct tctatcgcct
     2941 tcttgacgag ttcttctgat tcgaaatgac cgaccaagcg acgcccaacc ggtatcagct
     3001 cactcaaagg cggtaatacg gttatccaca gaatcagggg ataacgcagg aaagaacatg
     3061 tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc
     3121 cataggctcc gcccccctga cgagcatcac aaaaatcgac gctcaagtca gaggtggcga
     3181 aacccgacag gactataaag ataccaggcg tttccccctg gaagctccct cgtgcgctct
     3241 cctgttccga ccctgccgct taccggatac ctgtccgcct ttctcccttc gggaagcgtg
     3301 gcgctttctc atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag
     3361 ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct gcgccttatc cggtaactat
     3421 cgtcttgagt ccaacccggt aagacacgac ttatcgccac tggcagcagc cactggtaac
     3481 aggattagca gagcgaggta tgtaggcggt gctacagagt tcttgaagtg gtggcctaac
     3541 tacggctaca ctagaaggac agtatttggt atctgcgctc tgctgaagcc agttaccttc
     3601 ggaaaaagag ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggtggtttt
     3661 tttgtttgca agcagcagat tacgcgcaga aaaaaaggat ttcaagaaga tcctttgatc
     3721 ttttctacgg ggtctgacgc tcagtggaac gaaaactcac gttaagggat tttggtcatg
     3781 agattatcaa aaaggatctt cacctagatc cttttatagt ccggaatcgt cacacaaaaa
     3841 accaacacac agatgtaatg aaaataaaga tattttattg cggccatcgt gatggctaga
     3901 gtctaaactc gacctacctc cttagcgcgc ggtcgtgtac tgggccggcg gttgccgctg
     3961 tcggccagca cctcgacgca gccgtcggtc catacggctg cgctccggcg agcgatttgt
     4021 gtgcgaccga cggtgccggc cccgctgcgg acgatggcat cgcagcggcc ttgagcccag
     4081 gcagcatcgt cgaagttgcc gtccacgagg ctctggtaga gctgatccag gccgatgcgc
     4141 agcatgtagg ctcgcagacg agggctgccg gccagctcgg gatgccggcg ctcgaagtag
     4201 cgagtctgct gctccatgca agccagccag ggccgccaga agaagatgtt ggccacctcg
     4261 tactgactgt ccccgaacat agcttcggac cagtcgatta cggcggtgat gcggccgttg
     4321 tcggtcagga cgttgttgct gccgaagtcg gcatggacga ggtggcgcac ctcgggacag
     4381 tcttcggccc acagcatcag ttcgtccagg gcttgagcta cgctggcgga cacggtgtcg
     4441 tccatcacgg tctgccagtg gtagacatgg ggatcagcaa tggcgcaaat gaaatcccgc
     4501 caggttgtgt actggccgat gccttggggc ccgaatgggc cgaagccgct ggtttgactg
     4561 aggtcggcgg cggcaatagc atccatagct tcggcgacag gctgtaacac agcaggcagc
     4621 tctgtttcgg ggaggtcttg gagagtaacg ccttgggcgc gtctactgat gcagtatgtc
     4681 aggctctcgc tgaactcgcc gatgtccaac acttcgggga tgggtagtgc agcgctggcg
     4741 aagtggcggt acacgtagcg gtctttgtag aagccatcag cgcagctatt cacccgcagt
     4801 acatagccgc gtccgccgac atcgaagctg aaggctcggc tctcttcgcc ctccgacaac
     4861 tgcatcaggt cgctcacact gtcgaacttc tcgatgagaa atttttcaac gctggtagcg
     4921 gtgagttcgg gcttcttcat ggtggcgcta gtgtcagaag aatcgagctt tttgcaaaag
     4981 cctaggcctc caaaaaagcc tcctcactac ttctggaata gctcagaggc cgaggcggcc
     5041 tcggcctctg cataaataaa aaaaattagt cagccatggg gcggagaatg ggcggaactg
     5101 ggcggagtta ggggcgggat gggcggagtt aggggcggga ctatggttgc tgactaattg
     5161 agatgcatgc tttgcatact tctgcctgct ggggagcctg gggactttcc acacctggtt
     5221 gctgactaat tgagatgcat gctttgcata cttctgcctg ctggggagcc tggggacttt
     5281 ccacacccta actgacacac attccacagc tggttctttc cgcctcagaa ggtacctaac
     5341 caagttcctc tttcagaggt tatttcaggc catggtgctg cgc
//
