LOCUS       sequence-476614-        5386 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Express LgBiT-NRAS 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 5386)
  AUTHORS   Promega
  TITLE     Promega Plasmid Collection
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 5386)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: 
            "Unpublished"
COMMENT     Sequence Label: LgBiT-NRAS BiBiT-Ready Vector
FEATURES             Location/Qualifiers
     source          1..5386
                     /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"
     promoter        complement(1440..1458)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(1441..1458)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     enhancer        1578..1881
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     primer_bind     2036..2056
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     promoter        2089..2136
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             2168..2962
                     /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(2222..2241)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     2832..2851
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     rep_origin      complement(3120..3708)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3200..3219)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     polyA_signal    3834..3882
                     /note="synthetic polyadenylation signal"
     CDS             complement(3906..4943)
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
                     YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
                     ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
                     TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
                     GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
                     NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
                     EVGRV"
     RBS             3919..3927
                     /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(4974..5331)
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      4988..5123
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     enhancer        5140..5331
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     5288..5308
                     /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 ttacatcacc acacatggca atcccataca accctgagtc ccatcatcac tgctgttgag
      361 ttttttcatt cggtactggc gtatttctct taccagtgtg taaaaagcat cttcaacacc
      421 ctgtctggtc ttggctgagg tttcaatgaa tggaatcccg taactcttgg ccagttcgtg
      481 ggcttgtttt gtatcaactg tccttgttgg caaatcacac ttgtttccca ctagcaccat
      541 aggtacatca tccgagtctt ttactcgctt aatctgctcc ctgtagaggt taatatccgc
      601 aaatgacttg ctattattga tggcaaatac acagaggaag ccttcgcctg tcctcatgta
      661 ttggtctctc atggcactgt actcttcttg tccagctgta tccagtatgt ccaacaaaca
      721 ggtttcacca tctataacca cttgttttct gtaagaatcc tctatggtgg gatcatattc
      781 atctacaaag tggttctgga ttagctggat tgtcagtgcg cttttcccaa caccacctgc
      841 tccaaccacc accagtttgt actcagtcat ggcgatcgca ccgctcgagc ctccacctcc
      901 gctcccgcca ccaccggagc tcccactgtt gatggttact cggaacagca tggagccgtc
      961 gggggtgatc aggcgctcgt cgataatttt gttgccgttc cacagggtcc ctgttacagt
     1021 gatctttttg ccgtcgaaca cggcgatgcc ttcatacggc cgtccgaaat agttcagcat
     1081 gttcggcgta accccgtcga ttaccagtgt gccatagggc aggatcacct taaagtgatg
     1141 atcatccaca gggtacacca ccttaaacac ctcttcgatc tgggccattt ggtcggcgct
     1201 cagaccttca tacgggatga tgacatggat gtcgatcttc agggcatttt caccgctccg
     1261 gacaatcctt tggatcggag ttacggacac ggcgagattc tgcagcaaac tggacacacc
     1321 tccctgttca aggacttggt ccaggttgta ggcggctgtc tgttcccagt ccccaacgaa
     1381 atcttcgagt gtgaagacca tggtgagcgc tagctgcaaa aagaacaagt agcttgtatt
     1441 ctatagtgtc acctaaatgg gcgatctgac ggttcactaa accagctctg cttatatagg
     1501 cctccctggc gcgccactag ttattaatag taatcaatta cggggtcatt agttcatagc
     1561 ccatatatgg agttccgcgt tacataactt acggtaaatg gcccgcctgg ctgaccgccc
     1621 aacgaccccc gcccattgac gtcaataatg acgtatgttc ccatagtaac gccaataggg
     1681 actttccatt gacgtcaatg ggtggagtat ttacggtaaa ctgcccactt ggcagtacat
     1741 caagtgtatc atatgccaag tccgccccct attgacgtca atgacggtaa atggcccgcc
     1801 tggcattatg cccagtacat gaccttacgg gactttccta cttggcagta catctacgta
     1861 ttagtcatcg ctattaccat ggtgatgcgg ttttggcagt acaccaatgg gcgtggatag
     1921 cggtttgact cacggggatt tccaagtctc caccccattg acgtcaatgg gagtttgttt
     1981 tggcaccaaa atcaacggga ctttccaaaa tgtcgtaata accccgcccc gttgacgcaa
     2041 atgggcggta ggcgtgtacg gtgggaggtc aaatcgaatt ctaatactgt tgacaattaa
     2101 tcatcggcat agtatatcgg catagtataa tacgacaagg tgaggaacta aacccaggag
     2161 gcagatcatg attgaacaag atggattgca cgcaggttct ccggccgctt gggtggagag
     2221 gctattcggc tatgactggg cacaacagac aatcggctgc tctgatgccg ccgtgttccg
     2281 gctgtcagcg caggggcgcc cggttctttt tgtcaagacc gacctgtccg gtgccctgaa
     2341 tgaactgcag gacgaggcag cgcggctatc gtggctggcc acgacgggcg ttccttgcgc
     2401 agctgtgctc gacgttgtca ctgaagcggg aagggactgg ctgctattgg gcgaagtgcc
     2461 ggggcaggat ctcctgtcat ctcaccttgc tcctgccgag aaagtatcca tcatggctga
     2521 tgcaatgcgg cggctgcata cgcttgatcc ggctacctgc ccattcgacc accaagcgaa
     2581 acatcgcatc gagcgagcac gtactcggat ggaagccggt cttgtcgatc aggatgatct
     2641 ggacgaagag catcaggggc tcgcgccagc cgaactgttc gccaggctca aggcgcgcat
     2701 gcccgacggc gaggatctcg tcgtgaccca tggcgatgcc tgcttgccga atatcatggt
     2761 ggaaaatggc cgcttttctg gattcatcga ctgtggccgg ctgggtgtgg cggaccgcta
     2821 tcaggacata gcgttggcta cccgtgatat tgctgaagag cttggcggcg aatgggctga
     2881 ccgcttcctc gtgctttacg gtatcgccgc tcccgattcg cagcgcatcg ccttctatcg
     2941 ccttcttgac gagttcttct gattcgaaat gaccgaccaa gcgacgccca accggtatca
     3001 gctcactcaa aggcggtaat acggttatcc acagaatcag gggataacgc aggaaagaac
     3061 atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt
     3121 ttccataggc tccgcccccc tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg
     3181 cgaaacccga caggactata aagataccag gcgtttcccc ctggaagctc cctcgtgcgc
     3241 tctcctgttc cgaccctgcc gcttaccgga tacctgtccg cctttctccc ttcgggaagc
     3301 gtggcgcttt ctcatagctc acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc
     3361 aagctgggct gtgtgcacga accccccgtt cagcccgacc gctgcgcctt atccggtaac
     3421 tatcgtcttg agtccaaccc ggtaagacac gacttatcgc cactggcagc agccactggt
     3481 aacaggatta gcagagcgag gtatgtaggc ggtgctacag agttcttgaa gtggtggcct
     3541 aactacggct acactagaag gacagtattt ggtatctgcg ctctgctgaa gccagttacc
     3601 ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt
     3661 ttttttgttt gcaagcagca gattacgcgc agaaaaaaag gatttcaaga agatcctttg
     3721 atcttttcta cggggtctga cgctcagtgg aacgaaaact cacgttaagg gattttggtc
     3781 atgagattat caaaaaggat cttcacctag atccttttat agtccggaat cgtcacacaa
     3841 aaaaccaaca cacagatgta atgaaaataa agatatttta ttgcggccat cgtgatggct
     3901 agagtctaaa ctcgacctac ctccttagcg cgcggtcgtg tactgggccg gcggttgccg
     3961 ctgtcggcca gcacctcgac gcagccgtcg gtccatacgg ctgcgctccg gcgagcgatt
     4021 tgtgtgcgac cgacggtgcc ggccccgctg cggacgatgg catcgcagcg gccttgagcc
     4081 caggcagcat cgtcgaagtt gccgtccacg aggctctggt agagctgatc caggccgatg
     4141 cgcagcatgt aggctcgcag acgagggctg ccggccagct cgggatgccg gcgctcgaag
     4201 tagcgagtct gctgctccat gcaagccagc cagggccgcc agaagaagat gttggccacc
     4261 tcgtactgac tgtccccgaa catagcttcg gaccagtcga ttacggcggt gatgcggccg
     4321 ttgtcggtca ggacgttgtt gctgccgaag tcggcatgga cgaggtggcg cacctcggga
     4381 cagtcttcgg cccacagcat cagttcgtcc agggcttgag ctacgctggc ggacacggtg
     4441 tcgtccatca cggtctgcca gtggtagaca tggggatcag caatggcgca aatgaaatcc
     4501 cgccaggttg tgtactggcc gatgccttgg ggcccgaatg ggccgaagcc gctggtttga
     4561 ctgaggtcgg cggcggcaat agcatccata gcttcggcga caggctgtaa cacagcaggc
     4621 agctctgttt cggggaggtc ttggagagta acgccttggg cgcgtctact gatgcagtat
     4681 gtcaggctct cgctgaactc gccgatgtcc aacacttcgg ggatgggtag tgcagcgctg
     4741 gcgaagtggc ggtacacgta gcggtctttg tagaagccat cagcgcagct attcacccgc
     4801 agtacatagc cgcgtccgcc gacatcgaag ctgaaggctc ggctctcttc gccctccgac
     4861 aactgcatca ggtcgctcac actgtcgaac ttctcgatga gaaatttttc aacgctggta
     4921 gcggtgagtt cgggcttctt catggtggcg ctagtgtcag aagaatcgag ctttttgcaa
     4981 aagcctaggc ctccaaaaaa gcctcctcac tacttctgga atagctcaga ggccgaggcg
     5041 gcctcggcct ctgcataaat aaaaaaaatt agtcagccat ggggcggaga atgggcggaa
     5101 ctgggcggag ttaggggcgg gatgggcgga gttaggggcg ggactatggt tgctgactaa
     5161 ttgagatgca tgctttgcat acttctgcct gctggggagc ctggggactt tccacacctg
     5221 gttgctgact aattgagatg catgctttgc atacttctgc ctgctgggga gcctggggac
     5281 tttccacacc ctaactgaca cacattccac agctggttct ttccgcctca gaaggtacct
     5341 aaccaagttc ctctttcaga ggttatttca ggccatggtg ctgcgc
//
