LOCUS       sequence-476795-        4898 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Express SmBiT-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 4898)
  AUTHORS   Promega
  TITLE     Promega Plasmid Collection
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 4898)
  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            "Unpublished"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: SmBiT-NRAS BiBiT-Ready Vector
FEATURES             Location/Qualifiers
     source          1..4898
                     /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"
     polyA_signal    1961..2082
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1972..1991)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     protein_bind    complement(2197..2230)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     promoter        2240..2287
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             2319..3113
                     /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(2373..2392)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     2983..3002
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     rep_origin      complement(3271..3859)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3351..3370)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     polyA_signal    3985..4033
                     /note="synthetic polyadenylation signal"
     CDS             complement(4057..4455)
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
                     VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
                     KAIVKDSDGQPTAVGIRELLPSGYVWEG"
     regulatory      4452..4461
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     promoter        complement(4486..4843)
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      4500..4635
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     enhancer        4652..4843
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     4800..4820
                     /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 gactgagtac aaactggtgg tggttggagc aggtggtgtt
     1321 gggaaaagcg cactgacaat ccagctaatc cagaaccact ttgtagatga atatgatccc
     1381 accatagagg attcttacag aaaacaagtg gttatagatg gtgaaacctg tttgttggac
     1441 atactggata cagctggaca agaagagtac agtgccatga gagaccaata catgaggaca
     1501 ggcgaaggct tcctctgtgt atttgccatc aataatagca agtcatttgc ggatattaac
     1561 ctctacaggg agcagattaa gcgagtaaaa gactcggatg atgtacctat ggtgctagtg
     1621 ggaaacaagt gtgatttgcc aacaaggaca gttgatacaa aacaagccca cgaactggcc
     1681 aagagttacg ggattccatt cattgaaacc tcagccaaga ccagacaggg tgttgaagat
     1741 gctttttaca cactggtaag agaaatacgc cagtaccgaa tgaaaaaact caacagcagt
     1801 gatgatggga ctcagggttg tatgggattg ccatgtgtgg tgatgtaagt ttaaacgaat
     1861 tcgggctcgg tacccgggga tcctctagag tcgacctgca ggcatgcaag ctgatttggc
     1921 tgctgccacc gctgagcaag gccgcttcga gcagacatga taagatacat tgatgagttt
     1981 ggacaaacca caactagaat gcagtgaaaa aaatgcttta tttgtgaaat ttgtgatgct
     2041 attgctttat ttgtaaccat tataagctgc aataaacaag ttaacaacaa caattgcatt
     2101 cattttatgt ttcaggttca gggggagatg tgggaggttt ttttaagcaa gtaaaacctc
     2161 tacaaatgtg gtaaaatctt aattaagaat tctaatataa cttcgtatag catacattat
     2221 acgaagttat ggatccactg ttgacaatta atcatcggca tagtatatcg gcatagtata
     2281 atacgacaag gtgaggaact aaacccagga ggcagatcat gattgaacaa gatggattgc
     2341 acgcaggttc tccggccgct tgggtggaga ggctattcgg ctatgactgg gcacaacaga
     2401 caatcggctg ctctgatgcc gccgtgttcc ggctgtcagc gcaggggcgc ccggttcttt
     2461 ttgtcaagac cgacctgtcc ggtgccctga atgaactgca ggacgaggca gcgcggctat
     2521 cgtggctggc cacgacgggc gttccttgcg cagctgtgct cgacgttgtc actgaagcgg
     2581 gaagggactg gctgctattg ggcgaagtgc cggggcagga tctcctgtca tctcaccttg
     2641 ctcctgccga gaaagtatcc atcatggctg atgcaatgcg gcggctgcat acgcttgatc
     2701 cggctacctg cccattcgac caccaagcga aacatcgcat cgagcgagca cgtactcgga
     2761 tggaagccgg tcttgtcgat caggatgatc tggacgaaga gcatcagggg ctcgcgccag
     2821 ccgaactgtt cgccaggctc aaggcgcgca tgcccgacgg cgaggatctc gtcgtgaccc
     2881 atggcgatgc ctgcttgccg aatatcatgg tggaaaatgg ccgcttttct ggattcatcg
     2941 actgtggccg gctgggtgtg gcggaccgct atcaggacat agcgttggct acccgtgata
     3001 ttgctgaaga gcttggcggc gaatgggctg accgcttcct cgtgctttac ggtatcgccg
     3061 ctcccgattc gcagcgcatc gccttctatc gccttcttga cgagttcttc tgattcgaaa
     3121 tgaccgacca agcgacgccc aaccggtatc agctcactca aaggcggtaa tacggttatc
     3181 cacagaatca ggggataacg caggaaagaa catgtgagca aaaggccagc aaaaggccag
     3241 gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg ctccgccccc ctgacgagca
     3301 tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg acaggactat aaagatacca
     3361 ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg
     3421 atacctgtcc gcctttctcc cttcgggaag cgtggcgctt tctcatagct cacgctgtag
     3481 gtatctcagt tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt
     3541 tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc cggtaagaca
     3601 cgacttatcg ccactggcag cagccactgg taacaggatt agcagagcga ggtatgtagg
     3661 cggtgctaca gagttcttga agtggtggcc taactacggc tacactagaa ggacagtatt
     3721 tggtatctgc gctctgctga agccagttac cttcggaaaa agagttggta gctcttgatc
     3781 cggcaaacaa accaccgctg gtagcggtgg tttttttgtt tgcaagcagc agattacgcg
     3841 cagaaaaaaa ggatttcaag aagatccttt gatcttttct acggggtctg acgctcagtg
     3901 gaacgaaaac tcacgttaag ggattttggt catgagatta tcaaaaagga tcttcaccta
     3961 gatcctttta tagtccggaa tcgtcacaca aaaaaccaac acacagatgt aatgaaaata
     4021 aagatatttt attgcggcca tcgtgatggc tagagtttag ccctcccaca cataaccaga
     4081 gggcagcaat tcacgaatcc caactgccgt cggctgtcca tcactgtcct tcactatggc
     4141 tttgatccca ggatgcagat cgagaagcac ctgtcggcac cgtccgcagg ggctcaagat
     4201 gcccctgttc tcatttccga tcgcgacgat acaagtcagg ttgccagctg ccgcagcagc
     4261 agcagtgccc agcaccacga gttctgcaca aggtccccca gtaaaatgat atacattgac
     4321 accagtgaag atgcggccgt cgctagagag agctgcgctg gcgacgctgt agtcttcaga
     4381 gatggggatg ctgttgattg tagccgttgc tctttcaatg agggtggatt cttcttgaga
     4441 caaaggcttg gccatggtgg cgctagtgtc agaagaatcg agctttttgc aaaagcctag
     4501 gcctccaaaa aagcctcctc actacttctg gaatagctca gaggccgagg cggcctcggc
     4561 ctctgcataa ataaaaaaaa ttagtcagcc atggggcgga gaatgggcgg aactgggcgg
     4621 agttaggggc gggatgggcg gagttagggg cgggactatg gttgctgact aattgagatg
     4681 catgctttgc atacttctgc ctgctgggga gcctggggac tttccacacc tggttgctga
     4741 ctaattgaga tgcatgcttt gcatacttct gcctgctggg gagcctgggg actttccaca
     4801 ccctaactga cacacattcc acagctggtt ctttccgcct cagaaggtac ctaaccaagt
     4861 tcctctttca gaggttattt caggccatgg tgctgcgc
//
