LOCUS       sequence-479829-        6228 bp    DNA     circular SYN 07-AUG-2025
DEFINITION  Express cRaf-SmBiT 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 6228)
  AUTHORS   Promega
  TITLE     Promega Plasmid Collection
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 6228)
  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            "Unpublished"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: NanoBiT Assay Vector, cRaf
FEATURES             Location/Qualifiers
     source          1..6228
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        138..517
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        518..721
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     672..692
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     intron          857..989
                     /label=chimeric intron
                     /note="chimera between introns from human beta-globin and 
                     immunoglobulin heavy chain genes"
     primer_bind     972..991
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     primer_bind     1033..1052
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1033..1051
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(3171..3189)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     polyA_signal    3226..3347
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3237..3256)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     3291..3310
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     promoter        3592..3949
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        3592..3783
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(3615..3635)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      3800..3935
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     promoter        3964..4011
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             4043..4837
                     /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(4097..4116)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4707..4726
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4901..4949
                     /note="synthetic polyadenylation signal"
     rep_origin      complement(5188..5776)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5268..5287)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     misc_feature    5892..6175
                     /label=cer region
                     /note="ColE1-derived recombination site that helps to 
                     maintain plasmids as monomers"
ORIGIN
        1 tcaatattgg ccattagcca tattattcat tggttatata gcataaatca atattggcta
       61 ttggccattg catacgttgt atctatatca taatatgtac atttatattg gctcatgtcc
      121 aatatgaccg ccatgttggc attgattatt gactagttat taatagtaat caattacggg
      181 gtcattagtt catagcccat atatggagtt ccgcgttaca taacttacgg taaatggccc
      241 gcctggctga ccgcccaacg acccccgccc attgacgtca ataatgacgt atgttcccat
      301 agtaacgcca atagggactt tccattgacg tcaatgggtg gagtatttac ggtaaactgc
      361 ccacttggca gtacatcaag tgtatcatat gccaagtccg ccccctattg acgtcaatga
      421 cggtaaatgg cccgcctggc attatgccca gtacatgacc ttacgggact ttcctacttg
      481 gcagtacatc tacgtattag tcatcgctat taccatggtg atgcggtttt ggcagtacac
      541 caatgggcgt ggatagcggt ttgactcacg gggatttcca agtctccacc ccattgacgt
      601 caatgggagt ttgttttggc accaaaatca acgggacttt ccaaaatgtc gtaataaccc
      661 cgccccgttg acgcaaatgg gcggtaggcg tgtacggtgg gaggtctata taagcagagc
      721 tggtttagtg aaccgtcaga tcactagaag ctttattgcg gtagtttatc acagttaaat
      781 tgctaacgca gtcagtgctt ctgacacaac agtctcgaac ttaagctgca gaagttggtc
      841 gtgaggcact gggcaggtaa gtatcaaggt tacaagacag gtttaaggag accaatagaa
      901 actgggcttg tcgagacaga gaagactctt gcgtttctga taggcaccta ttggtcttac
      961 tgacatccac tttgcctttc tctccacagg tgtccactcc cagttcaatt acagctctta
     1021 aggctagagt attaatacga ctcactatag ggctagcgat cgccatggag cacatacagg
     1081 gagcttggaa gacgatcagc aatggttttg gattcaaaga tgccgtgttt gatggctcca
     1141 gctgcatctc tcctacaata gttcagcagt ttggctatca gcgccgggca tcagatgatg
     1201 gcaaactcac agatccttct aagacaagca acactatccg tgttttcttg ccgaacaagc
     1261 aaagaacagt ggtcaatgtg cgaaatggaa tgagcttgca tgactgcctt atgaaagcac
     1321 tcaaggtgag gggcctgcaa ccagagtgct gtgcagtgtt cagacttctc cacgaacaca
     1381 aaggtaaaaa agcacgctta gattggaata ctgatgctgc gtctttgatt ggagaagaac
     1441 ttcaagtaga tttcctggat catgttcccc tcacaacaca caactttgct cggaagacgt
     1501 tcctgaagct tgccttctgt gacatctgtc agaaattcct gctcaatgga tttcgatgtc
     1561 agacttgtgg ctacaaattt catgagcact gtagcaccaa agtacctact atgtgtgtgg
     1621 actggagtaa catcagacaa ctcttattgt ttccaaattc cactattggt gatagtggag
     1681 tcccagcact accttctttg actatgcgtc gtatgcgaga gtctgtttcc aggatgcctg
     1741 ttagttctca gcacagatat tctacacctc acgccttcac ctttaacacc tccagtccct
     1801 catctgaagg ttccctctcc cagaggcaga ggtcgacatc cacacctaat gtccacatgg
     1861 tcagcaccac cctgcctgtg gacagcagga tgattgagga tgcaattcga agtcacagcg
     1921 aatcagcctc accttcagcc ctgtccagta gccccaacaa tctgagccca acaggctggt
     1981 cacagccgaa aacccccgtg ccagcacaaa gagagcgggc accagtatct gggacccagg
     2041 agaaaaacaa aattaggcct cgtggacaga gagattcaag ctattattgg gaaatagaag
     2101 ccagtgaagt gatgctgtcc actcggattg ggtcaggctc ttttggaact gtttataagg
     2161 gtaaatggca cggagatgtt gcagtaaaga tcctaaaggt tgtcgaccca accccagagc
     2221 aattccaggc cttcaggaat gaggtggctg ttctgcgcaa aacacggcat gtgaacattc
     2281 tgcttttcat ggggtacatg acaaaggaca acctggcaat tgtgacccag tggtgcgagg
     2341 gcagcagcct ctacaaacac ctgcatgtcc aggagaccaa gtttcagatg ttccagctaa
     2401 ttgacattgc ccggcagacg gctcagggaa tggactattt gcatgcaaag aacatcatcc
     2461 atagagacat gaaatccaac aatatatttc tccatgaagg cttaacagtg aaaattggag
     2521 attttggttt ggcaacagta aagtcacgct ggagtggttc tcagcaggtt gaacaaccta
     2581 ctggctctgt cctctggatg gccccagagg tgatccgaat gcaggataac aacccattca
     2641 gtttccagtc ggatgtctac tcctatggca tcgtattgta tgaactgatg acgggggagc
     2701 ttccttattc tcacatcaac aaccgagatc agatcatctt catggtgggc cgaggatatg
     2761 cctccccaga tcttagtaag ctatataaga actgccccaa agcaatgaag aggctggtag
     2821 ctgactgtgt gaagaaagta aaggaagaga ggcctctttt tccccagatc ctgtcttcca
     2881 ttgagctgct ccaacactct ctaccgaaga tcaaccggag cgcttccgag ccatccttgc
     2941 atcgggcagc ccacactgag gatatcaatg cttgcacgct gaccacgtcc ccgaggctgc
     3001 ctgtcttcgt ttctctcgga ggaggtggct caggtggagg gggctccgtg accggctacc
     3061 ggctgttcga ggagattctg taaggccgcg actctagagt cgacctgcag gcatgcaagc
     3121 tgatccggct gctaacaaag cccgaaagga agctgagttg gctgctgcca ccgctgagca
     3181 ataactagca taaccccttg gggcggccgc ttcgagcaga catgataaga tacattgatg
     3241 agtttggaca aaccacaact agaatgcagt gaaaaaaatg ctttatttgt gaaatttgtg
     3301 atgctattgc tttatttgta accattataa gctgcaataa acaagttaac aacaacaatt
     3361 gcattcattt tatgtttcag gttcaggggg agatgtggga ggttttttta agcaagtaaa
     3421 acctctacaa atgtggtaaa atcgaatttt aacaaaatat taacgcttac aatttcctga
     3481 tgcggtattt tctccttacg catctgtgcg gtatttcaca ccgcatacgc ggatctgcgc
     3541 agcaccatgg cctgaaataa cctctgaaag aggaacttgg ttaggtacct tctgaggcgg
     3601 aaagaaccag ctgtggaatg tgtgtcagtt agggtgtgga aagtccccag gctccccagc
     3661 aggcagaagt atgcaaagca tgcatctcaa ttagtcagca accaggtgtg gaaagtcccc
     3721 aggctcccca gcaggcagaa gtatgcaaag catgcatctc aattagtcag caaccatagt
     3781 cccgccccta actccgccca tcccgcccct aactccgccc agttccgccc attctccgcc
     3841 ccatggctga ctaatttttt ttatttatgc agaggccgag gccgcctcgg cctctgagct
     3901 attccagaag tagtgaggag gcttttttgg aggcctaggc ttttgcaaaa agcttaatta
     3961 actgttgaca attaatcatc ggcatagtat atcggcatag tataatacga caaggtgagg
     4021 aactaaaccc aggaggcaga tcatgattga acaagatgga ttgcacgcag gttctccggc
     4081 cgcttgggtg gagaggctat tcggctatga ctgggcacaa cagacaatcg gctgctctga
     4141 tgccgccgtg ttccggctgt cagcgcaggg gcgcccggtt ctttttgtca agaccgacct
     4201 gtccggtgcc ctgaatgaac tgcaggacga ggcagcgcgg ctatcgtggc tggccacgac
     4261 gggcgttcct tgcgcagctg tgctcgacgt tgtcactgaa gcgggaaggg actggctgct
     4321 attgggcgaa gtgccggggc aggatctcct gtcatctcac cttgctcctg ccgagaaagt
     4381 atccatcatg gctgatgcaa tgcggcggct gcatacgctt gatccggcta cctgcccatt
     4441 cgaccaccaa gcgaaacatc gcatcgagcg agcacgtact cggatggaag ccggtcttgt
     4501 cgatcaggat gatctggacg aagagcatca ggggctcgcg ccagccgaac tgttcgccag
     4561 gctcaaggcg cgcatgcccg acggcgagga tctcgtcgtg acccatggcg atgcctgctt
     4621 gccgaatatc atggtggaaa atggccgctt ttctggattc atcgactgtg gccggctggg
     4681 tgtggcggac cgctatcagg acatagcgtt ggctacccgt gatattgctg aagagcttgg
     4741 cggcgaatgg gctgaccgct tcctcgtgct ttacggtatc gccgctcccg attcgcagcg
     4801 catcgccttc tatcgccttc ttgacgagtt cttctgagcg ggactctggg gttcgaaatg
     4861 accgaccaag cgacgcccaa cctgccatca cgatggccgc aataaaatat ctttattttc
     4921 attacatctg tgtgttggtt ttttgtgtga atcgatagcg ataaggatcc tctttgcgct
     4981 tgcgttttcc cttgtccaga tagcccagta gctgacattc atccggggtc agcaccgttt
     5041 ctgcggactg gctttctacc cggtatcagc tcactcaaag gcggtaatac ggttatccac
     5101 agaatcaggg gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa
     5161 ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc cgcccccctg acgagcatca
     5221 caaaaatcga cgctcaagtc agaggtggcg aaacccgaca ggactataaa gataccaggc
     5281 gtttccccct ggaagctccc tcgtgcgctc tcctgttccg accctgccgc ttaccggata
     5341 cctgtccgcc tttctccctt cgggaagcgt ggcgctttct catagctcac gctgtaggta
     5401 tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac cccccgttca
     5461 gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag tccaacccgg taagacacga
     5521 cttatcgcca ctggcagcag ccactggtaa caggattagc agagcgaggt atgtaggcgg
     5581 tgctacagag ttcttgaagt ggtggcctaa ctacggctac actagaagga cagtatttgg
     5641 tatctgcgct ctgctgaagc cagttacctt cggaaaaaga gttggtagct cttgatccgg
     5701 caaacaaacc accgctggta gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag
     5761 aaaaaaagga tttcaagaag atcctttgat cttttctacg gggtctgacg ctcagtggaa
     5821 cgaaaactca cgttaaggga ttttggtcat gagattatca aaaaggatct tcacctagat
     5881 ccttttatag tccggaaata caggaacgca cgctggatgg cccttcgctg ggatggtgaa
     5941 accatgaaaa atggcagctt cagtggatta agtgggggta atgtggcctg taccctctgg
     6001 ttgcataggt attcatacgg ttaaaattta tcaggcgcga ttgcggcagt ttttcgggtg
     6061 gtttgttgcc atttttacct gtctgctgcc gtgatcgcgc tgaacgcgtt ttagcggtgc
     6121 gtacaattaa gggattatgg taaatccact tactgtctgc cctcgtagcc atcgagataa
     6181 accgcagtac tccggccacg atgcgtccgg cgtagaggat cgagatct
//
