LOCUS       sequence-479919-        9165 bp    DNA     circular SYN 07-AUG-2025
DEFINITION  Express CRAF-LgBiT_BRAF-SmBiT Fusion Proteins in Mammalian Cells 
            under a CMV promoter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 9165)
  AUTHORS   Promega
  TITLE     Promega Plasmid Collection
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 9165)
  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            "Unpublished"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: CRAF:BRAF BiBiT Vector
FEATURES             Location/Qualifiers
     source          1..9165
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     polyA_signal    24..105
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     regulatory      3088..3097
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     promoter        complement(3134..3152)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(3135..3152)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     enhancer        3272..3575
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        3576..3779
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     3730..3750
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     intron          3915..4047
                     /label=chimeric intron
                     /note="chimera between introns from human beta-globin and 
                     immunoglobulin heavy chain genes"
     primer_bind     4030..4049
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     primer_bind     4091..4110
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        4091..4109
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     polyA_signal    6228..6349
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(6239..6258)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     protein_bind    complement(6464..6497)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     promoter        6507..6554
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             6586..7380
                     /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(6640..6659)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     7250..7269
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     rep_origin      complement(7538..8126)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(7618..7637)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     polyA_signal    8252..8300
                     /note="synthetic polyadenylation signal"
     CDS             complement(8324..8722)
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
                     VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
                     KAIVKDSDGQPTAVGIRELLPSGYVWEG"
     regulatory      8719..8728
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     promoter        complement(8753..9110)
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      8767..8902
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     enhancer        8919..9110
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     9067..9087
                     /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 ggtgtttcta gagtcgcggc cttagctgtt gatggttact
      301 cggaacagca tggagccgtc gggggtgatc aggcgctcgt cgataatttt gttgccgttc
      361 cacagggtcc ctgttacagt gatctttttg ccgtcgaaca cggcgatgcc ttcatacggc
      421 cgtccgaaat agttcagcat gttcggcgta accccgtcga ttaccagtgt gccatagggc
      481 aggatcacct taaagtgatg atcatccaca gggtacacca ccttaaacac ctcttcgatc
      541 tgggccattt ggtcggcgct cagaccttca tacgggatga tgacatggat gtcgatcttc
      601 agggcatttt caccgctccg gacaatcctt tggatcggag ttacggacac ggcgagattc
      661 tgcagcaaac tggacacacc tccctgttca aggacttggt ccaggttgta ggcggctgtc
      721 tgttcccagt ccccaacgaa atcttcgagt gtgaagacgg agccccctcc acctgagcca
      781 cctcctccga gagaaacgtg gacaggaaac gcaccatatc cccctgcctg gatgggtgtt
      841 tttggagaag cacaagcata tagactaaaa tcctctgttt ggaaaccagc ccgattcaag
      901 gagggttctg atgcactgcg gtgaattttt ggcaatgagc gggccagcag ctcaatagag
      961 gcgagaattt ggggaaagag tggtctctca tctcttttct ttttgaggca ctctgccatt
     1021 aatctcttca tggcttttgg acagttactc cgtaccttac tgagatctgg agacaggtat
     1081 cctcgtccca ccataaaaat tatctggtcc ctgttgttga tgtttgaata aggtaactgt
     1141 ccagtcatca attcatacag aacaattcca aatgcatata catctgactg aaagctgtat
     1201 ggatttttat cttgcattct gatgacttct ggtgccatcc acaaaatgga tccagacaac
     1261 tgttcaaact gatgggaccc actccatcga gatttcactg tagctagacc aaaatcacct
     1321 atttttactg tgaggtcttc atgaagaaat atattattac tcttgaggtc tctgtggatg
     1381 attgacttgg cgtgtaagta atccatgccc tgtgcagtct gtcgtgcaat atctataagt
     1441 ttgatcatct caaatttggt ctcaatgata tggagatggt gatacaagct ggagccctca
     1501 caccactggg taacaatagc cagttgtggc tttgtggaat agcccatgaa gagtaggata
     1561 ttcacatgtc gtgttttcct gagtactcct acttcatttt tgaaggcttg taactgctga
     1621 ggtgtaggtg ctgtcacatt caacattttc actgccacat caccatgcca ctttcccttg
     1681 tagactgttc caaatgatcc agatccaatt ctttgtccca ctgtaatctg cccatcagga
     1741 atctcccaat catcactcga gtcccgtcta ccaagtgttt tcattcgatt cctgtcttct
     1801 gaggatgaag atgacttcct ttctcgctga ggtcctggag atttctgtaa ggctttcacg
     1861 ttagttagtg agccaggtaa tgaggcaggg ggggtagcag acaaacctgt ggttgatcct
     1921 ccatcaccac gaaatccttg gtctctaatc aagtcatcaa tattgacagg ttctattgtg
     1981 tttatatgca cattgggagc tgatgaggat cggtctcgtt gcccaaattg atttcgatga
     2041 tcttcatctg ctggtcggaa gggctgtgga attggaatgg attttgaagg agacggactg
     2101 gtgagaattt ggggcccaat agagtccgag gcgggtgcgg aaggggatga tccagatgtt
     2161 agggcagtct ctgctaagga cgcctcttcc tgtggtattg ggtggtgttc aaagaacttg
     2221 gagacaaaca gcaaatcaag ttggtcataa ttaacacaca tcagtggaac ttctgtacta
     2281 caacgctggt gaaatttata accacatgtt tgacagcgga aaccctggaa aagcagcttt
     2341 cgacaaaagt cacaaaatgc taaggtgaaa aacgtttttc gtacaaagtt gtgtgttgta
     2401 agtggaacat tctccaacac ttccacatgc aattcttctc cagtaagcca ggaaatatca
     2461 gtgtcccaac caattggttt cttctctcca tcctgaattc tgtaaacagc acagcactct
     2521 gggattagac ctctcatcat cagtgctttc tttagactgt ctcggactgt aactccacac
     2581 cttgcaggta ccactgtcct ctgtttgttg ggcaggaaga ctctaacgat aggtttttgt
     2641 ggtgacttgg ggttgctccg tgccacatct gtgggatttt gaaaaactga aagagatgaa
     2701 ggtagcactg aaaggctaga agaggaagaa gatgtaacgg tatccattga tgcagagcta
     2761 gaaacagaaa aatcagttcc gttccccaga gattccaata actgttgttc tctttgttgg
     2821 agtgcatcta gcttgctggt gtattcttca taggcctcca gatatattga tggtggatta
     2881 tgctccccac caaatttgtc caatagggcc tctatatgtt cctgtgtcaa cttaatcatt
     2941 tgtttgatat tccacacctc ctccggaatg gcagggtccg cagccgaaga ggccgcggcg
     3001 ccggcgccgg cgccggcctc gggctccatg tccccgttga acagagcctg gcccggctcc
     3061 gcgccgccac caccgccacc gctcagcgcc gccatggcga tcgctagctg caaaaagaac
     3121 aagtagcttg tattctatag tgtcacctaa atgggcgatc tgacggttca ctaaaccagc
     3181 tctgcttata taggcctccc tggcgcgcca ctagttatta atagtaatca attacggggt
     3241 cattagttca tagcccatat atggagttcc gcgttacata acttacggta aatggcccgc
     3301 ctggctgacc gcccaacgac ccccgcccat tgacgtcaat aatgacgtat gttcccatag
     3361 taacgccaat agggactttc cattgacgtc aatgggtgga gtatttacgg taaactgccc
     3421 acttggcagt acatcaagtg tatcatatgc caagtccgcc ccctattgac gtcaatgacg
     3481 gtaaatggcc cgcctggcat tatgcccagt acatgacctt acgggacttt cctacttggc
     3541 agtacatcta cgtattagtc atcgctatta ccatggtgat gcggttttgg cagtacacca
     3601 atgggcgtgg atagcggttt gactcacggg gatttccaag tctccacccc attgacgtca
     3661 atgggagttt gttttggcac caaaatcaac gggactttcc aaaatgtcgt aataaccccg
     3721 ccccgttgac gcaaatgggc ggtaggcgtg tacggtggga ggtctatata agcagagctg
     3781 gtttagtgaa ccgtcagatc actagaagct ttattgcggt agtttatcac agttaaattg
     3841 ctaacgcagt cagtgcttct gacacaacag tctcgaactt aagctgcaga agttggtcgt
     3901 gaggcactgg gcaggtaagt atcaaggtta caagacaggt ttaaggagac caatagaaac
     3961 tgggcttgtc gagacagaga agactcttgc gtttctgata ggcacctatt ggtcttactg
     4021 acatccactt tgcctttctc tccacaggtg tccactccca gttcaattac agctcttaag
     4081 gctagagtat taatacgact cactataggg ctagcgatcg ccatggagca catacaggga
     4141 gcttggaaga cgatcagcaa tggttttgga ttcaaagatg ccgtgtttga tggctccagc
     4201 tgcatctctc ctacaatagt tcagcagttt ggctatcagc gccgggcatc agatgatggc
     4261 aaactcacag atccttctaa gacaagcaac actatccgtg ttttcttgcc gaacaagcaa
     4321 agaacagtgg tcaatgtgcg aaatggaatg agcttgcatg actgccttat gaaagcactc
     4381 aaggtgaggg gcctgcaacc agagtgctgt gcagtgttca gacttctcca cgaacacaaa
     4441 ggtaaaaaag cacgcttaga ttggaatact gatgctgcgt ctttgattgg agaagaactt
     4501 caagtagatt tcctggatca tgttcccctc acaacacaca actttgctcg gaagacgttc
     4561 ctgaagcttg ccttctgtga catctgtcag aaattcctgc tcaatggatt tcgatgtcag
     4621 acttgtggct acaaatttca tgagcactgt agcaccaaag tacctactat gtgtgtggac
     4681 tggagtaaca tcagacaact cttattgttt ccaaattcca ctattggtga tagtggagtc
     4741 ccagcactac cttctttgac tatgcgtcgt atgcgagagt ctgtttccag gatgcctgtt
     4801 agttctcagc acagatattc tacacctcac gccttcacct ttaacacctc cagtccctca
     4861 tctgaaggtt ccctctccca gaggcagagg tcgacatcca cacctaatgt ccacatggtc
     4921 agcaccaccc tgcctgtgga cagcaggatg attgaggatg caattcgaag tcacagcgaa
     4981 tcagcctcac cttcagccct gtccagtagc cccaacaatc tgagcccaac aggctggtca
     5041 cagccgaaaa cccccgtgcc agcacaaaga gagcgggcac cagtatctgg gacccaggag
     5101 aaaaacaaaa ttaggcctcg tggacagaga gattcaagct attattggga aatagaagcc
     5161 agtgaagtga tgctgtccac tcggattggg tcaggctctt ttggaactgt ttataagggt
     5221 aaatggcacg gagatgttgc agtaaagatc ctaaaggttg tcgacccaac cccagagcaa
     5281 ttccaggcct tcaggaatga ggtggctgtt ctgcgcaaaa cacggcatgt gaacattctg
     5341 cttttcatgg ggtacatgac aaaggacaac ctggcaattg tgacccagtg gtgcgagggc
     5401 agcagcctct acaaacacct gcatgtccag gagaccaagt ttcagatgtt ccagctaatt
     5461 gacattgccc ggcagacggc tcagggaatg gactatttgc atgcaaagaa catcatccat
     5521 agagacatga aatccaacaa tatatttctc catgaaggct taacagtgaa aattggagat
     5581 tttggtttgg caacagtaaa gtcacgctgg agtggttctc agcaggttga acaacctact
     5641 ggctctgtcc tctggatggc cccagaggtg atccgaatgc aggataacaa cccattcagt
     5701 ttccagtcgg atgtctactc ctatggcatc gtattgtatg aactgatgac gggggagctt
     5761 ccttattctc acatcaacaa ccgagatcag atcatcttca tggtgggccg aggatatgcc
     5821 tccccagatc ttagtaagct atataagaac tgccccaaag caatgaagag gctggtagct
     5881 gactgtgtga agaaagtaaa ggaagagagg cctctttttc cccagatcct gtcttccatt
     5941 gagctgctcc aacactctct accgaagatc aaccggagcg cttccgagcc atccttgcat
     6001 cgggcagccc acactgagga tatcaatgct tgcacgctga ccacgtcccc gaggctgcct
     6061 gtcttcgttt ctctcggagg aggtggctca ggtggagggg gctccgtgac cggctaccgg
     6121 ctgttcgagg agattctgta aggccgcgac tctagagtcg acctgcaggc atgcaagctg
     6181 atttggctgc tgccaccgct gagcaaggcc gcttcgagca gacatgataa gatacattga
     6241 tgagtttgga caaaccacaa ctagaatgca gtgaaaaaaa tgctttattt gtgaaatttg
     6301 tgatgctatt gctttatttg taaccattat aagctgcaat aaacaagtta acaacaacaa
     6361 ttgcattcat tttatgtttc aggttcaggg ggagatgtgg gaggtttttt taagcaagta
     6421 aaacctctac aaatgtggta aaatcttaat taagaattct aatataactt cgtatagcat
     6481 acattatacg aagttatgga tccactgttg acaattaatc atcggcatag tatatcggca
     6541 tagtataata cgacaaggtg aggaactaaa cccaggaggc agatcatgat tgaacaagat
     6601 ggattgcacg caggttctcc ggccgcttgg gtggagaggc tattcggcta tgactgggca
     6661 caacagacaa tcggctgctc tgatgccgcc gtgttccggc tgtcagcgca ggggcgcccg
     6721 gttctttttg tcaagaccga cctgtccggt gccctgaatg aactgcagga cgaggcagcg
     6781 cggctatcgt ggctggccac gacgggcgtt ccttgcgcag ctgtgctcga cgttgtcact
     6841 gaagcgggaa gggactggct gctattgggc gaagtgccgg ggcaggatct cctgtcatct
     6901 caccttgctc ctgccgagaa agtatccatc atggctgatg caatgcggcg gctgcatacg
     6961 cttgatccgg ctacctgccc attcgaccac caagcgaaac atcgcatcga gcgagcacgt
     7021 actcggatgg aagccggtct tgtcgatcag gatgatctgg acgaagagca tcaggggctc
     7081 gcgccagccg aactgttcgc caggctcaag gcgcgcatgc ccgacggcga ggatctcgtc
     7141 gtgacccatg gcgatgcctg cttgccgaat atcatggtgg aaaatggccg cttttctgga
     7201 ttcatcgact gtggccggct gggtgtggcg gaccgctatc aggacatagc gttggctacc
     7261 cgtgatattg ctgaagagct tggcggcgaa tgggctgacc gcttcctcgt gctttacggt
     7321 atcgccgctc ccgattcgca gcgcatcgcc ttctatcgcc ttcttgacga gttcttctga
     7381 ttcgaaatga ccgaccaagc gacgcccaac cggtatcagc tcactcaaag gcggtaatac
     7441 ggttatccac agaatcaggg gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa
     7501 aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc cgcccccctg
     7561 acgagcatca caaaaatcga cgctcaagtc agaggtggcg aaacccgaca ggactataaa
     7621 gataccaggc gtttccccct ggaagctccc tcgtgcgctc tcctgttccg accctgccgc
     7681 ttaccggata cctgtccgcc tttctccctt cgggaagcgt ggcgctttct catagctcac
     7741 gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac
     7801 cccccgttca gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag tccaacccgg
     7861 taagacacga cttatcgcca ctggcagcag ccactggtaa caggattagc agagcgaggt
     7921 atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa ctacggctac actagaagga
     7981 cagtatttgg tatctgcgct ctgctgaagc cagttacctt cggaaaaaga gttggtagct
     8041 cttgatccgg caaacaaacc accgctggta gcggtggttt ttttgtttgc aagcagcaga
     8101 ttacgcgcag aaaaaaagga tttcaagaag atcctttgat cttttctacg gggtctgacg
     8161 ctcagtggaa cgaaaactca cgttaaggga ttttggtcat gagattatca aaaaggatct
     8221 tcacctagat ccttttatag tccggaatcg tcacacaaaa aaccaacaca cagatgtaat
     8281 gaaaataaag atattttatt gcggccatcg tgatggctag agtttagccc tcccacacat
     8341 aaccagaggg cagcaattca cgaatcccaa ctgccgtcgg ctgtccatca ctgtccttca
     8401 ctatggcttt gatcccagga tgcagatcga gaagcacctg tcggcaccgt ccgcaggggc
     8461 tcaagatgcc cctgttctca tttccgatcg cgacgataca agtcaggttg ccagctgccg
     8521 cagcagcagc agtgcccagc accacgagtt ctgcacaagg tcccccagta aaatgatata
     8581 cattgacacc agtgaagatg cggccgtcgc tagagagagc tgcgctggcg acgctgtagt
     8641 cttcagagat ggggatgctg ttgattgtag ccgttgctct ttcaatgagg gtggattctt
     8701 cttgagacaa aggcttggcc atggtggcgc tagtgtcaga agaatcgagc tttttgcaaa
     8761 agcctaggcc tccaaaaaag cctcctcact acttctggaa tagctcagag gccgaggcgg
     8821 cctcggcctc tgcataaata aaaaaaatta gtcagccatg gggcggagaa tgggcggaac
     8881 tgggcggagt taggggcggg atgggcggag ttaggggcgg gactatggtt gctgactaat
     8941 tgagatgcat gctttgcata cttctgcctg ctggggagcc tggggacttt ccacacctgg
     9001 ttgctgacta attgagatgc atgctttgca tacttctgcc tgctggggag cctggggact
     9061 ttccacaccc taactgacac acattccaca gctggttctt tccgcctcag aaggtaccta
     9121 accaagttcc tctttcagag gttatttcag gccatggtgc tgcgc
//
