LOCUS       sequence-479921-        6600 bp    DNA     circular SYN 07-AUG-2025
DEFINITION  Express BRAF-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 6600)
  AUTHORS   Promega
  TITLE     Promega Plasmid Collection
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 6600)
  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            "Unpublished"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: CMV/BRAF-SmBiT/BlastR Vector
FEATURES             Location/Qualifiers
     source          1..6600
                     /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"
     regulatory      1187..1196
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     polyA_signal    3663..3784
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3674..3693)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     protein_bind    complement(3899..3932)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     promoter        3942..3989
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             4021..4815
                     /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(4075..4094)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4685..4704
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     rep_origin      complement(4973..5561)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5053..5072)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     polyA_signal    5687..5735
                     /note="synthetic polyadenylation signal"
     CDS             complement(5759..6157)
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
                     VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
                     KAIVKDSDGQPTAVGIRELLPSGYVWEG"
     regulatory      6154..6163
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     promoter        complement(6188..6545)
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      6202..6337
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     enhancer        6354..6545
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     6502..6522
                     /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 cgatcgccat ggcggcgctg
     1201 agcggtggcg gtggtggcgg cgcggagccg ggccaggctc tgttcaacgg ggacatggag
     1261 cccgaggccg gcgccggcgc cggcgccgcg gcctcttcgg ctgcggaccc tgccattccg
     1321 gaggaggtgt ggaatatcaa acaaatgatt aagttgacac aggaacatat agaggcccta
     1381 ttggacaaat ttggtgggga gcataatcca ccatcaatat atctggaggc ctatgaagaa
     1441 tacaccagca agctagatgc actccaacaa agagaacaac agttattgga atctctgggg
     1501 aacggaactg atttttctgt ttctagctct gcatcaatgg ataccgttac atcttcttcc
     1561 tcttctagcc tttcagtgct accttcatct ctttcagttt ttcaaaatcc cacagatgtg
     1621 gcacggagca accccaagtc accacaaaaa cctatcgtta gagtcttcct gcccaacaaa
     1681 cagaggacag tggtacctgc aaggtgtgga gttacagtcc gagacagtct aaagaaagca
     1741 ctgatgatga gaggtctaat cccagagtgc tgtgctgttt acagaattca ggatggagag
     1801 aagaaaccaa ttggttggga cactgatatt tcctggctta ctggagaaga attgcatgtg
     1861 gaagtgttgg agaatgttcc acttacaaca cacaactttg tacgaaaaac gtttttcacc
     1921 ttagcatttt gtgacttttg tcgaaagctg cttttccagg gtttccgctg tcaaacatgt
     1981 ggttataaat ttcaccagcg ttgtagtaca gaagttccac tgatgtgtgt taattatgac
     2041 caacttgatt tgctgtttgt ctccaagttc tttgaacacc acccaatacc acaggaagag
     2101 gcgtccttag cagagactgc cctaacatct ggatcatccc cttccgcacc cgcctcggac
     2161 tctattgggc cccaaattct caccagtccg tctccttcaa aatccattcc aattccacag
     2221 cccttccgac cagcagatga agatcatcga aatcaatttg ggcaacgaga ccgatcctca
     2281 tcagctccca atgtgcatat aaacacaata gaacctgtca atattgatga cttgattaga
     2341 gaccaaggat ttcgtggtga tggaggatca accacaggtt tgtctgctac cccccctgcc
     2401 tcattacctg gctcactaac taacgtgaaa gccttacaga aatctccagg acctcagcga
     2461 gaaaggaagt catcttcatc ctcagaagac aggaatcgaa tgaaaacact tggtagacgg
     2521 gactcgagtg atgattggga gattcctgat gggcagatta cagtgggaca aagaattgga
     2581 tctggatcat ttggaacagt ctacaaggga aagtggcatg gtgatgtggc agtgaaaatg
     2641 ttgaatgtga cagcacctac acctcagcag ttacaagcct tcaaaaatga agtaggagta
     2701 ctcaggaaaa cacgacatgt gaatatccta ctcttcatgg gctattccac aaagccacaa
     2761 ctggctattg ttacccagtg gtgtgagggc tccagcttgt atcaccatct ccatatcatt
     2821 gagaccaaat ttgagatgat caaacttata gatattgcac gacagactgc acagggcatg
     2881 gattacttac acgccaagtc aatcatccac agagacctca agagtaataa tatatttctt
     2941 catgaagacc tcacagtaaa aataggtgat tttggtctag ctacagtgaa atctcgatgg
     3001 agtgggtccc atcagtttga acagttgtct ggatccattt tgtggatggc accagaagtc
     3061 atcagaatgc aagataaaaa tccatacagc tttcagtcag atgtatatgc atttggaatt
     3121 gttctgtatg aattgatgac tggacagtta ccttattcaa acatcaacaa cagggaccag
     3181 ataattttta tggtgggacg aggatacctg tctccagatc tcagtaaggt acggagtaac
     3241 tgtccaaaag ccatgaagag attaatggca gagtgcctca aaaagaaaag agatgagaga
     3301 ccactctttc cccaaattct cgcctctatt gagctgctgg cccgctcatt gccaaaaatt
     3361 caccgcagtg catcagaacc ctccttgaat cgggctggtt tccaaacaga ggattttagt
     3421 ctatatgctt gtgcttctcc aaaaacaccc atccaggcag ggggatatgg tgcgtttcct
     3481 gtccacgttt ctcaaggcag ttcaggtggt ggcgggagcg gaggtggagg ctcgagcggt
     3541 gtgaccggct accggctgtt cgaggagatt ctgtaaggcc gcgactctag agtcgacctg
     3601 caggcatgca agctgatttg gctgctgcca ccgctgagca aggccgcttc gagcagacat
     3661 gataagatac attgatgagt ttggacaaac cacaactaga atgcagtgaa aaaaatgctt
     3721 tatttgtgaa atttgtgatg ctattgcttt atttgtaacc attataagct gcaataaaca
     3781 agttaacaac aacaattgca ttcattttat gtttcaggtt cagggggaga tgtgggaggt
     3841 ttttttaagc aagtaaaacc tctacaaatg tggtaaaatc ttaattaaga attctaatat
     3901 aacttcgtat agcatacatt atacgaagtt atggatccac tgttgacaat taatcatcgg
     3961 catagtatat cggcatagta taatacgaca aggtgaggaa ctaaacccag gaggcagatc
     4021 atgattgaac aagatggatt gcacgcaggt tctccggccg cttgggtgga gaggctattc
     4081 ggctatgact gggcacaaca gacaatcggc tgctctgatg ccgccgtgtt ccggctgtca
     4141 gcgcaggggc gcccggttct ttttgtcaag accgacctgt ccggtgccct gaatgaactg
     4201 caggacgagg cagcgcggct atcgtggctg gccacgacgg gcgttccttg cgcagctgtg
     4261 ctcgacgttg tcactgaagc gggaagggac tggctgctat tgggcgaagt gccggggcag
     4321 gatctcctgt catctcacct tgctcctgcc gagaaagtat ccatcatggc tgatgcaatg
     4381 cggcggctgc atacgcttga tccggctacc tgcccattcg accaccaagc gaaacatcgc
     4441 atcgagcgag cacgtactcg gatggaagcc ggtcttgtcg atcaggatga tctggacgaa
     4501 gagcatcagg ggctcgcgcc agccgaactg ttcgccaggc tcaaggcgcg catgcccgac
     4561 ggcgaggatc tcgtcgtgac ccatggcgat gcctgcttgc cgaatatcat ggtggaaaat
     4621 ggccgctttt ctggattcat cgactgtggc cggctgggtg tggcggaccg ctatcaggac
     4681 atagcgttgg ctacccgtga tattgctgaa gagcttggcg gcgaatgggc tgaccgcttc
     4741 ctcgtgcttt acggtatcgc cgctcccgat tcgcagcgca tcgccttcta tcgccttctt
     4801 gacgagttct tctgattcga aatgaccgac caagcgacgc ccaaccggta tcagctcact
     4861 caaaggcggt aatacggtta tccacagaat caggggataa cgcaggaaag aacatgtgag
     4921 caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg tttttccata
     4981 ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg tggcgaaacc
     5041 cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg cgctctcctg
     5101 ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga agcgtggcgc
     5161 tttctcatag ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc tccaagctgg
     5221 gctgtgtgca cgaacccccc gttcagcccg accgctgcgc cttatccggt aactatcgtc
     5281 ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact ggtaacagga
     5341 ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg cctaactacg
     5401 gctacactag aaggacagta tttggtatct gcgctctgct gaagccagtt accttcggaa
     5461 aaagagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt ggtttttttg
     5521 tttgcaagca gcagattacg cgcagaaaaa aaggatttca agaagatcct ttgatctttt
     5581 ctacggggtc tgacgctcag tggaacgaaa actcacgtta agggattttg gtcatgagat
     5641 tatcaaaaag gatcttcacc tagatccttt tatagtccgg aatcgtcaca caaaaaacca
     5701 acacacagat gtaatgaaaa taaagatatt ttattgcggc catcgtgatg gctagagttt
     5761 agccctccca cacataacca gagggcagca attcacgaat cccaactgcc gtcggctgtc
     5821 catcactgtc cttcactatg gctttgatcc caggatgcag atcgagaagc acctgtcggc
     5881 accgtccgca ggggctcaag atgcccctgt tctcatttcc gatcgcgacg atacaagtca
     5941 ggttgccagc tgccgcagca gcagcagtgc ccagcaccac gagttctgca caaggtcccc
     6001 cagtaaaatg atatacattg acaccagtga agatgcggcc gtcgctagag agagctgcgc
     6061 tggcgacgct gtagtcttca gagatgggga tgctgttgat tgtagccgtt gctctttcaa
     6121 tgagggtgga ttcttcttga gacaaaggct tggccatggt ggcgctagtg tcagaagaat
     6181 cgagcttttt gcaaaagcct aggcctccaa aaaagcctcc tcactacttc tggaatagct
     6241 cagaggccga ggcggcctcg gcctctgcat aaataaaaaa aattagtcag ccatggggcg
     6301 gagaatgggc ggaactgggc ggagttaggg gcgggatggg cggagttagg ggcgggacta
     6361 tggttgctga ctaattgaga tgcatgcttt gcatacttct gcctgctggg gagcctgggg
     6421 actttccaca cctggttgct gactaattga gatgcatgct ttgcatactt ctgcctgctg
     6481 gggagcctgg ggactttcca caccctaact gacacacatt ccacagctgg ttctttccgc
     6541 ctcagaaggt acctaaccaa gttcctcttt cagaggttat ttcaggccat ggtgctgcgc
//
