LOCUS       sequence-480606-        9549 bp    DNA     circular SYN 25-JUL-2025
DEFINITION  Express BRAF-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 9549)
  AUTHORS   Promega
  TITLE     Promega Plasmid Collection
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 9549)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Jul 25, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: 
            "Unpublished"
COMMENT     Sequence Label: BRAF:BRAF BiBiT Vector
FEATURES             Location/Qualifiers
     source          1..9549
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     polyA_signal    24..105
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     regulatory      3103..3112
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     promoter        complement(3149..3167)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(3150..3167)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     enhancer        3287..3590
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        3591..3794
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     3745..3765
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     intron          3930..4062
                     /label=chimeric intron
                     /note="chimera between introns from human beta-globin and 
                     immunoglobulin heavy chain genes"
     primer_bind     4045..4064
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     primer_bind     4106..4125
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        4106..4124
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      4136..4145
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     polyA_signal    6612..6733
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(6623..6642)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     protein_bind    complement(6848..6881)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     promoter        6891..6938
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             6970..7764
                     /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(7024..7043)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     7634..7653
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     rep_origin      complement(7922..8510)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(8002..8021)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     polyA_signal    8636..8684
                     /note="synthetic polyadenylation signal"
     CDS             complement(8708..9106)
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
                     VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
                     KAIVKDSDGQPTAVGIRELLPSGYVWEG"
     regulatory      9103..9112
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     promoter        complement(9137..9494)
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      9151..9286
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     enhancer        9303..9494
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     9451..9471
                     /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 gtgaagacac cgctcgagcc tccacctccg
      781 ctcccgccac cacctgaact gccttgagaa acgtggacag gaaacgcacc atatccccct
      841 gcctggatgg gtgtttttgg agaagcacaa gcatatagac taaaatcctc tgtttggaaa
      901 ccagcccgat tcaaggaggg ttctgatgca ctgcggtgaa tttttggcaa tgagcgggcc
      961 agcagctcaa tagaggcgag aatttgggga aagagtggtc tctcatctct tttctttttg
     1021 aggcactctg ccattaatct cttcatggct tttggacagt tactccgtac cttactgaga
     1081 tctggagaca ggtatcctcg tcccaccata aaaattatct ggtccctgtt gttgatgttt
     1141 gaataaggta actgtccagt catcaattca tacagaacaa ttccaaatgc atatacatct
     1201 gactgaaagc tgtatggatt tttatcttgc attctgatga cttctggtgc catccacaaa
     1261 atggatccag acaactgttc aaactgatgg gacccactcc atcgagattt cactgtagct
     1321 agaccaaaat cacctatttt tactgtgagg tcttcatgaa gaaatatatt attactcttg
     1381 aggtctctgt ggatgattga cttggcgtgt aagtaatcca tgccctgtgc agtctgtcgt
     1441 gcaatatcta taagtttgat catctcaaat ttggtctcaa tgatatggag atggtgatac
     1501 aagctggagc cctcacacca ctgggtaaca atagccagtt gtggctttgt ggaatagccc
     1561 atgaagagta ggatattcac atgtcgtgtt ttcctgagta ctcctacttc atttttgaag
     1621 gcttgtaact gctgaggtgt aggtgctgtc acattcaaca ttttcactgc cacatcacca
     1681 tgccactttc ccttgtagac tgttccaaat gatccagatc caattctttg tcccactgta
     1741 atctgcccat caggaatctc ccaatcatca ctcgagtccc gtctaccaag tgttttcatt
     1801 cgattcctgt cttctgagga tgaagatgac ttcctttctc gctgaggtcc tggagatttc
     1861 tgtaaggctt tcacgttagt tagtgagcca ggtaatgagg cagggggggt agcagacaaa
     1921 cctgtggttg atcctccatc accacgaaat ccttggtctc taatcaagtc atcaatattg
     1981 acaggttcta ttgtgtttat atgcacattg ggagctgatg aggatcggtc tcgttgccca
     2041 aattgatttc gatgatcttc atctgctggt cggaagggct gtggaattgg aatggatttt
     2101 gaaggagacg gactggtgag aatttggggc ccaatagagt ccgaggcggg tgcggaaggg
     2161 gatgatccag atgttagggc agtctctgct aaggacgcct cttcctgtgg tattgggtgg
     2221 tgttcaaaga acttggagac aaacagcaaa tcaagttggt cataattaac acacatcagt
     2281 ggaacttctg tactacaacg ctggtgaaat ttataaccac atgtttgaca gcggaaaccc
     2341 tggaaaagca gctttcgaca aaagtcacaa aatgctaagg tgaaaaacgt ttttcgtaca
     2401 aagttgtgtg ttgtaagtgg aacattctcc aacacttcca catgcaattc ttctccagta
     2461 agccaggaaa tatcagtgtc ccaaccaatt ggtttcttct ctccatcctg aattctgtaa
     2521 acagcacagc actctgggat tagacctctc atcatcagtg ctttctttag actgtctcgg
     2581 actgtaactc cacaccttgc aggtaccact gtcctctgtt tgttgggcag gaagactcta
     2641 acgataggtt tttgtggtga cttggggttg ctccgtgcca catctgtggg attttgaaaa
     2701 actgaaagag atgaaggtag cactgaaagg ctagaagagg aagaagatgt aacggtatcc
     2761 attgatgcag agctagaaac agaaaaatca gttccgttcc ccagagattc caataactgt
     2821 tgttctcttt gttggagtgc atctagcttg ctggtgtatt cttcataggc ctctagatat
     2881 attgatggtg gattatgctc cccaccaaat ttgtccaata gggcctctat atgttcctgt
     2941 gtcaacttaa tcatttgttt gatattccac acctcctccg gaatggcagg gtccgcagcc
     3001 gaagaggccg cggcgccggc gccggcgccg gcctcgggct ccatgtcccc gttgaacaga
     3061 gcctggcccg gctccgcgcc gccaccaccg ccaccgctca gcgccgccat ggcgatcgct
     3121 agctgcaaaa agaacaagta gcttgtattc tatagtgtca cctaaatggg cgatctgacg
     3181 gttcactaaa ccagctctgc ttatataggc ctccctggcg cgccactagt tattaatagt
     3241 aatcaattac ggggtcatta gttcatagcc catatatgga gttccgcgtt acataactta
     3301 cggtaaatgg cccgcctggc tgaccgccca acgacccccg cccattgacg tcaataatga
     3361 cgtatgttcc catagtaacg ccaataggga ctttccattg acgtcaatgg gtggagtatt
     3421 tacggtaaac tgcccacttg gcagtacatc aagtgtatca tatgccaagt ccgcccccta
     3481 ttgacgtcaa tgacggtaaa tggcccgcct ggcattatgc ccagtacatg accttacggg
     3541 actttcctac ttggcagtac atctacgtat tagtcatcgc tattaccatg gtgatgcggt
     3601 tttggcagta caccaatggg cgtggatagc ggtttgactc acggggattt ccaagtctcc
     3661 accccattga cgtcaatggg agtttgtttt ggcaccaaaa tcaacgggac tttccaaaat
     3721 gtcgtaataa ccccgccccg ttgacgcaaa tgggcggtag gcgtgtacgg tgggaggtct
     3781 atataagcag agctggttta gtgaaccgtc agatcactag aagctttatt gcggtagttt
     3841 atcacagtta aattgctaac gcagtcagtg cttctgacac aacagtctcg aacttaagct
     3901 gcagaagttg gtcgtgaggc actgggcagg taagtatcaa ggttacaaga caggtttaag
     3961 gagaccaata gaaactgggc ttgtcgagac agagaagact cttgcgtttc tgataggcac
     4021 ctattggtct tactgacatc cactttgcct ttctctccac aggtgtccac tcccagttca
     4081 attacagctc ttaaggctag agtattaata cgactcacta tagggctagc gatcgccatg
     4141 gcggcgctga gcggtggcgg tggtggcggc gcggagccgg gccaggctct gttcaacggg
     4201 gacatggagc ccgaggccgg cgccggcgcc ggcgccgcgg cctcttcggc tgcggaccct
     4261 gccattccgg aggaggtgtg gaatatcaaa caaatgatta agttgacaca ggaacatata
     4321 gaggccctat tggacaaatt tggtggggag cataatccac catcaatata tctggaggcc
     4381 tatgaagaat acaccagcaa gctagatgca ctccaacaaa gagaacaaca gttattggaa
     4441 tctctgggga acggaactga tttttctgtt tctagctctg catcaatgga taccgttaca
     4501 tcttcttcct cttctagcct ttcagtgcta ccttcatctc tttcagtttt tcaaaatccc
     4561 acagatgtgg cacggagcaa ccccaagtca ccacaaaaac ctatcgttag agtcttcctg
     4621 cccaacaaac agaggacagt ggtacctgca aggtgtggag ttacagtccg agacagtcta
     4681 aagaaagcac tgatgatgag aggtctaatc ccagagtgct gtgctgttta cagaattcag
     4741 gatggagaga agaaaccaat tggttgggac actgatattt cctggcttac tggagaagaa
     4801 ttgcatgtgg aagtgttgga gaatgttcca cttacaacac acaactttgt acgaaaaacg
     4861 tttttcacct tagcattttg tgacttttgt cgaaagctgc ttttccaggg tttccgctgt
     4921 caaacatgtg gttataaatt tcaccagcgt tgtagtacag aagttccact gatgtgtgtt
     4981 aattatgacc aacttgattt gctgtttgtc tccaagttct ttgaacacca cccaatacca
     5041 caggaagagg cgtccttagc agagactgcc ctaacatctg gatcatcccc ttccgcaccc
     5101 gcctcggact ctattgggcc ccaaattctc accagtccgt ctccttcaaa atccattcca
     5161 attccacagc ccttccgacc agcagatgaa gatcatcgaa atcaatttgg gcaacgagac
     5221 cgatcctcat cagctcccaa tgtgcatata aacacaatag aacctgtcaa tattgatgac
     5281 ttgattagag accaaggatt tcgtggtgat ggaggatcaa ccacaggttt gtctgctacc
     5341 ccccctgcct cattacctgg ctcactaact aacgtgaaag ccttacagaa atctccagga
     5401 cctcagcgag aaaggaagtc atcttcatcc tcagaagaca ggaatcgaat gaaaacactt
     5461 ggtagacggg actcgagtga tgattgggag attcctgatg ggcagattac agtgggacaa
     5521 agaattggat ctggatcatt tggaacagtc tacaagggaa agtggcatgg tgatgtggca
     5581 gtgaaaatgt tgaatgtgac agcacctaca cctcagcagt tacaagcctt caaaaatgaa
     5641 gtaggagtac tcaggaaaac acgacatgtg aatatcctac tcttcatggg ctattccaca
     5701 aagccacaac tggctattgt tacccagtgg tgtgagggct ccagcttgta tcaccatctc
     5761 catatcattg agaccaaatt tgagatgatc aaacttatag atattgcacg acagactgca
     5821 cagggcatgg attacttaca cgccaagtca atcatccaca gagacctcaa gagtaataat
     5881 atatttcttc atgaagacct cacagtaaaa ataggtgatt ttggtctagc tacagtgaaa
     5941 tctcgatgga gtgggtccca tcagtttgaa cagttgtctg gatccatttt gtggatggca
     6001 ccagaagtca tcagaatgca agataaaaat ccatacagct ttcagtcaga tgtatatgca
     6061 tttggaattg ttctgtatga attgatgact ggacagttac cttattcaaa catcaacaac
     6121 agggaccaga taatttttat ggtgggacga ggatacctgt ctccagatct cagtaaggta
     6181 cggagtaact gtccaaaagc catgaagaga ttaatggcag agtgcctcaa aaagaaaaga
     6241 gatgagagac cactctttcc ccaaattctc gcctctattg agctgctggc ccgctcattg
     6301 ccaaaaattc accgcagtgc atcagaaccc tccttgaatc gggctggttt ccaaacagag
     6361 gattttagtc tatatgcttg tgcttctcca aaaacaccca tccaggcagg gggatatggt
     6421 gcgtttcctg tccacgtttc tcaaggcagt tcaggtggtg gcgggagcgg aggtggaggc
     6481 tcgagcggtg tgaccggcta ccggctgttc gaggagattc tgtaaggccg cgactctaga
     6541 gtcgacctgc aggcatgcaa gctgatttgg ctgctgccac cgctgagcaa ggccgcttcg
     6601 agcagacatg ataagataca ttgatgagtt tggacaaacc acaactagaa tgcagtgaaa
     6661 aaaatgcttt atttgtgaaa tttgtgatgc tattgcttta tttgtaacca ttataagctg
     6721 caataaacaa gttaacaaca acaattgcat tcattttatg tttcaggttc agggggagat
     6781 gtgggaggtt tttttaagca agtaaaacct ctacaaatgt ggtaaaatct taattaagaa
     6841 ttctaatata acttcgtata gcatacatta tacgaagtta tggatccact gttgacaatt
     6901 aatcatcggc atagtatatc ggcatagtat aatacgacaa ggtgaggaac taaacccagg
     6961 aggcagatca tgattgaaca agatggattg cacgcaggtt ctccggccgc ttgggtggag
     7021 aggctattcg gctatgactg ggcacaacag acaatcggct gctctgatgc cgccgtgttc
     7081 cggctgtcag cgcaggggcg cccggttctt tttgtcaaga ccgacctgtc cggtgccctg
     7141 aatgaactgc aggacgaggc agcgcggcta tcgtggctgg ccacgacggg cgttccttgc
     7201 gcagctgtgc tcgacgttgt cactgaagcg ggaagggact ggctgctatt gggcgaagtg
     7261 ccggggcagg atctcctgtc atctcacctt gctcctgccg agaaagtatc catcatggct
     7321 gatgcaatgc ggcggctgca tacgcttgat ccggctacct gcccattcga ccaccaagcg
     7381 aaacatcgca tcgagcgagc acgtactcgg atggaagccg gtcttgtcga tcaggatgat
     7441 ctggacgaag agcatcaggg gctcgcgcca gccgaactgt tcgccaggct caaggcgcgc
     7501 atgcccgacg gcgaggatct cgtcgtgacc catggcgatg cctgcttgcc gaatatcatg
     7561 gtggaaaatg gccgcttttc tggattcatc gactgtggcc ggctgggtgt ggcggaccgc
     7621 tatcaggaca tagcgttggc tacccgtgat attgctgaag agcttggcgg cgaatgggct
     7681 gaccgcttcc tcgtgcttta cggtatcgcc gctcccgatt cgcagcgcat cgccttctat
     7741 cgccttcttg acgagttctt ctgattcgaa atgaccgacc aagcgacgcc caaccggtat
     7801 cagctcactc aaaggcggta atacggttat ccacagaatc aggggataac gcaggaaaga
     7861 acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt
     7921 ttttccatag gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt
     7981 ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc
     8041 gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa
     8101 gcgtggcgct ttctcatagc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct
     8161 ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta
     8221 actatcgtct tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg
     8281 gtaacaggat tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggc
     8341 ctaactacgg ctacactaga aggacagtat ttggtatctg cgctctgctg aagccagtta
     8401 ccttcggaaa aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtg
     8461 gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatttcaa gaagatcctt
     8521 tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg
     8581 tcatgagatt atcaaaaagg atcttcacct agatcctttt atagtccgga atcgtcacac
     8641 aaaaaaccaa cacacagatg taatgaaaat aaagatattt tattgcggcc atcgtgatgg
     8701 ctagagttta gccctcccac acataaccag agggcagcaa ttcacgaatc ccaactgccg
     8761 tcggctgtcc atcactgtcc ttcactatgg ctttgatccc aggatgcaga tcgagaagca
     8821 cctgtcggca ccgtccgcag gggctcaaga tgcccctgtt ctcatttccg atcgcgacga
     8881 tacaagtcag gttgccagct gccgcagcag cagcagtgcc cagcaccacg agttctgcac
     8941 aaggtccccc agtaaaatga tatacattga caccagtgaa gatgcggccg tcgctagaga
     9001 gagctgcgct ggcgacgctg tagtcttcag agatggggat gctgttgatt gtagccgttg
     9061 ctctttcaat gagggtggat tcttcttgag acaaaggctt ggccatggtg gcgctagtgt
     9121 cagaagaatc gagctttttg caaaagccta ggcctccaaa aaagcctcct cactacttct
     9181 ggaatagctc agaggccgag gcggcctcgg cctctgcata aataaaaaaa attagtcagc
     9241 catggggcgg agaatgggcg gaactgggcg gagttagggg cgggatgggc ggagttaggg
     9301 gcgggactat ggttgctgac taattgagat gcatgctttg catacttctg cctgctgggg
     9361 agcctgggga ctttccacac ctggttgctg actaattgag atgcatgctt tgcatacttc
     9421 tgcctgctgg ggagcctggg gactttccac accctaactg acacacattc cacagctggt
     9481 tctttccgcc tcagaaggta cctaaccaag ttcctctttc agaggttatt tcaggccatg
     9541 gtgctgcgc
//
