LOCUS       sequence-479918-        8841 bp    DNA     circular SYN 07-AUG-2025
DEFINITION  Express CRAF-LgBiT_CRAF-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 8841)
  AUTHORS   Promega
  TITLE     Promega Plasmid Collection
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 8841)
  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            "Unpublished"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:CRAF BiBiT Vector
FEATURES             Location/Qualifiers
     source          1..8841
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     polyA_signal    24..105
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     promoter        complement(2795..2813)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(2796..2813)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     enhancer        2933..3236
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        3237..3440
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     3391..3411
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     intron          3576..3708
                     /label=chimeric intron
                     /note="chimera between introns from human beta-globin and 
                     immunoglobulin heavy chain genes"
     primer_bind     3691..3710
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     primer_bind     3752..3771
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        3752..3770
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     polyA_signal    5904..6025
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5915..5934)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     protein_bind    complement(6140..6173)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     promoter        6183..6230
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             6262..7056
                     /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(6316..6335)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     6926..6945
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     rep_origin      complement(7214..7802)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(7294..7313)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     polyA_signal    7928..7976
                     /note="synthetic polyadenylation signal"
     CDS             complement(8000..8398)
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
                     VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
                     KAIVKDSDGQPTAVGIRELLPSGYVWEG"
     regulatory      8395..8404
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     promoter        complement(8429..8786)
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      8443..8578
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     enhancer        8595..8786
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     8743..8763
                     /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 acgaagacag gcagcctcgg ggacgtggtc
      841 agcgtgcaag cattgatatc ctcagtgtgg gctgcccgat gcaaggatgg ctcggaagcg
      901 ctccggttga tcttcggtag agagtgttgg agcagctcaa tggaagacag gatctgggga
      961 aaaagaggcc tctcttcctt tactttcttc acacagtcag ctaccagcct cttcattgct
     1021 ttggggcagt tcttatatag cttactaaga tctggggagg catatcctcg gcccaccatg
     1081 aagatgatct gatctcggtt gttgatgtga gaataaggaa gctcccccgt catcagttca
     1141 tacaatacga tgccatagga gtagacatcc gactggaaac tgaatgggtt gttatcctgc
     1201 attcggatca cctctggggc catccagagg acagagccag taggttgttc aacctgctga
     1261 gaaccactcc agcgtgactt tactgttgcc aaaccaaaat ctccaatttt cactgttaag
     1321 ccttcatgga gaaatatatt gttggatttc atgtctctat ggatgatgtt ctttgcatgc
     1381 aaatagtcca ttccctgagc cgtctgccgg gcaatgtcaa ttagctggaa catctgaaac
     1441 ttggtctcct ggacatgcag gtgtttgtag aggctgctgc cctcgcacca ctgggtcaca
     1501 attgccaggt tgtcctttgt catgtacccc atgaaaagca gaatgttcac atgccgtgtt
     1561 ttgcgcagaa cagccacctc attcctgaag gcctggaatt gctctggggt tgggtcgaca
     1621 acctttagga tctttactgc aacatctccg tgccatttac ccttataaac agttccaaaa
     1681 gagcctgacc caatccgagt ggacagcatc acttcactgg cttctatttc ccaataatag
     1741 cttgaatctc tctgtccacg aggcctaatt ttgtttttct cctgggtccc agatactggt
     1801 gcccgctctc tttgtgctgg cacgggggtt ttcggctgtg accagcctgt tgggctcaga
     1861 ttgttggggc tactggacag ggctgaaggt gaggctgatt cgctgtgact tcgaattgca
     1921 tcctcaatca tcctgctgtc cacaggcagg gtggtgctga ccatgtggac attaggtgtg
     1981 gatgtcgacc tctgcctctg ggagagggaa ccttcagatg agggactgga ggtgttaaag
     2041 gtgaaggcgt gaggtgtaga atatctgtgc tgagaactaa caggcatcct ggaaacagac
     2101 tctcgcatac gacgcatagt caaagaaggt agtgctggga ctccactatc accaatagtg
     2161 gaatttggaa acaataagag ttgtctgatg ttactccagt ccacacacat agtaggtact
     2221 ttggtgctac agtgctcatg aaatttgtag ccacaagtct gacatcgaaa tccattgagc
     2281 aggaatttct gacagatgtc acagaaggca agcttcagga acgtcttccg agcaaagttg
     2341 tgtgttgtga ggggaacatg atccaggaaa tctacttgaa gttcttctcc aatcaaagac
     2401 gcagcatcag tattccaatc taagcgtgct tttttacctt tgtgttcgtg gagaagtctg
     2461 aacactgcac agcactctgg ttgcaggccc ctcaccttga gtgctttcat aaggcagtca
     2521 tgcaagctca ttccatttcg cacattgacc actgttcttt gcttgttcgg caagaaaaca
     2581 cggatagtgt tgcttgtctt agaaggatct gtgagtttgc catcatctga tgcccggcgc
     2641 tgatagccaa actgctgaac tattgtagga gagatgcagc tggagccatc aaacacggca
     2701 tctttgaatc caaaaccatt gctgatcgtc ttccaagctc cctgtatgtg ctccatggcg
     2761 atcgctagct gcaaaaagaa caagtagctt gtattctata gtgtcaccta aatgggcgat
     2821 ctgacggttc actaaaccag ctctgcttat ataggcctcc ctggcgcgcc actagttatt
     2881 aatagtaatc aattacgggg tcattagttc atagcccata tatggagttc cgcgttacat
     2941 aacttacggt aaatggcccg cctggctgac cgcccaacga cccccgccca ttgacgtcaa
     3001 taatgacgta tgttcccata gtaacgccaa tagggacttt ccattgacgt caatgggtgg
     3061 agtatttacg gtaaactgcc cacttggcag tacatcaagt gtatcatatg ccaagtccgc
     3121 cccctattga cgtcaatgac ggtaaatggc ccgcctggca ttatgcccag tacatgacct
     3181 tacgggactt tcctacttgg cagtacatct acgtattagt catcgctatt accatggtga
     3241 tgcggttttg gcagtacacc aatgggcgtg gatagcggtt tgactcacgg ggatttccaa
     3301 gtctccaccc cattgacgtc aatgggagtt tgttttggca ccaaaatcaa cgggactttc
     3361 caaaatgtcg taataacccc gccccgttga cgcaaatggg cggtaggcgt gtacggtggg
     3421 aggtctatat aagcagagct ggtttagtga accgtcagat cactagaagc tttattgcgg
     3481 tagtttatca cagttaaatt gctaacgcag tcagtgcttc tgacacaaca gtctcgaact
     3541 taagctgcag aagttggtcg tgaggcactg ggcaggtaag tatcaaggtt acaagacagg
     3601 tttaaggaga ccaatagaaa ctgggcttgt cgagacagag aagactcttg cgtttctgat
     3661 aggcacctat tggtcttact gacatccact ttgcctttct ctccacaggt gtccactccc
     3721 agttcaatta cagctcttaa ggctagagta ttaatacgac tcactatagg gctagcgatc
     3781 gccatggagc acatacaggg agcttggaag acgatcagca atggttttgg attcaaagat
     3841 gccgtgtttg atggctccag ctgcatctct cctacaatag ttcagcagtt tggctatcag
     3901 cgccgggcat cagatgatgg caaactcaca gatccttcta agacaagcaa cactatccgt
     3961 gttttcttgc cgaacaagca aagaacagtg gtcaatgtgc gaaatggaat gagcttgcat
     4021 gactgcctta tgaaagcact caaggtgagg ggcctgcaac cagagtgctg tgcagtgttc
     4081 agacttctcc acgaacacaa aggtaaaaaa gcacgcttag attggaatac tgatgctgcg
     4141 tctttgattg gagaagaact tcaagtagat ttcctggatc atgttcccct cacaacacac
     4201 aactttgctc ggaagacgtt cctgaagctt gccttctgtg acatctgtca gaaattcctg
     4261 ctcaatggat ttcgatgtca gacttgtggc tacaaatttc atgagcactg tagcaccaaa
     4321 gtacctacta tgtgtgtgga ctggagtaac atcagacaac tcttattgtt tccaaattcc
     4381 actattggtg atagtggagt cccagcacta ccttctttga ctatgcgtcg tatgcgagag
     4441 tctgtttcca ggatgcctgt tagttctcag cacagatatt ctacacctca cgccttcacc
     4501 tttaacacct ccagtccctc atctgaaggt tccctctccc agaggcagag gtcgacatcc
     4561 acacctaatg tccacatggt cagcaccacc ctgcctgtgg acagcaggat gattgaggat
     4621 gcaattcgaa gtcacagcga atcagcctca ccttcagccc tgtccagtag ccccaacaat
     4681 ctgagcccaa caggctggtc acagccgaaa acccccgtgc cagcacaaag agagcgggca
     4741 ccagtatctg ggacccagga gaaaaacaaa attaggcctc gtggacagag agattcaagc
     4801 tattattggg aaatagaagc cagtgaagtg atgctgtcca ctcggattgg gtcaggctct
     4861 tttggaactg tttataaggg taaatggcac ggagatgttg cagtaaagat cctaaaggtt
     4921 gtcgacccaa ccccagagca attccaggcc ttcaggaatg aggtggctgt tctgcgcaaa
     4981 acacggcatg tgaacattct gcttttcatg gggtacatga caaaggacaa cctggcaatt
     5041 gtgacccagt ggtgcgaggg cagcagcctc tacaaacacc tgcatgtcca ggagaccaag
     5101 tttcagatgt tccagctaat tgacattgcc cggcagacgg ctcagggaat ggactatttg
     5161 catgcaaaga acatcatcca tagagacatg aaatccaaca atatatttct ccatgaaggc
     5221 ttaacagtga aaattggaga ttttggtttg gcaacagtaa agtcacgctg gagtggttct
     5281 cagcaggttg aacaacctac tggctctgtc ctctggatgg ccccagaggt gatccgaatg
     5341 caggataaca acccattcag tttccagtcg gatgtctact cctatggcat cgtattgtat
     5401 gaactgatga cgggggagct tccttattct cacatcaaca accgagatca gatcatcttc
     5461 atggtgggcc gaggatatgc ctccccagat cttagtaagc tatataagaa ctgccccaaa
     5521 gcaatgaaga ggctggtagc tgactgtgtg aagaaagtaa aggaagagag gcctcttttt
     5581 ccccagatcc tgtcttccat tgagctgctc caacactctc taccgaagat caaccggagc
     5641 gcttccgagc catccttgca tcgggcagcc cacactgagg atatcaatgc ttgcacgctg
     5701 accacgtccc cgaggctgcc tgtcttcgtt tctcaaggca gttcaggtgg tggcgggagc
     5761 ggaggtggag gctcgagcgg tgtgaccggc taccggctgt tcgaggagat tctgtaaggc
     5821 cgcgactcta gagtcgacct gcaggcatgc aagctgattt ggctgctgcc accgctgagc
     5881 aaggccgctt cgagcagaca tgataagata cattgatgag tttggacaaa ccacaactag
     5941 aatgcagtga aaaaaatgct ttatttgtga aatttgtgat gctattgctt tatttgtaac
     6001 cattataagc tgcaataaac aagttaacaa caacaattgc attcatttta tgtttcaggt
     6061 tcagggggag atgtgggagg tttttttaag caagtaaaac ctctacaaat gtggtaaaat
     6121 cttaattaag aattctaata taacttcgta tagcatacat tatacgaagt tatggatcca
     6181 ctgttgacaa ttaatcatcg gcatagtata tcggcatagt ataatacgac aaggtgagga
     6241 actaaaccca ggaggcagat catgattgaa caagatggat tgcacgcagg ttctccggcc
     6301 gcttgggtgg agaggctatt cggctatgac tgggcacaac agacaatcgg ctgctctgat
     6361 gccgccgtgt tccggctgtc agcgcagggg cgcccggttc tttttgtcaa gaccgacctg
     6421 tccggtgccc tgaatgaact gcaggacgag gcagcgcggc tatcgtggct ggccacgacg
     6481 ggcgttcctt gcgcagctgt gctcgacgtt gtcactgaag cgggaaggga ctggctgcta
     6541 ttgggcgaag tgccggggca ggatctcctg tcatctcacc ttgctcctgc cgagaaagta
     6601 tccatcatgg ctgatgcaat gcggcggctg catacgcttg atccggctac ctgcccattc
     6661 gaccaccaag cgaaacatcg catcgagcga gcacgtactc ggatggaagc cggtcttgtc
     6721 gatcaggatg atctggacga agagcatcag gggctcgcgc cagccgaact gttcgccagg
     6781 ctcaaggcgc gcatgcccga cggcgaggat ctcgtcgtga cccatggcga tgcctgcttg
     6841 ccgaatatca tggtggaaaa tggccgcttt tctggattca tcgactgtgg ccggctgggt
     6901 gtggcggacc gctatcagga catagcgttg gctacccgtg atattgctga agagcttggc
     6961 ggcgaatggg ctgaccgctt cctcgtgctt tacggtatcg ccgctcccga ttcgcagcgc
     7021 atcgccttct atcgccttct tgacgagttc ttctgattcg aaatgaccga ccaagcgacg
     7081 cccaaccggt atcagctcac tcaaaggcgg taatacggtt atccacagaa tcaggggata
     7141 acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg
     7201 cgttgctggc gtttttccat aggctccgcc cccctgacga gcatcacaaa aatcgacgct
     7261 caagtcagag gtggcgaaac ccgacaggac tataaagata ccaggcgttt ccccctggaa
     7321 gctccctcgt gcgctctcct gttccgaccc tgccgcttac cggatacctg tccgcctttc
     7381 tcccttcggg aagcgtggcg ctttctcata gctcacgctg taggtatctc agttcggtgt
     7441 aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg
     7501 ccttatccgg taactatcgt cttgagtcca acccggtaag acacgactta tcgccactgg
     7561 cagcagccac tggtaacagg attagcagag cgaggtatgt aggcggtgct acagagttct
     7621 tgaagtggtg gcctaactac ggctacacta gaaggacagt atttggtatc tgcgctctgc
     7681 tgaagccagt taccttcgga aaaagagttg gtagctcttg atccggcaaa caaaccaccg
     7741 ctggtagcgg tggttttttt gtttgcaagc agcagattac gcgcagaaaa aaaggatttc
     7801 aagaagatcc tttgatcttt tctacggggt ctgacgctca gtggaacgaa aactcacgtt
     7861 aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt ttatagtccg
     7921 gaatcgtcac acaaaaaacc aacacacaga tgtaatgaaa ataaagatat tttattgcgg
     7981 ccatcgtgat ggctagagtt tagccctccc acacataacc agagggcagc aattcacgaa
     8041 tcccaactgc cgtcggctgt ccatcactgt ccttcactat ggctttgatc ccaggatgca
     8101 gatcgagaag cacctgtcgg caccgtccgc aggggctcaa gatgcccctg ttctcatttc
     8161 cgatcgcgac gatacaagtc aggttgccag ctgccgcagc agcagcagtg cccagcacca
     8221 cgagttctgc acaaggtccc ccagtaaaat gatatacatt gacaccagtg aagatgcggc
     8281 cgtcgctaga gagagctgcg ctggcgacgc tgtagtcttc agagatgggg atgctgttga
     8341 ttgtagccgt tgctctttca atgagggtgg attcttcttg agacaaaggc ttggccatgg
     8401 tggcgctagt gtcagaagaa tcgagctttt tgcaaaagcc taggcctcca aaaaagcctc
     8461 ctcactactt ctggaatagc tcagaggccg aggcggcctc ggcctctgca taaataaaaa
     8521 aaattagtca gccatggggc ggagaatggg cggaactggg cggagttagg ggcgggatgg
     8581 gcggagttag gggcgggact atggttgctg actaattgag atgcatgctt tgcatacttc
     8641 tgcctgctgg ggagcctggg gactttccac acctggttgc tgactaattg agatgcatgc
     8701 tttgcatact tctgcctgct ggggagcctg gggactttcc acaccctaac tgacacacat
     8761 tccacagctg gttctttccg cctcagaagg tacctaacca agttcctctt tcagaggtta
     8821 tttcaggcca tggtgctgcg c
//
